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 of 264 lactating cows and 1056 quarters milk were collected.

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 negative staphylococci 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

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.

3.4.5 Isolation and Identification of Coagulase Negative Staphylococci 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 of Staphylococcus 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 of staphylococci 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 negative staphylococci.

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), 90180 (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<0.05).

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<0.05) is summarized in (Table 3). Our finding on prevalence of sub clinical mastitis on breeds of cow were high on exotic breeds than local zebu breeds of dairy cows (Table 4). Table 2. Prevalence of clinical and sub clinical mastitis at herd, cow and quarter levels in Jimma dairy farms Observation level Cow level Quarter level Herd level Clinical mastitis No examined 264 1056 48 Positive (%) 36(13.6) 144(13.6) 18(37.5) Sub clinical mastitis No examined 264 1056 48 CMT positive (%) 192 (72.7) 656 (62.1) 46 (95.8)

Table 3.prevalence of sub clinical mastitis at quarter level risk factors in Jimma dairy farms Quarters Left Front Left Rear Right Rear Right Front Teat Injury Yes No Status of subclinical mastitis No of examined Negative Positive 256 84 172(67.2) 255 255 252 17 994 100 91 87 7 355 155(60.8) 164(64.3) 165(65.5) 24 (70.8) 639 (64.3) Univariate analysis p-value OR CI (95%) 0.526 1.1 0.786-1.600 0.373 0.982 0.504 0.876 0.8 0.9 1.7 0.9 0.602-1.210 0.692-1.399 1 0.436-2.030

Table 4. Prevalence of sub clinical mastitis and associated risk factors on cow attributes Cow factor No of examined Age 3-5 >5 lactation 1-90 91-180 >180 Breed Local Exotic Parity Primiparious Multiparious Teat hygiene Clean Dirt Udder hygiene Clean Dirt Flank hygiene Clean Dirt 0.003 532 486 282 315 421 64 954 342 676 401 617 299 719 286 732 279 83 99 111 152 25 337 104 258 210 152 98 264 110 252 355 (66.7) 301 (61.9) 183 (64.9) 204 (64.8) 269 (63.9) 39 (60.9) 617 (64.7) 0.004 238 (69.6) 418 (61.8) 0.367 249 (62.1) 407 (66.0) 0.241 200 (66.8) 496 (68.9) 0.305 176 (61.5) 480 (65.6) 0.8 0.657-1.140 1.2 0.895-1.554 0.9 0.690-1.147 0.7 0.515-1.884 0.589 0.983 0.844 0.600 1.0 1.0 1 1.1 0.800-1.489 0.748-1.346 1 0.690-1.902 0.6 0.458-0.850 Status of sub clinical mastitis Negative Positive (%) Univariate analysis p-value OR CI (95%)

Tail hygiene Clean Dirt

0.647 306 712 113 249 193 (63.1) 463 (65.0)

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<0.05). The usual practice of body washing of dairy cows in the dairy farms had a variation on the prevalence of sub clinical mastitis. The prevalence of subclinical mastitis were decreased on dairy cows who washed every week than those cows washed at wit in 15 days and more than 30 days intervals , their association was statistically significant (p<0.05) Table 5. Table 5. Prevalence of sub clinical mastitis and related risk factors at the herd attributes in Jimma dairy farms herd factor No of examined Floor Concrete Wood Soil Body washing 7days 15-30days >30 days Water sources Pipe Ground River History of clinical mastitis Yes No 789 181 36 273 599 146 915 48 55 223 795 296 54 12 116 215 31 318 32 12 138 224 Status of sub clinical mastitis Negative Positive (%) 493 (62.5) 127(70.2) 48 (75.0) 157 (57.5) 384 (64.1) 115 (78.8) 597(65.2) 16(33.3) 43(78.2) 0.000 85 (71.8) 751(38.1) 3.6 0.187-0.344 0.198 0.936 0.055 0.001 0.000 0.005 1 0.6 0.9 1 0.4 0.5 1 0.367-1.231 0.500-1.894 1 0.284-1.671 0.383-0.843 Univariate analysis p-value OR CI (95%)

4.2 Prevalence of Coagulase Negative Staphylococci From the total of 1056 quarter and 264 lactating dairy cows examined the overall prevalence of coagulase negative staphylococci 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 negative staphylococci at quarter 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 Cow level Quarter level Herd level No of examined Clinical (%) 264 1056 48 3 (1.1 ) 36 (3.41) 5(10.4) Sub clinical (%) 53 (20.1) 204 (19.3) 30(62.5) 21.2 22.7 72.9 Types of mastitis Over all CNS prevalence (%)

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 idea about the clinical mastitis but none of them knew about the presence of sub clinical mastitis except looking for clinical mastitis cases.

Table7. Prevalence of CNS on cows related risk factors in Jimma dairy farms Cow factor Groups Status of Coagulase negative staphylococci No Age Parity Primiparous Multiparious Lactation 1-90 91-180 >180 Hygiene of teats Hygiene of udder Hygiene of flank Hygiene of tail Teat injury Clean Dirt Clean Dirty Clean Dirty Clean Dirty Yes No 349 707 288 331 437 412 644 310 46 295 761 316 740 28 232 86(24.6) 154(21.8) 55(19.1) 80(24.2) 105(24.0) 93(22.8) 147(22.6) 68(21.9) 172(23.1) 69(23.4) 171(22.5) 72(22.8) 168(22.7) 8(28.6) 1028(22.6) 0.977 0.456 1.0 1.4 0.734-1.375 0.597-3.156 0.692 0.851 0.93 1.0 0.682-1.289 0.766-1.449 0.136 0.806 0.054 0.924 1 0.7 0.9 0.98 1 0.479-1.007 0.689-1.337 0.734-1.324 3-5 >5 examined 852 204 CNS positive (%) 197(23.1) 43(21.1) 0.297 0.85 0.630-1.152 p-value 0.532 OR 0.88 CI (95%) 0.612-1.288 Univariate analysis

Table 8. Prevalence of Coagulase negative staphylococci in Jimma dairy farms on cow level risk factors Cow factor Group Age 3-5 >5 Parity Primiparious Multiparious Lactation 1-90 91-180 Hygiene of teats Hygiene of udder Hygiene of flank Hygiene >180 Clean Dirty Clean Dirty Clean Dirty Clean No of cows examined 45 11 13 43 15 17 24 30 26 51 5 13 43 10 46 54 2 Positive (%) 26(57.7) 4 (36.3) 10(76.9) 20 (46.0) 8 (53.3) 11(64.7) 11(45.8) 14(46.6) 16(61.5) 28 (54.9) 2 (40.0) 7 (53.8) 23 (53.4) 5 (50.0) 25 (54.3) 29 (53.7) 1(50.0) 0.721 0.150 0.351 0.335 0.904 0.737 0.590 0.731 1 2.8 1.3 1.7 1.1 0.8 0.7 1.7 1 0.693-10.916 0.343-4.696 0.571-5.181 0.172-7.337 0.207-3.048 0.152-2.918 0.101-28.701 0.067 4.4 0.860-22.055 p-value 0.200 OR 0.4 CI (95%) 0.109-1.590 Status of coagulase negative staphylococci Univariate analysis

of tail Dirty Teat injury Yes No

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).

Table 9. Prevalence of CNS and related herd risk factors in Jimma dairy farms Herd level Groups Prevalence of Coagulase negative staphylococci No Flooring type Watering sources Concrete Wood Soil Ground River Pipe History of clinical mastitis Yes No examined 819 185 52 48 56 952 15 41 CNS positive (%) 181(22.1) 44 (23.8) 15 (28.8) 11(22.9) 14(25.0) 215(22.6) 3(20.0) 27(65.8) p-value 0.479 0.456 0.261 0.561 0.880 0.802 0.002 OR 1 0.70 0.77 1.2 1.0 1 8.5 CI (95%) 1 0.3761.304 0.3871.533 0.622-2.404 0.413-2.809 1 2.21432.826 Univariate analysis

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 (p<0.25) Variables Age 3-5 >5 Parity Primiparious Multiparious Previous history of clinical mastitis Flooring type Yes No Concrete Wood Soil 7days 15-30days >30 days 0.000 0.3 0.202-0.385 0.699 0.9 0.697-1.274 Groups Multivariate logistic reggeration analysis P -value 0.665 OR 0.9 CI (95%) 0.660-1.304

0.428

1.0

0.928-1.192

Body washing frequency Hygiene of Udder

0.010

1.3

1.073-1.676

0.205

1.1

0.906-1.582

Clean Dirt CNS Multivariate analysis Stages of lactations 1-90 91-180 >180 History of clinical mastitis Parity Primiparious Multiparious 4.3 Bacteriological Isolates yes No

0.057

1.2

0.995-1.428

0.093

0.7

0.497-1.056

0.571

0.9

0.669-1.248

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

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 Clinical Bacillus cerus Cornybacterium bovis Streptococcus agalactia Streptococcus Staphylococcus aures dysagalactia Escherichia coli CNS Total 3 8 2 3 2 1 36 55 Sub clinical 21 20 0 0 5 3 204 254 36 33 2 3 10 5 240 329 10.9 10.0 0.6 0.9 3.0 1.5 72.9 100 Frequency Prevalence (%)

4.4 Antibiotic Susceptibility Test Coagulase negative staphylococci bacteria 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 No Tetracycline Streptomycin Ampicillin Amoxycillin Tylosin Trimetroprim Cefuroxime 80g 100g 33g 30g 150g 2-5g 30g 29 33 33 33 32 36 33 Susceptible (%) 80.6 91.7 91.7 91.7 89.9 100 91.4 No 7 3 3 3 4 0 3 Resistant (%) 19.4 8.3 8.3 8.3 11.1 0 8.6 Drug content Range of disk diffusion inhibition zone diameter (mm)

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

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 Karimuribo et 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<0.05) and (OR=1.3). Poor farm sanitary or drainage conditions result in contamination of cows environment by wastes. When a cow lays down and makes a direct contact with the environment, the teats, udder, flank and tail becomes dirty. Consequently, pathogens originating from the environment may get an access to colonize the teat canals and to cause infections. Keeping cows and farm in good hygienic conditions can help decrease in the incidence of intramammary infections caused by environmental pathogens as well

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 (p<0.05). This report is in agreement with observations by Biffa (2005). Previously infected cows may have greater risk of being re-infected. Repeated challenges of the mammary tissues with microorganisms together with other stress factors could put the mammary tissues at greater risks of re-infection of intramammary tissues. Compared to wooden and concrete floor types, soil floor types were found to be associated to increased prevalence of sub clinical mastitis (p>0.05) 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 negative staphylococci 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

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 and Headrick (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<0.05) were statistically significant. This report agrees with Timms and Schultzh (1984); Oliver (1997); Leavens et al., (1997); Chaffer et al., (1999) and Taponen et al., (2007). During early lactation lack of proper feed for the dairy cows may result lowered migration of neutrophils and delay in neutrophil migration may results with increase in prevalence of intramammary infections, lack of post milking teat disinfection, treatment of clinical cases, and

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 negative staphylococci 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.

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.

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

The study showed that the prevalence of sub clinical mastitis and coagulase negative staphylococci 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 (p<0.05). In Jimma dairy farms cows managed under soil (ground) flooring types had a high risk of getting sub clinical mastitis (p>0.05) as compared to wood and concrete floor types. Furthermore frequency of body washing of dairy cows in dairy farms have a significant association (p<0.05) with the prevalence of sub clinical mastitis, prevalence of sub clinical mastitis infection in dairy cows were decreased as frequency of body washing of cows increases. The late stages of lactation had a significant effect in the prevalence of coagulase negative staphylococci. The infection rate of CNS on Primiparious cows were relatively higher as compared to Multiparious cows, in addition dairy cows having injured teat had more likely to be affected by coagulase negative staphylococci and sub clinical intramammary infections than healthy teats. There was no significant difference observed on hygiene of teat, udder, flank and tail in the prevalence of CNS intramammary infections. The in vitro susceptibility tests for Coagulase negative staphylococci showed that CNS were highly for susceptible for Trimetroprim followed by Streptomycin, Ampicillin, Amoxycillin, Cefuroxime, Tylosin and Tetracycline.

7. RECOMMENDATIONS

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 negative staphylococci in Jimma.