seroprevalence of reproductive and respiratory …
TRANSCRIPT
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.
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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).
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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
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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
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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).
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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
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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
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(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
R.pdf
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).
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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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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