GENERAL INTRODUCTION

Rodents pose a threaten towards crops in fields and

stores. In addition, they may attack people and their domestic

animals spreading many infectious diseases via their endo-

and ectoparasites. The control of Norway rat (Rattus

norvegicus Berk.), the most prevailing species lives close to

man, depends mainly on rodenticides such as metal

phosphides, fluoroacetamide, hypercalcemics and the

worldwide commonly used coumarin-derived anticoagulants.

Constituting over 40% of all mammal species, Rodents

are the largest and most successful group of mammals

worldwide. They have a high rate of reproduction and a good

ability to adapt to a wide variety of habitats (Parshad 1999)

Although rodents are often only associated with

infrastructural damages, crop attacking and eating or spoiling

of stored food and products, the veterinary and zoonotic risks

of rodents are frequently underestimated. Wild rodents can be

reservoirs and vectors of a number of agents that cause serious

diseases for human and domestic animal; there are more than

20 transmissible diseases that are known to be directly

transmitted by rodents to humans, by the assistance of blood-

sucking parasites like fleas, ticks and mites (Khatoon et al.

2004). Wild rodents act as definitive and/or intermediate hosts

of many parasites, which are common to domestic animals,

and humans. Some rodent parasites are epidemiologically

2

important as they are prevalent parasites of humans and their

domestic animals. The eggs of parasites are passed out in

rodent droppings in fields, grain stores and amongst foodstuffs

in houses, and are responsible for disease spread (Khatoon et

al. 2004). As rodents live in a close proximity with human and

their animals and expose to the blood-sucking arthropods, the

possibility for transmission of parasites increases.

Controlling of rodents and their endo- and ectoparasites

has been done mainly using anticoagulant rodenticides. The

repeated use and application of such anticoagulant

rodenticides for long periods may result in the rapid

development of resistance to these compounds in wild rodent

species.

Resistance to anticoagulants can develop in a population

after 5-10 years sustained use of anticoagulant rodenticides.

No enough data exist on the baseline susceptibility of rodent

populations in Egypt to anticoagulants or their changing

patterns of susceptibility in areas of sustained use. Monitoring

systems for rodent populations and changes to poisoning

methods will assist Egypt rodent control groups in avoiding

the resistance-induced control problems now seen outside

Egypt. Sustained control of rodents is likely to be

substantially dependent on toxicants, and anticoagulant

poisons in particular, for the foreseeable future .

3

The aim of this work

This study was carried out to determine what the major

Norway rat parasites are, and to monitor its resistance to

warfarin anticoagulant rodenticide at some governorates of

Egypt. Therefore, the scope of the present work was to cover

the following points:

1- To study the Norway rat species population structure at

four different governorates.

2- To identify Norway rat helminthic parasites and to

determine their incidence and distribution at four

different governorates.

3- To identify Norway rat ectoparasites, and to determine

their prevalence and general indices that is useful to

understand the role of arthropod vectors as well as

mammalian reservoirs in the maintenance of various

diseases in the study areas.

4- To monitor the Norway rat resistance to warfarin (First

generation anticoagulant rodenticide) at four different

governorates by using the conventional method, non-

choice feeding test.

5- To monitor the Norway rat resistance to anticoagulants

rodenticides (warfarin) at four different governorates

through VKORC1 analysis using Polymerase Chain

Reaction (PCR) technique.

4

5

Part I: Endo and Ectoparasites of Rattus

norvegicus

INTRODUCTION

Norway rat, Rattus norvegicus (Berk. 1769), is a

cosmopolitan rodent species with a wide distribution in urban

and suburban-rural habitats. It is commonly found living near

sources of food and water, such as garbage and drainage

ditches, streams or sewers. Because of its high ability to

harbor many zoonotic agents, wild Norway rats play a

significant role as definitive and/or intermediate hosts for

vector-borne animal and human diseases (Easterbrook et al.,

2007).

Zoonotic disease or zoonosis are the diseases that can be

transmitted from either wild or domesticated animals to

humans. About 60% of all infectious disease agents affecting

humans are zoonotic in origin and most of the zoonotic

reservoir species are rodents (Taylor et al., 2001). Viral,

bacterial and protozoan pathogens responsible for zoonotic

diseases are excreted by rodent hosts or are transferred via the

bite of a bloodsucking arthropod and then enter the human

body via inhalation, swallowing or skin punctures (Ostfeld

and Holt, 2004). The most famous zoonotic disease associated

with rodent presence is probably the infection of rodent fleas

with bubonic plague caused by Yersinia pestis bacterium,

resulting in many millions of casualties worldwide.

6

Endoparasites of rodents play an important role in the

zoonotic cycles of many diseases, such as, schistosomiasis and

angiostrongyliosis. Parasites in rats, particularly helminthes,

belong to the four major groups; Nematoda, Cestoda,

Trematoda and Acanthocephala. Cestode and nematode

parasites in rat have been reported from all parts of the world.

Vampirolepis nana and Hymenolepis diminuta are commonly

found in rats and mice and they are potentially transmissible

(Zoonosis) to man. The occurrence of H. diminuta and V. nana

in certain rodents is of interest since the possibility exists that

rats and mice may serve as reservoir hosts and help in

dissemination of these worms to domestic animals and man

causing zoonosis (Jawdat and Mahmoud, 1980).

Also, rodents are suitable for hospitality of some groups

of arthropods that are known as ectoparasites. They are well -

adapted for living on the external surface of rodents bodies

(permanent or temporary). Rats are known to harbor four

groups of arthropod ectoparasites: fleas, ticks, mites and lice

(Ansari, 1953; Abu-Madi, et al., 2005).

Ectoparasitic arthropods as vectors of zoonotic

pathogens have an important role in causing diseases such as

anaplasmosis, ehrlichiosis, rickettsiosis, plague, lyme

borreliosis, viral encephalitis, tularemia, CCHF, zoonotic

leishmaniasis, murine typhus, etc. They can also transmit

disease to human by: feces, urine, saliva, milk and blood.

7

Among the ectoparasites infesting rats, the best known

and most dangerous to man is the rat flea, Xenopsylla cheopis

(Rothschild). This flea is the vector of Yersinia pestis, the

causative agent of plague, and Rickettsia typhi, the causative

agent of murine typhus. Rickettsial agents, such as

Anaplasma, Bartonella, Coxiella, Ehrlichia, and Rickettsia,

have been detected by molecular tools from Egyptian

ectoparasites, such as lice, fleas, and ticks (Reeves et al.,

2006).

8

9

REVIEW OF LITERATURE

Indo and ectoparasites associated with Rattus

norvegicus

Rodents (rats and mice) follow man wherever he goes

carrying with them many serious zoonotic diseases (El Shazly

et al., 1991). Historically, R. norvegicus has played a major

role in diseases transmission. This fact is still important in

today's world as it acts as a reservoir and transmits many

serious diseases of man and animals like plague,

hymenolepiasis, leishmaniasis, trichinosis, babesiosis and

toxoplasmosis. (Louisiana, 2000).

1. A brief about Rattus norvegicus (Berkenhout, 1769)

Rattus norvegicus is a cosmopolitan rat species that may

has many common names like brown rat, Norway rat, sewer

rat or burrowing rat. Its usual habitat is away from houses, in

drains or in burrows. It is fleshier than R. rattus with broad

head, blunt muzzle, small eyes, short ears which, when drawn

forward, do not touch each other. Fur is rough, grey brown

above and whitish grey on the abdomen. The tail is shorter

than the length of the body and head combined. The faecal

pellets are sausage shaped and usually occur in groups. It is a

commensal rat and not a true domestic rat (Nowak, 1999).

Thought to have originated in northern China, R.

norvegicus has now spread to all continents and is the

01

dominant rat in Europe and much of North America. It is a

common pest wherever humans live particularly in urban areas

and degraded environments (Banks et al., 2003).

Classification of Rattus norvegicus (according to

Nowak, 1999)

Kingdom: Animalia

Phylum: Chordata

Sabphylum: Vertebrata

Class: Mammalia Linnaeus, 1758

Subclass: Eutheria Parker and Haswell, 1897

Infraclass: Eutheria Gill, 1872

Order: Rodentia Bowdich, 1821

Suborder: Myomorpha Brandt, 1855

Family: Muridae Illiger, 1815

Subfamily: Murinae Illiger, 1815

Genus : Rattus Fischer, 1803

Rattus norvegicus (Berkenhour, 1767)

2. Endoparasites of rats

The ecology, in particularly the component community

structure, of helminth parasites in small rodent population has

been well documented in temperate regions of Europe (Abu-

Madi et al., 1998). In contrast, and despite the wealth of

information on species lists and taxonomy, there is little

00

comparable data for rodents living in tropics (Behnke et al.,

2000).

Rats and mice in Egypt are well-known to be the

definitive hosts (reservoir hosts) of several helminthes (Arafa,

1968; Monib, 1980; Wissa, 1980). It has been known from the

previous work that rats act as reservoir hosts for many

parasitic helminthes as Trematodes, Cestodes and Nematodes.

a. Trematode

The Echinoparyphium recurvatunz is a trematod parasite

of the small intestine especially the duodenum of the domestic

duck, and pigeons. This parasite has also been recorded in

rats, dogs, cats and man in Egypt, Malaysia and Indonesia

(Soulsby, 1982). E. recurvatunz parasite causes emaciation,

anemia and sometime weakness of the legs; this is explained

by the marked enteritis which observed on autopsy (Bowman,

1999).

Prohentistoman vivax is a well-known parasite of fish

eating birds and mammals like Rattus norvegicus. It has been

recorded to be infectious to Man (Chandler and Clork, 1961).

Schistosoma mansoni is a blood fluke occurs in the

mesenteric veins of man in Africa, South America and the

Middle East where humans are the most important definitive

host. However, a variety of animals have been found to be

02

naturally infected with S. inansoni since it has been recorded

in gerbils and Nile grass rats in Egypt, rodents in Southern

Africa and Zaire, Various species of rodents and wild

mammals and cattle in Brazil and Baboons, and rodents and

dogs in East Africa (Soulsby, 1982).

Mansour (1973) in Egypt, reported that 3 out of 22

Arvicanthis niloticus caught from Giza were naturally infected

with S. mansoni and S. haematobium. He added that on

experimental work this animal can serve as a natural reservoir

host. Also, El-Nahal et al., (1982) and Morsy et al., (1982)

reported the presence of the bilharzial worms or its antibodies

in some species of rodents. Likewise, Fedorko (1999) reported

S. japoniam in different rat species in Philippines in

association with different other endoparasites.

b. Cestode

Hymenolepis nana is essentially a parasite of rats

(rodents) but it also infects humans especially children. It is

distributed all over the world and it is the most common

cestode infecting humans in the tropics and subtropics, but

human infection is most prevalent in areas where temperature

is high and sanitary conditions are poor (Miyazaki, 1991;

Smyth, 1996; Roberts and Janovy, 2001).

H. nana has an alternate mode of infection consists of

internal autoinfection, where the egg release their hexacanth

03

embryo, which penetrate the villi continuing the infective

cycle without passage through the external environment. The

life span of adult worms is 4 to 6 weeks but internal

autoinfection allows the infection to persist for years. One

reason for the facultative nature of the life cycle and

autoinfection is that H. nana cysticercoids can develop at

higher temperatures than can those of other hymenolepidids

(Smyth, 1996; Andreassen, 1998).

Infection of H. nana to the rat occurs by taking in an

intermediate hosts or eggs. Transmission of eggs from one

patient to another is considered the main route for human

infection, but insect hosts could also serve as sources of

infection (Bowman, 1999).

As long as the number of worms of H. nana in the

intestine is small, no symptoms are noted. As the

autoinfection progress, damage to the intestinal mucosa would

result from the invasion of cysticorcoids causing cellular

infiltration consisted of polymorphnuclear leucocytes and

lymphocytes (Andreassen, 1998). Also attachment of scoleces

of adults to mucosa could cause changes in the form of

disintegration of the villi, ulcers and haemorrhage in some

parts of the mucosa and cellular infiltration of submucosa

leading to hypertrophy and thickening of submucosa in other

parts (Crompton, 1999).

04

In light infection of H. nana, usually no symptoms

appears and it can pass unnoticed. But in heavy infection

patients may complain of loss of appetite, nausea, vomiting,

abdominal pain and diarrhea may arise. Nervous symptoms

such as insomnia, vertigo, headache, dizziness, irritability and

epileptiform convulsion (Lioyd, 1998).

H. diminuta is a cosmopolitan worm that is primarily

parasite of rats (Rattus spp.). Beetles of the genera Tribolium

and Tenebrio serve as an intermediate host for H. diminuta.

When provided with a choice of rodent faeces with or without

the tapeworm's eggs, the beetles preferentially consume the

faeces containing the eggs (Pappas et al., 1995).

Human infection with rat tapeworm, H. diminuta, is

considered rare and usually accidental (Schantz, 1996;

Andreassen, 1998) and almost always occur in children (Tena

et al., 1998).

Rat nests almost always contain larvae and pupae of

fleas that frequently harbor cysticercoids in their

haemocoeles. As the cysticercoid persists also in adult fleas

parasitizing rats, infection may result when the fleas are taken

in by the animal. In other words, the life cycle of this cestode

can be maintained within a rat nest. Infected rats disseminate

eggs with the faeces, which may be ingested by insects that

would in turn serve as infectious sources for humans. Human

05

infection could occur by eating food containing infected

insects. Since rat fleas can parasitize humans, crushing such

fleas with finders may result in infection via fingertips

contaminated with cysticercoid (Miyazaki, 1991).

H. diminuta parasites in the upper middle part of the

small intestine. Autoinfection does not occur; as a result, the

number of worms inhabiting a human host is accordingly

small. Symptoms are therefore slight, if there; only such light

ones as reduced appetite, abdominal pain and diarrhea may

occasionally be encountered (Lioyd, 1998).

Cysticercus fasciolaris is the heabatic larval stage of

tapeworm Taenia taeniaeformis. It infects rabbits, black rat,

cotton rat and other wild rodents. The adult tapeworm is

usually found in small intestine of cats (rat eater) and wild

carnivorous and may be found accidentally in dogs. The

hepatic larval stage and the adult stage occur worldwide

(Wanas et al., 1993). Strobilocercus is embedded in the liver

parenchyma in a pea-sized nodule (Esch and Self; 1995). The

main interest behind this species lies in its larval stage which

does not form a cysticercus but a strobilocercus that may

induce sarcoma in host liver.

Reaching the liver in the intermediate host, the

strobilocercus develops and rapidly becomes infective after

30 days (Smyth, 1996). The strobilocercus, in the final host,

has only the scolex which develops in cat small intestine into

06

an adult tapeworm of about 60 cm long (Lioyd, 1998)

c. Nematode

Syphacia species, the natural oxyurid nematodes of rats,

are considered zoonotic parasites. Human infection is resulted

from accidental contamination of human food or drink with

droppings of infected rodents (Wescott, 1992). This occurs in

localities with highly infected rodent population and poor

sanitation (El-Shazly et al., 1994).

Inhabiting the caeca of domistic rats and mice, the

oxyurid Syphacia spp. is a common parasitic nematode with a

direct life cycle (Tattersall et al., 1994).

Aspiculuris tetraptera is a pinworm of rats and mice,

occurs in the large intestine. The cuticle is transversely

striated with broad cervical alae terminating abruptly at the

level of oesophageal bulb. when a narrow lateral flanges run

to the posterior extremity. The mouth is with three lips.

Oesophagus is club-shaped followed by a well-developed oval

bulb. The life cycle of A. tetraptera is direct. Eggs pass in

faeces and the infective stage is reached in about six days.

Infection is by ingestion of eggs and the prepatent period is

about 23 days. Negligible pathogenicity is associated with the

infection; it is not a zoonotic infection (Arafa, 1968).

07

Protospirura marsupialis is a spiruroid nematode of

rodents, inhabiting the stomach. It is large and

semitransparent. The body is attenuated anteriorly, without

lateral flanges, the mouth has two large trilobed lateral l ips,

each lobe bearing a papilla externally at the base, and three

teeth on its inner surface. There are cervical papillae anterior

to the nerve ring. Buccal capsule is long, cylindrical with very

long oesophagus which is divided into two parts. Females

measure 67.5-79.0 mm in length and 1.45-1.60 mm in breadth

with very short conical tail. Males are shorter than females

measuring 40-50 mm in length. Its posterior extremity is

spiral, with caudal alae well developed. Male has two unequal

spicules. It is not recorded to be of zoonotic importance

(Yamagoti, 1962; Wanas et al., 1993).

3. Ectoparasites of rats

The intimate association of commensal rodents with

man, and the role of ectoparasites in transmission of

pathogens to man led several workers to pay attentions to

study their host parasite fauna (Allam et al., 2002).

In Egypt, many scientists gave an account of the

parasite species of Acari found on rodents. Hoogstraal and

Traub (1956) studied the fleas of Egypt and Johnson (1960)

studied the sucking lice. Also, Abdou (1981) made a study of

08

the commensal and wild rodents and their ectoparasites in

Assiut area

Rifaat et al., (1969) studied the relative incidence and

distribution of the medically important ectoparasites in the

various geographical zones of the country. The rodents and

fleas were studied at Ismailia Governorate (Morsy et al.,

1982), Suez Governorate (Morsy et al., 1986), Sharkia

Governorate (Zeese et al., 1990) and South Sinai Governorate

(Shoukry et al., 1993).

Rodents reserve and transmit many serious diseases of

man and animals as plague, hymenolepiasis, leishmaniasis,

trichinosis, babesiosis and toxoplasmosis. Man is infected

with these diseases by contagion as well as by the arthropod-

ectoparasites of rodents (Hilton. 1998). Ectoparasites could be

from-rat-to-rat or from-rat-to-man vectors. Man becomes an

incidental host of disease when bitten by ectoparasites or

when ectoparasite faeces contaminate the bite wound

(Shoukry et al.. 1991).

Ectoparasites obtain some of their requirements, like

oxygen, from the physical environment, and to some extent,

are influenced by factors that affect their non-parasitic

associates. They are also dependent on their hosts for

nutritional requirements and for developmental and maturation

stimuli (Soliman et al., 2001a).

09

a. Fleas

In general, fleas are not very host specific, although

they have preferred hosts. Most can transfer from one of their

hosts to another or to a host of a different species. Their

common names (for example, rat flea or human flea) refer

only to the preferred host and do not imply that they attack the

host exclusively. At least 19 different species have been

recorded as biting humans (Harwood and James, 1997).

Fleas could transmit many zoonotic diseases from rat to

man. Plague (black death) is essentially a disease of rodents

from which it is contracted by humans through the bites of

fleas, particularly Xenopsylla cheopis (Ryckman, 1971). It is

caused by a bacterium, Yersinia pestis. The bacterium releases

two potent toxins that have identical serious effects. Some

animals such as rats and mice, are more sensitive to the toxins

than others (rabbits and dogs) (Lewis, 1993).

Yersinia pestis is widely distributed in rodents and

occurs across broad areas of every continent. The bacteria are

consumed by a flea along with its blood meal, and the

organisms multiply in the flea's gut, often to the extent that

passage of food through the proventricular teeth is blocked

(Hilton, 1998). When the flea next feeds, the new blood meal

cannot pass the obstruction, but is contaminated by the

bacteria and then regurgitated back into the bite wound. The

21

propensity of a particular flea species to have its gut blocked

by growth of Yersinia pestis is an important determinant of its

efficacy as a vector. Xenopsylla cheopis is a good vector

because it becomes blocked easily and feeds readily both on

infected rodents and humans (Roberts and Janovy, 2001).

The disease may exist in rodent populations in acute,

subacute, and chronic forms. Epidemics among humans

usually closely follow epizootics, with high mortality among

rats. When the rat dies, its fleas depart and seek greener

pastures (Allam et al., 2002).

The second important disease could transmitted from

rats to humans is murine typhus or flea-borne typhus. It is

caused by Rickettsia nzooseri or R. typhi and occurs in warmer

climate throughout the world. Murine typhus can infect a wide

range of small mammals but the most important reservoir is

Rattus norvegicus in which it causes slight disease symptoms.

Murine typhus can be transmitted from one rat to others by

Xenopsylla cheopis, Nosopsyllus fasciatus, Leptopsyllus

semis, Polyplax spinulosa (the rat louse); and the tropical rat

mite Ornithonyssus bacoti. In humans the disease is a rather

mild. But it may involves febrile illness of about 14 days, with

chills, severe headache, body pains, and rash. X. cheopis is

considered the primary vector transmitting the disease to

humans either through the bite or through contamination of

skin abrasions with flea faeces by scratching. Ingestion of

20

infected fleas and their faeces also can produce infection in

rats. The rickettsias proliferate in the midgut cells of the flea

but do not kill it. Rupture of the midgut cells releases the

organisms into the gut of the flea. (Farhang and Traub, 1985).

The incidence of murine typhus had been dropped

dramatically after the institution of a rat control program, use

of DDT, and increasing use of antibiotics (Roberts and

Janovy, 2001).

Lastly, Nosopsyllus fasciatus is a vector for

Trypanosoma Lewisi of rats. Ctenocephalides Canis, C. felis

and Pulex irritans serve as intermediate hosts of Dipylidium

caninum, a common tapeworm of cats and dogs. Nosopyllus

fasciatus and Xenopsylla cheopis can serve as vectors for the

rat tapeworm, Hymenolepis diminuta. The mouse tapeworm

Vampirolepis nana can develop in X. cheopis, C. felis, and P.

irritans; all of these fleas acquire the tapeworm as larvae

when they consume the eggs which pass in the faeces of the

vertebrate host, retaining the cysticercoid in their hemocoel

through metamorphosis to the adult. All these three species

can be transmitted to humans if the person inadvertently

ingests an infected flea (Robert and Janovy, 2001).

22

b. Lice

Lice are permanent ectoparasites on mammals including

rats and humans. Unlike fleas, lice are species-specific;

although rats may be infected with lice, those lice will not

cross over from one species of animal to another and if so; it

won't take long for it to realize this animal is not its food

source and will jumps onto a rat again (McArthur, 1999).

Hoplopleuro pacifica is the tropical rat louse

occurring on various species of rats throughout the world, it

is slender forms 1-2 mm in length with large paratergal plates

(Soulsby, 1982).

Polyplux spinulosa (the rat louse) is an anopluran

louse (Sucking louse) of rat causing restlessness, pruritus,

anaemia and debilitation in rats. Because lice are species-

specific, transmission to other animals or humans is not a

concern. P. spinulosa is a vector responsible for spread of

Haemobartonella muris (rickettsia, blood parasite) and

Rickettsia typhi between rats which may be passed to humans

via rat fleas (Hendrix, 1998; McArthur. 1999).

c. Mites

Mites are very important parasite on or in the skin,

the respiratory system or other organs of mammalian host.

Although some mites are not actually parasites of vertebrates,

they stimulate allergic reactions when they or their remains

23

come into contact with a susceptible individual (Bakr et al.,

1995).

Mites are temporally blood-sucking ectoparasites of

mammals (including rodents and human). Rat mites

frequently attack people living in rodent-infested-buildings.

Mites' bite may produce irritation, and sometimes painful

allergic dermatitis or mite respiratory allergy particulary in

children. This occurs especially in the absence of their natural

hosts. Rat mites are associated with groceries and warehouses

(Cook, 1997).

Animal in an environment infested with mites may be

anemic and exhibit a marked reproductive decline. The mite

can transmit rickettsial organisms in humans. Ornithonyssus

bacoti could transmit Yersinia pestis (the cause of plague),

Rickettsia typhi (the cause of murine typhus) and Coxrella

buinetii (the cause of Q fever). 0. bacoti is the intermediate

host of the filarial nematode of rodents Litomosoides

Allodermanyssus sanguineus may transmit Rickettsia akari

the cause of rickettsial pox of man (Hendrix, 1998).

Mites are transmitted to man by direct contact with an

infected animal, but also may arrive in contaminated bedding

or wood products (McArthur, 1999).

Rats may be infected with Radforia ensifera, the fur

mite of rats, which is not bloodsucker and is often endemic to

rats. Transmission between rats usually occurs by direct

contact. This species of mite is not known to infect humans

24

and it does not cause problems unless the infestation is heavy

or the rat is ill with another disease.

Burrowing mite of rats Notaedres inuitis is among the

ear mange mites. A skin scraping and a microscope are

needed to see these mites. They attack the ear pinnae, tail,

nose, and extremities. These mites are spread by direct

contact. Lesions caused by it are reddened crusty and itchy.

They may also infect other rodents, but are not known to

infect humans.

25

MATERIALS AND METHODS

1. Study Locations

Commensal Norway rats (Rattus norvegicus) were

collected from four governorates; Beni Suef (Wish-El-Bab

Village), Giza (El-Mansouria village), Qaliubiya (Tookh and

Beltan villages) and Behaira (El-Tayria village).

2. Collection and manipulation of rats

The study was carried out during the period from July

2012 to December 2013. Live Rats were captured using wire-

box traps of the usual spring-door type. Traps were distributed

in the evening at houses, poultry farms and drainage then

collected next morning. Bait materials were consisting of

tomato slices, fried fish or fried potato. Positive traps

provided with water using wet cotton and put in cloth bags

then transferred to laboratory for the study. The collected rats

were identified using the keys given by Arafa (1968) and

Osborn and Helmy (1980). Sex was determined by examining

the external genitalia of males and females and weight was

registered then a reference number was given to each

individual.

3. Examination of rats for endoparasites

a. Examination of intestinal parasites

The abdomen and chest of each rat were split opened

after killing. The lumen tract was then removed in one piece

26

and left in a separate petri-dish for some time in saline

solution to insure complete relaxation and easy removing of

the worm contents. Then it was slit opened in warm normal

saline. Freed helminthes if visible to the naked eyes were

picked out using a blunt forceps and transferred to petri-dishes

containing warm saline solution. The other contents were

evacuated into separate labeled jars full of water and were

taken thoroughly and left to sediment. The supernatant fluid

was decanted and the process of washing was repeated several

times with distilled water. Finally the sediment was placed in

a large petri-dish and examined for minute worms under a

stereomicroscope. Such worms were picked off using either a

wide mouthed pipette or a camel's hair-brush.

The mucous membrane of the stomach, on the other

hand, was examined under a dissecting stereomicroscope

utilizing a strong source of light of adherent worms and if

present could be picked out in warm normal saline.

Besides, careful searching for the smaller worms both in

the intestinal contents and scrapings of the mucosa was

carried out to extract the worms present inside.

Helminthes of large sized were easily spotted by the

naked eyes or by the aid of a hand lens. However, it should be

stated that some parasitic worms might have been missed due

to their minute size especially if they were scanty. In order to

27

overcome this difficulty, the mucosal surface of the

gastrointestinal tract was rubbed or lightly scraped to assure

complete transfer of worms to the container.

Worms were stirred vigorously for few minutes to allow

thorough relaxation, after which they were preserved in well

stoppered vials containing sufficient amount of glycerin-

alcohol (consists of 95 parts 70% alcohol and 5 parts glycerin)

and a label carrying the date, location and corresponding

serial number of each animal.

In the meantime the split opened abdomen and chest

were inspected for extra intestinal helminthes.

b. Examination of non-intestinal endoparasites

The liver, kidney, heart, lungs and reproductive organs

were inspected for cysts or worms which were then counted.

Particularly, liver was examined for cysts (e.g., Cysticercus

fascialaris) which dissected out and notched in warm normal

saline to free their worm contents.

c. Preparation of adult helminthes for examination

(according to Gardner et al., 1988)

1. Washing of adult helminthes

Before examining the worms, they were washed several

times in warm normal saline solution to separate them from

mucous and debris and to inspect their movement as

28

monitored while still living. Specimens preserved in glycerin-

alcohol were brought down to water (in descending grades of

alcohol 50% then 30% for 15 minutes each then to distilled

water several changes prior to staining).

2. Relaxation

By lifting the specimens in refrigerator for 2 h.

3. Fixation

Cestodes were roughly measured before being divided

into small pieces; head region, mature segments and gravid

segments and then gently compressed between two slides, and

fixed in 1% formalin for 24 h.

Nematodes were dropped in 70% hot alcohol (60°C)

then preserved in 70% alcohol containing 5% glycerine. For

studying the morphological feature of nematodes, they were

first cleared in lactophenol for 24h which was prepared from:

10 gm phenol, 10.6 ml glycerol, 8.2 ml lactic acid and 10 ml

distilled water. The worms were then mounted on glass slid

dipping in Canada balsam and left in an oven at 38°C to dry.

4. Staining

Cestodes were stained with acetic-acid alum carmine

formulated from: 20 gm. carmine, 25 ml acetic acid, 6 gm.

potassium alum and 100 ml distilled water.

29

The dye was boiled for an hour then cooled and the acid

was then added and left for ten days for maturation.

Thereafter, the solution was filtered. Working solution was 1

part of stock solution and 99 parts distilled water.

Half an hour was found sufficient to stain the

trematodes and small scolices of cestodes, while mature and

gravid segments were left for 2 hours. Helminthes were then

washed with water several times to remove the excess of the

stain.

5. Mounting

After staining, the specimens were dehydrated in

ascending grades of alcohol (30-50) % for half an hour each.

Destaining and differentiation of the over-stained specimens

were done in 1% acid alcohol (1 part of hydrochloric acid in

99 parts of 70% alcohol). The process was microscopically

checked until the specimens became well differentiated. The

specimens were then washed several times in 50% alcohol to

remove the residual hydrochloric acid. Specimens were then

dehydrated by passing through ascending grades of alcohol

70%, 95% and absolute alcohol half an hour each. Stained

specimens were then cleared in clove oil followed by two

washed of xylene. They were mounted in Canada balsam and

left in an oven at 38°C to dry for few weeks.

The detected helminth parasites were identified

according to Monib (1980) and El-Azzazy (1981).

31

4. Examination of rats for ectoparasites

a. Ectoparasites collecting

Rats skin with terminal parts of the four limps, tail and

head were put in modified tullgren funnel.

The ectoparasites received in petri dish filled with 70%

alcohol, were picked up with a moistened camel's hair brush

with the aid of a strong source of light. Then, the ectoparasites

were dropped in separate vials containing 70% alcohol and a

label comprising both the date, location and the corresponding

serial code number of each animal.

b. Ectoparasites' preparation, mounting and

identification

Arthropod ectoparasites preserved in 70% alcohol were

brought down to water in descending grades of alcohol 50-

30% 15 minutes each.

Fleas and lice were then removed to 10% potassium

hydroxide or lactophenol after puncturing the specimens on

the ventral side, and then left overnight until soft parts were

dissolve. The material was washed thoroughly in distilled

water slightly acidified with 10 drops of acetic acid to remove

the alkali and then treated with ascending grades of alcohol -

50%, 70%, 90% and 95% - 20 minutes each.

30

The individuals were then cleared in clove oil for 10

minutes. Mounting was performed in Canada balsam then left

to dry in oven at 38°C.

Mites, on the other hand, were mounted from 70%

alcohol after cleaning in water into Hoyer's medium.

Fleas species recorded were identified according to the

key given by Soulsby (1982), lice were identified according to

the key given by Johnson (1960) and mites were identified

according to Krantz (1978).

32

33

RESULTS AND DISCUSSIONS

1. Rattus norvegicus investigations

Rattus norvegicus was collected from four governorates:

Giza, Beheira, Beni Suef and Qaliubiya . the structure of its

population was studied, the whole number of Rattus

norvegicus live trapped was 83; 34 from Giza, 24 from

Beheira, 10 from Beni Suef and 15 from Qaliubiya .

Table 1. Rattus norvegicus population structure

Gov. No. Males' No. Females' No.

Mature Immature Total Mature Immature Total

Giza 34 12 7 19 10 5 15

Beheira 24 9 5 14 6 4 10

Bani-Suef 10 4 1 5 3 2 5

Qaliubiya 15 6 4 10 3 2 5

Total 83 31 17 48 22 13 35

Based on sex, the Norway rat population was consisted

of 48 male individuals and 35 female individuals. The male to

female ratio (sex ratio) was 1.37:1. The maturity status was

obtained, therefore, the population was divided into mature

individuals (53) and immature individuals (30), table (1).

This result showed that males' number is bigger than

females', and the reason behind may be that females stay in

borrows to lactate and to take care of offspring or to avoid the

harsh weather conditions during pregnancy and after giving

birth. While, on the other hand, males don‘t have all these

34

constrains; they usually explore and roam more than females.

This result is in accordance with that obtained by El-Bahrawy

and Al-Dakhil (1993) but it is in discrepancy with that of Soliman

et al. (2001b).

2. Parasites of R. norvegicus recorded

Rodents play an important role as hosts of parasites and

reservoirs of many zoonotic diseases. A total of twelve species

of parasites were found of which 11 were zoonotic including,

two Cestodes (Hymenolepis diminuta and Cysticercus

fasciolaris), three fleas (Xenopsylla cheopis, Echidnophaga

gallinacea and Ctenocephalides felis), two sucking lice (Hoplopleura

oenomydis and Polyplax spinulosa) and four mites (Ornithonussus

bacoti, Lealaps nuttalli, Liponyssoides sanguineus and Radfordia

ensifera).

a. Endoparasites

Indoarasites of rats, particularly helminthes, are

belonging to the four major groups; Nematoda, Cestoda,

Trematoda and Acanthocephala; Cestode and nematode

parasites in rat have been reported from all parts of the world.

In this study, we have just recorded two cestodes:

Hymenolepis diminuta and Cysticercus fasciolaris, which are

commonly found in rats and mice and they are potentially

transmissible (Zoonosis) to man and one non-zoonosis

nematode, Spirura talpae.

35

Helmenthic parasites pose a major part of this study, as

65 individuals out of 83 rats were infected with one or more

helminthic parasites with an infection rate of 78.31 %. This

rate of infection among small mammals is slightly higher than

that obtained by Arafa (1968) and Monib (1980). A reasonable

explanations for that could be the contact increase between

man and rats in recent years or the environmental

contamination increase or even the climatic changes that

favour parasitic transmission. These findings are in harmony

with those showing that wild small rodents rarely remain

uninfected (Behnke et al., 2001). Also, the high prevalence of

infection with helminthic parasites in the Norway rats might

be attributed to its high reproductive activity, high population

density and its omnivorous way of nutrition (Hrgović et al.,

1991).

1. Types of Infection of endoparasites

The type of infection of helminthic parasites varies

among individuals. Some individuals were infected with only

one helminthic parasite, 27 individuals (32.5%) and some

were double infected, 32 individuals (38.5%) while triple

infection was recorded in just 6 individuals (7.2%), table (2).

In a similar study of endoparasites of Norway rat, Rezan et al.

(2012) stated that Single parasitic infection was the highest

(52%), followed by double infection, 16%, and two cases of

triple infection (8%). No more than four helminthic species

36

were found in one host (Kataranovski, 2011).

Table 2. Types of Infection of endoparasites

Gov. Single (%) Double (%) Triple (%)

Giza 11 13 2

Beheira 8 9 2

Bani-Suef 3 5 1

Qaliubiya 5 5 1

Total 27 (32.5%) 32 (38.5%) 6 (7.2%)

2. Endoparasite species recorded

Three different species of helminthic parasites were

recorded in Rattus norvegicus examined from different

locations comprising tow cestodes , Hyminolipis diminuta and

Cysticercus faciularis, and one nematode Spirura talpae. No

new species were recorded in the given areas of this study.

a. Cysticercus fasciolaris:

Cysticercus fasciolaris is a larval and cystic stage of

Taenia taeniaeformis and it is a feline tapeworm. The

intermediate hosts of T. taeniaeformis are mouse, rat, cat,

muskrat, squirrel, rabbit, other rodent, bat, and human that

may catch the infection through contaminated water or feed

materials with infected cat faeces (Al-Jashamy, 2010).

The C. fasciolaris was found in the liver of Rattus

norvegicus in the form of whitish prominent single to multiple

parasitic cysts. The sizes of the cysts varied from 4 to 12 mm

in diameter. Each cyst contained a single live characteristic

strobilocercus larva. Mature C. fasciolaris showed obvious

37

scolex, long neck and pseudo-segmentation, larva revealed

armed rostellum characterized by double rows of hooks and

four suckers which were clearly obvious, Fig. 1.

Fig. 1. Cysticercus fasciolaris; (A) Rat's Liver (3x) showing

pea sized cyst (B) Strobilocercus larvae of Taeniae

taeniaeformes (100x) with rostellum armed with double row of

hooks.

Hymenolepis diminuta:

H. diminuta (Fig. 2) is a cosmopolitan worm that is

primarily parasite of rats. It has been reported in different

parts of the world including Kuwait (Zakaria and Zaghloul,

1982), Great Britain (Webster and Macdonald, 1995), Croatia

(StojĈeviĆ et al., 2004), Qatar (Abu Madi et al., 2005),

Argentina (Gomez-Villafañe et al., 2008) and Kuala Lumpur,

Malesia, Southeastern Asia (Paramasvaran et al., 2009). H.

diminuta parasites mainly in the upper middle part of the

small intestine.

38

Fig. 2. Hymenolepis diminuta from R. rattus intestine (A)

Unarmed scolex (100x) (B) Maturing proglottids (100x) (C)

Maturing proglottids (400x) with a median ovary and three

testes. (C) Gravid segments (400x) (E) Eggs teased apart from

gravid segments.

b. Spirura talpae

S. talpae is the only nematode species found during this

study. It was picked from the stomach where it was parasiting

with capacity of 1-4 larvae. According To Gbif Backbone

Taxonomy S. talpae is classified as follows:

Kingdom: Animalia

39

Phylum: Nematoda

Class: Secernentea

Order: Spirurida Chitwood, 1933

Family: Spiruridae Örley, 1885

Genus: Spirura Blanchard, 1849

Fig. 3. Spirura talpae; (A) Anterior end of male (100x),

(B)Posterior end of male (100x), (C) Anterior end of female

(100x), (D) posterior end of female (100x).

41

3. Infection prevalence based on host location

Location of infestation may affect the infection prevalence.

However, in this study, the infection percentage does not considerably

differ among locations.

There was no tangible difference among the cestodes

infection percentages in three locations, as it was 70.59%,

73.33% and 75% at Giza, Qaliubiya and Beheira

governorates; respectively, but at Bani-Suef, it was higher

(90%). Likewise, the nematode infection percentages were

41.18%, 33.33% and 40% at Giza, Beheira and Qaliubiya;

respectively, and it was slightly greater at Beni Suef (50%).

The combined infection percentages of both cestodes and

nematodes exhibited the same pattern, table (3). These results

could be supported with that obtained by Allymehr et al.,

(2012) who stated that the rate of rodent infection with

nematodes and cestodes differs among locations.

Table 3. Endoparasites Infection prevalence at study locations

Governorate Cestodes Nematodes Total

Endoparasites

Total

infected

No.

Infection

%

Total

infected

No.

Infection

%

Total

infected

No.

Infection

%

Giza 24 70.59 14 41.18 26 76.47

Beheira 18 75 8 33.33 19 79.17

Bani-Suef 9 90 5 50 9 90

Qaliubiya 11 73.33 6 40 11 73.33

40

4. Infection prevalence based on host sex

Sex is an internal host factors that may have impact of

intestinal helminth fauna of Norway rats. Sex-related

differences were noted in the prevalence of infection with

some endoparasites, e.g., Capillaria sp. and Trichuris muris,

was higher in males than in females, (kataranovski et al.,

2011).

Both Rattus norvegicus sexes were examined for their

endoparasites. Regarding cestodes, males were more infected

than females as 39/(83) males were infected (46.99%) versus

23/(83) females (27.71%). The prevalence percentage on

males was 81.25% (the percentage of males infected out of the

total number of males) while, it was 65.71% on females. This

indicates that the rate of the infection prevalence on males is

greater than that on females. Similarly, nematodes infection

was greater on males, 20 (24.1%) than that on female, 13

(15.66%). But the prevalence of infection of male's population

was close to that of female's; 41.67% for male's and 37.14%

for female's; respectively, table (4).

Such conclude is in concurrence with that found by

Abu-Madi et al., (2005) who maintain that the abundance of

infection and worm burdens were affected with the sex of the

host. They stated that "the worm burdens in adult rats were

almost twice as heavy in males compared with females".

42

42

Table 4. Infection prevalence of endoparasites based on host sex

Governorate

Males Females

Infected

males'

No.

Infected

males'

%

Infection

prevalence

%

Infected

females'

No.

Infected

females'

%

Infection

prevalence

%

Cestodes

Giza 15 44.12 78.95 9 26.47 60.00

Beheira 11 45.83 78.57 7 29.17 80.00

Bani-Suef 5 50.00 100.00 4 40.00 80.00

Qaliubiya 8 53.33 80.00 3 20.00 60.00

Total 39 46.99 81.25 23 27.71 65.71

Nematodes

Giza 7 20.59 36.84 7 20.59 46.67

Beheira 5 20.83 35.71 3 12.50 30.00

Bani-Suef 3 30.00 60.00 2 20.00 40.00

Qaliubiya 5 33.33 50.00 1 6.67 20.00

Total 20 24.10 41.67 13 15.66 37.14

43

Also, These results are in solidarity with those gained

by (Udonsi,1998; Kataranovski et al., 2011). Taking into

consideration the fact that infected males have larger home

range than uninfected males and that the home range of males

tend to overlap which could increase their chance to

disseminate the infection and to increase the exposure by

uninfected rats (Brown et al., 1994) while reproductive

females show a stronger site-specific organization which

could explain low rates of transmission (kataranovski et al.,

2011), we can come up with an acceptable justification of the

high rate of prevalence of helminthic infection of males

compared with females. Brown et al., (1994),

correspondingly, proposed that the infected rodents move

more often and faster than uninfected rodents which proved an

over spread distribution.

Also, the adverse impact of the male hormone

(testosterone) on immune defense functions may represent a

greater tendency of males for helminthic infection (Folstad

and Karter, 1992). In the same way, Udonsi (1998) suggested

that increased estrogen level in females may increase

resistance to infection.

On the contrary, Nur-syazana et al., (2013) and Viljoen

et al., (2011) have different point of view, they claim that sex

and reproductive status contribute little to the parasite

prevalence and abundances or have no influence on the macro-

44

parasites community structure as both sexes share the same

burrow system.

5. Infection prevalence based on host age

In many reported studies, both abundance and

prevalence of infestation of endoparasites are host age

dependent.

In this study, 44 individuals out of 83 (53.01%) were

cestode infected mature and the infected immature individuals

were only 18 (21.69%). The prevalence of infestation among

mature individuals was greater than that among immature

individuals as 83.02% of mature individuals were infected

versus 60% of immature individuals.

As to nematode infection, 28 out of 83 (33.73%) were

infected mature individuals while 5 (6.02%) individuals were

infected immature. The prevalence of nematode infection

among mature individuals was 52.83% but it was only 16.67%

among immature individuals.

These outcomes are in harmony with those of Abu-Madi

et al., (2005) that The abundance of infection and prevalence

of H. diminuta was influenced by the host age. Adults of both

sexes harbored heavier infection than juveniles. Reasons for

this may lie behind the fact that older rats have a longer

exposure time to potential infection (Easterbrook et al., 2007).

45

45

Table 5. Infection prevalence of Endoparasites in mature and immature rats

Mature Immature

Governorate

Infected

mature

(No.)

Infected

mature

(%)

Infection

prevalence

(%)

Infected

immature

(No.)

Infected

immature

(%)

Infection

prevalence

(%)

Cestodes

Giza 16 47.06 72.73 8 23.53 66.67

Beheira 14 58.33 93.33 4 16.67 44.44

Bani-Suef 6 60.00 87.71 3 30.00 100.00

Qaliubiya 8 53.33 88.89 3 20.00 50.00

Total 44 53.01 83.02 18 21.69 60.00

Nematodes

Giza 12 35.29 54.55 2 5.88 16.67

Beheira 7 29.17 46.67 1 4.17 11.11

Bani-Suef 3 30.00 42.86 2 20.00 66.67

Qaliubiya 6 40.00 66.67 0 0.00 0.00

Total 28 33.73 52.83 5 6.02 16.67

46

In contrast, this result contradicts that revealed by

Udonsi, (1998) who justified his findings that juveniles or

immature individuals have a greater need for food materials

necessary for growth which containing infective parasite

stages while they are still immunologically naïve. This is in

line with Nur-syazana et al., (2013) who indicated that neither

intrinsic (host age, host sex) nor extrinsic (season) factors

influenced the macro-parasites community structure.

b. Ectoparasites

Rodents in particularly, Rattus norvegicus are usually

infected by certain groups of arthropods; fleas, lice and mites.

In this study 77.2% of Rattus norvegicus were infested with at

least one ectoparasite. This high rate of infestation could be

supported by the relatively small home range of the Norway

rat in addition to its neighborhood to domestic animals which

might pose an important source of infestation.

1. Ectoparasite species recorded in this study

Results of our study revealed that 938 ectoparasites,

comprising:140 (14.93%) fleas, 234 (24.95%) lice and 564 (60.1%)

mites, (Fig. 4), are belong to 4 orders, 7 families, 9 genera and 9

species, Fig. 5. Ectoparasite species collected from 83 individuals of

live trapped Rattus norvegicus include:

47

Fleas (Insecta: Siphonaptera)

Pulicidae: Xenopsylla cheopis,

Echidnophaga gallinacea

Ctenocephalides felis

Lice (Insecta: Anoplura)

Hoplopleuridae: Hoplopleura oenomydis

Polyplacidae: Polyplax spinulosa

Mites (Acari: Mesostigmata)

Macronyssidae: Ornithonyssus bacoti

Laelapidae: Laelaps nuttalli

Dermanyssidae: Liponyssoides sanguineus

(Acari: Prostigmata)

Myobiidae: Radfordia ensifera

Fig. 4. Relative frequency of ectoparasites groups

48

F

ig. 5

. Arth

rop

od

e ecto

para

sites record

ed o

n R

attu

s norveg

icus. (1

)Xen

opsylla

cheo

pis, (2

) Cten

ocep

halid

es

felis, (3) E

chid

noph

aga g

allin

acea

, (4) P

olyp

lax sp

inu

losa

, (5) H

oplo

pleu

ra o

enom

ydis, (6

) Lip

on

yssoid

es

san

gu

ineu

s, (7) L

aela

ps n

utta

lli, (8) O

rnith

on

yssus b

aco

ti an

d (9

) Radfo

rdia

ensifera

49

2. Infection prevalence and general indices of ectoparasite

according to location

Location of infestation could be a key factor of infection

prevalence. In this study, the numbers of infected individuals vary

among locations and the different ectoparasites general indices as well.

Regarding to the flea infection, Giza governorate had

the highest infection percentage (50%) and the highest flea

index as well (2.56). on the other side, Beni Suef had the

lowest flea infection percentage (20%) and the lowest flea

index (0.5).

Lice and lice index had a certain pattern which is

different from that in fleas. Although Beni Suef governorate

had the highest lice infection percentage (50%), Giza

governorate had the highest lice index (3.76). this means that

the lice burden is higher in Giza than that in the other three

locations. In the same context, Beheira governorate had the

lowest lice infection percentage (25%), but its lice index

(2.46) is bigger than that of Beni Suef (2.1) and Qaliubiya

(1.73) table (6).

With regard to mite infection, Beni Suef governorate

came first (70%) followed by Beheira governorate(66.67%)

while Qaliubiya had the lowest percentage of infection

(40%). Mite indices were relatively high; since it ranged from

4.27 in Qaliubiya governorate to 11.3 in Beheira governorate,

table (6).

51

Table 6. Infection prevalence and general indices of

ectoparasite according to location

Flea Lice Mite

Gov. Total

infected

No, (%)

General

flea index

Total

infected

No, (%)

General

Lice

index

Total

infected

No, (%)

General

Mite

index

Giza 17, (50) 2.56 9, (26.47) 3.76 17, (50) 5.26

Beheira 6, (25) 1.16 6, (25) 2.46 16, (66.67) 11.3

Bani-Suef 2, (20) 0.5 5, (50) 2.1 7, (70) 5

Qalyobya 4, (26.67) 1.4 4, (26.67) 1.73 6, (40) 4.27

From the aforementioned data, it is obvious that the rate

and the indices of infestation of different ectoparasites vary

from one location to another. These findings are in accordance

with El Deeb et al., (1999) and Soliman et al., (2001b) that

the distribution of ectoparasites varied according to rodent

host and location. Also, Kia et al., (2009) stated "the

infestation rate to different ectoparasite depend on season,

size of rodents, host preference, sex of host, host age, location

of capture and co-evolution between rodent and

ectoparasites". Similarly, Telmadarraiy et al., (2007)

mentioned the Infection prevalence and general indices of

ectoparasite mainly depend on season, rodent species,

ectoparasite species, location, method of catch, and host

population dynamics. For instance, The indices of infestation

by the mites Laelaps nuttalli, the louse Polyplax spinulosa and

the flea Xenonpsylla cheopis, on Rattus norvegicus in Brazil

50

were related to seasonal period, sex of the host and area of

capture (Linardi et al., 1985).

In my point of view, location is the key factor affects

the Infection prevalence and general indices of ectoparasite

because location change involves many criteria like

geographical situation, ecological condition, rodent predators,

seasonal activities, human practices and sources of infection

that influence the ectoparasite prevalence and indices

3. Infection prevalence based on host sex

Rattus norvegicus population was divided into males and

females to find out if there is a variation of the infection prevalence of

different ectoparasites between both rat sexes. The male infection

prevalence percentage calculated as the percent of infected males'

number to the whole males' population.

Respecting fleas' infection, 19 infected male individuals

(22.89%) represented 39.58% of the whole males' population

(male infection prevalence percentage). Infected females were

10 individuals with a percentage of 12.05%. The prevalence of

infection among females was 28.57%. There no fleas were

recorded on females in Beni Suef (0%), while Giza was the

highest in both infected males and infected females

percentages, (29.41% and 22.59%); respectively. Also, the

flea infection prevalence was the uppermost in Giza since it

was 52.63% among males and 46.67% among females.

52

Table 7. Infection prevalence of the ectoparasites on both male and female hosts

Males Females

GOV Infected

males' No.

Infected

males' % Infection

prevalence%

Infected

females' No.

Infected

females' % Infection

prevalence%

Fleas

Giza 10 29.41 52.63 7 20.59 46.67

Beheira 4 16.67 28.57 2 8.33 20.00

Bani-Suef 2 20.00 40.00 0 0.00 0.00

Qaliubiya 3 20.00 30.00 1 6.67 20.00

Total 19 22.89 39.58 10 12.05 28.57

Lice

Giza 6 17.65 31.58 3 8.82 20.00

Beheira 2 8.33 14.29 4 16.67 40.00

Bani-Suef 1 10.00 20.00 4 40.00 80.00

Qaliubiya 3 20.00 30.00 1 6.67 20.00

Total 12 14.46 25.00 12 14.46 34.29

Mites

Giza 10 29.41 52.64 7 20.59 46.67

Beheira 9 37.50 64.29 7 29.17 70.00

Bani-Suef 4 40.00 80.00 3 30.00 60.00

Qaliubiya 5 33.33 50.00 1 6.67 20.00

Total 28 33.73 58.33 18 21.69 51.43

53

Regarding lice infection, a total of 12 male-individuals

(out of 83, the whole population) were infected with a

percentage of 14.46%. The infection prevalence among them

was 25% (12 out of 48 males). Infected females' number was

equal to that of males' (12, 14.46%) but the infection

prevalence among females (34.28%) was greater than that

among males.

Mite infection and prevalence was the greatest

comparing to other ectoparasites as 28 males (33.73%) and 18

females (21.69%) were infected. Also the prevalence of

infection among males (52.33%) and females (51.43%) was

the highest when compared with fleas and lice. There were no

differences of infection prevalence based on host sex.

General indices of ectoparasites based on host sex

Ectoparasites indices were calculated for both sexes for

determining if there is a relationship between the host sex and

the parasites' burden.

The flea index in males is bigger than that in females in

all governorates except for Giza but the total flea indices in

both males and females are equal (1.69). There was a big

difference between the male and female lice indices in Beheira

and Beni Suef governorates as they were 0.86 / 4.7 and 0.6 /

3.6; respectively, but the total lice index in males (2.85) was

almost bigger than that in females (2.77). With regard to mite,

54

the total mite index was approximately bigger in males than it

in females. But still there were some differences according to

locations, Table (8).

Table 8. General indices of ectoparasite based on host sex

Gov. Flea index Lice index Mite index

Male Female Male Female Male Female

Giza 2.26 2.93 4.42 2.93 6 4.33

Beheira 1.2 1 0.86 4.7 11.7 11.6

Bani-Suef 1 0 0.6 3.6 4.4 5.6

Qalyobya 1.6 1 2 1.2 6 0.8

Total 1.69 1.69 2.85 2.77 7.31 6.09

Overall outcome reflects that no host sex-associated

differences in the prevalence of infection were found for

ectoparasites. This result is in agreement with Nur-Syazana et

al., (2013) who did not find any strong independent effects of

host sex on the prevalence of ectoparasites although more

females were observed infested compared to males. But, at the

same time, this result contradicts the findings of Linardi et al.,

(1985), Botelho and Linardi (1994) and Kia et al., (2009) that

the ectoparasites preferentially infested male rodents, both in

wild and urban environments.

4. Infection prevalence based on host age:

We divided the host population into two groups, mature and

immature, to study the effect of the age on the infection prevalence of

ectoparasites.

55

Table 9. Infection prevalence of ectoparasite on mature and immature individuals

GOV Mature Immature

Infected

Mature No.

Infected

Mature %

Infection

prevalence%

Infected

Immature

No.

infected

Immature

%

Infection

prevalence %

Flea

Giza 12 35.29 54.55 5 14.71 41.67

Beheira 3 12.50 20.00 3 12.50 33.33

Bani-Suef 2 20.00 28.57 0 0.00 0.00

Qaliubiya 3 20.00 33.33 1 6.67 16.67

Total 20 24.10 37.74 9 10.84 30.00

Lice

Giza 9 26.47 40.91 0 0.00 0.00

Beheira 5 20.83 33.33 1 4.17 11.11

Bani-Suef 2 20.00 28.57 3 30.00 100.00

Qaliubiya 2 13.33 22.22 2 13.33 33.33

Total 18 21.69 33.96 6 7.23 20.00

Mite

Giza 14 41.18 63.64 3 8.82 25.00

Beheira 12 50.00 80.00 4 16.67 44.44

Bani-Suef 5 50.00 71.43 2 20.00 66.67

Qaliubiya 2 13.33 22.22 4 26.67 66.67

Total 33 39.76 62.26 13 15.66 43.33

56

A total of 20 (24.1%) mature individuals versus 9

(10.84%) immature individuals were infected with fleas. The

flea infection prevalence inside the mature population was

37.74% which was relatively higher than that inside the

immature population (30%). It means that mature individuals

are likely to be infected than immature individuals. Also, the

infection prevalence is likely to be the different between

mature and immature individuals with a slight tendency to be

higher in mature individuals.

Lice infection varied between mature and immature rats,

as a total of 18 mature individuals (21.69%) and 6 immature

individuals (7.23%) were infected. The infection prevalence of

lice inside the mature population (33.96%) was higher than

that inside immature population (20%). It is clear that

immature individuals are less likely to be infected.

Unlike fleas and lice, mite infection was higher and

more prevalent; as 33 mature individuals (39.76%) and 13

immature individuals (15.66%) were infected. When

comparing the infection prevalence between mature and

immature individuals, it was found that the infection

prevalence in mature individuals (62.26%) was greater than it

in immature individuals (43.33%).

57

General indices of ectoparasite based on host age:

General indices of the three main groups of arthropod

ectoparasites, fleas, lice and mites, were conducted for each age

stage as follows:

Generally, mature individuals tend to have bigger

ectoparasite index than immature individuals. As to flea index,

it was 1.96 in mature individuals versus 1.2 in immature

individuals, also lice index in mature individuals was three

times bigger (3.75) than it in immature individuals (1.17).

Likewise, the mite index was bigger in mature individuals

(7.15) than it in immature individuals (6.17). So it is

predictable for us to record high infection and high prevalence

of ectoparasite in mature individuals, while it tends to be low

in immature individuals, Table (10).

Table 10. General indices of ectoparasite based on host age

Gov. Flea index Lice index Mite index

Mature Immature Mature Immature Mature Immature

Giza 3.14 1.5 5.82 0 7.32 1.5

Beheira 1.27 0.89 3.27 1.1 14.53 5.89

Bani-Suef 0.43 0 1.7 3 2.85 9

Qalyobya 2.1 0.33 2 1.33 4.33 4.17

Total 1.96 1.20 3.75 1.17 7.15 6.17

Age is one of the key elements of a rodent host that may

affect the foraging choices of ectoparasites. The increased

prevalence and general infestation index of ectoparasites are

positively correlated to the increased densities of their hosts

(Anderson and Gordon, 1982). Randolph (1975); Thompson et

58

al., (1998) and Kia et al., (2009) stated that the catch rate and

infestation rate of different ectoparasite depend on host age.

Many important parameters in host–parasite dynamics, such as

infestation level of hosts and the consequent parasite

distribution among host individuals are often age-dependent

(Anderson and Gordon, 1982; Hudson and Dobson, 1997)

Juvenile rodents have larger surface to volume ratio and

thus, higher energy requirements for maintenance per unit

body mass (Kleiber, 1975). They also require additional

energy for somatic growth, maturation, and for mounting an

immune response. Thus, adult rodents under field conditions

usually represent a better nutritional resource than juveniles

(Buxton, 1984). Also, adult hosts show higher infestation

levels than juveniles because they have more time to

accumulate parasites (Hawlena et al., 2006).

59

Part II: Resistance of Rattus norvegicus to

warfarin, the first generation anticoagulant

INTRODUCTION

The best-known anticoagulant agent, warfarin, was

developed in the 1940s. Today, warfarin is used as a

rodenticide. It is added to grain meal in low concentrations

(usually between 0.005% and 0.1%) making the poisoned bait

product relatively safe for humans to handle. Warfarin causes

a slow death by gradual acting of internal bleeding. Within a

decade of the introduction of warfarin as a rodenticide, rats

and mice resistant to the poison were discovered. Among the

first resistant species described were Norway rats (Rattus

norvegicus), ship rats (R. rattus) and house mice (Mus

musculus). These initial discoveries were made in rural areas

of the United Kingdom and in other locations, not only in

Europe, but also in the United States, Asia, and Australia.

Decade ago, VKORC1 (vitamin K epoxide reductase

complex subunit 1), the target enzyme for coumarins, was

identified. VKORC1 is a key component of the vitamin K cycle

that reduces vitamin K epoxide and at the same time is

inhibited by warfarin. It was shown that mutations in VKORC1

confer resistance to anticoagulants of the Coumarin-type in

humans and rodents.

61

Because rodents carrying resistance mutations survive

poisoning, they are selected for survival in areas where

anticoagulant rodenticides are used. Genetic mutations

conferring resistance to anticoagulant rodenticides were

identified in both rats and mice. In rats and mice independent

mutations have arisen in different warfarin-resistance areas

throughout the world and affect different amino acid positions

of the VKORC1protein.

According to Rost et al., (2004), mutations in VKORC1

may cause a heritable resistance to warfarin, possibly by

preventing coumarin derivatives from interfering with the

activity of the reductase enzyme. So, resistance against

warfarin-like compounds poses a considerable problem for

efficacy of pest control.

60

REVIEW OF LITERATURE

1. Anticoagulant rodenticides

Control of Rattus norvegicus (Norway rat) depends

mainly on toxicants, either acute or chronic rodenticides to get

rid of its harmful in fields, houses and stores, and to

manipulate the diseases they carry. Acute poisons were used

for ages before the discovery of warfarin. It is well known that

R. norvegicus is very neophobic (being unfamiliar to new

items in their environment). Neophobic rats may eat a small

non-lethal dose of new bait. Survived rats learn to avoid the

bait that may consequently cause problems concerning the

rodenticides (Baert, 2012)

Anticoagulant rodenticides were first discovered in the

1940 s and have since become the most widely used toxicants

for commensal rodent control due to their convenience, safety,

and minimal impact on the environment. This new group of

rodenticides have been introduced as an alternative of acute

toxicants. Warfarin and related anticoagulant compounds

(coumarins) were massively used in the early 60s‘, and they

were a great choice to reduce or eradicate rat populations from

many area. Poisoned rodents die from internal bleeding as a

result of loss of the blood's clotting ability. Prior to death, the

animal exhibits increasing weakness due to blood loss.

anticoagulant baits are slow in action (several days following

62

the ingestion of a lethal dose), the target animal is unable to

associate its illness with the bait eaten. Therefore, bait

shyness does not occur. This delayed action also has a safety

advantage because it provides time to administer the antidote

(vitamin K1) to save pets, livestock, and people who may have

accidentally ingested the bait (Pelz et al., 2005)

There are two generations of anticoagulants; the first

generation anticoagulants: or multiple-feed rodenticides

(warfarin, pindone, diphacinone and clorophacinone). These

compounds are chronic in their action, requiring multiple

feedings over several days to a week or more to produce

death. First generation rodenticidal anticoagulants generally

have shorter elimination half-lives, require higher

concentrations (usually between 0.005% and 0.1%) and

consecutive intake over days in order to accumulate the lethal

dose, and less toxic than second generation agents. On the

other hand, second generation agents are far more toxic than

first generation. They are generally applied in lower

concentrations in baits — usually on the order of 0.001% to

0.005%. They are lethal after a single ingestion of bait and are

also effective against strains of rodents that became resistant

to first generation anticoagulants; thus, the second generation

anticoagulants are sometimes referred to as "superwarfarins.

63

a. Anticoagulant rodenticide

Where anticoagulants have been used over long periods

of time at a particular location, there is an increased potential

for a population to become somewhat resistant to the lethal

effects of the baits. Resistance to warfarin was first observed

in Scotland in 1958 (Boyle, 1960). Since then, resistant rats

have been reported all over the world, in Great Britain,

Denmark, Germany, Belgium, Finland and France, the USA,

Canada, Australia, and Japan (Mayumi et al., 2008). Warfarin-

resistance has led to failure of their control using warfarin as a

rodenticide. Rats and mice that are resistant to warfarin also

show some resistance to all first generation anticoagulants,

rendering control with these compounds less effective.

Bailey and Eason (2000) stated that resistance to

anticoagulants can develop in a population after 5-10 years

sustained use of anticoagulant rodenticides. No enough data

are existed on the baseline susceptibility of rodent populations

in Egypt to anticoagulants or their changing patterns of

susceptibility in areas of sustained use. Monitoring systems

for wild target populations and changes to poisoning methods

will assist Egypt rodent control groups in avoiding the

resistance-induced control problems now seen outside Egypt.

Sustained control of rodents on the mainland is likely to be

substantially dependent on toxicants and anticoagulant

poisons in particular for the foreseeable future.

64

b. Mode and site of action of anticoagulants

Coumarins act as a vitamin K antagonist and block the

vitamin K cycle in the liver, preventing the reduction of

vitamin K epoxide to vitamin K by vitamin K epoxide

reductase (VKOR). Vitamin K is an essential co-factor in the

activation of several vitamin K-dependant coagulation factors

through which it plays an important role in blood coagulation.

When coumarins bind with VKOR, intoxication with

anticoagulants will lead to a deficiency of vitamin K and

coagulation factors, causing coagulation disorders such as

spontaneous bleeding and eventually death (OldenBurG et al.,

2008).

Anticoagulants act by interfering with the synthesis of

prothrombin, disturbing the normal clotting mechanisms and

causing an increased tendency to bleed.

The anticoagulant action of rodenticides arises from

inhibition of vitamin K metabolism in the liver. Vitamin K is

essential for the production of several blood-clotting proteins

and, when greatly reduced in concentration, results in fatal

hemorrhaging. Vitamin K in its reduced form (vitamin K

hydroquinone) is a co-factor for a carboxylase active in the

production of proteins such as clotting factors II,VII, IX, and

X. During this process, vitamin K is oxidised to vitamin KO

and is then unavailable until recycled to vitamin K

hydroquinone by the enzyme vitamin K epoxide reductase

65

(VKOR). It is this enzyme that is inactivated by the action of

anticoagulants, which have a similar structure to vitamin K

and bind strongly to the enzyme, leaving it unavailable for the

recycling of vitamin KO (Oldenburg et al., 2000).

All anticoagulants work by inhibiting the generation of

an active form of vitamin K1 via inhibition of vitamin K1

epoxide reductase. The presence of vitamin K as a cofactor is

required to the activation of clotting factors II, VII, IX, and X.

The VKORC1 gene produces the enzyme vitamin K1 epoxide

reductase, an essential enzyme in the vitamin K cycle and the

one blocked by all anticoagulant rodenticides (Buckle, 1994)

Anticoagulants can inhibit two different enzymes of the

vitamine K cycle: the epoxyde reductase and the vitamine K

reductase (although some scientists consider these two

enzymes are, in fact a single protein). The epoxide reductase

is the rate-limiting step and inhibition by anticoagulants will

result in the accumulation of Vitamine K epoxide, which is

not active. The second step is not as critical, since other

pathways may lead to the activation of vitamine K, such as the

diaphorases. Inhibition of this vitamin K cycle results in a

decreased production of active coagulation factors which, in

turn, will result in coagulation disorders and hemorrhages

(Berny, 2011).

66

2. Vitamin K and blood coagulation

Vitamin K1 is found mainly in green leafy vegetables

such as kale, spinach, and broccoli while vitaminK2 is found

in liver, milk, cheese, and fermented soy products such as

Natto. Menadione is a chemically synthesized derivative used

for animal feed.

a. The role of Vitamin K on blood coagulation

Synthesis of prothrombin, factors VII, IX and X are

dependent on vitamin K. besides, three other proteins are

vitamin K-dependent, in addition to other non-plasma

proteins. calcium ions are essential for activation of all these

proteins. The characteristic feature of the vitamin K-

dependent proteins is that they contain a modified glutamic

residue which has an extra carboxy-group attached to the γ-

carbon. This carboxy-group is added at a post-translational

vitamin K-dependent process (Mayumi et al., 2008).

Calcium ions are required as a co-factor for the action

of all the vitamin K-dependent proteins and the γ-

carboxyglutamic acid residues form the high affinity calcium

binding sites in these proteins (Jackson, 1972). As mentioned

above, γ-carboxyglutamic acid is formed by a vitamin K-

dependent process. The carboxylation of the specific glutamic

acid residues in the N-terminal regions of these proteins

occurs as a post translational event, and unlike other

67

biological carboxylation reactions, there is no dependence on

biotin or high energy phosphate. The only requirements are

reduced vitamin K, molecular oxgen and carbon dioxide and

an enzyme present in liver microsomes (Suttie, 1985).

The mechanism by which the carbon is activated and

transferred to the γ-carbon is not fully understood. However,

it is thought that the reduced vitamin reacts with molecular

oxygen to form a peroxy intermediate (a peroxy radical)

which then reacts with carbon dioxide to form a

peroxycarbonate adduct of the vitamin which decomposes to

carboxlate the glutamic acid residues in the presence of the

carboxlase1 and the vitamin is converted to the epoxide.

Under physiological conditions, the epoxide is converted back

to the reduced from through the vitamin cycle (Olson et al.,

1984; Suttie, 1985).

b. Vitamin K cycle, site of action and target molecule

of warfarin

Vitamin K functions as a cofactor for the γ-carboxylase,

an enzyme that resides in the endoplasmic reticulum (ER)

membrane and participates in posttranslational γ-

carboxylation of newly synthesized vitamin K-dependent

proteins. The γ- carboxylase converts a limited number of

glutamic acid residues in the amino-terminal part of the

targeted proteins to γ-carboxyglutamic acid (Gla), calcium-

binding residues. Members of the vitamin K-dependent protein

68

family include the coagulation factors prothrombin; factors

VII, IX, X, protein S, protein C, and protein Z, as well as

several other proteins synthesized outside the liver. These

proteins include osteocalcin, matrix Gla protein, Gas6, protein

S, and some recently discovered proline-rich transmembrane

proteins (Wallin et al., 2001).

Before serving as a cofactor for γ-carboxylase, vitamin

K must be reduced to the hydroquinone (vitamin K1H2).

When one Gla residue in the targeted protein is formed, one

hydroquinone molecule is converted to the metabolite vitamin

K1 2,3-epoxide. The epoxide is reduced back to the

hydroquinone form of the vitamin by an integral membrane

protein complex of the ER, the vitamin K epoxide reductase

(VKOR). This cyclic conversion establishes a redox cycle for

vitamin K known as the vitamin K cycle. VKOR is the target

for the anticoagulant drug warfarin, (Wallin et al., 2001)

Vitamin K-dependent proteins require carboxylation for

activity. The amount of vitamin K in the diet is often limiting

for the carboxylation reaction. It has been commonly assumed

that vitamin K may also be provided by enteric bacteria;

however, if coprophagy is prevented, rats fed a vitamin K-free

diet develop severe bleeding problems in weeks. Of more

interest is the recent observation that vitaminK1 appears to be

taken up primarily in the liver while vitamin K2 appears to

69

preferentially accumulate in arteries and extra hepatic

locations (Stafford, 2005).

The production and activation of coagulation factors

VII, IX, X and prothrombin are dependent on the vitamin K

cycle. Post translational modification of glutamate to gamma

carboxyl glutamate is required for the activity of vitamin K-

dependent proteins (Stafford, 2005). The carboxylated Glu

residue is converted to a Gla amino acid and a reduced

vitamin K molecule is converted to vitamin K epoxide. Before

vitamin K can be reused in the vitamin K cycle, vitamin K

epoxide must be converted back to reduced vitamin K by

vitamin K 2,3-epoxide reductase (VKOR). Recently, Wajih et

al. identified the novel endogenous molecules that transfer the

electron to VKOR and regenerate the vitamin K cycle (Wajih

et al., 2005; Wajih et al., 2007).

Warfarin blocks the vitamin K cycle and inhibits the γ-

carboxylation of the vitamin K-dependent blood-clotting

factors. An inadequate supply of vitamin K blocks the

production of prothrombin and leads to hemorrhaging

(Thijssen et al., 1989).

3. Resistance to anticoagulants

Resistance is defined according to the European and

Mediterranean Plant Protection Organization as follows;

"Rodenticide-resistant rodents should be able to survive doses

71

of rodenticide that would kill ‗normal‘ or ‗susceptible‘

conspecifics‖ (EPPO, 1995). Greaves, (1994) describes

anticoagulant resistance as "a major loss of efficacy in

practical conditions where the anticoagulant has been applied

correctly, the loss of efficacy being due to the presence of a

strain of rodent with a heritable and commensurately reduced

sensitivity to the anticoagulant". Monitoring for resistance is

important to reveal the secret behind its spread and to manage

resistant populations (Buckle, 2006).

a. Techniques used in resistance detection in rodents

There are few relevant techniques for detection of

resistance to anticoagulants. They are either in vivo assays,

like, feeding test and blood clotting response test (BCR) or in

vitro assays, like, VKOR activity, CYP450 metabolism and

VKORC1 testing. Each technique has its pros and cons as

follow.

1. Feeding tests

Basically, it is a no-choice feeding test over 6 days with

a 50 ppm warfarin, bromadiolone or difenacoum bait for

instance. Rodents surviving the 5-day test period (+14 days

observation) are classified as resistant to the anticoagulant

tested. This method may involve some modifications i some

cases. Some authors consider that this test has several

limitations, especially with regards to local variations in the

70

resistance of the strain, which would need adaptation of the

exposure period to cover a wider range of susceptibility.

Unfortunately, this approach requires a large number of

animals and is ethically critical (Gill and McNicoll, 1991).

2. BCR testing

It first developed by Martin et al., (1979). In its present

form, the BCR can be conducted in two ways: The first

approach is to determine the rat capacity to use the vitamin K

epoxide substrate (1 mg/kg) as a vitamin K source in the

presence of an anticoagulant (warfarin) (5 mg/kg).

Determination of the clotting response (Prothrombin time)

24hours later is a good indicator of the resistance status. A

new modified methos relies on the administration of a low

dose (1 mg/kg) warfarin, with no vitamine K epoxyde and

investigation of the clotting capacity 24hours later. The recent

developments of this approach are based on the works by Gill

and McNicoll (1991) and Prescott and Buckle (2000), who

tested several protocols with various anticoagulants. The

second approach investigates the rat vitamin K deficiency

status (Martin et al., 1979), this approach has been less

developed.

3. Measuring the activity of the hepatic vitamin K

epoxide reductase (VKOR)

Several protocols may be used on liver microsomes or

any other enzyme system (Lasseur et al., 2007). This approach

72

provides a very good estimate of the enzyme activity and the

resistance status of a population. Hepatic VKOR as-sessment

is carried out in vitro by monitoring the activity of VKOR in

the presence and absence of the toxicant. Susceptible samples

show minimal VKOR activity when anticoagulant is present,

while enzyme activity in resist-ant samples remains above

20% of original levels (MacNicoll, 1993).

4. CYP metabolism

It is an in vitro approach, like the VKOR activity assay,

and requires microsoms and analytical devices to look at

warfarin metabolites produced. It is not a standard tool for the

monitoring of resistance so far (Ishizuka et al., 2006).

5. VKORC1 sequencing

Sequencing of VKORC1 appears as one of the most

interesting and cost effective tools till now. It can be applied

rapidly on large scale samples, even across a country. It can

also provide a good indication of the resistance level

conveyed by a given mutation. The PCR dependent test aims

at detecting mutation in VKORC1 gene that may confer

resistance to anticoagulants. Sequencing of VKORC1 only

requires a piece of animal tissue (tail, ear, fur may be used)

and does not necessitate live-trapping of rodents. This

approach may be simpler even further with the use of qPCR

and specific primers, especially when only one mutation is

73

expected or known to occur in a certain area (Grandemange et

al., 2010).

b. The mechanisms of anticoagulant rodenticide

resistance.

The resistance mechanism mainly involves VKORC1,

the molecular target for coumarin drugs. Mutation of VKORC1

may constitute the genetic basis of anticoagulation resistance

in R. losea (Wang et al., 2008). Resistant to anticoagulants

involves VKOR modification through point mutations of the

DNA. While still remaining functional, VCOR displays a

reduced affinity for the toxicant or the toxicant is more easily

replaced by the vitamin KO. This modification is inheritable

(Bailey and Eason, 2000).

In 2004, two research groups identified and reported a

novel molecule that contributes to VKOR activity in the rat

and named it vitamin K epoxide reductase complex subunit 1

(VKORC1) (Rost et al., 2004; Li et al., 2004).

Pelz et al. (2005) identified which part of the genetic

code of rats and mice carried the DNA sequence, or gene,

which alters when rodents become resistant to anticoagulants.

The gene they discovered produces the enzyme vitamin K1

epoxide reductase, a crucial enzyme in the vitamin K cycle

and the one blocked by all anticoagulant. The gene was given

the name VKORC1 and the sequence of amino-acids used in

its construction was decoded.

74

In the first report on VKORC1, Rost et al. (2004)

suggested that warfarin resistance in rats was attributable to

the Tyr139Phe mutation in the VKORC1 gene. The VKORC1

139 mutation in warfarin-resistant rats causes a structural

conformation of the VKORC1 protein, and this conformation

prevents warfarin blocking (Rost et al., 2005).

c. The chemical structure of VKORC1

The enzyme is mainly found in liver cells. It is seen as a

chain of 163 amino-acids which passes several times through

the membrane of the endoplasmic reticulum (ER). The amino-

acids are numbered in the chain (Tie and Nicchitta, 2005).

VKORC1 is an 18 kDa hydrophobic protein resident in the

endoplasmic reticulum membrane. Hydrophobicity plots and

secondary structure predictions suggest a topology of

VKORC1 protein that includes 3 to 4 a-helical transmembrane

segments (Goodstadt and Ponting, 2004), Fig. 6.

All anticoagulants target the site of the vitamin K

epoxide reductase enzyme complex (VKOR) and the binding

of anticoagulants to the VKOR inhibits the essential

production of prothrombin, thus destroying blood clotting

ability. Today, it is possible to differentiate between rats that

are either susceptible or resistant to anticoagulants by means

of a blood clotting response (BCR) test, where changes in

blood coagulation during anticoagulant exposure are

75

visualized (Martin, 1973; Gill, et al. 1993). The genetic

background behind this inheritable trait has been investigated

over the last three decades (Pelz, et al. 2005).

Fig. 6. The chemical structure of VKORC1

d. Mutations in VKORC1 conferring resistance to

warfarin

Mutated genes are given names which describe the

position of the mutated amino acid in the DNA sequence of

the enzyme, e.g. In the case of the common French resistance

mutation this is at position 139. The name of the original

(wild-type) amino-acid is tyrosine and that of the mutant

amino-acid is phenylalanine. These are put before and after

the position number, hence tyrosine139phenylalanine. The

76

names of the amino-acids are commonly abbreviated, i.e.

tyr139phe.

Since the establishment of a correlation between

mutations within the VKORC1 and anticoagulant resistance in

rodents, various mutations in VKORC1 have been identified as

conferring anticoagulant resistance in rats and mice (Pelz et

al., 2005; Pelz, 2007). Recently, missense mutation in a

protein of the VKOR complex, named VKORC1, was

identified as being related to warfarin resistance (Rost et al.

2004). The VKORC1 mutations are currently believed to be

the genetic basis of anticoagulant resistance, conferring

resistance to, at the very least, the first-generation

anticoagulant warfarin, (Rost et al., 2004; Pelz et al., 2005).

One particular VKORC1 mutation, a change in an amino acid

from tyrosine to cysteine in exon 3 at codon position 139

(Y139C) coincides with anticoagulant resistance in Danish

and German rats (Pelz, et al,. 2005).

VKORC1 polymorphisms in rats from warfarin-

resistance areas in Europe, Asia, North-and South-America

have been reported; England Ile821Ile; Hungary Ile821Ile;

Korea Ile821Ile; Indonesia Ile821Ile, Ile90Leu, Ser103Ser,

Ile107Ile, Thr137Thr, Ala143Val; USA, Santa Cruz

Arg12Arg, Ile90Leu, Leu94Leu, Ile107Ile, Thr137Thr,

Ala143Ala; USA, Chicago Ile821Ile; Argentina Arg12Arg,

77

Ile90Leu, Leu94Leu, Ile107Ile, Thr137Thr, Ala143Ala (Rost

et al., 2009).

More than 30 missense mutations in the gene VKORC1

in humans, rats and mice have been found, 16 of which have

been confirmed to confer a certain degree of resistance or

insensibility to warfarin. A change of amino acids at positions

120 to 139 is connected to the strongest degree of resistance

observed. The tyrosin-cystein substitution at position 139 in

VKORC1 is probably the most widespread mutation. It is

common in Denmark and northwestern Germany, and was

found in parts of Hungary, France and England. Other

widespread mutations are the tyrosin-phenylallanin

substitution at position 139, which is common in France and

Belgium and was also found in Korea, the leucin-glutamic

acid substitution at position 128 (―Scottish-type resistance‖,

Scottland, northern England and parts of France) and the

leucin-glutamic acid substitution at position 120 (Hampshire-

and Berkshire-resistance, Southern England, parts of France

and locally in Belgium. The well-known Welsh-type

resistance can be attributed to a tyrosin-serin substitution at

position 139, however, the effect upon the degree of resistance

seems to be less pronounced than in the other two

substitutions at position 139 mentioned above. The occurrence

of resistance described for the Chicago (Illinois, USA) area

seems to be due to an arginin-prolin substitution at position 35

that was also detected in a wild rat from central France. Again

78

the degree of resistance mediated by this mutation seems to be

relatively low (Pelz, 2008)..

More than 250 rats and mice from anticoagulant-

exposed areas in Europe, East Asia, South Africa and both

North and South America were screened for mutations in the

VKORC1 gene. Pre-screening revealed a panel of mutations

and SNPs (single nucleotide polymorphisms) in the VKORC1

gene. Three already described mutations could be detected in

rats trapped in different English counties: the Tyr139Cys,

Tyr139Ser and the Leu128Gln substitutions. All three

mutations confer a moderately reduced VKOR activity and are

resistant to warfarin inhibition to a variable degree. Arg33Pro

substitution was observed in two confirmed warfarin-resistant

rats from Nottinghamshire. A Phe63Cys substitution was

detected in rats from Cambridge, including two rats with an

additional Ala26Thr or a Tyr39Asn amino acid exchange.

While Ala26Thr has – similar to Ala26Ser – only a moderate

effect on VKOR activity with a reduction to approximately

56% of wild-type activity, the Phe63Cys and the Tyr39Asn

substitutions reduce the VKOR activity to about 30% of

normal. Since both amino acids Phe63 and Tyr39 are highly

conserved in vertebrates and also in the mosquito, a

substitution of these amino acids is expected to have an

influence on protein function. VKOR activity measurements

of the Glu67Lys variant (observed in six rats from Japan)

showed a reduced vitamin K epoxide turnover of about 33%

79

compared to the wild-type protein. The most drastic effect on

VKOR activity was observed for the Trp59Arg substitution.

Only 16% residual VKOR activity could be measured after

recombinant expression of this VKORC1 mutant (Rost et al.,

2009).

Tyr139Cys, Tyr139Ser, Tyr139Phe, Leu128Gln and

Leu128Ser mutations dramatically reduce VKOR activity. It is

suggest that mutations in VKORC1 are the genetic basis of

anticoagulant resistance in wild populations of rodents,

although the mutations alone do not explain all aspects of

resistance that have been reported. These mutations may

induce compensatory mechanisms to maintain blood clotting.

These findings provide the basis for a DNA-based field

monitoring of anticoagulant resistance in rodents. However,

the ability to maintain a functional blood clotting mechanism

under anticoagulant exposure can be attributed to a

physiological response of the individual rat, which may be

enhanced by a genetic change in the VKORC1. (Pelz et al.,

2005; Pelz, 2007).

81

80

MATERIALS AND METHODS

Warfarin resistance study

1. Determination of the susceptibility level of rats to

warfarin Using a laboratory feeding test(no-choice)

The study population consisted of 42 R. norvegicus,12 from

Giza, 12 from Beheira, 9 from Beni Suef and 9 from Qaliubiya . All

animals were healthy adults and females were not pregnant. They were

confined in individual cages and received the same food and water ad

libitum. All animals received humane care to be in good conditions as

urine and droppings were being removed daily. Resistance to warfarin

was assayed by feeding studies, the no-choice-feeding test developed

by the World Health Organization (WHO) was used with some

modifications: an acclimatization period, followed by a pretest diet

assessment of 7 days, then by a 6-day no-choice feeding schedule of

0.005% (50 ppm) warfarin-containing yellow corn. Diet consumption

was monitored and recorded daily), and 22 days of post-treatment

observation are maintained. Survival during the test with the amount

of active ingredient consumed greater than 10 mg/kg body weight,

were considered as evidence of resistance

2. VKORC1 analysis using Polymerase Chain Reaction

(PCR)

Livers were excised, rapidly frozen in liquid nitrogen, and stored

at -20 °C. The total RNA was isolated and reverse transcribed to

cDNA. VKORC1 was amplified using primers based on corresponding

sequences for Rattus norvegicus in Gen-Bank (Accession No.

82

NM_203335). The primers amplify a 511 bp fragment spanning the

whole ORF of VKORC1 mRNA. Nucleotide sequences for the sense

primer and antisense primer were 5'-GTGTCTGCGCTGTACTGTCG-

3' and 5'-CCTCAGGGCTTTTTGACCTT -3'; respectively. PCR

products were electrophoresed and visualized under UV light and the

picture taken with a gel documentation system The following DNA

fragment was sequenced by the Sanger method (Sanger and Coulson

1975).

Procedure:

a. RNA extraction

Total RNA purification protocol

Liver tissue samples were grind in a mortar using liquid nitrogen

then a 0.1 gm was measured immediately in a 1.5 ml tube. 300 μl lysis

buffer and 6 μl of 14.3Mβ-mercaptoethanol were added immediately

and 10 min vortex. 600 μl of diluted Proteinase K (10 μl of the

included Proteinase K diluted in 590 μl of TE buffer) were added.

Vortex to mix thoroughly was done and then incubated at 15-25°C for

10 min. After that; Samples were Centrifuged for 5 min at 12000 xg

and supernatant was transferred into new RNase free tube. 450 μl

ethanol (96-100%) were add and mixed by pipetting. Up to 700 μl of

lysate were transferred to the GeneJET RNA Purification Column

inserted in a collection tube then centrifuged for 1 min at ≥12000 x g.

The flow-through was discarded and the purification column was

placed back into the collection tube.

83

Once all of the lysate has been transferred, 700 μl of Wash

Buffer 1 were added to the GeneJET RNA Purification Column and

centrifuged for 1 min at ≥12000 x g. 600 μl of Wash Buffer 2

(supplemented with ethanol) were added to the GeneJET RNA

Purification Column and centrifuged for 1 min at ≥12000 x g. 250 μl of

Wash Buffer 2 were added to the GeneJET RNA Purification Column

and centrifuged for 2 min at ≥12000 x g.

50 μl of nuclease-free water, were added to the center of the

GeneJET RNA Purification Column membrane, then centrifuged for 1

min at ≥12000 x g to elute RNA. the purification column was discarded

and the purified RNA became ready for Using in downstream

applications or to be stored at -20°C until use.

b. Synthesis of cDNA from RNA

11 μl RNA were added in a 0.2ml tube that placed in ice then 1

μl Oligo (dT) primer was added to get 12 μl total volume. The 12 μl

mix was Incubated at 65oC for 5 min then the tube placed back on ice

to add: 4 μl (5x) reaction buffer, 1 μl RiboLock RNase Inhibitor, 2 μl

10mM dNTP Mix (nucleotides) and 1 μl RevertAid M-MuLV Reverse

Transcriptase. The 20 μl total volume was mixed gently and put into

the PCR machine at 42oC for 60 min then at 70

oC for 5 min.

PCR reactions

Gentl vortex and brief centrifugation of DreamTaq Green PCR

Master Mix (2X) were done after thawing. In a thin-walled PCR tube

84

placed on ice, the following components were added for each 25 μl

reaction: 12.5 μl DreamTaq Green PCR Master Mix (2X), 1 μl Forward

primer, 1 μl Reverse primer, 2 μl Template DNA and nuclease-free

water to get 25 μl total volume. PCR was performd using the

recommended thermal cycling conditions consisted of 94C for 5 min,

followed by 35 cycles of 94°C for 45 s, annealing 60°C for 45 s, and

72°C for 60 s and a final extension at 72°C for 10 min.

c. DNA electrophoresis

PCR products were electrophoresed on a 1.0% agarose gel

stained with ethidium bromide and visualized under UV light and the

picture taken with a gel documentation system The following DNA

fragments were cut and purified using a TI-ANgel Midi purification kit.

d. DNA Sequence

The DNA fragment was sequenced by the Sanger method (and

Coulson, 1975).

e. DNA analysis

Sequence alignments were performed using gene-bank data base

(http://www.ncbi.nlm.nih.gov). Coding region sequence and predicted

amino acid sequence of VKORC1 were deduced from nucleotide

sequences according to the coding frame in R. norvegicus. Mutation

and polymorphism screens were then performed with ClustalW

(Thompson et al. 1994).

85

RESULTS AND DISCUSSIONS

Warfarin resistance

Resistance can hinder management strategies with bad

consequences for stored products, constructions protection,

hygiene and animal health. In many parts of the world,

anticoagulants of the first generation are not an option for the

control of resistant Norway rats. The spread of resistant rats

and conditions supporting and reducing resistance should be

investigated in order to improve resistance management

strategies and avoid the misuse of anticoagulants.

1- Monitoring resistance to warfarin using feeding test

42 Norway rats were collected from four governorates (12 rats

from Giza, 12 from Beheira, 9 from Qaliubiya and 9 from Bani-Suef)

for resistance study. No significant deference between males and

females average body weight (P< 0.05). Out of the 42 individuals, 5

rats were survived the 28-days no choice feeding test (6-day no-choice

feeding schedule of 0.005% warfarin and 22 days of post-treatment

observation). The resistance rate was 11.9%. There were two resistant

individuals found in Bani-Suef, while the other three governorates have

one individual each. Four survivals were males and the fifth one was

female. They consumed amount of active ingredient was greater than

10 mg/kg body weight. There was no significance difference between

the total consumption of active ingredient of resistant and susceptible

individuals (p < 0.05), table (11).

86

Table 11. Warfarin feeding test results for resistance monitoring.

Animals

Body weight (g)

Mortality Total consumption of active ingredient (mg/kg)

(no) % Survived Died

Site Sex, No. Mean ± SD Range Mean Range Mean ± SD Rang

Giza

Male,6 256.50±50.68 178-340 5/6 83.33% 12.45 9.47±0.68 7.02-11.92

Female,6 238.33±61.79 145-303 6/6 100% --- 10.89±0.16 7.70-18.3

Beheira

Male,7 242.43±84.09 125-368 6/7 85.70% 11.55 10.54±1.79 7.24-12.14

Female,5 272.6±71.36 212-354 5/5 100% 11.88±1.81 10.16-14.38

Bani-Suef

Male,5 268.5±115.44 136-401 4/5 100% 10.77 10.27±5.03 5.98-18.38

Female,4 315.75±125.15 148-450 3/4 100% 10.00 8.36±2.28 6.58-11.69

Qaliubiya

Male,6 271.75±90.16 120-350 5/6 83.33% 13.88 13.60±3.44 9.93-19.21

Female,3 242.17±106.66 173-365 3/3 100% - 14.83±4.45 10.64-19.50

87

2. VKORC1 analysis using Polymerase Chain Reaction

This approach has also been associated with recent genetic

advances and the identification of the first gene involved in the

synthesis of the VKOR enzyme. This first gene (VKORC1) is clearly

located on the chromosome 1 of the rat, associated with the D1Rat219

microsatellite. The embedded in the endoplasmic reticulum protein has

three trans-membrane domains. Mutated forms are associated with

severe changes in VKOR activity (Rost et al, 2004). This small protein

(18kDa) with 3 exons and encoding a small trans-membrane protein

(163 AA) was computed and a suggested structure that has been

published (Tie et al, 2005).

VKORC1 gene of 35 samples, 5 resistant (feeding-test survivals)

and 23 susceptible (died during the feeding test) was extracted,

amplified, sequenced and analyzed for mutation. Besides, 7 specimens

trapped from suspected resistance area in Giza and sent to the lab.

directly (did not undergo the feeding test).

The total RNA was isolated and reverse transcribed to cDNA.

VKORC1 gene was amplified using specific primers based on

corresponding sequences for Rattus norvegicus in Gen-Bank. The

cDNA was amplified and the PCR products were run on a 1.0%

agarose gel (Fig. 7). The corresponding cDNA fragments were cut and

purified and then sequenced according to Sanger and Coulson (1975), as

shown in Fig. 8, 9. Finally, sequence alignments were performed.

88

Fig. 7. PCR products were subjected to electrophoresis on a

1.5% agarose gel. "s" tested to be susceptible individuals, "R"

tested to be resistant individuals. "M" represents the DNA

marker, (R6 did not undergo the feeding test).

89

Fig. 8. The DNA sequence of VKORC1 gene amplified using specific primers, the product length

about 550 pb from R. norvegicus

91

Fig. 9. DNA sequencing result; the DNA sequence of VKORC1 gene amplified using specific primers, the

product length about 550 pb from R. norvegicus

90

a. Analyses of VKORC1 for SNPs

The sequence obtained was aligned with Rattus norvegicus

vitamin K epoxide reductase complex subunit 1 (VKORC1) mRNA,

complete cds Sequence ID: gb|AY423047.1| (Fig. 10) and

polymorphism screening were carried out by sequence alignment.

b. Identification of the point mutation of VKORC1 gene

Nucleotide sequences were analyzed for SNPs (single nucleotide

polymorphism) or point mutations. Nucleotide substitution name takes

the form: (position) (original nucleotide) > (substituted nucleotide),

e.g., 87C > T means the original C nucleotide at the position 87 is

changed into T.

mRNA sequence is converted into correspondent amino-acids

(Fig. 11a), then aligned with vitamin K epoxide reductase complex

subunit 1 precursor (Rattus norvegicus) amino-acid Sequence

ID: ref|NP_976080.1| (Fig. 11b) and screened for mutations.

Amino-acid substitution or mutation name takes a certain form:

original amino acid (position) mutated amino acid, e.g., when the

original amino-acid Histidine (H) is substituted at the position 28 with

Tyrosine (Y), the mutation name will be H28Y. Mutations detected in

both resistant and susceptible individuals that involved nucleotide

alteration do not have the same effect; however, there were different

types of mutations detected as follows.

92

a 1

1

76

26

151

51

226

76

301

101

376

126

451

151

GACATGGGCACCACC TGGAGGAGCCCTGGA CGTTTGCGGCTTGCA CTATGCCTCGCTGGC CTAGCCCTCTCACTG

D M G T T W R S P G R L R L A L C L A G L A L S L TACGCACTGCACGTG AAGGCGGCGCGCGCC CGCAATGAGGATTAC CGCGCGCTCTGCGAC GTGGGCACGGCCATC

Y A L H V K A A R A R N E D Y R A L C D V G T A I AGCTGTTCCCGCGTC TTCTCCTCTCGGTGG GGCCGGGGCTTTGGG CTGGTGGAGCATGTG TTAGGAGCTGACAGC

S C S R V F S S R W G R G F G L V E H V L G A D S ATCCTCAACCAATCC AACAGCATATTTGGT TGCATGTTCTACACC ATACAGCTGTTGTTA GGTTGCTTGAGGGGA

I L N Q S N S I F G C M F Y T I Q L L L G C L R G CGTTGGGCCTCTATC CTACTGATCCTGAGT TCCCTGGTGTCTGTC GCTGGTTCTCTGTAC CTGGCCTGGATCCTG

R W A S I L L I L S S L V S V A G S L Y L A W I L TTCTTTGTCCTGTAT GATTTCTGCATTGTT TGCATCACCACCTAT GCCATCAATGCGGGC CTGATGTTGCTTAGC

F F V L Y D F C I V C I T T Y A I N A G L M L L S TTCCAGAAGGTGCCA GAACACAAGGTCAAA AAGCCCTGAGGT

F Q K V P E H K V K K P * G

b Query 16 CCTGGACGTTTGCGGCTTGCACTATGCCTCGCTGGCCTAGCCCTCTCACTGTACGCACTG 75

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

VKORC1 27 CCTGGACGTTTGCGGCTTGCACTATGCCTCGCTGGCCTAGCCCTCTCACTGTACGCACTG 86

Query 76 CACGTGAAGGCGGCGCGCGCCCGCAATGAGGATTACCGCGCGCTCTGCGACGTGGGCACG 135

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

VKORC1 87 CACGTGAAGGCGGCGCGCGCCCGCAATGAGGATTACCGCGCGCTCTGCGACGTGGGCACG 146

Query 136 GCCATCAGCTGTTCCCGCGTCTTCTCCTCTCGGTGGGGCCGGGGCTTTGGGCTGGTGGAG 195

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

VKORC1 147 GCCATCAGCTGTTCCCGCGTCTTCTCCTCTCGGTGGGGCCGGGGCTTTGGGCTGGTGGAG 206

Query 196 CATGTGTTAGGAGCTGACAGCATCCTCAACCAATCCAACAGCATTTTTGGTTGCATGTTC 255

|||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||

VKORC1 207 CATGTGTTAGGAGCTGACAGCATCCTCAACCAATCCAACAGCATATTTGGTTGCATGTTC 266

Query 256 TACACCATACAGCTGTTGTTAGGTTGCTTGAGGGGACGTTGGGCCTCTATCCTACTGATC 315

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

VKORC1 267 TACACCATACAGCTGTTGTTAGGTTGCTTGAGGGGACGTTGGGCCTCTATCCTACTGATC 326

Query 316 CTGAGTTCCCTGGTGTCTGTCGCTGGTTCTCTGTACCTGGCCTGGATCCTGTTCTTTGTC 375

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

VKORC1 327 CTGAGTTCCCTGGTGTCTGTCGCTGGTTCTCTGTACCTGGCCTGGATCCTGTTCTTTGTC 386

Query 376 CTGTATGATTTCTGCATTGTTTGCATCACCACCTATGCCATCAATGCGGGCCTGATGTTG 435

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

VKORC1 387 CTGTATGATTTCTGCATTGTTTGCATCACCACCTATGCCATCAATGCGGGCCTGATGTTG 446

Query 436 CTTAGCTTCCAGAAGGTGCCAGAACACAAGGTCAAAAAGCCCTGAGG 482

|||||||||||||||||||||||||||||||||||||||||||||||

VKORC1 447 CTTAGCTTCCAGAAGGTGCCAGAACACAAGGTCAAAAAGCCCTGAGG 493

Fig. 10. a) VKORC1 gene nucleotide sequence and amino-acid residues

sequence, Accession number NM_203335.2 b) Pairwise alignment between

VKORC1 and the gene sequence of a susceptible sample (the query)

93

a GRLRLALCLAGLALSLYALYVKAARARNEDYRALCDVGTAISCSR

VFSSRWGRGFGLVEHVLGADSILNQSNSIFGCMFYTLQLLLGCLR

GRWASILLILSSLVSVAGSLYLAWILFFVLYDFCIVCI

b Sample 7 GRLRLALCLAGLALSLYALYVKAARARNEDYRALCDVGTAISCSRVFSSRWGRGFGLVEH 66

GRLRLALCLAGLALSLYAL++KAARARNEDYRALCDVGTAISCSRVFSSRWGRGFGLVEH

VKORC1 9 GRLRLALCLAGLALSLYALHLKAARARNEDYRALCDVGTAISCSRVFSSRWGRGFGLVEH 68

Sample 67 VLGADSILNQSNSIFGCMFYTLQLLLGCLRGRWASILLILSSLVSVAGSLYLAWILFF 124

VLGADSILNQSNSIFGCMFYTLQLLLGCLRGRWASILLILSSLVSVAGSLYLAWILFF

VKORC1 69 VLGADSILNQSNSIFGCMFYTLQLLLGCLRGRWASILLILSSLVSVAGSLYLAWILFF 126

Sample 125 VLYDFCIVCI 134

VLYDFCIVCI

VKORC1 127 VLYDFCIVCI 136

Fig. 11. a) Amino-acids correspondent to VKORC1 nucleotide sequence.

b) Pairwise alignment between amino-acids of VKORC1 and the

sequence of a resistant sample

1. Silent mutations (synonymous)

Some variants do not alter the amino acid sequence of the

protein as the new triple codon gives the same amino-acid. Thus, they

are likely to represent no-effect polymorphisms. This kind of mutations

is called silent mutation (synonymous mutations). Since the genetic

code is degenerate, several codons produce the same amino acid.

Especially, third base changes often have no effect on the amino acid

sequence of the protein. These mutations affect the DNA but not the

protein.

94

Fig. 12. shows a silent mutation that resulting no change of

amino-acid sequence (I82I). There is a substitution at the position 246

where "A" nucleotide changed into "T". The triplet codon "ATA"

became "ATT" but still gives the same amino-acid Isoleucine. More

silent mutations detected are shown in table (12) and table (13).

Fig. 12. Synonymous mutation – part of susceptible sample sequence.

2. Neutral mutations

Mutation that alters the amino acid sequence of the protein but

does not change its function as replaced amino acid is chemically

similar or has little influence on protein function. e.g., I133L mutation

(Fig. 13 ) are neutral because Leucine and Isoleucine amino-acids are

close to each other and there is no much difference in their influence on

protein function. The aforementioned two neutral mutations detected in

a resistant individual from Giza.

95

Fig. 13. Neutral mutation – part of susceptible sample sequence.

3. Missense mutations:

Missense mutations substitute one amino acid for another

different one. There are some variants or substitutions of nucleotides

predicted to alter the protein structure and could lead to functional

impairment or change of VKORC1 activity. Mutation screening

revealed some missense mutations, e.g., V29G, in which Valine

changes into Glutamine, Fig. 14. More missense mutations detected are

shown in tables 12 and 13.

Fig. 14. Missense mutation – part of resistant sample sequence.

96

c. VKORC1 mutations and resistance to warfarin

Since it has been discovered in 2004 by Rost et al., VKORC1

has been the main subject of many studies to explain susceptibility and

resistance of rodents and human to anticoagulants. It was published that

there is a correlation between mutations within the VKORC1 and the

anticoagulant resistance in Rattus norvegicus. Many mutations have

been reccorded in VKORC1 as conferring anticoagulant resistance in

rats and mice (Lasseur et al., 2005 and Pelz, 2007).

VKORC1 of susceptible individuals was sequenced to make

comparison between susceptible and resistant rats. Two silent and one

neutral mutations were detected, I82I, P154P and I133L. The I82I

polymorphism was identified in both susceptible and resistant rats, i.e.

two resistant and 7 susceptible rats. Since it is silent mutation, it has no

effect on the amino acid level and this was considered irrelevant to

resistance. Similarly, the I133L mutation has no tangible effect as the

Isoleucine converted to Luecine which is close to it, table (12).

The I82I mutation was previously detected in many countries

and it is among the VKORC1 mutations recorded in genebank with

dbSNP rs# cluster id: rs66459411, Table (13). This variant occurred at

high frequency in rats from all continents. Thus it may be an ancestral

variant or may have arisen several times independently (Rost et al.,

2009).

97

P154P mutation was found in two resistant individuals co-

existed with V29G. The NCBI database for short genetic variations

(dbSNP) currently includes SNPs for VKORC1. The 26 recorded

mutations (Table 13) include an SNP at position 154 where Proline

changes into Leucine (P154L) under the dbSNP entry rs8143495,

Which is a missense mutation that is different from our detected

synonymous mutation (P154P).

Of the variants which do cause amino acid substitutions, are

H28Y, V29M and E155K, table (12). All these missense mutations

were recorded in resistant individuals. One of them was previously

recorded that involves Glutamic Acid substituted with Lysine at

position 155 by Grandemange et al., (2010) in France.

In this study we found V29 is likely to be mutated; as is was

mutated in 5 resistant individuals. Rost et al., (2004) found that the

mutation V29L resulted in warfarin resistance. Also, the mutation

H28Y was found in accompany with V29. However, we might not

attribute Rattus norvegicus warfarin resistance to it unless

conformation studies are carried out to assess its effect on VKOR

activity.

Yet, to establish monitoring technique of resistance to

anticoagulants based on VKORC1 mutations we suggest that future

studies need to consider larger numbers of rats randomly collected from

local populations. Besides, more screening should be done to determine

98

the prevailing mutation which if found considered as an indicator of

resistance.

Table 12. VKORC1 mutations (SNPs) recorded in Rattus

norvegicus.

Mu

tati

on

Wil

d

cod

on

⇒M

uta

nt

cod

on

Mu

tati

on

Po

siti

on

AA

WT

⇒A

A m

ut.

AA

P

osi

tion

Fu

nct

ion

No

. o

f

spec

imen

s

Susceptible I82I ATA⇒ATT 246 I⇒I 82 Silent 7

I133L ATT⇒CTT 397 I⇒L 133 Neutral 1

Resistant H28Y CAC⇒TAC 82 H⇒Y 28 Missense 1

H28Y CAC⇒TAT 82,84 H⇒Y 28 Missense 4

V29M GTG⇒ATG 85 V⇒M 29 Missense 1

V29Q GTG⇒GAG 86 V⇒Q 29 Missense 1

V29L GTG⇒TTA 85 V⇒L 29 Missense 1

V29G GTG⇒GGG 86 V⇒G 29 Missense 2

I82I ATA⇒ATT 246 I⇒I 82 Silent 2

P154P CCA⇒CCT 462 P⇒P 154 Silent 2

E155K GAA⇒AAA 463 E ⇒K 155 Missense 1

Table 13. VKORC1 mutations (SNPs) recorded in genebank data base.

Ch

r.

posi

tion

mR

NA

pos.

db

SN

P

rs#

clu

ster

id

Fu

nct

ion

db

SN

P

all

ele

Pro

tein

resi

du

e

Cod

on

pos.

Am

ino

aci

d p

os.

206361639 483 rs8143495 missense T Leu [L] 2 154

contig ref. C Pro [P] 2 154

206361671 451 rs66459407 synonymous C Ala [A] 3 143

synonymous T Ala [A] 3 143

contig ref. G Ala [A] 3 143

206361672 450 rs66459409 missense A Glu [E] 2 143

missense G Gly [G] 2 143

contig ref. C Ala [A] 2 143

206361679 443 rs66459405 missense C Leu [L] 1 141

missense T Phe [F] 1 141

contig ref. A Ile [I] 1 141

99

Ch

r.

posi

tion

mR

NA

pos.

db

SN

P

rs#

clu

ster

id

Fu

nct

ion

db

SN

P

all

ele

Pro

tein

resi

du

e

Cod

on

pos.

Am

ino

aci

d p

os.

206361684 438 rs66459399 missense T Phe [F] 2 139

contig ref. A Tyr [Y] 2 139

206361689 433 rs66459397 synonymous A Thr [T] 3 137

synonymous G Thr [T] 3 137

contig ref. C Thr [T] 3 137

206361717 405 rs66459395 missense A Gln [Q] 2 128

contig ref. T Leu [L] 2 128

206361741 381 rs66459393 missense A Gln [Q] 2 120

contig ref. T Leu [L] 2 120

206361766 356 rs66459391 missense A Met [M] 1 112

missense C Leu [L] 1 112

contig ref. G Val [V] 1 112

206361779 343 rs66459389 missense G Met [M] 3 107

synonymous T Ile [I] 3 107

contig ref. C Ile [I] 3 107

206361791 331 rs66459387 synonymous A Ser [S] 3 103

synonymous G Ser [S] 3 103

contig ref. T Ser [S] 3 103

206362664 302 rs66459385 Missense A Ile [I] 1 94

Missense G Val [V] 1 94

contig ref. T Leu [L] 1 94

206362676 290 rs66459383 Missense T Leu [L] 1 90

contig ref. A Ile [I] 1 90

206362698 268 rs66459411 Synonymous T Ile [I] 3 82

contig ref. A Ile [I] 3 82

206362745 221 rs66459381 Nonsense T [Ter[*]] 1 67

Missense C Gln [Q] 1 67

contig ref. G Glu [E] 1 67

206362756 210 rs66459379 Missense A Tyr [Y] 2 63

Contu. Table 13.

011

Ch

r.

posi

tion

mR

NA

pos.

db

SN

P

rs#

clu

ster

id

Fu

nct

ion

db

SN

P

all

ele

Pro

tein

resi

du

e

Cod

on

pos.

Am

ino

aci

d p

os.

Missense C Ser [S] 2 63

contig ref. T Phe [F] 2 63

206362769 197 rs66459377 Missense A Arg [R] 1 59

contig ref. T Trp [W] 1 59

206363714 188 rs66459375 Missense A Thr [T] 1 56

Missense G Ala [A] 1 56

contig ref. T Ser [S] 1 56

206363765 137 rs66459373 Missense A Asn [N] 1 39

contig ref. T Tyr [Y] 1 39

206363776 126 rs66459371 Missense C Pro [P] 2 35

contig ref. G Arg [R] 2 35

206363782 120 rs66459369 Missense C Pro [P] 2 33

contig ref. G Arg [R] 2 33

206363804 98 rs66459367 Missense C Pro [P] 1 26

Missense T Ser [S] 1 26

contig ref. G Ala [A] 1 26

206363819 83 rs66459365 missense C Pro [P] 1 21

missense T Ser [S] 1 21

contig ref. G Ala [A] 1 21

206363844 58 rs66459363 synonymous C Arg [R] 3 12

synonymous T Arg [R] 3 12

contig ref. G Arg [R] 3 12

Contu. Table 13.

010

GENERAL CONCLUSION

The presence of zoonotic ectoparasites that have medical and

veterinary importance confirms Rattus norvegicus as a reservoir for

different types of pathologies, which, therefore, constitutes a risk to the

public health. The information presented in this study enables us to

understand the major parasitic infections that Norway rat harbors and

transmits to people and domestic animals in Egypt. Periodical

surveillance and monitoring in local problem areas combined with

raising awareness help local authorities in the emergency situations

prevent rodent-borne diseases.

Polymorphisms in the vitamin K epoxide reductase complex

subunit 1 (VKORC1) gene and substitutions of amino acids in the

VKOR protein are the major cause for rodenticide resistance.

Monitoring resistance to anticoagulants should be periodically done to

avoid the use of ineffective rodenticides.

This work gives information about how it is important to

allocate mutations carried by some resistant rats. It is now possible to

monitor the resistance to warfarin by detecting only the mutation

repeatedly arose in resistant population. However this work need to be

supported with some complementary studies to measure the effect of

the new mutations on VKOR activity.

012

013

SAMMARY

Rodents are a group of the largest and most successful

groups of mammals; they have a high reproductive efficiency

and great ability to adapt over a wide environmental range.

Although rodents damages are mainly associated with

agricultural crops in stores and fields or farm animals'

attacking and the destruction of facilities, their health

problems are underestimated. Rodents can be reservoirs or

carriers for a number of dangerous pathogens of humans and

farm animals. Anticoagulant rodenticides are mainly used to

eliminate the rodents and undermine the chances of the spread

of diseases and associated parasites. The emergence of

resistance problems against anticoagulant rodenticides by

some members of the rodent threatens its usage in the

foreseeable future.

Norway rat was chosen as one of the important species

of rodents in Egypt to conduct the study . The first section

dealt with the study of its endo- and ectoparasites, while the

second section tackled the study of resistance to anticoagulant

rodenticides (warfarin). Four governorates were selected to

conduct the study, namely: Giza, Beheira, Qaliubiya and Beni

Suef. The present work covers the following points:

014

a. The study of the Norway rat endo- and ectoparasites

1- Studying the Norway rat species population structure at

four different governorates.

2- Identifying the Norway rat helminthic parasites and

determining their incidence and distribution at four

different governorates.

3- Identifying the Norway rat ectoparasites, and

determining their prevalence and general indices that is

useful to understand the role of arthropod vectors as

well as mammalian reservoirs in the maintenance of

various diseases in the study areas.

b. The study of the Norway rat resistance to warfarin

1- Monitoring the Norway rat resistance to warfarin (First

generation anticoagulant rodenticide) at four different

governorates by using the conventional method, non-

choice feeding test.

2- Monitoring the Norway rat resistance to anticoagulants

rodenticides (warfarin) at four different governorates

through VKORC1 analysis using Polymerase Chain

Reaction (PCR) technique.

The results obtained were as follows:

1. The study of the Norway rat endo- and ectoparasites

a. Rattus norvegicus investigations

Eighty three Rattus norvegicus were live trapped from four

governorates: 34 from Giza, 24 from Beheira, 10 from Beni Suef and

015

15 from Qaliubiya. Their population structure was studied to study the

effect of sex and age on parasits' infection. the sex ratio was 1.37

males:1 female. Based on age, the maturity status was 53 mature and

30 immature individuals.

b. Endoparasites

In this study, we have just recorded two cestodes:

Hymenolepis diminuta and Cysticercus fasciolaris, which are

commonly found in rats and mice and they are potentially

transmissible (Zoonosis) to man and one non-zoonosis

nematode, Spirura talpae. No new species were recorded

during the study.

Sixty five individuals out of 83 were infected with one

or more helminthic parasites with an infection rate of 78.31 %.

The type of infection of helminthic parasites varies

among individuals. Some individuals were infected with only

one helminthic parasite, 27 individuals (32.5%) and some

were double infected, 32 individuals (38.5%) while triple

infection was recorded in just 6 individuals (7.2%).

1. Infection prevalence of Endoparasites based on host

location.

Location of infestation could have a tangible effect on infection

prevalence. However, in this study, the rate of rodent infection

with nematodes and cestodes does not considerably differ

among locations.

016

There was no concrete difference among the cestodes

infection percentages in three locations, as it was 70.59%,

73.33% and 75% in Giza, Qaliubiya and Beheira; respectively,

but in Bani-Suef, it was higher (90%). Likewise, the nematode

infection percentages were 41.18%, 33.33% and 40% in Giza,

Beheira and Qaliubiya; respectively and it was slightly greater

in Bani Suef (50%). The combined infection percentages of

both cestodes and nematodes exhibited the same pattern.

2. Infection prevalence of endoparasites based on host

sex

Both Rattus norvegicus sexes were examined for their

endoparasites. Regarding cestodes, males were more infected

than females as 39/(83) males were infected (46.99%) versus

23/(83) females (27.71%). The prevalence percentage on

males was 81.25% (the percentage of males infected out of the

total number of males) while, it was 65.71% on females. This

indicates that the rate of the infection prevalence on males is

greater than that on females. Similarly, nematodes infection

was greater on males, 20 (24.1%) than that on female, 13

(15.66%). But the prevalence of infection of male's population

was close to that of female's; 41.67% for male's and 37.14%

for female's; respectively.

3. Infection prevalence of Endoparasites based on host age

In this study, 44 individuals out of 83 (53.01%) were

cestode infected mature and the infected immature individuals

017

were only 18 (21.69%). The prevalence of infestation among

mature individuals was greater than that among immature

individuals as 83.02% of mature individuals were infected

versus 60% of immature individuals.

As to nematode infection, 28 (33.73%) were infected

mature individuals while 5 (6.02%) individuals were infected

immature. The prevalence of nematode infection among

mature individuals was 52.83% but it was only 16.67% among

immature individuals.

c. Ectoparasites

Rodents in particularly, Rattus norvegicus are usually infected

with certain groups of arthropods; fleas, lice and mites. In this study

77.2% of Rattus norvegicus were infested with at least one ectoparasite.

Results of this study revealed that 938 ectoparasites, comprising: 140

(14.93%) fleas, 234 (24.95%) lice and 564 (60.1%) mites, are

belonging to 4 orders, 7 families, 9 genera and 9 species.

1. Infection prevalence and general indices of ectoparasite

according to location:

As to fleas' infection, Giza governorate had the highest infection

percentage (50%) and the highest flea index as well (2.56). On the

other side, Beni Suef had the lowest flea infection percentage (20%)

and the lowest flea index (0.5).

Although Beni Suef governorate had the highest lice infection

percentage (50%), Giza governorate had the highest lice index (3.76).

018

this means that the lice burden is higher in Giza than that in the other

three locations. In the same context, Behaira governorate had the

lowest lice infection percentage (25%), but its lice index (2.46) is

bigger than that of Beni Suef (2.1) and Qaliubiya (1.73), table (2).

With regard to mite infection, Beni Suef governorate came first

(70%) followed by Beheira Governorate (66.67%) while Qaliubiya had

the lowest percentage of infection (40%). Mite indices were relatively

high; since it ranged from 4.27 in Qaliubiya governorate to 11.3 in

Beheira governorate.

2. Infection prevalence of ectoparasites based on host sex

Nineteen infected male individuals (22.89%) represented

39.58% of the whole males' population. Infected females were 10

individuals with a percentage of 12.05%. The prevalence of infection

among females was 28.57%.

Regarding lice infection, a total of 12 male-individuals (out of

83, the whole population) were infected (14.46%). The infection

prevalence among them was 25% (12 out of 48 males). Infected

females' number was equal to that of males' (12, 14.46%) but the

infection prevalence among females (34.28%) was greater than that

among males.

Mite infection and prevalence was the greatest comparing to

other ectoparasites as 28 males (33.73%) and 18 females (21.69%)

were infected. Also the prevalence of infection among males (52.33%)

and females (51.43%) was the highest when compared with fleas and

019

lice. There were no differences of infection prevalence based on host

sex.

General indices of ectoparasites based on host sex:

The flea index in males is bigger than that in females in all

governorates except for Giza but the total flea indices in both males and

females are equal (1.69). There was a big difference between the

male/female lice indices in Beheira and Beni Suef as they were

0.86/4.7 and 0.6/3.6; respectively, but the total lice index in males

(2.85) was slightly higher than that in females (2.77). With regard to

mite, the total mite index was approximately bigger in males than it in

females. But still there were some differences according to locations,

table.

3. Infection prevalence of ectoparasites based on host age:

A total of 20 mature individuals versus 9 immature individuals

were infected with fleas. The flea infection prevalence inside the

mature population (37.74%) was relatively higher than that inside the

immature population (30%).

Lice infection varied between mature and immature rats, as a

total of 18 mature individuals (21.69%) and 6 immature individuals

(7.23%) were infected. The infection prevalence of lice inside the

mature population (33.96%) was higher than that inside immature

population (20%).

Unlike fleas and lice, mites' infection was higher and more

prevalent; as 33 mature individuals (39.76%) and 13 immature

001

individuals (15.66%) were infected. When comparing the infection

prevalence between mature and immature individuals, it found that the

infection prevalence in mature individuals (62.26%) was greater than it

in immature individuals (43.33%).

General indices of ectoparasites based on host age:

Generally, mature individuals tend to have bigger ectoparasite

index than immature individuals. Flea index was 1.96 in mature

individuals versus 1.2 in immature's, also lice index in mature

individuals (3.75) was three times bigger than it in immature's (1.17).

Likewise, the mite index was bigger in mature individuals (7.15) than it

in immature's (6.17).

Part II: Warfarin resistance study

The study of the Norway rat resistance to warfarin was done

through two methods. The first method involved the use of traditional

test known as no-choice feeding test while the second method, the

latest currently used, involves VKORC1 gene analysis to search for

mutations associated with resistance in some rodent individuals.

1-Feeding test (no-choice)

Forty two Norway rats were collected from four governorates

(12 from Giza, 12 from Beheira, 9 from Qaliubiya and 9 from Bani-

Suef). No significant deference between males and females average

body weight (P < 0.05). Out of the 42 individuals, 5 rats were survived

the 28-days no choice feeding test. The resistance rate was 11.9%.

There were two resistant individuals found in Bani-Suef, while the

000

other three governorates have one individual each. The survived rats

consumed amount of active ingredient greater than 10 mg/kg body

weight. There was no significance difference between the total

consumption of active ingredient of resistant and susceptible

individuals (p < 0.05).

2- VKORC1 sequencing using Polymerase Chain Reaction (PCR)

technique

VKORC1 gene of 35 samples, 5 resistant (feeding test survivals)

and 32 susceptible (died during the feeding test) was extracted,

amplified, sequenced and analyzed for mutation.

Analyses of VKORC1 for single nucleotide polymorphism

(SNPs)

The gene sequence obtained was aligned with Rattus norvegicus

vitamin K epoxide reductase complex subunit 1 (VKORC1) mRNA,

complete cds Sequence ID: gb|AY423047.1| and polymorphism

screening were carried out by sequence alignment.

mRNA sequence is converted into correspondent amino-acids,

then aligned with vitamin K epoxide reductase complex subunit 1

precursor (Rattus norvegicus) amino-acid Sequence

ID: ref|NP_976080.1| and screened for mutations. Three types of

mutation have been recorded as follows:

002

VKORC1 mutations and resistance to warfarin

Two silent and one neutral mutations have been detected, I82I,

P154P and I133L. The I82I polymorphism was identified in both

susceptible and resistant rats, 2 resistant and 7 susceptible rats. Since it

is silent mutation, it has no effect on the amino acid level, and this was

considered irrelevant to resistance. Similarly, I133L mutation has no

tangible effect as the Isoleucine converted to Luecine.

The I82I was previously detected in many countries and it is

among the VKORC1 mutations recorded in genebank with dbSNP rs#

cluster id: rs66459411.

P154P mutation was found in two resistant individuals co-

existed with V29G. The NCBI Database for Short Genetic Variations

(dbSNP) currently includes SNPs for VKORC1 of which an SNP at

position 154 where Proline changes into Leucine (P154L) under the

dbSNP entry rs8143495.

E155K, V29G, V29L, V29M, V29Q and H28Y are of the

mutations that involved amino acid substitutions. All these missense

mutations were recorded in resistant individuals. One of them was

previously recorded that involves Glutamic Acid substituted with

Lysine at position 155 by Grandemange et al., (2010) in France. Rost et

al., (2004) found that the mutation V29L resulted in warfarin

resistance. In this study we found that V29 is likely to be mutated; as is

was mutated in 5 resistant individuals. Also, the mutation H28Y has

been found in an accompany with V29.

003

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