International Journal of Veterinary Science & Technology

Research article
Study of Prevalence, Associated Risk Factors and
Causative Bacteria of Bovine Mastitis in Ethiopia -
Desiye Tesfaye Tegegne*, Shimels Tikuye Yalew, Bezina Arega Emeru, Yohannes Equar
Messele, Gebremeskel Mamu Werid, Shelema Kelbessa Bora and Mossisa Dire Babura
Animal Biotechnology Research Program, National Agricultural Biotechnology Research Center, Ethiopian
Institute of Agricultural Research, Ethiopia
*Address for Correspondence: Desiye Tesfaye Tegegne, Animal Biotechnology Research Program, National
Agricultural Biotechnology Research Center, Ethiopian Institute of Agricultural Research, P. O. Box 249, Holeta,
Ethiopia, Tel: +251-911-113-398; ORCiD: orcid.org/0000-0003-3722-6743;
E-mail:
Submitted: 02 December 2019; Approved: 10 February 2020; Published: 13 February 2020
Cite this article: Tegegne DT, Yalew ST, Emeru BA, Messele YE, Werid GM, et al. Study of Prevalence,
Associated Risk Factors and Causative Bacteria of Bovine Mastitis in Ethiopia. Int J Vet Sci Technol. 2020;4(1):
001-006.
Copyright: © 2020 Tegegne DT, et al. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
International Journal of
Veterinary Science & Technology
ISSN: 2640-4397

International Journal of Veterinary Science & Technology
SCIRES Literature - Volume 4 Issue 1 - www.scireslit.com Page -002
ISSN: 2640-4397
Abstract
Bovine mastitis has a negative impact through economic losses in the dairy sector across the globe. A cross sectional study was carried out from
September 2015 to July 2016 to determine the prevalence of bovine mastitis, associated risk factors and isolation of major causative bacteria in lactating
dairy cows in selected districts of central highland of Ethiopia. A total of 304 lactating cows selected randomly from five districts were screened by California
Mastitis Test (CMT) for subclinical mastitis. Based on CMT result and clinical examination, over all prevalence of mastitis at cow level was 70.62% (214/304).
Two hundred fourteen milk samples collected from CMT positive cows were cultured for isolation of major causative bacteria. From 214 milk samples,
187 were culture positive and the most prevalent isolates were Staphylococcus aureus 42.25% (79/187) followed by Streptococcus agalactiae 14.43%
(27/187). Other bacterial isolates were included Coagulase Negative Staphylococcus species 12.83% (24/187), Streptococcus dysgalactiae 5.88% (11/187),
Escherichia coli 13.38% (25/187) and Entrococcus feacalis 11.23% (21/187) were also isolated. Moreover, age, parity number, visible teat abnormalities,
husbandry practice, barn floor status and milking hygiene were considered as risk factors for the occurrence of bovine mastitis and they were found
significantly associated with the occurrence of mastitis (p < 0.05). The findings of this study warrants the need for strategic approach including dairy extension
that focus on enhancing dairy farmers’ awareness and practice of hygienic milking, regular screening for subclinical mastitis, dry cow therapy and culling of
chronically infected cows.
Keywords: Bovine mastitis; Bacteria; Ethiopia; Prevalence; Risk factors
INTRODUCTION
Ethiopia is believed to have the largest livestock population in
Africa. This livestock sector has been contributing considerable
portion to the economy of the country and still promising to rally
round the economic development of the country [1]. It is eminent
that livestock products and by products supply provide mainly the
needed animal protein that contributes to the improvement of the
nutritional status of the people [2]. Even though Ethiopia is the
most populous country in cattle than any African country; the per
capital milk consumption was 16 kg, which was lower than other
countries in the region [3]. This is partly due to the low genetic milk
production potential of the indigenous zebu cattle [2]. To increase
milk production cross breeding of indigenous zebu with exotic
breeds particularly with Holstein Friesian is widely practiced which
resulted in a larger portion of the dairy cattle population. However,
this market oriented dairy production, a rapidly growing system in
many African countries, is subjected to diseases of intensification
including mastitis and reproductive disorders [4].
Mastitis is one of the most important disease affecting dairy
cows and it is a multi-factorial disease with worldwide distribution
which incurs serious economic losses to dairy industry [5]. The
disease results in decreased production, discarded milk and medical
treatments as well as a higher level of premature culling of affected
animals [6]. Milk and milk by-products are considered to contribute
to the social and economic development in rural areas where the
dairy production from cattle is one of the major sources of income in
many households [7]. Dairy products also provide essential food and
nutrition for people in these areas [8]. A number of previous reports
from different part of Ethiopia indicated that mastitis is a serious
problem in dairy industry [9]. Bovine mastitis can reduce milk yield,
increase culling rate, incur treatment cost, and occasionally result in
death from severe infection [2]. Moreover, mastitis had been known
to cause a great deal of loss or reduction of productivity, to influence
the quality and quantity of milk yield, and to cause culling of animals
at an unacceptable age [8]. Generally, as with most infectious disease,
mastitis risk factors depends on three components that is exposure
to the microbes, cow defense mechanism and environment and
management factors [10]. The economic loss due to the disease
is considerable and can be crucial, especially for small-scale dairy
farmers in Ethiopia. Therefore, the objectives of this study were
to determine the prevalence of bovine mastitis, isolate the major
bacteria that cause bovine mastitis and to determine the various risk
factors associated with the occurrence of mastitis in Ethiopia.
MATERIALS AND METHODS
Animal characteristics
The crossbred dairy cows (Borana × Holstein breed) found in the
study area is managed under extensive system as a source of milk,
meat and drought power. Small holder dairy farms in most areas are
housed always and provided feed in their stall.
Study areas
This research was conducted in central highlands of Ethiopia
that fall in the administrative territory of Oromia National Regional
State. So, Oromia Special Zone of Surrounding Addis Ababa is found
in the central part of the Oromia Regional State, surrounding the
capital city-Addis Ababa. For the purpose of this study five (Holeta,
Menagesha, Burayu, Sululta and Sebeta) major towns were selected
based on number of commercial dairy farms [2].
Study population and husbandry practice
Study animals were cross breed lactating dairy cows owned by
government and other commercial dairy farms in central highland
of Ethiopia. The animals were often managed under both semi-
intensive and intensive management system. They are often provided
with some supplementary diet in addition to the natural pasture and
agricultural by-products and some are maintained usually in separate
stalls, a short distance from each other in a house.
Study design and Sample size determination
Cross-sectional study was conducted from September 2015 to
June 2016 in lactating cross breed dairy cows in the study area. The
sample size was determined at 95% confidence interval, 5% precision
and from previous studies in the study area by [11] with an expected
prevalence of 34.4%. Thus, the sample size was calculated based on
formula of [12] and the minimum sample size was 346 animals.
However, due to the lack of cooperation in some farm owners, only
303 lactating dairy cows were included for this study.
Study methodology
The study methodology involved reviewing farm documents,
farm inspection, animal examination and laboratory investigation.
Farm records with respect to animals’ parity, past disease history,
production performance were reviewed. Relevant information
related to the previous health history of the mammary quarters was
obtained from case record book. Other available farm documents
were also read.

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Farm inspection: Farm inspection was practiced to assess the
housing conditions, feeding practices and milking practices. The
housing condition was qualified as poor when there is bad smell,
feed trough and gutter (for waste drainage) were dirty, animals flank,
udder and belly were soiled. The housing condition was qualified
as good when none of the above indicated defects were observed.
Milking practice was investigated through close observation at the
time of milking.
Preparation of udder and teats for milk sample collection: The
udder, especially the teats were cleaned and dried before milk sample
collection. Dust, particles or other filth was removed by brushing the
surface of the teats and udder with a dry towel. The teats were washed
with tap water and dried. Then, the teats were disinfected with cotton
soaked in 70% ethyl alcohol.
Sampling method and Sample handling: Milk samples were
collected by standard milk sampling techniques from all lactating
cows with clinical and subclinical mastitis. To reduce contamination
of the teat ends during sample collection, the near teats were sampled
first followed by the far once. Approximately 10 ml of paired milk was
collected from each quarter (one for CMT and one for bacteriological
examination) into labeled sterile screwed cap universal bottle after
discarding the first three milking streams. Samples were placed
in ice box and transported to the Animal Biotechnology Research
Laboratory under National Agricultural Biotechnology Research
Center and processed as soon as possible without any delay [13].
California Mastitis Test (CMT): Subclinical mastitis cases were
diagnosed based on CMT results and the nature of gel formation
(milk and CMT reagent), which shows the presence and severity
of the infection [6]. Before sample collection for bacteriological
examination, milk samples were examined for visible abnormalities
and were screened by the CMT. From each quarter of the udder, a
squirt of milk sample was dropped in each of the strip cups on the
CMT paddle and an equal amount of CMT reagent was added to each
cup and mixed gently. The test result was interpreted based on the
thickness of gel formed by CMT reagent and milk mixture and scored
as 0 (negative), T (trace), 1 (weak positive), 2 (distinct positive) and 3
(strong positive). Finally, quarters with CMT score of 1 or above were
judged as positive for sub-clinical mastitis; otherwise negative [13].
Bacteriological isolation and identification: Milk samples from
subclinical quarters were bacteriological examined according to the
procedures employed by [13]. Briefly, before inoculating into primary
culture medium, the milk samples were centrifuged so as to increase
the bacterial load. A loop full of centrifuged mastitic milk sample
was taken from each infected quarter and inoculated separately on
to blood agar base enriched with 5% ovine blood using quadrant
streaking method. The inoculated plates were incubated aerobically
at 37°
C for 24 to 48 hours, after which presence or absence of bacterial
growth, colony morphology, color and hemolytic characteristics were
recorded on primary culture. Prior to further biochemical tests, the
isolated bacteria on blood agar were sub-cultured into nutrient agar.
Each culture was subjected to gram staining to determine their shape,
and gram reaction. Catalase test using 3% Hydrogen per oxide (H2
O2
)
was performed to identify catalase positive and catalase negative
bacteria. Mannitol Salt Agar (Oxiod,UK) and purple base agar
(Difco) with 1% maltose were used to differentiate Staphylococcus
species and incubated at 37°
C and examined after 24-48 hours for
mannitol and maltose fermentation respectively. Tube coagulase test
using rabbit plasma was used to identify the coagulase positive and
coagulase negative Staphylococcus species. Enterobactericae species
were identified using Oxidase test, SIM medium (Oxiod, UK) for
sulfur production, indole test after addition few drops of kovacs
reagent and motility test, TSI (Triple Sugar Iron) (Oxiod, UK) to
detect sugar fermentation, sulfur and gas production, MacConkey
agar (Oxiod, UK) for lactose fermentation and colony characteristics
and Simmon’s citrate agar (Oxiod, UK) to differentiate bacteria based
on citrate utilization [14].
Data entry and analysis
Data were coded, cleaned and entered into Microsoft Excel
computer software. Statistical analysis was carried out using SPSS
version 20. Data were analyzed descriptively using descriptive
statistics in the first step; thereafter association of the different
variables with interest of outcome was analyzed using a Chi-squared
(χ2
) test. The association was considered significant when odds ratio
was greater than one and p-value was less than 0.05.
RESULTS
Prevalence of bovine subclinical mastitis and its
associated risk factors
From a total of 303 samples collected from dairy farms of the
study area and were screened by CMT, which yielded an overall
prevalence of 70.62% that is 187 animals examined had infection
in their udders as evidence of mastitis. This result showed that the
association between the occurrence of mastitis in the selected cows
and different potential risk factors. Accordingly, mastitis prevalence
showed significant variation among different age groups, husbandry
practice, visible teat lesion, parity number, barn floor status and milk
hygiene (p < 0.05) (Table 1).
Study animals were grouped into three as younger than 6 years,
age range of 6 and 10 years and older than 10 years. From 50, 87 and
166 cows were examined with age in these age groups, 28 (56%),
50 (57.47%) and 136 (81.97%) were found positive for mastitis,
respectively. The result showed that the prevalence of mastitis was
significantly higher (81.97%) in cows older than 10 years followed by
cows in the age range of 6 to 10 years and lowest in cows younger than
6 years. The statistical analysis also showed that there exist highly
significant differences among the three age groups (p < 0.05) in the
occurrences of mastitis (Table 1).
Both parity number and visible teat abnormalities were found to
be significantly (p < 0.05) associated with the occurrence of mastitis.
The highest prevalence of mastitis was observed 45 (90.00%) in cows
with parity of more than 6, followed by less than 3 and between 3 and
6 parity (Table 1). The highest prevalence of mastitis was observed
99 (91.66%) in cows with two teat blind, followed by teat laceration/
lesion one teat blind (Table1).
Cows managed intensively (p < 0.05) was highly susceptible to
mastitis when compared to those managed semi intensively with
respective number and percent of 79 (84.04%) and 135 (64.59%)
among the totally examined cows, respectively (Table 1). The
prevalence of mastitis was higher 196 (96.07%) in cows on farms
with poor barn floor status condition and lower 8 (8.08%) in farms
with good conditions barn floor status condition. Statistically, highly
significant association was observed between mastitis and barn floor
status of the farms visited (p < 0.05) (Table1).
Furthermore, mastitis prevalence was found to be higher 174
(93.04%) in only washed during milking and lower 40 (34.48%) in

ISSN: 2640-4397
washed and dried with towel (Table1). Statistical analysis showed the
existence of significant (p < 0.05) association between the occurrence
of mastitis and milking hygiene (Table1).
Generally, in the present study, age, parity number, visible teat
abnormalities, husbandry practice, barn floor status and milking
hygiene were considered as risk factors for the occurrence of mastitis
and they were found significantly associated with the occurrence of
mastitis (p < 0.05) (Table1).
In district wise, total cows examined in Farm A, Farm B, Farm C,
Farm D and Farm E were 154,13,27,79 and 30, out of these 59.09 %
, 84.61% ,100%, 72.15% and 93.33% cows were positive for mastitis,
respectively (Table 2).
Bacterial isolation
From a total of 214 milk samples cultured, 187 (87.38%) were
culture positive. Analysis of bacteriological examination of milk
samples was made to identify the main etiological agents involved
in the bovine mastitis. Major causative bacterial were identified
on the basis of their cultural, staining characteristics and different
biochemical test. The highest prevalent bacteria was found to
be Staphylococcus aureus 79 (42.25%) followed by Streptococcus
agalactiae 27 (14.43%), Escherichia coli 25(13.38%), Coagulase
Negative Staphylococcus species 24 (12.83%), Entrococcus feacalis 21
(11.23%) and Streptococcus dysgalactiae (5.88%) in that order (Table
3).
DISCUSSION
Bovine mastitis remains a serious and common disease in
animals with significant economic losses in dairy industry worldwide
[15]. Currently, in Ethiopia especially in the central highlands where
most of the dairy farms are found, the information on prevalence, its
associated risk factors and major mastitis causing bacteria species is
scarce. Therefore, the objectives of the current study was to determine
the prevalence, its major causative bacteria species and risk factors
associated with the occurrence of bovine subclinical mastitis.
The overall prevalence of bovine subclinical mastitis was 70.62 %
at cow level in this study area. The present findings was comparable
to the findings of [9,16-18], who got 69.8% in dairy farms of Addis
Ababa and its vicinity, 71% in dairy farms of Holeta town , 71.1%
from Holeta and 74.7% around Addis Ababa, respectively. The finding
was higher than previous reports of [19-22] who reported 40.4% in
southern Ethiopia, 38.2% in Adami-Tulu central Ethiopia, 39.8% in
and around Addis Ababa, and 39.7% in Chaffa valley in north eastern
Ethiopia, respectively. However, it is lower than the reports of [23]
who indicated that 81.0% were positive for mastitis at cows’ level in
Holeta, central highland of Ethiopia, The variation in prevalence and
incidence of mastitis between the different studies might be partly
due to different types of diagnostic tests, sampling procedures and
associated risk factors such as age, number of parity, visible teat
abnormalities, husbandry practice, barn floor status, milking hygiene,
and breed of the animals included in the studies.
Among the risk factors considered; age, number of parity, visible
teat abnormalities, husbandry practice, barn floor status and milking
hygiene were found to be statistically significant (p < 0.05) with the
occurrence of mastitis. The result showed that the prevalence of
mastitis was significantly higher (81.97%) in cows older than 10 years
followed by cows in the age range of 6 to 10 years and lowest in cows
younger than 6 years. There was statistically significant difference
Table 1: Association between some selected risk factors and occurrence of bovine mastitis.
Risk factors Category Number of examined Number of positive Prevalence (%) X2
P - Value
Age group
(Years)
< 6
6-10
>10
50
87
166
28
50
136
56
57.47
81.97
22.63 0.000
Parity number
< 3
3-6
> 6
67
186
50
48
121
45
71.64
65.05
90
11.86 0.003
Visible teat abnormalities
Normal
One teat blind
Two teat blind
Teat laceration
135
46
108
14
74
31
99
10
54.81
67.39
91.66
71.42
39.55
0.000
Husbandry practice
Intensive
Semi-intensive
94
209
79
135
84.04
64.59
11.82 0.000
Barn ﬂoor status
Poor
Good
204
99
196
8
96.07
8.08
194.95 0.000
Milk Hygiene
Only washed
Washed and dried with towel
187
116
174
40
93.04
34.48
118.36 0.000
Table 2: Prevalence of bovine mastitis at cow level in diﬀerent districts in central
highland Ethiopia.
Districts
No. of examined
cows
No. of positive
cows
Prevalence (%)
Farm A 154 91 59.09
Farm B 13 11 84.61
Farm C 27 27 100
Farm D 79 57 72.15
Farm E 30 28 93.33
Table 3: The frequency of bacteria isolated from bovine mastitis in selected
districts of central highland of Ethiopia.
Bacterial species Total number of isolates Prevalence (%)
Staphylococcus aureus 79 42.25
CNS*
24 12.83
Streptococcus agalactiae 27 14.43
Streptococcus dysgalactiae 11 5.88
Entrococcus feacalis 21 11.23
Escherichia coli 25 13.38
Total 187 100
*
CNS = Coagulase Negative Staphylococcus species

ISSN: 2640-4397
among different age groups. This finding is in broad agreement with
reports made by different authors in different parts of the country
[24]. Who reported age considered as potential risk factor to mastitis
and older cows were more affected by mastitis than younger cows, this
finding is also agreed with previous reports on mastitis in Southern
Ethiopia in holeta area and it’s surrounding [17]. The increase
in prevalence rate with the advancing age may be due to gradual
suppression of immune system of the body, structural changes in
udder and teats and repeated exposure to milking practices.
The highest prevalence of subclinical mastitis was observed
(90.00%) in cows with parity of more than 6, followed by less than
3 and 3-6 parity. The highest prevalence of mastitis was observed
(91.66%) in cows with two teat blind, followed by teat laceration/
lesion (71.42%) and one teat blind (67.39%). This is in agreement
with [21,25,26] who identified parity as risk factor to mastitis in the
study conducted at different parts of Ethiopia. This might be due to
the increased opportunity of infection with time and the prolonged
duration of infection, especially in a herd without mastitis control
program and associate with the ability of the immune system of an
animal to defend infection causing agents.
Intensively managed cows present a higher risk for the
development of mastitis (84.04%), followed by semi-intensive
(64.59%), with least risk among extensively managed animals. This
result was line with previous reports on mastitis in [27]. Housing
increases the risk of mastitis because of the confinement of the
animals, and the multiplication of pathogens in the litters elevates teat
challenge, and consequently mastitis. Mastitis prevalence increases
in herds housed under poor stable and drainage conditions. This is
much more evident for coliform mastitis [28]. Several factors in the
environment affect the exposure of a cow to microorganisms. Sources
of environmental exposure are manure, bedding, feeds, dirt, mud and
water. A good example of this is Escherichia coli, which is present
in the environment of the cow. Several studies have indeed linked
the cleanliness of the barn, and the colony count in the bedding with
the incidence of clinical mastitis [29]. Prevalence of mastitis was
significantly (p < 0.05) associated with milking hygienic practice.
Cows at farms with poor milking hygiene standard (96.07%) are
severely affected than those with good milking hygiene practices
(8.08%). This result agreed with the report of [27,30]. This might
be due to high stocking density, dirty bedding or ground, infected
utensils, poor ventilation and high humidity.
The result obtained from bacteriological analysis of the samples
revealed that from the total of 214 strong CMT positive samples, 187
(87.38%) were culture positive which is higher than who reported
proportions of 18% [31]. In this study the predominant organisms
isolated from mastitis to be Staphylococcus aureus followed by
Streptococcus agalactiae. The predominance and primary role of S.
aureusisolateinbovinemastitishasalsobeenreportedinotherstudies
[14,17,31]. Different researches finding indicate that, Staphylococcus
aureus was the most frequently isolated bacteria as per the reports of
[16,32] in dairy farms of Holeta and Selalle towns, respectively.
The preponderance of contagious mastitis in this study may be
ascribed to the lack of proper milking procedure before milking,
during the time of milking and post milking [14]. For instance
absence of pre and post teat dipping using antiseptics, washing of
milker’s hands and using teats secretion as a lubricant of teats at
the time of milking which is often practiced in the study area might
contributed to the spread of these pathogens from infected teats
to healthy ones. In the present study indicated that environmental
bacteria like Escherichia coli was isolated in high proportion (9.4%).
This is in congruent with the reports of [32,33] who found 7.5% of the
total isolates. In contrast, this figure is higher than isolates reported
by [16,27] who reported 4.57% and 0.75% in different parts of
Ethiopia, respectively. The presence of environmental bacteria might
be an implication of unhygienic milking practice and contamination
of cows’ teats and environment with their dung in the study area.
CONCLUSION AND RECOMMENDATIONS
The current study showed that an overall prevalence of 214
(70.62%) from total of 303 cows examined bovine mastitis was
recorded in the study area. This indicates that mastitis is a serious
problem across herds in this study areas. The present study revealed
that mastitis has great economic significance associated with reduced
production and productivity of cows suffering from mastitis in study
area. Among the risk factors considered, age, number of parity, visible
teat abnormalities, husbandry practice, barn floor status and milking
hygiene were found to be statistically significant (p < 0.05) with the
occurrence of mastitis. In this study the predominant organisms
isolated from mastitis to be Staphylococcus aureus followed by
Streptococcus agalactiae. So, it is a problem for individual particular
and the country in general. In line with above facts the following
recommendations are forwarded: Awareness creation should be
given to the dairy herds on the impacts of bovine mastitis and its
associated risk factor, all quarters of the udder of each cow should be
periodically checked for the timely treatment and prevention, good
record keeping practice on the general herd health of dairy farms,
adequate housing with proper sanitation and ventilation should be
regularly maintained, Since the bacteria isolated from cows’ milk
samples in the present study are types that cause both contagious and
environmental mastitis, correct and good milking techniques should
be applied.
ACKNOWLEDGEMENTS
I greatly appreciate the contribution made by the Ethiopian
Institute of Agricultural Research, National Agricultural
Biotechnology Research Center in funding this project and the staff
of the Animal Biotechnology Research Program, for assisting during
the bench work which has led to the success of this work. Authors
also thankful to the editor and an anonymous reviewer for their
constructive comments that helped to improve the quality of this
paper.
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