PREVALENCE, ASSOCIATED RISK FACTORS AND MAJOR
CAUSATIVE AGENTS OF BOVINE MASTITIS IN SELECTED
DAIRY FARMS IN AND AROUND DIRE DAWA
MSc THESIS
HAGOS GEBRESELASSIE WELDEMARIAM
NOVEMBER 2015
HARAMAYA UNIVERSITY, HARAMAYA
Prevalence, Associated Risk Factors and Major Causative Agents of
Bovine Mastitis in Selected Dairy Farms in and around Dire Dawa
A Thesis Submitted to the Postgraduate Program (School of Animal and
Range Sciences)
HARAMAYA UNIVERSITY
In Partial Fulfilment of the Requirements for the Degree of MASTER
OF SCIENCE IN AGRICULTURE (ANIMAL PRODUCTION)
BY
Hagos Gebreselassie Weldemariam
November 2015
Haramaya University, Haramaya
ii
POSTGRADUATE PROGRAM DIRECTORATE
HARAMAYA UNIVERSITY
As Thesis Research advisors, we here by certify that we have read and evaluated this thesis
Prepared, under our guidance by Hagos Gebreselassie Weldemariam titled “Prevalence,
Associated Risk Factors and Major Causative Agents of Bovine Mastitis in Selected
Dairy Farms in and Around Dire Dawa.” We recommend that it can be submitted as it is
fulfilling the Thesis requirement.
Yitbarek Getachew (PhD)
Major Advisor
_______________
Signature
Mengistu Urge (PhD)
Co–Advisor
_______________
Signature
As members of the Board of Examiners of the MSc Thesis Open Defense Examination, we
Certify that we have read, evaluated the Thesis prepared by Hagos Gebreselassie
Weldemariam and examined the candidate. We recommend that the Thesis be accepted as
fulfilling the Thesis requirement for the Degree of Master of Science in Agriculture
(Animal Production).
______________________
_________________
Chairperson
Signature
______________________
_________________
Internal Examiner
Signature
______________________
_________________
External Examiner
Signature
Final approval and acceptance of the Thesis is contingent upon the submission of its final
copy to the Council of Graduate Studies (CGS) through the candidate’s department or
school graduate committee (DGC or SGC).
iii
DEDICATION
Dedicated to my Mother Weizero Letebirhan Abebe who sacrificed much to bring me up to
this level but not lucky to see the final fruits of my effort.
iv
STATEMENT OF THE AUTHOR
First, I declare that this thesis is the result of my own work and that all sources or materials
used for this thesis have been duly acknowledged. This thesis is submitted in partial
fulfilment of the requirements for MSc degree at Haramaya University and to be made
available at the University’s Library under the rules of the Library. I confidently declare
that this thesis has not been submitted to any other institutions anywhere for the award of
any academic degree, diploma, or certificate. Brief quotations from this thesis are
allowable without special permission, provided that accurate acknowledgement of source
is made. Requests for permission for extended quotation from or reproduction of this
manuscript in whole or in part may be granted by the head of the School/Director of the
Graduate Program when in his or her judgment the proposed use of the material is in the
interests of scholarship. In all other instances, however, permission must be obtained from
the author.
Name: Hagos Gebreselassie Weldemariam
Signature_________
Place: Haramaya University, Haramaya
Date of Submission: -----------------------
v
BIOGRAPHICAL SKETCH
The author was born in Ginner town, central zone of Tigray Region, in June 21, 1983 from
his father Ato Gebreselassie Weldemariam and his mother W/o Letebirhan Abebe. He
attended his primary and secondary school at Myssesela and Werei Secondary School,
respectively. After completing secondary school with good results of the Ethiopian schools
Leaving Certificate Examination (ESLC), he joined Addis Ababa University in 2004
academic year and graduated with DVM (Doctor of Veterinary Medicine) in 2009. After
completing his study, he was employed in Army foundation of Hurso Agriculture
Development. He joined Haramaya University in 2012 to pursue his graduate studies in
MSc in Animal Production. Currently, he is assigned and working as a veterinarian and
supervisor in Army Foundation Hurso Agriculture Development. The author is married and
blessed with one child.
vi
ACKNOWLEDGEMENT
First of all, I would like to thank the Almighty God and my wife, Sister Abirehet
Hailemariam who gave me the opportunity to pursue my graduate study at Haramaya
University where I gained much. I would like to thank many people and organizations who
supported me in accomplishing this thesis work. My greatest thank and heartfelt
appreciation goes to my major advisor Dr. Yitbarek Getachew and co-advisor Dr.
Mengistu Urge for their valuable guidance, suggestions and provision of related study
materials in both proposal development and thesis writing. I greatly acknowledge Dire
Dawa Regional Laboratory technician Ermias Demssie and his work mates for their
unreserved and valuable support in sample collection and laboratory work. Without their
assistances, the completion of this paper would have been hardly possible. I would like
also to express my sincere appreciation to Army Foundation for giving me chance to
pursue my postgraduate study and covering study expenses. I would like also to forward
my appreciation to the manager of Hurso Agriculture Development, Major Zeray Desalegn
for his contribution in moral and materials.
vii
TABLE OF CONTENTS
DEDICATION
IV
STATEMENT OF THE AUTHOR
V
BIOGRAPHICAL SKETCH
VI
ACKNOWLEDGEMENT
VII
TABLE OF CONTENTS
VIII
LISTS OF TABLES
X
LISTS OF FIGURES
XI
ACRONYMS AND ABBREVIATIONS
XII
ABSTRACT
XIII
1. INTRODUCTION
1
2. LITERATURE REVIEW
4
2.1. Definition of Mastitis
4
2.2. Clinical and Subclinical forms of Mastitis
4
2.3. Prevalence of Bovine Mastitis
5
2.4. Prevalence of Bovine Mastitis in Ethiopia
6
2.5. Pathogenesis of Bovine Mastitis
7
2.6. Aetiology of Bovine Mastitis
8
2.6.1. Staphylococcus Species
2.6.2. Streptococcus Species
2.6.3. Coliforms Bacteria
2.7. Economic Importance of Mastitis
8
9
10
10
2.8. Diagnosis of Mastitis
12
2.8.1. California Mastitis Test (CMT)
2.8.2. Culture
2.9. Classification of Mastitis Pathogens
12
12
13
2.9.1. Contagious Mastitis (cow-associated)
2.9.2. Environmental Pathogens (Mastitis)
2.10. Mastitis and Associated Risk Factors
13
14
14
2.10.1. Age and parity of the cow
2.10.2. Inherited features of the cow
2.10.3. Breed and milk yield
2.10.4. Stage of lactation
2.10.5. Mammary regression
2.10.6. Milking Machine
2.10.7. Nutrition
15
16
16
16
17
17
18
viii
TABLE OF CONTENTS (Continued)
2.10.8. Weather and climate
2.10.9. Milking hygiene and procedures
3. MATERIALS AND METHODS
18
19
20
3.1. Study Area
20
3.2. Study population
20
3.3. Study Design, Sample Size and Sampling Method
21
3.4. Data Collection
22
3.4.1. Questionnaire
3.4.2. Clinical examination of udder and milk
3.4.3. California Mastitis Test (CMT)
3.4.4. Milk Sample Collection, Handling and Transportation
3.4.5. Microbiological Culture (Etiological Factors)
3.5. Data Analysis
22
22
23
23
24
25
4. RESULTS
26
4.1. Description of the Study Population and Dairy Farms
26
4.1.1. Study Animals
4.1.2. Farming System
4.2. Prevalence of Mastitis
26
26
26
4.2.1. Animal level
4.2.2. Quarter level
4.2.3. Types of Dairy Production Systems and Mastitis Prevalence
4.3. Associated Risk Factors
26
27
27
28
4.3.1. Host Factors
4.3.2. Environmental Factors
4.3.3. Microbiological Culture (Aetiological Factors)
5. DISCUSSIONS
29
30
31
32
6. SUMMERY, CONCULISION AND RECOMMENDATIONS
36
7. REFERENCES
37
8. APPENDEXES
47
ix
LISTS OF TABLES
Table
page
Table 1. prevalence of subclinical mastitis at animal level
27
Table 2. Clinical, blind and CMT scores of each quarters of lactating cow in sample farms
27
Table 3. Prevalence of bovine mastitis related to production systems in and around Dire
Dawa.
27
Table 4. Prevalence of bovine mastitis in small and medium farms
28
Table 5. Association between the occurrence of subclinical mastitis and risk factors
29
Table 6. Association between subclinical mastitis and Environmental Factors
30
Table 7. Frequency distribution of isolated bacteria from mastitic dairy farms
31
x
LISTS OF FIGURES
Figure
Page
1. Map of the study area
21
2. Bar graph for the prevalence of subclinical mastitis in the study area
28
xi
ACRONYMS AND ABBREVIATIONS
BMSCC
Bulk Milk Somatic Cell Count
CM
Clinical Mastitis
CMT
California Mastitis Test
CNS
Coagulase Negative Staphylococci
CPS
Coagulase Positive Staphylococci
CSA
Central Statistics Agency
DDPAIA
Dire Dawa Provisional Administration Investment Agency
DNA
Dinuclotide Nucleic Acid
DNP
Days Not Pregnant
DVM
Doctor of Veterinary Medicine
ETB
Ethiopian Birr
FVM
Faculty of Veterinary Medicine
HF
Holstein Friesian
IMI
Intramammary Infection
KPa
Kilo Pascal
MS
Micro- Soft
MWT
Modified White Side Test
NMC
National Mastitis Council
PCR
Polymerase Chain Reaction
SAS
Statistical Analysis System
SCC
Somatic Cell Count
xii
Prevalence, Associated Risk Factors and Major Causative Agents of
Bovine Mastitis in Selected Dairy Farms in and around Dire Dawa
ABSTRACT
A cross-sectional investigation was conducted from August, 2014 to December, 2014 to
determine the prevalence of bovine mastitis, assess the association of some putative risk
factors with occurrence of mastitis in lactating cows and identify the major bovine mastitis
causative agents in selected dairy farms in and around Dire Dawa. A total of 403 lactating
cows (331 high grade Holstein Friesian and 72 indigenous zebu breeds) were examined
clinically. California Mastitis Test (CMT) was used to detect clinical and subclinical
mastitis. The overall prevalence of mastitis at cow and quarter level was 91.8% and
70.85%, respectively. Three hundred twenty (79.4%) cows were positive for subclinical
mastitis while only 50 (12.4%) had clinical mastitis. Among the total (1612) quarters
investigated, 20 quarters (1.24%) were blind. The prevalence of mastitis was significantly
(P<0.05) associated with age (X2= 30.38, P=0.000), breed (X2=3.93, P=0.047), milk yield
X2=13.99, P=0.001), and previous record of mastitis (X2=12.23, P=0.000). Adult cows,
Holstein breed, high milk yield and mastitis history were determined as attributing factors
for higher prevalence of mastitis in the dairy farms. A total of 27 bacterial isolates were
recovered from 27 pooled milk samples and gram-positive cocci were the most common
pathogens. The pathogens isolated were Staphylococcus species (66.67%), Streptococcus
species (14.81%), Coli-forms (Escherichia coli (11.11%) and Bacillus species (7.41%)).
The present study showed that mastitis is a serious problem in dairy farms in and around
Dire Dawa city. Contagious were the major causes of mastitis. Therefore, hygienic milking
practice, screening and culling of chronically infected and older cows with repeated
mastitis records, cow therapy and awareness creation among dairy farm owners should be
practiced to reduce the risk of mastitis.
Keywords: Clinical Mastitis, Hygienic milking, Prevalence, Subclinical Mastitis
xiii
1. INTRODUCTION
Ethiopia holds large potential for dairy development mainly due to its large livestock
population and relatively favourable climate for improved high-yielding animal breeds.
The country enjoys diverse topographic and climatic conditions and milk production takes
place across all agro-ecological zones. In the highlands, milk is mainly produced by small
scale mixed farmers, while in the lowlands, pastoralist production systems are
predominant. There are also intensive and commercial dairy farms concentrated in and
around major cities and towns of the country. The majority of cows kept are indigenous
breeds, with a limited number of farmers keeping few crossbred grade dairy animals
(Holloway et al., 2000).
Dairy provides rural farmers with a way to increase assets, a method to diversify income
and nutrition. Dairy is also an important tool to address poverty, enhance agricultural
development, and create employment opportunities beyond an immediate household or
smallholder dairy operation. Dairy is a development tool because it widens and sustains
pathways out of poverty through securing assets of the poor, improving smallholder
productivity and increasing market participation by the poor (ILRI, 2007). Hence,
development of the dairy sector in Ethiopia can contribute significantly to poverty
alleviation, improved nutrition and household income (Mohamed et al., 2004). But, the
dairy sector has not been fully exploited and promoted (Tangka et al., 2002).
In Ethiopia, lack of modern animal husbandry and management, limited skilled manpower
in dairy technology and marketing, inadequate distribution systems and limited packaging
choices have affected the sector. Severe shortages of animal feed supplies and the high cost
of running a dairy farm are becoming the major bottlenecks to dairy development.
Transportation cost is the other additional extra costs paid by regional farm holders, as they
are buying majority of the feeds from Addis Ababa. Low productivity of cattle because of
their genetic makeup, increased running and investment cost per unit (CSA, 2007) and
disease, mainly udder inflammation, is becoming a serious problem.
Mastitis (inflammation of mammary gland) is among important health problems in dairy
cattle. It has been considered as one of the most important threats affecting the dairy
2
industry. Mastitis remains to be the most economically damaging and zoonotic potential
disease for dairy industry and consumers worldwide irrespective of the species of animal
(Ojo et al., 2009). It is the major concern of the dairy industry worldwide for a number of
reasons, such as mastitis has deleterious effects on milk composition, yield, and quality of
dairy products, it is considered a welfare concern due to the pain cows experience,
especially during an episode of acute, severe mastitis (Kemp et al., 2008; Leslie and
Petersson-Wolfe, 2012). Mastitis is the most common production limiting and costly
disease. Factors which contribute to the economic impacts of mastitis include milk
production losses, diagnostic costs, treatment costs, discarded milk, and increased risk of
other diseases and culling of dairy animals (Halasa et al., 2007).
In Ethiopia, the available information indicates that bovine mastitis is one of the most
frequently encountered diseases of dairy cows. According to Lemma et al. (2001) of the
major diseases of crossbred cows in Addis Ababa milk shed, clinical mastitis was the
second most frequent disease next to reproductive diseases, in which 171 cows out of 556
were found to be affected. According to Hussein et al. (1997) the prevalence of clinical
and subclinical mastitis in the central regions of Ethiopia are found to be 5.3% and 19% on
cow basis and 1.9% and 7.4 % on quarter basis, respectively. In a study conducted at Repi
and Debre-Zeit dairy farms, out of 186 lactating cows, forty (21.5%) were clinically
affected and 71 (38%) sub-clinically infected (Workineh et al., 2002). Bishi (1998) noted
that the economic losses from clinical and subclinical mastitis in Addis Ababa milk shed to
be approximately 270 Ethiopian birr (ETB) per lactation. According to Mungube (2001)
the estimated economic losses in terms of milk production loss, treatment cost, withdrawal
and culling losses due to mastitis in the peri-urban areas of Addis Ababa were about
210.8 ETB/cow/lactation of which milk production loss contributed 38.4%. In other study
conducted in the same study area (Mungube et al., 2005) a milk production loses of 17.2%
was reported due to quarter infected with mastitis. In the country, mastitis has long been
known (Tamirat, 2007, Bitew et al., 2010). However, the information available on the
magnitude, risk factors and causative agent of the disease is inadequate. Such information
is important when designing appropriate strategies that would help to reduce its prevalence
and effects (Biffa et al., 2005). Most studies in Ethiopia were carried out in Addis Ababa
and its surroundings, which are not representative of other regions of the country (Almaw
et al., 2009).
3
Dairy farming in Dire Dawa Administration is an emerging employment sector of the
youth, university graduates and women. As most of the farm owners are new to the sector,
they are deficient of the basic knowledge of mastitis control and prevention strategies and
these could favour mastitis to be prevalent. Moreover, heat, humidity and draught are the
prevailing climatic conditions of Dire Dawa as heat and humidity increases, so does the
bacterial multiplication as well as the load of pathogens in the environment. Most of the
dairy farms are established around the home of owners due to lack of land and also milking
practice is manual, which increases mastitis disease transmission (Yonus, 2011). These all
mentioned above are the most important risk factors which contribute to the increase in
intramammary infection in the dairy farms (Ranjan et al., 2011). Therefore, it was
hypothesised that the poor practise in the emerging dairy industry and climatic conditions
favour the presence and persistence of mastitis in the study area.
Therefore, the purpose of the present study is to generate up-to-date information with the
following objectives:

To determine the prevalence of clinical and subclinical mastitis

To identify the major mastitis causative agents and associated risk factors
associated with the prevalence of mastitis
4
2. LITERATURE REVIEW
2.1. Definition of Mastitis
Mastitis is inflammation of the parenchyma of the mammary gland, which can result from
cow exposure to a variety of infectious agents. It is characterized by an array of physical
and chemical changes in milk, pathologic changes to the glandular mammary tissue, and at
times systemic changes within the affected animal. Mammary infections are divided into
two categories, sub-clinical and clinical. Cases of sub-clinical mastitis are described as the
presence of an infection without visually evident sign of local inflammation or systemic
involvement (Erskine, 2011). Detection of sub-clinical mastitis can be accomplished by
determining the somatic cell count (SCC) in milk of individual cows using a California
Mastitis Test (CMT) or automated cell count methods. Somatic cells include lymphocytes,
macrophages, polymorphonuclear cells and some epithelial cells, all of which reflect the
inflammatory response in the udder to an intramammary infection (IMI). Somatic cell
counts to monitor for sub-clinical mastitis can be conducted at the quarter, cow, and herd
levels (Schukken et al., 2003).
2.2. Clinical and Subclinical forms of Mastitis
Mastitis in both clinical and subclinical forms is a frustrating, costly and extremely
complex disease that results in a marked reduction in the quality and quantity of milk
(Harmon, 1994). Annual losses in the dairy industry due to mastitis was approximately two
billion dollars in USA and 526 million dollars in India, in which subclinical mastitis are
responsible for approximately 70% of these dollars losses (Varshney and Naresh, 2004).
The invisible changes in subclinical mastitis can be recognized indirectly by several
diagnostic methods including the California Mastitis test (CMT), the Modified White Side
Test (MWT), SCC, PH, chlorine and catalase tests. These tests are preferred to be screening
tests for subclinical mastitis as they can be used easily, yielding rapid as well as satisfying
results (Lesile et al., 2002).
5
In clinical mastitis (CM), there are visible changes to the normal appearance of milk,
which could include a colour change, a consistency change, or the presence of flakes, clots,
and/or blood. Physical changes to the udder may be present in CM cases ranging from
warmth, diffuse swelling, and pain to gangrene in severe cases. Chronic mastitis can result
in local fibrosis and atrophy of mammary tissue. When only local signs are evident a case
of mastitis is considered mild or moderate. A case of mastitis is considered severe when
systemic signs of an inflammatory response are apparent including fever, anorexia, and
shock (Erskine, 2011).
2.3. Prevalence of Bovine Mastitis
The prevalence of mastitis pathogens varies from herd to herd. However, bacterial
pathogens appear to be the most prominent contributor to worldwide mastitis (Wilson et
al., 1997). The prevalence of subclinical mastitis in dairy herds is often surprising to
producers. Moreover, subclinically infected udder quarters can develop clinical mastitis
and the rate of new infections can be high. Cows with subclinical mastitis are those with no
visible changes in the appearance of the milk and/or the udder, but milk production
decreases by 10 to 20% with undesirable effect on its constituents and nutritional value
rendering it of low quality and unfit for processing (Holdway, 1992). Although there are no
visible or palpable external changes, the infection is present and inflammation is occurred
in the udder (Blowey and Edmondson, 1995).
Clinical mastitis is less likely in younger animals. Reduction in clinical mastitis has been a
major success over the past 35 years, in countries with a developed dairy industry
(Poelarends et al., 2001). Most mastitis occurs as a low grade infection, a subclinical state,
which affects 10-15% of the cows, increasing milk leucocytes content, reducing milk
production and increasing milk bacterial content. These all contribute to reduced milk
value as a food and in monetary terms (Barbano, 2004). The prevalence of such infections
is a significant risk to uninfected animals in the herd as many mechanisms exist to expose
the animals to new infection. Most commonly these include the common lying areas in
housing or at pasture, the milking machine and successive contact of different cows or
teats by the milker preparing the teats for milking. The prevalence of Coagulase-negative
6
staphylococci (CNS) mastitis is higher in primiparous cows than in older cows (Tenhagen
et al., 2006).
Contagious mastitis is primarily transmitted at milking time and the milking process affects
the patency of the teat orifice which can increase the risk of development of environmental
mastitis. Mammary quarter infection prevalence ranges between 28.9-74.6% prepartum
and 12.3-45.5% at parturition (Fox, 2009). Coagulase-negative staphylococci (CNS) are
the most prevalent cause of subclinical intramammary infections in heifers. Coagulasepositive staphylococci (CPS) in some studies are the second most prevalent pathogens,
while in other studies the environmental mastitis pathogens are more prevalent. The risk
factors or subclinical mastitis appear to be season, herd location and trimester of pregnancy
(Fox, 2009).
2.4. Prevalence of Bovine Mastitis in Ethiopia
In Ethiopia, the available information indicated that bovine mastitis is one of the most
frequently encountered diseases of dairy cows. According to Lemma et al. (2001) of the
major diseases of crossbred cows in Addis Ababa milk shed, clinical mastitis was the
second most frequent disease next to reproductive diseases, in which 171 cows out of 556
were found to be affected. Generally, the prevalence of clinical and subclinical mastitis in
different parts of Ethiopia range from 1.2 to 21.5% and 19 to 46.6%, respectively (Kerro
and Tareke, 2003).
These limited studies showed that bovine mastitis is among the problems hindering dairy
productivity in Ethiopia and this requires the development of methodologies of control
program under the prevailing husbandry system. However, according to Hussein et al.
(1997) so far efforts have been concentrated only on the treatment of clinical cases. On the
other hand, losses from mastitis have been attributed mainly to decreased milk production
from subclinical mastitis (DeGraves and Fetrow, 1993).
Kassa et al. (1999) carried out a survey of mastitis in dairy herds of the Ethiopian central
high lands. Out of 10, 908 quarters examined from 2,681 cows, they found prevalence of
clinical mastitis, non-functional or blocked quarters and subclinical mastitis to be 1.2%,
7
3.8% and 38.9% on cow basis, respectively. According to Hussein et al. (1997) the
prevalence of clinical and subclinical mastitis are found to be 5.3% and 19% on cow basis
and 1.9% and 7.4 % on quarter basis, respectively, in the central regions of Ethiopia. In a
study conducted at Repi and Debre-Zeit dairy farms, out of 186 lactating cows, forty
(21.5%) were clinically affected and 71 (38%) subclinically infected (Workineh et al.,
2002). The overall prevalence in this study was 59.7%. Bishi (1998) reported mastitis
prevalence rates of 34.3% and 5.3% at cow level in Addis Ababa region, for subclinical
and clinical mastitis, respectively.
Generally, the prevalence of clinical and sub clinical mastitis in different parts of Ethiopia
range from 1.2 to 21.5% and 19 to 46.6%, respectively (Workineh et al., 2002). Clinical
(4.9%) and sub clinical (45.5%) was reported in Bahir Dar, Ethiopia (Mekuria, 1986). In
the same study area after ten years, a prevalence of 40% sub clinical mastitis was reported
(Shirmeka, 1996). Even though the disease has been known locally in Ethiopia, it has not
been studied systematically, making information available on the prevalence of disease and
associated with economic loss inadequate.
2.5. Pathogenesis of Bovine Mastitis
Mastitis is a complex and multi factorial disease, the occurrence of which depends on
variables related to the animal, environment and pathogen. Among the pathogens, bacterial
agent are the most common one, the greatest share of which resides widely distributed in
the environment of dairy cows, hence a common threat to the mammary. There is evidence
that pathogens use various mechanisms to impinge upon cell death pathways. A number of
pathogens are armed with an array of virulence determinants, which interact with key
components of a host cell’s death pathways or interfere with regulation of transcription
factors monitoring cell survival. These virulence factors induce cell death by a variety of
mechanisms, which include, pore-forming toxins, which interact with the host cell
membrane and permit the leakage of cellular components, toxins that express their
enzymatic activity in the host cytosol, effect or proteins delivered directly into host cells by
a highly specialized type-III secretary system, super antigens that target immune cells and
other modulators of host cell death (Weinrauch and Zychlinsky, 1999).
8
2.6. Aetiology of Bovine Mastitis
The primary cause of mastitis is a wide spectrum of bacterial strains. However, incidences
of viral, algal and fungal-related mastitis were also reported (Pyorala, 2003). Over 135
different microorganisms (bacterial, algal or fungal) have been isolated from bovine IMI,
but the majority of infections are caused by staphylococci, streptococci, and gram-negative
bacteria (Watts, 1988). Microorganisms that cause mastitis are generally classified as
either contagious or environmental based upon their primary reservoir and mode of
transmission. Staphylococcus aureus and Streptococcus agalactiae are contagious
pathogens and are commonly transmitted among cows by contact with infected milk.
These pathogens are of particular importance because they cause mainly subclinical forms
of IMI that are often difficult to detect. Primary environmental pathogens include different
types of bacteria: species of streptococci other than Streptococcus agalactiae
(Streptococcus species), coliform species (Escherichia coli, Klebsiella species,
Enterobacter species) and pseudomonas species.
2.6.1. Staphylococcus Species
Staphylococcus aureus is one of the most prevalent contagious mastitis pathogen that
colonizes the teats during damage to the skin surface. It produces many enzymes and
toxins and penetrates deep into the mammary tissue and resists phagocytosis. Some of the
Staphylococcus aureus strains have antibiotic resistance and can cause the problem with
the treatment (Petersson-Wolfe et al., 2010). The result of Staphylococcus aureus
infections is a decrease in milk yield and increase somatic cell count. Transmission of
Staphylococcus aureus infections occurs mainly through contaminated milking machines,
udder wash equipment, and the hands of milking machine operators. It can survive outside
of the cow for a shorter period of time. Infections caused by Staphylococcus aureus are
mostly sub-clinical with periodic flare-up of clinical symptoms. Chronic infection of
heifers can serve as a source of new infection in the herd. The frequency of the
Staphylococcus aureus infections is related to age of the cow. Culling, grouping and dry
cow therapy helps fight Staphylococcus aureus infections in a herd (Syensk Mjölk, 2003).
9
The chronic and subclinical forms predominate and on a herd basis, are the most important.
Staphylococcus aureus bacteria produce toxins that destroy cell membranes and can
directly damage milk-producing tissue. White blood cells (leukocytes) are attracted to the
area of inflammation, where they attempt to fight the infection. Initially, the bacteria
damage the tissues lining the teats and gland cisterns within the quarter, which eventually
leads to formation of scar tissue. The bacteria then move up into the duct system and
establish deep-seated pockets of infection in the milk secreting cells (alveoli). This is
followed by the formation of abscesses that wall-off the bacteria to prevent spread but
allow the bacteria to avoid detection by the immune system. The abscesses prevent
antibiotics from reaching the bacteria and are the primary reason why the response to
treatment is poor (Petersson-Wolfe et al., 2010).
2.6.2. Streptococcus Species
Streptococcus dysgalactiae is also one of the contagious pathogens. It can spread
throughout a herd from a single infected animal. The infected udder is the most important
reservoir for this bacterium. They are transmitted to uninfected quarter mainly at milking
time. Contaminated milking machines, udder wash cloths, and the hands of machine
operator also transmits these bacteria (NMC, 1996). Breakdowns of contagious mastitis are
usually due to the introduction of infected animals to the herd, or the employment of men
who carry infection with them. The infections are mainly sub-clinical (NMC, 1996) and
there are most frequent in the younger age groups.
Streptococcus dysgalactiae is generally characterized as an environmental pathogen, but
also may have characteristics of a contagious organism and appears to spread from cow to
cow. This pathogen is generally responsive to teat dipping and dry cow therapy, but new
infections can occur in a herd when no other udder infections by this organism are present
(Harmon, 1996). Teat damage caused by contamination with sand or grit, or poorly
operating milking machines is prime reasons for Streptococcus dysgalactiae mastitis.
Streptococcus uberis is an important environmental pathogen particularly because it is
ubiquitous in the dairy environment. Identification of Streptococcus uberis is currently
based on observation of the cultural and morphological characteristics, biochemical tests
determination, and enzyme activity (Khan et al., 2003). On the other hand, several
10
commercial microbial identification systems have also been used to differentiate
Streptococcus uberis from other species of streptococci and enterococci isolated from
bovine mastitis, and more recently, molecular tools such as PCR-based protocols have
been proposed to provide an accurate identification of Streptococcus uberis isolates
(Reinoso et al., 2011).
2.6.3. Coliforms Bacteria
Among coliforms bacteria, E. coli is the most frequently isolated from bovine milk in cows
belonging to dairy farms with intensive systems of milk production. E. coli is a member of
Enterobacteriaceae family. Its primary importance is its ability for lactose fermentation.
Over 700 antigenic types or serotypes of E. coli have been recognised based on O, H, and
K antigens. Two classes of coliforms have to be distinguished: strains that are harmless
(non-pathogenic strains) and strains that cause a wide variety of typical clinical infections
(pathogenic strains). Millions of non-pathogenic E. coli bacteria are living in the humans
and animals normal intestinal microflora. E. coli is ubiquitous in the cow’s environment
because is massively excreted with the faeces. E. coli causes infection and inflammation of
the mammary gland in dairy cows mainly around parturition and during early lactation
striking local and sometimes severe systemic clinical symptoms. Clinical signs vary from
very severe, even fatal forms, or mild mastitis, where cows have only local signs in the
udder. During mastitis, the host defense status is a factor determining the outcome of the
disease. Particularly, during E. coli mastitis, the neutrophil is a key factor in the cows’
defence against IMI. However, virulence of the involved bacterial strain may also play a
role. Most of the pathogenic E. coli strains posses several kinds of pathogenic mechanisms
and virulence factors. A non-specific but potent factor that is important during the
pathogenesis of E. coli is the endotoxin or lipopolysaccharide, which is responsible for
most pathophysiological effects (Tormo et al., 2005).
2.7. Economic Importance of Mastitis
Mastitis is one of the most important diseases that cause economic loss in dairy industry
worldwide (Bachaya et al., 2011). The mean annual incidence is 41.6 cases per 100 cows
and affected cows suffered a mean of 1.5 cases and 16.4% of quarters suffered at least one
11
repeat case (Bradley and Green, 2001). Bennett et al. (1999) estimated the total economic
impact of clinical mastitis to be £119 per cow-case in Great Britain.
More than $130 million is lost by the Australian dairy industry ($A200/cow/year) every
year due to poor udder health resulting in reduced milk production that is mainly
associated with mastitis. A herd without an effective mastitis control programme may
witness morbidity as high as 40% with infection, on an average of two quarters of the
mammary gland. Of the various clinical manifestations, subclinical mastitis is
economically the most important due to its long term effects on milk yields (Zafalon et al.,
2007). Huge economic losses are also incurred due to unmarketable milk or milk-products
contaminated with antibiotic residues originating from treatment in the developing nations
as well as from the use of antibiotics as growth promoters particularly in dairy feedlots in
the developed world. The prolonged use of antibiotics in the treatment of mastitis has led
to the additional problem of emergence antibiotic resistant strains, hence the constant
concern about the resistant strains entering the food chain (Virdis et al., 2010).
Clinical and subclinical mastitis both severely affect milk yield and milk quality. Milk
production of cows bearing mastitis is significantly lower than that of healthy cows.
Furthermore, the nutritional quality is lower and the somatic cells count (SCC) is
substantially higher (Schukken et al., 2009). The SCC of milk is regarded as the industry’s
standard indicator for the general quality of produced milk. It is determined as the total
count of white blood cells per millilitre of milk. Normal milk is believed to have SCC of
approximately 200,000 cells/ml or less. An infection in the mammary gland of the udder
causes a large influx of somatic cells, predominantly polymorphonuclear neutrophils,
which can increase the SCC of milk up to 1 million cells/ml (Madouasse et al., 2010).
Subclinical mastitis commonly contributes a more substantial part to high SCC’s within a
herd and is usually a reliable indication of the development of clinical mastitis (Van den
Borne et al., 2011).
Mastitis causes a loss of over 1.7 billion dollars a year in the USA alone (Sahoo et al.,
2012). It is characterised by an increase in somatic cells, especially leukocytes, in the milk
and by pathological changes in the mammary tissue (Ranjan et al., 2010).
12
2.8. Diagnosis of Mastitis
Clinical mastitis is recognized by the appearance of abnormal milk, gland swelling and /or
illness. Subclinical mastitis is characterized by normal milk and hence requires indirect
tests to detect.
2.8.1. California Mastitis Test (CMT)
The California Mastitis Test (CMT) remains the only reliable screening test for subclinical
mastitis that can be easily used at the cow side. The CMT was developed to test milk from
individual quarters but also been used on composite and bulk milk samples. The CMT
involves mixing and swirling equal parts of bromocresol violet reagent and milk in a
plastic paddle with a compartment for each quarter (Quinn et al., 1999). The test results are
interpreted subjectively as either a negative, trace, 1+, 2+ or 3+ inflammatory response
based on the viscosity of the gel formed by mixing the reagent with milk.
Fresh unrefrigerated milk can be tested using the CMT for up to 12 hours. Reliable
readings can be obtained from refrigerated milk for up to 36 hours. If stored milk is used,
the milk must be thoroughly mixed prior to testing because somatic cells tend to segregate
with milk fat. The CMT reaction must be scored within 15 seconds of mixing because
weak reactions will disappear after that time. The degree of reaction between the detergent
and the DNA of nuclei is a measure of the numbers of somatic cells in milk. The threshold
for CMT scores depend on the objective of the study. If it is used to minimize the rate of
false negatives, the test should be read as negative versus positive with trace scores
regarded as positive. If the CMT is to be used in culling decisions, a threshold with a lower
rate of false positives may be desirable (Larsen, 2000).
2.8.2. Culture
The microbiological examination of both individual cow and bulk tank culture are
elements of mastitis control. Most mastitis control programs include the use of individual
cow cultures to determine which mastitis pathogens are present on the farm. Culturing can
be used in a targeted fashion for specific control programs such as segregation plans for
13
contagious mastitis or for surveillance to detect the presence of new or emerging pathogen.
Culturing is also used to evaluate treatment efficacy and to establish susceptibility patterns
to aid in the development of rational treatment strategies (Larsen, 2000).
2.9. Classification of Mastitis Pathogens
Classically, mastitis pathogens have been classified as contagious pathogens and
environmental pathogens (Burvenich et al., 2003). The contagious pathogens are adopted
to survive within the host particularly within the mammary gland. They are capable of
causing subclinical infections, which are typically manifest as an elevation in the somatic
cell count (leukocytes, predominantly neutrophils and epithelial cells) of milk from the
affected quarter; they are typically spread from cow to cow at or around the time of
milking (Radostits et al., 1994). In contrast, the environmental pathogens are best
described as opportunistic invaders of the mammary gland, not adapted to survival within
the host; typically they invade, multiply, engender a host immune response and are rapidly
eliminated.
The
major
contagious
pathogens
comprise
Staphylococcus
aureus,
Streptococcus dysgalactiae and Streptococcus agalactiae; the major environmental
pathogens comprise the Enterobacteriacae (particularly E. coli) and Streptococcus uberis
(Dogan et al., 2006).
2.9.1. Contagious Mastitis (cow-associated)
Contagious mastitis is defined as IMI transmitted directly from cow to cow (Erskine,
2001). Incidence of contagious mastitis depends on the dose and type of microbes to which
a cow is exposed as well as physical barriers and the innate and acquired defense
mechanisms. Although many different types of bacteria may cause mastitis, those of
greatest interest are the pathogens commonly found on dairy farms and those for which the
prevalence of IMI is high. The main contagious organisms are Streptococcus agalactaiae,
Staphylococcus aureus, Corynebacterium bovis and Mycoplasma species. Staphylococcus
aureus is generally considered to be the most prevalent cause of IMI. It has been estimated
that, depending on the breed and geographical location of the herd, between 7-40% of all
cows are infected with Staphylococcus aureus at any given time.
14
2.9.2. Environmental Pathogens (Mastitis)
Mastitis caused by the environmental pathogens is traditionally considered to occur
sporadically without long lasting effects within the host. The contagious pathogens, on the
contrary, are able to persist within the host for prolonged periods of time causing
continuous occurrence of mastitis and spreading between quarters and cows (Passey et al.,
2008). More recently, DNA fingerprinting data suggested that some E. coli strains have
adapted to survive within the udder and cause recurrent mastitis. There is, however, no
apparent single factor which is responsible for the persistence of E. coli infections in the
udder (Almeida et al., 2011).
The main environmental organisms are gram-negative bacteria, which include the
coliforms and environmental streptococci. The gram-negative bacteria include Escherichia
coli, Klebsiella species, Enterobacter species, Citrobacter species, Seratia, Pseudomonas
species, Proteus and Actinomyces pyogenes. The environmental streptococci include
Streptococcus uberis, Streptococcus dysgaladiae, and Streptococcus equinus. The majority
of infections caused by coliform pathogens result in acute mastitis when compared to
infections caused by contagious pathogens and environmental streptococci, but the
infections are generally of shorter duration (less than seven days). The exception to this is
Actinomyces pyogenes, which generates large and persistent production losses (Smith and
Hogan, 1993).
2.10. Mastitis and Associated Risk Factors
Many risk factors of mastitis related to environment, the microflora and the animal have
been investigated in spite of wide control efforts with little reasonable results. However,
the bovine mammary gland infection (mastitis) is a frequent and important problem among
livestock herds in most of the countries. The occurrence of mastitis is influenced by
managemental and different environmental factors like housing of animals, type of milking
and milking utensils, and type of feeding, hygienic quality of water, health of lactating
animals and execution of various preventive procedures. Incidence of mastitis changes
with season, its rate has been reported to be highest during the winter (Nyman et al., 2007).
15
Breen et al. (2009) investigated risk factors which were related with milk leukocyte count
in different quarters of cattle. The following individual risk factors, teat end callosity,
hyperkeratosis of quarters, body state, udder and leg hygiene and capacity of milk quality
and production were assessed. Significant association with an increased risk of milk
leukocyte count was found with increasing lactation number and lactation stage than
contamination of skin of quarters and udder. Results suggested that energy status,
individual quarter and different factors at animal level play important role to measure intramammary infections by measuring milk somatic cell in subsequent lactations. The large
number of predisposing factors that contribute to the emergence of mastitis in dairy cattle
may be physiological, genetic, pathological or environmental (Sordillo, 2005) described
below:
2.10.1. Age and parity of the cow
Increasing parity increased the risk of clinical mastitis in cows (Kavitha et al., 2009),
although the reason for this association is not clear. Sharma et al. (2007) conducted a study
on 500 lactating cows of different age, parities and stage of lactation belonging to different
organized or un-organized dairy farms. Older cows (>10 years) are at more risk (44.6%),
particularly for subclinical mastitis (38.6%), than younger cows (23.6%) in which clinical
mastitis was predominant. Cows with many calves (>7) have about 13-times greater risk
(62.9%) of developing an udder infection than those with fewer (3) calves (11.3%).
It has been demonstrated that occurrence of mastitis in infected quarters increases with age
in cows (Harmon, 1994), being the highest at 7 years of age. This may be due to an
increased cellular response to intramammary infection or due to permanent udder tissue
damage resulting from the primary infection. Efficient innate host defence mechanisms of
the younger animals are one possibility that makes them less susceptible to infection
(Dulin et al., 1988). However, at least one study conducted using 4133 cattle including
both cross-bred and non-descriptive breeds revealed the highest risk of occurrence of
mastitis to be between the ages of 4-6 years, followed by the age group between 2-4 years,
with the least occurrence noted between 6-8 years of age (Mahajan et al., 2011).
16
2.10.2. Inherited features of the cow
Various genetic traits may also have a considerable impact upon the susceptibility of the
animal to mastitis. These genetic traits include the natural resistance, teat shape and
conformation, positioning of udders, relative distance between teats, milk yield and fat
content of milk. High milk yielders with higher than average fat content are reported to be
more susceptible to mastitis (Grohn et al., 1990). The conformation of the udder and shape
of the teat are inherited characteristics that may also affect susceptibility to mastitis. Cows
with elongated teats are more vulnerable to mastitis infection than cows with inverted teat
ends (Seykora and Mc Daniel, 1985). Broad udders, lower hind-quarters and teats placed
widely help the infectious agent and should be selected against it (Thomas et al., 1984).
2.10.3. Breed and milk yield
Risk of mastitis varies from breed to breed. High yielding cows are generally considered to
be more susceptible to intramammary infection e.g. Holstein Frisian (HF), Jersey or HF
and Jersey cross bred dairy cows are more susceptible to mastitis than Desi (Zebu) breeds
of cows (Compton et al., 2007). It might be due to more resistance to disease and they are
low milk producer than cross bred cows. Increased risk of clinical mastitis in Friesian
compared with Jersey and Ayrshire heifers (Compton et al., 2007).
2.10.4. Stage of lactation
The incidence of mastitis is reported to be higher immediately after parturition, early
lactation and during the dry period, especially the first 2-3 weeks (Fadlelmula et al., 2009)
due probably to increased oxidative stress and reduced antioxidant defence mechanisms
during early lactation (Sharma et al., 2011). An increase in somatic cell numbers or count
(SCC) which is mainly neutrophils is observed immediately after parturition, which
remains high for a few weeks irrespective of the presence or absence of infection. This
increased SCC is the cow’s natural first line of defence to prepare for the onset of the new
lactation. Relatively recent studies have revealed that cows in late lactation always show a
higher than average SCC than that seen at other stages of the lactation period (Peeler et al.,
2000), potentially representing increased subclinical infection, leading to a fall in milk
production.
17
2.10.5. Mammary regression
There are significant functional changes in the udder during the early and late lactation and
dry period, which affect the cow’s susceptibility to infections. Lactating cows under stress
show premature mammary regression. Such a condition compromises udder’s natural
defence mechanisms (Giesecke et al., 1994; Capuco et al., 2003) leading to invasion of the
teat canals by potential pathogens. The same condition prevails during the healing process
of lesions because the resistance to causal agents remains less effective.
2.10.6. Milking Machine
Extraneous factors such as the milking habits of farmers and faulty milking machines
favour the pathogens to gain access to mammary gland and proliferate, potentially leading
to mastitis (Mein et al., 2004). In farms where machines are employed for milking, it is
important to maintain physiologically optimal pressure [50 kPa for most machines],
because pressures in excess of this may lead to injury in the teat. Fluctuations in the
pressure due to inadequate vacuum reserve must be avoided to prevent occurrence of
mastitis. Proper installation as well as the correct maintenance of milking machines is
important to avoid an inadequate vacuum level, teat and tissue damage and incomplete
milking. The vacuum level created by the vacuum pump is another important factor for
complete and high quality milking. Experiments have shown that a teat subjected to a
vacuum level of 10.5-12.5 inches at the time of peak milk flow results in rapid, complete
and high quality milk yield, and the teat suffers minimum physical pressure (Jones, 2009).
Two-chambered teat cups are found to be better than single chambered teat cups in regard
to achieving complete milking as well as fewer incidences of teat injuries (Mein and
Schuring, 2003). Sanitary milking habits are important to avoid the spreading of bacteria or
their proliferation. Faulty milking equipment due to poor installation or maintenance can
cause tissue trauma, teat damage, poor milking out, erratic vacuum levels and can also
transmit infectious agents at milking time.
18
2.10.7. Nutrition
The quality and plan of nutrition appears to be an important factor that influences clinical
manifestation of mastitis in heifers and cows (Heinrichs et al., 2009) although no
relationship between the incidence of mastitis and either high energy or high protein feed
in cows has been reported (Rodenburg, 2012). Vitamin E is one of the important
supplements in dairy feed to boost the immune response of cows (Spears and Weiss, 2008)
as it has been reported to enhance the neutrophil function as well as the phagocytic
properties of neutrophils after parturition. Vitamin E is often combined with selenium,
which acts as an anti-oxidant by preventing oxidative stress. A number of investigations
have demonstrated that neutrophils of selenium fed cows are more effective at killing
mastitis causing microorganisms than those not supplemented with selenium (Underwood
and Suttle, 1999).
There are several factors both infectious and non-infectious, that can cause bovine mastitis;
and there is an increase in the evidence that nutritional factors are associated with mastitis
in cows and heifers (Heinrichs et al., 2009). Despite all the measures taken by dairy
producers to prevent intramammary infections in their herds, it is not always clearly
understood that there is a strong relationship between nutrition and susceptibility to
mastitis. When the consumption of minerals (selenium) and vitamins (vitamin E) by dairy
cows is not optimal, it can have negative effects on immunity. Animals become more
susceptible to diseases such as mastitis, because a depressed immune system is not able to
fight off bacteria that invade the udder.
2.10.8. Weather and climate
The incidence of mastitis is greatly influenced by the weather conditions and prevailing
climatic conditions. Heat, humidity, cold and draught are the important predisposing
factors (Reneau, 2012). A higher incidence of mastitis has been reported to occur
particularly during summer rainy months (Sentitula et al., 2012). As heat and humidity
increases, so does the bacterial multiplication as well as the load of pathogens in the
environment. Conversely, an alternative study has reported a higher incidence of coliform
19
mastitis during the cold months of the year when the temperature was reported to be less
than 21°C (Ranjan et al., 2011).
Housing is also a factor that aggravates the incidence of mastitis, first due to excess
numbers of animals in a limited space; and also because of the use of bedding material that
easily allows for bacterial survival and growth, which over exposes animals and challenges
their immune defense mechanisms (Hogan and Smith, 2003). Prevention of intramammary
infections leads to better milk quality, which in turn is beneficial for the dairy industry
because high quality milk means extended shelf life, increased cheese yield, and increased
consumption of dairy products.
2.10.9. Milking hygiene and procedures
Moisture, mud, and manure present in the environment of the cow are the primary sources
of exposure for environmental mastitis pathogens, and hygiene scores of cows provide
visible evidence of exposure to these potential sources. Milking hygiene reduce the
pathogenic organisms from inhabiting the immediate environment or skin of the animals
and minimizing their spread during milking process. The practice of regular teat dipping is
not much more common at house hold level but washing the udder with clean water and
drying with individual cow towel, using strip cups and disinfecting the teat with germicide
are some of the milking procedures used to minimize the occurrence of mastitis in the
dairy farms. In addition to these, milking of heifer first, healthy cows second and mastitic
cows last are also good practices to minimize the intramammary infection. Therefore,
prevalence of mastitis in cows is more at unorganized dairy farms as compared to
organized dairy farms. Udder hygiene significantly associated with the risk of
environmental pathogen intramammary infection in cows and milking procedures
(Compton et al., 2007).
20
3. MATERIALS AND METHODS
3.1. Study Area
The study was conducted in and around Dire Dawa (Fig 1) from August, 2014 to
December, 2014. (DDPAIA, 2005) reported that Dire Dawa is located in eastern part of
Ethiopia between 9 27’ E’ and 49’N latitude and between 41 38’ and 19’ E longitude
occupying about 133,000 hectare of land and the distance from Addis Ababa is 515
kilometre. The climatic condition of Dire Dawa Administrative Region seems to be greatly
influenced by its topography, which lies between 950–1250 metre above sea level, and
which is characterized by warm and dry climate with a relatively low level of precipitation.
The region has two rainy seasons; that is, a small rain occurs from March to April, and a
main shower of rain that extends from August to September. The aggregate average annual
rainfall that the region gets from these two seasons is about 604 mm. On the other hand,
the region is believed to have an abundant underground water resource. The monthly
average maximum and minimum temperatures of the study area are 32.40C and 19.10C,
respectively and the mean annual relative humidity is 48.2 %.
3.2. Study population
There are 31 small (3-29 cows) and medium (30-52 cows) private dairy farms in and
around Dire Dawa. The study population were, therefore, selected dairy farms in the study
area. Most of the dairy farms are established around the home of owners due to lack of
land and appropriate allocation of land by the city administration. For this reason, disease
transmission may be very high and mastitis is one of the most prevalent economically
important diseases (Yonus, 2011). In the current study, the study animals were lactating
dairy cows that are found in selected dairy farms in and around Dire Dawa. About 403
lactating cows (331 high grades Holstein Friesian and 72 local (zebu)) breed were
investigated.
21
Figure 1. Map of the study area
3.3. Study Design, Sample Size and Sampling Method
A cross sectional study was conducted. Dairy farms were purposively selected based on
accessibility. Simple random sampling technique was followed to select the dairy farms
but the lactating cows in the selected dairy farms were sampled using cluster sampling
method. The desired sample size was calculated according to the formula given by
Thrusfield (2005).
n= 1.962Pexp (1-Pexp)
d2
Where:
22
n = required sample size
Pexp = expected prevalence
d
= desired absolute precision
Previously subclinical mastitis prevalence was reported as 36% in Holstein and 6% in local
breeds (Adugna, 2008). As there was variation of mastitis prevalence among the breeds
and thus breed specific sample size was calculated as follows:
High grade Holstein breed: n= 1.962*0.36 (1-0.36) = 354
(above 80% blood level)
0.052
Local breed: n= 1.962*0.06 (1-0.06) = 87
(100% local)
0.052
3.4. Data Collection
3.4.1. Questionnaire
Questionnaire was compiled to evaluate the effect of potential risk factors on the
occurrence of mastitis. Farm owners having dairy cows of varied breed and cow number
were interviewed through a one stop visit. Data on each sample cow was collected in a
properly designed format. Risk factors considered were breed, age, parity, and stage of
lactation, previous mastitis history, udder washing, towel usage, udder drying, barn
washing, blind teat, good milking procedure, hand milking, housing and lesion on the
udder skin or teat. The factors were categorized into host factors, management and
environmental factors. The host factors to be considered were age, parity, inherited
features of the cow, breed, milk yield, stage of lactation and mammary regression. The
management factors were: nutrition, milking hygiene, milking machine. Housing and
climate were considered as environmental factors.
3.4.2. Clinical examination of udder and milk
Clinical findings like abnormalities of secretions, abnormalities of size, consistency and
temperature of mammary gland were examined by visual inspection and palpation. Pain
23
reaction upon palpation, changes in the milk (blood tinged milk, watery secretions, clots,
pus), and change in consistency of udder were considered as indications of the presence of
clinical mastitis. The milk was examined for its colour, odour, consistency and other
abnormalities.
3.4.3. California Mastitis Test (CMT)
The reaction involved in the CMT is the disintegration of leukocytes when milk is mixed
with the reagent (Babaei et al., 2007). According to the visible reactions, the results were
classified in four scores: 0 = negative or trace 1 = weak positive, 2 = distinct positive and 3
= strong positive. Mammary glands without clinical abnormalities and with apparently
normal milk that was bacteriologically negative and negative on the California mastitis test
were considered to be normal milk, while those that were bacteriologically positive and
with the CMT positive were considered to have subclinical mastitis.
The California Mastitis Test was carried out as described by Quinn et al. (2004). A squirt
of milk, about 2 ml from each half was placed in each of 2 shallow cups in the CMT
paddle. An equal amount of the commercial CMT reagent was added to each cup. A gentle
circular motion was applied to the mixtures in a horizontal plane for 15 seconds. The result
of the test was indicated on the basis of gel formation. The interpretation (grades) of the
CMT was evocated and the results graded as 0 for negative and trace 1, 2 and 3, for
positive (Quinn et al., 2002).
3.4.4. Milk Sample Collection, Handling and Transportation
Aseptic procedures for collecting CMT positive quarter milk samples as described by
Hogan et al. (1999) and Quinn et al. (2004) were followed. About 27 pooled milk samples
were collected from 15 dairy farms and five
kebeles in and around Dire Dawa. This
means the samples were collected from each lactating cow and were mixed in one
container for microbiological culture. Each milk sample was collected under aseptic
conditions in a sterile screw caped bottle containing anticoagulant which numbered to
identify the particular dairy farm. All milk samples were sent directly to the laboratory,
within an hour for routine cultural techniques. The time for milk sample collection was
before milking. Udders and especially teats were cleaned and dried before sample
24
collection. Each teat end was scrubbed vigorously with cotton alcohol pads. A separate
pledged of cotton was used for each teat. The first stream of milk was discarded and 10 ml
of milk from each cattle in a given farm was pooled into clean container. After collection,
the sample was placed in an icebox and transported to Dire Dawa regional laboratory for
cultural examination.
3.4.5. Microbiological Culture (Etiological Factors)
To prepare media for bacterial culture, the manufacturer´ s instructions were followed. All
glass wares used for the preparation of media were first sterilized using appropriate
equipment like autoclave, hot air oven, the appropriate amount of dehydrated media were
weighed out of using sensitive balance and the required amount of distilled water was
added to the powder media. Dehydrated media containing agar were dissolved in heating
mantle until it boils and frothy appearance was settled (removed), then the media were
sterilized by autoclave at 121C for 15 min holding time, and cooled in water bath at 50C
before poured into the Petri dishes. Some media like blood agar requires addition of blood
after it is cooled to 50C since RBC do not tolerate higher temperature (Quinn et al., 2002).
The common media used during the study were blood agar and Mac Conkey agar. Milk
samples were cultured onto 10% sheep blood agar and Mac Conkey agar plates according
to Coulon et al. (2002). Culturing of milk sample collected from dairy farms, in search for
mastitis producing organisms in standard of examination for mastitis (Radostits et al.,
2007).
The inoculated plates were incubated aerobically for 24-48 h at 37°C. The plate was
examined for growth, morphology activity. Suspected colonies were sub-cultured on a new
plate for further investigation. For primary identification of bacteria, once a pure culture is
obtained, a gram-stained smear from the culture was used to categorize the bacteria based
on gram-reaction and cellular morphology. Staphylococcus species, streptococcus species
and E. coli were the targets, because, these pathogens are the major microorganisms which
cause bovine mastitis (Potter, 2008). Suspected colonies were identified morphologically
and microscopically. Cultures with fine bacterial growth were considered as positive and
cultures with no visible growth were taken as negative, but polluted cultures with disturbed
media were considered as contaminated (Shakoor, 2005).
25
3.5. Data Analysis
The collected data during the study periods were entered into MS- Excel spread sheet and
descriptive statistics was used to illustrate the various variables in the production system
including husbandry and management variables. For data analysis, SPSS version 20 was
used. The chi-square (χ2) test was used to assess the association among the hypothesised
risk factors namely the breed, age, udder washing, udder drying, farm hygiene
management system and stage of lactation with the occurrence of the disease. Finally,
logistic regression analysis was used to determine the predicting factor of mastitis. In all
the analysis, confidence level was held at 95% and statistical analysis was consider
significant at p<0.05.
26
4. RESULTS
4.1. Description of the Study Population and Dairy Farms
4.1.1. Study Animals
The average age of the lactating dairy cows was 8.5 years (ranged between 2 and 15 years)
and the milk yield ranged between one and 35 litres per day (average 18). The average
parity of the lactating cows was 6.5 (ranged between one and 12).
4.1.2. Farming System
The dairy farms practised both extensive and intensive dairy production systems. Number
of animals per farm ranges from one to fifty one (1-51) lactating cows in the farms
surveyed. The extensive dairy production system ranged from one to three lactating cows
but the intensive dairy production system ranged from two to fifty one lactating cows. The
feeding status of the dairy farms was quite different between extensive and intensive dairy
production systems. In extensive dairy production system no concentrate feeding; it was
only roughage feeding but in intensive dairy production system, there was relatively
concentrate and roughage feeding practice. The milking practice of all dairy farms was
hand milking (manual). The establishment of the dairy farms (farm age) ranges from five
to thirty six (5-36) years.
4.2. Prevalence of Mastitis
4.2.1. Animal level
The animal level prevalence of mastitis is presented in Table1. The dairy farms were
classified based on numbers of lactating cows. During the study period, 403 lactating cows
were clinically examined from 20 smallholder and medium dairy farms in and around Dire
Dawa. The prevalence of bovine mastitis in the study area was 50/403 (12.4%) and
320/403 (79.4%) for clinical and subclinical mastitis, respectively. Among 50 clinical
mastitic cases 2 of them were negative on CMT. The prevalence of subclinical mastitis in
27
different breeds was 81.27% in Holstein, 70.8% in Zebu and the overall prevalence was
79.4%.
Table 1. Prevalence of Subclinical Mastitis at animal level
Animals
Holstein
Zebu
Overall
Number of cows examined
331
72
403
Mastitis +ve
269
51
320
Prevalence (%)
81.27
70.80
79.40
4.2.2. Quarter level
All quarters of the lactating cows were examined, out of 1592 quarters examined 777
(48.80%) were positive for subclinical mastitis and 50 (3.14%) were detected with clinical
mastitis. Among the quarters examined 28.27% were negative and 1.24% was blind. The
strong positive and trace positive at quarter level were 3.27% and 16.52%, respectively
(Table 2).
Table 2. Clinical, blind and CMT scores of each quarters of lactating cow in sample farms
CMT Scores
RF
RH
LF
LH
Negative (0)
96
94
144
116
Trace (+)
72
72
67
52
Positive (2+)
204
203
167
203
Strong positive (3+)
16
11
11
14
Clinical
10
17
10
13
Blind
5
6
4
5
Total
403
403
403
403
RF= Right Front; RH= Right Hind; LF= Left Front; LH= Left Hind
Prevalence%
450(28.27)
263(16.52)
777(48.80)
52(3.27)
50(3.14)
20(1.24)
1612
4.2.3. Types of Dairy Production Systems and Mastitis Prevalence
Table 3. Prevalence of bovine mastitis related to production systems in and around Dire
Dawa.
Intensive
Extensive
Total
No. of cows
331
72
403
Clinical
40 (12.08%)
10 (13.89%)
50 (12.4%)
Subclinical
269 (81.4%)
51 (70.8%)
320 (79.4%)
Total
93.35%
84.72%
91.8%
28
Table 4. Prevalence of Bovine Mastitis in small and medium farms
Small farm
Medium farm
Total
Examined cows
289
114
403
Clinical
32 (11.07%)
18 (15.79%)
50 (12.4%)
Subclinical
224 (77.5%)
96 (84.21%)
320 (79.4%)
Overall
88.57%
100.00%
91.8%
The prevalence of bovine mastitis was 11.07% clinical, 77.5% subclinical in small farm
and 15.79% clinical, 84.21% subclinical in medium farm. The overall bovine mastitis
prevalence was 88.57% and 100.00% in small and medium farms, respectively.
Prevalence
82
80
78
76
74
72
70
68
66
64
Prevalence
Holstein
Zebu
Overall
The prevalence of subclinical mastitis in the study area
4.3. Associated Risk Factors
Many factors were considered as potential risks for the occurrence of bovine mastitis in the
current study. The potential risk factors were classified as host factors (breed, age, milk
yield, lactation stage, previous mastitis history, parity and teat lesion) and environmental or
management factors (udder washing, towel usage, udder drying, barn washing and concrete
floor).
29
4.3.1. Host Factors
Age of animal was significantly associated (P=0.000) with prevalence of mastitis. The
condition was four time less likely (OR: 0.24) to be detected in young animals (66.67%)
compared to adult cows (89.08%). Breed was significantly associated with mastitis
prevalence. The prevalence of mastitis was 81.2% in Holstein and 13.89% in zebu cattle.
Holstein breeds had 1.78 [1.0, 3.1] higher odds to be positive for CMT compared to zebu
breed. Significantly higher presence of mastitis was noted in high milk yielding cows (P=
0.001). Cows with previous clinical mastitis history were 3.6 time more likely to be
positive on CMT (Table 5).
Table 5. Association between the occurrence of subclinical mastitis and risk factors
Host Factors
Animal
examined
Mastitis
Positive
(%)
Breed
HF
Zebu
331
72
174
229
P-value
OR[95%CI]
0.047
1.78[1.0,
3.2]
30.38
0.000
0.24[0.14,
0.41]
13.99
0.001
0.42
0.51
2.27
0.32
12.23
0.000
0.344
0.557
269 (81.2)
51 (70.8)
Age
Young
Adult
Chisquare
value
3.93
116(66.67)
204(89.08)
Milk Yield
(<10 lit) Low
138
100(72.46)
(>10 lit) Medium 184
152(82.61)
(>20 lit) High
81
75(92.59)
No. parity
1-5
330
260(78.79)
>=6
73
60(82.19)
Lactation
stage
Early
164
128(78.05)
Mid
129
108(83.72)
Late
110
26(23.63)
History of
mastitis
Yes
98
90(91.83)
No
305
230(75.41)
Teat lesion
Present
25
21(84)
Absent
378
299(79.10)
OR= Odds ratio, CI=Confidence interval
3.66[1.7,
7.9]
30
The mastitis positive cows were 78.04% in early, 83.72% in mid and 76.36% (X2=2.27,
P=0.32) in late lactation stages for subclinical mastitis. From the 320 subclinical mastitis
positive cows 21(6.00%) cows had teat lesion but 299 (93.44%) were free from teat lesion.
4.3.2. Environmental Factors
Presence or absence of good milking practices and environmental factors that were
hypothesised to favour the existence of mastitis in a farm were cross checked but all
environmental and husbandry factors considered in the study were not significantly
associated with mastitis prevalence. The detail information on mastitis prevalence Vis –aVis the actors is provided in Table 6.
Table 6. Association between subclinical mastitis and Environmental Factors
Environmental Factors
Animal
examined
Mastitis
Positive
(%)
Udder
Washing
Yes
No
331
72
Towel
Usage
Yes
No
152
251
Chisquare
value
3.94
269(81.27) 51(70.80)
2.1
P-value
OR[95%CI]
0.047
1.78 [1.00,
3.18]
0.148
115(75.66)
205(81.67)
Udder
Drying
2.1
0.148
Yes
No
152
251
115(75.66)
205(81.67)
Yes
No
265
138
214(80.75) 0.86
106(76.81)
0.353
1.49
0.222
Barn
Cleaning
Floor Type
Concrete 316
Soil
87
255(80.69)
65(74.71)
OR= Odds ratio, CI=Confidence interval
Cows which were washed their udder had high mastitis prevalence than that of not washed,
because, using of common source of washing water (bucket) and common towel usage.
31
4.3.3. Microbiological Culture (Aetiological Factors)
All samples were positive for different bacterial species in microbiological culture. The
bacteria isolated with high prevalence were staphylococcus species 18/27 (66.67%),
streptococcus species 4/27 (14.81%), E. coli 3/27 (11.11%), and bacillus species 2 /27
(7.41%).
Table 7. Frequency distribution of isolated bacteria from mastitic dairy farms
Bacterial isolates
Staphylococcus species
Streptococcus species
E. coli
Bacillus species
Total
Frequency
18
04
03
02
27
Prevalence (%)
66.67
14.81
11.11
7.41
100.00
Among the samples examined, 18 samples were positive for Staphylococcus species. This
took the highest percent of the mastitis causative agents. But Bacillus species were the
least prevalent mastitis causative agents.
32
5. DISCUSSIONS
This study showed overall prevalence of bovine mastitis in lactating dairy cows to be
higher than that of the previous reports in and around Dire Dawa. Adugna (2008) reported
the prevalence to be 36% in Holstein and 6% in local breeds in Dire Dawa. The high
prevalence in the present study might be attributed to the poor management and increasing
environmental temperature. In the present study, like some previous studies (Almaw et al.,
2008; Getahun et al., 2008), the majority of the cases of mastitis were subclinical. Farmers
have good knowledge about clinical mastitis due to the observed visible changes, thus they
timely treat the disease, but subclinical mastitis is not easy to be identified by farmers and
its silent effect is critical to dairy producers.
The prevalence of clinical type of bovine mastitis is higher than the previous findings of
Bedada and Hiko (2011) and is lower than Workineh et al. (2002), who reported the
prevalence rate of 10.3 and 21.5%, respectively in different parts of Ethiopia. The clinical
mastitis prevalence in high grade Holstein breed in this study is higher than that reported
by Bishi (1998) who reported 5.3% prevalence in Addis Ababa. Mastitis is a complex
disease and the difference in results could be due to difference in management system,
topographic and climate condition between the farms.
Subclinical mastitis was high in both breeds compared to clinical mastitis. The prevalence
of subclinical mastitis of this finding is in agreement with 80.6% reported by Dabash et al.
(2012), and it is somewhat comparable with the prevalence rate of 89.5% reported by
Argaw and Tolosa (2008). However, the prevalence of sub clinical mastitis in the present
study is far higher than previous reports of Moges et al. (2012 and Mekibib et al. (2010)
who noted a prevalence rate of 30.6 and 25.22%, respectively in different areas of
Ethiopia. The high prevalence rate is due to improper milking hygiene, lack of post
milking teat dipping and poor housing facilities in the present dairy farms studied.
The quarter level clinical mastitis prevalence (3.1%) recorded in the current study is lower
than the finding of Haftu et al. (2012), but the subclinical mastitis prevalence is higher than
that reported by Haftu et al. (2012). This could be due to poor hygiene and management
system. The prevalence of blind teat in this work is higher when compared with that
33
reported by Haftu et al. (2012) and Bitew et al. (2010). The blind quarters observed in this
study are an indication of a serious mastitis problem on the farms and the absence of
culling that should have served to remove a source of mammary pathogens for the cows.
Association of mastitis occurrence with parity was evaluated and found statistically not
significant (P>0.05). The reported in the current study is contradicts with the previous
reports (Tamirat, 2007; Mekibib et al., 2010; Haftu et al., 2012). This is attributed to the
increased opportunity of infection with time and the prolonged duration of infection,
especially in a herd without mastitis control program (Radostits et al., 2007).
Age of animal was highly significantly associated (P=0.000) with prevalence of mastitis.
The condition was four time less likely (OR: 0.24) to be detected in young animals
(89.1%) compared to adult cows (66.7%). Breed was significantly associated with mastitis
prevalence. The prevalence of mastitis was 81.2% in Holstein and 13.89% in zebu cattle.
Holstein breeds had 1.78 [1.0, 3.1] higher odds to be positive for CMT compared to zebu
breed. Significantly higher presence of mastitis was noted in high milk yielding cows (P=
0.001). Cows with previous clinical mastitis history were 3.6 time more likely to be
positive on CMT.
Among the risk factors considered to have effect on the occurrence of mastitis, udder
washing was found to be statistically significant (P < 0.05). In the present study, parity and
lactation stage were not found to increase the occurrence of mastitis significantly (p >
0.05). According to Erskine (2001) primiparous cows have more effective defense
mechanism than multiparous cows. The prevalence of subclinical infection decreases as the
stage of lactation progresses. These infections are generally the result of contagious
mastitis and caused by an inability of mastitis control rather than a physiologic effect
(Erskine, 2001). In this study, it was observed that cows with skin lesions on their teats
and/or udder had a high prevalence of mastitis. Similar observation has been recorded by
Abdurahman (2006) in eastern Ethiopia. Mulei (1999) also noted that mammary gland
quarters with teat lesions were 7.2 times more likely to have bacterial organisms isolated
from them than those without any teat lesions in the Kiambu district of Kenya.
34
The association between concrete floor and high prevalence of subclinical mastitis
recorded in this study is due to absence of barn cleaning and poor barn hygiene. Cows with
previous history of mastitis were found more likely to be mastitic. This observation is
supported by the findings of Biffa et al. (2005). Prevalence of mastitis was significantly
(P<0.05) associated with milking hygiene practice. The results revealed that animals with
poor hygiene of milking process had a high prevalence of mastitis. Poor hygiene of
milking process also was identified as a risk factor for occurrence of bovine mastitis in
another study in Ethiopia (Abdurahman, 2006). This might be due to absence of udder
washing, milking of cows with common milkers’ which have cuts and chaps on their
hands, and using of common udder cloths, which could be vectors of spread specially for
contagious mastitis
The common isolated genera of bacteria in the present study (Staphylococcus,
Streptococcus, Bacillus, and Escherichia) agree with the findings of Abdurrahman (2006),
Kalla et al. (2008), and Abera et al. (2010) who noted Staphylococcus, Streptococcus, and
Escherichia as major mastitogens. Radostits et al. (2000) asserted that Satphylococcus
aureus is well adapted to survive in the udder and usually establishes a mild subclinical
infection of long duration, from which it shed in milk, facilitating transmission to healthy
animals, mainly during milking procedures. Bacterial isolates were recorded from all the
403 clinically and sub clinically affected lactating cows. All the investigated dairy farms
were positive for different bacterial species. This indicates that dairy farms have similar
problems in hygiene and husbandry practices which did not able to minimize the mastitis
causing micro-organisms.
Staphylococcus species were the predominant pathogens constituting 66.67% of all
bacterial isolates in the current study. This does not agree with that reported by Mekibib et
al. (2010) percentage of which is lower than the current study. The relative high prevalence
of Staphylococcus species in the current study show the absence of dry cow therapy and
low culling rate of chronically infected animals in the study area. Streptococcus species
were also found prevalent with 14.81% share of the total isolates. The finding in the
present study is in agreement with that noted by Bitew et al. (2010) at Bahir Dar and its
environs (13.9%). However, Hawari and Al-dabbas (2008) reported 26.2% relative
35
frequency of Streptococcus species in Jordan and Atyabi et al. (2006) who found 33.54%
at farms around Tehran.
E. coli occurred with the prevalence of 11.11% of the isolates. The findings is different
from the previous reports by Mekibib et al. (2010) at Holeta (4.6%) and Sori et al. (2005)
in and around Sebeta (0.75%), but is comparable with the report of Hawari and Al-dabbas
(2008) in Jordan (15.6%). E. coli is an environmental contaminant and its high prevalence
in the present report could be related to hygienic status practiced at the study site. Bacillus
species were the least prevalent bacteria species (7.41%) from the bacterial isolates. The
reason could be bacillus species are not common causative agents of bovine mastitis.
The present study shows high prevalence of bovine mastitis with Staphylococcus and
Streptococcus species to be the dominant bacterial isolates in the study area. Based on the
results of this study, we recommend implementation of strict fortnight mastitis control
program, improved milking hygiene, prevention of skin lesions, culling of chronic mastitis
carriers, and treating of clinically infected cows in the study area.
36
6. SUMMERY, CONCULISION AND RECOMMENDATIONS
In developing countries such as Ethiopia, cows in the dairy farms are maintained in
inadequate hygienic environment, poor animal health service, and lack of proper attention
to health of the mammary gland. Mastitis has been known to cause a great loss of
productivity through poor milk quality, reduced milk yield, and due to culling of cows. The
overall prevalence of mastitis at cow and quarter level in the current study was 91.8% and
70.85%, respectively. Three hundred twenty (79.4%) cows were positive for subclinical
mastitis while only 50 (12.4%) had clinical mastitis. Among the total (1612) quarters
investigated, 20 teats (1.24%) were blind. The present study shows high prevalence of
bovine mastitis with Staphylococcus and Streptococcus species to be the dominant
bacterial isolates in the study area. Based on the results of this study, we recommend
implementation of strict fortnight mastitis control program, improved milking hygiene,
prevention of skin lesions, culling of chronic mastitis carriers, and treating of clinically
infected cows in the study area.
Based on the above conclusion, the following points are forwarded as recommendations
 To reduce the prevalence of the disease, different epidemiological factors that interplay in
mastitis occurrence should be studied.
 Extension service including health education must be launched to create public awareness
about the disease, since dairy is becoming the common enterprise and owners are new to
the business and not knowledgeable about udder health.
 Implementation of better management practices with introduction of comparatively
mastitis tolerant animals.
 Proper milking procedure with post milking teat disinfection, prompt treating of mastitis
positive cows, segregation of diseased cows and culling incurable cows should be
encouraged in the study area to reduce the prevalence of bovine mastitis.
37
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8. APPENDEXES
8.1. ANNEXES
Annex 1. Questionnaire format
Owners Name: _______________Address______________
Date of sample Collection___________________
1. Cow History:
Breed_____age___calving date____parity_____previous history of mastitis___milk
yield___
Teat Lesion: present_______absent_____
Gross milk quality: watery____bloodtinged____clots/flakes___ normal___
Sample collected from: RR___RF____LF___LR_____
CMT score: RR___RF____LF___LR_____
2. Milking practice:
Manual______________ or using milking machine_______________
Do you wash udder before milking? yes____ no____
Do you dry after washing? yes____ no____
Do you use the same cloth for all teats? yes____ no____
Do you practice milking mastitic cows last? yes____ no____
Do you wash your hand before milking and after milking one cow? Yes___ no___
3. Feeding Status:
concentrate_______,
grass________,
legumes__________,
feeding
interval______,
concentrate: roughage ratio__________, kilogram per day__________
4. Housing:
Floor concrete______stone_____soil______slopy_____leveled_____
Roof: metal sheet______ grass_____
Wall: concrete_________mud______other______
Manure removal: daily______weekly____monthly____other (specify) ______
5. Do you practise good milking procedures? Yes___ no____
6. Have you ever used teat dipping after milking? Yes___ no___ if yes, what type?
48
Annex 2. Interpretation of CMT Findings
Source: Quinn et al. (1999)
Score
Interpretation
Visible reaction
0
Negative
Milk fluid and normal
T (Trace)
Trace
Slight precipitation
1
Weak positive
Distinct precipitation but no gel formation
2
Distinct positive
Mixture thickens with gel formation
3
Strong positive
Viscosity greatly increased .strong gel that is
cohesive with a convex surface