faculty of the veterinary medicine chidiebere.pdf · 2015-08-28 · 2 seroprevalence and risk...
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Digitally Signed by: Content manager’s
Name
DN : CN = Weabmaster’s name
O= University of Nigeria, Nsukka
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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
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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
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
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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
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(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.
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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
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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
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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
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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,
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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.
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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.
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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
REFERENCES
Abbas, B. and Agab, H., (2002). A review of camel brucellosis. Preventive Veterinary Medicine
55, 47–56.
Abbas, B., and Tilley, P., (1990). Pastoral management for protecting ecological balance in
Halaib District, Red Sea Province, Sudan. Nomadic Peoples 29, 77–86.
Abdullah, A., Narin A. E.M., Savino, D and Sprague, K. (1984). Paleomagnetic study of some of
the dikes from the Esh El Mellaha Range, Eastern Desert, Egypt. Journal of African
Earth Sciences 2: 267 – 275.
Abubakar M.; Mansoor. M. and Arshed, M. J., (2012). Bovine brucellosis; Old and New
Concepts with pollistan Perspective. Parkistan Veterinary Journal, 32 (10):30.
Abukakar M.; Arshed M. J.; Hussain, M.; Ehtisham-ul-Haq and Q All, (2010). Serological
evidence of Brucella abortus prevaience in Punjab province, Pakistan a cross-sectional
study. Transbound Emerg Dis, 57.443-447.
Acha N.P., and Szyfers, B. (2003). Zoononses and communicable disease common to man and
animals. Third ed, Vol.1. Pan American Health Organization (PAHO), Washington, DC.
Adams L.G. (2002). – The pathology of brucellosis reflects the outcome of the battle between the
host genome and the Brucella genome. Vet. Microbiol., 90 (1-4), 553-561.
Adamu, N.B.; Okoh, A,E.J and Telta, D.A. (2007). Bovine brucellosis in Biu and Gwoza areas
of Borno State of Nigeria and CN public health implications. J. life and Environmental
sciences, 15:88-96.
Adedoyin F.J, Adesokan H. K, Otuh P. I, Omobowale T.O, Abiola J. O, Stack J. A, and Cadmus
S.I.B (2012). Sero-Prevalence of Brucellosis in Household and Stray Dogs in
71
Ibadan, South Western Nigeria. Book of abstract: 49th
annual congress of the Nigerian
Veterinary Medical Association- Ekiti State, Nigeria. P. 103.
Adekolu, E.O (1989). A Serological Survey of Human brucellosis in Kainji lake Population of
Nigeria. Trop Vet. 7:141-140.
Adesiyun, A. A and Oni, O., (1990). Sero-Prevalence of Brucella aborlus agglutinins in
abattoir workers and animals in three Nigerian cities. Bull Anim. Hlth Prod. Afri. 38.
203-204.
Adesiyun A A, Adbullahi S.U, and Adeyanju JB, (1986). Prevalence of Brucella abortus and
brucella canis antibodies in dogs in Nigeria. Journal of Small Animal Practice 27:31-37.
Ajogi, F. I. (1997). Seroprevalence of brucellosis in slaughtered cattle in four Northern states of
Nigeria. Tropical Vet. 15:21-24.
Ajogi, I.; Adesiyun, A. A and Esuruoso, G. O (1998). Settling the Nomadiciin Wase and Wawa-
Zange grazing reserve in Sudan Savannah Zone of Nigeria 11: The prevalence of bovine
brucellosis. Nig. Vet. J. 1: 80- 84.
Alausa, K. O (1984). Human brucellosis in Nigeria. Inter. J. Clinc Microbiol., 1: 5 -11.
Alton G. G.; Jones L. M.; Angus R. D and Verger J. M. (1988). Techniques for brucellosis
diagnosis. Laboratory Institute Nationale de laresearche agronomique, Paris. Pp 187.
Alton, G. G.; Maw, J; Rogerson, B. A. and Pherson G. G. (1975b). The serological diagnosis of
Bovine brucellosis: an evaluation of the Compliment Fixation Test, Serum Agglutination
Test and Rose Bengal Plate Test. Vet. J. 51: 57-63.
Alton, G.G., Jones, L.M and Pietx, D.E. (1975a). Laboratory techniques in Brucellosis. Second
edition; FAO/WHO, Geneva, Monograph Series No. 55.
Alursurp T.N. (1974). Failure to demonstrate brucella infection in naturally exposed ewe. Vet.
Rec. 94; 183-166.
72
Arellano – Reynoso B, Lapaque N, and Salcedo S, (2005). Cyclic beta – 1,2-glucan is a Brucella
virulence factor required for intacellular survival. Nat Immun; 6:618-63.
Asanda, N.N. and Agbede, S. A (2001). Brucellosis in Cattle and Human in Adamawa State.
Sokoto Journal of Veterinary Science. 3:34 – 38.Available at www.ivis.org; updated
1999.(Accessed 1st August, 2012).
Aulakh H. K.; Patil, P. K.; Sharma, S.; Kumar, H.; Mahajan, V. and K. S. Sandhu., (2008). A
Study on the Epidemiology of Bovine Brucellosis in Punjab (India) Using Milk-ELISA.
Acta Vet. Brno, 77:393-399.
Azwai, S.M., Carter, S.D., Woldehiwet, Z., MacMillan, A., (2001). Camel brucellosis:
evaluation of field sera by conventional serological tests and ELISA. Journal of Camel
Practice and Research 8, 185–193.
Baba M.M, Sarkindared S.E and Brisibe F. (2001). Serological evidence of brucellosis among
predisposed patients with pyrexia of unknown origin in the Northern Eastern Nigeria,
Cent. Eur J Pub. Health, 9:158-161.
Bale, J.O.; Nuru S.; and Addo, P. B., (1982). Serological Study of sheep and goat brucellosis in
Northern Nigeria. Bull. Anim. Hlth. Prod. Afr. 30: 73 – 79.
Bale, J.O. and Nuru S. (1982). Serological Study of brucellosis in local fowls in northern
Nigeria, Journal of Animal Production Research; 1:53:55.
Bale, J.O., (1980). Serological and bacteriological studies of ovine and caprine brucellosis in
Northern Nigeria. M.Sc. Thesis, Ahmadu Bello University, Zaria.
73
Bale, O.O.J., Nuru, S., Addo, P.B. and Adeyinka, I.A. (2003) Bacteriological investigation of
sheep and goat‟s milk for brucellosis in government farms in northern Nigeria. Nigerian
Journal of Animal Production, 30(1):109-116.
Banerjee, K. and Bhalty, M. A. (1970). A survey of bovine brucellosis in Northern Nigeria (A
preliminary communication). Bull.epiz,Dis.Afri, 18: 333-335.
Behzadi MA and Mogheiseh A, 2011. Outbreak investigation of brucellosis at a Kennel in iran.
Pak Vet J, 31:379-380.
Bel, K.J. and Lescelles, A.K. (1973). The use of the antigolboulin test in the diagnosis of bovine
brucellosis. Res. Vet. Sci. 14:239-244.
Bello-Onaighise, G., Vaikosen, S.E and Evivie, S. E (2012). Abortion cases in pig darms in
Benin City and some surrounding communities in Edo State, Nigeria. Nigerian Journal of
Agriculture, Food and Environment. 8(4): 37 – 42.
Bercovich, Z. (1998) Maintenance of Brucella abortus free herds: A review with emphasis on
the epidemiology and the problem in diagnosing brucellosis in areas of low prevalence.
Veterinary quarterly 20:81-88.
Bertu W. J, Ajogi I, Bale J.O.O , Kwaga J. K. P. and Ocholi R. A. (2010). Sero-epidemiology of
brucellosis in small ruminants in Plateau State, Nigeria. African Journal of Microbiology
Research, 4(19):1935-1938.
Bertu W.J (2006) Brucellosis in small ruminants. M.Sc Thesis Submitted to the Department of
Veterinary Public Health and Preventive Medicine. Ahmadu Bello University Zaria.
Biol. Stand., 56:371-383.
Braun W, and Bonestell A.E., (1947). Independent variations of characteristics in Brucella
abortus variants and their detection. Am J Vet Res, 8: 386-90.
74
Brew S.D., L.L. Perrett, J.A Stack, A.P Macmillan, and N.J. Staunton, (1999). Human exposure
to Brucella recovered from sea mammals. Vet. Rec., 144:483.
Blasco, J. M., (1990). Brucella ovis. In Neilson K. and Duncan, J. R editors. Animal Brucellosis.
Boca Raton (FL): CRC Press: 351 – 370.
Briones, G., Inon de Iannino, N., Roset, M., Vigliocco, A., Paulo, P.S., Ugalde, R.A., (2001).
Brucella abortus cyclic beta-1,2-glucan mutants have reduced virulence in mice and are
defective in intracellular replication in HeLa cells. Infect. Immun. 69, 4528–4535.
Brisibe, F.; Nawatte, D. R and BOT, C. J. (1993). Serological prevalence of brucellosis in sheep
and humans beings in Maidughari Metrolopolis. Trop. Vet., 11: 27 – 33.
Brooks, W. C., (2006) Brucellosis in dogs. Veterinary Information Network Inc. Online at:
http://www.VeterinaryPartner.com (accessed June 2010)
Brower, A., et al., (2007) Investigation of the spread of Brucella canis via the U.S.interstate dog
trade. Int J Infect Dis,. 11(5): p. 454-8.
Bruce D. 1893. Sur une nouvelle forme de fievre. Ann Inst Pasteur. 4:289- 304
Bruce D., 1887. Note on the discovery of a microorganism in Malta fever. Practicioner . 39:161-
70.
Brunner, A. and Gillespie, J (1966). Infectious diseases of domestic animals. 2nd
edition. Ithaca,
New York. Cornel University Press. 241 – 251.
Buddle, M. B. and Boyes, B. W. (1953). A Brucella mutant causing genital infection in sheep
in Newzealand. Aust. Vet. J. 29: 145 – 153.
75
Butter, J.E.; Seawright, G.T, Mc Given P. I and Gilsdorf M. (1981). Class and sub-class antibody
response of Brucella abortus strain 19-vaccinated and field strain challenged cattle:
evidence for predominant Ig G, system. Plenum Press, New York 770- 798.
Cadmus S I B.; Ijagbone I. F.; Oputa, H.E.; Adesokan, H. K. and Stack J. A (2006). Serological
survey of brucellosis in livestock animals and workers in Ibadan, Nigeria. African
Journal of Biomedical Research 9:163-168.
Cadmus S. I. B.; Adesokan, H. K.; Ajala, O. O. Odetokun, W. O.; Perret L. L. and Stack, J. A.;
(2011). Seroprevalence of Brucella abortus and B. canis in household dogs in
Southwestern Nigeria: a preliminary report. J. S. Afr. Vet. Assoc. (82) 1: 56 -7.
Cadmus S.I.B; Adesokan H.; Adedokan B.O. and Stack J.A. (2010). Seroprevalence of bovine
brucellosis in trade cattle slaughtered in Ibadan, Nigeria, from 2004-2006. Journal of
South African Veterinary Association. 81:50-53.
Cadmus S.I.B; Adesokan H.K., Ajala O.O., Odetokun W.O., Perrett L.L. and Stack J.A. (2012).
Seroprevalence of Brucella abortus and B. Canis in household dos in south western
Nigeria. A preliminary report. Journal of South African Veterinary Association . 82(1).
Carmichael L. E (1967). Canine brucellosis: isolation, diagnosis, transmission. Proc Annu Meet
US Anim Health Assoc , 71:517-527.
Carmichael L. E and Joubert J. C (1988). Transmission of Brucella canis by contact exposure.
Cornell Vet , 78:63-73.
Carmichael L. E and Greene C. E., (1990). Canine brucellosis, in Greene CE (ed): Infectious
Diseases of the Dog and Cat. Philadelphia, WB Saunders, pp 573–584.
76
Carmichael L. E, Zoha S. J. and Flores-Castro R., (1984). Problems in the serodiagnosis of
canine brucellosis: dog responses to cell wall and internal antigens of Brucella canis. Dev
Biol Stand 56:371-383.
Carmichael L. E, and Kenney R. M., (1970). Canine brucellosis: the clinical disease,
pathogenesis, and immune response. J Am Vet Med Assoc 156:1726-1734.
Carmichael L. E., (1976). Canine brucellosis: An annotated review with selected cautionary
comments. Theriogenology 1976, 6:105-116.
Carmichael L. E.; Greene C. E (2006). Canine brucellosis (2006). In: Greene CE, editor.
Infectious Diseases of the Dog and Cat. 3. Philadelphia: Elsevier Saunders: pp.369-
381.
Carmichael L. E.; Zoha S. J. and Flores-Castro R., (1984b). Problems in the serodiagnosis of
canine brucellosis: dog responses to cell wall and internal antigens of Brucella canis. Dev
Biol Stand 56:371-383.
Carmichael L. E.; Zoha S. J. and Flores-Castro R., (1984a). Biological properties and dog
response to a variant (M-) strain of Brucella canis. Dev Biol Stand , 56:649-656.
Carmichael L.E and Greene C. E., (1990,). Canine brucellosis, in Greene CE (ed): Infectious
Diseases of the Dog and Cat. Philadelphia, WB Saunders, pp 573–584.
Carmicheal L.E and Kenney, R. M (1968). Canine abortion caused by Brucella cains. Journal of
American Veterinary Medical Association, 152:605-616.
Carmicheal R. E, (1966). Abortion in zoo beagles. Journal of American Veterinary Medical
Association, 149:1126.
Cathleen. J.E and Sheem, E.J (1986). Transmission of bovine brucellosis from dam to calf. J.Am.
Vet. Rec. 188:867- 869.
77
Chauhan H. C.; Chandel, B. S. and Shah, N. M, (2000). Seroprevalence of brucellosis in
bufalloes in Gujrat. Indian Vet. J; 77: 1105 – 6.
Christopher S. Umapathy B. L and Ravikumar, K. L (2010). Brucellosis: review on the recent
trends in pathogenicity and laboratory diagnosis. J Lab Physicians 2:55-60.
Chukwu, C. C (1987b). Studies on seroprevalence of bovine brucellosis in Enugu and Nsukka,
Nigeria. Zariya Vet. 2: 25
Cloeckaerta.; Verger, J. M.; Grayon M.; Paquet J, Y.; Garin-Bastuji B.; Foster, G and Godforiod,
J., (2001). Classification of Brucella spp. Isolated from marine mammals by DNA polymorphism
at the omp2 locus. Microbes Infect 3:729 -738.
Collard, P. (1962). Antibodies against brucellae in the Sera of healthy persons in various parts of
Nigeria. West African Medical Journal. 89:172-177.
Cook, O, R., Cambell, R. W and Barrow. G (1988). Brucellosis in Northern Queensland rodents.
Aust. Vet. J. 42:5-8.
Cook, W. E., Williams, E. S., Throne, E. T., Kreeger, T. J., Stout, G., Bardsley, K., Edwards, H.,
Schuring, G., Colby, L. A., Enright, F., and Elzer, P. H., (2002). Brucella abortus strain
RB51 vaccination in elk. I. Efficacy of reduced dosage. J. Wildl. Dis. 38: 18 -26.
Cooper C. W (1991). The epidemiology of human brucellosis in a well defined urban population
in Saudi Arabia. J. Trop. Med. Hyg. 94:416–422.
Corbel M. J. (1973). The direct fluorescent Antibody Test for the detection of Brucella abortus
in bovine abortion material. Journal of Hygiene 71: 123.
78
Corbel M. J. (1984). International community on bacterial-sub committee on the taxonomy of
brucella. Minutes of a meeting of 10th
August, 1982. Buston M.A int’ l. J. Syst. Bacteriol.
34: 366 – 367.
Corbel, M.J. (1988). Brucellosis: Fertility and infertility in the veterinary practice (ed) by Laing
J.A; Morgan, W. J.B and Wagner, W. C. Bailitere Tindall, London. 189-221.
Crow, A., (2012). Knowledge, Attitude and Practices of liencesed Dog breeders in Kansas
regarding canine brucellosis.
http://www.Krex.kstate.edu/dspace,bitstram/handle/2097/15801/Crow%20Report.po?seq
uence. Accessed 18th
September, 2013.
Cuningham, B. and Dolan, L. A (1978). The use of Rose Bengal plate test in animal vaccinated
with K45/20A vaccine. Irish veterinary Journals 177:182.
Currier, S. A. (1989). The fluorescent Antibody Test dectection of B. abortus in bovine
materials. Journal of Hygiene 71:123.
Dalling, T., Robertson, A., Boddie, G., Spruell, J., (1988). Diseases of camels. In: The
International Encyclopedia of Veterinary Medicine. Edinburgh, U.K.; W. Green and Son.
pp. 585.
Dauglas, I. D (2006). Seroepidemiological studies on bovine brucellosis in Benue State. A Ph. D
thesis submitted to the department of Veterinary Public Health and Preventive Medcine,
University of Nigeria Nsukka, Nigeria. Pp. 125 – 126.
Davis, D.S: Templetion J.W;. Ficht, T.A;. Williams J. and Kopec J.D (1990). Brucella abortus in
ELK and captive Bison I. Serological bacteriology. Pathogens and transmission to cattle.
Journal of wild life diseases. 26:360-170.
79
De - Masis, F.; Di Girolama, A.; Petrini,A.; Pizzigallo, E and Giovani. A. (2005). Correlation
between animal and human brucellosis in Italy during the period 1997 – 2002. Clin.
Microbial Infect 11: 632 – 636.
Deutsch H. F.; Morton J. I (1957) Dissociation of human serum macroglobulins.
Science , 125:600-601
Edu. C.E (2005). Seroprevalence of Brucella infection in pregnant women in Jos, Plateau State.
Global Journal of Medicine. (in press).
England, T., Kelly, L., Jones, R.D., MacMillan, A. and Wooldridge, M., (2004). A simulation
model of brucellosis spread in British cattle under several testing regimes. Preventive
Veterinary Medicine 63, 63–73.
Esuruoso G.O., 1974. Bovine brucellosis in Nigeria. Veterinary Record, 98:54.
Esuruoso, G.O. 1979, Current situation in Nigeria and a preliminary evaluation of probable costs
and benefits of a proposed brucellosis control programme for the country. Vet. Epid. And
Economic PP. 644-649.
Evans, A. C. (1918). Further studies on Bacterium abortus and related bacteria 11: A comparism
of Bacterium abortus with Bacterium broncheosepticus and with the organism which
causes Malta fever. J. Infect. Dis. 22:580.
Ewalt D. R and Bricker, B. J., (2000).Validation of the Abbreviated Brucella AMOS PCR as a
Rapid Screening Method for Differentiation of Brucella abortus Field Strain Isolates and
the Vaccine Strains, 19 and RB51. J Clin Microbiol.; 38(8): 3085–3086
Ewalt D. R. ; Payeur J. B.; Martin, B. M.; Cummins D. R. and Miller W. G., (1994).
Characteristics of a Brucella spp., from a bottlenose dolphin (Tursiops truncatus). J. Vet
Diagn Invest 6:448 – 452.
80
Eze, E.N. (1977). Fate of Brucella in traditionally soured milk. Bulletin Animal Health and
Production in Africa, 23:1
Falade, S. (1977). Absence of brucella antibodies in dogs. Bulletin of Animal Health and
Productionn in Africa, 25(1):97.
Falade, S. (1978). A Comparism of three Serological tests in the diagnosis of caprine Brucellosis.
Research in Veterinary Science. 24:376-377.
Falade, S.; Ojo. M. O. and Sellers, K. C., (1974). A serological survey of caprine brucellosis in
Nigeria. Bull. Epiz. Dis, Afr., 22: 335 – 339.
Falade, S., (1974). Brucella agglutinating antibodies in the sera of persons dwelling in Ibadan
and the Surrounding districts. Nigerian Veterinary Journal 3: 21-23.
FAO/OIE/WHO (1995). Animal Production Year Book, No.34 FAO Rome.
FAO/OIE/WHO, 2006. Brucellosis in Human and Animals. Produced by World Health
Organization in collaboration with the food and Agricultural Organization of the
United Nations and World Health Organization for Animal Health.
WHO/CDS/EPR/2006.7. PP.1-71.
FAO/WHO (1986).World Health Expert committee on brucellosis.
http://www.integratedveterinarypathologybyalexandrabrower.com/uploads/1/4/1/0/14103
113/brower_neis_outreach_b._cains_presentation.pdf.
Fernando P.P.; Klaus N.; Luis E. S and Wei L. Y (2010). Diagnosis of Brucellosis. The Open
Veterinary Science Journal, 4: 46-60
Forbes L. B and Tessaro, S. V., (1996). Infection of cattle with Brucella abortus biovar 1
isolated from a bison in Wood Buffalo National Park. Can Vet J, 37: 415-419.
81
Forbes L.B (1990). Brucella abortus infection in 14 farm dogs. J. Anim. Vet. Med. Ass.
196.911-916.
Forbes, L. B.; Nielson, O.; Measures, L. and Ewalt, D. R. (2000). Brucellosis in ringed seals and
harp seals from Canada. J. Wildl. Dis 36:595 – 598.
Foster G.; Osterman B. S.; Godfroid J, et al., (2007). Brucella ceti sp. Nov. and Brucella
Pinnipedialis sp. Nov. for Brucella strain with cetaceans and seals as their preferred
hosts. Int J Syst Evol Microbiol 57:2688-93.
Godfroid J.; Cloeckaert A.; Liautard J. P, et al., (2005) from the discovery of the Malta fever’s
agent to the discovery of a marine reservoir, brucellosis has continuous been a re-
emerging zoonosis. Vet Res. 36:313-26.
Godfroid, J., Nielsen, K. and Saegerman, C., (2010). Diagnosis of brucellosis in livestock and
wildlife. Cro. Med. J. 51, 296-305.
Gugong, V. T.; Ajogi I.; Junaidu K.; Okolocha E. C., Ngbede E. O.; Hambolu S. E. and Maurice,
N. A., (2012). Avian influenza (H5 subtypes) antibodies in village chickens in four local
government areas of Kaduna State, Nigeria. Vet. World 5(12): 713 – 717.
Gul, S. T. and Khan, A., (2007). Epidemiology and epizootology of brucellosis: A review.
Pakistan Vet. J., 27(3): 145-151.
Gupta, R. S. (2005). Protein signatures distinctive of alpha protobacteria and its sub groups and a
model for alpha protobacterial evolution. Crit. Rev. Microbiol. 31: 101 – 135.
Gyuranecz Miklo´ s, Levente Szeredi, Zsuzsanna Ro´ nai, Be´la De´nes, La´szlo´ Dencso´´, A´
da´m Da´n, Nimro´d Pa´ lmai, Zso´ fia Hauser, Erzse´bet Lami, La´ szlo´ Makrai, Ka´ roly
82
Erde´lyi, Szila´rd Ja´nosi (2011).Detection of Brucella canis–induced reproductive
diseases in a kennel. J Vet Diagn Invest 23:143–147.
Hadju S. (1971). A study of the hetrogenicity of brucella antibodies 11: the proving of brucella
post infection antibodies by means of gel filtration on Sephadex G. 200. Veterinary
Bulletin 4:1631-1632.
Halle, P.D. and Ajogi, I. (1997). Burcellosis: A review of recent developments. Israel J. Vet.
Med. 52:125-131.
Hamidy M.E.R., Amin. A.S., 2002. Detection of Brucella spp. in the milk of infected cattle,
sheep, goats and camels by PCR. Veterinary Journal 163:299-305.
Hollelt R.B., (2006). Canine brucellosis: outbreaks and compliance. Theriogenology, 66:575-
587.
Hungerford, T. G. (1975). Brucellosis of cattle. In disease of livestock, 8th
edition. MC Graw-
Hill Book Company Sydney. 189-202.
Iowa State University (2012). Center for food securityand public health: Canine brucellosis
Brucella canis.http://www.cfsph.iastate.edu/Facesheet/pdfs/brucellosis_canis.pdf.
(Accessed 30th
August, 2012).
Jenny G. G. and Berman, D. T. (1962). Staining intracellular brucella organism by means of
fluorescent antibody. AM . J. Vet. Res. 23:596
Johnson C. A and Walker R. D (1992). Clinical signs and diagnosis of Brucella canis infection.
Compend Contin Educ Pract Vet 14(6):763–772.
Junaidu, A. U.; Oboegbulem S. I. and Salihu, M. D. (2008). Seroprevalence of brucellosis in
prison farm in Sokoto State, Nigeria. Asian Journal of Epidermiology 1(1): 24 – 28.
83
Junaidu, A. U; Oboegbulem, S. I; Garba, H.S; Daneji, A. I. and Gulumbe, M.L.
(2004).Brucella infection in Animals and Man in a Private Farm in Tureta L.G.A.,
Sokoto State, Nigeria. Sokoto Journal of Veterinary Science (in press).
Junaidu, A.U., Oboegbulem, S.I., Sharubutu, G.H., and Daneji, A.I., (2006). Brucellosis in
camels (Camelus dromedaries) slaughtered in Sokoto, Northwestern Nigeria. Animal
Production Research Advances 2, 158–160.
Kaamfer, p.; Scholtz, H. C.; Huber, B.; Thummes, K.; Busse H. J.; Maas, E. W. and Falsen, E
(2006). Description of pseudobacterium kiredjaniae spnov. Int. Syst. Evolmicrobial 57:
755 - 760.
Kazi, M. R. A.; Bahanur, R. M.; Saddiqur R. M.; Jae-Cheol H and Jin-ho P (2005). Prevalence of
Brucella antibodies in sera of cows in Bangledish. J. Vet. Science 6(3): 223 – 226.
Keid, L.R., Soares, M., Viera, N., Megid, J., Salgado V., Vasconcellos, S., Da costa, M.Gregon,
F. and R. chizenhain, L. (2007). Disagnosis fo canine brucellosis: Compariism between
serological and microbiological tests and a PCR based on primers to 16s-23s rDNA
interspacer. Veterinary Research communications, 31:954-965.
Kerwin S. C, Lewis D. D, Hribernik T. N, Partington B, Hosgood G, and Eilts B.
(1992). Diskospondylitis associated with Brucella canis infection in dogs: 14 case (1980-
1991). J Am Vet Med Assoc , 201:1253-1257.
Kudi, A.C.; Kalla, D.J.U.; Kudi. N.C and Yusuf H. (1997). Serological survey of brucellosis in
traditionally managed domestic fowl in northern Guinea savannah. Nigeria. World
Poultry Science Journal 53:287-289.
84
Labrune, P.; Jabir, B and Magny J. F. (1990). Recurrent enterocolitis-like symptoms as a
possible presenting manifestation of neonatal Brucella melitensis infection. Acta Paediatr
Scand 1990; 79: 707-709
Lapaque N, Moriyon I, and Moreno E, (2005). Brucella lipopolysaccharide acts as a virulence
factor. Curr Opin Microbiol; 8:60-6
Ledbetter E. C, Landry M. P, Stokol T, Kern T. J and Messick J. B, (2009). Brucella canis
endophthalmitis in 3 dogs: clinical features, diagnosis, and treatment. Vet
Ophthalmol. , 12:183-191.
Lopez-Goni I, Guzman-Verri C, and Manterola L, (2002) Regulation of Brucella virulence by
the two-component system BvrR/BvrS. Vet Microbiol; 90:329-39.
Lubani M. M.; Dudin K. I.; Sharda D. C.; AbuSinna N. M.; Al- Shab T.; Al-Refeai A. A.; Labani
S. M. and Nasrallah A (1988). Neonatal brucellosis. Eur. J. Pediatr. 147: 520 – 522.
Lucero, N. E., Escobar, G. I., Ayala, S. M. and Jacob, N. (2005). Diagnosis of human brucellosis
caused by Brucella canis. J. Med. Microbiol. 54, 457-461.
Luchsinger D.W. and Anderson R.K. (1967). – Epizootiology of brucellosis in a flock of sheep.
J. Am. vet. med. Assoc., 150 (9),1017-1021.
Maadi H, M Moharamnejad and M Haghi, 2011. Prevalence of brucellosis in cattle in Urmia,
Iran. Pak Vet J, 31: 81-82.
Maloney G E, and Fraser W R (2004). Brucellosis, Omaha, Nebraska, e Medicine, 2004 Online
at: http://www.emedicin.com/emerg/topic883,htm (Accessed 1 October, 2006).
Mateu-de-Antonio EM, Martin M, Casal J (1994). Comparison of serologic tests used in canine
brucellosis diagnosis. J Vet Diagn Invest , 6:257-259.
85
Mc Donald, W.L., Jamaludin, R; Mackereth, G., Hansen, M. Humphery, S., Short P., Taylor, T.,
Swingler, J., Dawson, C.E, Whitmore, A.M., Stubberfielf, E., Perrett L.L. and Simmons,
G., (2006). Characterization of Brucella s. Stain an a marine-mammal type despite
isolation from patient with spinal osteomylities in New Zealand. Journal of Clincial
Microbiology, 444363-4370.
Meador V. P., and Deyoe B. L., (1989). Intracellular localization of Brucella abortus in bovine
placenta. Vet. Pathol., 26: 513 – 5.
Moore J. A, and Kakuk T., (1967). Male dogs naturally infected with Brucella canis. J Am Vet
Med Assoc , 155:1352-1358.
Moreno E, and Moriyon I. In: Dworking M, Falkow S, Rosemberg E, Schleifer KH, and
Stackebrandt E, Eds. (2001). The Prokaryotes, Springer: New York (Electronic
version).
Morgan, W. J. B., (1968). The serological diagnosis of bovine brucellosis. Veterinary record.
80:612-615.
Morgan, W. J. B.; Mackinon, D. J.; Gill K. P. W.; Gover S. G. M and Norris P. W. (1978).
Standard laboratory techniques for the diagnosis of brucellosis 2nd
edition MAF. Central
Veterinary Laboratory, Weybridge, England.
Morgan. W.J. B.; Mackinon D.J. and Cullen, G.A (1969). The Rose Bengal Plate Agglutination
test in the diagnosis of brucellosis. Veterinary Records. 85:636-641.
Moriyon, I., Grillo, M.J., Monreal, D., Gonzalez, D., Marin, C., Lopez-Goni, I., Mainar-Jaime,
R.C., Moreno, E., Blasco, J.M., (2004). Rough vaccines in animal brucellosis: structural
and genetic basis and present status. Vet. Res. 35, 1–38.
86
Musa, M.T., Eisa, M.Z., M El Sanousi, M., Abdel Wahab, E.M., and Perrett, L., (2008).
Brucellosis in Camels (Camelus dromedarius) in Darfur, Western Sudan. Journal of
Comparative Pathology 138, 151–155.
MZCP (1998). The Mediterranean zoonoses control programme report on the 3rd
workshop on
human and animal brucellosis. Am. J. Vet. Res. 30: 1811 – 1816.
Njoku, A.J (1995). Brucellosis or Malaria. The Nigerian Journal of Parasitology 16:73-78.
Nuru, S. and Dennis, S. M. (1975). Bovine brucellosis in Northern Nigeria. A serological survey.
J. Nig. Vet. Med. Ass. 4: 3 – 8.
Ocholi R. A; Kwaga, J. K.; Ajogi, I.; Bale, J and Bertu, W.J., (2004). Epidemiology, Problems
and prospect for control of brucellosis in Nigeria. Vom Journal of Veterinary Science No.
1:78-86.
Ocholi R.A (1990). Prevalence of brucellosis antibodies in fulani cattle herd in Kaduna State.
M.Sc thesis, Ahmadu Bello University, Zaria 18-25.
Ocholi, R. A.; Kwaga, J. K. P.; Ajogi, I. and Bale, J.O.O. (2005). Abortion due to Brucella
abortus in sheep in Nigeria. Revue scientifique et technique Office international des
Epizooties, 24(3): 973-979.
Ocholi, R.A., Kalejaiye, J.O and Okewole, P.A (1993). Brucellosis in Nigeria. A review.
Tropical Veterinarian. 11:15-26.
Office International des Epizooties (OIE), (2009). Porcine Brucellosis. In:Manual of standards
for diagnostic tests and vaccines,(World Organisation for Animal Health), Paris, France.
Ogundipe, G.A.T.; Onyemi, M.O. and Ijagbone I. F. (1994). Serological prevalence of Brucella
abortus agglutinations in slaughtered cattle in Ibadan. Tropical Veterinarian 12:158-161.
87
Oko A.E.J (1980). Abortion in sheep near Kano State, Nigeria. Journal of Tropical Anim
Health Production 12, 11-15.
Oko, A. E. J., Alexiev, I. and Agbonlahor, D. E (1978). Brucellosis in dogs in Kano, Nigeria.
Tropical Animal Health and Production. 10:219-220.
Onunkwo, J. I., Njoga, E. O., Nwanta, J. A., Shoyinka, S. V. O. and Onyenwe, I. W. and Eze, J.
I. (2011). Serological survey of Porcine Brucella infection in South East, Nigeria. Nig.
Vet. J. 32, 60-62.
Onunkwo, J.I (2005). Seroepidimiological survey of brucella infection in slaughter goats in
Nsukka Agricultural area, Enugu State M.Sc thesis submitted to the Department
veterinary Public Health and Preventive Medicine, University of Nigeria Nsukka, 22-38.
Palomarez – Ressendiiz E. ; Arellano – Reynoso B.; Hernandez – Castro r.; Tenorio – Gutierrez
V.; Salas – Tellez E.; Suraz –Guemes F. and Efren Diaz – Aparicio (2012). Immunogenic
Response of Brucella canis virB10 and virB11 Mutant in Murine Model. Front Cell
Infect. Microbiol. 2:35.
Pappas G, Papadimitriou P, Akritidis N, Christous L, Tsianos E V (2006). The new global map
of human brucellosis. Lancet Infectious Diseases 6(2):91-99.
Poester, F. P.; Nielsen, K.; Samartino, L. E. and Yu, W. L. (2010). Diagnosis of Brucellosis. The
Open Veterinary Science Journal, 4:46 – 60.
Polt S. S.; Dismukes, W. E.; Flint, A and Schaefer J (1982) Human brucellosis caused by
Brucella canis: clinical features and immune response. Ann Intern Med 1982, 97:717-
719.
Prior, M.G (1976). Isolation of brucella abortus from 2 dogs in contact with bovine brucellosis.
Can J. Cop. Med 40:117-118.
88
Radosits, O. Blood, D.C; Gray CC (1995). Veterinary Medicine: A text book of the diseases of
cattle, pigs, sheep, goats and horses. Bailliere. Tindal, London pp 56-57.
Radostits, W., Gay, C.C., Hinchcliff, K.W., Constable, P.D., (2007). Veterinary Medicine, tenth
ed. Elsevier Saunders, London, pp. 389–390.
Rajish Chahota; Mandesp Sharma; Kotach, R.C; Sabhaoh Veerma; Singh, M.M. and Arsani,
R.K. (2003). Brucellosis outbreak in an organized Dairy Farm Involving Cows and in
contact Human beings. Veterinarsle ARCHIV. 8:95-102.
Ramos T. R. R.; Junior J. W. P.; Sobrinho P. A. M.; Santana V. L. A.; Guerra N. R.; Melo L. E.
H, et al. (2008). Epidemiological aspects of an infection by Brucella abortus in risk
occupational groups in the microregion of Araguaina, Tocantins. Braz J Infect
Dis;12:133–8.
Ray, W. C., (1979). Brucellosis (due to Brucella abortus and suis). In: Steele J, ed. Handbook
Series in Zoonoses. Section A: Bacterial, Rickettial and Mycotic Diseases. Roca Raton,
Florida, USA: CRC Press, Inc., 99-127.
Rikin, E. U. (1988b). Brucellosis of cattle in Nigeria. Proposal for a control programme under
intensive and extensive husbandry system. Acta. Vet. Scand. Suppl. 84:94 – 97.
Robinson, A. 2003. Guidelines for coordinated human and animal brucellosis surveillance In
FAO animal production and health paper 156.
Ross H. M.; Foster G.; Reid R. J.; Jahans, K.L and MacMillian, A. P., (1994). Brucella species
infection in sea-mammals. Vet Rec 134:359.
89
Ross H. M.; Jahans, K.L.; MacMillian, A. P.,Reid R. J.; Thompson P. M and Foster G., (1996).
Brucella species infection in North Sea seal and cetacean populations. Vet Rec 138: 647
– 648.
Ruben S. M.; Dillon P. J.; Schreck R.; Henkel T., Chen C. H.; Maher M.; Baeuerle P. A and
Rosen C. A (1991). Isolation of a rel-related human cDNA that potentially encodes the
65-KD subunit of NF-kappa B. Science 251(5000): 1490 – 1493.
Rumley R. L. and Chapman S.W., (1986)Brucella canis: an infectious cause of prolonged fever
of undetermined origin. South Med J, 79:626-628.
Saleem. S. F. and Mohsen, A., (1997). Brucellosis in fish. Vet. Med-Czeh 42:5 – 7.
Sati, S.N. (2002). Seroepidimiological survey of Brucella abortus infection in Fulani breeding
herds and trade cattle in the middle belt and southeast. M.Sc thesis submitted to the
Department of veterinary pathology and Microbiology, University of Nigeria Nsukka –
pp.1-8.
Scheftel Johni (2003). Brucella canis: Potential for zoonotic transmission. Compendium
Continued Edu. Practicing Vet: 25 (11) 846-853
Schelling E, Diguimbaye C, Daoud S, Nicolet J, Boerlin P, Tanner M, Zinsstag J. (2003).
Brucellosis and Q-fever seroprevalences of nomadic pastoralists and their livestock in
Chad. Preventive Veterinary Medicine. 2003;61:279–293.
Schnurrennerger, P.R; Walker, J.F and Martin I.R.L (1975). Brucella infection in Illenosi
veterinarian J. Am. Vet. Med. Ass. 167:1084-1089.
Scholtz, H.C Hubalek, Z; sedlacek, Vergnaud, G; Tomaso, H; Aldahouk, S; Melzer, F; Kamfer,
P. Neubauer H; Cloekaert, A and Maquart, M (2008). Brucella macroti sp. Isolated from
the common vole Microtus arvalis. Int. J. syst evol microbial 58, 375-382.
90
Seifert, H. S. H., (1996). Contact diseases caused by Aerobic rod-brucellosis in tropical animal
health. Kluwar Academic Publishers, Dodretch, Neitherland. 356 – 368.
Seleem, M. N.; Boyle . S. M. and Sriranganathan N., (2010). Brucellosis: A re- emerging
zoonosis. Veterinary Microbiology 140 (2010) 392 – 398.
Seleem. M.N; Jain, N.; Pothayee, N,; Ranjan, A.; Riffle J.S and Sriranganathan N. (2009).
Targeting Brucella Melitensis with polymeric nano particles containing srepotmycin and
doxocycline. FEMS microbiology letters 294,24-31.
Serikawa T, and Muraguchi T., (1979). Significance of urine in transmission of canine
brucellosis. Nihon Juigaku Zasshi , 41:607-616.
Serikawa T, Takada H, Kondo Y, Muraguchi T, and Yamada J (1984). Multiplication of
Brucella canis in male reproductive organs and detection of autoantibody to spermatozoa
in canine brucellosis. Dev Biol Stand , 56:295-305.
Shin. S. and Carmicheal L.E, (1999). Canine brucellosis caused by Brucella canis.
http://www.IV/S.org/Advances/infect_dis_Carmicheal/shin/ivis.pdf(Accessed 30th
August, 2012).
Shin. S. and Carmicheal L.E, 1999. Canine brucellosis caused by Brucella canis.
http://www.IV/S.org/Advances/infect_dis_Carmicheal/shin/ivis.pdf (Accessed
30th
August, 2012).
Smeak D. D, Olmstead M. L and Hohn R. B., (1987). Brucella canis osteomyelitis in two dogs
with total hip replacements. J Am Vet Med Assoc , 191:986-990.
Smith, H. A; Jones, T. C; Hunt, R. D (1972). Veterinary Pathology. 4th
edition. Lea and Ferbiger,
Philadephia, U. S. A. 594 – 598.
91
Sohn, A. H.; Probert, W.S Glaser, C.S; Gupta N.; Bollen A. W.; Wongi J. D.; Grace E. M and
Mc Donald W.C. (2003) Human neurobrucellsis with inercerebral granuloma caused by
a marine nammal Brucella sp. Emerg infect. Dis.g (2003).
Solonitsuin, M.O., (1949). Brucellosis in camels. Veterinarya, Moscow 26, 16–21.
Spink W. W and Morisset R., (1970). Epidemic canine brucellosis due to a new species: Brucella
canis. Trans Am Clin Climatol Assoc 81:43-50.
Stoner, H. G. and Lackman, D. B. (1957). A new specie of Brucella isolated from the desert
wood rat (Neotome Lepida). Am. J. Vet. Res. 18: 947 – 949.
Tabak, F.; Hakko, E.; Mete B.; Ozaras, R.; Mert, A and Oziturk R.; (2008). Is family screening
necessary in brucellosis infection? British Vet. Journal. 198-190.
Taul, L. K.; Powell, H. S.; and Baker, O. E (1967). Canine abortion due to unclassified Gram –
negative bacterium. Vet. Med. Small Anim. Clin., 73: 543 - 4
Teshome, H., Molla, B., Tibbo, M., (2003). A seroprevalence study of camel brucellosis
Theriogenology 1976, 6:105-116.
Thrusfield M. V.,( 2005). Criteria for Success of Questionnaire. In: Veterinary Epidemiology.
3rd Ed, Blackwell Science, Oxford, UK, pp: 189-213.
Vinayak A, Greene C. E, Moore P. A, and Powell-Johnson G. (2004). Clinical resolution of
Brucella canis-induced ocular inflammation in a dog. J Am Vet Med Assoc., 224:1804-
1807.
Wadood, F.; Ahmad, M.; Khan, A. Gul, S. T. And Rehman, N (2009). Seroprevalence of
brucellosis in horses in and around Faisalabad. Pakistan Vet. J., 2009, 29(4): 196-198.
Wanke, M. M. (2004) “Canine Brucellosis.” Animal Reproduction Science, 82 :195-207.
92
Wedimann, H (1991). Survey of means now available for combating brucellosis in cattle
production in the tropics. Animal research and development 33:98-111.
Whatmore, A. M.; Perret. L. L. and Macmillian A. P. (2009). A characteristic of the genetic
diversity of brucella by multilocus seguence. Bimco Milla Centre, Microbial. 7: 34.
Williams, K. P.; Sobral B. W and Dickerman A. W (2007). A robost species tree for the Alpha
protobacteria. J. Bact. 187: 4578 – 4586.
Wilson, R.T., (1984). The Camel. Longman, New York, ISBN 0-582-77512-4.
White P. G, and Wilson J. B (1951). Differentiation of smooth and non-smooth colonies of
brucellae. J Bacteriol., 61: 239-40.
Yagupsky P and Baron, Ej, 2005. Laboratory exposures to Brucellae and implication for
bioterrorism. J. Emerg infectious Dis, 5: 821-842.
Yanagi, M. and Yamasato, K., (1993). Phylogenetic analysis of the family Rhizobiaceae and
related bacteria sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS
Microbiol. Lett. 107, 115 -120.
Young, W. K., Edwards L. D., and Searson J. E., (1989). Evaluation of three LPS- specific
monoclonal antibodies for the diagnosis of bovine brucellosis. Res. Vet. Sci. 46: 413 – 5.
Zaki, R., (1948). Brucella infection among ewes, camels and pigs in Egypt. Journal of
Comparative Pathology 58, 145–151.
Zowghi E. and Ebadi A. (1988). Abortion due to Brucella abortus in sheep in Iran. Rev. sci.
tech. Off. int. Epiz., 7 (2), 379-382
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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