1

SEROPREVALENCE OF REPRODUCTIVE AND RESPIRATORY DISEASES

AND THEIR POTENTIAL RISK FACTORS IN HORO, BONGA AND MENZ

SHEEP IN ETHIOPIA.

By:

AZEB G/TENSAY

Sep, 2016

Ethiopia

I

AKNOWLEDGEMENTS

I am very much indebted to Dr. Tesfaye Sisay, Addis Ababa University, for his patience,

motivation; continuous guidance who helped me in all times of my research work. I

would like to express my deepest gratitude and sincere thanks to Barbara Wieland, ILRI

Addis Ababa, for her immense and priceless support and devoting her precious time in

guiding, reading and correcting this paper. Barbara was the key person who created the

opportunity to do the laboratory analysis especially for the reproductive diseases.

I would like to express my deepest respect and most sincere gratitude to Mourad Rekik,

Ayenalem Haile and Barbara Rischkowsky from ICARDA for giving me the chance to

work with them, and also for their supervision and valuable comments they provided,

which helped me to get best experience.

The National Animal Health and Disease Investigation Center (NAHDIC) at Sebeta and

the National Veterinary Institute (NVI) serology laboratory technicians are heartfully

acknowledged for availing laboratory facilities to undertake ELISA tests.

This work could not have been done without the kind co-operation and assistance of

livestock owners, staff members of Bonga, Debre Berhan and Bako research centers who

assisted me in the collection of sample, interview livestock owners and language

translation. At last but not least, I thank driver Eshetu Zerihun for his kind cooperation.

Thank you all for your help.

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TABLE OF CONTENTS

Page

AKNOWLEDGEMENTS ................................................................................................. I

TABLE OF CONTENTS ................................................................................................ II

LIST OF TABLES .......................................................................................................... IV

LIST OF FIGURES ......................................................................................................... V

LIST OF ANNEXES ....................................................................................................... VI

ABBREVATION ........................................................................................................... VII

ABSTRACT .................................................................................................................. VIII

1. INTRODUCTION ..................................................................................................... 1

2. LITERATURE REVIEW ......................................................................................... 6

2.1. Productivity and Reproductive Performance of Small Ruminants in

Ethiopia………………………………………………………………………………...6

2.2. Reproductive Disease………………………………………………………....11

2.2.1. Chlamydia (Enzootic Abortion of Ewes)..................................................... 11

2.2.2. Coxiella Burnetti ......................................................................................... 21

2.2.3. Toxoplasma ................................................................................................. 29

2.2.4. Border Disease Virus .................................................................................. 39

2.2.5. Brucella ....................................................................................................... 46

2.3. Respiratory Disease………………………………………………..………….53

2.3.1. Pasteurella…………………………………………………………………...53

2.3.2. PPR (Peste Des Petits Ruminants) ............................................................. 58

3. MATERIALS AND METHODS ............................................................................ 64

3.1Description of the Study Area ............................................................................... 64

3.2. Study Animals and Study Methods………………………………..………...66

3.3. Sample Size Determination…………………………………………………..66

3.4. Sample collection and Laboratory Analysis………………….……………..67

3.4.1. Questionnaire .............................................................................................. 67

3.4.2. Serum sample collection ............................................................................. 68

3.4.3. Laboratory analysis .................................................................................... 68

3.5. Data analysis………………………………………...………………………...70

III

4. RESULTS ................................................................................................................. 72

4.1. Reproductive Performance and Problems……………………………...…...72

4.2. Reproductive Disease……………………………………..…………………..76

4.3. Respiratory Disease…………………..……………………………………….83

5. DISCUSSION ........................................................................................................... 88

5.1. Reproductive Performance and Problems……………………………...…...88

5.2. Reproductive Disease……………………………………..…………………..89

5.3. Respiratory disease……………………….…………………………………..97

6. CONCLUSION AND RECOMMENDATIONS................................................. 101

6.1. Reproductive Performance and Problems…………………………...…….102

6.2. Reproductive Disease………………………………………..……..………..102

6.3. Respiratory Disease…………………………………….……………………105

7. REFERENCE......................................................................................................... 108

8. ANNEXES .............................................................................................................. 132

IV

LIST OF TABLES

Page

Table 1: Prevalence of PPR in the seven surveyed regions in Ethiopia ........................... 60

Table 2: Reproductive performances by region ................................................................ 72

Table 3: Reproductive problems by region ....................................................................... 73

Table 4: Reproductive problems by CBBP /non-CBBP ................................................... 74

Table 5: Reproductive performance by CBBP/non-CBBP ............................................... 75

Table 6: Chlamydia prevalence in the three regions by different risk factors .................. 76

Table 7: Bonga region Chlamydia prevalence by different risk factors ........................... 77

Table 8: Multivariate logistic regression of Chlamydia ................................................... 78

Table 9: Q-fever prevalence in the three regions by different risk factors ....................... 78

Table 10: Menz region Coxiella burnetii prevalence by different risk factors ................. 79

Table 11: Multivariable logistic regression for region and Age ....................................... 80

Table 12: Seroprevalence of Toxoplasma gondi by different risk factors ........................ 81

Table 13: Seroprevalence of Toxoplasma gondi in Horro region ..................................... 81

Table 14: Multivariable logistic regression for region and sex ........................................ 82

Table 15: Pasteurella serotypes by region ....................................................................... 84

Table 16: Pasteurella serotypes between CBBP/non-CBBP ............................................ 85

Table 17: Seroprevalence of PPR by different risk factors............................................... 86

Table 18: Seroprevalence of PPR in Menz ....................................................................... 86

Table 19: Multivariable logistic regression for PPR ........................................................ 87

V

LIST OF FIGURES

Page

Figure 1: Pathways for Toxoplasma gondii infection ................................................................... 35

VI

LIST OF ANNEXES

Page

Annex: 1 Questionnaire format ....................................................................................... 132

Annex: 2 Sample lists of rams in each Household ......................................................... 138

Annex: 3 Determination of age with different numbers of erupted permanent incisors 139

Annex: 4: Chlamydia procedures .................................................................................... 139

Annex: 5 Q-fever procedures ......................................................................................... 140

Annex: 6 Toxoplasma procedures ................................................................................. 141

Annex: 7 Pasturellosis procedure of IHA (indirect heamaglutination test) ................... 142

Annex: 8 PPR procedures .............................................................................................. 144

Annex: 9 Reproductive performances in the three regions ............................................ 145

Annex: 10 Huma clinical signs related with Q-fever ..................................................... 145

Annex: 11 Q-fever risk factors from Questionnaire ...................................................... 147

Annex: 12 Toxoplasma risk factors from questionnaire ................................................ 148

Annex: 13 PPR risk factors from questionnaire ............................................................ 148

Annex: 14 Source of animal by different risk factors for Chlamydia............................ 148

Annex: 15 Role of HH members in SR production by region ....................................... 149

Annex: 16 Reproductive performances with reproductive diseases .............................. 150

Annex: 17 Month of pregnancy for abortion by region ................................................. 153

Annex: 18 The cause of lamb mortality in 2014/15 ...................................................... 153

VII

ABBREVATION

AGID Agar Gel Immunodiffusion

ANOVA Analysis of Variance

BD Border Disease

BID (bis in die) twice a day

BVDV Bovine Viral Diarrhoea Virus

CBBP Community Based Breeding Programmes

ELISA Enzyme-Linked Immuno Assay

FAO Food and Agriculture Organization

MZN Ziehl-Neelsen stain

OEA Ovine enzootic abortion

OIE World Organisation for Animal Health

SPSS Statistical Package for Social Sciences

VN Virus Neutralisation

VIII

ABSTRACT

A cross sectional study was conducted from April 2015 to November 2016, with the

objectives; to assess the relation of reproductive performance with the disease, to

determine the seroprevalence of reproductive diseases and respiratory diseases, compare

health of rams in community based breeding programs (CBBP) with rams not in CBBPs,

and to assess the potential risk factors in Horro, Bonga and Menz regions, Ethiopia. A

total of 448 sheep were tested for Chlamydia, Coxiella burnetii, Toxoplasma, Brucella,

Border disease, Pasturella and PPR by using serological diagnostic tools. From those

diseases the animals were tested negative for Border Disease Virus and Brucella. The

current study result shows the reproductive performance of sheep were significantly

different (p<0.05) between regions and not significant as compare to CBBP and non-

CBBP household members. The overall seroprevalence of for Chlamydia (259/447,

57.9%), for Q-fever (170/447, 38%), for Toxoplasma (177/445, 39.8%), for Pasturella M.

hemoletica serotype A (338/360, 93.9%), P. multocida PA (240/360, 66.7%) and B.

trihalosi serotype T (356/360, 98.9%) and for PPR (50/448, 11.2%) respectively. Region,

age, sex, farm in CBBP, ram in CBBP were taken as potential risk factors for the

occurrence of the diseases in sheep. The odds of acquiring PPR infection was

significantly higher in Menz (Odds Ratio [OR] = 10.9, 95% CI: 3.75-31.6; P = 0.00) than

Bonga. The odds of acquiring Chlamydia infection was significantly higher in Bonga

(Odds Ratio [OR] = 3.25, 95% CI: 1.99-5.32; P = 0.000) than Menz and in rams enrolled

in CBBPs (OR= 2.16, 95% CI: 1.05-4.41; P = 0.034). Similarly, the odds of acquiring Q-

fever infection was significantly higher in Menz (Odds Ratio [OR] = 7.57, 95% CI: 4.08-

14.03; P = 0.000) compared to Bonga and in adults (above 3year) (OR= 3.54, 95% CI:

1.54-7.72; P= 0.002) compared to young (6month-1year) age groups. The odds of

acquiring Toxoplasma infection was significantly higher in Horro (Odds Ratio [OR] =

4.41, 95% CI: 2.63-7.4; P = 0.000) compared to Menz. The results of the present study

indicate that the seroprevalence of the reproductive disease is high in sheep. The higher

seroprevalence shows a possibility to reduce reproductive performance and since the

included pathogens are zoonotic, potentially a risk to humans who have close contact

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with diseased animals. The high prevalence can be explained through the lack of vaccines

for these diseases, lack of awareness, unrestricted animal movement and the laboratory

technique used to diagnose prevalence of the diseases. Interpretation of the pasteurella

result is more difficult since it was impossible to get reliable information on past

vaccination history and specificity of the pasteuralla diagnosis seems questionable.

However, this is the first report to determine the prevalence of Q-fever, Chlamydia and

Border disease from sheep of Ethiopia and the results highlight that these diseases may

play an important role in the poor reproductive performance of small ruminants in

Ethiopia. The findings are the first baseline, but given the small scope of the project,

further epidemiological studies are warranted to unravel the impact in food animals as

well as the risk of transmission to humans, create awareness to the animal owners, and

inform preventive and control measures.

Key words: CBBP, ELISA, Ram, Reproductive disease, Risk factors, Sheep,

Seroprevalence, Ethiopia.

1

1. INTRODUCTION

In Ethiopia, farmers rear sheep mainly for sale and consumption. Sheep owners gain a

vast range of products and services such as meat, milk, skin, wool, manure, gifts,

religious rituals, etc. (Hirpa and Abebe, 2008). Sheep are also a means of risk mitigation

against crop failures, property security and monetary saving in addition to many other

socioeconomic and cultural functions (Gatenby, 2002). Sheep contribute 21% of the total

ruminant livestock meat output of the country, with the annual national mutton

production estimated to be at 77 thousand metric tons (Sebsibe, 2008).

The total pouplation of small ruminant in Ethiopia is 29.3 million sheep and 29.1 million

goats (CSA, 2015). Ethiopia harbours a huge and diverse sheep population and this

genetic diversity is a requisite for the present and future livelihoods of the large

population of rural poor farmers (Abegaz, 2007). Sheep are living banks for their owners

and serve as source of immediate cash and insurance against crop failure especially

where land productivity is low and unreliable due to erratic rainfall, severe erosion, frost,

and water logging problems (Tibbo, 2006).

There is a need to improve sheep productivity through breeding, conservation and

sustainable utilization to meet the protein demand by the ever increasing human

population and to improve the livelihoods of poor livestock keepers and alleviate poverty

among the rural poor dwellers. Presently, for sustainable genetic improvement and

conservation of farm animal genetic resources, development of community-based

strategies which take into consideration the need, knowledge and aspiration of local

community are being advocated (Wollny, 2003).

The reproductive performance of small ruminants is considered to be low with annual

lambing and kidding rates of 1.2 for ewes and 1.5 for does (Tsedeke, 2007). The average

carcass weight of Ethiopian sheep and goat is 10kg, which is the second lowest in Sub-

Saharan Africa (FAO, 2004). They are said to be late in age at first lambing/kidding.

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They are characterized by low fertility, prolificacy and weaning rate and mature body

weight is about 30-40kg.

Problems associated with sheep and goat reproduction represent an important economic

loss in terms of lost milk yield and meat production and in lower stock replacement rate.

The major reproductive problems include abortions, stillbirths, low or no milk

production, mastitis, uterine infections, delivery problems and lamb/kid mortality. Many

of the above problems are associated with systemic diseases that lower the overall

performance of the animal, while others specifically cause fetal mortality, abortion or

male infertility. Although, there are a number of factors affecting the normal reproductive

process, the infectious agents are considered to be the most important problems causing

significant economic losses at the herd level (Kebede, 2011).

The important role infectious diseases on reproductive performance is well known,

nevertheless few studies on specific reproductive pathogens in small ruminants have been

conducted.

Chlamydia, an obligate intracellular gram-negative bacterium, is known to cause a

variety of diseases in animals and humans (Rohde et al., 2010). Chlamydiaceae have a

single genus Chlamydia that includes nine species; among them C. abortus and C.

pecorum can cause diseases in sheep (Rohde et al., 2010); (Stephens et al., 2009). In

particular, C. abortus is recognized as a major cause of abortion and lamb loss throughout

the world, especially in the intensively managed farms (Longbottom et al., 2013);

(Entrican and Wheelhouse, 2006). C. abortus usually causes ulcerationof endometrial

epithelium resulting in placental infection if infection was acquired during the early

stages of that pregnancy. More typically, infection acquired during late gestation will

result in abortion in the following gestation andthe symptoms caused by C. abortus also

include epididymitis,pneumonia, arthritis, and conjunctivitis (Rekiki et al., 2002);

(Zhong, 2009).Recentreports described the presence of C. abortus DNA in the eyes of

ewes (Gerber et al., 2007); (Polkinghorne et al., 2009). C. abortus not only causes

economic loss in the sheep industry, but also induces abortions in humans due to contact

with aborting sheep or goats (Pospischil et al., 2002).

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Coxiella burnetii is an important intracellular pathogen that has been implicated in cases

of Q fever, a zoonotic worldwide disease with acute and chronic stages. Ruminants

(cattle, sheep and goats) can act as primary reservoirs of C. burnetii and a variety of

species like humans, small rodents, dogs, cats, birds, fish, reptiles and arthropods may be

infected (Ioannou et al., 2009; Ioannou et al., 2011). There is a list of symptoms

commonly seen with acute Q fever in human that the combination of them varies from

person to person; high fevers (up to 40°C - 40.5°C), severe headache, general malaise,

myalgia, chills and/or sweats, nonproductive cough, nausea, vomiting, diarrhea, ab-

dominal and chest pain are the most important signs. The most common clinical signs in

animals are pneumonia, abortion, still birth and delivery of weak offspring that can lead

to economic losses (Arricau-Bouvery and Rodolakis, 2005).

Toxoplasmosis is the most common disease complication next to tuberculosis among

HIV seropositive admissions and deaths (Dawit and Shishay, 2014). However, despite

having such an adverse health effect similar to salmonellosis and campylobacteriosis,

toxoplasmosis is still a neglected and underreported disease (Kijlstra and Jongert, 2008).

Sheep and goats play an important role in the epidemiology of toxoplasmosis. They have

big potential to spread the tissue cysts of T. gondii to humans through consumption of

raw or undercooked meat and /or offal (Kijlstra and Jongert, 2008).

Border disease virus (BDV) is an important pathogen in sheep and goat production.

Border disease is a form of infectious abortion in sheep (Nettleton et al., 1998). On a

national scale it is not as important as other causes of abortion, but on individual farms it

can cause serious losses. The disease is characterised by barren ewes, abortion, stillbirths

and the birth of small, weak lambs, a variable percentage of which show tremor,

abnormal body conformation and hairy fleeces ("hairy-shaker" or "fuzzy" lambs). The

cause of border disease is a virus serologically related to bovine virus diarrhoea (BVD)

virus (Edwards et al., 1995).

4

Brucella can cause epididymitis, orchitis and impaired fertility in rams. Initially, only

poor quality semen may be seen; sperm motility and concentration may be decreased, and

individual sperm are often abnormal. Later, palpable lesions may occur in the epididymis

and scrotum. Epididymitis may be unilateral or, occasionally, bilateral. The testes may

atrophy. Palpable lesions are often permanent, although they are transient in a few cases.

Some rams shed B. ovis for long periods without clinically apparent lesions. B. ovis can

also cause abortions and placentitis in ewes, but this appears to be uncommon. Infected

ewes may give birth to weak lambs that die soon after birth. Systemic signs are rare in

adult ewes and rams. http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_ovis.pdf

However there are other health challenges affecting productivity in small ruminants.

Peste des petits ruminants (PPR) is one of the diseases of major economic importance

and imposes a significant constraint upon sheep and goat production owing to its high

mortality rate. It is an acute, highly contagious and frequently fatal disease of sheep and

goats caused by PPR virus (PPRV), a member of genus morbillivirus of family

Paramyxoviridae (Zahur et al., 2009). PPR is widespread in Africa, Arabia, Middle East

and in some geographical areas of Asia, including much of the Indian subcontinent.

Furthermore, because of outbreaks in Morocco and the existing commercial trade

between Morocco and both Algeria and Spain, the situation raised huge concern owing to

the increased risk of introduction of the disease into free zones in northern Africa and

into Europe (FAO, 2009; Khalafalla et al., 2010).

Pneumonic pasteurellosis is one of the most economically important infectious diseases

of small ruminants (Prabhakar et al., 2012). M. haemolytica, B. trehalosi, and P.

multocida are common commensal organisms of the upper respiratory tract (tonsils and

naso-pharynx) of apparently healthy sheep and goats. They are distributed worldwide,

and diseases caused by them are common in all ages, although the prevalence of

serotypes may vary by region and flock (Shayegh et al., 2009; Sherrill, 2012).

This project is part of the CGIAR research program on Livestock and Fish implemented

by the International Center for Agricultural Research in the Dry Areas (ICARDA) and

5

International Livestock Research Institute (ILRI). The program has promoted the

development of community based breeding programs CBBP. Once these CBBPs are up

and running, detailed and up to date information is needed regarding to their health

performance of CBBP compared to non-CBBP households that have the selected highly

performed breeds in the regions.

So far a lot of work has been done in selecting superior rams but no works was done on

the effect of reproductive disease on the reproduction and production and in general on

diseases that might be transmitted through breeding rams. This study was implemented in

three geographical locations where CBBPs are operating, namely Menz, Bonga and

Horro, which are the source of local sheep breeds in Ethiopia. In this study it’s important

to compare the reproductive disease in geographical location (altitude), management

(husbandry) practice, breed, age and flocks size, CBBP membership. Therefore, the aim

of this research paper was;

To assess health status and risk factors for infectious diseases, with a focus on

reproductive diseases, in breeding sheep

The specific objectives were;

To determine seroprevalence of common sheep diseases in the selected areas

To determine seroprevalence of reproductive diseases in the selected areas

To assess the potential risk factors (determinants) associated with reproductive

diseases and disease easily spread through breeding rams

To compare health of rams in the Community Based Breeding Programmes

(CBBP) with health of rams in other households

To assess the risk of CBBP rams in spreading disease between households

6

2. LITERATURE REVIEW

2.1. Productivity and Reproductive Performance of Small Ruminants in Ethiopia

I. Productivity of Small Ruminants

Small ruminants are found widely distributed across the different agro-ecological zones

of the country (EARO, 2000). According to Gizaw et al. (2008) the sheep types in

Ethiopia are classified into four major groups based on their physical characteristics:

short fat-tailed, long fat- tailed, thin-tailed and fat-rumped sheep and also based on DNA

differences, it has been classified into nine genetically distinct breeds Simien Short fat-

tailed, Menz, Washera, Horro, Arsi-Bale, Bonga, Afar, Black head Somali and Gumz

breed.

Sheep production in Ethiopia is based on indigenous breeds except Awassi-Menz cross-

breeds that contribute less than 1% of the population. Despite low level of productivity

due to several technical (genotype, feeding and animal health), institutional,

environmental and infrastructural constraints (Tibbo, 2006), indigenous sheep breeds

have great potential to contributing more to the livelihoods of the people in low-input,

smallholder crop livestock and pastoral production systems (Kosgey and Okeyo, 2007).

Sheep and goats are relatively cheap and are often the first asset acquired by the

community. The increased domestic and international demand for Ethiopian sheep and

goats has established them as important sources of Inland Revenue as well as foreign

currency. This increased demand also creates an opportunity to substantially improve

food security of the population and to alleviate poverty. Farmers prefer sheep that have

brown coat colors and are valued as good or excellent breeds (Mengesha and Tsega,

2012).

7

Although Ethiopia is the second in Africa and sixth

in the world in sheep populations

(Demelash et al., 2006), indigenous sheep are poor in performances. Ethiopian

indigenous sheep are characterized by slow growth, late maturity and low production

performances. The mean carcass production of such sheep is estimated as around 10 kg

(FAO, 2009), which is low as compared to the average of sub-Saharan countries with

annual off take rates of around 33% (EPA, 2002). The productivity of local sheep is low

with high mortality of lambs (Tibbo, 2006). The low productivity of indigenous flocks

can partially be attributed to the low management standards of the traditional production

systems. However, provision of vaccination, improved feeding, clean water and night

time enclosure relatively improves the production performance of indigenous sheep.

Lebbie et al. (1992) and Getachew et al. (2010) from Zimbabwe and Ethiopia

respectively reported that to improve the sheep production, selection and evaluation of

the best animals should be concentrated on the traditional sector. Generally, livestock

improvement programs targeting smallholder farmers need to incorporate existing

traditional herding, breeding practices, trait preferences and the multiple roles of sheep.

The minimum and maximum average matured weights of sheep were also reported as

21.6±9.3 and 41.5±2.0 kg, respectively (Abebe, 2010) in Ethiopia. The dressing

percentage and carcass weights of Ethiopian sheep were reported to be 42.5% and 11.0

kg (Berhe, 2010) and 55.55% and 18 kg (Wood et al. 2010) from Bristol, respectively.

Sandip (2011) reported, from India that the dressing percentage of the Shahabadi Sheep

ewes were 39%, which is low. Moreover, Berhe (2010) reported that average carcass

weight of Ethiopian sheep was 10-12 kg and the annual mortality loss of sheep is also

estimated around 14-16%.

II. Breeding program of small ruminants

Research results in Ethiopia indicated the existence of high phenotypic diversity for

morphological characters on sheep found in the country (Solomon, 2008) and the

significant within and between breed variation on growth and survival in Menz and Horro

8

sheep breeds and moderate heritability for growth traits for Menz, Horro and Afar sheep

breeds (Markos, 2006; Solomon et al., 2007).

Different sheep and goat breeds have developed in a wide range of environments and

have consequently evolved a variety of reproductive strategies to suit these environments.

Local breeds of sheep and goats in tropical conditions are either non-seasonal breeders or

exhibit only a weak seasonality of reproduction. Females ovulate and exhibit estrus

almost the whole year round, even though short periods of anovulation and anestrous are

detected in some females. http://www.esgpip.org/HandBook/Chapter5.html

In the Ethiopian highlands, most conception in sheep and goats occurs during or

following the periods of the short rains in March through May. A study conducted in the

central highlands (Ada District) reported that most lambing and kidding occurred during

the heavy rains (August–September), indicating that most of the conception occurred

during or following the small rains in March–May.

http://www.esgpip.org/HandBook/Chapter5.html

An important feature of a genetic improvement program is that the effects of selection

accumulate over time. The economic benefits of selection also accumulate. Breeding

programs should therefore be seen as investments for sustainable improvements of

animal stock and its potential to produce food or other goods.

http://www.esgpip.org/HandBook/Chapter6.html

In Ethiopia, animals to be used for breeding purposes should be selected carefully and

superior animals should be identified accurately. Sheep and goats can be selected based

on records of performance and visual appraisal. Selection based on records is the best

way to achieve good results. Additional visual appraisal of the selected animals is

advantageous. Visual appraisal of a contemporary group of animals may be considered

where record keeping is not practical or is nonexistent. Visual identification of superior

animals is less successful compared to selection based on records. Differences among

9

animals of the same age from similar dams (parity, age, condition) kept under similar

management serve as indicators of genetic variability that can be exploited in a breeding

program. Performance records are more important for selection of animals (breeding

schemes) which involve the selection of superior animals from among a group. The

interest of the farmer or the breeder could be performance of an animal at a certain age.

In this case reliance on memory is of little value and very often not practical. It is often

necessary to keep simple pedigrees such as sire and dam, so that the performance of

parents can be related to that of their offspring. This is essential for selection schemes.

For crossbreeding, recording the breeds involved might be sufficient unless there is an

additional requirement to avoid future inbreeding because of a small number of animals

or a small geographic area. http://www.esgpip.org/HandBook/Chapter6.html

Many attempts to improve indigenous sheep genotype based on pure breeding using

technologies proved in developed world were also failed due to poor participation of

farmers, interruption of high governmental or other institutional subsidy, small flock size,

single sire flocks, lack of animal identification, lack of performance and pedigree

recording, low level of literacy and organizational shortcomings (Sölkner et al., 1998;

Kosgey et al., 2006).

Good reproductive performance is a prerequisite for any successful genetic improvement

and it determines production efficiency (Zewdu, 2008). Reproductive performance

depends on various factors including age at first lambing, litter size, lambing interval and

the life time productivity of the ewe and life time lamb crop (Amelmal, 2011).

Age at puberity

According to the Amelmal (2011) Age at sexual maturity (puberty) was 11.05+1.6,

10.88+1.7 and 9.5+1.4 months for males and 11.13+2.7, 10.8+1.9 and 9.5+1.4 months for

females in Tocha, Mareka and Konta, respectively. The sexual maturity (puberty) in local

sheep in Illu Abba Bora and Gumuz female sheep was reported to be 5-8 and 7.21+1.75

months, respectively (Dhaba, 2013; Solomon, 2007). The result of Tsedeke (2007) for

10

age at puberty of local Alaba sheep were 6.7 and 6.9 months for male and female

respectively.

Age at first service

Age at first service for Bonga breeds were 7.51+2.14 and 9.3+2.2 months and for Horro

breeds were 7.1+3 and 7.8+2.4 months for males and females, respectively (Zewdu,

2008).

Age at first lambing

Age at first lambing is based on breed, husbandry and management practices and has

wide variation among African sheep. In most traditional systems, first lambing occurs at

450-540 days when ewe weights are 80-85 percent of mature size and Poor nutrition,

disease or parasitic burdens and genotype limit early growth and it can put obstacle for

early maturity for giving first birth. Year and season of birth in which the ewe lamb was

born influence the age at first lambing through their effect on feed supply and quality

during different seasons (Mukasa-Mugerwa and Lahlou-Kassi, 1995).

Lambing interval

According to Solomon (2007) in association with Gumuz breed had an average lambing

interval of 6.64+1.13 months so the breed can produce three lambing in two years even

under the traditional management system but the work of (Belete, 2009) and Zewdu

(2008) indicates that lambing interval of Bonga and Horro ewes were around 8 and

7.8+2.4 month respectively. Among other breeds of sheep in Ethiopia that had short

lambing interval were Menz (8.5 month) and Afar sheep (9 month) Tesfaye (2008).

Reproductive life span and life time lamb crop

Long reproductive life span in tropical (unfavorable) condition is one of the adaptation

traits of tropical livestock. According to Zewdu (2008) the average reproductive life span

of Horro and Bonga ewes was 7.9+3.1 years and 7.4+2.7 years, respectively. Long term

reproductive performance (long living, high fertility, ability to produce more offspring)

of dams should be given more importance in selection programs. According to Zewdu

11

(2008) on an average a Bonga and Horro ewe delivers 12.2+1.80 and 15.3+4.3 lambs in

their life time and for Gumuz sheep (13.5+1.76 lambs) in Metema areas (Solomon,

2007). The local ewe produce on average 8.57+3.7 (Tocha), 8.62+4.1 (Mareka) and

10.78+4.7 (Konta) lambs in thier life time (Amelmal, 2011).

2.2. Reproductive disease

2.2.1. Chlamydia (Enzootic Abortion of Ewes)

Background

Ovine chlamydiosis (enzootic abortion of ewes [EAE] or ovine enzootic abortion [OEA])

is caused by the bacterium Chlamydophila abortus. Chlamydophila abortus, formally

called Chlamydia psittaci, is a non-motile, coccoid, obligate intracellular parasite.

Taxonomically, the family Chlamydiaceae has been divided into two genera and nine

species based on sequence analysis of the 16s and 23s rRNA genes (Everett et al., 1999).

The genus Chlamydia includes C. trachomatis (humans), C. suis (swine) and C.

muridarum (mouse and hamster). The genus Chlamydophila includes C. psittaci (avian),

C. felis (cat), C. abortus (sheep, goat and cattle), C. caviae (guinea-pig), the former

species C. pecorum (sheep and cattle) and C. pneumoniae (humans). The terms

‘chlamydiosis’ and ‘chlamydia (e)’ are used to refer to members of the genus Chlamydia

in general. However, a binomial of the generic and specific names is used when referring

to a particular chlamydial species.

Epidemiology

C. abortus is recognized as a major cause of reproductive loss in sheep and goats

worldwide, although the disease does not appear to occur in Australia or New Zealand

(Aitken et al., 2007). In countries of Northern Europe, OEA is the most common

infectious cause of abortion in lowland flocks that are intensively managed during the

lambing period. In the United Kingdom, OEA accounts for approximately 44% of all

12

diagnosed infectious cases of abortion. The organism can also infect cattle, pigs, horses,

and deer, although such infections are thought to be less common.

Clinical sign

In sheep the disease is usually manifested as abortion in the last 2 to 3 weeks of gestation,

while the goats can abort at any stage of pregnancy, but most abortions are during the last

2 to 3 weeks of gestation (Matthews, 1999; Nietfeld, 2001). C. abortus has affinity for

placental tissues. In rams and bucks, C. abortus can cause orchitis and seminal

vesiculitis, resulting in the shedding of the organism in semen (Appleyard et al., 1985).

It is one of the most important causes of reproductive failure in sheep and goats

(Rodolakis et al., 1998; Aitken, 2000). Chlamydial abortion in late pregnancy causes

serious reproductive wastage in many sheep-rearing areas of the world, particularly

where flocks are closely congregated during the parturient period (Aitken & Longbottom,

2007; Longbottom & Coulter, 2003).

Abortion typically occurs in the last 2–3 weeks of pregnancy with the appearance of

stillborn lambs and grossly inflamed placentas. Infection can also result in the delivery of

full-term stillborn lambs and weak lambs that generally fail to survive beyond 48 hours.

It is also not uncommon in multiple births for an infected ewe to produce one dead lamb

and one or more weak or healthy lambs. Infection is generally established in a ‘clean’

flock through the introduction of infected replacements and results in a small number of

abortions in the first year, which is followed by an ‘abortion storm’ in the second year

that can affect up to around 30% of ewes.

Infected animals show no clinical illness prior to abortion, although behavioral changes

and a vulval discharge may be observed in ewes within the last 48 hours of pregnancy.

Pathogenesis commences around day 90 of gestation coincident with a phase of rapid

fetal growth when chlamydial invasion of placentomes produces a progressively diffuse

inflammatory response, thrombotic vasculitis and tissue necrosis. Milder changes occur

in the fetal liver and lung and, in cases in which placental damage is severe; there may be

evidence of hypoxic brain damage (Buxton et al., 2002). Abortion probably results from

13

a combination of impairment of materno-fetal nutrient and gaseous exchange, disruption

of hormonal regulation of pregnancy and induced cytokine aggression (Entrican, 2002).

Infected females shed vast numbers of infective C. abortus at the time of abortion or

parturition, particularly in the placenta and uterine discharges and at subsequent lambing

(Papp et al., 1994), thus providing an infection source in the flock. Aborted ewes do not

usually abort again from C. abortus infection. Recent evidence suggests that the

proportion of infected ewes is reduced at the subsequent breeding season and only low

levels of chlamydial DNA are detected during the periovulation period and at lambing, so

that this would not have significant impact on the epidemiology (Livingstone et al., 2009;

Gutierrez et al., 2011).

Chlamydial abortion also occurs in goats and, less frequently, cattle, pigs, horses and deer

may be affected. In sheep, abortion in late pregnancy with expulsion of necrotic fetal

membranes are key diagnostic indicators, with care being needed to distinguish the

diffuse pattern of necrosis from that caused by Toxoplasma gondii (cotyledons only).

Distinction from other infectious causes of abortion such as brucellosis, coxiellosisor

other bacterial pathogens (Campylobacter, Listeria, Salmonellacan be achieved by

microscopy and/or

culture.http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.07.07_ENZ_ABO

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Transmission

The main source of C. abortus in the environment is placentas and foetal fluids of

affected animals. During the lambing season, elementary bodies remained infectious for

several days (Papp et al., 1994). It was documented that ingestion was the main route of

infection (Wilsmore et al., 1986). Few reports suggested inhalation as another route of

transmission (Jones and Anderson, 1988). Based on experimental findings, venereal

transmission was suggested as a less common route of transmission (Appleyard et al.,

1985). Development of clinical signs due to C. abortus infection depends on the time of

infection. Sheep and goats infected 5–6 weeks before parturition can develop clinical

14

disease during the current pregnancy (Morgan et al., 1988). Animals infected during the

last 4 weeks of gestation can develop latent infection and may develop clinical signs in

the next gestation (Wilsmore et al., 1990). It was found that infected and latently infected

sheep and goats may shed C. abortus in their reproductive tract for up to 3 years post

infection (Morgan et al., 1988).

Diagnosis

Identification of the agent

A. Smears

Where the clinical history of the flock and the character of lesions in aborted placentae

suggest enzootic abortion, a diagnosis can be attempted by microscopic examination of

smears made from affected chorionic villi or adjacent chorion. Several staining

procedures are satisfactory, for example, modified Machiavello, Giemsa, Brucella

differential, or modified Ziehl–Neelsen stains (Stamp et al., 1950). In positive cases

stained by the latter method and examined under a high-power microscope, large

numbers of small (300 nm) coccoid elementary bodies are seen singly or in clumps

stained red against the blue background of cellular debris. Under dark-ground

illumination, the elementary bodies are pale green. If placental material is not available,

smears may be made from vaginal swabs of females that have aborted within the previous

24 hours, or from the moist fleece of a freshly aborted or stillborn lamb that has not been

cleaned by its mother, or from the abomasal content of the aborted or stillborn lamb. In

general, such preparations contain fewer organisms than placental

smears.http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.07.07_ENZ_ABO

R.pdf

In terms of morphology and staining characteristics, C. abortus resembles the rickettsia

Coxiella burnetii. Care must be taken to differentiate between these two organisms in

cases lacking a good history or evidence of chlamydial-induced placental pathology.

Antigenic differences between C. abortus and Coxiella burnetii can be detected

15

serologically. Fluorescent antibody tests (FATs) using a specific antiserum or

monoclonal antibody may be used for identification of C. abortus in smears.

B. Antigen detection

Several chlamydial genus-level antigen-detection tests are available commercially. A

comparative assessment of several such assays, on non-ovine material, indicated that

those using enzyme-linked immunosorbent assay (ELISA) methodology were more

sensitive than kits employing a FAT (Wood & Timms, 1992). Under the test conditions

used, a kit that detects chlamydial lipopolysaccharide (LPS) was judged to be the most

sensitive of the rapid ELISA-based systems investigated. Though occasionally yielding

false-positive results, particularly with avian faecal samples, the kit also gave satisfactory

results with ovine placental samples (Wilsmore & Davidson, 1991) although it should be

noted that it does not differentiate between C. abortus and other chlamydial species that

may contaminate the samples. In histopathological sections, antigen detection can be

performed using commercially available anti-Chlamydia antibodies directed against LPS

or MOMP (major outer membrane protein) (Borel et al., 2006).

C. DNA

Amplification of chlamydial DNA by polymerase chain reaction (PCR) and real-time

PCR provide alternative approaches for verifying the presence of chlamydiae in

biological samples without resorting to culture. PCR is highly sensitive for this purpose,

but has the attendant risk of cross-contamination between samples or environmental

contamination of samples in the field, so appropriate measures must be taken to avoid

this happening. Biotechnology in the diagnosis of infectious diseases and vaccine

development. Another potential problem is in the production of false negatives resulting

from PCR-inhibitory substances in the samples. Methods for discriminating between

amplified DNA sequences originating from C. abortus and C. pecorum have been

described (DeGraves et al., 2003; Everett & Andersen, 1999; Jee et al., 2004; Laroucau

et al., 2001; Thiele et al., 1992). In the last few years, real-time PCR has become the

preferred method in diagnostic laboratories for its rapidity, high throughput and ease of

standardisation platform have (Sachse et al., 2009). Recently, DNA microarray

16

hybridisation assays using the ArrayTube been developed and hold much promise for the

direct detection and identification of organisms from clinical samples (Borel et al., 2008;

Sachse et al., 2005). PCR assays in combination with restriction fragment length

polymorphism analysis have been developed with potential to differentiate naturally

infected from vaccinated animals (DIVA) (Laroucau et al., 2010; Wheelhouse et al.,

2010).

D. Isolation of the agent

Chlamydophila abortus can be isolated in embryonated chicken eggs or in cell culture,

the latter being the method of choice for isolation of new strains. The causative agent of

chlamydiosis is zoonotic and thus isolation and identification procedures should be

carried out under biosafety level 2

conditionshttp://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.07.07_ENZ_A

BOR.pdf

Tissue samples, such as diseased cotyledons, placental membranes, fetal lung or liver or

vaginal swabs that may be subject to any delay before isolation procedures begin should

be maintained in a suitable transport medium in the interim period. For optimal recovery

such samples should be stored frozen, preferably at –80°C, or otherwise at –20°C. The

most satisfactory medium is sucrose/phosphate/glutamate or SPG medium (sucrose [74.6

g/litre], KH2PO4 [0.512 g/litre], K2HPO4 [1.237 g/litre], L-glutamic acid [0.721 g/litre])

supplemented with 10% fetal bovine serum, antibiotic (streptomycin and gentamycin are

suitable, but not penicillin), and a fungal inhibitor (Spencer & Johnson, 1983). A

tissue:medium ratio of 1:10 is commonly employed. Alternatively, approximately 1 g of

tissue is ground with sterile sand in 8 ml of transport medium.

Chicken embryos: Test samples are prepared as 10% suspensions in nutrient broth

containing streptomycin (not penicillin) (200 µg/ml); 0.2 ml of suspension is inoculated

into the yolk sac of 6–8-day old embryos, which are then further incubated at 37°C.

Infected embryos die between 4 and 13 days after inoculation. Smears prepared from

their vascularised yolk sac membranes reveal large numbers of elementary

17

bodies.http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.07.07_ENZ_ABO

R.pdf

Cell cultures: Chlamydophila abortus of ovine origin can be isolated in a variety of cell

types, but McCoy, Buffalo Green Monkey (BGM) or baby hamster kidney (BHK) cells

are most commonly used. For confirmatory diagnosis, cultured cell monolayers are

suspended in growth medium at a concentration of 2 × 105 cells/ml. Aliquots of 2 ml of

the suspension are dispensed into flat-bottomed glass Universal bottles, each containing a

single 16 mm cover-slip. Confluent cover-slip monolayers are achieved after incubation

for 24 hours at 37°C. The growth medium is removed and replaced by 2 ml of test

inoculum, which is then centrifuged at 2500 g for 30 minutes on to the cover-slip

monolayer to promote infection. After further incubation for 2–3 days, the cover-slip

monolayers are fixed in methanol and stained with Giemsa or according to the method of

Gimenez (Arens & Weingarten, 1981; Gimenez, 1964). After methanol fixation, infected

cultures contain basophilic (Giemsa) or eosinophilic (Gimenez) intracytoplasmic

inclusions. Similar procedures are used in culturing C. abortus for antigen preparation.

FAT techniques can also be used and are equally effective.

Serological tests

A. Complement fixation test

Complement fixation (CF) is the most widely used procedure for detecting infection

(sheep and goats are generally tested within 3 months of abortion or parturition). The test

will also detect evidence of vaccination. Infection is evident principally during active

placental infection in the last month of gestation and following the bacteraemia that often

accompanies abortion. Consequently, paired sera collected at the time of abortion and

again at least 3 weeks later may reveal a rising CF antibody titre that will provide a basis

for a retrospective diagnosis. Antigenic cross-reactivity between C. abortus and C.

pecorum, as well as with some Gram-negative bacteria (e.g. Acinetobacter), can give rise

to low false-positive CF test results. Thus, titres less than 1/32 in individual animals

should be considered to be nonspecific for C. abortus, although they could also be due to

18

a low grade infection with C. abortus. Ambiguous results can be investigated further by

western blot analysis using purified elementary bodies (Jones et al., 1997).

Antigen is prepared from heavily infected yolk sac membranes obtained from chicken

embryos that have been inoculated in the same manner as those used to isolate the

organism from field material. The preparation of the antigen should be carried out in a

biosafety cabinet with the appropriate biosecurity precautions to prevent human infection.

Chopped and ground membranes are suspended in phosphate buffer, pH 7.6, at the rate of

2 ml per g membrane. After removal of crude debris, the supernatant fluid is centrifuged

at 10,000 g for 1 hour at 4°C, the deposit is resuspended in a small volume of saline, and

a smear of this is examined to ensure a high yield of chlamydiae. The suspension is held

in a boiling water bath for 20 minutes, or is autoclaved, and sodium azide (0.3%) is added

as a preservative. Antigen may also be prepared from cell cultures infected with C.

abortus. Infected monolayers are suspended in phosphate buffer, pH 7.6, and the cells are

disrupted by homogenisation or ultrasonication. Gross debris is removed and subsequent

procedures are as for the preparation of antigen from infected yolk sacs. In either case,

CF tests with standardised complement and antisera will establish the optimal working

dilution for each batch of

antigen.http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.07.07_ENZ_AB

OR.pdf

B. Other tests

The serological responses to C. abortus and C. pecorum can be resolved by indirect

micro-immunofluorescence, but the procedure is too time-consuming for routine

diagnostic purposes. ELISAs developed independently by several research groups have

not been adapted for general diagnostic work, partly because of difficulties associated

with the use of particulate antigens. However, a novel ELISA that incorporates a stable,

solubilised antigen has been used to test experimental and field samples, and has given

results that, though lacking species specificity, have a higher sensitivity than the CF test

(Anderson et al., 1995); (Jones et al., 1997).

19

Other tests using monoclonal antibody technology in a competitive ELISA (Salti-

Montesanto et al., 1997) and recombinant antigen technology in indirect ELISAs

(Longbottom et al., 2002) have been developed and shown to be more sensitive and

specific than the CF test in differentiating animals infected with C. abortus from those

infected with C. pecorum. However, these tests are currently mainly used as research

tools, and have not been developed commercially. A number of commercially available

serological tests have been evaluated and compared with these ‘in-house’ tests with

variable results (Jones et al., 1997; Vretou et al., 2007, Wilson et al., 2009). None of the

serological tests that is available can differentiate vaccination titres from those acquired

as a result of natural infection (Borel et al., 2005).

Treatment

If OEA is suspected to be present in a flock/herd, the administration of a long-acting

oxytetracycline preparation (20 mg/kg body weight intramuscularly) will reduce the

severity of infection and losses resulting from abortion (Longbottom and Coulter,

2003);(Aitken et al.,2007). It is important that treatmentis given soon after the 95th to

100th day of gestation, the point at which pathologic changes start to occur. Further doses

can be subsequently given at 2-week intervals until the time of lambing. Although such

treatment reduces losses and limits the shedding of infectious organisms, it does not

eliminate the infection nor reverse any pathologic damage already done to the placenta,

thus abortions or the delivery of stillborn or weakly lambs can still occur, and the shed

organisms are a source of infection for other naive animals.

In humans, early therapeutic intervention is important. Severely ill patients require

supportive therapy, including fluids, oxygen, and measures to combat toxic shock.

Tetracyclines, erythromycin, and clarithromycin are administered orally or parenterally,

depending on clinical severity (Longbottom and Coulter, 2003).

Prevention and Control

20

During an OEA outbreak the primary aim is to limit the spread of infection to other naive

animals (Longbottom and Coulter, 2003, Aitken et al., 2007). The major sources of

infection are the placental membranes, dead fetuses, coats of live lambs/kids born to

infected mothers, and vaginal discharges. Thus, affected animals should be identified and

isolated as quickly as possible and all dead fetuses, placental membranes, and bedding

should be carefully disposed of; lambing pens must be cleaned and disinfected

(Longbottom and Coulter, 2003). Pregnant women and immunocompromised individuals

are advised not to work with sheep, particularly during the lambing period, and should

avoid all contact with possible sources of infection, including work clothing. It may cause

a flu-like illness and human abortion. The bird strains of chlamydia may cause

pneumonia in humans (Longbottom and Coulter, 2003; Winter and Charnley, 1999).

Basic hygiene procedures, including thorough washing of hands and the use of disposable

gloves, are essential when handling potentially infected materials. Ewes that have aborted

are considered immune to further disease, although this immunity is not sterile. Ewes

may become persistently infected carriers and might continue to excrete infectious

organisms at next estrus, (Papp et al., 1994; Papp and Shewen, 1996) thus providing

anopportunity for venereal transmission of infection via the ram, although more recent

evidence using quantitative real-time PCR suggests that the risks of this are low

(Livingstone et al., 2009).

Although antibiotic treatment can be used in exceptional circumstances to reduce

abortion losses, it should not be used routinely to control infection. Instead it is better to

use a combination of flock/herd management and vaccination (Longbottom and Coulter,

2003). Flock/herd management aims to keep animals “clean” by keeping the flock/herd

“closed,” through breeding own replacement animals or by buying them in from OEA-

free accredited sources, such as those that participate in the various United Kingdom

Premium Health Schemes (Longbottom and Coulter, 2003; Entrican et al., 2001).

If there is any doubt regarding the status of replacements or for animals bought from non-

accredited sources, these should be vaccinated before entering them into the flock/herd.

21

In most of Europe, there is currently available an attenuated (“live”) vaccine based on a

temperature-sensitive mutant strain (C. abortus strain 1B) (Rodolakis, 1983), that is

available from 2 commercial companies. The vaccines must be administered at least 4

weeks before mating and cannot be used in combination with antibiotic treatment.

Inactivated vaccines can also be prepared from organisms grown in hens’ eggs or cell

culture (Jones et al., 1995). These vaccines are safe for administration during pregnancy.

Both types of vaccines confer good protection from abortion, but do not completely

eradicate the shedding of infectious organisms at parturition, and some vaccinated

animals still abort as a result of wild-type infections. Recently the live vaccine has been

detected in the placentas of vaccinated animals that have aborted as a result of OEA,

suggesting a role for the vaccine in causing disease in some animals (Wheelhouse et al.,

2010).

However, this requires further investigations to determine the proportion of animals

affected in an outbreak. Despite these findings, the importance of continuing the

vaccinations is stressed, as this is still the most effective way to protect from disease

(Wheelhouse et al., 2010). Vaccine development research to produce the next-generation

OEA vaccine continues to progress. This treatment is likely to be a subunit vaccine,

based on protective recombinant antigens identified through comparative genomic and

proteomic approaches, and which is capable of eliciting the required mucosal and

systemic cellular and humoral responses (Longbottom and Livingstone, 2006).

2.2.2. Coxiella Burnetti

Background

The febrile illness ‘Query fever’ (Q fever) was first reported in 1935, among workers in

slaughterhouses in Australia (Angelakis et al., 2012). In Europe, cases of Q fever in

humans were first reported from soldiers in the Balkan region including Bulgaria in 1940

(Astobiza et al., 2010), and subsequently in Germany shortly after World War II

(Anderson et al., 2009), and in the Netherlands in 1956 (Astobiza et al., 2012). Initial

22

hypotheses about potential exposures and infectious pathways emerged following the

development of illness in experimental animals (guinea pigs) via feeding of ticks

(Arricau‐Bouvery et al., 2003), collected from febrile livestock in Nine Mile, United

States. Investigations into cases of atypical pneumonia subsequently revealed the

importance of aerosol transmission.

Epidemiological linkages with animals were later identified, and infection was found in a

broad range of hosts (Alsaleh et al., 2011; Anderson et al., 2013). It was initially thought

that Q fever was primarily an occupational risk (for people who worked closely with

animals) however this was subsequently expanded, with risk groups also including people

with a specific health status (pregnancy, cardiac diseases, immune-compromised). Blood

donation was identified as a potential source of infection. In domestic ruminants, as in

people, C. burnetti infection and Q fever (the disease) are not the same. C. burnetti

infection is usually subclinical (i.e. the animal is infected with C. burnetii but without

clinical signs). Q-fever which develops in a subset of infected animals, presents as late

abortion and reproductive disorders (Alsaleh et al., 2011; Astobiza et al., 2012).

Ethiology

Q fever is caused by the aerobic intracellular organism Coxiella burnetii. Although

Coxiella was historically considered to be a Rickettsia, gene-sequence analysis now

classifies the genus Coxiella in the order Legionellales, family Coxiellaceae, with

Rickettsiella and Aquicella (Seshadri et al., 2003).

The organism exists in 2 different antigenic phases. In nature, C. burnetii exists in phase I

form, which is virulent. However, when cultivated in nonimmunocompetent cell cultures

or hens’ eggs, the organism mutates irreversibly to the phase II form, which is less

virulent (Quevedo Diaz and Lukacova, 1998). C. burnetii has 2 different morphologic

forms, a large and a small form. In addition, an endospore-like structure is observed in

the large form which is highly resistant to environmental degradation, such as high

temperatures, ultraviolet light, and osmotic shock (Mearns, 2007). In the mammalian

23

host, monocyte-macrophages are the only known target cells of the bacteria (Maurin and

Raoult, 1999).

Coxiella Burnetti has been isolated from a wide variety of wild and domestic animals.

Coxiella burnetii is common in the environment, where it can persist for years as a

spore‐like form that is resistant to heat, drying and many disinfectants (Guidance for a

Coordinated Public Health and Animal Health Response, 2013).

Epidemiology

In Animals

Coxiella burnetii is a worldwide zoonosis that occurs in all geographic and climatic

zones, with the exception of Antarctica and possibly New Zealand (Hilbink et al., 1993).

However, in many countries Q fever is not a reportable disease, so it is difficult to know

exactly where it occurs.

Coxiella burnetii infection has been documented in a broad range of animals including

almost all mammals that have been tested, as well as birds and ticks (Maurin and Raoult,

1999). Studies of feral and domestic cats have found that prevalence (based on either

PCR or antibodies) can range from 8.5% in client‐owned animals, to 41.7% in stray cats

(Cairns et al., 2007; Porter et al., 2011). Goats, sheep, and cattle are the domestic species

most clinically affected by C. burnetii infection, and are most often implicated in

transmission to humans (Berri et al., 2005; Porter et al., 2011). Coxiella burnetii infection

in animals is reportable to animal health agencies in 44 states.

Coxiella burnetii infection is common in U.S. dairy cattle herds. A 2007 national dairy

study that included testing of bulk tank milk samples reported that 77% of 528 operations

in the U.S. were positive for C. burnetii (NAHMS, 2007). In a similar bulk tank milk

study of U.S. veterinary‐school‐associated dairy herds, 22 of 24 were positive for C.

burnetii antibodies using an immunofluorescence assay (McQuiston et al., 2005). In

addition, a three‐year study of dairy herds in the Northeast U.S. reported a herd level

prevalence (using PCR) of more than 94% (Kim et al., 2005).

24

Animals may become infected by direct contact with infected animals and contaminated

environments and/or from inhalation of aerosolized bacteria. Birth products (including

placenta, fetuses, and amniotic and allantoic fluids), excreta, and milk are the most likely

sources of infection (Heydel and Willems, 2011; Porter et al., 2011). Because the

spore‐like form of the bacterium can survive for years in the environment and travel long

distances as an aerosol, dry, windy conditions may contribute to animal exposure and

disease transmission. The bacteria have also been found in ticks, which may also serve as

a source of infection for animals (Astobiza et al., 2011; Toledo et al., 2009).

Duration, quantity and route of shedding can vary by host species. Goats commonly shed

C. burnetii in birth products, feces and milk (Rodolakis et al., 2007; Rousset et al., 2009).

Goats have also been shown to shed the organism in vaginal mucus, even nulliparous

animals and goats delivering healthy‐appearing live‐born kids (Alsaleh et al., 2011; Roest

et al., 2011).

C. burnetii is a pathogen known to be resistant to disinfectants and environmental factors,

and Brouqui et al. (2007), reported that C. burnetii could survive for years in animal

faeces for 8-9 months in sand and 19 months in dried tick faeces. Studies done in rural

areas have all indicated that poor hygiene could be an exacerbating factor in the spread of

C. burnetii (Lyytikäinen et al., 1998).

A 2007 national dairy study that included testing of bulk tank milk samples reported that

77% of 528 operations in the U.S. were positive for C. burnetii (NAHMS, 2007). In

addition, a three‐year study of dairy herds in the Northeast U.S. reported a herd level

prevalence (using PCR) of more than 94% (Kim et al., 2005). Coxiella burnetii has

received international attention in recent years, primarily due to a large‐scale outbreak in

the Netherlands from 2007 to 2010 involving more than 4,000 human cases and the

euthanasia of 50,000 goats, one of the primary reservoirs for the bacterium (Van der

Hoek et al., 2010).

25

The serological prevalence of C. burnetii infection in farm animals varies by host species,

geographic area and time, whereby it also should be noted that different serological cut-

offs were used in different studies. Within-herd prevalence estimates for cattle were up to

20.8% in Bulgaria, 15.0% in France, 19.3% in Germany, 21.0% in the Netherlands, for

goats up to 40.0% in Bulgaria, 88.1% in France, 2.5% in Germany, 7.8% in the

Netherlands, and for sheep up to 56.9% in Bulgaria, 20.0% in France, 8.7% in Germany,

and 3.5% in the Netherlands respectively. Herd prevalence estimates, whereby a herd is

considered positive when at least one animal in the herd was serologically-confirmed,

were higher than within-herd prevalence. Herd prevalence for cattle was up to 73.0%, in

France, and up to 37.0 % in the Netherlands. For goats it was 40.0% in France and 17.8%

in the Netherlands while for sheep values of 89.0% in France, and 14.5% in the

Netherlands were respectively found. Regional differences were observed: up to four-fold

among farm animals in different areas of Bulgaria (Cairns et al., 2007), and higher in

some rural German regions (CDC, 2011; CDC, 2009).

In humans as many as 50% of human C. burnetii infections are asymptomatic;

symptomatic infections most commonly present as a non‐specific febrile illness that may

occur in conjunction with pneumonia or hepatitis (CDC, 2012; Maurin and Raoult, 1999).

Untreated, acute Q fever has a low case fatality rate (<2%) and, when treated, the

case‐fatality rate is negligible (Maurin and Raoult, 1999).

Clinical sign

In animals, C burnetii infections are generally asymptomatic. Except for abortion,

stillbirth, and the delivery of weak offspring, clinical signs in ruminants are rare.

However, C burnetii may induce pneumonia, conjunctivitis, and hepatitis (Arricau-

Bouvery and Rodolakis, 2005). The abortion rate can range from 3% to 80% of pregnant

females (Berri et al., 2001). High abortion rates are rarely observed, although abortion

storms in some herds have been described (Sanford et al., 1994). Stress, resulting from

overcrowding or poor nutrition, may play an important role in an infected goat aborting.

In the majority of cases, abortion or stillbirth occurs at the end of the gestation period,

without specific clinical signs, only when placental damage has been severe. Aborted

26

fetuses appear normal, but infected placentas exhibit intercotyledonary fibrous thickening

and discolored exudates that may be mineralized (Sanford et al., 1994; Moore et al.,

1991).

Coxiella burnetii infection in livestock species is generally asymptomatic. Goats and

sheep are the species in which abortions, stillbirths, and early neonatal mortality have

been most frequently documented (Berri et al., 2007); (Hatchette et al., 2003; Porter et

al., 2011). Abortion in cattle due to C. burnetii infection has been reported, and more

research is needed to determine the organism’s role in infertility, metritis and

endometritis (López‐Gatius et al., 2012). Coxiella burnetii can cause adverse pregnancy

outcomes in cats, and contact with infected cats has been associated with human infection

(Cairns et al., 2007; Komiya et al., 2003).

In humans, acute Q fever may not be promptly diagnosed, because of nonspecific initial

clinical signs such as fever, pneumonia, headache, and weakness, and the time between

onset of clinical signs and therapy may be greater than 2 months. Chronic infection may

result in severe granulomatous hepatitis, osteomyelitis, and valvular endocarditis with

high case fatality rates (Fournier et al., 1998).

Acute Q fever is characterized by sudden onset of fever to 104º‐105º F, chills, profuse

sweating, severe headache with retro orbital pain, weakness, nausea, vomiting, diarrhea,

non‐productive cough, and abdominal or chest pain (Heydel and Willems, 2011). Chronic

Q fever most commonly presents as endocarditis occurring weeks to years after an acute

infection (Fennolar et al., 2001). Other manifestations include vascular infections and

infections of the bone, liver or reproductive organs (Maurin and Raoult, 1999).

Regardless of species, the highest numbers of organisms are shed in conjunction with an

adverse pregnancy event (abortion, stillbirth, or neonatal weakness).

Transmission

Infected sheep are more likely to shed C. burnetii in birth products, vaginal discharge,

and feces, and are less likely than cattle to shed the organism persistently in milk. Cattle

may shed the organism in milk for weeks to months after calving (Guatteo et al., 2012).

27

Coxiella burnetii has been detected in the feces, milk, urine, and vaginal discharge,

semen, and birth products of infected animals (Arricau‐Bouvery et al., 2003; Masala et

al., 2004). Regardless of species, the highest numbers of organisms are shed in

conjunction with an adverse pregnancy event (abortion, stillbirth, or neonatal weakness).

Diagnosis

Current alternatives to diagnose C. burnetii infection in ruminants include serologic

analysis, organism isolation by cell culture (eg, shell vial culture) or live animal

inoculation, and immunohistochemical and PCR-based detection. For instance, a single

touchdown PCR could be used to detect C. burnetii from genital swabs, milk, and fecal

samples (Berri et al., 2000). In the acute phase of the infection, C. burnetii can be

detected in lungs, spleen, liver, and blood (Maurin and Raoult, 1999).

Placental smear or impression of placentas could be stained, for instance using a modified

Ziehl-Nielsen procedure. Coxiella is stained as acid-fast rod-like organisms, observed

extra- and intracellularly (Mearns, 2007). Because C. burnetii can be shed heavily at the

time of normal lambing/kidding, isolation of the organisms as a sole procedure is not

considered enough to confirm the diagnosis as the cause of abortion (Hatchette et al.,

2001).

Several serologic tests are available, such as complement fixation test, enzymelinked

immunosorbent assay (ELISA), and a fluorescent antibody test (Kovacova et al., 1998).

However, carrier animals may also have an antibody titer increase in late pregnancy

(Smith and Sherman, 2009). In addition; laboratory animal inoculation and isolation in

embryonated eggs are other possible diagnostic techniques. For Q fever diagnostics, it

has recently been recommended to use PCR and immunofluorescence tests of Coxiella on

parturition products and vaginal secretions at abortion (Berri et al., 2000; Arricau

Bouvery et al., 2003).

A definitive diagnosis of Q fever in animals is based on the observation of the occurrence

of abortions and/or stillbirths, confirmation of the presence of the aetiological agent (i.e.

28

polymerase chain reaction (PCR), isolation, staining, immunofluorescence assay tests are

positive) and positive serological findings in the herd (McCauley et al., 2007).

Treatment

If Q fever is suspected, aborting animals and other animals in late pregnancy should be

treated with tetracycline. The regime consists in 2 injections of oxytetracycline (20 mg/kg

BW) during the last month of gestation, although this treatment does not totally suppress

abortions and shedding of C burnetii at lambing (Berri et al., 2005). Antibiotic treatment

is mainly used to minimize shedding of the organisms in the placenta and birth fluids

rather than to eliminate it, but its efficacy has not been evaluated (Kaza, 1999). Placentas

and aborted fetuses should be destroyed properly, and aborted animals should be isolated.

In addition, materials such as bedding and straw contaminated with birth fluids and other

secretions from affected animals should bedestroyed.

Although it may not be practical or possible to eliminate the risk of Q fever in a typical

farm setting, the risk for spread can be decreased by 1) proper sanitation – good hygiene,

especially when working with parturient animals; 2) segregated kidding/lambing areas; 3)

removal of risk material from birthing areas (birthing products/fluids, contaminated

bedding, manure); 4) good manure management; 5) control of ticks on livestock; and 6)

restriction of moving peri-parturient animals (close to birthing or giving birth within the

past two weeks) off the farm.

http://agr.wa.gov/FoodAnimal/AnimalHealth/Diseases/QFeverManagementPractices.pdf

In human acute Q fever usually clears up within a few weeks with no treatment. If you

have symptoms, your doctor will likely prescribe antibiotics. The antibiotic tetracycline

(doxycycline) is often used to treat Q fever. Patients usually recover promptly when

treatment is started without delay. Chronic Q fever requires specific antibiotic treatment,

multiple follow-up tests and possibly surgery. http://www.disabled-

world.com/health/query-fever.php

29

Prevention and Control

Given the ubiquitous nature of C. burnetii and its persistence in the environment,

complete eradication of the bacteria from an infected farm would be nearly impossible.

Nevertheless, transmission can be reduced with good hygiene and other management

practices that reduce environmental load, such as immediately removing and disposing of

aborted fetuses, dead newborns, and placentas. Coxiella burnetii is shed in the milk of

infected animals; therefore, their milk should not be consumed raw or sold unpasteurized

direct to consumers. Pasteurizing milk at 145° F (63° C) for at least 30 minutes or at 161°

F (72° C) for 15 seconds is sufficient to destroy C. burnetii, as well as other pathogens

that can be present in raw milk.

http://www.fda.gov/Food/ResourcesForYou/StudentsTeachers/ScienceandTheFoodSuppl

y/ucm

A vaccine (Q-Vax®) is available to protect people against Q fever. Vaccination is

recommended for all people who are working in, or intend to work in, a high-risk

occupation. Work places at risk should have a vaccination program in place.

http://www.disabled-world.com/health/query-fever.php

2.2.3. Toxoplasma

Background

Toxoplasma gondii is among the best studied parasites due to its medical and veterinary

importance. Up to and including the twentieth century, fifteen thousand original articles

and 500 reviews had been published on the subject (Tenter et al., 2000). From the year

2000 through the current year (2013), over eight thousand articles with the terms

“toxoplasma” or “toxoplasmosis” were indexed in PubMed, the National Center for

Biotechnology Information U.S. (NCBI).

The parasite was discovered over a century ago (Dubey, 2008a), reported its presence in

the tissues of an African rodent, Ctenodactylus gundi, which had been used to study

30

leishmaniasis. Concurrently, in Brazil, Alfonso Splendore (1908), had also detected the

parasite in rabbits, but the parasite was appointment by Nicolle and Manceaux in 1909

(Dubey, 2008a). About thirty years after its discovery, the parasite was first isolated in

animals (Sabin and Olitsky, 1937) and in humans (Wolf et al., 1939) because it is not

only a parasite that affects animals but also a human disease of public health importance,

a zoonosis. Twenty years after its isolation, T. gondii was finally recognized worldwide

as an important cause of abortion in sheep (Hartley and Marshall, 1957). Since this time,

several studies have been published on the issue, noting the significant facts that the

parasite is capable of being transmitted to humans through the consumption of

undercooked meat (Villena et al., 2012) and that it can cause economic losses to farmers

by causing pathologic abortion in small ruminants (Bispo et al., 2011).

Ethiology

Toxoplasmosis is caused by the obligate intracellular protozoan parasite Toxoplasma

gondii (Tenter et al., 2000; Dubey, 2010). Toxoplasma gondii belongs to the Kingdom

Animalia, Phylum Apicomplexa, Class Protozoa, Subclass Coccidian, Order Eucoccidia,

Family Sarcocystidae and Genus Toxoplasma (Dubey, 2010). The protozoan phylum

Apicomplexa contains pathogens of substantial medical and veterinary importance

including Plasmodium, Toxoplasma, Cryptosporidium, Eimeria, Neospora and Theileria

species (Blake et al., 2011). Toxoplasma gondii is a tissue cyst-forming coccidian

parasite (Innes, 2010).

Epidemiology

The prevalence of T. gondii in cats in Ethiopia was high. Ethiopian cats live outdoors,

hunt, feed on scraps and garbage-thus more exposed to the parasite (Dubey et al., 2012).

In a study from California, USA, the annual environmental burden per square meter was

estimated to be in the range of 94 to 4671 oocysts, based on a low prevalence (0.9 %) of

oocysts in cat feces (Dabritz et al., 2007). Therefore, if we assume 17.51 % oocyst

shedder cats in Ethiopia, and 100 million oocysts per shedder (Robert-Gangneux and

Dardé, 2012) the environmental burden in urban residential areas where cats abound is

apparently high.

31

Wild and domestic cats play a central role in the epidemiology of T.gondii infections by

shedding resistant oocysts in the environment, hence serving as a significant source of

infection for food animals and humans (Gajadhar et al., 2006). The infection can be

maintained for an indefinite period of time in small rodents through cannibalism or

scavenging (Marquardt et al., 2000).

Toxoplasmosis is an economically important disease in animal husbandry globally as it is

a major cause of reproductive failure by leading to early embryonic death and resorption,

fetal death and mummification (Dubey, 2009), abortion, stillbirths, and neonatal death in

small ruminants (Marquardt et al., 2000; Dubey, 2010).

Considerable geographical differences exist in prevalence of toxoplasmosis. Differences

in the epidemiology of the infection in various geographical areas and between

population groups within the same area may be explained by differences in exposure to

the two main sources of the infection: the tissue cyst (in meat of animals) and the oocyst

(in soil contaminated by cat feces) (Remington et al., 2006). Cultural habits with regard

to food probably are the major cause of the differences in frequency of T.gondii infection

from one country to another, from one region to another in the same country, and from

one ethnic group to another in the same region (Remington et al., 2006). Higher

prevalence of toxoplasmosis in warm and moist areas compared to cold and dry areas was

attributed to the longer viability of T.gondii oocysts in moist or humid environments (Van

der Puije et al., 2000).

In humans, the occurrence of toxoplasmosis is higher in Africa (greater than 50%), the

Middle East, parts of Southeast Asia, Latin America and parts of Eastern and Central

Europe than in the United States and most Western European countries (Pappas et al.,

2009). A decreasing trend of T.gondii seroprevalence was reported in the United States

and some European countries over the last decades (Pappas et al., 2009). The incidence

of ocular disease caused by T.gondii is higher in Africa and South America compared to

32

that of Europe. In South America, ocular toxoplasmosis due to virulent strains is

associated with high burden of visual disability (Peterson et al., 2012).

Previously, the seroprevalence of toxoplasmosis in humans in France was found to be as

high as 75-80%. This high prevalence of infection was attributed to a preference for

eating raw or undercooked meat (Miller et al., 2009). However, in recent years the

seroprevalence has decreased markedly in pregnant women: from 83% in 1965 to 54.3%

in 1995, 43.8% in 2003 to 37.0% in 2010. This drastic reduction has been attributed to

intervention measures through a national program to prevent congenital toxoplasmosis

since 1978 (Nogareda et al., 2013).

Clinical sign

Manifestation of the clinical signs of toxoplasmosis in animals, as well as in humans,

depends primarily on the immune response of the infected host and on the virulence of

the sample of T. gondii (Amendoeira et al., 1999). According to Millar et al. (2008), farm

animals such as sheep are more susceptible to infection when compared with other

species.

According to Buxton et al. (1998), the clinical signs of toxoplasmosis are observed when

pregnant sheep are infected for the first time. Typical clinical signs include the

production of stillborn and/or weak lambs, in addition to mummified fetuses. Abortion in

sheep is cited by many researchers from different regions of the world (Van den Brom et

al., 2012) as a principal signs of infections by T. gondii. Additionally, this symptom is the

primary cause of economic losses in the sheep industry due to the high prevalence of

parasite infection that exists (Moreno et al., 2012).

In addition to the clinical signs that the reproductive agent T. gondii can cause, other

clinical signs of toxoplasmosis in animals are fever, dyspnea, and neurological signs

(Soccol et al., 2009). The signs described above and that are associated with the clinical

and physical examination of the animal, in addition to complementary diagnostic

methods, can contribute to the identification of the disease.

33

The clinical spectrum of T. gondii infection varies from an asymptomatic state to severe

illness. The parasite can affect the host’s lymph nodes, eyes, central nervous system,

liver, and heart (Alvarado-Esquivel et al., 2013). Primary infections with T. gondii

acquired during pregnancy are usually asymptomatic for the pregnant woman but can

lead to serious neonatal complications. Screening of T. gondii infections during antenatal

care should be considered as the main strategy to minimize congenital toxoplasmosis

(Mwambe et al., 2013).

Transmission

Not only the domestic cat but also all species of cats can excrete T. gondii non-sporulated

oocysts after ingesting the infective stage of the parasite, which consists of bradyzoites;

bradyzoites are present in the tissue cysts of the intermediate host, or as sporozoites,

which are formed inside the sporocysts after the sporulation of the oocysts (Dubey,

2010).

These non-sporulated oocysts are eliminated by the definitive host into the environment,

undergo a change in their structure and become potentially infectious, able to sporulate

one to five days after excretion (Tzanidakis et al., 2012). Tachyzoites are observed only

in acute infections and systemic disease states and are present primarily during congenital

transmission (Dubey, 2010). The principal routes of transmission for toxoplasmosis

through intermediate hosts are as follows: transplacentally (vertical/congenital) and

horizontally by the ingestion of tissue cysts contained in raw or undercooked animal

tissues and the ingestion of food or water contaminated with sporulating oocysts.

According to Dubey (2009), the ingestion of undercooked beef and lamb is a major

source of infection for humans.

Recent studies have drawn attention to the consumption of raw milk and unpasteurized

sheep meat that might contain tachyzoites if the animal is in the acute phase of the

disease. Camossi et al. (2011) detected DNA from T. gondii in seven milk samples from

20 sheep that had been naturally infected by the parasite, thus demonstrating that milk

34

can also be a route of infection for humans. However, for this to occur, no lesion needs to

be present in the oral cavity of the host because the tachyzoites show little resistance to

the action of gastric juices and are therefore destroyed in a short time when ingested

orally, while the bradyzoite forms are resistant to the enzymes present in gastric juices

(Prado et al., 2011).

Vertical transmission of T. gondii during pregnancy affects the intermediate hosts (sheep

and other animals) and even the definitive hosts. The pathogenesis of abortion develops

when the parasite proliferates in the placenta and reaches the fetus.

When there is no abortion, congenital lesions are initiated and are considered irreversible

(Dubey, 1994). There is yet another likely route of infection by T. gondii that has been

studied, treating it as a venereal or sexual parasite. Although the subject has been rarely

reported, the first papers on the subject were published for sheep in 2010 (Moraes et al.,

2010a; Moraes et al., 2010b; Moraes et al., 2010c; Lopes et al., 2013a).

35

Figure 1: Pathways for Toxoplasma gondii infection

Source: (Tenter et al., 2000)

Diagnosis

Diagnosis of T. gondii in sheep can be made by means of direct tests, such as

histopathology, immunohistochemistry, PCR and bioassay, as well as by means of

indirect tests (serum) based on the detection of anti-T. gondii antibodies, or by a

combination of these methods (Dubey, 2010).

For establishing a serological survey of T. gondii, serological tests are essential because

they reports the actual situation and the degree of infection in the animals studied (Braga-

Filho et al., 2010). Moreover, various serological tests exist that can be used for the

detection of both IgG and IgM (Pereira et al., 2012; Silva et al., 2013).

36

The indirect fluorescent antibody test (IFAT) is the most commonly used and is therefore

considered as the gold standard for diagnosis, as cited by various authors (Silva et al.,

2003; Soraes et al., 2009; Ueno et al., 2009). However, the modified agglutination test

(MAT) is also widely used for the diagnosis of toxoplasmosis in animals and humans

because it detects IgG with the additional advantage of not requiring a specific conjugate,

and it also does not require sophisticated equipment for diagnosis (Dubey, 2010). In

sheep, the MAT has been used for diagnostic serology in France (Dumètre et al., 2006),

Spain (Mainar-Jaime and Barberán, 2007), Egypt (Shaapan et al., 2008), the United

States (Dubey et al., 2008b), Iran (Raegui et al., 2011), and Brazil (Silva et al., 2013),

among other countries.

The Elisa test has been widely used for the serological diagnosis of T. gondii in sheep

(Soccol et al., 2009, Andrade et al., 2013, García-Bocanegra et al., 2013, Gebremedhin et

al., 2013). An Elisa kit might represent a valuable tool for collecting information on

toxoplasmosis infections during sheep production, and additionally, for diagnosis in

slaughter houses, helping to control this widespread zoonosis.

Other tests, such as the latex agglutination test (LAT) described by Gondim et al. (1999)

and the Sabin-Feldman reaction (RSF) described by Larsson et al. (1980), also detect the

anti-T. gondii antibody in serum independent of a specific conjugate and can be used for

diagnosis in animals and people.

The bioassay in mice is one of the primary methods used to detect T. gondii cysts in

tissue for confirming suspected cases of infection by the parasite. It is considered to be a

very sensitive diagnostic test, but it is also very costly, difficult and slow (Rosa et al.,

2001; Tsutsui et al., 2007). One study evaluated the presence of T. gondii in commercial

cuts of pork (ham, loin, rib and shoulder) through bioassay and PCR in experimentally

inoculated animals; the bioassay test was more sensitive than PCR (Tsutsui et al., 2007).

37

The first report of the detection of T. gondii DNA was conducted in the 1980s by Burg et

al. (1989) using the B1 gene. Since then, studies have been performed with tissues such

as brain, cardiac and skeletal muscle, and liver (Esteban-Redondo and Innes, 1998;

Asgari et al., 2011) as well as blood (Spalding et al., 2003) for identification of the

parasite using PCR.

Samples of brain, tongue, and liver and from neck, intercostals and femoral muscle from

78 sheep and goats were tested in Iran using nested PCR, and the researchers detected T.

gondii DNA in 21.8% of the tongue, 19.2% of the brain and 17.9% of the muscle tissue

samples. The data confirmed the high rate of toxoplasmosis infection in small ruminants

in the country. In addition, the researchers warned the population after studying the

infection of the human population in the region by the parasite (Asgari et al., 2011)

Hematological and biochemical parameters, although not conclusive, can assist in the

diagnosis of T. gondii. However, studies of these parameters in sheep naturally infected

with T. gondii are scarce in the literature consulted (Silva et al., 2011). The data

published for sheep indicate that chronic parasitism influences changes in hematological

and biochemical parameters, primarily lymphopenia, neutrophilia and decreased values

of alanine transaminase (ALT).

Treatment

The parasite has been shown to be destroyed by certain antibiotic treatments. These can

include sulphonamide which is often given orally. Infected ewes may be given certain

medicines advised by the veterinarian two months before lambing to prevent the

pregnancy complications which arise from infection.

http://www.netvet.co.uk/sheep/toxoplasmosis.htm

Prevention and control

Diagnosis by means of laboratory tests, when it is rapid and reliable, can be a control

measure because it confirms the toxoplasmosis infection in the herd and can be

38

implemented to reduce the impact of infection and protect the economic viability of the

livestock (Dubey, 2010).

Freezing meat in a domestic freezer for at least one night before consumption by animals

and/or humans seems to be an easy and economical method for reducing the chances of

transmission of T. gondii (Dubey, 2008a). Disinfectants can be used to destroy T. gondii,

however, there are few options, including ethanol and acetic acid (concentration 95%/5%

for 24 hr) and ammonium hydroxide (5% for 30 min). Another way to destroy the oocysts

is by high pressure processing (Lindsay et al., 2008). In addition, education and public

health programs are fundamental to disease control (Foulon, 1992). Control measures and

prevention are essential for the control of toxoplasmosis in humans and animals, thus

avoiding unnecessary losses and outbreaks caused by lack of sanitary management for

animals.

The most effective method of preventing T. gondii infection in sheep is to vaccinate them

against the disease. Like natural infection, vaccination produces a solid immunity and

therefore sheep can be given long-lasting protection by the use of a single injection. The

vaccine (ToxovaxTM), which is licensed for use in certain countries in Europe including

the UK, is marketed by Intervet (Rodger and Buxton, 2006).

It was developed in New Zealand and work was later carried out at Moredun to establish

its efficacy and safety in sheep. ToxovaxTM is one of only two parasitological vaccines

in the world. Replacement ewe lambs can be vaccinated from 5 months of age and non-

pregnant, healthy ewes may be vaccinated at any time, apart from the 3 week period

before tupping (do NOT vaccinate pregnant sheep). ToxovaxTM is a live vaccine which

is relatively fragile, needs to be handled with care and should not be administered by

susceptible people (pregnant women or immunocompromised people (Rodger and

Buxton, 2006).

Research has shown that a significant reduction in lamb losses due to toxoplasmosis can

be achieved by feeding the coccidiostat decoquinate (Deccox - Alpharma Ltd) during

39

pregnancy. It should be added to the feed to provide 2 mg/ kg body weight/day from mid-

pregnancy (Rodger and Buxton, 2006). Decoquinate is most effective if it is already

being fed to susceptible ewes at the time they encounter infection rather than after

infection is established. It is not suited to management systems in which supplementary

feed is not given (Rodger and Buxton, 2006).

2.2.4. Border Disease Virus

Background

Border disease is a congenital viral disease of sheep and goats and was first reported in

1959 in the Border region of Wales and England. The disease is characterised by barren

ewes, abortion, stillbirth and the birth of small, weak lambs showing tremor, abnormal

body conformation and hairy fleeces (Nettleton et al., 1998). Monies et al. (2004)

documented in lambs persistently infected with Border disease virus (BDV) an enteric

disease characterised by diarrhoea and illthrift.

Serological investigations have shown a worldwide distribution of Border disease virus.

Seroprevalence rates vary in sheep from 5 to 50% depending on country or region in-

vestigated (Nettleton et al., 1998). Serological investigations in Austria have shown a

mean flock prevalence of 62.9% and a mean individual prevalence of 29.4% with marked

regional differences (Krametter-Froetscher et al., 2007a). Clinical cases of the disease

have been reported from several European countries (Schaarschmidt et al., 2000; Braun

et al., 2002; Monies et al., 2004). Although in Austria typical clinical Border disease has

to date not been reported, Krametter-Froetscher et al. (2007b) described the first cases of

sheep persistently infected with Border disease virus. These sheep have been identified

during an epidemiological study car ried out in the alpine region of Austria and they were

clinically healthy. Krametter-Froetscher et al. (2008) were able to prove seroconversion

of susceptible calves after contact to persistently Border disease virus infected sheep.

Etiology

40

Border disease is caused by infection of the fetus in early pregnancy with a pestivirus

(Flaviviridae) closely related to the viruses of classical swine fever and bovine viral

diarrhea/mucosal disease. Surviving lambs are persistently viremic, and the virus is

present in their excretions and secretions, including semen. Cattle, goats, and pigs are

also susceptible to infection with border disease virus. Persistently infected individuals

have been demonstrated in all of the aforementioned species from natural in-utero

infection. Transmission of border disease virus to cattle can occur from commingled

grazing with persistent or acutely infected sheep. Acute infections in immunocompetent

animals are usually transient and subclinical and result in immunity to challenge with

homologous but not heterologous strains of virus.

http://www.merckvetmanual.com/mvm/generalized_conditions/congenital_and_inherited

_anomalies/border_disease.html

Epidemiology

In naive flocks exposed to bovine viral diarrhea virus (BVDV), up to 50% or more of

lambs born may be affected with border disease. Thereafter, the prevalence declines,

although the disease may become endemic when “recovered” lambs are retained for

breeding. The virus is most commonly introduced into susceptible flocks by the addition

of persistently infected sheep or pregnant ewes carrying an infected fetus. However,

sheep can also acquire infection from transiently or persistently infected cattle. For

practical purposes, it should be assumed that sheep and cattle are equally susceptible to

all strains of border disease virus and bovine viral diarrhea virus, even though at least

four phylogenetic groups of pestiviruses have been identified in domestic ruminants.

http://www.merckvetmanual.com/mvm/generalized_conditions/congenital_and_inherited

_anomalies/border_disease.html

Clinical sign

Affected flocks probably are recognized first at lambing time by an increase in the

number of barren ewes and in the birth of undersized lambs with excessively hairy and

sometimes excessively pigmented fleece. Skeletal abnormalities that may be seen in

newborn lambs include a decreased crown-rump length, shortened tibia and radius, and a

41

shortened longitudinal axis of the cranium. Some lambs exhibit involuntary muscular

tremors, particularly of the trunk and hindlegs. The tremors are reduced at rest and

exacerbated by purposeful movement. In others, skeletal defects such as dropped pasterns

and mandibular brachygnathia may predominate. Affected lambs have a poor survival

rate. In survivors, nervous signs gradually disappear within 3–4 months. Even in the

absence of typical hairy-shaker lambs, outbreaks of low fertility in ewes and poor

viability and ill-thrift in lambs may be associated with border disease virus infection. In

severe cases, abnormal development of the cerebrum may be seen at necropsy, resulting

in hydrocephalus, hydranencephaly, porencephaly, or microcephaly. Cerebellar

hypoplasia or cerebellar dysplasia may also occur. Otherwise, the characteristic lesions

are microscopic and involve the white matter of the CNS. There is a deficiency of myelin

and an increase in interfascicular glial cells, in which myelin-like lipid droplets may

accumulate. These changes are most obvious in the newborn and gradually resolve.

http://www.merckvetmanual.com/mvm/generalized_conditions/congenital_and_inherited

_anomalies/border_disease.html

Diagnosis

Identification of the agent (the prescribed test for international trade)

There is no designated OIE reference laboratory for BDV, but the reference laboratories

for BVDV or CSFV will be able to provide advice. One of the most sensitive proven

methods for identifying BDV remains virus isolation. Direct immunofluorescence or

other immunohistochemical techniques on frozen tissue sections as well as antigen-

detecting ELISA and conventional and real-time RT-PCR are also valuable methods for

identifying BDV-infected animals (OIE Terrestrial Manual, 2008).

I. Virus isolation

It is essential that laboratories undertaking virus isolation have a guaranteed supply of

pestivirus-free susceptible cells and fetal bovine serum (FBS), or equivalent, that contain

no anti-pestivirus activity and no contaminating virus. It is important that a laboratory

quality assurance programme be in place. The virus can be isolated in a number of

primary or secondary ovine cell cultures (e.g. kidney, testes, lung). Ovine cell lines for

42

BDV growth are rare. Semicontinuous cell lines derived from fetal lamb muscle (FLM),

whole embryo (Thabti et al., 2002) or sheep choroid plexus can be useful, but different

lines vary considerably in their susceptibility to the virus. Ovine cells have been used

successfully for the isolation and growth of BD viruses and BVDV types 1 and 2 from

sheep. In regions where sheep may become infected with BVD viruses from cattle, a

virus isolation system using both ovine and bovine cells could be optimal. Several bovine

cell cultures may be suggested, including testicular, embryonic tracheal or turbinate cells,

or a susceptible continuous kidney cell line. However, bovine cells are insensitive for the

primary isolation and growth of some BD viruses, so reliance on bovine cells alone is

inadvisable.

From live animals, serum can be tested for the presence of infectious virus, but the most

sensitive way to confirm pestivirus viraemia is to wash leukocytes repeatedly (at least

three times) in culture medium before co-cultivating them with susceptible cells for 5–7

days. Cells are frozen and thawed once and an aliquot passaged onto further susceptible

cells grown on cover-slips, chamber slides or plastic plates. The cells are stained, 3–

4days later, for the presence of pestivirus using an immunofluorescence or

immunoperoxidase test. Tissues should be collected from dead animals in virus transport

medium (10% [w/v]). In the laboratory, the tissues are ground, centrifuged to remove

debris, and the supernatant passed through 0.45 μm filters. Spleen, thyroid, thymus,

kidney, brain, lymph nodes and gut lesions are the best organs for virus isolation (OIE

Terrestrial Manual, 2008).

Semen can be examined for the presence of BDV, but raw semen is strongly cytotoxic

and must be diluted, usually at least 1/10 in culture medium. As the major threat of BDV-

infected semen is from PI rams, blood is a more reliable clinical sample than semen for

identifying such animals. There are many variations in virus isolation procedures. All

should be optimised for maximum sensitivity using a standard reference virus preparation

and, whenever possible, recent BDV field isolates (OIE Terrestrial Manual, 2008).

Immunohistochemistry

43

Viral antigen demonstration is possible in most of the tissues of PI animals (Braun et al.,

2002). This should be done on acetone-fixed frozen tissue sections (cryostat sections) or

paraffin wax embedded samples using appropriate antibodies. Panpestivirus-specific

antibodies with NS2-3 specificity are suitable. Tissues with a high amount of viral

antigen are brain, thyroid gland and oral mucosa. Skin biopsies have been shown to be

useful for invivo diagnosis of persistent BDV infection.

Enzyme-linked immunosorbent assay for antigen detection

The first ELISA for pestivirus antigen detection was described for detecting viraemic

sheep. This has now been modified into a double MAb capture ELISA for use in sheep

and cattle. Two capture MAbs are bound to wells in microtitre plates, and two other

MAbs, conjugated to peroxidase, serve as detector MAbs (Entrican et al., 1994). The test

is most commonly employed to identify PI viraemic sheep using washed, detergent-lysed

blood leukocytes. The sensitivity is close to that of virus isolation and it is a practical

method for screening high numbers of blood samples. As with virus isolation, high levels

of colostral antibody can mask persistent viraemia. The ELISA is more effective than

virus isolation in the presence of antibody, but may give falsenegative results in viraemic

lambs younger than 2 months old. The ELISA is usually not sensitive enough to detect

acute BDV infections on blood samples. As well as for testing leukocytes, the antigen

ELISA can also be used on tissue suspensions, especially spleen, from suspected PI sheep

and, as an alternative to immunofluorescence and immunoperoxidase methods, on cell

cultures. Several pestivirus ELISA methods have been published and commercial kits are

now available for detecting BDV. ELISAs employing MAbs recognising epitopes on the

conserved non-structural NS2-3 should recognise all strains of BDV. ELISAs relying on

MAbs recognising epitopes on structural proteins, such as Erns, that are used for BVDV

detection in cattle are unsuitable for the diagnosis of BDV viraemia in sheep.

II. Serological tests

Antibody to BDV is usually detected in sheep sera using VN or an ELISA. The less

sensitive agar gel immunodiffusion (AGID) test may also be used. Control positive and

negative reference sera must be included in every test. These should give results within

44

predetermined limits for the test to be considered valid. Single sera can be tested to

determine the prevalence of BDV in a flock, region or country. For diagnosis, however,

acute and convalescent sera are the best samples for confirming acute BDV infection.

Repeat sera from one animal should always be tested alongside each other on the same

plate (OIE Terrestrial Manual, 2008).

III. Enzyme-linked immunosorbent assay

An MAb-capture ELISA for measuring BDV antibodies has been described. Two

panpestivirus MAbs that detect different epitopes on the immunodominant nonstructural

protein NS 2/3 are used to capture detergent-lysed cell-culture grown antigen. The results

correlate qualitatively with the VN test (Fenton et al., 1991).

Antigen is prepared as follows: Use eight 225 cm2 flasks of newly confluent FLM cells;

four flasks will be controls and four will be infected. Wash the flasks and infect four with

a 0.01–0.1 m.o.i. (multiplicity of infection) of Moredun cytopathic BDV. Allow the virus

to adsorb for 2 hours at 37°C. Add maintenance media containing 2% FBS (free from

BDV antibody), and incubate cultures for 4–5 days until CPE is obvious. Pool four

control flask supernatants and separately pool four infected flask supernatants. Centrifuge

at 3000 g for 15 minutes to pellet cells. Discard the supernatants. Retain the cell pellets.

Wash the flasks with 50 ml of PBS and repeat the centrifugation step as above. Pool all

the control cell pellets in 8 ml PBS containing 1% Nonidet P40 and return 2 ml to each

control flask to lyse the remaining attached cells. Repeat for infected cells. Keep the

flasks at 4°C for at least 2 hours agitating the small volume of fluid on the cells

vigorously every 30 minutes to ensure total cell detachment. Centrifuge the control and

infected antigen at 12,000 g for 5 minutes to remove the cell debris. Supernatant antigens

are stored at –70°C in small aliquots (Fenton et al., 1991).

IV. Agar gel immunodiffusion test

The AGID test was first used to demonstrate an immunological relationship between BD,

BVD and CSF viruses. The Oregon C24V strain of BVDV grown on calf testis cells has

been used to detect antibody in sheep. Suitable antigen can be prepared using medium

45

harvested from cells showing early CPE. Concentration of the medium approximately

100-fold by dialysis against polyethylene glycol (PEG) is required. Alternatively, PEG

6000 can be added to sonicated virus/cell suspensions at the rate of 8% (w/v). After

constant stirring overnight at 4°C, the precipitate is removed by centrifugation at 1800 g

for 1 hour. The supernatant is decanted thoroughly and the precipitate resuspended to 1%

of the original virus/cell culture volume in distilled water. The resuspended precipitate is

centrifuged at 286,000 g for 2 hours and the supernatant withdrawn for use as antigen.

The precipitate is discarded (OIE Terrestrial Manual, 2008).

Treatment

There is no effective treatment for persistently infected lambs.

http://www.merckvetmanual.com/mvm/generalized_conditions/congenital_and_inherited

_anomalies/border_disease.html

Prevention and Control

Bulk tank milk samples can be tested for antibodies to BVDV to screen for the presence

of virus within dairy sheep flocks. Serology should be performed on the dams of affected

lambs. Most should have high levels of antibody and be immune to further challenge with

the same strain of virus in subsequent pregnancies. Those that do not have antibody titers

should be screened for virus to identify any that are persistently infected. Recovered

lambs should not be retained for breeding but can be mixed with replacement stock well

before breeding season to maximize opportunities for the latter to become infected and

develop immunity before subsequent matings. There is no effective vaccine. BVDV

vaccines for cattle cannot be recommended for use in sheep, because border disease

viruses most commonly isolated from sheep are antigenically distinct from BVDV most

common in cattle.

http://www.merckvetmanual.com/mvm/generalized_conditions/congenital_and_inherited

_anomalies/border_disease.html

46

2.2.5. Brucella

Background

Brucellosis is a zoonotic infection caused by the bacterial genus Brucella. The bacteria

are transmitted from animals to humans by ingestion through infected food products,

direct contact with an infected animal, or inhalation of aerosols. The disease is an old one

that has been known by various names, including Mediterranean fever, Malta fever,

gastric remittent fever, and undulant fever. Humans are accidental hosts, but brucellosis

continues to be a major public health concern worldwide and is the most common

zoonotic infection (Pappas et al., 2006).

Etiology

Brucellosis results from infection by various species of brucella, a gram negative,

facultative intercellular rod in the family Brucellanceae (Seifert, 1996). Six species occur

in humans and animals: Brucella bortus, B. melitensis, B. suis, B. ovis, B. canis and B.

nentomae. Brueclla abortus usually causes brucellosis in cattle, bison and buffalo.

Brucella melitensis is the most important species in sheep and goats, and B. suis in pigs.

B. ovis can cause infertility in rams. B. neotomae is found in American wood rats. In

humans, brucellosis can be caused by B. abortus, B. melitensis, B. suis and rarely by B.

canis. Seven biovars have been identified for B. abortus, three for B. melitensis and five

for B. suis. Brucella melitensis (biovars 1, 2 or 3) is the main causative agent of caprine

and ovine brucellosis. Sporadic cases caused by B. abortus have been observed, but

clinical disease is rare (OIE, 2000). Brucella ovis affects only sheep especially rams

causing epididymitis. In rams, the disease is characterized by epididymitis, orchitis and

impaired fertility (Nicoletti, 1998: Spickler, 2003).

Epidemiology

The factors influencing the epidemiology of Brucellosis infection in any geographic

location can be classified in to factors associated with the transmission of the disease

between herds and factors influencing the maintenance and spread of infection with in the

herd. Brucellosis caused by B. melitensis occurs in sheep and goat raising regions of the

47

world with exception of North America, Australia and Newzealand. Factors associated

with brucellosis include host factor (age, sex and breed), agent and extrinsic factors

(environmental factors) including management and ecology (Walker, 1999).

Brucellosis is found worldwide but is well controlled in most developed countries.

Clinical disease is still common in Africa, the Middle East, central and South East Asia,

South America and some Mediterranean countries (OIE, 2000). Brucella species vary in

their geographic distribution. B. melitensis is particularly common in Latin America,

central Asia, the Mediterranean, and around the Arabian Gulf. This species does not seem

to occur in northern Europe, south East Asia, Australia or New Zealand. It is rare in the

United States. B. ovis is seen in Australia, New Zealand and many other sheep raising

regions, including the United States (FAO, 2002).

In Ethiopia prevalence rate of brucellosis in sheep and goats in central highlands is

reported to be 1.5% in sheep and 1.3% in goats and prevalence of 4.8% from Afar and

9.7% from Somali region was reported (Kumat, 1997; Ashenafi et al., 2007). The disease

has also been reported from Addis Ababa, Debreberhan and Abomssa abattoirs using rose

Bengal plate test (Teshale et al., 2007).

Clinical signs

The primary clinical manifestations of brucellosis are related to the reproductive tract.

The biggest problem of brucella infection is the uncertain incubation period, which may

vary between 15 days to months and years depending on invasion site, infective dose and

other factors (Weidmann, 1991; FAO, 2002). Clinically, the disease is characterized by

one or more of the following signs: abortions, retained fetal membranes, orchitis,

epididymitis and rarely, arthritis with excretion of the organisms in uterine discharges

and in milk (OIE, 2000). Sheep and goats with brucellosis often abort during the final

trimester. Goat flocks may have an abortion storm after the disease is contracted. The

abortion problem appears to be more in goats than in sheep. In goats and rarely in sheep,

a systemic disease with fever, depression, weight loss, diarrhea, mastitis, lameness,

hygroma and orchitis in males’ can occur (Pugh, 2002). Goats that have aborted once are

48

not likely to abort for a second time (Nicoletti, 1998; FAO, 2002). Retention of fetal

membrane may or may not occur. Goats shed brucella in milk for years, but sheep may

shed during one or more location period (FAO, 2002). In non-pregnant sheep and goats,

infection is localized on the udder and supramammary lymphnode (Seifert, 1996). B. ovis

causes a genital infection of ovine livestock manifested by epididymitis, infrequent

abortions and increased lamb mortality (OIE, 2000).

B. ovis can cause epididymitis, orchitis and impaired fertility in rams. Initially, only poor

quality semen may be seen; sperm motility and concentration may be decreased, and

individual sperm are often abnormal. Later, palpable lesions may occur in the epididymis

and scrotum. Epididymitis may be unilateral or, occasionally, bilateral. The testes may

atrophy. Palpable lesions are often permanent, although they are transient in a few cases.

Some rams shed B. ovis for long periods without clinically apparent lesions. B. ovis can

also cause abortions and placentitis in ewes, but this appears to be uncommon. Infected

ewes may give birth to weak lambs that die soon after birth. Systemic signs are rare in

adult ewes and rams. http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_ovis.pdf

Transmission

In most circumstances, the primary route of transmission of brucella is the placenta, fetal

fluids and vaginal discharges expelled by infected ewes when they abort or have a full-

term parturition and ingestion of contaminated feed and water during overcrowding as

well as contact through intact skin and conjunctiva (FAO, 2002: OIE, 2000). Lambs may

be infected while in the uterus or by sucking infected milk of their mother (Nicoletti,

1998; FAO, 2002).

As goats ingest contaminated feed and water, the organisms enter through mucus

membranes and localize themselves in the udder, uterus, testes, spleen and lymph nodes.

In sheep, the organisms appear to be transmitted orally from ram to ram or ram to ewe,

but not from ewe to ewe (Pugh, 2002). Transmission between rams occurs via passive

venereal infection and by direct ram to ram transfer during the breading season

(Radostitis et al., 2007). Direct ram to ram transmission during non breeding periods is

49

quite frequent and has been suggested to take place by several routes, including the rectal

mucosa. Infected ewes may excrete B. ovis in vaginal discharges and milk and

accordingly, ewe-to-ram and lactating ewe-to-lamb transmission could also be a

determinant mechanism of infection (OIE, 2000). The organism can survive on pasture

for several months but transmission by fomites is believed to have no practical

significance (Radostitis et al., 2007).

B. ovis is often transmitted from ram to ram by passive venereal transmission via ewes.

Ewes can carry this organism in the vagina for at least two months and act as mechanical

vectors. Some ewes become infected, and shed B. ovis in vaginal discharges and milk.

Rams often become persistently infected, and many of these animals shed B. ovis

intermittently in the semen for 2 to 4years or longer. B. ovis can also be transmitted by

direct non-venereal contact between rams. Ram-to-ram transmission is poorly understood

and may occur by a variety of routes, including oral transmission. Shedding has been

demonstrated in the urine as well as in semen and genital secretions.

http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_ovis.pdf

Diagnosis

Diagnosis of brucellosis is made possible by direct demonstration of the causal organism

using staining, immune florescent antibody, culture, animal inoculation and polymerase

chain reaction (PCR) and indirectly by demonstration of antibodies using serological

techniques (Alton, 1975; Corbel, 1997; Quinn et al., 1999). However, achievement of a

reliable diagnosis of brucellosis is a tedious process, since isolation is affected by a

number of factors, such as high fastidious nature of brucella, presence of a lesser number

of viable organisms in the sample and delay in sample submission (leading to

putrefaction). In addition, a prolonged incubation period may lead to failure in its

isolation (konrad, 1981; Verma et al., 2000). Thus in situations where bacteriological

examination is not practicable, diagnosis of brucella infection must often be based on

serological methods (OIE, 2000).

Clinical examination

50

B. ovis infections should be considered when rams develop epididymitis and testicular

atrophy, or poor semen quality is seen. Some but not all rams have palpable lesions.

http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_ovis.pdf

Laboratory tests

Microscopic examination of semen or smears stained with the Stamp's modification of

the Ziehl-Neelsen method can be useful for a presumptive diagnosis. Brucella species are

not truly acid-fast, but they are resistant to decolorization by weak acids, and stain red

against a blue background. Brucellae are coccobacilli or short rods, usually arranged

singly but sometimes in pairs or small groups. This test is not definitive. Other organisms

such as Chlamydophila abortus and Coxiella burnetii can resemble Brucella in this test.

Brucella melitensis can also be confused with B. ovis. Immunostaining is sometimes used

to identify B. ovis in semen.

http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_ovis.pdf

In routine tests, anti brucella antibodies are detected in serum. The most widely used

serum-testing producers for the diagnosis of brucella infections in sheep and goats are the

buffered brucella antigen tests (BBAT), i.e. the card and Rose Bengal (RB) plate

agglutination tests which are essentially the same, and the compliment fixation test

(CFT). The bulk milk ring test, which has been very useful in cattle, is ineffective in

small ruminants. In small ruminants, the Rose Bengal plate Test (RBPT) and the

complement fixation test (CFT) are the most widely used methods and are the only

prescribed tests (FAO, 1986; OIE, 2000).

Microscopic examination and culture methods

Specimen of fetal stomach, lung, liver, placenta, cotyledon and vaginal discharges are

stained with Gram stain and modified Ziehl Neelsen stains. Brucella appears as small red-

colored, coccobacili in clumps. Blood or bone marrow samples can be taken cultured in

5-10% blood agar is used. To check up bacterial and fungal contamination; Brucella

selective media are often used. The selective media are nutritive media, blood agar based

with 5% sero-negative equine or bovine serum. On primary isolation it usually requires

51

the addition of 5-10% carbon dioxide and takes 3-5 days incubation at 37oC for visible

colonies to appear (Quinn et al., 2002).

Rose Bengal Plate Test (RBPT)

It is a spot agglutination technique. It does need special laboratory facilities and is simple

and easy to perform. It used to screen sera for Brucella antibodies. The test detects

specific antibodies of the IgM and IgG type. Although the low PH (3.6) of the antigen

enhances the specificity of the test and temperature of the antigen and the ambient

temperature at which the reaction takes place may influence the sensitivity and specificity

of the RBPT test (WHO, 1997; Nielson et al., 2001).

Complement fixation test (CFT)

This test detects specific antibodies of the IgM and IgG1 type that fixe complement. The

CFT is highly specific but it requires highly trained personnel as well as suitable

laboratory facilities. It measures more antibodies of the IgG1 type than antibodies of the

IgM type (Georgios et al., 2005).

ELISA tests

The ELISA tests offer excellent sensitivity and specificity whilst being robust, fairly

simple to perform with a minimum of equipment and readily available from a number of

commercial sources in kit form. They are more suitable than the CFT for use in smaller

laboratories and ELISA technology is now used for diagnosis of a wide range of animal

and human diseases. Although in principle ELISAs can be used for the tests of serum

from all species of animal and man, results may vary between laboratories depending on

the exact methodology used. Not all standardization issues have yet been fully addressed.

For screening, the test is generally carried out at a single dilution. It should be noted,

however, that although the ELISAs are more sensitive than the RBPT, sometimes they do

not detect infected animals which are RBPT positive. It is also important to note that

ELISAs are only marginally more specific than RBPT or CFT (WHO, 1997). There are

also other serological diagnostic tests that use for diagnosis of brucellosis such as SAT,

PCR, and so on (Georgios et al., 2005).

52

Treatment

In animal the treatment of animal brucellosis has not been fully successful because of the

intra cellular localization of brucellosis with in phagocytic cells of the reticuloendothelial

system, in lymph nodes, liver, spleen, mammary glands, and reproductive organs.

Therefore, brucellosis is protected from antibodies, complement and antibiotics (OIE,

1992). Thus treatment of infected sheep and goat with antibiotics is not recommended

because of the high treatment failure rate, cost, potential risk of maintaining infected

animals, and antibiotic residues in cheese production (Walker 1999; FAO, 2002).

Tetracycline and dihydrostreptomycine have been used to treat B. ovis infection in rams

with variable results. Once palpable epididymal lesion is present treatment is not

beneficial (Walker, 1999).

In humans Doxycycline (six weeks) plus streptomycin (two or three weeks) regimen is

more effective regimen than doxycycline plus rifampicin (six weeks) regimen. Since it

needs daily intramuscular (IM injection, access to care and cost are important factors in

deciding between two choices. Quinolone plus rifampicin (six weeks) regimen is slightly

better tolerated than doxycycline plus rifampicin, and low quality evidence did not show

any difference in overall effectiveness.

http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0049388/

Brucellosis can be difficult to treat in humans. Antibiotics commonly used to treat

brucellosis include: tetracycline, streptomycin, doxycycline and rifampin. The treatment

can include more than one kind of antibiotic and can be taken for many weeks to prevent

the disease from returning. Recovery can take weeks, even months. Patients who receive

treatment within one month of the start of symptoms can be cured of the disease.

http://www.webmd.com/a-to-z-guides/brucellosis-symptoms-treatment?page=3

Prevention and control

The control and prevention measure to be adopted should be realistically based on

through understanding of local and regional variations in animal husbandry practices,

social customs, infrastructure and epidemiological patterns of the disease (Mustofa and

53

Nicoletti, 1993). Control and eradication of brucellosis can be achieved through reduction

of the disease to the lowest possible level by vaccination and application of test and

slaughter policy (FAO/WHO, 1986). In countries with a low prevalence of infection

slaughter of the entire flock is generally the control measure of choice. In small

commercial flocks, culling of infected rams and replacement with B. ovis free rams may

be the most economical approach (Pugh, 2002). In many countries in which caprine and

ovine brucellosis in prevalent, the disease in controlled by an intensive vaccination

program that is very effective. The killed vaccine occasionally used in sheep (B. ovis)

appears to have poor efficacy. Attenuated strain of B. melitensis can be given

subcutaneously in kids and lambs 3 to 8 month of age. This vaccine may cause abortion

in pregnant animals (Pugh, 2002; Radostits et al., 2007).

2.3.Respiratory Disease

2.3.1. Pasteurella

Background

Pasteurella and Mannheimia organisms are β-hemolytic, gram-negative, aerobic, non-

motile, non-spore forming coccobacilli in the family Pasteurellaceae. This family tends to

inhabit the mucosal surfaces of the GI, respiratory, and genital tract of mammals. Many

are known as opportunistic secondary invaders. Some species show preferences for

specific surfaces and hosts. Updating of phylogenetic data has resulted in renaming based

on gene sequence analysis. As a result, P. haemolytica biotypes A and T were reclassified

as M. haemolytica (biotype A) and P. trehalosi (biotype T).

http://www.merckvetmanual.com/mvm/generalized_conditions/pasteurellosis_of_sheep_

and_goats/overview_of_pasteurellosis_of_sheep_and_goats.html

More recently, P. trehalosi has been reclassified as Bibersteinia trehalosi. Each isolate

of M. haemolytica and B. trehalosi is designated with a biotype and serotype. M.

haemolytica A2 is the most common strain isolated from sheep and goat respiratory

pasteurellosis, although A6 and A13 have been reported in sheep. M. haemolytica A2 is

54

routinely reported from cases of mastitis in sheep. B. trehalosi T3, T4, T10, and T15 have

been most often associated with the systemic or septicemic form of pasteurellosis

affecting lambs. These serotypes have been regrouped to B. trehalosi biotype 2, and a

new biotype 4 has been added. B. trehalosi is often isolated from the lungs of sheep,

goats, and cattle, but pathogenicity is variable and may be incidental. P. multocida has

also been reported as a cause of pneumonic pasteurellosis in sheep and goats and has

been isolated in herd outbreaks of septic arthritis. M. haemolytica is the most commonly

isolated bacteria in clinical cases, followed closely by B. trehalosi, with P.

multocida seen less frequently.

http://www.merckvetmanual.com/mvm/generalized_conditions/pasteurellosis_of_sheep_

and_goats/overview_of_pasteurellosis_of_sheep_and_goats.html

Etiology

P. haemolytica (currently M. haemolytica), P. multocida and P. trehalosi (currently

Bibersteinia trehalosi) are the three bacteria most commonly cause pasteurellosis in small

ruminants. The disease pneumonic pasteurellosis, observed in sheep and goats is

commonly caused by P. haemolytica and rarely caused by P. multocida. P. haemolytica

was first identified and recognized in 1932 (De Alwis, 1992), while P. multocida was

first discovered by Perroncito in 1878 and named after Louis Pasteur who first isolated

and described this Gram-negative bacterium as the cause of fowl disease in 1880 and

subdivided into four subspecies that include Multocida, Gallicida, Septica and recently

described Tigris (Sherrill, 2012).

Epidemiology

Pneumonic pasteurellosis is one of the most economically important infectious diseases

of small ruminants with a high prevalence occurs throughout the world (Prabhakar et al.,

2012). It was first described in Iceland and subsequently has been reported in many

countries such as Australia, USA, Britain, Norway, South Africa, Somalia and Ethiopia

(Habashy et al., 2009). M. haemolytica, B. trehalosi, and P. multocida are common

commensal organisms of the upper respiratory tract of apparently healthy sheep and

goats. They are distributed worldwide, and diseases caused by them are common in all

55

ages, although the prevalence of serotypes may vary by region and flock (Shayegh et al.,

2009; Sherrill, 2012).

Clinical Signs

B. trehalosi mainly causes septicemia and systemic pasteurellosis in sheep <2 month old.

The systemic form of pasteurellosis caused by B. trehalosi is characterized by fever,

listlessness, poor appetite, and sudden death in young sheep. The organism is thought to

move from the tonsils to the lungs and pass into the blood. This results in septicemia and

localization of the infection in one or more tissues such as the joints, udder, meninges, or

lungs. P. multocida has been reported to be isolated from polyarthritis in young lambs. M.

haemolytica has been reported from cases of mastitis, especially in sheep. All of these

bacteria can cause severe fibrinonecrotic pneumonia in sheep and goats. The disease is

characterized by acute onset of illness, very high fevers, dyspnea, anorexia, and often

death.

http://www.merckvetmanual.com/mvm/generalized_conditions/pasteurellosis_of_sheep_

and_goats/overview_of_pasteurellosis_of_sheep_and_goats.html

Transmission

The transmission of the disease is by direct contact and aerosol from the diseased to

healthy animals. Most of these infectious organisms are spread by direct contact with

body fluids (such as saliva, nasal discharge), contaminated feeders, troughs, and

equipment (Brogden et al., 1998). The worst epidemics occur during the rainy season, in

animals in poor physical condition, stress thought to increase susceptibility to infection,

and closely kept flock and wet conditions seem to contribute to the spread of the disease.

Infection of humans is generally associated with some form of animal contact, most

commonly a dog and cat bite or scratch (Gerardo et al., 2001).

Diagnosis

History of earlier outbreaks, a recent failure to vaccinate, clinical signs and the

remarkable lesions like dark red/purple areas and firm to the touch are evident mainly in

the anterior and cardiac lobes of the lung may be suggestive diagnosis. Giemsa/Gram

56

stained blood smear, bacteriological identification (Culture and biochemical tests),

molecular (such as PCR) and serological (Such as ELISA and IHA), Histopathological

and Immuno-histochemical (IHC) diagnostic methods are confirmatory diagnosis of the

disease which have been indicated (Bell, 2008; Kopcha, 2012; Zafer et al. 2013).

Identification Methods of Serotypes

I. Serological Methods

According to Sridhar (2006), serotyping is based on fact that strains of same species can

differ in the antigenic determinants expressed on the cell surface. Surface structures such

as lipopolysaccaride, membrane proteins, capsular polysaccharides, flagella and fimbriae

exhibit antigenic variations. Strains differentiated by antigenic differences are known as

'serotypes'. Serotyping is used in several gram negative and gram positive bacteria. The

advantage of serotyping is most strains are typeable, they have good reproducibility and

ease of interpretation though some have ease of performance, while the disadvantage is

some auto-agglutinable (rough) strains are untypeable, some methods of serotyping are

technically demanding, there is dependency on good quality reagent from commercial

sources, in-house preparation of reagents is difficult process, serotyping has poor

discriminatory power due to large number of serotypes, cross-reaction of antigens and

untypeable nature of some strains.

Serotyping is performed using several serologic tests such as coagulation test (CA),

indirect haemagglutination test (IHA) and enzyme linked immune-sorbent assay

(ELISA). CA and indirect haemagglutination test are accessible tests at any diagnostic

laboratory, and they have been proven to be reliable and suitable for serotyping clinical

isolates of a variety of gram-positive and gram-negative organisms (Del Río et al., 2003).

IHA have been employed successfully for the identification of serological types of M.

haemolytica and P. multocida and it is most common method (Sawada, et al., 1982).

ELISA is easy to perform, cost-effective without particular equipment, high sensitivity

and specificity (Peterson et al., 1997).

57

II. Molecular Methods

Polymerase Chain Reaction (PCR)

There are common PCR types used in Pasteurella serotyping such as conventional PCR,

multiplex PCR and Real time PCR (Terry et al., 1998; Ranjan et al., 2011). A multiplex

PCR assay is a rapid alternative to the conventional capsular serotyping system and used

for capsular types determination and it is highly specific and its result correlated well

with conventional. But real time PCR is highly sensitive than this type (Ranjan et al.,

2011). The advantages of the PCR compared with other tests include better speed,

sensitivity, specificity and simplicity. It does not require culture or laboratory animals

and is, therefore, safer as a result of the avoidance of handling live bacteria (Gautam et

al., 2004).

Filed alternation gel electrophoresis (FAGE)

This technique is also known as ‘pulsed field gel electrophoresis’ and it is a method of

fingerprinting with high specificity and precision. The major drawbacks of this technique

are the requirements of highly purified intact DNA and specialized and expensive

electrophoresis equipment, which is generally not available in normal diagnostic

laboratories (Ranjan et al., 2011).

Treatment

Early identification of respiratory disease and introduction of effective antibiotic therapy

is necessary. Death losses are high in severely affected animals. Antimicrobial

susceptibility patterns of M. haemolytica, B. trehalosi, and P. multicoda have shown

resistance to penicillins (all three organisms), sulfadimethoxine (P. multocida), and

tetracyclines (B. trehalosi). Ampicillin, ceftiofur, danofloxacin, enrofloxacin, florfenicol,

trimethoprim-sulfamethoxazole, and tulathromycin would be expected to have good

efficacy. Treatment is frequently unrewarding unless begun very early in the disease

process because of rapid progression of lung damage and endotoxin release. Parenteral

fluids and anti-inflammatory agents are important adjuncts to antibiotic therapy.

Administering prophylactic antibiotics to at-risk lambs may be beneficial.

58

http://www.merckvetmanual.com/mvm/generalized_conditions/pasteurellosis_of_sheep_

and_goats/overview_of_pasteurellosis_of_sheep_and_goats.html

Prevention and Control

Principles for prevention of disease and the utilization of preventive measures faster

optimal health and welfare, enhance productivity and economic efficiency, and assure

abundant, safe, and wholesome food. Control and prevention lies with correction of the

predisposing factors whenever practical (Kopcha, 2012). Treatment of pasteurellosis

using antibiotics can be very effective in control of the disease. Whenever possible,

treatment should be based on bacterial culture and sensitivity, especially in flock

outbreaks, when valuable animals are involved, or in acute or chronic cases when initial

therapeutic attempts have failed (Sherrill, 2012).

Chloramphenicol, sulfamethoxazole and tetracycline were effective drugs whereas

gentamycin and vancomycin were totally inactive against the isolates of Mannheimia

haemolytica and Pasteurella multocida. Measures such as, improving management

practices by providing optimal sanitation and air quality in housing, minimizing

transportation stress, providing good quality hay and water, and supplement as

appropriate should be taken into account to reduce the prevalence (Maru et al., 2013).

2.3.2. PPR (Peste Des Petits Ruminants)

Background

Peste des Petits ruminants (PPR), also known as goat plague, is a highly contagious and

infectious viral disease affecting domestic and wild small ruminants (Furley et al., 1987).

Since its first description in Cote d’Ivoire in 1942, the disease is steadily progressed over

time throughout across Africa, the Middle East and Asia (Libeau et al., 2014). The

infection has long been considered as caused by a variant rinderpest virus, adapted to

small ruminants. The recognition of PPR virus as a novel member of the Morbillivirus

genus occurred only in the late 70s by using more sensitive laboratory techniques (Gibbs

et al., 1979).

59

PPR was clinically suspected for the first time in Ethiopia in 1977 in a goat herd in the

Afar region, east of the country (Roeder et al., 1994). Clinical and serological evidence of

its presence has been reported by (Taylor et al., 1984) and later confirmed in 1991 with

cDNA probe in lymph nodes and spleen specimens collected from an outbreak in a

holding near Addis Ababa (Roeder et al., 1994). During the nineties, several small

serological surveys were conducted, mainly east of an imaginary line that would run

parallel to the Rift valley and pass through Addis Ababa.

Ethiology

Peste des Petits Ruminants (PPR) is a severe and highly infectious viral disease of small

ruminants. The PPR virus (PPRV) belongs to the genus Morbillivirus in the family

Paramyxoviridae. It is closely related to the rinderpest virus of bovines and buffaloes,

distemper virus of dogs and other wild carnivores, human measles virus and

Morbilliviruses of marine mammals (Jones et al., 1993; Yayehrad, 1997).

Epidemiology

The disease is currently endemic in most of Africa, the Middle East, South Asia and

China (Felix, 2013). Nowadays the disease is recognized as responsible for mortality and

morbidity across most of the sub-Saharan African countries situated north of the equator,

in the Arabian Peninsula, in India and in numerous other countries in Asia (Diallo, 2003;

Ethiopia and molecular studies on virulence, 2005; Shaila et al., 2005). Although

nationwide sero-surveys have been conducted in countries such as the sultanate of Oman,

Turkey, Jordan and India, information on the frequency and distribution of PPR is often

lacking when control or eradication campaigns are initiated (Taylor et al., 1990; Lefevre

et al., 1991; Ozkul et al., 2002; Singh et al., 2004).

In 1996 Gelagay found that 14.6% of sheep sampled along 4 roads from Debre Berhan to

Addis Ababa were seropositive (Gelagay, 1996). In 1997 Yayerade found up to 100% of

seropositive individuals in groups of adult male sheep and animals that survived

suspected outbreaks. Although these studies provide very limited and potentially biased

information about the frequency and distribution of PPR in Ethiopia, they clearly suggest

60

that the virus has been circulating extensively among the small ruminant population of

Ethiopia during the nineties. Based on the reported morbidity and mortality of the

infection and the size and structure of the small ruminant sector it is likely that PPR

became one of the most economically important livestock diseases in the country

(Abraham et al., 2005; Gopilo, 2005).

Table 1: Prevalence of PPR in the seven surveyed regions in Ethiopia

Regions Number of samples collected in each

region and % of the whole survey

Prevalence with 95% CI

Afar 1653 (12.1%) 15.3% (13.6–17.0)

Amhara 5992 (43.9%) 4.6% (4.0–5.1)

Benishangul

Gumuz

729 (5.3%) 8.0% (6.0–9.9)

Oromia

2290 (16.8%) 1.7% (1.2–2.2)

SNNPR

1622 (11.9%) 1.8% (1.1–2.4)

Somali

465 (3.4%) 21.3% (17.6–25.0)

Tigray

900 (6.6%) 15.3% (13.6–15.9)

Total

13651 (100%) 6.4% (6.0–6.8)

Number of samples collected and prevalence of PPR in each of the surveyed regions. In

brackets: % of the whole survey they represent and 95% confidence intervals (Waret-

Szkuta et al., 2008).

61

Clinical Signs

In small ruminants, infection by PPRV is characterized by sudden depression, fever,

nasal and ocular discharge, diarrhoea and occasionally death (Diallo, 2003).

Most cases of PPR are acute, with a sudden fever that may last for 5-8 days before the

animal either dies or begins to recover. The characteristic signs begin with a clear

discharge from the nose that becomes grey and sticky. The discharge from the nose may

remain mild or may progress to severe inflammation of the mucous membrane of the

nose characterized by the presence of exudates that crust over, blocking the nostrils

causing respiratory distress. The nasal mucous membranes may develop small areas of

erosion. The conjunctiva may be congested with matted eyelids. The mucous membranes

in the mouth may also be eroded. Concurrently, animals will most likely have profuse,

non-hemorrhagic diarrhea resulting in severe dehydration, which may progress to

emaciation, difficult breathing and die within 5-10 days. Bronchopneumonia with

coughing is common late in the disease. Abortion may be seen in pregnant animals. The

prognosis of acute PPR is usually poor. The severity of the disease and outcome in the

individual is correlated with the extent of the mouth lesions. Prognosis is good in cases

where the lesions resolve within 2 to 3 days. It is poor when extensive necrosis and

secondary bacterial infections result in a fetid odor from the animal’s mouth. Respiratory

involvement is also a poor prognostic sign (Zewdie, 2009).

Transmission

Transmission of PPR requires close contact. The virus is present in eye, nose, and mouth

discharges as well as feces. Most infections occur through inhalation of aerosols from

sneezing and coughing animals. Animals may be infectious during the incubation period.

There is no known carrier state (Zewdie,

2009).http://www.esgpip.org/pdf/Technical%20Bulletin%20No.20.pdf

Diagnosis

Differential diagnosis include: rinderpest, contagious caprine pleuropneumonia,

bluetongue, Pasteurellosis, contagious ecthyma, foot and mouth disease, heartwater,

62

coccidiosis, Nairobi sheep disease and mineral poisoning. The case history, geographic

location and the combination of clinical signs can help differentiate some of these

diseases (Zewdie, 2009).

To confirm PPR outbreak some laboratory tests have to be carried out at the National

Animal Health Diagnostic & Investigation Center or at the nearby Animal Health

Regional Laboratory development agents should inform the nearby animal health

personnel about the outbreak so that appropriate samples should be taken. For PPR

diagnosis swabs of the mucous membrane of eye, nose, mouth and rectal discharges

should be collected. Whole blood must be collected in heparinized tubes. Samples may

also be taken of the spleen, large intestine and lungs. These samples should be

transported under refrigeration (Zewdie, 2009).

Treatment

There is no treatment for PPR but it helps to give antibiotics to stop secondary bacterial

infections (Zewdie, 2009).

Prevention and Control

Control of PPR in endemic areas relies mainly in vaccination (Diallo, 2004). In 1989 a

homologous vaccine that induces lifelong immunity in both sheep and goats was

developed (Diallo et al., 1989; Couacy-Hymann et al., 1995; Diallo, 2003).

Barns, tools and other items that have been in contact with the sick animals must be

cleansed and disinfected with common disinfectants (phenol, sodium hydroxide 2%,

virkon) as well as alcohol, ether, and detergents. The virus can survive for long periods of

time in chilled or frozen tissues. New animals should be quarantined for three weeks

before allowing them to mix with the flock. In a case of PPR outbreak, animals with signs

of PPR should be isolated immediately and sheep and goats around the outbreak area

should be vaccinated as soon as possible. Vaccine for PPR is effective. Vaccinate before

start of the rainy season. In endemic areas sheep and goats should be vaccinated annually.

63

Vaccine for PPR is produced by the National Veterinary Institute. Carcasses of dead

animals and contaminated items should be buried or burned (Zewdie, 2009).

64

3. MATERIALS AND METHODS

3.1.Description of the study area

The study was carried out in Bonga (Southern Nations, Nationalities and Peoples’

Regional State of Ethiopia), Horro (Oromia Regional State of Ethiopia), and Menz

(Amhara Regional State of Ethiopia) regions. Two villages were selected purposively

from each region and the regions were chosen for the reason that they are known as the

centre/origin for Bonga, Horro and Menz sheep breeds. The villages were categorized by

CBBP members and not-CBBP member households. Those household members of CBBP

have criteria to select superior ram to serve, controlled matting, ram castration, the

selected ram rotates some household’s. In contrary, those households of not-CBBP

members they don’t get the program facility above mentioned. The total sheep population

by zone is Kaffa 420,378, Kelem Wellega 113,514 and 820,947 North Shewa Zone

(CSA, 2015).

Menz

Menz is one of the former provinces in north Shewa administrative zone. Currently Menz

area is divided into four Woredas namely; Menz Gera Mider, Menz Mama Mider, Menz

Keya Gebriel and Menz Lallo Mider. In Menz area the survey conducted in Mollaleand

Mehalmeda. These villages were selected based on their potential for sheep production

and the areas believed to be the main habitat of Menz sheep breed (Markos, 2006). The

area is located at an altitude above 2800 to 3200 m.a.s.l and about 280 km north of Addis

Ababa. The area is characterized by bi-modal rainfall with main rainy season (June to

September) and erratic and unreliable short rainy season (February to March). Based on

the meteorological data obtained from Debre Berhan Agricultural Research Centre from

the year 1985 to 2005, the annual rainfall at Mehal Meda town (the capital of the Menz

Gera woreda) was about 900 mm and the minimum and maximum average temperatures

were 6.8oC and 17.6oC, respectively. The production system of Menz can be

characterized as a mixed crop-livestock system. The cool highland parts of Menz are

65

believed to be the main habitat of Menz sheep. The potential of the area for sheep

production is documented (MOA, 1998; Abebe, 1999).

About 98% of the population in the area depends on agriculture. The farming system of

the study area is largely characterized by mixed crop-livestock production system. Crop

production is limited due to severe frost, poor soil fertility and unreliable rainfall. Thus,

the area is characterized as one of the drought prone areas of the Amhara National

Regional State (CSA, 2005). However, our GPS shows in Menz (3037-3117 m.a.sl).

Based on the 2007 national census conducted by the Central Statistical Agency of

Ethiopia (CSA), this woreda has a total population of 85,129, of whom 42,102 are men

and 43,027 women; 6,513 or 7.65% are urban inhabitants. The majority of the inhabitants

practiced Ethiopian Orthodox Christianity, with 99.67% reporting that as their religion.

https://en.wikipedia.org/wiki/Menz_Mam_Midir

Bonga

Bonga is a town and separate woreda in southwestern Ethiopia. Located southwest

of Jimma in the Keffa Zone of the Southern Nations, Nationalities and Peoples

Region upon a hill in the upper Barta valley, it has a latitude and longitude of

7°16′N 36°14′ECoordinates: 7°16′N 36°14′E with an elevation of 1,714 meters above sea

level. It is surrounded by Ginbo woreda.Adiyo Kaka located at 509 km south west of the

capital Addis Ababa. Adiyo Kaka is located in 36o 47’E longitude and 7o 26 ’N latitude

with altitude ranging from 500 to 3500 meters. Adiyo Kaka have themaximum and

minimum annual temperature is 36oC and 3oC, respectively (SUDCA, 2007). The study

was conducted on two selected villages of Boka and Shutta. However, our GPS shows in

Bonga (2532-2543 m.a.sl).

Horro

Horro is located at about 315 km from Addis Ababa (9º 34´N latitude and 37º 06´ E

longitude) in the Horro Guduru Wollega zone, Oromia Regional State, West Ethiopia.

The district has two major agro-ecologies: highland and midland. Horro has diverse crops

66

and livestock resources due to its favorable production environments. That is why it is

selected as one of the Agricultural Growth Program (AGP) districts (Duguma et al.,

2012).Its main rainy season occurs between May and September and the dry season lasts

from October to April. The altitude ranges from 1800 to 2835 (HARDO, 2006).Horro

Guduru Welega is one of the zones of the Oromia Region in Ethiopia. It is named after

the former province of Welega, whose eastern part lay in the area Horro Guduru Welega

now occupies. The study was conducted on two selected villages of Laku and Gitilo.

However, our GPS shows Horro with altitude of (2684-2688 m.a.s.l.).

http://www.oromiyaa.com/english/index.php?option=com_content&view=category&layo

ut=blog&id=121&Itemid

3.2.Study animals and study methods

Rams aged above six months used for service and ewes were included in the study. A

cross-sectional study design was implemented to determine the seroprevalence of the

reproductive diseases, respiratory disease and to assess the potential risk factors that

facilitate for the occurrence of the disease in the study area from April 2015-November

2016. The households were initially stratified by participating in CBBP or not-CBBP

then 20 households from each stratum in each village were selected by simple random

sampling. Individual animals were sampled by sex strata randomly from each household.

In each region 40 households were selected for sampling of animals from the selected

villages a total of 120 households was sampled. From each selected households 3-4

animals were sampled from the total flocks.

3.3.Sample size determination

Total sample size will be estimated according to Thrusfield, (2005) by using 95%

confidence interval and 5% precision. As there is no comprehensive previous study on

sero-prevalence of common sheep disease, 50% expected prevalence was considered

during sample size estimation. Therefore, the total sample was 384 animals but to

67

increase the precision the total sample was increased to 450. Hence a total of 150 animals

were sampled from each study sites, in general 450 sera sample was collected from the

three regions.

3.4. Sample collection and laboratory analysis

3.4.1. Questionnaire

To make use of any existing sources of information, both secondary and primary

information were used in the study. Secondary information was collected from district

offices of agriculture, recorded data and the district veterinarians. Key informant

interview and visual observation were used to collect the primary data. Expert of

livestock extension, elders and veterinarians were used as key informants of the study

during field data collection.

A total of 120 households were selected randomly and interviewed individually during

blood sample collection by using structured questionnaire. Body condition, breed, age,

sex, altitude (location), CBBP and Ram-CBBP were considered as major risk factors to

the occurrence of the disease and properly recorded on the prepared format. CBBP is

abbreviation of Community Based Breeding program. The sampled households can be

CBBP members and the others are not-CBBP members. Ram-CBBP is selected rams for

service that are found in the CBBP members because some rams found not selected in the

CBBP member households.

A questionnaire was prepared (Annex: 1), the questionnaire was pre-tested before

administration and some re-arrangement, reframing and correcting in accordance with

respondent perceptions were done. The questionnaire was administered to the randomly

selected household heads or representatives by a team of enumerators recruited and

trained for the purpose with close supervision by the researcher. Based on the

questionnaire the following information was captured: Reproductive performances like

age at first puberty, lambing interval, litter size (number of lambs born per ewe per

lambing) and lambing season, Breeding selection criteria and castration practices, Feed

situation, like major feed sources, supplementation, grazing method and water source,

68

Major diseases of sheep in the area, Responsibility of sheep related works in the

household.

3.4.2. Serum sample collection

Serum was the sample of interest to determine the prevalence of these diseases. Whole

blood was collected from the jugular veins of randomly selected rams and ewes into 10

ml sterile vacutainer tubes and stored overnight at room temperature for serum collection.

The serum was transferred into a sterile cryovial bearing the identification number,

location, breed and village then transported in an icebox to laboratory for refrigeration

and then stored at -20°C until the laboratory analysis was done in National Veterinary

Institute (NVI), Bishoftu and at NAHDIC in Sebeta. According to Desta (2009), age of

the animals was determined by observing different numbers of erupted permanent

incisors (Annex: 3). The sample list that has household and animal information is

indicated in (Annex: 2).

3.4.3. Laboratory analysis

After the serum was separated it was stores at -20°C until the laboratory analysis was

done in National Veterinary Institute (NVI), Bishoftu and (National Animal Health

Diagnostic and Investigation) NHADIC. Brucella, Pasturella, PPR and CCPP laboratory

analysis was done in NVI and Chlamydia, Q-Fever, Toxoplasma and Border disease

laboratory analysis was done in NHADIC that was funded by ILRI.

Chlamydia

The type of laboratory test employed was Indirect ELISA test protocol. IDEXX

Chlamydiosis Total Ab Test demonstrated 100% specificity and 89% to 95% sensitivity

in a trial of 81 caprine samples across three naturally infected herds, one experimentally

infected herd, and two known negative herds from france (Schalch et al., 1998). The type

of laboratory test employed was Indirect ELISA test protocol used in NAHDIC at

01/09/2015 andthe procedure is indicated in (Annex: 4).

69

Q-fever

The type of laboratory test employed was Indirect ELISA test protocol used in NAHDIC

06/09/2015 and the procedure is indicated in (Annex: 5). Indirect serological assays are

often used for individual diagnosis and herd screening, including IFA, CFT, and ELISA.

Because of their ease of use, reliability, and scalability, ELISA test kits have become the

choice tool for veterinary diagnosis and large-scale routine herd monitoring. The IDEXX

Q-Fever Ab Test, available from IDEXX Laboratories, has shown excellent specificity

and high sensitivity consistently competitive with the complement fixation test.

https://www.idexx.com/pdf/en_us/livestock-poultry/abortive-disease-information-

brochure.pdf

Toxoplasma

The type of laboratory test employed was Indirect ELISA test protocol used in NAHDIC

at 11/09/2015 and the procedure is indicated in (Annex: 6). Diagnosis of T. gondii in

sheep can be made by means of direct tests, such as histopathology,

immunohistochemistry, PCR and bioassay, as well as by means of indirect tests (serum)

basedon the detection of anti-T. gondii antibodies, or by a combination of these methods

(Dubey, 2010).

Border disease

A total of 445 blood sera were tested using Indirect ELISA test protocol (BVD IDEEX

kit) in NAHDIC at 11/09/2015. After the laboratory analysis the OD value was calculated

in percentage. The cut off value were results ≥45% taken as positive and <45% OD value

were negative results.

Brucellosis

Rose Bengal precipitation Test (RBPT): Serum of 75μl was mixed with 25μl of antigen

on an enamel plate to produce a zone approximately of l to 2 cm in diameter. The

mixture was rocked gently for four minutes at ambient temperature and then

observed for agglutination. Any visible reaction was graded as positive and otherwise

negative. RBT is useful for screening sheep sera for antibodies to B. melitensis and B.

70

ovis (brucella species). The test was conducted at NVI, Bishoftu, according to the test

procedure recommended by OIE (2004).

Pasturellosis

A total of 360 sera samples were selected from the total 448 samples. Equal samples were

selected from each regions and the clear and pure serum was selected for testing. The

selection of samples was due to the expense of the laboratory test. The type of laboratory

test employed was Indirect Haemagglutination (IHA) test protocol used in NVI as

indicated in (Annex: 7) and an agglutination rate of >50% was taken as positive. Each

sample was tested for M. haemolytica type A1, A2, A7, P. multocida type A and all B.

trehalosi types based on the source of the reference serotypes were (CIRAD-EMVT,

France).

PPR

The type of laboratory test employed was Competitive ELISA (C-ELISA) test protocol

(Libeau et al., 1995) used in NVI as indicated in (Annex: 8). after the laboratory test the

OD value was calculated in percentage. The cut off value were results below or equal to

50% taken as positive. From 50 - 60% results were taken as suspected results and above

60% were strong negative results.

3.5.Data analysis

The data genereated were stored in a Microsoft excel spreadsheet and analysed using

Statistical Package for Social Sciences (SPSS version 22). Seroprevalence was calculated

by dividing the total number of sheep tested positive by ELISA by the total number of

sheep tested. Similarly, flock level was calculated as the number of flocks with at least

one positive animal divided by the total number of flocks tested. Logistic regression

(univarient and multivarient) was used to compute the odds ratio associated with potential

risk factors by using Epiinfo (Epiinfo version 3.4). Variables with more than two

categories were transformed into indicator (dummy) variables. Potential risk factors

71

included in the model were selected based on the existing literature. Non-collinear

variables that presented P-value of < 0.15 in univariable analysis were included in the

multivariable logistic regression model at animal level. Flocks containing at least one

seropositive animal were considered positive. The 95% confidence level was used and

results were considered significant at P ≤ 0.05.

72

4. RESULTS

4.1. Reproductive Performance and Problems

I. Herd characteristics of production parameter

The present study shows except the Lambing interval all other variables of

reproductive performance were significantly different (p<0.05) for different regions

(Table 2).

Table 2: Reproductive performances by region

Variables Region N Mean Std. Deviation P-value

Age puberity (mon) Horro 39 6.71 3.15 .000

Bonga 40 6.67 1.38

Menz 41 11.22 4.63

Age first service

(mon)

Horro 39 7.23 3.43 .000

Bonga 40

41

6.58 1.15

Menz 12.51 4.14

Age first lambing

(mon)

Horro 39 13.89 4.95 .000

Bonga 40 12.50 1.85

Menz 41 18.95 4.17

Lambing interval

(mon)

Horro 39 7.74 4.26 .425

Bonga 40 8.05 1.63

Menz 41 8.58 2.20

Lamb born perlifetime Horro 39 13.17 4.79 .000

Bonga 40 11.70 1.74

Menz 41 8.41 2.12

Age at weaning (mon) Horro 39 4.23 .95 .000

Bonga 40 4.02 .94

Menz 41 4.95 1.04

Twining rate (%) Horro 39 30.51 16.21 .000

Bonga 40 48.70 27.62

Menz 41 1.68 4.62

Total 120 26.72 26.91

73

In Horro region 41% of the respondents replied that abortion seen in their sheep flock and

higher at the third month of pregnancy (Annex: 17). In all regions lamb mortality occurs

frequently as compare to dystocia, stillbirth and abortion. About 25.8% respondents

replied that the cause was due to diarrhea and 17.5% due to Coughing (Annex: 18). Out

of 120 respondents only 6 were reported for sheep dystocia and the cause of the dystocia

were due to large foetus size 67%, foetus come in abnormal position 17% and first

parturition and twinning 16% (Table 3).

Table 3: Reproductive problems by region

Menz

Horro

Bonga

Total

Variables Frequency % Frequency % Frequency % Frequency %

P-

value

Lamb

mortality 27 65.9 29 74.4 22 55 78 65 .195

Dystocia 2 4.9 4 10.3 0 0 6 5 .112

Stillbirth 1 2.4 7 17.9 4 10 12 10 .069

Abortion 5 12.2 16 41 3 7.5 24 20 .000

I. Role of HH members in SR production between regions

In Horro the burden of responsibility for sheep purchased and sell was on both male and

females (elders) and cleaning barn was the responsibility of females and girls. In Bonga

sheep purchase and sell was done by male but cleaning the sheep barn was done by

females. However, in Menz sheep purchase and sell was done only by males as compare

to females. Incontrary sheep watering, feeding and barn cleaning were the responsibility

of females (Annex: 15).

II. Reproductive performance and problems in CBBP/non-CBBP

households

All the reproductive problems were not significantly different by CBBP and non-CBBP

members. The reproductive performance of sheep was not significant as compare to

CBBP and non-CBBP household members (see Table 4 and 5).

74

Table 4: Reproductive problems by CBBP /non-CBBP

CBBP

Non-CBBP

Total

Variables Frequency % Frequency % Frequency % P-value

Lamb

mortality 50 63.3 28 68.3 78 65 .58

Dystocia 3 3.8 3 7.3 6 5 .40

Stillbirth 9 11.4 3 7.3 12 10 .48

Abortion 16 20.3 8 19.5 24 20 .92

75

Table 5: Reproductive performance by CBBP/non-CBBP

Horro

Bonga

Menz

Variables CBBP N Mean

Std.

Deviation N Mean

Std.

Deviation N Mean

Std.

Deviation

P-

value

Age of puberity (mon) 0 10 6.70 2.06 14 7.14 1.70 17 11.41 3.84 .263

1 29 6.72 3.48 26 6.42 1.14 24 11.08 5.20

Age first service

(mon)

0 10 7.20 2.62 14 6.73 1.73 17 13.18 3.19 .185

1 29 7.24 3.72 26 6.50 0.71 24 12.04 4.72

Age first lambing

(mon)

0 10 13.70 3.77 14 13.07 2.13 17 19.53 3.57 .219

1 29 13.97 5.36 26 12.19 1.65 24 18.54 4.58

Lambing interval

(mon)

0 10 7.80 3.65 14 7.86 1.66 17 9.47 2.21 .305

1 29 7.72 4.52 26 8.15 1.64 24 7.96 2.01

Lamb born per

lifetime

0 10 13.20 4.18 14 11.00 1.71 17 7.12 1.50 .016

1 29 13.17 5.06 26 12.08 1.67 24 9.33 2.04

Age at weaning (mon) 0 10 4.00 0.82 14 4.36 0.93 17 5.12 1.17 .187

1 29 4.31 1.00 26 3.85 0.92 24 4.83 0.96

Twining rate (%) 0 10 30.50 19.07 14 50.57 27.30 17 1.71 3.29 .703

1 29 30.52 15.49 26 47.69 28.29 24 1.67 5.45

Total

39 30.51 16.21 40 48.70 27.63 41 1.68 4.63

76

4.2. Reproductive Disease

There was no significant association between the production parameters (reproductive

performance) and the reproductive disease. With the exception of Coxiella burnetii had

significantly higher prevalence in lamb mortality for Coxiella burnetii positive herds than

negative herds (Annex: 16).

I. Chlamydia

A. Seroprevalence of Chlamydia

The overall seroprevalence of Chlamydia in the current study was 57.9% animal level

and 89.2% flock level. Significantly higher prevalence was recorded in Bonga 74.5%

than Horro 52% and Menz 47.3% region (Table 6).

Table 6: Chlamydia prevalence in the three regions by different risk factors

Variables

No.tested Positive Prevalence (%) P-value

Region Bonga 149 111 74.5 0.00

Horro 152 79 52

Menz 146 69 47.3

Sex Male 129 68 52.7 0.094

Female 318 191 60.1

Age 6mon-1yr 70 46 65.70 0.468

1yr-2yr 84 46 54.8

2yr-3yr 90 49 54.4

above 3yr 203 118 58.1

CBBP No 143 78 54.5 0.185

Yes 304 181 59.5

Total

447 259 57.9

Variables

No.tested Positive Prevalence (%) P-value

Ram-CBBP No 53 22 41.5 0.025

77

Yes 76 46 60.5

Total 129 68 52.7

B. Seroprevalence of Chlamydia in Bonga region

Significantly higher prevalence was seen in Boka village 81.8% than Shutta village

66.7% (Table 7).

Table 7: Bonga region Chlamydia prevalence by different risk factors

Variables

No.tested Positive Prevalence (%) P-value

Village Boka 77 63 81.8 0.026

Shutta 72 48 66.7

Sex Male 38 28 73.7 0.526

Female 111 83 74.8

Age 6mon-1yr 47 34 72.30 0.831

1yr-2yr 22 17 77.3

2yr-3yr 33 25 75.8

above 3yr 47 35 74.5

CBBP No 45 36 80 0.211

Yes 104 75 72.1

Total

149 111 74.5

Variables

No.tested Positive Prevalence (%) P-value

Ram-CBBP No 10 7 70 0.53

Yes 28 21 75

Total

38 28 73.7

C. Univariable logistic regression of Chlamydia

The logistic regression was used to test the strength of associations between the risk

factors and the prevalence of the disease. Bonga region had 3.26 times more odds of

Chlamydia seropositive than Menz. Ram-CBBP had 2.16 times more odds of Chlamydia

78

seropositive than Ram not-CBBP. In this analysis Menz region and Ram non-CBBP were

used as a reference category.

D. Multivarient logistic regression

Finally region and Ram-CBBP were risk factors for the occurrence of Chlamydia in the

current study areas (Table 8).

Table 8: Multivariate logistic regression of Chlamydia

95 % CI (OR)

Variables

Odds Ratio Lower Upper P-value

Region Horro/Menz 1.2077 0.7664 1.903 0.4161

Bonga/Menz 3.2597 1.9946 5.3272 0.000

Ram-CBBP Yes/No 2.1606 1.0578 4.4132 0.0345

II. Coxiella burneiti

A. Seroprevalence of Coxiella burnetii

The overall seroprevalence of Q-fever in animal level was 38% and flock level was

68.3%. Significantly higher prevalence was seen in Menz region (54.1%) but lower

prevalence was seen in Bonga region (12.1%). Significantly higher prevalence in adults

than young age groups (Table 9).

Table 9: Q-fever prevalence in the three regions by different risk factors

Variables

No.tested Positive Prevalence (%) P-value

Region Bonga 149 18 12.1 .000

Horo 152 73 48.0

Menz 146 79 54.1

Sex Male 129 42 32.6 .079

Female 318 128 40.3

Age 6mon-1yr 70 9 12.9 .000

1yr-2yr 84 24 28.6

2yr-3yr 90 38 42.2

above 3yr 203 99 48.8

CBBP No 143 51 35.7 .274

79

Yes 304 119 39.1

Total 447 170 38.0

Variables

No. tested Positive Prevalence (%) P-value

Ram-CBBP No 53 13 24.53 0.104

Yes 76 29 38.2

Total

129 42 32.6

B. Prevalence of Coxiella burnetii by Menz region

Significantly higher (P<0.05) prevalence was seen in female 66.7% than male 33.9% and

higher prevalence was recorded in Ram-CBBP 52.4% than Ram not-CBBP 22.9%. The

logistic regression shows within menz region female had 3.89 times more odds of Q-

fever seropositive than males and in Rams-CBBP had 3.71 times more odds of Q-fever

seropositive than Rams-nonCBBP. In this analysis male and Rams-nonCBBP were used

as a reference category (Table 10).

Table 10: Menz region Coxiella burnetii prevalence by different risk factors

Variables

No. tested Positive Prevalence (%) P-value

Village Mehalmeda 68 41 60.3 0.161

Molale 78 38 48.7

Sex Male 56 19 33.9 0.00

Female 90 60 66.7

Age 6mon-1yr 5 0 0 1.20

1yr-2yr 38 9 23.7

2yr-3yr 27 15 55.6

above 3yr 76 55 72.4

CBBP No 58 33 56.9 0.583

Yes 88 46 52.3

Total

146 79 54.1

Variable

No. tested Positive

Prevalence

(%) P-value

Ram-CBBP No 35 8 22.9 0.024

80

Yes 21 11 52.4

Total

56 19 33.9

C. Univariable logistic regression of Coxiella burnetii

The logistic regression was used to test the strength of associations between the risk

factors and the prevalence of the disease.Menz region had 8.58 times more odds of Q-

fever seropositive than Bonga region. >3year age groups had 6.45 times more odds of Q-

fever seropositive than 6mon-1year age groups. In this analysis Bonga region and 6mon-

1year age groups were used as a reference category.

D. Multivarient Logistic regression

Multivariable logistic regression analysis for significantly associated risk factors was

conducted simultaneously. Ram-CBBP, Sex and CBBP were removed from the model

and finally age and region were the only risk factors for the occurrence of Q-Fever in the

study areas (Table 11).

Table 11: Multivariable logistic regression for region and Age

Risk factors

Odds Ratio (95% CI) P-Value

Age 1yr-2yr/6mon-1yr 1.349 0.5437, 3.3452 0.5188

2yr-3yr/6mon-1yr 3.3 1.3822, 7.8763 0.0072

above 3yr/6mon-1yr 3.458 1.548, 7.725 0.0025

Region Horo/Bonga 5.894 3.2281, 10.7606 0.000

Menz/Bonga 7.571 4.0844, 14.0355 0.000

III. Toxoplasma

A. Prevalence of Toxoplasma gondi by different risk factors

The overall flock and animal level seroprevalences of T.gondi was 70.8% and 39.8%

respectively. Higher prevalence was recorded in Horro 56.3% than Bonga 41.2% and

Menz 21.2% region (Table 12).

81

Table 12: Seroprevalence of Toxoplasma gondi by different risk factors

Variables

No. tested No. positive Prevalence (%) P-value

Region Bonga 148 61 41.2 0.000

Horro 151 85 56.3

Menz 146 31 21.2

Sex Female 318 145 45.6 0.000

Male 127 32 25.2

Age 6mon-1yr 68 21 30.9 0.001

1yr-2yr 84 21 25

2yr-3yr 90 37 41.1

above 3yr 203 98 48.3

CBBP Yes 304 138 45.4 0.000

No 141 39 27.7

Total

445 177 39.8

Variables

No. tested No. positive Prevalence (%) P-value

Ram-CBBP Yes 76 23 30.3 0.108

No 51 9 17.6

Total

127 32 25.2

B. Prevalence of T.gondi in Horro region

Sex, age and CBBP had significant variation with Toxoplasma gondi in Horro region

(Table 13).

Table 13: Seroprevalence of Toxoplasma gondi in Horro region

Variables

No. tested No. positive Prevalence (%) P-value

Village Gitilo 76 45 59.2 .467

Laku 75 40 53.3

Sex Female 117 74 63.2 .001

Male 34 11 32.4

Age 6mon-1yr 17 6 35.3 .021

1yr-2yr 24 9 37.5

2yr-3yr 30 17 56.7

82

above 3yr 80 53 66.3

CBBP Yes 112 69 61.6 .026

No 39 16 41

Total

151 85 56.3

Variables

No. tested No. positive Prevalence (%) P-value

Ram-CBBP Yes 27 8 29.6 .505

No 7 3 42.9

Total

34 11 32.4

C. Univarient logistic regression

During statistical analysis, for all risk factor the lowest prevalence was used as a

reference category. The univarient logistic regression showed that the risk of infection in

Horro (OR= 4.77, 95% CI; 2.86 - 7.96) and Bonga (OR= 2.60, 95% CI; 1.55-4.35) was

significantly higher than in Menz region.

D. Multivarient Logistic regression

Multivariable logistic regression analysis for significantly associated risk factors was

conducted simultaneously. By comparingthe more complex model with the less complex

model using backward elimination region, CBBP and sex were the major risk factors for

the occurrence of T. gondi infection (Table 14).

Table 14: Multivariable logistic regression for region and sex

Risk factors

Odds Ratio 95% CI P-Value

Region Bonga/Menz 2.41 1.43, 4.05 0.0009

Horo/Menz 4.412 2.63, 7.40 0.000

Sex Female/Male 2.192 1.36, 3.52 0.0012

CBBP Yes/No 2.1742 1.41,3.35 0.0004

83

IV. Border disease

A total of 445 samples were tested for border disease and all the samples were tested

negative.

V. Brucella

A total of 448 samples were tested by Rose Bengal Plate test and all the samples were

tested negative.

4.3. Respiratory Disease

I. Pasteurella

Eight serotypes in both sexes of sheep distributing orderly in the study area were

identified. The extent to which selected serotypes were only considered was depending

up on their current importance in some parts of our country.

A. Serotypes distribution by region

From 360 sheep samples, 93.9%, 66.7% and 98.9% of them were infected with M.

haemolytica serotypes, P.multocida serotype A and B. trehalosi serotypes, respectively.

The eight serotypes were associated with the species of the animal as assessed by uni-

variate analysis. M. haemolytica serotype A2 as well as B. trehalosi serotype T3, T4 and

T10 were significantly (P<0.05) associated with Horro/Bonga and Menz/Bonga (Table

15).

84

Table 15: Pasteurella serotypes by region

Bonga Horro Menz Total (n= 360) Horro/Bonga

Menz/Bonga

(n= 117) (n= 132) (n= 111)

Uni-variate

Uni-variate

Serotypes Positive (%) Positive (%) Positive (%) Positive (%) OR (95% CI) P-value OR (95% CI) P-value

A1 98 (83.8) 98 (74.2) 74 (66.7) 270 (75) 0.55 (0.29 - 1.04) 0.0691 0.38 (0.20 - 0.72) 0.0032

A2 46 (39.3) 48 (36.4) 74 (66.7) 168 (46.7) 0.88 (0.52 - 1.47) 0.6315 3.08 (1.79 - 5.30) 0.000

A7 74 (63.2) 86 (65.2) 67(60.4) 227 (63.1) 1.08 (0.64 - 1.82) 0.7544 0.88 (0.51 - 1.51) 0.6538

Total A 113 (96.6) 121 (91.7) 104 (93.7) 338 (93.9) 0.38 (0.12 - 1.24) 0.1149 0.52 (0.14 - 1.84) 0.3163

PA 84 (71.8) 78 (59.1) 78 (70.3) 240 (66.7) 0.56 (0.33 - 0.96) 0.0367 0.92 (0.523 - 1.64) 0.7997

T3 62 (53) 84 (63.6) 92(82.9) 238 (66.1) 1.55 (0.93 - 2.57 ) 0.0894 4.29 (2.32 - 7.92) 0.000

T4 105 (89.7) 127 (96.2) 103 (92.8) 335 (93.1) 2.90 (0.99 - 8.50) 0.052 1.47 (0.57- 3.74) 0.4181

T10 77 (65.8) 103 (78) 80 (72.1) 260 (72.2) 1.84 (1.05 - 3.23) 0.0326 1.34 (0.76 - 2.35) 0.3082

T15 78 (66.7) 81 (61.4) 72 (64.9) 231 (64.2) 0.79 (0.47 - 1.33) 0.385 0.92 (0.53 - 1.59) 0.7744

Total T 115 (98.3) 131 (99.2) 110 (99.1) 356 (98.9) 2.27 (0.20 - 25.45) 0.5037 1.91 (0.17 - 21.39) 0.5985

85

A= Mannhaemia haemolytica serotype A, PA = Pasteurellamultocida serotype A, T =

Bibersteinia trehalosi serotype T, OR = Odds Ratio, CI = Confidence Interval

B. Serotypes distribution with in CBBP and non-CBBP households

There was no significant variation between CBBP and non-CBBP groups for all

serotypes in the current study (Table 16).

Table 16: Pasteurella serotypes between CBBP/non-CBBP

CBBP/Non-CBBP

Non-CBBP (n=119) CBBP (n= 241) Uni-variate

Serotypes Positive (%) Positive (%) OR (95% CI) P-value

A1 89 (74.8) 181 (75.1) 1.01 (0.61 - 1.68) 0.9484

A2 68 (57.1) 100 (41.5) 1.43 (0.59 – 3.45) 0.4211

A7 67 (56.3) 160 (66.4) 1.53 (0.97 - 2.40) 0.0628

Total A 110(92.4) 228(94.6) 1.43(0.59-3.45) 0.4211

PA 77 (64.7) 163 (67.6) 1.13 (0.71 - 1.81) 0.5793

T3 79 (66.4) 159 (66.0) 0.98 (0.61 -1.56) 0.9382

T4 109 (91.6) 226 (93.8) 1.38 (0.60 -3.17) 0.4458

T10 82 (68.9) 178 (73.9) 1.27 (0.78 -2.06) 0.3243

T15 83 (69.7) 148 (61.4) 0.69 (0.43 -1.10) 0.1216

Total T 118(99.2) 238(98.8) 0.67(0.06-6.52) 0.732

C. Multivariate Logistic regression

Uni-variate analysis result indicated that of the risk factors studied; only region and age

of the animals were significantly associated to the infection of the major serotypes (M.

haemolytica serotype A1, A2, P. multocida PA, B. trehalosi serotype T3, T4, T10 for

region Menz had 0.38 times lower Odds ratio than Bonga to pasturella infection where as

Menz had 3.08 times higher Odds of pasturella infection than Bonga. In addition with in

age groups M. haemolytica serotype A1, A2, B. trehalosi serotype T10, T15)

respectively. Serotype T15 in adults (above one year) had higher Odds ratio as compare

to young age groups (6mon-1year). Ram-CBBP had significant variation with in ram-

CBBP and ram not-CBBP.

86

II. PPR

A. Seroprevalence of PPR

The overall seroprevalence of PPR virus antibody for non-vaccinated sheep in the current

study was 11.2%. Higher prevalence was seen in Menz with 22.8% as compare to Bonga

2.6% and Horro 8.6% regions (Table 17).

Table 17: Seroprevalence of PPR by different risk factors

Variables

No. tested Positive Prevalence (%) P-value

Region Bonga 152 4 2.6 0.00

Horro 151 13 8.6

Menz 145 33 22.8

Sex Male 131 12 9.2 0.387

Female 317 38 12

Age 6mon-1yr 72 2 2.8 0.025

1yr-2yr 84 7 8.3

2yr-3yr 90 10 11.1

above 3yr 202 31 15.3

CBBP No 141 16 11.3 0.932

Yes 307 34 11.1

Total

448 50 11.2

Ram in CBBP No 53 5 9.4 .929

Yes 78 7 9

Total

131 12 9.2

B. Seroprevalence of PPR in Menz

Significantly higher seroprevalence was seen on female 29.2% than male 12.5% (Table

18).

Table 18: Seroprevalence of PPR in Menz

Variables

No. tested Positive Prevalence (%) P-value

Village Mehalmeda 68 14 20.6 .558

Molale 77 19 24.7

Sex Male 56 7 12.5 .019

Female 89 26 29.2

87

Age 6mon-1yr 5 0 0 .035

1yr-2yr 38 4 10.5

2yr-3yr 27 5 18.5

above 3yr 75 24 32

CBBP No 57 9 15.8 .107

Yes 88 24 27.3

Total

145 33 22.8

Ram in CBBP No 35 5 14.3 .602

Yes 21 2 9.5

Total

56 7 12.5

C. Univariable logistic regression

The logistic regression was used to test the strength of associations between the risk

factors and the prevalence of the disease. Menz region had 10.9 times more odds of PPR

seropositive than Bonga. Adult age groups had 6.34 times more odds of seropositivity

than young age groups.

D. Multivariable logistic regression of PPR

Multivariable logistic regression analysis for significantly associated risk factors was

conducted simultaneously. Sex, age and CBBP/Non-CBBP were excluded from final

model as they didn’t have significant association after the effect of Region was removed.

Region was the only risk factor for the occurrence of PPR in the study areas and sex was

the only risk factor in Menz region. Breed was removed from the model due to its multi-

colinearity with Region (Table 19).

Table 19: Multivariable logistic regression for PPR

Risk factor

Odds Ratio (95% CI) P-Value

Region Horo/Bonga 3.4855 (1.10, 10.94) 0.0325

Menz/Bonga 10.9018 (3.75, 31.66) 0.00

88

5. DISCUSSION

5.1. Reproductive Performance and Problems

The reproductive performance of sheep (Age puberity, Age first service, Age first

lambing, Lamb born perlifetime and Age at weaning in months) were significantly

different by location/breed. This might be due to difference in Genetic, management

difference and variation in availability and quality of feed resource across the difference

seasons (DBARC, 2006; Abate, 2016).

The current study shows that Age of puberity in Horro, Bonga, Menz were 6.7+3.15,

6.6+1.38, 11.2+4.6 respectivelly. Menz breed were slow in reproductive maturity as

compare to Horro and Bonga breeds. That was lower than reports of Amelmal (2011)

11.13+2.7, 10.8+1.9 and 9.5+1.4 months for female sheep in Tocha, Mareka and Konta,

respectively. The current result was in agreement with Tsedeke (2007) age of puberity in

local Alaba sheep were 6.7 and 6.9 months for male and female sheep.

Age at first service in Horro, Bonga and Menz were 7.2+3.4, 6.5+1.1, 12.5+4.1

respectivelly. It was lower as compare to Zewdu (2008) age at first service for Bonga

breed was 9.3+2.2 months and for Horro breeds 7.8+2.4 months for females. Age at first

lambing in Horro, Bonga and Menz were 13.8+4.9, 12.5+1.8, 18.9+4.1 respectivelly. In

Horro and Bonga breed similar result was reported by Zewdu (2008) the mean age at first

lambing was 13.3+1.7 and 14.9+3.1 months respectivelly. However, small mean age at

first lambing for Menz breed 15.22 month was reported by Abebe (1999).

89

Lambing interval in Horro, Bonga and Menz were 7.7+4.2, 8+1.6, 8.5+2.2 months’

respectivelly. The current result was in agreement with (Belete, 2009) and (Zewdu, 2008)

indicates that lambing interval of Bonga and Horro ewes were around 8 and 7.8+2.4

month respectively. However, high lambing interval in Menz 8.5 month was seen

(Tesfaye, 2008).

Lamb born perlife time in Horro, Bonga and Menz ewe were 13.1+4.7, 11.1+1.7, 8.4+2.1

respectivelly. Similar result was reported for Gumuz sheep (13.5+1.76 lambs) in Metema

areas (Solomon, 2007) and according to Amelmal (2011) the local ewe produce on

average 8.57+3.7 (Tocha), 8.62+4.1 (Mareka) and 10.78+4.7 (Konta) lambs in their life

time. In contrast, high mean of lamb born perlife time was reported by Zewdu (2008) on

an average a Bonga ewe delivers 12.2+1.80 and Horro ewe delivers 15.3+4.3 lambs in

their life time.

Respondents reported that the reproductive problems seen in Horro, Bonga and Menz

were (lamb mortality) 74.4%, 55%, 65%; (Abortion) 41%, 7.5%, 12.% and (Still birth)

17.9%, 10%, 2.4% respectively. From the respondent’s response the reproductive

problems were higer in Horro region as compare to other regions, this could be associated

with higher prevalence of toxoplasma gondi in the herd of Horro region.

The reproductive performance of sheep with in the regions was not significant as

compare to CBBP and non-CBBP household members. This might be associated with

those CBBP/non-CBBP households had the same breed of animals and the superior

selected rams of CBBP were served for non-CBBP groups secretly that could be the

reason to get almost the same reproductive performance. All the reproductive problems

were not significantly different by CBBP and non-CBBP members. Even if, it was not

significant most respondents of non-CBBP households had reproductive problems as

compare to CBBP member households.

5.2. Reproductive Disease

90

I. Chlamydia

The isolation of the microorganism is difficult and the diagnosis is mainly based on

serological methods as agent isolation remains a difficult and time-consuming task.

https://www.idexx.com/livestock-poultry/ruminant/chlamydia.html

The present study is the first detailed study that investigated the distribution of

Chlamydophila abortus infection in sheep on selected geographic distribution of

Ethiopia. In the present study, the overall Chlamydiaseroprevalence in animal level was

57.9% and flock level was 89.2%. The current study showed that Chlamydial abortion

was prevalent in small ruminants in the study areas. The seroprevalence of

Chlamydophila abortus in small ruminants varies widely from one country to another and

within regions, ranging from 2.9% in China (Zhuo et al., 2012) to 31.1% in Mexico

(Jimenez-Estrada et al., 2008).

In the present study the overall seroprevalence of C. abortuswas 57.9% which was higher

than reports 3.8% in Nigeria (Abubakar, 2015); 25.6% in Iran (Esmaeili et al., 2015);

18.65% in Tibetan sheep China (Si-Yuan et al., 2014); 8% in Namibia (Samkange et al.,

2010). The different prevalence observed was probably due to differences in animal-

welfare, sanitation, climates and husbandry practices, ecology and breed.

The prevalence difference between regions was in Bonga (74.5%), Horro (52%) and

Menz (47.3%) regions. Significantly higher prevalence was seen in Bonga region as

compare to the other regions that could be due to Ecology/topography. Bonga topography

is midland as compare to Menz region. The other reason for higher prevalence of C.

abortus in Bonga might be sheep housing. In Menz and Horro regions sheep housing was

a separate sheep house but in Bonga region 60% of the respondents respond that

adjoining house with poor sanitation. Bonga have mean lamb mortality rate of (2.8+1.72)

as compare to Menz (0.39+0.51) per year. In addition, Boka village had higher

prevalence as compare to Shutta this might be due to source of animals; 10% of the

91

respondents said that they purchase sheep from market as compare to 5% Shutta they

purchase sheep from market that could be the source of the disease. Based on Aitken et

al. (2007), reports that OEA is the most common infectious cause of abortion in lowland

flocks that are intensively managed during the lambing period. This study revealed that

the geographical origin of sheep is one of the risk factors associated with C.

abortus seroprevalence.

Significantly higher prevalence was seen in Ram-CBBP (60.5%) as compare to Ram not-

CBBP (41.5%) this might be due to the rams in non-CBBP households were used only in

one house but the selected rams found in CBBP groups were used as share for many

households. The shared rams could have great chance to get the infection from diseased

flock (Wilsmore et al., 1986). Rams found in the CBBP were a source of disease to the

other households.

In the present study, the C. abortus seroprevalence in male and female sheep was 52.7%

and 60.1%, respectively. However, there was no significant difference in C.

abortus seroprevalence between genders (p=0.094), which is consistent with the studies

of Huang et al. (2013), in which they reported negative association between sex and C.

abortus prevalence in Tibetan sheep in Tibet, implying that gender may not be a crucial

factor for C. abortus infection.

All writers strongly believe that CFT, which has been used extensively to diagnose

enzootic ovine abortion, is still a gold standard for serological diagnosis at Ab titre ≥

1:40. Other serological tests like ELISA and IFA have similar problems of false positive

and false negative results (Griffiths et al., 1996). However, the IDEXX Chlamydiosis

Total Ab Test demonstrated 100% specificity and 89% to 95% sensitivity in a trial of 81

caprine samples across three naturally infected herds, one experimentally infected herd,

and two known negative herds from France (Schalch et al., 1998).

II. Coxiella burnetii

92

In the present study the overall prevalence of C. burnetii was 38%, this study was higher

than reports of Abiri et al. (2016) 17.3% Aborted sheep fetuses using PCR in Iran,

Masala et al. (2003) 10% sheep C. burnetii in analyzed fetuses by PCR in Italy, Rizzo et

al. (2016) 16.3% sheep in Northwest Italy, Schimmer et al. (2011) 21.4% on dairy goat

farms in Netherlands. The prevalence difference might be due to difference in ecology,

difference in diagnostic method used, and season of sample collected on April the

bacteria can easily transmitted aerosol as reported by Maurin and Raoult (1999). Even if

the sample type and diagnostic method was different nearly similar prevalence was seen

in the report of Cantas et al. (2011) 33% sheep abortion cases were positive for C.

burnetii with the Trans and CB PCRs test of foetal abomasal content in North Cyprus.

The present research shows significantly different (P<0.05) prevalence between regions,

higher prevalence reported in Menz (54.1%) and lower prevalence was seen in Bonga

(12.1%) regions. This difference might be due to animal movement in Menz (9.8%),

Horro (7.7%) and Bonga (7.5%) respondent’s said that their animal source was purchased

respectively. Similar study shows geographical distribution in the occurrence of C.

burnetii abortion among ruminants of the reported sample abortion cases in the

Northbound Region (35%), Border Region (53%) and Karpas Region (42%) respectively

were caused by C. burnetii in North Cyprus (Cantas et al., 2011) and Asadi et al. (2012)

also reports significantly higher prevalence in Central Iran followed by Southwest and

Western Iran. This could be due to favorable climatic conditions for aerosol transmission

of C. burnetii in this area.

The seroprevalence of C. burnetii was increased significantly with age 6mon-1year

(12.9%), 1year-2year (28.6%), 2year-3year (42.2%) and above 3year (48.8%),

respectively. Similar study reported by Schimmer et al. (2011) younger than 1year

(5.8%), between 1-3years (15.7%) and older than 3years (26.1%) for goats in

Netherlands.

93

In the logistic regression analysis Menz region had 8.58 times significantly higher Odds

ratio than Bonga region. There was 6.45 times significantly higher Odds of ratio in the

>3year than 6mon-1year age group. In multivarient logistic regression age and region

were the only risk factors for the occurrence of Q-fever in the study areas. Additionally,

in multivarient logistic regression sex and Ram-CBBP were taken as a risk factor for the

occurrence of C. burnetii in Menz region. This might be due to male to female ratio was

not proportional, female animals were higher than male in each households this is due to

the owners could have only one or two breeding rams. In ram-CBBP had 3.71 times Odds

ratio as compare to Ram-non CBBP, this could be due to the selected rams were used for

breeding and rotate the house that don’t have rams as compare to rams not-CBBP

households and can be used to spread the disease.

Respondents that have dog in their house had significantly (P<0.05) higher herd

prevalence (77%) as compare to respondents that didn’t have dog (54.3%). Therefore, the

presence of dog in the farm was used as a risk factor for the occurrence of C. burnetii in

the region. A similar study done in North Cyprus shows presence of carnivore on farm

had 3.3 Odds ratio with (p=0.01) (Cantas et al., 2011).

Significantly higher prevalence was reported by sheep flock size. As the flock size

increase the prevalence become higher. This might be due to the pathogen transmites by

aerosole and close contact facilitates the disease transmission.

The higher prevalence of C. burnetii in Menz region can be a risk for the public health

that has close contact with the animals. Clinical signs such as Coughing 17(27.9%),

headache 14(23.0%) and diarrhea 10(16.4%) was seen in the respondents (Annex: 10).

Therefore, it’s important to do further researches in isolating the agents in detail and

assess the public health. Similar studies was done by Heydel and Willems (2011), Acute

Q fever is characterized by sudden onset of fever to 104ºF -105ºF, chills, profuse

sweating, severe headache, weakness, nausea, vomiting, diarrhea, non-productive cough,

and abdominal or chest pain. Additionally, similar result was reported by Abiri et al.

(2016) in Iran.

94

III. Toxoplasma

The overall seroprevalence of T.gondiin animal leve and flock level seroprevalence were

39.8% and 70.8% respectively. The current result was in the range of prevalence

estimated in previous studies in Ethiopia, which ranged from 11.9% in Central Ethiopia

(Bekele and Kassali, 1989) and 52.6% in Nazareth (Negash et al., 2004).

Higher prevalence was reported in Horro (56.3%) region than Bonga (41.2%) and Menz

(21.2%). This difference might be due to Horro region is in the midland as compare to

Menz region highland that have lower prevalence. This could be related with high lamb

mortality in the region; Horro (3.37+3.31) mean lamb mortality as compare to Menz

0.39+0.51 mean lamb mortality; the disease could have an impact on the occurrence of

lamb mortality in the region. Horro region (2684-2688 m.a.s.l.), Bonga (2532-2543

m.a.sl) and in Menz (3037-3117 m.a.sl). This result was in agreement with (Negash et al.,

2004) in Nazareth, Gebremedhin et al. (2014) in Central Ethiopia. Higher prevalence was

observed in warm and moist areas than in cold or hot dry areas. The high rate of T. gondii

infection suggests a high environmental contamination with oocysts (Deconinck et al.,

1996).

Lower prevalence was reported as compare to the current study by Gebremedhin et al.

(2014), 20% in Central Ethiopia, 31.45% in selected districts of SNNPR (Gebremedhin

and Gizaw, 2014), 22.1% in Rio Grande do Norte (Andrade et al., 2013) and 29.9% in

Mexico by (Alvarado-Esquivel et al., 2013).

In contrary, higher prevalence was reported 56% in Nazareth by (Negash et al., 2004),

54.7% in Nazareth (Negash et al., 2004), 49.3% in Spain by (García-Bocanegra et al.,

2013), 53.7% in Greece by (Anastacia et al., 2013), Zimbabwe (67.9%) (Hove et al.,

2005) and Egypt (47.5%) reported by Barakat et al. (2009). The wide variation in the

seroprevalence of T. gondii infection seen between the present study and aforementioned

studies might be due to difference in sample size, agro-ecology, and climate, season of

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sample collected, cat density, animal management, type of serological tests used and the

cut off value (Opsteegh et al., 2010b).

Significantly higher prevalence (P<0.05) was seen in female (45.6%) than male sheep

(25.2%). Similar reports was seen in Negash et al. (2004), Zewdu et al. (2013),

(Gebremedhin et al., 2013)which indicated the role of gender as an important variable for

infection by T. gondii (high infection in females than males). This might be attributed to

the management system in that ewes are retained in the farm for longer periods for

breeding purpose than males. Few rams are retained for mating while the majority are

culled and sold for cash purpose. In contrary a study done by Gebremedhin and Gizaw

(2014), reports that gender has no significant association with the risk of T.gondi

infection.

Significantly different association (P<0.05) was seen between age groups, adult sheep

had 2.08(1.16-3.74) times exposed than young sheep. Similar study also reported

significantly higher prevalence in adult than young sheep by Carneiro et al., 2009;

Ramzan et al., 2009; Dubey, 2010; Gebremedhin et al. (2014) in Central Ethiopia, adult

age group were 8.55(2.79-26.15) times more exposed than young age in selected districts

of SNNPR (Gebremedhin and Gizaw, 2014). The significantly high prevalence in adult

sheep than young sheep is due to high chance of exposure to the source of infection as the

age increases and suggests that most sheep acquire the infection post-natal (Dubey,

2010); (Gebremedhin et al., 2013). According to Katzer et al. (2011) study done in

Scotland, the major source of infection for adult sheep is likely to be through the

consumption of sporulated oocysts from the environment.

Significant association was seen in CBBP (45.4%) and non-CBBP (27.7%) households.

This might be due to the proportion of animal in the non-CBBP were small due to lack of

cooperation for the sampling.

The presence of cats is crucial in the life cycle of T.gondii and is significantly associated

with increased seroprevalence in many reports (Dubey and Beattie, 1988; Dubey, 2010).

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However, the presence of cat in each house hold was not related with the occurrence of

Toxoplasma gondi. Significantly higher (P<0.05) prevalence was recorded in with

absence of cat 46.2% in the house than presence of cat 31.8% in the house. Similar study

was done by Mengesha et al. (1984); Guebre-Xabier et al. (1993) reported the absence of

a significant association between T.gondi seropositivity and the presence of cat in

households. This might be expected that in those households reporting absence of cats at

their home, stray and wild cats from neighboring areas might visit their areas and access

sheep grazing land. The current study didn’t isolate oocysts or study seroprevalence in

cats. Probably cats in some households are not infected at all and in this case presence of

cats and their numbers is not directly related to seroprevalence of T. gondi.

IV. Border Disease Virus

Border Disease Virus infection has not yet been reported in Ethiopia, and no study has

been performed on the epidemiology of BDV. The current study shows 0% prevalence to

Border Disease in all the study areas. The current result was lower than reports of Yazici

et al. (2012) 48.12% with cELISA in Turkey and 44.4% prevalence from sera sample in

China and BDV strain was classified as BDV-3 (Mao et al., 2015).

V. Brucella

The current study shows 0% prevalence or very low prevalence to brucella in all the three

regions. The current result was lower than reports of Ashenafi et al. (2007), with 11.6%

in Afar region; Megersa et al. (2010), with 1.56% in Borena; Bekele et al. (2011) 1.5% in

small ruminants at Jijiga District; 1.17% and 1.88% sheep and goats at Yabello district

(Dabassa et al., 2013); 0.48% and 3.09% sheep and goats at pastoral area of Oromia and

Somali (Tsehay et al., 2014); 1.63% and 1.86% sheep and goat at Debre Ziet and Modjo

export abattoirs (Tsegay et al., 2014) and 2.5% in North Kordofan State Sudan (Abdallah

et al., 2015). This might be due to the difference in animal management system;

unrestricted animal movements may have enhanced the spread of infection; the mixing of

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animals at market places and watering points and population density of small ruminant in

the area (Tsehay et al., 2014).

5.3. Respiratory disease

I. Pasturella

The predominant serotypes identified in this study were B. trehalosi serotype T4 (93.1%),

T10 (72.2%), T3 (66.1%) and T15 (64.1%). This finding was higher than the findings of

Hussein and Elsawi Mohamed (1984), who reported B.trehalosi serotype T3 (12.7%), T4

(11.5%) and T10 (10.5%), while B.trehalosi serotype T15 was not detected in sheep at

Sudan; Tesfaye and Abebe (2003), who reported B.trehalosi serotype T3 (14%), T4 (8%)

and T15 (4%) in sheep of Quana district of Wollo in Northern part of Ethiopia. Similarly,

the same authors reports lower prevalence of T4 (8%), T3 (4%) and T15 (2%) in Gimba

district, Wollo Northern part of Ethiopia. The authors did not detect B.trehalosi serotype

T10 in both districts of Wollo. However, Kirka and Kaya (2005) reported B.trehalosi

serotype T4 (8.3%) was the only serotype present in sheep in Turkey. The most probable

explanation for the discrepancy of the current findings from elsewhere could be due to

weather condition, nature of the pathogen host immunity, overcrowding in a limited

space, poor management, rough handling and distant transport or shipping (Brogden et

al., 1998).

The prevalence of M.haemolytica serotype A1 was predominant (75%), A7 (63.1%) and

A2 (46.7%) serotypes studied in this work were higher than the findings in Wollo areas

of Ethiopia (Tesfaye and Abebe, 2003), A1 (12%), A7 (6%) and A2 (4%) and in Sudan

(Hussein and Elsawi Mohamed 1984), A2 (17%),A1 (14.5%) and A7 (10%). It was also

higher than the finding of the same authors in Gimba district of Wollo in sheep of

Northern part of Ethiopia A1 (16%), A2 and A7 (2%). Lower prevalence was also

reported for A2 (36.1%), A7 (5.9%) and A1 (5.3%) in sheep of Northern Nigeria

(Odugbo et al., 2003). The prevalence of A2 and A7 (20.8%) and A1 (4.1%) was lower

than the current findings in sheep of Turkey (Kirkan and Kaya, 2005) and A1 (33.1%),

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A7 (31.8%) and A2 (28.5%) in sheep of Farta and Lay-Gayint districts of South Gonder,

Northwest Ethiopia (Yeshwas et al., 2013).

In the current study, P. multocida type A (66.7%) was lower in prevalence than the

M.haemolytica serotypes (93.9%) and B.trehalosi serotypes (98.9%) respectivelly.

Similar findings were reported by Biruk et al. (2013), lower P. multocida type A (2.3%),

M.haemolytica serotypes (26.4%) and B.trehalosi serotypes (7.4%) in Asella. However,

this finding was higher than the 10% prevalence in sheep of Debre- Brehan in Central

part of Ethiopia (Gelagay et al., 2004); 6.1% in sheep of Turkey (Guler et al., 2013) and

6.6% in sheep of Farta and Lay-Gayint districts of South Gonder, Northwest Ethiopia

(Yeshwas et al., 2013). Therefore, this prevalence difference was most probably due to

difference in environmental condition, immunity of the animals, and circulation of

particular serotypes in the area and the reaction of animals to different levels of stress.

The reaction of animals to stress is rather variable even within individual animals of the

same species (Brogden et al. 1998; Mohamed and Abdelsalam, 2008).

Region was a significant risk factor for majority of the serotypes (M. haemolytica

serotype A1 and A2, B. trehalosi serotype T3, T4 and T10, P. multocida PA). However,

the other serotypes were not taken as a risk factor with in the region. Similar study done

in Farta and Lay-Gayint districts of South Gonder, Northwest Ethiopia, shows different

serotype were distributed by location (Yeshwas et al., 2013). Age group was taken as a

risk factor in some of the serotypes (M. haemolytica serotype A1 and A2, B. trehalosi

serotype T10 and T15). The disease was higher in adults than young age groups except

M. haemolytica serotype A1. In another study in Ireland, M. haemolytica serotypes (A1

up to A14) and B. trehalosi serotypes (T4, T10 and T15) were isolated from different age

groups in sheep and cattle (Ball et al., 1993). This finding was incontrary to (Ball et al.,

1993), majority of the pasteurellosis prevalence was higher in young age groups (<1 year)

than adult ages. These differences might be due to adaptability of the serotypes and

immune response of the animals. Region, age, sex, CBBP and Ram-CBBP had been

evaluated as a risk factor for pasteurellosis in this study. Of these, region, age and Ram-

CBBP were a risk factors using multivariable logistic regression analysis.

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II. PPR

A diagnostic technique, which is simple, rapid, specific and sensitive is preferred for

intensive surveillance of a disease. C-ELISA test is one such test for screening of

antibodies to various morbilliviruses (Abd El-Rahim et al., 2010; OIE, 2013). There was

no information on the specificity and sensitivity of the PPR C-ELISA in the kit manual.

Therefore, estimates by other laboratories that employed similar anti-N protein

monoclonal antibody were accepted. The sensitivity of 93.4% and specificity 98.5% was

reported (Choi et al., 2005).

Significantly higher prevalence was reported in Menz 22.8% than Horro 8.6% and Bonga

2.6%. This difference might be due to difference in grazing type, in menz most of

animals were used communal grazing as compare to bonga that have a private grazing

management. In agreement with this study, Abubakar et al., (2009) reported continued

year round circulation of the virus enhanced by frequent animal-to-animal contacts. This

difference also could be due to animal movement in Menz (9.8%), Horro (7.7%) and

Bonga (7.5%) respondent’s said that their animal source was purchased respectively.

Similar, introduction of new animals purchased from live animal market have been

implicated as a source of the disease in India (Singh et al., 2004a; Mehmood et al., 2009).

From these findings varied prevalence of PPR could be due to the geographical

difference; these findings are agreed with the results reported by Shuaib (2011) and Muse

et al. (2012).

The overall seroprevalence (11.2%) was lower than the finding in previous studies

carried out in the country (29.5%) by Megersa et al., (2011), 13% in Afar, Borena, East

shewa, Gambella and Jijiga in sheep (Abraham et al., 2005), 27% in Eastern Amhara

Region by (Alemu, 2014), 22.4% in Turkey by Özkul et al., (2002); 33% in India by

Singh et al. (2004a); 26% in Bangladish by Banik et al., (2008); 32.8% in India by

Balamurugan et al. (2012); 22.1% in Tanzania by Kivaria et al. (2013); 34.2% in

Pakistan by Munir et al. (2012a) and 43.7% in Sudan Salih et al. (2014).

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However this report was higher than 6.8% by Abraham et al. (2005) and 6.4% by Waret-

Szkuta et al. (2008). A significant difference observed in seroprevalence of PPRV among

study areas could be due to the variation in small ruminant population, flock size,

movement of sheep and goat flocks, outbreak, introduction of new animals, seasonal

grazing and management system. Prior study by Waret-Szkuta et al. (2008) pointed out

that there is large variation between regions and woredas of the country.

Even if it was not significant higher prevalence was seen in female than male. Similar

study done by Waret-Szkuta et al. (2008); Khan et al. (2008); Salih et al. (2014),

observed a significantly higher seroprevalence of PPR in females compared to males.

This could be related to the physiological differences where females reveal some degree

of predominance infection as a result of production and reproduction related stresses

(Megersa et al., 2011).

With respect to age category, the highest prevalence of PPR was observed in adults

compared to young age. These findings are in agreement with previous reports from

Ethiopia (Waret-Szkuta et al. 2008); Pakistan (Abubakar et al., 2011); Turkey (Ozkul et

al., 2002) and India (Singh et al. 2004a); where they reported high prevalence in adults. It

has been documented that sheep and goats exposed to natural infection to PPRV at a very

young age may carry antibodies for 1-2 year following exposure and remains positive for

a long time (Dhar et al., 2002; Ozkul et al., 2002; Singh et al., 2004a). Therefore, adult

animals might be more vulnerable to PPR infections as compared to younger animals.

CBBP/non-CBBP households and Ram selected by CBBP and not selected by CBBP had

equal chance of positivity to PPR. The disease distributed equally in the membered or not

membered and used as a risk fator.

Flock size was not significant with the positivity of PPR and mixed flock (sheep and

goat) doesn’t have effect on the positivity of PPR. Incontrary, a study by Al-Majali et al.

(2008) showed an association of large flock sizes and mixed farming with PPR

seropositivity in Jordan. Raising sheep along with goats was also found to be a risk factor

for PPR seropositivity by other investigators (Anderson and McKay, 1994).In

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multivarient logistic regression region was the only risk factor for the occurrence of PPR.

In addition, Sex was also the risk factor for the positivity of PPR in Menz region.

6. CONCLUSION AND RECOMMENDATIONS

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6.1. Reproductive Performance and Problems

Ethiopia has a diverse indigenous sheep population and owned by smallholder farmers as

an integral part of the livestock sub-sector for trade and meat consumption in household.

However, the annual meat production and off-take is very low as compared to the huge

population and genetic source of country. The reproductive performance of sheep in the

current study showed variation among breed and location. From the questionnaire the

reproductive disease of sheep in the current study had equal effect on CBBP/non-CBBP

household members.

Therefore, based on the above conclusive remarks the following recommendations are

forwarded:

- On station (longitudinal study design) for evaluation of reproductive performance

and problems of indigenous sheep should be done, to get clear and exact results.

- The CBBP groups should give priority to test ram health as a critera during

selection of rams to avoid the transmission of disease by the selected rams.

6.2. Reproductive Disease

I. Chlamydia

It could be concluded from this study that C. abortus infection is highly prevalent in

sheep in the current study areas. The C. abortus infection rate in Bonga area is almost

double of the rate of menz area. By multivarient logistic regression region and Ram-

CBBP were the main risk factors for C. abortus seroprevalence. The higher prevalence

has possibility for loss of production, reproduction performance failure and a risk of

infection to human beings that have close contact with the animals. The present higher

prevalence can be related with: There was no vaccination program in the country. The

laboratory technique used to diagnose the prevalence of Chlamydia was not

recommended by OIE and due to the kit was expensive and can’t perform the Ab titer

103

procedure. Therefore, it is recommended that there should need further study in

Chlamydophila abortus with a more advanced diagnostic methods like detecting the

agent up to strain level, antibody tititer should be done, sample should be collected from

adult females during lambing and find active cases to confirm the agent correctly. The

present study is the first report that gives a warning to the responsible stakeholders to

control the disease transmission as early as possible.

Therefore, based on the above concluding remarks the following recommendations are

forwarded:

Possible control measures to alleviate the community problem should be adopted

by the responsible stakeholders like Ministry of Livestock and Fish, ILRI and

ICARDA.

Further detailed study on the economic impact and potential risk factors of C.

abortus infection on small ruminants by using a more advanced diagnostic

methods.

Create awareness to the society and efficient management measures to prevent

and control C. abortus infection in the study areas.

II. Coxiella burnetii

This study established the presence of C. burnetii in sheep in different regions of Ethiopia

with higher prevalence in Menz region than other study areas. Multivarient logistic

regression shows Region and Age were the only risk factors for the occurrence of C.

burnetii in the study areas. The flock level prevalence 68.3% reflectes high circulation of

C. burnetii within a farm and a risk for environmental contamination and spread. Dog and

flock size also taken as risk factors in the herd level. This implies reproductive failure in

animals and zoonotic effect to human being. The results of this study yielded baseline

information that may be useful to set up future epidemiologic, flock management and

public health policies for the prevention and control of C. burnetii in Ethiopia.

104

Therefore, based on the above concluding remarks the following recommendations are

forwarded:

Future researches needs to be performed on larger sample sizes from other regions for

better understanding the potential risk factors of C. burnetii.

Further researches in isolating the agents in detail and assess the underestimated

public health issue in regions with high prevalence.

Among the most important management schemes to control the transmission of C.

burnetii in infected herds, it can be useful to provide a special location for the par-

turition, proper sanitation, good manure management etc.

The diagnostic methods used leads to have higher prevalence. Therefore, it’s better to

use as international office of epizootics (OIE) recommends PCR as one of the most

effective methods for C. burnetii diagnosis for isolation of the bacteria.

During selection of rams for breeding it’s important to consider reproductive disease

test as a selection criteria.

III. Toxoplasma

The overall prevalence shows higher prevalence of Toxoplasmosis in the study areas.

This implies high lamb mortality and weak lamb birth due to the disease that leads to low

production performance, cause of considerable reproductive wastage in sheep, zoonotic

transmission to human being causes multiple diseases in humans and impact on the

economy in general. The current study shows the major risk factors for toxoplasmosis is

region, sex and CBBP. However cat was not taken as a risk factor. Therefore it is

advisable to control the disease.

Therefore, based on the above concluding remarks the following recommendations are

forwarded:

Further studies by using advanced diagnostic methods to confirm the effect of the

disease.

It is important to educate or create awareness to the animal owners about the effect of

the disease on both animal and human.

105

Further detailed epidemiological study to assess the risk factors of the disease on

human being, small ruminant and cats is warranted for eventual control of the disease

IV. Border disease

The current study shows zero prevalence to BDV in all the study areas. The test was done

for the first time and it was important to know the current status of the disease to

concentrate on the reproductive disease that results reduction in reproductive

performance of the small ruminants.

V. Brucella

Most researchers were belived that brucella was the main reproductive disease for

reproductive failure. The current study shows the reproductive problems were mostly the

cause of the newly entered diseases rather than brucella. Therefore, it is important for the

researchers to concentrate on other reproductive diseases.

6.3.Respiratory disease

I. Pasturella

The current study shows higher pasturella prevalence with regard to region (location) and

age groups. Region and age were arisk factors using multivariable logistic regression

analysis. Region was a significant risk factor for majority of the serotypes (M.

haemolytica serotype A1, A2, P.Multocida PA and B. trehalosi serotype T3, T4 and

T10). Age group was also a risk factor for B. trehalosi serotype T10, T15 and M.

haemolytica A1, A2 infection. There was a cross infection to serotypes. There was also a

cross reaction of the serotypes during laboratory analysis. Therefore, the monovalent

killed P. multocidabiotype A-vaccine might not be protective to the diverse serotypes

affecting single animal

Based on the above concluding remarks, the following recommendations are forwarded:

106

All Pasteurella serotypes, virulence factors and their pathogenicity should be

investigated in future study

Multivalent vaccine that effectively prevent the major identified circulating

serotypes in the area should be developed

Detailed epidemiological and risk factor study should be conducted for each

serotypes in sheep and other livestock species.

Interpretation of the pasteurella results is more difficult since it was impossible

to get reliable information on past vaccination history and the specificity of the

pasteuralla diagnosis seems questionable. Therefore, it’s better to evaluate the

procedure for the future.

II. PPR

Prevalence of PPR was higher in Menz as compare to Horro and Bonga. Region, sex and

age were significantly associated with the positivity of PPR in the current study areas.

Region and sex were the only risk factors for the occurence of PPR. In addition, the study

has identified the source of introduction of PPR to be newly purchased animals and

communal grazing. Therefore, the first level of control is the restriction of movement of

animals from endemic areas, with rigorous quarantine and surveillance procedures if a

total ban is not practical to prevent the spread of the disease and the transmission of the

virus to different localities.

Therefore, based on the above conclusive remarks the following recommendations are

forwarded:

Further detail epidemiological research is needed by using advanced diagnostic

methods to identify the potential risk factors of PPR, so that to develop effective

control strategies for PPR in large area of the country.

There should be further studies to identify the gene sequences and lineage of the

PPR virus isolated in this study so that we could better understand the recent

molecular epidemiology of the disease.

107

Responsible stakeholders should have effert to perform PPR vaccination of small

ruminants for the control of the disease should be encouraged and applied more

strictly and strategically to reported areas using the homologous PPR vaccine that

is recommended by the OIE.

Create awareness to the animal owners on the disease effect and advantage of

vaccination for proper control method.

108

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131

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Research in Veterinary Science, 94: 43-48.

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Zhuo, G.H., Shang, C.C., Zhuo, Y.Q., Gao, M., Fan, G.Y., Tian, T.T., Yao, Y.L., Chen, D.K. and

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Website related

http://www.fao.org/ag/againfo/programmes/en/empres/gemp/avis/B103

brucellosis/tools/0_geo_world-distribution.html

http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_ovis.pdf

http://www.nzsheep.co.nz/uploads/documents/Brucella%20ovis%20info%20for%20farm

ers_2012.pdf

http://www.webmd.com/a-to-z-guides/brucellosis-symptoms-treatment?page=3

http://www.esgpip.org/pdf/Technical%20Bulletin%20No.20.pdf

http://www.disabled-world.com/health/query-fever.php accessed on April, 2016

http://www.fda.gov/Food/ResourcesForYou/StudentsTeachers/ScienceandTheFoodSuppl

y/ucm 215846.htm

http://www.netvet.co.uk/sheep/toxoplasmosis.htm

http://www.merckvetmanual.com/mvm/generalized_conditions/pasteurellosis_of_sheep_

and_goats/overview_of_pasteurellosis_of_sheep_and_goats.html

http://www.merckvetmanual.com/mvm/generalized_conditions/congenital_and_inherited

_anomalies/border_disease.html

132

8. ANNEXES

Annex: 1 Questionnaire format

Date:_______________

Household level questionnaire format to determine the reproductive performance in

sheep flocks, common reproductive disease seroprevalence and risk factors in Horo,

Bonga and Menze rams

Region _______________________ Zone _____________________

Districts ______________________ PA/ Kebelle________________

Village ______________ Household no.____________

Altitude (GPS) ______________

I. Demographic Characteristics of the Households

1. Name of household head: _____________________________Sex:

______Age______

2. Family size? 1. Male____________2. Female_____________3. Total___________

3. Level of education of the household head?

1. Illiterate 2. Read and write 3.Elementary school 4. High school 5. College and

University

II. Livestock holding

Type of animal Quantity (no.) Holding Source of

animals Own Share/Ribi

Sheep

Below 3 months

133

3-6 month males

3-6 month females

6-12 females

Intact males (6months and above)

Females

Castrates

Goat

Cattle

Horse

Donkey

Mule

Poultry

Dogs

Cats

III. Indicate members of household responsible for sheep management in this year

(Tick accordingly)

Activities Children (<15yrs) Adult ((≥ 15yrs)

Boys Girls Hired Males Females Hired

1. Sheep purchasing

2. Selling sheep

3. Help during parturition

4. Grazing

5. Stall-feeding/zero grazing

6. Watering

7. Caring for sick animals

8. Barn cleaning

9. Others (specify)

134

IV. Performance of sheep population in the area

Reproductive parameters Tick (√)

1. Seasonality of mating (seasonal =1; continuous=2)

2. Average age at sexual maturity/puberty

3 Age at first service

4. Average no. of services to conceive

5. Average age at 1st lambing

6. Average lambing interval

7 Average lamb born per life time

8. Age at weaning

9 Twining rate/ frequency

10 Peak lambing season/months

V. Sheep reproduction performance/ management of genetic resources

4. What are the selection criteria for breeding sheep?

1. Size

2. Color

3. Tail type

4. Twining rate

5. Age at first lambing

6. Pedigree

5. What is the source of ewe (replacement stock) at your home?

1. Raised at home

2. Purchased

3. Relative gift

4. Others (specify) __________________

135

6. Source of rams within the last 12 months

1. Own ram/breed

2. Own (bought)

3. Donated

4. Borrowed

5. Neighbors

7. Do you castrate rams? Yes No

8. If yes, why?

1. Fattening

2. Control breeding

3. Better temperament

4. Better price

5. Others (Specify)_____

9. If yes, at what age?

1. < 3 month

2. 3- 6 months

3. 6 – 12 months

4. > 6 months

11. Is there lamb mortality in your home? Yes No

12. If yes, how many lambs died within 12 months? ______________________________

13. What is the cause of lamb mortality?

________________________________________

14. Is there a birth problem (dystocia) in ewes? Yes No

15. If yes, what is the cause/reason of dystocia?

__________________________________

16. Is there still birth in your home? Yes No

17. If yes, at which month? ___________________________________

18. Is there any occurrence of abortions? Yes No

136

1. If yes, how many times? _____________________________

2. At what month of pregnancy? _________________________

VI. Housing of sheep

19. Where is sheep housing; 1, dwelling house 2, adjoining house, 3, sheep house 4,

field

VII. Sheep feed and their seasonal distribution

Major feed

Age sex Availability by Season Source Storage

of feed

Communal

grazing

Sep-Nov Dec-

Feb

Mar-

May

June-

Aug

Private grazing

Supplementation

Hay

Crop stubble

Crop residues

Improved forages

Other specify

VIII. Water sources of sheep and its seasonality

Season Distance from

dwelling

house (km)

Frequency of watering

Once per day Every

2days

Every

3days

Every

4days

Source

of water

Dry season

Wet season

137

Short rainy season

* 1 hour travel = 5 Km

IX. Sheep diseases commonly seen

20. Major diseases and other associated reasons of sheep death within 12 months

Local

name

Death

date

Birth

date

Major

death

reasons

sex No of animals

affected by this

disease

No of animals

died by this

diseases

Remark

21. Which of the following clinical signs have you observed in your flock over the last

six months?

Clinical sign Neve

r

Rarel

y

Often Alway

s

Age group and % of affected

Coughing

Diarrhea

Circling

Other

neurological

signs

Mastitis

Lameness

Dull/tired

Eye discharge

Nose discharge

Salivation

Ectoparasites

Weight loss

Rough hair

Pale mucous

membrane

Slow growth

Other

Rarely= occurs sometimes Often= occurs many times at short intervals

22. Have your sheep received any treatment/vaccine in the last six months? If yes, please

specify.

138

Treatment/Vaccinat

ion

At which

year

At which month and

date

How many

times

Specify the

name

FMD

Pasturellosis

Sheep pox

Anthrax

CCPP

PPR

Yes, but, I don’t

know the name

None

Antibiotics

Antihelminthics

Acaracide

Other

Investigated and completed by

Name _________________________ Date ____________________

Annex: 2 Sample lists of rams in each Household

Sample list

Sample

ID

Animal

ID

(eartag)

Age Sex Breed Ram

in

CBBP

BCS Health history 6 months

1.1

1.2

139

Household ID…………… Village………………….

Name…………………….. Date……………………

Annex: 3 Determination of age with different numbers of erupted permanent incisors

No. of permanent incisors Estimated age range

0 pair Less than 1 year

1 pair 1-1½ years

2 pair 1½-2years

3 pair 2½-3years

4 pair More than three years

Broken mouth Aged

Source: Desta (2009)

Annex: 4: Chlamydia procedures

1. Predilute samples and positive and negative controls 1:400 in a tube using the wash

solution.

2. Dispense 100 μl of prediluted samples and positive and negative controls into the

appropriate wells of the microtiter plate. Final dilution=1:400.

3. Cover the microtiter plate with a lid and incubate for 60 minutes (+5 min.) at +37°C

(+3°C).

4. Wash each well with approximately 300μl of wash solution three times. Empty liquid

contents of all wells after each wash. Following the final aspiration, firmly tap

residual wash fluid from each plate onto absorbent material. Avoid plate drying

between washes and prior to the addition of the next reagent.

5. Dispense100 μl of the conjugate into each well.

1.3

1.4

140

6. Cover and incubate the microtiter plate for 60 minutes (+5 min.) at +37°C (+3°C) in a

humid chamber.

7. Repeat step 4

8. Dispense 100 μl of TMB substrate N.12 into each well.

9. Incubate at 18-26°C for 15 minutes (+1min.).

10. Stop the color reaction by adding 100 μl stop solution N.3 per well. The stop solution

should be dispensed in the same order and at the same speed as the substrate.

11. Read the results using a photometer at a wavelength of 450nm.

Note: The IDEXX has instrument and software systems available that calculate means and

% values and provide data summeries.

The OD of the positive control (PCx̄) and the OD of the samples (sample A450) are corrected

by subtracting the OD of the negative control (NCx̄).

S/P % = 100×sample A450-NCx̄

PCx̄ - NCx̄

Interpretation of results:-

<30% - Negative, ≥30% to <40% - Suspect, ≥40% - Positive

Annex: 5 Q-fever procedures

All reagents must be allowed to come to 18-25°C before use. Mix reagents by gentle

inverting or swirling.

1. Obtain coated plates and record the sample position. If using partial plates, remove

those wells sufficient for samples to be tested. Place the remaining wells, along with

the desiccant, in the extra zip lock bag provided and return to 2-8°C.

2. Dispense 100 μl of diluted negative control (NC) in to duplicate wells.

3. Dispense 100 μl of diluted positive control (PC) in to duplicate wells.

4. Dispense 100 μl of diluted samples in to appropriate wells.

5. Mix the content of the wells by gently tapping the plate or use a microplate shaker.

6. Cover the microplate and incubate for 60 minutes (+5 min.) at +37°C (+3°C). The

plates should be tightly sealed or incubated in a humid chamber using plate covers to

avoid any evaporation.

7. Remove the solution and wash each well wih approximately 300 μl of wash solution

3 times. Avoid plate drying between plate washings and prior to the addition of the

141

next reagent. Tap each plate onto absorbent material after the final wash to remove

any residual wash fluid.

8. Dispense 100 μl of the conjugate into each well.

9. Cover the microplate and incubate for 60 minutes (+5 min.) at +37°C (+3°C). The

plates the plates should be tightly sealed or incubated in a humid chamber using plate

covers to avoid any evaporation. Repeat step 7.

10. Dispense 100 μl of TMB substrate N.12 in to each well.

11. Incubate at 18-26°C for 15 minutes (+ 1min.) away from direct light.

12. Dispense 100 μl of stop solution N.3 in to each well.

13. Read the results using a photometer at a wavelength of 450 nm.

Calculation:

S/P % = 100× Sample A(450) - (NCx̄) A (450)

PCx̄ - NCx̄

Interpretation of results:-

S/P %= <30% - Negative, 30% ≤ S/P % <40% - Suspect, ≥40% - Positive

Annex: 6 Toxoplasma procedures

All reagents must be allowed to come to 18-26°C before use. Reagents should be mixed

by gentle swirling or vortexing.

1. Predilute samples and positive and negative controls 1:400 in a tube using wash

solution.

2. Dispense 100 μl of prediluted samples and positive and negative control in to the

appropriate wells of the microtiter plate. Final dilution = 1:400.

3. Mix the contents with in each well by gently shaking the microtiter plate briefly (a

microtiter plate shaker can be used).

4. Cover the microplate (with a lid, aluminium fooil or adhesive plate cover) and

incubate for 60 minutes (+5 min.) at +37°C (+3°C).

5. Wash each well with approximetly 300 μl of wash solution three times. Remove

liquid contents os all wells after each wash. Following the final elimination, firmly

tap residual wash fluid from each plate on to absorbent material. Avoid plate drying

between washes and prior to the addition of the next reagent.

142

6. Dispense 100 μl of the conjugate in to each well.

7. Cover the microplate (with a lid, aluminium foil or adhesive plate cover) and incubate

the microtiter plate for 60 minutes (+5 min.) at +37°C (+ 3°C) in a humid chamber.

8. Repeat step 5.

9. Dispense 100 μl of TMB substrate N.12 in to each well.

10. Incubate at 18-26°C for 15 minutes (+1 min).

11. Stop the color reaction by adding 100 μl stop solution N.3 per well. The stop solution

should be dispensed in the same order and at the same speed as the substrate.

12. Read the results using a photometer at a wavelength of 450 nm.

Calculation

The OD of the positive control (PCx̄) and the OD of the samples (Sample A450) are

corrected by subtracting the OD of the negative control (NCx̄).

PCx̄ - NCx̄

S/P % = 100× Sample A450 -NCx̄

PCx̄ - NCx̄

Interpretation of results:-

S/P %= <20% - Negative, ≥20% to <30% - Suspect, ≥30% to <100% - Weak positive,

≥100%Positive

Annex: 7 Pasturellosis procedure of IHA (indirect heamaglutination test)

According to Sawada et al. (1982) the procedures are indicated as follows:

Antigen Preparation

Extract the antigens by heat extraction method followed by centrifugation.

Seed reference strains on tryptose-serum agar and incubate at 37°C for 18 -20

hours (four serotypes deal with at a time)

Alternatively, culture the serotypes in tryptose-serum broth and incubated for 18-

20 hours at 37°C

Harvest the growth in PBS in proportion of 20 to 30 colonies in 10 ml PBS

143

Centrifuge cultures at 2000 rpm for 20 minutes and re suspend the sediment in

equal volume of PBS

Heat this suspension in water bath at 60°C for an hour to kill viable organisms,

centrifuge at 5,500 rpm for 15 minutes at 4°C by refrigerated centrifuge

Discover clear supernatant fluid and used as capsular antigen extract

Sensitisation of SRBC

Draw blood from the jugular vein of sheep freely flowing into a syringe

containing Alsever`s solution, take 75 ml sheep blood in 125ml Alsever`s

solution. Add small amount of crystalline penicillin to avoid bacterial

contaminants. Store at +4oC at least one day overnight, the blood can be used for

about 2 weeks

Wash three times in PBS by centrifugation at 2000 rpm for 10 minutes

Add 100 µl of packed (PCV) RBCs to 10 ml of each antigen

Add 50 µl of 50% gluteraldehyde and homogenise by gentle shaking and

incubated for one hour at 37°C with periodical shaking

Centrifuge at 2,000 rpm for 10 minutes and wash two times in PBS by

centrifugation

Finally add 10 ml of PBS to the final sediment and made up to 1% suspension

Test procedures

For screening positive sera, add 95 µl of 1XPBS into micro plate (control) rows

A1-A12, C1-C12, E1-E12, G1-G12, and add 5 µl of test sera in the same wells

from the pre plates. The final dilution is 1/10th. Transfer 50 µl diluted test sera in

the (test samples) rows B1-B12, D1-D12, F1-F12, H1-H12, and add 50 µl of

sensitized SRBCs to respective wells. Add 50 µl of unsensitized 1% SRBC to the

control micro plate rows in parallel and incubate in moist chamber for one hour at

37°C

144

Add 100 µl 1/10 dilutions in PBS to the first rows of the plate in

duplicatesTransferring 50µl to the other wells (1:10, 1:20, 1:160, etc…) make a

serial double fold dilution and discard the final 50µl dilution sera

Add Control tests, in which sensitised and unsensitised SRBC's to respective

positive and negative sera parallel in every test

Cover the plates with micro plate sealer to prevent evaporation and incubated at

370C in moist chamber for 60 minutes with constant agitation

Complete and coarse agglutination of red cells indicates a positive reaction; small

button of deposited cells are a negative reaction.

50% agglutination rate is taken as positive

Annex: 8 PPR procedures

Competitive ELISA based on the use of MAb anti-nucleoprotein and a recombinant

nucleoprotein produced in Baculovirus was used as described by supplier manual

(CIRAD). Briefly, the plate was coated with PPR antigen by adding 50 µl diluted in

phosphate buffered saline (PBS) in 1/1300 dilution rate and incubated for 1 h at

37°C.Then, the plates were washed three times in washing buffer and blot dried. About

45 µl of blocking buffer was added to all wells, and then 5 µl of blocking buffer was

further added to the monoclonal control wells, 55 µl of blocking buffer to conjugated

control wells, 5 µl of test sera to test wells, 5 µl of strong positive, weak positive and

negative sera to control wells, and 50 µl of monoclonal antibody diluted 1/100 in

blocking buffer to all wells except the conjugate control wells and incubated 1 hour at

37°C. Then, the plates were washed three times and blot dried. 50 µl of antimouse

conjugate 1/1000 in blocking buffer was added and incubated at 37°C for 1 hour. Then,

the plate was washed three times with washing buffer. 50 µl substrate/chromogen

solutions was added and kept for 10 min in the dark place. Finally, stop solution was

added and read with ELISA reader at 492 nm. The ODs of all samples including the

controls were calculated and are expressed as the percent inhibition (PI) as follows: PI=

145

100-[(OD of the wells/OD of the monoclonal wells]. Those less than or equal to 50%

were considered as positive results.

Annex: 9 Reproductive performances in the three regions

Region

Age of

puberity

(mon)

Age

first

service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born per

life time

Age at

weaning

(mon) Twining rate

Menz Mean 11.21 12.51 18.95 8.58 8.41 4.95 1.68

Median 12.00 12.00 18.00 8.00 8.00 5.00 0.00

Std. Deviation 4.63 4.14 4.17 2.20 2.12 1.04 4.62

Horro Mean 6.71 7.23 13.90 7.74 13.17 4.23 30.51

Median 6.00 6.00 12.00 7.00 12.00 4.00 25.00

Std. Deviation 3.15 3.44 4.95 4.26 4.79 0.95 16.21

Bonga Mean 6.67 6.58 12.50 8.05 11.70 4.03 48.70

Median 6.50 6.00 12.00 8.00 12.00 4.00 50.00

Std. Deviation 1.38 1.15 1.85 1.63 1.74 0.94 27.62

Total Mean 8.24 8.82 15.16 8.13 11.05 4.40 26.73

Median 7.00 7.00 13.00 7.50 10.00 4.00 25.00

Std. Deviation 3.95 4.15 4.76 2.90 3.72 1.05 26.91

Annex: 10 Huma clinical signs related with Q-fever

Clinical sign Frequency %

Coughing 17 27.9

Diarrhea 10 16.4

Headache 14 23.0

fungus(itching) 3 4.9

Gastritis 3 4.9

Stomacache 2 3.3

eye discharge 2 3.3

chest pain 1 1.6

Anemia 1 1.6

dull/tired 1 1.6

heart problem 1 1.6

146

Hypertension 1 1.6

Itching 1 1.6

tooth problem 1 1.6

Vomiting 1 1.6

147

Annex: 11 Q-fever risk factors from Questionnaire

Univarient Logistic

Regression

Risk factors

No. HH Positive

Prevalence

(%)

P-

value Variables Odds Ratio (95% CI)

P-

Value

Dog presence Yes 74 57 77 0.009 Yes/No 2.8165 1.27,6.22 0.0106

No 46 25 54.3

Sheep

(Flock size) ≤10 32 16 50 0 11_20/≤10 1.6 0.65,3.89 0.3007

10_20 52 32 61.5

21_30/≤10 7 1.36,35.92 0.0197

21_30 16 14 87.5

>30/≤10 4852719.36 0,>1.0E12 0.9589

>30 20 20 100

Cat presence Yes 53 41 77.4 0.59

No 67 41 61.2

Goat contact Yes 22 13 59.1 0.302

No 98 69 70.4

Total

120 82 68.3

148

Annex: 12 Toxoplasma risk factors from questionnaire

Variables

No. HH Positive Prevalence (%) P-value

Cat Yes 53 35 66 .304

No 67 50 74.6

Dog Yes 74 54 73 .513

No 46 31 67.4

Goat contact Yes 22 15 68.2 .762

No 98 70 71.4

Sheep Flock size ≤10 32 22 68.8 .722

10_20 52 35 67.3

21_30 16 12 75

>30 20 16 80

Total

120 85 70.8

Annex: 13 PPR risk factors from questionnaire

Variables

N Positive Prevalence (%) P-value

Sheep flock ≤10 32 9 28.1 .906

11_20 52 16 30.8

21_30 16 6 37.5

>30 20 7 35

Goat contact No 98 34 34.7 .132

Yes 22 4 18.2

Total

120 38 31.7

Annex: 14 Source of animal by different risk factors for Chlamydia

Variables

Own (%) Purchased (%) P-value

Region Menz 90.2 9.8 .920

Horro 92.3 7.7

Bonga 92.5 7.5

Village Mahalmeda 85 15 .823

Molale 95.2 4.8

Gitilo 94.7 5.3

Laku 90 10

Boka 90 10

Shutta 95 5

CBBP Yes 96.2 3.8 .013

No 82.9 17.1

149

Total 120 110 (91.7) 10 (8.3)

Annex: 15 Role of HH members in small ruminant production by region

0

20

40

60

80

100

Res

po

nd

ents

(%

)

Work responsibility in Horo

boys

girls

males

females

hired

0

20

40

60

80

100

Res

ponden

ts (

%)

Work responsibility in Bonga

boys

girls

males

females

hired

150

Annex: 16 Reproductive performances with reproductive diseases

Horro

Chlamydia

Age of

puberity

(mon)

Age first

service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born per

life time

Age at

weaning

(mon)

Twining

rate

Negative Mean 6.17 7.33 14.0 6.83 13.50 4.83 38.33

Std.

Deviation 1.33 2.88 2.9 0.98 6.57 1.17 12.91

N 6 6 6 6 6 6 6

Positive Mean 6.82 7.21 13.88 7.91 13.12 4.12 29.09

Std.

Deviation 3.39 3.57 5.27 4.61 4.53 0.89 16.51

N 33 33 33 33 33 33 33

Total Mean 6.72 7.23 13.90 7.74 13.18 4.23 30.51

Std.

Deviation 3.15 3.44 4.95 4.27 4.80 0.96 16.21

N 39 39 39 39 39 39 39

Q-fever

Age of

puberity

(mon)

Age first

service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born per

life time

Age at

weaning

(mon)

Twining

rate

Negative Mean 6.38 6.25 12.38 7.63 13.50 4.13 33.75

Std.

Deviation 0.74 1.16 2.62 1.92 6.35 0.99 16.64

N 8 8 8 8 8 8 8

Positive Mean 6.81 7.48 14.29 7.77 13.10 4.26 29.68

Std.

Deviation 3.53 3.78 5.36 4.71 4.44 0.96 16.28

N 31 31 31 31 31 31 31

Total Mean 6.72 7.23 13.90 7.74 13.18 4.23 30.51

Std.

Deviation 3.15 3.44 4.95 4.27 4.80 0.96 16.21

0.0

20.0

40.0

60.0

80.0

100.0

Res

po

nd

ents

(%

)Work responsibility in Menz

boys

girls

males

females

hired

151

N 39 39 39 39 39 39 39

Toxoplasma

Age of

puberity

(mon)

Age first

service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born per

life time

Age at

weaning

(mon)

Twining

rate

Negative Mean 6.20 5.60 12.80 6.60 12.80 5.00 34.00

Std.

Deviation 0.45 1.14 2.39 0.55 4.76 0.71 10.84

N 5 5 5 5 5 5 5

Positive Mean 6.79 7.47 14.06 7.91 13.24 4.12 30.00

Std.

Deviation 3.37 3.60 5.23 4.55 4.87 0.95 16.92

N 34 34 34 34 34 34 34

Total Mean 6.72 7.23 13.90 7.74 13.18 4.23 30.51

Std.

Deviation 3.15 3.44 4.95 4.27 4.80 0.96 16.21

N 39 39 39 39 39 39 39

Bonga

Chlamydia

Age

puberity

(mon)

Age first service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born

per life

time

Age at

weaning

(mon)

Twining

rate (%)

Positive Mean 6.68 6.58 12.50 8.05 11.70 4.03 48.70

Std.

Deviation 1.38 1.15 1.85 1.63 1.74 .947 27.63

N 40 40 40 40 40 40 40

Total Mean 6.68 6.58 12.50 8.05 11.70 4.03 48.70

Std.

Deviation 1.38 1.15 1.85 1.63 1.74 0.95 27.63

N 40 40 40 40 40 40 40

Q-fever

Age of

puberity

(mon)

Age first service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born

per life

time

Age at

weaning

(mon)

Twining

rate (%)

Negative Mean 6.77 6.66 12.58 7.81 11.62 4.15 52.23

Std.

Deviation 1.68 1.27 1.65 1.55 1.65 0.97 28.43

N 26 26 26 26 26 26 26

Positive Mean 6.50 6.43 12.36 8.500 11.86 3.79 42.14

Std.

Deviation 0.52 0.94 2.24 1.7431 1.96 0.89 25.77

N 14 14 14 14 14 14 14

Total Mean 6.68 6.58 12.50 8.05 11.70 4.03 48.70

Std.

Deviation 1.38 1.15 1.85 1.63 1.74 0.95 27.63

N 40 40 40 40 40 40 40

Toxoplasma

Age of

puberity

(mon)

Age first service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born

per life

time

Age at

weaning

(mon)

Twining

rate (%)

Negative Mean 7.400 6.22 13.70 8.50 11.20 4.00 36.00

152

Std.

Deviation 1.9551 0.47 2.63 1.78 1.40 1.15 29.51

N 10 10 10 10 10 10 10

Positive Mean 6.433 6.70 12.10 7.90 11.87 4.03 52.93

Std.

Deviation 1.0726 1.29 1.35 1.58 1.83 0.89 26.12

N 30 30 30 30 30 30 30

Total Mean 6.675 6.58 12.50 8.05 11.70 4.03 48.70

Std.

Deviation 1.3847 1.15 1.85 1.63 1.74 0.95 27.63

N 40 40 40 40 40 40 40

Menz

Chlamydia

Age

puberity

(mon)

Age first

service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born per

life time

Age at

weaning

(mon)

Twining

rate (%)

Negative Mean 11.43 13.43 19.57 8.86 8.86 4.86 4.29

Std.

Deviation 3.78 3.21 3.10 3.02 1.95 1.21 9.32

N 7 7 7 7 7 7 7

Positive Mean 11.18 12.32 18.82 8.53 8.32 4.97 1.15

Std.

Deviation 4.84 4.33 4.39 2.05 2.17 1.03 2.90

N 34 34 34 34 34 34 34

Total Mean 11.22 12.51 18.95 8.59 8.41 4.95 1.68

Std.

Deviation 4.63 4.15 4.17 2.20 2.12 1.05 4.63

N 41 41 41 41 41 41 41

Q-fever

Age

puberity

(mon)

Age first

service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born per

life time

Age at

weaning

(mon)

Twining

rate (%)

Negative Mean 11.00 11.00 17.25 8.25 7.75 5.25 0.00

Std.

Deviation 2.00 2.00 2.87 2.63 1.26 1.26 0.00

N 4 4 4 4 4 4 4

Positive Mean 11.24 12.68 19.14 8.62 8.49 4.92 1.86

Std.

Deviation 4.85 4.30 4.28 2.19 2.19 1.04 4.84

N 37 37 37 37 37 37 37

Total Mean 11.22 12.51 18.95 8.59 8.41 4.95 1.68

Std.

Deviation 4.63 4.15 4.17 2.20 2.12 1.05 4.63

N 41 41 41 41 41 41 41

Toxoplasma

Age

puberity

(mon)

Age first

service

(mon)

Age first

lambing

(mon)

Lambing

interval

(mon)

Lamb

born per

life time

Age at

weaning

(mon)

Twining

rate (%)

Negative Mean 10.75 12.35 18.85 9.15 7.90 5.10 0.25

Median 11.00 12.00 18.00 9.00 8.00 5.00 0.00

Std.

Deviation 4.82 4.34 4.51 2.03 2.36 1.12 1.12

N 20 20 20 20 20 20 20

Positive Mean 11.67 12.67 19.05 8.05 8.90 4.81 3.05

153

Std.

Deviation 4.52 4.05 3.93 2.27 1.79 0.98 6.14

N 21 21 21 21 21 21 21

Total Mean 11.22 12.51 18.95 8.59 8.41 4.95 1.68

Std.

Deviation 4.63 4.15 4.17 2.20 2.12 1.05 4.63

N 41 41 41 41 41 41 41

Annex: 17 Month of pregnancy for abortion by region

Menz

Horro

Bonga

Total

Pregnancy (mon) Frequency % Frequency % Frequency % Frequency %

P-

value

Two 1 20 3 18.8 0 0 4 16.7 .487

Three 4 80 7 43.8 3 100 14 58.3

Four 0 0 5 31.3 0 0 5 20.8

Five 0 0 1 6.3 0 0 1 4.2

Annex: 18 The cause of lamb mortality in 2014/15

Variables No. of respondents % of respondents

Diarrhea 31/78 25.8

Coughing 21 17.5

Drought 17 14.2

Sudden death 6 5.0

Circling 6 5.0

lamb not suckling dam milk 6 5.0

swelling of head 5 4.2

swelling of neck 2 1.7

shallow breathing 2 1.7

Bloat 2 1.7