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Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan 1 THE GLOBAL CHICKEN: RECENT REMARKABLE EVENTS AND FUTURE THREATS AND OPPORTUNITIES Corrie Brown, DVM, PhD, DACVP Josiah Meigs Distinguished Professor University of Georgia, Athens, GA 30602, USA [email protected] ABSTRACT Chickens have the distinction of being the oldest domesticated food animal and also the most populous. The red junglefowl was domesticated in Asia, as early as 7000BC, becoming the common barnyard animal we know. Today there are at least 19 billion chickens in the world, one for every three humans. Chicken crosses many cultural boundaries with ease, it is the one meat that can be found universally in the cuisines of every nation. Intensive production has allowed chickens to expand beyond the traditional backyard bird to assume a huge role in agribusiness. Globally, there are roughly 87M metric tons of broiler meat produced annually, with 12% of that designated for export. The World Organization for Animal Health (OIE) lists 13 diseases of poultry requiring international notification and potential suspension of export capacity. Of these, two deserve special attention for their negative impacts – highly pathogenic avian influenza (HPAI) and Newcastle disease (ND). HPAI has emerged in recent years as a very serious threat to poultry production globally because of the propensity of certain strains to infect and occasionally kill humans. The ND virus continues to circulate and decimate chicken populations it infects. Sustainable solutions to these two diseases represent huge opportunities for our profession. Although much smaller in numbers overall than their commercial cousins, village chickens are the main staple of smallholders throughout the world and provide a very important part of animal source food, so essential in supplying the micronutrients needed for growth and overall health. Village chickens also form a key part of the microeconomy, as the extra income from selling eggs or meat, usually done by women, is invested back into the family. As such, village chickens represent a marvelous opportunity to enhance the livelihoods and health of the rural poor. However, these roaming chickens continue to pose a serious threat to the commercial sector in every country, as has been evidenced many times with both HPAI and ND. Resolving the conflict, these chickens present to their commercial cousins will be essential. Key Words: Trade; economy; highly pathogenic avian influenza, Newcastle disease ORIGINS OF CHICKEN Chickens have the distinction of being the oldest domesticated food animal, with the red junglefowl becoming part of the human backyard as early as 7000BC in Asia. It was probably first domesticated for cockfighting, the world’s oldest continual sport. The red junglefowl was likely crossed with what is today known as the grey junglefowl of South Asia, which gave it the yellow skin we know, and the familiar barnyard animal of today.

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Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

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THE GLOBAL CHICKEN: RECENT REMARKABLE EVENTS AND

FUTURE THREATS AND OPPORTUNITIES

Corrie Brown, DVM, PhD, DACVP

Josiah Meigs Distinguished Professor

University of Georgia, Athens, GA 30602, USA

[email protected]

ABSTRACT

Chickens have the distinction of being the oldest domesticated food animal and also the most

populous. The red junglefowl was domesticated in Asia, as early as 7000BC, becoming the common

barnyard animal we know. Today there are at least 19 billion chickens in the world, one for every

three humans. Chicken crosses many cultural boundaries with ease, it is the one meat that can be

found universally in the cuisines of every nation.

Intensive production has allowed chickens to expand beyond the traditional backyard bird to

assume a huge role in agribusiness. Globally, there are roughly 87M metric tons of broiler meat

produced annually, with 12% of that designated for export. The World Organization for Animal

Health (OIE) lists 13 diseases of poultry requiring international notification and potential suspension

of export capacity. Of these, two deserve special attention for their negative impacts – highly

pathogenic avian influenza (HPAI) and Newcastle disease (ND). HPAI has emerged in recent years as

a very serious threat to poultry production globally because of the propensity of certain strains to

infect and occasionally kill humans. The ND virus continues to circulate and decimate chicken

populations it infects. Sustainable solutions to these two diseases represent huge opportunities for

our profession.

Although much smaller in numbers overall than their commercial cousins, village chickens are the

main staple of smallholders throughout the world and provide a very important part of animal source

food, so essential in supplying the micronutrients needed for growth and overall health. Village

chickens also form a key part of the microeconomy, as the extra income from selling eggs or meat,

usually done by women, is invested back into the family. As such, village chickens represent a

marvelous opportunity to enhance the livelihoods and health of the rural poor. However, these

roaming chickens continue to pose a serious threat to the commercial sector in every country, as has

been evidenced many times with both HPAI and ND. Resolving the conflict, these chickens present to

their commercial cousins will be essential.

Key Words: Trade; economy; highly pathogenic avian influenza, Newcastle disease

ORIGINS OF CHICKEN

Chickens have the distinction of being the oldest domesticated food animal, with the red

junglefowl becoming part of the human backyard as early as 7000BC in Asia. It was probably first

domesticated for cockfighting, the world’s oldest continual sport. The red junglefowl was likely

crossed with what is today known as the grey junglefowl of South Asia, which gave it the yellow skin

we know, and the familiar barnyard animal of today.

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Chickens also win the contest for the most populous food animal. The number of chickens in the

world have tripled over the last 30 years, so that today there are almost 22 billion chickens in the

world, one for every three humans.

Poultry is the second most commonly consumed meat in the world (33% of total), following

closely after pork, which comprises 34%. And certainly poultry is found in more places than pork. In

fact, chicken is the one meat that can be found universally in the cuisines of every nation. It crosses all

cultural boundaries with ease. Virtually every country has some dish they proclaim as their own, and

that contains chicken – Southern Fried Chicken, Coq Au Vin, Chicken Parmigiana, Kung Pao Chicken,

Tandoori Chicken, Chicken Kiev, etc.

HOW CHICKEN INTENSIFICATION CAME ABOUT

How did we get chicken from the small backyard flocks, overseen predominantly by smallholders,

and constituting a minority of the global livestock species populations, to 22 billion today? In the

1950’s, farmers began to realize that they could raise a bird for meat only, rather than the dual

purpose bird that scavenges about the yard. They built houses, supplied the birds with some grain,

and then began selective breeding so that they would reach market weight at the earliest possible

moment. As a result, profits were excellent, and affordability superb, allowing both consumers and

producers to great advantage. This deliberate breeding has allowed the time to market for broilers to

drop considerably over the last 20 years, from a couple of months down to only 5 weeks.

Intensification of the poultry industry reached its maximum growth in the 1990’s and early

2000’s, as can be seen from the massive expansion in numbers, according to FAOSTAT (Fig. 1).

Much of this expansion in intensive poultry production took place in the developing world. Brazil

provides a stellar example, where a country that is considered to be an emerging market, was able to

modernize and intensify its poultry production to the point where, since 2014, Brazil is now the

leading exporter of chicken globally. This translates into considerable foreign cash flowing into the

country, helping to diversify other industries and creating overall a more robust national economy.

Most of the growth of the industry in the developing world has been done through investments

by foreign-owned companies. Historically, foreign direct investment (FDI) has often been oriented

toward food. And of all the agricultural commodities, poultry production lends itself the best to FDI.

International investors look for opportunities to invest that will provide good value for dollar given,

and poultry production in the developing world fits perfectly. There is availability of inexpensive

labor, the setup costs are attainable, and the turnover of product into the marketplace is rapid. As a

result, huge monetary flows went from the developed to the developing world, helping to expand

intensive poultry production.

Global FDI took a slight downturn with the worldwide recession in 2008, but now is picking up

again. As FDI continues to grow, it is predominantly South-South flows of funds, mostly due to

emerging markets in Asia investing in developing nations.

GLOBAL TRADE

Chickens might just serve as the “mascot” of globalization. The advent of complex

interdependence represented by increasing trade among countries has chicken at the forefront of

cross-border traffic and international efforts at development.

Intensive production has allowed chickens to expand beyond the traditional backyard bird to

assume a huge role in agribusiness. Globally, there are roughly 87M metric tons of broiler meat

produced annually, with 12% of that designated for export, totaling more than 20B USD (Fig. 2). This

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represents huge value for exporting countries but also has large risks. Disease incursions can halt

trade precipitously, greatly endangering the economic enterprise.

ECONOMIC OPPORTUNITIES AND FOOD SECURITY PROVIDED BY CHICKENS

Poultry is widely considered as the livestock of the poor. Chickens kept by smallholders are

referred to as “village” chickens, and are perfectly suited to the environment and the local economic

situation. Input resources are minimal, mostly just kitchen leftovers, but those who also raise grain

may supply the birds with small amounts of grain as well.

Fig. 1: Estimates of numbers of chickens in the

world, demonstrating and almost tripling of

populations over 30 years.

Fig. 2: Dollar value of chicken meat that is

exported from one country to another,

globally, demonstrating a ten-fold increase

over 25 years.

Globally, most rural households have some poultry. Although much smaller in numbers overall

than their commercial cousins, village chickens are the main staple of smallholders throughout the

world and provide a very important part of animal source food, so essential in supplying the

micronutrients needed for cognitive growth and overall health.

There are probably a billion people in the world who qualify as “smallholders.” Many consider

the term “smallholder” to mean those farmers that are not yet intensified, or those farmers that have

yet to move out of poverty. However, smallholders have persisted for centuries, and are likely to

persist long into the future. Within the smallholder system, the household is the main unit, and

poultry offer huge value-added for minimal input.

Village chickens form a key part of the microeconomy, as the extra income from selling eggs or

meat, usually done by women, is invested back into the family, with much of the income going to

children’s schooling or medical costs. It is estimated that 2-5% of annual household income across

subSaharan Africa is from poultry and poultry products. Also the high quality protein that chickens

and their eggs provide is invaluable for supplying the animal source food so essential in helping to

ensure adequate micronutrients for optimal health and cognitive development. As such, village

chickens represent a marvelous opportunity to enhance the livelihoods and health of the rural poor.

Most development programs aimed at decreasing rural poverty have at least a partial focus on

smallholder poultry.

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Across the world, the numbers of village chickens are only a small fraction of total chicken

populations, as they are far outnumbered, on a global scale, by their commercially-reared cousins.

However, depending on the specific country, village chickens may actually be the overwhelming

majority. This is generally true for most of the low-income food-deficit countries. In Nigeria, for

instance, village chickens are thought to constitute 77% of the national flock. And in Ethiopia, Uganda,

and Malawi, more than 80% of the chickens raised in these countries are “village” rather than

“commercial.”

Unfortunately, there are often considerable losses in the village birds. Predators claim many and

illnesses constantly threaten. Diseases are particularly hard to control, as biosecurity is really not

feasible for these roaming scavengers. As a result, village chickens continue to pose a serious threat

to the commercial sector in every country, as has been evidenced many times with both highly

pathogenic avian influenza (HPAI) and Newcastle disease (ND). Resolving the conflict these chickens

present to their commercial cousins will be essential.

DISEASES THREATENING THE INDUSTRIES

Robust and continuing trade in animals and animal products is dependent on freedom from

disease. The World Organization for Animal Health (OIE) lists 13 diseases of poultry requiring

international notification and potential suspension of export capacity. Of these, two deserve special

attention for their negative impacts – HPAI and ND. HPAI has emerged in recent years as a very

serious threat to poultry production globally because of the propensity of certain strains to infect and

occasionally kill humans. The ND virus continues to circulate and decimate chicken populations it

infects. Sustainable solutions to these two diseases represent huge opportunities for our profession.

These two diseases are covered in more detail below.

In addition, there are emerging diseases which cause consternation and can temporarily interrupt

commerce.

Avian leucosis (subgroup J) virus (ALV-J) arose in the late 1990’s, creating myeloid neoplasms.

Unfortunately some breeding companies had their genetic stock contaminated with ALV-J, resulting in

the disease being exported to more than 50 countries. There were major losses of broiler breeders

worldwide.

Fowl adenovirus serotype 4, the cause of hydropericardium syndrome, was first described in

Pakistan in the 1990’s and has now spread to many parts of the world. There can be heavy losses,

with a sudden onset of mortality up to 80%. In addition to causing illness and death, the virus is

known to be immunosuppressive because of its damaging effects on lymphoid tissues.

The highly pathogenic avian influenza global pandemic (H5N1) that began in 2003 gave great

insight into economic problems engendered as a result of a trade-limiting and/or public health-threat

disease. Direct losses were especially high in Southeast Asian countries. It is estimated that the

outbreaks in 2003-2004 resulted in losses of 17% of the total bird populations in Vietnam,

representing 44M birds, and 14.5% in Thailand, or 29M birds. Thailand lost its position as the 5th

largest global exporter of poultry, as, during the first quarter of 2004, poultry exports fell 75%.

Similar decreases were seen in Hong Kong and China. There were also great losses in other parts of

the economy as well, for instance, tourism dropped, as travelers became wary of journeying to

locales where there might be affected birds. Although research has shown that infection of humans

requires very close contact with affected birds, often multiple birds, nevertheless, the 40% mortality

rate in humans associated with infection has made many public health officials very cautious. This

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virus continues to circulate, greatly hampering trade and creating economic hardships as well as

straining public health resources in affected countries.

Costs and threats regarding the smallholders as a result of H5N1 were less rigorously quantified.

As mandatory slaughter was instituted in many areas, it had a marked negative impact on the

smallholder. It wiped out the contribution of poultry to the family income as well as to food security.

Sale value was markedly reduced. For instance, in Vietnam, a country hit hard by H5N1,

confinement/eradication method resulted in a 10-25% decrease in the poorest families’ annual

incomes.

The H5N1 experience also showed the world how the character and shape of the outbreak

curves can differ according to the relative proportions of village vs. commercial chickens. In East and

Southeast Asia, the Middle East, and Africa, there were high casualty rates in the poultry industry,

extensive spread, and prolonged outbreaks, largely due to the presence of outdoor birds that kept

the outbreaks percolating. Western European countries such as Germany and France also have some

outdoor birds, but not typical smallholder operations, more like niche markets, and they were able to

ban outdoor poultry production, which greatly abbreviated their outbreak experiences. So, it was

apparent that controlling the outbreaks required monitoring among the outdoor birds, a very

challenging endeavor in resource-poor countries.

Another strain of HPAI, H5N2, entered the US in December 2014, with migratory birds infecting

backyard poultry kept in the Pacific Northwest. The virus subsequently spread to the Midwest and

decimated poultry production in some states. As a result, during the first half of 2015, US exports

dropped by 14%, as various trading partners placed sanctions on American-origin poultry and poultry

products. The value of the US poultry industry fell by $390M. A total of 49M birds succumbed to the

disease or were euthanized in the face of spread. This caused increased prices in chicken and very

notably eggs, which doubled in price during the first few months of the outbreak. Thanksgiving

turkeys, a large November-based market in the US, are predicted to be offered at extremely high

prices, as a result of the major turkey producers losing much of their stock during the H5N2

outbreaks over the summer.

Newcastle disease, an avian paramyxovirus, has a global distribution. There are only very few

areas of the world that can claim freedom from the disease, and even these areas have periodic

incursions. This disease may be responsible for more morbidity and mortality, and failure of

industries, and also enhanced food insecurity, than any other disease of chickens.

When Newcastle disease enters a house of chickens, the morbidity rate is close to 100% and the

mortality can be as much as 90%.

Recent outbreaks in countries with advanced animal health infrastructure demonstrate how an

incursion can be extremely costly. An outbreak in the US in 2002-2003 took the lives of three

million birds with estimated industry losses of $5B. Regionalization of the country, according to OIE

guidelines, saved the national chicken industry from total implosion, as the disease remained confined

to the most western part of the US, allowing the chicken production on East and Midwest to

continue relatively unimpaired.

Finding hard data about the impact of ND on village chickens is more challenging. It is estimated

to be the main cause of loss of chickens throughout Africa. Where data exist, for instance, in Chad,

annual losses to smallholder farmers from ND are estimated at 65-100% annually. Unfortunately the

current vaccines for ND may protect against severe disease but overwhelming challenge exposure

will still result in birds becoming ill and dying. Also, vaccinated birds, when infected, still shed the

field virus in their feces when infected, and so the virus can continue to spread. A question often

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asked is about the prevalence of ND. It is known that, in contrast to HPAI, ND is more likely to be

in the region constantly, not just in an outbreak form. A serologic study of unvaccinated birds

demonstrates that the virus is probably constantly or intermittently present throughout the year in

areas.

CONCLUSIONS

In summary, poultry has become the most global of all livestock commodities, with tremendously

burgeoning numbers of commercial birds which undergo a significant amount of international

movement. At the same time, poultry remains the stronghold of the smallholder, supplying much

needed boosts to food security, nutrition, and household economies. Disease agents don’t care

which sector – smallholder or commercial – they infect and the resulting damage to both can be

severe. Ensuring that both sectors are maintained in a sustainable and profitable way is essential.

REFERENCES

Adler J and A Lawler, 2012. How the chicken conquered the world. Smithsonian, 43: 40-47.

Antipas BB, K Bidjeh and ML Youssouf, 2012. Epidemiology of Newcastle disease and its economic

impact in Chad. Euro J Experim Biol, 2: 2286-2292.

Asthana M, R Chandra and R Kumar, 2013. Hydropericardium syndrome: current state and future

developments. Archive Virology 158: 921-931. doi: 10.1007/s00705-012-1570x

Bagust TJ, 2013. Emerging pathogens of poultry diseases. Poult Develop Rev, FAO, 101-102.

Birol E, D Asare-Marfo, G Ayele, A Mensah-Bonsu, L Ndirangu et al., 2010. The impact of avian flu on

livelihood outcomes in Africa: evidence from Ethiopia, Ghana, Kenya and Nigeria. Afr J Agric Res

Econ, 8: 275-288.

Otte J, D Roland-Holst and D Pfeiffer, 2006. HPAI control measures and household incomes in

Vietnam. Pro-Poor Livestock Policy Initiative, www.fao/ag/pplpi.html

Rushton J, R Viscarra, E Guerne Bleich and A McLeod, 2005. Impact of avian influenza outbreaks in

the poultry sectors of five South East Asian countries (Cambodia, Indonesia, Lao PDR, Thailand,

Viet Nam) outbreak costs, responses and potential long term control. World’s Poult Sci J, 61:

491-514. doi: 10.1079/WPS200570.

Serrão E, J Meers, R Pym, R Copland, D Eagle et al., 2012. Prevalence and incidence of Newcastle

disease and prevalence of avian influenza infection of scavenging village chickens in Timor-Lesté.

Prev Vet Med, 104: 301-308.

Sonaiya EB, 2007. Family poultry, food security and the impact of HPAI. World’s Poult Sci J, 63:132-

138. doi: 10.1079/WPS2006135

Taha FA, 2007. How Highly Pathogenic Avian Influenza (H5N1) Has Affected World Poultry-Meat

Trade. A Report from the Economic Research Service, LDP-M-159-02.

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VECTOR VACCINES FOR POULTRY: THEIR ADVANTAGES AND LIMITATIONS

COMPARED TO CLASSICAL VACCINES

Michel Bublot

Merial S.A.S., 254 rue Marcel Mérieux 69007 Lyon, France

ABSTRACT

Vector vaccines have been developed and licensed for poultry since the mid-1990s. Avian vector

vaccines are bivalent vaccines since the vector is itself a vaccine strain. The current avian licensed

vector vaccines are based on 3 types of vectors: fowlpox, Newcastle disease (ND) virus and

herpesvirus of turkey (HVT), the latter type being the most widely used. The most successful is an

HVT-IBD that confers protection against both Marek’s disease and infectious bursal disease (IBD). It

combines major advantages over conventional IBD vaccines including safety, efficacy and a single

hatchery administration. In contrast to vector vaccines against other diseases, the efficacy of HVT-

IBD outperformed that of classical IBD vaccines, including the immune complex IBD vaccines. HVT-

ND vaccines are safe and induce a long duration of protection but a slow onset and a poor local

immunity. The use of live ND vaccine is still required in complementation of HVT-ND vaccines.

HVT-ILT vaccines are bringing safety to ILT vaccines, but their efficacy is clearly lower than the

classical live CEO vaccines. HVT-AI induces a broad level of protection but a slow onset of immunity.

These HVT-based vectors are currently not fully compatible and poultry veterinarians need to know

how each type of vaccine is working, their performances and limitations in order to set up a

vaccination program including both vector and classical vaccines in line with the field situation.

Key Words: Vector vaccine, modified-live vaccine, immune-complex vaccine, IBD, ND, ILT

Introduction

The quality and performances of vaccines has improved a lot since the pioneered work of

Edward Jenner and Louis Pasteur in the 18th and 19th century, respectively. Industrial production of

classical vaccines based on modified-live or inactivated pathogen agents initiated in the second half of

the 20th century. In 1973, the cloning a foreign DNA fragment into a bacteria plasmid using restriction

enzyme was the start of the rapidly evolving genetic engineering area which led to the modern

biotechnology. Thirteen year later (1986), the first “biotech vaccine” was licensed against human

hepatitis B; it was a subunit vaccine produced in yeast and it replaced the first generation vaccine

made from plasma of infected patients. In the veterinary field, a vaccinia-vectored vaccine was first

licensed in 1994 as baits to vaccinate wildlife against rabies (Brochier et al., 1991). Since then, many

new biotech vaccines have been developed, especially for veterinary applications (Meeusen et al.,

2007). In poultry, viral vector vaccine technology is the most successful technology applied for

development of new vaccines. This minireview will focus on the description of currently licensed

vector vaccines for poultry. Their advantages and limitations compared to classical vaccines will be

discussed.

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1. The mechanisms of action of vector vaccines

A vector vaccine is a vaccine that utilizes one organism (the vector) as a carrier to generate

protection against a second organism. Since the pioneer work on the vaccinia vector, numerous

studies have shown the potential of vector vaccines in veterinary medicine (Brun et al., 2008). Avian

vector vaccines are often bivalent vaccines since the vector is usually itself a vaccine strain. There are

2 major components in vector vaccines: the vector and the foreign sequence inserted into its

genome. This inserted sequence will allow expression of a “protective gene” of the targeted pathogen

agent during in vivo replication of the vector. The resulting “protective protein” will then induce an

immune response against this agent that will protect the vaccinated host from subsequent challenge

(Fig. 1). The immune response includes humoral, cellular as well as mucosal immunity if the vector

replicates in mucosal tissues. The inserted sequence also contains sequences that will drive optimal

expression of the protective gene such as promoter and poly-adenylation signal. The protective

gene(s) of most viruses are known but those from bacteria or parasites remain to be identified. This

is one of the reasons why vector vaccines usually target viral and not bacterial or parasitic diseases.

Fig. 1: Schematic representation of the mechanisms of action of the HVT-IBD vector vaccine as an

example of bivalent vector vaccine protecting against both infectious bursal disease (IBD) and Marek’s

disease (MD). The vector vaccine infects the target cells of the vaccinated host soon after vaccination.

It then starts its replication cycle during which it expresses the foreign protective protein (in this

example of HVT-IBD, the IBDV VP2 protein). This foreign protein as well as the vector proteins will

induce an immune response that will protect the vaccinated chicken against both IBD and MD. Vector

virus-infected cells will also release new vector virus progeny that will infect new target cells. These

successive replication cycles will further increase foreign protective protein and vector production,

and consequently, the immune response against both IBD and MD.

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The choice of the vector is critical since it will have an impact on many vaccine properties

including the route and the timing of vaccine administration, the persistence of the vaccine and

protective gene expression, the efficacy of the vaccine in presence of maternally-derived antibodies

(MDA), the dose of the vaccine, the type of immunity induced by the vaccine, the possibility to

associate the vector vaccine with other vaccines, and the manufacturing and stability of vaccine. Large

DNA viruses such as poxvirus and herpesvirus are vectors of choice since their genome can be easily

manipulated and they can afford insertion of relatively large foreign sequences that are stably

integrated into their genome. In mammals, non-replicative vectors such as the canarypox have been

developed (Poulet et al., 2007), but in avian, all licensed vector vaccines are currently based on

replicative vectors. Replicative vectors allow using a lower dose and only one administration.

Fowlpox (FP) and herpesvirus of turkey (HVT) are the most common vectors used in poultry but

Newcastle disease virus (NDV) has also been recently developed. The viral vector vaccines may

contain a certain amount of the protective protein either in or outside its viral structure, but it is

usually considered that it is the de novo expression of the protective gene during in vivo vector

replication that will induce most of the protective immune response and not the protective protein

physically present in the vaccine.

2. Fowlpox-based vector vaccines

Licensed fowlpox (FP)-based vector vaccines have been developed against Newcastle disease

(ND), avian influenza (AI), infectious laryngotracheitis (ILT) and Mycoplasma gallisepticum (Mg). They

are bivalent vaccines since they also protect against fowlpox disease. The FP-NDV was the first

vector vaccine to be licensed in poultry in 1994 (Taylor et al., 1990) in the USA followed by the FP-AI

(H5 subtype) (Swayne et al., 1997). The latter (TROVAC®-AIV H5*) expresses the hemagglutinin

(HA) gene from a 1983 highly pathogenic (HP) Irish H5N8 isolate and it has been used mainly in

Mexico infection since 1998 to protect chickens against low pathogenic (LP) H5N2. This vaccine can

be administered by the sub-cutaneous route to one-day-old chicks at the hatchery at the same time

as Marek’s disease vaccine. It was shown to induce a broad immunity against multiple HP and LP AI of

H5 subtypes, including H5N1 isolates that emerged in the early 2000s (Bublot et al., 2006, Bublot et

al., 2010). The immune response is directed against the HA only, and detection of infection in

vaccinated birds can then easily be performed using serologic tests detecting the antibody response

against other AI proteins such as the nucleoprotein; it is therefore compatible with a DIVA

(differentiating infected from vaccinated animals) strategy. Since their emergences, these H5N1 (and

H5-based reassortants with other neuraminidase (NA) subtypes) isolates evolved antigenically and

this fowlpox-based vaccine progressively lost its ability to protect well against these new isolates.

Interestingly, the FP-AI vaccine induces a strong priming response, which can be boosted by the

administration of an inactivated AI vaccine. This heterologous prime-boost vaccination scheme was

found to induce a broad protection even against HPAI H5N1 antigenic-drift variants such as those

which emerged in Indonesia in 2007 and in Egypt in 2008, against which both the FP-AI and

heterologous inactivated vaccines alone were poorly protective (Swayne et al., 2015). The recent

study of anti-vector and anti-insert MDA interferences on AI immunogenicity and efficacy of the FP-

AI and inactivated vaccines indicated that the anti-vector (anti-FP) MDA had limited effects on FP-AI

immunogenicity; the anti-insert (anti-AI) MDA had the highest negative impact on the inactivated

vaccine but it also interfered with FP-AI. However, the FP-AI priming in birds with AI MDA could

clearly overcome the strong MDA interference on inactivated vaccine (Richard-Mazet et al., 2014).

This prime-boost regimen has been applied in the field in Mexico and in Egypt. It was also shown to

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be immunogenic in ducks, a species in which the fowlpox is not thought to replicate (Steensels et al.,

2009, Bublot et al., 2010). A FP-AI was also developed in China in 2005 to control HPAI H5N1

infections (Chen and Bu, 2009).

The efficacy levels induced by the FP-ILT and FP-Mg against ILT (Johnson et al., 2010, Vagnozzi et

al., 2012) and Mg challenge (Ferguson-Noel et al., 2012), respectively, were found to be relatively low

compared to classical vaccines. This poor performance may be due to the lack of induction of

mucosal immunity induced by the fowlpox vector, and, for Mg, to the poor protective ability of the

Mg gene inserted into the fowlpox vector.

3. Herpes of turkey-based vector vaccine

The HVT vector has advantages over FP: It is suitable and safe for both in ovo and subcutaneous

hatchery administration and to provide a life-long immunity. The long duration of immunity is likely

the result of persistence of the HVT vector in the vaccinated chickens: it is thought that the

protective gene inserted into the HVT vector is expressed either continuously during latency or

frequently during regular episodes of reactivation. This vector can also hasten the maturation of the

chicken embryo immune response when administered in ovo (Gimeno et al., 2015). The generation of

the first HVT vector vaccine was reported in 1992 in a NDV model (Morgan et al., 1992), but the first

licensed HVT-based vector was an infectious bursal disease vaccine (HVT-IBD designated

VAXXITEK® HVT+IBD*) that was launched in Brazil in 2006 and that became the most used vector

vaccine; more than 65 billion birds were vaccinated so far in at least 75 countries worldwide (Darteil

et al., 1995, Bublot et al., 2007, Le Gros et al., 2009). This live vector vaccine expresses the IBDV gene

coding for VP2, the external capsid protein of IBDV. It combines major advantages over modified-live

(MLV) or immune complex (ICx) conventional IBD vaccines including safety, efficacy and a single

hatchery administration. It is the only live IBD vaccine that does not induce bursal lesions and

therefore, has no immunosuppressive effect. The IBD MLV contain either attenuated intermediate (I)

or less attenuated intermediate plus (I+) IBDV strain. They are administered in the drinking water

either once or twice when mean MDA levels reached the level low enough so that they can replicate.

This level is lower for I-based MLV but for both types of MLV, it is below the level at which very

virulent or variant IBD strains replicate (Fig. 2A). The immune complex (ICx) vaccine is administered

at the hatchery but the I+ IBDV strain present in this vaccine will not replicate until MDA reach a

certain level (Jeurissen et al., 1998), which is also below the level that inhibits wild type IBDV

replication (Fig. 2B). There is therefore a period of time at which the birds are sensitive to wild type

IBDV infection and are not yet protected by these MLV or ICx vaccines; this period is called the

“immunity gap” (Fig. 2). In contrast to these conventional vaccines, the HVT-vectored IBD vaccine

starts to replicate soon after hatchery administration, even in birds with high levels of passive anti-

IBDV MDAs. The early HVT vector replication and IBDV VP2 gene expression allow the early

induction of active IBDV antibody titers that will progressively compensate the decrease of passive

MDA, the overall antibody levels remaining above the protective threshold (Fig. 2C) (Prandini et al.,

2008). The vector vaccine technology is therefore the only one that can solve the issues of safety

and immunity gap observed with MLV and ICx IBD vaccines. The HVT-IBD vector vaccine as well as

the ICx allowed to move the IBD vaccination from the farm to the hatchery where vaccination could

be performed in better conditions. The convenience and success of hatchery vaccination has

increased the demand for hatchery vaccines with long duration of immunity against other diseases.

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Fig. 2A

Fig. 2B

Fig. 2: Comparison of immune response induced by three types of IBDV vaccines: (A) intermediate

(I) or intermediate plus (I+) MLV vaccine administered by drinking water, (B) immune complex (ICx)

vaccine containing an I+ MLV administered at the hatchery, and (C) HVT-IBD vector vaccine

administered at the hatchery. Immunity gap will be observed with the I, I+ and ICx vaccines but not

with the HVT-IBD vector vaccine. Other abbreviations used in this figure: Ab antibody, MDA

maternally-derived antibodies, wt wild type, vvIBDV very virulent infectious bursal disease virus.

HVT-vectored ND vaccines were licensed soon after HVT-ND. The NDV gene inserted into the

vector is the gene coding for the fusion (F) protein, which plays an important role in NDV entry.

Surprisingly, this vaccine was shown to induce HI antibody titers which usually target the

hemagglutinin-neuraminidase (HN) NDV protein. The anti-F antibodies likely prevent the contact

between NDV HN hemagglutination sites and red blood cells by steric hindrance (Palya et al., 2012).

These HVT-ND vaccines do not induce respiratory reactions. The onset of immunity induced by

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HVT-ND vaccine was delayed compared to NDV MLV and the efficacy level in birds with MDAs was

lower than in SPF chickens (Morgan et al., 1993). However, the duration of immunity induced by

HVT-ND vaccine was live long (up to 72 week-of-age in layers) (Palya et al., 2014). The HVT-ND has

a systemic replication which does not induce a good mucosal immunity and the protection against

tracheal replication induced after intra-ocular NDV challenge was found to be low (Morgan et al.,

1992). Live ND vaccines need therefore to be used in addition of HVT-ND to compensate their slow

onset of immunity and their poor local immunity induction. The ND killed vaccine applied before the

laying period was also found to boost the antibody titers and to better reduce the NDV shedding

after challenge (Palya et al., 2014).

The licensed HVT-ILT vaccines contain either the glycoprotein D and glycoprotein I (Johnson et

al., 2010) or a truncated form of glycoprotein B (Esaki et al., 2013). These HVT-ILT vaccines were

shown to be safer than the conventional chicken embryo (CEO) or tissue culture (TCO) MLV

vaccines. However, their efficacy level was lower than that induced by the conventional vaccines

(Johnson et al., 2010, Vagnozzi et al., 2012). As for HVT-ND, their onset of immunity and local

protection against ILT replication in the trachea were relatively weaker than MLV vaccine in

conventional chickens (Johnson et al., 2010, Vagnozzi et al., 2012, Esaki et al., 2013).

An HVT-avian influenza (AI) vaccine has also recently been licensed. It contains the hemagglutinin

gene from a highly pathogenic AI (HPAI) H5N1 strain isolated in Hungary in 2006 (Rauw et al., 2011).

As for FP-AI, this HVT-AI vaccine is compatible with the DIVA strategy. It was shown to induce a

good level of clinical protection (from 60 to 100%) against a wide panel of H5 strains including

antigenic variants in SPF and/or in chickens with MDA (Rauw et al., 2011, Soejoedono et al., 2012,

Kilany et al., 2014, Kapczynski et al., 2015, Kilany et al., 2015). Both HI antibody titers and cell-

mediated immunity contributed to protection (Kapczynski et al., 2015). AI MDA may delay the onset

of immunity but do not seem to impact the duration of immunity (Rauw, 2015). The boost with an

AI inactivated vaccine could further increase the protection and antibody levels and decrease viral

load in oropharyngeal swabs after challenge (Rauw et al., 2012, Soejoedono et al., 2012, Kilany et al.,

2015).

HVT vector vaccines do not spread from vaccinated chickens to non-vaccinated ones. It is

therefore very important to keep the cold chain during transportation, to store and prepare the

vaccine using the standard operating procedures and to assure correct administration of the vaccine

to all chicks. Any missed birds at vaccination will not have a second chance to get this vaccine and

will not be protected. This is a key factor of success for the application of this type of vaccine.

Another important aspect to consider is the health status of the vaccinated chicks. Indeed, a

functional immune system is required to get optimal immunity from vector vaccines. Early infection

with immunosuppressive viruses such as chicken infectious anemia virus will likely impaired vaccine

potency and it is therefore very important to control the early infection and vertical transmission in

young chicks by vaccinating the breeders against these agents.

HVT vector vaccines are fully compatible with Marek’s disease serotype 1 (CVI988) or serotype

2 (SB-1) vaccines. In contrast, combination of an HVT vector vaccine with parental HVT or with

another HVT vector vaccine usually leads to the decrease of protection against at least one of the

target diseases. The mechanism of this interference is not known but it may be due, at least in part,

to the competitive replication and infection of target cells. Interference between some HVT vector

pairs may be more pronounced than others. In particular, the association of the HVT-AI with an

HVT-ND has been reported to have minor negative impact on AI and ND immunogenicity: it delayed

the onset of immune response against AI and ND (Rauw, 2015). At the present time, no HVT vector

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has a claim of compatibility with another HVT vector. Additional research is needed to evaluate if

the interference observed between specific HVT vector pairs is sufficiently low to be acceptable in

some field epidemiological situations.

4. Newcastle disease virus-based vector vaccine

NDV vector vaccines (Ge et al., 2007) have been used against H5 HPAI in China since 2006

(Chen and Bu, 2009) and against H5N2 LPAI in Mexico since 2008 (Sarfati-Mizrahi et al., 2010). They

are bivalent, protecting against both ND and AI and are compatible with the DIVA strategy. The AI

HA expressed by the NDV vector was shown to be present at the surface of the NDV (Veits et al.,

2006). One of their major advantages is that they are administered by mucosal route either by eye

drop or by mass administration using spray or drinking water. The mucosal administration and

mucosal replication of this vector allows the induction of a local immunity, and therefore, such vector

may be ideal for respiratory diseases. Although most chickens are vaccinated with NDV MLV by

spray at the hatchery, all but one (Lardinois et al., 2012) NDV-AI vaccination publications reported

vaccination of 1 to 3 week-old chickens. Data comparing immunogenicity of NDV-AI when

administered at 1 day-of-age or at 1 week-of-age or later are still missing to evaluate if the chicken’s

adaptive immune system is mature enough at hatch to provide optimal AI immune response using this

type of vector. Anti-NDV and anti-AI MDA were shown to interfere on NDV-AI immunogenicity but

at variable levels (Sarfati-Mizrahi et al., 2010, Faulkner et al., 2013, Lambrecht et al., 2015).

Interestingly, anti-NDV MDA had positive impact on AI protection whereas anti-AI MDA decreased

AI protection (Lambrecht et al., 2015). NDV-AI was also found to be immunogenic in ducks and a

FP-AI priming at 1 or 2 day of age followed by a NDV-AI boost 15 days later fully protect SPF

Muscovy ducks 12 weeks later (Niqueux et al., 2013) indicating that the prime-boost vaccination

scheme performed with two different vector vaccines may be very efficient and still compatible with

the DIVA strategy. Encouraging data have also been recently published on the use of the NDV vector

as a vaccine for ILT (Kanabagatte Basavarajappa et al., 2014, Zhao et al., 2014) but these candidates

have not been licensed so far.

5. Future challenges on the development of new vector vaccines

The development of vector vaccines against avian diseases during last two decades has been a

success story, especially the HVT vector has significantly changed the industry. The integrated

poultry industry has clearly seen an advantage to use potent vaccines at the hatchery and to decrease

vaccine administrations at the farm. The challenge today is to develop polyvalent vector vaccines that

can be administered concomitantly at the hatchery without interference. One of the possible

solutions is to insert the protective gene(s) from different pathogen agents into one vector. Inserting

several genes into one vector may potentially lead to genetic instability and to decreased efficacy

compared to vector with single insert. The development of a double HVT-ND+ILT has recently been

reported (Morsey et al., 2015). Another strategy could be the use of other vectors that do not

interfere with the existing ones. One potential example could be the generation of the CVI988

(Rispens) MDV serotype 1 strain as a vector as previously described (Sakaguchi et al., 1998). In

addition, there would clearly be a benefit to create other vectors that are able to induce a strong

mucosal immune response and compatible with existing vaccines.

Another challenge is the induction of a broad protection against pathogenic agents that show a

great antigenic variability, such as avian influenza. One of the possible solutions is to modify the

inserted gene sequence so that it better matches that of circulating strains such as described in (Giles

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and Ross, 2011); other solutions for antigen design leading to an increase of the broadness of

protection are also being evaluated (Schussek et al., 2014). MDA interference on vector vaccines is

also an issue; additional studies should be done to better understand the mechanisms of interference

and to design ways to minimize its impact. As described above, the prime-boost strategy using either

two different vector vaccines or one vector and one classical vaccine have shown promising results in

terms of broadening the immune response and of overcoming MDA interference (Niqueux et al.,

2013, Richard-Mazet et al., 2014, Schussek et al., 2014).

There are also many pathogen agents including viruses such as the infectious bronchitis virus,

bacteria and parasites for which the vector vaccine technology has been disappointing. This is likely

due to the unavailability or poor protective ability of the protective gene identified for these agents.

Vaccinomics technologies should facilitate the finding and the design of better protective genes in the

future (Schussek et al., 2014).

Conclusion

The vector vaccine technology, and in particular the HVT vector, has changed ways to control

some of the major poultry diseases. The HVT-IBD vaccine is the only vaccine that can combine

hatchery administration, absence of vaccine-induced bursal lesions and its consequent

immunosuppression, and excellent IBD protection levels in birds with MDA without any immunity

gap. This technology has pushed back vaccine administration from the field to the hatchery where

vaccination can be better controlled. HVT-ND, HVT-AI and HVT-ILT are providing excellent safety

and duration of protection after one hatchery administration, but they do not provide a rapid onset

of protection, nor a good mucosal immunity. Optimal protection may need concomitant or booster

vaccination with MLV and/or inactivated vaccines. Fowlpox and NDV vectored vaccines have also

shown interesting features, which when combined with other vaccines may clearly induce broader

immunity. Future research on vector vaccines will need to be focused on solving the current

problems observed with the use of theses vaccines and in particular, their compatibility and their

induction of a strong mucosal immune response, a rapid onset and a broad protection in chicks with

high levels of MDA. The solution may come from the development of newly designed vector vaccines

but it may also include the combination of biotech and conventional vaccines. The challenge for the

poultry veterinarian will be to know how each vaccine works in order to design the optimal

vaccination program that will give protection linked to the epidemiologic area.

Acknowledgement

I thank Marcus Remmers for the critical reading of the manuscript. *TROVAC and *VAXXITEK

are registered trademarks of Merial in the USA and elsewhere.

References

Brochier, B, MP Kieny, F Costy, P Coppens, B Bauduin, et al., 1991. Large-scale eradication of rabies

using recombinant vaccinia-rabies vaccine. Nature, 354: 520-522.

Brun, A, E Albina, T Barret, DA Chapman, M Czub, et al., 2008. Antigen delivery systems for

veterinary vaccine development. Viral-vector based delivery systems. Vaccine, 26: 6508-6528.

Bublot, M, RJ Manvell, W Shell and IH Brown, 2010. High level of protection induced by two fowlpox

vector vaccines against a highly pathogenic avian influenza H5N1 challenge in specific-pathogen-

free chickens. Avian Dis, 54: 257-261.

Page 15: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

15

Bublot, M, N Pritchard, FX Le Gros and S Goutebroze, 2007. Use of a vectored vaccine against

infectious bursal disease of chickens in the face of high-titred maternally derived antibody. J

Comp Pathol, 137 Suppl 1: S81-84.

Bublot, M, N Pritchard, DE Swayne, P Selleck, K Karaca, et al., 2006. Development and use of fowlpox

vectored vaccines for avian influenza. Ann N Y Acad Sci, 1081: 193-201.

Bublot, M, A Richard-Mazet, S Chanavat-Bizzini, FX Le Gros, M Duboeuf, et al., 2010. Immunogenicity

of poxvirus vector avian influenza vaccines in Muscovy and Pekin ducks. Avian Dis, 54: 232-238.

Chen, H and Z Bu, 2009. Development and application of avian influenza vaccines in China. Curr Top

Microbiol Immunol, 333: 153-162.

Darteil, R, M Bublot, E Laplace, JF Bouquet, JC Audonnet, et al., 1995. Herpesvirus of turkey

recombinant viruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce

protection against an IBDV virulent challenge in chickens. Virology, 211: 481-490.

Esaki, M, L Noland, T Eddins, A Godoy, S Saeki, et al., 2013. Safety and efficacy of a turkey herpesvirus

vector laryngotracheitis vaccine for chickens. Avian Dis, 57: 192-198.

Faulkner, OB, C Estevez, Q Yu and DL Suarez, 2013. Passive antibody transfer in chickens to model

maternal antibody after avian influenza vaccination. Vet Immunol Immunopathol, 152: 341-347.

Ferguson-Noel, N, K Cookson, VA Laibinis and SH Kleven, 2012. The efficacy of three commercial

Mycoplasma gallisepticum vaccines in laying hens. Avian Dis, 56: 272-275.

Ge, J, G Deng, Z Wen, G Tian, Y Wang, et al., 2007. Newcastle disease virus-based live attenuated

vaccine completely protects chickens and mice from lethal challenge of homologous and

heterologous H5N1 avian influenza viruses. J Virol, 81: 150-158.

Giles, BM and TM Ross, 2011. A computationally optimized broadly reactive antigen (COBRA) based

H5N1 VLP vaccine elicits broadly reactive antibodies in mice and ferrets. Vaccine, 29: 3043-3054.

Gimeno, IM, NM Faiz, AL Cortes, T Barbosa, T Villalobos, et al., 2015. In Ovo Vaccination with

Turkey Herpesvirus Hastens Maturation of Chicken Embryo Immune Responses in Specific-

Pathogen-Free Chickens. Avian Dis, 59: 375-383.

Jeurissen, SH, EM Janse, PR Lehrbach, EE Haddad, A Avakian, et al., 1998. The working mechanism of

an immune complex vaccine that protects chickens against infectious bursal disease. Immunology,

95: 494-500.

Johnson, DI, A Vagnozzi, F Dorea, SM Riblet, A Mundt, et al., 2010. Protection against infectious

laryngotracheitis by in ovo vaccination with commercially available viral vector recombinant

vaccines. Avian Dis, 54: 1251-1259.

Kanabagatte Basavarajappa, M, S Kumar, SK Khattar, GT Gebreluul, A Paldurai, et al., 2014. A

recombinant Newcastle disease virus (NDV) expressing infectious laryngotracheitis virus (ILTV)

surface glycoprotein D protects against highly virulent ILTV and NDV challenges in chickens.

Vaccine, 32: 3555-3563.

Kapczynski, DR, M Esaki, KM Dorsey, H Jiang, M Jackwood, et al., 2015. Vaccine protection of

chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT

vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian

influenza virus. Vaccine, 33: 1197-1205.

Kilany, W, G Dauphin, A Selim, A Tripodi, M Samy, et al., 2014. Protection conferred by recombinant

turkey herpesvirus avian influenza (rHVT-H5) vaccine in the rearing period in two commercial

layer chicken breeds in Egypt. Avian Pathol, 43: 514-523.

Kilany, WH, MK Hassan, M Safwat, S Mohammed, A Selim, et al., 2015. Comparison of the

effectiveness of rHVT-H5, inactivated H5 and rHVT-H5 with inactivated H5 prime/boost

Page 16: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

16

vaccination regimes in commercial broiler chickens carrying MDAs against HPAI H5N1 clade

2.2.1 virus. Avian Pathol, 44: 333-341.

Lambrecht, B, A Lardinois, O vandersleyen, M Steensels, N Desloges, et al., 2015. Stronger

interference of Avian Influenza than Newcastle Disease Virus specific maternal derived antibodies

with a recombinant NDV-H5 vaccine. Avian Dis, In press.

Lardinois, A, M Steensels, B Lambrecht, N Desloges, M Rahaus, et al., 2012. Potency of a recombinant

NDV-H5 vaccine against various HPAI H5N1 virus challenges in SPF chickens. Avian Dis, 56: 928-

936.

Le Gros, FX, A Dancer, C Giacomini, L Pizzoni, M Bublot, et al., 2009. Field efficacy trial of a novel

HVT-IBD vector vaccine for 1-day-old broilers. Vaccine, 27: 592-596.

Meeusen, NT, J Walker, A Peters, PP Pastoret and G Jungersen, 2007. Current status of veterinary

vaccines. Clinical Microbiology Reviews, 20: 489-510.

Morgan, RW, J Gelb, Jr., CR Pope and PJ Sondermeijer, 1993. Efficacy in chickens of a herpesvirus of

turkeys recombinant vaccine containing the fusion gene of Newcastle disease virus: onset of

protection and effect of maternal antibodies. Avian Dis, 37: 1032-1040.

Morgan, RW, J Gelb, Jr., CS Schreurs, D Lutticken, JK Rosenberger, et al., 1992. Protection of

chickens from Newcastle and Marek's diseases with a recombinant herpesvirus of turkeys vaccine

expressing the Newcastle disease virus fusion protein. Avian Dis, 36: 858-870.

Morsey, M, L Gergen, S Cook, B Ledesma, W Cress, et al., 2015. Double recombinant HVT-based

vaccines for simultaneous protection against Newcastle disease, Marek’s disease and Infectious

Laryngeotracheitis. XIXth World Veterinary Poultry Association Congress, Capetown, South

Africa.

Niqueux, E, O Guionie, M Amelot and V Jestin, 2013. Prime-boost vaccination with recombinant H5-

fowlpox and Newcastle disease virus vectors affords lasting protection in SPF Muscovy ducks

against highly pathogenic H5N1 influenza virus. Vaccine, 31: 4121-4128.

Palya, V, I Kiss, T Tatar-Kis, T Mato, B Felfoldi, et al., 2012. Advancement in vaccination against

Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces

challenge virus shedding with the absence of vaccine reactions. Avian Dis, 56: 282-287.

Palya, V, T Tatar-Kis, T Mato, B Felfoldi, E Kovacs, et al., 2014. Onset and long-term duration of

immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in

commercial layers. Vet Immunol Immunopathol, 158: 105-115.

Poulet, H, J Minke, MC Pardo, V Juillard, B Nordgren, et al., 2007. Development and registration of

recombinant veterinary vaccines. The example of the canarypox vector platform. Vaccine, 25:

5606-5612.

Prandini, F, M Bublot, FX Le Gros, A Dancer, L Pizzoni, et al., 2008. Assessment of the immune

response in broilers and pullets using two ELISA kits after in ovo or day-old vaccination with a

vectored HVT + IBD vaccine (VAXXITEK® HVT+IBD). Zootecnica International, Sept2008: 40-

50.

Rauw, F, 2015. Evaluation of the compatibility between rHVT-F and rHVT-H5 ND and AI vaccines

regarding immunogenicity and efficacy when administrated simultaneously to day-old chickens.

Interference with MDA. 19th World Veterinary Pourlty Association Congress Capetown, South

Africa.

Rauw, F, V Palya, Y Gardin, T Tatar-Kis, KM Dorsey, et al., 2012. Efficacy of rHVT-AI vector vaccine

in broilers with passive immunity against challenge with two antigenically divergent Egyptian clade

2.2.1 HPAI H5N1 strains. Avian Dis, 56: 913-922.

Page 17: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

17

Rauw, F, V Palya, S Van Borm, S Welby, T Tatar-Kis, et al., 2011. Further evidence of antigenic drift

and protective efficacy afforded by a recombinant HVT-H5 vaccine against challenge with two

antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains. Vaccine, 29: 2590-2600.

Richard-Mazet, A, S Goutebroze, FX Le Gros, DE Swayne and M Bublot, 2014. Immunogenicity and

efficacy of fowlpox-vectored and inactivated avian influenza vaccines alone or in a prime-boost

schedule in chickens with maternal antibodies. Vet Res, 45: 107.

Sakaguchi, M, H Nakamura, K Sonoda, H Okamura, K Yokogawa, et al., 1998. Protection of chickens

with or without maternal antibodies against both Marek's and Newcastle diseases by one-time

vaccination with recombinant vaccine of Marek's disease virus type 1. Vaccine, 16: 472-479.

Sarfati-Mizrahi, D, B Lozano-Dubernard, E Soto-Priante, F Castro-Peralta, R Flores-Castro, et al.,

2010. Protective dose of a recombinant Newcastle disease LaSota-avian influenza virus H5

vaccine against H5N2 highly pathogenic avian influenza virus and velogenic viscerotropic

Newcastle disease virus in broilers with high maternal antibody levels. Avian Dis, 54: 239-241.

Schussek, S, A Trieu and DL Doolan, 2014. Genome- and proteome-wide screening strategies for

antigen discovery and immunogen design. Biotechnol Adv, 32: 403-414.

Soejoedono, RD, S Murtini, V Palya, B Felfoldi, T Mato, et al., 2012. Efficacy of a recombinant HVT-H5

vaccine against challenge with two genetically divergent Indonesian HPAI H5N1 strains. Avian

Dis, 56: 923-927.

Steensels, M, M Bublot, S Van Borm, J De Vriese, B Lambrecht, et al., 2009. Prime-boost vaccination

with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin

ducks challenged with Asian H5N1 HPAI. Vaccine, 27: 646-654.

Swayne, DE, JR Beck and TR Mickle, 1997. Efficacy of recombinant fowl poxvirus vaccine in protecting

chickens against a highly pathogenic Mexican-origin H5N2 avian influenza virus. Avian Dis, 41:

910-922.

Swayne, DE, DL Suarez, E Spackman, S Jadhao, G Dauphin, et al., 2015. Antibody titer has positive

predictive value for vaccine protection against challenge with natural antigenic-drift variants of

H5N1 high-pathogenicity avian influenza viruses from Indonesia. J Virol, 89: 3746-3762.

Taylor, J, C Edbauer, A Rey-Senelonge, JF Bouquet, E Norton, et al., 1990. Newcastle disease virus

fusion protein expressed in a fowlpox virus recombinant confers protection in chickens. J Virol,

64: 1441-1450.

Vagnozzi, A, G Zavala, SM Riblet, A Mundt and M Garcia, 2012. Protection induced by commercially

available live-attenuated and recombinant viral vector vaccines against infectious laryngotracheitis

virus in broiler chickens. Avian Pathol, 41: 21-31.

Veits, J, D Wiesner, W Fuchs, B Hoffmann, H Granzow, et al., 2006. Newcastle disease virus

expressing H5 hemagglutinin gene protects chickens against Newcastle disease and avian

influenza. Proc Natl Acad Sci U S A, 103: 8197-8202.

Zhao, W, S Spatz, Z Zhang, G Wen, M Garcia, et al., 2014. Newcastle disease virus (NDV)

recombinants expressing infectious laryngotracheitis virus (ILTV) glycoproteins gB and gD

protect chickens against ILTV and NDV challenges. J Virol, 88: 8397-8406.

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KEY ASPECTS TO BE CONSIDERED ON THE MONITORING AND TESTING OF

FLOCKS VACCINATED WITH RHVT-ND

Roberto Soares, DVM, MAM, ACPV

Ceva Animal Health, Malaysia

ABSTRACT

Newcastle disease virus (NDV) causes illness in many avian species and typically manifests in

respiratory and gastrointestinal or nervous system (or both) symptoms. The most severe form of

Newcastle disease (ND) can result in mortality rates exceeding 90% in susceptible chicken flocks.

Different strategies have been used to control ND, but vaccination is by far the most popular

approach. Numerous conventional live and inactivated ND vaccines have been used for many decades

in routine vaccination protocols in the poultry industry. Nevertheless, this condition still causes many

problems around the world, especially due to interference of maternally derived antibody (MDA) and

problems with vaccine application in the field. ND vaccines can also cause post-vaccination reaction

and interference with Infectious Bronchitis vaccines. Such drawbacks with conventional vaccines led

to the development of live viral vector vaccines, also known as recombinant vaccines. A particular

success story has been the development of a recombinant vaccine using as a vector the Turkey

Herpesvirus (HVT) to express the Newcastle disease virus fusion protein. The HVT possess a large

DNA where foreign gens, such as F gen from NDV, can be safely inserted into nonessential region of

the HVT genome along with an appropriate promoter, without disturbing the its infectivity. The

recombinant HVT ND (rHVT ND) has been demonstrating strong protective efficacy to different

NDV genotypes in many chicken producers around the world. This vaccine provide protection after

replication of HVT virus, which stimulation immune response neutralizing antibodies to virus F

protein, one of the major functional glycoprotein on the NDV surface and by stimulating the cellular

immune response. The assessment rHVT ND vaccine replication and take is done through molecular

technique (RT-PCR) and immune response (HI and ELISA). After the introduction of a rHVT ND

vaccine into the market, we have been monitoring the onset and duration of immunity to NDV in

many flocks under different vaccination protocol in field and laboratory conditions. Data will be

presented and discussed showing key aspects to be considered on monitoring and testing Broilers

and Layers flocks vaccinated with rHVT ND.

Key Words: Newcastle Disease virus, recombinant HVT vaccines, monitoring

Introduction

Newcastle disease virus (NDV; avian paramyxovirus serotype I) causes severe clinical disease in

many avian species, which typically is manifested in respiratory and/or gastrointestinal and/or

neurological clinical signs and lesions. The most severe forms of Newcastle disease (ND) can result in

mortality rates exceeding 90% in susceptible poultry flocks.

Different strategies have been used to control ND, but sanitary policy, biosecurity and

vaccination are by far the most popular approaches. Numerous conventional live and inactivated

(killed) ND vaccines have been used for many decades in routine vaccination protocols for broilers

and long living birds. Nevertheless, the disease still causes huge losses around the world. These

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conventional vaccines very often fail especially due to the interference of maternally derived

antibodies (MDA) and improper vaccine application in the field. ND vaccines can also cause post-

vaccination reaction and interference with Infectious Bronchitis vaccines. Such drawbacks with

conventional vaccines led to the development of safer live viral vector vaccines, also known as

recombinant vaccines. A particular success story has been the development of a recombinant vaccine

using the Turkey Herpesvirus (HVT) as a vector in which the F protein gene (fusion protein) of NDV

has been inserted. The rHVT-F has demonstrated to be highly efficacious in protecting against strong

challenges with different NDV genotypes. Additionally, this type of vaccine proved to be safe (Morgan

et al., 1992), less sensitive to MDA (Morgan et al., 1993) and induces life-long immunity (Reddy et al.,

1996, Palya et al., 2014). Due to that, rHVT-F vaccine has been rapidly introduced into the Broiler,

Breeder, and Layer vaccination programs worldwide. Although serology has been widely used to

assess the “take’ of classical ND vaccines (Live attenuated and inactivated), with the introduction of

new generation of ND vaccine using recombinant technology, sometimes questions arise regarding

how to monitor the “take” of this type of vaccine. The aim of this article is to share key aspects that

should be considered for monitoring of broiler and layer flocks vaccinated with rHVT-F.

Monitoring rHVT ND vaccine

Following its injection, either by in-ovo or subcutaneously on the first day of age, rHVT-F vaccine

replicates in the host cells, inducing progressive but strong humoral and cellular immune responses

against the F protein expressed on cell surface. The monitoring of the rHVT-F vaccine can be

accomplished by detection of rHVT-F vaccine virus using PCR and/or by seroconversion using HI.

Detection of rHVT-F vaccine virus

After inoculation, MD vaccine viruses, including HVT, can be recovered by PCR from different

tissues. The Spleen and feather pulp (FP) samples have been wide used to detect MD virus replication

pattern and vaccine take (Fabricant et al., 1982; Baigent and Davison, 1999; Baigent et al., 2005). Since

rHVT-F vaccine is a HVT based recombinant vaccine, its replication follows the same pattern as

ordinary HVT (Reddy et al., 1996). Therefore, spleen and FP can be used to confirm vaccination in

flocks that received rHVT-F vaccine.

We recently evaluated the take of rHVT-F in spleen and feather samples from 4 weeks old

Broiler flocks (unpublished data). In this trial we were able to recover rHVT-F from 100% of spleen

sample, however only 69.4% of FP samples were positive. Due to the low recovering of rHVT-F

vaccine from FP, spleen has been selected as preferential sample for monitoring rHVT-F vaccine

“take”.

The assessment of vaccine “take” in spleen samples from commercial Layers after rHVT-F

vaccination was done by using HVT specific real-time PCR (Palya et al., 2014). The rHVT-F was

detected in 70% to 100% of spleen samples between 2 and 4 weeks post-vaccination. Moreover, the

rate of positivity increased as the birds get older reaching 100% between 4 and 6 weeks post-

vaccination. After week 4, the expression of the F gene was evidenced in most of the birds until 74

weeks, which was the end of test period. These results confirmed the life-long persistence of the

virus in vaccinated birds and thereby ensuring strong and long lasting immunity to ND through the

continuous antigenic stimulation.

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Monitoring Antibody Immune Response to rHVT-F

As it was abovementioned, the F glycoprotein expressed by rHVT-F vaccine has been successfully

protecting chickens against virulent NDV challenge. The F protein elicits neutralizing antibodies,

which are capable to prevent NDV cell penetration and cell-to-cell spreading. This immune response

can be measured by hemagglutination inhibition (HI) and by ELISA.

Palya et al. (2012), evaluated the onset of immunity in Broilers vaccinated with rHVT-F vaccine by

in ovo and subcutaneous inoculation at hatch. The antibody immune response was assessed by HI and

by ELISA (BioCheck). The HI test results clearly detected specific antibody response at 4-6 weeks of

age, reaching a HI titer close to 5 log2 at 6 weeks. ELISA test was able to show some antibody

response when compared to unvaccinated group but, in most of the cases, the measured ELISA

values remained negative (below the positive threshold given for the kit by the manufacturer).

In other trial, this time with commercial Layers vaccinated with rHVT-F alone or in combination

with live attenuated ND vaccine, (Palya et al., 2014), detection of onset and duration of antibody

response was assessed using HI test and ELISA. The results showed onset of antibody response

clearly detectable by HI test at 4 and 6 weeks in the pullets vaccinated with the combination but not

in the pullets vaccinated by the rHVT-F alone. ELISA clearly detected antibody response in the pullets

vaccinated with the combination at 6 weeks, but not at 4 weeks. The antibody response induce by a

single vaccination at hatch, whatever the program, could be detected for 74 weeks with both assays.

Conclusion

The recombinant rHVT-F vaccine expressing the F protein applied in the hatchery induces

homogenous and solid immunity against MD and ND by stimulating cellular and humoral immune

responses. However, many external factors may interfere with the efficacy of this vaccine such as

inappropriate storage, poor preparation and/or administration of the vaccine. Therefore, it is very

important to monitor vaccine take and seroconversion following vaccine application in the hatchery.

The real-time PCR has demonstrated to be a sensitive mean to assess the replication of rHVT-F in

the spleen of Broilers and Layers at around 4 weeks of age. Antibody response could be measured

by HI and ELISA test, however, HI test showed to be more sensitive than ELISA to detect early

seroconversion.

References

Baigent S, V Nair and R Currie, 2006. Real-time quantitative PCR for Marek’s disease vaccine virus in

feather samples: applications and opportunities. Dev Biol (Basel), 126: 271-281.

Baigent SJ, LJ Petherbridge, K Howes, LP Smith, RJ Currie et al., 2005. Absolute quantitation of

Marek’s disease virus genome copy number in chicken feather and lymphocyte samples using

real-time PCR. J Virol Methods, 123: 53-64.

Baigent SJ, LP Smith, RJ Currie and VK Nair, 2005. Replication kinetics of Marek’s disease vaccine

virus in feathers and lymphoid tissues using PCR and virus isolation. J Gen Virol, 86: 2989-2998. Fabricant J, BW Calnek and KA Schat, 1982. The early pathogenesis of turkey herpesvirus infection in

chickens and turkeys. Avian Dis, 26: 257-264.

Morgan RW, J Gelb Jr, CR Pope and PJ Sondermeijer, 1993. Efficacy in chickens of a herpesvirus of

turkeys recombinant vaccine containing the fusion gene of Newcastle disease virus: onset of

protection and effect of maternal antibodies. Avian Dis, 37: 1032-1040.

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Morgan RW, J Gelb Jr, CS Schreurs, D Lutticken, JK Rosenberger et al., 1982. Protection of chickens

from Newcastle and Marek’s diseases with a recombinant herpesvirus of turkeys vaccine

expressing the Newcastle disease virus fusion protein. Avian Dis, 36: 858-870.

Palya V, I Kiss, T Tata´r-Kis, T Mato, B. Felföldi et al., 2012. Advancement in vaccination against

Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces

challenge virus shedding with the absence of vaccine reactions. Avian Dis, 56: 282-287.

Palya V, T Tata´r-Kis, T Mato, B Felföldi and Y Gardin, 2014. Onset and long-term duration of

immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in

commercial layers. Vet Immunol Immunop, 158: 105-115.

Rauw F, Y Gardin, V Palya, S Anbari, S Lemaire et al., 2010. Improved vaccination against Newcastle

disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old.

Vaccine, 28: 823-833.

Reddy SK, JM Sharma, J Ahmad, DN Reddy, JK McMillen et al., 1996. Protective efficacy of a

recombinant herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek’s diseases

in specific-pathogen-free chickens. Vaccine, 14: 469-477.

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NEW CASTLE DISEASE: ITS IMPACT AND CONTROL IN PAKISTAN

Hanif Nazir Chaudhary

Bio-POUL International,

Defence Housing Authority, Lahore Cantt, Pakistan

ABSTRACT

Newcastle Disease is one of the major disease threat to all types and ages of the commercial

poultry as well as back yard game and wild birds in Pakistan. In the past we did not have many choices

to protect the birds from but now the focus is on proper diagnosis. The protection do not start from

vaccination. It starts with good bio-security vaccination and then most importantly regular titre

monitoring. Since couple of years problem is seen commonly in long living birds like breeders and

layers where the titer monitoring is there but a better interpretation need more attention. If the

mean titer looks fine and there are birds falling below protection threshold those birds get problem

and not only suffer mortality but also production losses. In majority of cases the mortality continues

because of egg peritonitis and at the same time the chick quality suffers as well. It’s important to

regularly monitor titers day 1,25 and slaughter age in broilers to achieve Log 2 titers above 5 in

broilers at day 25 while as in long live birds all birds every 4 week monitoring should be above 7 log 2

HI titers and if that's not the case an immediate decision of re vaccination becomes important. Not

doing so flock gets hit by ND and variable losses can occur.

INTRODUCTION

Not a new disease but from last year’s last years the disease has shown its new phase never seen

before. It caused unprecedented mortalities in broilers and production losses occurred both in layers

and breeders.

In the beginning like always there was debate on the confirmation but soon everyone agreed that its

nothing else but ND. In this regard PPA disease control committee got involved with field scientists

as well as public sector research organizations along with leading private sector diagnostic labs. There

30 weeks

old

breeder

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were series of meetings particularly in Lahore and Islamabad the most affected areas. The research

scientists presented their experimental work and lab trials confirming that existing vaccines do

protect this challenge of ND which is not the same in field conditions. Getting information from

international scientists through their experience from similar situation from other countries was part

of the National Disease Control Committee particularly that of regional countries of Asia Pacific it

was realized that we need to focus on.

Monitoring antibody titers

It was concluded that if the problem have to be protected well then the titers at any stage should

not go lower than 5 which is a high asking rate for broilers but became possible by incorporating

killed vaccines along with just only using live vaccines in broilers.

This gave a boost in titers and not only protected the vaccinated flocks but also started

education in infection load by not allowing disease virus to get passages to get more pathogenicity.

Today when you are reading these lines the disease has come at large under control except may be

some rare sporadic cases.

Courtesy Dr Kamal

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Keeping the titers high

Since the disease has reduced but we must not forget that ND is an endemic problem which may

erupt once it finds favorable conditions. One of the favorable conditions may be that we forget our

objective of keeping titers high and start compromising on low titers by reducing vaccines to cut the

cost. Cutting cost can be more attractive when market of the broiler is down and broiler flocks are

sold at breakeven or even lower than production cost.

Profit and loss is part of the business and very important to look at but nothing is more costly

when disease comes and losses go out of control. We must keep vaccination programs aiming high

titers by both live and killed vaccines.

In long live birds layers and breeders its important to monitor antibody titers at every 4 weeks

frequency and make sure lowest titers of any sample should not be below Log 2 at 8. Companies

have multiple flocks so can keep a track of past flocks and a baseline can be developed against existing

vaccination program.

Immunosuppression

This used to be less attentive problem in the past but now became more important because

birds cannot built good titers if Gumboro subclinical or clinical affect the flock and damage immune

organs. Science is progressing and thanks to the new inventions. We were handicapped in the past to

choose from live vaccines which were mild, intermediate, Intermediate plus and even hot vaccines.

We were having support of killed vaccines but it remained always a debate to choose from safety

RANGE AVG RANGE AVG RANGE AVG RANGE AVG RANGE AVG

1 1st day 4 to 7 5.9

2 4th 5 to 12 10.06 7 to 11 9.5 6 to 11 7.92 543-2266 1199 5 to 9 7.83

3 8th 5 to 12 10.07 10 to11 10.93 7 to 11 10.4 8 to 10 8.79 8 to 9 8.75

4 13th 7 t0 11 10.47 11 to 11 11 9 to 11 10.77 8 to 10 9 9 to 10 9.5

5 17th 9 t0 11 10.67 8 to 11 10.5 8 to 11 10.7 7 to 9 8.16

6 21st 8 to 11 10.2 8 to 11 10.37 7 to 11 10.5 8 to 10 9.09

7 26th 8 to 11 10.07 9 to 11 10.08 7 to 10 9.07 4to7 (9-11)5.9 (10.25)

8 30th 7 t0 11 9.47 8 to 11 9.92 10 to 11 10.83 7 to 9 8.58

9 34th 8 to 11 9.73 7 to 11 9.17 10 to 11 10.8 8 to 9 8.66

10 39th 6 to 10 7.53 6 to 10 8.1 10 to 11 10.9

11 43rd 6 to 10 7.67 9 to 11 10.67 10 to 11 10.8

12 47th 10 t0 11 10.8 9 to 11 10.57 10 to 11 10.8

13 52nd 9 t0 11 10.6 9 to 11 10.67 10 to 11 10.67

14 56th 9 to 11 10.7 9 to 11 10.17 10 to 11 10.73

15 60th 10 t0 11 10.87 9 to 11 10.67 10 to 11 10.8

16 65th 10 t0 11 11 9 to 11 10.7 10 to 11 10.8

17 69th 8 to 11 10.6 10 to 11 10.67 10 to 11 10.9

18 73rd 10 to 11 10.9 10 to 11 10.63

19 77+

Flock 3 Flock 4

Culled Culled

ND Titers monitoring profileFlock 5S.NO,

AGE

WEEKS Flock 1 Flock 2

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efficacy dilemma. If that was settled then cost issue was popping up strongly along with vaccines

handling issue.

With the vector vaccines now available in Pakistan most of the practical issues have been

addressed and this timely availability of this vaccine helped controlling this havoc of ND by proper

attention towards immunosuppression.

Points to note

We often get confused with the interpretation of titers which is not an easy subject to

understand. Protection is not only linked with the level of antibodies you measure with tests like HI

tests. The nature has given birds and animals multiple layers of protection just like the army that the

formation of troops is deployed in several layers of defense. The titers in SPF chicks coming from SPF

parents respond different to vaccines while as the commercial chicks coming from vaccinated parents

behave altogether different. Titers of different ages have different meanings.

The birds receiving live and killed vaccines at day old show higher titers after 30 days Challenge

of ND at 1,5,10,15.20,30,40,50 days of age. Live and killed vaccination combination at day old gave full

protection at day 20. Protection against ND challenge remained high like SPF chicks trial in the group

which got live and killed ND vaccine at day old.

Conclusion

Bio-security is fundamental prerequisite to control all diseases where present time ND is no

exception. I am not going into lengthy debates and just focus on vaccination. A minimum of 2-3 live

vaccines and 1-2 killed vaccines are important to protect this ND we have been suffering from recent

past along with Vector Gumboro vaccine to protect immunosuppression due to Gumboro.

Its worth mentioning that all companies do not produce same quality of vaccines. Definitely its

our duty to find out who is the best and not just who is cheap. During the outbreak time while

analyzing the facts we often found use of poor source vaccines. The unfortunate part of this disease

season was everything was sold by everyone on the name of ND as one of the reason was top

companies ran out of stock and farmer found shelter under a source could not save him. Being a

present day farmers it’s our duty to arrange vaccine 1st and then go for chicks.

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RESPIRATORY DISEASES OF POULTRY WITH SPECIAL ATTENTION

TO AVIAN INFLUENZA

Hafez M. Hafez1* and El-Sayed M. Abdelwhab2

1Institute of Poultry Diseases, Free University Berlin, Germany; 2Institute of Molecular Virology and

Cell Biology, Federal Research Institute for Animal Health, Friedrich-Loeffler-Institut, Germany

*Corresponding Author: [email protected]

ABSTRACT

Respiratory diseases of poultry are associated with severe economic losses, due to high

mortality, high medication cost, drop in egg production in layer and breeder flocks and in many

cases low fertility and hatchability. Several pathogens are incriminated as possible cause either

alone or in synergy with different other micro-organisms or accompanied with non-infectious

factors such as climatic conditions and management related problems.

Worldwide the emerging and re-emerging respiratory diseases and/or infections of poultry are:

Infectious Bronchitis, Infectious Laryngotracheitis, Avian Metapneumovirus, Ornithobacterium

rhinotracheale and Fowl cholera infections. In addition, Avian Influenza, Newcastle disease and

Mycoplasma infections appear to cause problem in some countries.

Influenza A viruses are members of the Orthomyxoviridae family. At present 18 H subtypes

and 9 N subtypes are known. Currently, only the viruses of H5 and H7 subtype have been shown

to be highly pathogenic for poultry, but not all H5 and H7 viruses are highly pathogenic. However,

it has been proven that highly pathogenic avian influenza viruses emerge in domest ic poultry from

low pathogenic progenitors. Since December 2003, epidemic influenza has devastated the poultry

industry. From 2014 till now about 35 countries reported Influenza outbreaks tens of millions of

birds were culled or died from the disease. In addition, infection with subtype H9N2 accompanied

with high economic losses affected the poultry in several countries.

The final diagnosis of respiratory infections can only be reached by isolation and identification

of the causative agent and /or by detection of DNA or RNA using PCR. In addition, serological

examinations for detection of antibodies can be also carried out.

Disease prevention and control focuses primarily on: prevent the introduction and spread of

infectious diseases. This includes biosecurity as well as Eradication policy such as in case of HPAI

and sometimes by industry in case of vertically transmitted infections. Vaccination is regarded as

one of the most beneficial control measure. However, vaccinal breaks can be observed in some

vaccinated flocks due to incomplete immunization coverage, or vaccine failure, but were also

associated with immune escape mutants from the current vaccine strains. Antimicrobials are

important and essential tools to control bacterial infectious diseases. Generally, therapy or

vaccination alone is of little value, unless they are accompanied with improvements in all aspects of

management and biosecurity.

Key Words: Influenza, Hygiene, Vaccination, Therapies

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Introduction

Respiratory diseases of poultry remain of major economic and public health importance. Many

pathogenic microorganisms are present to a limited degree under most management conditions. If

conditions favourable for multiplication of the specific pathogen exist, an active disease outbreak

may occur in apparently healthy flocks. The severity and course of any respiratory disease is

influenced by virulence of the agent, immune status of the birds and management.

Respiratory diseases of poultry are associated with severe economic losses, due to high

mortality, high medication cost, drop in egg production in layer and breeder flocks and in many

cases low fertility and hatchability. In breeder flocks attention must be paid to prevent infections

with vertically transmitted agents. Early recognition and monitoring programmes are essential in

managing the infections and minimizing the economic impacts. Many of these diseases once

introduced into a geographic area, can explode into an epidemic and may have a significant negative

effect on the national and international trade.

Several pathogens are incriminated as a possible cause either alone (mono-causal) or in

synergy with different other micro-organisms (multi-causal) or accompanied by non-infectious

factors such as climatic conditions and management related problems (Table 1).

Table 1: Some possible cause of respiratory disease in poultry

Non infectious Infectious

Management Viral agents

Litter quality IB, ILT, ND, Influenza A, aMPV,

Stocking density PMV3, Pox

Ventilation rate Bacterial agents

Temperature ORT, P. multocida, Mycoplasma, E. coli

High ammonia level Chlamydia, Haemophilus, Bordetella

High dust concentration Streptococci, Staphylococci

Feed Mycotic agents

High dust content Aspergillus fumigatus

Vitamin A deficiency Parasites

Syngamus, Cryptosporidium

These infectious agents can be introduced and spread in poultry farms by different routes. It

occurs either by the vertical and/or horizontal route. At early days of age, the main disease problems

are related to vertically transmitted infections such as Mycoplasma, salmonella, E. coli or improper

hatchery management. Those and other infectious agents can also be transmitted horizontally

(laterally) by direct contact between infected and non-infected susceptible birds, and through indirect

contact with contaminated feed, water, equipment, environment and dust through ingestion or

inhalation. The severity of clinical signs, duration of the disease and mortality are extremely variable

and are influenced by type, virulence and the pathogenicity of the infectious agent, immune status and

age of the birds as well as by many environmental factors such as poor management, inadequate

ventilation, high stocking density, poor litter conditions, poor hygiene, high ammonia level, concurrent

diseases and the type of secondary infections.

The diagnosis of the disease complexes usually is not a straightforward business. Basically the

diagnosis consists of case history as well as management and environmental investigation on spot. In

addition, clinical investigations and post-mortem examination are an important step toward disease

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diagnosis. However, clinical signs and necropsies are mostly not the final step of the diagnosis. The

final diagnosis can be reached by laboratory diagnosis. Presumptive diagnosis of infections therefore

must be confirmed by isolation and identification of the causative agent. Further possibilities are the

detection of DNA or RNA using PCR. Serological examinations for detection of antibodies can be

carried out. In general, however, many factors such as governmental regulation, goal of examinations,

cost benefit analysis, equipment facilities, availability of reagents and experiences of the staff are

influenced and to some instance limit the choice of the laboratory methods (Hafez and Hess, 1999).

The emerging and re-emerging respiratory diseases and or infections of poultry mostly include

Infectious Bronchitis (IB), Infectious Laryngotracheitis (ILT), Avian Metapneumovirus (aMPV),

Ornithobacterium rhinotracheale (ORT) and Fowl cholera (FC) infections. In addition, Avian Influenza

(AI), Newcastle disease (ND) and Mycoplasma infections appear to cause poultry health problems in

some countries.

Prevention and control of respiratory diseases focused primarily on dedicated planning and sound

management practices, which prevent the introduction and spread of infectious diseases. This

includes managing the environment by supplying adequate ventilation and heat to maintain bird

comfort, to keep the litter in good condition, insure supplying fresh feed and water of diseases with

good quality, and limiting exposure to infectious agents through biosecurity, cleaning and disinfection.

Eradication policy and killing of infected flocks and/or contact flocks by legislations in cases of

suspicion or confirmed outbreaks with a considerable public health and/or economic impacts such as

in case of highly pathogenic avian influenza (HPAI) and ND in the EU as well as in case Salmonella

Enteritidis or Salmonella Typhimurium in breeder flocks (Hafez, 2005). In addition, sometimes by

industry in case of vertically transmitted infections such as mycoplasma in breeder flocks. In all case

knowledge about micro-organism, sensitivity to physical and chemical agents, mode of transmission

and method of isolation and /or detection are essential.

Vaccination is regarded as one of the most beneficial biopharmaceutical interventions, due to

its ability to induce protection against infectious diseases through activation of the immune system.

However several considerations should be taken in account before using vaccines such as:

governmental regulations, epidemiological situation in the area and /or on the farm, goal of

vaccination, availability of the vaccine and cost benefit analysis. Progressive vaccine production

technologies such as recombinant, subunit, reverse genetic and nucleic acid vaccines can significantly

reduce the cost of vaccines, ensure better efficacy and allow easy and rapid intervention to face the

steady mutation of the microorganisms. Furthermore, the development of efficient vaccines against

bacterial infections will lead to a reduction of the use of antibiotics and subsequently of the

development of resistant bacteria.

Antimicrobials are important and essential tools to control bacterial infectious diseases, if the

above mentioned measures did not prevent the infection in aim to insure health of the flock and to

enhance the welfare and to reduce the economic losses. In such cases, therapy should be considered

as the last effective weapon, but treatment without accurate diagnosis, critical selection of the

product, accurate dosage, adequate duration and monitoring treatment is unacceptable. In addition, if

necessary as in case of treatment failure, corrective action should be taken.

In the space available, it is not possible to review extensively the entire field of respiratory

diseases. Instead, this paper is limited to Avian influenza.

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Avian Influenza

Avian influenza (AI) is a highly contagious disease of many kinds of poultry, wild and cage birds. It

is characterised by marked variations in morbidity, mortality, signs and lesions. In addition, the

infection causes periodically epidemics in humans, horses, pigs, seals, whales, and variety of birds. AI

viruses are members of the Orthomyxoviridae family. Within the family there are four types of

influenza: A, B, C and D. Types B and C affect only humans and type D in cattle. Type A virus, the

only known to infect birds. The RNA virus is enveloped, heat labile, sensitive to ether, chloroform

and different chemical disinfectants (Swayne et al., 2013).

The AI viruses are RNA negative-sense, single-stranded, enveloped viruses contain genomes

composed of eight separate segments encode for at least 11 viral proteins. Avian influenza A viruses

are divided into subtypes on the base of the antigenic relationships of the surface glycoproteins

haemagglutinin (HA) and neuraminidase (NA). The haemagglutinin and neuraminidase are repectivelly

important in the attachment and release of the virus from the host cells (Palese and Shaw, 2007). To

date, 18 H and 11 N subtypes of avian influenza viruses (AIV) have been detected. All AIV subtypes

except H17N10 (Tong et al., 2012) and H18N11 (Tong et al., 2013), which recently detected in bats,

are known to infect birds.

Based on their pathogenicity for poultry, AI viruses are divided into low pathogenic (LPAIV)

resulting in mild or asymptomatic infections and highly pathogenic (HPAIV) causing up to 100%

morbidity and mortality. Accorrding ot the European Union Council directive (EC, 2005), Highly

pathogenic avian influenza (HPAI): means an infection of poultry or other captive birds caused by: (a)

avian influenza viruses of the subtypes H5 or H7 with genome sequences codifying for multiple basic

amino acids at the cleavage site of the haemagglutinin molecule similar to that observed for other

HPAI viruses, indicating that the haemagglutinin molecule can be cleaved by a host ubiquitous

protease; or (b) avian influenza viruses with an intravenous pathogenicity index in six-week old

chickens greater than 1.2. low pathogenic avian influenza (LPAI): means an infection of poultry or

other captive birds caused by avian influenza viruses of any subtype including H5 or H7 that do not

come within the above mentioned definition of HPAI.

Generally only few strains of H5 or H7 subtypes fulfilled the defined criteria of high pathogenicity

which potentially evolve from low virulent precursors (Lupiani and Reddy, 2009). However, HPAIVs

are responsible for magnificent economic losses in poultry industry and pose a serious threat to

public health (Webster et al., 1992; Peiris et al., 2007).

Constant genetic and antigenic variation of AIV is an important feature for continuous evolution

of the virus (Brown, 2000). Gradual antigenic changes due to acquisition of point mutations known as

“antigenic drift” are commonly regarded to be the driving mechanism for influenza virus epidemics

from one year to the next. However, possible “antigenic shift or reassortment” of influenza virus

occurs by exchange genes from different subtypes is relatively infrequent, however it results in severe

pandemics (Ferguson et al., 2003).

There is little or no evidence of vertical transmission (egg-borne infection). However, eggshell

surfaces can be contaminated with the virus. The disease transmitted horizontally by direct contact

with infected birds or indirectly through contaminated equipments. In addition, the infection can

easily be spread by people (contaminated shoes, clothes), crates egg flats, and egg cases vehicles. Wild

and domesticated waterfowl are the major natural reservoir of influenza viruses. Representatives of

all of the different subtypes of influenza A viruses have been isolated from birds, particularly from

aquatic species such as ducks, geese, and gulls (Alexander, 2000). They may be infected with more

than one type without clinical signs, excrete the virus for long periods and mostly do not develop

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detectable antibodies. A marked similarity between the subtypes prevalent in the waterfowl

population and those infecting poultry were reported several times. The continuing spread of H5N1

appears to be related to two factors: spread through movement of poultry (legal as well as illegal) and

spread through wild migratory birds (Liu et al., 2005). Free-ranging backyard chickens, illegal

transportation of domestic birds, and cockfighting also have been shown to contribute to spread of

the virus (Tiensin et al., 2005). Kilpatrick et al. (2006) investigated the pathways by which the virus has

and will spread between countries. They integrated data on phylogenetic relationships of virus

isolates, migratory bird movements, and trade in poultry and wild birds to determine the pathway for

52 individual introduction events into countries and predict future spread. The results show that 9 of

21 of H5N1 introductions to countries in Asia were most likely through poultry, and 3 of 21 were

most likely through migrating birds. In contrast, spread to most (20/23) countries in Europe was most

likely through migratory birds. Spread in Africa was likely partly by poultry (2/8 introductions) and

partly by migrating birds (3/8). The obtained results predict that H5N1 is more likely to be

introduced into the Western Hemisphere through infected poultry and into the mainland United

States by subsequent movement of migrating birds from neighbouring countries, rather than from

eastern Siberia. These results highlight the potential synergism between trade and wild animal

movement in the emergence and pandemic spread of pathogens and demonstrate the value of

predictive models for disease control.

The severity of clinical signs, course of the disease, and mortality in poultry after infection with

AIV are extremely variable from highly acute to a very mild or even inapparent form with few or no

clinical signs. Clinical signs may include high mortality, ruffled feathers, depression, diarrhoea, sudden

drop in egg production, cyanosis of comb and wattles, oedema and swelling of head, blood-tinged

discharge from nostrils, respiratory distress, incoordination and pin-point haemorrhages mostly seen

on the feet and shanks.

Lesions at post mortem may include swelling of the face. Removing skin from the carcass will

show a clear straw-coloured fluid in the subcutaneous tissues. Blood vessels are usually engorged.

Haemorrhage may be seen in the trachea, proventriculus, and throughout the intestines. Pancreatic

necrosis and nephritis are also not uncommon. Young broilers may show signs of severe dehydration

with other lesions less pronounced or entirely absent.

The final diagnosis should be based on laboratory examination and consideration of the exsisting

legislations. Usually based on isolation and identification of the virus and /or detection of the RNA

using PCR. In addition, serological examinations for detection of antibodies can be also carried out.

Disease prevention and control

The control based mostly on enforcement of biosecurity measures, surveillance, culling of

infected flocks, and preventive vaccination, were used in some countries to reduce the economic

losses. The community measures for the control of HPAI are based first on the depopulation of the

infected flocks, in accordance with community legislation on animal welfare. Once the presence of

HPAIV has been officially confirmed all poultry and other captive birds on the holding shall be killed

without delay under official supervision. The killing shall be carried out in such a way as to avoid the

risk of spread of avian influenza, in particular during transport. If an outbreak occurs, it is necessary to

prevent any further spread of infection by carefully monitoring and restricting movements of poultry

and by tightening biosecurity measures at all levels of poultry production, by cleaning and disinfecting

the infected holding, by establishing protection based on a minimum radius of 3 kilometres around

the infected holding itself contained in a surveillance zone based on a minimum radius of 10

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kilometres and, if necessary, by vaccination (EC, 2005). In some countries and places, culling of flocks

infected with LPAI H5 or H7 were applied too.

In accordance with Directive 2005/94/EC (EC, 2005), vaccination against avian influenza is

generally prohibited in the EU. However, under certain circumstances a member state can introduce

emergency vaccination as a short term measure or may also introduce preventative vaccination in

poultry or other captive birds as a long term measure. The Commission shall immediately examine

and approve the vaccination plan.

The vaccination strategy should allow differentiation between infected and vaccinated animals.

Products of vaccinated poultry, such as meat and table eggs, can then be placed on the market in

accordance with the relevant Community legislation.

The major advantages of the vaccine to control AIV in poultry are to reduce shedding of the

virus, morbidity, mortality, bird-to-bird transmission and to limit decrease in egg production. In most

of countries inactivated adjuvanted homologous /or heterologous vaccines are used. For both

homologous and heterologous vaccines, the degree of clinical protection and the reduction in viral

shedding are improved by a higher antigen mass in the vaccine. For heterologous vaccines the degree

of protection is not strictly correlated to the degree of homology between the haemagglutinin genes

of the vaccine and challenge strains. It was expected to continue for the next 10 years (Swayne, 2012;

Spackman and Swayne, 2013).

During the outbreak of HPAI H5N2 in 1994-1995 Mexico applied large-scale vaccination

campaign using inactivated homologous H5N2 vaccines (Garcia et al., 1998; Lee et al., 2004). Recently,

inactivated H7N3 vaccines were used in Mexico to control the ongoing outbreaks of HPAI H7N3

since 2012 (Kapczynski et al., 2013). Also, Pakistan used inactivated H7N1 vaccines to control HPAI

H7N1 outbreaks in 1995 (Naeem and Hussain, 1995) and in 2003-2004 (Naeem & Siddique, 2006).

Also, the inactivated H7N3 and H7N1 vaccines as a part of the intervention plan were used in Italy

against H7N1 and H7N3 in 2000-2002, respectively (Capua and Marangon, 2007). In USA against

LPAIV H7N2 in 2003 (Capua and Alexander, 2004) and in North Korea against H7N7 in 2005

(Swayne, 2012).

After the re-emergence of the Asian H5N1 in 2003, vaccines were used in many countries to

reduce the economic losses of the disease. Occasionally, vaccines for HPAI H5N1 have been used for

short periods in Cote d’Ivoire, France, Kazakhstan, Mongolia, Netherlands, Pakistan, Russia and

Sudan. Currently, four countries use different H5N1 vaccines in poultry: China since 2004, Indonesia

since 2004, Viet Nam since 2005 and Egypt since 2006 (Abd-Abdelwhab and Hafez, 2015).

Furthermore, 10 countries used inactivated H9N2 vaccines in poultry.

Canada and USA used vaccines to control H1 and H3 swine influenza viruses in turkeys.

Germany, South Africa and USA used vaccines to control H6 outbreaks and also USA used H2 and

H4 vaccines (Swayne, 2012). The use of bivalent H5-H7 and H7-H9 inactivated vaccines were used in

Italy (Capua and Alexander, 2004) and Pakistan (Capua and Marangon, 2007), respectively.

Several novel vaccines have been developed with high efficiency such as recombinant fowl pox

viruses expressing the H5 or H7 antigen or other vectors; infectious laryngotracheitis virus.

Additional Examples include, DNA vaccines, Subunit vaccines, vaccines based on reverse genetics,

Adenovirus-, baculovirus-, Newcastle disease-vectored vaccine and Newcastle disease virus–based

bivalent live attenuated vaccine were described and summarized by (Abdelwhab et al., 2014,

Abdelwahab and Hafez, 2015). So far, only H5- expressing viral vectored vaccines have been used in

the field (i.e.: in China, Egypt and Mexico) to control HPAI H5N1 and H5N2 outbreaks in poultry

(Bublot et al., 2007; Swayne, 2012).

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Vaccinal breaks were observed in vaccinated flocks in some countries. Vaccinal break, defined as

sub-optimal vaccinal protection of a flock and can have several causes. The efficacy of vaccine is very

much dependent on the quality of the product as well as the quality of the manufacturing process and

quality control procedures. In addition, the antigen concentration is very important. According to

Gardin (2007) the reduction of the antigen content leads to a reduction of the capacity of the vaccine

to reduce the shedding, although antibody response to vaccination remains almost identical and

although the monitoring of antibody response in the fields is useful to check the quality of the

vaccination, but is not a very accurate and sensitive way to evaluate the level of protection.

Inappropriate storage, handling and improper administration are further factors. The quality of

the vaccines application is crucial since all non injected chickens are not protected, and improperly

injected chicks will be poorly protected. Using post-vaccination necropsy (residue of oil at the site of

injection) or serological testing demonstrated, that it is not uncommon to see as much as 20% or

30% or even more of chickens that were not injected (Gardin, 2007).

Several challenges facing the efficiency of the vaccine to control the HPAIV outbreaks have been

reported and summarized by (Abdelwahab and Hafez, 2015) as follow:

1) Vaccine is HA subtype specific and in some regions where multiple subtypes are co-circulating

(i.e., H5, H7 and H9), vaccination against multiple HA subtypes is required (Suarez and Schultz-

Cherry, 2000).

2) Vaccine-induced antibodies hinder routine serological surveillance and differentiation of infected

birds from vaccinated ones requires more advanced diagnostic strategies (Suarez, 2005).

3) Vaccination may prevent the clinical disease but can’t prevent the infection, this lead to “silent”

circulation of the virus in vaccinated flocks poses a potential risk of virus spread among poultry

flocks and spillover to humans (Capua and Alexander, 2006; Hafez et al., 2010).

4) Immune pressure induced by vaccination on the circulating virus in an area increases the

evolution rate of the virus and accelerates the viral antigenic drift to evade the host-immune

response (Lee and Song, 2013).

5) After emergence of antigenic variants, the vaccine becomes useless and/or inefficient to protect

the birds and periodical update of the vaccine is required (Abdelwhab et al., 2011; Grund et al.,

2011; Kilany et al., 2011).

6) Vaccine-induced immunity usually peaks three to four weeks after vaccination and duration of

protection following immunization remains to be elucidated (Swayne and Kapczynski, 2008).

7) Maternally acquired immunity induced by vaccination of breeder flocks could interfere with

vaccination of young birds (Maas et al., 2011; Abdelwhab et al., 2012).

8) Other domestic poultry (i.e., ducks, geese, turkeys), zoo and/or exotic birds even within the

same species (i.e., Muscovy vs. Pekin ducks) respond differently to vaccination which have not yet

been fully investigated compared to chickens (Cagle et al., 2011).

9) Co-infections or prior infection with immunosuppressive pathogens or ingestion of mycotoxins

can inhibit the immune response of AIV-vaccinated birds (Sun et al., 2009; Hegazy et al., 2011).

10) For the recombinant vaccines, keeping cold-chain is pivotal, it can cause or aggravate respiratory

tract infections and reassortment with wild type viruses can not be totally excluded (Abdelwahb

and Hafez, 2015).

11) Factors related to vaccine manufacturing, quality, identity of vaccine strain, improper handling

and/or administration can be decisive for efficiency of any AIV vaccine (Swayne and Kapczynski,

2008).

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Therefore, presence of new alternative and complementary strategies target different AIV

serotypes/subtypes/drift-variants should be encouraged. Several possible alternative approaches for

control of AIV in poultry particularly against the HPAI (H5N1) subtypes were described and

summarized (Abdelwhab and Hafez, 2012).

Conclusions

In conclusion avian influenza infections in poultry are associated with severe economic losses,

early recognition and monitoring programmes are essential in managing the infections and a universal

solution for prevention and control of avian influenza does not exist. Generally, one of the above

mentioned measures alone is of little value, unless they are accompanied by improvements in all

aspects of management and bio-security. In countries in which the infection is endemic and when

other control measures such as stamping out, movement restriction of poultry and bio-security

cannot stop the spread of the infection poultry flocks should be vaccinated using a vaccine of high

quality.

Finally, since the success of any control program depends on the hygiene practices of the

personnel, it is essential to incorporate education programs about micro-organisms and their modes

of transmission, as well as awareness of the reasons behind such control programs for all people

involved throughout the poultry production chain.

References

Abdelwhab EM and HM Hafez, 2012. Insight into Alternative Approaches for Control of Avian

Influenza in Poultry, with Emphasis on Highly Pathogenic H5N1. Viruses, 4, 3179-3208;

doi:10.3390/v4113179.

Abdelwhab EM and HM Hafez, 2015. Control of Avian Influenza in Poultry with Antivirals and

Molecular Manipulation. Epidemiology II Theory, Research and Practice Publisher: Concept Press

Ltd. ISBN: 978-1-922227-75-1.

Abdelwhab EM, C Grund, MM Aly, M Beer, TC Harder et al., 2011. Multiple dose vaccination with

heterologous H5N2 vaccine: immune response and protection against variant clade 2.2.1 highly

pathogenic avian influenza H5N1 in broiler breeder chickens. Vaccine, 29: 6219-6225.

Abdelwhab EM, C Grund, MM Aly, M Beer, TC Harder et al., 2012. Influence of maternal immunity on

vaccine efficacy and susceptibility of one day old chicks against Egyptian highly pathogenic avian

influenza H5N1. Vet Microbiol, 155: 13-20.

Abdelwhab EM, J Veits and TC Mettenleiter, 2014. Prevalence and control of H7 avian influenza

viruses in birds and humans. Epidemiology and Infection, 1-25.

Alexander DJ, 2000. A review of avian influenza in different bird species. Proceedings of the ESVV

Symposium on Animal Influenza Viruses, Gent 1999. Vet. Microbiol, 74: 3-13.

Brown EG, 2000. Influenza virus genetics. Biomed Pharmacoth, 54: 196-209.

Bublot M, N Pritchard, JS Cruz, TR Mickle, P Selleck et al., 2007. Efficacy of a fowlpox-vectored avian

influenza H5 vaccine against Asian H5N1 highly pathogenic avian influenza virus challenge. Avian

Dis, 51(1 Suppl): 498-500.

Cagle C, TL To, T Nguyen, J Wasilenko, SC Adams et al., 2011. Pekin and Muscovy ducks respond

differently to vaccination with a H5N1 highly pathogenic avian influenza (HPAI) commercial

inactivated vaccine. Vaccine, 29: 6549-6557.

Page 34: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

34

Capua I and S Marangon, 2007. The use of vaccination to combat multiple introductions of Notifiable

Avian Influenza viruses of the H5 and H7 subtypes between 2000 and 2006 in Italy. Vaccine, 25:

4987-4995.

Capua I and DJ Alexander, 2004. Avian influenza: recent developments. Avian Pathol, 33: 393-404.

Capua I and DJ Alexander, 2006. The challenge of avian influenza to the veterinary community. Avian

Pathol, 35: 189-205.

DH Lee and CS Song, 2013. H9N2 avian influenza virus in Korea: evolution and vaccination. Clin Exp

Vaccine Res, 2: 26-33.

EC, 2005. COUNCIL DIRECTIVE 2005/94/EC of 20 December 2005 on Community measures for

the control of avian influenza and repealing Directive 92/40/EEC. Off J Eur Union, L: 10-17.

Ferguson NM, AP Galvani, RM Bush, 2003. Ecological and immunological determinants of influenza

evolution. Nature, 422: 428-433.

Garcia A, H Johnson, DK Srivastava, DA Jayawardene, DR Wehr et al., 1998. Efficacy of inactivated

H5N2 influenza vaccines against lethal A/Chicken/Queretaro/19/95 infection. Avian Dis, 42: 248-

256.

Gardin Y, 2007. Vaccination against H5N1 highly pathogenic avian influenza: some questions to be

addressed. Proceedings of the 56th Western Poultry Disease Conference, March 26-29, 2007.

Las Vegas, Nevada, USA, pp: 80-83.

Grund C, EM Abdelwhab, AS Arafa, M Ziller, MK Hassan et al., 2011. Highly pathogenic avian

influenza virus H5N1 from Egypt escapes vaccine-induced immunity but confers clinical

protection against a heterologous clade 2.2.1 Egyptian isolate. Vaccine, 29: 5567-5573.

Hafez HM and M Hess, 1999. Modern techniques in diagnosis of poultry diseases. Archiv für

Gefluegelkunde, 63: 237-245.

Hafez HM, 2005. Governmental regulations and concept behind eradication and control of some

important poultry diseases. World’s Poult Sci J, 61: 569-581.

Hafez HM, A Arafa, EM Abdelwhab, A Selim, SG Khoulosy et al., 2010. Avian influenza H5N1 virus

infections in vaccinated commercial and backyard poultry in Egypt. Poult Sci, 89: 1609-1613.

Hegazy AM, FM Abdallah, LK Abd-El Samie and AA Nazim, 2011. The relation between some

immunosuppressive agents and widespread nature of highly pathogenic avian influenza (HPAI)

post vaccination. J Amer Sci, 7: 66-72.

Kapczynski DR, M Pantin-Jackwood, SG Guzman, Y Ricardez, E Spackman et al., 2013.

Characterization of the 2012 highly pathogenic avian influenza H7N3 virus isolated from poultry

in an outbreak in Mexico: pathobiology and vaccine protection. J Virol, 87: 9086-9096.

Kilany WH, EM Abdelwhab, AS Arafa, A Selim, M Safwat et al., 2011. Protective efficacy of H5

inactivated vaccines in meat turkey poults after challenge with Egyptian variant highly pathogenic

avian influenza H5N1 virus. Vet Microbiol, 150: 28-34.

Kilpatrick AM, AA Chmura, DW Gibbons, RC Fleischer, PP Marra et al., 2006. Predicting the global

spread of H5N1 avian influenza. Proceedings National Academy of Sciences of the USA, 103:

19215-19216.

Lee CW, DA Senne and DL Suarez, 2004. Effect of vaccine use in the evolution of Mexican lineage

H5N2 avian influenza virus. J Virol, 78: 8372-8381.

Liu J, H Xiao, F Lei, Q Zhu, K Qin et al. 2005. Highly pathogenic H5N1 influenza virus infection in

migratory birds. (Brevia) Science 309(5738), 1206. Highly Pathogenic H5N1 Influenza Virus

Infection in Migratory Birds.

Page 35: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

35

Lupiani B and SM. Reddy, 2009. The history of avian influenza. Comp Immunol Microbiol Infect Dis,

32: 311-323.

Maas R, S Rosema, D van Zoelen and S Venema, 2011. Maternal immunity against avian influenza

H5N1 in chickens: limited protection and interference with vaccine efficacy. Avian Pathol, 40: 87-

92.

Naeem K and N Siddique, 2006. Use of strategic vaccination for the control of avian influenza in

Pakistan. Dev Biol (Basel), 124: 145-150.

Naeem K and M Hussain, 1995. An outbreak of avian influenza in poultry in Pakistan. Vet Rec, 137:

439.

Palese P and ML Shaw, 2007. Orthomyxoviridae: The viruses and their replication. In Fields Virology,

5th ed; Knipe DM, PM Howley, Eds; Lippincott Williams & Wilkins: Philadelphia, PA, USA pp:

1647-1689.

Peiris JS, DM de Jong and Y Guan, 2007. Avian influenza virus (H5N1): a threat to human health. Clin

Microbiol Rev, 20: 243-267.

Spackman E and DE Swayne, 2013. Vaccination of gallinaceous poultry for H5N1 highly pathogenic

avian influenza: current questions and new technology. Virus Res, 178: 121-132.

Suarez DL, 2005. Overview of avian influenza DIVA test strategies. Biologicals, 33: 221-226.

Suarez DL and S Schultz-Cherry, 2000. Immunology of avian influenza virus: a review. Dev Comp

Immunol, 24: 269-283.

Sun S, Z Cui, J Wang and Z Wang, 2009. Protective efficacy of vaccination against highly pathogenic

avian influenza is dramatically suppressed by early infection of chickens with reticuloendotheliosis

virus. Avian Pathol, 38: 31-34.

Swayne DE and D Kapczynski, 2008. Strategies and challenges for eliciting immunity against avian

influenza virus in birds. Immunolog Rev, 225: 314-331.

Swayne DE, 2009. Avian influenza vaccines and therapies for poultry. Compar Immunol, Microbiol

Infec Dis, 32: 351-363.

Swayne DE, 2012. Impact of vaccines and vaccination on global control of avian influenza. Avian Dis,

56(4 Suppl): 818-828.

Swayne DE, DL Suarez and LD Sims, 2013. Influenza. In: Swayne DE, JR Glisson, LR McDougald, LK

Nolan, DL Suarez and V Nair (Ed). Diseases of Poultry. 13th Edition. John Wiley & Sons, Inc, USA

ISBN: 978-0-470-95899-5. pp: 181-281.

Tiensin T, P Chaitaweesup, T Songserm, A Chaising, W Hoonsuwan et al., 2005. Highly pathogenic

avian influenza H5N1, Thailand 2004. Emer Infec Dis, 11: 1664-1672.

Tong S, X Zhu, Y Li, M Shi, J Zhang et al., 2013. New world bats harbor diverse influenza a viruses.

PLoS Pathogens, 9: e1003657.

Tong S, Y Li, P Rivailler, C Conrardy, DA Castillo et al., 2012. A distinct lineage of influenza A virus

from bats. Proc Natl Acad Sci USA, 109: 4269–4274.

Webster RG, WJ Bean, OT Gorman, TM Chambers and Y Kawaoka 1992. Evolution and ecology of

influenza A viruses. Microbiolog Rev, 56: 152-179.

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PATHOGENESIS OF AVIAN AIRSACCULITIS: CON-INFECTION OF CHLAMYDIA

PSITTACI WITH H9N2, ORT AND ASPERGILLUS FUMIGATUS CONTRIBUTES TO

SEVERE PNEUMONIA AND HIGHLY MORTALITY IN SPF CHICKENS

Jun Chu1, Qiang Zhang1, Zonghui Zuo1, Peng Zhao1, Tianyuan Zhang1,

Zhiqiang Shen2, Guanggang Qu2 and Cheng He1*

1Key Lab of Animal Epidemiology and Zoonosis of Ministry of Agriculture, China; College of

Veterinary Medicine, China Agricultural University, Beijing 100193, China; 2Shandong Binzhou

Academy of Animal Science & Veterinary Medicine, Shandong 256600, China

*Corresponding Author: [email protected]

ABSTRACT

Since 2007, outbreak of airsacculitis is characterized as the uncontrollable respiratory distress in

broilers, pigeons and hens and causes a huge loss to Chinese poultry industry. However, the

pathogenesis is unclear. In current study, the sera were collected to detect antibodies against

Ornithobacterium rhinotracheale (ORT), Chlamydia psittaci (C. psittaci) and Avian metapeumovirus (aMPV)

from the recovery birds. SPF chickens inoculated with the clinical isolates from the diseased birds

were explored to identify pathogenesis between airsacculitis and multi-infection.

A total of 1693 serum samples were collected and detected using commercial ELISA kits. In the

survey, 550 samples were detected to be positive for ORT (80.3%), 241 sera were identified as

positive for aMPV (65.5%) while 364 samples were found to be positive for C. psittaci

(68.4%).Interestingly, chickens aged 1-day-old was found to be 90.1-100.0% positive for C. psittaci,

66.7-83.3% positive for aMPV and 29.5-73.5% positive for ORT, respectively. Higher seroprevalence

of C. psittaci and aMPV were found in the breeding species as compared to those of commercial

chickens.

In the artificial experiment, eighty SPF chickens aged 21-day-old were randomly divided into 8

groups. Post inoculated with C. psittaci, ORT and H9N2 by throat/ intranasal drop, chickens were

given Aspergillus fumigatus (A. fumigatus). Mortality, body weight gain, lesion scores and cytokines were

evaluated. Consequently, 37.5% mortality was observed in the birds with C. psittaci, ORT, H9N2 and

Aspergillus fumigatus at same time while 25% mortality was found in the combination with C. psittaci,

ORT and A. fumigatus infection. Also, airsacculitis was replicated in two groups, while other groups

had respiratory diseases without mortality.

Our seroprevalences survey indicates that ORT, C. psittaci and aMPV are prevalent in poultry.

Combination of C. psittaci, ORT, H9N2 and A. fumigatus contribute to the replication of poultry air

sacculitis. The early infection of C. psittaci plays a leading role and induces secondary infections to

H9N2 and ORT, triggering the injury of air sacs and lungs.

Key Words: Air sacculitis, seroprevalence, co-infection, SPF chickens

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LARYNGOTRACHEITIS IN CHICKENS IN PAKISTAN

Ahmed Din Anjum

Department of Pathobiology, Riphah College of Veterinary Sciences, Lahore, Pakistan

ABSTRACT

Respiratory diseases are one of the most common problems in today's commercial poultry and

fancy birds. Amongst these, Infectious laryngotracheitis (ILT) is a highly contagious disease of

chickens. Field outbreaks of ILT in layer flocks, pheasants and peafowl are reported here. Outbreaks

in White Leghorn layers were seen between 12 and 18 weeks of age. Majority of the affected birds

had conjunctivitis, sticky nose, tracheal rales and gasping. The flocks also exhibited decrease in feed

consumption. Morbidity reached almost 100% in some flocks and mortality ranged between 10 and

70 per cent. At necropsy, nasal cavity contained copious amounts of clear thick mucus to yellowish

exudate. Lesions were most consistently found in the larynx and upper trachea. Histologically, severe

necrotizing tracheobronchitis and occasionally basophilic intranuclear inclusion bodies were seen in

the tracheal epithelium. Vaccination, eye drop, in the face of these outbreaks mostly induced

adequate protection around 4th day post vaccination. In conclusion, the outbreaks of ILT in layer

flocks and in fancy birds should be taken as an emerging threat. There may be more ILT cases which

are incorrectly diagnosed due to the nondescript clinical signs of this disease. Along with biosecurity

measures, ILT vaccine may be considered in the regular vaccination schedules. Vaccination early in an

outbreak of ILT can be an effective tool to decrease mortality.

Key Words: Infectious laryngotracheitis, Clinical signs, Lesions, Vaccination

Introduction

Respiratory diseases are one of the most common, world-wide, problems in today's commercial

poultry and fancy birds. Amongst these, Infectious laryngotracheitis (ILT) is a highly contagious viral

respiratory disease of chickens. Clinically, ILT may present as a mild, acute or peracute disease and is

characterised by severe dyspnoea, neck extension, conjunctivitis and severe production losses (Bagust

et al., 2000). It is a disease of economic significance for the poultry industry because it induces high

mortality and drops in egg production and egg size in layers and delayed growth, poor feed

conversion and increased condemnations in broilers (Humberd et al., 2002; Kirkpatrick et al., 2006;

Anonymous, 2008).

Until 1980-1990’s, it was occasionally seen in breeder flocks in Northern areas in Pakistan

(Anjum, 1991). This presentation describes the field outbreaks of ILT in commercial layers, pheasants

and peafowl in Punjab province including Sargodha, Faisalabad and Lahore. Outbreaks in White

Leghorn layers were seen between 12 and 18 weeks of age.

Materials and Methods

The study is based on investigation of field outbreaks of respiratory disease in commercial

poultry and wildlife. Flock history (acute), disease trend (rapidly spreading among pen-mates),

symptoms (conjunctivitis, neck extension during an inspiratory effort with open beak to inhale more

air) and characteristic lesions (conjunctivitis, tracheitis) in birds were taken as suggestive of ILT.

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Blood samples were collected from few flocks at the start of the respiratory signs and three

weeks later. Sera were separated and antibody titres were determined using commercial ELISA kits

(Biocheck, Holland).

Clinical Description

The ILT outbreaks were seen in layer flocks in Sargodha, Faisalabad and Lahore, during the

months of March through May 2015. The clinical presentation of field cases of ILT was variable from

mild to severe.

The affected pullets, initially exhibited signs of ocular edema, conjunctivitis and ocular discharge

followed by nasal discharge, moist rales and open-mouth breathing. Severely affected birds frequently

emitted a cawing sound - high pitched squawk. In pullets, the morbidity was upto 90 per cent and the

mortality was variable from 5-15 per cent.

In laying flocks, initially there was reduced feed intake. Many of the affected birds were sitting

with their eyes closed and head between feathers. There was frothy discharge from eyes or sticky

eyes. Later, some of the affected birds developed swelling of the infra-orbital sinuses. Mortality was

less than 10 per cent. Clinical signs were accompanied by 10-15 per cent egg drop. Egg production

returned to its previous rate in around four weeks. Egg quality was not affected.

At postmortem examination, nasal cavity contained copious amounts of clear thick mucus. There

was mild congestion to numerous petechial haemorrhages on the mucosal surface of the larynx and

trachea. Very few birds had caseous exudate or diptheritic casts occluding the lumen of upper

trachea. There was general body congestion (dark muscles) but viscera appeared normal.

In case of more than one houses at any farm, spread from house to house took around a seven

to 10 days. Vaccination, eye drop, in the face of these outbreaks mostly induced adequate protection

around 4th day post-vaccination. Clinical signs and mortality tended to disappear in around three to

four weeks through entire flock. Clinical recovery in some flocks prolonged to 7-8 weeks and

immunosuppression was suspected for this prolonged recovery.

At certain wildlife facilities, pheasants were most affected whereas peafowl and turkeys did not

show clinical disease. Clinical signs and lesions in pheasants were similar to those seen in the layer

birds. There was manifold increase in antibody titre against ILT without any vaccination with ILT virus

(Table 1).

Table 1: Antibody titre against ILT in affected flocks.

Flock

First

Sample

Second sample (21 days later)

Mean titre Range CV (%)

1 259 3341 662-6900 55.04

2 216 3070 870-5300 45.36

3 58 3513 1083-6200 44.05

4 197 3686 1461-6700 48.26

Discussion

Until the year 2000, breeder flocks were mainly kept in Northern areas to take advantage of

weather. With the start of environmentally controlled houses, breeder flocks are now also raised in

plain areas in Punjab. In Pakistan, to date, preventive vaccination for ILT is mainly practiced only for

breeding stocks. Three vaccine types are available viz., chick embryo origin (CEO), tissue culture

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origin (TCO), and a pox-vectored recombinant vaccine. The CEO vaccines are usually more invasive

and have the capability of reverting to virulence and causing full-blown ILT signs. The TCO does not

spread significantly or revert to virulence. In case of the pox-vectored recombinant vaccine, if a bird

has had previous exposure to pox prior to being given this vaccine, immunity to ILT will be minimal

Vaccinated birds or birds recovered from natural infection are immune for around a year. Parental

immunity is passed on to the progeny but maternal antibodies do not protect offspring against ILTV

infection or interfere with vaccination (Davison, et al., 2006; Vagnozzi, et al., 2012; Coppo, et al.,

2013).

During 1990’s outbreaks of ILT were limited to Northern areas in Pakistan (Anjum, 1991). This

study describes field outbreaks of ILT in Punjab. A rising antibody titre in paired sera against ILT

(Table-1) confirms the disease. These outbreaks may be an evidence of increasing occurrence of the

disease in Pakistan in recent years. A priori, this spread may be related to emergence of pathogenic

strains from vaccines used in breeder flocks. Noteworthy, once a bird is infected with ILT virus, it

becomes a life-long carrier and can shed the ILT virus during times of stress, infecting other birds

(Hughes, 1991). Even the vaccine strains can shed virus for 10-11 days after inoculation.

Guy et al. (1991) reported an increased virulence of modified-live ILT vaccine virus following

bird-to-bird passage. Recently, Lee et al. (2012) showed that current ILT vaccines are mildly

pathogenic and increase in virulence by bird-to-bird passage, from vaccinates to non-vaccinates, in the

field. Through uncontrollable “back passage” the vaccine viruses may regain enough virulence to

cause an outbreak of ILT (vaccinal laryngotracheitis, also known as VLT) (WSDA, 2011).

Furthermore, the recombinant vaccines do not induce mortality and do not cause an increase in feed

conversion or in mortality, but they do allow field viruses to replicate in the trachea and conjunctiva,

and in consequence, vaccinated-challenged chickens do get infected with field viruses (Vagnozzi, et al.,

2012).

Clinical signs and lesions are very similar to those reported in other studies (Kirkpatrick et al.,

2006). Other than morbidity, mortality and reduced performance, ILT is also suspected to synergize

the impact of pathogens that have normally little impact such as Mycoplasma synoviae. Outbreaks can

occur in broilers resulting in a marked reduction in feed intake and growth rate (VanderKop, 1993;

Linares et al., 1994). However, ILT in broilers was not under study during this period. Vaccination in

the face of an outbreak in egg-type layers can be an effective tool to reduce the severity, mortality

and longevity of the disease. However, never use the vaccine unless the diagnosis is definitely

confirmed.

References

Anjum AD, 1991. Emerging Diseases of Poultry in Pakistan. Proceedings of the 3rd International

Congess organized by Pakistan Veterinary Medical Association on 28-29 November 1990 in

Islamabad. pp: 205-213.

Aonymous, 2008. Avian infectious laryngotracheitis - Chapter 2.3.3. OIE Terrestrial Manual, pp. 456-

463.

Bagust TJ, RC Jones and JS Guy, 2000. Avian ILT. Rev Sci Tech, 19: 483-492.

Coppo MJ, AH Noormohammadi, GF Browning and JM Devlin, 2013. Challenges and recent

advancements in Infectious Laryngotracheitis Virus vaccines. Avian Pathol, 42: 195–205.

Davison S, EN Gingerich, S Casavant and RJ Eckroade, 2006. Evaluation of the efficacy of a live

fowpox-vectored infectious laryngotracheitis/avian encephalomyelitis vaccine against ILT viral

challenge. Avian Dis, 50: 50–54.

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40

Guy JS, HJ Barnes and L Smith, 1991. Increased virulence of modified-live ILT vaccine virus following

bird-to-bird passage. Avian Dis, 35: 348-355.

Hughes CS, RA Williams, RM Gaskell, FT Jordan, JM Brandbury et al., 1991. Latency and reactivation

of infectious laryngotracheitis vaccine virus. Arch Virol, 121: 213-218.

Humberd J, M García, SM Ribler, RS Resurrección and TP Brown, 2002. Detection of Infectious

Laryngotracheitis Virus in formalin-fixed, paraffin-embedded tissues by Nested polymerase chain

reaction. Avian Dis, 46: 64-74.

Kirkpatrick NC, A Mahmoudian, CA Colson, JM Devlin and AH Noormohammadi, 2006. Relationship

between mortality, clinical signs and tracheal pathology in infectious laryngotracheitis. Avian

Pathol, 35: 449–453.

Lee SW, PF Markham, MJ Coppo, AR Legione, JF Markham et al., 2012. Attenuated vaccines can

recombine to form virulent field viruses. Science, 337: 188.

Linares JA, AA Bickford, GL Cooper, BR Charlton and PR Woolcock, 1994. An outbreak of infectious

laryngotracheitis in California broilers. Avian Dis, 38: 188-192.

Vagnozzi A, G Zavala, SM Riblet, A Mundt and M Garcia, 2012. Protection induced by commercially

available live-attenuated and recombinant viral vector vaccines against infectious laryngotracheitis

virus in broiler chickens. Avian Pathol, 41: 21–31.

VanderKop MA, 1993. Infectious laryngotracheitis in commercial broiler chickens. Can Vet J, 34: 185.

WSDA, 2011. Vaccine-like Infectious Laryngotracheitis (ILT). Washington State Department of

Agriculture, extension paper.

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41

INFECTIOUS BRONCHITIS: CURRENT DISEASE SCENARIO IN PAKISTAN

Khalid Naeem*, Saba Rafique, Naila Siddique and Mohammad Athar Abbas

National Reference Lab for Poultry Diseases,

National Agricultural Research Centre,

Park Road, Islamabad, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Infectious Bronchitis Virus (IBV) is considered to be primarily involved in various in infections of

respiratory tract and urogenital tract in chickens of different age groups in this country. The sero-

prevalence data from non-IBV vaccinated back-yard poultry from different areas in the country have

revealed a wide distribution of IBV antibodies, which reflected that around 70% of the tested chicken

possessed positive IBV ELISA antibody titers, ranging from 453 to 16747. Whereas the normal base-

line post-vaccination mean antibody titers in IBV vaccinated laying flocks usually ranges from 4000 to

8000, some extremely high mean titers range of 12,000-18,000 has been recorded. This is reflection

of the fact that such birds were exposed to the field IBVs despite routine vaccination. Furthermore,

multiple field isolates of IBV earlier identified by RT-PCR also have been characterized using

restriction fragment length polymorphism (RFLP), where the results indicated the distribution of

these isolates into various distinct groups (genotypes), being different from the known IBV vaccine

strains being used in this country. Similarly, a variety of clinical picture observed in the field has

shown some links to the condition of co-infection between IBV and ORT or AIV H-9. Furthermore,

some of the available data regarding the sequence analysis of spike gene of local isolates indicate

continuous emergence of IBV variants in the field which obviously requires readdressing of the issues

of vaccine selection and adoption of more effective vaccination schedules.

Key Words: Infectious Bronchitis Virus, Sero-prevalence, ELISA, RT-PCR

Introduction

Avian infectious bronchitis virus (IBV) causes a highly contagious and economically significant

disease in chickens. The disease is characterized by respiratory signs but in young chickens severe

respiratory distress commonly occurs while in layers it causes decrease in egg production. Both the

chickens and pheasants are considered natural host of IBV where the virus cause disease. The virus is

transmitted by the air-borne route, direct chicken to chicken contact and indirectly through

mechanical spread. It can persist in the birds and faeces for several weeks or months.

The IB virus is a member of the genus Coronavirus, family Coronaviridae, Order Nidovirales. IBV and

other avian coronaviruses of turkeys and pheasants are classified as group 3 coronaviruses, with

mammalian coronaviruses comprising groups 1, 2 and 4. Group 4 is the more recently identified

severe acute respiratory syndrome (SARS) coronavirus (Cavanagh, 2003). IBV is an enveloped,

positive sense single stranded RNA virus containing an un-segmented genome approximately 27.6 kb

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in size. The virion has four structural proteins: nucleocapsid protein (N), membrane glycoprotein (M)

small envelope protein (E), and glycosylation spike glycoprotein (SP) (Su et al., 2011).

Many IBV serotypes have been described probably due to the frequent point mutations that

occur in RNA viruses and also due to recombination events in nature. For this reason, the

characterization of virus isolates existing in the field is very important. The spike (SP) protein is one

of the major structure proteins of IBV proteins that is cleaved into two smaller proteins namely SP1

and SP2. SP1 gene contains three hypervariable regions that are responsible for the induction of

neutralizing and serotype specific antibodies (Haqshenas et al., 2005).

In some of the earlier reported studies from Pakistan, antibodies to several "American" and

"European" IB variants have been demonstrated (Ahmed et al., 2007). A recent study has revealed the

sero-prevalence of IBV in backyard poultry and duck, indicating these species may act as a reservoir

for IBV or related viruses (Rahim et al., 2015). As no IBV vaccination is used in these species, such an

alternative reservoir would have major implications for vaccination and control programs for IBV

prevention in commercial poultry in this country. On the basis of this information some additional

work is ongoing here at NRLPD-NARC regarding the isolation, serotyping and genotyping of the IBVs

from the field so that a suitable vaccination program using common field serotype as vaccines can be

adopted to protect against the locally prevalent distinct IBVs.

During 2013-15 a comprehensive serological evaluation of the flocks with or without the usage

of IBV vaccines has been conducted. The sero-prevalence data from non-IBV vaccinated back-yard

poultry from different areas in the country have revealed a wide distribution of IBV antibodies, which

reflected that around 70% of the tested chickens possessed positive IBV ELISA antibody titers, ranging

between 450 to 16747 (Rahim et al., 2015). Whereas for IBV the normal base-line post-vaccination

mean antibody titres at the time of laying usually ranges in this country from 3000 to 7000. However,

in such vaccinated flocks some extremely high antibody titre ranges of 12,000-21,000 have also been

recorded among the flocks showing production losses with or without some changes in egg quality. In

some cases, such conditions have also led to the detection of IBVs from the selected flocks. All this

reflected that such birds were exposed to the field IBVs despite routine vaccination. Furthermore,

multiple field isolates of IBV identified here by RT-PCR have also been characterized using restriction

fragment length polymorphism (RFLP), where the results indicated the distribution of these isolates

into few distinct groups (genotypes), being different from the known IBV vaccine strains in use in this

country (Rafique et al., 2015). Similarly, a variety of clinical pictures observed in the field has shown

the involvement of ORT or AIV-9, later confirmed by the detection of these organisms through

serological and/or PCR evaluation.

In Pakistan, traditionally IBV Mass type strains have been used for vaccination, however, at

different times variants of D-274, D-1466, 4/91 and GA 98 have also been used. Unfortunately, at

many places use of any or all of these vaccines did not protect the flocks from subsequent IBV

infections. The IBV recovered from such cases have been grouped into 4/91-like variants, QX-like

variants and a group of Pak Isolates of unique variation (Rafique et al., 2005). Additional work is being

carried out to further characterize these new IBV isolates.

Based on the routine NRLPD-NARC surveillance data regarding PCR-based detection of IBVs, it

has been found that 4/91 vaccine viruses are persisting in broiler breeder flocks even up to 8-9 weeks

post vaccination. In this regard, some earlier studies examining re-isolated IBV vaccine viruses have

shown that selection of vaccine subpopulations as well as mutations in the spike glycoprotein can be

detected after only one infectious cycle (McKinley et al., 2008). Additional studies specifically

examining the mechanism for the persistence of IBV 4/91 vaccine under local field conditions in this

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country are required, as it is well known that the longer IBV persists in the field the more

opportunity it has to undergo genetic drift and shift resulting in new variant viruses, which may be the

cause of current vaccine failure. In order to achieve higher level of protection of commercial layers

and parent stock during the laying period, the use of inactivated IBV vaccines after a priming with live

IBV vaccines has been generally practiced to be effective against homologous Mass-strain challenges,

but for increasing the level of protection against heterologous challenges (variants), development of

killed vaccines from the most common circulating variant for use in the laying period could be an

appropriate solution for overcoming the prevailing IBV infections among the vaccinated flocks.

Conclusion

It is pointed out that the presence of IBV infection in commercial poultry among the flocks

vaccinated with multiple serotypes of IBV is indicator of independent evolution of IBV in Pakistan and

persistence of divergent stains currently circulating in the country. It is very critical to complete

genetic characterization of circulating IBV viruses to study the genetic relatedness among viruses and

vaccine strains. This will guide us for best vaccines selection and improve our effort to control the

disease.

References

Ahmed Z, K Naeem and A Hameed, 2007. Detection and sero-prevalence of infectious bronchitis

virus strains in commercial poultry in Pakistan. Poult Sci, 86: 1392-35.

Cavanagh D, 2003. Severe acute respiratory syndrome vaccine development experiences of

vaccination against avian infectious bronchitis coronavirus. Avian Pathol, 32: 567–582.

Haqshenas G, K Assasi and H Akrami, 2005. Isolation and molecular characterization of infectious

bronchitis virus isolate Shiraz IBV, by RT PCR and restriction enzyme analysis. Iranian J Vet Res,

6: 9-15.

McKinley T, AH Deborah and MW Jackwood, 2008. Avian coronavirus infectious bronchitis

attenuated live vaccines undergo selection of subpopulations and mutations following vaccination.

Vaccine, 26: 1274-1284.

Rafique S, K Naeem, N Siddique, MA Abbas, AA Shah et al., 2015. Sequence analysis of 793B-like

Infectious bronchitis virus isolated from Pakistan. Turkish J Biol ((Submitted).

Rahim A, K Naeem, N Siddique, A Hameed and S Rafique, 2015. Determination of Avian Infectious

Bronchitis Virus sero-prevalence among commercial and backyard poultry. Intl J Poul Sci

(Submitted).

Su JL, ZT Zhou, ZS Guo, QR Xu, YC Xiao et al., 2011. Identification of five bronchitis virus (IBV)

strains isolated in China and phylogenetic analysis of the S1gene. Afr J Microbiol Res, 6: 2194-

2201.

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IMMUNE ENHANCER PROPOLIS AND POULTRY PROPOLIS VACCINE

Zhiqiang Shen* and Guanggang Qu,

Shandong Binzhou Animal Science and Veterinary Medicine Academy, No 169, Huanghe 2nd Road,

Binzhou, Shandong, China

*Corresponding Author: [email protected]

ABSTRACT

The propolis is a type of natural material and wasp product formed by mixture of cells tissue

fluid and gelatinoid collected by bees from collagen plants axillary bud and lingual gland, wax

secretions of bees, and possesses broad spectrum biologic activities and immunologic enhancement.

The propolis is acknowledged as the greatest “natural antibiotics” all over the world, with the

properties of anti-inflammatory, anti-bacterial, oxidative stability, anti-aging, anti-virus, cytoactive,

reinforce energy, pull oneself together, relieve fever, detoxification and immunity enhancement. The

contents in propolis, including flavones, enzymes, alcohols, lipids and acids, have wide immunological

effects.

Professor Shen Zhiqiang, as the leader of a research group, has been researching vaccines for

livestock and poultry by using nanometer propolis over 20 years, is the first one to apply the natural

propolis with the function of a broad spectrum of biological activities instead of conventional chemical

propolis to develop vaccines. The group established an innovation technology platform with

independent innovation and intellectual property rights for the nano-propolis adjuvant vaccine

development and industrialization.

Series of the nano-propolis adjuvant vaccines developed by the team can fully trigger the body's

immune defense system, including the cellular immune system, humoral immune system, red blood

cells immune system and macrophage complement immune system to produce specific and non-

specific immunity. The vaccines are green, safe, fast, efficient and long-duration. Furthermore, the

vaccines can meet the requirement of food safety standards, are easy for storage, transportation and

application, and play an important role in animal disease prevention and control in China.

Key Words: Propolis; Adjuvant; Vaccine; Immune system

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TREATMENT OF AVIAN TIBIAL DYSCHONDROPLASIA USING TRADITIONAL

CHINESE MEDICINES BY HSP90 INHIBITION

Muhammad Kashif Iqbal1§, Jingying Liu1§, Fazul Nabi1,2, Muhammad Shahzad3 and Jiakui Li1*

1College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, PR China; 2Faculty of Veterinary & Animal Sciences, Lasbela University of Agriculture, Water and Marine

Sciences, Uthal 90150, Balochistan, Pakistan; 3University College of Veterinary & Animal Sciences, The

Islamia University of Bahawalpur 63100, Pakistan

§These authors equally contributed in this study

*Corresponding Author: [email protected]

ABSTRACT

Tibial dyschondroplasia (TD) mainly occurring in fast growing avian species is an important

tibiotarsal bone disorder that contributes a great economic loss in poultry industry. TD is

characterized by a-vascular and non-mineralized growth plate and is attributed to abnormal

differentiation of chondrocytes and lameness. Heat-shock protein 90 (Hsp90) is a proangiogenic

factor in animal tissues; however; its expressions increase in case of chicken TD. The

Epigallocatechin-3-gallate (EGCG) and Apigenin are traditional Chinese medicine (TCM) which are

well known for their Hsp90 inhibitory activities. In this experiment, TD was induced by dietary thiram

and the TD-affected birds were treated with EGCG and Apigenin. The histological study of growth

plates was carried out with H&E staining, and the mRNA expression of Hsp90 was examined by

reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Results showed

that as compared to control group, TD had displayed the changes in chondrocytes differentiation

with lack of blood vessels; and an increased expression of Hsp90 was observed significantly (P<0.05)

resulting in the development of TD and lameness. However, on administering the EGCG and

Apigenin to TD-affected birds, the normal chondrocytes columnar organization was restored with

vascularization and decreased Hsp90 expression activity (P<0.05) which ultimately abrogated the

lameness. Our results suggest that Hsp90 is the key factor in the development of TD, and EGCG and

Apigenin have a novel effect on Hsp90 inhibition properties thus reducing the lameness and leg

deformity in chicken broiler. Ours findings are a first time approach towards the treatment of TD in

broiler chicken through TCM.

Key Words: Tibial dyschondroplasia; Hsp90; traditional Chinese medicine; EGCG, Apigenin

INTRODUCTION

Tibial dyschondroplasia (TD, a long bone disorder in fast growing birds demonstrates the

presence of a-vascularized and non-mineralized cartilage in the growth plate (Shahzad et al., 2015)

bringing changes in normal differentiation of chondrocytes normally associated with cartilage

vascularization and mineralization (Pines et al., 1998).

Heat shock protein 90 (Hsp90) among its clients plays a major role in angiogenesis. In cancer

therapy, several Hsp90 inhibitors have been developed to inhibit vascularization. The inhibition of

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Hsp90 by synthetic Geldanamycin has been recently studied in TD where the down regulation of

Hsp90 brought the angiogenesis in the TD-affected area.

Many natural plants and extracts rich in polyphenols, flavonoids and carotens as antioxidants have

been recognized to treat many diseases. Being extensively utilized in animals, such plants have been

found better than synthetic drugs due to their lower cytotoxicity and side effects. Traditional Chinese

medicines (TCM) have been used for centuries in China to cure various diseases. Among those, many

medicines have been employed to promote angiogenesis to treat several bone related conditions

depicting a positive effect on the treatment (Yang et al 2014).

Epigallocatechin-3-gallate (EGCG), a main component of green tea is found to have the curative

effects in inflammatory and oxidative stressed conditions. EGCG is a novel Hsp90 inhibitor and

protective against various types of cancer (Yin et al 2009, Clement Yuri 2009, Song et al 2005) by

suppressing the expression of Hsp70 and HSP90 in vivo and in vitro cultures (Tran et al., 2010).

Apigenin, a member of flavone family is present in vegetables, fruits, spices and herbs; and have

the anti-inflammatory, antioxidant and anti-cancerous properties. This compound has been reported

to inhibit the Hsp90 expressions through hypoxia-inducible factor 1 alpha (HIF-1 alpha) (Shukla et al.

2010, Fang et al., 2005).

In this study, we evaluated the efficiency and safety of TCM (EGCG and Apigenin) in un-

vascularized avian growth plates in thiram-induced avian tibial dyschondroplasia; and investigated their

effect as Hsp90 inhibitors in TD-affected growth plate.

MATERIALS AND METHODS

Experimental birds: The experiment was conducted concerning all national legislations and

protection of animal welfare under the approval of committee of Huazhong Agricultural University

Wuhan, China. Three hundred one-day-old male broiler chicks average weighing 48±0.5 g were

purchased from commercial hatchery and maintained under standard hygienic conditions.

TD establishment and treatment with TCM: The chicks were weighed and divided equally into

two groups: a control group (n=150) which received a standard normal diet, and thiram group kept

on the same diet as to the control group (n=150) but with the addition of 40 mg/kg of tetra methyl

thiuram disulphide (Thiram) to induce TD til the end of experiment. TD was evaluated and scored

according to Pines et al (2005). After disease induction, when more than seventy five percent birds

started showing the lameness signs, half of the birds from the thiram group were separated into two

separate groups designated as the EGCG and Apigenin treatment groups. These groups were fed

with the thiram-containing diet and treated with TCM; the birds of EGCG group were treated with

EGCG @ 10 mg/kg/d intra-peritoneally (i.p) (Tianjin Shilan Technology Co.Ltd. China) and birds in

Apigenin group were given Apigenin @ 5mg/kg/d i.p (Wuhan Dinghui Chemical Co.LTD) intra-

peritoneally (i.p). Normal saline was administered to control group.

Broiler chicks from all groups were euthanized by cervical dislocation on day 7 & 14 and growth

plates were dissected out for further analysis. Tibiotarsal bones from each group were fixed in 4%

paraformaldehyde, frozen in liquid nitrogen and stored at -70oC.

Hematoxylin & eosin (H&E) staining: Growth plate samples were fixed overnight in 4%

paraformaldehyde in PBS at 4oC and the bone tissue was decalcified in 10% EDTA. After dehydration

in ethanol, clearing in xylene and embedding in paraffin wax, the growth plate histological slices were

cut into 5 um thickness to prepare the histological slides and stained by H & E staining method.

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RNA extraction and reverse transcription quantitative real time PCR (RT-qPCR):

Growth plates were disrupted and homogenized in TRIzol reagent (Invitrogen, Carlsbad, California

USA) to extract the total RNA which was transcribed to cDNA with cDNA kit (TransGen Biotech

Co.Ltd Beijing, China). RT-qPCR was performed with specific Hsp90 primers from Gallus gallus

sequence as: Forward CCTCCTCCATACGTGATGTGTCA and Reverse

GCCTGGGCATTGATGAAGATG. Quantification of gene expression was performed by Step One

Real-Time PCR system (Applied Biosystems, CA, USA) with SYBR green kit (Takara Dalian, China).

All samples were run with following thermal parameters: 95oC for 30 sec, 40 amplification cycles at

95oC for 8 sec, 59oC for 30 sec and 72oC for 30 seconds. The delta CT method was used to quantify

the expressions and the differences between gene expression levels were analyzed by t test. The

differences were considered significant at P<0.05.

Statistical analysis: All data was analyzed by one way ANOVA following the Tukey’s test and

presented as means ± standard error of means (SEM). The differences were considered statistically

significant at P<0.05.

RESULTS

Effect of EGCG on thiram-induced TD-affected growth plates: Morphologically, the birds

started showing signs of lameness after two days of TD induction (Fig. 1). On slaughtering, an un-

vascularized mass on proximal side of tibiotarsal bones was observed in TD-affected birds (Fig. 1). In

EGCG treatment group, the growth plate width and lameness signs started to reduce which was

more obvious on day 14 as compared to thiram-fed group. In H&E staining, a significant difference

was observed between normal and TD-affected growth plate with rough column organization and less

blood vessels in chondrocytes in later group (Fig. 2). The EGCG administration resulted in a new

blood vessel formation with a normal and proper chondrocyte disclosure in the hypertrophic region

of growth plate (Fig. 2). In qRT-PCR, the levels of Hsp90 expressions was found up-regulated

significantly (P<0.05) in thiram-fed TD birds as compared to control group; however, a significant

reduction in Hsp90 expressions was observed in EGCG administered group bringing its level

returned to the normal values in the region (Fig. 3).

Effect of apegenin on thiram-induced TD-affected growth plate: To cure TD, the anti-Hsp90

treatment was studied by using Apigenin as an Hsp90 inhibitor. After the administration of Apegenin

to the thiram-induced TD chicks, the affected growth plate size and signs of lameness were abrogated

on day 14. At this stage, the chicks were able to stand and walk properly (Fig. 4). On H&E staining of

TD-affected cartilage cells (thiram-fed group), the hypertrophic zone of growth plate depicted a

decline in blood vessels and improper chondrocytes differentiation; however, the Apigenin

administration cured the chicks from TD and by bringing about the massive blood vessels in the

hypertrophic area (Fig. 5). In parallel to that, a significant increase in Hsp90 expressions was observed

(P<0.05) in TD-affected birds as compared to control group; however in contrary, the Apegenin

administration caused a reduction in Hsp90 expressions in growth plate (Fig. 6).

DISCUSSION

Chicken broiler long bone defects ultimately lead to gait abnormality and pose a serious animal

welfare issue resulting into great economic loss. The mechanism of endochondral ossification occurs

in growth plates. The chondrocytes are surrounded in extracellular matrix (ECM) which is a

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reservoir of various growth factors and several enzymes involved in chicken endochondral

ossification (Shahzad et al., 2014).

Fig. 1: Effect of EGCG on growth plate size and lameness. Chicks and tibial growth plates were

photographed. Lameness and the avascular, distended growth plate with tibial dyschondroplasia and

the recovery from lameness and normal size growth plate size after EGCG treatment. GP=growth

plate; TD=Tibial dyschondroplasia.

Fig. 2: Growth plate sections stained with hematoxylin and eosin staining. After EGCG treatment,

appropriate chondrocyte disclosure and new blood vessels formations. HZ, Hypertrophic Zone.

Arrow indicates blood vessels in the hypertrophic zone.

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Fig. 3: Real-time quantitative PCR analysis of Hsp90 in growth plate was evaluated before and after

administration of EGCG in TD affected birds. The results are expressed in arbitrary units as the

means ±SE. Control group set to one thus equivalent to the N-fold difference. abcletters indicate

significant difference (P <0.05).

Tibial Dyschondroplasia is described by the presence of a dull white non-mineralized cartilage mass

that extends into the metaphysis of the proximal tibiotarsus bone. (Leach and Nesheim 1965).

In this study, TD was induced by dietary 40 mg/kg of thiram in feed. Thiram an agricultural

fungicide has experimentally taken much attention due to its toxicity in poultry farming by causing

lameness in birds (Rath et al., 2005). After TD induction, the chicks were administered with TCM

medicines (EGCG and Apigenin); alongside thiram-feed was continued till the end of experiment.

Both medicines were found to start recovery after two days of their administration and a complete

healing was observed within a week. A complete whole recovery was seen by the end of experiment

with a decrease in growth plate lesion and lameness in TD-afflicted birds.

An alternative therapy is a worth approach because of less toxic side effects towards treatment.

Traditional Chinese medicines (TCM) have been used in clinical practice for thousands of years and

many herbs have been demonstrated for the treatment of bones and joint diseases. Recently, various

modern techniques have provided insight to meet such interests; and modern science has been

looking for their validation, especially in the control and treatment of chronic diseases. (Zhang et al.,

2012, Leung, 2001). The basic aim of present study was the treatment of TD by using TCM; EGCG

and Apigenin by employing their ability to inhibit Hsp90 activity in avian growth plates.

Hsp90 inhibitors are widely used to prevent the vascularization in cancer; however, the previous

reports have confirmed the role of Hsp90 in chicken growth plate where the Hsp90 inhibition has

brought the vascularization in TD-affected area with the abrogation of lameness. We have also

reconnoitered the therapeutic benefits of TCM by targeting the Hsp90 in TD. The results revealed

that the administration of TCM had brought massive blood vessels in TD-afflicted area by inhibiting

the Hsp90 expression and resulted into the re-establishment of normal growth plate morphology.

Herzog et al. (2011) and Genin et al. (2012) recently reported the Hsp-90 inhibition activity by a

synthetic drug which resulted in the abrogation of lameness in affected growth plate.

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Fig. 4: Effect of Apigenin growth plate size and lameness. Lameness and the large size growth plate in

the chicks with TD. Apigenin treatment resulted in normal growth plate size and recovery from TD.

GP= growth plate; TDL= TD lesion.

Fig. 5: Hematoxylin and eosin staining of growth plate histological sections of normal , TD and

Apigenin treated groups. TD afflicted growth plate was with irregular columnar arrangement of the

chondrocytes. Apigenin administration causes the appropriate columnar arrangement with emergent

huge blood vessels in the hypertrophic zone. Arrow indicates blood vessels. Columnar arrangements

of chondrocytes can be seen in parenthesis. PZ=Proliferative zone, HZ=Hypertrophic zone.

Hsp90 is a chaperone protein that aids other proteins and stabilizes them against stress. In our

experiment, by administering TCM, the mRNA expressions of Hsp90 were restored to their normal

levels in thiram-induced birds and the lameness started subsiding with an increase in blood supply in

the affected area. TD is characterized through cell death, lack of blood supply, and damage of blood

capillaries in the chondrocyte of growth plate (Rath et al., 2005). In present study, the increased

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expression of Hsp90 in TD lesions was in accordance with the previous reports (Herzog et al., 2011;

Genin et al., 2012; Shahzad et al., 2015).

In our experiment, the administration of EGCG and Apigenin increased blood supply in the

hypertrophic zone of growth plate and brought the normal differentiation. Thiram induction

promoted chicken growth plate abnormal endochondral calcification and chondrocyte proliferation

and disturbed the development of normal cartilage (Tian et al., 2009; Shahzad et al., 2014). In

conclusion, this is the first study describing the use of traditional Chinese medicine, EGCG and

Apigenin in chicken broiler to cure the lameness in broiler chicken by inhibiting the Hsp90 activity.

Fig. 6: Hsp90 mRNA expression was analyzed in chicken epiphyseal growth plates of proximal tibiae

isolated from normal, TD induced, Apegenin treated chicks. Data expressed in arbitrary units as the

mean±SE. Control group set to one thus equivalent to the N-fold difference. abLetters indicate

significant difference (P<0.05).

Acknowledgment: The study was supported by the Research Fund for the Doctoral Program of

Higher Education of China (No. 20120146110017) and The National Natural Science Foundation of

China (No.31460682).

REFERENCES

Clement Y, 2009. Can green tea do that? A literature review of the clinical evidence. Prev Med 49:

83-87.

Fang J, C Xia, Z Cao, JZ Zheng, E Reed et al., 2005. Apigenin inhibits VEGF and HIF-1 expression via

PI3K/AKT/p70S6K1 and HDM2/p53 pathways. Faseb J, 19: 342-353.

Genin O, A Hasdai, D Shinder and M Pines, 2012. The effect of inhibition of heat-shock proteins on

thiram-induced tibial dyschondroplasia. Poult Sci, 91: 1619-1626.

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Herzog A, O Genin, A Hasdai, D Shinder and M Pines, 2011. Hsp90 and angiogenesis in bone

disorders-lessons from the avian growth plate. Am J Physiol Regul Integr Comp Physiol, 301:

R140-147.

Leach RM Jr and MC Nesheim, 1965. Nutritional, genetic and morphological studies of an abnormal

cartilage formation in young 405 chicks. J Nutr, 86: 236-244.

Leung PC, 2001. Evidence-based alternative medicine Hong Kong Med J, 7: 332-334.

Pine M, A Hasdai and M Monsonego-Ornan, 2005. Tibial dyschondroplasia – tools, new insights and

future prospects World’s Poult Sci J, 61: 285-297.

Pines M, V Knopov, O Genina, S Hurwitz, A Faerman et al., 1998. Development of avian tibial

dyschondroplasia: gene expression and protein synthesis. Calcif Tissue Int, 63: 521-527.

Rath NC, MP Richards, WE Huff, GR Huff and JM Balog, 2005. Changes in the tibial growth plates of

chickens with thiram-induced dyschondroplasia. J Comp Pathol, 133: 41-52.

Shahzad M, J Liu, J Gao, Z Wang, D Zhang et al., 2015. Differential expression of extracellular matrix

metalloproteinase inducer (EMMPRIN/CD147) in avian tibial dyschondroplasia. Avian Pathol, 44:

13-18.

Shahzad M, J Gao, P Qin, J Liu, Z Wang et al., 2014. Expression of Genes encoding matrilin-3 and

cyclin-I during the impairment and recovery of chicken growth plate in tibial dyschondroplasia.

Avian Dis, 58: 468-473.

Shukla S and S Gupta, 2010. Apigenin: a promising molecule for cancer prevention. Pharm Res 27:

962-978.

Song JM, KH Lee and BL Seong, 2005. Antiviral effect of catechins in green tea on influenza virus.

Antiviral Res, 68: 66-74.

Tran PL, SA Kim, HS Choi, JH Yoon and SG Ahn, 2010. Epigallocatechin-3-gallate suppresses the

expression of HSP70 and HSP90 and exhibits anti-tumor activity in vitro and in vivo. BMC

Cancer, 10: 276.

Tian WX, WP Zhang, JK Li, DR Bi, DZ Guo et al., 2009. Identification of differentially expressed

genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia. Avian

Pathol, 38: 161-166.

Velada I, F Capela-Silva, F Reis, E Pires, C Egas et al., 2011. Expression of genes encoding extracellular

matrix macromolecules and metalloproteinases in avian tibial dyschondroplasia. J Comp Pathol,

145: 174-186.

Yang Y, A Chin, L Zhang, J Lu and RW Wong, 2014. The role of traditional Chinese medicines in

osteogenesis and angiogenesis. Phytother Res, 28: 1-8.

Yin, Z, EC Henry and TA Gasiewicz, 2009. (-)-Epigallocatechin-3-gallate is a novel Hsp90 inhibitor.

Biochemistry, 48: 336-345.

Zhang D, J Zhang, C Fong, X Yao and M Yang, 2012. Herba epimedii flavonoids suppress osteoclastic

differentiation and bone resorption by inducing G2/M arrest and apoptosis. Biochimie, 94: 2514-

2522.

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ENTERIC DISORDER IN POULTRY: NEVER-ENDING STORY

Hafez Mohamed Hafez, MVSc., Dr.med.vet; Dr. med vet habil.

Institute of Poultry Diseases, Free University Berlin, Germany

Koenigsweg 63, 14165 Berlin, Germany

[email protected]

ABSTRACT

The enteric health of growing poultry is imperative to success of the production. The basic role

of poultry production is turning feed stuffs into meat. Any changes in this turning process, due to

mechanical, chemical or biological disturbance of digestive system (enteric disorders) is mostly

accompanied with high economic losses due to poor performance, increased mortality rates and

increased medication costs.

Several pathogens (viruses, bacteria and parasites) are incriminated as possible cause of enteric

disorders either alone (mono-causal), in synergy with other micro-organisms (multi-causal), or with

non-infectious causes such as feed and /or management related factors. In addition, excessive levels of

mycotoxins and biogenic amines in feed lead to enteric disorders. The severity of clinical signs and

course of the disorders are influenced by several factors such as management, nutrition and the

involved agent(s). Under field conditions, however, it is difficult to determine whether the true cause

of enteric disorders, is of infectious or non-infectious origin.

The effect of antimicrobial growth promoters (AGP) on gut flora results in improvement of

digestion, better absorption of nutrients, and a more stable balance in the microbial population and

reduce the intestinal stress. However, AGP can also increase the prevalence of drug-resistant

bacteria. In recent years and since the ban of use of antimicrobial growth promoters in several

countries the incidence of intestinal disorders especially those caused by clostridial infection was

drastically increased. Field observations in several countries in Europe showed that poultry industry

faced several problems in after banned of AGP`s. Based on „Precautionary Principle” the EU has

decided to ban the use growth-promoting antibiotics in feed of food producing animals completely by

January 2006. The impact of the ban of the antibiotics used as growth promoters has been seen on

the performances (body weight and Feed conversion rate) as well as on the rearing husbandry (Wet

litter and ammonia level), Animal welfare problem (Foot pad dermatitis) and general health issues on

the birds (enteric disorders due to dysbacteriosis and Clostridial infections). The present review

described in general the several factors involved in enteric disorders in poultry.

Key Words: Enteric diseases, Necrotic enteritis, Poultry

Introduction

The basic role of poultry production is turning feed stuffs into meat. Broilers and meat turkeys

are very efficient at both growth and feed conversion rate. Any slight alteration from the optimal

condition is mostly accompanied by disruption of the growth process and all over performance. To

reach the maximal potential of development, considerable demands should be placed on good

intestinal health.

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Enteric disorders are one of the most important groups of diseases they affect poultry and are

continuing to cause high economic losses in the many areas world- wide due to increased mortality

rates, decreased weight gain, increased medication costs, and increased feed conversion rates. Several

pathogens (viruses, bacteria and parasites) are incriminated as possible causes of enteric disorders

either alone (mono-causal), in synergy with different other microorganisms (multi-causal) or with

non-infectious causes such as feed and /or management related factors (Table 1). Under field

conditions, however, it is difficult to determine weather the true cause of enteric disorders in poultry

is of infectious or non-infectious origin (Hafez, 2011).

Table 1: Some possible causes of enteric disorders in poultry

Non - Infectious Infectious

Feed Viral agents

Structure Reo, Astro, Entero, Rota,

Palatability Coronavirus enteritis, HE

Energy content ND, Influenza A

Pellet quality Bacterial agents

Management Salmonellas, E. coli,

Available feed space Clostridia

Available water space Mycotic agents

Distribution of feeders Candida

Distribution of waterers Parasites

Air quality Coccidia, Histomonas,

Temperature Hexamitia, Ascaridia

Stocking density

Infections with Clostridium perfringens

Infections with Clostridium perfringens (C. perfringens) in poultrycan cause several clinical

manifestations and lesions include necrotic enteritis, necrotic dermatitis, cholangiohepatitis as well

as gizzard erosion. However, subclinical infection can take place too. In addition, C. perfringens

type A has been showed to cause food poisoning in humans (Løvland and Kaldhusdal, 2001;

McClane et al., 2006; Novoa-Garrido et al., 2006).

C. perfringens is a Gram-positive, non-motile, spore-forming anaerobic bacterium which

iswidespread in soils, feed, litter and the intestinal tract of diseased and healthy birds. C. perfringens

grows extremely rapidly, with a generation time of 8-10 min, and growth is accompanied by abundant

gas production (Bryant and Stevens, 1997). The bacterial spores are very resistant to heat,

desiccation, acids and many chemical disinfectants (Willis, 1977).

C. perfringens is divided into 5 biotypes A, B, C, D, and E based on the synthesis of four major

lethal toxins: alpha, beta, epsilon, and iota. Along with these four major toxins, enterotoxin (CPE) and

beta2 (CPB2) toxins are considered as important toxins for enteric diseases. However, it is not clear

whether CPE and CPB2 are involved in C. perfringens-associated avian enteric diseases (Crespo et al.,

2007). The infections in poultry are mostly caused by C.perfringens type A, and to a lesser extent by

type C (Engström et al., 2003). Because C. perfringens type A is highly prevalent in the intestines of

healthy animals, controversy exists about its real pathogenic role (McClane et al., 2006). Additionally,

it was shown that strains isolated from necrotic enteritis outbreaks did not produce more alpha toxin

compared to isolates from the gut of clinically healthy broilers (Gholamiandehkordi et al., 2006).

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Timbermont et al. (2009) examined the ability of C. perfringens isolates from both healthy and diseased

poultry, and from calf hemorrhagic enteritis cases, producing different concentrations of alpha toxin

in vitro, to induce necrotic enteritis in broilers. The obtained results revealed that induction of

necrotic lesions in the broiler gut is not associated with the ability to produce alpha toxin in vitro.

Moreover, the results also suggest that the virulence of C. perfringens strains is to some extent host

specific since two C. perfringens strains isolated from calf hemorrhagic enteritis were not able to

produce necrotic lesions in chickens. Keyburn et al. (2008) were able to identify a novel toxin (netB)

in a C. perfringens type A strains isolated from chickens suffering from necrotic enteritis. According to

the authors this novel toxin is the first definitive virulence factor to be identified in avian C. perfringens

strains capable of causing necrotic enteritis. However, netB strain could be also found in healthy

chickens and turkeys (Gad et al., 2011a) as well as in other animal species such bovine (Martin, 2010).

On the hand, Martin (2010) reported that the majority (58%) of chickens with NE were caused by C.

perfringens isolates that were NetB positive. Under experimental condition they found that only

strains that possess NetB were capable of producing NE regardless of the source of the isolate. NetB

negative strains including those isolated from cases of NE were unable to produce NE in the disease

model. Martin and Smyth (2009) also found a strong correlation between the detection of the cpb2

gene and netb gene. However, when interpreting the results it has to be kept in mind that the

presence of the gene of a toxin does not necessarily mean that the toxin is produced, as it was shown

for netb toxin (Abildgaard et al., 2010) or cpb2 (Crespo et al.,2007).

Necrotic enteritis (NE)

NE is an acute disease caused by C. perfringens when proliferates to high numbers in the small

intestine and produces toxins responsible for damaging the intestinal lining. The disease has been

observed in several domestic and wild birds worldwide. Recently several reviews were published

(Van Immerseel et al., 2004; Opengart, 2008, Hafez, 2011). Beside clinically manifested disease,

subclinical infections may take place and are mostly accompanied with reduction of performance. The

most important source of infection in poultry appears to be contaminated feed, litter, water and the

environment. In addition, some reports about the possible vertical transmission have been published

(Köhler et al., 1974; Craven et al., 2003). Recently, Martin (2010) were able to demonstrate under

experimental condition, that factors such as co-infection with Eimeria species, genotype of chicken

and the strain of C. perfringens were the most critical factors involved in disease development, while

other factors such as age of chickens, contact with litter and protein content of the diet played a

lesser role.

After experimental infection, the first mild clinical signs are evident approximately 24 to 36

hours after administration of a pure C. perfringens culture to broiler chickens. The clinical signs

appear suddenly; apparently healthy birds may become acutely depressed and die within hours.

Mortality ranges between 2 and 10%. Affected birds show ruffled feathers, marked depression, in-

appetence, tendency to huddle, watery droppings and diarrhoea.

On autopsy dehydration is the most common finding. Breast muscles are dark red and gizzards

are full of litter. Severe inflammation in the duodenum and jejunum is the most predominant finding,

but in some instances the entire length of the intestinal tract is involved.

The intestine is distended thin walled and filled with gas and contains dark offensive fluid. The mucosa

is covered with green or brown diphteroid membrane, which can be easily separated from the lining.

As the condition progresses, areas of necrosis can be recognized from outside of the intestine.

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The presence of C. perfringens in the intestinal tract or inoculation of the animals with high doses

of C. perfringens, however, does generally not lead to the development of necrotic enteritis. One or

several predisposing factors may be required to elicit the clinical signs and lesions. It appears that

some dysfunctions of the alimentary tract are necessary predisposing cause of infection. Intestinal

stasis, intestinal distension, coccidiosis, salmonellosis, crop mycosis and haemorrhagic enteritis (HE)

may predispose the birds to infection. Also consumption of diets high in energy, protein and fish meal

as well as the consumption of high fibre litter and wheat based diet (Kaldhusdal and Skjerve, 1996;

Kocher, 2003 Williams, 2005). In addition, Siegel et al. (1993) reported that genetic susceptibility

could be an additional factor, which can influence the course of infection.

Cholangiohepatitis

Cholangiohepatitis causes severe economic losses due to high liver condemnation rate on the

processing and downgraded of the slaughtered carcasses. Clostridium perfringens is usually isolated in

association with the disease. The hepatitis characterized by an enlarged firm liver sometimes with a

slightly knobby surface and a medium tan colour. Histopathological lesions consist of hyperplasia of

the bile duct, fibrinoid necrosis, cholangitis and occasionally focal granulomatous inflammation

(Onderka et al., 1990; Løvland and Kaldhusdal, 1999; Sasaki et al., 2000).

Gizzard erosions

Gizzard erosions has been observed in commercial broiler chickens and several non-infectious

factors such as mycotoxin-contaminated feed, vitamin B6 and E deficiency, inadequate levels of

sulphur-containing dietary amino acids, high levels of dietary copper, pelleted feed as well as inclusion

of certain fish meals in the diets and were discriminated as possible cause. Ono et al. (2003) reported

on Outbreaks of adenoviral gizzard erosion in slaughtered broiler chickens in Japan and Novoa-

Garrido et al. (2006) found a significant association between gizzard lesions and increased caecal C.

perfringens counts in broiler chickens.

Diagnosis

A presumptive diagnosis may be made from the case history, clinical signs, lesions and staining

fresh smears of upper part of the intestinal tract with Gram stain showing an abundant number of

clostridia organisms. This should be confirmed by the isolation of the causative agent. For isolation

several media are available such as sheep blood agar supplemented with neomycin or tryptose-sulfite-

cycloserine agar (TSC). The identification can be carried out using biochemical tests. In addition, PCR

was developed to detect of alpha toxin (Heikinheimo and Korkeala, 2005) as well as a real-time PCR

for quantitative detection of C. perfringens in gastrointestinal tract of poultry (Wise and Siragusa,

2005). Also ELISAs for direct detection of C. perfringens major toxins and enterotoxin are

commercially available.

Treatment

Treatment with antibiotics such as penicillin, amoxicillin, ampicillin, erythromycin,

dihydrostreptomycin and tetracyclin provided a satisfactory clinical response. Penicillin’s are

known to be particularly active against C. perfringens. Resistance to penicillin is very rare and β-

lactamase has not been demonstrated. Three days is the minimum duration of treatment, however

longer applications may be required. Recently, Gad et al. (2011b) were determined the minimum

inhibitory concentrations of 16 antibiotics for 100 Clostridium perfringens isolates collected between

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2008 and 2009 from commercial turkey flocks using a commercially available broth micro- dilution

test kit. No isolates were resistant against β- lactam antibiotics (amoxicillin, oxacillin, and penicillin),

lincospectin, tylosin, doxycyclin, tetracycline, enrofloxacin, trimethoprim/sulfamethoxazole,

lincomycin, and tilmicosin. A low frequency of resistance was detected against erythromycin and

tiamulin with 5 and 20%, respectively. Spectinomycin, neomycin and colistin showed the highest

incidence of resistance with 74, 94 and 100%, respectively. Similar results were also obtained by

testing from strain collect from layery flocks. No isolates were resistant against β-lactam antibiotics

(amoxicillin, oxacillin, and penicillin), lincospectin, tylosin, doxycyclin, tetracycline, enrofloxacin,

trimethoprim/ sulfamethoxazole, lincomycin, and tilmicosin. A low frequency of resistance was

detected against erythromycin and tiamulin with 17.4% and 19.6% respectively. However, most of the

isolated (67.4%) were partially sensitive to erythromycin. Spectinomycin, neomycin, and colistin

showed the highest incidence of resistance with 87.0%, 93.5%, and 100% respectively (Gad et al.,

2012)

According to Brennan et al. (2000) administration of dietary Tylan® for seven consecutive days

following confirmation of an NE field outbreak reduced the NE mortality and lesion score and

improved overall growth as well as feed conversion in broilers. The optimum dose of Tylan to

control NE was 100 ppm. No resistance to the ionophorous anticoccidial drugs such as Narasin has

been found (Martel et al., 2004). Brennan et al. (2001) reported that Narasin, when administrated at

70 ppm in feed from Day 0 to 41 prevents morbidity, mortality and suppression of growth and feed

conversion associated with NE in broilers.

Vaccination

Active and passive immunity using vaccination against C. perfringens and its toxins appears to

offer protection. Heier et al. (2001) found out that broiler flocks with high titres of maternal

antibodies against C. perfringens alpha-toxin had lower mortality during the production period than

flocks with low tiers. Also Løvland et al. (2004) use toxoids vaccines based on C. perfringens type A

and C toxoids to vaccinate breeder flocks. The IgG responses in vaccinated parent hens were distinct

and the levels of antibodies to C. perfringens alpha - toxin in progeny of the vaccinated hens was high

enough to protect the progeny against subclinical C. perfringens associated necrotic enteritis. On the

other hand several recent investigations showed that immunity to NE after oral infection using

virulent strain and subsequent treatment is much better than using avirulent C. perfringens strains

and they identified immunogenic secreted proteins apparently uniquely produced by virulent C.

perfringens isolates and concluded that there are certain secreted proteins beside to alpha-toxin, that

are involved in immunity to NE in broiler chickens (Thompson et al., 2006; Kulkarni et al., 2007).

Further additional study showed the ability of oral immunisation against C. perfringens in broiler

chickens using an attenuated Salmonella vaccine vector (Kulkarni et al., 2008).

Alternatives to AGPs

There are several approaches to overcome the detrimental effects on poultry performance after

a withdrawal of antimicrobial grpwth promotors. Investigations indicate that competitive exclusion,

prebiotics, probiotics, enzymes and acids can impact the incidence and severity of NE in poultry. The

data suggest that these products may provide the poultry industry with an alternative management

tool that has the potential to promote better intestinal health and decrease monetary losses due to

C. perfringens (McReynolds et al., 2009). At this moment it is difficult to evaluate novel strategies

developed to antibiotic-free feeding concepts. Combination of different approaches is necessary to

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enhance the performance and reduced health status of the birds. The practical relevance of these

approaches may vary between the different areas in the world. Currently, a wide range of novel feed

additives are commercially available. The suggested modes of action are summarized in Table 2 as

described by (Langhout et al., 2003).

Table 2: Different groups of non-antibiotic feed additive and their suggested mode of action

Additive Possible mode of action

Probiotics Introduction of desirable bacteria into the gastrointestinal tract

Prebiotics Promotion of the growth of desirable bacteria in the gastrointestinal

tract

Enzymes Elimination of the anti-microbial effects of carbohydrates

Immune stimulating

products

Reducing sub-clinical infections via an improved development of the

immune system

Acids Inhibition of the growth of bacteria

Essential oils Inhibition of the growth of bacteria, improving the development of

the immune system, improving the palatability of the diet

On the other hand (Thiery, 2005) tried many alternatives to growth promoters in turkey feed. Most

of results of the additives he tried were very disappointing. They sometimes show a slight effect at

one period, but no effect at the end of the rearing period, sometimes even showing a negative effect

(Table 3).

Table 3: Experience with different groups of non-antibiotic feed additive in meat Turkey

Prebiotics One result with MOS at 3rd weeks of age on the feed conversion rate, but

nothing after

Probiotic Sometimes a slight effect on FCR at one point.

Once an interesting result on the decreasing of the

E. coli population in the gut.

Essential oils and spices Slight or no effect for a competitive cost.

Nevertheless, the association of some plant extracts with other selected

additives may improve the droppings quality as well as FCR.

Acidifiers Nothing at all (single or blended, encapsulated or not…)

Clays Improve the quality of the pellets

Conclusions

Implementation of several approaches such as improvement of management, feed formulation

and use of alternative products to modulate the intestinal flora led to an improvement of the

situation. Limiting exposure to infectious agents through biosecurity, vaccination, supportive therapy,

cleaning and disinfection are essential. In addition, early recognition in managing the enteric disorders

is very important. Finally, use of an effective anticoccidial drug in the ration is helpful to minimise the

effect of enteritis. Since, recent investigations showed that the use of some alternative products might

be able to reduce the intestinal colonization with pathogenic bacterial agents. This could be an

additional tool to reduce enteric disorders in future.

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References

Abildgaard L, TE Sondergaard, RM Engberg, A Schramm, O Højberg, 2010. In vitro production of

necrotic enteritis toxin B, NetB, by netB-positive and netB-negative Clostridium perfringens

originating from healthy and diseased broiler chickens. Vet Microbiol, 144: 231-235.

Brennan JJ, G Moore, S Poe, G Vessie, J Wilson et al., 2000. Efficacy of dietary tylosin phosphate

(Tylan) for control of necrotic enteritis in broiler chickens. 89th Meeting Poultry Science Assoc,

Montreal, Canada (Abs.).

Brennan JJ, R Bagg, DA Barnum, J Wislon and P Dick, 2001. Efficacy of Narasin in the prevention of

necrotic enteritis in broiler chicks. Avian Dis, 45: 210-214.

Bryant AE and LS Stevens, 1997. The Pathogenesis of Gas Gangrene. Academic Press, San Diego,

USA, pp: 186-187.

Craven SE, NA Cox, JS Bailey and DE Cosby, 2003. Incidence and tracking of Clostridium perfringens

through an integrated broiler chicken operation. Avian Dis, 47: 707-711.

Crespo R, DJ Fisher, HL Shivaprasad, ME Fernandez-Miyakawa and FA Uzal, 2007. Toxinotypes of

Clostridium perfringens isolated from sick and healthy avian species J Vet Diag Invest, 19: 329-

333.

Engström BE, C Fermer, A Lindberg, E Saarinen, V Båverud et al., 2003. Molecular typing of isolates of

Clostridium perfringens from healthy and diseased poultry. Vet Microbiol, 94: 225-235.

Gad W, R Hauck, M Krüger and HM Hafez, 2011a. Prevalence of Clostridium perfringens in

commercial turkey and layer flocks. Archiv für Geflügelkunde, 75: 74-79.

Gad W, R Hauck, M Krüger and HM Hafez, 2011b. Determination of antibiotic sensitivities of

Clostridium perfringens isolates from commercial turkeys in Germany in vitro. Archiv für

Geflügelkunde, 75: 80-83.

Gad W, R Hauck, M Krüger and HM Hafez 2012. In vitro determination of antibiotic sensitivities

of Clostridium perfringens isolates from layer flocks in Germany Arch. Geflügelk., 76: 234-238.

Gholamiandehkordi A, R Ducatelle, M Heyndrickx, F Haesebrouck and F Van Immerseel, 2006.

Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry

flocks with different disease status. Vet Microbiol, 113:143-152.

Hafez HM, 2011. Enteric diseases of poultry with special attention to Clostridium perfringens. Pak

Vet J, 31: 175-184.

Heier BT, A Løvland, KB Soleim, M Kaldhusdal and J Jarp, 2001. A field study of naturally occurring

specific antibodies against Clostridium perfringens alpha toxin in Norwegian broiler flocks. Avian

Dis, 45: 724-732.

Heikinheimo A and H Korkeala, 2005. Multiplex PCR assay for toxinotyping Clostridium

perfringens isolates obtained from Finnish broiler chickens. Letters Appl Microb, 40: 407-411.

Kaldhusdal M and E Skjerve, 1996. Association between cereal contents in the diet and incidence of

necrotic enteritis in broiler chickens in Norway. Prev Vet Med, 28: 1-16.

Keyburn AL, JD Boyce, P Vaz, TL Bannam, ME Ford et al., 2008. NetB, a new toxin that is associated

with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog 4: e26. doi: 10.1371

/journal. ppat.0040026.

Kocher A, 2003. Nutritional manipulation of necrotic enteritis outbreak in broilers. Recent Adv

Anim Nutr (Australia), 14: 111-116.

Köhler B, S Kölbach and J Meine,1974. Untersuchungen zur nekrotischen Enteritis der Hühner. 2.

Mitteilung: Mikrobiologische Aspekte. Monatsh für Veterinärmedizin, 29: 385-391.

Page 60: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

60

Kulkarni RR, VR Parreira, S Sharif and JF Prescott, 2007. Immunization of Broiler Chickens against

Clostridium perfringens -Induced Necrotic Enteritis. Can Vet J, 14: 1070-1077.

Kulkarni RR, VR Parreira, S Sharif and JF Prescott, 2008.Oral immunization of broiler chickens against

necrotic enteritis with an attenuated Salmonella vaccine vector expressing Clostridium

perfringens antigens. Vaccine, 26: 4194-4203.

Langhout P and P Wijtten, 2003. The use of antimicrobial, enzymes, prebiotics, probiotics, essential

oils and organic acids in broilers; a review. In: Latin American Poultry Congress, 18., 2003, Santa

Cruz de La Sierra, 2003. Proceedings. Santa Cruz de La Sierra: XVIII Congresso Latino

Americano de Avicultura. CD-Rom. anghout P, 2007. Broilers nutrition optimisation. Afma

Matrix, 16: 33-37.

Løvland A and M Kaldhusdal, 1999. Liver lesions seen at slaughter as an indicator of necrotic enteritis

in broiler flocks. FEMS Microbiol Med Microbiol, 24: 345-351.

Løvland A, M Kaldhusdal, K Redhead, E Skjerve and A Lillehaug, 2004. Maternal vaccination against

subclinical necrotic enteritis in broilers. Avian Path, 33: 81-90.

Martel A, LA Devriese, K Cauwerts, K De Gussem, A Decostere et al., 2004. Susceptibility of

Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials. Avian Path,

33: 3-7.

Martin TG and JA Smyth, 2009: Prevalence of netB among some clinical isolates of Clostridium

perfringens from animals in the United States. Vet Microbiol, 136: 202-205.

Martin TG, 2010. The importance of the strain of Clostridium perfringens in the development of

necrotic enteritis of poultry. Dissertations Collection for University of Connecticut. Paper

AAI3402008. http://digitalcommons.uconn.edu/dissertations/AAI34 02008. Accessed on January

1, 2010.

McClane BA, FA Uzal, MEF Miyakawa, D Lyerly and T Wilkins, 2006. The Enterotoxic Clostridia. In:

The Prokaryotes: A handbook on the biology of bacteria. Dworkin M and Falkow S eds. Springer,

pp: 763- 778.

McReynolds J, C Waneck, J Byrd, K Genovese, S Duke et al., 2009. Efficacy of multistrain direct-fed

microbial and phytogenetic products in reducing necrotic enteritis in commercial broilers. Poult

Sci, 88: 2075-2080.

Novoa-Garrido M, S Larsen and M Kaldhusdal, 2006. Association between gizzard lesions and

increased caecal Clostridium perfringens counts in broiler chickens. Avian Path, 35: 367-372.

Onderka DK, CC Langevin and JA Hanson, 1990. Fibrosing cholehepatitis in broiler chickens

induced by bile duct ligations or inoculation of Clostridium perfringens. Can J Vet Res, 54: 285-

290.

Ono M, Y Okuda, S Yazawa, I Shibata, S Sato et al., 2003. Outbreaks of adenoviral gizzard erosion in

slaughtered broiler chickens in Japan. Vet Rec, 153: 775-779.

Opengart K, 2008. Necrotic enteritis. In: Diseases of Poultry.12th Ed, Saif YM, Fadly AM, Glisson JR,

McDougald LR, Nolan LK and Swayne DE, Eds. Iowa State University Press. Iowa, USA, pp:

872- 879.

Sasaki J, M Goryo and K Okada, 2000. Cholangiohepatitis in chickens induced by bile duct ligations

and inoculation of Clostridium perfringens. Avian Path, 29: 405-410.

Siegel PB, AS Larsen, CT Larsen and EA Dunnington, 1993. Resistance of chickens to an outbreak of

necrotic enteritis as influenced by major histocompatibility genotype and background genome.

Poult Sci, 72: 1189-1191.

Page 61: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

61

Thompson DR, VR Parreira, RR Kulkarni and JF Prescott, 2006. Live attenuated vaccine-based

control of necrotic enteritis of broiler chickens. Vet Microbiol, 113: 25–34.

Thiery P, 2005. How to deal with the removal of the antibiotic growth promoters (AGP) in Turkeys,

consequences for the producer- The French experience. In: Turkey production: prospects on

future developments (Ed. Hafez, H.M). Mensch & Buch Verlag, Berlin-Germany. ISBN: 3-89820-

993-8. pp: 63-69.

Van Immerseel F, J De Buck, F Pasmans, G Huyghebaert, F Haesebrouck et al., 2004. Clostridium

perfringens in poultry: an emerging threat for animal and public health. Avian Path, 33: 537-549.

Williams RB, 2005. Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated

disease management by maintenance of gut integrity. Avian Path, 34: 159-180.

Willis AT, 1977. Anaerobic-bacteriology: clinical and laboratory practice. (3 ED) pp: 360.

Butterworths, London,

Thompson DR, VR Parreira, RR Kulkarni and JF Prescott, 2006. Live attenuated vaccine-based

control of necrotic enteritis of broiler chickens. Vet Microbiol, 113: 25–34.

Timbermont l, A Lanckriet, AR Gholamiandehkordi, F Pasmans, A Martel et al., 2009. Origin of

Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers.

Comp Immunol Microbiol Infect Dis, 32: 503–512

Wise MG and GR Siragusa, 2005. Quantitative Detection of Clostridium perfringens in the Broiler

Fowl Gastrointestinal Tract by Real-Time PCR. Appl Environ Microbiol, 71: 3911-3916.

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HA AND NA GENE SEQUENCE ANALYSIS OF 13 H9N2 SUBTYPE AVIAN

INFLUENZA VIRUSES ISOLATED IN SHANDONG, CHINA

Guanggang Qu1,4, Zhiqiang Shen1*, Wu Ai2, Bing Huang2, Changjiang Wang3, Yuexing Wu3, Maofeng

Li3, Jishan Liu1, Jinlong Chen3 and Feng Wei1,4

1Binzhou Animal Science and Veterinary Medicine Academy of Shandong Province, Binzhou 256600,

PR China; 2Institute of poultry, Shandong Academy of Agricultural Sciences, Jinnan 250023, PR China; 3Shandong Lvdu Bio-Technology Co., Ltd, Binzhou 256600, PR China; 4Shandong Binzhou

Research,Development and Promotion Center for Livestock and Poultry Propolis VaccineS,

Binzhou 256600, PR China

*Corresponding Author: [email protected]

ABSTRACT

In order to understand the genetic variation and epidemic regularity of avian influenza virus

subtype H9N2 in Shandong Province, this research collected 13 strains of H9N2 subtype isolated

from different chicken farms in Shandong. HA and NA gene sequence of H9N2 virus amplification

was operated by RT-PCR to determine sequence. Homology analysis and genetic evolution analysis

were applied to analyze the HA and NA gene complete sequences of avian influenza viruses. Results

showed that 12 of the isolates HA genes belonged to S2-like, which is a classical prevalent strain in

Shandong. The homology of nucleotide sequences (amino acid sequences) between the isolates and

CK /SD /S2/2005 is 92.0%-96.3% (93.9%-97.7%). However, the relationship of the isolates and the

BJ94 strain which is the primary strain isolated in China is far, implying the evolution of HA gene of

AIV. While the homology of NA gene of SD 02 isolate is far from that of other 12 isolates and the

reference strains.

Key Words: Avian influenza virus, H9N2 subtype, HA gene, NA gene, genetic evolution analysis

INTRODUCTION

Avian influenza is a highly viral contagious disease caused by type A orthomyxovirus, which has

multiple serum subtypes with frequent genetic variation. According to the surface glycoproteins:

hemagglutinin (HA) and neuraminidase (NA), these viruses are divided into subtypes (Guan et al.,

1996). At present, 17 HA (H1-H17) and 10 NA (N1-N10)subtypes have been recognized (Zhang

et al., 2013). All subtypes were identified initially from avian species, except for the H17N10 subtype,

which was isolated from bats (Tong et al., 2012). Based on the difference in pathogenicity and

virulence, avian influenza viruses can be divided into two distinct groups (Capua and Alexander,

2014), which are highly pathogenic avian influenza viruses (HPAIV) and low pathogenic avian influenza

viruses (LPAIV). The HPAIV with high fatality rate resulted in serious economic losses in the poultry

industry. However, the harmful effect caused by LPAIV cannot be overlooked. Among the LPAIV, the

H9N2 subtype has been most studied because of its pandemic potential and its successful

transmission to humans (Seifi et al., 2010). In 1966, the H9N2 subtype avian influenza virus was

isolated initially from turkey population in America and it was mainly found in aquatic birds and wild

ducks of North America then in China, H9N2 AIV was firstly isolated from chickens population in

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1994 (Chen et al., 1994; Li et al., 2003; Liu et al., 2003a,2003d), and since then this subtype of avian

influenza virus has been widely spread in China's major poultry breeding areas. Infection of H9N2

often results in respiratory symptoms, decrease of egg laying rate, immunosuppression and leading to

infection by other pathogens, despite its low pathogenic characteristic, which caused tremendous

economic loss to Chinese poultry industry (Chen et al., 2012). Furthermore, it may lead to potential

crises of anthropozoonosis because of the fact that H9N2 virus contains internal genes needed for

the human transmission of the H5N1 subtype (Guo et al., 2000).

The complete genome of the H9N2 virus is composed of eight antisense single-stranded RNA

fragments encoding at least 11 kinds of protein, of which hemagglutinin (HA) and neuraminidase (NA)

are two important elements of the surface of the virus. HA gene and NA gene are two most frequent

mutations in the genome of avian influenza viruses, which are closely related to the virulence and

transmission of the virus. HA, the main surface antigen and protective antigen of influenza virus, plays

a key role in virus adsorption and membrane-spanning process, and it can stimulate the body to

produce antibodies. The amino acid sequence of HA cleavage site and potential glycosylation sites are

the key factors to determine the pathogenicity and virulence of avian influenza viruses. As one of the

major glycoproteins on the surface of the avian influenza virus, NA plays an important part in the

diffusing of the virus which can avoid accumulation of the virus when budding with the function of

cracking the connection between hemagglutinin and cell surface sialic acid, on the other hand, NA has

the effect on the cutting ability of HA gene, resulting in the different pathogenicity of different avian

influenza subtypes to some extent.

In this study, the HA and NA genes of 13 H9N2 AIV strains isolated from chickens from

Shandong provinces in 2015 were analyzed with other publicly available sequences of H9N2 virus , to

predict the trend of H9N2 virus evolution in Shandong provinces in China.

MATERIALS AND METHODS

Sample collection: The H9N2 viruses used in this study were isolated from the lung samples of

sick chickens from Chicken Farms in Shandong Province in 2005. All lung samples were stored at

−80ºC until used for molecular diagnosis and virus isolation.

Virus detection: The frozen lung samples were homogenized with cold phosphate-buffered saline

(PBS) (pH 7.2) and then centrifuged at 12000 rpm, 4ºC for 15 min to remove the solid debris. The

homogenates was collected for viral RNA extraction by using a QIAamp Viral RNA Mini Kit (Qiagen

Inc., Valencia, CA, USA) in accordance with the manufacturer’s instructions. Total RNA was eluted

from the column in a final volume of 60 μl and stored at −80ºC. RT-PCR detection was performed by

using PrimeScript™ One Step RT-PCR Kit (TAKARA, Dalian, China) with the specific primers (Table

1) directed to the matrix (M) gene.

Virus isolation: 100 μl the homogenates positive by RT-PCR containing 1000 U/ml of penicillin and

1000 μg/ml of streptomycin were inoculated into the allantoic cavity of 9-11days old specific-

pathogenic-free (SPF) chicken eggs and the eggs were incubated for 48–72 h at 37ºC and harvested

according to the standard protocols described in the WHO Manual on Animal Influenza Diagnosis

and Surveillance (Webster et al., 2005). The hemagglutination assay was performed following a

previously described method (Reed and Muench, 1938; Webster et al., 2005).

Primer design and sequence determination: According to the HA and NA gene sequences of

AIV were downloaded from GenBank, to find relatively conservative region, the upstream and

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downstream primers were designed by Primer 5.0. And the primers were synthesized by Shanghai

Generay Biotech Co., Ltd. The upstream primer of HA gene: F: 5’-ACTCAAGATGG

AAGCACTATCAC-3’.The downstream primer of HA gene: R: 5’-GGTGTTTTT

GCCAATTATATACA-3’.The upstream primer of NA gene: F: 5’-AGCRAAAG

CAGGAGTAAAAATGAAT-3’.The downstream primer of NA gene: R: 5’-AGTAGA

AACAAGGAGTTTTTTCTAAAA-3’.

Sequence analysis: Virus RNA was extracted according to the QIAamp Viral RNA Mini Kit (Qiagen

Inc., Valencia, CA, USA) guidelines. Reverse transcription followed by PCR was conducted using the

specific primers for HA and NA following the M-MLV reverse transcription manual (New England

Biolabs, Inc, Ipswich, MA, USA). The PCR was performed in a volume of 25 μL, with the reaction

mixture containing buffer mixture 12.5 μL, primer mixture (10 μM) 2 μL, template cDNA 3 μL,

DiH2O.7.5 μL and conducted as follows: 94 ℃ for 5 min followed by 35 cycles at 94 C for 45 s, 50ºC

for 45 s, 72ºC for 2 min with a final extension of 72ºC for 10 min.The product was purified using a

PCR purification kit (Promega, Madison, WI, USA) and ligated into the PMD18-T vector (TAKARA).

The recombinant plasmid was extracted from the by the Wizard_ Plus SV Minipreps

(Promega, Madison, WI, USA) and sequenced by Shanghai Sangon Biotech Co.,Ltd. The sequencing

results were submitted to NCBI to BLAST with sequences in GenBank, and Phylogenetic analyses

were performed with other influenza virus sequence data available in GenBank using software

Lasergene 7.0 (DNAStar).

RESULTS

Identification and Isolation of H9N2: To identify the virus using RT-PCR, the results showed 13

samples were H9N2 subtype positive. In order to isolate the viruses, 9~11 days old SPF chicken

embryos were inoculated with the extraction of pathologic specimen and the allantoic fluid was

harvested at 72h. RT-PCR test results showed that these isolates were H9N2 subtypes. The HA test

was done for the 13 ofs strains virus (Table 1).

Table 1: H9N2 influenza viruses sequenced in the present study

Strains Genotype Amio acids of RBS in HA gene Cleavage site HA

titer 183 190 226 228

SD 01 S I G R PARSSR GL 10

SD 02 N I G R PSRSSR GL 9

SD 03 N I G R PSRSSR GL 8

SD 04 N I G R PSRSSR GL 10

SD 05 N I G R PSRSSR GL 9

SD 06 N I G R PSRSSR GL 9

SD 07 N I G R PSRSSR GL 6

SD 08 N I G R PSRSSR GL 8

SD 09 N I G R PSRSSR GL 11

SD 10 N I G R PSRSSR GL 8

SD 11 N I G R PSRSSR GL 11

SD 12 N I G R PSRSSR GL 9

SD 13 N I G R PSRSSR GL 11

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Genetic evolution analysis: By RT-PCR assay, HA and NA genes with a length of 1.7 kb and 1.4

kb separately from 13 isolates, were amplified and the size of the specific segments were consistent

with the expected results (Fig.1). Homology comparison were operated for HA and NA genes of the

13 isolates and reference H9N2 subtypes downloaded from GenBank to draw phylogenetic trees,

respectively.

Fig. 1: RT-PCR amplification of HA and NA genes

The phylogenetic tree of HA gene segments showed that SD 01 isolate belonged to BJ94-like

strain; and the other 12 viruses belonged to the S2-like sub lineage, implying that this kind of

genotype may be the dominant H9N2 strain in Shandong Province. The phylogenetic tree of NA

genes showed that all of the NA genes of the isolates belonged to Y280-like subgroup except for the

one of SD 02 which keep aboriginal and was far from other isolates and reference strains in

relationship. Unanimously, the NA protein sequences of 12 H9N2 isolates had a deletion of 3 amino

acids in 62-65th site except that of SD 02.

HA genes sequence and nucleotide (amino acids) homology analysis: The HA genes of 13

H9N2 isolates were sequenced. Results showed that 12 of them had an open reading frame (ORF)

with length of 1683 bp, encoding 560 amino acids and the rest one had an ORF with length of 1680

bp, encoding 559 amino acids. The homologies comparison result of HA suggested that 12 of the

isolates except for SD 01 strain shared higher homology with reference strains isolated in 2011-2013

than these isolated in the early years, while SD01 strain was highly identical to the vaccine strain SD-6

isolated in 1996. The H9N2 viruses we isolated in 2015 shared nucleotide (amino acid) homologies

of 92.5-100% (94.1-100%), but only homologies of 89.3%-91.6% (90.2%-93.2%) with the vaccine strain

SD-6.

1.7k bp 1.4k bp

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Fig. 2: The phylogenetic tree of HA genes

Amino acid sequence analysis of HA cleavage site and receptor binding site: To investigate

the molecular characteristics of H9N2 influenza viruses in Shandong province, the deduced amino

acid sequences of the HA protein of isolated strains were aligned. The analysis of key amino acid

residues of HA showed that the amino acid sequence near the cleavage site was PSRSSR/G and

contains an A to S substitution at 334th amino acid residues (Table 1), compared to the315- PARSSR/G-

321 motif of most early BJ/94-like viruses. It was proved that the leucine residue at position 226 in

HA is related to the specificity of human virus-like receptor, while our sequence showed the position

226 were G not L among the 13 isolates. Deficiency of basic amino acids in the cleavage site sequence

implied that these isolates were low pathogenic avian influenza viruses.

In HA sequence of avian influenza (Fig. 2), the X which is consistent with the sequence of NXT/S,

is considered to be a potential glycosylation site that can affect the binding ability of HA receptor and

virulence of the virus. The SD 02 strain shares 8 potential glycosylation sites which has a quantity

advantage than other isolates (Table 2).

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Table 2: Potential glycosylation site of HA

Viruses Potential glycosylation site

29 141 145 218 298 305 313 492

NST NVS NGT NRT NTT NVS NCS NGT

SD 01 + + + - + + - +

SD 02 + + + + + + + +

SD 03 + + - + + + + +

SD 04 + + - - + + + +

SD 05 + + - + + + + +

SD 06 + + - + + + + +

SD 07 + + - - + + + +

SD 08 + + - - + + - +

SD 09 + + - + + + + +

SD 10 + + - - + + + +

SD 11 + + - + + + + +

SD 12 + + - - + + + +

SD 13 + + - + + + + +

Amino acid deficiency analysis of NA: Amino acid deficiency is very common in the H9N2

subtypes, and only part of the H9N2 strains encodes a complete NA amino acid sequence (Fig. 3). All

of the NA amino acid sequence of H9N2 isolates except SD 02 has a lack of 3 amino acids in 63-65th

site.

DISCUSSION

H9N2 subtype virus has been an important one that is influencing the poultry industry since it

was first identified in China in 1994 and it exist in chickens, ducks and a variety of other birds. In

addition, the fact that more and more of the H9N2 strains with receptor binding capacity can infect

people implies that we should pay close attention to the genetic evolutionary characteristics and

molecular epidemiology of this subtype of avian flu virus.

In the present study, we isolated and identified 13 H9N2 subtypes of avian influenza, and analyzed

the genetic evolution of the HA and NA genes of these viruses with the available sequence data

downloaded from NCBI. Based on the epidemic region of the H9N2 subtype AIV, it is divided into

Eurasian pedigree and the American pedigree. The Eurasian lineage is prevalent in Eurasia, especially

in East Asia and Southeast Asia, and the American pedigrees are mainly in the North America. Most

of China’s epidemic H9N2 strains belong to Eurasian lineages except for Y439 occurred in Hong

Kong and CK HL35 (DQ064366) are American pedigree. There’s no American pedigree H9N2

subtypes found in Shandong Province.

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Fig. 3: The phylogenetic tree of NA genes

The result of phylogenetic analysis of HA and NA genes of H9N2 strains showed that the HA

cleavage site sequence of the 13 isolated strains was PSRSSR/GL despite of an A to S substitution.

According to the characteristics of HA cleavage site of the virus, all of the 13 H9N2 isolates belonged

to the low virulence strains with no consecutive basic amino acids sequences (Liu et al., 2003). It is

generally believed that the 191st amino acid of HA receptor binding site is the most conservative one

and the amino acid of 13 H9N2 isolates in this site was Amine (N) in our study. The amino acid of

234 site usually changes first when the H9N2 subtype to adjust to a new host (Matrosovich et al.,

2000). The amino acid of 12 strains of the isolates in our study was leucine (L) in that site, coinciding

with the H9N2 subtype of Hong Kong human resource, except for SD 01 strain with glutamine (Q) in

that site.

Guo et al. (2002) believe that the deficiency of 3 amino acids in the 63-65th site of NA amino acid

sequence is a useful genetic marker (Guo et al., 2002), and Liu think that it’s a mark of Chinese H9N2

subgroup. The 12 H9N2 strains have this deficiency in our study except SD 02 isolates. Whether this

deficiency of amino acids has an effect on the virulence of H9N2 still needs to be studied.

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REFERENCES

Capua I and DJ Alexander, 2004. Avian influenza: recent developments. Avian Pathol, 33: 393-404.

Chen F, ZQ Yan, J Liu, et al., 2012. Phylogenetic analysis of hemagglutinin genes of 40 H9N2 subtype

avian influenza viruses isolated from poultry in China from 2010 to 2011. Virus genes, 45: 69-75.

Guan Y, KF Shortridge, S Krauss et al., 1999. Molecular characterization of H9N2 influenza viruses:

were they the donors of the “internal” genes of H5N1 viruses in Hong Kong?. Proceedings of the

National Academy of Sciences, 96: 9363-9367.

Guo XF, M Liao, CA Xin, 2002. Cloning and sequencing of HA and NA gene of A/ Chicken/ Guangxi/

99(H9N2). J Anim Husb Vet Med, 33: 486-491.

Guo YJ, S Krauss, DA Senne et al. 2000. Characterization of the pathogenicity of members of the

newly established H9N2 influenza virus lineages in Asia. Virology, 267: 279-288.

Liu H, X Liu, J Cheng et al. 2003. Phylogenetic analysis of the hemagglutinin genes of twenty-six avian

influenza viruses of subtype H9N2 isolated from chickens in China during 1996-2001. Avian Dis,

47: 116-127.

Matrosovich M, A Tuzikov, N Bovin et al., 2000. Early alterations of the receptor-binding properties

of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J

Virol, 74: 8502-8512.

Seifi S, K Asasi, A Mohammadi, 2010. Natural co-infection caused by avian influenza H9 subtype and

infectious bronchitis viruses in broiler chicken farms. Vet Arhiv, 80: 269-281.

Tong S, Y Li, P Rivailler et al., 2012. A distinct lineage of influenza A virus from bats. Proceedings of

the National Academy of Sciences, 109: 4269-4274.

Zhang Q, J Shi, G Deng et al., 2013. H7N9 influenza viruses are transmissible in ferrets by respiratory

droplet. Science, 341: 410-414.

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MYCOTOXICOSIS: A PERSISTENT THREAT TO POULTRY INDUSTRY

Muhammad Zargham Khan*, Sheraz Ahmed Bhatti, Aisha Khatoon,

Ahrar Khan and Muhammad Kashif Saleemi

Department of Pathology, University of Agriculture Faisalabad, Pakistan

*Corresponding author: [email protected]

ABSTRACT

Fungi are ubiquitously present in the agricultural products and by-products. Mycotoxins, the

secondary metabolites of toxigenic species of fungi are universally present in the agricultural

products. So far more than 400 mycotoxins have been identified. However, only few of these bear

significance from injurious effects in the animals and human consuming them. The most important

mycotoxins from the food animal and human diseases point of view include aflatoxins, ochratoxins,

DON, DAS, T-2, zerealenone etc. Animals fed on mycotoxin contaminated feeds not only may

suffer from injurious effects of these mycotoxins but also pass these metabolites into the animals food

products in a variety of dairy and agricultural products. Most of the mycotoxins grow in the field

conditions on the crops still standing in the fields. The concentration of these mycotoxins in cereals

and grains vary according to the climatic conditions. Still other mycotoxins are produced by the fungi

under storage conditions under optimal conditions of humidity and temperature. Such fungi are

known as storage fungi. Aflatoxins and ochratoxins are the most frequently know storage fungi. The

present presentation discusses the prevalence of different mycotoxins present in the feeds and foods

and their impact upon the animals and human health. Different strategies to alleviate the injurious

effects of mycotoxins will also be discussed.

Key Words: Mycotoxins, Agricultural products, Poultry Feed.

INTRODUCTION

The word “mycotoxin” originated from the combination of a Latin word “toxicum” and Greek

word “mykes” meaning poison and fungus (Turner et al., 2010). The term ‘mycotoxin’ is usually used

for the secondary metabolites of the fungi that easily colonize agricultural crops and also during the

post-harvest stages (Richard, 2007). Contributing factors to the contamination of food and feed stuffs

with mycotoxins include moisture content, environmental temperature and the activity of insect

(Coulombe, 1993). Up until now, more than 400 mycotoxins with toxic potential have been identified

(Kabak et al., 2006), however, only a few of them have distinct toxic effects. They are usually

genotypically specific. A variety of fungal species can produce the same mycotoxin, for example,

ochratoxin is produced by Penicillium verrucosum in the temperate regions of the world while in

tropical regions of the world Aspergillus ochraceus is the principal ochratoxin producing specie (Kabak,

2009; Thrane, 1989).

Mycotoxins pose a potential threat to the health of humans and the domestic animals. These are

extremely variable in their physical and biological properties and toxic effects. A high concentration

of mycotoxins in feed and food can impose a health risk to animals and humans leading to economic

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losses (Dinis et al., 2007). However, assessment of the adverse health effects have been complicated

by many factors, including the intake levels, toxin species, age of target animals, duration of exposure,

mechanisms of action and metabolism. In addition, there is a lack of research on the availability of

reliable and efficient methods for detection and quantitation of i) mycotoxins, ii) difference in animal

species sensitivity, iii) errors in sample collection. Inefficient analytical methods used and the co-

existence of a variety of mycotoxins and their interactions need attention of researchers (Whitlow &

Hagler Jr., 2002).

The toxic syndromes resulting from mycotoxin intake are known as mycotoxicoses (Richard et

al., 2003). The principal target organs in case of mycotoxicosis are liver, kidney, lungs, the endocrine,

nervous and the immune system (Abdulkadar et al., 2004). Ingestion of mycotoxin contaminated feed

in farm animals resulted in their residual presence in products like milk, meat, cheese and eggs leading

to exposure of customers to mycotoxins (Ramos and Hernandez, 1996).

Aflatoxins

Aspergillus flauvs and A. parasiticus are the two main species of fungi known to produce aflatoxins

(AF) as their secondary metabolites and to the lesser extent A. nomius is also aflatoxigenic (Frisvad,

2005; Richard, 2009). A large variety of feed and foodstuffs such as dried fruits, cereals, nuts and

spices have been found contaminated with AF (Diener et al., 1987). Out of 18 different types of

isolated aflatoxins, AFB1, AFB2, AFG1 and AFG2 are of great importance and common natural

contaminant of food and feedstuff (Battilani et al., 2008). In milking animals AFB1 and AFB2 are

converted into their toxic metabolites called aflatoxin M1 (AFM1) and M2 (AFM2), respectively.

Poultry birds are susceptible to AFB1 toxicity. Significant adverse health effects, including death

have been observed by feeding of AFB1 contaminated feed. Significant morphologic alterations were

observed in liver, which became enlarged, friable and lighter in color (Quezada et al., 2000). The

toxicity of AFB1 in broiler birds occurs in dose and time related manner. Most of the research work

conducted so far, indicated that severity of the toxicity enhanced with prolongation of duration of

feeding contaminated diet and the level of contamination (Oliveira et al., 2002; Hussain et al., 2008;

Khan et al., 2014).Hussain et al. (2008) also reported decrease in body weight due to prolonged

duration of exposure to the AFB1 contaminated diet. The exposure of the broiler birds with 0.3

mg/kg of AFB1 resulted in decreased total serum protein levels (Raju and Devegowda, 2000).

Recently published epidemiological reports suggested a close relation between the AF

contaminated feed and incidence of Newcastle disease outbreak. In general, the lower doses of AFB1

may adversely affect the immune system of the bird whereas higher levels of contamination elicit a

negative effect on the performance parameters. The threshold dose of AFB1 to induce the negative

influence on cell mediated and humoral immune responses has been reported to be approximately

0.4 and 1.0 mg/ kg, respectively (Yunus et al., 2011). Therefore, chronic intake of contaminated feed

containing the lower concentration of aflatoxins might pose a severe risk to animal health, increasing

susceptibility to infections or reducing vaccination efficacy.

Ochratoxins

Ochratoxin (OT) was discovered in 1965 as a toxic secondary metabolite of Aspergillus ochraceus.

However, its toxic potential was realized after isolation from sorghum seed strain K-804 in South

Africa (Scott, 1965). It was first reported by Shotwell et al. (1969) as a natural contaminant of corn.

There are three members of this family known as OTA, OTB and OTC. Among these the most toxic

and commonly detected type in foodstuff is OTA (Atkins & Norman, 1998; Peraica et al., 1999). The

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cereal commodities (wheat, maize, barley and oats) are the primary cause of ochratoxin

contamination of food and foodstuffs in addition to groundnuts, dried fruits and coffee beans, which

has been infected by the OT producing fungi like A. ochraceus, A. niger, A. carbonarius, P. verrucosum, P.

virridicatum etc. (Bennett & Klich, 2003).

The International Agency for Research on Cancer (IARC) categorized AF as a possible source of

human carcinogen and placed it into the category ‘group 2B’. The toxic effects resulting from the

exposure of OTA included nephrotoxicity, hepatotoxicity, immunosuppression and carcinogenic

effects on animals and humans (Anonymous, 1993). Different species exhibited different sensitivity

levels when exposed to acute OTA toxicity (O'Brien and Dietrich, 2005). Pigs are particularly

sensitive to OTA because of the long serum half-life and tissue accumulation. This was sustained by

high protein affinity and enterohepatic and renal recirculation. Poultry species eliminate OTA faster

than mammals, leading to a lower accumulation level. The OTA half-life in plasma of pigs was 20 to 30

times higher than that of poultry, thus resulted in higher incidence of ochratoxicosis in pigs (Duarte et

al., 2011).The toxicity of OTA in the poultry birds has been reported by several researchers. The

lethal dose of OTA (LD50) varies in different poultry species and depends on the age and route of

administration. LD50 value for broiler birds at first day of age was 2.14 mg/kg and at three weeks of

age, it was 3.6 mg/kg body weight (Peckham et al., 1971). Following chronic exposure to lower levels

of OTA, the kidneys were primarily affected, causing mycotoxic nephropathy in pigs and chickens

(Stoev et al., 2012). Several pathological changes could be observed, varying from desquamation and

focal degeneration of tubular epithelium cells to peritubular fibrosis and thickening of the basal

membrane (O'Brien and Dietrich, 2005). This lead to renal insufficiency, but not to tumor promotion

in poultry and mammals. In addition, OTA was hepatotoxic, teratogenic and immunotoxic (Duarte et

al., 2011).

STRATEGIES TO COMBAT MYCOTOXINS INDUCED INJURIOUS EFFECTS

A survey conducted by FAO in 2001, specified that almost 25% of world annual crops are

contaminated with mycotoxins (Anonymous, 2001). Keeping in view the contamination level and

deleterious effects of mycotoxins a number of strategies have been deployed to reduce the growth of

mycotoxigenic fungi, to detoxify contaminated feed and to lower the systemic availability once

mycotoxins are ingested by the animal.

Contamination of the crops with mycotoxins might occur in pre-harvest stage when the crops

are standing in the field or during storage and processing (postharvest stage). Approaches for

preventing mycotoxicosis in animals may therefore, be divided into pre-harvesting and post-harvesting

strategies. The mycotoxins control during pre-harvest stage is difficult and their contamination can

possibly be reduced by the development of the resistant crops by genetic modifications and breeding.

Specific mycotoxin contamination can be reduced significantly by the application of certain techniques,

although the complete elimination of mycotoxins is currently not achievable (Kabak et al., 2006). The

most important strategy to bear in mind for pre-harvesting is the application of good agricultural

practices. Appropriate good agricultural practices include controlling the insect’s infestation, crop

rotation, elimination of crop residues, irrigation, soil cultivation and proper use of chemicals.

Postharvest storage conditions are essential to prevent the growth of mold and consequently the

mycotoxins (Schrodter, 2004). The moisture contents during storage should be kept less than 15% to

avoid the development of hotspots with high moisture levels which favor the mold growth (Jard et al.,

2011). Before storage, visibly damaged or infected grains should be removed. This method is

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however, not exhaustive or very specific (Jard et al., 2011) and multiple reduction strategies should be

combined.

Several chemical detoxification methods have also been described. Any method adopted for

detoxification must not affect the nutritive value of the commodity and also must not result in the

development of toxic products during the mycotoxin inactivation process. The wide varieties of

chemical decontamination processes include radiation, oxidation, reduction, ammonization,

alkalization, acidification and deamination (Kabak et al., 2006).

Many of the previously described methods for the detoxification of agricultural commodities have

restricted application due to the associated problems including incomplete detoxification and the

inapplicability in practice. An alternative approach was the inclusion of mycotoxin detoxifying agents

in the feed to decrease the bioavailability of toxins. This method presently has been the most

commonly used one (Jard et al., 2011; Kolosova and Stroka, 2011). These detoxifiers can be divided

into two classes, namely mycotoxin binders and mycotoxin modifiers. The two classes have different

modes of action. Mycotoxin binders adsorb toxin in the gut, resulting in the excretion of toxin-binder

complex in feces, whereas mycotoxin modifiers transform the toxin into non- toxic metabolites

(Anonymous, 2009).

Mycotoxin Binders

Mycotoxin binders (adsorbing or sequestering substances) are the compounds having large

molecular weight and are able to bind the mycotoxin during its passage through the digestive tract of

the animal. The complex formed between toxin and binder is eliminated from the body through feces,

thus preventing its absorption from gut into the circulation. Mycotoxin binders have been divided

mainly into silica based inorganic compounds and carbon based organic polymers (Anonymous, 2009).

Inorganic Binders

The ability of the inorganic binder to bind the mycotoxin depends on physical and chemical

properties of the both adsorbent and the mycotoxin. Physical properties of the adsorbent i.e. surface

area, charge distribution, total charge and pore size play a vital role during the binding process. The

properties of the mycotoxin such as polarity, charge distribution, shape and solubility are important

factors playing a significant role. Generally speaking, the binding capacity increased with surface area

and chemical affinities between both binder and mycotoxin (Avantaggiato et al., 2005; Huwig et al.,

2001; Kabak et al., 2006).

Bentonite Clay

Aluminosilicate minerals (clays) have been the most studied and the largest class of mycotoxin

binders used to alleviate the deleterious effects of mycotoxins through adsorption. There are two

different subclasses of aluminosilicates i.e. phyllosilicate and tectosilicate. Phyllosilicates include the

bentonite, montmorillonite, smectite, kaolinite and illites while tectosilicates include the zeolite

(Anonymous, 2009). The structure of montmorillonite consists of layers of octahedral and

tetrahedral aluminum and silicon, respectively, which are coordinated with oxygen atoms. Bentonite

is generally impure clay consisting mostly of montmorillonite. Zeolites possess a three dimensional

structure and contains tetrahedrons of SiO4 and AlO4. In these minerals, the replacement of the

tetravalent silicon with the trivalent aluminum lead to the deficit of positive charge, so the inorganic

cations like sodium, potassium and calcium balance this deficiency. In the hydrated sodium calcium

aluminosilicate (HSCAS) the naturally occurring sodium ions were replaced by the calcium ions and

protons (Huwig et al., 2001). HSCAS is a heat processed and purified montmorillonite clay. It was

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developed by Phillips et al. (1988) and commercialized as NovaSil®. Clay products, including

bentonites, zeolites and HSCAS are the most common feed additives effective in binding polar

mycotoxins, such as aflatoxins (Kabak et al., 2006). However, OTA and fusarium mycotoxins, such as

fumonisins, zearalenone (ZON) and trichothecenes do not bound to these clays because of their

fairly non-polar properties (Avantaggiato et al., 2005; Kabak et al., 2006; Phillips et al., 2008). HSCAS

has a lamellar interlayer structure in which the planar AFB1 can be bound. The interaction is based on

the negative charge of the clay with the partly positive charged dicarbonyls of AFB1 (Phillips et al.,

2008).

Silicate binders such as bentonite have been found effective to ameliorate the toxic effects of

mycotoxins (Indresh et al., 2013). A significant increase in the feed intake, body weight and FCR was

observed when chicks were offered a feed added with 0.5% of sodium bentonite in the presence of

aflatoxin. A significant increase was observed in the relative weight of the liver, heart and gizzard 5.34,

0.72 and 2.05 percent, respectively. A 40% mortality was observed on feeding aflatoxin contaminated

feed which was reversed with the incorporation of 0.5% sodium bentonite (Pasha et al., 2007). Similar

results were also published by Eraslan et al. (2004) and Rosa et al. (2001). During an in-vitro study to

check the efficacy of sodium bentonite of Argentina origin, a high binding ability was observed from

the aqueous solution of AFB1 (Rosa et al., 2001). A slight protection in broiler birds was observed

against aflatoxicosis when 0.3% of sodium bentonite was added to the feed contaminated with

aflatoxin (5 mg/kg). The body weight gain and feed intake were non-significantly different from the

control group, however, the histolopathological and biochemical data revealed that the protective

effect against the toxic effect of aflatoxin was not up to the mark. The addition of 0.5% of

montmorillonite to the broiler diet significantly decreased the deleterious effect of AFB1 when added

to the feed at a dose rate of 200 ppb (Desheng et al., 2005). The addition of 3 g modified

montmorillonite nanocomposite to diet amended with 0.1 mg AFB1/kg indicated a significant

protective effect on the relative weight of the organs, hematological and biochemical values.

However, a significant decrease in the body weight gain and feed intake was observed in the birds fed

AFB1 contaminated diet without incorporation of binder, compared to the control group (Shi et al.,

2006). Ramos and Heranadez (1997) reported that aluminosilicates like HSCAS are efficient to

detoxify the AF due to its polar nature so reduced the chances for the development of aflatoxicosis.

Dietary addition of acid bentonite (1%, 10%) and HSCAS (1%) to the OTA contaminated diet

(1mg/kg) had no effect on the blood and tissue levels of toxin in pigs (Plank et al., 1990). The use of

HSCAS in case of OTA toxicity did not improve the performance parameters of the broiler birds and

the depression of humoral immune response of broiler chicks by feeding OTA at 2 mg/kg feed with

or without aluminosilicates against NDV (Santin et al., 2002b). Addition of 0.5% HSCAS to the diet

made from moldy corn did not ameliorate the negative effects on average daily weight gain and FCR

of the broiler birds (Liu et al., 2011). Garcia et al. (2003) conducted a study to access the binding

ability of two commercial binders (Zeotek & Mycofix) against OTA (567 ppb) toxicity in the broiler

birds. The binders were not much effective in reducing the detrimental effects on the plasma

proteins, albumins, globulins and uric acid levels in the blood. Similarly the use of clay based binder

having calcium bentonite in its composition at a dose rate of 4 kg/ton reduced the level of OTA in

three fish feed samples from 15, 6 and 6 µg/kg to 1, zero and zero, respectively (Abdelaziz et al.,

2010). The use of diatomaceous earth significantly reduced the toxic effects of OTA for most of the

studied parameters except the relative weight of liver in laying hens when compared to the control

group (Denli et al., 2008).

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Activated charcoal

Another sorbent of interest is activated charcoal (AC), also called active carbon. AC is an

insoluble powder and formed by an activation process of pyrolysis of various organic compounds to

develop an extremely porous structure (Galvano et al., 2001). The surface area, structure and pore

size of the mycotoxin played a critical role in the binding ability of AC. The surface-to-mass ratio of

AC varied from 500 to 3500 m2/g. AC effectively bound a wide variety of drugs and toxic substances.

Since the 19th century, it has been effectively used to treat severe intoxication problems (Huwig et al.,

2001). AC has been proven an effective adsorbent of DON, ZON, AFB1, FB1 and OTA (Huwig et al.,

2001; Avantaggiato et al., 2004; Devreese et al., 2012).

The preliminary study conducted by Decker and Corby (1980) suggested that AFB1 (1 mg) was

efficiently adsorbed during an in vitro study at neutral pH by the AC (100 mg). Edrington et al. (1997)

reported a slight protective effect of super AC against aflatoxicosis in the broiler birds. The birds

were offered a diet amended with 4 mg/kg of aflatoxin for up to 21 days of age, with or without the

addition of 0.5 % of super AC. A significant decrease in the body weight gain was observed in the

birds fed with aflatoxin alone, while a moderate protection was observed with the addition of super

AC. The addition of AC to aflatoxin contaminated diet tended to improve the body weight gain and

feed utilization (Teleb et al., 2004; Dalvi and McGown, 1984). Galvano et al. (1996) reported the

protective effect of charcoal to reduce the residues of aflatoxin in the milk of cow, however; this

protection was not less compared to the protection provided by the clay-based binders. Similarly,

Diaz et al. (2004) stated that the adding 45 g of AC in ration on a daily basis had no significant

reduction on the residues of aflatoxin in milk while the addition of binders having clay or esterified

glucan at a dose of 225 and 10g, respectively to each cow per day, lead to a significant reduction in

residues of aflatoxin in milk. Similarly, the experiments conducted on rats (Abdel-Wahhab et al.,

(1999), mink (Bonna et al., 1991) and turkey poult (Edrington et al., 1996) suggested that the clay

based binders were more efficient compared to the charcoal in case of aflatoxin toxicity. Jindal et al.

(1994) reported a moderate ameliorative effect of AC (200 ppm) against the toxic effects induced by

AFB1 (0.5 ppm) in the broiler birds fed the amended feed from day1 to 42 of age. There was a

significant reduction in the inhibitory effect of AFB1 on the body weight and feed intake of the birds.

Serum biochemical parameters were also improved, but no significant effect was observed on the

cholesterol levels. Ademoyero and Dalvi (1983) observed a considerable reduction of toxic injury to

the liver caused by AFB1. Kutlu et al. (2001) conducted a six week experiment in the broiler birds

and reported a significant increase in feed intake, body weight gain and improved FCR at different

levels of wood charcoal (0, 25, 50 and 100 g/kg) up to 28 days of age. However, no significant effect

was observed at 49 days of age except on the FCR, ash contents of carcass and carcass weight and

yield. Similarly, in the second experiment conducted on the laying hens for a 7 week period with an

initial age of 34 weeks, the dietary addition of wood charcoal at an inclusion rate of 0, 10, 20 and 40

g/kg feed did not significantly affect the performance parameters and egg quality. However, a

significant reduction in the quantity of broken eggs was observed in a dose dependent manner.

In case of OTA toxicity inclusion of 1% AC to the feed of the broiler birds did not result in

significant protection. Addition of 1% AC to the pig diet amended with 1mg OTA/kg feed resulted in

a slight decrease in the blood OTA level, however, tenfold dosage resulted in a 50% to 80% reduction

of OTA levels in both blood and tissue (Plank et al., 1990). Liu et al. (2011) reported that at 1%

inclusion rate of AC made from willow tree in the diet formulated from less moldy corn did not

improve the average daily weight gain and FCR of the broiler birds during first three weeks of age.

The inclusion rate of 2% resulted in lower average daily weight gain, poor FCR, more leg problem and

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higher mortality in the broiler birds possibly due to dilution or binding of the nutrients. Describing

the results of a feeding trial Rotter et al. (1989) reported that addition of 10,000 ppm of AC in feed

contaminated with OTA (4µg/kg) had no effect on OTA toxicity. Addition of 0.24g/L of activated

carbon to the sweet wine effectively reduces up to 70% of OTA.

Mycotoxin Modifiers

Another strategy to control mycotoxicoses in animals is the application of microorganisms and

their enzymes, called mycotoxin modifiers or mycotoxin bio-transforming agents. These products

degrade or biotransform mycotoxins into less toxic metabolites. Mycotoxin modifiers can be divided

into four classes: bacteria, yeasts, fungi and enzymes. These modifiers act on the mycotoxins during

their passageway through the intestinal tract of the animal thus preventing absorption of mycotoxins

into the circulation. Effective use of mycotoxin modifiers depends upon certain properties of these

substances. These properties include their ability to rapidly degrade the toxic substance into nontoxic

compound, preserve the organoleptic and the nutritive value of the feed, safety of use and stability

during the intestinal passage at different levels of pH. One has to consider different practicable and

economical aspects for selecting mycotoxin modifiers (Awad et al., 2010; Kolosova and Stroka, 2011).

The key point for the successful modifier is that the microorganism should survive and adopt the

environment during its passage in the animal’s gut (Zhou et al., 2008).

Trichosporon mycotoxinivorans

The microorganisms isolated from the animals gut contents are usually appropriate for the

development of good modifier which will act in the animal’s intestines. Among different

microorganisms, so far tested for inactivation of OTA, only Trichosporon mycotoxinivorans (TM) has

been thoroughly investigated regarding its ochratoxin degrading abilities and feasibility for its

commercial use. This yeast, primarily derived from the hindgut of the lower termites

Mastotermesdarwiniensis, was isolated and characterized previously by Molnar et al. (2004). This yeast

was capable of modifying ZON and OTA into non-toxic metabolites.

A study by Politis et al. (2005) demonstrated that inclusion of TM (105 CFU/g) in the diet

alleviated the immunotoxic effects of OTA (0.5 mg/kg) in broiler chickens and also has the ability to

degrade zearalenone when used as a feed additive (Vekiru et al., 2010). Addition of TM at dose rate

of 1 & 2 kg/ton attenuated the harmful effects of dietary OTA (0.5 & 1mg/kg) on serum liver enzymes

and also pathomorphological and histological changes in the internal organs of broiler birds (Hanif et

al., 2008). A significant reduction in the residue of OTA in serum, kidney and liver was also reported

by Hanif et al. (2012) when TM was added at dose rate of 1 & 2 kg/ton in OTA (500 ppb and 1000

ppb) contaminated broiler diet. Similarly, the use of clay based binder having Eubacterium and TM in

its composition significantly improved the FCR compared to the groups received only OTA

contaminated diet (Hanif et al., 2008). Trichosporon mycotoxinivorans proved an efficient toxin modifier

in case of OTA toxicity as it converts the OTA to non-toxic OTα and phenylalanine (Schatzmayr et

al., 2006; Molnar et al., 2004).

Other potential OTA degrading yeast species included Phaffiarhodozyma and Xanthophyllomyces

dendrorhous (Peteri et al., 2007) but the responsible enzymes have not well been characterized and

their practical application up at present is limited. Styriak and Conkova (2002) reported that two out

of several tested Saccharomyces cerevisiae strains were able to degrade 25 or 50% of fumonisins B1

(FB1) after 5 days of incubation, which is, therefore, unusable in practice.

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Milk Thistle seeds

The seeds of Milk thistle (Sylibum marianum), a medicinal herb, has been extensively used in folk

medicine for treating liver diseases. Certain active ingredients found in the seed of this plant possess

numerous medicinal properties. Earlier in 1960, a German scientist isolated a flavonoid ‘silymarin’

from MTS. Chemically, it is composed of four flavonoids- silybin, isosilybin, silydianin, and silychristin.

Silybin is the major component constituting 50 to 70% of silymarin and exhibit greater biological

activities (Dumari et al., 2014).

The bioactive extract from Silybum marianum seed, silymarin, contains a mixture of

flavonolignans and a residual fraction that has not been defined chemically in structural details

(Skottova et al., 2003). Silymarin is used in humans for the treatment of numerous liver disorders

characterized by degenerative necrosis and functional impairments (Luper, 1998). Kalorey et al.

(2005), reported that silymarin improved body weight and feed intake in the presence of aflatoxin B1

in feed, while it had no effect on the feed conversion ratio (Tedesco et al., 2004). Similarly, Gowda

and Sastry (2000) confirmed a significant improvement of silymarin on body weight gain and

attributed its effects to antioxidant activity in the protein synthesis stimulation by the bird’s enzymatic

system. The higher weight gain was reported by Chakarverty and Parsad (1991), in silymarin

supplemented group. Kalorey et al. (2005) reported the protective role of silymarin against

aflatoxicosis on the weight of bursa of Fabricius. As evident from some researches, aflatoxins reduced

lymphoid organs weight (thymus, bursa and spleen) in aflatoxicosis (Tedesco et al., 2004). Silybum

marianum was more efficient to protect the spleen against adverse effects of aflatoxin as compared

with the synthetic toxin binders (Kalorey et al., 2005). Regarding ochratoxicosis Khatoon et al. (2013)

reported improvement of different immunological responses lymphoproliferative response to avian

tuberculin and phagocytic index of circulating macrophages by silymarin in white Leghorn cockerels.

Distillery Yeast Sludge

Distillery yeast sludge (DS), a by-product of sugarcane molasses based distillery industry, is

considered a waste in East Asian countries but chemically this waste has high contents of protein

which is far higher than what found in ordinary cereals. This product can be effectively used as an

alternative feed source and studies have reported that up to 50% of it as an alternative feed source is

safe and can provide better results instead of ordinary feed (Rameshwari and Karthikeyan, 2005).

Mujahid et al. (2012) conducted a study in which they found the efficient protective efficacy of 1-2%

yeast sludge to protect aflatoxin induced alteration in poultry while Hashmi et al., (2006) reported

the efficacy of 1% yeast sludge in ochratoxin A treated birds receiving up to 200 ppb levels.

Moreover; Saccharomyces cerevisiae, being the active yeast found in sludge has the ability to adsorb

aflatoxins up to 90% in short time interval (Murthy and Devegowda, 2004). Mannan oligosaccharide

which is a derivative of yeast has also been evaluated for its protective efficacy against different

mycotoxins and has been proved highly beneficial in providing protection in birds (Baptista et al.,

2004).

CONCLUSION

The rate of poultry feed mycotoxin contamination is likely to increase in line with the trend

witnessed in preceding years. The unwanted effects of mycotoxins can be prevented with an

appropriate mycotoxin binder. Different substances including binding clays, activated charcoal,

distillery sludge, Trichosporon mycotoxivorans and milk thistle seeds extracts are the promising

substance for use as mycotoxin adsorbing or inactivating agents. The combination of adsorption and

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biotransformation technologies can be used effectively to deactivate the major groups of mycotoxins

found in intensive poultry production and to prevent economic losses associated with the

contamination of these mycotoxins.

REFREENCES

Abdelaziz M, W Anwer and AH Abdelrazek, 2010. Field Study on the Mycotoxin Binding Effects of

Clay in Oreochromis niloticus Feeds and Their Impacts on the Performance as Well as the Health

Status throughout the Culture Season. Interdisciplinary Bio Central, 2: 1-6.

Abdel-Wahhab MA, SA Nada and Amra, 1999. Effect of aluminosilicates and bentonite on aflatoxin-

induced developmental toxicity in rat. J Appl Toxicol, 19: 199-204.

Abdulkadar, WHA, AA Al-Ali, MA Al-Kildi and HJ Al-Jedah, 2004. Mycotoxins in food products

available in Qatar. Food Control, 15: 543-548.

Ademoyero AA and RR Dalvi, 1983. Efficacy of activated charcoal and other agents in the reduction

of hepatotoxic effects of a single dose of aflatoxin B1 in chickens. Toxicol Lett, 16: 153-157.

Anonymous, 1993. Monographs on the Evaluation of Carcinogenic Risks to Humans: Some Naturally

Occurring Substances, Food Items and Constituents, Heterocyclic Aromatic Amines and

Mycotoxins. International Agency for Research on Cancer, Geneva, 56: 489-521.

Anonymous, 2001. Manual on the application of the HACCP system in mycotoxin prevention and

control. FAO Food and Nutition Paper No. 73, Rome, Italy.

Anonymous, 2009. Commision Regulation 386/2009/EC of 12 May 2009 amending Regulation (EC)

No 1831/2003 of the European Parliament and of the Council as regards the establishment of a

new functional group of feed additives. Official Journal of the European Union L 118, 66.

Atkins D and J Norman, 1998. Mycotoxins and food safety. Nutr Food Sci, 5: 260-266.

Avantaggiato G, M Solfrizzo and Visconti, 2005. Recent advances on the use of adsorbent materials

for detoxification of Fusarium mycotoxins. Food Addit Contam A, 22: 379-388.

Avantaggiato G, R Havenaar and A Visconti, 2004. Evaluation of the intestinal absorption of

deoxynivalenol and nivalenol by an in vitro gastrointestinal model, and the binding efficacy of

activated carbon and other adsorbent materials. Food ChemToxicol, 42: 817-824.

Awad WA, K Ghareeb, J Bohm and Zentek, 2010. Decontamination and detoxification strategies for

the Fusarium mycotoxin deoxynivalenol in animal feed and the effectiveness of microbial

biodegradation. Food Addit Contam A, 27: 510-520.

Baptista AS, J Horii, MA Calori-Domingues, EM da Gloria, JM Salgado et al., 2004. The capacity of

manno-oligosaccharides, thermolysed yeast and active yeast to attenuate aflatoxicosis.World J

Microbiol Biotechnol, 20: 475-481.

Battilani P, C Barbano and A Logrieco, 2008. Risk assessment and safety evaluation of mycotoxins in

fruits. In: Barkai-Golan, R. and Paster, N. (Eds) Mycotoxins in fruits and vegetables. Elsevier, San

Diego, CA, USA, pp. 1-26.

Bennett JW and M Klich, 2003. Mycotoxins. Clin Microbiol Rev, 16: 497-516.

Chakarverty A and JParsad, 1991. Study on the effect of Milk Thistle extract on the performance of

broiler chicks. Indian Poult Adv, 24: 37-38.

Coulombe RA, 1993. Biological action of mycotoxins. J Dairy Sci 76: 880-891.

Dalvi RR and C McGowan, 1984. Experimental induction of chronic aflatoxicosis in chickens by

purified aflatoxin B1 and its reversal by activated charcoal, Phenobarbital and reduced

glutathione. Poult Sci, 63: 485-491.

Page 79: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

79

Decker WJ and DG Corby, 1980. Activated charcoal adsorbs aflatoxin B1. Vet Hum Toxicol, 22: 388-

389.

Denli M, JC Blandon, ME Guynot, S Salado and JF Perez, 2008. Efficacy of a New Ochratoxin-Binding

Agent (OcraTox) to Counteract the Deleterious Effects of Ochratoxin A in Laying Hens. Poult

Sci, 87: 2266-2272.

Desheng Q, L Fan, Y Yanhu and Niya, 2005. Adsorption of aflatoxin B-1 on montmorillonite. Poult

Sci, 84: 959-961.

Devreese M, AOsselaere, J Goossens, V Vandenbroucke, SD Baere, et al., 2012. New bolus models

for in vivo efficacy testing of mycotoxin-detoxifying agents in relation to EFSA guidelines assessed

using deoxynivalenol in broiler chickens. Food Addit Contam A, 29: 1101-1107.

Diaz DE, WM Hagler, JT Blackwelder, JA Eve, BA Hopkins, et al., 2004. Aflatoxin binders II: Reduction

of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed.

Mycopathologia, 00: 1-8.

Diener UL, RJ Cole, TH Sanders, GA Payne, LS Lee, and MA Klich, 1987.Epidemiology of aflatoxin

format ion by Aspergillus flavus.Annu Rev Phytopathol, 25: 249-270.

Dinis AMP, CM Lino and AS Pena, 2007.Ochratoxin A in nephropathic patients from two cities

ofcentral zone in Portugal. J Pharmaceut Biomed Anal 44: 553–557.

Duarte SC, CM Lino and A Pena, 2011. Ochratoxin A in feed of food-producing animals: an

undesirable mycotoxin with health and performance effects. Vet Microbiol, 154: 1-13.

Dumari MA, H Sarir, OF Makki, N Afzali, 2014. Effect of milk thistle (silybum marianum l.) on

biochemical parameters and immunity of broiler chicks fed aflatoxin b1 after three weeks. Iran J

Toxicol, 8: 1098-1103.

Edrington TS, LF Kubena, RB Harvey and GE Rottinghaus, 1997. Influence of a superactivated

charcoal on the toxic effects of aflatoxin or T-2 toxin in growing broilers. Poult Sci, 76: 1205-

1211.

Edrington, TS, AB Sarr, LF Kubena, RB Harvey and TD Phillips. 1996. Hydrated sodium calcium

aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of

aflatoxin M1 in turkey poults. Lack of effect by activated charcoal on aflatoxicosis. Toxicol Lett,

89:115-122.

Eraslan G, M Akdogan, E Yarsan, D Essiz, F Sahindokuyucu, et al., 2004. Effects of aflatoxin and

sodium bentonite administered in feed alone or combined on lipid peroxidation in the liver and

kidneys of broilers. B Vet I Pulawy, 48: 301-304.

Frisvad JC, P Skouboe and RA Samson, 2005. Taxonomic comparison of three different groups of

aflatoxin producers and a new efficient producer of aflatoxin B1, strerigmatocystin and 3-O-

methylsterigmatocystin, Aspergillus rambellii sp. nov. Syst Appl Microbiol, 28: 442-453.

Galvano F, A Pietri, B Fallico, T Bertuzzi, S Scire, et al., 1996. Activated carbons: in vitro affinity for

aflatoxin B1 and relation of adsorption ability to physicochemical parameters. J Food Protect, 59:

545-550.

Galvano F, A Piva, ARitieni and G Galvano, 2001. Dietary strategies to counteract the effects of

mycotoxins: a review. J Food Prot, 64: 120-131.

Garcia AR, E Avila, R Rosiles and VM Petrone, 2003. Evaluation of two mycotoxin binders to reduce

toxicity of broiler diets containing ochratoxin A and T-2 toxin contaminated grain. Avian Dis, 47:

691-699.

Gowda SK and VRBSastry, 2000. Neem (Azadirachtaindica) seed cake in animal feeding-scope and

limitation-Review. Asian Australas J Anim Sci, 13: 720-728.

Page 80: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

80

Hanif NQ, G Muhammad, K Muhammad, I Tahira and GK Raja, 2012. Reduction of ochratoxin A in

broiler serum and tissues by Trichosporon mycotoxinivorans. Res Vet Sci, 93: 795-797.

Hanif NQ, G Muhammad, M Siddique, A Khanum, T Ahmed, et al., 2008. Clinico-pathomorphological,

serum biochemical and histological studies in broilers fed ochratoxin A and a toxin deactivator

(Mycofix Plus). Br Poult Sci, 49: 632-642.

Hashmi I, TN Pasha, MA Jabbar, M Akram and AS Hashmi, 2006 Study of adsorption potential of

yeast sludge against aflatoxins in broiler chicks J Anim Plant Sci 16: 12-14.

Hussain Z, MZ Khan and Zl Hassan, 2008. Production of Aflatoxins from Aspergillus Flavus and Acute

aflatoxicosis in young broiler chicks. Pak J Agri Sci, 45: 95-102.

Huwig A, S Freimund, O Kappeli and H Dutler, 2001. Mycotoxin detoxication of animal feed by

different adsorbents.Toxicol Lett 122: 179-188.

Indresh HC, Devegowda G, Ruban SW and MC Shivakumar, 2013. Effects of high grade bentonite on

performance, organ weights and serum biochemistry during aflatoxicosis in broilers. Vet World,

6: 313-317.

Jard G, T Liboz, F Mathieu, AGuyonvarch and A Lebrihi, 2011. Review of mycotoxin reduction in

food and feed: from prevention in the field to detoxification by adsorption or transformation.

Food Addit Contam A, 28: 1590-1609.

Jindal N, SK Mahipal and NK Mahajan, 1994. Toxicity of aflatoxin B1 in broiler chicks and its

reduction by activated charcoal. Res Vet Sci 56: 37-40.

Kabak B, 2009. Ochratoxin A in cereal-derived products in Turkey: occurrence and exposure

assessment. Food Chem Toxicol, 47: 348-352.

Kabak B, AD Dobson and I Var, 2006. Strategies to prevent mycotoxin contamination of food and

animal feed: a review. Crit Rev Food Sci Nutr, 46: 593-619.

Kalorey DR, NV kurkure, IS Ramgaonkar, PS Sakhare, S Warke et al., 2005. Effect of polyherbal feed

supplement “Growell” during induced aflatoxicosis, ochratoxicosis and combined mycotoxicoses

in broilers. Asian Australas J Anim Sci, 18: 375-383.

Khan WA, MZ Khan, A Khan, ZU Hassan, S Rafique, et al., 2014. Dietary vitamin E in White Leghorn

layer breeder hens: a strategy to combat aflatoxin B1-induced damage, Avian Pathol, 43: 389-395.

Khatoon A, MZ Khan, A Khan, MK Saleemi and I Javed, 2013. Amelioration of Ochratoxin A-induced

immunotoxic effects by Silymarin and Vitamin E in White Leghorn Cockerels. J Immunotoxicol

10: 25-31

Kolosova A and Stroka, 2011. Substances for reduction of the contamination of feed by mycotoxins: a

review. World Mycotoxin J, 4: 225-256.

Kutlu HR, L Unsal and M Gorgulu, 2001. Effects of providing dietary wood (oak) charcoal to broiler

chicks and laying hens. Anim Feed Sci Technol, 90:213-226.

Liu YL, GQ Meng, HR Wang, HL Zhu, YQ Hou, et al., 2011. Effect of three mycotoxin adsorbents on

growth performance, nutrient retention and meat quality in broilers fed on mould-contaminated

feed. Br Poult Sci, 52: 255-263.

Luper S, 1998. A review of plants used in the treatment of liver disease: Part I. Altern Med Rev, 3:

410–421.

Molnar O, G Schatzmayr, E Fuchs and H Prillinger, 2004.Trichosporon mycotoxinivorans sp. nov., A New

yeast species useful in biological detoxification of various mycotoxins. Sys Appl Microbiol, 27:

661-671.

Mujahid H, AS Hashmi, AA Anjum, A Waris and Y Tipu, 2012 Detoxification potential of ochratoxin

by yeast sludge and evaluation in broiler chicks J Plant Anim Sci, 22: 601-604.

Page 81: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

81

O'Brien E and DR Dietrich, 2005. Ochratoxin A: the continuing enigma. Crit Rev Toxicol, 35: 33-60.

Oliveira CAF, JF Rosmaninho, P Butkeraitis, B Correa, TA Reis, et al., 2002. Effect of Low Levels of

Dietary Aflatoxin B1 on Laying Japanese quail. Poult Sci, 81: 976-980.

Pasha TN, MU Farooq, FM Khattak, MA Jabbar and AD Khan, 2007. Effectiveness of sodium bentonite

and two commercial products as afalatoxin absorbents in diets for broiler chickens. Anim Feed

Sci Tech, 132: 103-110.

Peckham JC, B Doupnik and OH Jones, 1971. Acute toxicity of ochratoxins A and B in chicks. Appl

Microbiol, 21: 292-494.

Peraica M, AM Domijan, R Fuchs, A Lucic and B Radic, 1999. The occurrence of ochratoxin A in

blood in general population of Croatia. Toxicol Lett, 110: 105-112.

Peteri Z, J Teren, C Vagvolgyi, and J Varga, 2007. Ochratoxin degradation and adsorption caused by

astaxanthin-producing yeasts. Food Microbiol, 24: 205-210.

Phillips TD, E Afriyie-Gyawu, J Williams, H Huebner, NA Ankrah, et al., 2008. Reducing human

exposure to aflatoxin through the use of clay: a review. Food Addit Contam A, 25: 134-145.

Phillips TD, LF Kubena, RB Harvey, DR Taylor and ND Heidelbaugh, 1988. Hydrated sodium calcium

aluminosilicate: a high affinity sorbent for aflatoxin. Poult Sci, 67: 243-247.

Plank G, J Bauer, A Grunkemeier, S Fischer, B Gedek and H Berner, 1990. The protective effect of

adsorbents against ochratoxin A in swine. Tierarztl Prax, 18: 483-489.

Politis I, K Fegeros, S Nitsch, G Schatzmayr and D Kantas, 2005. Use of Trichosporon mycotoxinivorans

to suppress the effects of ochratoxicosis on the immune system of broiler chicks. Br Poult Sci,

46:58-65.

Quezada T, H Cuellar, F Jaramillo-Juarez, AG Valdivia and JL Reyes, 2000. Effects of aflatoxin B (1) on

the liver and kidney of broiler chickens during development. Comp Biochem Physiol C Toxicol

Pharmacol, 125: 265-272.

Raju MV and G Devegowda, 2000. Influence of esterified-glucomannan on performance and organ

morphology, serum biochemistry and haematology in broilers exposed to individual and

combined mycotoxicosis (aflatoxin, ochratoxin and T-2 toxin). Br Poult Sci, 41: 640-650.

Rameshwari KS and S Karthikeyan, 2005. Distillery yeast sludge as an alternative feed resource in

poultry Int J Poult Sci, 4: 787-789

Ramos AJ and E Hernandez, 1996.In vitro aflatoxin adsorption by means of a montmorillonite

silicate.A study of adsorption isotherms. Anim Feed Sci Technol, 62: 263-269.

Ramos AJ and E Hernandez, 1997. Prevention of aflatoxicosis in farm animals by means of hydrated

sodium calcium aluminosilicate addition to feedstuffs: A review. Anim Feed Sci Technol, 65: 197-

206.

Richard E, N Heutte, V Bouchart and D Garon, 2009. Evaluation of fungal contamination and

mycotoxin production in maize silage. Anim Feed Sci Technol, 148: 309-320

Richard JL, 2007. Some major mycotoxins and their mycotoxicoses—an overview. Int J Food

Microbiol, 119: 3-10.

Richard JL, GA Payne, AE Desjardin, C Maragos, WP Norred, et al., 2003. Mycotoxins, risks in plant,

animal and human systems. CAST Task Force Report 139. Council for Agricultural Science and

Technology. Ames, Iowa, USA, p. 101–103.

Rosa CAR, R Miazzo, C Magnoli, M Salvano, SM Chiacchiera, et al., 2001. Evaluation of the efficacy of

bentonite from the south of Argentina to ameliorate the toxic effects of aflatoxin in broilers.

Poult Sci, 80: 139-144.

Page 82: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

82

Santin E, AC Paulillo, PC Maiorka, AC Alessi, EL Krabbe et al., 2002. The effects of

ochratoxin/aluminosilicate interaction on the tissues and humoral immune response of broilers.

Avian Pathol, 31: 73-79.

Schatzmayr G, F Zehner, M Taubel, D Schatzmayr, A Kilmitsch, et al., 2006. Microbiologicals for

deactivating mycotoxins. Mol Nutr Food Res, 50: 543-551.

Schrodter R, 2004. Influence of harvest and storage conditions on trichothecenes levels in various

cereals. Toxicol Lett, 153: 47-49.

Scott DB, 1965. Toxigenic fungai isolated from cereal and legume crops. Mycopath Mycol Appl, 25:

213-222.

Shi YH, ZR Xu, JL Feng and CZ Wang, 2006. Efficacy of modified montmorillonite nanocomposite to

reduce the toxicity of aflatoxin in broiler chicks. Anim Feed Sci Tech, 129: 138-148.

Shotwell OL, CW Hesseltine and ML Goulden, 1969. Ochratoxin A: Occurrence as natural

contaminant of a corn sample. Appl Microbiol, 17: 765-766.

Skottova N, R Vecera, K Urbanek, P Vana, D Walterova et al., 2003. Effects of polyphenolic fractions

of silymarin on lipoprotein profile in rats fed cholesterolrichdiets. Pharmacol Res, 47: 17–26.

Stoev SD, D Gundasheva, I Zarkov, T Mircheva, D Zapryanova, et al., 2012. Experimental mycotoxic

nephropathy in pigs provoked by a mouldy diet containing ochratoxin A and fumonisin B1. Exp

Toxicol Pathol, 64: 733-741.

Styriak I and E Conkova, 2002. Microbial binding and biodegradation of mycotoxins. Vet Hum

Toxicol, 44: 358-361.

Tedesco D, C Domeneghini, D Sciannimanico, MTameni, S Steidler et al., 2004. Efficacy of silymarin

phospholipid complex in reducing the toxicity of aflatoxin B1 in broiler chicks. Poult Sci, 83:

1839-1843.

Teleb HM, AA Hegazy and YA Hussein, 2004. Efficiency of kaolin and activated charcoal to reduce

the toxicity of low level of Aflatoxins in broilers. Sci J King Faisal Univ, 5, 1425.

Thrane U, 1989. Fusarium: Mycotoxins, Taxonomy and Pathogenicity, In J. Chelowski (Ed), Elsevier,

Amsterdam, p. 199

Turner NW, S Subrahmanyam and SA Piletsky, 2010. Analytical methods for determination of

mycotoxins: a review. Anal Chim Acta, 632: 168-180.

Underhill KL, BA Rotter, BK Thompson, DB Prelusky and HL Trenholm, 1995. Effectiveness of

cholestyramine in the detoxification of zearalenone as determined in mice. Bull Environ Contam

Toxicol, 54: 128-134.

Vekiru E, C Hametner, R Mitterbauer, J Rechthaler, G Adam, et al., 2010. Cleavage of zearalenone by

Trichosporon mycotoxinivorans to a novel nonestrogenic metabolite. Appl Environ Microbiol, 76:

2353–2359.

Whitlow LW and WM Hagler Jr, 2002.Mycotoxins in feeds. Feedstuffs, 74: 1-10.

Yunus AG, E Razzazi-Fazeli and J Bohm, 2011. Aflatoxin B1 in affecting broiler’s performance,

immunity, and gastrointestinal tract: a review of history and contemporary issues. Toxins, 3: 566-

590.

Zhou T, J He and J Gong, 2008. Microbial transformation of trichothecene mycotoxins. World

Mycotoxin J, 1: 23-30.

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MOLECULAR CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS ISOLATED

FROM RECENT OUTBREAKS IN JORDAN AND STRATEGIES FOR ITS CONTROL

Mohammad Q. Al-Natour1*, Nadim M. Amarin1 Hisham Al –Maa’itah1 and Ilaria Capua2

1Department of Pathology and Public Health, Faculty of Veterinary Medicine, Jordan University of

Science and Technology, P.O. Box 3030, Irbid 22110, Jordan; 2OIE/FAO Reference Laboratory for

Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Viale

dell'Università, 10, 35020, Legnaro, Padova, Italy

*Corresponding author: [email protected]

ABSTRACT

A total of 46 different poultry flocks were investigated for ND virus isolation and

characterization. Samples were collected from Broiler (n=23), Layer (n=5), Broiler breeder (n=3),

Ostriches (n=1), Turkeys (n=1), Peacock (n=1), Ducks (n =2), and backyard local chicken flocks

(n=10). The majority of these flocks experiencing signs and lesions typical of airsacculitis.

Following the inoculation of embryonated fowl’s SPF eggs 39 haemagglutinating agents were

isolated, identified and then fully characterized, as described in EU Council Directive 92/66/EEC

(CEC, 1992). Thirty five out of the 39 isolates reacted with antiserum against APMV-1. The other 4

strains were inhibited by APMV-2 antiserum. Nineteen of the 39 isolates showed intracerebral

pathogenicity index (ICPI) ranging from 0.8 to 1.8. Fusion protein cleavage site amino acid sequence

analysis of these APMV-1 isolates indicated the presence of multiple basic amino acids at the C-

terminus of the F2 region and phenylalanine at the N-terminus of the F1 region {(112-117)

RRQKR*F} confirming the velogenic nature of the viruses. Sixteen out of the 35 APMV-1 isolate

were identifies as Lentogenic – B1 group according to their ICPI that ranged between 0.2 and 0.3,

thus indicating that the viruses were not virulent. Fusion protein cleavage site amino acid sequence

analysis of these APMV-1 isolates indicated the absence of multiple basic amino acids at the C-

terminus of the F2 region and phenylalanine at the N-terminus of the F1 region, {GRQGR*L}

confirming the lentogenic nature of the viruses.

APMV-1 and APMV-2 (Yucaipa) viruses were identified in 39 (85%) of the 46 examined flocks.

The prevalence rate of NDV strains for velogenic (41%), Lentogenic (35%) and Yucaipa (9%) among

the examined 46 flocks. The information reported herein appears to be that APMVs circulating

efficiently in Jordanian poultry industry and it is the first report regarding APMV-2 (Yucaipa)

circulation in Jordan. Biosecurity measures should be strictly implemented along with sound

vaccination programs and good managemental practices at the farm level and backyard flocks. The

control strategies will be discussed.

Key Words: Newcastle disease virus, Yucaipa, Poultry flocks, Jordan.

Introduction

Newcastle disease (ND) is an important viral disease of poultry affecting more than 241 different

avian species around the world (Kaleta and Baldauf. 1988). The disease may be devastating in

commercial poultry with significant economic losses because of its high mortality and the negative

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effect on international trade. Virulent NDV isolates (mesogens and velogens) are notifiable agents

that require reporting to the OIE (OIE, 2000). Newcastle disease is in List-A in the OIE and its

control is the subject of legislation in many parts of the world (CEC, 1992).

Newcastle disease virus (NDV) belongs to the family Paramyxoviridae, subfamily;

Paramyxovirinae of the genus Avulavirus (ICTV. 2012). NDV is a single-stranded RNA negative-sense,

non-segmented, enveloped. Although 11 serotypes of avian paramyxoviruses (APMV-1 to APMV-11)

have been recognized (Briand et al., 2012; Fornells et al. 2012; Miller et al. 2010(a)), APMV-1 remains

the most important pathogen for poultry known as Newcastle disease virus. The pathogenicity of the

disease ranged from asymptomatic form (low virulence) to virulent which is devastating disease with

high mortality in susceptible birds. According to clinical signs in chicken it is classified as: (1)

asymptomatic enteric, (2) lentogenic, (3) mesogenic, (4) velogenic (Suarez, 2013).

Current definition of NDV according to the OIE is ‘‘Newcastle disease is defined as an infection of

birds caused by a virus of avian paramyxovirus serotype 1 (APMV-1) that meets one of the following

criteria for virulence: (a) the virus has an intracerebral pathogenicity index (ICPI) in day-old chicks

(Gallus gallus) of 0.7 or greater. Or (b) multiple basic amino acids have been demonstrated in the virus

(either directly or by deduction) at the C-terminus of the F2 protein and phenylalanine at residue

117, which is the N-terminus of the F1 protein. The term ‘multiple basic amino acids’ refers to at

least three arginine or lysine residues between residues 113 and 116. Failure to demonstrate the

characteristic pattern of amino acid residues as described above would require characterization of

the isolated virus by an ICPI test’’ (OIE, 2008, OIE. 2012). Currently, there are multiple NDV lineages

circulating worldwide that are genetically highly diverse (Aldous et al., 2003; Capua et al., 2002; Gould

et al., 2001; Miller et al., 2010b). Because DIVA strategies do not exist for ND, it is difficult to

differentiate vaccinated from infected animals. This is confusing situation in some endemic countries

that still use live mesogenic vaccine strains that are defined as virulent due to their cleavage sites

and high ICPI values (Wu et al., 2010).

The Jordanian poultry industry is growing fast and it comprises 53% of the total animal industry.

Therefore, it is considered to play an important role in the country economy. Different types of

poultry farms in Jordan including Grandparents, parents, breeders, layer, broiler, turkeys, ducks,

ostrich, quail, and different backyard birds of different bird species including Pigeons, Peacocks, in

addition to cage birds, game birds and avian species in Zoos. Avian viral diseases are of significant

importance to the poultry industry worldwide including Jordan. Newcastle disease outbreaks cause

significant economic losses to farmers and thus the economy. Therefor this paper presents our

findings regarding NDV in Jordan.

Materials and Methods

Investigated Flocks

Since 2003, suspected ND samples were obtained from different poultry sources; farm visits,

necropsy or sample submission to our laboratories for virus isolation attempts. These include

multiple outbreaks in vaccinated and no-vaccinated broiler, layer, broiler breeder, ostriches, turkeys,

peacock, ducks, and backyard local flocks.

Samples collection

Samples were collected aseptically from either freshly dead birds or after performing euthanasia

for birds before necropsy. Trachea, bronchi, lungs, livers and spleen samples were collected from 5-

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10 birds/flock for Newcastle Disease virus isolation attempts. Collected samples in sterile universal

tubes stored at -20 C, and then processed for viral identification according to (OIE, 2012).

Serology and virology

All serological and virological investigations were done in accordance to EU Directive 92/

66/EEC. The HA and HI tests were conducted to detect antibodies to all avian paramyxoviruses

APMV 1-9 using 4 haemagglutination units. Virus isolation was performed in five 9 to11 day-old

emberyonating specific pathogen free (SPF) fowl’s eggs as described previously (CEC, 1992).

Extraction of viral RNA from one sample tissues, Primers used and RT-PCR assays were according to

(Ababneh et al., 2012).

Partial nucleotide sequence of suspected NDV isolates

A partial amino acids sequence at the cleavage site 112-117 was determined to all suspected

NDV isolates (CEC 1992). In addition the nucleotide sequence was done to the 375 region of the

NDV fusion (F) protein gene and the geogroup were determined according to Aldous et al. (2003).

All our NDV suspected samples were sequenced in Italy except one sample was sequenced in Jordan

(Ababneh et al., 2012).

Results and Discussion

Investigated Flocks

A total of 46 different poultry flocks with different age were investigated for ND virus isolation

and characterization. Samples were collected from Broiler (n=23), Layer (n=5), Broiler breeder

(n=3), Ostriches (n=1), Turkeys (n=1), Peacock (n=1), Ducks (n =2), and backyard local chicken

flocks (n=10). Some flocks were vaccinated with NDV vaccines once or more than once depending

on age, while other birds were not vaccinated or their vaccination history in question. Mortality

rates ranged from 1.5-25%, or 50% in vaccinated birds and 80-90% in non-vaccinated flocks. Clinical

signs vary from mild respiratory with or without nervous signs (torticollis) to sever respiratory

distress. Some birds show depression, ruffled father, green watery feces, blue comb and airsacculitis

with difficulty in breathing. Lesions ranged from mild trachietis with air sacculitis to congestion and

extensive hemorrhages in the trachea, caecal tonsils proventriculus and other parts of the digestive

system.

It is important to emphasis that clinical signs and gross lesions are not pathognomonic for ND

diagnosis. Therefore, laboratory diagnosis is very important and will allow differentiating between

velogenic ND and Highly pathogenic avian influenza.

Serology and virology

Following the inoculation of embryonated fowl’s SPF eggs 39 haemagglutinating agents were

isolated, identified and then fully characterized. Four of the HA positive isolates were identified by

monoclonal antibody as APMV2 “Yucaipa Viruses” Table 1”. All the four Yucaipa were identified in 4

broiler flocks. Other studies reported APMV-2 Yucaipa-like viruses isolation in China from the

imported Gouldian Finch (Chloebia gouldiae), while isolates from the same APMV serotype were

isolated from domestic and wild birds in Costa Rica. In general, APMV-2 was isolated from a variety

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of species including robin, eagle, pheasant, parrot, canary, domestic poultry, and waterfowl (Suarez,

2013).

Twelve isolates originated from broiler flocks and 4 from backyard flocks were identified as

APM1 (Lentogenic – B1 group) based on their intracerebral pathogenicity index (ICPI) of 0.1 to 0.3,

indicating that these isolates were not virulent. The amino acid sequence at the cleavage site 112-117

of these isolates was glycine, arginine, glutamine, glycine, arginine, and leucine (Table 1). The fusion

protein cleavage site amino acid sequence analysis indicated absence of multiple basic amino acids at

the C-terminus of the F2 region and phenylalanine at the N-terminus of the F1 region (GRQGR*L)

confirming the lentogenic nature of the viruses. These are considered vaccine strains as they were

isolated from vaccinated birds with La Sota ND vaccines. It was indicated by Aldous et al. (2003) that

all lentogenic isolates were grouped together with the vaccinal La Sota strain in genetic lineage 2.

Table 1: Avian Paramtxoviruses isolated from broiler and backyard flocks in Jordan

Flock Number

of Isolates

Typing Molecular

pathotype

(F protein)

ICPIa Serum Amino acid sequence at

cleavage site (112-117)

Broiler 4 PMV2 (Yucaipa) Not applied Not applicable -

Backyard 4 PMV1(Newcastle) GRQGR*LA Lentogenic – B1

groupb

0.1

Broiler 12 PMV1(Newcastle) GRQGR*LA

Lentogenic – B1

groupb

0.2- 0.3

Total 20 aICPI = Intracerebral Pathogenicity Index; AAmino acid symbols: G=glycine, R=arginine, Q=glutamine,

and L=leucine; Basic amino acids shown in bold. bTypization by E. W. Aldous et al., (see phylogenetic

tree) as Lentogenic – B1 group. ^ All the lentogenic isolates were grouped together with the vaccinal

La Sota strain in genetic lineage 2 (Aldous et al., Avian Pathology, 2003, 32:239-257).

Velogenic APMV1 was identified in 19 flocks including broiler breeders, layer, broiler, turkeys,

Peacock, Ostriches and Backyard flocks (Table 2). These isolates showed intracerebral pathogenicity

index (ICPI) ranging from 0.8 to 1.8. The amino acid sequence at the cleavage site 112-117 of these

isolates was (RRQKR*F): arginine, arginine, glutamine, lysine, arginine, and phenylananine (Table 2).

This Fusion protein cleavage site amino acid sequence analysis of these APMV-1 isolates indicated the

presence of multiple basic amino acids at the C-terminus of the F2 region and phenylalanine at the N-

terminus of the F1 region confirming the velogenic nature of the viruses.

Phylogenetic analysis of the NDV Jordanian isolates carries motif in the cleavage site of the

fusion protein which is consistent with motif present in most velogenic NDV isolates of the 5d

lineage; class II (Rui et al., 2010). A Phylogenetic tree of the Jordanian isolate compared with other

NDV sequences is shown in (Fig. 1).

The majority of velogenic NDV strains Africa and Asia are of lineage 5d (Berhanu et al., 2010;

Bogoyavlenskiy et al., 2009). In particular, in China most isolated NDV strains are of lineage 5d (Liu et

al., 2007). The Jordanian NDV isolate (Chicken/Jordan/Jo11/2011) had a nucleotide similarity in the

sequenced fragment of fusion gene of 99.4% to the Chinese strain SG/Liaoning/2009 and 95.3% to the

NDV of lineage 5d. The Chinese strain SG/Liaoning/2009 was isolated from a broiler chicken (age 22

days) in 2009 with a MDT of 45 h. The nucleotide similarity in the sequenced fragment of the fusion

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Table 2: Avian Paramtxoviruses-1 (Velogenic Newcastle) isolates from different poultry flocks

Flock Numbe

r of

Isolates

Typing Molecular

patotype

(F protein)

ICPIa Serum Amino acid sequence at

cleavage site (112-117)

Backyard 6 PMV1 (Newcastle) RRQKR*FA Velogenic 0.8- 1.6

Broiler 2 PMV1 (Newcastle) RRQKR*FA Velogenic 1.7

Layer 5 PMV1 (Newcastle) RRQKR*FA Velogenic

Breeder 3 PMV1 (Newcastle) RRQKR*FA Velogenic 1.7

Turkey 1 PMV1 (Newcastle) RRQKR*FA Velogenic 1.7

Peacock 1 PMV1 (Newcastle) RRQKR*FA Velogenic Not done

Ostriches 1 PMV1 (Newcastle) RRQKR*FA Velogenic 1.8

Total 19 a ICPI = Intracerebral Pathogenicity Index; A Amino acid symbols = F = phenylananine, G = glycine,

K= lysine, Q = glutamine, R = arginine; Basic amino acids shown in bold.

Fig. 1: Phylogenetic tree of the nucleotide sequences of the partial fusion gene fragment of NDV

isolated and the references strains from GenBank database. Sequences were aligned by using BioEdit

(v 7.0.5.3) and MUSCLE (v 3.7) programmes. Maximum likelihood (ML) phylogenetic analysis with

bootstrap values for n = 100 replicates was performed using PhyML phylogenetic interface. The

Jordanian NDV strain (NDV-Chicken/Jordan/Jo11/2011) is closely related to the Chinese strain

China/SG/Liaoning/2009 and belongs to the lineage 5d.

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gene of the Jordanian NDV to other NDV lineages was in the range of 83–90.6% with the lowest

similarity to NDV lineage 2 and the highest to NDV lineage 5c. Two Israeli NDV strains

(Peacock/Israel/IS 903/2008 and Chicken/Israel/IS 875/2008) were compared to the Jordanian NDV

isolate; the nucleotide similarity between these isolates was 94.7% and both Israeli NDV strains were

of the lineage 5d.

The Jordanian NDV isolate had a MDT of 46 h, which confirms the velogenic nature of this

isolate. According to the World Animal Health Information Database (WAHID) Interface

www.web.oie.int/wahis/public.php?page=dis- ease_immediate_summary, (accessed 05.09.11), recent

NDV outbreaks were reported in Honduras (1 outbreak), Mexico (2 outbreaks), Peru (3 outbreaks),

Sweden (1 outbreak) and Israel had 84 outbreaks. The most recent NDV outbreaks in Israel started

in mid-December 2010. The high nucleotide similarity between the Chinese strain SG/Liaoning/2009

and the Jordanian NDV isolate suggests that the source of the Jordanian NDV isolates may came

from China, as NDV outbreaks of South African NDV strains, have also been demonstrated to be

closely related to Chinese strains (Abolnik et al., 2004). Wild birds are considered to be the natural

reservoir of NDV and were blamed for certain NDV outbreaks (Burridge et al., 1975). Also they may

play a role in the evolution and the transmission of NDV to domestic fowl (Jindal et al., 2009; Kim et

al., 2007) and they might be responsible for the introduction of the Chinese NDV strain to Jordan.

Prevention and control strategies

Each method of NDV spread in prevention policies regardless if the control is applied at

international, national or at farm level needs to be considered in prevention of susceptible birds from

infection. Reduction of the number of susceptible birds can be achieved by vaccination.

The importance of biosecurity and preventing domestic poultry from contacting other birds

needs reinforcement. High levels of biosecurity on the farm certainly minimize the risk of

introduction of any poultry diseases in the poultry house. Biosecurity must be designed taking into

account requirements for each location. Water and feed quality and pest and litter management are

areas that need to be tightly controlled. Routine disinfection and cleaning procedures and post-

outbreak protocols should be in place prior to an ND outbreak. Biosecurity measures also should be

employed in backyard bird situations.

Prevention of wild birds to gain access to poultry houses is probably still the main source for

virus introduction. Training programs for poultry worker on the implementation of biosecurity

practices and sound vaccination programs should be employed. Providing Quarantine stations for

imported cage birds to ensure they were not infected and shedding the viruses. Mandatory

vaccination of racing pigeons should be implemented.

In many countries ND is often controlled by identifying and culling infected birds while

simultaneously restricting the movement of birds and bird products within a defined area surrounding

the infected birds. Disposing of infected carcasses and litter without further disseminating the virus is

problematic.

Ring vaccination around an outbreak may be employed according to country control policy. Since

all APMV serotypes are known or likely to have wild bird reservoirs that can spill over to poultry,

vaccination of wild bird species should also be considered as they continue to harbor virulent NDV

worldwide. Vaccines should decrease or prevent virus shedding from vaccinated birds especially in

endemic countries as seen in avian influenza vaccine development.

Vaccination policies vary with each country. Preventative or prophylactic vaccination is allowed in

most countries of the world. Estonia, Finland, and Sweden currently do not allow preventative

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vaccination of chickens. Finland also prohibits the rearing of poultry outside for portions of the year

when they are more likely to mingle with wild birds (Suarez, 2013).

Conclusion

It appears to be that APMVs circulating efficiently in Jordanian poultry industry and it is the first

report regarding APMV-2 (Yucaipa) circulation in Jordan. Newcastle is a devastating disease and

requires continuous surveillance programs to be implemented. The presence of multiple NDV strains

in the Far East and highly transmissible nature of the virus can complicate and increase the cost of

attempts to prevent the spread of infection to the Middle East and other parts of the world.

Biosecurity measures should be strictly implemented along with sound vaccination programs and

good managemental practices at the farm level and backyard flocks. Reporting system should be

implemented and needs to be upgraded by the Governmental and the private industry in Jordan.

Acknowledgments

The authors like to thank Jordan University of Science & Technology, Veterinarians in Jordan Ministry

of agriculture and Dr. Ilarea Capua research team OIE/FAO Reference Laboratory for Newcastle

Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università,

10, 35020, Legnaro, Padova, Italy.

References

Ababneh MK, AE Dalab, SR Alsaad, MB Al-Zghoul and MQ Al-Natour, 2012. Molecular

characterization of a recent Newcastle disease virus outbreak in Jordan. Res Vet Sci, 93: 1512-

1514.

Abolnik C, RF Horner, SP Bisschop, ME Parker, M Romito et al., 2004. A phylogenetic study of South

African Newcastle disease virus strains isolated between 1990 and 2002 suggests epidemiological

origins in the Far East. Arch Virol, 149: 603-619.

Aldous EW, JK Mynn, J Banks, DJ Alexander, 2003. A molecular epidemiological study of avian

paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial

nucleotide sequence of the fusion protein gene. Avian Pathol, 32: 239-256.

Berhanu A, A Ideris, AR Omar and MH Bejo, 2010. Molecular characterization of partial fusion gene

and C-terminus extension length of haemagglutininneuraminidase gene of recently isolated

Newcastle disease virus isolates in Malaysia. Virol J, 7: 183.

Bogoyavlenskiy A, V Berezin, A Prilipov, E Usachev, O Lyapina et al., 2009. Newcastle disease

outbreaks in Kazakhstan and Kyrgyzstan during 1998(2000), 2001, 2003. 2004, and 2005 were

caused by viruses of the genotypes VIIb and VIId. Virus Genes 39: 94-101.

Burridge MJ, HP Riemann and WW Utterback, 1975. Methods of spread of velogenic viscerotropic

Newcastle disease virus in the Southern Californian epidemic of 1971–1973. Avian Dis, 19: 666–

678.

Briand FX, A Henry, P Massinand and V Jestin, 2012. Complete genome sequence of a novel avian

paramyxovirus. J Virol, 86: 7710.

Capua I, PM Dalla, F Mutinelli, S Marangon and C Terregino, 2002. Newcastle disease outbreaks in

Italy during 2000. Vet Rec, 150: 565–568.

CEC, 1992. Council Directive 92/66/EEC of 14 July 1992 introducing Community measures for the

control of Newcastle disease. Off J Eur Comm, L260, pp: 1-20. European Union (EU).

Page 90: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

90

Fornells LA, TF Silva, I Bianchi, CE Travassos, MH Liberal et al., 2012. Detection of paramyxoviruses

in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast. Vet Microbiol,

156: 429–433.

Gould AR, JA Kattenbelt, P Selleck, E Hansson, A Della-Porta et al., 2001. Virulent Newcastle disease

in Australia: molecular epidemiological analysis of viruses isolated prior to and during the

outbreaks of 1998–2000. Virus Res, 77: 51–60.

International Committee on Taxonomy on Viruses. 2012. Virus Taxonomy: Ninth Report of the

International Committee on Taxonomy of Viruses. Academic Press, Waltham, MA.

Jindal N, Y Chander, AK Chockalingam, de M Abin, PT Redig et al., 2009. Phylogenetic analysis of

Newcastle disease viruses isolated from waterfowl in the upper midwest region of the United

States. Virol J, 6: 191.

Kim, LM, DJ King, PE Curry, DL Suarez, DE Swayne et al., 2007. Phylogenetic diversity among low-

virulence Newcastle disease viruses from waterfowl and shorebirds and comparison of genotype

distributions to those of poultry-origin isolates. J Virol, 81: 12641–12653.

Kaleta EF and C Baldauf, 1988. Newcastle disease in free living and pet birds. In: Newcastle Disease.

D.J. Alexander, ed. Kluwer Academic Publishers, Dordrecht, Netherlands; Boston,

Massachusetts, USA, 197–246.

Liu H, Z Wang, Y Wu, D Zheng, C Sun et al., 2007. Molecular epidemiological analysis of Newcastle

disease virus isolated in China in 2005. J Virolog Meth, 140: 206–211.

Miller PJ, EL Decanini and CL Afonso, 2010(b). Newcastle disease: evolution of genotypes and the

related diagnostic challenges. Infection, Gen Evol, 10: 26–35.

Miller PJ, EL Decanini and CL Afonso, 2010. Newcastle disease: evolution of genotypes and the

related diagnostic challenges. Infec, Gen Evol, 10: 26–35.

Miller PJ, CL Afonso, E Spackman, MA Scott, JC Pedersen et al. 2010(a). Evidence for a new avian

paramyxovirus serotype 10 detected in Rockhopper penguins from the Falkland Islands. J Virol,

84:11496–11504.

OIE, 2000. Biological Standards Commission. Report of the meeting of the OIE Standards Commission.

OIE, Paris.

OIE, 2008. Manual of diagnostic tests and vaccines for terrestrial animals. Available from:

<http://www.oie.int/eng/normes/mmanual/A_summry.htm>.

OIE, 2012. Manual of diagnostic tests and vaccines for terrestrial animals: mammals, birds and bees.

Biological Standards Commission, Vol. 1, Part 2, Chapter 2.03.14. OIE, Paris. 1–19.

Rui Z, P Juan, S Jingliang, Z Jixun, W Xiaoting et al., 2010. Phylogenetic characterization of Newcastle

disease virus isolated in the mainland of China during 2001–2009. Vet Microbiol 141: 246–257.

Suarez DL, 2013. Newcastle Disease, Other Avian Paramyxoviruses, and Avian Metapneumovirus

Infections Chapter 3 in: Diseases of Poultry, Thirteenth Edition. David E Swayne. pp: 89-138. ©

2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

World Animal Health Information Database (WAHID) Interface

www.web.oie.int/wahis/public.php?page=dis- ease_immediate_summary, (accessed 05.09.11),

Wu S, W Wang, C Yao, X Wang, S Hu et al., 2010. Genetic diversity of Newcastle disease viruses

isolated from domestic poultry species in Eastern China during 2005–2008. Arch Virol, 2: 253–

261.

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RESEARCH PROGRESS ON A NOVEL DUCK FLAVIVIRUS DISEASE

Sufang LV, Guangjun GUO, Guanggang Qu, Feng LI, Ling MO and Zhiqiang SHEN*

Shandong Binzhou Animal Science & Veterinary Medicine Institute, Binzhou 256600, China; Shandong

Lvdu Bio-industry Co., Ltd., Binzhou 256600, China

*Corresponding Author: [email protected]

ABSTRACT

A severe egg drop disease caused by the infection of a novel duck flavivirus outbroke successively

in many provinces in southeastern China since 2010. It was identified as a duck Tembusu virus

(DTMUV) and was named by Chinese Association of Animal Science and Veterinary Medicine in the

first symposium on waterfowl disease control, which was presumed to be a mosquito-borne flavivirus

of the Ntaya virus subgroup in the genus Flavivirus, family Flaviviridae. Currently, a large number of

studies have been conducted on the epidemiology, clinical symptoms and pathological changes,

etiology, and rapid diagnoses of the virus. The disease remains a constant threat to the duck industry.

In order to provide reference for subsequent in-depth study, in this paper, research progress on the

disease was summarized based on previous studies. Furthermore, the potential infection or

asymptomatic infection in humans should be evaluated as soon as possible.

Key words: Duck; Duck flavivirus; Research progress

INTRODUCTION

Since April 2010, a novel duck disease outbroke successively in Zhejiang, Fujian, Guangdong,

Guangxi, Jiangsu, Jiangxi, Anhui, Henan, Hebei, Shandong and Beijing, especially in most duck farms in

mid-eastern China. The disease mainly led to a serious decline in egg laying rate with rapid

propagation and wide range of infection. Infected ducks get a high fever at the early stage and exhibit

certain neurological symptoms at the late stage, such as paralysis, tumbling, astasia and ataxia. The

course of disease lasts 1-2 months. Resistant ducks can gradually recover laying capability, with an

elimination rate of approximately 10-30%. The disease causes serious economic losses to the duck

industry in China. According to previous studies, a new flavivirus was isolated from the tissues of

infected ducks, which was named as BYD (Bai Yang Dian) virus based on the geographical

nomenclature principle [1]. Infection of duck flavivirus will cause severe decline in egg laying rate and

ovarian inflammation in ducks. Therefore, the disease is also known as duck egg-drop syndrome, duck

infectious ovaritis, duck flavivirus infection and duck Tembusu virus infection. In the first symposium

on waterfowl disease control held by Chinese Association of Animal Science and Veterinary Medicine

in 2011, the disease was named as duck Tembusu virus disease. The pathogen is indentified as a novel

flavivirus ——duck Tembusu virus, which is a mosquito-borne flavivirus of the Ntaya virus subgroup

in the genus Flavivirus, family Flaviviridae. In this paper, the epidemiology, clinical symptoms and

pathological changes, etiology, diagnoses and detection of duck Tembusu virus disease was

summarized, which provided scientific basis for the prevention and control of this disease.

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Epidemiological Analysis

Under natural conditions, duck flavivirus (DFV) can infect all varieties of wild ducks and egg-

laying ducks (such as Shaoyang duck, Shanma duck, Jinyun duck and Jinding duck) except Muscovy

duck, and the susceptible animal populations tend to expand. Duck Tembusu virus disease possesses

rapid spread speed and wide propagation range, which outbreaks successively from south to north,

showing certain epidemicity. Once propagated, laying ducks in the farm will all be infected with the

disease, which outbreaks rapidly and can generally spread throughout the duck population within 3-7

d. According to previous studies, ducks with early onset of disease exhibit relatively low mortality,

while those with late onset exhibit high mortality and serious disease condition. In general, older

meat-type breeding ducks are susceptible to the disease and recover slowly, which can recover laying

capability two weeks later post-recovery and the laying rate can achieve 90% of the original level after

a month. So far, the pathogenesis and propagation mechanism of the disease are unclear yet.

According to clinical observations, horizontal propagation through the respiratory tract is an

important pathway. Most members in genus Flavivirus can be propagated by mosquitoes. Therefore,

duck Tembusu virus may be propagated through hemophagous bugs such as mosquitoes. However,

the disease was still highly prevalent by the end of 2010, indicating that its propagation is not simply

dependent on mosquitoes (Su et al., 2011). In clinical practices, the detection rate of duck Tembusu

virus in theca folliculi of infected ducks reaches the highest, suggesting that the virus may also be

propagated vertically. Duck Tembusu virus has been isolated from cloacal swabs, which indicates that

the virus may be discharged through excrement, thus polluting the environment, feeds, equipments,

eggs, egg trays, water and conveyances.

In addition to the ducks, Tembusu virus can also infect geese, sparrows, pigeons and other

species. Huang et al. (2012) reported that a flavivirus disease occurred continuously in a goose farm of

Jiangsu Province since April 2010; the brains, lungs, livers, hearts and ovaries all exhibited different

degrees of hemorrhage, congestion, swelling and spleen necrosis after dissection; a goose flavivirus

strain JS804 was isolated from the issues of diseased geese. Therefore, flavivirus can infect both ducks

and geese, which may be one of the reasons for the rapid spread and high pathogenicity of flavivirus

diseases. Liu et al. (2012) analyzed liver, brain, kidney, spleen and ovarian samples from 245 diseased

chickens and 57 diseased geese with NS5 gene-targeting RT-PCR method and found that 56.1% of

chicken samples and 38.6% of goose samples were infected with Tembusu virus, which led to a

significant decline in egg laying in animal regression tests. Lin et al. (2012) investigated the

susceptibility of chickens to duck flavivirus and found that duck flavivirus in chickens could not form

detectable viremia due to the low viral replication load; the reproductive system of chickens was not

the target organ of flavivirus, thus the infected chickens exhibited no significant clinical symptoms or

gross lesions. Tang et al. (2012) isolated flavivirus from dead house sparrows in a duck farm of

Shandong Province that could induce serious egg-drop disease in ducks. A pathogenicity test was

conducted by injecting the isolated flavivirus into ducks; results showed that the isolated virus was a

virulent strain, which revealed that house sparrows played an important role in the spread of viruses

among various species.

Clinical Symptoms and Pathological Changes

In clinical practices, duck Tembusu virus disease leads to rapidly decreased food intake, greatly

reduction in egg laying rate, significant drop of yield or even death. The egg laying rate could decline

from 90% to below 10% within 4-5 d. The disease occurs mainly in egg-laying ducks and breeding

duck. Previous studies reported that duck Tembusu virus disease could occur in 3-21-days old ducks

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(Yun et al., 2012). All varieties of ducks can be infected with Tembusu virus except Muscovy duck.

Egg-laying ducks infected with Tembusu virus exhibit increasing temperature, reducing food intake

and increasing water intake, mainly exhaust yellow-green loose stools on the 3rd d after the onset;

younger multi-batch breeding ducks are more susceptible to the disease; egg-laying ducks bred by

more than 60 weeks exhibit later onset of the disease and more serious disease condition; the

mortality rate is positively correlated with the control of secondary bacterial infection, which mainly

ranges between 3%-10%. In the incubation period, breeding ducks exhibit onset of the disease at

around 10 weeks old; disease cases first occurred in drakes, showing fervescence, depression, loss of

appetite and beak cyanosis, with the mortality rate lower than 5%. Commercial ducks exhibit onset of

the disease at around 20 days old and exhaust white watery stools.

After necropsy observations, diseased ducks mainly present ovarian dysplasia, increased

congestion in fallopian tube and hemorrhagic secretions, follicular degeneration, follicular hyperemia

and hemorrhage, a large amount of blutene chloaides on heart surface, pale myocardium, commonly

inner membrane bleeding, occasionally outer membrane bleeding, cord necrosis, liver swelling and

yellowing, mottled and marble-like spleens with occasional extreme swelling and cracking and surface

bleeding or needle-like white spotty necrosis, meningeal congestion and hemorrhage, cerebral edema,

hyperemia or bleeding, and varying degrees of pancreatic lesions in small intestines, kidneys and

pancreas.

In addition, diseased ducks exhibit various histopathological changes, including severe hepatic

steatosis, expanding bile capillary filled with bile pigments, inflammatory cells infiltrating around blood

vessels, decreased leukomonocytes in liver, intestinal villi shedding with loose lamina propria and a

large amount of inflammatory cells, significantly expanding oviduct vessels, a large amount of

erythrocytes in vessels, a large number of inflammatory cells infiltrating in epithelial tissue with loose

lamina propria, a large amount of degenerated and necrotic inflammatory cells in strata subserosum

and muscle cell layer with significant haemorrhage symptom, myocardial hemorrhage, necrosis of

myocardial fibers with occasional calcification and inflammatory cells infiltrating around fibers,

epicardial structural disorder with a large number of degenerated and necrotic inflammatory cells,

focal necrosis in pancreas, renal tubular epithelial cell swelling, cerebral vasodilation with a large

number of erythrocytes, swelling, degeneration and necrosis of nerve cells infiltrated with

inflammatory cells, significant "satellite phenomenon", inflammatory cells infiltrating in meninx,

softening and necrosis in cerebellum, partial necrosis in cerebral cortex, glandular gastric epithelial

cell degeneration and exfoliation, and a large number of inflammatory cells in the glandular cavity.

Etiological Analyses

Pathogens and taxonomic status

According to various literatures and reports, based on electron microscopy, analysis of physical and

chemical characteristics, molecular biology identification and artificial infection test, in accordance

with the international classification standards for Flavivirus members, approximately 1 kb sequence in

the 3' end of NS5 gene shares above 84% homology with the nucleotide sequence of the same species

of viruses, which indicates that the pathogen causing the serious egg-drop disease in China is a novel

duck flavivirus (DFV) that belongs to mosquito-borne flavivirus of the Ntaya virus subgroup in the

genus Flavivirus, family Flaviviridae. Flavivirus is a large group of single-strand RNA viruses, including

Japanese encephalitis virus, yellow fever virus, dengue virus, Ntaya virus and more than 70 members,

which have common antigenic determinants. Flavivirus strains can be divided into eight serum

subgroups and nine single serotypes. The incubation period of Flavivirus in mammals lasts about 12-18

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h and the duration of virus proliferation exceeds 3-4 d, which commonly causes non-dominant

infection in mammals and birds. Under natural conditions, Flavivirus is mainly spread by arthropods.

Biological characteristics of the pathogen

DFV has typical morphology of flavivirus. The virions are spherical, enveloped and 40-50 nm in

size, covered with spikes on the surface, which are mainly replicated in the cytoplasm of infected

cells. DFV is sensitive to chloroform, ether and deoxycholate that can be inactivated by 5%

chloroform, which is also sensitive to acid and alkali, with the optimal pH of 6-9. DFV is not resistant

to heat that can be inactivated by heating at 56 ℃ for 20 min or heating at 60 ℃ for 15 min. DFV can

not agglutinate erythrocytes of chickens, ducks, geese, pigeons, pigs, rabbits, rats and humans. DFV

can be propagated in duck embryos, chicken embryos, duck embryo fibroblasts (DEFs) and some

continuous cell lines such as Vero, human embryonic kidney cells (HEK293), vertebrate cells (DF-

1/BHK-21) and mosquito cells (C6/36), causing significant cytopathy. The diseased cells become

round, float and disintegrate, which can not be propagated in chicken embryo fibroblasts (CEFs) (Yun

et al., 2012). Compared with chicken embryos, DFV is rapidly propagated in duck embryos with

significant lesions. Therefore, duck embryos are more suitable for the isolation of DFV. Liao et al.

(2011) inoculated SPF chicken embryos with the isolated strain and found that the virus was highly

lethal to chicken embryos; its pathogenicity to chicken embryos (10-4.4 ELD50/0.2 ml) and to duck

embryos (10-4.6 ELD50/0.2 ml) was similar.

Molecular structure characteristics of the pathogen

DFV nucleic acid belongs to unsegmented infectious single-strand positive strand RNA consisting

of approximately 10 990 nucleotides containing a single open reading frame (ORF), 5' untranslated

region (UTR) and 3' untranslated region (UTR). The genomic structure is 5'-UTR-Cv-Ci-PrM-M-E-

NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3'. The 5'-UTR is 142 nt in length, containing a I-type

m7GpppNp cap structure. The 3'-UTR is 618 nt in length without poly (A) tail structure. UTR is the

initiation site for replication of flavivirus genome that affects the replication, translation and

pathogenicity of DFV. The 3'-UTR contains partial conserved sequences. The single open reading

frame consists of approximately 3425 aa and encodes 11 proteins. The structural proteins are located

on the 5' end, including capsid protein C, membrane proteins PrM and M, and envelope protein E. C

proteins contain a large amount of basic amino acids and are involved in viral assembly process; E

proteins can promote the fusion between virus and host cells, and induce the production of

neutralizing antibodies. There are seven non-structural proteins, including NS1, NS2A, NS2B, NS3,

NS4A, NS4B and NS5. Specifically, NS1 is the major immunogen in virus infection process (Yan et al.,

2011; Liu et al., 2012; Liu et al., 2012; Tang et al., 2012; Yun et al., 2012). At present, little information

is available on molecular biology of DFV. The functions of various genes and the mechanism of virus

infection still require further investigation.

Liu et al. (2012) analyzed genome sequence and domain of the isolated strain and found that the

C-terminal of NS3 protein contained conserved RNA helix domain (D285-E286-A287-H288), and C-

terminal of NS5 protein contained RNA-dependent RNA polymerase domain (G667-D668-D669).

Yun et al. (2012) predicted secondary structure of three isolated strains and found that conserved

sequence of 3' untranslated region was RCS3-CS3-RCS2-CS2-CS1 (the sequence played an important

role in virus replication process), with a typical number of cysteine residues in flavivirus: the number

of PrM proteins, E proteins, NS1 proteins were 6, 12 and 12, respectively (similar to the study of Liu

et al. (2012), and the number of potential N-linked glycosylation sites were 2, 1 and 3, respectively

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(different from the study of Liu et al. (2012)). Currently, the function of these sites is unclear. Some

scholars believe that these sites may play a certain role in virus replication, virulence, maturation and

release of virions, which provides important reference for subsequent research of virus replication

and pathogenesis. Li et al. (2013) cloned full-length cDNA of JXSP strain and obtained a large number

of viruses by transfecting cDNA into BHK-21 cells. The growth characteristics in BHK-21 cells and

toxicity to ducklings and BALB/c mice were similar to parental virus. The cloning of stable infectious

full-length cDNA provides valuable reference for further investigating the genetic characteristics of

the virus.

Diagnosis and Detection Methods

In general, duck flavivirus disease can be diagnosed preliminarily based on the onset age,

symptoms and pathological changes in clinical practices. By subsequent etiological and serological

diagnosis in the laboratory, duck flavivirus disease can be distinguished from other common duck

diseases including avian influenza virus and egg-drop syndrome which can also lead to egg laying

abnormality. Generally, duck Tembusu virus can not agglutinate erythrocytes of chickens, ducks,

pigeons and Bab1 / c mice. Therefore, it can be identified by 1% chicken erythrocyte hemagglutination

test and hemagglutination-inhibition test. At present, neutralization test, enzyme-linked

immunosorbent assay (ELISA), and molecular biology diagnostic technology are commonly used for

the diagnosis of DFV in laboratory.

Antibody detection method

Conventional serological method can be used to detect the level of virus-specific antibodies in

serum samples. Neutralization test targeting acute stage and recovery stage is one of the most

important methods for serological diagnosis. In addition, ELISA is also a commonly used method in

serological detection, which has been widely used. Li et al. (2012) developed E protein-specific

monoclonal antibody of FX2010 strain and established an ELISA method to block virus neutralizing

antibodies in serum that was rapid, sensitive and highly specific, with a coincidence rate of 70.6% in

serum neutralizing antibody test using chicken embryos. Ji et al. (2011) for the first time established

an indirect ELISA method for detecting DFV serum antibodies using purified DFV FX2010 strain as a

coating antigen and optimized various detection conditions; results showed that the optimal coating

concentration was 1.675 μg/hole, the optimal coating condition was placing at 37 ℃ for 2 h and at 4

℃ overnight, the optimal dilution for serum and secondary antibodies was 1:200 and 1:2000

respectively, and the threshold criteria was 0.432; according to the application results, the established

method exhibited high specificity, sensitivity and stability. Hao et al. (2012) established an indirect

ELISA method for rapid detection of DFV antibody using recombinant E protein as a coating antigen,

which provided technical means for serological monitoring of the disease.

Antigen detection method

With the continuous deepening of DFV studies, related molecular biology methods have been

constantly established and improved. Wan et al. (2011) designed primers based on NS5 gene

sequence of flaviviruss and established a RT-PCR method. Tang et al. (2012) established semi-nested

RT-PCR rapid detection method but results showed that RT-PCR and semi-nested RT-PCR exhibited

inadequate sensitivity and easily led to false positive results. Huang et al. (2012) designed two pairs of

primers according to gene sequence of goose flaviviruss JS804 strain and established a nested RT-PCR

method for detection of avian flavivirus, which exhibited strong specificity and 1 000 times higher

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sensitivity than conventional RT-PCR. The established method was applied to detect clinical samples

and results showed that the positive rate was 58.14%, while that of conventional RT-PCR was only

17.44%. Hao et al. (2012) and Zhang et al. (2012) established nested PCR detection systems with 1

000 times higher sensitivity than conventional RT-PCR. Yun et al. (2012) established a real-time RT-

PCR method with strong specificity and the minimum detectable value of RNA was 1 copies/reaction;

with the established method, the experimental process including nucleic acid extraction could be

completed within 2 h, suggesting that it was an ideal method for clinical diagnosis and epidemiological

investigation. Based on the isolated duck Tembusu-like BYD virus and other viruses with close

genetic relationship, Wang et al. (2011) designed six primers according to E protein gene and

established a one-step RT-LAMP detection method for gene amplification monitoring using SYBR1

green fluorescent protein as a marker. Based on a series of clinical detection and verification, RT-

LAMP method was simple and fast, which could be completed within 50 min.

Compared with the sensitivity of conventional RT-PCR (190 copies/μl), the minimum detection

limit for RNA of RT-LAMP method was 2 copies/μl. Therefore, RT-LAMP method was more suitable

for detection in basic laboratories or common laboratories without special equipments. Jiang et al.

(2012) established RT-LAMP method and SYBR Green1 real-time quantitative RT-PCR method for

laboratory evaluation and detection of clinical samples. Results showed that these two methods

possessed good specificity and high sensitivity, with the minimum detection limit of 1 × 10 -4 and 1×10-

3 PFU/reaction, respectively. The fluorescence method was more suitable for quantitative analysis.

The study also found that the spleen might be a major target organ for virus replication, which

provided a powerful tool for the diagnosis and epidemiological investigation of Tembusu virus. Yan et

al. (2011) and Gao et al. (2013) accurate, rapid, quantitative TaqMan real-time quantitative RT-PCR

methods. The RT-PCR method established by Yan et al. (2011) showed 100 times higher sensitivity

than conventional RT-PCR method; the method established by Gao et al. (2013) could detect 1.9

TCID50 viral nucleic acids at least. Yan et al. (2012) established a real-time RT-LAMP method and a

real-time RT-PCR method for comparison; results indicated that these two methods exhibited similar

sensitivity and good specificity with the minimum detection limit of 0.01 ELD50; in addition, the

reproducibility of RT-PCR method was slightly higher than that of RT-LAMP method. Overall, RT-

LAMP method was more convenient and easier with good sensitivity and high specificity, which could

be popularized and applied in resource-limited grassroots. Tang et al. (2012) also established an RT-

LAMP method for rapid detection; SYBR Green1 fluorescent dye was added and incubated at 64℃

for 45 min. Results showed that the minimum detection limit for RNA was 10 copies/μl; the

established method shared no cross reactions with other similar viruses, which was more convenient,

time-saving and labor-saving, with good specificity. Gao et al. (2012) amplified NS3 gene fragment of

DFV with RT-PCR technology and prepared digoxigenin-labeled nucleic acid probes; results indicated

that the probes had good specificity and the minimum detection limit for RNA was 100 μg/L. The

liver, lung, spleen, theca folliculi and cloacal swabs of suspected flavivirus-infected ducks were

detected; results indicated that the detection rate in theca folliculi was the highest, which provided a

reliable method for the studies of epidemiology and etiology of DFV.

Prevention and Control Measures

Currently, it is difficult to prepare inactivated vaccines of DFV due to its low reproducibility in

chicken and duck embryos and extremely low titer. No genetically engineered vaccines have been

developed for the prevention and control of DFV. Some research institutes produced vaccines using

the isolated virus and achieved some results. Recovered ducks will no longer be infected with the

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disease. Therefore, infected ducks can be treated by injecting egg yolk antibody of recovered ducks.

In our laboratory, specific serums of diseased ducks were collected one week after the onset for

treating DFV disease. Results showed significant prevention and control effect; injecting the specific

serums could rapidly relieve the symptoms of diseased ducks, improve appetite, and accelerate the

recovery of egg-laying capacity. Based on pathogenicity test and whole-genome sequence analysis,

Wan et al. (2011) selected highly pathogenic and stably propagated WR strain from five DFV strains

and developed inactivated oil emulsion vaccine; results showed that the developed inactivated oil

emulsion vaccine was safe, non-toxic and side effect-free, exhibiting good immune protection effects

on DFV virulent strain. Xu et al. (2012) confirmed that immunizing mice with E gene-encoding

recombinant plasmid DNA could induce effective immune responses, which laid foundation for

subsequent development of DNA vaccine. Han et al. (2012) investigated the formation and fluctuation

laws (virusemia period) of virusemia in Pekin ducklings and sheldrakes infected with duck flavivirus,

which provided reference for evaluation of vaccine potency and determination of the specific blood

collection time after infection. Pekin ducklings infected with flavivirus exhibited short-term (1-8 d)

virusemia at the early stage, the virus load in duck blood reached the maximum at 3.8 d post-infection

(91 h). The positive isolation rate of flavivirus from chicken embryos inoculated with serum samples

of female Pekin ducklings was significantly higher than that of male Pekin ducklings; virusemia in SPF

ducks (sheldrakes) reached the peak at 4 d post-infection.

At present, flavivirus disease is mainly prevented and controlled by strengthening the feeding and

management, standardizing common vaccine immunization programs, implementing all-in / all-out

system, forbidding mixed feeding of chickens, ducks and geese, appropriately adding antibiotics to

reduce secondary (concurrent) bacterial infection, and using detoxifying, heat-clearing and

dehumidifying herbal preparations to improve immunity. Specifically, the farm should be constructed

far away from the highway, slaughterhouses and live animal markets; stocking density should be

reduced, ventilation and insulation should be strengthened; the surrounding environment should be

cleaned and disinfected regularly; mosquitoes should be repelled by establishing mosquito-proof

window; no breeding poultry can be introduced from infected areas; all-in / all-out system should be

implemented; the daily feeding and management should be strengthened by selecting high-quality

feeds; the poultry house and warehouse should be managed strictly to prevent the invasion of house

sparrows; diseased, dead poultry and feces should be removed by innocent treatment.

References

Gao F, KX Yu, XL Ma et al., 2013. Development and application of real-time RT-PCR assay for duck

flavivirus. Chin J Vet Sci, 33: 16-19.

Gao XH, YX Diao, Y Tang, et al., 2012. Preparation of digoxigenin-labeled DNA probe for detection

of duck flavivirus and its application. Chin J Vet Sci, 32: 525-528.

Han CH, J Lin, YH Liu, et al., 2012. Study on the viremia changes of duck infectious by duck

hemorrhagic ovarian inflammation virus (Flavivirus). Proceedings of The 16th Symposium Poultry

Health Branch, Chinese Association of Animal and Veterinary Science, 10:116.

Hao MF, 2012. Development and application of the nested PCR assay for duck tembusu virus and

Recombinant Protein indirect ELISA Assa. Taian: Shandong Agricultural University: 41-44.

Huang X, K Han, D Zhao, et al., 2012. Identification and molecular characterization of a novel

flavivirus isolated from geese in China. Res Vet Sci, pii: S0034-5288(12)00347-5.

Huang XM, DM Zhao, YZ Liu et al., 2012. Establishment and application of a nested RT-PCR method

for detection of avian flavivirus. Anim Husb Vet Med, 44:1-5.

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98

Ji XW, LP Yan, PX Yan, et al., 2011. Establishment of an indirect ELISA for detection of antibody

against duck Tembusu virus. Chin J Prev Vet Med, 33: 630-634.

Jiang T, J Liu, YQ Deng et al., 2012. Development of RT-LAMP and real-time RT-PCR assays for the

rapid detection of the new duck Tembusu-like BYD virus. Arch Virol, 157: 2273-2280.

Li S, L Zhang, Y Wang et al., 2013. An infectious full-length cDNA clone of duck Tembusu virus, a

newly emerging flavivirus causing duck egg drop syndrome in China. Virus Res, 171: 238-241.

Li X, G Li, Q Teng et al., 2012. Development of a blocking ELISA for detection of serum neutralizing

antibodies against newly emerged duck Tembusu virus. PLoS One, 7: e53026.

Liao M, XD Mu, Y Geng, et al., 2011. The primary study on virus isolation of duck infectious egg

failings. Chin J Anim Infec Dis, 19: 22-26.

Lin J, CH Han, YH Liu et al., 2012. Study on the susceptibility of chicken for duck hemorrhagic ovarian

inflammation virus (Flavivirus). Proceedings of The 16th Symposium Poultry Health Branch,

Chinese Association of Animal and Veterinary Science.

Liu M, C Liu, G Li et al., 2012. Complete genomic sequence of duck flavivirus from China. J Virol, 86:

3398.

Liu M, S Chen, Y Chen et al., 2012. Adapted tembusu-like virus in chickens and geese in China. J Clin

Microbiol, 50: 2807-2809.

Liu P, H Lu, S Li et al., 2012. Genomic and antigenic characterization of the newly emerging Chinese

duck egg-drop syndrome flavivirus: genomic comparison with Tembusu and Sitiawan viruses. J

Gen Virol, 93: 2158-2170.

Su J, S Li, X Hu et al., 2011. Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related

flavivirus. PLoS One, 2011,6(3):e18106.

Tang Y, Y Diao, C Yu et al., 2012. Characterization of a tembusu virus isolated from naturally infected

house sparrows (Passer domesticus) in Northern China. Transbound Emerg Dis, 20.

Tang Y, Y Diao, C Yu et al., 2012. Rapid dsetection of Tembusu virus by reverse-transcription, loop-

mediated isothermal amplification (RT-LAMP). Transbound Emerg Dis, 59: 208-213.

Tang Y, Y Diao, X Gao et al., 2012. Analysis of the complete genome of Tembusu virus, a flavivirus

isolated from ducks in China. Transbound Emerg Dis, 59: 336-343.

Tang Y, YX Diao, XH Gao et al., 2012. Development of a semi-nested RT-PCR assay for detection of

duck flavivirus. Chin J Vet Sci, 32: 517-520.

Wan CH, SH Shi, GH Fu et al., 2011. Development and immune effects determination of duck

flavivirus oil emulsion inactivated vaccine. Poult Husb Dis Cont, 10: 20-22.

Wan CH, SH Shi, LF Cheng et al., 2011. Establishment of RT-PCR for detecting duck hemorrhagic

ovaritis causing abrupt egg-laying reduction in ducks. F J Agri Sci, 26: 10-12.

Wang YL, XY Yuan, YF Li et al., 2011. Rapid detection of newly isolated Tembusu-related Flavivirus

by reverse-transcription loop-mediated isothermal amplification assay. Virol J, 8: 553.

Xu DW, GX Li, XS Li et al., 2012. Construction and immunogenicity of DNA vaccine encoding E gene

of duck Tembusu virus. Chin J Pre Vet Med, 34: 305-308.

Yan L, P Yan, J Zhou et al., 2011. Establishing a TaqMan-based real-time PCR assay for the rapid

detection and quantification of the newly emerged duck Tembusu virus. Virol J, 8: 464.

Yan L, S Peng, P Yan et al., 2012. Comparison of real-time reverse transcription loop-mediated

isothermal amplification and real-time reverse transcription polymerase chain reaction for duck

Tembusu virus. J Virol Meth, 182: 50-55.

Yan P, Y Zhao, X Zhang et al., 2011. An infectious disease of ducks caused by a newly emerged

Tembusu virus strain in mainland China. Virology, 417:1-8.

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Yun T, D Zhang, X Ma et al., 2012. Complete genome sequence of a novel flavivirus, duck tembusu

virus, isolated from ducks and geese in china. J Virol, 86: 3406-3407.

Yun T, W Ye, Z Ni et al., 2012. Identification and molecular characterization of a novel flavivirus

isolated from Pekin ducklings in China.Vet Microbiol, 157: 311-319.

Yun T, Z Ni, J Hua et al., 2012. Development of a one-step real-time RT-PCR assay using a minor-

groove-binding probe for the detection of duck Tembusu virus. J Virol Methods, 181: 148-154.

Zhang L, BX Hu, SG Yan et al., 2012. Development and application of the nested PCR assay for duck

flavivirus. F J Agri Sci, 27: 124-129.

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TOXICOPATHOLOGICAL EFFECTS OF SUB LETHAL DOSES OF

THIAMETHOXAM IN COCKERELS

Shafia Tehseen Gul*, Muhammad Farooq, Ahrar Khan and Maqbool Ahmad1,

Riaz Hussain2 and Shoaib Niaz

Department of Pathology, 1Department of Theriogenology, University of Agriculture, Faisalabad-

38040, Pakistan; 2University College of Veterinary and Animal Science, Islamia University of

Bahawalpur, Bahawalpur-63100, Pakistan.

*Corresponding Author: [email protected]

ABSTRACT

Thiamethoxam belonged to the sub-class of the nicotinoid insecticide, is a second-generation

neonicotinoid. It provides excellent control of a wide variety of commercially important insect pests

on a variety of crops. The aim of present project was to find out the toxic effects of sub lethal doses

of thiamethoxam in cockerels. A total of 40 cockerels having age about 14 weeks were procured

from local market and divided into five equal groups. Group A was kept as a control and other four

were treated with thiamethoxam. Different doses (mixed in distilled water) were administered

through crop tubing, containing 250, 500, 750 and 1000 mg/kg of body weight to group B, C, D and E,

respectively on daily basis. Four birds from each group were euthanized at 15th and 30th day of

experiment. The blood samples with anticoagulant were collected for hematological parameters like

erythrocyte (RBC) and leukocyte counts (TLC), packed cell volume and hemoglobin concentration

(Hb. Conc.). Different organs like liver, kidney, proventriculus and intestine were also collected for

histopathology. The data thus collected were analyzed through ANOVA and different group means

were compared by Duncan’s multiple range tests using M-stat statistical software package. A

significant decrease in the numbers of RBC was observed in thiamethoxam treated birds as compared

to the control group. In Group D lowest RBC count was noted. The hematocrit values and Hb.

Conc. of groups B, C and D were significantly lower than control group and lowest values were

observed in group D. TLC decreased significantly in group C and D as compared to control group. In

the present study, the specific clinical signs of toxicity were observed in thiamethoxam treated birds.

Typical gross lesions were observed in kidneys, liver and intestine. Relative weight of the liver and

kidneys was increased significantly in groups C and D as compared to control group at day 15 and

30th of trial. Highest relative weights of liver and kidneys were observed in group D. Marked

Microscopic lesions were also noticed in liver, kidney and intestines of treated birds. It was concluded

that sub lethal doses of thaimothaxam can induce toxicity in cockerels.

Keywords: Thiamethoxam, Toxicopathology, Cockerels

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EXPERIMENTAL ADMINISTRATION OF CHLORPYRIFOS AND ARSENIC IN

BROLIER CHICKS: TOXICOPATHOLOGICAL EFFECTS

Muhammad Zishan Ahmad1,2, Ahrar Khan1*, Maqbool Ahmad3,

Muhammad Imran Arshad4 and Sami-Ullah5

1Department of Pathology, University of Agriculture, Faisalabad, Pakistan. 2School of Forensic

Sciences. The University of Faisalabad, Pakistan, 3Department of Theriogenology; 4Institute of

Microbiology, University of Agriculture, Faisalabad, Pakistan. 5Department of Applied Statistics,

University College of Agriculture, University of Sargodha, Pakistan.

*Corresponding Author: [email protected]

ABSTRACT

Arsenic (As) is the major contaminant of water leading to deterioration of drinking water quality

in Pakistan. Arsenic results in acute and chronic toxicity that can be exacerbated by exposure to

environmental contaminants. The pesticides are the major contaminants, which are frequently

present in various feeds and food stuffs. In Pakistan the use of insecticides increased in last decade,

which is dangerous to human health. Organophosphate (OP) insecticides the chlorpyrifos (CPF) due

to its broad spectrum activity has a major role agriculture farming. The purpose of present study was

to investigate the concurrent feeding of pesticide CPF in broiler birds and its effects on health

biomarkers. A total of 150 broiler chicks were used in the present study. Arsenic was fed at a dose

rate of 50 mg/kg orally in combination with CPF. The CPF was reconstituted in corn oil as vehicle (1

ml/kg) and it was fed orally through stomach tube at a dose of 5, 10 and 20 mg/kg body weight of

birds for 2 weeks. Birds in control group received corn oil 1ml/kg only. After 2 weeks of feeding, the

birds showed signs of toxicity including salivation, lacrimation, gasping, convulsions, frequent

defecation and tremors in (CPF-20 mg/kg+ As-50mg/kg BW) group. Significant decrease in body

weight was also observed in treated groups as compared with control group. Alterations in

hematological parameters i.e. total erythrocyte counts; hemoglobin concentration, hematocrit and

total leukocyte were observed in high dosed treated group (CPF-20 mg/kg+ As-50mg/kg BW) than

control birds or other low dosed fed birds. Significant decrease in acetylcholinesterase (AChE)

activity and higher levels of serum alanine aminotransferase (ALT) were found in (CPF-20 mg/kg+ As-

50mg/kg BW) fed birds compared to control birds. Moreover, histopathological changes including

necrotic and degenerative changes were observed in various internal organs of As-CPF exposed

birds. It is concluded that the co-exposure of chlorpyrifos and arsenic induced toxico-pathological

changes in broiler birds.

Key Words: Arsenic, Environmental Intoxication, Broiler Birds, Health Biomarkers

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POST MORTEM BASED STUDY ON BROILER DISEASES IN TRADITIONALLY

MANAGED FARMS OF AZAD KASHMIR

Zaheer Hanif1, Asim Shamim2* and Azar Hayat1

1Ittfaq Poultry Disease Diagnostic Laboratory and Clinic® Private Limite, Muzaffarabad, Azad

Kashmir, 2Department of Pathobiology, Faculty of Veterinary and Animal Sciences, The University of

Poonch, Rawalakot, Azad Kashmir

*Corresponding Author: [email protected]

ABSTRACT

Broiler rearing is a source of income for farmers and its consumption is cradle of protein for

consumers. Diseases of viral, bacterial and protozoal origin effect broiler production and economics

of business. The present study designed to evaluate the frequency of diseases in broiler rearing

traditionally in and around of Muzaffarabad, capital of Azad Kashmir for the period of one year from

January, 2014 to December, 2014. For the said purpose, a random sampling technique was applied for

the collection of samples. A total of 1000 broiler were collected from more than hundred farms

which is located at different places of Muzaffarabad, Azad Kashmir. Broiler were kept in separate

polythene bags which were properly labelled and brought to the Ittfaq poultry disease diagnostic

laboratory and clinic® located in the heart of Muzaffarabad city for clinical examination. Postmortem

was performed in order to get clear picture of diseases. The following diseases were observed and

recorded during the study period in decreasing trend on the basis of their clinical manifestation and

lesions present on their affected parts including, Infectious Bursal Disease, Collibacilois, Enteritis,

Chronic Respiratory Disease, Coccidiosis, New Castle Disease and Hydropericardium Syndrome.

The frequency of observed diseases were significantly different at each farm levels, in different age

groups and seasonally. However, ratio of viral origin diseases was significantly higher in wet season,

whereas bacterial diseases pattern was hig--h in dry season in the study area. Timely vaccination,

treatment and proper management for better health and production are recommended.

Key Words: Broiler, Post-Mortem, Diseases, Muzaffarabad, Azad Kashmir

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CLINICO-HEMATO-BIOCHEMICAL STUDIES OF NEWCASTLE DISEASE IN

ARSENIC INTOXICATED BROILER CHICKS

Hafiz Iftikhar Hussain1, Ahrar Khan1*, Cheng He2, Adeel Sattar1, M. Zargham Khan1, Shen Zhiqiang3

Jiakui Li4 and Aisha Khatoon1

1Department of Pathology, University of Agriculture, Faisalabad, Pakistan; 2Key Lab of Animal

Epidemiology and Zoonosis of Ministry of Agriculture; College of Veterinary Medicine, China

Agricultural University, Beijing, China; 3Shandong Binzhou Animal Science and Veterinary Medicine

Academy, Shandong 256600, China; 4College of Veterinary Medicine, Huazhong Agricultural University,

Wuhan, PR China

*Corresponding Author: [email protected]

ABSTRACT

This study was planned to evaluate the clinical, hematological and biochemical alterations of ND in

arsenic intoxicated broilers. Total 240 one-day old broiler chicks were purchased from a local hatchery

and kept on basal feed and water available ad libitum. Vaccination schedule for broilers was followed.

After seven days of acclimatization, the birds were divided randomly into eight equal groups. The

treatment was carried out in groups as, group 1 acted as control while groups 4, 6, 7, and 8 were given

the selected dose of disodium hydrogen arsenate from day 7 to 42. Groups 2, 5, 7 and 8 were

challenged by field isolated Newcastle disease virus at day 24th of experiment. Normal vaccination

schedule will be carried out in group 3, 5, 6 and 8. The dilution of arsenic (disodium hydrogen arsenate)

was administered orally through crop tubing. Slaughtering was done on experimental days 7th, 14th, 21st,

28th and 35th for the collection of blood and organs. Some physical parameters, hemato-biocemical and

histopathological studies were carried out. Data obtained were analyzed statistically.

Clinical signs exhibited by treated birds were salivation, frequent defecation, gasping, lacrimation,

convulsion and tremor. These clinical signs were more severe in arsenic intoxicated and NDV

challenged groups. Feed intake, absolute and relative weight gain significantly decreased in treated

groups. Gross lesions observed were shrunken liver, swollen kidneys, congested lungs and hemorrhagic

intestine. Microscopically necrosis of hepatocytes, cytoplasmic vacuolization, mononuclear cell

infiltration and hemorrhages in liver were observed. Congestion was present in intestine and sloughing

of epithelium was common. In kidney necrosis of tubular epithelium, cytoplasmic vacuolization, cellular

infiltration and atrophy of glomeruli were present. Different hematological parameters, TLC, TEC, Hb,

PCV, MCV, and MCHC were decreased in arsenic intoxicated and NDV challenged groups while the

condition was more severe with their combine exposure. Biochemical parameters like total serum

protein, albumin and globulin decreased significantly in treated groups. Serum creatinine, urea, ALT and

LADH were significantly increased from that of control group. The results so obtained clearly

demonstrated that arsenic treatment induce adverse effects on clinical, hematological and biochemical

parameters in broiler chicks and NDV challenge enhanced these conditions.

Key Words: Arsenic Intoxication, Broiler Chicks, Newcastle Disease virus Challenge, Hemato-

Biochemical Studies

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THE NUTRITION OF BROILERS IN MOROCCO AND THE HAND

ENCOUNTERED PROBLEMS – A CASE STUDY

Mohamed HACHIMI

Laboratoire de Biologie et Santé, Équipe de Recherche en Environnement et Parasitologie, UFR

Doctorale Parasitologie comparée : Applications médicales et vétérinaires, Faculté des Sciences,

Université Ibn Tofaïl, BP 133, 14000 Kénitra, Morocco

*Corresponding Author: [email protected]

ABSTRACT

The interest accorded to the animal feeding quality is a result of the food crises involvement.

Many stakeholders have developed during last decades a quality system and traceability tools to

enhance the risks control and monitoring at the level of the producing units. The coccidiosis, whose

pathogen belongs to the genus Eimeria parasites is prevalent in the intensive livestock and poultry are

among the major constraints hampering the development of the poultry sector in Morocco. A

parasitological study on 149 breeding of flesh chicken in the Gharb region demonstrated 19.5% of

suspected cases of coccidiosis. The location of the lesions and the microscopic aspect of the oocysts

involved three coccidial species, Eimeria necatrix, E. maxima and E. tenella. The breeding success

requires good control of medication and optimal hygienic measures. Our study aimed to explore

cecal and intestinal coccidiosis and to conduct a prospective epidemiological study on the parasitic

disease in chickens farmed in the region of Gharb. The coccidiosis increased the consumption index,

retarded growth and generated lots heterogeneous. The main causes of the development of the

disease were poor control conditions and food quality. The results of the parasitological chicken

breeding study, based on the location of lesions and the appearance of microscopic oocysts allowed

considering the involvement of three different coccidian species on all 290 samples examined: Eimeria

maxima, Eimeria necatrix and Eimeria tenella. The results presented concluded that coccidiosis is a real

economic threat to poultry. HACCP (Hazard Analysis Critical Control Point), or Hazard Analysis,

Critical Control Points (ADMPC) is an effective method for controlling hazards in this sector.

Key Words: Quality, Crisis, Traceability, Control, Morocco, Coccidiosis, Eimeria, Gharb, HACCP

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PROGRESSION OF IBH-HPS IN EXPERIMENTALLY INFECTED BROILERS

Shahid Ali1,*, Muhammad Shahid Mahmood2, Muhammad Asif Zahoor1 and Zeeshan Nawaz1

1Department of Microbiology, Government College University, Faisalabad-Pakistan. 2Institute of

Microbiology, University of Agriculture, Faisalabad-Pakistan.

*Corresponding Author: [email protected]

ABSTRACT

Infectious diseases are the leading cause of morbidity and mortality in domestic and commercial

poultry throughout the world. Among these, Inclusion Body Hepatitis-Hydro-Pericardium Syndrome

(IBH-HPS) is considered as one of the fatal diseases which cause high mortality in broilers. In the

current study, well isolated, purified and sequenced fowl adenovirus serotype-4 (Accession number:

DQ 264728) was used to induce experimental infection in broilers. The birds (15 days of age) were

divided into three groups consisting of 30 birds each i.e. group A: inoculated subcutaneously, group B:

inoculated through drinking water whereas group C was kept as control. All the birds were housed

under same controlled conditions and environment. Non-medicated feed and water was supplied ad

libitum throughout the experiment. Infected groups showed clinical signs of the disease as early as 5

days post infection (dpi). In group B mortality started 7 dpi which continued up to 11 dpi, whereas

birds in group A showed mortality 10 dpi which continued up to 14 dpi. All the remaining birds were

sacrificed at 15 dpi. Liver and kidney tissues were collected from all birds for the identification of

IBH-HPS virus using agar gel precipitation test (AGPT) and reverse passive haemagglutination assay

(RPHA). Mortality in group B was 83% and in group A was 60%, whereas no morbidity and mortality

has been noticed in control group.

Altogether, it is concluded from the results of present findings that IBH-HPS is highly contagious

disease of broilers which causes high mortality through oral route as compared to subcutaneous

route of infection. Furthermore, AGPT and RPHA are good diagnostic tools for identification of re-

isolated IBH-HPS virus from infected birds.

Key Words: Inclusion Body Hepatitis, Hydro-pericardium syndrome, Fowl Adenovirus, Broilers

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ANTIVIRAL ACTIVITY OF VIRO CARE GZ-08 AGAINST NEWCASTLE DISEASE

VIRUS IN POULTRY AND ITS IN-VITRO CYTOTOXICITY ASSAY

Abuzar Muhammad Afzal*, Muhammad Hidayat Rasool, Abu Bakar Siddique and Muhammad Saqalein

Department of Microbiology, Government College University, Faisalabad, Pakistan

*Corresponding Author: abuzar_336@ yahoo.com

ABSTRACT

Newcastle disease (ND), regarded as one of the most important disease of poultry throughout

the World caused by Newcastle Disease Virus (NDV); it causes huge economic losses to poultry

industry of Pakistan. Regardless of vaccination, other prevention and control measures are necessary

to prevent ND outbreaks. Natural resources were exploited to obtain antiviral compounds in several

latest studies. In this study, the antiviral activity of Viro Care GZ-08™ (a commercial product made by

Shigadry with earth a Japanese company) was checked in-vitro, in-ovo and in-vivo. The cytotoxicity assay

of the product was performed using Vero cell line. Based on overall results, from in-vitro, in-ovo and in-

vivo trials it was found that the stock solution and 1:2 dilution of GZ-08™ did have some antiviral

activity but at the same time these concentrations are cytotoxic too. During in-vivo trials it was

observed that stock solution and 1:2 dilution of GZ-08™ shown better results when it was used in

combination with conventional vaccine. As the concentration decreased, cytotoxicity lost but with

the loss of antiviral activity as well. Based on these findings, it may be endorsed that GZ-08™

sanitizer/spray can be used as antiviral agent to clean or disinfect some non-living surfaces against

different viruses in general and NDV in particular. However, for in-vivo use of GZ-08™ in poultry

against NDV, its dose adjustment is necessary in order to get maximum antiviral activity with least

toxicity.

Key Words: Newcastle disease, Viro Care GZ-08™, Cytotoxicity, Vero cell line

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IMMUNO-PATHOLOGICAL STUDIES OF NEWCASTLE DISEASE IN ARSENIC

INTOXICATED BROILER CHICKS

Adeel Sattar1, Ahrar Khan1, Hafiz Iftikhar Hussain1, Riaz Hussain2, Jiakui Li3, Muhammad Kashif

Saleemi1, Cheng He4, Shafia Tahseen Gul1 and Shen Zhiqiang5

1Department of Pathology, University of Agriculture, Faisalabad, Pakistan.2University College of

Veterinary and Animal Science, The Islamia University of Bahawalpur-63100, Pakistan; 3College of

Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; 4Key Lab of Animal

Epidemiology and Zoonosis of Ministry of Agriculture; College of Veterinary Medicine, Agricultural

University, Beijing, China; 5Shandong Binzhou Animal Science and Veterinary Medicine Academy,

Shandong 256600, China

*Corresponding Author: [email protected]

ABSTRACT

This study was carried out on 240 one-day old broiler chicks. After seven days of acclimatization,

the birds were divided in to eight equal groups. Group 1 was kept as control and groups 4, 6, 7 and 8

were given the selected dose of disodium hydrogen arsenate from 7 to 35 days @50 mg.kg-1 BW.

Groups 2, 5, 7 and 8 were challenged by field isolated ND virus at day 24. The normal vaccination

schedule was carried out in Groups 3, 5, 6 and 8. The birds were closely monitored for clinical signs.

Randomly selected six birds from each group were slaughtered humanely on 7th, 14th, 21st, 28th and

35th days. Serum samples were collected for determination of cellular and humoral immune response

and morbid tissues (bursa, spleen and thymus) were collected for histopathology. Different

immunological parameters were studied. The collected data were analyzed statistically.

The arsenic treated groups exhibited more prominent signs as compared to control and

vaccinated groups. Grossly thymus was hemorrhagic and bursa was regressed in arsenic treated birds.

Microscopically, bursa revealed increased interfollicular connective tissue, lymphoid cells depletion,

vacuolation in bursal epithelium and lymphoid cells in both cortex and medullary region. In challenged

groups, increased fibrosis and inter-follicular connective tissue proliferation was observed. In spleen

there was severe congestion, cytoplasmic vacuolation in some areas, disorganization of white and red

pulp, mild to moderate necrosis and hyperplasia of reticular cells was observed. In thymus severe

congestion, vacuolation of cytoplasm in modularly region and necrosis of monocytes were noticed.

The absolute and relative weight of spleen was increased significantly, whereas decreased in case of

bursa and thymus due to arsenic intoxication. The antibody titers against ND were decreased due to

arsenic treatment and response to SRBCs was also lowered in treated groups with respect to control

group. The phagocytic ability and lymphoproliferative response was decreased in case of arsenic

treatment and challenged groups with respect to control group. It can be concluded that arsenic is

capable of inducing immunotoxicity and histopathological alterations in broiler chicks and ND virus

has capabilities of increasing the harmful effects of arsenic either alone or in combination.

Key Words: Arsenic Intoxication, Broiler Chicks, Newcastle Disease virus Challenge, Immuno-

Pathological Studies

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DETECTION OF AVIAN POX VIRUS FROM FIELD CASES OF PIGEONS AND ITS

PATHOGENESIS IN EXPERIMENTALLY INDUCED INFECTION

Tayyaba Shafiq1, Farzana Rizvi1*, Tahir Habib Rizvi2, ShafiaTahseen Gul1, Muhammad Numan3,

Mudasser Habib4 and Nouman Amjad1

1Department of Pathology, University of Agriculture, Faisalabad, Pakistan. 2District Head Quarter

Hospital, Faisalabad, Pakistan. 3Livestock Production Research Institute, Okara, Pakistan. 4Nuclear

Institute for Agriculture and Biology, Faisalabad, Pakistan.

*Corresponding Author: [email protected]

ABSTARCT

Pigeon pox is an infectious and contagious disease of young birds, spreads horizontally and

outbreaks have been reported in several countries. A field survey of 20 pigeon farms suspected for

pigeon pox disease was carried out in and around district Faisalabad. Samples from skin lesions of

diseased birds were collected and subjected to Polymerase chain reaction (PCR) for confirmation.

Experimental infection in pigeons was induced by confirmed isolated virus. For this purpose, twenty-

birds of approximately four weeks of age were randomly selected and divided into two equal groups..

The pox virus was inoculated through wing web puncture method in pigeons of 1st group and they

were observed till the gross lesions development on skin. However, the 2nd group was kept as

control. Then these birds from both groups were euthanized humanely to collect blood samples with

and without anticoagulant for hematological and serum parameters. Tissue samples from skin lesions

were collected for histopathology. A significant increase in total leukocyte count, total protein and

globulin concentration was observed in the diseased pigeons as compared to healthy pigeons.

However, a non-significant difference was observed in red blood cell count, hemoglobin

concentration, packed cell volume and serum albumin concentration among pox infected and healthy

pigeons. Microscopically, several changes like hperkeratosis and hyperplasiain stratum corneum,

lymphocytic infiltration in stratum spinosum, bollinger bodies and vacuolization in the epithelial cells

of skin tissues were observed.

Key Words: Pigeon Pox, Wing Web Puncture, Bollinger bodies, Vacuolization.

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109

COMPARATIVE PROTECTIVE EFFECT OF GLYCYRRHIZINIC ACID AND PURIFIED

IMMUNOGLOBULINS (IgG) IN NEWCASTLE DISEASE INFECTED BROILER

CHICKS

Nouman Amjad1, Farzana Rizvi1, Muhammad Kashif Saleemi1, Muhammad Numan1,3

Tayyaba Shafiq1 and Tahir Habib Rizvi2

1Department of Pathology, University of Agriculture, Faisalabad, Pakistan. 2District Head Quarter

Hospital, Faisalabad, Pakistan. 3Livestock Production Research Institute, Okara, Pakistan.

*Corresponding Author: [email protected]

ABSTRACT

Newcastle Disease (ND) included in list A of World Organization for Animal Health and cause of

economic disasters of poultry. Vaccination is major source of prevention but outbreaks in vaccinated

flocks lead to huge economic losses. Certain biochemical and herbal are agents used to reduce the

devastating effects of ND, such as antiviral peptides and antibodies combination against certain viral

proteins. Immunoglobulins (IgG) were raised in broiler chicks by inoculating La Sota vaccine and IFA.

IgG were purified by successive precipitations with Ammonium Sulphate [(NH4)2 SO4], and its

efficacy was compared with antiviral agent Glycyrrhizinic Acid (VIUSID®). A total of 75 day old chicks

were divided in to five equal groups i.e. A, B, C, D and E. At age of 15 days, all groups were

inoculated with ND Virus except group E. Group A and B were treated with IgG 2 ml/bird, I/M 6

hours post infection (PI) and IV 5 days PI, respectively. Group C was treated with Glycyrrhizinic Acid

(VIUSID®) @ 1ml/liter of drinking water. Birds of groups D and E were designated as Positive and

Negative control respectively. Birds of group A showed lowest morbidity and mortality, while group

D showed highest morbidity and mortality. Group D was kept as positive control without treatment,

showed maximum clinical signs. During postmortem examination hemorrhages in proventriculus,

intestinal ulcers and congested trachea were observed. All groups showed high values of GMT, group

A showed high values due to treatment of purified IgG while group D showed high values of GMT

due to onset of disease. This trial concluded that purified IgG were used to decrease the lethal effects

in initial phase of disease. Glycyrrhetinic Acid (VIUSID®) reduced the mortality but unable to stop

mortality in infected flock.

Key Words: Newcastle Disease, Purified Immunoglobulins, Glycyrrhetinic Acid, GMT

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A NOVEL ANTIVIRAL EXTRACT OF RHIZOMA DRYOPTERIS CRASSIRHIZOMAE

BY ENHANCING IMMUNE SYSTEM IN SPF CHICKEN MODEL

Qiang Zhang, Jun Chu, Tianyuan Zhang and Cheng He

Key Lab of Animal Epidemiology and Zoonosis of Ministry of Agriculture, China. College of

Veterinary Medicine, China Agricultural University, Beijing 100193, China.

ABSTRACT

Rhizoma Dryopteris Crassirhizomae (RDC) is used widely in traditional medicine, prescriptions and

recorded both in Chinese Pharmacopoeia and Chinese Veterinary Pharmacopoeia. It fortifies general

vitality, improves digestion and builds up the body's defense against viruses. However, active extracts

for veterinary medicines are unavailable due to lack of further investigation. Our current study aims

to evaluate immune response post administration of Rhizoma Dryopteris Crassirhizomae extract in SPF

chicken model. One hundred and fifty three-week-old SPF chickens were randomly divided into 6

groups. Birds orally received 750 mg/kg, 500 mg/kg and 250 mg/kg of RDC granulations for 14 days.

Meanwhile chickens were administered with 100 mg/kg of Levamisole as the positive control while

birds were given intramuscularly 100 mg/kg of cyclophosphamide for 5 times with one-day interval as

the negative control. Chickens given physiological saline were included as the health control. Prior to

treatment, all above groups received one dose of attenuated vaccine against Newcastle Diseases

Virus (NDV) and Infectious Bronchitis Virus (IBV). Post administration RDC granulation, body weight,

and immune organ index, antibody levels, lymphocyte proliferation, monocytes-macrophage activity,

cytokines and NK cell activity were monitored on day 7, day 14 and day 21. With respect to whole

immune system booster, higher fabricius index were induced in the chickens with 250-500mg/kg of

RDC granulations from day 7 to day 14 while an increasing thymus index was determined in the

500mg/kg of RDC group on day 14 in comparison with the positive control group.As for humoral

boosting, both RDC groups and Levamisole were able to induce high positive NDV and IBV specific

antibodies as compared to the negative control group and healthy control. More important,

lymphocytes proliferation index increased significantly in the birds with 750 mg/kg of RDC

granulations as compared to that of Levamisole group. Also, significant increase in macrophage and

NK cell activity was also detected in the SPF chickens administered with 250 mg/kg of RDC

granulations. Based on the standard module associated with immune enhancing herbs, RDC

granulations are able to boost the immune system by both humoral and cellular response in chickens.

Our studies indicated that the RDC granulation is a promising antiviral herb extract against poultry

viral diseases.

Key Words: Rhizoma Dryopteris Crassirhizomae, immune system booster, humoral response, cellular

response, chickens

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PRIMARY INFECTION WITH CHLAMYDIA PSITTACI EXACERBATES RESPIRATORY

DISEASES OF AVIAN INFLUENZA VIRUS H9N2 BY SUPPRESSING IMMUNE

RESPONSE

Jun Chu1, Qiang Zhang1, Tianyuan Zhang1, E Han1, Peng Zhao1, Cheng He1 and Yongzheng Wu2

1Key Lab of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary

Medicine, China Agricultural University, Beijing 100193, China; 2Unit of Innate Defense

& Inflammation, INSERM U874, Institut Pasteur, 75015 Paris, France

ABSTRACT

Both Chlamydia psittaci (C. psittaci) and avian influenza H9N2 subtype virus pose a huge threat to

avian respiratory distress in poultry industry and are potential zoonotic agents. Clinically, H9N2 and

C. psittaci were isolated and identified frequently from the diseased chickens with severe respiratory

diseases. However, an association between con-infection with above two pathogens and pathogenesis

is unknown, resulting in the delay of the effective control strategy.

In Experiment 1, SPF chickens were administered intratracheally with highly virulent C. psittaci HJ

strain and low virulent C. psittaci CB3 strain, respectively. Meanwhile, all birds received intra-nasally

with one dose of the attenuated vaccine against Newcastle disease virus (NDV). Post inoculation, body

weight, immune organ index and T cell subsets were significantly elevated at days 7 and decreased at

days 10, whereas NDV-specific antibody levels were found lower in the chickens with C. psittaci HJ

group. Experiment 2: Birds were divided into 5 groups, Group A intratracheally received C. psittaci HJ

and H9N2 simultaneously; Group B were administered with C. psittaci HJ, then received H9N2 after 3

days later; Group C received H9N2 firstly then inoculated with C. psittaci HJ after 3 days later; Group

D and Group E received C. psittaci HJ or H9N2 alone, respectively. Consequently, lower body weight

gain, immune organ index and higher lesion score were detected in C. psittaci/ H9N2 group and C.

psittaci+H9N2 group in comparison with H9N2 alone. Moreover, 65%, 80% and 90% birds survived

in the combination of C. psittaci/ H9N2 group, C psittaci+H9N2 group and H9N2/ C. psittaci group as

compared to 100% survival rate in C. psittaci group and H9N2 group. Our studies indicate that

primary infections with C. psittaci contributes to severe respiratory disease by H9N2 infection

because of suppression of humoral immunity and due to alteration in Th1 balance.

Key Words: C. psittaci, H9N2, Co-infection, Respiratory disease, Immunosuppression

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112

THE ADJUVANT EFFECT OF BETA-1,3/1,6 GLUCAN ON NDV INACTIVATED

VACCINE

Tianyuan Zhang, Qiang Zhang, Jun Chu, Zonghui Zuo, Zhenhai Han, Jia Li,

Er Han and Cheng He

Key Lab of Animal Epidemiology and Zoonosis of Ministry of Agriculture, China. College of

Veterinary Medicine, China Agricultural University, Beijing 100193, China

ABSTRACT

β-Glucans from fungal and yeast sources have been widely used in enhancing protective immunity

against infectious agents. Moreover, β-glucan improves the antibody response in poultry immunized

by the avian influenza A H5N1 and H5N2 vaccines. However, the mechanism of the beta-1,3/1,6

glucan is unclear due to lack further research. Our research aims to evaluate both humoral response

and cellular response post immunization with the inactivated vaccine Newcastle Diseases Virus

(NDV).

Prior to vaccination, the inactivated NDV antigens were blended with 1%,2%,5%,10% of

beta-1,3/1,6 glucan and then emulsified with mineral oil into water-oil form vaccines. Sixty 21-day-old

SPF chickens were randomly divided into 6 groups. Chickens were administrated with the above

NDV inactivated vaccine with beta-glucan as the adjuvants while birds inoculated with the commercial

NDV inactivated vaccine as the positive control and the chickens received physiological saline as

negative control. Body weight gain, immune organ index, NDV antibodies, lymphocyte proliferation

and cytokines were monitored post immunization two weeks. With respect to whole immune system

boosting, spleen, thymus and bursa indices were significantly increased on day 14 and on day 21. Post

challenge with virulent NDV strain, all the birds were survived in the vaccines with 1% and 2% beta-

1.3/1,6 glucan as compared to 80% live birds with the commercial vaccine. In terms of humoral

response, the significant increasing NDV antibodies were also detected in the chickens inoculated the

vaccine with 2% beta-1.3/1,6 glucan compared to those of the commercial vaccine on two-time

points. As for lymphocyte subsets and cytokines, CD3+ T cells, CD4+T cells,IL-2,IL-6, IFN-γand IL-10

were significantly increased in the birds with 2% Β-1,3/1,6 glucan in comparison with other vaccines

with the beta glucans and the commercial vaccine. It may be concluded that beta-1,3/1,6 glucan is a

promising adjuvant in the inactivated vaccine against poultry viral diseases.

Key Words: beta-1,3/1,6 glucan, Newcastle Diseases Virus, adjuvant, Inactivated vaccine

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113

MOLECULAR DETECTION OF CHICKEN INFECTIOUS ANEMIA IN DAY OLD

BROILER CHICKS FROM FAISALABAD PAKISTAN

Saif-ur-Rehman, Muhammad Kashif Saleemi*, Muhammad Zargham Khan, Ahrar Khan, Bilal Aslam1,

Aisha Khatoon, Zain-ul-Abidin2 and Asim Shahzad

Department of Pathology; 1Institute of Physiology, Pharmacology, and Pharmacy, University of

Agriculture, Faisalabad; 2Veterinary Research Institute, Zarar Shaheed Road Lahore Cantt, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Chicken infectious anemia (CIA) is an emerging immunosuppressive infectious disease of poultry

in Pakistan. It results in high mortality, poor growth, immunosupression and poor response to vaccine

in young birds. In Pakistan, information regarding disease status of CIA in different types of birds

including commercial layers, broilers and parent stock is not yet available. Therefore, present field

study was designed to investigate the molecular epidemiology of chicken infectious anemia in day old

broiler chicks, because the detection of CIA in young chicks may also represent the status of disease

in parent flocks. For this purpose, a total 254 blood samples were collected from different farms and

hatcheries located in Faisalabad, Punjab, Pakistan. These samples were analyzed for chicken anemia

virus (CAV) through PCR, using specific primers (CAV1 & CAV2) of highly conserved VP-2 coding

gene. Total 38 (14.96 %) samples from 17 farms were found positive for CAV through PCR assy. The

hematological parameters like RBC, Hb and PCV of all samples were also determined and the

hematological values of CAV positive birds showed (RBC(X106/µl) 1.99 ± 0.37, Hb (g/dl) 5.88 ± 0.77,

PCV (%) 18.74 ± 2.97) severe anemia. The results of present study suggested that CIA is prevalent in

Pakistan. Further epidemiological and molecular investigations are required to design and implement

control strategies for this important immunosuppressive disease.

Key Words: Chicken Infectious anemia, Immunosuppression, PCR, Broilers, Epidemiology

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114

USE OF ETHNO-VETERINARY PRACTICES IN POULTRY DISEASES

Aamir Sharif1, Tanveer Ahmad2 and Jahan Zeb Ansari1

1Government Poultry Farm, Bahawalpur, Livestock and Dairy Development Department, Punjab,

Lahore – Pakistan, 2Department of Clinical Medicine and Surgery,

University of Agriculture, Faisalabad – Pakistan.

*Corresponding Author: [email protected]

ABSTRACT

In developing countries like Pakistan, the poultry sector is encountered with the high incidence of

infectious diseases. The use of chemical medicines is costly, have toxic effect in birds, pose the

serious threat of microbial resistance and involve risk of hazards of drug residues in poultry meat and

eggs. The use of ethno-veterinary medicine is one of the alternatives for treatment and control of

poultry diseases. The use of herbal extracts, homemade remedies, use of different parts of plants like

bark, stem, root, fruits, leaves, seeds, alone or in combination with other substances are the cheap,

economical, accessible and efficient substitutes of chemical medicines. The commonly used ethno-

veterinary practices include the use of leaves or bark of Azadirachta indica for endoparasites,

Kalanchoe crenata for coccidiosis, leaves of Carica papaya (pawpaw) for diarrhoea and endo-parasitism,

Piper guineese (pepper) for cough, use of Allium sativum (garlic) as repellent and use of Khaya

senegalensis (mahogany), Solanum nofiflorum (wild garden egg), Vernonia amygdalina (bitter leaf) and

Capsicum frutescens (pepper) for the treatment of Newcastle disease. It is required that more findings

regarding validation of ethnoveterinary medicines be documented by the researchers for

substantiating the results for potential and multipurpose uses of ethno-veterinary practices in the

treatment and control of poultry diseases.

Key Words: Ethno-veterinary practices, Poultry, Diseases, Treatment, Control

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115

JAPANESE QUAIL (Coturnix coturnix Japonica) GROW BETTER ON FEED

SUPPLEMENTED WITH NEOMYCIN RATHER THAN ORGAINC ACID

Umar Farooq*, Muhammad Farooq Khalid, Muhammad Khalid Bashir, Muhammad Amer Shehzad,

Muhammad Arslan, Muhammad Auon and Pervez Akhtar

University of Agriculture Faisalabad Sub-Campus, Toba Tek Singh, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Organically produce poultry products are consumer’s choice and there is also a huge demand to

eliminate use of antibiotic growth promoters from the animal feed. Hence the demand for scientific

research to find suitable natural growth promoter is intensifying. In this regard the present study was

planned to investigate the use of natural vs antibiotic growth promoter in Japanese quail diet. Mixed

sex Japanese quail chicks (n=280; 1 week old) were housed in an open sided house with floor pens

containing sand as a litter material. The following treatments were used: group A (2.5 g Neomycin

per kg feed), B (2.5 g organic acid comprised of formic acid, lactic acid, propionic acid, fatty acid,

propyleneglycol, oregano oil) and C (Control). Each treatment was applied to two replicates of 45

chicks each. Quails were reared under uniform conditions of humidity, temperature (33-38 ◦C),

ventilation, 18/6 hours light/dark cycle, ad-libitum feed (Table 2) and water supply. The data were

recorded on 14, 21 and 28 of age and following parameters were studied: Body weight, feed

conversion ratio, dressing percentage and giblet weights digestibility coefficients for CP (crude

protein), EE (ether extract), CF (crude fiber), and NFE (nitrogen free extract), and nutritive value of

TDN % (total digestible nutrients), DCP % (digestible crude protein), ME (metabolizable energy) and

mortality. The results showed significantly (P < 0.05) higher body weight (121.4±15) for Neomycin

compared with both, i.e., organic acid (112.2±11) and control (110.4±13). However, no statistical

difference was observed for any other parameter, i.e., FCR, dressing percentage, giblets weight,

digestibility coefficients and mortality. We concluded that use of Neomycin in quail diet is beneficial

for its growth.

Key Words: Growth promoters, Growth rate, Broiler quails

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116

INHIBITION OF CHICKEN RECOMBINANT FREEZE-DRIED INTERFERON TO NDV

F48E10 IN CHICKEN EMBRYOS1

Guo Shijin1,2, Wang Yanping1,2, Fu Shijun1, Zhang Zhimei1,2, Xu Qianqian1,2 and Shen Zhiqiang1,2*

1Shandong Binzhou Animal Science and Veterinary Medicine Academy, Shandong 256600, China; 2Shandong Lvdu Ante Veterinary Drug Co., Ltd., Shandong 256600, China

*Corresponding Author: [email protected].

ABSTRACT

This study was to investigate the inhibiting effect of chicken recombinant freeze-dried interferon

against Newcastle Disease Virus (NDV) F48E10 through vaccinating 9-day-old chicken embryo allantoic

cavity. After 24 hours of the reorganization, the 10 day chicken embryos established their own

immune system and then were infected with the NDV F48E10 to study the protection effect of

interferon to chick embryo in vivo. The Newcastle disease virus F48E10 (Virulent virus) was diluted

100 times with normal saline under aseptic conditions and inoculated in 10 pieces 10-day-old SPF

chicken embryo, 0.2 ml per egg. All the embryo died within 36-48 hours and the allantoic fluid was

collected. The hemagglutination titer of the recovery disease was 28. The virulence of Newcastle

disease (ELD50) showed that there was no chicken embryo death within 24-48 hours. In 48-72hours

mortality was 5, 4 and 1 in groups of titer 10-7, 10-8 and 10-9respectively. According to the Reed-

Muench method calculating median lethal dose, ELD50 was -8.5/ml, when the virus was diluted in 10-

8.5, 0.1 ml of each chicken embryo inoculation could make 50% of the chicken embryo death. Antiviral

activity increased with the increase of the inoculation content of interferon, the protection ratio

could reach 90% when the inoculation content was 1.28 mg/chicken embryos, when the inoculation

dose of more than 1.28 mg, the protection ratio of recombinant interferon on chicken embryo

decreased. The results showed that the recombinant F48E10 had a good anti F48E10 effect in a certain

range, and the protective ratio of chicken with 1.28mg/ chicken embryos was 90%.

Key Words: Chicken recombinant freeze-dried interferon; Chick embryo; F48E10; Protection ratio

1 This work was financially supported by the Shandong Modern Agricultural Technology & Industry

System, China (SDAIT-13-011-10) and Shandong Technical Innovation Project (201210916001).

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PREVENTIVE EFFECT OF QINGWEN BAIDU GRANULES ON THE ARTIFICIAL

CHICKENS INFECTIOUS BRONCHITIS2

GUO Shi-jin1,2,WANG Yan-ping1,2, FU Shi-jun1, ZHANG Zhi-mei1,2, YANG Li-mei1,2,ZHOU Chun-

feng1,2, XU Qian-qian1,2 and SHEN Zhi-qiang1,2*

1Shandong Binzhou Animal Science and Veterinary Medicine Academy, Shandong 256600, China;

2Shandong Lvdu Ante Veterinary Drug Co., Ltd., Shandong 256600, China

*Corresponding Author: [email protected]

ABSTRACT

Chicken infectious bronchitis (IB) is an acute respiratory and highly contagious disease, which

affected the egg production and egg quality. IBV strains of serotype and mutation as well as the strong

vaccine limitations are one of the large-scale farms frequent diseases. Therefore, rapid and effective

treatment of the IB is of great significance for large-scale farms. Qingwen Baidu granules are

composed of many kinds of traditional Chinese medicine such as rhizoma coptidis, buffalo horn,

rehmanniae, gypsum and radix scrophulariae by the combination process of traditional and modern

technology and provided by Shandong Lvdu Ante Veterinary Drug Co., Ltd (No. 20120210). In this

study, seven hundred 21-day-old Hy-line variety brown chickens were infected artificial by IB of

standard IBV M41 strain and then divided into seven groups at random. Before or after inoculation,

different dosages of Qingwen Baidu granules were added into drinking water to observe the clinical

symptom, incidence rate, pathological alterations and fatality rate, and with Qingwen Baidu powder as

positive control drug. The results showed that addition of 0.25‰ Qingwen Baidu granules in the

drinking water could protect the chickens from IBV strains M41 attacking significantly compared with

that of positive control group, the disease incidence of granule prevention group was only 9.0%,

which was significantly lower than the positive control group (P<0.01). 0.25‰ Qingwen Baidu

granules in drinking water can effectively cure the artificial chickens IB by standard M41 strain of IBV,

and the curative rate was 98.00%, and significantly higher than that of Qingwen Baidu powder treated

group and low dose treatment group and positive control group (P<0.01). In conclusion, the

recommended dose for preventing the chicken infectious bronchitis was 0.25‰, the curing dose was

0.5‰ in drinking water for 5 d.

Key Words: Qingwen Baidu granules; Artificial chickens infectious bronchitis; M41; Preventive

treatment

2 This work was financially supported by the Shandong Modern Agricultural Technology & Industry

System, China (SDAIT-13-011-10) and Shandong Technical Innovation Project (201210916001).

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PROTECTIVE EFFECTS OF BERBERINE IN DITHIO-CARBAMATE INDUCED-LIVER

OXIDATIVE STRESS AND TOXICITY IN CHICKEN BROILERS

Muhammad Shahzad1*, Riaz Hussain1, Zhi Wang2, Mudassar Iqbal1 and Jiakui Li2*

1University College of Veterinary & Animal Sciences, The Islamia University of Bahawalpur

2College of Veterinary Medicine, Huazhong Agricultural University Wuhan, China

*Corresponding Authors: [email protected], [email protected]

ABSTRACT

Dithio-carbamates are organic compounds being used as fungicide, pest and rodent repellents in

agricultural fields and thus occur as contaminants in products being employed for litter material and

poultry feed. Berberine, generally administered as chloride is an isoquinoline alkaloid of the

protoberberine type being used as an antifungal, antioxidative, anti-inflammatory and antitumor agent.

A study was conducted to investigate the protective effects of berberine chloride in dithio-carbamate

(thiram) induced-liver oxidative stress and toxicity in chicken broilers. One hundred and fifty

commercial chicken broilers were allocated into three groups: control group, thiram-induced group

(50mg/kg) and berberine (25mg/kg/day) treated group. Serum samples were collected on day 07, 11

and 14 post-hatch to determine the ALT (alanine aminotransferase), AST (aspartate

aminotransferase) and ALP (alkaline phosphatase) activity. The liver samples were collected at the

end of trial to determine the activity of SOD (superoxide dismutase), GSH-Px (glutathione

peroxidase) and MDA (malondialdehyde) contents. The results depicted that thiram increased the

level of serum ALT, AST and liver MDA contents while decreased the serum ALP and liver

antioxidant enzymes (SOD, GSH-Px); however, by treating the birds with berberine chloride, the

level of transferases (ALT, AST) and MDA contents were in normal range and the activity of ALP and

both antioxidants were detected close to normal as compared to control group. Moreover, different

stages of degeneration were also observed in liver histology in thiram-induced birds which became

normal on treating with berberine. In conclusion, our findings suggest that the oxidative imbalance

and damage to liver induced by dithio-carbamate can be restored by using berberine in chicken

broilers.

Key Words: Dithio-carbamate, Berberine, Liver toxicity, Antioxidants

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APPLICATION OF CHICKEN LITTER IN AGRICULTURE: PROPER MANAGEMENT

PRACTICE IS NECESSARY TO AVOID ENVIRONMENTAL CONTAMINATIONS BY

PATHOGENS

Muhammad Waseem* and Muhammad Asif Zahoor

Department of Microbiology, Government College University, Faisalabad, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

In agriculture, poultry manure is being used as organic fertilizers to improve the soil structure

and fertility. In spite of as nutrients source for crop production, chicken litter may also contains a

variety of pathogens; either host-specific or zoonotic, such as prions, viruses, bacteria, protozoa, and

helminthes. These pathogens are difficult to eradicate from poultry production facilities because some

are endemic to poultry and may have a resistant stage in their life cycle (e.g., a cyst or spore) that

enhances their survival in the environment and facilitates transmission to other animals or humans

through ingestion of fecal-contaminated water or food. Survival times in manure vary according to

pathogens, the medium and environmental conditions resulting in use of different manure

management practices depending upon the pathogens anticipated. Composting of manure, especially

when properly aerated, is an effective management practice that can generate the heat needed to

inactivate a number of pathogens, including Salmonella, Campylobacter, E. coli, and protozoa. Ultraviolet

light promotes die-off, and spreading manure on the surface during land application can promote

greater die off through exposure to UV light and desiccation. However, a small population of

pathogenic cells may survive or regrow in the finished compost products under favorable conditions.

Physical, chemical, and biological treatments can be alternative ways for pathogen inactivation, but

may not always lead to the complete elimination of foodborne pathogens in chicken litter. Based on

the hurdle concept, each kind of treatment can be used in combination alongwith other disinfection

strategies to potentiate microbial lethality.

Key Words: Chicken, Zoonotic, Pathogens, Food Borne, Litter treatment

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IMMUNE FUNCTION OF CHINESE FORMULA QINGWEN BAIDU GRANULES IN

BROILERS

Guo Shi-jin1, 2, Fu Shi-jun1, Xu Qian-qian1,2, Zhang Zhi-mei, Wang Yan-ping1,2 and Shen Zhi-qiang1,2*

1Shandong Binzhou Animal Science and Veterinary Medicine Academy, Shandong-256600, China

2Shandong Lvdu Ante Veterinary Drug Co., Ltd., Shandong-256600, China

*Corresponding Author: [email protected]

ABSTRACT

This study was designed to investigate the effects of Qingwen Baidu granules on the antibody

level, immune organ index and the lymphocyte transformation of broilers. Hy-line variety white cocks

of 30 days were used to evaluate the antibody titer of Newcastle Disease in each group, and MTT

method to determine the T lymphocyte proliferation, and organ weighing methods to measure the

immune organ index 21 days after immunization. Qingwen Baidu granules were provided by Shandong

Lvdu Ante Veterinary Drug Co., Ltd (No. 20120210). The birds were divided into 3 groups: (1) test

drug group birds were administered Qingwen Baidu granules @ 0.5 g/l dosage for 3 days through

drinking water at; (2) a positive control was treated with levamisole (1%) and a negative control

group without any treatment. After treatment, Live (Clone 30) ND Vaccine was administered by eye

drops/nasal inhalation.

The valence of ND antibody of test drug group was significantly higher than those of positive

control group and negative control group (P<0.05). However, there was no significant difference

between positive control group and negative control group, which indicated that Qingwen Baidu

granules could prolong the residue time in the body and improve the lymphocyte conversion ratio.

The test drug group and the positive control group both improved the lymphocyte conversion ratio

(P<0.05), and enhanced the cellular immune function. These results indicated that the Qingwen Baidu

granules could raise the disease resistance, improve the serum ND antibody level and peripheral

blood lymphocyte proliferation, enhance the cellular immune function, and elevate the immune organ

index and growth, in order to raise the immune function. Results indicated that Qingwen Baidu

granules can significantly potentiate cellular and humoral immunity. In conclusion, the Chinese

Qingwen Baidu granules can improve serum ND antibody level, improve peripheral blood lymphocyte

proliferation, enhance cellular immune function and elevate immune organ index and growth.

Key Words: Qingwen Baidu, Immunity, Broiler, ND antibody, Lymphocyte

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EFFECTS OF ELECTROLYTES IMBALANCE ON IMMUNITY AGAINST

NEWCASTLE DISEASE VIRUS INFECTION

Muhammad Hassan Mushtaq*1, Farrukh Ali Khan1, Aqeel Javeed1, Amjad Riaz1,

Hussain Filza2 and Amjad Khan1

1University of Veterinary and Animal Sciences, Lahore, Pakistan, 2Medical University of Vienna,

Vienna, Austria

*Corresponding Author: [email protected]

ABSTRACT

Sodium (Na) and chloride (Cl) function with phosphate and bicarbonate to maintain optimum pH of

the body. The minimum level of Na in poultry rations is 0.15%. Low Na level affect ration consumption,

while high level has laxative effect. The recognition of Na towards causing edema has shown the

importance of balance of electrolytes in the body. Taking in consideration the importance of Na salts in

broiler ration, present project was designed to observe the effect of excessive dietary Na salts on weight

gain, FCR, serum Na concentration, edematous lesions and on the immune status of the broiler against

NDV infection. For this purpose 100 broiler chicks were randomly divided into 4 groups. Group A, B, C

and D were fed on diet with 0.36% NaCl, 0.36% sodium bicarbonate, 0.18% NaCl and 0.18% sodium

bicarbonate and 0.18% sodium salts (routine) respectively. On day 8 and 28 ND vaccine was

administered to all groups. All the birds were weekly weighed to calculate FCR. Blood samples were

collected on days 14, 28 and 42 day age to determine the antibody titer against ND virus through

Haemagglutination Inhibition (HI) Test and for the estimation of serum sodium concentration through

spectrophotometry. Results showed that birds of group A had better feed conversion ratio and weight

gain as compared others, whereas birds of group D had poor FCR as compared to the birds of group B

and C. On analysis of serum Na concentration by spectrophotometer, the birds of group A had

maximum Na concentration and birds of group D had lowest serum Na concentration. Statistical analysis

showed a significant (P<0.05) difference in the serum Na levels of all groups except within group B and C.

The highest GMHI titer against ND virus was observed in sera of birds of group D and the lowest in the

sera of birds from group A. No edematous lesions were observed in birds of any group. It is also

interesting that electrolytes imbalance may influence not only performance but also the immune response

of broilers against ND virus.

Key Words: NDV, Vaccine, FCR, Immune response.

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A CROSS-SECTIONAL SURVEY ON PARASITES OF BACKYARD POULTRY IN

PUNJAB PAKISTAN

Ghazala Nawaz1, Muhammad Nawaz Malik2, Muhammad Hassan Mushtaq*3, Fraz Munir Ahmad4, Ali

Abdullah Shah5, Farooq Iqbal6, Shinawar Waseem Ali7, Aqeel Javed8 and Amjad Khan9

1Veterinary research institute Lahore, Pakistan, 2Project Director (Diagnostic Laboratories) L&DD,

Punjab, Pakistan, 3Department of Epidemiology and Public Health, University of Veterinary and Animal

Sciences, Lahore, Pakistan, 4Assistant Disease Investigation Officer Rahim Yar Khan, Punjab Pakistan, 5Pathobiology PMAS Arid Agriculture University, Rawalpindi, Pakistan, 6Department of livestock

production and management, PMAS Arid Agriculture University, Rawalpindi, Pakistan, 7Institute of

agriculture sciences, Punjab University, Lahore, Pakistan, 8Department of Pharmacology, University of

Veterinary and Animal Sciences, Lahore, Pakistan, 9Department of Epidemiology and Public Health,

University of Veterinary and Animal Sciences, Lahore, Pakistan.

*Corresponding Author: [email protected]

ABSTRACT

In Pakistan, backyard poultry rearing amongst rural communities is one of the important venture

and essential part of the mixed farming system. Same is the case in many developing Asian and African

countries. Very few studies have been conducted regarding the prevalence of endoparasites in free

ranging poultry in Pakistan. The present cross sectional survey was designed to compute

endoparasites prevalence in backyard poultry in rural communities’ small holder farming system in

Punjab province. This is the first in its nature in Pakistan as a model survey conducted parallel to mass

vaccination against Newcastle disease virus in Poultry. The study area was divided into three

zones/regions, i.e., northern, southern and central Punjab for spatial distribution pattern of

endoparasites to unveil the most infested areas. A total of 17,061 villages and 55,586 rural households

were visited and faecal samples (n=242106) were collected and were tested for different egg/ova type

and parasitic worm loads. Cumulative higher prevalence of 86.69% was recorded in southern,

followed by central and northern Punjab, respectively. While at specie level 47.88% samples were

found positive for nematode and 38.8% for coccidian parasites in southern Punjab. Furthermore the

highest burden of parasitic infestation of 100% prevalence rate was estimated in the backyard poultry

population of district Khanewal in the southern Punjab. It was concluded that southern and central

Punjab rural communities were infested the most, therefore, prioritization regarding control

strategies should be focused on these areas.

Key Words: Backyard poultry, Rural, Coccidian, Nematodes, Pakistan.

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REGULATION EFFECTS OF PROTOCATECHUIC ACID ON CELL APOPTOSIS

CAUSED BY INFECTIOUS BURSAL DISEASE

Changbo Ou1 and Cheng He2*

1College of Animal science, Henan Institute of Science and Technology, Xinxiang 453003, Henan,

China, 2Key Lab of Animal Epidemiology and Zoonosis of Ministry of Agriculture, China. College of

Veterinary Medicine, China Agricultural University, Beijing 100193, China

*Corresponding Author: [email protected]

ABSTRACT

Infectious Bursal Disease Virus (IBDV), one of important avian pathogens, could cause immuno-

suppressions in 3- or 6-week-old chickens and bursal B lymphocytes apoptosis. In our previous

studies, protocatechuic acid (PCA) could effectively increase survival rates and improve bursal injury

of chickens artificially infected with IBDV. Therefore, the current study was to investigate the

regulation effects of PCA on bursal damage caused by IBDV infection. Thirty-five 21-day-old SPF

chickens were randomly divided into two groups with the PCA group 20 and infection group 15.

These chickens were inoculated intraocularly with 0.2 ml of 102.5 EID50 of IBDV strain CJ801. The

birds were orally administered with 20 mg/kg body weight of PCA and saline water respectively, for 5

days and then monitored daily for 10 days uptill 24 hours post infection. Bursae were collected from

chickens on days 3, 6 and 9 after infection for immunohistochemistry. These paraffin sections were

then analysed for protein determination of Bax and Bcl-2. TdT-mediated dUTP nick end labeling

(TUNEL) was also used to quantify apoptosis of bursa lymphocytes. The immunohistochemistry

results displayed that the ratio of Bax/Bcl-2 in the PCA group significantly increased (P<0.01)

compared to that of the infection group. However, the pro-apoptotic protein Bax and anti-apoptotic

protein Bcl-2 in the infection group simultaneously reduced from days 3 to 9. The TUNEL results

showed that the apoptotic percentage of lymphocytes in the PCA group was non significantly lower

than that of the infection group at third day, but the apoptotic percentage increased a little in the

PCA group at day 9. These results indicated that PCA could promote cell apoptosis at the early stage

of IBDV infection, while it delayed cell apoptosis at the later stage of IBDV infection.

Key Words: Protocatechuic acid, Infectious bursal disease virus, Apoptosis, Bax, Bcl-2

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PREPARATION AND EXPERIMENTAL EVALUATION OF BIVALENT INACTIVATED

AVIAN INFLUENZA (H9N2) AND THERMOSTABLE NEWCASTLE DISEASE

VACCINE AGAINST CHALLENGE IN BROILERS

Rabia Sabir and M Shahid Mahmood*

Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Avian Influenza virus (AIV) and Newcastle Disease virus (NDV) are responsible for causing Avian

Influenza (AI) and Newcastle Disease (ND) respectively in birds, both of which detrimentally affect

the poultry birds in terms of significant economic losses. Biosecurity and immunization are the

promising ways to combat viral infection. Bivalent vaccines provide protective immunity against these

two major diseases and are more economical than monovalent vaccines.

The goal of current research was production of bivalent montanoid adjuvanted-inactivated

Newcastle Disease (ND) and Avian Influenza (AI) vaccine. Research was carried out in three phases.

Primarily the antigens (I2strain of Newcastle Disease, H9N2 serotype of Avian Influenza) were verified

quantitatively and qualitatively using biological and serological assays. Second phase of research was

preparation of bivalent AI-ND vaccine. Quality control tests such as safety and sterility tests were

executed according to standard protocols. In third phase of research broiler chicks were grouped as

follows: Group A were immunized with bivalent inactivated vaccine prepared during the study, Group

B received commercially available bivalent inactivated vaccine and Group C received no vaccine (PBS).

Later on, 35 days old birds were challenged with AIV and NDV and at 14, 21, 28 and 40 days of

vaccination, sera were collected from each group and antibody titers were determined through HI

and ELISA.

Results of HI test and ELISA indicated that maximum antibody titers of self-made bivalent vaccine

were achieved at 28 days of age that was 128 and 1.784 for AI; 124.8 and 1.951 for ND respectively.

However, antibody titers at day 40 (after virus challenge) were 73.60 and 1.619 for AI; 73.60 and

1.650 for ND, respectively. Similarly results of commercially available bivalent vaccine were at 28 days

of age was 56 and 1.123 for AI; 36.80 and 1.235 for ND respectively. However, antibody titers at day

40 (after virus challenge) were 22.40 and 0.825 for AI; 22.40 and 0.704 for ND, respectively.

Research data was statistically analyzed through T-test and the results for HI test of self-made

bivalent vaccine were significant however, the results were highly significant for ELISA. Towards the

end, it was concluded that bivalent vaccine prepared during this study yielded better and long lasting

antibody titers as compared to commercially available bivalent vaccine for ND and AI.

Key Words: Newcastle Disease virus, Avian Influenza virus, Broilers, Bivalent vaccine, ELISA.

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BLADDER BLOCKAGE AND TESTICULAR CHANGES IN MALE OSTRICH

(STRUTHIO CAMELUS) ASSOCIATED WITH ASPERGILLOSIS

Riaz Hussain1, Fazal Mahmood2, Sajid Hameed1 and Mudassar Iqbal1

1University College of Veterinary and Animal Science, The Islamia University of Bahawalpur-63100,

Bahawalpur; 2Department of Pathology, Faculty of Veterinary Sciences, University of Agriculture,

Faisalabad-38040, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Huge economic losses occur in terms of poor growth, decrease production and mortality in different

species of birds including ostrich throughout the worlds due to aspergillosis. The present report

elaborates the simultaneous occurrence of blocked bladder associated with penile protrusion,

proventriculus impaction and testicular changes in a five years old male ostrich (Struthio camelus) died

of aspergillosis infection. Prior to death different clinical signs such as anorexia, dysponea and

coughing were observed. Necropsy of male ostrich revealed significant enlargement of urinary

bladder impacted with yellow color and amorphous material. Spherical grayish white raised areas of

caseous necrotic foci of variable diameter were sparsely spread over the air sacs. Multiple solitary

well circumscribed nodules with hard consistency, hanging with fibrous threads in air sacs were

packed with yellow cheesy material. Grossly the testes were smaller in size and hard in consistency.

Histologically, the testes were significantly atrophied; there were increased connective tissue

proliferation with chronic inflammatory cell infiltration. Moreover, seminiferous tubules were lined by

one to two layers of cells exhibiting degenerative and necrotic changes. Some tubules showed

obliterated lumen and multinucleated giant cells with engulfed necrotic cells were also observed.

Aspergillus samples were collected and stained with lactophenol, Giemsa’s and florescent stain

revealed prominent hyphae and sporangium, histologically lungs tissue revealed multiple areas of

caseous necrosis. Proventriculus was full of gravel, iron bars corn cobs and plastic bottles.

Key Words: Ostrich, Aspergillosis, Bladder, Testes, Histopathology

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SERUM BIOCHEMICAL AND HISTOPATHOLOGICAL CHANGES INDUCED BY

CONCURRENT EXPOSURE OF ARSENIC AND COPPER SULPHATE IN ADULT

MALE BIRDS

Abdul Ghaffar1*, Riaz Hussain2 and Ahrar Khan3

1Department of Life Sciences (Zoology); 2University College of Veterinary and Animal Sciences, The

Islamia University of Bahawalpur- 63100; 3Department of Pathology, University of Agriculture,

Faisalabad-38040, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

The present experimental study was conducted to determine the clinico-heamatological and

serum biochemical impacts induced by concurrent oral administration of arsenic and copper sulphate

in adult male birds. After seven days of acclimatization a total of 28 adult male birds were randomly

divided into 7 groups each having four birds. Arsenic and copper sulphate alone and in different

combinations was given to birds for 30 days. Blood samples were collected from each bird at days 10,

20 and 30 of the experiment. Various clinical signs like decreased feed intake, body weight, ruffled

feather, depression, dullness, ocular discharge, open mouth breathing, diarrhea and pale comb were

observed at higher levels of arsenic and copper sulphate. Absolute and relative weight of liver testes,

kidneys, spleen, lungs trachea and thymus were significantly different in treated birds as compared to

control group. Serum biochemical parameters such as aspartate aminotransferase, alkaline phosphate,

creatine-kinase, cholesterol, triglyceride and malondialdehyde concentrations were significantly (P<

0.05) higher in different treated groups at different experimental days. From the results of this study

it can be concluded that arsenic and copper sulphate alone at higher levels and in combination even at

lower levels poses serious clinico-biochemical effects in avian species.

Key Words: Birds, Copper sulphate, Arsenic, Serum Biochemistry

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EFFECTS OF DIFFERENT LEVELS OF DAP AND ARSENIC ON SOME HAEMATO-

BIOCHEMICAL AND HISTOPATHOLOGICAL CHANGES IN LAYERS

Riaz Hussain1*, Abdul Ghaffar2, Ahrar Khan3 and Muhammad Shahzad1

1University College of Veterinary and Animal Sciences; 2Department of Life Sciences (Zoology), The

Islamia University of Bahawalpur- 63100; 3Department of Pathology, University of Agriculture,

Faisalabad, Pakistan-38000

*Corresponding Author: [email protected]

ABSTRACT

In present experimental study some hemato-biochemical and histopathological effects of DAP

and Arsenic were observed in layers birds. For this purpose a total of 72 chicks of same age and

weight were purchased from local hatchery and were kept under similar conditions. After one week

of acclimatization all the birds were randomly divided into six equal groups (A-F) having twelve birds

each. Different levels of diammonium phosphate and arsenic in combinations were given to birds

orally for 39 days. Four birds from each group were killed at days, 13, 26 and 39 of the experiment

for collection of blood. A significant decrease in hematological parameters such as erythrocyte

counts, hemoglobin concentration and hematocrit values were recorded as compared to control

group. A significant increase in serum cholesterol and creatinine phospho kinase concentration was

also recorded at higher level of DAP and arsenic. Histopathological examination of different tissues

exhibited various microscopic changes in thymus, kidneys, bursa, liver and kidneys. Moderate to

severe congestion in thymus, increased urinary space and tubular degenerations in kidneys,

vaccuolation in bursa and liver were the prominent features. The results of this study shows that

different levels of DAP and arsenic poses adverse impacts even at low levels in combination in birds.

Key Words: Birds, Arsenic, Diammonium phosphate, Blood, Serum, Histology

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SEROLOGICAL SCREENING OF BROILER AND LAYER FLOCKS FOR MYCOPLASMA

SYNOVIAE INFECTION IN PAKISTAN

Madiha Kiran, Syed Ehtisham-ul-Haque*, Usman Waheed and Muhammad Younus

Department of Pathobiology, College of Veterinary and Animal Sciences Jhang-35200, Pakistan

Corresponding Author: [email protected]

ABSTRACT

Mycoplasma synoviae (MS) is an economically significant and second most important avian

Mycoplasma pathogen for the worldwide poultry production. It has been associated with subclinical

upper respiratory tract infection and infectious synovitis in broiler, layer and breeder flocks. The

most effective control measure is to maintain MS free breeder flocks through good biosecurity and

regular monitoring with serology. The present study was carried out to monitor MS infection in the

poultry farms located in Districts Jhang and Layyah of Punjab, Pakistan by serological technique i.e.

rapid serum agglutination (RSA) test. A total of 104 sera samples were collected from broiler and

layer birds with the history of respiratory disease and analyzed for antibodies against MS. RSA was

performed using SPAFAS MS Plate Antigen (Charles River Lab., CT, USA) for Plate Agglutination

Test. Mycoplasma synoviae reagent serum (SPMS0312) (Charles River Lab., CT, USA) served as

control positive sera. Results of RSA revealed 51% positivity for MS. In conclusion, serological

screening is the best tool to detect the infection in a flock. This was a preliminary study and the

positive results will be further confirmed through molecular detection of MS. The present study was

completed under financial grant by International Foundation for Science (IFS), Stockholm, Sweden

Grant Ref. Number. B/5365-1 on “Development of Loop-mediated isothermal amplification (LAMP)

assay: a simple and cost effective diagnostic test for the molecular detection of economically

important Mycoplasma pathogens of chickens” granted to Chief Investigator Dr. Syed Ehtisham-ul-

Haque.

Key Words: Mycoplasma synoviae, Serological screening, RSA, Pakistan

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CHEMICAL COMPOSITION, BIOLOGICAL ACTIVITY AND APPLICATION IN

ANIMAL SCIENCE OF PROPOLIS- A REVIEW

Shijun Fu, Shijin Guo, Guanggang Qu and Zhiqiang Shen*

Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China

*Corresponding Author: [email protected]

ABSTRACT

Propolis is a resinous hive product collected by honeybees from various plant sources. Several

groups of researchers have focused their attention on the biological activity of propolis and its active

principles. Many scientific articles are published every year in different international journals related

to the plant sources and extraction technology of propolis. This review article compiles recent

findings concerning the plant origin, extraction technology, main constituents, bioactivity, applications

in animal husbandry and quality control of propolis.

Key Words: Propolis; bioactivity; application; animal science

INTRODUCTION

Propolis is the generic name for the resinous product of complex composition collected by

honey bees from bud and exudates of various plants (Banskota et al., 2001; Sforcin, 2007). More than

300 constituents have been identified so far, among which phenolic compounds, including flavonoids,

are major components (Hegazi et al., 2000). Propolis has attracted researchers’ interest in the last

decades because of several biological and pharmacological properties, such as immunomodulatory

(Sforcin, 2007), antitumor (Khalil, 2006), antimicrobial (Bankova et al., 2000), antitrypanosomal

activities (Da Silva et al., 2004; Syamsudin et al., 2009), antioxidant (Ozguner et al., 2005) and

angiogenesis (Ahn et al., 2009). This review describes recent findings concerning the plant origin,

extraction technology, main constituents, bioactivity, applications in animal science as well as quality

control of propolis.

Plant Origin and Extraction Technology

Plant Origin

To understand what causes the differences in chemical composition, it is necessary to keep in

mind the plant origin of propolis. For propolis production, bees use materials resulting from a variety

of botanical processes in different parts of plants (Bankova, 2005). Poplar and Baccharis are well

known as the source plants of European and Brazilian propolis, respectively. With further research,

there are several new plant sources continued to be found. For instance, the propolis from Okinawa

and Okayama, Japan, contain some prenylflavonoids not seen in other regions such as Europe and

Brazil. The plant origins of Okinawa and Okayama propolis are Macaranga tanarius (Kumazawa et al.,

2008) and Rhus javanica var. chinensis (Murase et al., 2008), respectively.

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Extraction Technology

Propolis cannot be used as a raw material; it must be purified by extraction with solvents. This

process should remove the inert material and preserve the polyphenolic fractions (Gomez-Caravaca

et al., 2006). Various extraction techniques were applied to extract the biologically active constituents

of propolis. Extraction with ethanol is particularly suitable to obtain dewaxed propolis extracts rich in

polyphenolic components and this is the most commonly used solvent (Park and Ikegaki, 1998; Pietta

et al., 2002; Popova et al., 2004). In addition, extraction with pure water (Woisky and Salatino, 1998)

(these extracts are likely to contain phenolic acids which are very soluble in water), methanol (Cao et

al., 2004), hexane and acetone (Pereira et al., 2000) and chloroform (Negri et al., 2003) have also

been used.

Recently, several modern extraction methods such as ultrasound, microwave and supercritical

carbon dioxide (SC-CO2) have been developed for the fast and efficient extraction. Chen et al.

(2009b) applied supercritical carbon dioxide (SC-CO2) extraction and obtained 3,5-diprenyl-4-

hydroxycinnamic acid (DHCA) from propolis. Traditional maceration extraction, ultrasound

extraction, and microwave assisted extraction were employed to compare their efficiency (Trusheva

et al., 2007). The results shown that microwave assisted extraction was very rapid but led to the

extraction of a large amount of non-phenolic and non-flavonoid material; ultrasound extraction gave

the highest percentage of extracted phenolics. Compared to the maceration extraction, microwave

assisted extraction and ultrasound extraction methods provided high extraction yield, requiring short

timeframes and less labour; ultrasound extraction was shown to be the most efficient method based

on yield, extraction time and selectivity.

Main Constituents and Activity of Propolis

Main Constituents of Propolis

Propolis is a complex mixture of substances collected by honeybees from buds or exudates of

plants (resin), beeswax and other substances, such as pollen and sugars (Teixeira et al., 2010). The

chemical composition of the propolis significantly depends on the collecting location, time and plant

source (Park et al., 2002; Bankova, 2005; Melliou et al., 2005; Alencar et al., 2007). As a consequence,

more than 300 components have been identified so far, among which phenolic compounds, including

flavonoids, are major components (Hegazi et al., 2000; Khalil, 2006).

Due to the different climate, there are different plant distributions in temperate, subtropical and

tropical regions. Consequently, the plant sources of propolis source are quite different. Leaf-buds of

Populus nigra (black poplar) are sources of propolis resin in temperate regions (Bankova et al., 1992).

Propolis resin from Europe and China contain predominantly flavonoids and secondarily phenolic acid

esters (Bankova, 2000). Iranian propolis has been shown to contain aromatic acids (benzoic and

benzenepropanoic), esters of caffeic and phenylethyl-trans-4-coumaric acids, flavonoids (pinocembrin,

chrysin), among other constituents (Mohammadzadeh et al., 2007).

The seasonal variations in the chemical composition of propolis have also been demonstrated.

Brazilian propolis contents of all compounds varied along the year (Teixeira et al., 2010). Chemical

composition of Brazilian propolis detected a pattern, according to which diterpenes started appearing

in summer and reached a maximum in autumn, being absent along other seasons (Bankova et al.,

1998).

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Bioactivity of Propolis

Antimicrobial activity of propolis

More and more publications are appearing which combine antimicrobial and other biological

studies with chemical analyses of the tested propolis samples. Although different propolis origins lead

to distinct chemical composition, propolis is the defense of bees against infections, therefore, the

antibacterial and antifungal activity of propolis are present (Bankova, 2005).

Propolis antimicrobial properties have been widely investigated and demonstrated. Propolis

extract showed in vitro antibacterial activity, inhibition of cell adherence and inhibition of water-

insoluble glucan formation (Koo et al., 2000). Propolis also showed antiviral (Huleihel and Isanu, 2002;

Gekker et al., 2005), antifungal (Sforcin et al., 2001; Salomão et al., 2004), antiparasite (Salomao et al.,

2004; Freitas et al., 2006) and antitrypanosomal activities (Da Silva et al., 2004).

Immunoregulation and antiproliferative activity of propolis

In vitro and in vivo assays demonstrated that propolis may activate macrophages, increasing their

microbicidal activity. Propolis enhances the lytic activity of natural killer cells against tumor cells

(Sforcin et al., 2007). Besides, caffeates of the Netherlands propolis were considered to be active

constituent’s antiproliferative activity (Banskota et al., 2002).

Antioxidant activity of propolis

Propolis with strong antioxidant activity contained antioxidative compounds such as kaempferol

and phenethyl caffeate. Propolis exerts its antioxidative effect where it is assumed to accumulate,

such as on the kidney, where it is excreted, and on the gastrointestinal tract, where propolis

influences these tissues even from the outside of the cell (Sun et al., 2000). Ethanol extracts of

propolis from Argentina, Australia, China, Hungary and New Zealand had relatively strong antioxidant

activities, and were also correlated with the total polyphenol and flavonoid contents (Kumazaw et al.,

2004). Caffeic acid phenethyl ester exhibits a protective effect on mobile phone-induced and free

radical mediated oxidative renal impairment in rats (Ozguner et al., 2005).

Application of Propolis in Animal Science

Application of Propolis as Feed Additive

Dietary supplementation of laying hens exposed to heat stress with propolis (5 g/kg diet) can

attenuate heat stress-induced oxidative damage and increase growth performance and digestibility,

improve eggshell thickness and egg weight (Tatli Seven, 2008, 2009). Propolis supplementation as

alternative to antibiotics in broilers in heat stress conditions may be used as redound to performance

and digestibility (Tatli Seven and Seven, 2008). Addition of propolis at 3g/kg in the laying hens diet

resulted in significant increases in the serum IgG and IgM levels and erythrocyte count, significant

decrease in the peripheral blood T-lymphocyte percentage. Hemoglobin and hematocrit values and

total leucocyte and differential leucocytes counts were not influenced by propolis supplementation

(Çetin et al., 2010). Supplementation of lambs with propolis improved weight gain, feed utilization,

percentage of dressed meat, meat digestibility and tenderness (Bonomi, 2002a). Weight gain and feed

consumption of pregnant sows with propolis supplementation were improved (Bonomi, 2003). The

live weight gain, feed utilization, carcass yield, meat digestibility and tenderness of young bulls fed with

mixed feeds with propolis were improved (Bonomi and Bonomi, 2002). The addition of 30 ppm

propolis to feed improved egg production in laying hens. Speed of growth and use and digestibility of

feed were enhanced by the inclusion of 40-60 ppm propolis in feed for hen, turkeys, and by 20-40

ppm in feed for guinea fowl, ducks, broilers and rabbits (Bonomi, 2002b).

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Therapeutic Properties of Propolis

It is known that propolis possesses antimicrobial, antioxidative, antiulcer and antitumor activities.

Therefore, propolis has attracted much attention in recent years as a useful or potential substance

used in medicine and cosmetics products. Furthermore, it is now extensively used in foods and

beverages with the claim that it can maintain or improve human health (Khalil, 2006; Syamsudin et al.,

2009). Recently, propolis had gained popularity application in veterinary such as treatment of young

cattle dermatophytosis (Cam et al., 2009).

Application of propolis as vaccine adjuvant

Propolis can stimulate higher antibody production, suggesting its use in vaccines, as an adjuvant

(Sforcin, 2007). Phenolic compounds such as artepillin C and the derivatives of cinnamic acid besides

other flavonoid substances were abundant in the propolis extract, and they could be the main

substances with adjuvant action (Fischer et al., 2007). In recent years, it has been used as an adjuvant

for mammals, poultry and fish. Propolis could stimulate leucocyte activity and antibody titre in

vaccinated fish and increased the survival rate following challenge (Chu, 2006). Because of unique

ultrastructure, vaccines with propolis as adjuvant have many advantages such as high stability, slowly

release in the body and long storage stage (Shen et al., 2000).

Quality Control of Propolis

Pesticide Residues in Propolis

The most important contaminants in propolis are the substances used for control of bee pests.

Chemical protection of beehives is commonly carried out by treatment with different kinds of

pesticide (Bogdanov, 2006). Therefore, monitoring of pesticide residues in propolis is of particular

concern to consumer safety. A method based on matrix solid-phase dispersion (MSPD) was

developed to determine bifenthrin, buprofezin, tetradifon, and vinclozolin in propolis using gas

chromatography-mass spectrometry in selected ion monitoring mode (dos Santos et al., 2008). Chen

et al. (2009b) employed gas chromatography-electron capture detection using double column series

solid-phase extraction for the simultaneous determination of 17 organochlorine pesticides in propolis.

An analytical method in propolis was developed and validated for the determination of four

tetracyclines by high performance liquid chromatography (Zhou et al., 2008).

Toxicity Analysis of Propolis

Products containing propolis have been used increasingly as dietary supplements. Although

reports of allergic reactions are not uncommon, propolis is relatively non-toxic, with a no-effect level

(NOEL) in a 90-mouse study of 1400 mg/kg body weight/day (Burdock, 1998). Subchronic toxicity

study of oral propolis extract indicated that no significant behavioral and clinical toxicity has been

seen in male rats (Mohammadzadeh et al., 2007).

Propolis’s systemic toxicity is rarely reported and hence may be underestimated. It is also a

potent sensitizer and should not be used in patients with an allergic predisposition, in particular an

allergy to pollen (Menniti-Ippolito et al., 2008). Besides, propolis could induce acute renal failure and

emphasizes the need for vigilance and care when propolis is used as a medicine or dietary supplement

(Li et al., 2005).

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Conclusions

Propolis has a wide range of biological activity and pharmacological effects, and has attracted

considerable attention from both scientists and entrepreneurs. The chemical composition of the

propolis significantly depends on the collecting location, time and plant source. Dietary

supplementation of animal with propolis can increase growth performance and digestibility.

Furthermore, propolis is a useful substance used in veterinary medicine and acts as an excellent

adjuvant. It needs to be noted that pesticide residues in propolis should be paid more attention

during furture applications.

With the continuous development of science and technology, the chemical composition,

pharmacological effects, extensive applications and quality control of propolis will be constantly

studied.

Acknowledgments

This work was supported by the Shandong Science and Technology Development Project

(2009GG10009001) of China. The authors wish to express their gratitude to Professor Shirley Wang

for her valuable guidance and suggestions of this paper.

References

Acikgoz Z, B Yucel and O Altan, 2005. The effects of propolis supplementation on broiler

performance and feed digestibility. Archiv für Geflügelkunde, 69: 117-122.

Ahn MR, K Kunimasa, S Kumazawa, T Nakayama, K Kaji et al. 2009. Correlation between

antiangiogenic activity and antioxidant activity of various components from propolis. Mol Nutr

Food Res, 63: 643-651.

Alencar SM, TLC Oldoni, ML Castro, ISR Cabral, CM Costa-Neto et al., 2007. Chemical composition

and biological activity of a new type of Brazilian propolis: red propolis. J Ethnopharmacol, 113:

278-283.

Bankova V, G Boudourova-Krasteva, S Popov, JM Sforcin and SRC Funari, 1998. Seasonal variations of

the chemical composition of Brazilian propolis. Apidologie, 29: 361–367.

Bankova V, SL de Castro and MC Marcucci, 2000. Propolis: recent advances in chemistry and plant

origin. Apidologie, 31: 3-15.

Bankova V, Dyulgerov A, Popov S, Evstatieva L, L Kuleva et al., 1992. Propolis produced in Bulgaria

and Mongolia-phenolic compounds and plant-origin. Apidologie, 23: 79–85.

Bankova V, 2005. Recent trends and important developments in propolis research. Evid-based Compl

Alt, 2: 29-32.

Banksota AH, Y Tezuka and SH Kadota, 2001. Recent progress in pharmacological research of

propolis. Phytother Res, 15: 561-571.

Banskota AH, T Nagaoka, LY Sumioka, Y Tezuka, S Awale et al., 2002. Antiproliferative activity of the

Netherlands propolis and its active principles in cancer cell lines. J Ethnopharmacol, 80: 67-73.

Bogdanov S, 2006. Contaminants of bee products. Apidologie, 37: 1-18.

Bonomi A, BM Bonomi, A Mazzotti and A Sabbioni, 2002a. The use of propolis in light lamb feeding.

La Rivista di Scienza dell’Alimentazione, 31: 65-75.

Bonomi A and BM Bonomi, 2002. The use of propolis in feeding young bulls. La Rivista di Scienza

dell’Alimentazione, 31: 91-103.

Bonomi A, 2002b. Propolis in the feed of small species. Informatore Agrario, 58: 41-43.

Bonomi A, 2003. Use of propolis in the feeding of sows. Rivista di Suinicoltura, 2: 101-106.

Page 134: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

134

Burdock GA, 1998. Review of the biological properties and toxicity of bee propolis (propolis). Food

Chem Toxicol, 36: 347-363.

Cam Y, AN Koç, S Silici, V Günes, H Buldu et al., 2009. Treatment of dermatophytosis in young cattle

with propolis and Whitfield’s ointment. Vet Rec, 165: 57-58.

Cao YH, Y Wang and Yuan, Q 2004. Analysis of flavonoids and phenolic acid in propolis by capillary

electrophoresis. Chromatographia, 59: 135-140.

Çetin E, S Silici, N Çetin and BK Güçlü, 2010. Effects of diets containing different concentrations of

propolis on hematological and immunological variables in laying hens. Poult Sci, 89: 1703-1708.

Chen CR, YN Lee, MR Lee and Chang, CM 2009a. Supercritical fluids extraction of cinnamic acid

derivatives from Brazilian propolis and the effect on growth inhibition of colon cancer cells. J

Taiwan Instit Chem Eng, 40: 130-135.

Chen F, LZ Chen, Q Wang, JH Zhou, XF Xue and J Zhao, 2009b. Determination of organochlorine

pesticides in propolis by gas chromatography-electron capture detection using double column

series solid-phase extraction. Anal Bioanalytical Chem, 393: 1073-1079.

Chu WH, 2006. Adjuvant effect of propolis on immunisation by inactivated Aeromonas hydrophila in

carp (Carassius auratus gibelio). Fish Shellfish Immun, 21: 113-117.

Da Silva CIB, K Salomao, M Shimizu, S Bankova, AR Custodio et al., 2004. Antitrypanosomal activity

of Brazilian propolis from Apis mellifera. Chem Pharm Bull, 52: 602-604.

dos Santos TFS, A Aquino, HS Dórea and S Navickiene, 2008. MSPD procedure for determining

buprofezin, tetradifon, vinclozolin, and bifenthrin residues in propolis by gas chromatography-

mass spectrometry. Anal Bioanalyt Chem, 390: 1425-1430.

Fischer G, MB Cleff, LA Dummer, N Paulino, AS Paulino et al., 2007. Adjuvant effect of green propolis

on humoral immune response of bovines immunized with bovine herpesvirus type 5. Vet

Immunol Immunop, 116: 79-84.

Freitas SF, L Shinohara, JM Sforcin and S Guimarães, 2006. In vitro effects of propolis on Giardia

duodenalis trophozoites. Phytomedicine, 13: 170-175.

Gekker G, SX Hu, M Spivak, JR Lokensgard and PK Peterson, 2005. Anti-HIV-1 activity of propolis in

CD4+ lymphocyte and microglial cell cultures. J Ethnopharmacol, 102: 158–163.

Gomez-Caravaca AM, M Gomez-Romero, D Arraez-Roman, A Segura-Carretero and A Fernandez-

Gutierrez, 2006. Advances in the analysis of phenolic compounds in products derived from bees.

J Pharmaceut Biomed, 41: 1220-1234.

Hegazi AG, FK Abd El Hady and FA Abd Allah, 2000. Chemical composition and antimicrobial activity

of European propolis. Z Naturforsch, 55: 70-75.

Huleihel M and V Isanu, 2002. Anti-herpes simplex virus effect of an aqueous extract of propolis. Isr

Med Assoc J, 4: 923–927.

Khalil ML, 2006. Biological activity of bee propolis in health and disease. Asian Pac J Cancer Prev, 7:

22-31.

Koo H, BP Gomes, PL Rosalen, GM Ambrosano, YK Park et al., 2000. In vitro antimicrobial activity of

propolis and Arnica montana against oral pathogens. Arch Oral Biol, 45: 141-148.

Kumazaw S, T Hamasaka and T Nakayama, 2004. Antioxidant activity of propolis of various

geographic origins. Food Chem, 84: 329-339.

Kumazawa S, J Nakamura, M Murase, M Miyagawa, MR Ahn et al., 2008. Plant origin of Okinawan

propolis: honeybee behavior observation and phytochemical analysis. Naturwissenschaften, 95:

781-786.

Page 135: Proceedings zafar-corrected

Proceedings of the “International Seminar on Poultry Diseases” 14-15 Dec, 2015, Department of Pathology, University of Agriculture, Faisalabad, Pakistan

135

Melliou E, E Stratis and I Chinou, 2007. Volatile constituents of propolis from various regions of

Greece-Antimicrobial activity. Food Chem, 103: 375-380.

Menniti-Ippolito F, G Mazzanti, A Vitalone, F Firenzuoli and C Santuccio, 2008. Surveillance of

suspected adverse reactions to natural health products: the case of propolis. Drug Saf, 31: 419-

423.

Mohammadzadeh S, M Shariatpanahi, M Hamedi, R Ahmadkhaniha, N Samadi et al., 2007. Chemical

composition, oral toxicity and antimicrobial activity of Iranian propolis. Food Chem, 103: 1097–

1103.

Murase M, Kato A Sun, T Ono, J Nakamura et al., 2008. Rhus javanica var. chinensis as a new plant

origin of propolis from Okayama, Japan. Biosci, Biotech, Biochem, 72: 2782-2784.

Negri, G, MLF Salatino and A Salatino, 2003. Green propolis: unreported constituents and a novel

compound from chloroform extracts. J Apicult Res, 42: 39-41.

Ozguner F, F Oktem, A Ayata, A Koyu and HR Yilmaz, 2005. A novel antioxidant agent caffeic acid

phenethyl ester prevents long-term mobile phone exposure-induced renal impairment in rat:

Prognostic value ofmalondialdehyde, N-acetyl-β-D-glucosaminidase and nitric oxide

determination. Mol Cell Biochem, 277: 73-80.

Park, YK, SM Alencar and CL Aguiar, 2002. Botanical origin and chemical composition of Brazilian

propolis. J Agri Food Chem, 50: 2502-2506.

Park YK and M Ikegaki, 1998. Preparation of water and ethanolic extracts of propolis and evaluation

of the preparations. Biosci, Biotech, Biochem, 62: 2230-2232.

Pereira AS, M Norsell, N Cardoso and FR Aquino Neto, 2000. Rapid screening of polar compounds

in Brazilian propolis by High-Temperature High-Resolution gas chromatography-mass

spectrometry. J Agric Food Chem, 48: 5226–5230.

Pietta PG, C Gordana, and AM Pietta, 2002. Analytical methods for quality control of propolis.

Fitoterapia, 73: S7-S20.

Popova M, V Bankova, D Butovska, V Petkov, B Nikolova-Damyanova et al., 2004. Validated methods

for the quantification of biologically active constituents of poplar-type propolis. Phytochem

Analysis, 15: 235-240.

Salomão K, AP Dantas, CM Borba, LC Campos, DG Machado et al., 2004. Chemical composition and

microbicidal activity of extracts from Brazilian and Bulgarian propolis. Lett Appl Microbiol, 38:

87-92.

Sforcin JM, JA Fernandes, CAM Lopes, SRC Funari and V Bankova, 2001. Seasonal effect of Brazilian

propolis on Candida albicans and Candida tropicalis. J Venom Anim Toxins, 7: 139-144.

Sforcin JM, 2007. Propolis and the immune system: a review. J Ethnopharmacol, 113: 1-14.

Shen ZQ, XX Zhang, CW Lin, JS Liu, KL Xu et al., 2002. Development of preparation technology,

safety and potency tests of the vaccine of propolis adjuvant inactivated vaccine against Newcastle

disease in poultry. Chin J Prev Vet Med, 22: 275-277.

Sun F, S Hayami, S Haruna, Y Ogiri, K Tanaka et al., 2000. In vivo antioxidative activity of propolis

evaluated by the interaction with vitamins C and E and the level of lipid hydroperoxides in rats. J

Agric Food Chem, 48(5): 1462-1465.

Syamsudin Dewi RM and Kusmardi, 2009. Immunomodulatory and in vivo antiplasmodial activities of

propolis extracts. Am J Pharmacol Toxicol, 4: 75-79.

Tatli Seven P, S Yilmaz, I Seven, IH Cerci, MA Azman et al., 2009. Effects of propolis on selected

blood indicators and antioxidant enzyme activities in broilers under heat stress. Acta Vet Brno,

78: 75-83.

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Tatli Seven P, 2008. The Effects of dietary Turkish propolis and vitamin C on performance,

digestibility, egg production and egg quality in laying hens under different environmental

temperatures. Asian-Austral J Anim Sci, 21: 1164-1170.

Tatli Seven P and I Seven, 2008. Effect of dietary Turkish propolis as alternative to antibiotic on

performance and digestibility in broilers exposed to heat stress J Appl Anim Res, 34: 193-196.

Teixeira ÉW, D Message, G Negri, A Salatino and PC Stringheta, 2010. Seasonal variation, chemical

composition and antioxidant activity of Brazilian propolis samples. Evid-based Compl Alt, 7: 307-

315.

Trusheva B, D Trunkova and V Bankova, 2007. Different extraction methods of biologically active

components from propolis: a preliminary study. Chem Cent J, 1: 13-16.

Woisky RG and A Salatino, 1998. Analysis of propolis: some parameters and procedures for chemical

quality control. J Apicult Res, 37: 99-105.

Zhou JH, XF Xue, Y Li, JZ Zhang, F Chen et al., 2009. Multiresidue determination of tetracycline

antibiotics in propolis by using HPLC-UV detection with ultrasonic-assisted extraction and two-

step solid phase extraction. Food Chem, 115: 1074-1080.

Li YJ, JL Lin, CW Yang and CC Yu, 2005. Acute renal failure induced by a Brazilian variety of propolis.

Am J Kidney Dis, 46: 125-129.

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DETECTION OF SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) IN KIT GENE

IN DUCKS (ANAS PLATYRHYNCHOS DOMESTICUS) AND ANALYSIS OF THEIR

RELATIONSHIP WITH DUCK FEATHER COLOR

Li Xuechan1*, Muhammad Shahzad2*, Zahid Iqbal3 and Li Shijun1

1Key Lab of Agriculture Animal Genetics, Breeding, and Reproduction of Ministry of Education,

Huazhong Agricultural University, Wuhan, People’s Republic of China; 2University College of Veterinary

& Animal Sciences, The Islamia University of Bahawalpur, Pakistan; 3National Reference Laboratory of

Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, People’s Republic of China

*Corresponding authors: [email protected], [email protected]

ABSTRACT

Plumage color control is essential for the uniform appearance of birds in poultry industry. White

plumage is the most favorable color for meat-type commercial bird producers not only because ducks

with unpigmented feathers are easy to clean, but also genes involved in melanogenesis may have

pleiotropic effects on other phenotypes. It is well established that in avian species, KIT gene is related

with melanocyte growth and development but the mechanism is not clear yet. Our previous study

found that KIT gene expression in black and white bulbs have very significant differences, suggesting

that there might be some KIT gene mutation correlated with duck feather color variation. In this

study, through the duck KIT gene fragment sequencing, two SNPs have been found to be restricted

genotype which later was confirmed through PCR. In this study, Enshi Ma duck, Jingjiang Ma duck,

Liancheng white duck, Baigai duck, Yingtaogu duck and Muscovy were used for genotyping and

determining the allelic frequency. Statistical analysis indicated the presence of an intermediate

difference between these two SNPs; however, no relation had been observed in these SNPs with the

plumage color.

Key Words: Duck, Feather, SNPs, KIT gene

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AVIAN REO-VIRAL INFECTIONS: AN EMERGING VIRAL THREAT TO POULTRY

INDUSTRY OF PAKISTAN

Bahar-e-Mustafa, Sibtain Ahmad*, Muhammad Tariq and Umair Hassan Khan

University of Agriculture, Faisalabad-Sub campus Toba Tek Singh, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Avian reoviruses are associated with the diseases in several species of the wild birds like geese,

ducks and turkeys but currently in commercial poultry they are posing a serious threat by viral

arthritis. These viruses belong to family Reoviridae and specifically genus is Orthoreovirus. They

derive their name from the respiratory enteric orphan as firstly they were observed from the

respective tracts in humans but they were not associated with any of the diseases. These viruses are

RNA in nature (double stranded) and possess a segmented genome (10 segments), icosahedral

symmetry and a double capsid. Due to these characteristics of the virus, it is liable to mutate its

genome frequently and therefore, numerous serotypes exist. Viral arthritis is particularly problem of

the broilers and it affects the joints of the birds. Several complications associated with the disease e.g.

poor FCR, decreased uniformity in the growth and body weights and moreover, the reduced quality

of the carcass. In case of breeder, the onset of the disease prior to the production leads to the

reduced production and hatchability. Virus has also got capability to be vertically transmitted to the

next generation. All of these factors make this disease economically important and emphasize the

rapid diagnosis and control of the disease. The disease can be readily diagnosed on the basis of the

molecular methods e.g. RT-PCR and serological assays such as ELISA and IFA. However control of

the disease is much important and should be implanted by the scrupulous biosecurity and vaccination

by the homologous virus.

Key Words: Reovirus, Viral Arthritis, Vertical Transmission, Poultry

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LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) BASED DETECTION

OF POULTRY DISEASES: CURRENT SCENARIO AND FUTURE PERSPECTIVES

Sibtain Ahmad*, Bahar-e-Mustafa and Riaz Mustafa

University of Agriculture, Faisalabad-Sub campus Toba Tek Singh, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Newcastle disease (ND), Infectious Bursal disease (IBD) and Infectious Bronchitis (IB) are the

most disastrous viral diseases of poultry industry. Various techniques employed for the diagnosis of

these diseases are although, useful yet it takes too long time to diagnose. Moreover, it is difficult to

employ them under field circumstances. So there arises the importance of LAMP as a diagnostic

technique that may be easily carried out under field conditions and takes less time to diagnose a

disease. Loop-mediated isothermal amplification (LAMP) was developed by Japanese scientists

Notomi and his colleagues in 2000. This technique encompasses both of the requirements i.e. it is

quick to perform and can be very efficiently adapted to the field conditions provided appropriate

primers are designed. Being isothermal the test is so simple to carry out that it can be performed in a

water bath/ hot plate at 60◦C in a much less time (almost 45-60 mins.) than by other tests. As this

technique is less subjective and has no requirements of expensive laboratory facilities, this approach

can improve chicken health and welfare by improving the diagnosis of already prevalent diseases and

emerging diseases of economic importance.

Key Words: Isothermal, Viral Diseases, Molecular Diagnosis, Health.

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MYCOTOXINS: A HIDDEN THREAT TO WILD AND FANCY BIRDS

Bahar-E-Mustafa1, Umair Hassan Khan1*, Waseem Abbas1 and Muhammad Saad Zubair2

1University of Agriculture, Faisalabad, Sub-Campus Toba Tek Singh; 2Government College University,

Faisalabad, Pakistan.

*Corresponding Author: [email protected]

ABSTRACT

Under certain circumstances (temperature and moisture), several species of the fungi produce

several toxins collectively known as mycotoxins. Several pet birds are highly susceptible to these

mycotoxins and depend upon the certain physiological status of the birds. Birds in stress are

particularly prone to develop characteristic signs of mycotoxicosis. Studies have shown that ducklings

have been more susceptible to the aflatoxins than the commercial poultry indicating role of specie

variation. Efforts should be made to avoid offering birds with damaged or broken seeds as they may

contain higher levels of mycotoxins. Corn and peanuts are notorious for having higher levels of

mycotoxins. Until now four major types of mycotoxins have been identified which are dangerous to

poultry and they include aflatoxin, trichothecenes, deoxynevalenol (DON) and ochratoxin. Aflatoxins

are produced by the Aspergillus parasiticus. These toxins inhibit the protein and nucleic acid synthesis.

These are potent hepatotoxin and it causes anorexia, depression, reduced growth in birds.

Characteristic lesions include enlarged and friable liver, enlarged pancreas and spleen. It is also

associated with a severe drop in the immune status of the birds. Trichothenenes are produced by the

Fusarium spp. And this has deleterious effects on the mucous membranes of the birds, producing

several ulcerative lesions. Moreover, it is also associated with the flaccid paralysis of neck and wings

of the birds. This toxin is also associated with the development of the contact dermatitis, gangrene of

peripheral organs, poor feather growth and even nervous disorders. These signs are also occasionally

observed in the wild birds exposed to the peanuts. Ochratoxins are produced by the Aspergillus have

been associated with the renal and liver failure in birds, bone marrow suppression and nervous

manifestations. This toxin has also been associated with the reduced functioning of immune system.

Key Words: Mycotoxins, Fancy birds, Aflatoxin, Ochratoxin, Trichothecenes, Wild birds

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HEMATOLOGICAL AND MUTAGENIC CHANGES INDUCED BY CONCURRENT

ARSENIC AND COPPER SULPHATE IN ADULT POULTRY MALES

Abdul Ghaffar1*, Riaz Hussain2 and Ahrar Khan3

1Department of Life Sciences (Zoology); The Islamia University of Bahawalpur- 63100; 2University

College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur- 63100, Pakistan;

3Department of Pathology, Faculty of Veterinary Science, University of Agriculture, Faisalabad,

Pakistan-38040, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

The present experimental study was conducted to determine the clinico-hematological and

mutagenic impacts induced by concurrent oral administration of arsenic and copper sulphate in adult

male birds. After acclimatization, male birds were randomly divided into equal seven groups. All the

experimental birds received arsenic and copper sulphate alone and in different combinations for 30

days. Blood samples were collected from each bird at days 10, 20 and 30 of the experiment. Various

clinical signs like decreased feed intake, body weight, ruffled feather, depression, dullness, ocular

discharge, open mouth breathing, diarrhea and pale comb were observed at higher levels of arsenic

and copper sulphate. In treated birds the values of total erythrocytes counts, leukocyte counts,

hemoglobin concentration and mean corpuscular hemoglobin concentration were significantly

decreased while packed cell volume and mean corpuscular volume increased. Results showed that

frequency of erythrocytes with micronuclei, blabbed, lobed, notched and cells with nuclear remnants

were significantly increased. From the results of this study it can be concluded that arsenic and

copper sulphate alone at higher levels and in combination even at lower levels pose serious clinico-

hematological and mutagenic effects in adult male birds.

Key Words: Birds, Copper sulphate, Arsenic, Hematology, Micronuclei, Nuclear remnants

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EFFECTS OF DIFFERENT LEVELS OF DAP AND ARSENIC ON SOME HAEMATO-

BIOCHEMICAL AND HISTOPATHOLOGICAL CHANGES IN LAYERS

Riaz Hussain1*, Abdul Ghaffar2 and Ahrar Khan3

1University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur- 63100,

Pakistan; 2Department of Life Sciences (Zoology). The Islamia University of Bahawalpur- 63100; 3Department of Pathology, Faculty of Veterinary Science, University of Agriculture, Faisalabad,

Pakistan-38040, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

In present experimental study some hemato-biochemical and histopathological effects of DAP

and Arsenic were observed in layers birds. For this purpose a total of 72 chicks of same age and

weight were purchased from local hatchery and were kept under similar conditions. After one week

of acclimatization all the birds were randomly divided into six equal groups (A-F) having twelve birds

each. Different levels of diammonium phosphate and arsenic in combinations were given to birds

orally for 39 days. Four birds from each group were killed at days, 13, 26 and 39 of the experiment

for collection of blood. A significant decrease in hematological parameters such as erythrocyte

counts, hemoglobin concentration and hematocrit values were recorded as compared to control

group. A significant increase in serum cholesterol and creatinine phospho kinase concentration was

also recorded at higher level of DAP and arsenic. Histopathological examination of different tissues

exhibited various microscopic changes in thymus, kidneys, bursa, liver and kidneys. Moderate to

severe congestion in thymus, increased urinary space and tubular degenerations in kidneys,

vaccuolation in bursa and liver were the prominent features. The results of this study showed that

different levels of DAP and arsenic poses adverse impacts even at low levels in combination in birds.

Key Words: Birds, Arsenic, Diammonium phosphate, Blood, Serum, Histology

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143

THE ANTIVIRAL ACTIVITY FROM ELEVEN SELECTED CHOLISTANI

PLANTS AGAINST INFECTIOUS BURSAL DISEASE VIRUS

AND INFECTIOUS BRONCHITIS VIRUS

Mirza I Shahzad1*, Amna Aslam2, Sabeeha Parveen2, Hina Ashraf3, Zahid Kamran1, Nargis Naz1, Syeda S

Zehra1, and Muhammad Mukhtar3

1University College of Veterinary and Animal Sciences, The Islamia University of Bahwalpur;

2Department of Life Sciences, The Islamia University of Bahawalpur; 3Cholistan Institute of Desert

Studies, The Islamia University of Bahawalpur, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

In the recent past there has been a tremendous increase in the use of plant based health

products in developing as well as developed countries resulting in an exponential growth of herbal

products globally. An upward trend has been observed in the research on herbals. Herbal medicines

have a strong traditional or conceptual base and the potential to be useful as drugs in terms of safety

and effectiveness leads for treating different diseases. World Health Organization has made an

attempt to identify all medicinal plants used globally and listed more than 20,000 species. Present

study is based on evaluation of antiviral potential of methanolic extracts of Achyranthes aspera,

Haloxylon recurvum, Haloxylon salicornicum, Oxystelma esculentum, Ochthochloa compressa, Neurada

procumbens, Panicum antidotale ,Salsola baryosma, Suaeda fruticosa, Sporobolos icolados and Solanum

surattense. These plants are well reported for their antibacterial, antifungal, anticancerous,

antiperiodic, diuretic, purgative, laxative, antiasthmatic, hepatoprotective, anti-allergic and various

other important medicinal properties. According to literature these plant are rich source of

phytochemical and pharmacological compounds. But their antiviral activity especially against poultry

pathogens like Infectious Bronchitis Virus (IBV) and Infectious Bursal Disease Virus (IBDV) was not

reported before. In this study antiviral compounds from dried whole plants were extracted in

methanol and later concentrated by rotary evaporator. The concentrated drug was air dried and

finally dissolved in autoclaved water, filtered through 0.22µ syringe filter and use in antiviral assay

against different viruses in 7-11 days chicken embryonated eggs. To check antiviral activity the drug

was mixed with live virus in varying concentrations and propagated into 7-11 days embryonated eggs.

Eggs were opened after 48-72 hours in sterile conditions and allantoic fluid was collected. HA test

was performed to quantify the titer of IBV and IHA test for IBDV after each passage and after

challenge with drug. Almost all plant extracts were effective against IBV except S. surattense, which

has shown slight decrease in HA titer as compared to control. Some plant extracts were very

effective like O. compressa and S. icolados, which kept the HA titer of virus at 0. Similarly other plants

control the virus in varying degree and kept their titers at 8 in case of H. salicornicum, N. procumbens

and S. baryosma, 16 in case of A. aspera, H. recurvum and P. antidotale, 32 in case of O. esculentum and

64 in case of S. fruticosa.

Key Words: Antiviral Activity, IBDV, IBV, Cholistan, Medicinal Plants, Pakistan

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144

EFFECT OF CHICORY (CICHORIUM INTYBUS) LEAVES EXTRACT

ON GROWTH, NUTRIENT DIGESTIBILITY, HEMATOLOGY

AND IMMUNE RESPONSE OF BROILERS

Saima Liaqat,, Muhammad Shazad Sarwar, Farah Ali1 and Riaz Hussain1

University College of Veterinary and Animal Sciences,

The Islamia University Bahawalpur 63100-Pakistan

*Corresponding Author: [email protected]

ABSTRACT

The present study was conducted to evaluate the effects of Chicory (Cichorium intybus) leaves

extract on growth, nutrient digestibility, hematology and immune response in broiler chicks. Two

separate trials were conducted in this research project, first was performance and the second was

digestibility trial. For this purpose one hundred fifty day old broiler chicks were randomly divided

into five equal groups (A-E) each having 15 birds in performance trial. Group A (positive control) was

offered diet supplemented with an antibiotic growth promoter and coccidiostat. Group B, C, D and E

(negative control) were offered diet without supplementation of any coccidiostat and antibiotic

growth promoter and given water supplemented with chicory leaves extract @ 10ml/liter; extracted

in distilled water at three different pH levels i.e. 3 pH (HCl), 7 pH (distilled water) and 12 pH

(NaOH), respectively. Group E (negative control) was given water without supplementation of

chicory leaves aqueous extract. The blood samples with and without anticoagulant (EDTA) were

collected from all the birds for hematological, immune response and serum biochemical analysis. The

digestibility trial was simultaneously conducted on twenty-five individually caged birds to check the

digestibility of crude protein, crude fat and crude fiber. Significant results were recorded for weight

gain, feed conversion ratio, serum metabolites, immune response against Newcastle disease and

digestibility of crude protein among others. The results of present study revealed that use of chicory

extracts in broiler production is recommended as an in expensive but efficient alternative antibiotic

growth promoter without residual effects.

Key Words: Broiler, Chicory leaves, Hematology, Immune response

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145

STUDY ON SELECTION OF RIEMERELLA ANATIPESTIFER STRAINS AND

OPTIMIZATION OF HIGH DENSITY FERMENTATION OF THE ISOLATES

WANG Yan, MIAO Li-zhong, ZHUANG Jin-qiu, FU Qiang and WEI Feng

Shandong Binzhou Animal Science & Veterinary Medicine Academy, Binzhou, Shandong, 256600,

China

ABSTRACT

Riemerella anatipestifer disease, namely the infectious serositis of duck, is one of the most

important infectious diseases which caused duck farming economic loss; it’s widely distributed in the

world country and duck feeding area. This research is based on the 11 strains of Riemerella

anatipestifer separated from a duck farm, through a series of tests in the dominant serotype strains

with strong pathogenicity and good immunogenicity as the study object, through the contrast test to

determine the medium which have an enrichment effect, using single factor test and research the

orthogonal test of Riemerella anatipestifer liquid culture medium formula, and discussed the optimum

medium composition of Riemerella anatipestifer growth, and the optimization results are applied to

the high density fermentation, optimization of fermentation conditions, lay a good foundation for

vaccine development of high-quality, efficient next. The main research contents are as follows. The

results showed that: 11 isolated strains of bacteria can be divided into three serotypes, infection of

type I and type II; cytotoxicity test indicated that the 11 isolates of 14 day old Cherry Valley duckling

pathogenic differences, from 4/10 to 10/10 range; immunogenicity test results showed that isolates

JX-2, JX-6 and the JX-10 based vaccine protection against challenge infection rate can reach more

than 80%. It should consider the use of containing the strains of 3 serotypes made polyvalent vaccine

vaccination on the farm. This study compares the effect of four medium enrichment The results show

that, the improved yeast broth enrichment effect is the best in the four kinds of medium, the results

of single factor experiment and orthogonal test showed that when the selected 20% yeast extract 5%;

pig stomach digestion solution 10%; 8% soybean peptone; get horse serum 5% the bacterial

concentration was the highest. High density fermentation conditions optimization results display:

0.1L/S.L ventilation; speed 150 R / min; medium pH of 7.4 the number of live bacteria and bacteria

concentration was highest. The optimization of fermentation process of formulation and optimization,

mass production of 3 batches of Riemerella anatipestifer semi-finished antigen in GMP workshop,

living bacteria number is about 30% higher than that before optimization, thus the research provides

experimental basis for the study of high density fermentation production of Riemerella anatipestifer

antigen.

Key Words: Riemerella anatipestifer, Strain selection, Medium optimization, Big pots fermentation

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146

EFFICACY OF TOXIN BINDER IN REDUCING INDUCED AFLATOXIN B1 AND

OCHRATOXIN A IN BROILER CHICKENS

Muhammad Umer Zahid1, Anjum Khalique1, Saima1, Jibran Hussain2, Aftab Ahmad3, Muhammad

Hassan Mushtaq*4, Aqeel Javeed5 and Amjad Khan4

1Department of Animal Nutrition, University of Veterinary and Animal Sciences, Ravi Campus,

University of Veterinary and Animal Sciences, Lahore.2Department of Poultry Production, University

of Veterinary and Animal Sciences, Lahore.3Department of Microbiology, University of Veterinary and

Animal Sciences, Ravi Campus, University of Veterinary and Animal Sciences, Lahore.4Department

of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore. 5Department of Pharmacology and Toxicology, University of Veterinary and Animal Sciences, Lahore.

*Corresponding Author: [email protected]

ABSTRACT

Aflatoxin B1 and Ochratoxin A (OTA) two of the most prevalent and lethal forms of aflatoxons,

is a growing problem affecting poultry industry and as well as a serious hazard for public health

consuming infected poultry meat. Efficacy of toxins binder in ameliorating induced Aflatoxin B1 and

ochratoxin A was evaluated in broilers. The most commonly avaialable commercial mycotoxin

binders were evaluated in vivo. Birds were distributed randomly into 8 groups each containing 20

birds. Each group was raised on different dietary treatment during the study period. Data were

collected regarding production performance (feed intake (g), body weight gain (g), FCR, Mortality),

toxin binding ability (fecal sample), and incidence of disease and carcass characteristics (dressing %

age, keel and shank length, giblet weight (g), bursa, spleen and thymus weight (g). Generalized linear

model was used to evaluate the combine impact of different variables. However supplementation of

mycotoxin binder feed supplement proved amelioration with significant (P<0.05) impact in the tested

mycotoxicosis in broilers. Analysis of data revealed significantly (P<0.05) higher dressing %, thymus

and bursa weight in birds fed on 1g/kg toxin binder, 220ppb ochratoxin + 1g/kg toxin binder and

200ppb aflatoxin + 100 ppb ochratoxin + 1g/kg toxin binder respectively. Feed consumption ratio

was observed unaffected in all the groups fed on different combination of dietary treatments. Non-

significant differences among different treatment groups might show the admirable efficacy of the

toxin binder. It was concluded that mycotoxin binders had a significant effect in plunging the level of

Aflatoxin B1 and ochratoxin A in broilers.

Key Words: Broiler, Toxin binder, Aflatoxin, Ochratoxin

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147

ASSOCIATION OF SELECTED PATHOGENICITY GENES OF ESCHERICHIA COLI

WITH GROSS AND HISTOPATHOLOGICAL LESIONS OF COLIBACILLOSIS

IN BROILERS

Abdul Wadood Jan, M. Tariq Javed, Sameen Qayoom Lone, Aisha Khatoon, Zafar Iqbal Qureshi, Aziz-

ur-Rehman and M. Sohaib Aslam

Department of Pathology, Faculty of Veterinary Science,

University of Agriculture, Faisalabad, Pakistan

ABSTRACT

Escherichia coli (E. coli) infections are widely reported in the poultry sector across the globe. The

studies on pathogenicity genes of E. coli are available but no effort has been made to find an

association of pathogenicity genes with gross and microscopic lesions in broilers. The present study

was conducted on commercial broilers to detect different pathogenicity genes (fimC, tsh, iucD, papC,

fyuA, irp2, ECO) of E. coli and associated lesions in broilers. The lesions were noted with different

levels of severity in various body organs of birds infected with E. coli. In overall, the liver

histopathology showed inflammation, congestion, degenerative changes with vacuolation in the

cytoplasm. Heart histopathology showed degenerative changes in the muscles along with

accumulation of inflammatory cells. Spleen histopathology showed depletion of lymphocytes with

necrotic changes in the splenic nodules and intestine showed necrotic and inflammatory changes in

the intestine with sloughing of epithelium. The genes detected were fimC (92%), tsh (80%), iucD (72%),

fyuA (60%), papC (48%) and irp2 (32%) in cases, respectively. The results showed that there was

positive correlation between fyuA gene with liver gross lesions (P<0.05). The fimC, tsh, iucD, fyuA,

papC and irp2 genes were detected in 64, 56, 56, 36, 36 and 28% cases showing severe microscopic

changes in liver. The same genes were detected in 56, 48, 44, 36, 36 and 28% cases showing severe

microscopic changes in heart; in 24, 16, 20, 4, 16 and 28% cases showing severe microscopic changes

in spleen; in 24, 16, 20, 4, 16 and 28% cases, respectively showing severe microscopic changes in

intestine. It can be concluded from the present results that there is an association of some genes with

the induction of lesions in different body organs of broilers and further studies are required to

strengthen these findings.

Key Words: E. coli, Broilers, Pathogenicity genes, Pathology

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148

ISOLATION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA)

FROM LIVE COCKERELS (PAKISTAN)

Zaytoon Zaheer*, Iftikhar Hussain, and Sajjad ur Rahman

Institute of Microbiology, University of Agriculture Faisalabad, Pakistan.

*Corresponding Author: [email protected]

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA) has been isolated several times from raw

poultry meat or carcasses; however, these were mainly the human-associated strains. Hence, the

possible human involvement in contamination of poultry carcasses by the slaughterhouse workers

may not be ruled out in such cases. During this study efforts were focused on the isolation of MRSA

from the live poultry. Samples were collected with the help of sterile swabs from oropharynx of 30

live cockerels. The swabs were inserted into the oropharynx of the birds and rotated for nearly 15-

30 seconds. The sample swabs were enriched in the mannitol-salt broth at 370C for 24 hours. The

enriched swabs were subsequently cultured at 370C for 24 hours on the Staph-110 medium. Upon

observing the results of macroscopic morphology (round, convex, opaque), microscopic morphology

(gram positive, cocci arranged in the form of clusters) and biochemical test (negative indole test,

positive methyl red, mannitol fermentation, catalase and coagulase test with rabbit plasma within 4

hours), 25 samples were found to be positive for Staphylococcus aureus. The hemolysis pattern of

these 25 S.aureus isolates was confirmed by culturing them on the blood agar. On the blood agar 15

isolates displayed β-hemolysis and 10 isolates displayed α/β hemolysis. These 25 S.aureus isolates

were then cultured on the chromagar and incubation was carried out at 370C for 24 hours. Four out

of 25 S.aureus isolates produced red to mauve colored, glistening, convex, round and mucoid colonies

of MRSA on the chromagar. The four MRSA isolates were further confirmed by performing

methicillin- disc susceptibility test on the Muller-Hinton agar. After confirming methicillin resistance of

the isolates latex agglutination test was performed to detect the aberrant protein of MRSA called

penicillin binding protein 2a (PBP2a), encoded by mecA gene of the positive MRSA isolates. All of the

MRSA isolates showed a positive catalase test, positive tube coagulase test with the rabbit plasma

within 4 hours, red to mauve colored colonies on the chromagar, β-hemolysis on the blood agar,

methicillin resistance in the disc susceptibility testing against methicillin disc and a positive latex

agglutination test for the detection of PBP2a.

Key Words: Methicillin-resistant Staphylococcus aureus, MRSA, live poultry, latex agglutination test.

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149

PATHOGENESIS OF CLOSTRIDIUM PERFERINGENS (NECROTIC ENTERITIS) IN

EXPERIMENTALLY INFECTED BROILER CHICKENS

Arif Mahmood1*, Muti ur Rehman Khan1, Mushtaq Ahmad2, M. Zahid Khan1 and Mustafa Ahmed1

1Department of Pathology; 2Department of Theriogenology, University of Veterinary and Animal

Sciences, Lahore, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Clostridium perferingens-induced disease is considered as the most lethal bacterial enteric

disease of poultry which causes multiple lesions in small intestine containing tightly adhered pseudo

membrane. The present study was designed to observe development of lesions in C. perferingens

induced enteritis in chicken being fed with normal or high energy diet. The day old chicken hatchlings

(n=105) were procured and divided into three groups (n=35 each): control (cont), clostridium (cols)

and clostridium with wheat (colsw). All the birds were vaccinated using standard protocol against

other diseases. Con and cols groups were fed normal feed while colsw group was fed with wheat

mash as high energy diet to favor bacterial growth. Cols and colsw groups were fed orally 1 mL

(3×1010 CFU/ml) culture of C. perfringens overnight to induce the infection in birds. Birds (n=8) were

sacrificed from each group after every 10 days and random samples of intestine were procured for

histo-pathological examination to determine villus size in intestine. the responses in birds challenged

orally with C. perfringens could be placed into two categories: (1) no apparent pathological changes

in the intestinal tissue and (2) sub-clinical inflammatory responses with focal, multi-focal, locally

extensive, or disseminated distribution throughout various sections of duodenum, jejunum, and ileum.

In birds that responded with intestinal lesions, hyperemia and occasional hemorrhages were the main

gross changes. The data were analyzed using one way ANOVA. The results indicate that none of the

challenge trials produced overt clinical signs of NE and mortalities associated with oral exposure of

C. perfringens. It was observed that length and height of cont group was higher (P<0.05) the cols and

colsw groups at 10, 20, 30 and 40 day of examination. It was noted that distinctly pronounced

pathological lesions developed more in colsw group at 30 and 40th day of examination. The

architecture of villi gradually decreased (P<0.05) in groups having oral administration of clostridium

while size remained same in control group. It can be concluded that C. perferingens reduced the size

of villi owing to lesser intestinal function; and this lethal effect of C. peferinges is favored by presence

of high energy component in diet of chicken.

Key Words: Clostridium perfringens, Broiler chicken, Intestinal villi, Wheat mash

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150

GROWTH PROMOTING AND POSSIBLE TOXICOPATHOLOGICAL EFFECTS OF

SANGUINARINE (SANGROVIT®) IN GROWING BROILERS

Abdul Qayyum1, M. Kashif Saleemi*, M. Zargham Khan, Ahrar Khan, Aisha Khatoon, Farzana Rizivi,

Asim Sultan1, Zahid Hussain1, Tanvir Ahmed2 and M. Sohail Sajjid3, Zain-ul-Abidin4

Department of Pathology, University of Agriculture, Faisalabad; 1Department of Livestock and Dairy

Development Punjab, Pakistan; 2Department of Clinical Medicine and Surgery, University of

Agriculture, Faisalabad; 3Department of Parasitology, University of Agriculture, Faisalabad; 4Veterinary

Research Institute (VRI), Zarar Shaheed Road, Lahore, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Sanguinarineis a plant extract obtained from Macleayacordata plant belonging to family

Papaveracae. It is used as antibacterial growth promoter for poultry, livestock and pig industry. The

present experimental study was planned to investigate the growth promoting and possible

pathological effects (if any) of Sanguinarine available as commercial product Sangrovit®. One hundred

day old broiler chicks were procured from commercial hatchery and divided into 5 equal groups, i.e.,

A-E. The commercial feed and water was offered to the chicks ad libitum. The group E was kept as

control group, while group A Sangrovit @ 1 gm/10 lit drinking water (DW) 24 hours daily, group B

Sangrovit @ 1 gm/10 lit for 12 hours daily, group C 50 mg/kg feed, group D 1 gm/5 lit DW 24 hours

daily. The duration of experiment was 42 days. Physical and some hemato-biochemical and

pathological parameters were studied. The data thus obtained was subjected to analysis of variances

(ANOVA) test and group means were compared by Duncan’s multiple range test (DMR).

Birds administrated with Sangrovit 1gm/5 lit drinking water 24 hours were depressed, less

attractive towards feed, water and loose drooping were observed and this situation remained for two

weeks. Mortality in group A and D was 25 and 35%, respectively. Feed intake of group D was

significantly lower than the control group E. The body weight of group B and C were significantly

higher than the control group while group D showed lower body weight as compared to control

group. In serum biochemical parameters total protein and globulin were significantly higher in groups

C and D as compared to control group. Urea of groups B, C and D were significantly higher than

control group. ALT was lower in C group while AST was lower in groups A, C and D. cholesterol of

groups A and C were significantly higher and group D was significantly lower than the control group.

Grossly the kidneys of the group D were swollen. Microscopically mild to moderate degree of

congestion was present in the liver of group D. In kidneys of group A mild degree of congestion was

present throughout parenchyma, while in group D urinary spaces were condensed and hazy in

appearance. In the duodenum goblet cell secretion was higher in groups B and C, these changes were

observed in dose related manner. From the above mentioned findings of the present study it is

concluded that Sangrovit should be used @ 1gm/10 lit through drinking water 12 hours daily or

50mg/kg feed. At this dose it is an excellent replacement of antibiotic growth promoters (AGPs).

Key Words: Broilers, Sanguinarine (Sangrovit®), Toxicopathological effects

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151

EMERGING THREAT OF NECROTIC ENTERITIS IN POULTRY BIRDS: A REVIEW

Fayyaz-ul-Hassan1, Muhammad Kashif Saleemi2*, Masood Akhtar3, Zain-ul-Abidin4 and Shahid Rafique5

1National University of Science and Technology Islamabad; 2Department of Pathology, University of

Agriculture Faisalabad; 3Faculty of Veterinary Science, Bahauddin Zakariya University Multan; 4Veterinary Research Institute (VRI), Zarar Shaheed Road, Lahore; 5Animal Sciences Division, Pakistan

Agricultural Research Council Islamabad, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Necrotic enteritis (NE) is an emerging economically significant problem of broiler industry

caused by a bacterium Clostridium Perfringens. NE is one of the top ranked intestine damaging bacterial

disease of poultry birds. Under normal conditions, the bacteria live harmlessly in the gut but

whenever there are drastic changes in the environment of gut, it quickly leads to proliferation of

bacteria. C. perfringens possess novel toxins such as alpha and β toxins which are considered key

virulence factors for the pathogenesis of NE. Moreover. It is clearly known that Necrotic enteritis is

produced under specific conditions only by specific strains of C. perfringens. Favorable environment

for the growth of C. perfringens is produced by mucosal damage inducing factors such as parasitism

(coccidiosis) high fiber diets, poor hygienic and housing conditions in addition to toxins. Moreover,

excessive use of antibiotic growth promoters (AGP) enhance the capability of C. perfringens to induce

disease. C. perfringens possess plc gene that encode for the Alpha toxin. A toxoid vaccine using alpha

toxin produced antibody response which was transferred to the progeny as well and resulted into

partial protection from NE. These toxoid vaccines are still in debate and needs a deep insight of

mechanisms involving the role of alpha toxin in development of immunity and pathogenesis. This

review has three purposes. First, it is designed to summarize the currently available information about

necrotic enteritis in chicken. Second, it is aimed to elaborate the pathogenesis of necrotic enteritis at

molecular level. Finally, future prospects of vaccination against necrotic enteritis and other possible

novel methods for the control of necrotic enteritis are suggested.

Key Words: Chicken, Necrotic Enteritis, Clostridium, Alpha Toxin

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152

INCREASING IN VIVO ISOLATION OF PHOSPHORUS FROM MYO-INOSITOL-

HEXA-PHOSPHORIC ACID (IP6) OF SORGHUM USING PHYTASE ENZYME

Asad Sultan*, Sarzamin Khan, Muhammad Subhan Qureshi and N Imtiaz

Department of Poultry Science, The University of Agriculture, Peshawar-Pakistan 25210

*Corresponding Author: [email protected]

ABSTRACT

Enormous amount of nutrients losses occur due partial digestion and presence of certain anti-

nutritional factors in different poultry feed ingredients. Phosphorus is mainly leached from poultry

wastes due to presence of phytate bound P in cereal grains and lack of phytase enzyme in

monogastrics. An effective strategy to improve nutrient utilization is the application of feed enzymes

that target different anti-nutritional factors. This could effectively enhance nutrients bioavailability.

Sorghum, a less common cereal has higher amount of phytate compared to other cereal and has poor

nutritive value. In this study the impact of phytase was assessed in improving the availability of

nutrients. Three bioassay diets with and without enzymes of sorghum (918 g/kg, sole source of

protein) in mash form were prepared to observe phosphorus and nitrogen availability by broilers at

day-21. Diets in mash form were prepared with (Acid insoluble ash; AIA) as an indigestible marker.

Three diets, a control (PH-0) and to other were added phytase enzyme (10000 FTU/g) at level of

0.01% (PH-1) and 0.015% (PH-2). Feed, digesta and faecal samples were collected, processed and

analyzed using standard lab protocols for nitrogen, phosphorus and phytate. Ileal nitrogen digestibility

was significantly enhanced (1.2 and 2.9 %, respectively) and faecal nitrogen loses were significantly

reduced (42%) by birds in PH-2 group. Ileal phosphorus digestibility coefficient in treated groups were

0.51 (PH-1) and 0.53 (PH-2), respectively, to non-treated control group (PH-0; 0.40). Similarly

enzymes supplemented groups had higher phytate digestibility (0.0.72 and 0.76, respectively). pH of

the bedding material in phytase treated group was similar and numerically lower (6.6) to control

group (7.4). These findings revealed that feed enzymes improved nutrient digestibility of sorghum and

potentially can minimize nutrient losses to soil.

Key Words: Sorghum, Broilers, Phytase, Nutrient Digestibility

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153

A PRELIMINARY ASSESSMENT OF THE NUTRIENT DIGESTIBILITY AND

APPARENT METABOLIZABLE ENERGY OF WHEAT, SORGHUM AND MAIZE BY

MIGRATORY DEMOISELLE CRANE (ANTHROPOIDES VIRGO)

Sarzamin Khan*, Asad Sultan, Mohammad Numan Khan and Rafiullah Khan

Department of Poultry Science, The Agriculture University Peshawar, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Anthropoides virgo are on the verge of extinct due to diverse ecological factors. Raising demoiselle

cranes in captivity to propagate their population needs a better understanding of their nutrient

requirements and digestibility. In present study total tract nutrient digestibility and apparent

metabolizable energy of wheat, sorghum and maize was assessed by adult demoiselle cranes. Thirty

six adult demoiselle cranes were fed whole wheat, sorghum or maize in three replicated (n=12; 3

birds/replicate) groups (WC, SC and MC) in standard size pens fitted with rubber mats on floor for

faeces collection. All birds were fed these grains for 10 days including 3 days of adaptation. During

last seven days grain intake and faeces output was measured. Gross energy and other nutrients of

grains and feces were determined using standard lab procedures and digestibility coefficients and

apparent metabolizable energy were calculated. Digestibility coefficient of dry matter (0.72) and crude

protein (0.69) and apparent metabolizable energy (14.13MJ kg-1) was significantly higher for wheat

followed by maize and sorghum. Calcium and phosphorus digestibility coefficient was greater for

sorghum and maize compared to wheat. Cranes digested fat more from maize in comparison to

other type of cereal grains. These findings revealed that crane digestibility pattern for cereal grains is

different and need further work to accurately assess their nutrients requirements.

Key Words: Crane, Cereal Grains, Digestibility, Apparent Metabolizable Energy

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154

MYCOTOXINS: TRANS-GENERATIONAL IMMUNOTXIC COMPOUNDS

Zahoor Ul Hassan1,* and Muhammad Zargham Khan2

Department of Animal Health, The University of Agriculture, Peshawar; 2Department of Pathology,

Faculty of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan

*Corresponding author email: [email protected]

ABSTRACT

Among the many toxicities, immune-modulatory effects of aflatoxin B1 (AFB1) and ochratoxin A

(OTA) are well documented in avian and mammalian spps. In this study, we summarize the findings of

two independent experiments, in which immunological status of chicks hatched from the mycotoxins

contaminated eggs was assessed. In experiment 1, breeder hens were exposed to OTA and AFB1

contaminated feed. The graded doses of mycotoxins were given alone and in combination. The

hatching eggs were collected and incubated under standard conditions to have progeny chicks. The

hatchlings were maintained on mycotoxins free diet and assessed for their immunological status using

macrophage function assay, immune-localization of antibodies bearing cells in spleen and bursa,

antibodies titers against sheep RBC and carbon clearance assay by circulatory macrophages. In

experiment 2, different levels of OTA were placed on to the air cells of the hatching eggs prior to

incubation. These eggs were incubated till hatching. The chick obtained from the eggs were evaluated

for the humoral and cell mediated immune responses. The chicks obtained in the experiment 1,

showed a severe depression in the immune responses both for humoral and cell mediated immunity.

An ameliorative immuno-toxicological responses was noted in the chicks from the hens co-exposed

to two mycotoxins. As in experiment 1, the chicks hatched from the eggs in experiment 2, also

showed immuno-suppression. The findings of these studies clearly showed that immunotoxic

activities of mycotoxins are not limited to the directly exposed animals, but these are transferred to

the progeny of exposed spps, at least in the avian.

Key Words: Mycotoxins, Breeder Hens, Immune System, Progeny Chicks

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PATHOLOGICAL EFFECTS OF IN OVO VACCINATION AGAINST NEWCASTLE

DISEASE IN CHICKEN AND COMPARISON OF ITS IMMUNE RESPONSE WITH

POST-HATCH VACCINATION

Aisha Khatoon1*, Javaria Nazeer1, Muhammad Zargham Khan1, Ahrar Khan1, Muhammad Kashif

Saleemi1, Zain ul Abidin2 and Bilal Aslam3

1Department of Pathology, University of Agriculture, Faisalabad, Pakistan;

Veterinary Research Institute, Zara Shaheed Road Lahore; Institute of Physiology and Pharmacology,

University of agriculture, Faisalabad, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

New castle disease (ND) is an endemic and prevalent disease in Pakistan. Prevention of disease is

mainly practiced by vaccination. Killed and live attenuated vaccines are being used through drinking

water or oculo-nasal route. In ovo vaccination for ND is not being carried out in Pakistan and this

study was designed to investigate the pathological effects of in ovo vaccination of different strains of

ND and comparison of its immunogenic potential with post hatch vaccination against NDV. Total 150

embryonated eggs were divided in five groups A, B, C, D, and E. Group A was kept as negative

control (unvaccinated against ND). Group B (shamed group) injected with 0.1ml normal saline. The

embryonated eggs of groups C, D and E were in ovo vaccinated with 0.1 ml of Lasota, Mukteswar and

Hitchner B1 strains at day 18 of incubation, respectively. After hatching group B was given vaccination

at day 7 and 21 while group C, D and E received booster vaccination of ND with Lasota strain

through oculo nasal route only at day 21. Group A remained unvaccinated. Development of

immunity following pre-hatch or post-hatch vaccination was examined through HI test by collecting

the serum samples from all the birds in all the groups on day 1st, 7th, 14th, 21st, and 28th of age. Six

birds from each group were slaughtered at 1st day of age and day 35 of the experiment. Organs were

observed and collected for any pathological lesions. Results of the experiment revealed hatchability in

all groups above 90%. All the birds showed normal behavioral and clinical signs. Lymphoid organs of

all the birds including spleen, thymus and bursa of Fabricius were normal in gross appearance.

Absolute and relative weights of bursa, spleen and thymus were significantly high in Group D as

compared to control and other vaccinated groups. At day 1 and 7 highest antibody titers against ND

were observed in group D followed by C and E. at day 14, 21, 28 and 35 titers were highest in group

D followed by C, B, E and A. it can be concluded that in ovo vaccination gives better immunological

responses as compared to post hatch vaccination and there are no detrimental effect of this

procedure on chicks.

Key Words: Newcastle disease, In ovo, Immunity, Antibody titers, Post hatch

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AMELIORATIVE EFFECTS OF CARNITINE AND VITAMIN E UPON OCHRATOXIN

INDUCED IMMUNO-TOXICOLOGICAL EFFECTS IN WHITE LEGHORN

COCKERELS

Sheraz Ahmed Bhatti*, Muhammad Zargham Khan, Ahrar Khan, Muhammad Kashif Saleemi, Aisha

Khatoon and Muhammad Noman Naseem

Department of Pathology, University of Agriculture, Faisalabad, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

This study aimed to evaluate the effect of different dietary levels of ochratoxin A (OTA), in the

presence and absence of carnitine and vitamin E, on the humoral immune responses of White

Leghorn cockerels. Day old birds were divided into 12 groups having 20 birds each and were offered

diets contaminated with OTA (1.0 mg/kg and 2.0 mg/kg feed) alone and concurrently with carnitine

(1.0 g/kg feed) and/or vitamin E (0.2 g/kg feed) for 42 days. The humoral immune responses were

accessed by lymphoproliferative response to avian tuberculin, carbon clearance assay and antibody

response to the SRBCs. The dietary addition of OTA alone suppressed the humoral immune

responses, however, the dietary exposure of birds to 1.0 mg/kg OTA in the presence of carnitine

and/or vitamin E ameliorated the toxic effects of OTA. The ameliorative response was absent in the

birds fed 2.0 mg/kg OTA in the presence and absence of carnitine and vitamin E. The relative weight

of the bursa of Fabricius and thymus of the birds exposed to the higher dietary level of OTA alone or

in combination with carnitine and vitamin E was reduced and microscopically, degenerative changes

were observed in the lymphoid organs. Both carnitine and vitamin E partially ameliorated the toxic

effect of OTA on the immune responses of the White Leghorn cockerels.

Key Words: Ochratoxin, Immunotoxicity, Carnitine, Vitamin E

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MEAL WORM (Tenebrio molitor) AS POTENTIAL ALTERNATIVE SOURCE OF

PROTEIN SUPPLEMENTATION IN BROILER

Ibrar Hussain, Sarzamin Khan*, Asad Sultan, Naila Chand and Rafiullah Khan

Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences,

The University of Agriculture, Peshawar, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Present study determined the effect of meal worm supplementation on feed intake, body weight gain,

feed conversion ratio (FCR), dressing percentage, and mortality, acceptability and HI antibody titer

against Newcastle disease in broilers. Meal worm larvae were produced in the lab, killed with

concentrated salt solution and dried in the oven at 40 0C for 24 hrs. Proximate analysis, amino acid

and mineral profile of the dried meal worm meal was conducted before use. A total of 120 day old

broiler chicks were divided into 4 groups (A, B, C and D) of three replicates each containing ten

birds. The study used 4 treatments at inclusion levels of meal worm A (50g), B (100g), C (150g) and

D (Control) and continued for four weeks. Proximate composition of worm meal showed crude

protein value (45.83%), crude fats (34.2%) and ash content (3.51%), essential amino acid as lysine

(4.51±0.3) and Methionine (1.34±0.4) with substantial amount of calcium 4.1gm/kg and phosphorus

7.06gm/kg. No significant effect was found (P≥0.05) on the mean feed intake. Body weight gain was

significantly higher in all supplemented groups. Overall FCR was significantly (p ≥ 0.05) higher for

control group. Compared with other groups the decreasing trend of FCR was declining as (1.99±0.01

to 1.75±0.01) with the increasing level of meal worm meal. Dressing percentage was significantly

(P≤0.05) higher for supplemented groups as compared to control. Non-significant differences were

observed in acceptability, hemagglutination antibody titer against Newcastle disease and mortality

among groups. It was concluded that meal worm meal could be safely used in broiler ration for better

performance without any loss to antibodies titer and acceptability of chicken meat.

Key Words: Worm meal, Proximate Analysis, Amino Acid Analysis, Broilers, Overall Performance

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PATHOGENESIS AND MOLECULAR CHARACTERIZATION OF MYCOPLASMA

GALLISEPTICUM IN NATURALLY INFECTED BROILER CHICKENS

Azmat Ullah1, Umar Saddique1*, Zahoor ul Hassan1, Muhammad Kamal Shah1, Hanif Ur Rehman1,

Shakoor Ahmed Qureshi1 and Sadeeq ur Rahman1,2

1Department of Animal Health, Faculty of Animal Husbandry and Veterinary Sciences,

The University of Agriculture, Peshawar-Pakistan; 2College of Veterinary Sciences and AH Abdul Wali

Khan University, Garden Campus, Mardan, Khyber Pakhtunkhwa-Pakistan

*Corresponding author: [email protected]

ABSTRACT

The current study was designed to get incite on pathogenesis and molecular characterization of

locally isolated Mycoplasma gallisepticum (MG) from naturally infected broiler chicken. For this

purpose, a total of 250 tracheal swab samples from broiler chicken suspected of respiratory

infections were processed for culturing onto pleura-pneumonia like organism (PPLO) medium for MG

identification. Phenotypically, typical fried egg and nipples like colonies with positive glucose

fermentation and inability to hydrolyze arginin were further subjected to DNA extraction for

molecular identification using polymerase chain reaction (PCR). The results indicated that 64/250

(25.6 %) were confirmed MG positive using PCR. Interestingly, the data indicated that more (50%) of

adult (>20 days of age) birds were MG-culture positive as compared (46%) to younger (<20 days of

age) chicken. Similarly, PCR indicated that 38/120 (31.6%) of MG-culture-positive were adults and

26/130 (20.0%) were younger indicating older birds are apparently dominant carriers of MG.

Histopathology of lungs of MG-infected birds revealed emphysema, leukocytic infiltration and

thickening of interlobular septa. There was focal necrosis and infiltration of leukocytes in the liver and

sloughing of tracheal epithelium was the predominant feature of finding. The data revealed that MG is

more prevalent in older age broiler chicken with systemic manifestation.

Key Words: PPLO, PCR, DNA, Mycoplasma gallisepticum, Histopathology

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MOLECULAR EPIDEMIOLOGY AND PATHOLOGY OF CHICKEN INFECTIOUS

ANEMIA IN LAYER CHICKS IN FAISALABAD PAKISTAN

Muhammad Saleem1, Aisha Khatoon1*, Muhammad Zargham Khan1, Muhammad Kashif Saleemi1,

Zain ul Abidin2 and Zia ud Din Sindhu 3

1Department of Pathology, University of Agriculture, Faisalabad; 2Veterinary Research Institute, Zara

Shaheed Road Lahore; 3Department of Parasitology, University of agriculture, Faisalabad.

*Corresponding Author: [email protected]

ABSTRACT

Chicken anemia virus (CAV) is the causative agent for chicken infectious anemia (CIA) disease in

poultry which is an economically important disease. CIA is responsible for anemia,

immunosuppression, decrease weight gain and low production. CAV is emerging worldwide and has

got considerable attention. In Pakistan only one outbreak report is available on CAV implicating study

of disease at wider scale. This study was designed to check the prevalence of chicken anemia virus

through PCR in layer chicks of less than 7 day of age in district Faisalabad. And it is the first

epidemiological study on CAV in layer chicks in Pakistan. For this purpose, 245 samples of different

organ (liver, spleen and thymus) and blood from live birds were collected from five tehsils of

Faisalabad. The average values of hemoglobin and pack cell volume were 4.72g/d and 18.35%

respectively. Out of 245 pooled samples 65 were found positive for PCR assay. An overall prevalence

of 26% was found in district Faisalabad. Histopathologically, CAV positive birds showed moderate to

severe congestion of blood vessel in liver. Thymus and spleen of CIAV positive birds showed marked

lymphocytic depletion. It was concluded that there was high prevalence of disease. The prevalence of

CIAV was significantly different among different areas of Faisalabad, in different age group and with

respect to different environmental conditions, while there was no significant difference of prevalence

in cockerels and females. This study shows that there is need to study this disease at much wider

scale in order to access the prevalence of disease in country and to reduce the economic losses

occurring due to chicken infectious anemia.

Key Words: Chicken infectious anemia, Layer chicks, PCR, Prevalence

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EFFECT OF LACTIC ACID BACTERIA ON MUCOSAL FRONT AGAINST E.coli

INFECTION IN POULTRY BIRDS.

M. Abubakar Siddique*, Sajjad ur Rahman, Babar Hayat, Ahsan Naveed, Hafiz Sohaib Mazhar, Aitezaz

Ahsan, Hassan Zafar, Fakhar Hayat, Noorulain, Naila Afzal and Faisal Rasheed

Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan.

*Corresponding author: [email protected]

ABSTRACT

Poultry is a well-developed sector of agriculture industry in Pakistan. Poultry industry plays a

major role in the GDP of Pakistan. Many food borne pathogens play role in causing different digestive

problems in poultry and influence the production of eggs and meat. In poultry industry extensive

antibiotics are used to control these pathogens for the improvement of meat and egg production

.Present study was conducted to evaluate the impact of lactic acid producing bacteria on the immune

status against E.coli infection in poultry birds. Lactic acid bacteria i.e. lactobacillus fermentum was

isolated from conventional yoghurt sample. Out of 20 samples 13 samples were positive for

lactobacillus fermentum which were identified on the basis of their morphological characteristics as it is

gram positive rod shaped bacteria with small white round colonies on MRS agar. Lactobacillus

fermentum was furthered identified on the basis of their biochemical tests as it was catalase negative

and sugar fermentation tests. After identification three concentrations were maintain that were 104,

105,106 cfu/ml. A trial was conducted on the poultry birds. They were divided into four groups A, B,

C and D. Different concentration of probiotics which were Control, 104, 105and106 cfu/ml were given

to each group respectively. Birds were kept for 15 days. At day 7 and 15 plasma were collected from

respective groups of poultry. Macrophages were collected from the peritoneal cavity of poultry birds.

Macrophages migration inhibition factor assay were performed invitro. The results of this assay

showed that group administered with high probiotic concentration i.e 106 cfu/ml showed that immune

response was increased more effectively against E.coli as compared to other poultry groups. Because

% inhibition of macrophages was 64, 52, 44 and 31% for D, C, B and A, respectively. The results

showed that the group with high % inhibition of macrophages show significantly high cell mediated

immune response against E.coli.

Key Words: Poultry, lactic Acid Bacteria, E.coli, Mucosa, Macrophages, Migration Inhibition Factor,

Mucosal immunology

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IMMUNOLOGICAL CROSS REACTIVITY AMONG SALMONELLA TYPHIMURIUM

ISOLATES FROM POULTRY AND NON-AVIAN ORIGIN

Hafiz Sohaib Mazhar*, Sajjad ur Rahman, Babar Hayat, Ahsan Naveed, Abubakar Saddique, Aitezaz

Ahsan, Hassan Zafar, Fakhar Hayat, Aqsa Bukhari, Naila Afzal and Faisal Rasheed

Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan.

*Corresponding author: [email protected]

ABSTRACT

Salmonella is transferred to the human by animals and their byproducts contaminated with meat,

eggs, and dust, and also by contaminated water. In present study the immunological cross reactivity

was determined among isolates of Salmonella recovered from poultry birds, other animals and human

enteritis cases. Total of 40 samples were processed. Complete identification showed that 60%

Salmonella typhimurium was isolated from droppings of enteric cases of poultry birds, 60% from cattle,

60 % from sheep, 60% from dog, 40 % from cat, 40% from human and 20% from horse, and 40% from

honey bee. Agar gel precipitation test and Serum Neutralization Assay were performed to find out

common antigenic moieties among Salmonella typhimurium isolates. In serum neutralization assay,

antigen from poultry birds exhibited maximum cross reactivity with antiserum from sheep and honey

bee 1:16, besides homologous cross as 1:64. Antiserum from human, cattle and cat presented

comparatively less affiliation with poultry antigen with titer 1:8 followed by antiserum from horse and

dog as 1:4. Poultry antigen displayed strong precipitation bands (+ + +) in agar gel precipitation test

with sheep and cattle antiserum while medium strength precipitation bands (+ + -) were observed

with human and honey bee antiserum followed by no immunological cross reactivity with cat dog and

horse antiserum. Concludingly, the poultry antigen showed maximum cross reactivity with sheep and

honey bee while poultry antiserum displayed maximum affiliation with human antigen.

Key Words: Poultry, Salmonella typhimurium, Serum Neutralization Assay, AGPT, Interspecies Cross

reactivity, Antibiotic Susceptibility

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COMPARATIVE EFFICACY OF COMMERCIALLY AVAILABLE ANTIMICROBIALS

AGAINST LOCAL ISOLATES OF MYCOPLASMA GALLISEPTICUM

Farida Tahir1, Umer Saddique1, Shakoor Ahmad1, Zahoor Ul Hassan1, Sadeeq Ur Rehman2, Muhammad

Kamal Shah1*, Hamayun Khan1, Murad Ali Khan1, Sajjad Ali Shah1 and Hanif Ur Rehman3

1Department of Animal Heath, the University of Agriculture Peshawar; 2College of Veterinary

Sciences & Animal Husbandry, Abdul Wali Khan University, Mardan; 3Veterinary Research Institute,

Peshawar, Pakistan

*Corresponding Author: [email protected]

ABSTRACT

Avian mycoplasmosis is an important respiratory disease causes heavy economic losses in poultry

industry throughout the country. The present study was carried out in different farms of the district

Peshawar, Khyber Pakhtun Khwa, Pakistan to investigate the different antibiotic efficacy against PCR

confirmed local isolates of Mycoplasma gallisepticum. Five different commercially available

antimicrobials like Enrofloxacin, Tylosin, Gentamycin, Oxytetracycline and Sulphonamides were

tested in vitro by gel diffusion assay and micro broth dilution method for zone of inhibition and

minimum inhibitory concentration (MIC) respectively. Drugs sensitivity results showed that

Enrofloxacin was the most efficacious drug having the least MIC of 0.002±0.0001 mg/ml and

maximum zone of inhibition 17±02 mm among the tested drugs followed by Sulphonamide

0.02±0.001 mg/mI and 15±1.6 mm zone of inhibition. Interestingly Tylosin, Oxytetracycline and

Gentamycin showed resistant against all isolates of mycoplasma gallisepticum. This resistance might be

due to indiscriminative uses of theses antibiotics in the study area. Our findings suggest that

Enrofloxacin is the drug of choice for the treatment of Mycoplasma gallisepticum.

Key Words: Mycoplasma gallisepticum, Gel diffusion, Antibiotics, Micro broth dilution, MIC

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PREPARATION OF OIL BASED VACCINE AGAINST PIGEON PARAMYXOVIRUSES

Muhammad Samiullah1, Farzana Rizvi2*, Muhammad Noman Naseem2 and Muhammad Imran2

1Livestock Dairy Development Department, Punjab, Pakistan. 2Department of Pathology, University of Agriculture, Faisalabad, Pakistan.

*Corresponding author: [email protected]

ABSTRACT

Pigeons are one of few domesticated bird species which are raised by the humans for their

homing ability and for purposes such as food (meat), entertainment/hobby (racing), and for treatment

of various diseases. The study was conducted to prepare an oil-emulsified formalized PPMV-1 vaccine

using local field isolate and vaccine was evaluated in experimental conditions. A mesogenic strain of

PPMV-1 was used to prepare the vaccine and propagated in embryonated eggs. HA titer of the

pooled AAF was measured as 1:2048. Regarding the physical properties, the color of the vaccine was

milky-white and the flow time of the vaccine was 3.0 seconds. The vaccine remained stable for 20

weeks at room temperature. For the safety test vaccine was inoculated intramuscularly in pigeons.

The birds remained healthy, only a small granulomatous lesion was seen by the fourth day of

inoculation. These results suggested that the vaccine is quite safe and could be evaluated in the

experimental trial. This evaluation was based upon the ability of the vaccine to produce a humoral

antibody response against the field isolate of PPMV-1. For this reason, vaccine was inoculated in

pigeons and humoral immune response was determined against PPMV-1. Maximum GMT was

increased upto 139.58 at 21 days post-vaccination and it was 279.17 in single shot and 234.75 at

double shot vaccination. Vaccine was also evaluated by challenge protection. Mortality in pigeons with

single vaccinated was 10% while it was 7.5% in pigeon vaccinated with booster dose. From this trial it

can be concluded that PPMV-1 (double dose) vaccine was helpful in protection of pigeons against

Newcastle disease.

Key Words: Pigeon, Newcastle Disease, Oil-emulsified formalized vaccine, GMT

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DETECTION OF PIGEON PARAMYXOVIRUSES FROM NEWCASTLE DISEASE

OUTBREAKS IN PIGEONS

Muhammad Samiullah1,2, Farzana Rizvi2*, Muhammad Noman Naseem2 and Muhammad Imran2

1Livestock Dairy Development Department, Punjab, Pakistan 2Department of Pathology, University of Agriculture, Faisalabad, Pakistan

*Corresponding author: [email protected].

ABSTRACT

Pigeons are universally kept by humans on all parts of the world since centuries along with

chickens. Newcastle disease locally called Jholah in Punjab is worldwide distributed disease causing

sever outbreaks in pigeon lofts throughout the world. The project comprised of different phases.

Seroprevelance of Jholah was observed in Faisalabad, Lahore, Karachi, Peshawar and Rawalpindi. Virus

from diseased pigeons was isolated and identified and its pathotyping was done. Monoclonal

antibodies against APMV-1 & PPMV-1 were procured from Defra, the department for Enviorment,

food and Rural Affairs, UK. Theses monoclonal antibodies were used to develop a rapid diagnostic

test for the diagnosis of Jholah in field. Clinical signs and lesions in pigeons were recorded during the

study. The clinical signs observed among naturally infected pigeons were wing paralysis, blindness,

shivering of head and neck i.e., tortticollis and greenish mcucoid diarrhea. Postmortem lesions

observed in infected pigeons were ulcers in intestine and hemorrhages in proventriculus, enlarged

spleen and airsacculitis. Hhistopathological studies showed edema in proventriculus and emphysema

in aliveoli, pulmonary congestion and edematous fluid in air sacs and lymphatic infiltration and

hyperplasia in spleen.

Key Words: Pigeon, Newcastle Disease, Jholah, Monoclonal antibodies

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ASSOCIATION OF SELECTED PATHOGENICITY GENES OF ESCHERIACHIA COLI

WITH GROSS AND HISTOPATHOLOGICAL LESIONS IN CASES OF

EARLY CHICK MORTALITY

Sameen Qayoom Lone, M. Tariq Javed, Abdul Wadood Jan, Farzana Rizvi, Zafar Iqbal Qureshi,

Aziz-ur-Rehman and M. Sohaib Aslam

Department of Pathology, Faculty of Veterinary Science, University of Agriculture Faisalabad, Pakistan

ABSTRACT

Avian pathogenic E. coli (APEC) strains cause diseases in birds at various ages. It can cause

extensive mortality in poultry flocks leading to great economic losses. Recent reports showed that

the APEC pathogenicity is associated with certain virulence genes are located within the bacterial

genome and/or their ColV plasmids. Identification and characterization of these genes are essential to

implementing efficient disease control and prevention systems. The aim of this study was to identify

the virulence associated genes in cases of early chick mortality. Study included the poultry farms

within Faisalabad region suspected for of Escherichia coli infections. The postmortem findings of

various organs collected from morbid poultry birds of age ten or less than ten days revealed that

gross lesions on liver were present in all the confirmed cases of E. coli infections. Out of which 25%

showed severe gross lesions, 45.83% showed moderate and 29.17% showed mild lesions. On gross

examination, 79.17% lungs showed abnormality, 87.50% cases of heart, out of which 45.83%, 33.33%

and 8.33% were mild, moderate and severe lesions, respectively. Study showed that in 79.17% cases

lesions were present in spleen out of which 50%, 20.83% and 8.33% were mild, moderate and severe,

respectively. Results showed that in 87.50% cases gross lesions were present in the intestine, out of

which 4.17%, 66.67% and 16.67% were mild, moderate and severe, respectively. The genes fimC,

papC, iucD, fyuA and tsh were positive at the rate of 41.67%, 16.67%, 54.17%, 20.83% and 37.50%,

respectively. However, no irp2 gene was detected from the samples positive for E. coli infections. The

gene fimC was detected in 58.33% of cases which showed severe microscopic changes in liver while

gene tsh was detected in 37.50% and iucD was detected in 54.17% cases each showing severe

microscopic changes in liver. The gene fyuA was detected in 20.83% and papC gene was detected in

16.67% cases each showing severe microscopic changes in liver. The genes fimC, tsh, iucD, fyuA and

papC were not detected in 41.67, 62.50, 45.83, 79.17 and 83.33% of cases, though microscopic

changes of variant degree were seen in liver.

Key Words: E. coli, early chick, broiler, pathogenicity genes,