PREVALENCE AND RISK FACTORS ASSOCIATED WITH HELMINTH-BOVINE

TUBERCULOSIS CO-INFECTION AMONG SELECTED CATTLE HERDS IN OYO

STATE

BY

VICTOR OLUWATOYIN AKINSEYE

BSc (ILORIN); MSc (IBADAN)

MATRIC NO: 154687

A THESIS IN THE DEPARTMENT OF VETERINARY PUBLIC HEALTH AND

PREVENTIVE MEDICINE

SUBMITTED TO THE FACULTY OF VETERINARY MEDICINE, IN PARTIAL

FULFILLMENT OF THE REQUIREMENTS FOR THE CONVERSION
EXAMINATION FROM MASTER OF PHILOSOPHY (M.PHIL) TO DOCTOR OF
PHILOSOPHY (PH.D) OF THE UNIVERSITY OF IBADAN

MAY 2016

1
 CERTIFICATION

I certify that this research work was carried out by AKINSEYE, VICTOR OLUWATOYIN
under my supervision in the Department of Veterinary Public Health and Preventive Medicine,
University of Ibadan, Ibadan, Nigeria.

........................................
Supervisor

Prof S.I.B Cadmus

D.V.M., M.V.P.H., Ph.D. (Ibadan)
Department of
Veterinary Public and Preventive Medicine,
University of Ibadan, Ibadan

2
LIST OF ABBREVIATIONS

TB Tuberculosis

BTB Bovine tuberculosis

M. tb Mycobacterium tuberculosis

M. bovis Mycobacterium bovis

HIV/AIDS Human immunodeficiency virus/Acquired immune deficiency syndrome

WHO World Health Organisation

NTB Neglected tropical diseases

GIT Gastrointestinal tracts

Th2 T-helper cell 2

Th1 T-helper cell 1

SICCT Single intradermal comparative cervical test

TST Tuberculin skin test

PPD Purified protein derivative

IFN-γ Interferon gamma

TNF-α Tumour necrosis factor alpha

IL Interleukin

MtbC Mycobacterium tuberculosis complex

BCG Bacillus Calmette-Guerin

RD1 Region of difference

TGFβ Transforming growth factor beta

T-regs Regulatory T-cells

3
 ABSTRACT

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is a disease of major public

health importance responsible for millions of illness and death annually. Bovine TB (BTB) and

helminth infections are both diseases of debilitating and socioeconomic importance with dire

consequences on both livestock production and public health. Furthermore, there are evidence of

immunological interplay existing in animals co-infected with BTB and helminths resulting in

reduced sensitivity of immunological diagnostic tests of TB in cattle.

We conducted a cross sectional study among cattle herds in Itesiwaju (rural) and Ido (peri-urban)

Local Government Areas (LGA) of Oyo State between January 2013 and February 2014 to

determine the prevalence of BTB-helminth co-infection. Animals were selected based on

purposive sampling method. Cattle were screened for BTB using the single comparative

intradermal tuberculin test (SCITT) while faecal samples of cattle were examined for helminths

using standard sedimentation, floatation and copro-culture techniques. Data were analysed using

STATA 12, all tests were two tailed and stastical significance was set at p≤0.05.

Overall, 288 cattle from 35 herds were screened. An individual animal prevalence of 8.3%

(BTB), 55.9% (helminth) and 4.9% (helminth-BTB co-infection) were obtained while herds

prevalence of 50.0%, 90.0% and 36.7% were recorded for BTB, helminth and helminth-BTB co-

infection respectively. Relatively higher (55.2%) rate of cattle were screened from the peri-

urban, while majority of the animals screened were Bunaji (88.5%), female (60.4%) and adult

(74.0%). There was no significant association between BTB infection and location (OR=1.3;

95%CI:0.5–3.2), breed (OR=1.3; 95%CI:0.3–6.1), sex (OR=1.3; 95%CI:0.6–3.3), age (OR=0.2;

95%CI:0.1–1.0) as well as helminth burden (OR=1.4; 95%CI:0.5 – 3.6). Equally, no significant

association was observed between helminth infection and location (OR=0.7; 95%CI:0.4–1.1),

4
breed (OR=1.1; 95%CI:0.4–2.7), sex (OR=0.9; 95%CI:0.5 – 1.5), age (OR=0.8; 95%CI:0.4 –

1.4). However, location and breed were identified as risk factors for the occurrence of helminth–

BTB co-infection among cattle screened with animals in the rural area (OR=1.5; 95%CI:1.0–2.2)

and Balami breed (OR=15.3; 95%CI:1.2 – 199.5) of cattle being more likely to be co-infected.

From the 161 (55.9%) cattle positive for helminths infection, a total of 14 helminths from

different genera, including 10 nematodes, three trematodes and one cestode were found.

Our findings reveal that helminth-BTB co-infection exists among cattle herds. Also, BTB and

helminth infections are prevalent among cattle herds screened. Given the fact that helminth

infection has been implicated in negatively impacting on the diagnosis of BTB, it therefore

becomes imperative to investigate the role of helminths in the accurate diagnosis of bovine

tuberculosis among cattle herds and its consequences in the control of this disease in Nigeria.

Keywords: Bovine tuberculosis, helminth infection, diagnosis, control, tuberculin screening,

positive reactors, Ibadan

DEDICATION

This research work is dedicated to my loving parents Mr. and Mrs. F.K. Akinseye

5
ACKNOWLEDGMENT

6
I wish to express my profound gratitude to my supervisor; Professor S.I.B. Cadmus for his

encouragement and mentorship so far has greatly contributed to the materialization of this

research work.

My sincere appreciations go to all staff and members of the Department of Veterinary Public

Health and Preventive Medicine: the Acting HOD Dr Olayinka O. Ishola, Professor G.A.T.

Ogundipe, Professor S. A. Agbede, Professor Olanike K. Adeyemo, Dr. O. O. Babalobi, Dr I I.

G. Adeyemi, Dr. I. F. Ijagbone, Dr. O. B. Adedeji, Dr Victoria O. Adetunji, Dr B. O. Olugasa,

Dr. I. Olatoye, Dr. Veronica E. Adetunji, Mr B. Ojomo, Mrs Beatrice Kolawole, Mr. Olayemi

Okunlade, Mr. Folorunsho and others too numerous to mention.

Many thanks to the past and present members of the Tuberculosis and Brucellosis Research

Laboratory, Dr. H.K. Adesokan, Dr Tayo Falodun, Dr. Charity A. Agada, Dr. Joe Akwoba

Ogugua, Dr Dupe Ayoola, Dr Bukola Adedipe, Dr Dupe Hambolu and Dr A.M Adebayo; I

remain grateful for the imprints that you have made in my life.

I want to appreciate in a special way the Ajayis: Prof S.O Ajayi, Mrs G.I.B. Ajayi, Deji Ajayi,

Tobiloba Ajayi and Tomisin Ajayi for your great impact in my life and encouragement during

the course my research work. May the lord continue to bless you and reward you accordingly.

Also my wonderful friends who supported me materially and morally Mr Ibukun Afolami, Mr

Tolulope Omolekan, Mr Temitope Adedeji.

I also appreciate the moral support of Rev. Fr. Eddy Emokpo who supported me with lots prayers

and advice during the course of this work.

Words cannot express my thanks and appreciation to my loving Parent Mr and Mrs F.K.

Akinseye, my siblings: Mr Abiodun Akinseye, Ben Akinseye Yetunde Akinseye for your prayers

7
encouragement and moral support. I also want to appreciate my fiancée Miss Anthonia Akinsete

for your encouragement and prayer, I love you my baby.

Finally, my profound gratitude goes to God Almighty, the Eternal, Invincible most faithful God

for the journey so far. May your name be blessed in my life both now and forever, Amen.

8
TABLE OF CONTENTS

CONTENT PAGE

Title page i

Certification ii

List of abbreviation iii

Abstract iv

Dedication vii

Acknowledgement viii

Table of content ix

List of tables xiii

List of figures xiv

List of plate xv

CHAPTER ONE 1

INTRODUCTION 1

1.1 Introduction 1

1.2 Justification 3

1.3 Aim 3

1.4 Objectives 4

1.5 Research Questions 4

CHAPTER TWO 5

LITERATURE REVIEW 5

2.1 Historical background 5

2.2 Global burden and distribution 6

9
2.3 Aetiology 7

2.4 Bovine Tuberculosis (BTB) 7

2.5 Mycobacterium bovis 11

2.6 Characteristics of Mycobacterium bovis 11

2.7 Epidemiology of bovine tuberculosis 13

2.7.1 Environmental and animal reservoirs of M. bovis 13

2.7.1.1 Environmental reservoir 13

2.7.1.1.1 Locations 13

2.7.1.1.2 Physiological characteristics for environmental survival 13

2.7.1.2 Animal reservoir 14

2.7.1.2.1 Wildlife as a source of M. bovis 14

2.7.1.2.2 Physiological characteristics for host adaptation 14

2.7.1.2.3 Spread in domestic livestock 16

2.7.2 Transmission 16

2.7.2.1 Animal–to–animal transmission 16

2.7.2.2 Animal–to–human transmission 18

2.7.2.3 Human-to-animal transmission 20

2.7.2.4 Human-to-human transmission 21

2.7.3 Bovine tuberculosis in the animal population 22

2.7.4 Factors responsible for the persistence of bovine tuberculosis

in Nigeria 24

2.7.4.1 Bovine tuberculosis control in Nigeria 24

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2.7.4.2 Eating habit and standard of living 25

2.7.4.3 Diagnosis 26

2.7.4.5 HIV/AIDS-associated TB due to M. bovis 27

2.7.4.6 Illiteracy 27

2.7.5 Diagnosis of BTB 28

2.7.5.1 Tuberculin test 29

2.8 Immunology of tuberculosis 30

2.9 Helminth 33

2.9.1 Definition 33

2.9.2 Aetiology 33

2.9.3 History 33

2.9.4 Geographic distribution 34

2.9.5 Biodiversity 34

2.9.6 Nematyhelminthes 34

2.9.7 Platyhelminthes 35

2.9.7.1 Trematodes 35

2.9.7.2 Cestodes 35

2.9.8 Predilection sites 36

2.9.9 Life-cycles 36

2.9.9.1 Direct life-cycles 36

2.9.9.2 Indirect life-cycle 36

2.9.10 Developmental phases 37

2.9.10.1 Nematodes 37

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2.9.10.2 Cestodes 37

2.9.10.3 Trematodes 37

2.9.11 Route of entry 42

2.9.12 Pathogenesis 42

2.9.13 Clinical signs 43

2.9.14 Diagnosis 44

2.9.15 Immunology of helminth 44

2.9.16 Immune interplay of helminth-TB co-infection 45

2.9.17 Potential impact of helminth infection on TB diagnosis 46

CHAPTER THREE 47

MATERIALS AND METHODS 47

3.1 Study Area 47

3.2 Study design 50

3.3 Sample size and Sampling 50

3.4 Single Comparative Intradermal Tuberculin Test (SCITT) 51

3.5 Coprological examination of feacal sample for

helminth eggs 52

3.5.1 Helminth infection 52

3.5.1.1 Floatation technique 52

3.5.1.2 Sedimentation technique 53

3.6 Copro-culture 53

3.7 Statistical Analysis 54

CHAPTER FOUR 55

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RESULTS 55

4.1 Results of distribution of all the variables of cattle

screened 55

4.2 Results of the BTB infection among cattle screened 57

4.3 Results of the gastrointestinal helminth infection among

cattle screened 59

4.4 Diversity of helminth species recovered from the cattle

screened 61

4.5 Results of the helminth-BTB co-infection among cattle

screened 62

4.6 Results of the of logistic regression analysis of variables

significant at 10% level in bivariate analysis 64

CHAPTER FIVE 68

DISCUSSION 68

CONCLUSION 71

REFERENCES 72

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List of Tables

Table 1 Distribution of cattle screened according to all the variables tested 55

Table 2 Prevalence of BTB infection (measured by tuberculin test) among

cattle herds according to location, breed, sex, age and burden

of helminth infection 57

Table 3 Prevalence of gastrointestinal helminth in relation to location, sex,

breed and age of cattle 59

Table 4 Prevalence of helminth-BTB co-infection among cattle herds 62

Table 5 Results of logistic regression analysis of variables significant at

10% level with the main outcome measure (C.I status) in bivariate analysis 64

14
List of Figures

Figure 2.1 Pathway depicting the immunological interplay between

Th1, Th2 and Treg : a classical scenario in helminth and TB

co-infection in cattle

32

Figure 2.2 Nematode cycle: egg- larvae (L1-L4) – adult 38

Figure 2.3 Cestode cycle: egg- metacestode- adult 39

Figure 2.4 Trematode cycle: egg- miracidium- sporocyst- rediae-

cercariae- (metacercariae)- adult 40

Figure 2.5 Life cycle of helminth (possible route of re-infection) 41

Figure 3.1 Map of Oyo State, showing the local government areas

where the study was carried out 49

Figure 3.2 Individual and herd prevalence of BTB, helminth as well as

helminth-BTB co-infection among cattle screened 66

Figure 3.3 Venn diagram showing infection rates among cattle screened 67

15
Lists of Plates

Plate 1 Herds near the (Bororo camp) camp: In typical Bororo camps

herds are kept nearby the camps in lands where the next season’s

crops are to be planted. 48

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 CHAPTER ONE

INTRODUCTION

1.1 Introduction

Tuberculosis (TB) continues to be a major challenge globally as it is responsible for several

morbidities and mortalities among millions of people annually (WHO, 2014). It ranks alongside

HIV as a leading cause of death from an infectious disease worldwide, (WHO, 2015). In 2014,

9.6 million new TB cases and 1.5 million deaths were reported globally, out of which 1.1 million

were HIV-negative and 0.4 million were HIV-positive (WHO, 2015). Globally, from 9.6 million

incidence of TB, which ranges from 9.1 million – 10.0 million, equivalent to 133 cases per 100

population, Africa region accounts for 28% of the total burden (WHO, 2015) with Nigeria

having an estimated 570,000 cases of TB in 2013 (WHO, 2015).

Bovine tuberculosis (BTB), caused by Mycobacterium bovis, is a zoonotic disease which

primarily affects cattle. The disease is associated with loss of productivity and restrictions in

international exports in countries where it is endemic; and therefore of great economic

importance (Cadmus et al., 2006). It is a lingering concern in most of the developing countries;

however, in most developed countries, eradication measures such as test-and-slaughter policy

and compulsory pasteurization of milk have led to effective control of the problem (Ayele et al.,

2004; Amanfu, 2006). Bovine TB is one of the seven most neglected endemic zoonoses in the

world, particularly in developing countries (WHO, 2006 very old!!!!!). Bovine TB has serious

public health consequences and estimated to be responsible for approximately 10% of human TB

in Africa (Cosivi et al., 1998). Though several diseases abound in Africa, Nigeria has the highest

17
burden of negleted tropical diseases (NTDs) coupled with an un-estimated number of BTB

(Hotez et al., 2009). Of these NTDs, those caused by helminths are prevalent worldwide,

especially in sub-Saharan Africa where control measures are largely non-existent.

Helminths adversely affect the health status of animals leading to substantial economic losses for

the livestock industry. Aside damages inflicted on the health and productivity of animals,

infestation of helminth is also responsible for loss in body weight, poor reproductive

performance, digestive disturbance, and emaciation for longer period (Radostits et al., 1994).

Tuberculosis is the second major co-infection whose prognosis is associated with parasitemia

(Zvi et al., 2010). Bovine TB and helminth infections are both of significant economic and

public health importance; coincidentally, they share the same ecological niche, in the developing

world. Helminths are among the most widespread infectious agents that have affected, and still

affect both livestock and human populations, particularly in marginalized, low-income and

resource-constrained regions of the world. It is estimated that over one billion people in

developing regions of sub-Saharan Africa, Asia and the Americas are infected with one or more

species of helminths (Hotez et al., 2011). The gastrointestinal tracts (GIT) of animals harbour a

variety of helminths, which are known to cause major constraints to ruminants’ productivity on a

clinical and subclinical level (Martinez- Gonzalez, 1998).

Accurate diagnosis of BTB will facilitate effective epidemiological investigation of the disease

in endemic areas and this will in turn provide platform for its control and possible eradication.

While the diagnosis of many diseases including BTB requires detection of the host’s immune

response to the causative agent, helminth parasites impair their host’s immune response. Hence,

they increase susceptibility to infection and equally compromising the sensitivity of

immunologically based diagnostic tests in determining the prevalence of such diseases [Claridge

18
et al., 2012]. Reports showed that helminth infection may bias the immunological responses

towards a Th2 (eosinophilic) type and may reduce the sensitivity of immunological diagnostic

tests for TB in cattle which are Th1 dependent (Flynn et al., 20011). Thus, a biased immune

response caused by Th2, induced helminth infection, might impact negatively on the effort at

accurately diagnosing BTB in this part of world where both diseases are endemic.

1.2 Justification of Study

The burden of BTB seems unabated in cattle population and its endemicity persists especially in

developing countries. Its diagnosis and hence control have been challenging given the available

evidence showing that helminth infection stimulates strong Th2 immune response which

suppresses Th1 anti-TB response. Coincidentally, helminth infection shares the same

“developing world” niche with BTB infection among cattle. The conventional ante-mortem BTB

diagnostic test (single intradermal comparative cervical tuberculin test-(SICCTT) measures a

delayed type hypersensitivity response to the tuberculin antigen-purified protein derivative

(PPD). This test is dependent on functional antigen specific T-cells and their capacity to secrete

IFN-γ (a Th1 induced cytokine). There is therefore the need to investigate the burden as well as

risk factors associated with the occurrence of helminths – BTB co-infection among cattle herds

in Nigeria where these diseases are endemic.

1.3 Aim

To determine the prevalence of helminth-BTB co-infection and its associated risk factors among

cattle herds in selected parts of Oyo State, south- western Nigeria.

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1.4 Objectives

 To determine the current prevalence of helminth, BTB and helminth-BTB co-infection in

cattle herds in selected parts of Oyo State.

 To investigate the association between location, breed, sex as well as age of cattle and

helminth-BTB co-infection.

 To determine the relationship between helminth burden and BTB (using tuberculin test)

in cattle

1.5 Research Questions

 What is the prevalence of helminth, BTB and helminth-BTB co-infection among cattle

herds in Oyo State?

 What are the roles of location, breed, sex and age of cattle in susceptibility to helminth-

BTB co-infection among cattle herds?

 What is the relationship between helminth burden and tuberculin test in cattle?

20
CHAPTER TWO

LITERATURE REVIEW

2.1 Historical background

Tuberculosis (TB) has a long history. It has existed before the beginning of recorded history and

has left its impact on all aspects of human life including music, art, and literature; and has greatly

influenced the advancement in biomedical sciences and healthcare. Its causative agent,

Mycobacterium tuberculosis, may have killed more persons than any other microbial pathogen

(Daniel, 2006). Tuberculosis has infected humans and animals since ancient times. The disease

was described in Italian writing 2000 years or more before Christ was born and was also found to

be evident in Neolithic man from various skulls and other bones recovered from different parts

of the world (Salo, 1994).

The disease was prevalent in Egypt and Rome (Zink 2003, Donoghue 2004); it had been present

in America before Columbus (Salo 1994, Konomi 2002, Sotomayor 2004), and in Borneo before

any European contact (Donoghue 2004). The earliest DNA-based documentation of the presence

of M. tuberculosis complex organisms was accomplished in a sub-chondral articular surface

from an extinct long-horned Pleistocene bison from Wyoming, US, which was radiocarbon-dated

at 17,870 +/- 230 years before the present (Rothschild 2001). German microbiologist, Robert

Koch discovered the causative organism, the tubercle bacillus in 1882; in 1890 he developed the

tuberculin test for the diagnosis of the disease. In 1924, a vaccine called Bacillus Calmete Guerin

(BCG) for individuals exposed to the disease was developed.

21
Tuberculosis in cattle, which was also called ‘Pearl-disease’, attracted attention thousands of

years ago and the early meat inspection regulations in various countries were concerned with this

form of the disease (Salo 1994). The danger of eating meat from TB-infected cattle existed in

Mosaic laws and the German regulations banning the sale of TB meat (Collins and Grange,

1983).

When Robert Koch, a German bacteriologist unveiled the mystery of TB in March 1882 with the

discovery of the tubercle bacillus, he believed that the same organism caused the bovine and

human TB. Commenting on the source of the disease in human beings; he (1882) wrote that,

“one of these sources and certainly the most important, is the expectorant of individuals with TB,

another is tuberculous domestic animal, notably cattle”. This statement led to considerable

increase in interest in the prevention of infection by inspection of meat and heat treatment

(pasteurization) of milk.

2.2 Global burden and distribution

In 2014, there were estimated 9.6 million incidence cases of TB (ranging between 9.1 million –

10.0 million) equivalent to 133 cases per 100 population globally (WHO, 2014). The absolute

number of incident cases is falling slowly, at an average rate of 1.5% per year between 2000 and

2014 and 2.1% between 2013 and 2014 (WHO, 2014). In all, most of the estimated number of

cases in 2013 occurred in Asia (58%) and the African Region (28%); smaller proportions of

cases occurred in the Eastern Mediterranean Region (8%), the European Region (3%) and the

Region of the Americas (3%) (WHO, 2014). The 22 Highest Burden Countries (HBCs) that have

been given highest priority at the global level since the year 2000 accounted for 83% of all

estimated incident cases worldwide. The six countries that stand out as having the largest number

22
of incident cases in 2013 were India (2.0 million–2.3 million), Indonesia (0.7 million−1.4

million), China (0.86 million–1.0 million), Nigeria (340 000−870 000), Pakistan (370 000−650

000), and South Africa (400 000−510 000). India Indonesia and China alone accounted for a

combined total of 43%of global cases in 2014 (WHO, 2015). Of the 9.6 million incident cases,

an estimated 1.0 million were children and 3.2 million (range, 3.1–3.4 million) occurred among

women. Also, 6 million TB illness cases and 1.5 million death was reported globally, out of

which 1.2 million were HIV-negative and 0.4 million were HIV-positive (WHO, 2015).

2.3 Aetiology

The disease is caused by Mycobacterium tuberculosis and any member of the so-called

Mycobacterium tuberculosis complex (MtbC); these include Mycobacterium tuberculosis,

Mycobacterium africanum, Mycobacterium bovis (along with the M. bovis-derived bacillus

Calmette-Guerin [BCG] vaccine strains), Mycobacterium microti, Mycobacterium bovis subsp.

caprae (M. caprae), and “Mycobacterium tuberculosis subsp. canettii” (Brosch et al., 2002;

Mostowy et al., 2002). The most common species of Mycobacterium associated with the disease

in humans is M. tuberculosis, although an unknown proportion of cases are due to M. bovis

(Cadmus, 2003).

2.4 Bovine Tuberculosis (BTB)

Bovine TB (BTB) is an infectious disease of cattle caused by Mycobacterium bovis and is

characterized by the formation of tubercles in any tissue/organ of the animal. It is zoonotic, being

transmitted to humans by an aerogenous route and/or through consumption of infected milk and

other cattle products.

23
BTB is a disease of both economic and zoonotic importance and has resulted in the adoption of

country-wide control programmes in western countries. Developing countries, in contrast, know

little of the disease’s prevention, particularly as the contribution of BTB to human TB has

received limited attention. Major constraints, including lack of resources to study the disease

pattern and implement control measures, have led to the current situation in Africa (Sharp and

Daborn, 1995).

The disease is a chronic contagious respiratory disease of cattle which spreads horizontally

within and between species, by the aerosol and ingestion (O'Reilly and Daborn, 1995). It is

occurring in almost all developed and developing nations of the world. The incidence of disease

is not only higher in the developing nations but in the absence of any national control and

eradication program, it is also increasing worldwide particularly in the Asian, African and Latin

American countries (Bonsu et al., 2001). Mycobacterium bovis, the cause of BTB and M.

tuberculosis, the cause of classical human TB, are genetically and antigenically very similar and

cause identical clinical disease in humans (Dankner et al., 1993). There is considerable and

continuing public health significance of M. bovis infection in humans and animals and the

disease has emerged as a major zoonotic problem in many African countries (WHO, 1994). In

humans, M. bovis is the major cause of extra-pulmonary tuberculosis like tuberculosis of

gastrointestinal tract and tuberculosis of cervical and mesenteric lymph nodes, the peritoneum,

and the genito-urinary tract (Bonsu et al., 2001; Dakner et al., 1993). Bovine tuberculosis, a

chronic infectious disease of animals is characterized by the formation of granulomas in tissues

and organs, more significantly in the lungs, lymph nodes, intestine and kidney etc. It is caused by

slowly growing non-photochromogenic bacilli members of the MtbC: M. bovis and M. caprae

species. However, M. bovis is the most universal pathogen among mycobacteria and affects

24
many vertebrate animals of all age groups including humans although, cattle, goats and pigs are

found to be most susceptible, while sheep and horses are showing a high natural resistance

(Radostits et al., 2007; Thoen et al., 2006).

BTB has been significantly widely distributed throughout the world and it has been a cause of

great economic loss in animal production. In developed countries, BTB in animals is a rarity

with occasional severe occurrences in small groups of herds. In developing countries, however,

such as in 46% of African, 44% of Asian and 35% of the South American and the Caribbean

countries, sporadic occurrences and (particularly in Africa 11%) enzootic occurrences of BTB

have been reported (Cosivi et al., 1998). Apart from being the most important disease of

intensification with a serious effect on animal production, it also has a significant public health

importance (O’Reilly and Daborn, 1995). Although, the direct correlation between M. bovis

infection in cattle and human populations is not well known (Collins and Grange, 1983; Cosivi et

al., 1995), zoonotic BTB is present in most developing countries where surveillance and control

activities are often inadequate or unavailable. The actual impact of animal BTB on human health

is generally considered low in developed and developing countries, which may be based on the

rare identification of M. bovis isolates from human patients (Amanfu, 2006). In addition, the

occurrence of BTB due to M. bovis in humans is difficult to determine accurately because of

technical problems in isolating the micro-organism (Collins and Grange, 1983). In most

developed countries of the world, the disease in farmed animals is now relatively well controlled

and supplementary precautions of regulated meat inspection and milk pasteurization have

minimized the risk of human infection from M. bovis. Where human TB caused by M. bovis is

encountered in countries of the developed world, it is relatively rare and estimated to be at

around <1% of all tuberculosis cases (Grange, 2001). In such instances, infection is often seen in

25
the elderly, who have or have had agriculture associations, and disease has probably arisen from

reactivation of dormant lesions. The precise epidemiology of this disease in animal and human

populations in these countries has not been established and the contribution of M. bovis to human

tuberculosis in most instances therefore remains largely unknown. However, the correlation

between the prevalence of M. bovis infection in humans and in local cattle populations in Africa

highlights the potential threat of BTB to humans in such countries (Daborn et al.,1996).

Currently, BTB in humans is becoming increasingly important in developing countries, as

humans and animals are sharing the same micro-environment and dwelling premises, especially

in rural areas. At present, due to the association of mycobacteria with the HIV/AIDS pandemic

and in view of the high prevalence of HIV/AIDS in the developing world and susceptibility of

AIDS patients to tuberculosis in general, the situation changing is most likely (Amanfu, 2006).

Prevalence data on BTB infection in Africa is scarce. There is, however, sufficient evidence to

indicate that it is widely distributed in almost all African countries and even is found at high

prevalence in some animal populations (WHO, 1994; Ayele et al., 2004; Zinsstag et al., 2006a).

Thus BTB is still a great concern in many developing countries and Nigeria is one of those

where BTB is considered as a prevalent disease in cattle populations. Currently TB ranks

alongside HIV/AIDS, as being responsible for the deaths of more people each year than any

other single infectious disease, with more than 6 million new cases and 1.5 million deaths per

year and it is responsible for one-third of all deaths of HIV-infected individuals in Africa

(Zinsstag et al., 2006a; WHO, 2015). In Nigeria, the endemic nature of the human tuberculosis

has long been documented (Alhaji, 1976).

2.5 Mycobacterium bovis

26
Mycobacteriumbovis is commonly known as bovine tubercle bacillus and was first distinguished

from other mycobacteria through the work of Smith (1898) and Koch (1901). They are short to

moderately long rods. On primary isolation, growth is very poor on glycerol-containing media,

although repeated subculture permits adaptation to growth on such media. Furthermore, freshly

isolated cultures of M. bovis are microaerophilic; inocula dispersed into liquid, semisolid or solid

agar media grow in the medium but not on the surface, as distinguished from M. tuberculosis

which is highly aerobic (Schmiedel and Gerloff, 1965). On repeated subculture, M. bovis will

adapt to aerobic growth. Dilute inocula on egg media yield small, rounded, white colonies, with

irregular edges and a granular surface after 21 days or more of incubation at 37oC. Colonies on

transparent oleic acid albumin agar are thin, flat, generally corded; not easily emulsified in

absence of a detergent.

2.6 Characteristics of Mycobacterium bovis

M. bovis is a slow-growing non-photochromogenic acid-fast bacillus. In general, the bovine

tubercle bacillus is short (1-3um long) relatively plump and not as beaded as the human type but

shows solid staining. M. bovis is Gram positive while its cell wall composition is similar to

Gram-negative organisms (Kotani et al, 1959). The wax D-component which plays a decisive

role in inducing delayed type hypersensitivity of M. bovis differs from that of M. tuberculosis in

lacking peptides, a fact which apparently makes it unsuitable for use as an adjuvant (Alhaji,

1976). English workers frequently use the terms eugonic (heaped up growth indicates an aerobe)

and dysgonic (a flat sheet of growth indicates a microaerophil) in referring to the ease with

which mammalian bacilli can be cultivated, the first term being equivalent in general to the

human and the latter to the bovine type. The human type develops a little faster than the bovine

on cultures (Hagan and Bruner 1951). M. bovis grows slowly on solid or liquid media than M.

27
tuberculosis, especially on primary isolation. M. bovis can be differentiated from the other

species of the genus Mycobacterium based on series of biochemical tests. It is microaerophilic,

nitrate reduction negative or weakly positive, niacin production negative, weakly catalase

positive, susceptible to thiophen 2-carboxylic acid hydrazide (5mg/1) and resistant to

pyrazinamide (60mg/1).

Inclusion of a dye, usually malachite green, in egg-based media enables colonies of

mycobacteria to be more readily seen against a contrasting background and also tends to inhibit

the growth of certain contaminants in primary cultures. The growth of M. bovis is inhibited by

glycerol. Theobald Smith (1905) as cited by Hagan and Bruner (1951) early pointed out a growth

character by which the mammalian strains generally may be differentiated from each other. This

depends upon the fact that the human type utilises glycerol very actively whereas the utilisation

by the bovine type is limited. When cultivated upon a broth containing 3% glycerol, human types

produce enough acid to maintain a terminal acidity, whereas bovine types leave the medium

alkaline. Smith tested his fully developed cultures with phenolphthalein. If the reaction is acid,

there is a strong probability that the stain belongs to the human type; if alkaline, that it is of the

bovine type. If the original glycerol content of the medium is below 1%, both types will produce

an alkaline terminal reaction (Smith, 1905 as cited by Hagan and Bruner, 1951). An egg medium

with pyruvate replacing glycerol is favourable for growth, but the bacilli can be grown either on

egg or agar base medium. The colonial morphology of M. bovis varies with the medium for

instance, primary colonies on Middlebrook’s 7 H – 10 medium, are colourless, flat, irregular,

rough and dull, while on Stonebrink’s medium they are white, moist and convex resembling

those of M. avium.

2.7 Epidemiology of bovine tuberculosis

28
2.7.1 Environmental and animal reservoirs of M. bovis

2.7.1.1 Environmental reservoir

2.7.1.1.1 Locations

M. bovis is considered to be an obligate intracellular pathogen whose most efficient way of

infection is direct animal contact (Pallock and Neil, 2002). However, experimental evidence has

shown that M. bovis can survive for long periods outside an animal host in an environment

directly or indirectly contaminated by discharges of infected animals, suggesting other possible

ways of transmission. Yet in cattle, the natural host of M. bovis and the main source of human

spread, transmission via the oral route or even the respiratory route by inhalation of dust particles

in fields where no wildlife reservoirs are implicated in transmission to livestock, would play a

less important role. This is because the excretion of the organisms in faeces even from heavily

infected cattle occurs irregularly and at a low frequency (Menzies and Neil, 2000). There are no

records of human infection by M. bovis coming from a direct environmental source, revealing

that this way of transmission is not the most important one for this pathogen.

2.7.1.1.2 Physiological characteristics for environmental survival

The success of tubercle bacilli as pathogens comes mainly from its ability to persist in the host

for long periods and cause disease by overcoming host immune responses (Flynn and Chan,

2001). Nevertheless, the possibility of surviving for long periods in the environment is explained

by the mycobacterial impermeable cell wall (Brennan and Nikaido, 1995) and slow growth

(Gonzalez-y-Merchand et al1997). In contrast, other features render these species more sensitive

to environmental survival, like a more enhanced pH sensitivity of the tuberculosis complex

compared to Mycobacterium avium-intracellulare complex (MAC) species (Chapman and

29
Bernard 1962 and Cotter and Hill, 2003). Genomic comparisons between MAC and M.

tuberculosis complex members will not only allow to explaining differences in virulence

determinants between these two mycobacterial complexes but also the disparities in

environmental survival factors.

2.7.1.2 Animal reservoir

2.7.1.2.1 Wildlife as a source of M. bovis

Domestic and non-domestic animals may be considered either as maintenance (or reservoir)

hosts or non-maintenance (or spill-over) hosts for bovine tuberculosis. In reservoir host species,

infection can persist through horizontal transfer in the absence of any other source of M. bovis

and may as well be transmitted to other susceptible hosts. In contrast, spillover hosts become

infected with M. bovis but the infection only occurs sporadically or persists within these

populations if a true maintenance host is present in the ecosystem. If the source of infection is

removed, the prevalence for this disease is reduced and it can only be maintained in the long

term by re-infection from another source (Haydon et al., 2002).

2.7.1.2.2 Physiological characteristics for host adaptation

Although the course of infection, clinical signs and development of disease can vary within

different host species, it can be presumed that certain essential physiological characteristics are

common for successful infection in any susceptible host. The analysis of the complete genome

sequence of M. bovis (Garnier et al., 2003) provides a means to dissect these characteristics.

To begin with, the cell wall protects the bacteria from harsh environments but also promotes

intracellular persistence (Brennan and Nicado, 1995).

30
The ability to infect and persist in the macrophage by inhibiting phagosome-lysosome fusion,

creating a privileged compartment and remaining sequestered away from the terminal endocytic

organelles, is central to the success of the pathogen (Goren et al., 1976; Raja, 2004). The

presence of acidic, glycine-rich proteins (PE and PPE families) also found in M. leprae (Cole et

al., 2001) and M. marinum (Ramakrishman et al., 2000) whose genes are involved in virulence

are worth mentioning. Another important genetic factor implicated in the attenuation of the M.

bovis BCG strain is the lack of the RD1 locus (Ramakrishman et al., 2000), which is involved in

a novel described secretion system (Ramakrishman et al., 2000).

Latency is another important aspect of tubercle bacilli pathogenesis. The molecular basis for the

persistence phenotype and the pertinent host immune mechanisms that contribute to the

maintenance of tuberculosis latency are just beginning to be understood. The bacillus releases

peripheral cell wall lipids into their host cells, which induce the granulatomous response. This

represents active manipulation of the host’s response to ensure the maintenance of the infection.

The granuloma appears as a balance structure that walls off the infection and limits its

metastasis. However, the very prison that limits spread could well restrict the capacity of the host

to activate the macrophages required to kill the bacteria (Russell, 2003).

2.7.1.2.3 Spread in domestic livestock

Within domesticated animals, cattle, farmed buffalo and goats are considered reservoir hosts of

M. bovis, while pigs, cats, dogs, horses and sheep are considered spillover hosts (Cousins, 2003;

Biet et al., 2005). The realisation that wildlife is infected with M. bovis may result in apparent

failure programmes to eradicate the infection from cattle (Delahay et al., 2002). Knowledge of

31
wildlife tuberculosis through appropriate surveillance programmes in feral animal populations

may be important in the research strategies for the total elimination of livestock tuberculosis.

2.7.2 Transmission

Tuberculosis is primarily a respiratory disease but it can also spread to other parts of the body.

The primary route of transmission of infection within and between species is by the airborne

route and is facilitated by close, prolonged contact between infected and healthy humans or

animals through the exchange of respiratory secretions (O’Reilly and Daborn 1995 and Kempf et

al., 2005). However, other routes of transmission such as congenital and vertical transmission

have been recorded (Collins and Grange, 1983 and Neil et al., 1994). Transmission of

tuberculosis can be animal-to-animal, animal-to-human, human-to-animal as well as human-to-

human (Collins and Grange 1987)

2.7.2.1 Animal–to–animal transmission

In cattle as well as in other animal hosts, the route of transmission of M. bovis can be deduced by

the pattern of lesions observed in slaughtered animals. Animals with lesions restricted to the

thoracic cavity are presumed to have been infected by the inhalation of aerosols, while those with

lesions in mesenteric lymph nodes are thought to have acquired the infection by ingestion

(Pollock and Neil, 2002). In field cases of cattle, the majorities of lesions are found in the upper

and lower respiratory tract and associated lymph nodes. Thus, it is considered that the inhalation

of M. bovis is the most probable route of infection (Neil et al., 1994). In fact, the development of

tuberculosis lesions which invade the airways is thought to be required to facilitate active

excretion and aerosol spread of M. bovis (Menzies and Neil, 2000). Respiratory excretion and

inhalation of M. bovis is considered to be the main route through which cattle-to-cattle

32
transmission occurs in bovines. Droplets of contaminated water, eructation while ruminating

infected pastures or inhalation of contaminated dust particles can also be an alternative way of

aerogenous infection. This is, in fact, suspected to be the most likely way cattle could get

infected in a contaminated environment by badger excretions (Phillips et al., 2003). Ingestion of

M. bovis directly from infected animals or from contaminated pastures, water or fomites is

considered secondary to respiratory spread, as deduced from the minor presence of mesenteric

lesions in cattle cases (Menzies and Neil 2000). Congenital infections and vertical transmission

to calves as well as genital transmission are uncommon in regions where intensive eradication

programmes operate.

Intensive livestock farming promotes close contact between animals, favouring the spread of M.

bovis (Alhaji 1979; O’Reiley and Daborn, 1995; Shirima, 2003). Extensive livestock farming,

however, especially transhumance with no housing system, raises the question as to how bovine

TB transmission can take place. Close contact between animals occurs for example at water

points such as ponds, wells, and streams. In Africa, grazing animals usually gather at night for

protection from predators. Vaccination and artificial insemination centers, dipping tanks, auction

stations, market places and transportation are the commonest animal gathering places, and again

are sites where transmission of infection could easily occur. Due to the high ambient temperature

in tropical zones, animals tend to concentrate under trees or other shaded areas for parts of the

day, preferring to graze early in the morning and late in the afternoon (Ayele et al., 2004).

Possibly the most dangerous spots for nose-to-nose or mouth-to-mouth contact between animals

are salt supplementing points. Therefore, while extensive farming is safer than zero level grazing

systems to prevent disease transmission, some of the above situations stimulate the dangers of

intensive farming in relation to disease transmission (Ayele et al., 2004).

33
2.7.2.2 Animal-to-human transmission

In industrialized countries, the incidence of tuberculosis due to M. bovis in human is almost at

zero level as a result of pasteurisation of milk and milk products and eradication of bovine

tuberculosis in cattle population (Radostits, et al., 1995). However, in developing countries

especially Africa, the disease in animals can be widely distributed in regions where control

measures are not applied or are conducted sporadically and pasteurisation is rarely practised

(Cosivi et. al., 1998), and hence transmission to humans. The direct correlation between M.

bovis infection in cattle and disease in the human population has been well documented in

industrialized countries as well as in developing countries (Cosivi et al., 1998, Cook et al., 1996

and Ameni and Erhikum, 2007). Pulmonary TB due to M. bovis is more common in rural

dwellers, as a result of inhalation of dust particles or bacteria-containing aerosols shed by

infected animals, while urban dwellers acquire the infection via the gastrointestinal route and

develop extra-pulmonary TB (Daborn et al., 1996). In countries with a relatively high prevalence

of bovine TB in cattle, abattoir and farm workers are the groups most exposed to infection

(Ayele et al., 2004).

Infection of humans may occur by the inhalation of aerosols or through the consumption of

contaminated milk. The aerosols are the result of animal excretion but can also be produced by

handling lesioned carcasses (Neil et al., 1986). This route of infection leads to respiratory

tuberculosis.

Current economic and social globalization has created greater opportunities for the spread of

zoonotic disease such as TB. When considering the revival of TB in countries previously

declared to be free of the disease, it is worth noting the statement by Grange: ‘we are now

34
learning the hard way that none are safe until all are safe’ (Grange, 1994). Also enhancing

human infection with bovine tuberculosis is the menace of HIV/AIDS. The incidence of human

tuberculosis increased globally in 2003, but incidence, prevalence, and death rates were

approximately stable or decreased in all countries except Africa. Out of the 15 counties in the

world with the highest rates of tuberculosis, 13 are in Africa. It is estimated that 2.4 million

Africans become infected and 540 000 die annually from the disease (Mitka, 2005, OIE, 2006;

Raviglione et al., 1995). An HIV infection result in humans becoming much more susceptible to

all forms of tuberculosis; and it is estimated that 50% or more of new cases are related to prior

HIV infection (Raviglione et al., 1995). This not only poses a risk for other humans but also

results in cows being exposed to far higher levels of M. tuberculosis and other Mycobacteria than

was previously the case. Mixed infections of different M. tuberculosis strains in the same patient

have also been demonstrated (Warren et al., 2004). The cycle of tuberculosis infection between

HIV positive workers and cows, has yet to be fully explored.

The primary sources of infection for humans are consumption of unpasteurised milk and close

association between humans and animals (Coetzer and Tustin, 2005). Rural inhabitants and some

urban dwellers in Africa still consume unpasteurised and sour milk potentially infected with M.

bovis. Milk-borne infection is the main cause of non-pulmonary TB in areas where bovine TB is

common and uncontrolled. The agro-pastoral system of farming in Africa also exposes the

farmer to the mycobacteria which may be present in the faeces excreted by infected animal often

used as manure to fertilize the farmlands. Traditionally in Africa, cows suckle their calves and

are milked at the same time. Usually the cow is separated from the calf for about 12 hours, either

at night or, more usually, during the day and milked as the calf is returned to her. An infected

cow could produce milk containing mycobacteria, or cough infected droplets in the direction of

35
the milker. Milk is seldom pasteurised in pastoral societies, and, even if soured, can still contain

infective levels of mycobacteria [Coetzer and Tustin, 2005; Kazwala, 1996]. Animals in

traditional African farming systems are seldom culled and there is a greater chance for chronic

tuberculosis in old cows, particularly those subjected to stress (Michel et al., 2004).

2.7.2.3 Human-to-animal transmission

Fritshe et al., (2004) recently reported a scenario of tuberculosis in cattle exposed to a patient

infected with M. bovis, where the strain isolated in the cattle and the patient where identical. The

human patient, a 72-year old farmer was reported to have been exposed and contaminated from a

farm during childhood. The role of humans in infecting cattle with bovine TB was reviewed by

Torning in 1965 (van Soolingen et al., 1994). Sjögren and Hillerdal (1978) cited several

examples of human-to-cattle transmission, and stressed the potential danger that patients with

smear-positive pulmonary TB due to M. bovis may pose to animals. However, reports of human

infection of cattle are rare (O’Reilly and Daborn, 1995).The genitourinary tract in humans is a

site of non-pulmonary TB due to M. bovis; genitourinary TB may appear to be of little

importance to epidemiologists in studying human infection, but this route of infection from man

to cattle is well-documented. Grange and Yates reported that farm workers urinating in cowsheds

may represent a source of infection for animals (Grange and Yates, 1994). An analogous

situation is thought to occur in rural Africa, where patients with genitourinary TB may urinate on

pasture; animals craving salt preferentially graze on this grass and may succumb to infection. In

the same vein, people also live very close to the animals. Due to hand milking, the contact

between cow and human is also much closer and there is a high possibility that droplet-mediated

transmission of tuberculosis could occur – an infected human could exhale contaminated

particles into the bucket of milk or at the cow.

36
2.7.2.4 Human-to-human transmission

Mycobacterium bovis is pathogenic for humans, but its pathogenicity may be less than that of M.

tuberculosis. Person to person spread of M. bovis has been recorded, but the relative contribution

of this mode of transmission to the overall problem of tuberculosis has not been investigated in

detail. Human TB caused by M. bovis as a result of human-to-human transmission was reported

in the Netherlands in 1994 (van Soolingen et al., 1994). Evidence of transmission of M. bovis

between humans is considered rare and largely anecdotal, and the rate of transmission seems

insignificant compared to animal-to-animal or animal-to-human infection (O’ reilly and Daborn,

1995). Though this mode of transmission is considered less efficient than that of M. tuberculosis

(van Soolingen, 2001), transmission among HIV-infected humans, where immunosuppression

increases the susceptibility of the host organism to infection, may be different. Mycobacterium

bovis has been isolated from HIV-infected individuals in some industrialised countries, with an

additional serious complication of high primary resistance to isoniazid, streptomycin and

pyrazinamide (Guerrero et al., 1997). In France, there was a report of human-human

transmission of tuberculosis caused by M. bovis in immunocompetent patients (Sunder et al.,

2009).

2.7.3 Bovine tuberculosis in the animal population

In Nigeria, the presence of bovine tuberculosis can be traced back to 1932 (Alhaji, 1976). Past

studies have confirmed the prevalence and the endemicity of the tuberculosis in indigenous cattle

population (Alhaji 1976; Ayanwale, 1989). Prevalence rates of 0.49% and 1.29% have been

reported in slaughtered cattle in Sokoto State by Dusai and Abdullahi (1994) and Ajogi et al.

(1995) respectively. In similar studies conducted in Oyo and Osun States, south-west Nigeria as

37
well as in Maiduguri north-eastern part of the country, reported prevalence rates of 1.43%,

1.22% and 1.26% were respectively obtained(Ogundipe and Ajiboye, 1997, Ishola et al., 2001

and Egbe-Niyi et al., 2000). Wekhe and Berepubo (1989) reported a prevalence of 8.2% in a

study carried out in an eastern abattoir of the country; while Cadmus (2006) confirmed the

prevalence of 8.8% in Bodija abattoir in the west.

There has been a rise in the prevalence and the existence of foci of infection of the disease as

demonstrated by the tuberculin results of Alhaji (1976); Ayanwale (1989); Shehu (1988);

Cadmus et al. (2004); Abubakar (2007); Okaiyeto et al. (2008); Cadmus et al. (2009) and

Ibrahim et al. (2012) where individual prevalence rates of between 1% and16.65% as well as

herd prevalence of 45.45% were reported. The isolation of the causative agent along with other

mycobacteria species in fresh and sour milk have been reported as well across the country

(Alhaji 1976; Shehu 1988; Okolo 1992; Abubakar 2007; Cadmus and Adesokan 2007; Ofukwu

2008 and Okaiyeto et al. 2008).

Various organs especially lungs and lymph nodes of suspected tuberculous cattle from abattoirs

and slaughtered houses have yielded M. bovis in most of the cases with a small number of other

mycobacteria such as M. tuberculosis and M. africanum (Cadmus et al., 2006, 2007 and

Abubakar 2007). Mycobacterium bovis have been isolated from the nasal secretions of tuberculin

positive cattle by Ayanwale (1989) while Ibrahim et al., (2010) in a study conducted in Jigawa

State ascertained that tuberculin reactivity is significantly associated with respiratory signs hence

re-emphasizing inhalation as the route of transmission. This has a grave public health implication

seeing the disease can be transmitted to humans through the inhalation of cough sprays.

38
The disease was reported in other animal species in Nigeria. A rare case of tuberculosis of the

skin was reported in a horse which was presented to the Veterinary Teaching Hospital of the

Usmanu Dandodiyo University Sokoto with a main complain of circumscribed deep seated

wound located on the cheek (Garba et al, 2001). Mycobacterium bovis was isolated from a horse

The infection resulted in cross-infection to the horse boy who reacted positively to tuberculin

test.

2.7.4 Bovine tuberculosis in humans in Nigeria

The occurrence of BTB due to M. bovis in humans is difficult to determine accurately in Africa

because of technical problems in isolating the organism (Collins and Grange, 1983). Currently,

BTB in humans is becoming increasingly important in developing countries like Nigeria as

humans and animals are sharing the same micro-environment and dwelling premises especially

in rural areas (Abubakar, 2007). The association of TB with the HIV/AIDS pandemic and in

view of the high prevalence of the human TB due to M. bovis is likely to change (Amanfu,

2006).

Rural inhabitants and some urban dwellers in Nigeria still consume unpasteurized and soured

milk potentially infected with M. bovis. A study conducted in four states in Northern Nigeria by

Idrisu and Schnurrenberger (1977) reported that one of the 10 mycobacteria isolated from

sputum-positive cultures was M. bovis. In addition, Idigbe et al. (1986) reported that 4 (3.9%) of

102 M. tuberculosis complex isolates from sputum samples of patients with pulmonary TB, were

M. bovis. Other studies in Nigeria reveal a range of prevalence in different states of the country.

Garba et al. (2004) in an investigation to determine the species of Mycobacterium involved in

human tuberculosis in Sokoto State reported that 39 of the 106 samples yielded Mycobacterium

39
on culture; and on further characterization, eight were M. bovis and 4 atypical mycobacteria and

were principally from cases of extra-pulmonary tuberculosis, thus giving a prevalence of 7.55%.

In a related study in Jos, Plateau State, Mawak et al. (2006) discovered that 10 out of the 65

isolates from sputum of patients with persistent bronchopneumonia were M. bovis. In Ibadan,

south-western Nigeria, M. bovis was isolated from a child with cervical lymphadenitis (Cadmus

et al., 2004). Also in another study to establish baseline strains causing tuberculosis in Nigeria

conducted in Ibadan, Cadmus et al. (2006) established that 13% of the disease in the humans

studied was caused by M. africanum and M. bovis rather than M. tuberculosis. Furthermore, in a

bacteriological screening of cattle marketers in Akinyele International Cattle Market in Oyo

State for the prevalence of Mycobacterium; 26 out of 70 people screened (37.14%) tested

positive for tuberculosis by cultural isolation and biochemical characterization showed that five

(5) of the isolates were M. bovis giving a prevalence of 7.14%, however, deletion analysis

confirmed only two isolates as M. bovis (Adesokan, 2008).

2.7.4 Factors responsible for the persistence of bovine tuberculosis in Nigeria

2.7.4.1 Bovine tuberculosis control in Nigeria:

The control of BTB in Nigeria is regulated by the Federal Ministry of Agriculture. However, the

control policy of the Animal Disease (Control) Decree of 1988 is poorly or inadequately

implemented (Abubakar et al., 2011). This is largely due to politico-economic reasons and high

cost of sustainable testing and slaughter of infected animals and the subsequent compensation to

the farmers. In addition to these is the problem of social unrest due to political instability and

ethnic wars especially between the Fulani herders and local farmers (Ayele et al., 2004 and

Abubakar et al., 2011).

40
Also contributing to this occurrence is the lack of quarantine and communication networks at the

various control posts which enhances animal smuggling across state boundaries (Ayele et al.,

2004). This is also buttressed by the fact that most control posts have turned into revenue

collection points rather than checking points for disease surveillance. Most abattoirs are under-

staffed lacking sufficient veterinary expertise. The uncooperative attitudes of butchers coupled

with illiteracy, during meat inspection as a result of little or no compensation by the government

for condemned carcasses due to bovine tuberculosis is also a major bottle-neck in the control of

the disease in most African countries especially Nigeria.

2.7.4.2 Eating habit and standard of living

Majority of the cattle in Nigeria are of dual purpose and are with the pastoralists; who own them

as a sign of wealth, cohabit with them and also feed on their milk. The cattle are slaughtered only

during ceremonies. Therefore, the risk of transmission of the disease from cattle to humans

abound as there has been association between the keeping of cattle and the occurrence of both

pulmonary and extra pulmonary tuberculosis in man (Grange and Yates 1994; Ameni and

Erkihun 2007).

The primary sources of infection for humans are consumption of unpasteurised milk and close

association between humans and animals (Coetzer and Tustin, 2005). Rural inhabitants and some

urban dwellers in Nigeria still consume unpasteurised and soured milk potentially infected with

M. bovis (Abubakar, 2007 and Cadmus et al., 2008). Milk-borne infection is the main cause of

non-pulmonary TB in areas where bovine TB is common and uncontrolled. The agro-pastoral

system of farming in Africa also exposes the farmer to the mycobacteria which may be present in

the faeces excreted by infected animal often used as manure to fertilize the farmlands. An

infected cow could produce milk containing mycobacteria, or cough infected droplets in the

41
direction of the milker. Milk is seldom pasteurised in pastoral societies, and, even if soured, can

still contain infective levels of mycobacteria [Coetzer and Tustin, 2005; Kazwala, 1996].

Animals in traditional African farming systems are seldom culled and there is a greater chance

for chronic tuberculosis in old cows, particularly those subjected to stress (Michel et al., 2004).

Additionally, prior to sale, cattle are fattened in close proximity to the farmers’ home and after

being sold in at the market; they are being slaughtered in nearby abattoirs where butchers use

minimal protective clothing and process diseases carcasses with bear hands (Cadmus et al.,

2008).

The following factors contribute to the risk of contracting bovine tuberculosis: family owner of

cattle, previous ownership of livestock, history of working with animals, living with relative that

owns cattle and consumption of unpasteurized milk and raw or poorly cooked meat (Abubakar et

al., 2011).

2.7.4.4 Diagnosis

Diagnosis of tuberculosis in Nigeria most times ends at the smear level (Abubakar et al., 2011)

and as a result of that, infection by M. bovis cannot be differentiated from that caused by M.

tuberculosis. Consequently, cases of treatment failures or relapse often occur as M. bovis is

resistant to pyrazinamide which is widely used for the treatment of infections of M. tuberculosis

complex in humans (Krauss et al., 2003). There are very few laboratories that carry out culture

and isolation in Nigeria, and oftentimes it is mostly done for research purposes not for routine

diagnosis. The inability to identify the specific species of the Mycobacterium causing human

tuberculosis in the country has resulted in the lack of understanding of the extent of the

involvement of BTB in the tuberculosis epidemic in the country.

42
A major setback is the failure of the National TB control program to recognize the importance of

BTB as a major public health problem, coupled with lack of collaboration between the

veterinary and human doctors with regard to the control of the disease (Abubakar, 2007).

2.7.4.5 HIV/AIDS-associated TB due to M. bovis

Reports indicate that the incidence of tuberculosis in humans has risen as result of HIV/AIDS

epidemic (WHO, 2005 and Shitaye et al., 2007). As the result of the HIV/AIDS epidemic, there

has been increase in the incidence of BTB in humans as well (Cosivi et al., 1998, Ayele et al.,

2004 and Zinsstag et al., 2006).

Tuberculosis and other mycobacterial infections are major opportunistic infections in HIV/AIDS

infected individuals (Grange et al., 1994; Raviglione et al., 1995), while HIV/AIDS is a major

predisposing factor for tuberculosis including reactivation of the disease. The current spreading

pandemic of HIV/AIDS infection in developing countries, especially where BTB is prevalent in

domestic and wild animals poses an additional serious public health threat (Grange and Yates,

1994; Grange et al., 1994; Cosivi et al., 1995; Pavlik et al., 2002; Ayele et al., 2004).

2. 7.4.6 Illiteracy

The inability to read and write as well as the failure to utilize modern methods of communication

(Ayele et al., 2004) is a major problem Nigeria; and the limited knowledge of the rural dwellers,

herders, farmers and the general public about the epidemiology of BTB, make prevention and

control programs difficult and often impossible to apply (Shitaye et al., 2007).

2.7.5 Diagnosis of BTB

43
Many methods exist for the diagnosis of BTB in cattle. Some of these methods include: In vivo

test like tuberculin screening, the cellular test based on the quantification of gamma interferon as

well as In vitro methods like post-mortem diagnosis of macroscopic lesions of tuberculosis,

microscopic examination of lesion, Mycobacterial culture, biochemical characterization and

molecular methods (OIE, 2009). Bovine tuberculosis can be diagnosed in live cattle on the field

with the tuberculin skin test. A strong skin-based immune response to bovine tuberculin is

consistent with infection. In many instances this is performed in a comparative manner, using

avian tuberculin in addition to bovine. The magnitude of the avian response is taken into

consideration when determining positive or negative status. All skin tests are two step procedures

involving tuberculin injection on day one, and a reading of the skin response 72 hours later.

Presumptive testing may be carried out using histopathology and/or the microscopic

demonstration of acid-fast bacilli, where direct smears from clinical samples or tissues (usually

collected at post mortem) may be stained with the Ziehl Neelsen stain, a fluorescent acid-fast

stain or immunoperoxidase techniques. Confirmatory testing involves isolation of M. bovis on

selective culture media, which are incubated for eight weeks. The organism can be confirmed

with biochemical tests or polymerase chain reaction (PCR) assays (including spoligotyping

which can both confirm and type bacteria). Blood-based tests for immune responses to M. bovis

include the lymphocyte proliferation and gamma-interferon assays and serological tests.

Detection of bTB in Nigeria is carried out most commonly on the basis of tuberculin skin testing,

abattoir meat inspection and rarely on bacteriological techniques. However, the current status of

bTB at the national level is unknown because screening of cattle by the tuberculin skin test in

Nigeria is sporadic (Abubakar, 2007).

44
2.7.5.1 Tuberculin Tests

The primary screening test for BTB in live cattle is performed using one of the variants of the

TST: the caudal fold test (CFT), the (mid) cervical intradermal test (CIT), or the comparative

cervical test (CCT). The TST represents the OIE prescribed test for international trade and

constitutes a delayed type hypersensitivity test (Anon, 2008a). It measures dermal swelling

primarily because of a cell-mediated immune response (CMI) three days (72 hours) after

intradermal injection of purified protein derivative (PPD) in the skin of the caudal fold (CFT) or

neck (CIT), respectively. The skin of the neck is regarded to be more sensitive to a tuberculin-

related hypersensitivity reaction than the skin of the caudal fold. To compensate for this

difference, higher doses of PPD may be used in the caudal fold. Because animals are frequently

exposed to or infected with various non-tuberculous mycobacteria, cross-reactive responses to

PPD-B may occur as many antigens contained within PPD-B are shared between non-

tuberculous and tuberculous mycobacteria. The CCT is used to differentiate between animals

infected with M. bovis and those sensitized to PPD-B as a result of exposure to other

mycobacteria. Responses to bovine and avian tuberculins are compared according to OIE

guidelines or those developed by national eradication programs (e.g. use of a scattergram in the

USA). Historically, the TST has been the primary ante-mortem test available to support bTB

eradication campaigns. Advantages of the TST and reasons for its wide use are low costs, high

availability, long history of use and, for a long time, the lack of alternative methods to detect

bTB. Still, this test has many known limitations including difficulties in administration and

interpretation of results, need for a second-step visit, low degree of standardization, and

imperfect test accuracy (De la Rua-Domenech et al., 2006). Performance of TST techniques may

vary because of differences in tuberculin doses, PPD preparations, site of application, and

45
interpretation schemes. In fact, disparate performances have been found in many international

studies (Monaghan et al., 1994): 68–96.8% sensitivity and 96–98.8% specificity for CFT, 80–

91% sensitivity and 75.5–96.8% specificity for CIT, 55.1–93.5% sensitivity and 88.8–100%

specificity for CCT. Recent data from some countries or regions indicate a markedly reduced

sensitivity of TST in certain situations. A study in Northern Ireland showed that as few as 59%

of animals with confirmed M. bovis infection were detected by CCT in some herds (Welsh, M.,

and McNair, J., unpublished results). Similarly, herd (n = 42) sensitivity of CCT in fighting bulls

in Camargue, France, was 58% (10.6% sensitivity for individuals, n ‡ 9000) with more than 80%

of bTB cases detected upon subsequent slaughter inspection (Keck, N., unpublished results).

Twenty-eight of the 42 herds contained at least one culture-positive animal and 142 animals had

tuberculous lesions upon slaughter inspection. Immunosuppression caused by stress (handling of

fighting bulls) or anergy because of an advanced stage of bTB possibly account for the low

sensitivity of TST in some circumstances. These data may represent the lower end of the

performance spectrum of TST and not average values. Still, a low sensitivity constitutes a serious

problem for the sustainable success of control and eradication programs.

2.8 Immunology of tuberculosis

Upon the inhalation of Mtb, the alveolar macrophages are among the first cells to encounter the

microorganism in the host-pathogen relationship that decides outcome of infection (Figure 2.1).

Within 2 to 6 wk of infection,cell-mediated immunity (CMI) develops, and there is an influx of

lymphocytes and activated macrophages into the lesion resulting in granuloma formation. The

exponential growth of the bacilli is checked and dead macrophages form a caseum. Despite the

potential of macrophages to kill pathogens, Mtb can escape this fate. Resting macrophages allow

some degree of Mtb replication, whereas activated cells can either suppress or kill the bacteria

46
(Hirsch et al., 1997). TB antigen presentation by dendritic cells, potentially aided by neutrophils

(Stewart et al., 1999), in the draining lymph nodes induces a local immune response which

results in the recruitment of various cell types to the site of infection, including monocytes,

macrophages, neutrophils and dendritic cells (Figure 2.1). Together, these cells form a primary

granuloma which, while enclosing the infection, may also permit Mtb growth until T cell are

recruited to the infection site (Figure 2.1). Protection against active TB is known to require a

clearly delineated Th1 response, mediated by IFN γ, IL-2, and TNF-α (Flynn et al., 2011;

Wangoo et al., 2001; Shevach et al., 2001) which may clear infection or drive it into an immune-

mediated containment, or latency. Specifically, individuals defective in genes encoding for IFN

γ or the IFN γ receptor are susceptible to fulminant mycobacterial infections, including Mtb

(Iwashiro et al., 2001). IFN γ is important in macrophage activation with the resulting

stimulation of nitric oxide synthase and L-arginase production, an essential step for proper

defense against TB infection (Wangoo et al., 2001; Shevach et al., 2001).

47
 Mycobacterium
bovis
Helminth
Inhalation

Chronic helminth
Helminth infection
infection induce a
induce a dominant Alveolar
regulatory network
Th2 reponse Macrophage
i.e. suppressive
with ingested
regulatory T cells
M. bovis
(T-reg)

M. bovis
IL-10 IL-4, 1L-5, infected
TGF-β IL-9, IL-13 Macrophage

Local immune
response and
IFN-γ
recruitment of
immune cells

Granuloma
formation MONOCYTES
MACROPHAGES
Induction NEUTROPHILS
of T-cells
DENDRITIC CELLS

TNF-α
Fig 2.1: Pathway depicting the immunological interplay between Th1,Th2 & Treg : a classical scenario in
helminth and TB co-infection in cattle

48
2.9 Helminthosis

2. 9.1 Definition

Helminths are multicellular pathogens that infect vast number of human and animal hosts,

causing widespread of chronic disease and morbidity (Crompton and Nesheim, 2002).

2.9.2 Aetiology

Helminths or worms cause a wide range of health problems to both man and animals (Colley et

al., 2001). Helminthosis, in large part, is caused by members of the phyla Nematyhelminthes and

Platyhelminthes (Kennedy and Harnett, 2001).

2.9.3 History

In Nigeria, gastrointestinal nematode infection was predicted as an obstacle to expansion of

cattle industry in a study conducted by Lee (1955) in south western states of Nigeria where rainy

season is prolonged. Records compiled by Sprent (1946) listed thirteen (13) species of

nematodes from the alimentary tract of cattle in Nigeria. A checklist was also compiled by

Schillhorn van Veen et al. (1975) comprising 16 different species of gastrointestinal nematodes

of cattle in northern Nigeria. Historically, gastrointestinal helminth infections have been

associated with great economic losses to farmers throughout the world, these loses manifest

through morbidity in acute cases and in chronic infection reduced weight gains, reduced food

conversion, abortion, infertility, reduced meat and milk production (Ogunrinade, 1984; Karki,

1987; Beriajaya et al., 1995). The negative impact of helminth infections on livestock

productivity in tropical countries has long been established. Reports by Ndarathi et al. (1989)

and Olusi (1997), Edosomwan and Ewarami (2012) contained recent appraisals of this problem.

The eggs of intestinal helminths can be found in the mummified feces of humans dating back

49
thousands of years (Cox, 2002; Hotez, 2008), can also be recognized in many of the

characteristic clinical features of helminth infections from the ancient writings of Hippocrates,

Egyptian medical papyri and the Bible (Cox, 2002; Hotez, 2008).

2.9.4 Geographic distribution

Helminths are eukaryotic parasites that infect approximately one-third of the world's population

and are one of the most common infections affecting poor people living in the developing world

(Fritsche et al., 1993). Gastrointestinal parasites are known to be widespread in Nigeria and limit

ruminant production in many areas of the country (Keyyu et al., 2005).

2.9.5 Biodiversity

Parasitic helminths represent an extreme in the spectrum of pathogens, as large multicellular

animals derived from free-living metazoan ancestors. Although commonly grouped together, the

helminths in fact comprise two very distantly related taxa that diverged 600 million or more

years ago (Knoll and Carroll, 1999): the roundworms or Nematyhelminthes and flatworms or

Platyhelminthes (flukes and tapeworms).

2.9.6 Nematyhelminthes

Nematyhelminthes have long thin unsegmented tube-like bodies with anterior mouths and

longitudinal digestive tracts. A major class of this group is the nematoda, they have a fluid-filled

internal body cavity (pseudocoelum) which acts as a hydrostatic skeleton providing rigidity (so-

called ‘tubes under pressure’). Worms use longitudinal muscles to produce a sideways thrashing

motion. Most species are dioecious. The reproductive system consists of long tubules that serve

as ovaries or testes. In females, the reproductive tubule (ovary) is usually double. Males are

50
smaller than females and have one or two hardened spicules on their posterior ends that guide

sperm to the female’s genital pore. Species identification is often based on spicule structure.

2.9.7 Platyhelminthes

Platyhelminthes (flatworms) are flattened from the dorsal to ventral surfaces. The classes of this

phylum include trematodes and cestodes.

2.9.7.1 Trematodes

Trematodes (flukes) have small flat leaf-like bodies with oral and ventral suckers and a blind

sac-like gut. They do not have a body cavity (acoelomate) and are dorsoventrally flattened with

bilateral symmetry. They exhibit elaborate gliding or creeping motion over substrates using

compact 3-D arrays of muscles. Most species are hermaphroditic (individuals with male and

female reproductive systems) although some blood flukes form separate male and female adults.

The suckers hold the animal in place. Flukes obtain food by absorbing it through their outer

covering, called the cuticle. Flukes are given common names according to the tissue of the

definitive host in which the adults live (for example, liver fluke).

2.9.7.2 Cestodes

Cestodes (tapeworms) are intestinal parasites with long flat ribbon-like bodies with a single

anterior holdfast organ (scolex) and numerous segments called proglottids. They do not have a

gut and all nutrients are taken up through the tegument. They do not have a body cavity

(acoelomate) and are flattened to facilitate perfusion to all tissues. Segments exhibit slow body

flexion produced by longitudinal and transverse muscles. All tapeworms are hermaphroditic and

each segment contains both male and female organs.

51
2.7.8 Predilection sites

Species belonging to both Nematyhelminthes and Platyhelminthes phyla occupy numerous

niches within their mammalian hosts, ranging from intestinal lumen to intravascular and even

intracellular sites (Littlewood and Bray, 2001).

2.7.9 Life-cycles

All parasitic worms are obligate parasites, that is, they cannot complete their life cycle without

spending some time on their host. Some helminth parasites are very host specific, i.e. they are

able to complete their life cycle only on that particular host species or closely related ones (e.g.

Toxocara vitullorum which infests cattle). Other species can develop on many different host

species (e.g. liver flukes: Fasciola hepatica). Many helminth species have complex life cycles,

alternating stages on the host and stages off the host. The duration of each development stage

depends strongly on ecologic and climatic conditions. Understanding the life cycle is very

important in establishing and managing preventative measures. There are two major types of life

cycles, depending basically on the number of host:

2.9.9.1 Direct life cycles

This has a single host that is called definitive or final host, where the worms reach maturity and

reproduce. Roundworms often have direct life cycles.

2.9.9.2 Indirect life cycles

This has at least one obligate intermediate host in addition to the final host. Typical intermediate

hosts are often small invertebrates such as snails, insects, crustaceans, etc. The worms complete

certain development stages inside these intermediate hosts, with or without harming them. In

some species the immature stages can multiply asexually inside the intermediate hosts. The final

hosts get infected mostly by ingestion of infected intermediate hosts. Flukes (trematodes) and

52
tapeworms (cestodes) often have indirect life cycles. Some species have two obligate

intermediate hosts (e.g. Dicrocoelium dendriticum).

2.9.10 Developmental phases

Helminths form three main developmental stages: eggs, larvae and adults. Adult worms infect

definitive hosts (those in which sexual development occurs) whereas larval stages may be free-

living or parasitize invertebrate vectors, intermediate or paratenic hosts.

2.9.10.1 Nematodes

Nematodes produce eggs that embryonate in utero or outside the host. The emergent larvae

undergo four metamorphoses (moults) before they mature as adult male or female worms.

2.9.10.2 Cestodes

Cestodes eggs released from gravid segments embryonate to produce six-hooked embryos

(hexacanthoncospheres) which are ingested by intermediate hosts. The oncospheres penetrate

host tissues and become metacestodes (encysted larvae). When eaten by definitive hosts, they

excyst and form adult tapeworms.

2.9.10.3 Trematodes

Trematodes have more complex life-cycles where ‘larval’ stages undergo asexual amplification

in snail intermediate hosts. Eggs hatch to release free-swimming miracidia which actively infect

snails and multiply in sac-like sporocysts to produce numerous rediae. These stages mature to

cercariae which are released from the snails and either actively infect new definitive hosts or

form encysted metacercariae on aquatic vegetation which is eaten by definitive hosts.

53
Figure 2.2: Nematode cycle: egg- larvae (L1-L4) – adult Commented [DA1]: Indicate L1-L4

54
Fig. 2.3: Cestode cycle: egg- metacestode- adult

55
Fig. 2.4: Trematode cycle: egg- miracidium- sporocyst- rediae- cercariae- (metacercariae)-

adult

56
Figure 2.5: Life cycle of helminth (possible route of re-infection)

57
2.9.11 Route of entry

The word helminth is derived from a Greek word meaning worm (Faust et al., 1970). Infective

stages of parasitic worms have several routes of entry into their hosts. Entry through natural

opening is mostly through the mouth, quite seldom through the anus, nose or other opening.

Ingestion through the mouth is mostly passive, such that infective stages are ingested with

contaminated food. Skin entry could be active when agent penetrates by its own effort or

passively through a bite of a vector (insects). A special case is transplacenta transmission when

worm is transmitted from dam to young, it could also occur via milk.

2.9.12 Pathogenesis

Well-nourished, healthy animals often tolerate considerable worm burdens without major harm,

i.e. without developing a disease, somehow in a natural balance. But if they lose condition (e.g.

due to stress, droughts, malnutrition, etc.) the worm infestation turns into an excessive burden

and the disease breaks out. Unlike other pathogens (viruses, bacteria, protozoa and fungi),

helminths do not proliferate within their hosts. Helminths grow, moult, mature and then produce

offspring which are voided from the host to infect new hosts. Worm burdens in individual hosts

(severity of infection) are therefore dependent on intake (number of infective stages taken up).

Worms develop slowly compared to other infectious pathogens so any resultant diseases are slow

in onset and chronic in nature. Although most helminth infections are well tolerated by their

hosts and are often asymptomatic, subclinical infections have been associated with significant

loss of condition in infected hosts. Helminth infection can result in losses in productivity through

a reduction of feed intake and feed conversion efficiency, loss of blood and even death. Toxins

are produced by mature worms that destroy red blood cells, which result to unthrifty anemic

58
conditions generated. While immature worms migrating through the body tissues of animals

open the way for bacteria and fungi to enter, this causes serious diseases.

2.9.13 Clinical signs

Helminths are responsible for substantial loss of productivity in the livestock industry, although

helminthic infections are often asymptomatic, severe pathology can occur (Tibbo et al., 2006;

Nwosu et al., 2007). Many potential helminthic infections are eliminated by host defenses; others

become established and may persist for prolonged periods, even years. In many cases the same

individual may be infected by more than one parasite (Mckenzie, 2005; Hotez et al., 2006). Their

harmful effects on these animals range from gastro-enteritis, anorexia, abdominal distention,

diarrhea and emaciation. In addition, helminthosis adversely affects ruminants e.g. hematological

and biochemical disturbances (Iqbal et al., 1993; Hayat et al., 1996).Helminths cause serious

clinical diseases characterized by high morbidity and mortality. Clinical signs of infection vary

considerably depending on the site and duration of infection. Larval and adult nematodes lodge,

migrate or encyst within tissues resulting in obstruction, inflammation, oedema, anaemia, lesions

and granuloma formation. Infections by adult cestodes are generally benign as they are not

invasive, but the larval stages penetrate and encyst within tissues leading to inflammation, space-

occupying lesions and organ malfunction. Adult flukes usually cause obstruction, inflammation

and fibrosis in tubular organs, but the eggs of blood flukes can lodge in tissues causing extensive

granulomatous reactions and hypertension. All of which result in serious economic losses to the

farmer and the nation in general (Junaidu and Adamu, 1997). Similarly, they constitute a major

impediment to efficient and profitable livestock production (Akerejola et al., 1999; Jithendran

and Bhat, 1999).

59
2.9.14 Diagnoses

Helminths are studied in parasitology because they cause infectious diseases and most are

diagnosed by microscopic examination of eggs or larvae. Eggs may have striations (lines), a

spine, or an operculum (hatch by which the larva leaves). Although faecal examination has a

high specificity, its sensitivity is believed to be low, due to the long pre-patent period,

intermittent egg excretion and over dispersion of the fluke population (Vercruysse and

Claerebout, 2001).

2.9.15 Immunology of helminth

Helminths induce a strong Th2 host response that promotes mucus secretion, collagen deposition

and wound healing mechanisms that are critical for helminth expulsion (Bethony et al., 2006).

Yet despite the induction of such protective Th2 responses, helminths are often able to persist in

the host for a long time, resulting in chronic infection (Allen and Maizels, 2011). Persistence in

the host is achieved, in part, by the induction of immunoregulatory pathways that are favorable

to helminth survival. It has been observed that during helminth infection, expanding populations

of regulatory T-cells (T-regs) produce cytokines like transforming growth factor β (TGFβ) and

interleukin 10 (IL-10) (Figure 3), that can down-modulate both Th1 and Th2 inflammatory

responses (Babu et al., 2006; Satoguina et al., 2002; King et al., 1993; Gillan et al., 2005). T-

regs interfere with effector T cell activation (Shevach et al., 2001; Oldenhove et al., 2003), and

not surprisingly therefore, excessive exposure to parasitic helminth infection has been associated

with generalized immune hyporesponsiveness (Turner et al., 2008), rather than exacerbation of

existing conditions like TB.

60
Th2 and immunoregulatory cytokines like IL4, IL10 and TGF generated during helminth

infection may act as potent inhibitors of the Th1 responses to TB infection (Rook, 2007; Redford

et al., 2011; Hirch et al., 1997) (Figure 3).

2.9.16 Immune interplay of helminth-TB co-infection

It has been reported that TB infection induce Th1 immune response and helminth induce Th2

response. Both Th1 and Th2 cells were shown to negatively influence each other both in vitro

and in vivo: IFN-γ inhibits Th2 responses while IL-4 inhibits Th1 responses (Elias et al., 2006).

It is becoming increasingly clear that helminth infections, in addition to stimulating vigorous Th2

responses, can induce suppressive T cell populations known as regulatory T cells (Tregs)

(Shevach et al., 2001). Tregs produce inhibitory cytokines (IL-10 and TGF-β) that suppress Th1

type responses and interfere with effector T cell activation (Shevach et al., 2001). Initially it was

thought that Tregs are important primarily in the control of pathogenic T cells and autoimmune

responses (Shevach et al., 2001). Recently however, it has become apparent that Tregs could be

induced to regulate responses to pathogens (Hesse et al. 2004; Maizels et al. 2004). Reports have

shown the involvement of Tregs in helminth induced immunosuppression (Satoguina et al. 2002;

Pearce & McKee 2004). In experimental nematode infection, it has been observed that there was

enhancement of Treg function associated with up regulated Th2 responses (Gillan & Devaney

2005). Such enhanced Treg function associated with helminth infection may suppress Th1

responses directed against unrelated antigens and/or pathogens (Oldenhove et al., 2003).

61
2.9.17 Potential impact of helminth infection on TB diagnosis

Intact host immune responses are needed for reliable TB diagnostic tests. Thus, attenuated

immune responses caused by helminth infection may reduce the reliability of diagnostic tests

such as the intradermal tuberculin test, a delayed-type hypersensitivity reaction to TB first

described by Robert Koch in 1890. Consistent with this, Stewart et al. (1999) reported an

association between onchocerciasis worm (skin microfilariae) density, down-modulation of

cellular responses to PPD and skewing of the immune response towards a Th2 profile. Further

evidence that worm burden may affect immune responses comes from a deworming trial (Elias et

al., 2001). Among the helminth-infected animals, those treated with albendazole, but not the

placebo-treated group, showed an increase in cell-mediated proliferation and IFNγ production to

PPD. However, no significant effect of deworming was recorded in the skin reaction – possibly

reflecting the lower sensitivity of the skin test compared to cellular responses. Of note, even in

utero helminth exposure may affect downstream immune response to PPD. In one study, children

born to uninfected mothers produced 10-fold higher PPD-specific IFNγ compared to children

from worm-infected mothers (Malhotra et al., 1999). Not all studies have suggested an

association between helminthes and PPD reactivity.

62
 CHAPTER THREE

3.0. MATERIAL AND METHODS

3.1. Study Area

This study was carried out in two Local Government Areas (LGA) of Oyo State namely, Ido and

Itesiwaju LGAs (Figure 3.1). The two local governments are located between latitude 8′30′ N

and 7′15′ N; and 3′00′ E and 4′00′ E (Figure 3.1). Though Oyo State is situated in the forest belt

in Nigeria, derived savannah areas stretch southwards from west of Oyo close to the coast in

Benin and Togo Republics. The relatively open land created an area of low humidity thereby

reducing disease risks to the zebu cattle, the major breed reared by the Fulani herdsmen in

Nigeria. Coupled with this ecological factor is the cultural/religious factor: Islam is wide spread

in Yoruba land. Thus, the settlement of the Fulani pastoralists has a long history in Oyo State.

The Fulani pastoralists were well established in savannah north of Yoruba land and dates back to

1830s and 1850s (Blench, 1999).

63
Plate 1: Herds near the Fulani Bororo camp. In typical Fulani Bororo camps, herds are

kept near the camps in lands where the next season’s crops are to be planted.

64
Figure 3.1: Map of Oyo State, showing the local government areas where the study was

carried out

65
Unlike most rural parts of Oyo State, the areas used for this study have large population of cattle

herds owned by Fulani herdsmen due to milk collection centers established by the Federal

Government in conjunction with private organization. Majority of cattle sampled in these study

sites belong to Fulani herdsmen who are believed to have migrated there from the northern part

of Nigeria in search of pasture and water, and have become domiciled, over a long period of

time.

3.2 Sample design

A cross-sectional study was used.

3.3 Sample size and sampling

To calculate the required number of animals to sample in order to ascertain the prevalence of co-

infection among cattle, we adopted the formula earlier described by Thrusfeild, 1997, using an

expected prevalence (Pexp) of 79.0% (Risco et al., 2014), and a desired absolute precision (d) of

5% and a confidence level of 95%.

1.962 . 𝑃. (1 − 𝑃)
𝑛=
𝑑2

1.962 𝑋 0.79 𝑋 (1 − 0.79)

𝑛=
0.052

𝑛 = 256

This gave a total sample size of 256. For each animal screened for BTB, faecal samples were

also collected per rectum into well-labeled sterile polythene bags and in ice packs to the

66
Parasitology Laboratory, Department of Veterinary Microbiology and Parasitology, Faculty of Commented [D2]: I believe our lab was used for this!!!!

Veterinary Medicine, University of Ibadan, where they were examined for helminth eggs. In

each herd, about 10% of the animals were randomly selected. Herds were purposively selected

based on cooperation of herdsmen and availability of animals. Each animal was assigned a

number at the beginning of the SCITT testing and data such as breed, sex, age and body

condition score (BCS) were obtained. The body conditions of the animals were scored according

to the modification of guidelines earlier explained by Nicholson and Butterworth (1986). In all,

288 animals from 30 herds were screened for helminth and BTB.

3.4 Single Comparative Intradermal Tuberculin Test (SCITT)

The SCITT was done by intradermally injecting the animals with both avian and bovine purified

protein derivatives (PPD) in the middle neck region (on the left side for consistency) as

described by the World Organization for Animal Health standards [OIE, 2009]. Briefly, two sites

of about 2 cm2 diameter, approximately 12 to 15 cm apart, were shaved using a sterile surgical

blade after which the skin thickness was measured using a manual caliper. Thereafter, exactly

0.1 ml containing 30,000 IU/ml of bovine PPD (Bovine tuberculin PPD, Prionics Lelystad BV,

Lelystad, The Netherlands) and 0.1 ml with 25,000 IU/ml of avian PPD (Avian tuberculin PPD,

Prionics Lelystad BV, Lelystad, The Netherlands) were intradermally injected with the use of

two separate sterile syringes (one for each) into the shaved area. At site of each injection, the

formation of a bleb confirmed a successful procedure.

After 72 hours, the measurement of the shaved area in the screened animals was done using

caliper. The initial and final measurement was carried out by the same person in order to avoid

error which may arise from individual measurement variations. The interpretation of the

67
measurements was done in accordance with the guidelines stipulated by World Organization for

Animal Health Standards (OIE, 2009). A positive reaction is recorded if increase in skin

thickness at the injection site of bovine PPD (Bfinal – Binitial) was at least 4mm more than that

at the avian site (Afinal – Ainitial). A herd is considered positive if at least one animal in the

herd is a positive reactor.

3.5 Coprological examination of faecal sample for helminth eggs

3.5.1 Helminth infection

This was done according to the protocols earlier described by Thienpoint (Thienpoint, 1979) and

Khin-Khin (Khin-Khin, 2007). Eggs were identified on the basis of their morphological features

as described by Soulsby (Soulsby, 1982), based on:.

• Standard floatation and sedimentation technique

• Copro-culture

3.5.1.1 Floatation technique

• Approximately 3g of faeces was put into a beaker.

• 50ml of saturated NaCl was added to the faeces.

• Faeces and saturated NaCl were thoroughly mixed together with a stirring rod.

• Resulting suspension was filtered through a tea strainer, into another beaker.

• Faecal suspension was dispensed into a test tube which was placed in a test tube rack.

• Faecal suspension in test tube rack was gently topped, leaving a convex meniscus at the

top of the tube.

• Coverslip was placed on top of the test tube and allowed to stand for 20minutes.

68
• Coverslip was carefully lifted with the drop of fluid adhering to it, and placed on a glass

slide.

• Prepared sample was viewed under the microscope at (x10 and x40) magnification.

Principle: floatation technique is a qualitative test for the detection of nematode and cestode eggs

in faeces. It is based on the separation of eggs from faecal materials and concentrating them by

means of a floatation fluid with an appropriate specific gravity.

3.5.1.2 Sedimentation technique

• Approximately 3g of faeces was put into a beaker.

• 50ml of tap water was added to the faeces.

• Faeces and water were thoroughly mixed together with a stirring rod.

• Resulting suspension was filtered through a tea strainer, into another beaker.

• Faecal suspension was dispensed into a test tube which was placed in a test tube rack.

• Faecal suspension was allowed to sediment for 15minutes.

• Supernatant was decanted carefully.

• Sediment was pipetted to a glass slide and covered with a coverslip.

• Prepared sample was viewed under the microscope at (x10 and x40) magnification.

Principle: sedimentation technique is a qualitative method for detecting trematode eggs in the

faeces. Most trematode eggs are relatively large and heavy compared to nematode eggs.

3.6 Copro-culture

• The culture of larvae from faeces of infected cattle was carried out in test tubes.

69
• A thin layer of faeces (1-2mm) was spread on the middle third part of a piece of filter

paper (110mm).

• This piece was put into a 15ml conical tip test tube containing about 3ml of distilled

water.

• Filter paper was pushed to about 1cm from the bottom, while the unsoiled side of the

filter paper is kept towards the opening of the test tube and fixed with cotton wool.

• The culture was kept for 10days in an incubator at 370C.

• Culture was checked every day in order to top water when due.

• Freed larvae move towards the water and are pipetted.

• Further examination is carried out by viewing with a microscope.

3.7 Statistical Analysis

Data collected were entered into excel sheets and analysis were performed using the statistical

software package STATA version12. Data were analyzed to determine the association between

infections and the locations, breed, age, sex and other variables. Group differences were tested

using chi-square statistics for categorical variables. A multi-variable adjusted logistic regression

was carried out using all the variables that were statistically significant at the 10% level with the

main outcome measures (co-infections status) in bivariate analysis. All tests were two-tailed and

statistical significance was set at p<0.05.

70
CHAPTER FOUR

RESULTS

4.1 Results of distribution of all the variables of cattle screened

In all, 288 cattle from 30 herds were screened (12 from rural and18 from peri-urban). Greater

number (55.2%) of cattle was screened in the peri-urban area. Majority (88.5%) of the cattle

screened were Bunaji breed, cows (60.4%) as well as cattle older than three years (74.0%)

(Table 1). An individual animal prevalence of 55.9%, 8.3% and 4.9% was recorded for

helminths, BTB and helminth-BTB co-infection, , while herd prevalence of 90%, 50% and

36.7% were recorded for helminth, BTB and helminth-BTB co-infection, respectively.

Furthermore, prevalences of 35.8% and 20.1% were respectively recorded for single and

multiple helminth infections.

71
Table 1: Distribution of cattle screened according to all the variables tested (N = 288)

VARIABLES CATEGORY FREQUENCY PERCENTAGE

Location Rural 129 44.8

Peri-urban 159 55.2

Breed Bunaji 255 88.5

Sokoto Gudali 19 6.6

Balami 14 4.9

Sex Male 114 39.6

Female 174 60.4

Age ≥ 3years 213 74.0

< 3years 75 26.4

Helminth Status Positive 161 55.9

Negative 127 44.1

Tuberculin Test Positive reactor 24 8.3

Negative reactor 264 91.7

Co-infection Status Co-infected 14 4.86

Non-co-infected 274 95.1

Burden of Helminth No infection 127 44.1

Infection

Single infection 103 35.8

Multiple infection 58 20.1

72
4.2: Results of the BTB infection among cattle screened

Cattle screened in the rural area are more likely to be infected (positive reactors) when compared

to those in the peri-urban (OR=1.3; 95%CI:0.5 – 3.2). Again, Bunaji breed of cattle (OR=1.3;

95%CI:0.3 – 6.1), female cattle (OR=1.3; 95%CI:0.6 – 3.2) as well as cattle older than three

years (OR=1.3; 95%CI:0.6 – 3.3) showed more likelihood to be positive reactors (Table 2).

73
 Table 2: Prevalence of BTB infection (measured by tuberculin test) among cattle herds
according to location, breed, sex, age and burden of helminth infection
VARIABLE CATEGORY TUBERCULIN TEST OR P-value
RESULT
Pos. reactors Neg. reactors
n (%) n (%)
Location

Rural 14 (10.85) 115 (89.15) 1.3 0.52 – 3.22 0.74

Peri-urban 10 (6.29) 149 (93.71) 1

Breed

Bunaji 21 (8.24) 234 (91.76) 1

Sokoto Gudali 2 (10.53) 17 (89.47) 1.3 0.28 – 6.06 0.93

Balami 1 (7.14) 13 (92.86) 0.9 0.11 – 6.88 0.72

Sex

Male 8 (7.02) 106 (92.98)

Female 16 (9.20) 158 (90.80) 1.3 0.55 –3.27 0.66

Age

≥ 3years 22 (10.33) 191 (89.67)

< 3years 2 (2.67) 73 (97.33) 0.2 0.05 – 0.98 0.06

Burden of helminth

infection

No infection 9 (7.09) 118 (92.91)

Single 10 (9.71) 93 (90.29) 1.4 0.55 – 3.61 0.63

infection

Multiple 5 (8.62) 53 (91.38) 1.2 0.39 – 3.87 0.94

infection

74
4.3: Results of the gastrointestinal helminth infection among cattle screened

Among the cattle screened, those in the rural are more likely to be infected with gastrointestinal

helminth when compared to those in the peri-urban area (OR=1.4; 95%CI:0.9 – 2.5). However,

Sokoto Gudali (OR=1.1; 95%CI:0.4 – 2.7), female cattle (OR=0.9; 95%CI:0.5 – 1.5) as well as

cattle older than three years (OR=0.8; 95%CI:0.4 – 1.4) are equally likely to be infected when

compared to Bunaji breed, male cattle and cattle younger than three years of age, respectively

(Table 3).

75
Table 3: Prevalence of gastrointestinal helminths in relation to location, sex, breed and age

of cattle

VARIABLE CATEGORY HELMINTHS OR P-value

Positive Negative

Location

Rural 79 (61.2) 50 (38.8) 1.4

Peri-urban 82 (51.6) 77 (48.4) 1 0.9 – 2.5 0.13

Breed

Bunaji 144 (56.5) 111 (43.5) 1

Sokoto Gudali 11 (57.9) 8 (42.1) 1.1 0.4 – 2.7 0.91

Balami 6 (42.9) 8 (57.1) 0.5 0.2 – 1.7 0.47

Sex

Male 65 (57.0) 49 (43.0) 1

Female 96 (55.2) 78 (44.8) 0.9 0.5 – 1.5 0.85

Age

≥ 3years 122 (57.3) 91 (42.7) 1

< 3years 39 (52.0) 36 (48.0) 0.8 0.4 – 1.4 0.51

76
4.4 Diversity of helminth species recovered from the cattle screened

In all, 14 different species of gastrointestinal helminths were recovered from the cattle screened:

10 nematodes, three trematodes and one cestode.

NEMATODES (10)

• Trichostrongylus sp

• Oesophagostomum sp

• Ostertagia sp

• Bunostomum sp

• Cooperia sp

• Haemonchus sp

• Chabertia sp

• Toxocara sp

• Strongyloides sp

• Nematodirus sp

TREMATODES (3)

• Dicrocoelium sp

• Paramphistomum sp

• Fasciola sp

CESTODE (1)

• Moniezia sp

77
4.5: Results of the helminth-BTB co-infection among cattle screened

The results of the helminth-BTB co-infection show that cattle screened in the rural area are more

likely to be co-infected when compared to those in the peri-urban (OR=3.3; 95%CI:1.1 – 50.0)

(Table 4). Furthermore, though not significant, Sokoto Gudali (OR=1.1; 95%CI: 0.1 – 9.1) and

Balami (OR=1.6; 95%CI: 0.2 – 12.9) breeds showed high likelihood of being co-infected when

compared to Bunaji breed of cattle (Table 4). In the same vein, cow (OR=1.2; 95%CI: 0.4 – 3.6)

and cattle older than three years of age (OR=3.0; 95%CI:0.6 – 33.3) were more likely to show

co-infection when compared to bulls and cattle younger than three years of age, although these

differences were not significant (Table 4).

78
Table 4: Prevalence of helminth-BTB co-infection among cattle herds (N = 288; Herds=30)

VARIABLE CATEGORY CO-INFECTION STATUS OR 95% CI P-value

Positive Negative
n (%) n (%)

Location

Rural 10 (7.75) 119 (92.25) 3.3 1.14 – 50.00 0.04

Peri-urban 4 (2.52) 155 (97.48) 1

Breed

Bunaji 12 (4.71) 243 (95.29) 1

Sokoto Gudali 1 (5.26) 18 (94.74) 1.1 0.14 – 9.14 0.65

Balami 1 (7.14) 13 (92.86) 1.6 11 – 2.9 0.02

Sex

Male 5 (4.39) 109 (95.61) 1

Female 9 (5.17) 165 (94.83) 1.2 0.38 – 3.64 0.98

Age

≥ 3years 13 (6.10) 200 (93.90) 5 0.62 – 33.33 0.18

< 3years 1 (1.33) 74 (98.67) 1

79
4.6: Results of the of logistic regression analysis of variables significant at 10% level in

bivariate analysis

The final multivariate adjusted logistic regression analysis identified location (figures ???) where

animals were sampled and breed (figures) of cattle as significant risk factors for the occurrence

of helminth-BTB co-infection among cattle screened. Animals screened in the rural area were

two times more likely to be co-infected with helminth-BTB when compared to those screened in

the peri-urban, while Balami breed of cattle were three times more likely to be co-infected when

compared to Bunaji breed (Table 5).

80
Table 5: Results of logistic regression analysis of variables significant at 10% level with

the main outcome measure (C.I status) in bivariate analysis.

VARIABLE OR 95%CI P-value

Location

Rural 1.8 1.4 – 3.3 0.01

Peri-urban 1

Breed

Bunaji 1

Sokoto Gudali 2.8 0.3 – 26.9 0.37

Balami 15.3 1.2 – 19.5 0.03

81
 Bovine TB Infection
Helminth infection
Helminth-BTB Co-infection 90%

90
80
70 55.9%
60 50%
50 36.7%
40
30
20 8.3% 4.9%
10
0
Individual Animal Herd Prevalence
Prevalence

Figure 3.2: Individual and herd prevalence of BTB, helminth as well as helminth-BTB co-infection
among cattle screened

82
Figure 3.3: Venn diagram showing infection rates among cattle screened

*C-I = CO-INFECTION

83
CHAPTER FIVE

DISCUSSION

The results of this study recorded an individual animal prevalence of 4.9% and herd prevalence

of 36.7% for helminth–BTB co-infection among the cattle herds screened. This, to the best of our

knowledge is one of the first herd studies on helminth-BTB co-infection in Ibadan, south-western

Nigeria, although concurrent BTB with other infections in cattle have been reported in Nigeria

(Cadmus et al., 2008; Danbirni et al., 2010). The prevalence of 4.9% reported in this study is

higher than 2.8% reported by Adedipe et al. (2014) in a study carried out among slaughtered

cattle, Ibadan, south-western Nigeria.

Our findings identified location as a risk factor associated with the co-infection of helminth-BTB

among cattle herds screened; with cattle in the rural area being more likely to be co-infected than

those in the peri-urban (OR=1.3;95%CI: 1.0 – 1.5). This could be attributed to the fact that

most of the herds sampled in the rural area were larger in size compared to those in the peri-

urban. Larger herd size increases the chances of exposure and interaction with other herds, and

this has been implicated as a major risk factor for susceptibility of animals to infectious diseases

(Berhe et al., 2007; Unger et al., 2003).

In the same vein, breed was observed as a risk factor associated with co-infection among the

cattle herd screened. The Balami breed of cattle showed the highest likelihood of being co-

infected when compared to Bunaji (OR=1.6; 95%CI: 1.2 – 19.5). Again, female animals showed

higher likelihood of being co-infected when compared to males, while older animals (≥ 3years)

were five times more likely to be co-infected than young animals (< 3years). This may be

attributed to study type, sampling techniques and sources of animals (Asante-Poku et al., 2014).

84
Also genetic factor has been implicated in the susceptibility or resistance of animals to infections

(Refai et al., 2002; Martinez et al., 2010).

Furthermore, our findings reveal 8.3% individual animal and 50% herd prevalence of BTB

infection using the SCITT. The individual animal prevalence recorded in this study is higher than

2.0% and 2.7% reported by Ibrahim et al. (2012) and Zeweld, (2013) respectively. However, it

is lower than 10.5% reported by Cadmus et al. (2004);14% by Ameni et al. (2007); 15.08% by

Yohanna et al. (2008); 14.3% by Thakur et al.(2010) and 11.4% by Biru et al. (2014)

respectively (Cadmus et al., 2004; Ameni et al., 2007; Yohanna et al., 2008; Thakur et al., 2010

Biru et al., 2014). These variations in prevalence can be attributed to several factors such as: age

of animals; breed of animals screened; herd size of animals; farming practices in terms of stock

densities; pasture systems and contact between animals; geographical divergence and sources of

animals; sampling techniques; individual differences in interpretation of tests and the number of

animals sampled (Asante-Poku et al., 2014). Coupled with this is the lack of a BTB control

policy in Nigeria which has consequently given rise to sheer transmission of the disease among

cattle from various herds in the country (Cadmus et al., 2010).

Moreover, although not significant, our findings showed that animal screened in the rural area

were more likely to be positive reactors when compared to those from peri-urban. In the same

vein, Sokoto Gudali breed of cattle, female animals as well as older (≥ 3years) animals showed

more likelihood of being more positive reactors. Our findings did not record any significant

association between positive reactivity and helminth burden (measure by tuberculin screening

test) among animals screened. There was no significant difference in the prevalence of BTB

between animals with single helminth burden and those with multiple infections.

85
Again, our study recorded an individual animal and herd prevalences of gastrointestinal helminth

among cattle screened to be 55.9% and 90.0% respectively. This further reaffirms the fact that

cattle are not routinely dewormed in this part of the world and, as such, there is a serious public

health concern, owing to the fact that some of these helminths are zoonotic. The prevalence

reported in this study is lower than 62.1% reported by Elele et al. (2013) in a study conducted in

Port Harcourt, south-south Nigeria (Elele et al., 2013). Furthermore, the prevalence recorded in

the present study is higher than 46.1% reported in Ibadan, south-west (Adedipe et al., 2015),

47.4% in Benin City, south-south (Edosomwan and Shoyemi, 2012), and 50.8% in Ebonyi State,

south-east (Nwigwe et al., 2013), respectively. The difference observed could be attributed to

several factors such as the periods or seasons during which the investigations were carried out by

the authors as well as the sources of cattle sampled in the various regions. However, based on the

most prevalent helminths found, our findings showed different helminth profiles when compared

to those reported by Hailu et al. (2011), Mir et al. (2013) and Nwigwe et al. (2013), who

reported trematodes as the most prevalent in studies carried out in India, Ethiopia and Eastern

Nigeria, respectively as against nematodes in our study. The difference observed could however

be associated with the differences in geographical and/or climatic conditions and ecology, since

presence of trematode infection is dependent on availability of the intermediate hosts which

might differ in quantity/population in the various study sites (Mir et al., 2013) ..

The results obtained in this study should be put in the context of the fact that it is a part/initial

step of a larger study which is aimed at investigating the role of helminth in the accurate and/or

precise diagnosis of BTB. Going by relatively high prevalence of bTB, helminth and helminth- Commented [D3]: Tell us which to use1111

bTB co-infections recorded in this study, this study has been able to establish a baseline data for

further studies which are aimed at investigating the impact (negative/positive) of a pre-existing

86
helminth burden on the diagnosis of bTB in helminth-bTB co-infected cattle in a natural setting

(i.e. extensive Fulani cattle herds).

CONCLUSION

The results of this study reveal that helminth-BTB co-infection is prevalent among cattle herds

screened in Oyo State. Again, our findings showed that bovine BTB and helminth infections

exist among the cattle herds. Going by the fact that pre-existing helminth infection has been

reported in various experimental studies to be capable of negatively influencing the diagnosis of

Bbovine TB, there is the need to investigate the potential role of helminths on BTB infection

and its diagnosis among cattle herds, from the natural host scenario in Nigeria. This will enable

effective control of the scourge of BTB among cattle population considering the endemicity of

both BTB and helminth infections in this study area coupled with lack of control measures in

developing countries like Nigeria.

87
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