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3. MATERIALS AND METHODS

3.1 Study Area

Jimma is located in Oromia regional state about 355km south-west of capital city of Ethiopia. It

has an agro-ecological setting of highlands 15% midlands 67% and lowlands 18% and

found at on latitude of 70 40N and 36o 10S and longitude of 36o 50 E having an

elevation of 1750 metres above sea level. The study area receives a mean annual rainfall of

about 1530 millimeters. The mean annual minimum and maximum temperature ranges

from 11.40C and 26.8C respectively.

3.2 Study Design

A cross sectional study was conducted to determine the prevalence and herd level risk factors of

intramammary infection caused by coagulase negative staphylococci in Jimma dairy herds and its

associated risk factors from June 2011to December 2012. There are 50 small holder dairy farms

found in Jimma town. All of them were registered by dairy cooperative in Jimma town and of these

48 dairy farms were included in the study. The milk sample was collected from all volunteer farms

found in the study frame and from all lactating dairy cows found in the dairy farms. All dairy farms

in Jimma are small holder and under the same management systems.

3.3 Sampling Methods and Sample Size Determination

The sample size was determined from the 50 small holder dairy farms which are registered by

dairy cooperatives in Jimma town. The sampling frame from the study site indicated that the farms

were small holder dairy farms having an average of four lactating cows each. In addition, of the

registered dairy farms all volunteer farms in Jimma were included in the study. Accordingly, all the

lactating cows from the 48 dairy farms were considered for this study which consisted of a total of264 lactating cows and 1056 quarters milk were collected.


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3.4 Study Methodology

3.4.1 Questionnaire Survey

A questionnaire survey was conducted to obtain basic information on different potential risk

factors that influence the occurrence of sub clinical mastitis and coagulase negativestaphylococci

induced intramammary infections. regarding cow and farm attributes such as parity of cow, stage

of lactation ,age , and hygiene of teats, udder, flank and tails of dairy cows, breed of cows, teat

injury ,previous history of clinical mastitis and frequency of body washing; farm floor status

(concrete ,wood and soil (ground) and sources of watering(tap water ,ground, river ).

3.4.2 Preparation of Teat and Udder

Milk sample collection was carried out according to the procedures given by National Mastitis

Council (1990). The udder and teats were washed thoroughly with tap water then the teat ends are

swabbed with cotton soaked in 70% ethyl alcohol. Separate pledged cotton was used for each teat

then allowed till it dries. Always the collections of milk sample were done before milking by the

owners.

3.4.3 Collection of Milk Sample

As per the National Mastitis Council (1990) guidelines, after preparation of teats, the first 3-4

streams milk was discarded from each quarter. Then 25 ml of milk sample was collected from each

quarter in to a sterilized and labeled screw capped test tubes. The collected milk then transported in

icebox to Jimma University, College of Agriculture and Veterinary Medicine of school of

veterinary medicine Microbiology laboratory and cultured immediately or stored at 4 C until

cultured.

3.4.4 California Mastitis Test


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The California mastitis test was used to detect the presence of sub clinical mastitis and it was

carried out according to procedures given by NMC (1990). After the milk sample arrived to the

Jimma University College of Agriculture and school of Veterinary Medicine microbiology

laboratory a squirt of milk about 2 ml from each quarter of the udder was placed in each of four

shallow of California Mastitis Test paddle. Then an equal amount of the California mastitis test

reagent was added to the California mastitis test paddle and then a gentle circular motion for 15

seconds was applied in a horizontal plane to mix the milk. A gel formation within a few seconds an

indication of a positive samples; The result was scored 0 as negative and T, 1, 2, and 3 as positive

based on the degree of gel formation. The determination of degree of California mastitis test was

based on the degree of gel formation and the sample were categorized as negative if there is no gel

or precipitation formation. If at least one quarter was positive for California mastitis test then the

cow was considered as positive for mastitis infection.


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3.4.5 Isolation and Identification of Coagulase NegativeStaphylococci

After the CMT test the samples which are positive for subclinical mastitis was kept for

overnight in a refrigerator at 4C then the milk sample was thawed for 3-5 hr at room

temperature. A 0.1 ml of milk was inoculated aseptically in to sterile Blood Agar Plates

enriched with 7% heparinized sheep blood. Then after the inoculated milk sample

incubated at 37C for 24 hr under aerobic culture conditions. After 24 hr the blood agar

plates was examined for the presence ofStaphylococcus colonies and hemolytic pattern on

the surface of blood agar plates and the haemolysis on the blood agar plate were

determined. Then gram staining was carried out to detect and characterize the species of

bacteria whether it is Gram positive bacteria or gram negative bacteria. In order to get pure

culture the Gram positive cocci bacterial colonies was sub cultured on nutrient agar plates

and incubated at 37C for 24 to 48 hr. From the sub cultured gram positive bacterial

colonies slide Catalase test was applied after the colonies identified by gram staining and

Catalase test as staphylococcus species the colonies was inoculated on Manitole Salt Agar

(MSA) plates and incubated at 37C for 24 hrs. After 24 hrs it was examined for the

growth and change in the colour of the Manitole Salt Agar medium. The presence of

growth and change of pH in the media (red to yellow colour) were regard as presence of

the salt tolerant staphylococci bacteria. Finally, the staphylococci bacteria inoculated on

DNAse media and incubated for 24hr, 37oC under aerobic condition. The media was

examined by adding 20% HCl on the DNAse media then the media was checked for the

presence of black dark clear zone produced by precipitation of bacterial DNAse due to the

reaction produced with the 20% HCl on the DNAse media. After identifying the presence

ofstaphylococci colonies by the above techniques, the coagulase test was carried out. In

the coagulase test the tube coagulase test was performed in sterile tubes by adding 0.5 ml

of citrated rabbit plasma then staphylococci colonies obtained from blood agar were

inoculated in to the tubes then it was incubated at 37C. Evaluation of clotting was taken at

30 min intervals for the first 4 hr of the test and then after 24 hr incubation. the reaction

were considered as positive if any degree of clotting from a loose clot to a solid clot that is

immovable when the tube is inverted was visible within the tube and no degree of clotting

was taken as coagulase negativestaphylococci.


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3.4.6 The Antibiotic Susceptibility Test

The Antibiotic susceptibility test was performed by using McFarland standards of disk diffusion

method on Mueller Hinton Agar. A bacterial colony obtained from the sub cultured on nutrient

agar medium incubated at 37C under aerobic conditions for 24 hr were used for the susceptibility

testing , the McFarland standard 0.5 was fixed by mixing 1-2 bacterial colonies with 5ml of saline

solution. After mixing of bacterial colony in saline solution turbidity of the bacterial measured at

0.5 McFarland. Then a loop of the suspension was inoculated on the Mueller Hinton agar plate by

using cotton swabs until it covers the whole surface of agar. Using antibiotic applicator antibiotic

discs were dispensed on the already inoculated Mueller Hinton agar plate and incubated

aerobically at 37C for 24 hr. Tetracyclin (80g), Streptomycin (100g), Ampicillin (33g),

Amoxycillin (30g), Tylosin (150g), Trimetroprim (2-5g), and cefuroxime (30g) were

antibiotics used for sensitivity test. The results were recorded as resistant and susceptible

depending on the measurement of the inhibition zone. Digital caliper was used to measure the

inhibition zone and interpretation of results was in accordance of National council of clinical and

laboratory standards (NCCLS) guidelines.

3.4.7 Data Analysis

Questioner survey and laboratory findings data were stored and coded in to Microsoft office excel

2007. Explanatory variables were categorized as follows: lactation 1-90 days (early lactation), 90-

180 (mid lactation) and greater than 180 (late lactation), and parity is categorized as Primiparious

for parity 1 and Multiparious cows for more than one parity. The prevalence of coagulase negative

staphylococci and sub clinical mastitis and the possible association of the disease with the different

risk factors like stage of lactation, parity, age, farm facilities, milking procedures, and others were

analyzed by descriptive statistic, and odds ratio, Univariate and multivariate logistic reggeration

were performed by using SPSS software version 16.0. The logistic reggeration was used to

determine whether there is a significant difference association between the probabilities of

occurrence of CNS and subclinical mastitis in response to the risk factors in one or more

categories. The difference was statistically significant if the p-value was less than 0.05 (p


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4. RESULTS

4.1 Prevalence of Sub clinical mastitis and Associated Risk Factors

The prevalence of sub clinical mastitis in Jimma small holder dairy farms were 62.1% on quarter

level, 72.7% at cow level and 95.8% on herd level. Out of 1056 quarters examined 38 (3.56 %)

were blocked. Prevalence of clinical mastitis in Jimma dairy farms at quarter level was 144

(13.6%) and 36 (13.6%) at cow level (Table 2). The distribution of SCM among quarter positions

and injured teats had a high risk of getting sub clinical mastitis as compared to non injured teats,

their association was statistically insignificant (p


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Table 3.prevalence of sub clinical mastitis at quarter level risk factors in Jimma dairy farms

Quarters Status of subclinical mastitis Univariate analysis

No of examined Negative Positive p-value OR CI (95%)

Left Front 256 84 172(67.2) 0.526 1.1 0.786-1.600

Left Rear 255 100 155(60.8) 0.373 0.8 0.602-1.210

Right Rear 255 91 164(64.3) 0.982 0.9 0.692-1.399

Right Front 252 87 165(65.5) 0.504 1.7 1

Teat InjuryYes

No

17

994

7

355

24 (70.8)

639 (64.3)

0.876 0.9 0.436-2.030

Table 4. Prevalence of sub clinical mastitis and associated risk factors on cow attributesCow factor Status of sub clinical mastitis Univariate analysis

No of

examined

Negative Positive (%) p-value OR CI (95%)

Age

3-5

>5

532

486

279

83

355 (66.7)

301 (61.9)

0.003 0.6 0.458-0.850

lactation

1-90

91-180>180

282

315421

99

111152

183 (64.9)

204 (64.8)269 (63.9)

0.589

0.9830.844

1.0

1.01

0.800-1.489

0.748-1.3461

Breed

LocalExotic

64954

25337

39 (60.9)617 (64.7)

0.600 1.1 0.690-1.902

Parity

PrimipariousMultiparious

342676

104258

238 (69.6)418 (61.8)

0.004 0.7 0.515-1.884

Teat hygiene

Clean

Dirt

401

617

210

152

249 (62.1)

407 (66.0)

0.367 0.9 0.690-1.147

Udder hygiene

CleanDirt

299719

98264

200 (66.8)496 (68.9)

0.241 1.2 0.895-1.554

Flank hygiene

CleanDirt

286732

110252

176 (61.5)480 (65.6)

0.305 0.8 0.657-1.140


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Tail hygiene

CleanDirt

306712

113249

193 (63.1)463 (65.0)

0.647 0.9 0.716-1-231

Sub clinical mastitis in Jimma dairy farms was increased on cows managed under soil (ground)

flooring types than dairy farms having wood and concrete floor types , soil floor systems had a

significant association with the prevalence of sub clinical mastitis (p


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4.2 Prevalence of Coagulase NegativeStaphylococci

From the total of 1056 quarter and 264 lactating dairy cows examined the overall prevalence of

coagulase negativestaphylococci at the quarter level was 22.72% and 22.2% on the cow level and

72.9% on the herd level. Of the total 1056 quarters examined 38 (3.6%) quarter were blind.

Among the total cows examined the presence of CNS bacteria at quarter level in clinical mastitis

cases were 36(3.41%) and sub clinical mastitis 156(14.8%) at quarter level (Table 6). The

prevalence of coagulase negativestaphylococci atquarter positions were 74(7.01%), 71(6.72%),

48(4.54%) and 47(4.45%) on Left front, Left rear, Right rear and Right front respectively.

Table 6. Prevalence of CNS intramammary infection on clinical and sub clinical mastitis

Observation

level

No of

examined

Types of mastitis Over all CNS

prevalence (%)

Clinical (%) Sub clinical (%)

Cow level 264 3 (1.1 ) 53 (20.1) 21.2

Quarter level 1056 36 (3.41) 204 (19.3) 22.7

Herd level 48 5(10.4) 30(62.5) 72.9

4.2 Associated Risk Factors on the Occurrence of CNS

From the total of 1056 quarter and 264 dairy cows examined 240 coagulase negative staphylococci

were isolated at quarter level. The prevalence of CNS in age groups, parity and stages of lactations

were summarized in table 7. Association of ages, parity, teat, udder, flank and tail hygiene and

presence of teat injury with the prevalence of coagulase negative staphylococci at cow level was

statistically insignificant (p>0.05). Animal with previous history of clinical mastitis had a

prevalence of CNS intramammary infection 43 (18.5%) whereas those cows with no previous

history of clinical mastitis had 197(23.9%). In Jimma small holder dairy farms, almost all farms

used udder preparation before milking and washing of their hands prior to milking but none of

them uses separate towel for drying of teats after milking. Although 99 % of owners have an ideaabout the clinical mastitis but none of them knew about the presence of sub clinical mastitis except

looking for clinical mastitis cases.


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Table7. Prevalence of CNS on cows related risk factors in Jimma dairy farms

Cow

factor

Groups Status of Coagulase negative

staphylococci

Univariate analysis

No

examined

CNS positive (%) p-value OR CI (95%)

Age 3-5

>5

852

204

197(23.1)

43(21.1)

0.532 0.88 0.612-1.288

Parity

Primiparous

Multiparious

349

707

86(24.6)

154(21.8)

0.297 0.85 0.630-1.152

Lactation 1-90

91-180

>180

288

331

437

55(19.1)

80(24.2)

105(24.0)

0.136

0.806

0.054

1

0.7

0.9

1

0.479-1.007

0.689-1.337

Hygieneof teats

CleanDirt

412644

93(22.8)147(22.6)

0.924 0.98 0.734-1.324

Hygiene

of udder

Clean

Dirty

310

46

68(21.9)

172(23.1)

0.692 0.93 0.682-1.289

Hygiene

of flank

Clean

Dirty

295

761

69(23.4)

171(22.5)

0.851 1.0 0.766-1.449

Hygiene

of tail

Clean

Dirty

316

740

72(22.8)

168(22.7)

0.977 1.0 0.734-1.375

Teat

injury

Yes

No

28

232

8(28.6)

1028(22.6)

0.456 1.4 0.597-3.156


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Table 8. Prevalence of Coagulase negativestaphylococci in Jimma dairy farms on cow

level risk factors

Cow

factor

Status of coagulase negative staphylococci Univariate analysis

Group No of cows

examined

Positive (%) p-value OR CI (95%)

Age 3-5

>5

45

11

26(57.7)

4 (36.3)

0.200 0.4 0.109-1.590

Parity Primiparious

Multiparious

13

43

10(76.9)

20 (46.0)

0.067 4.4 0.860-22.055

Lactation 1-90

91-180

>180

15

17

24

8 (53.3)

11(64.7)

11(45.8)

0.721

0.150

0.351

1

2.8

1.3

1

0.693-10.916

0.343-4.696Hygiene

of teats

Clean

Dirty

30

26

14(46.6)

16(61.5)

0.335 1.7 0.571-5.181

Hygiene

of udder

Clean

Dirty

51

5

28 (54.9)

2 (40.0)

0.904 1.1 0.172-7.337

Hygiene

of flank

Clean

Dirty

13

43

7 (53.8)

23 (53.4)

0.737 0.8 0.207-3.048

Hygiene

of tail

Clean

Dirty

10

46

5 (50.0)

25 (54.3)

0.590 0.7 0.152-2.918

Teat injury Yes

No

54

2

29 (53.7)

1(50.0)

0.731 1.7 0.101-28.701

Herd level risk factors associated with the prevalence of coagulase negative staphylococci like

floor type and sources of watering ,the infection of coagulase negative staphylococci were

increased on cows kept under soil floor types as compared to concrete and wood floor types and

not significantly associated (p>0.05) with CNS mammary infection (Table 9).


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Table 9. Prevalence of CNS and related herd risk factors in Jimma dairy farms

Herd

level

Groups Prevalence of Coagulase negative

staphylococci

Univariate analysis

No

examined

CNS

positive (%)

p-value OR CI (95%)

Flooring

type

Concrete

Wood

Soil

819

185

52

181(22.1)

44 (23.8)

15 (28.8)

0.479

0.456

0.261

1

0.70

0.77

1

0.3761.304

0.3871.533

Watering

sources

Ground

River

Pipe

48

56

952

11(22.9)

14(25.0)

215(22.6)

0.561

0.880

0.802

1.2

1.0

1

0.622-2.404

0.413-2.809

1

History of

clinical

mastitis

Yes

No

15

41

3(20.0)

27(65.8)

0.002 8.5 2.214-

32.826


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Risk factors associated with the prevalence of coagulase negative staphylococci intramammary

infection offered to the final multivariate analysis (p-value < 0.25) table 10.

Table10. Risk factors offered to the final multivariate logistic reggeration analysis (p5

0.665 0.9 0.660-1.304

Parity

Primiparious

Multiparious

0.699 0.9 0.697-1.274

Previous history of

clinical mastitis YesNo

0.000 0.3 0.202-0.385

Flooring type Concrete

Wood

Soil

0.428 1.0 0.928-1.192

Body washing

frequency 7days15-30days

>30 da s

0.010 1.3 1.073-1.676

Hygiene of

Udder Clean

Dirt

0.205 1.1 0.906-1.582

CNS Multivariate analysis

Stages of lactations

1-9091-180

>180

0.057 1.2 0.995-1.428

History of clinical

mastitisyes

No

0.093 0.7 0.497-1.056

Parity

PrimipariousMultiparious

0.571 0.9 0.669-1.248

4.3 Bacteriological Isolates

Along with coagulase negative staphylococci isolates, different other types of bacterial pathogens

were isolated from bovine milk involved in the causes of intramammary infections in Jimma dairy


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herds. Among the different species of bacteria which is responsible for mastitis infections in dairy

cows Bacillus cerus was the second frequently isolated pathogens followed by Cornybacterium

bovis, Staphylococcus aures,Escherichia coli, S. dysagalactia and S. agalactia (Table 11).

Table 11. Prevalence of bacterial isolates in clinical and sub clinical mastitis

Bacterial isolates Types of Mastitis Frequency Prevalence

(%)

Clinical Sub clinical

Bacillus cerus 3 21 36 10.9

Cornybacterium bovis 8 20 33 10.0

Streptococcus agalactia 2 0 2 0.6

Streptococcus 3 0 3 0.9

Staphylococcus aures 2 5 10 3.0

Escherichia coli 1 3 5 1.5

CNS 36 204 240 72.9

Total 55 254 329 100


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4.4 Antibiotic Susceptibility Test

Coagulase negativestaphylococcibacteria isolated from intramammary infection cases of Jimma

dairy herds were tested for susceptibility against seven common antimicrobial drugs. CNS

were (100%) sensitive to Trimetroprim. Sensitivity to other antimicrobial agents were as

follows; followed by (91.7%) Streptomycin, (91.7%) Ampicillin, (91.7%) Amoxycillin,

(91.4%) Cefuroxime, (89.9%) Tylosin and Tetracycline (80.6%) were summarized in table 12.

Table12. Antibiotic susceptibility patterns of CNS bacteria for different antimicrobial

agents

Antimicrobial

agent

Drug content Range of disk diffusion inhibition zone diameter (mm)

No Susceptible (%) No Resistant (%)

Tetracycline 80g 29 80.6 7 19.4

Streptomycin 100g 33 91.7 3 8.3

Ampicillin 33g 33 91.7 3 8.3

Amoxycillin 30g 33 91.7 3 8.3

Tylosin 150g 32 89.9 4 11.1

Trimetroprim 2-5g 36 100 0 0

Cefuroxime 30g 33 91.4 3 8.6

5 .DISCUSSION

In agreement with several other previous reports (Kassa et al., 1997; Hussein, 1999; Workineh et

al., 2002; Kerro and Tareke, 2003), the present study indicated sub clinical mastitis to be the most


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frequently encountered forms of bovine mastitis in several dairy farms. This may be attributable to

the prevailing lack of knowledge and attention among dairy producers meaning a shortage of

proper management interventions and hence increased incidence of the condition. The occurrence

of mastitis induced blind mammary quarters, which has a direct impact on milk production with a

consequent influence on food security. The present finding showed that the prevalence of blind

quarter were 3.6%. The prevalence of blind quarters may be due to lack of screening and treatment

of sub clinical mastitis and also inadequate follow up of clinical and chronic cases together with

persistent challenges of the mammary glands by microbial pathogens could be the main

predisposing factors to quarter blindness.

The quarter level prevalence of sub clinical mastitis in the present study was 62.1%. This is

comparable with the 62.9% reported by Kerro and Tareke (2003) in Southern Ethiopia. However,

this prevalence was higher than those reported from; the central highlands (kelay et al.,(2010)

22.3%; Nesru et al., (1997) 19%; Abaineh and Sintayehu (2001) 34.6% and Sori et al., (2005)

36.7%), commercial farms in Ethiopia (Workineh et al., (2002) 45.4%) as well as other similar

reports (Bishi (1998) 34.30%; Shirmeka (1996) 40% and Almaw (2004) 34.4%). This variation

may be attributable to poor husbandry practices and other risk factors prevailing among Jimma

dairy herds. In Tanzania the prevalence of sub clinical mastitis in small holder farms reported by

Kivaria et al., (2004) and Karimuriboet al., (2008) were 90.3 and 75.9% at cow and 84.5 and

46.2% at quarter level, respectively. This report were higher in the prevalence of sun clinical

mastitis as compared with present study in Jimma small holder dairy farms , the prevalence were at

62.1% quarter level and 72.7% at cow level. The differences in results could be due to differences

in management systems between farms and also stage of lactation, parity, breed of dairy cows.

Frequency of body washing of cows in dairy farms was significantly associated with the

prevalence of sub clinical mastitis (p


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as contagious pathogens. Cow with previous history of clinical mastitis had an increased

prevalence of sub clinical mastitis than cows with no history of clinical infection and their

association is statistically significant (p0.05) in this study. Soil floor types are much difficult to

clean and hence pose greater risk of contamination and intramammary infection. Injured teats had a

high prevalence of sub clinical mastitis in contrast with non injured teats (p>0.05), functional

closure of the teat keratin as determined by Williamson (1995) he found that it has a strong

protective effect against new infection by both minor and major pathogens. When a teat get

injured it may lack this protective effect as a result of this it may increased in the prevalence of sub

clinical mastitis. The association to sub clinical mastitis prevalence of other cow, quarter and herd

level risk factors (age; parity; stage of lactation; teat injuries; breed; hygiene of teats, udder, flank

and tail; quarter positions) was found to be insignificant (P > 0.05) in the present study. This is in

contrast to the significant association reported by Almaw (2004); Biffa (2005) and Kelay (2008).

Coagulase negative staphylococci were long considered minor pathogens (Pyorala, 2009).

However, many studies from the developed world revealed CNS to be a highly prevalent cause of

mammary infection having significant impact on dairy farms; by increasing the bulk tank somatic

cell count as well as drug resistance pattern (Schukken, 2003). In contrast, prevalence of CNS

intramammary infection, their drug resistance pattern as well as risk factors were not studied in the

Ethiopian context.

Our finding showed the prevalence of coagulase negativestaphylococci in Jimma dairy herds were

22.72% at the quarter level 38.6% at the cow level and 72.9% at the herd level. This finding on the

prevalence of coagulase negative staphylococci were 22.72% at the quarter level nearly in

agreement with reports of Haile (2010) 23.16%. But higher than reports of Sori et al.,

(2010),18.8%; Lakew et al.,(2009),17.3%, and 19% Mekonen et al., 2011). This may be due to the

higher occurrence of injured teats and presence of higher proportions of Primiparious cows in the


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case of Jimma dairy herds. On the contrary, the current quarter level prevalence was lower than

those reported from Bahir-Dar (Bishi et al., 1998) 54% and the Addis Ababa milk shed (Mekbib et

al., (2009), 30.06%; Bitew et al .,(2010), 51.9%: Hussein et al.,(1999), 42% : Almaw

(2004),49.63% and 26.9% Mungube (2001) in Addis Ababa milk shed. The variation observed

between the prevalence of CNS on many researches done in Bahir-Dar and in Addis Ababa as

compared with the present findings conducted in Jimma dairy herds may be due to disparities of

parity and stages of lactations and poor husbandry practices in the dairy farms as well as types of

stalls in farms may expose the teats for injury, according to many studies on CNS bacteria they

stated that ,its an opportunistic pathogens and when ever there is a lowered immunity of mammary

glands there will be an increase in the prevalence of mammary gland infections by coagulase

negative staphylococci. The present finding were lower than study done in developed countries

reported by Taponen et al., (2008), 37.5% in Finland , Trinidad et al.,(1990), 50%; Devriese and

De Keyser (1980), 58.06%; Krukowski et al., (2000), 36.6% and Edwards et al., (1987), 55%. This

could be due to the variation with the herd size and difference in lactation stags and parity of dairy

cows.

Even though higher than Gillespie andHeadrick (2005),11.3%, in Netherlands ; Sampimon et al.,

(2009),10.8% ; Pitkala et al., (2004) 16.6%; Osteras et al., (2006),3.3% ;Hashemi et al.,

(2011),12.5%; Shpigel et al ., (1997),7.7%; Aarestrup (1995),4.1% and Haltia et al.,(2006) 8.3%

in Estonia. The difference among the prevalence of coagulase negative staphylococci may be due

to as a result of absence in post teat dipping, lack of treating dairy cows at dry periods and absence

of proper consideration for udder health. But the prevalence of coagulase negative staphylococci at

the cow level of present finding were higher than Haltia et al.,(2006) in Estonia 4.5% in cow level

and lower than 34.4% Sampimon et al.,(2010). This variation may be due to several factors like

management practices in dairy farms and other factors may play a role. Risk factors associated

with the prevalence of coagulase negative staphylococci intramammary infections, late stage of

lactation (p


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absence of treating dairy cows during dry periods. Teat canal keratin provides an important

protective effect by physically blocking the teat canal space and by producing antibacterial factors

(Hogan et al., 1988; Dingwell et al., 2004). Injured teats may lack this protective effect against

pathogens and this conditions may increased the likelihood of getting Coagulase negative

staphylococci infection on quarter positions.

Relatively the prevalence of Coagulase negative staphylococci were increased in Primiparious

cows as compared to more than one parity or Multiparious cows both in quarter and cow levels.

The likelihood of getting intramammary infection caused by coagulase negative staphylococci on

Primiparious cows were (OR=4.4).This is in agreement with Matthews et al., (1992), Poelarends et

al., (2001) and Tenhagen et al., (2006), the variation in exposure rate may be associated with loss

of premature keratin plug in teat canals are common in heifers and in Primiparious cows ,keratin

plugs are physical protective barrier for mastitis causing pathogens and loss of keratin plugs can

results the teat canal may remains open as a result of this CNS can get access to enter in to

mammary glands and absence of treating dairy cows during dry periods and few weeks before

parturition may increase infections than more than one parity (Hogan et al., 1988; Dingwell et al.,

2004). Present finding revealed that, coagulase negative staphylococci intramammary infection

more frequently occurred in subclinical mastitis than mild clinical mastitis cases. This in

agreement with reports by Jarp (1991) and Taponen et al., (2006) and attributed to the fact that

CNS causes mild udder infection. Cows having teat injury (OR=1.4) had higher risk of getting

CNS infection as compared to non injured teats at quarter level and at the cow level (OR=1.7).

This could be due to loss of healthy skin which is a physical barrier for different pathogens. So

when it gets injured teats skins may serve as harboring mastitis causing pathogens and the teat

canal may remain open as a result of physical damage on the teat and CNS bacteria then easily

penetrate the secretory tissue. Cows with no previous history of clinical mastitis (OR=1.4) at

quarter level and (OR=8.5) cow level showed a higher prevalence of mammary infection caused by

coagulase negativestaphylococci than those with a history of clinical infection, this could be due

to the increase in the occurrence of new intramammary infections in dairy farms and also it may be

as a result of, coagulase negative staphylococci induced intramammary infection mostly doesnt

show clinical mastitis however, it involved in sub clinical forms of mastitis in many dairy farms.


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The prevalence of coagulase negative staphylococci were moderately increased on dairy cows

managed under soil floor types as compared to concrete and wood floor types this may be due to

this may be due to soil ground could have a higher contamination of cows environments by urine

and feaces this may increase the risk of getting sub clinical intramammary infections as compared

to wood and concrete, according to different reports CNS freely lives in the environments (Harmon

et al., 1995).Other risk factors such as cow, quarter and herd level risk factors (age, parity, stage of

lactation, teat injuries, hygiene of teats ,udder ,flank and tail) was found to be insignificant (P >

0.05) in the present study, This is in contrast to the significant association reported by Taponen

(2009); Schukken (2003) and Sampimon (2010). The variation in age, parity as well as husbandry

practice may contribute in significant effect on listed risk factors.

This study shown that coagulase negative staphylococci isolates were highly susceptible for

Trimetroprim drugs (95%) followed by Ampicillin and amoxicillin and Cefaqunine (91.7%) and

Tylosin (89.1%) and Tetracycline (80.6%). The susceptibility of Coagulase negative staphylococci

bacteria to tetracycline was different as compared with Almaw (2001) who reported susceptibility

to tetracycline at 90.9% and to Streptomycin 81.81%. He reported for Streptomycin is lower as

compared to the findings of the present study 91.4% as well as from that of Sori et al., (2011) who

reported 100% which is also differs from present finding 81.6%. The results of the present study in

resistance of CNS to oxytetracycline was higher than Pitkala et al., (2004) 9% in Finland and 16%

in the Netherlands. Sampimon et al., (2009) reports the resistance of coagulase negative

staphylococci for penicillin were 14% and this is higher than the current studys findings. Hawari

and Fowzi (2008) reported the sensitivity of CNS for tetracycline was 52.8% which was lowest as

compared to these findings. It has been reported that the least sensitivity of CNS was to Ampicillin

(Dhakal et al., 2007: Kumar and Sharanu, 2002).According to Basappa et al., (2001), the

susceptibility of CNS for Ampicillin and Amoxacillin were 36.7% and 29.4%, which was lower

than the current findings. This might be due to the development of beta lactamase by CNS bacteria

due to ubiquitous miss use of drugs. Turgutol (2006) reported the susceptibility of Trimetroprim

and oxytetracycline were 62.2% and 68.45 respectively, lower as compared to present finding.


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Although some variability in antimicrobial susceptibility of bacteria were observed within and

among herds, in general, susceptible of CNS to Trimetroprim, Ampicillin, Tetracycline, Tylosin,

Cefaqunine, and amoxicillin was higher and this report agrees with McDougall,1998; Pyorala ,

1998; Taponen et al.,2006; Sol et al., 2000; Taponen et al., 2003; Rainard et al., 1990).

6. SUMMERY AND CONCLUSION


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The study showed that the prevalence of sub clinical mastitis and coagulase negativestaphylococci

as the causes of intramammary infections in Jimma dairy farms were illustrated. Our study

showed that prevalence of sub clinical mastitis in Jimma dairy farms involved in the causes of

bovine mastitis were increased as compared to clinical mastitis intramammary infections The

prevalence of sub clinical mastitis were higher on dairy cows with previous history of clinical

mastitis compared with non infected cows with clinical mastitis before, their association were

statistically significant (p0.05) as compared to wood

and concrete floor types. Furthermore frequency of body washing of dairy cows in dairy farms

have a significant association (p


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It is essential to create awareness for the smallholder dairy producers regarding the presence of

subclinical intramammary infections in the study area. The owners of dairy herds should be

advised on proper milking techniques, improved sanitation, and effective use of teat dipping and

dry period therapy. There should be improvement in management practice and udder health

monitoring, environmental conditions and avoiding of teat injury to reduce the prevalence of sub

clinical and CNS intramammary infections in Jimma dairy farms. It is effective to use drugs like

Trimetroprim, Cefuroxime, Tylosin, Amoxicillin, Ampicillin and streptomycin for the treatment of

intramammary infections caused by coagulase negativestaphylococci in Jimma.

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