INTERNATIONAL JOURNAL OF VETERINARY AND ANIMAL MEDICINE (ISSN:2517-7362)

Mastitis is an important disease of intensification which is caused by a wide range of microorganisms and induces distinct lesions in the mammary tissues. A cross sectional study was carried out from November 2014 to March 2015 to investigate sub-clinical mastitis, putative risk factors and characterize the phenotypic drug sensitivity patterns of strains isolated from subclinical cases. Accordingly, clinical examination was conducted on lactating cows and a total of 94 cows were screened by California mastitis test. About 58.5% of cows had subclinical mastitis and the quarter level infection was 31%. The chi-square analysis indicated that lactating cows in the last lactation stage were more prone to mastitis infection than in the early lactation phase and this was statistically significant (P < 0.05). Milk samples were collected from subclinical cases and subjected to bacteriological culture and disc diffusion test. The dominant isolated bacteria were Staphylococcus auras (29.78%), Staphylococcus epidermis (12.76%), Streptococcus species (12.76%) and Escherichia coli (10.63%). The phenotypic characterization of antimicrobial sensitivity test showed that the isolates were resistant to penicillin while two isolates were resistant to Suphamethoxazole. All the isolates were sensitive to Gentamycin and relatively to Tetracycline. In conclusion, the study revealed that subclinical mastitis is a great problem in the dairy farm. Thus, early diagnosis and advanced screening test should be adopted to select the antibiotic of choice for the treatment of mastitis.

Bacterial Isolates; Cases; Dairy Farm; Sensitive; Sub-Clinical Mastitis; Virulent

Bovine mastitis is one of the most important diseases of intensification which affects the dairy industry and results in considerable economic losses for dairy producers [1]. It is the inflammation of the parenchyma of mammary tissues which is characterized by changes in the milk, discoloration, clots, udder swelling, pain and pathological changes in the glandular tissues as well as an increased leukocytes number in the infected cells [2]. Bovine clinical and subclinical mastitis in the dairy farm is primarily caused bacterial and fungal infections. Staphylococcus aurous, Staphylococci agalatiae, Streptococci dysgalactiae and agalactiae and Strebtoccocci uberis are the common cause of clinical and subclinical infections in the dairy industry [3]. In mastitis infected cows, the production potential is reduced by 15% and 30% in infected quarter. Eschericia coli and klebiella species are also responsible for mastitis infection. Staphylococcus aurous, Staphylococci agalatiae are contagious pathogens and attached to the udder of lactating cows. Staphylococcus aurous is mainly responsible for clinical and subclinical form of mastitis in lactating cows [4]. Other coliform bacteria that occur in the environment are also the source of infection.

Besides this, there is a risk of suffering from consumption of milk contaminated by bacterial infection [5]. The disease is also associated with huge loss of milk yield, poor quality of milk, discarding of milk and antimicrobial treatment as well as reduced the productive life of milking cows [6]. It is important to conduct identification of subclinical mastitis infected cows in the dairy farm in order to apply intervention options. California mastitis tests based screening of infected cows; Culturing and conducting individual cow based somatic cell count are important alternative methods to make diagnostic and treatment of infected cows [7]. Multiple antimicrobial therapies are used to mitigate mastitis infection in the dairy industry. Frequent and inappropriate usage of these drugs lead to the development of antimicrobial resistance [8]. The emergence and spread of bacterial antibiotic resistance gene is favored by the induction of mobile genetic elements that carriers multidrug resistant virulent pathogens in the farm [9]. Studies conducted in the different areas of Ethiopia, indicated there is high prevalence of mastitis infection in small scale dairy cattle industry. The prevalence ranges from 16% to 80 % for subclinical infections in different dairy farms [10].

The occurrence of mastitis infection and antimicrobial resistance in the dairy farm showed that the situation needs urgent prevention and effective treatment interventions. Adoption of other control practices such as post milking disinfection, culling of chronically infected cows, and segregation of infected cows should be adopted in the dairy farms. There is dearth of studies on subclinical mastitis causing pathogens and phenotypic characteristics of strains isolated from subclinical cases. Therefore, the objectives of these studies were:

• To investigate bovine intra-mammary infection and potential risk factors at Holeta research dairy farm
• To profile and characterize the phenotypic drug resistance patterns of isolated strains from cases of subclinical mastitis
  

Study area

Holeta Agricultural Research Center (HARC) is located at 34Km west of Addis Ababa at an elevation of 2400 m.a.s.l in the central high land of Ethiopia. The area is characterized by mild subtropical weather, with average minimum and maximum annual temperature of 6.3 and 22.1°C, respectively. The animals were under intensive and semi intensive management systems. The area also experience bimodal rainfall pattern with a long rainy season extends from June to September while the short rainy season extends from March to April [11].

Study cows and sample size determination 

The study animals were Holstein-Fresian, and Boran - HolsteinFresian crossbreed lactating cows that were included in the study. Lactating cow parameters such as breed, age, parity and production potential and the farm parameters such as management system and hygiene protocols were recorded during the study period. The sample size was determined based upon the number of lactating cows and occurrence of sub clinical mastitis cases in the farm. Accordingly, a total of 94 lactating cows were screened for subclinical mastitis using California Mastitis Test (CMT).

Farm based study design and screening methods 

Cross sectional study was carried out to determine the infection rate of mastitis in the farm from December 2014 to April 2015 at cow and quarter level. Lactating cows infected by subclinical mastitis was screened by California mastitis test and the reactions were graded according to [11]. Clinical parameters and observational farm parameters were collected to evaluate the infections status during the study period.

Milk sampling and identification of bacterial strains

In this study, milk samples were collected from clinically infected and positively reacted cows to California mastitis test method. Milk samples were aseptically collected and processed by milk sampling techniques [12] to isolate and identify bacterial pathogens. The collected milk samples were cultured and sub cultured on various media. The inoculated media were then incubated at 37°C for 24 hours. Primary and secondary biochemical tests were done by using biochemical procedures. Culturing and biochemical tests were performed to characterize the causative agents from this mastitis infection in the farm.

Antimicrobial susceptibility test  

An in-vitro antimicrobial sensitivity test was conducted using Kirby- Bauer disk diffusion method and Muller Hinton agar [13] in order to screen the most effective drugs for the treatment of intra-mammary infection in the study farm. These screened and used antimicrobials were Tetracycline (30µg), Penicillin G (10u), Erythromycin (15µg), Gentamycin (10µg) and Sulphamethoxazole Trimethophrim (25µg). The antibiotic impregnated paper or discs (Oxoid UK) were applied on the surface of inoculated agar plates by an aseptic procedure. The bacterial suspensions in the sterile saline water was done by making the turbidity equal to Mac Far land 0.5 turbidity standards and incubate at 37°C for 2 hours. MuellerHinton agar which was used as plating medium was inoculated with bacterial suspension by using sterile cotton swab. Then antibiotic impregnated paper disk (Oxoid UK) were applied and pressed on to the plate with forceps. Plates were incubated at 37°C for 24 hours. The diameter zone of growth inhibition was classified as sensitive, intermediate and resistant to different antibiotics according to national committee for clinical laboratory standard break points to interpret the inhibition zones.

Data analysis

The collected data were entered to Microsoft excel spread sheet and analyzed by Stata (2013). The association of risk parameters and mastitis infection was done by chi square test. A p value < 5% were considered as statistically significant in the analysis.

Intra-mammary Infection in the dairy farm

The farm based CMT screening test indicated, out of the total 94 lactating cows examined and screened for subclinical infection, about 58.5% of the cows were found to be infected in the farm. Out of 390 teats examined, 31% (N = 122) of the teats were infected and positive for subclinical mastitis (Figure 1).

Risk factor analysis of sub clinical mastitis infection at Holeta dairy farm

The risk factor analysis indicated that mastitis infection was high in the late lactation stage (32%) which was statistically significant (P < 0.05). Lactating cows in the medium productive stage were prone to infection as compared to highly productive cows. The result indicated that there was no significant difference between the blood levels of cows to subclinical mastitis (Table 1).

The study indicated that farm level factors were significantly varied between mastitic and mastitis free cows. The infection was significantly higher (p < 0.05) in poorly hygienic cows than good hygienic lactating cows (Table 2).

Identification and characterization of bacterial species from subclinical mastitis  

All 55 CMT positive milk samples were cultured and about 29.78% (N = 14) harbored S. aureu, 12.7% (N=6) S. epidermidis and 10.6% (N=5) E. coli. The other pathogens were detected with high to moderate frequency (Table 3).epidermidis

Antimicrobial susceptibility patterns and phenotypic characterization  

An in vitro antibiotic susceptibility test was performed and the susceptibility pattern indicated E. coli, S. epidermidis, E. faecalis, P. aeruginosa and K. pneumoniae were resistant to Penicillin and Susceptibility to Gentamycin were observed in all isolates. P. aeruginosa and K. pneumonia isolates were resistant to the Sulphamethoxazole and intermediate sensitivity to Erythromycin was detected in E. faecalis and E. coli. Majority of the isolates were highly susceptible to compound Tetracycline except S. uberis which was highly resistant to this compound (Table 4).

The antimicrobial susceptibility results revealed that the highest resistance was observed for Tetracycline, Gentamycin and sulphamethoxazole that revealed multidrug resistance patterns (Table 5). 

In Ethiopia, subclinical mastitis is an important disease of intensification and a big challenge for the dairy development. In the present study, the overall prevalence of subclinical mastitis infection in the dairy farm was 58.5%. This result was lower than the study of [14] who reported 71% in exotic dairy cows in Holeta areas. The finding was higher than reports of [15,16] who reported the prevalence of subclinical mastitis in and around Batu town was 35% and 36.7% respectively. This result was also in line with the study of [17] who reported 53.3% in dairy farms of Bangladesh. This variation could be due to differences in breed, management practices, Age variability, and the types of strains involved and susceptibility of the host. The quarter level subclinical mastitis infection was about 31%. This investigation was lower than the findings of [18] and higher than the findings of [19] who reported the prevalence of 19.9% in infected quarters. This is might be due to the fact that hind quarters are more prone to contamination and the amount of milk produced enforces the opening of teat canal and allows the entry of environmental pathogens.

The risk factor analysis indicated that subclinical mastitis is high in the late lactation stage (32%) and this was statistically significant (P < 0.05). Lactating cows in the medium productive stage are highly prone to infection as compared to highly productive cows. This concept was in agreement with the findings of [15] who stated that , cow parity, stage of lactation and farm hygiene were found to be statistically significant (P < 0.05 and responsible for the occurrence of mastitis. The present work also stated that mastitis infection is higher in young aged lactating cows (33%) than Adult cows (27%). The prevalence was relatively higher than the previous reports of [19] who reported 22.82% in young cows and 53.5% in Adult cows. The probable reason could be due to correlation of infection and cow age increment. The current result also showed that lactating cows with poor hygienic condition were more prone to mastitis than those cows with good hygienic milking practices. This work was similar to the findings of [19] who indicated that poorly hygienic cows were more infected than in good hygienic cows. This could be related with absence of washing the udder, milking personnel and using of common cloth for milking cows [2]. The study also showed that infected and cultured milk harbored, S. aurous, E. coli and S. uberis and the isolation rate was about 29.78, 10.63% and 4.25% respectively. This finding was relatively similar to the previous report of [19] who reported 17.13% of S. aurous and 12.1% of prevalence of S. uberis reported by [20] in Holeta town. The variation could be due to cow management, teat dipping practices before and after milking, milking of infected animals lastly and application of dry cow therapies and treating mastitis cases at the early phase.

The in vitro antibiotic susceptibility test indicated that Gentamycin and Sulfamethazole drugs were the most effective drugs against subclinical mastitis infection. Among the isolated strains, E. coli, S. epidermidis, E. faecalis, P. aeruginosa and K. pneumoniae were resistant to Penicillin. P. aeruginosa and K. pneumonia isolates were resistant to Sulphamethoxazole and intermediate sensitivity to Erythromycin was detected in E. faecalis and E. coli. Majority of the isolates were highly sensitive to compound Tetracycline except S. uberis. The current finding was similar to the reports of [21] who indicated that E. coli isolate was sensitive to gentamycin compound. This could be justified by the fact that the development of drug resistance is due to repeated therapeutic or indiscriminate or extensive use of multiple drugs in the farm for either therapeutic or prophylactic purposes. Bacterial strains that have MAR index > 0.2 are originated from an environment where several antibiotics are frequently used in the dairy industry [22-24].

Bovine mastitis is a complex production disease in the study dairy farm and its adverse effect on the production potential and productivity of dairy sub sector is very high. This was investigated through clinical examination, CMT screening, Microbiological analysis and phenotypic evaluation. Multiple pathogens were responsible for the occurrence of mastitis and some pathogens emerged and resistant to commonly used antimicrobial agents. The dominant pathogens were S. aureus, 29.78% (N= 14), S. epidermidis 12.7% (N = 6) and E. coli infection which accounted for 10.6% (N = 5). An in vitro antimicrobial susceptibility evaluation indicated that E. coli, S. epidermidis, E. faecalis, P. aeruginosa and K. pneumoniae were resistant to Penicillin but E. coli was sensitive to Erythromycin. The majority of isolates were highly sensitive to compound Tetracycline except S. uberis. In conclusion, frequent screening of cows, dry cow therapy, pre-post milking teat disinfection and culling of chronically infected cows could be adopted as well as potent drugs should be practiced in the farm against the circulating isolates.

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