ISOLATION, IDENTIFICATION AND ANTIMICROBIAL PROFILE OF CORYNEBACTERIUM BOVIS FROM SELECTED DAIRY FARMS IN BISHOFTU, CENTRAL ETHIOPIA

From January to May 2018, a cross-sectional study was undertaken on lactating dairy cows in Bishoft u town to isolate Corynebacterium bovis , determine the prevalence and risk factors, and evaluate the eff ectiveness of several antibiotics in lactating dairy farms. Study animals were selected randomly from selected dairy farms in the area. Collecting milk samples from mastitic cows, cultivating, and then performing an antibiotic sensitivity test were the procedures followed. A total of 384 lactating dairy cows were examined with inspection and California Mastitis Test (CMT), in which 86 of them were found to be CMT positive. Accordingly, prevalence was 3.9% and 18.5% for cows aff ected by clinical and subclinical mastitis respectively. Th e prevalence of mastitis showed statistically signifi cant diff erence between, lactation stage, breed, age and washing ( p > 0.05). However, there was no statistically signifi cant diff erence noted in animal husbandry practice ( p > 0.05). A total of 384 lactating dairy cows were examined with inspection and CMT, in which 86 of them were found to be CMT positive. Out of the 86 mastitis positive samples (sample indicates milk from one cow) sent production of acid from glucose and a requirement for enriched basal media. C. bovis isolates have revealed a higher sensitivity to the kanamycin and streptomycin (71.4% each). A certain resistance has been noted to oxytetracycline (71.4%) and nalidixic acid (42.8%). Higher number of isolates showed moderate sensitivity or resistance to amoxicillin (51.1%). Regarding to multidrug resistance, the study refl ects that only one isolate (14.3%) shows multidrug resistance to four drugs namely kanamycin, amoxicillin, nalidixic acid and oxytetracycline. Th is study demonstrated that mastitis due to C. bovis is rare in lactating dairy farms in Bishoft u. Some of the risk factors for mastitis can be addressed by practical management of dairy cows. Farm owners should selectively use the antibiotics to which the bacteria do not show resistance, such as streptomycin and kanamycin.


INTRODUCTION
Ethiopia has high livestock production potential and is a home for around 61.6 million cattle (55.23% female and 44.77% male), 69.7 million sheep and goats, 11.7 million equines, 48.2 million poultry and 3.8 million camels. However, the country is not benefi ting from the livestock sector as its production potential. Along with drought, feed and water shortage, and genetic factors of the animals, animal diseases are the most common constraints to the sector in particular and the country in general. Livestock diseases can cause death of animals, loss of weights, slow down growth, poor fertility performance, decrease in physical power and the likes (CSA, 2020). Bovine mastitis is the sec-ond most frequent disease next to reproductive disorders and one of the major causes for economy failure in Ethiopia. It aff ects both the quantity and quality of milk. Mungube (2001) calculated the cost of mastitis in Addis Ababa's urban and peri-urban districts to be 210.8 Ethiopian Birr per cow per lactation. Aside from the fi nancial implications, there is a risk that bacterial contamination of milk from infected cows will make it unfi t for human consumption by causing food poisoning or providing a pathway for disease transmission to humans. Brucellosis and tuberculosis can be transmitted to humans in this way (Radostits et al., 2000).
In Ethiopia, mastitis prevalence rate was 85.6% and 81.2% using CMT and somatic cell count (SCC), respectively (Husien et al., 1999). An overall prevalence of 30.2% and 5.5% was obtained for subclinical and clinical mastitis, respectively, in a study conducted in urban and per-urban dairy production system in and around Addis Ababa. In addition, 43 and 75% prevalence rates of bovine mastitis were reported in diff erent parts of Ethiopia (Mekibib et al., 2010).
Mastitis induced via pathogenic microorganisms generally come from two sources: the environment such as Escherichia coli, Enterobacter and Klebsiella acquired by exposure of the teat to contaminated environment, or the animal itself like Staphylococcus aureus and Streptococcus agalactiae, Coagulase negative Staphylococcus, Micrococcus species, Corynebacterium species, Bacillus species, Pasteurella species, Mycoplasma etc. (Workineh et al., 2002). Corynebacterium bovis is the most frequently isolated Corynebacterium spp. from bovine intramammary infections (IMI) (Woodward et al., 1990). C. bovis readily colonizes the teat canal of dairy cows and has been used as an indicator of milking hygiene. It is not uncommon for C. bovis to be isolated from over 60% of quarter milk samples in herds where post milking teat antisepsis is not used. Indeed, the rate of new C. bovis IMI was about 30 times higher than that of Streptococcus agalactiae under experimental challenge settings. However, rather than real IMI, this high infection rate was thought to be attributable to teat canal colonization and subsequent contamination of milk samples (Woodward et al., 1990). Th is high reliance on presumptive identifi cation has limited the ability of most mastitis microbiology laboratories to recognize Corynebacterium spp. (Hogan et al., 1999).
In recent years, antimicrobial resistance has been a growing concern worldwide (WHO, 1997(WHO, , 2000. Acquired antimicrobial resistance in bacteria is an increasing threat in human as well as in veterinary medicine. Hence, monitoring antimicrobial susceptibility in pathogenic as well as in commensal bacteria in animals is recommended by OIE (Acar and Rostel, 2001). Such monitoring generates data of importance for therapeutic decisions and provides information on trends in resistance that might be cause for interventions regarding antimicrobial use. Mastitis is one of the most costly diseases for the dairy industry (Kossaibati and Esslemont, 1997) and antimicrobials are important parts of therapy of the disease. Susceptibility tests of milk samples submitted to state diagnostic laboratories that use the disk-diff usion method have demonstrated remarkable agreement but vary from results of a small survey processed using broth dilution (Constable and Morin, 2003).
Ethiopia holds large potential for dairy development due to its large livestock population the favorable climate for improved high-yielding animal breeds, and the relatively disease free environment for livestock. Given the considerable potential for smallholder income and employment generation from high value dairy products, development of the dairy sector in Ethiopia can contribute signifi cantly to poverty alleviation and nutrition in the country (Ahmed, et al., 2003). Various authors have indicated that mastitis is a major problem in Ethiopia. However, works on pathogen specifi c mastitis particularly that of C. bovis and their eff ect on milk production is insuffi cient. Th erefore, the objectives of this study were: To isolate and characterize C. bovis and test its antimicrobial susceptibility from dairy cows with mastitis, and to estimate prevalence of mastitis in lactating dairy cows in Bishoft u dairy farms.

Study Area
Th e study was conducted in Bishoft u town from January to May 2018. Bishoft u is located at 9ºN and 40ºE, in Oromia National Regional State about 47 km southeast of the capital city of Ethiopia, Addis Ababa. Th e altitude is about 1850m above sea level. It experiences a bimodal pattern of rainfall with the main rainy season extending from June to September (of which 84% of rain is expected) and a short rainy season from March to May with an average annual rainfall of 800mm. Th e mean annual minimum and maximum temperatures are 12.3 o C and 27.7 o C, respectively, with an overall average of 18.7ºC. Th e highest temperatures recorded in May and the mean relative humidity is 61.3%. Bishoft u is the center of Ada a Liben woreda. Th e Woreda has a total land area of about 1610.56 Km 2 and is divided into three agro-ecological zones: the midland (94%), highland (3%) and lowland (3%) (ADARDO, 2011).

Study Animals
Th e study populations included milking cows found in Bishoft u, selected from smallholder (< 5 cows), medium sized (5-16 cows) and large sized (>16 cows) private and government-owned dairy farms. Th e sampling animals were selected randomly from the selected dairy farms in the town.

Study Design
A cross-sectional study was carried out from January 2018 to May 2018 and bacteriological analysis of milk samples from mastitis infected dairy cattle found in Bishoft u town was performed.

Sample size
Th e total numbers of study animals required for the present study were calculated based on the formula given by Th rusfi eld (1995). As there was no previous information available in the study area, 50% expected prevalence was taken for sample size determination. Moreover, 5% level of precision and 95% of confi dence interval were used to calculate the sample size. Accordingly, a total of 384 animals were considered as sample size during the study period.

Milk Sample Collection
Composite samples of approximately 10 ml of fresh milk were collected from each cow before milking using sterile tight-seal sampling bottles to avoid leakage and contamination. From each farm, one sample per animal was taken by mixing the milk from all quarters. Before beginning with sample collection, loose dirt, bedding, and hair from the udder and teats were brushed with a hand and excessively dirty udder, the teats were washed with lukewarm water thoroughly dried with a towel, and disinfected with 70% ethyl alcohol. Before collecting milk samples from each quarter, the fi rst two streams of milk were discarded, and the milk and udder were examined for evidence of clinical mastitis. Between milking two cows, hands were cleansed in a sanitizing solution, and gloves were used if contagious diseases were anticipated. (Quinne et al., 1999). Th e sample was taken and stored in an ice box before being transported to the Microbiology Laboratory of the College of Veterinary Medicine and Agriculture for bacterial culture and isolation.

CMT Screening
Abnormal milk, milk clots, gland swelling, and cow disease are the signs of clinical mastitis, while CMT was used to identify subclinical mastitis. CMT was carried out according to the method described by Quinne et al., (1999). Each of the four shallow cups in the CMT paddle received roughly 2 ml of milk sample from each quarter. On each cup, an equal amount of CMT reagent was added, and the mixtures were gently rubbed together in a horizontal plane for no more than 15 seconds. Th e existence of subclinical mastitis was revealed by the coagulation and viscosity of the mixture.

Bacteriological techniques
Positive milk samples (clinical and sub-clinical) were collected from cows and cultured bacteriologically following standard microbiological procedures (Quinn et al., 1994). For primary isolation, cultivation, and detection of bacterial hemolytic reaction, blood agar (BBL R , Becton Dickinson, USA) was prepared. Bacteria were isolated by streaking one standard loop of milk (0.01ml) over the surface of blood agar supplemented with 5% sheep blood. Th e inoculated blood agar plates were placed in an incubator at 37°C for 48 h. Identifi cation of Corynebacteria isolated from bovine mammary glands has been largely based on colony morphology, hemolysis, and growth requirements. Th e presence of small, white and non-hemolytic colonies on 5% sheep blood agar aft er 48 h of incubation at 37°C indicates C. bovis. Furthermore, C. bovis tends to grow nicely only in regions of visible milk fats because it requires oleic acid. From subculture blood agar plates, colonies were streaked onto nutrient agar (Oxoid, Hampshire, England) for better colony characterization, biochemical checks and sample preservation. Th e presumptive Corynebacterium colonies, Gram positive, catalase positive and oxidase positive bacilli that passed via Gram stain, catalase and oxidase checks have been similarly purifi ed by sub culturing onto nutrient agar and the plates were incubated aerobically at 37°C for 24 h.
Th e diff erentiation between Corynebacterium and other mastitis-causing microorganisms were performed using a tube triple sugar iron (TSI) test and Methyl Red broth. Corynebateria can overcome the buff ering potential of the media by producing massive quantities of a stable acid end product from glucose fermentation, hence lowering the PH.
A suspension with a density equivalent to that of a 0.5 McFarland standard inoculums was prepared in 0.9% saline to achieve a fi nal density of the standard. Th e suspension was applied onto the surface of the Mueller-Hinton agar plates with a swab, and antibiotic disks were applied onto the surface of the inoculated Mueller-Hinton agar plates using aseptic technique (Quinne et al., 1994). Th e results were recorded aft er 24 h of incubation at 37°C and interpreted according to the guideline (NCCLS, 2012).

Data Management and Analysis
Th e data obtained from this study were compiled, entered to Microsoft Excel work sheet and analyzed with Statistical Package for Social Science (SPSS) 20. Descriptive statistics such as percentage, frequency and cross tabulation distribution were used to describe the nature and characteristics of generated data on the rate of bacterial isolation and resistant patterns of the bacterial isolates. A confi dence level of 95% was used to interpret statistical associations. Categorical variables were compared by using chi-square tests. P -Value was calculated and p > 0.05 was taken as statistically signifi cant.

RESULTS
A total of 384 lactating dairy cows were examined with inspection and CMT for detection of clinical and subclinical mastitis, respectively. Of the total lactating cows examined, overall mastitis prevalence in the area was 22.4% (68/384). Th e results showed that the prevalence rates of clinical and subclinical mastitis were 3.9% and 18.5%, respectively (Table 1). Th e result showed that the eff ect of lactation stage was statistically significant (p > 0.05) for the prevalence of bovine mastitis, and the infection rate was high in animals in early (49.1%) and late (68.4%) lactation stage as compared to the mid lactation stage (3.1%). Animals managed in semi-intensive husbandry practice showed high rate of infection (27.8%) as compared to those managed intensively (21.8%). Th e infection rate was lower in crossbred dairy cows (7.1%) than in those of Holstein Friesian (75%) and local breeds (11.8%). Th e other variable in the study was the age, and the study revealed the following values for dairy cows < 3 years (1.2%), 4-8 years (11.4%) and those > 8 years (81.9%), thus, as the age increases the incidence of mastitis also increases. In the present study, from selected potential risk factors the breed (p > 0.05), stage of lactation (p > 0.05), and age (p > 0.05) had statistically signifi cant eff ect, but animal husbandry practice had no signifi cant eff ects on the prevalence of mastitis (p > 0.05) ( Table 2). In the majority of mastitis, bacteriological techniques are used for phenotypic characterization to presumptively identify C. bovis. Basically, the organisms that exhibit a small white non-hemolytic colony type aft er 48 h of incubation in the area where butterfat was deposited on agar surface were presumptively considered to be C. bovis.
Out of 86 CMT positive samples, 57 (66.3%) organisms were identifi ed presumptively as coryneform bacteria and had similar biochemical reactions and were consistent with the C. bovis reference strain. All or almost all were small, circular with regular edges, white to cream in color, and non-hemolytic aft er 48 h incubation on blood agar enriched with 5% sheep blood. Colonies usually appear on the fi rst strike, which is due to the lipophilic nature of the bacterium.
All strains were Gram positive, catalase positive and oxidase positive. Th e remaining 29 strains were identifi ed as coryneforms based on Triple Iron Sugar (TSI) test. Th e study showed that coryneform bacteria ferment glucose and cause acid production, but no acid production was detected using carbohydrates lactose and sucrose as substrates (Table 3). Th e isolates identifi ed as coryneform bacteria were further identifi ed based on Methyl Red and Voges-Proskauer (MR-VP) test. Gas production was detected in 2 isolates. Key: (+) = positive, (-) = negative Information on the susceptibility of coryneform bacteria to antimicrobial agents is scarce. Th e study of the frequency of susceptibility of C. bovis (n = 7) to antibiotics revealed higher sensitivity to kanamycin and streptomycin (71.4% each). A certain resistance has been noted to oxytetracycline (71.4%) and nalidixic acid (42.8%). Higher number of isolates showed moderate sensitivity or resistance to amoxicillin (51.1%). Regarding to multidrug resistance, the study refl ects that only one isolate (14.3%) shows multidrug resistance pattern. Susceptibility and resistance patterns of each bacterial isolates are shown in Table 4. From the total C. bovis, 14.3% were resistant to one drug, 57.14% to two drugs and 14.3% were to more than two drugs.

DISCUSSION
Th is study showed the overall prevalence of mastitis associated with Corynebacterium bovis in lactating dairy farms to be 1.8%, which is lower than most of the previous reports in Ethiopia and is in agreement with the bovine mastitis reported by Dabash et al. (2014) with the prevalence of bovine mastitis of 2% in North Showa of Ethiopia. Th is fi nding is also comparable with the fi ndings of Belayneh et al., (2014) in East Showa zone, Akaki district, Ethiopia (1.2%); Moges et al., (2011) in and around Gonder (2.4%) and Oalekish et al. (2013) in northern Jordan (3.9%). Th is fi nding slightly diff ered from earlier investigations. In other regions in Ethiopia, Lidet et al. (2013) reported 0.52% isolation rate. Th e high isolation rate in this study could be associated with lowered resistance of the cow due to teat injury.
Clinical mastitis rate was low in all breeds as compared to subclinical mastitis. Th e prevalence of clinical mastitis in the present study (3.9%) was comparable to the reports from diff erent dairy farms: 6.3% in Bahir Dar (Gizat et al., 2007), and 7.9% in commercial farms in Ethiopia (Abaineh and Sintayehu, 2001). However, the present fi nding is by far lower than the reports of Kerro and Tarekegn (2003) in local, Friesian and Jersey cows in Sothern Ethiopia (12.1%), and 14.2% in lactating cows in smallholder farms in Tanzania (Kivaria et al., 2004). Mastitis is a complex disease and the diff erence in results could be due to variations in herd size, management practices, proportion of exotic gene inheritance, and agro-climates. Other risk factors might also have contributed to the observed diff erences in prevalence rates of mastitis among the fi ndings of various authors.
Signifi cant diff erences between the high-grade Holstein-Friesian, Holstein indigenous zebu crossbred and local zebu might be associated with their high milk yield. Radostits et al. (2007) stated that high-yielding cows are more susceptible to mastitis than the low-yielding ones.
Signifi cant eff ect of stage of lactation on the prevalence of mastitis was confi rmed in this study, being 49.15%, 3.1% and 68.4% in early, mid and late lactation, respectively, also reported by Nesru (1999), Mungube et al. (2005) and Kerro and Tarekegn (2003) in Ethiopia. Th e former two authors reported high prevalence of subclinical mastitis in cows at early and late stage of lactation as it is the case in this fi nding, while the last two reported higher prevalence at early stage of lactation. Th e variations in the eff ect of lactation stages between diff erent studies could be related to the disparities in age, parity and breed of the sampled animals.
Th e present study was also undertaken to determine the resistance pattern of bovine mastitis due to C. bovis to commonly used antimicrobials in the study area and to provide information to concerned animal health professionals. Th e selection of the types of antimicrobial agents was based on clinical considerations including frequent use of the drug in the study area and availability. Oxytetracycline was commonly used antimicrobial for the treatment of mastitis in the study area.
Th e poor inhibitory eff ect of oxytetracycline against C. bovis strains identifi ed in this study is in agreement with the data reported by Oalekish et al. (2013). Th e latter reported sensitivity to oxytetracycline in 12% of C. bovis isolates from subclinical mastitis in northern Jordan. In our study, C. bovis isolates showed most susceptibility to kanamycin and streptomycin (71.4% each). Nibret et al. (2011) obtained comparable results, where kanamycin was eff ective against C. bovis (100%). In the present study, unlike Oalekish et al. (2013), streptomycin was eff ective against C. bovis (71.4%). Out of the seven isolates investigated in the present study, only one isolate (14.3%) showed multidrug resistance to kanamycin, amoxicillin, nalidixic acid and oxytetracycline.
In general, streptomycin and kanamycin showed very good effi cacy, nalidixic acid and ceft riaxone showed moderate effi cacy, whereas oxytetracycline and amoxicillin showed poor effi cacy in almost all isolates.

CONCLUSION AND RECOMMENDATIONS
Bovine mastitis is an infl ammatory response of the mammary gland. It has a major impact on animal production, animal welfare and milk quality. Mastitis is one of the biggest problems for dairy industry because of high morbidity and signifi cant economic losses. Even though, the prevalence of C. bovis is lower than that of other common pathogens, it causes mostly subclinical mastitis and has substantial impact on the dairy industry. Milk from cows with subclinical mastitis accidentally mixed into bulk milk enters the food chain, and poses a threat to human health. Th e low prevalence of C. bovis did not restrict the organism to show resistance to antibiotics such as oxytetracycline, nalidixic acid and others, emphasize the need for serious and immediate attention towards consumers of raw milk. On the other hand, some antibiotics such as kanamycin and streptomycin were found to be eff ective against C. bovis. Th e appropriate and principled use of those antibiotics might help in the control and limitation of resistance against those strains. Based on the above fi nding and closing remarks, the following recommendations are forwarded: • Future work need to be done in the fi eld of isolation, characterization and prevalence of coryneform bacteria and consequently, the responsible important strains could be identifi ed. • Antimicrobial susceptibility test should be conducted at regular intervals to understand the development of resistance against the commonly used antibiotics. • Proper hygienic and improved management practices should be introduced at farm level.

Author's Contribution:
MY made contributions to conception and design of the study, involved in data collection, data curation, biochemical test and culturing the bacteria, data analysis and draft ing the manuscript. JT revised the manuscript critically and together with MY prepared the fi nal draft of the manuscript. Both authors read and approved the fi nal manuscript.