Pathogens isolated from clinical mastitis in Murrah buffaloes and their antibiogram

Aim: To determine the etiology of clinical mastitis in Murrah buffaloes and to develop an antibiogram of organisms isolated. Materials and Methods: A total of 564 quarter milk samples of 144 buffaloes suffering from clinical mastitis received in Veterinary College Central Laboratory were inoculated on blood agar, MacConkey’s lactose agar and sabouraud dextrose agar. Bacteria isolated were characterized to the species level and subjected to in-vitro antimicrobial sensitivity testing. Results: Out of 564 quarters examined for mastitis, 320 (56.73%) quarters were found culturally positive showing isolation of Staphylococcus aureus 140 (38.04%), Streptococcus dysgalactiae 112 (30.43%), Streptococcus agalactiae 13 (3.53%), Escherichia coli 74 (20.10%) and Corynebacterium pyogenes 29 (7.88%). On carrying antibiogram staphylococci and streptococci revealed high sensitivity towards chloramphenicol, gentamicin, amikacin and enrofloxacin. Streptococci showed high sensitivity towards ceftriaxone and cefaperazone also. E. coli was found highly sensitive to chloramphenicol and gentamicin. C. pyogenes was sensitive to the majority of antibiotics. Conclusion: S. aureus was the most predominant bacteria isolated from mastitis cases and irrespective of the isolates chloramphenicol was found to be most sensitive when tested in-vitro.


Introduction
India ranks first in milk production with 121.8 million tonnes of milk production [1] of which more than 55% comes from buffaloes.Haryana has the world's best dairy type buffalo, the Murrah, capable of milk yields as high as 35 kg a day.Mastitis is an inflammation of mammary glands causing heavy economic losses to the dairy industry throughout the world.These losses in India have been increased from Rs. 6053.21 crores [2] to Rs. 7165.51 crores per annum [3].
At present it has been established that mastitis can be caused by over 250 microorganisms [4] and detection of these pathogens is considered essential for the definitive diagnosis of mastitis.Treatment with antibiotics is one of the most important components to control mastitis.Due to indiscriminate use of antibiotics, antibiotic resistance has been developed against most of the commonly used drugs leading to treatment failure.Therefore, definitive diagnosis of etiological agents and their antimicrobial sensitivity prior to treatment will help in selecting suitable and cost effective antibiotic to treat an animal properly.
Therefore, the present study was planned to determine the etiological agent(s) responsible for causation of clinical mastitis in Murrah buffaloes and to determine their antimicrobial sensitivity to institute proper line treatment and adoption of control measures.

Ethical approval
State project on mastitis is under operation in College Central Laboratory, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar.The milk samples used in the current study were directly received from animal owners.As per University rules for these samples approval of Institutional Animal Ethics Committee is not required.

Place of study
The study was conducted in College Central Laboratory, College of Veterinary Sciences, LUVAS, Hisar.

Source of milk samples
A total of 564 quarter milk samples were received directly from animal owners of 144 buffaloes suffering from clinical mastitis in a sterile container from different parts of Haryana and nearby villages of Rajasthan.

Bacteriological examination
Immediately after receiving, the milk samples were inoculated in 0.01 ml volume on 5% sheep blood agar (BA), MacConkey's lactose agar (MLA) and sabouraud dextrose agar plates with the help of a 4 mm diameter platinum loop.The plates were incubated aerobically at 37°C for 24-48 h.Sub-cultures of the resulting growth were made on BA for purification of isolates and identified on the basis of Grams reaction, morphology and colony characteristics.All the isolates were characterized up to species' level following standard microbial procedures of National Mastitis Council [5].
The Gram-positive cocci were subjected to catalase test to differentiate between staphylococci and streptococci.To rule out possibility of micrococci, all the catalase positive cocci were subjected to oxidase test and oxidation fermentation test.On the basis of coagulase test, haemolysis on 5% BA and Staph™ latex test kit (HiMedia, Mumbai), staphylococci were identified up to species' level.
The organisms that on preliminary examination were found to be streptococci, were further characterized for different species using Strep™ latex test kit (HiMedia, Mumbai, India) as per instructions of the manufacturer.Other organisms like Corynebacterium pyogenes were identified on the basis of colony characteristics, Grams staining reaction, morphology and catalase test, whereas Escherichia coli were differentiated on the basis of growth on eosin methylene blue agar, MLA and IMViC test.

Antimicrobial susceptibility testing
Different strains of various organisms isolated from udder infections were subjected to in-vitro drug sensitivity testing, using 14 antimicrobials by disc-diffusion method [6].With the help of a platinum loop, a small amount of test culture was transferred into a tube of brain heart infusion broth and incubated for 2-5 h at 37°C, so as to obtain turbidity.With the help of a sterile cotton swab, the broth culture was then evenly spread by smearing over the surface of BA/Mueller-Hinton agar plates.The antimicrobial discs were placed on the agar and gently pressed.These were then, incubated at 37°C for 24 h.The sensitivity was observed on the basis of zone size interpretation chart, provided by the manufacturer.The results were recorded as sensitive, intermediate and resistant.Different antimicrobials used were ampicillin (25 mcg), ceftriaxone (30 mcg), amoxicillin (10 mcg), streptomycin (25 mcg), cloxacillin (30 mcg), chloramphenicol (30 mcg), neomycin (30 mcg), tylosin (15 mcg), enrofloxacin (10 mcg), cefoperazone (75 mcg), amikacin (30 mcg), penicillin-G (10 units), gentamicin (30 mcg), and oxytetracycline (30 mcg) (Hi media).
Variable sensitivity was shown by E. coli (74) isolates with maximum sensitivity towards chloramphenicol (94.59%) and least towards cloxacillin (8.10%).In the present study sensitivity of E. coli isolates towards gentamicin, ceftriaxone, enrofloxacin and amikacin were in the range of 74.32-82.43%.However, sensitivity to other antibiotics was comparatively low.Only 17.56% isolates were sensitive to tylosin.

Discussion
Prevalence of different microorganisms Among the various microorganisms isolated, S. aureus was the most prevalent followed by Streptococcus spp., which was in accordance with reports of several workers [7][8][9][10][11][12][13]. Sumathi et al. [14] also reported staphylococci as the most important and prevalent mastitis causing organism globally, including India.High prevalence of S. aureus points to poor milking time hygiene as this pathogen is mainly spread during and between two milking via milkers' hands and towels.S. aureus is also of public health importance as enterotoxigenic strains can cause food poisoning in human beings.Moreover, the existence of high concentration of S. aureus in milk also indicates the relatively poor quality of milk and milk products.These results also show that in India among streptococci, S. agalactiae and S. dysgalactiae are the most frequently encountered organisms followed by other species of streptococci.In developed countries like USA and UK, a dramatic decrease in the incidence of contagious mastitis pathogens has been achieved by implementing five-point mastitis control programme.In these countries infections caused by the S. agalactiae and S. dysgalactiae have been eradicated or reduced to very low levels, and the effect on staphylococcal infection is similar though the decline in infection level is slow.However over the same period there was no change in the prevalence of mastitis pathogens as no controls measures were adopted in India.In contrast to our study, Sumathi et al. [14], Dubal et al. [15], Ranjan et al. [16], Harini and Sumathi [17], Kurjogi and Kaliwal [18], Abd-Elrahman [19] and Jeykumar et al. [20] observed E. coli being second in prevalence following staphylococci.However, Marimuthu et al. [21] found 21% of organisms isolated to be Bacillus spp.which was totally absent in our study and other studies mentioned earlier.Isolation of E. coli is indicative of poor hygienic conditions of animal environment as E. coli infects the udder via teat canal from the environment.Isolation of C. pyogenes is indicative of poor care and managemental practices.C. pyogenes alone establishes itself readily in mammary tissue causing subclinical mastitis.The situation worsens when C. pyogenes infect the udder along with other bacteria leading to chronic mastitis ultimate loss of the quarter.

Antibiogram of S. aureus isolates
Results of antibiotic sensitivity pattern for S. aureus isolates showed amikacin to be the most sensitive among all the antibiotics tested which was in accordance with Ranjan et al. [16]; higher than reported by Pankaj et al. [8], whereas on the lower side as compared with Kurjogi and Kaliwal [18].However, S. aureus was the least sensitive against penicillin-G in our study that was similar to studies reported by Mohanty et al. [10] and Ranjan et al. [16] conducted around the country.The resistance of S. aureus to penicillin-G in our study may be attributed to the production of beta lactamase, an enzyme that inactivates penicillin and closely related antibiotics.The probable reason for the development of resistance by these organisms to penicillin-G is conventional, prolonged and indiscriminate usage of this drug in the field conditions.The reasons for higher sensitivity against penicillin-G reported by Pankaj et al. [8] and Charaya et al. [9] might be that both these studies were conducted at organised farm where rational use of antibiotics is done.

Antibiogram of streptococci
Chloramphenicol has been found most sensitive (97%) towards Streptococcus isolates irrespective of the species.Antibiogram pattern reported by Pankaj et al. [8] revealed higher sensitivity towards penicillin-G, neomycin, cloxacillin, gentamicin, amoxicillin, ceftriaxone and cefoperazone.However, few studies [8,16,18] showed lesser sensitivity of Streptococcus isolates towards penicillin-G, gentamicin, streptomycin, ampicillin and amikacin as compared to present study.While comparing sensitivity of enrofloxacin, we found Ranjan et al. [16] and Mohanty et al. [10] to be on higher side whereas Pankaj et al. [8] and Abd-Elrahman [19] on lower side.

Antibiogram of C. pyogenes isolates
In our study more than 80% C. pyogenes isolates revealed sensitivity towards all the antibiotics except penicillin-G.Similar to our study, Dutta et al. [22] also recorded high sensitivity towards ampicillin but contrary to our study they found less sensitivity towards cloxacillin.

Conclusion
In conclusion, the present study indicated considerable prevalence of the disease and pathogens from clinical mastitis in Hisar and its surroundings.Appropriate treatment and control strategies should be formulated to eradicate or reduce major pathogens S. aureus, S. dysgalactiae, S. agalactiae and E. coli.Mastitis is the single largest cause of antimicrobial use in dairy farms.Extensive and indiscriminate use of antibiotics against these bacteria results into emergence of drug resistant mutants which cause treatment failure in animals have also become a potential health risk for humans, as they are able to cause direct and indirect transmission of infection.Therefore, establishing an antibiogram of pathogens is very important from the clinical and economic points of view.Therefore, dairy farm owners need to be advised to get antimicrobial susceptibility testing prior to the institution of proper line of treatment.

Table - 1
: Relative frequency of organisms isolated from 564 milk samples of 144 buffaloes.