JIMMA UNIVERSITY

COLLEGE OF AGRICULTURE AND VETERNARY MEDICINE

SCHOOL OF VETERINARY MEDICINE

DEPARTMENT OF VETERNAY MICROBIOLOGY

SEROPREVALENCE OF BOVINE BRUCELLOSIS AND ITS ASSOCIATED RISK

FACTORS IN ESSERA DISTRICT, DAWURO ZONE, SOUTH WEST ETHIOPIA

MSc THESIS PROPOSAL

BY: AREGA ABERA AHSA

MAJOR ADVISOR: Dr. DERARTU TESFAYE (DVM, MSc ASSISTANT PROFESSOR)

CO-ADVISOR: Dr. NURRADIS IBRAHIM (PhD)

JANUARY, 2024
JIMMA, ETHIOPIA
 SEROPREVALENCE OF BOVINE BRUCELLOSIS AND ITS ASSOCIATED RISK
FACTORS IN ESSERA DISTRICT, FROM DAWURO ZONE

BY: AREGA ABERA AHSA

MSc THESIS PROPOSAL

Submitted to the Jimma University College of Agriculture and Veterinary

Medicine Department of Veterinary Micirobiology (Post Graguate) Partial Fulfillment of
the Requirements for the Degree of Master of Science in Veterinary microbiology

MAJOR ADVISOR: Dr. DERARTU TESFAYE (DVM, MSc ASSISTANT PROFESSOR)

CO-ADVISOR: Dr. NURRADIS IBRAHIM (PhD)

JANUARY, 2024

JIMMA, ETHIOPIA
 APPROVAL SHEET

Program of Study MSc in Veterinary Microbiology

Title Seroprevalence of Bovine Brucellosis and Its Associated Risk Factors in Essera
District, Dawuro Zone, South West Ethiopia

I have completed my thesis research work as per the approved proposal and it has been evaluated
and accepted by my advisors. Hence, I hereby kindly request the department to allow me to
submit proposal.

Candidate signature Date

Arega Abera, ID No RM 0619/15 ------------------ ---------------
We the proposal advisors have evaluated the content of the proposal and found it to be
satisfactory, executed according to the approved proposal, written according to the standard and
format of the university and is ready to be submitted. Hence, were recommended the proposal to
be submitted.
Name of Principal Advisor Signature Date
Dr. Derartu Tesfaye (DVM, MSc Assistant Professor) -------------- --------------

Co-advisor Signature Date

Dr. Nurradis Ibrahim (PhD ----------------- -------------------

Decision/suggestion of Department Graduate Council (DGC)

Chair person, DGC Signature Date

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

Chair person, CGS Signature Date

------------------------------------ ------------------------- --------------------
Table of Contents pages
ACKNOWLEDGEMENTS..........................................................................................................5
LIST OF TABLES.........................................................................................................................6
LIST OF FIGURE.........................................................................................................................7
ABBREVIATIONS........................................................................................................................8
ABSTRACT..................................................................................................................................11
1. INTRODUCTION.....................................................................................................................1
1.1. Background.......................................................................................................................................1
1.2. Statement of Problems......................................................................................................................2
1.3. Objectives...........................................................................................................................................4
1.3.1. General Objectives.........................................................................................................4
1.3.2. Specific objectives..........................................................................................................4
2. LITERATURE REVIEW.........................................................................................................5
2.1. Historical Overview of Brucellosis..................................................................................................5
2.2. Brucella species and Their Host Preferences.................................................................................5
2.2.1. Characteristics of Brucella Organisms...........................................................................6
2.2.2. Growth and biochemical properties...............................................................................6
2.2.3. Antigenic characteristics................................................................................................7
2.2.4. Molecular characteristics................................................................................................8
2.3. Brucellosis in Animals: Clinical Characteristics............................................................................9
2.4. Pathogenesis.....................................................................................................................................10
2.5. Diagnosis of Bovine Brucellosis.....................................................................................................11
2.5.1. Bacteriological detection methods...............................................................................11
2.5.2. Serological diagnosis....................................................................................................12
2.5.3. Molecular biology techniques......................................................................................14
2.6. Epidemiology of Brucellosis in Animals.......................................................................................14
2.6.1. Epidemiology of bovine brucellosis in Ethiopia..........................................................16
2.7. Mode of Transmission......................................................................................................................16
2.8. Treatment, Prevention and Control...................................................................................................17
2.8.1. Classification of endemic areas based on prevalence..................................................17
2.8.2 .Characterization of Brucella species............................................................................18
 2.8.3. Application of farm Biosafety measures......................................................................18
2.8.4. Application of veterinary extension.............................................................................19
3. MATERIALS AND METHODS............................................................................................20

3.1. The Study Area...............................................................................................................................20

3.2. Study design....................................................................................................................................21
3.3. Study Animals................................................................................................................................21
3.4. Sampling method and sample size determination.......................................................................22
3.5. Data collection...............................................................................................................................22
3.5.1. Questionnaire survey....................................................................................................22
3.6. Blood sample collection and laboratory tests...............................................................................22
3.7. Serological tests...............................................................................................................................23
3.7.1. Rose Bengal plate test (RBPT)....................................................................................23
3.7.2. Complement Fixation Test (CFT)...............................................................................23
3.7.3. Indirect ELISA............................................................................................................23
3.8. Data Analysis...............................................................................................................................24
4. Work and Budget Plan...........................................................................................................25

5. REFERENCES.......................................................................................................................29

6. Annex.......................................................................................................................................36
ACKNOWLEDGEMENTS
My first thanks go to a head of all things, praise be to the higher most Almighty GOD for his
save, mercy and support in my life.

I would like to express my grateful thanks and sincerest appreciation to my advisors Dr Derartu
Tesfaye and Dr Nurradis Ibrahim as their family relationship, intellectual guidance, helpful
advice, brilliant assistance, very hard work, committed strength and devotion of time in all my
works, and also I would like to express my grateful thanks and appreciation to Dr -Nezif
provision of materials and showing (shaping) direction on tittle how I could prepare my MSc
thesis proposal..
LIST OF TABLES

Table 1 .Work plan...................................................................................................................................25

Table 2. budget plan..................................................................................................................................26
Table 3. Laboratory equipment’s.............................................................................................................27
Table 4 .Stationary materials..................................................................................................................28

LIST OF FIGURE
Figure 1. Map of study area .....................................................................................................................21
ABBREVIATIONS

AMOS Abortus-Melitensis-Ovis-Suis

BAT Blood Agglutination Test

CFT Complement Fixation Test

CO2 Carbon dioxide

CSA Central Statistical Agency

DNA Deorxybonueclic Acid

ELISA Enzyme-linked Immune Sorbent Assay

EWAEPFCO Essera Woreda Agriculture, Environmental Protection, Forestry and

Cooperative Offices

EWFEDO Essera Woreda Finance and Economy Development Office

FAO Food and Agriculture Organization

GDP Gross Domestic Product

MHC Major Histocompatibility Complex

MZN Modified Ziehl-Neelsen

NRAMP Natural Resistance Associated Monocyte Protein

ORF Open Reading Frames

PCR Polymerase Chain Reaction

RBT Rose Bengal Test

RES Reticuloendothelial System

RLPS Rough lipopolysaccharide

SAT Serum Agglutination Test

S-LPS Smooth lipopolysaccharide

WHO World Health Organization

ABSTRACT
Ethiopia is a resourceful country with estimated cattle population of 59.5 million (CSA, 2017).
The livestock subsector has an enormous contribution to a national economy and livelihoods of
many Ethiopians and still promising to rally round the economic development of the country.
However, trans-boundary and zoonotic animal diseases such as bovine brucellosis constrain the
livestock sector of the country and affect livelihoods via their impact on animal health, animal
food production, availability and quality Brucellosis is one of the oldest and most widespread
zoonotic diseases, affecting food production in the tropics and subtropics and caused by
different species of the Genus Brucella. The Brucella species are facultative intracellular
pathogensthat can survive, multiply, and persist within phagocytic cells of the host resulting in
lifetime carriage of the organisms. The mode of transmission of the bacteria varies with the
epidemiological area, the animal reservoir and the occupational exposed groups. Factors such as
animal diseases, poor genetics, inadequate animal health services, nutritional deficiencies, and
management issues have all contributed to this situation. Also the objectives are to determine the
seroprevalence of bovine brucellosis and to assess the possible its risk factors in semi-intensive
farm system of the Essera woreda, Dawuro zone
1. Introduction

1.1. Background
Ethiopia is a resourceful country with estimated cattle population of 59.5 million (Belachew,
2019).The livestock subsector has an enormous contribution to a national economy and
livelihoods of many Ethiopians and still promising to rally round the economic development of
the country. The subsector contributes about 16.5% of the national Gross Domestic Product
(GDP) and 40% of the agricultural GDP excluding the values of draught power, manure and
trans-port of people and products(Zinchenko, 2017)

However, trans-boundary and zoonotic animal diseases such as bovine brucellosis constrain the
livestock sector of the country and affect livelihoods via their impact on animal health, animal
food production, availability and quality. Bovine brucellosis has a great impact on both animal
and human health as well as tremendous socio-economic impact in developing countries where
rural income relies largely on livestock breeding and dairy products(Lokamar et al., 2020) .
Brucellosis is considered by Food and Agriculture Organization (FAO), World Health
Organization (WHO) and World Organization for Animal health (OIE) as one of the most
widespread zoonosis in the world .According to OIE, it is the third most important zoonotic
disease in the world after rabies and anthrax. The disease affects cattle, swine, sheep, goats,
camels and dogs. It may also infect other wild ruminants and marine mammals.

Brucellosis is one of the oldest and most widespread zoonotic diseases, affecting food
production in the tropics and subtropics and caused by different species of the genus
brucella(Akeberegn and Dawit, 2018). The six classical species are B. abortus in cattle, B.
melitensis in goats, B. suis in pigs, B. canis in dogs, B. ovis in sheep, neotomae in rat. Brucella
abortus, B.melitensis, B. suis and to some extent, B. canis, are responsible for the majority of
infections in animals and humans. Brucella species are facultative intracellular pathogensthat
can survive, multiply, and persist within phagocytic cells of the host resulting in lifetime
carriage of the organism(Dabral, 2014). Then, ultimately, they become sequestered within
monocytes and macrophages of the reticuloendothelial system (RES), such as the lymph nodes,
liver, spleen, and bone marrow. Diseased animals shed the pathogen in uterine discharge,

1
vaginal discharge, and milk, and these bacteria can spread within the herd through ingestion of
contaminated material(Rosales and Ametaj, 2021).

The mode of transmission of the bacteria varies with the epidemiological area, the animal
reservoir and the occupational exposed groups. A precise diagnosis of Brucella spp. infection is
important for the control of the disease in animals and consequently in man. The transmission of
Brucella is usually through direct contact with an infected animal or fetus or indirect contact
with contaminated objects. Various risk factors for bovine brucellosis have been identified,
including herd size, cattle age and sex, management practices, interaction with wildlife,
environmental factors, and the use of different cattle breeds.

Epidemiology of Brucellosis in Animals and other bovidae, Brucella is usually transmitted from
animal to animal by contact following an abortionDadar et al. (2021). Pasture or animal barn
may be contaminated and the organisms are probably most frequently acquired by ingestion but
inhalation, conjunctival inoculation, skin contamination and udder inoculation from infected
milking cups are other possibilities. Brucellosis is a zoonotic infection that commonly affects
cattle in Ethiopia, causing significant negative economic impact(Tulu, 2022) .Also it is one of
the most widespread but neglected zoonotic diseases in developing countries including Ethiopia
where it is an endemic and growing problem causing public health impacts(Mersha et al., 2021).
Key factors such as animal diseases, poor genetics, inadequate animal health services, nutritional
deficiencies, and management issues have all contributed to this situation. One significant
animal disease that affects both cattle and humans is brucellosis, which has a high
seroprevalence in areas where people live in close proximity to livestock(Islam et al., 2013).
Various authors have conducted serological evaluations to determine the prevalence of Brucella
infections in Ethiopian cattle across different regions of the country(Legesse et al., 2023).

1.2. Statement of Problems

The most significant economic losses are usually incurred following bovine brucellosis(Kiros et
al., 2016). For instance, the serological prevalence of bovine brucellosis in Central America

2
during the last 10 years has been estimated between 4 and 8%, with higher prevalence in dairy
herds and with losses calculated at US dollar 25 million per year. In Ethiopia, information on
losses specifically through brucellosis in the different types of production system is
sparse(Yohannes et al., 2013). In Ethiopia, the first cases of brucellosis reported in the 1970s.
Since the first report of brucellosis the disease has been noted as one of the important livestock
diseases in the country. The individual animal level prevalence of bovine brucellosis in the study
area was 2.7% and the herd-level prevalence was 25.8%. Higher prevalence was observed in
larger herd sizes than the small and medium herds in smallholder dairy farms in Hawassa
town(Abera et al., 2019). So to narrow this gap the study will be conducted seroprevalence of
bovine brucellosis in Essera District Dawuro zone South West Ethiopia Region.

Brucellosis is a public health problem with adverse health implications both for animals and
human beings as well as economic implications for individuals and communities. Management,
animal movement, wide ranges of host, herd size, commingling of different animal species is
risk factors for animal brucellosis. The possible risk factors for human brucellosis are feeding
behavior occupational exposure, contact with diseased animals or their products and
discharges(Pereira et al., 2020).

The inadequate studies (the surveys) like lack information, missed sample collection, improper
use of sampling techniques and procedure, poor transportations and storage(Lippi et al., 2011).
So far conducted on brucellosis are not sufficient to show the exact national picture and
significance except highlighting the existence of the disease in very limited areas of the country
which were selected not based on strategic national disease survey approach but on personal
preference and motives of the investigators or researchers. Moreover, most of the studies so far
conducted were based on serological diagnostic technique; such as RBPT, CFT, I-ELISA,SAT
and BAT most of which were not according to OIE recommendation for international trade for
their sensitivity and specificity(Adam, 2018). The overall infection risk is also influenced by the
pattern of Brucella spp. present; as B.melitensis often represents a more serious public health
hazard than B. abortus (Mitiku and Desa, 2020).

Although the livestock sector in Ethiopia has a significant contribution to the national economy,
productivity (meat and milk) per animal is very low, majorly due to technical constraints and
disease like brucellosis. Crossbreeding indigenous cattle with high yielding exotic cattle is the

3
main policy established by the Ethiopian government to bridge the gap between supply and
demand for dairy products(Abera, 2016).
Brucellosis was categorized under tier one zoonotic diseases. The disease is known to be an
endemic and a growing problem in domestic livestock herds in Ethiopia causing significant loss
of productivity through abortion, prolonged calving, kidding, or lambing interval, low herd
fertility, and comparatively low milk production in farm animals as well as chronic and febrile
illness in humans. However, there are no feasible intervention mechanisms currently undergoing
in Ethiopia(Edao, 2021).
Additions to this Bovine Brucellosis was not studied and had no real information still in the
study areas of Dawuro Zone in Essera Woreda of Southwest Ethiopia Regional State by on semi-
intensive individual farm management systems. Including this year and consecutive for last five
years there has been increasing in abortion, decreasing milk production ,infertility and repeated
breeding occurred in cattle population(Eshete et al., 2023). Especially increasing in milking
cows but there was no documented information on status of bovine brucellosis in study area.
There are many small and medium dairy cattle owners growing and supply raw milk and milk
products (cheese, butter .etc ) and raw meat or flesh for the communities in Essera town and
other surrounding woreda of zone. The demand for consumption of milk .milk products and
meat in the area also increasing time by time this may leads to zoonotic diseases like
brucellosis(Douphrate et al., 2013). So, this study will be narrowed the gap and provided more
information on seroprevalence of bovine brucellosis and its potential risk factors that predispose
the animals in the study area. Therefore objectives of the current study will be:

1.3. Objectives

1.3.1. General Objectives

To determine the seroprevalence of bovine brucellosis in semi-intensive farm system of the
Essera woreda, Dawuro zone

1.3.2. Specific objectives

 To determine the seroprevalence of bovine brucellosis
 To assess the possible risk factors associated with bovine brucellosis in the study area.
 To assess the knowledge of farmers about bovine brucellosis in the study area.

4
2. LITERATURE REVIEW

2.1. Historical Overview of Brucellosis

Brucellosis is an ancient disease that can possibly be traced back to the 5th plague of Egypt
around 1600 BC. Recent examination of the ancient Egyptian bones, dating to around 750 BC,
showed evidence of sacroiliitis and other osteoarticular lesions, common complications of
brucellosis.(Seleem et al., 2010).
Eighteen centuries later, Sir David Bruce isolated Micrococcus melitensis (now B.melitensis)
from the spleen of a British soldier who died from a febrile illness (Malta fever) common among
military personnel stationed on Malta, an island not far away from Herculaneum The zoonotic
nature of the brucellosis was accidentally demonstrated in 1905 by isolating B. melitensis from
goat’s milk used for the production of soft cheese in Malta(Godfroid et al., 2005). In 1953,
B.ovis was identified as a cause of epididymitis in rams In the last 15 years 3 new non-classical
species of Brucella has been identified.

2.2. Brucella species and Their Host Preferences

Secondary hosts play a minor role in the maintenance and spread of a particular Brucella species.
Brucella abortus mainly infects cattle and is the main cause of contagious abortion. However,
sheep, goats, dogs, camels, buffaloes as well as feral animals may also contract B. abortus
infections.(Abdulzahra et al., 2022).Although sheep do not easily become infected with B.
abortus, they may become carriers and excrete brucellae for up to 40 months once they have
acquired the infection. This organism is a Gram negative, facultative intracellular pathogen, and
contains three biovars. All these biovars can cause disease in small ruminants, but their
geographic distribution varies. Isolation of B. abortus from swine ,horses and camels in areas
with enzootic brucellosis clearly indicates that these species may acquire infection with B.
abortus.(Behroozikhah et al., 2012).

2.2.1. Characteristics of Brucella Organisms

Brucellaea are facultative intracellular bacteria comprising of various species belonging to order
Rhizobials and family alpha proteobactereacea. Brucellaea are a small (0.6x0.6 to 1.5 μ m) gram
negative, non-motile, non-spore forming, rod shaped (cocco bacillary) bacteria. They are
partially acid fast positive because they are not decolorized by 0.5% of acetic acids in Modified

5
Ziehl-Neelsen (MZN) staining techniques. In MZN stained smears, the organisms appear as
cluster of red staining coccobacilli because they retain the carbol fuchsine(Markey et al., 2013).
The genus Brucella comprises of ten species based on their difference in host specificity. Of
these, six of them are called classical Brucella species affecting terrestrial animals. These include
of Brucella melitensis (goats, sheep, biovars 1-3), B. abortus (cattle, biovars 1-6 and 9), B. suis
(pigs, reinder’s and hares, biovars 1-5), B. ovis (sheep), B. canis (dogs) and B. neotomae (desert
wood rats) Recently, four additional new Brucella species has been identified(Khan, 2017).
These are B. ceti and B. pinnipedialis, which were recently identified in marine mammals with
Cetaceans (dolphin, porpoise, and whale species) and Pinnipeds (various seal species) as
preferred hosts respectively. B. microti was isolated from the common vole and B. inopinata
was. isolated from a breast implant wound of a female patient (Nymo et al., 2011).

2.2.2. Growth and biochemical properties

Brucellaea are aerobic, but some strains require an atmosphere containing 5-10% carbon dioxide
(CO2) for growth, especially on primary isolation. The optimum pH for growth varies from 6.6
to 7.4, and culture media should be adequately buffered near pH 6.8 for optimum growth. The
optimum growth temperature is 36-38°C, but most strains can grow between 20°C and 40°C.
Brucellaea require biotin, thiamin and nicotinamide for growth. The growth is improved by
serum or blood, but haemin (V-factor) and nicotinamide-adeninedinucleotide (X-factor) are not
required.

The growth of most Brucella strains is inhibited on media containing bile salts, tellurite or
selenite. Growth is usually poor in liquid media unless culture is vigorously agitated(Teske et al.,
2007). Growth in static liquid media favors dissociation of smooth-phase cultures to non-smooth
forms. Continuous and vigorous aeration will prevent this, provided a neutral pH is maintained.
In semi-solid media, CO2-independent Brucella strains produce a uniform turbidity from the
surface down to a depth of a few millimeters, while cultures of CO2-requiring strains produce a
disk of growth a few millimeters below the surface of the medium (OBONYO, 2018).

On suitable solid media Brucellaea colonies are visible after 2 days of incubation. After 4 days of
incubation, Brucellaea colonies are round, 1-2 mm in diameter, with smooth (S) margins, translucent and
a pale honey color when plates are viewed in the daylight through a transparent medium. When viewed

6
from above, colonies appear convex and pearly white. Later, colonies become larger and slightly darker 2
(Haque, 2008). Smooth Brucellaea cultures, such as B. melitensis cultures, have a tendency to undergo
variation during growth, especially with subcultures, and dissociate to rough (R) forms, and sometimes
mucoid (M) forms. Colonies are then much less transparent with a more granular, dull surface (R) or a
sticky glutinous texture (M), and range in color from matt white to brown in reflected or transmitted light.
Intermediate (I) forms between S, R and 9.

M forms may occur in cultures undergoing dissociation to the non-smooth state. Changes in the
colonial morphology are generally associated with changes in virulence, serological properties
and phage sensitivity(Nielsen, 2018). The metabolism of Brucellaea is oxidative and Brucellaea
cultures show no ability to acidify carbohydrate media in conventional tests. The Brucella
species are catalase positive and usually oxidase positive, and they reduce nitrate to nitrite
(except B. ovis and some B. canis strains). The production of H2S from sulphur containing
amino-acids also varies. B. melitensis does not produce H2S. Urease activity varies from fast to
very slow. Indole is not produced from tryptophane and acetylmethycarbinol is not produced
from glucose.

2.2.3. Antigenic characteristics

The outer cell membrane of the virulent and zoonotic species such as B. abortus, B. meletensis
and B. suis is made of smooth lipopolysaccharide (S-LPS) motifs. The S-LPS contains an O15
polysaccharide (OPS) that is chemically defined as a homo polymer of 4,6-dideoxy-4-
formamide-alpha-D-mannose, linked via 1,2-glycosidic linkages (Nielson, 2002) whereas the
non-smooth species such as B. ovis and B. canis lack the OPS on their LPS and have a rough
lipopolysaccharide (R-LPS) instead. The lack of the OPS rendered these rough species more
immunogenic following infection of the hosts. It is believed that the S-LPS are able to evade
innate immunity and to be a less potent inducer of inflammatory cytokines (Levinson, 2016).
These activities protect the bacterium against the initial immune response and enhance its ability
to survive in the host. Once inside a phagocytic cell, the S-LPS species are able to prevent the
infected cell from antigen presentation to T helper cells via the major histocompatibility complex
II (MHC II). The S-LPS enables the bacteria to hinder apoptosis by the infected cell as well.
These evasion techniques add to the S-LPS Brucella spp. virulence in comparison to the R-LPS
strains. The latter are unable to inhibit the host immune response and are greatly impacted by the

7
innate immune system, and are thus prevented from having a more severe effect on the host
(Brown et al., 2013).

2.2.4. Molecular characteristics

Over 40 Brucella phages have been reported to be lytic for Brucella members. All phages are
specific for the genus Brucella, and are not known to be active against any other bacteria that
have been tested. Thus, lysis by Brucella phages is a useful test to confirm the identity of
Brucella spp. and for speciation within the genus. The Brucella phages currently used for
Brucella typing are: Tbilisi (Tb), Weybridge (Wb), Izatnagar1 (Iz1) and R/C. The three former
phages are used for differentiation of smooth Brucella species. R/C is lytic for B. ovis and B.
canis (Banai and Corbel, 2010).

The genome of most Brucella species consists of two circular chromosomes or replicons of 1.1
and 2.2 Mb The genome of B. melitensis strain 16 M contain 3,294,935 (base pairs) bp
distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197
open reading frames (ORFs). The complete sequencing of the B. melitensis genome was
achieved in 2002. Natural plasmids have not been detected .Sequencing and annotation of the
genomes of B. suis, B. melitensis, and B. abortus has been completed; the majority of the open
reading frames share greater than 99 percent sequence identity between species(Bialer et al.,
2021).

2.3. Brucellosis in Animals: Clinical Characteristics

Brucellosis is a sub-acute or chronic disease which may affect many species of animals. In cattle,
sheep, goats, other ruminants and pigs the initial phase following infection is often not apparent.
In sexually mature animals the infection localizes in the reproductive system and typically
produces placentas followed by abortion in the pregnant female, usually during the last third of
pregnancy, and epididymitis and orchitis in the male. Clinical signs are not pathognomonic and
diagnosis is dependent upon demonstration of the presence of Brucella spp. either by isolation of
the bacteria or detection of their antigens or genetic material, or by demonstration of specific
antibody or cell-mediated immune responses(Al-ouqaili, 2006). Characteristic but not specific
signs of brucellosis in most animal hosts are abortion or premature births and retained placenta.
In some areas, abortion is relatively uncommon. In some parts of Africa, hygromas and
abscesses are the major clinical signs in nomadic or semi-nomadic cattle herds infected with B.
8
arbortus biovar 3. There is lowered milk production due to premature births. Interference with
fertility is usually temporary and most infected animals will abort only once and some are
unaffected.

The udder is often permanently infected, especially in the case of cows and goats. Shedding of
organisms in milk is frequent. Localized infections in sheep result in orchitis or epididymitis in
the case of B. melitensis and B. ovis. In goats, cattle, swine and dogs similar complications may
follow infection with B. melitensis, B. abortus, B. suis and B. canis respectively. Arthritis may
also be a rare sign in B. melitensis-infected sheep and goats. In horses, local abscess formation in
bursae may be the only clinical sign and infection in this species is often asymptomatic. Camels
infected with B. melitensis shed the organisms in milk and in some countries this is a serious
public health problem. Clinical signs of brucellosis in camels appear to be very rare (Wakene and
Mamo, 2017).

The severity of the disease depends upon many factors such as previous vaccination, age, sex
and management such as herd or flock size and density. Abortions are more prevalent in
unvaccinated animals and numbers of organisms shed are much greater. The bacteria are found
in tissues and fluids associated with pregnancy, the udder and the lymph nodes which drain the
relevant areas. Most infections result from ingestion of bacteria either from diseased animals or
contaminated feedstuffs(Sapkota et al., 2007). However, infection may also be acquired by
respiratory exposure and by contamination of abraded skin and mucosal surfaces. Natural
breeding transmits infection in swine and dogs and, to a lesser extent, sheep and goats. Persistent
bacteraemias are also more common in the first two species. Bacteraemia occurs during the
course of infection in other species but is usually intermittent and of short duration(Warren,
2001).

2.4. Pathogenesis
An important aspect of Brucella infection is its ability to persist and replicate within phagocytic
cells of the reticuloendothelial system as well as in non-phagocytic cells such as trophoblasts.
This ability involves a temporary fusion of the Brucella-containing vacuole with the lysosome,
and subsequent exclusion of the lysosomal proteins soon after internalization by the phagocytes,
the Brucella-containing vacuole interacts with early and late endosomes. The majority of

9
phagocytosed Brucella is destroyed by bactericidal action of free radicals of oxygen, nitric oxide
and enzymes inside phagolysosomes(Adem and Duguma, 2020). However, a certain number of
bacteria resist these bactericidal mechanisms, and after transient fusion with the lysosome can
actively exclude lysosomal proteins and redirect intracellular trafficking of Brucella-containing
vacuole to the endoplasmic reticulum, where the organism is capable of replicating Replication
leads to release of the bacteria from the cells, thus resulting in a bacteremic phase.

The Brucella virulence factor is thought to play a significant role in these intracellular survival
events. The Brucella virulence factor is a secretory pump that selectively pumps proteins and
macromolecules across membranes and is critical in pathogenesis and virulence of Brucella
infections.(Coloma-Rivero et al., 2021). It helps ensure the survival of the bacteria in the
phagolysosome and establishing the infection Lipopolysaccharide is another virulence factor of
Brucella that contributes to initial survival of bacteria in macrophages. The localization of
Brucella spp. in the reproductive tract leads to colonization of the chorionic trophoblasts of the
placenta in pregnant livestock. This affinity for the chorionic trophoblastic cells is due to the
presence of steroid hormones and erythritol which is an important substance present in allantoic
fluids that stimulates the replication of Brucella species(Anderson, 1985).

10
The stimulation of Brucella spp. seen in the presence of erythritol is due to the preferential use of
erythritol by Brucella spp. as an energy and carbon source, even in the presence of glucose and
other metabolites. The reason for the preferential use of erythritol is due to its ease of uptake by
the bacteria, as compared to glucose. This makes erythritol more readily available to the bacteria
for energy 13 consumption(Anderson, 1985). Growth of Brucella inside the trophoblasts also
become prominent when the concentration of steroid hormones of PGF2α, estrogen and cortisol
and erythritol is higher and acts together during late gestation of ruminants which results in late
term abortion or birth of weak offspring in case of female animals and epididymitis and orchitis
in male animals. The resulting placentitis caused by replicating bacteria results in ulceration of
the chorioallantoic membrane while sparing the endometrium of the uterus. The resulting
pathology leads to late gestation abortions in naïvely infected livestock(Schlafer and Foster,
2016) .

2.5. Diagnosis of Bovine Brucellosis

2.5.1. Bacteriological detection methods

The isolation and identification of Brucella offers a definitive diagnosis of brucellosis and may
be useful for epidemiological purposes and to monitor the progress of a vaccination programme.
It should be noted that all infected materials present a serious hazard, and they must be handled
with adequate precautions during collection, transport and processing. Smears of placental
cotyledon, vaginal discharge or fetal stomach contents may be stained using modified Ziehl-
Neelsen (Stamp) or Kosters‟ methods. The presence of large aggregates of intracellular, weakly
acid-fast organisms with Brucella morphology is presumptive evidence of brucellosis. Care must
be taken as other infectious agents such as Coxiella burnetii or Chlamydia may superficially
resemble Brucella(Adam, 2018).

Isolation may be performed by culturing body tissues or secretions like blood, milk and virginal
discharge. Brucella species can also be cultured from pus, cerebrospinal fluid, and pleural, joint
and ascetic fluids. Growth of the bacteria in culture media is an unequivocal proof of infection.
The identification of Brucella species in culture depends on a great deal of phenotypic traits such
as: CO2 requirement, phage typing and biochemical tests, which, among other problems, involve
time, bio-safety, trained personnel and somewhat ambiguous results(Kaltungo et al., 2014).

11
Broth or agar can be prepared from powder media for culture(Kaltungo et al., 2014) of Brucella
organisms. Due to the low Brucella load in 14 the blood and other body fluids, broth or a
biphasic medium are preferable for their culture. However, for other specimens, solid media with
2.5% agar facilitates the recognition of colonies and discourage bacterial dissociation. Optimum
pH for growth of Brucella varies from 6.6 to 7.4, and culture media should be adequately
buffered near pH 6.8 for optimum growth. The optimum growth temperature is 36-38°C.
However, most strains grow between 20 and 40°(Lip et al., 2020).

The most widely used selective medium is the Farrell’s medium which is prepared by the
addition of six antibiotics to a basal medium(De Miguel et al., 2011). On suitable solid media,
Brucella colonies can be visible after 2–3-days of incubation. After 4 days‟ of incubation,
Brucella colonies are round, 1–2 mm in diameter, with smooth margins. They are translucent and
a pale honey colour when plates are viewed in the daylight through a transparent medium. When
viewed from above, colonies appear convex and pearly white. Later, colonies become larger and
slightly darker. Smooth (S) Brucella cultures have a tendency to undergo variation during
growth, especially with subcultures, and to dissociate to rough (R) forms. Colonies are then
much less transparent, have a more granular, dull surface, and range in colour from matt white to
brown in reflected or transmitted light(Reddy et al., 2007).

2.5.2. Serological diagnosis

The most efficient and cost-effective method is usually screening all samples using a cheap and
rapid test which is sensitive enough to detect a high proportion of infected animals. Samples
positive to screening are then tested using more sophisticated, specific confirmatory tests for the
final diagnosis to be made. It is absolutely essential that only internationally recognized tests using
antigens standardized against the 2nd International anti-B. Abortus Serum is used. Appropriate
quality control sera should be included with each batch of tests, and tests should be repeated if
the quality control criteria are not met. Serological results must be interpreted against the
background of disease incidence, use of vaccination and the occurrence of false positive
reactions due to infection with other organisms.(Fierz, 2004). The RBT is one of a group of tests
known as the buffered Brucella antigen tests which rely on the principle that the ability of IgM
antibodies to bind to antigen is markedly reduced at a low pH. The RBT and other tests such as
the buffered plate agglutination tests and the card test play a major role in the serological
12
diagnosis of brucellosis worldwide. The RBT is a simple spot agglutination test where drops of
stained antigen and serum are mixed on a plate and any resulting agglutination signifies a
positive reaction. The test is an excellent screening test but may be oversensitive for diagnosis in
individual animals, particularly vaccinated ones. The procedure can be automated but this
requires custom-made equipment(Barbuddhe et al., 2020).

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 RBT, sometimes they do not detect infected animals which are RBT positive. It is also
important to note that ELISAs are only marginally more specific than RBT or CFT (Legesse et
al., 2023).

Complement fixation test (CFT) is another commonly used serological methods. The sensitivity
and specificity of the CFT is good, but it is a complex method to perform requiring good
laboratory facilities and trained staff(McGiven et al., 2003). If these are available and the test is
carried out regularly with good attention to quality assurance, then it can be very satisfactory. It
is essential to titrate each serum sample because of the occurrence of the prozone phenomenon
whereby low dilutions of some sera from infected animals do not fix complement. This is due to
the presence of high levels of non-complement fixing antibody isotypes competing for binding to
the antigen. At higher dilutions these are diluted out and complement is fixed. Such positive
samples will be missed if they are only screened at a single dilution. In other cases,
contaminating bacteria or other factors in serum samples fix or destroy complement causing a
positive reaction in the test, even in the absence of antigen. Such “anti-complementary” reactions
make the test void and a CFT result cannot be obtained. In addition to these commonly used
sero-diagnostic methods, supplementary tests such as Milk ring test, Fluorescence polarization

13
assay, Intradermal test, Serum agglutination test (SAT) and milk ELISA have been used seldom
for diagnosis of bovine brucellosis(Barbuddhe et al., 2020).

2.5.3. Molecular biology techniques

The polymerase chain reaction (PCR) is a recent and promising technique that allows for rapid
and accurate diagnosis of bovine brucellosis without the limitations of the conventional
methodology. Several genus-specific PCR systems using primer pairs that target 16S RNA
sequences and genes of different outer membrane proteins have been developed. The first
species-specific multiplex PCR was called Abortus-Melitensis-Ovis-Suis (AMOS-PCR) assay,
which is used to identify and differentiate B. abortus biovars 1, 2 and 4, B. melitensis, B. ovis and
B. suis biovar 1. The PCR is based on the polymorphism arising from species specific
localization of the insertion sequence IS711 in the Brucella chromosome. A Bruce-ladder
multiplex PCR assay was also developed for identification and differentiation of Brucella species
and vaccine strains in a single step(Scarpellini et al., 2002).

2.6. Epidemiology of Brucellosis in Animals

In cattle and other bovidae, Brucella is usually transmitted from animal to animal by contact
following an abortion. Pasture or animal barn may be contaminated and the organisms are
probably most frequently acquired by ingestion but inhalation, conjunctival inoculation, skin
contamination and udder inoculation from infected milking cups are other possibilities. The use
of pooled colostrums for feeding newborn calves may also transmit infection. Sexual
transmission usually plays little role in the epidemiology of bovine brucellosis. However,
artificial insemination can transmit the disease and semen must only be collected from animals
known to be free of infection. In sheep and goats, B. melitensis is nearly always the infecting
species. B. ovis can also infect sheep but is of little significance in relation to human disease. The
mode of transmission of B. melitensis in sheep and goats is similar to that in cattle but sexual
transmission probably plays a greater role(Rossetti et al., 2022).

The transmission of disease is facilitated by commingling of flocks and herds belonging to

different owners and by purchasing animals from unscreened sources. The sharing of male
breeding stock also promotes transfer of infection between farms. Transhumance of summer
grazing is a significant promoting factor in some areas as is the mingling of animals at markets

14
or fairs. In cold climates, it can be the custom to house animals in close space and this also
facilitates transmission of infection. Swine brucellosis is transmitted by direct contact with
recently aborted sows, by ingestion of contaminated food or exposure to a contaminated
environment. However, sexual transmission is particularly important. Brucellosis may be
introduced on to farms through the communal use of boars or by purchase of infected animals.

For all species, embryo transfer is safe provided that recommended procedures are followed. B.
canis can be a major problem in dog breeding kennels Polymorphism of the natural resistance
associated monocyte protein (NRAMP) gene has been shown to influence substantially
susceptibility to brucellosis in cattle and pigs. However, management practices are far more
important in determining the risk of infection. Latent or in apparent infections can occur in all
farm animal species. These usually result from infection in utero or in the early post-natal period.
Such animals can retain the infection for life and may remain serologically negative until after
the first abortion or parturition(McAllister, 2016). Latent infection has been estimated to occur in
the progeny of about 5% of infected cows. The extent of the problem in other species is not
known, but latency has been documented in sheep(Dubey et al., 2006).

Acquired immunity has a substantial effect on susceptibility. Vaccination of cattle with B.

abortus strain 19 or RB 51, or sheep and goats with B. melitensis Rev 1 can reduce susceptibility
a thousand fold or more to the homologous species. B. abortus strain 19 does not protect cattle
against B. melitensis. However, there is little information on the use of Rev1 vaccine in cattle.
The efficacy of this vaccine against the B. melitensis strains prevalent in some areas has also
been questioned .Vaccines must be obtained from a reliable, internationally approved source. It
is possible that strains of B. melitensis exist which can circumvent the immunity induced by this
vaccine (El Idrissi et al., 2001). However, it is at least as probable that variations in vaccine
quality have affected protection rates. For the present, Rev 1 vaccine is the most effective
vaccine available against B. melitensis and in many countries has given very good results. Its use
is 19 recommended when uncontrolled B. melitensis infection exists in ruminant populations.

15
2.6.1. Epidemiology of bovine brucellosis in Ethiopia
Country of Ethiopia particularly associated with cattle in both intensive and extensive
management systems. These prevalence studies in animals and human were largely confined to
serological surveys and commonly targeted bovine brucellosis, occasionally sheep and goats and
rarely camels (Gutema Wegi, 2020). So far, attempts to identify Brucella species in the country
were unsuccessful; the distribution and proportion of their natural hosts was also not studied
exhaustively. This is largely attributed to the degree of laboratory development and lack of
consumables for laboratory tests.

The most significant economic losses are usually incurred following bovine brucellosis. In
Ethiopia, information on losses specifically through brucellosis in the different types of
production systems is sparse, with the exception of Tariku (1994) who reported an annual loss
from brucellosis estimated to be 88,941.96 Ethiopian Birr ($5231 equivalent) among 193 cattle,
largely due to reduced milk production and abortions Both husbandry systems as well as
environmental several institutionally conditions greatly influence the spread of Brucella
infections(Yohannes et al., 2013).Ethiopia has owned commercial dairy farms, mostly situated in
and around Addis Ababa and in some regional towns. These farms have been the focus of most
Brucella surveys, potentially producing a bias in reported findings.

2.7. Mode of Transmission

Transmission is by contact with recently aborted animals or with food or environment
contaminated by abortions or excreta. Sexual transmission is also an important means of spread
and males can excrete the organisms in large numbers in their semen. Urinary excretion also
occurs and is a potential hazard to humans. However, in some countries where B. canis is present
in the dog population, overt human disease caused by this organism seems to occur
infrequently(Djokic et al., 2023). It should be remembered that dogs can acquire infection with
B. abortus, B. melitensis or B. suis from aborted ruminants or swine, usually by ingesting fetal or
placental material. They can then excrete these bacteria and may present a serious hazard to
humans and domestic livestock. B. suis biovar 4 causes brucellosis in caribou and reindeer. The
epidemiology is similar to that of bovine brucellosis. Transmission to people can occur through
the usual routes. However, ingestion of raw or undercooked reindeer bone marrow has also been
implicated as a source of human infection(Corbel, 2006).

16
In cattle, sheep, goats and swine, susceptibility to brucellosis is greatest in sexually mature
animals. Young animals are often resistant, although it should be noted that latent infections can
occur and such animals may present a hazard when mature. Breed may also affect susceptibility,
particularly in sheep. The milking breeds seem to be the most susceptible to B. melitensis. Breed
differences in susceptibility have not been clearly documented in cattle although genetically
determined differences in susceptibility of individual animals have been demonstrated(Clark and
García, 2017).

2.8. Treatment, Prevention and Control

As the source of human brucellosis is direct or indirect exposure to infected animals or their
products, prevention must focus on various strategies that will mitigate infection risks. To our
knowledge, there has been no national program proposed for prevention and control of
brucellosis in Ethiopia. Similarly at regional levels, no strategy is in place to control brucellosis.
This is largely a result of lack of facilities and budget to run such a program. Moreover, many
responsible bodies may not recognize the significance of brucellosis given the contradictory and
sometimes low prevalence data. However, at this time, it is crucial to define geographical extent
of the problem and then allocate resources and funds to initiate prevention and control strategies
in this country(Yach et al., 2004). These strategies have been proposed as follows.

2.8.1. Classification of endemic areas based on prevalence

This will enable instigation of appropriate control method in endemic areas. Identification of low
and high prevalence areas will greatly facilitate the implementation of appropriate control
programs, and should ideally be combined with other strategies like accurate 22 livestock census
data and a livestock identification system (either simple ear notches or more sophisticated ear
labeling system) Vaccine storage and quality control systems are also a priority coupled with
surveillance systems and post-vaccination surveillance to identify the remaining foci of infection
(the efficacy of post-vaccination surveillance is reliant upon existing records combined with
reliable livestock identification (Pal et al., 2017). In areas where the disease is less prevalent (for
example seroprevalence of less than 1%), test and cull policy with compensation may be
recommend. For areas with high and moderate prevalence (>5%) under well-organized farming
systems, we may recommend test and segregation policy by which animals with brucellosis will

17
be isolated and products consumed after pasteurization, with animals being disposed of properly
at the end of their productive live.

2.8.2. Characterization of Brucella species

One of the most successful methods for prevention and control of livestock brucellosis is through
vaccination. In different parts of the world both live vaccines, such as B. abortus S-19, B.
melitensis Rev-1, B. suis S-2, rough B. melitensis strain M111 and B. abortus strain RB51 and
killed vaccines, such as B. abortus 45/20 and B. melitensis H.38 are available. Each vaccine has
been reported to have its own advantages and disadvantages with protection following localized
persistence of live vaccines preferred by most and showing efficacy in small ruminants(Heegaard
et al., 2011).

Use of the RB51 attenuated live vaccine has recently gained popularity for control of brucellosis
in cattle, but on a cautionary note, the failure of this strain to induce serological reactivity,
coupled with its inherent resistance to rifampicin, might complicate detection and management
of zoonotic infection spilling into humans with occupational risk factors for acquiring
brucellosis. Currently, despite huge research efforts, no vaccine has been approved for the
prevention of human brucellosis. Treatment regimes for human brucellosis require combination
of antibiotics. These have recently been compared using meta-analysis. Currently, vaccination
against animal brucellosis has yet to be explored in Ethiopia(Sibhat et al., 2022).

2.8.3. Application of farm Biosafety measures

Implementation of measures to reduce the risk of infection through personal hygiene, adoption of
safe working practices, protection of the environment and food hygiene should minimize risks of
further infection. Under appropriate conditions, Brucella organisms can survive in the
environment for prolonged periods. Their ability to withstand inactivation under natural
conditions is relatively high compared with most other groups of non-sporing pathogenic
bacteria. B. abortus is inactivated by pasteurization and its survival outside the host is largely
dependent on environmental conditions(Russell and Gould, 2003).

The pathogen may survive in aborted fetus in the shade for up to eight months, for two to three
months in wet soil, one to two months in dry soil, three to four months in faeces, and eight

18
months in liquid manure tanks. For example, in nomadic populations where people travel in
search of green pasture and water, the proper handling and burying of abortion materials to
prevent contamination of water sources and pasture is of paramount importance. Furthermore,
the common practice of feeding abortion materials to dogs should be avoided as this increases
the risk of transmission to other animals(Wiebe and Howard, 2009).

2.8.4. Application of veterinary extension

The development of a national veterinary extension services in the country, is essential to
promote awareness about brucellosis, its impact on livestock production and zoonotic risks,
would provide a valuable prevention measure. This would help to unify both community/dairy
cattle producers to control and eliminate brucellosis. Currently, many dairy cattle producers hide
or dispose of animals with a history of abortion, potentially facilitating disease transmission
between farms and regions. This seriously undermines efforts of controlling and preventing the
disease(Brown and Gilfoyle, 2010).

19
3. MATERIALS AND METHODS

3.1. The Study Area

The study will be conducted Essera Woreda (District), Dawuro Zone Southwest Region of
Ethiopia from January 2024 to September 2024. Essera District is located in Dawuro Zone
South-west Region from Addis Ababa at 522, 575 and 584 kms from Addis Ababa through
Hosanna, Shashamene and Jimma roads respectively. Also Essera District located in Regional
City Town of Bonga 274km. The district have 29 kebeles (25 rural and 4 Urban cities kebele)
and total population of Essera district is 117,213 of male are 61373(52.36%),and female
55,840(47.64%) are female(EWFEDO,2023). This district falls into three agro-ecological
regions (kola, woyna dega and dega), of which kola whose elevation ranges from 1000m-1500m
above sea level, woynadega whose elevation ranges from 1500m- 2000m above sea level, and
dega whose elevation ranges from 2000-3000m above sea level.

The district covers a total area of 1043.1 km2 and lies between 6.7-7.020 latitude and 36.7 to
37.10 longitudes. The rainfall is a bimodal type: the short rainy season is between (February to
March) and the long between (May to September). The average annual rainfall ranges between
1401–1800 mm (Tonamo et al., 2015). The agricultural production system in the area was mixed
crop and livestock production system with the land use plan of the area, 38.4% is cultivated land,
13.39% grazing land 16.81% forest bushes and shrubland, 17.09 % cultivable, and 14.31% is
covered by others (Tonamo et al., 2015). The total animal populations of district is 871073 and
in each species (cattle 497,986(495785 and 2201) local and cross breed respectively) ,sheep
57607,goats 33716,equine 26898 (horse 14415,mule 8491 and3992),chicken 138751 and
beehives 116,115 data from Essera Woreda Agriculture,Environmental protection and Forestry
and Cooperative Offices (EWAEFCO,2023).

20
Figure 1 Map of study area .Source: www. Google Map of EWRDO (2013)

3.2. Study design

A cross-sectional study design will be carried out between February 2024 and September 2024 to
screening cattle for bovine brucellosis on using a pre-tested questionnaire and serological test.

3.3. Study Animals

The study consisted of dairy cattle that will be managed under the semi-intensive production
system. The cattle under study comprised of the cross breeds and local indigenous Zebu cattle.
Animals of both sexes and different age group greater than six months will be included in the
study .Dairy cows will be kept under cut grass and supplement feeding on night and natural
grazing on daytime, The local-breed animals mainly will be increased in number .than Holstein
Frisian and Jersey breed.in study.

3.4. Sampling method and sample size determination

The study will be included household dairy farms in Essera District of selected kebele around
Balle, Guza and Offa. Total cattle population of Balle kebele is 9200, Guza is 6250 and Offa
kebele is 6600. From those 384 sample size, Balle 160 head of cattle, Guza 109 head of cattle
and Offa 115 head of cattle will be selected and sampled from in their dairy cattle population

21
potential(EWAEPFCO 2023). The herds will be selected using random sampling techniques, and
then simple random sampling method will be used to sample each individual herds based on the
number of herds present in each households. The sample size will be calculated based on the
formula given below as described by Thrusfield, (2007).
N = (1.96)2P(1-P) ………………………………….. Equation 1
d2
Where n = sample size, Pexp = expected prevalence, and d = absolute precision.
Based on the expectation that disease prevalence will be 50% with 95% confidence level and
5% absolute precision, a sample size of 384 local and crossbreed dairy from requirement of 384
cattle. However, to account for non-response or missing values and avoid bias, sample will be
over took by 10%–20%, as suggested by(Volken, 2013).Therefore, we will be used a total cattle
of 461 in this study A proportionate number of animals will be then sampled from each kebele
based on its dairy cattle population.

3.5. Data collection

3.5.1. Questionnaire survey

A total of 100 farm owners will be interviewed using semi-structured questionnaire. Around
Balle 42.Guza 28 and around Offa 30 owners questionnaire survey with open and closed
questions will be used among the farmer (owners) whose farms will be tested.

3.6. Blood sample collection and laboratory tests

Animals will be restrained by owners and approximately 10 ml of blood sample will be collected
from the jugular vein of each individual animal using vacutainer tubes with 18-20-gauge
hypodermic needles. Each sample from each animal will be labeled by using codes describing
the specific animal and herd/farm and will be left at room temperature for a moment to allow
coagulation, then will be transferred to another sterile cryovial tube and will be transported
respecting the cold chain and finally stored at −20°C until serological testing will be analyzed in
the laboratory.

22
3.7. Serological tests

3.7.1. Rose Bengal plate test (RBPT)

It will be employed as a screening test on the serum samples for the presence of Brucella
agglutinins. The protocol of RBPT as recommended by OIE will be used as screening test for the
presence of Brucella antibody will be in the sampled sera. The sera and antigen will be removed
from the refrigerator 30 min before the test and allowed to reach room temperature. Equal
volumes of test sera will be added to 30 μl of RBPT antigen. The mixture will be thoroughly
mixed, shaken for 4 min and finally agglutination will be observed for positive sera. In the event
of ambiguous results, a repeat test will be conducted.

3.7.2. Complement Fixation Test (CFT)

The CFT test will be used to confirm the results of the RBPT test on serums that will be positive
for the standard Brucella antigen. Titration will be used to assess the reagent preparation, which
will be done in accordance with the World Organization for Animal Health's recommended
protocols (OIE, 2009). Strongly reacting sera, more than 75% fixation of complement (3+) at a
dilution of 1:5, or at least 50% fixation of complement (2+) at a dilution of 1:10 and higher, will
be regarded as positive; absence of fixation/complete hemolysis will be considered negative.

3.7.3. Indirect ELISA

I-ELISA technique will performed using an I-ELISA brucellosis kit following the
recommendations of the manual supplied with the kit. Briefly, medium binding capacity, 96 flat-
bottom wells in polystyrene plates, will be coated with 100 µl of hot water/hot phenol-extracted
B. abortus smooth lipopolysaccharide at a dilution of 1.0 µg/ml in a 0.06 M carbonate buffer (pH
9.6) and incubated overnight at 4 C. The plates will be then washed 3 times and test and control
sera will be added to the microplate wells at a dilution of 1:200. The plates will be incubated for
1hr at 370.c all the test sera will be tested in duplicate, whereas control sera will be tested in
quadruplicate.

23
3.8. Data Analysis
Data will be obtained from questionnaire survey and laboratory results will be recorded and
stored in Microsoft Excel for Windows 2010 and transferred to Statistical Package for the Social
Sciences (SPSS) version 20. Information on PA, sex, age, body condition Putative biological and
environmental factors believed to be associated with Brucella infection will be recorded. Parity,
history of abortion and retained fetal membrane will be collected. Data will be coded and
analyzed using descriptive and analytical statistics as appropriate. Logistic Regression analysis
was employed to identify the association of seropositivity with the potential risk factors. The
units of analysis will be individual cattle and herd. Animal level seroprevalence will be
computed by the number of positive animals will be divided by the total number of animals
tested and for herd level seroprevalence the number of positive herds will be divided to the total
number of individual herds tested.

24
 4. Work and Budget Plan
Table 1 Work plan

No Activities Months 2024

Jan Feb Mar Apr May June July Aug Sep Oct Nov

1 Proposal preparation   

2 Proposal defense  

3 Sample collection & 

questionnaires

4 Sample take to laboratory 

5 Laboratory work   

  

6 Sample separation   

7 Data developing /organization   

8 Data analyzing   

9 Paper writing  

10 Report comment  

25
 11 Thesis submission 

12 Thesis presentation 

Table 2 budget plan

N/o Qualifications Quantity/unit Durations Types of payment /cost Total

in days in sum
number

Peridiu Sample Transport & bed

m

1 Sample taking/ collection units Amt days In one sum round one sum

2 Researcher No 1 1month 200 6000 5 1125 5625 11625

Assistant /labor cost “ 6 20 days 1500 9000 - - 9000

Laboratory technicians “ 4 1 months 2000 8000 - - 8000

Total sum 23000 5625 28625

26
Table 3 Laboratory equipment’s

No. Equipment Measurement Quantity In single In total Remark

1 Laboratory equipment

Rose Bengal kit with its No 1 90000 90000

inclusions materials

Vactunaries tube No 15 550 8250

Needle Pack 2 520 1040

Savlon Liter 2 100 200

Alcohol “ 4 155 620

Cotton Roll 1 200 200

Glove surgical Box 10 100 1000

Glove long arm Box 1 300 300

Guan No 2 500 1000

Forceps &scissors No 4 40 160 each

27
 Mask Pack 2 50 100

Total 102,870

4 Stationary materials

No. Items Units Quantity Prices in single Prices in total Remark

1 Clip board No 1 160 160

2 Paper Pad 4 180 720

3 Ruler No 2 50 100

4 Pen Pack ½ 660 330

5 Printing Page 1600 2,50 4000

6 Photocopy Page 1600 1.5 2400

Total 7710

Budget summary
1 Sample collection and transport cost---------------------------------------28625birr

2 Equipment cost ---------------------------------------------------------------102,870birr

3 Stationary costs----------------------------------------------------------------7710 birr

28
Total ------------------------------------------------------------------------------139205

 Source of budget Jimma University

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AW, T.-C. & BLAIR, I. 2010. Occupational infections. Infectious Diseases, 715.

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6. Annex

Jimma University College of Agriculture and Veterinary Medicine Department of Veterinary

Microbiology

Questionnaires formats for sero prevalence of bovine brucellosis and its associated risk factors
in Essera District Dawuro Zone.

The following data will be collected on animal attributes: breed (local/cross), sex (male/female),
age and reproductive status, parity, stage of abortion (first trimester, second trimester and third
trimester), history of abortion and retained fetal membrane and breeding systems. Based on its
biological relevance, age will be stratified into three categories (0.5-<3 years, ≥3-6 years and >6
years) (Asgedom et al., 2016). The reproductive status will be also categorized (replacement
heifers, pregnant cows, lactating cow, dry and bulls). Besides, information on farms such as:
herd size will be categorized into [small scale (≤4 heads of cattle), medium scale (≥5-8 heads of
cattle) and large scale (≥8 heads of cattle)] and other management factors will be collected. The
presence of calving pens (No/Yes), waste disposal methods (placenta, aborted material and dead
animal) will be categorized into (burying, burning and open dump). Hygienic status of the
farms will be categorized as (clean and not clean) based on manure disposal, drainage and barn
ventilation. Farmer’s awareness about brucellosis (No/Yes) will be assessed.

I Information’s

1 Farmers name------------------------------------sex----------age-------kebele------woreda-------------
Zone---------------Region---------------

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2. Marital status A) Married B) Divorced C) Widowed D) Single

3. Educational statuses?

A illiterate B write and read C primary D secondary E higher level.

II Cattle information’s

1. Herd size A) large (≥8 heads of cattle) B) medium (≥5-8 heads of cattle) C) small(≤4
heads of cattle)

2. Breed: - A) local B) cross C) both

3. Reproductive status

A) Heifer B) milking cow C) not milking cows D) pregnant E) bull

4. What is main reproductive problem in your herd?

A) No B) Abortion C) Retained placenta membrane

5. Age of your animal? A) Young (0.5-<3 years) B) adult ( ≥3-6 years ) C) old (>6
years)

6. How many offspring your cow borne? (Parity status) A) <3 B) 4-6 C) >6

7. In what stage your cow abort pregnancy?

A) Early (1-3month) B) mid (4-6month) C) late (.>6month

8. Where did you get replacement stock? A) Owners source B) government C) market

9. What about hygienic of your cow’s barn? A) Poor B) Medium C) Good

10. What types of farm you use? A) Intensive B) extensive C) semi-intensive

11 Did your cow ever affected in retained fetal membrane? A yes B no

12 what types of mating practice your cow use? A natural B AI C both

13 Is there parturition pen in your herd? A yes B no

14 Do you separate your cow during parturition? A yes B no

15 have you cleaning your calving area after parturition? A yes B no

16 Is your cows repeat in breeding? A yes B no

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17 Did your house common with your cattle? A yes b no

18 have you awareness about zoonosis of bovine brucellosis? A yes b no

19 Did you have awareness of zoonotic diseases transmission when drinking raw milk?
A yes B no

20 Did you ever contact with aborted fetus? A yes B No

21 Did you ever contact with fetal membrane? A yes B No

22 Did you ever drinking raw milk? A yes B No

39