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Digitally Signed by: Content manager’s

Name

DN : CN = Weabmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

Fred Attah

ANYAOHA, CHIDIEBERE OHAZURIKE

PG/M. Sc/10/52913

SEROPREVALENCE AND RISK FACTORS OF BRUCELLA

INFECTION IN DOGS IN ENUGU AND ANAMBRA STATES.

Faculty of the Veterinary Medicine

Department of Veterinary Public Health & Preventive

Medicine

2

SEROPREVALENCE AND RISK FACTORS OF

BRUCELLA INFECTION IN DOGS IN ENUGU

AND ANAMBRA STATES.

BY

ANYAOHA, CHIDIEBERE OHAZURIKE

PG/M. Sc/10/52913

A RESEARCH DISSERTATION SUBMITTED TO THE

DEPARTMENT OF VETERINARY PUBLIC HEALTH AND

PREVENTIVE MEDICINE, FACULTY OF VETERINARY

MEDICINE IN PARTIAL FUFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN

VETERINARY PUBLIC HEALTH AND PREVENTIVE MEDICINE,

UNIVERSITY OF NIGERIA, NSUKKA.

FEBRUARY, 2015

3

DECLARATION

I, Dr. Anyaoha Chidiebere Ohazurike hereby declare that this work was carried out by me and

has not been submitted somewhere else.

------------------------------------------------- --------------------------------

Dr. Anyaoha Chidiebere Ohazurike Date

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CERTIFICATION

We certify that Dr. Anyaoha, Chidiebere Ohazurike carried out this research work in the

Department of Veterinary Public Health and Preventive Medicine at the University of Nigeria,

Nsukka. The work presented herein is original and has not been previously reported or submitted

anywhere else.

--------------------------------------- -----------------------------

Prof. J. A. Nwanta Date

(Supervisor)

--------------------------------------- -----------------------------

Prof. B. M. Anene Date

(Supervisor)

-------------------------------------- ------------------------------

Prof. A. O. Anaga Date

(Head of Department)

-------------------------------------- -------------------------------

Prof. C. O. Nwosu Date

(Dean, Faculty of Vet. Med.)

------------------------------------- -----------------------------

Date

5

(External Examiner)

DEDICATION

To God Almighty, who has protected me and kept me alive till today.

To my lovely parents, Mr. & Mrs C. O. Anyaoha, for their encouragement and support through

the course of this work.

To my beloved brothers and sisters, for their inspiration and encouragement.

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ACKNOWLEDGEMENT

I wish to express my profound gratitude and appreciation to my Supervisors, Professor J.A.

Nwanta and Prof. B.M. Anene for their understanding, masterly guidance and encouragement

throughout the execution of the laboratory work and writing of this research project. I also wish

to thank them for assisting me in providing additional literature materials and proper correction

of the work.

The cooperation and understanding received from the Head of the Department of Veterinary

Public Health and Preventive Medicine, Prof. A.O. Anaga is highly appreciated.

I am also particularly grateful to Prof. S.I. Oboegbulem and his wife for their in-depth assistance

in the course of this work. I also appreciate his contributions, corrections and his fatherly advice

towards hard work and achieving good results and making sure that what is worth doing is worth

doing well. His kind personal financial assistance towards the purchase of an extra

Immunocomb® Canine Brucellosis Antibodody Test Kit used in this study is highly appreciated.

Thanks, to all the staff of the Department of Veterinary Public Health and Preventive Medicine,

starting with the Head of the Department and all the lecturers that tutored me in one way or the

other; including: Prof. J. U. Umoh, Dr. R. N. Chizoba and Dr. E. C. Okolocha. I also wish to

appreciate the secretary of the Department Mrs. A. C. Uzommadu and her colleague Mrs

Uzoamaka Onoja for their friendship and cooperation throughout the duration of my study.

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I am particularly grateful to Dr. J. I. Onunkwo, Dr. (Mrs) O. Okorie Kanu, and Dr (Mrs) E. V.

Ezenduka for their kind assistance and their words of encouragement.

I wish to give special thanks to the Chief Laboratory Technologist of the Department, Mrs S. N.

Ikechukwu, and her colleagues Mrs. R. O. Ugwuani and Mr. Nonso Mgbeokwere who helped me

in the laboratory where I worked.

I would like to thank Dr. Sati Ngulukun for his magnanimity in procuring, shipping and

supplying us the Brucella abortus SAT and RBPT antigens used in this work.

I wish to acknowledge my course mates and laboratory mates, Dr. Lucy Umeh, Dr. Uche

Ekwueme, for their cooperation, understanding and assistance throughout our stay at Nsukka.

Thanks to my friends on campus, particularly Dr. Udeani, Dr. Anyanwu, Dr. Tehetna, Dr.

Obodoechi Linda, Dr. Nnenna, Chioma Ohams, Solange, Jackson, Bernard, Agnes, and Nungu

for the interesting times we had together.

I am also highly indebted to my friends, particularly Dr. Dozie Okoye and Dr. Edu Umeodnagu ,

Dr. Eugene Ibe, Dr. Etuokuwu and Dr. Cosmos Okeke (Kossy Vet.), for the moral and liberal

support during the process of sample collection.

Special appreciation is extended to my parents Mr. and Mrs. C.O. Anyaoha and my brothers and

sisters, Chidozie, Emeka, Chinemerem, Kelechi, John and Blessing for their love, understanding,

sacrifice and patience throughout the study.

Lastly, I wish to thank God Almighty for his love, care, strength, guidance and protection, for

seeing me through this work.

8

Dr. Chidiebere .O. Anyaoha

August 2014.

TABLE OF CONTENTS

Title Page

Declaration page -- -- -- -- -- -- -- -- -- i

Certification page -- -- -- -- -- -- -- -- -- ii

Dedication page -- -- -- -- -- -- -- -- -- iii

Acknowledgement -- -- -- -- -- -- -- -- -- iv

Table of contents -- -- -- -- -- -- -- -- -- vi

List of Tables -- -- -- -- -- -- -- -- -- -- x

List of figures -- -- -- -- -- -- -- -- -- -- xi

Abstracts -- -- -- -- -- -- -- -- -- -- xii

CHAPTER ONE: INTRODUCTION -- -- -- -- -- -- 1

1.1 Background of the Study -- -- -- -- -- -- -- 1

1.2 Statement of the Problem -- -- -- -- -- -- -- 3

1.3 Research Questions -- -- -- -- -- -- -- -- 5

1.4 Aims and Objective of the Study -- -- -- -- -- -- 5

1.5 Significance of the Study -- -- -- -- -- -- -- 6

CHAPTER TWO: LITREATURE REVIEW

2.1: Historical review -- -- -- -- -- -- -- -- 7

2.2 Aetiology -- -- -- -- -- -- -- -- -- 8

2.3 Classification -- -- -- -- -- -- -- -- -- 8

2.4 Epidemiology -- -- -- -- -- -- -- -- -- 9

2.4.1 Distribution of Brucellosis -- -- -- -- -- -- -- 9

2.4.2 Brucellosis in Nigeria -- -- -- -- -- -- -- -- 9

2.4.3 Canine Brucellosis -- -- -- -- -- -- -- -- 10

2.4.4 Bovine Brucellosis -- -- -- -- -- -- -- -- 10

9

2.4.5 Caprine Brucellosis -- -- -- -- -- -- -- -- 11

2.4.6 Brucellosis in Sheep -- -- -- -- -- -- -- -- 12

2.4.7 Brucellosis in Horses -- -- -- -- -- -- -- -- 12

2.4.8 Brucellosis in Camels -- -- -- -- -- -- -- -- 13

2.4.9 Brucellosis in Pigs -- -- -- -- -- -- -- -- 13

2.4.10 Brucellosis in Chickens -- -- -- -- -- -- -- 14

2.4.11 Brucellosis in Marine Animals -- -- -- -- -- -- 14

2.4.12 Brucellosis in Humans -- -- -- -- -- -- -- 15

2.5 Growth Media and Growth Characteristics of Brucella Organisms -- -- 16

2.6 Transmission of Brucellosis -- -- -- -- -- -- -- 17

2.7 Reservoir of Brucella canis and Brucella abortus -- -- -- -- 19

2.8 Resistance and Survival of Brucella Organisms in the Environment -- -- 20

2.9 Zoonotic Potential -- -- -- -- -- -- -- -- 20

2.10 Pathogenesis -- -- -- -- -- -- -- -- -- 21

2.11 Pathology -- -- -- -- -- -- -- -- -- 23

2.12 Immunity -- -- -- -- -- -- -- -- -- 24

2.12.1 Humoral Immunity and Globulin Production During Infection -- -- 24

2.12.2 Cell Mediated Immunity -- -- -- -- -- -- -- 24

2.13 Diagnosis of Brucella Infections -- -- -- -- -- -- 25

2.13.1 Clinical Signs -- -- -- -- -- -- -- -- -- 25

2.13.2 Direct Smear Examination -- -- -- -- -- -- -- 26

2.13.3 Identification and typing -- -- -- -- -- -- -- 26

2.13.4 Animal Innoculation -- -- -- -- -- -- -- -- 27

2.13.5 Serological Diagnosis -- -- -- -- -- -- -- -- 28

2.13.5.1 Rose Bengal Plate Test (RPBT) -- -- -- -- -- -- 28

2.13.5.2 Serum AgglutinationTest (SAT) -- -- -- -- -- -- 29

2.13.5.3 Rapid Slide Agglutination Test (RSAT) -- -- -- -- -- 29

2.13.5.4 Agar Gel Immunodiffusion Test (AGID) -- -- -- -- -- 30

2.13.5.5 Agglutination Tests -- -- -- -- -- -- -- -- 30

2.13.5.6 Other Serological Tests -- -- -- -- -- -- -- 30

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2.14 Treatment of Brucellosis in Animals and Humans -- -- -- -- 31

2.15 Public Health Importance -- -- -- -- -- -- -- 32

2.16 Economic Importance of Brucellosis -- -- -- -- -- -- 33

2.17 Control, Prevention and Eradication of Brucellosis -- -- -- -- 34

2.17.1 Vaccines -- -- -- -- -- -- -- -- -- 35

2.17.2 Control and Prevention of Brucellosis in Humans -- -- -- -- 35

CHAPTER THREE: MATERIALS AND METHODS -- -- -- -- 37

3.1. Study design -- -- -- -- -- -- -- -- -- 37

3.2. Area of study -- -- -- -- -- -- -- -- -- 37

3.3 Map of Enugu State -- -- -- -- -- -- -- -- 38

3.4 Map of Anambra State -- -- -- -- -- -- -- 39

3.5 Study Duration -- -- -- -- -- -- -- -- 40

3.6 Study Population -- -- -- -- -- -- -- -- 40

3.7 ` Sample Size Determination -- -- -- -- -- -- -- 40

3.8 Sampling Technique -- -- -- -- -- -- -- -- 40

3.9 Sample Collection -- -- -- -- -- -- -- -- 41

3.10 Sample Analysis -- -- -- -- -- -- -- -- 41

3.10.1 Serological Tests -- -- -- -- -- -- -- -- 41

3.10.2 `Test Procedure -- -- -- -- -- -- -- -- 42

3.10.2.1 Procedure for Rose Bengal plate test (RBPT) -- -- -- 42

3.10.2.2 Procedure for Serum Agglutination Tests (SAT) -- -- -- -- 42

3.10.2.3 Procedurefor the ImmunoComb® Canine Brucellosis Antibody Test Kit Specific

for B. canis Antigen -- -- -- -- -- -- -- -- 43

3.11 Statistical Analysis -- -- -- -- -- -- -- -- 44

CHAPTER FOUR: RESULTS -- -- -- -- -- -- -- 45

4.1 Distribution of Dogs Based on States and Sources where samples were collected 45

4.2 Prevalence of Brucella abortus Antibodies based on the Sources of Dogs -- 47

4.3 Prevalence of Brucella canis Antibodies based on the Sources of the Dogs -- 49

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4.4 Antibody Titre Levels of B. canis Positive Dogs Screened in Anambra and Enugu

States -- -- -- -- -- - -- -- -- 51

4.5 Antibody titre Levels of Brucella canis Positive Dogs based on the Sources

of Dogs -- -- -- -- -- -- -- -- -- -- 53

4.6 Sex Distribution of Brucella canis Antibody Positive Dogs -- -- -- 55

4.7 Antibody Titre Levels of B. canis Positive Dogs According to Sex -- -- 57

4.8 Age Distribution of Brucella canis Antibody Positive Dogs (Clinical and

Household dogs) -- -- -- -- -- -- -- -- 59

4.9 Breed distribution of Brucella canis Antibody Positive Dogs -- -- 61

CHAPTER FIVE: DISCUSSION -- -- -- -- -- -- -- 63

Discussion -- -- -- -- -- -- -- -- -- 63

CHAPTER SIX: CONCLUSION AND RECOMMENDATION -- -- 68

6.1 Conclusion -- -- -- -- -- -- -- -- -- 68

6.2 Recommendation -- -- -- -- -- -- -- -- 69

REFERENCES -- -- -- -- -- -- -- -- -- 70

APPENDICES

Appendix I Sources Where Samples Where Purposively Collected -- -- 93

Appendix II Animal Profile -- - -- -- -- -- -- 94

Appendix III Sample Size Determination -- -- -- -- -- -- 96

Appendix IV Odds Ratio And (95% C.I) From Multivariate Logistic Regressions

Comparing Seropositive And Seronegative Dogs For Brucella Canis

Antibodies In Anambra And Enugu States In Southeast, Nigeria -- -- 97

Appendix V Risk Factors associated with the presence of Brucella canis antibodies

in dogs presented at the Clinics and Household dogs sampled in

Southeast Nigeria-- -- -- -- -- -- -- -- 98

Appendix VI Components of the Canine Brucellosis Antibody Test Kit Used -- 99

Appendix VII Harvested Serum Samples Ready to be analyzed -- -- -- -- -- 100

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Appendix VIII Immuno-Comb Showing Positive and Negative Serum Samples-- 101

Appendix IX Dog Market/Slaughter Point at Igboukwu Market-- -- -- --- 102

Appendix X Dog Market/Slaughter Point at Orie Orba Market -- -- -- --- 103

Appendix XI Dogs Ready To Be Slaughtered For Meat At Orie Orba Market--- 104

LIST OF TABLES

Table Title Page

4.1 Distribution of Dogs Based on States and Sources where samples were collected 46

4.2 Prevalence of Brucella abortus Antibodies based on the Sources of Dogs - 48

4.3 Prevalence of Brucella canis Antibodies based on the sources of the Dogs screened 50

4.5 Antibody titre Levels of Brucella canis Positive Dogs based on the Sources

of Dogs-- -- -- -- -- -- -- -- -- -- 54

4.6 Sex Distribution of Brucella canis Antibody Positive Dogs -- -- -- 56

4.7 Antibody Titre Levels of B. canis Positive Dogs According to Sex -- -- 58

4.8 Age Distribution of Brucella canis Antibody Positive Dogs (Clinical and

Household dogs) -- -- -- -- -- -- -- -- 60

4.9 Breed Distribution of Brucella canis Antibody Positive Dogs -- -- 62

LIST OF FIGURES

Figure Title Page

1. Antibody titer levels of B. canis positive dogs screened in Anambra and Enugu States -- 52

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ABSTRACT

A cross-sectional survey was used to assess the seroprevalence of Brucella canis and Brucella

abortus antibodies in dogs in Anambra and Enugu States, Nigeria. The objectives of the study

were to determine the seroprevalence of B. canis and B. abortus in dogs presented to major vet

clinics, dogs for slaughter in selected areas and apparently healthy owned dogs in visited

households in Enugu and Anambra States; to determine the sex, age and breed distribution of B.

canis and B. abortus antibodies in screened dogs in both states and the possible risk factors for

brucella infections in the study area. Five major veterinary clinics, two each in Enugu and

Onitsha metropolis, one in Nsukka Urban; 2 major slaughter points in each state; and households

with dogs that have history of infertility or abortion were selected by purposive sampling

method. The study population was made up of 3 groups: dogs presented at veterinary clinics,

household dogs with history of infertility or abortion and, dogs slaughtered for meat at markets.

Visits were made to the purposively selected veterinary clinics, households, and slaughter points,

once every other week for six months. A total of 123 dogs made up of 65 clinic dogs, 34

slaughter dogs and 24 household dogs were screened. Profiles of the dogs presented at the clinics

and household dogs were also collected. Blood was collected from each dog and processed for

serology. For B. abortus antibody assay, the serum was subjected to Rose Bengal plate test

(RBPT) and Serum agglutination test (SAT). With the SAT, a titer value of 40IU/ml and above

was regarded as positive. For B. canis antibody identification, Solid Phase Immunoassay

technique using Immunocomb®

Canine Brucellosis Antibody Test Kit was used. Titer value of

1:200 (IFA titer) and above was regarded as positive. Chi-square statistic and odds ratio were

used to analyze the data obtained. Out of the 123 dogs, screened, none was positive for Brucella

abortus antibodies using both the Rose Bengal Plate Test (RBPT) and the Serum Agglutination

Test (SAT). Thirty-four (27.7%) of the dogs screened were positive for B. canis antibodies using

the Solid Phase Immunoassay Technique. Twenty-two out of 65 (18%) dogs presented to

veterinary clinics, 8 out of 24 household dogs (6.5%), and 4 out of the 14 slaughter dogs (3.3%)

were positive for B. canis antibodies. There was a strong association (p<0.05) between infection

and sex with the infection being significantly higher (p<0.05) in female than in male dogs.

Prevalence was significantly higher (p<0.05) in foreign breeds than in mixed and local breeds.

There was no association (p>0.05) between the infection and the antibody titer levels of the

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different categories of dogs. Dogs presented at the clinics and household dogs with titer levels

1:600 and above had history of recent abortion or infertility. There was significant association

(p<0.05) between the presence of Brucella canis antibodies and free roaming of dogs. This study

provides the first serological evidence of B. canis infection but found no evidence of antibodies

to B. abortus in dogs in Enugu and Anambra States. This shows that B. canis is endemic in both

states and can be considered as a new emerging disease, underscoring the need for further studies

including isolation of bacteria and DNA extraction . Female dogs, exotic breeds of dogs and free

roaming of dogs are at a higher risk of brucella infection in the study area. Therefore, preventive

and controlling measures are strongly recommended.

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SEROPREVALENCE AND RISK FACTORS OF

BRUCELLA INFECTION IN DOGS IN ENUGU AND

ANAMBRA STATES.

BY

ANYAOHA, CHIDIEBERE OHAZURIKE

PG/M. Sc/10/52913

DEPARTMENT OF VETERINARY PUBLIC HEALTH AND

PREVENTIVE MEDICINE,

FACULTY OF VETERINARY MEDICINE

UNIVERSITY OF NIGERIA,

NSUKKA.

FEBRUARY, 2015

16

CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

Brucellosis is considered by the Food and Agriculture Organization (FAO), the World Health

Organization (WHO) and the Office of International des Epizootics (OIE) as one of the most

wide spread zoonoses in the world (Schelling et al., 2003). According to OIE, it is the second

most important zoonotic disease in the world after rabies. The disease affects cattle, swine,

sheep, goats, camel and dogs. It may also infect other ruminants and marine mammals (Corbel,

1988; Radostits et al., 1995; Abubakar et al., 2012). Synonyms of brucellosis include: Undulant

fever, Malta fever, Mediterranean fever, Enzootic abortion, Epizootic abortion, Contagious

abortion, and Bang’s disease. It is an important Zoonotic disease and causes significant

reproductive loss in sexually mature animals (Forbes and Tessaro, 1996; Wadood et al., 2009).

The disease is manifested by late term abortions, weak calves, still births, infertility and

characterized mainly by placentitis, epididymitis and orchitis, with excretion of the organisms in

uterine discharges and milk (England et al., 2004). In females the most prominent sign is

abortion after 45-55 days of gestation in about 75% of the cases; early embroyonic death and

resorption, or abortion 10-20 days after mating may occur in some cases (Shin and Carmicheal,

1999). The organism occurs in the fetus, placenta, fetal fluids and vaginal discharges after an

abortion or stillbirth. This organism can be found in vaginal discharges for 4 to 6 weeks after

abortion; it is also shed in normal vaginal secretion; particularly, during estrus, as well as in milk

(Iowa State University, 2012). In males, the main sign is epididymitis of one or both testes and

infertility (Shin and Carmiceal, 1999). B. Canis infection are found in semen for up to two

months after infection and also found in urine, saliva, nasal and occur in secretions, and feces.

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Worldwide, nine species of the genus Brucella have been recognized and the genus Brucella

contains a group of very closely related bacteria. The first member of the group, Brucella

melitensis, affects primarily sheep and goats. The second member of the group, B. abortus,

affects primarily cattle B. suis affects pigs, B. Ovis affects sheep, B. canis affects dogs while the

other members include B. neotomae, B.microti, B. ceti and B. pinnipedialis (Corbel, 1988; Sati,

2002; Dauglas, 2006; Foster et al., 2007; Scholtz et al., 2008). Cross transmission of brucellosis

can occur between cattle, swine, sheep and goats and other species including dogs, horses, bison,

rein deer and camels (FAO, 2003). Dogs can be infected by four of the six species of Brucella

(Brucella canis, Brucella abortus, Brucella melitenses and Brucella suis, excluding Brucella ovis

and Brucella neotomae) (Hollet, 2006). Outbreaks of canine abortions had been reported in 1963,

but it was not until 1966 -1967 that B. canis was isolated from tissues and vaginal discharges of

dogs. (Carmicheal, 1966; Carmicheal et al., 1967, Taul et al., 1967).

Brucellosis was first recognized as a disease affecting human-beings on the Island of Malta in

the 19th

and early 20th

centuries when Brucella melitensis was first isolated from the spleen of a

resident of the Island of Malta who died from a disease locally known as Malta fever (Brunner

and Gilespire, 1966). In Nigeria, an outbreak of brucellosis was first reported in a government

cattle farm located in Zaria in 1934. Serum plate agglutination test showed positive reactors of

about 15% of the total animals in the farm (Banerjee and Bhalty, 1970).

Brucellosis as a zoonoses poses serious human health hazards worldwide (Hamidy et al., 2002;

Maloney and Fraser, 2004; Cadmus et al; 2006). Ruminants, pigs, horses, dogs and donkeys play

an important role in the transmission of this disease to man (Cadmus et al., 2006). In human, the

infection is acquired by consumption of contaminated food of animal origin, and through aerosol

(Gul and Khan, 2007). Infection can also result through contact with infected aborted materials

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such as aborted fetuses, placenta membranes or fluids and other vaginal exudates

(FAO/OIE/WHO, 2006).

Brucellosis has a considerable impact on animal and human health, as well as wide socio-

economic impacts, especially in countries in which rural income relies largely on livestock

breeding and dairy products (Maadi et al., 2011). It also causes morbidity and considerable loss

of productivity (Pappas et al., 2006). The disease is important from economic point of view; it is

one of the most devastating trans-boundary animal diseases and also a major barrier for trade

(Gul and Khan, 2007). Occupationally, Brucella can gain entry into humans especially

veterinarians, animal handlers, butchers, abattoir workers through abraded skins, mucous

membranes, conjunctiva, respiratory and gastro intestinal tracts.

1.2 Statement of the Problem

Dogs are the closest domestic animals to humans in Nigeria thus its interactions with human the

populace may lead to an increase in the risk of infection (Cadmus et al., 2012).

In dogs, the infection is insidious and many are asymptomatic (Ewalt and Bricker, 2000); with

the infected dogs shedding the organisms via urine, vaginal secretions, ejaculates, fetuses and

feces and carrier dogs can be a source of infection and transmission to households (Brooks,

2006).

The increase in dog ownership in Nigeria is associated with some risk factors that render them

vulnerable to brucellosis. Firstly, many exotic breeds are imported and are not screened before

entry into the country (Cadmus et al., 2011). Secondly, some household dogs are fed with fetuses

from cows and ruminants from slaughtered cattle with a history of bovine brucellosis from

abattoirs (Cadmus et. al, 2010). In addition to these factors, some household dogs roam around

19

freely, placing them at greater risk of exposure to brucellosis. Also non-screening of mating dogs

used for breeding predisposes to the high risk of infection among breeding kennels.

In both clinical and household dogs; as well as in Kennel dogs, abortion was recognized but not

considered to be from infections with Brucella. Abortion due to trypanosomosis and other causes

had also apparently been confused with Brucella abortion.

Increased consumption of dog meat has led to a spike in the slaughter of unscreened apparently

healthy dogs for meat, hence an increase in risk of infection to the handlers, butchers, etc.

Symptoms in human brucellosis can be highly variable, ranging from non-specific, flu-like

symptoms (acute form) to undulant fever which may progress to a more chronic form and can

also produce serious complications affecting the musculoskeletal, cardiovascular, and central

nervous systems, arthritis, orchitis and epidiymitis and may be confused with a wide range of

other diseases (Baba et. al, 2001). The cost of treatment and work days lost to ill health,

combined with the loss of productivity in the animal husbandry, leads to decreased availability of

food that adversely affects the health and economic well being of the population (Chauhan et al.,

2000; Baba et al, 2001).

Although brucellosis is a notifiable disease in Nigeria, the incidence, prevalence and distribution

of the disease is difficult to determine as the system of disease surveillance and reporting is

fragmentary and inefficient (Ocholi et al., 2004). Serological prevalence rate between 0.20% and

79.70% have been reported in animals and humans in various parts of the country (Esuroso,

1974; Chukwu, 1987b; Ocholi, 1993; Onunkwo et al., 2005; Junaidu et al; 2008); much

emphasis is laid on cattle, sheep and goat; the same cannot be said of dogs.

It is also regarded as a disease of public health significance as it is zooonotic in nature, thereby

making occupationally risk persons like butchers, abattoir workers, livestock owners and rearers,

20

veterinarians as well as other humans susceptible (Yagupsky and Baron, 2005; Belizadi and

Mogheiseh, 2011). Thus, there is need to know more about the disease.

1.3 Research Questions

1. Are B. canis and B. abortus present in dogs presented in major Veterinary clinics in

Enugu and Anambra States?

2. Are B. canis and B. abortus present in slaughter dogs in areas purposively selected for

screening in Enugu and Anambra States?

3. Are B. canis and B. abortus present in apparently healthy owned dogs not presented to

clinics?

4. What is the overall prevalence rate of Brucella infection in dogs in the study area?

5. What is the age, sex and breed distribution of Brucella infection in dogs in the study

area?

6. Are there possible risk factors that may influence infection rate in dogs in the study area?

1.4 Aim and Objectives of the Study.

The aim of this study is to conduct a seroprevalence survey of Brucella canis and Brucella

abortus infections in dogs in purposively selected areas of Enugu and Anambra States and to

determine the possible risk factors that may influence infection.

The specific objectives are:

1. To determine the seroprevalence of B. canis and B. abortus in dogs presented to major

veterinary clinics in Enugu and Anambra States.

21

2. To determine the seroprevalence of B. canis and B. abortus in dogs for slaughter in

selected areas in both states.

3. To determine the prevalence of B. canis and B. abortus in apparently healthy owned dogs

in visited households in both states.

4. To determine the overall prevalence rate of Brucella infections in the study area.

5. To determine the sex, age and breed distribution of B. canis and B. abortus in screened

dogs in Enugu and Anambra States.

6. To determine the possible risk factors that may influence the infection rates in dogs in the

study area.

1.5 Significance of the Study

This work will provide information on the current seroprevalence of Brucella antibodies in dogs

in these States as well as the prevalent species.

The study will also provide information on sex, breed, and age distribution as well as some risk

factors that may influence Brucella infection in dogs therefore contributing to the epidemiology

of Brucella infection in dogs in Anambra and Enugu States.

The result will also be used to provide an appropriate recommendation to dog owners,

veterinarians, butchers, cooks, public health authorities and government to institute urgent

measures towards the control of the disease.

22

CHAPTER TWO

2.0 LITREATURE REVIEW

2.1 Historical Review

Recent examination of the ancient Egyptian bones, dating to around 750BC, shows

evidence of sacroiliitis and other osteoarticular lesions, common complications of brucellosis

(Pappas and Papadimitirous, 2006). Brucellosis became a problem for the British Garrison in

Malta (Malta fever) with substantial morbidity and mortality among the soldiers. Dr David

Bruce, a military medic, was sent to try to deal with the problem. He coordinated a team of

scientific personnel which succeeded in 1887 in isolating Micrococcus melitenses from the

spleen of British soldier as a causative agent from raw goat milk consumed by the military

personnel (Bruce, 1887; Bruce, 1893).

In 1897, a Danish veterinarian, Dr. Frederic Bang identified an intracellular microorganism

described as Bacillus abortus as the cause of abortion in cattle. The disease was named after him,

“Bang’s disease”. Bang’s discovery was of interest only to veterinarians, dairy farmers and meat

producers. It had no impact on physicians and none of them made any correlation between

Bang’s disease and Malta fever as the organism in Malta fever, was described as “Micrococcus”

and Bangs disease as “Bacillus”. Twenty one years later an American microbiologist, Alice

Evans reported the close relationship between Bacillus abortus and Micrococcus melitenses

(Evans, 1918). Evans also identified Bacillus abortus in cow milk; she suggested a new name for

Malta fever and called it ‘Brucellosis’. Brucella suis was isolated in 1914 from aborted swine

fetuses (Smith et al., 1972). Brucella ovis was discovered by Buddle and Boyles, (1953) as an

organism affecting rams causing sterility. Stoener and Lackeman, (1957) isolated Brucella

neotome from desert wood rat (Neotoma lepida) in U.S.A. Carmicheal, (1966) an American

23

veterinarian from Cornell University first reported Brucella canis as the cause of abortion in

beagles. Brucella canis was first isolated in dogs by Carmicheal and Kenney in 1968. In 1910, a

British team led by Dr. David Bruce first isolated and confirmed brucellosis in Africa in the

Ankota district of Western Uganda by the isolation of Brucella melitensis from goats and man

(Smith et al., 1972).

2.2 Aetiology

Brucellosis is caused by Gram negative bacteria of the genus Brucella, which are facultative

intracellular cocobacilli that belong to the α2- proteobacteriacea (Yanagi and Yamasato, 1993).

Nine Brucella species are currently recognized, seven of them that affect terrestrial animals are:

B. abortus, B. melitenses, B. suis, B. ovis, B. canis, B. neotomae and B. microti (Scholz et al;

2008) and two that affect marine mammals are: B. ceti and B.pinnipedalis (Foster et al. 2007). B.

melitenses, B. suis and B.abortus are considered the most pathogenic species for humans and

have small ruminants, pigs and cattle as preferred hosts respectively (Godfroid et al., 2005).

B.ceti and B. pinnipedialis, can also cause human brucellosis (Foster et al; 2007). Importantly, B.

canis, a pathogen of dogs has a comparatively low zoonotic potential, while B. neotomae and B.

ovis that infect desert rats and sheep respectively, are not associated with human disease. Nine

biovas are recognized for B. abortus (Alton et al; 1988), three for B. melitenses and five for B.

suis. The remaining species have not been differentiated into biovars (Seleem et al; 2010).

[ 2.3 Classification

The genus Brucellae belongs to the order Rhizobiales within the class alpha-protobacteria

(Gupta, 2005; Williams et al; 2007; Whatmore et al., 2009). Although Brucella represents

24

intracellular animal pathogens, it shares close relationship with soil organisms. The family

Brucella consists of the genera Brucella, Mycoplasma and Octobacterium (Kaamfer et al.,

2006).

2.4 Epidemiology

2.4.1 Distribution of Brucellosis

Brucellosis has been reported from United States (particularly in Southern States), Canada,

Central and South America, some European Countries, Tunisia, Nigeria, Madagascar, Malaysia,

India, Korea, Japan and China; Brucellosis is probably found throughout most of the world;

however, New Zealand and Australia appear to be free of this organism (Iowa State University,

2012).

Dogs are the only species known to be affected by B. canis, however antibodies to this organism

have been found in other carnivores. Experimental infections can be established in domesticated

livestock and chimpanzees; however, these species are considered highly resistant to natural

exposure (Iowa State University, 2012). There are reports that dogs can also be infected with

other Brucella species: B. melitenses, B. abortus.

2.4.2 Brucellosis in Nigeria

Investigations and reports have shown that brucellosis is endemic in Nigeria (Halle and Ajogi,

1997). Evidence of, as well as frank out breaks of disease have occurred in cattle (Esuruoso,

1974; Nuru and Dennis, 1975; and Ajogi 1997) human beings (Banarjee and Bhalty, 1970;

Alausa 1984) sheep and goats (Falade et al., 1974; Bale et al., 1982; Brisibe et al., 1993) camels

25

(Oko, 1979). Initially there were doubts as to the presence of canine brucellosis in Nigeria when

Falade (1977) found no antibodies in dogs. The first confirmed reports of brucella in dogs were

cultural recovery of Brucellea canis from a case of abortion in an imported female boxer (Oko,

et al., 1978). Recent studies suggest increasing trend in the prevalence of the disease (Ocholi et

al., 2004); but due to poor disease reporting system in Nigeria, the distribution of the disease is

not documented. Subsequently, disease surveillance has not been very effective leading to poor

determination of the incidence and the prevalence of Brucellosis in Nigeria.

2.4.3 Canine Brucellosis

Brucellosis posses serious human health hazards worldwide (Hamidy et al., 2002, Maloney and

Fraser, 2004; Cadmus et al., 2006) while some countries have eliminated or substantially

reduced the disease by extensive eradication programmes, it remains endemic in many areas of

the world, including Nigeria (Cadmus et al, 2010). The canine brucella was first recognized in

1966 as the cause of abortions and reproductive failures, and it has since been recognized in

several countries (Shin and Carmichael 1999). However, with the current rise in dog breeding

using imported dog species and the attendant random movement of dogs through trade, and

increased slaughter of dogs as meat in the country, the spread of brucellosis among dogs in the

country is most likely to increase. This is because puppies or adult dogs from infected kennels

may be sold to non-infected kennels (Bertu, 2006).

2.4.4. Bovine Brucellosis

B. abortus the cause of bovine brucellosis, is transmitted by contact with the placenta, fetus and

placental, fetal and vaginal fluids from infected animals. Animals are infectious after either

abortion or full-term parturition. B. abortus may also be found in the milk, semen, feces and

26

hygroma fluids. Shedding in milk can be prolonged or lifelong or may be intermittent

(Bercovich, 1998). Other natural hosts are horses and humans but are not considered important in

the maintenance of the disease. Infection with B. abortus, occurs rarely in pigs, sheep and goats

(Sati, 2002). Infection occurs in cattle of all ages but persists in sexually matured animals; with

females being more susceptible than males (Hungerford, 1975). Pregnant females are more likely

to be more infected than non pregnant females and males because of a high concentration of

erythriol in gravid uterus which sustains growth of the organism (Sati, 2002). Congenital

infection may occur in calves born from infected dams, infection occurring in utero and may

remain latent in the calf and the animal may remain serologically negative until first trimester of

which it may begin to shed the organism to the environment. Isolation of the organism in dogs

has been found in a farm where cattle were serologically positive to brucellosis (Radostis et al.,

1995). Infection has been recorded in other animals such as bison, elk, heeves, deer, coyotes and

other wild ruminants (Radostis et al., 1995).

2.4.5 Caprine Brucellosis

Nigeria has a large population of domestic ruminants with goats estimated at 25.5 million being

the most numerous, followed by sheep and cattle with 14.5 and 12.5 million respectively

(FAO/OIE/WHO, 1995). Brucellosis in goat has been reported in various parts of Nigeria

(Falade, 1974, Bale, 1980; Ogundipe et al., 1994) all revealing that prevalence rates vary

between 2.1-14.5%. Recent studies showed a prevalence of 22.93% in Sokoto State (Junaidu et

al., 2008) and Adamu, 2012 showed a prevalence of 5.6% (Adamu et al., 2007).

27

2.4.6 Brucellosis in Sheep

Brucellosis in small ruminants (sheep and goats) has been reported in Northern Nigeria (Bale et

al., 2003). Brucellosis in sheep and goats is usually caused by B. melitensis. Infection with B.

abortus is rare, although the association of B. abortus with abortion in sheep has been

demonstrated in several countries through isolation of the organism (Luchsinger and Anderson,

1967; Zowghi and Ebadi, 1988; Ocholi et al., 2005). While a broad host range generally exists

for Brucella species, Brucella infection follows a very strict, host-related hierarchy of

pathogenicity (Adams, 2002). Thus, goats are the natural hosts of B. melitensis and sheep are

preferred hosts of the pathogen (Ocholi et al., 2005). Prevalence rates vary throughout and even

within the same geographical zones operating different husbandry techniques (Eze, 1977; Bale et

al., 2003). Recent studies suggest increasing trend in the prevalence of the disease (Ocholi et al.,

2005). A recent sero-epidermiological survey of brucellosis in sheep reveals a prevalence rate of

14.3% in Bauchi State (Ocholi et al., 2005) and a prevalence of 14.5% (Bertu et al., 2010) in

Plateau State. This further proves the confirmation of the existence of ovine brucellosis in

Nigeria.

2.4.7 Brucellosis in Horses

Indigenous horses have been used by the army and police force in Nigeria for ceremonial

parades, by mounted troops during special occasions to welcome dignitaries, and control crowds.

They are accorded special attention due to the immense role they play in polo games, cultural

festivals and security. The risk of disease transmission passed by these horses to riders, handlers,

and the general public may be significant. Reports on equine brucellosis are rare (Ocholi et al.,

2004) despite the endemicity of this disease in Nigeria.

28

2.4.8 Brucellosis in Camels

Brucellosis is a serious zoonotic disease affecting man and all domestic animals including

camels (Radostits et al., 2007). The camel plays vital socio-economic roles and supports the

survival of millions of people in the semi dry and arid zones of Asia and Africa. Although

camels were considered in the past, and for a fairly long time, as resistant to many disease

causing factors (Zaki, 1948; Dalling et al., 1988), it has been proved that they are susceptible to

common disease causing pathogens affecting other animal species (Wilson 1984; Abbas and

Tilley, 1990; Abbas and Agab 2002). However, camel brucellosis, has not received proper

attention from researches and scientists. Brucellosis was reported in camels as early as 1931

(Solonitsunin, 1949); since then the disease has been reported from all camel – keeping

countries: Libya with 4% (Azwai et al., 2001), Ethiopia with 5.7% (Teshome et al; 2003) and

Nigeria with 11.4% (Junaidu et al., 2006). Camels are not known to be primary hosts of

Brucella, but they are susceptible to both B. abortus and B. metitensis (Cooper, 1991). The

appearance of brucellosis in camels, depends on the Brucella species being prevalent in other

animals sharing their habitat, (cross-transmission between species) and on the husbandry system

(Musa et al., 2008). Camels play an important role in the epidemiology of brucellosis because

the disease may spread through milk (camel milk) especially to those, living in dry and arid

zones.

2.4.9 Brucellosis in pigs

Because of differences in cultural and religious beliefs, pig production in Nigeria is limited only

to a few states; from the middle belt to the southern parts, therefore, this limits the existence of

this disease to this region in pigs. Prevalence rate by Cadmus et al., (2006) in Ibadan, Onunkwo

29

et al., (2011) in South East Nigeria, implies that the prevalence of brucellosis among swine

population in Nigeria is on the increase. Previous studies have also stated that pigs infected with

Brucella remain so for life and continue to shed the organism (Lucero et al, 2005; OIE, 2009;

Godfroid et al., 2010). This indicates that these infected animals will still remain infected and

thus pose a threat to humans and other livestock if effective control measures are not adopted

(Bello-Onaghise et al., 2012).

2.4.10 Brucellosis in Chickens

Chickens are kept in most part of Nigeria due to their nutritional and economic importance (Baba

et al., 2001; Junaidu et al., 2006). Chickens have been reported to be susceptible to brucella

infection (Abdallah et al., 1984) causing a decrease in egg production in infected hens with the

recovery of brucella from the egg shell, egg yolk and white droppings and internal organs of

infected birds (Abdallah et al., 1984). A recent survey carried revealed 0.67% prevalence in

Kaduna state (Gugong et al., 2012), with antibodies to Brucella demonstrated in the local

chickens indicating that there is avian brucellosis in the locality sampled and in Nigeria in

general. Also in another survey where B. melitensis and B. abortus antigen were used, there was

a prevalence of 3.0% of B. melitensis in the number of birds sampled (Junaidu et al., 2006).

2.4.11 Brucellosis in Marine Animals

There is little information on the effects of brucellosis in marine mammals. Prior to the first

reports, in 1994, of brucellae isolations from stranded harbor seals (Phoca vitulina), harbor

porpoises (Phocoena phocoena) and common dolphins (Delphinus delphis) on the coast of

Scotland (Ross et al., 1994); and from an aborted fetus of a captive bottlenose dolphin (Tursiops

30

truncates) in California (Ewalt et al; 1994); brucellosis was not only unrecognized, but was not

even suspected in marine mammals. However, studies carried out indicated isolation of Brucella

from cetaceans and pinnipeds inhabiting seas and oceans of Europe and North America (Ross et

al; 1996; Forbes et al; 2000; Cloeckaert et al., 2001) or kept in captivity (Ross et al; 1994). In

the recent study concerning experimental infections of Nile Catfish with B. melitenses biovar 3,

the bacteria were successfully cultured from visceral organs, suggesting that these fishes are

susceptible to Brucellae (Saleem and Moshen 1997). However, in one case, exposure of a

laboratory worker to marine mammal brucellae revealed that such bacteria may also be

pathogenic to humans (Brew et al., 1999).

2.4.12 Brucellosis in Humans

Brucellosis in humans is hardly diagnosed in hospitals in Nigeria despite suggestions that the

magnitude of infections may be greater than appreciated (Njoku 1995; Rajis et al., 2003). This

absence may be due to other diseases with similar clinical signs that are endemic and hence often

diagnosed. Such diseases are typhoid fever, malaria and pasteurellosis with signs which include

acute recurring fever chill, headache, fatigue, night sweat and anorexia (Rajis et al., 2003). The

first case of brucellosis in a human being was in 1962 (Collard, 1962) when Brucella antibodies

were demonstrated in the sera obtained from healthy persons. In a recent study carried out in

Makurdi, North Central Nigeria, a prevalence rate of 7.6% was obtained in the study area, and

this agrees with other findings of other workers who reported high seroprevalence of the range of

6% to 28% among hospital patients in Nigeria (Collard 1962; Falade 1978; Asanda and Agbede,

2001; Edu, 2005). Also other studies revealed that Brucella abortus was the main cause (77.2%)

of human brucellosis (Ocholi, et al., 1993; Asanda and Agbede, 2001; Junaidu et al; 2004). Most

31

cases of human brucellosis were occupational hazards occurring amongst workers in the

livestock industry. Epidermiological studies have revealed significantly higher prevalence of

infection among occupationally exposed person (Bertu, 2006).

2.5. Growth Media and Growth Characteristics of Brucella Organism

There is a range of commercially available culture media for growing Brucella. The most

common basal media in use are: Triptcase soy (BBL®), Bacto Tryptose (Difco®), Triptic soy

(Gibco®), Tryptone soya (Oxoid®). The powder media can be used to prepare either broth or

agar medium. For culturing blood and other body fluids, it is preferred to use broth or a biphasic

medium (Castañeda), mainly because Brucella is often present in small numbers. Most Brucella

strains, particularly B. abortus biovar 2 and B. ovis, grow better in media containing 5-10% of

sterile (equine or bovine) serum free from Brucella antibodies (Fernando et al., 2010). During

the primary culture, it is very important to add antibacterial and antifungal agents to suppress the

growth of contaminants. Such agents are: 100 mg of cycloheximide (fungistat), 25,000 units of

bacitracin (active against gram-positive bacteria) and 6,000 units of polymyxin B (active against

gram-negative bacteria) (Sati, 2002; Fernando et al., 2010).

After 48-72h of incubation at 37°C, Brucella colonies are 0.5 to 1.0 mm in diameter with a

convex and circular outline. Smooth strains are transparent and pale yellow, resembling droplets

of honey with a shiny surface when observed in transmitted light. Rough colonies are more

opaque with a granular surface. Dissociation of Brucella can be detected by the emulsification of

a colony in 0.1% w/v aqueous acriflavine (Braun and Bonestell, 1947). Smooth colonies produce

a yellow uniform suspension whereas rough colonies produce granular agglutinates. Colonial

variation can be detected also by examining the plates under oblique light after staining the

32

colonies with crystal violet (White and Wilson, 1951). . Smooth colonies appear translucent and

pale yellow and rough colonies are stained with red, purple or blue with opaque and granular

appearance. Colonial morphology, staining, slide agglutination with anti-Brucella serum (smooth

or rough), urease, catalase and oxidase tests are the basis for a culture to be identified as

belonging to the genus Brucella. Once a culture has been identified as Brucella, it is important to

classify the species and the biovars. This further classification should be done in specialized or

reference laboratories. These tests are cumbersome and include carbon dioxide requirement

(CO2), production of hydrogen sulphide (H2S), dye sensitivity (thionin and basic fuchsin), phage

lysis, agglutination with A, M or R specific antisera and in some cases it is necessary to use the

oxidative metabolic method. This latter test is time consuming and hazardous to laboratory

personnel. For these reasons it should be performed only by international reference laboratories.

2.6 Transmission of Brucellosis

Dogs are most commonly infected by contact with vaginal discharges at estrous or after

abortions, or by ingesting infected placentas or fetuses. At abortion, the placenta and the

discharges can contain up to 1010

colony forming units (cfu) per mL, and the oral infection dose

is 2 × 106

cfu. Experimentally, 104

cfu has been sufficient for conjunctival infection (Carmichael,

1976; Serikawa and Muraguchi, 1979; Carmichael et al, 1984a). Thus, 1 ml placental tissue or

vaginal discharge is equal to approximately 100, 000 infectious doses, and the bitches can have a

vaginal discharge for up to 6 weeks after an abortion. Dogs can also be infected at mating. It

should be observed that chronically infected dogs can be serologically negative and negative on

blood culture, although B. canis can be detected from the prostate, epididymis and semen, or in

female dogs in vaginal secretion, and thus they can still infect other dogs at mating or via

33

artificial insemination (Carmichael et al, 1984b; Keid et al, 2007). Environmental infection is

possible, especially from areas where dogs often urinate (Serikawa and Muraguchi 1979), or

where vaginal discharges are deposited. Dogs living together are at risk of infecting each other

bitches (Carmichael and Joubert, 1988). It was suspected that contaminated urine was an

important source of infection in these cases, especially from male dogs. The concentration of

bacteria in urine and semen is highest from 1 to 4-6 months after infection (Carmichael and

Joubert, 1988). B. canis can also be spread on fomites. In conditions of high humidity, low

temperatures, and no sunlight, Brucella spp. can remain viable for several months in water,

aborted fetuses, feces, equipment and clothing. Brucella species can withstand drying,

particularly when organic material is present, and can survive in dust and soil. Survival is longer

when the temperature is low, particularly when it is below freezing (Iowa State University,

2012).

Transmission of Brucella abortus is mainly through the oral route because animals tend to lick

aborted fetuses, placentas and vaginal discharges of animals that aborted (Cunningham and

Dolan, 1978). Ingestion of contaminated water and feed with Brucella abortus have been also

reported (Bercovich, 1998). Infection can also occur invitro or when calves and young animals

born to a healthy mother are fed colostrums or milk from infected cows (Cathleen and Sheem,

1986).

Transmission in humans is principally by contact with infected materials such as carcasses

aborted fetuses, placentas, vaginal discharges, manure, semen and urine (Sati, 2002). Man to

man transmission is very rare (Currier 1989). Other routes of infection in man include inhalation

of air droplets containing the organism (Currier, 1989). Accidental self inoculation with strain 19

of Brucella abortus vaccine is another possible way of contacting the disease (Weidman, 1991).

34

Because person to person transmission rarely occurs, infected persons do not pose a threat to

their surroundings; therefore, eradication of the disease from the natural animal reservoirs leads

to a dramatic decrease in the incidence of human infection (Cook, et al., 2002). Only a few cases

have been reported of transmission by blood transfusion and bone marrow transplants (Young,

1989). Possible breast milk transmission to infants (Lubani et al., 1988; Meador et al., 1989;

LaBrune et al., 1990) and possible veneral transmission from infected laboratory workers to their

spouse (Ruben et al., 1991) have also been documented.

2.7 Reservoirs of Brucella canis and Brucella abortus.

Only domestic and wild canids appear to be highly susceptible to B. canis infection. Cats are

moderately susceptible. Guinea pigs, mice, rats and non-human primates have been

experimentally infected, but the disease is relatively mild in these species (Forbes, 1990). Dogs

play a significant role in the epidemiology of bovine brucellosis. Naturally Brucella abortus

infection in dogs has been reported and outbreaks of cattle brucellosis in association with

infected dogs have been reported (Prior, 1976; Forbes, 1990). Sheep and goats do not easily

become infected with Brucella abortus (Alursurp, 1974), but once acquired; they become

carriers and continue to excrete the organism (Oko, 1980; Corbel, 1988). Studies also indicate

that wild life animals such as buffalos, wild pigs, bears, foxes, rodents, bison and elks are

susceptible to Brucella abortus and may serve as reservoirs for cattle (Cook et al., 1988: Davies

et al., 1990). Birds can also be a reservoir of Brucella abortus for cattle and humans especially in

Nigeria (Bale and Nuru, 1982; Kudi et al., 1997).

35

2.8 Resistance and survival of Brucella organisms in the environment

The survival of brucellae organism in the environment plays an important role in the

epidermiology of the disease. Temperature, humidity and pH influence the organism ability to

survive (Bercovich 1998). Alteration of the environment by people can change the exposure

potential for animals and increase the prevalence or even the occurrence of brucellosis in these

species of animals. Brucellae do not generally multiply outside the host animal, but they may

persist in the environment for variable periods of time depending on protection from heat,

extremes in pH and drying (Corbel, 1988). Brucellae, may persist nearly indefinitely if frozen

and protected from the environment in aborted fetuses or placentas. At cool temperature (10-15

oC) the organism may persist for months if kept moist, but at high temperatures (45-50

oC) the

organism will die in a few hours. Brucella can also survive in bovine feces for at least 1 year, in

liquid manure and frozen soil for up to 2 ½ years. Contaminated straw remains infected for more

than one month. Brucellae is sensitive to a wide range of disinfectants including strong acids and

alkali, hypchlorite, iodosphors, 1% lysol and formaldehyde (Corbel, 1988). Brucellae show high

sensitivity to a wide range of antibiotics and chemotherapeautic agents including penicillins,

aminoglycosides, chloramphenicol, tetracycline and aminocydotol. However, most strains of the

organism are resistant to nancomycin, bacitracin, polymyxins, metronidazole and antifugal

agents (Corbel, 1988).

2.9. Zoonotic potential of brucellosis

Five out of the nine known Brucella species can affect humans and the most pathogenic and

invasive species for human is B. Melitensis followed in descending order by B. suis, B. abortus

and B. canis (Acha and Szyfres 2003). The zoonotic nature of the mairne Brucella (B. ceti) has

36

been documented (McDonald et a.l, 2006; Sohn et al., 2003). B. melitensis, B suis and B abortus

are listed as potential bio-weapons by the centers for Disease Control and Prevention in the

USA. This is due to the highly infectious nature of all the three species as they can be easily

aerosolized. Moreover, outbreak of brucellosis would be difficult to detect because the initial

symptoms are easily confused with those of the influenza (Seelem et al., 2009). In places where

brucellosis is endemic, humans can get infected via contact with the infected animals for

consumption of their products. Some specific occupational groups including farm workers

veterinarians, ranchers and meat packaging employees are considered at higher risk (Tabak et al.,

2008). B. abortus and B. suis infection usually affect occupational groups while B. melitensis

infections occuring more frequently than the other Brucella species in the general population

(Acha and Szyfres, 2003; De-Masis et al., 2005) through the consumption of sheep or goat milk

containing B. melitensis (De-Masis et al., 2005). In human brucellosis, person to person

transmission rarely occurs, infected persons do not pose a threat to their surroundings.

Eradication of the disease from the natural reservoirs leads to a dramatic decrease in human

infection (De-Masis et al., 2005).

2.10 Pathogenesis

The pathogenic potential of Brucella spp is highly dependent on its ability to enter and survive

within host cells. Brucella does not have classic virulence factors such as exotoxins, capsule, or

endotoxic, lipopolysacharide (LPS) (Moreno and Moriyon, 2001). The major virulence

mechanisms of Brucella are those required for host cell invasion and intacellular survival and

replication (Lopez et al., 2002; Arellano et al., 2005; Lapaque et al., 2005). Brucella organisms,

enter the body via the mucosal membranes of the orophaynx, genital tract or conjunctiva

37

(Carmicheal and Kenney, 1970). The bacteria are then phagocytised by macrophages and other

phagocytic cells, and transported via the blood to lymph nodes, spleen and genital organs, where

they multiply (Carmicheal and Kenney, 1970). Hyperplasia of the lymphoid tissue is seen

throughout the body (Spink and Morisset, 1970). Bacteremia occurs 1 to 4 weeks after the

infection and persists for at least 6 months, and then reoccurs intermittently for up to 5 years.

(Carmicheal et al., 1984)

Brucella organism may invade and cause complications in the eye, kidney, and meninges. After

an extremely variable duration of bacteremia, dogs infected with brucella may spontaneously

recover and show decreasing serum agglutination titer. However, utilization of such dogs for

breeding is not recommended. When dogs recover, they are reported to be immune to re-

infection for 3 months up to 4 years. (Carmicheal 1976; Carmicheal and Greene, 1990).

Bacteremia may persist for varying periods of time depending on the host and Brucella species.

In goats infected with B. melitenses, bacteremia is detectable in 10-20 days and may persist more

than 300 days, although the duration is generally less than 2 months. In cattle infected with B.

abortus the onset of bacteremia is variable – from a few days up to 2 months or more and may

last 5 months or more. In swine infected with B. suis, bacteremia occurs early and generally lasts

60-90 days but may persist for more than 3 years (Carmicheal and Greene, 1990). Bacteremia

with B. canis has been shown to be dose dependent. The organism has been detected with a

range of 1-4 weeks post-experimental exposure; in some cases the bacteremia was still detectable

at 1,120 days (Carmicheal and Greene, 1990).

38

2.11 Pathology

Lymphadenopathy of the retropharyngeal or inguinal lymph nodes or generalised

lymphadenopathy sometimes occurs (Carmichael and Kenney, 1970). Discospondylitis,

endocarditis and polygranulomatous dermatitis has been reported (Kerwin et al., 1992; Iowa

State University, 2012). Dogs that were experimentally infected with B. canis often showed

recurrent uveitis for several months after the infection (Carmichael, 1976) and there are several

case reports describing ocular signs, such as endophthalmitis and anterior uveitis, in dogs

infected with B. canis (Vinayak et al., 2004, Ledbetter et al., 2009). Male dogs are reported to

have enlarged scrotums often accompanied by surface dermatitis with possible discomfort at the

time of ejaculation. Lymphadenopathy and splenomegally are observed in both male and female

dogs. Often the male dog will have scrotal swelling; however, if chronically infected, testicular

atrophy is usually observed. Osteomyelitis related to hip prostheses has been described in two

dogs (Smeak et al., 1987). Chronic meningitis and non-suppurative encephalitis has been

associated with bacteria belonging to the genus Brucella (Carmichael and Kenney, 1970). In the

testicles, tubuli are fibrosed (Moore and Kakuk, 1967). Lesions in infected lymph nodes are

generally serous or suppurative lymphadenitis with accumulations of macrophages and

neutrophils. Brucella abortus and B. melitenses generally do not cause gross abscesses, whereas

B. suis frequently result in abscess formation (Ray, 1979; Blasco, 1990). The presence of

erythritol in the uterus of cattle, goats and sheep, and swine favours the growth of Brucella.

Proliferation of brucellae results in necrosis of the maternal and fetal placental membranes,

resulting in the death and eventual expulsion of the fetus. Abortion in sow can occur any time

during pregnancy, and fetal death can occur without subsequent expulsion of the fetus; hence

dead and live pigs can be born in the same litter (Ray, 1979; Alton, 1990; Blasco, 1990).

39

2.12 Immunity

2.12.1 Humoral Immunity and immunoglobulin production during infection

Natural hosts defenses against Brucella play an important role in protecting animals against

infection. Humoral immune response in animals are similar in most species affected by Brucella.

Antibodies produced in immune response to Brucella infection include IgA, IgM, IgG1 and IgG2

(Hadju, 1971; Bel and Lescelles, 1973; Butter et al., 1981). IgM are produced first following

infection with virulent strains of Brucella organism or parenteral vaccination with Brucella

abortus strain 19 (Corbel, 1988). They are usually in the first and second week of exposure.

IgG1, IgG2 and IgGA thereby follow. Appearance of immoglobulins in the serum of various

animal species probably follows patterns similar to those observed in cattle following

vaccination and natural infection. In vaccinated cattle the IgG begins to recede soon after

reaching a peak and, depending on dose and age of the animal when vaccinated, may not be

present in measurable quantities after a few months.

2.12.2 Cell-mediated immunity

Brucella are facultative intracellular parasites and can survive and multiply within

polymorphonuclear and mononuclear phagocytes as well as other cell types (Radostits et al.,

1995). When there is infection with Brucella organism, the polymorphous and mononuclear cells

internalize the bacteria and destroy them due to intracellular bactericidal activity (Corbel, 1988;

Sati, 2002). This process of activation of mononuclear and polynuclear cells for phagocytosis is

stimulated by interleukins released by T-lymphocytes responding specifically to the antigens

(Corbel, 1984).

40

2.13 Diagnosis of Brucella Infections

2.13.1 Clinical signs

Clinical signs are associated principally with the reproductive tract. In females, the most

prominent clinical sign is late-pregnancy abortion, after 45-55days of gestation in about 75% of

the cases, (Shin and Carmicheal,1999), and this was the first sign described to be related to

infection with Brucella canis when the bacterium was first recognized in 1966 (Carmichael,

1967). Aborted pups are partially autolysed and show signs of a generalized bacterial infection,

such as subcutaneous edema, abdominal haemorrhages, and degenerative lesions in liver,

kidneys, spleen and intestines. The bitch continues to excrete a brownish or green-gray discharge

for 1-6 weeks after the abortion (Carmichael and Kenney, 1968). Sometimes early embryonic

death and resorption occurs or abortion 10-20 days after mating (Shin and Carmicheal, 1999).

Weak pups may die within a few hours, but other times may survive up to a month. Seemingly

normal pups can also be born, and develop the disease later (Carmichael and Kenney, 1970).

Before puberty, a generalized lymphadenopathy is the most common clinical sign. Brucellosis

does not affect the estrous cycle, and bitches that have aborted can give birth to normal litters in

subsequent pregnancy, or have intermittent reproductive disturbances (Carmichael and

Kenney, 1970).

In male dogs, epididymitis and prostatitis are the most common clinical signs. In the acute stage,

the epididymis increases in size and is painful. Frequent licking may lead to scrotal edema and

dermatitis. In the chronic phase the epididymis may become small and hard, and the testicle

atrophic. Chronically infected dogs are often oligo- or azoospermic, and infertile. Sperm defects

that can be seen include tail defects, loose heads and distal droplets (Carmichael, 1976). The

testicular damage leads to production of auto-antibodies towards the sperm cells. These

41

antibodies can be detected in serum and seminal plasma from three months after the infection

(Serikawa et al., 1984). From 4 months after infection the sperm cells autoagglutinate and

probably contribute to male infertility (Serikawa et al., 1984).

Most often there are no general clinical signs, and infected dogs do not usually have a fever.

Sometimes the dogs have a dull coat, or show decreased exercise tolerance, but this is not

common (Wanke, 2004).

2.13.2 Direct Smear Examination

The organism can be demonstrated through stained smears prepared from fetal membranes, fetal

stomach contents, vaginal swabs, semen, vaginal discharges, fetal tissues including the lungs,

liver, and spleen, colostrums or milk samples, blood, samples of tissues collected during post

mortem examination like supramammary lymphnodes, illac lymphodes and retropharyngeal

lymphnodes, etc. The most common methods in use are the modified Ziehl-Neelsen and the

mollified Köster staining methods (Jenny and Berman, 1962; Corbel, 1973; Morgan et al., 1978).

2.13.3 Identification and Typing

Brucellae are cocobacilli or short rods, usually arranged singly but sometimes in pairs or small

groups. However, they are resistant to decolorization by weak acids (Poester et al., 2010). After

48-72 hours of incubation, at 37 oC, Brucella colonies are 0.5 to 1.0 mm in diameter with a

convex and circular outline. Smooth strains are transparent and pale yellow, resembling droplets

of honey with a shiny surface when observed in transmitted light, while rough colonies are more

opaque with granular surface (Braun and Bonesteal, 1947). Colonial morphology, staining, slide

agglutination with anti-Brucella serum (smooth or rough), urease, catalase and oxidase tests are

42

the basis for a culture to be identified as belonging to the genus Brucella. After identification of

a culture as brucella, further classification can be done in specialized reference laboratories.

These tests are cumbersome and include carbon dioxide (CO2) requirement, production of

hydrogen sulphide (H2S), dye sensitivity (thionin and basic fuchsin), phage lysis, agglutination

with A, M or R specific antisera and in some cases it is necessary to use the oxidative metabolic

method (Poester et al., 2010)

Another approach is the typing of Brucella strains for epidemiological investigations, including

taxonomic studies as well as tracing back strains to their origins. The strategy for the

development of these tests is based on the observation that most organisms (prokaryotic and

Eucaryotic) contain strings of tandem repeat sequences classified as microsatellites and mini-

satellites distributed throughout their genomes that may affect protein expression (Poester et al.,

2010).

2.13.4 Animal Inoculation

Another method for diagnosing Brucella is by animal inoculation. Even though B. canis is only

known to be important in dogs, antibodies to this organism have been reported occasionally in

wild canids including foxes and coyotes, as well as in racoon. Experimental infections have been

established in chimpanzees, mice, rabbits and guinea pigs. Guinea pigs are more susciptble than

Sheep, swine and cattle were reported to be highly resistant to experimental infection by oral and

conjuctival inoculation; however, two field infections of B. canis have been reported in cattle

(Iowa State University 2012). After oral inoculation, three out of 14 experimentally infected cats

developed bacteremia, but agglutinating antibodies were not detected (Iowa State University,

2012).

43

2.13.5 Serological Diagnosis

A definitive diagnosis of Brucella infection requires simultaneous application of different

laboratory techniques due to the lack of a single, highly reliable diagnostic method (Gyuraneoz

et al; 2011). Rose Bengal plate test (RBPT) and serum Agglutination test (SAT) are commonly

used. Both tests detect certain levels of antibody produced against Brucella organism present in

serum, semen and milk in a chronic disease. The serum may contain varying levels of antibodies

in form of IgM, IgG1, IgG2 and IgGA at different stages of infection (Onunkwo, 2005). Several

factors including long and variable incubation period, vaccination status and type of exposure

during infection may influence the quantity of antibody produced making serological diagnosis

difficult (Smith et al., 1972).

2.13.5.1 Rose Bengal Plate Test (RBPT)

This test is also called Agglutination test; and was introduced in 1957 by Rose and Roekpe to

differentiate specific Brucella agglutinins from non - specific factors (Alton et al; 1975b, Sati,

2002). The antigene used in RBPT method consists of Brucella cells stained with Rose Bengal

and suspended in buffer at Ph 3.65±0.05. RBPT is a screening test and samples that test positive

are further subjected to other tests such as Serum Agglutination Test, Complement Fixation Test,

Enzyme Linked Immunorbent Assay (ELISA) (Ocholi, 1990). The RBPT detects antibodies of

IgG1, IgG2 and IgM which are produced during early infections (Ocholi, 1990; Onunkwo,

2005). Different researches have different ratings for RBPT and SAT. Some researchers gave

credence to RBPT while others rate SAT high more than RBPT (Morgan et al., 1969; Kazi et al.,

2005).

44

2.13.5.2 Serum Agglutination Test (SAT)

Serum agglutination test referred to as the Standard Tube Brucella Agglutination Test (STT) is

commonly used for diagnosis of acute brucellosis (Bercovich, 1998). However 2-

mercaptoethanol (2ME) and Compliment Fixation Test (CFTs) are used for chronic brucellosis

where active infection continues even though agglutination titers return to low levels (Acha and

Szyfers, 2003). Serum Agglutination Test measures agglutination antibodies against IgM, IgG2,

IgA. Morgan (1968), recorded that the SAT may be negative in the early infection and chronic

infections. This limitation results from the test not detecting IgG1, a relevant antibody type

involved in early infection. Some of the limitations of SAT include:

1. The test detects non specific agglutinins and cross reacting antibodies as well as specific

antibodies arising from Brucella infection and vaccination.

2. In the incubative stage of the disease, the test may be the last of all the serological tests to

detect diagnostically significant titers.

3. In the chronic stage, the agglutinin titers tend to wane, often becoming negative when

those of some other tests remain positive.

2.13.5.3 Rapid Slide Agglutination Test (RSAT)

This test is commonly used, although a drawback to this method is the risk of false positive

samples. By treating the sample with 2 mercapto –ethanol (2-ME) the number of false positive

samples decreases (Mateude – Antonio et al., 1994; Keid et al.,2007) because IgM is dissociated

and IgM cross-reacts with other bacteria more commonly than IgG (Deutsch and Morton, 1957)

45

2.13.5.4 Agar Gel Immunodiffusion Test (AGID)

The Agar Gel Immunodiffussion (AGID) Test has been described as suitable if a chance

infection is suspected, because more chronic infections are positive using an AGID than using

other Tests (Carmicheal et al., 1984). However, the AGID is not used by many laboratories

today because of its low sensitivity and because it requires trained personnel and special media

(Hollelt, 2006).

2.13.5.5 Agglutination Tests

Agglutination test generally cannot be used efficiently for the diagnosis of infection with

Brucella canis and Brucella ovis which are rough species of Brucella. (Poester et al., 2010). As

the whole cell antigens autoagglutinate, precipitin tests using soluble antigens are used instead

(Poester et al., 2010).

2.13.5.6 Other Serological Tests

An array of other serological test for the diagnosis of brucellosis in dogs and other animal

species including man are available. Such tests including their development, test, procedure, use

and limitations have been described (Alton et al., 1975 a &b; Morgan et al; 1978; Seifert 1996;

Poester et al; 2010). Some of these tests include;

1. Compliment Fixation Test

2. Enzyme Liked Immunosorbent Assay (ELISA)

3. 2 – Mercap- Ethanol Test (2-MET).

4. Radio Immunodiffussion Test (RID)

5. Radio Immunassay Test (RIA)

46

6. Flourescent Antibody Test (FAT)

7. Antiglobulin Test (AGT) or Coomb’s test

8. Heat Inactivation Test (HIT)

9. Primary Binding Assay (PBs)

10. Fluorescence Polarization Assay (FPA)

11. Milk Ring Test (MRT).

No serological test has been shown to be reliable in routine diagnosis of swine brucellosis. While

in the diagnosis of ovine and caprine brucellosis the Rose Bengal plate agglutination, CF and

indirect ELISA tests are usually recommended for screening flocks and individual animals

(Robinson, 2003)

2.14 Treatment of Brucellosis in Animals and Humans

Treatment of canine brucellosis in dogs is generally not recommended because affected dogs

may continue to be a source of infection for other dogs and people in contact with them (Johnson

and Walker, 1992).Treatment is not recommended for dogs in breeding facilities because

antibiotics have limited effect and therapy is often unrewarding. Extensive use of antibiotics will

reduce bacteremia but cannot reliably eliminate the intracellular organism from the body.

Relapses are common, spaying or neutering the animal can reduce but not eliminate the

transmission risk (Wanke, 2004). Even in uncomplicated cases, treatment of canine brucellosis is

difficult, and relapses and treatment failures are common, especially in males (even neutered)

(Scheftel, 2003). Current recommendations for treatment of spayed and neutered pet dogs consist

of 4 to 8 week course of doxycycline (5 to 10mg/kg PO bid) together with streptomycin (5 to

10mg/kg IM bid) on the first and fourth weeks. Gentamycin, 2 to 4 mg/kg Sc or IM bid, may be

47

substituted for streptomycin if the drug is unavailable. (Carmicheal and Green, 1990). Antibiotic

therapy is described but no good long term studies have demonstrated complete remission with

antibiotic therapy (Hollett, 2006). The only reliable method of complete elimination of canine

brucellosis involves isolation, testing and euthanasia of positive animals (Crow, 2012).

The World Health Organisation (WHO) issued recommendations for the treatment of human

brucellosis in 1986 (FAO/WHO, 1986), suggesting the use of doxycycline, 100mg twice daily

for six weeks combined with rifampicin, 600-900mg daily for 2-3 weeks.

2.15 Public Health Importance

Worldwide prevalence of brucellosis in human populations has been studied and reviewed. The

Mediterranean Basin, South and Central America, Eastern Europe, Asia, Africa, the Caribbean

and Middle East are considered as high risk countries (Abubakar et al., 2012). From public

health view point, brucellosis is considered to be an occupational disease that mainly affects

farm labourers, slaughter house workers, butchers, shepherds, laboratory workers and

veterinarians (Yagupsky and Baron, 2005; Behzadi and Mogheiseh, 2011).

The slaughter house workers are more prone to acquire infection as compared to other

occupations because they are exposed to carcasses and viscera of infected animals and get

infected through cuts and wounds and splashing of infected blood and other fluid in the

conjuctiva (Ramos et al., 2008).

Transmission typically occurs through contact with infected animals, materials with skin

abrasions, inhalation of aerosols or ingestion of contaminated or unpasteurized food products

(Christopher et al., 2010). Symptoms in humans can be highly variable, ranging from non-

specific, flu-like symptoms (acute form) including intermittent fever, chills, night sweat,

48

headache, malaise, diarrhea with stomach cramps which may progress to a more chronic form

and can also produce serious complications affecting the musculoskeletal, cardiovascular, and

central nervous systems and other problems like arthritis, orchitis and epididymitis (Alusurp,

1974). According to Schnurrenberger et al., (1975), larger proportion of veterinary graduates

show serological evidence to Brucella infection than undergraduates and this was attributed to

the regular coming in contact the practicing veterinarians have with infected animals than student

veterinarians.

A history of exposure to dogs is usually needed to raise suspicion of infection with canine

brucellosis in humans (Rumley and Chapman, 1986). The infection may be under diagnosed due

to its rather unspecific symptoms. In Nigeria, high Brucella antibody titers have been reported

among occupationally- exposed persons (Adekolu, 1989; Adesiyun and Oni, 1990). Positive

blood cultures confirm the diagnosis, but as the patients have been treated with antibiotics in

many cases, the risk of a false negative culture is increased (Polt et al., 1982).

2.16 Economic Importance of Brucellosis

Brucellosis causes economic losses from abortion, temporary infertility, chronic metritis,

decreased milk production by dairy cows, death from acute metritis, sterility, increased cost of

animal replacement and low sale values of infected animals (Rikin, 1988b; Ajogi et al., 1998;

Sati, 2002). The dog breeding industry has grown significantly since B. canis was first

recognized in 1966 and because of this, B. canis, has become a major source of economic loss in

both large and small dog breeding facilities as well as households as a single outbreak can lead to

the euthanasia of hundreds of dogs (Brower et al., 2007).

49

Human losses include illness, physical incapacitation, loss of man power, economic losses due to

medical cost (Onunkwo, 2005). Although in Nigeria, no economic losses in terms of monetary

value as a result of canine brucellosis in dog have been estimated, economic loss due to bovine

brucellosis in Nigeria was estimated between N148.8m and N224m US Dollars excluding losses

in human productivity (Esuruoso 1979; Ocholi et al., 2004).

2.17 Control, Prevention and Eradication of Brucellosis

Currently control of canine brucellosis within a kennel typically relies on prevention of

infection and euthanasia of infected dogs (Carmicheal and Greene, 2006). In kennels that do not

elect to treat infected dogs, repeated post-treatment testing is necessary before the dog can be

considered to be free of infection. Treatment is usually reserved for pet animals early in the

course of infection, typically involving castration or ovariohysterectomy in combination with

antibiotic, therapy (Carmicheal and Greene, 2006). Dogs from kennels in which B. canis has

been diagnosed should not be used for breeding. Dogs from endemic areas should be kept

isolated until tested free of B. canis to avoid further spread of the disease. This is recommended

for natural mating, artificial insemination with fresh, chilled or frozen semen, and when

introducing new dogs into the kennels. For kennels in endemic areas, it is often recommended

that breeding animals are tested annually, and that all new dogs are tested before being

introduced into the kennel. Serologic tests can be negative up to 4 weeks after infection and at

least 12 weeks must pass to be sure of detecting antibodies in an infected animal (Carmicheal et

al., 1984).

50

2.17.1 Vaccines

Brucella infections in animals have an important economic impact especially in developing

countries as they cause abortion in the pregnant animals, reduce milk production and cause

infertility. In regions with high prevalence of the disease, the only way of controlling and

eradicating these zoonoses is by vaccination of all susceptible hosts and elimination of infected

animals (Briones et al., 2001). The most commonly used vaccines against bovine brucellosis are

B. abortus Strain 19 and the recently USDA approved Strain RB51; the later unlike Strain 19

does not interfere with serological diagnosis (Moriyon et al., 2004). The use of B. abortus Strain

19 vaccine leads to the production of antibodies whose persistence depends mainly on the age of

the animals at the time of vaccination. B. melitensis Strain Rev1 vaccine although highly

infectious to human, is considered as the best vaccine available for the control of ovine and

caprine brucellosis, especially when administered at the standard dose by conjunctival route.

Vaccination alone will not eradicate Brucella as the immunity produced by Brucella vaccines are

not absolute and can be circumvented by increasing the level of infection (Morgan, 1968).

Live human vaccines B. abortus Strain 19-BA and strain 104M are being used only in the former

Soviet Union and China, respectively (Acha and Szyfres, 2003). The development of a live

vaccine for the control and eradication of canine brucellosis is of great importance. Nevertheless,

currently, there is no information that describes an appropriate candidate that can be used as a

vaccine against B. canis (Palomares –Resendiiz et al., 2012).

2.17.2 Control and Prevention of Brucellosis in Humans

In many developing countries where brucellosis infection is prevalent the animal/human

relationship is close and high percentages of people are involved in animal agriculture as an

51

occupation. In developed countries where effective control and eradication programs have been

instituted, very low incidence of the disease in animals, leads to a corresponding low incidence

of the disease in man (MZCP, 1998). Consistent with exposure potential, farm workers and

animal health personnel should take precautions when working with animals that abort or give

birth and should refrain from drinking raw milk unless its source is known to be uninfected with

Brucella or other pathogens excreted in milk. Laboratory workers should use safety cabinets

when handling brucella cultures or tissues. Persons handling specimens for laboratory

submission should wear gloves and submit samples in leak-proof and crush-proof containers.

Vaccinating people in high risk agricultural occupation areas has been attempted in Russia and

China (FAO/WHO, 2006).

52

CHAPTER 3

MATERIALS AND METHODS

3.1. Study design

The study is a cross-sectional survey using purposive sampling technique to screen dogs for

Brucella antibodies

3.2. Area of study

This study was carried out in Enugu and Anambra States of Nigeria.

Enugu State is located between latitude 5o55N and 7

055N and longitude 6

053E and 7

055E. It

covers a total land area of about 802, 295km2 and has a population of 2.5million with a

population density of 240 persons per square kilometer (NPC, 2006). It is bounded in the South

by Abia and Imo States, in the East by Ebonyi State, in the North-East by Benue State, in the

North-west by Kogi State and in the West by Anambra State. Enugu State is made up of 3

Senatorial Zones and 17 Local Government Areas. The senatorial zones are: Enugu-East, Enugu-

West and Enugu-North. Tropical forests and savannah predominates the area ecologically. The

wet season lasts from April to October while the dry season lasts from October to early April.

The indigenous people of Enugu State are predominantly Igbo-speaking and are involved in two

major farm activities, crop and livestock. Dogs are kept as part of the culture of the people for

breeding, hunting and for security. Dog meat is eaten by a section of the population.

Anambra State is located between latitude 6o20N and longitude 7

o00E. It covers a total land

area of about 4, 844km2. According to National Population Commission (2006), it has a

population of about 3, 902, 051 with a population density of about 840 persons per square

kilometer. It is bounded by Delta State to the West, Enugu State to the East, Imo State to the

West and Kogi State to the North. The state is made up of three Senatorial Zones and 21 Local

Government Areas. The zones comprise of Anambra- North, Anambra- Central and Anambra-

South. The indigenous people of Anambra State are predominantly Igbo-speaking and are

involved mainly in trading, with the State having two seasons (dry and wet) and a tropical rain

forest. Like in Enugu State, dog keeping and dog eating is a common feature of some of the

indigenous population.

53

3.3 Map of Enugu State

Source : Local Government Areashttp://www.google.com.ng/url?

54

3.4 Map of Anambra State

Source: http://www.informationng.com/2013/07/state-inec-discovers-polling-booths-in

shrines-forests-in-anambra.html/anambra-state-map

55

3.5 Study duration

This study lasted for 24 weeks (February – July, 2013).

3.6 Study population

The study was purposively targeted towards 3 groups: (a) Dogs presented at veterinary clinics (b)

Household dogs with history of infertility or abortion and (c) Dogs at slaughter points in the

markets.

3.7 Sample Size Determination

The required sample size was determined using the following formular:

2

2

d

pqZn = (Thrusfield, 2005)

Where

n = Desired sample size

Z2 = 1.96 (normal distribution) from table

p = Prevalence rate from the average of previous studies

d = Desired absolute precision of ± 5% with 95% Confidence Interval

q = 1 – p

In this study, according to Adedoyin (2012), a prevalence rate of 7.94% was used for sample size

determination. Using the formular above, a sample size of 112 was calculated.

However, based on the number of Canine Brucellosis Antibody Test Kit®

bought which was 11

packs (each pack screens 12 dogs), 123 dogs were screened in this study.

3.8 Sampling technique

Five major veterinary clinics, two each in Enugu and Onitsha metropolis, one in Nsukka Urban;

2 major slaughter points in each State; and households with dogs that have history of infertility

or abortion were selected by purposive sampling method. Visits were made to the purposively

selected veterinary clinics, households, and slaughter points, once every other week for six

months.

56

A total of 123 dogs made up of 65 clinic dogs, 34 slaughtered dogs and 24 household dogs were

screened.

Profile of the dogs presented at the clinics and household dogs were also collected. Information

requested included: sex, age, and breed, history of infertility / abortion in female dogs, and some

possible risk factors / management practices for Brucella infection in dogs.

3.9 Sample collection.

Collection of blood

Aseptically, 5mls of blood sample was collected from the cephalic vein of each animal, after a

proper restraint, using sterile syringe and needles. The blood was kept in a slanting position for

about 30minutes to allow for proper clotting. It was then centrifuged at 3000rpm for 5minutes.

The sera were afterward harvested one after the other using separate syringes into labeled bijoux

bottles and stored at - 20oC until they were analyzed.

3.10 Sample analysis

3.10.1 Serological tests

(A) For B. abortus identification, two tests were used:

i) Rose Bengal Plate Test (RBPT): Antigen was procured from Central Veterinary Lab.,

New Haw, Weybridge Surrey, England.

ii) Serum Agglutination Test (SAT)

SAT specific for B abortus antigen was further diluted as described by the manufacturer

(Onderstepoort Biological Product (LTD) Republic of South Africa). One ml of antigen was

dissolved in 99ml phenol saline.

(B) For B. canis identification: ImmunoComb® Canine Brucellosis Antibody Test Kit specific

for B. canis antigen (Biogal Galed Labo., Kibbatz Galed 19240, Isreal) was used following the

manufacturer’s instructions.

57

3.10.2 Test procedure.

3.10.2.1 Procedure for Rose Bengal Plate test (RBPT)

The RBPT was carried out as described by Morgan et al (1978) and Alton et al (1975).

Using a pipette, 1 drop (0.03ml) of the test serum was placed on one spot of the slide using

another pipette. An equal volume of RBPT B. canis antigen was placed close to the test serum on

the slide. Using an applicator stick, the antigen and the test serum were mixed thoroughly, the

slide was then hand-rocked for about 4 minutes after which the slide was examined for

agglutination under a good source of light. Formation of pink granules (agglutination) was

recorded as positive while absence of pink granules (agglutination) was recorded as negative.

Interpretation of the test was as follows

++++100% agglutination (background is clear)

+++75% agglutination (background slightly turbid)

++50% agglutination (background moderately turbid)

+25% agglutination (background very turbid)

-No agglutination (remain homogenous)

3.10.2.2 Procedure for Serum Agglutination Tests (SAT)

The Serum Agglutination Test was carried out as described by Morgan et al., (1978) and Alton et

al., (1975).

Five sterile test tubes were arranged in rows in a test tube rack labeled 1-5. A volume 0f 0.8ml

of phenol saline was added into the first tube and 0.5ml of the phenol saline was added into the

2nd

to the 5th

test tube. Then 0.2ml of the test serum sample was placed into the first tube, mixed

thoroughly without frothing and 0.5ml of the mixture in the first test tube was transferred to the

next test tube and mixed. Serial dilution was carried on to the last test tube (tube 5) and 0.5ml of

the mixture from the 5th

test tube was discarded. One ml of the concentrated solution was added

to 99ml of phenol saline to form a 1% solution and 0.5ml of the reconstituted Serum

Agglutination Test. B. abortus antigen will then be added to each of the 5 test tubes using a

dropping pipette. The serum dilutions in the 5 tubes became 1:10, 1:20, 1:40, 1:80 and 1:160

representing agglutination of >12.5 IU/ml, >25 IU/ml, >50 IU/ml, >100 IU/ml, and >200 IU/ml

58

respectively (Morgan et al., 1978; Sati, 2002). The tubes were incubated at a temperature of

370C for 20-24hours. The degree of agglutination and clearance was read under a black

background against a light source. Titers of 1:40 (50 IU/ml) and above were taken as diagnostic

for B. abortus (Morgan et al., 1978; Sati, 2002). The results of this test were interpreted as

follows

++++100% agglutination (background is clear)

+++75% agglutination (background slightly turbid)

++50% agglutination (background moderately turbid)

+25% agglutination (background very turbid)

-No agglutination (remained homogenous)

3.10.2.3 Procedure for the ImmunoComb® Canine Brucellosis Antibody Test Kit Specific

for B. canis antigen (Biogal Galed Laboratories., Kibbatz Galed 19240, Isreal) used was

based on the solid phase immunoassay principle:

The test was carried out by dropping 5ul of each serum sample into each sample wells of the

multi-compartment developing plate (wells A) after opening the aluminium cover with the

tweezers. Then, the Immunocomb on which purified B. canis antigen is attached was removed

from its protective wrapping and fixed into well A so that antibodies from the samples if present,

will bind to the antigen on the comb. The conjugate was incubated for 5 minutes. After

incubation in well A, the developing plate was removed from the incubator and the rows of well

B were opened and the comb put into well B and incubated for 2 minutes so that unbound

antibodies were washed off. The Immunocomb was then transferred into wells of row C, row D,

row E, and row F and incubated for 5 minutes, 2 minutes, 2 minutes and 5 minutes respectively.

This was to allow for the colour reaction process to develop. Upon completion of colour

development in row F, the comb was moved back to row E for 2 minutes for colour fixation to

take place after which the comb was removed and allowed to dry. Upon drying, the

Immunocomb was aligned and compared with the Immunocomb scale, and shades of grey colour

that matches the positive reference spot, were regarded as positive.

Titre levels of 1:200 and above was regarded as positive.

59

3.11 Data Analysis

Using Statistical Package for Social Sciences (SPSS) 17.0, Chi-square (χ2) statistic, odds ratio

and 95% confidence interval were used to determine if there were significant association

between Brucella antibody prevalence and sex, age, breed, history of infertility/ abortion in

female dogs, and some possible risk factors/ management practices for Brucella infection in

dogs.

60

CHAPTER FOUR

RESULTS

4.1 Distribution of dogs based on States and sources where samples were

collected.

Over the period of the study, a total of 123 dogs were screened. Table 4.1 summarizes the

sources/ categories of the dogs screened. Sixty-eight dogs were screened in Enugu State while 55

dogs were screened in Anambra State. Veterinary clinics contributed 65 of the total dogs

screened while 34 and 24 dogs were screened from Slaughter points and households respectively.

61

Table 4.1: Distribution of dogs based on States and sources where samples

were collected.

Sources Number of dogs

Anambra State Enugu State Total

Veterinary Clinics 37 28 65

Slaughter House/ Market 6 28 34

Household 12 12 24

Grand Total 55 68 123

62

4.2: Prevalence of Brucella abortus antibodies based on the sources of the dogs

screened.

Of the 123 sera samples screened for Brucella abortus antibodies, none 0(0%) was positive

using both the Rose Bengal Plate Test (RBPT) and the Serum Agglutination Test (SAT) (Table

4.2).

63

Table 4.2: Prevalence of Brucella abortus antibodies based on the sources of

the dogs screened.

Source Number screened Number positive

RBPT SAT

Vet. Clinics 65 0 (0.00) 0 (0.00)

(Clinical dogs)

Slaughter points 34 0 (0.00) 0 (0.00)

Households 24 0 (0.00) 0 (0.00)

Total 123 0 (0.00) 0 (0.00)

Legend: RBPT=Rose Bengal Plate Test

SAT= Serum Agglutination Test.

64

4.3: Prevalence of Brucella canis antibodies based on the sources of the dogs.

Out of 123 dogs from the different sources screened, 34 were positive for B. canis antibodies

using the Immunocomb®

Canine Brucella Antibody Test Kit. This gave an overall

seroprevalence rate of 27.7 percent. Out of 65 dogs screened in the Veterinary Clinics (Clinical

dogs) in Anambra and Enugu States, 22 (18%) were positive for B. canis (Table 4.3). Four

(3.3%) out of 34 Slaughter dogs screened were positive. Of the 24 dogs screened from

Households, 8 (6.5%) were positive. There was no association between infection rate and

sources/categories of the dogs screened (χ2 = 5.925, P>0.05).

65

Table 4.3: Prevalence of Brucella canis antibodies based on the sources of the

dogs screened.

Source Number screened Number positive (%)

Immunocomb® B. canis Antibody

Test Kit

Vet. Clinics 65 22 (18)

(Clinical dogs)

Slaughter points 34 4 (3.3)

Households 24 8 (6.5)

Total 123 34 (27.7)

(χ2 = 5.925, P>0.05)

66

4.4: Antibody titer levels of B. canis positive dogs screened in Anambra and

Enugu States.

Out of the 34 B. canis positive dogs, 16 (47.1%) had a titer level of 1:200 (IFA Titer); 10(29.4%)

had titer level of 1:400 (IFA Titer), 4(11.8%) had a titer level of 1:600 (IFA Titer); while the

remaining 4(11.8%) had a titer level of 1:800 (IFA Titer) (Figure 1)

67

Figure 1: Antibody titer levels of B. canis positive dogs screened in Anambra

and Enugu States.

68

4.5: Antibody titer levels of Brucella canis positive dogs based on the sources

of dogs.

The antibody titer levels of B. canis positive dogs based on sources/ categories of screened dogs

are shown in Table 4.5. Ten (29.4%) of 16 positive dogs with antibody titer 1:200 (IFA Titer) are

dogs from veterinary clinics; 4 (11.8%) are slaughter dogs and 2 (5.9%) are household dogs.

Seven (20.6%) of 10 positive dogs with titer 1:400 (IFA Titer) are dogs from veterinary clinics; 3

(8.8%) are slaughter dogs with none from household dogs. Five (14.7%) of 8 dogs with titer

1:600 (IFA Titer) and above are dogs from veterinary clinics; 2 (5.8%) were household dogs and

only one (2.9%) is a slaughter dog. Chi- Square analysis showed no association between

antibody titer levels and sources of the dogs

(χ2 = 3.767; p>0.05). Two household dogs and 2 of the 5 dogs from veterinary clinics with titer

1:600 (IFA Titer) and above had history of recent abortion or infertility.

69

Table 4.5: Antibody titer levels of Brucella canis positive dogs based on the

sources of dogs

Titer level

Total

Dogs from

Vet. Clinics (%)

Slaughter

dogs (%)

Household

dogs (%)

1 : 200

16 10 (29.4) 4 (11.8) 2 (5.9)

1 : 400

10 7 (20.6) 3 (8.8) 0 (0)

1 : 600

4 3 (8.8) 0 (0) 1 (2.9)

1 : 800 4 2 (5.9) 1 (2.9) 1 (2.9)

Total 34 22 (64.7) 8 (23.5) 4 (11.8)

(χ2 = 3.767; p>0.05)

70

4.6: Sex distribution of Brucella canis antibody positive dogs.

The female dogs had a seroprevalence of 22 percent (Table 4.6) while the male dogs had a

seroprevalence of 6%. There was a strong association (p<0.05) between the infection of Brucella

canis and sex of the dogs screened (Table 4.6).

71

Table 4.6: Sex distribution of Brucella canis antibodies of dogs sampled in

South-East Nigeria.

Sex Number sampled Number positive (%)

Male 45 (36.6) 7 (5.9)a

Female 78 (63.4) 27 (21.9)b

Total 123 (100.0) 34 (27.6)

(χ2 = 5.174, p<0.023, df = 1)

72

4.7: Antibody titer levels of B. canis positive dogs according to sex.

From the 27 positive female dogs, 13(38.2%), 8(23.5%), 3(8.8%) and another 3(8.8%) had

antibody titer levels of 1:200(IFA Titer), 1:400(IFA Titer), 1:600(IFA Titer) and 1:800 (IFA

Titer) respectively. In males out of the 7 positive dogs, 3(8.8%), 2(5.9%), 1(2.9%) and 1(2.9%)

had titer levels of 1:200(IFA Titer), 1:400(IFA Titer), 1:600(IFA Titer) and 1:800(IFA Titer)

respectively. Chi-square analysis showed that there is no association (P>0.05) between titer level

and sex (Table 4.7).

73

Table 4.7: Antibody Titer Levels Of B. Canis Positive Dogs According To Sex.

Titer level Total (%) Female (%) Male (%)

1:200

16 13 (38.2) 3 (8.8)

1:400

10 8 (23.5) 2 (5.9)

1:600

4 3 (8.8) 1 (2.9)

1:800 4 3 (8.8) 1 2.9)

Total 34 (100) 27 (79.4) 7 (20.6)

(P>0.05; χ2= 0.130)

74

4.8: Age Distribution of Brucella Canis Antibody Positive Dogs (Clinical And

Household Dogs).

Dogs below 1 year old had a seroprevalence of 3.4%, dogs 1-<3 years of age had seroprevalence

of 10.1%, while those 3-<5years and 5 years and above, had seroprevalence of 15.7% and 4.5%

respectively. Thus 23 out of 66 dogs aged between 1 year and below 5 years were positive,

giving a seroprevalence of 34 percent. There was no association (p>0.05) between Brucella canis

infection and age in the dogs screened (Table 4.8).

75

Table 4.8: Age Distribution Of Brucella Canis Antibody Positive Dogs

(Clinical And Household Dogs).

Age (years) Number sampled Number positive (%)

<1year 11 3 (3.4)

1-<3 years 32 9 (10.1)

3-<5 years 34 14 (15.7)

5 & above 12 4 (4.5)

Total 89 30 (33.7)

(χ2 = 1.500, P>0.05)

76

4.9: Breed Distribution of Brucella Canis Antibody Positive Dogs.

Twenty-six out of 76 exotic breed of dogs were positive for B. canis antibody; this gives a

seroprevalence of 21.1% (Table 4.9). Mixed and local breeds each had seroprevalence of 3.3%.

Chi-square analysis revealed there was a strong association (P<0.05) between infection and the

breeds of dogs, with the infection being higher in exotic breeds of dogs.

77

Table 4.9: Breed Distribution Of Brucella Canis Antibody Positive Dogs.

Breed Number sampled Number Positive (%)

Exotic* 76 26 (21.1)a

Mixed 13 4 (3.3)b

Mongrel 34 4 (3.3)

b

Total 123 34 (27.6)

(Χ2 = 0.04; p<0.05)

• * Total no of dogs sampled= Rottweiler 28 (36.8%); Mastiff 20 (26.3%); Caucasian

13 (17.1%); Alsatian 13 (17.1%); Boer bull 1 (1.3%); Persian 1 (1.3%)

78

63

CHAPTER FIVE

DISCUSSION

Brucella abortus antibody was not detected in any of the 123 dogs screened; this means that out

of the 34 Brucella positive dogs, no antibody for B. abortus was found using both the Rose

Bengal Plate Test (RBPT) and Serum Agglutination Test (SAT). The zero prevalence suggests

that B. abortus, though important in livestock, is not of major importance in the epidemiology of

canine brucellosis in the study area. The apparent absence of B. abortus antibody may be

attributed to the type of management system practiced in the South-East. Dogs are either housed

or caged and though some dogs may roam freely in the neighborhood, they do not usually come

in contact with ruminants like, cattle, sheep, and goats. The dog profiles also showed that most

clients feed their dogs cooked household foods only. Therefore, the likelihood of being infected

with B. abortus is rare; as most infections of B. abortus in dogs is by dogs coming in close

contact with aborted tissues of infected farm animals through the consumption of these infected

materials like aborted fetuses and feces, placental, fetal and vaginal fluids. The findings of this

study on B. abortus is in contrast to the works of Cadmus et al., (2011) and Adedoyin et al.,

(2012) who reported seroprevalence of 5.46% and 2.48% respectively in household dogs in

Ibadan, South-West Nigeria.

The result of the present study suggests a high seroprevalence (27.7%) of Brucella canis in dogs

presented to veterinary clinics, apparently healthy slaughter dogs and those in the screened

households. None of the dogs screened in the study area were vaccinated, as vaccination of dogs

against brucellosis is not routinely carried out in Nigeria. Also, there is no information of any

vaccine against B. canis (Palomares –Resendiiz et al., 2012). Therefore, demonstration of

antibodies to brucella in dogs in the study area is suggestive of natural exposure to the organism.

64

It may also indicate lack of brucellosis control programme in animals. The seroprevalence of B.

canis in this study was relatively high when compared to other studies in Nigeria. This may be

attributed to the higher sensitivity of the diagnostic technique (Biogals Immunocomb®

Canine

Brucella Antibody Test Kit) used which is 99% sensitive and specific to the detection of

Brucella canis antibodies. Cadmus et al., (2011) and Adedoyin et al., (2012) reported a

seroprevalence of 0.27% in household dogs and 3.11% in household and hunting dogs

respectively, using B. canis Rapid Slide Agglutination Test (RSAT). However, the present result

was lower than the prevalence of 59.43% reported by Cadmus et al., (2012) in dogs used for

hunting in an indigenous community in Ibadan, South-Western Nigeria. The hunting dogs may

have high exposure potential to brucella.

Female dogs had a higher seroprevalence (22%) than male dogs (6.0%). A major contributing

factor to higher rates in females, could be that a single male dog, if infected and is used in mating

with different females, can transmit the infection through infected semen (Cadmus et al.,2011).

Also it may be due to the fact in this region, most dog owners in the area sampled prefer to keep

more female dogs than males (as they pay male dog owners for their male dogs to mate the

female dogs) for the purpose of additional income through the sale of their puppies. This

increases the chances of more females getting infected during mating. However, Radostits et al.,

(2007), have shown that erythritol, a polyhydric acid found in higher concentration in the

placentas and fetal fluids of females than in seminal vesicles and testis of males can be

responsible for females being more susceptible than males. This result is in agreement with the

findings of Cadmus et al., (2011) who reported a prevalence of 6.17% in females and 4.9% in

65

males. But in another study a slightly higher rate in males (29.6%) than in females (26.7%) was

recorded by Adesiyun et al., (1986).

The decrease in the positive samples as titer level increased maybe due to the fact that infection in

dogs with higher titer levels, may have entered into the chronic phase thereby making these

infected dogs not to be productive thus leading to the disposing or selling off of these non

producing females as complained by the owners while those with lower titer levels, are more in

number because the infection may not have started manifesting its characteristics in them.

Dogs presented at the clinics had the highest number of positive samples in the different titer

levels more than the other categories of dogs sampled; and this may be attributed to the fact that

more samples were gotten from clinical cases as most dogs brought to the Clinics came as a result

of either illness, routine check up or vaccination.

The different titer levels were also higher in females more than in males and this may be

attributed to the fact that female dogs where sampled more in the study. Also, Radostits et al.,

(2007), have shown that erythritol, a polyhydric acid found in higher concentration in the

placentas and fetal fluids of females than in seminal vesicles and testis of males can be

responsible for females being more susceptible than males. In addition to this, due to the

asymptomatic nature of dogs infected with B. canis, most male dogs used for breeding are

unscreened, and carry the infection for a long period of time, shedding it in the environment

through urine and semen; this may result in bitches being mated severally by the same infected

male dog as a result of repeated unsuccessful breeding, thereby increasing the infection load in

females.

66

Prevalence was lower among the young animals screened in this study compared to the older

ones. Usually young animals are protected by maternal immunity and thus are less susceptible to

infections. This shows that the infection increases with age. The high prevalence seen in older

animals is demonstrative of the chronic nature of brucellosis as it has been shown to increase with

age, and most affected animals carry the infection throughout their lives (Radiostits et al., 2007).

The reason for the increase in prevalence with respect to increase in age may be attributed to the

fact that the bacteria localizes mainly in the reproductive tracts, especially in pregnant animals.

There is also evidence that the mammary gland may even be more favoured for localization than

the reproductive tract (Abubakar et al., 2012). Age-wise prevalence studied by Aulakh et al.,

(2008) and Abubakar et al., (2010) showed that the incidence is high in sexually mature animals.

Therefore, increase in age, increases exposing probability to infection in dogs. However, the

result does not agree with the study carried out by Cadmus et al., (2011), where he reported more

prevalence in dogs less than one year old than in adult dogs.

There is a strong association between the infection rate and breeds of dogs screened; with the

infection occurring more in the exotic breeds of dog than the mixed and local (mongrel) breeds.

This may be related to the fact that exotic breeds of dogs are better cared for by the owners in the

two states, and therefore constituted the dominant breed (61.8%)) in the population sampled

(76/123) because it gives much more income from the sale of its puppies than the other breeds

thus, the chances of the disease occurring is more in these breeds. Behzadi and Mogheiseh,

(2011), argued that detection of canine brucellosis in exotic dogs may indicate a new source of

infection from abroad as these dogs may be imported from countries and regions where the

disease is endemic and due to lack of screening of dogs before mating, may be a source of

67

infection to uninfected dogs. The higher prevalence among the exotic breeds is in agreement with

the findings of Behzadi and Mogheseh (2011), were they got a prevalence of 19.35% in exotic

breeds of dogs. It is also in agreement with the findings of Cadmus et al., (2011) who got 50.55%

in Alsatian breeds of dogs.

There is a strong association (p<0.05) between B. canis infection and some risk factors such as

sex, breed and dogs moving freely in the neighbourhood. This was further supported by the

multivariate logistic regression (Appendix IV).

Some risk factors such as: dogs more than one year and above, dogs being used for breeding, dogs

sharing rooms with the members of the household, female dogs with history of infertility and

abortion, and male dogs used for mating, all gave a considerable higher positive number and

increases the chances of brucella infection in dogs in the study area even though not significant

(Appendix IV).

68

CHAPTER SIX

CONCLUSION AND RECOMMENDATIONS

6.1 Conclusion

The zero seroprevalence of B. abortus antibodies in dogs in this study does not totally mean the

non-existence of the disease in the localities studied but may infer that B. abortus in dogs does

rarely occur in this region, except when the dogs are in close contact with infected farm animals;

through the consumption of their infected materials like aborted fetuses and feces, placental, fetal

and vaginal fluids from infected animals as proven by previous researchers.

Also, the present study has shown a Brucella canis antibodies seroprevalence of 27.6 % in the

study area. This means that the general public is at risk and this calls for serious interventions

considering the zoonotic implications of the disease as infected dogs can be a source of infection

not only to animals but also to humans especially those that are in close contact with these

animals due to the nature of their work / occupation.

Some factors such as sex, breed, age, dogs moving freely in the neighbourhood, dogs being used

for breeding and male dogs used for mating increases the chances of brucella infection in dogs in

the study area and therefore should be considered in the epidemiology of canine brucellosis in

the study area.

The Immunocomb®Canine Brucellosis Antibody Test Kit used for this study has proven to be a

reliable test method (99% sensitive and specific) in the diagnosis of Brucella canis in dogs.

69

6.2 Recommendations

i. Management practices in keeping dogs is a risk factor for transmission of Brucella canis

infection in the study area, thus dogs should be restricted within cages in a fenced

residence.

ii. Dog breeds is an associated risk factor to seroprevalence of brucellosis, exotic breeds of

dogs imported into Nigeria should be screened against the infection.

iii. Control measures should be instituted by dog owners and kennel attendants through

constant cleaning of the environment and kennels in the study area to stamp out or

eradicate the infections among dogs and to avoid spread to other uninfected dogs and

possible transmission to humans because of its zoonotic implication.

iv. Hygienic measures such as proper disposal of aborted fetuses, placenta and other

contaminated materials and disinfection of kennels, premises should be strictly observed

by the dog owners especially those that keep dogs as pet and allowed them into their

living rooms so as to avoid being infected with infected dog secretions especially in

children.

v. Butchers, animal health workers, meat handlers and laboratory workers, including

veterinarians should endeavor to wear protective clothings, hand gloves and face masks

to avoid direct contact and inhalation of contaminated aerosols in the clinics and kennels.

vi. States and Federal Ministries of Health should create a Veterinary Public Health Unit that

will be handling zoonotic diseases as is found in other developed countries, for the

prevention, surveillance and control of zoonoses in general and brucellosis in particular.

70

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93

APPENDIX I

SOURCES WHERE SAMPLES WHERE PURPOSIVELY COLLECTED

A) Clinic dogs

I) Anambra State. (a) Apex Veterinary Clinic Onitsha . . . . . . . . . 24

(b) Omega Veterinary Clinic Onitsha. . . . . . . . 13

Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37

II) Enugu State. (a) VTH Nsukka . . . . . . . . . . . . . . . . . . . . . . . . . 7

(b) Enugu State Vet. Clinic Uwani . . . . . . . . . . 9

(c) Animal Vision World Clinic Enugu . . . . . . 12

Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Sub- Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

B) Slaughter dogs.

I) Anambra State (a) Igboukwu Market . . . . . . . . . . . . . . . . . . . 6

II) Enugu State (b) Orba Market . . . . . . . . . . . . . . . . . . . . . . . 28

Sub- Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

C) Household dogs

I) Anambra State (Onitsha metropolis) . . . . . . . . . . . . . . . . . . . . . . . 12

II) Enugu State (Enugu metropolis). . . . . . . . . . . . . . . . . . . . . . . . . . 12

Sub- Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Grand Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

94

APPENDIX II

ANIMAL PROFILE

Client details

1. Location of clinic (city/town) _______________________________

2. Location of owner (city/town) ______________________________

Animal profile, (please tick where appropriate)

3. No of dog(s) in the household ________ (a) male [ ] (b) female [ ]

If more than one, please complete the following for each dog.

4. Breed (a) Exotic [ ]

(b) Mixed [ ]

(c) Local [ ]

5. Sex: (a) Male [ ] (b) Female [ ]

6. Age (a) less than 1 year [ ]

(b) 1 to 3 years [ ]

(c) 3 to 5 years [ ]

(d) Above 5 years [ ]

7. Any other domestic animal(s) in the household (tick which ones apply)

(a) Goat [ ] (b) Sheep [ ] (c) cat [ ] (d) Others, please indicate

________________

8. (a) Do your dog most of the time, move freely in the neighborhood [ ]

95

(b) Do your dog roam freely (i) at night [ ] (ii) all the time [ ]

9. Feeding habits of Dog: (a) scavenging all the time [ ]

(b) Both household feed and scavenging [ ]

(c) Household feed only [ ]

10. Purpose of keeping dogs (tick where applicable):

(a) Companion for children and other members of the family [ ]

(b) Guard/breeding [ ]

(c) Hunting [ ]

11. Do your dog share rooms with members of the household:

(a) Yes [ ]

(b) No [ ]

For female dog:

12. (i) Any history of abortion? (a) Yes [ ] (b) No [ ]

(ii) If yes, number of times (a) Once [ ] (b) Twice [ ] (c) More [ ]

13. If more please state ____________________________________________

For male dog:

14. Has the male dog ever been used for mating: (a) Yes [ ] (b) No [ ]

96

APPENDIX III

SAMPLE SIZE DETERMINATION

Formula:

n = 2

2

d

PqZ

n = the desired sample size (when population is > 10,000)

z = the standard normal deviation, usually set at 1.96 (or more simply at 2.0) which

corresponds to the 95% confidence interval.

P= the proportion in the target population estimated to have a particular

characteristics,

q = 1.0 - p.

d= degree of accuracy desired, usually set at 0.05 or occasionally at 0.02.

n =

n= 112

(1.96)2(0.0794)(0.92)

(0.05)2

97

APPENDIX IV: Odds ratio and (95% C.I) from Multivariate Logistic Regressions comparing

seropositive and seronegative dogs for Brucella canis antibodies in Anambra and Enugu States in

Southeast, Nigeria (n =123; np =34)

Antibody of Brucella canis

Positive Negative

OR 95% confidence OR 95% confidence

Interval interval

Characteristics Total no Total no Lower Upper Lower Upper

Sampled positive bound bound bound bound

(%) Sex Male 45 7(15.6) 0.449 0.213 0.947 1.292 1.053 1.584

Female 78 27(34.6) - - - - - -

Moving freely

In the neigbourhood

Yes

35

22(62.9)

4.243

2.132

8.446

0.436

0.279

0.680

No 54 8(14.8) - - - - - -

Feeding habits of dog Scavenging - - - - - - -

Both 27 11(40.7) 1.325 0.738 2.396 0.854 0.600 1.217

HF only 62 19(30.6) - - - - - -

Purpose of keeping dogs

Companion/security

breeding 87 30(34.5) - - - 0.655 0.563 0.763

Hunting 2 0(0.0)

Dog share rooms with

Household Yes 22 6(27.3) 0.761 0.358 1.618 1.133 0.829 1.548

No 67 24(72.7) - - - - - -

Abortion history Yes 46 16(34.8) 0.957 0.485 1.888 1.025 0.701 1.498

No 22 8(36.4) - - - - - -

Male dog used

For mating Yes 16 5(31.3) 1.563 0.234 10.423 0.859 0.496 1.488

No 5 1(20.0) - - - - - -

*p<0.05, n = total no of sampled dogs, np = total no of positive dogs, HF= Household food.

98

APPENDIX V: Risk Factors associated with the presence of Brucella canis antibodies in dogs presented at the Clinics and

Household dogs sampled in Southeast Nigeria (Total number sampled =123, number positive = 34). (*= p<0.05)

Parameter Total no

sample

Total no positive (%) Chi-

square

value

P.

value

Sex

Male

Female

45

78

7 (15.6)

27 (34.6)

5.183

0.023*

Age Less than 1 year

11

3(27.3)

1.5

0.682

1 - < 3years 32 9(28.1)

3 - < 5years 34 14(41.2)

5 and above 12 4(33.3)

Breed Exotic

76

26(32.4)

5.988

0.050*

Mixed Mongrel 13 4(30.8)

Local 34 4(11.8)

Other animals in the household

Sheep

1

1(100.0)

5.264

0.072

Others 6 4(66.7)

None

Animal move freely in the neighbourhood

Yes

No

Feeding habits of dog

Scavenging all the time

Both scavenging and household

Household food only

Purpose of keeping dogs

Companion for children/security

Breeding

Hunting

Dog sharing rooms with the household Yes

No

History of infertility/abortion in females

Yes

No

Male dog used for mating

Yes

No

82

35

54

-

27

62

-

87

2

22

67

46

22

16

5

25(30.5)

22(62.9)

8(14.8)

-

11(40.7)

19(30.6)

-

30(34.5)

0(0.0)

6(27.3)

0(35.8)

16(34.8)

8(36.4)

5(31.3)

1(20.0)

21.934

-

0.858

-

-

1.04

-

0.542

-

0.016

-

0.236

-

0.000*

-

0.354

-

-

0.308

-

0.462

-

0.898

-

0.627

-

99

APPENDIX VI

COMPONENTS OF THE CANINE BRUCELLOSIS ANTIBODY TEST KIT USED.

(a) A multi-compartment development plate (b) Twelve capillary tubes (c) One

disposable tweezers (d) Immuno-comb

(a)

(c)

(d)

(b)

100

APPENDIX VII

HARVESTED SERUM SAMPLES READY TO BE ANALYZED

101

APPENDIX VIII

IMMUNO-COMB SHOWING POSITIVE SERUM SAMPLES

Negative. Positive.

102

APPENDIX IX

DOG MARKET/SLAUGHTER POINT AT IGBOUKWU MARKET.

103

APPENDIX X

DOG MARKET/SLAUGHTER POINT AT ORIE ORBA MARKET.

104

APPENDIX XI

DOGS READY TO BE SLAUGHTERED FOR MEAT AT ORIE ORBA MARKET