MATERIALS AND METHODS

Source of Milk Samples
Milk samples (100) were collected from cattle and buffaloes suffering from clinical mastitis from different locations in and around Udgir during January 2008 to December 2008. Relevant history of individual animal was recorded.

Isolation and Identification
The bacteria were isolated in pure culture on Nutrient Agar and the Candida were isolated on Corn Meal Agar (CMA). Further identification of bacteria was done on the basis of colony characters, staining (Grams) reaction, growth patterns on Blood Agar and Mac Conkey Agar (i.e., hemolysis and lactose fermentation, respectively) followed by motility, morphology, arrangements and biochemical tests (Indol, MR, VP, Citrate and Catalase). Candida was identified by colony characters on CMA and staining patterns.

Antimicrobial Susceptibility Testing
Minimal Inhibition Concentration (MIC) values of the bacterial organisms were analyzed for twelve different antimicrobials namely amoxicillin (30 mcg), ampicillin (25 mcg), ceftriaxone (30 mcg), cephotaxim (30 mcg), chloramphenicol (30 mcg), cloxacillin (10 mcg), ciprofloxacin (30 mcg), doxycycline (10 mcg), enrofloxacin (10 mcg), gentamycin (30 mcg), oxytetracycline (30 mcg) and streptomycin (25 mcg). The disc diffusion method as described previously (Bauer et al., 1966) was employed and the interpretation was made as per the interpretation chart provided by the manufacturer of discs depending on the diameter of zone of inhibition of bacterial growth.

Table 1:	Prevalence of mastitic agent from bovines in and around Udgir

Table 2:	Percent sensitivity of bovine mastitic agents to different antibiotics

RESULTS

The data analysis of this study showed that the age of all recorded cases ranged in between 5 to 8 years. Amongst hundred milk samples from clinical mastitis, 63% samples revealed bacterial growth and 7% samples revealed fungal (Candida) growth. No growth was evident in 30% samples.

Table 1 shows prevalence of mastitic agents in bovines in and around Udgir. The study indicated that the major prevalent pathogens associated with bovine mastitis in and around Udgir were Escherichia coli (40.00%) followed by Staphylococcus aureus (34.29%), other bacteria (15.71%) and Candida (10.00%) amongst isolated pathogens. The overall prevalence of Escherichia coli in bovine mastitis recorded during this study was 28.00% followed by Staphylococcus aureus (24.00%), other bacteria (11.00%) and Candida (7.00%). Other bacteria include Pasturella (1.43%), Pseudomonas (1.43%), Bacillus (2.87%), Proteus (1.43%) and mixed bacteria (8.57%).

The in vitro antibiogram studies were conducted on 63 samples which exhibited bacterial growth and antifungal therapy was advised for cases showing fungal mastitis. The in vitro antibiogram studies (Table 2) revealed ciprofloxacin to be the most effective drug (69.84%) followed by gentamycin (68.25%), enrofloxacin (58.73%), chloramphenicol (31.75%) and cephotaxim (23.81%). The sensitivity to cloxacillin observed was the lowest (1.59%) followed by ampicillin (3.18%), streptomycin (4.76%), oxytetracycline (7.94%), amoxicillin (9.52%), doxycycline (9.52%) and ceftriaxone (14.29%).

DISCUSSION

The age of all recorded cases ranged in between 5 to 8 years. Sharma et al. (2007) and Sharma and Prasad (2002) reported high prevalence of mastitis between 5 to 7 years and above 7 years of age. Increasing evidence of mastitis with advanced ages was recorded by Hawari Azmi and Fowzi (2008). They also noticed that the incidence of clinical and subclinical mastitis was increased with the sixth years old in some herds. Dhakal et al. (2007) found more incidence of mastitis in first and second calving which decreased with increased numbers of calving. The finding of the present study is in accordance with the observations of above workers with little variations which might be because of different geographical attributes and individual variation in susceptibility.

In this study the mastitic agents were isolated from 70% cases, while no growth was evident in 30%. The failure of pathogens to grow in vitro in high percentage of samples may be because of premedication of the animals with antibiotics, non-bacterial causes and the type of media that did not support the growth of whole range of bacteria associated with mastitis.

The study revealed Escherichia coli (40%) and Staphylococcus aureus (34.29%) as the major pathogens causing bovine mastitis. Sharma and Prasad (2002) recorded 54.50 per % incidence of Staphylococcus in bovine mastitis, while Shrirame et al. (2002) recorded more prevalence of Staphylococcus (72.35%). The predominant bacterial isolates recovered by Sumathi et al. (2008) from bovine mastitis were Staphylococcus aureus and Escherichia coli which are in accordance with the present findings. Hawari Azmi and Fowzi (2008) revealed that Staphylococcus aureus (40.6%) and Coliforms (26.1%) were the chief aetiological agents responsible for clinical mastitis. They reported the incidence of Proteus sp. 1.4% Pseudomonas sp. 4.3%, mixed 7.3% and others 5.8% in clinical mastitis. These findings support the findings of present study.

The findings of present study showed the highest incidence of Escherichia coli (40.00%), which is presumably due to the fact that Escherichia coli is the commonest environmental contaminants, which is closely associated with hygiene. It becomes pathogenic whenever the hygienic conditions of the animal or environment become poor. Moreover, the existence of high concentration of Escherichia coli in milk also indicates the relatively poor quality of milk, related with substandard hygiene of farm management, milk collection and processing system. Staphylococci, Pseudomonas and mixed growth were the second after Escherichia coli. Their presence was also an indication of sub-standard dairy farming. Higher incidence of Escherichia coli mastitis may be due to poor hygienic conditions, as Escherichia coli originates from the cow’s environment and infect the udder via the teat canal (Sumathi et al., 2008).

The hygiene at milking is of paramount important in control of these infections because the infections are spread during milking process (Harmon, 1993).

Gentamicin, enrofloxacin, ciprofloxacin and chloramphenicol are not commonly used for treatment of mastitis in the area of study resulting in higher efficacy of these drugs. Ciprofloxacin proved to be the drug of choice in this study. Few workers found highest sensitivity of mastitic agents to enrofloxacin, gentamycine (Dhakal et al., 2007; Kumar and Sharma, 2002) and chloramphenecol (Rao et al., 1989) and least sensitivity to ampicillin (Dhakal et al., 2007) and cloxacillin (Rao et al., 1989). Similar antibiogram patterns were reported by Sumathi et al. (2008) and Choudhuri (2000), while Anakalo et al. (2004) reported Staphylococcus aureus and Coagulase Negative Staphylococcus (CNS) as the major mastitis-inducing pathogens suggestive of a possible development of resistance from prolonged and indiscriminate usage of beta-lactam antibiotics.

The results of Hawari Azmi and Fowzi, (2008) are not in agreement to the present findings. They found that the 64% isolates, isolated from mastitic milk were sensitive to Tetracycline and 52.8% to Ampicillin.

Amoxicillin, ampicillin, cloxacillin, streptomycin and oxytetracycline are commonly used antibiotics in bovine mastitis. The mastitic bacteria showed resistance to these commonly used antibiotics. Indiscriminate and frequent use of these antibiotics in animals could be the reason for their ineffectiveness against mastitic bacteria. Since, streptomycin has been extensively used along with penicillin for treating mastitis; it may have led to the development of high resistance in bacteria against this antibiotic.

Moreover, due to lack of prophylactic agents, chemotherapy continues to play a major role in therapeutic management of the disease. For success of treatment the antibiotic sensitivity test play a major role. Recently higher antibiotics have been introduced in treatment of both sub-clinical and clinical mastitis (Sharma et al., 2007). It is believed that infections with Staphylococcus aureus respond poorly to therapy with antimicrobial agents, whether given parenterally or via the intramammary route (Moon et al., 2006). The emergence of antibacterial resistance among pathogens that affect animal health is of growing concern in veterinary medicine. Antimicrobial-resistant pathogens in animals have been incriminated as a potential health risk for humans from possible transmission as foodborne pathogens. Particularly, mastitis is the single greatest cause of antibacterial use on dairy farms (Moon et al., 2006).

Because the quality of the milk cannot be improved following extraction from the cow, production of high quality milk requires an efficient mastitis control program. Cows with a high prevalence of mastitis are incapable of producing high quality milk until the inflammation and infection in the udder are brought under control. This has severe economic implications for the milk producer, as the milk is no longer marketable and other animals are easily infected. Treatment and decrease in milk volume also cause considerable losses (Anakalo et al., 2004). Therefore, establishing an antibiogram of pathogens is very important from the clinical and economic points of view.

The findings of this study showed the use of higher antibiotics as most effective antibiotic therapy for control of bovine mastitis in and around Udgir area. Since, this study reported overall 7.00% prevalence of fungal mastitis, during antibacterial treatment, the fungal cause shall also be taken into consideration. Further, antibiogram of mastitic milk is suggested before starting the treatment so as to get maximum efficacy of used antibiotics and to prevent the development of resistance to antibiotics due to indiscriminate and frequent use.

Systematic records regarding the epidemiology of bovine mastitis including status of infection, antibiogram studies and treatment patterns would provide useful management information to the producer, farmer and veterinarian. This has been evident from countries where records have been documented regularly internationally (Anakalo et al., 2004). Thus, there is a need to routinely investigate and record the epidemiology of bovine mastitis and antibiogram sensitivity of bacterial isolates in various parts of India.