PREVALENCE OF SUB CLINICAL MASTITIS IN COWS: ITS ETIOLOG OGY AND ANTIBIOGRAM

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1 Indian J. Anim. Res., 46 (4) : , 2012 AGRICULTURAL RESEARCH COMMUNICATION CENTRE ccjournals.com / indianjournals.com nals.com PREVALENCE OF SUB CLINICAL MASTITIS IN COWS: ITS ETIOLOG OGY AND ANTIBIOGRAM Pankaj, Anshu Sharma*, Rajesh Chhabra and Neelesh Sindhu College Central Laboratory, College of Veterinary Sciences, Lal Lajpat Rai University of Veterinary & Animal Sciences, Hisar , India Received: Accepted: ABSTRACT Study was planned to determine the prevalence of sub clinical mastitis in crossbred and indigenous cows and to characterize etiological agent/s involved along with their antimicrobial sensitivity testing. Milk samples from 364 quarters of 95 lactating cows at an organized farm were screened. The overall quarter wise and animal wise prevalence on the basis of cultural examination was and 39.83%, respectively espectively.. According to International national Dairy Federation criteria, % quarters of cows were suffering from sub clinical mastitis on account of having somatic cell count (SCC) more than 5,00,000 per ml of milk and. The prevalence of latent mastitis (SCC < 5 x 10 5 /ml and ) and non-specific mastitis ( x 10 5 /ml of milk and negative) was observed as and 4.67 %, respectively espectively.. A total of 150 organisms were e recover ecovered ed out of 145 quarters. These were % coagulase staphylococci and % were coagulase negative staphylococci followed by Streptococcus dysgalactiae (22.66%), Streptococcus agalactiae (6.66 %) and Streptococcus uberis (2.66%), and (3.33%) quarters revealed mixed infections of Staphylococcus spp. and Streptococcus spp. The antibiogram of isolates revealed 100 % sensitivity to Cloxacillin, Ceftriaxone and Cefoperazone and high ( %) sensitivity towards Enrofloxacin, Cephalexin, Gentamicin and Lincomycin. Key words: Sub clinical mastitis, Cow, Prevalence, Antimicrobial sensitivity. INTRODUCTION The inflammation of mammary gland parenchyma is characterized by physical and chemical, usually associated with bacteriological changes in milk and pathological alterations of glandular tissue (Radostits et al. 2000). In clinical mastitis there is gross abnormality in milk. Sub clinical mastitis (SCM) exists without any gross signs and symptoms therefore, goes unnoticed most of the times and is responsible for heavy economic loss to dairy industry (Dua,2001; Wadhwa et al., 2003).Therefore, to develop proper prevention and control strategies, it is imperative to know the prevalence and etiology of sub clinical mastitis at the earliest. Further, the extensive and indiscriminate use of antibiotics in the treatment and control of mastitis may lead to undesirable presence of drug residues in milk, emergence of antibiotic resistant bacteria and necessary withholding period. So to avoid indiscriminate use of antibiotics and to reduce the cost of treatment practice of antibiotic sensitivity testing should be adopted before initiation of treatment. In India the limited information available on the prevalence of SCM in cows is invariably based on a single parameter which does not fulfill International Dairy Federation (IDF) criteria which is based on cultural examination and somatic cell count (SCC) of milk. Therefore, the study was planned to determine the prevalence of sub-clinical mastitis in crossbred and indigenous cows following IDF criteria and to characterize etiological agent/s involved along with their antibiotic sensitivity testing. MATERIALS AND METHODS The milk samples collected aseptically from 364 quarters of 95 lactating cows at an organized crossbred dairy herd of Holstein Fresien x Hariana, *Corresponding author s anshusharma_dryahoomail.com

2 Vol. 46, No. 4, 2012 Holstein Fresien x Hariana x Jersey, and indigenous cows of Hariana breed were shaken thoroughly and 0.01 ml of the milk sample was streaked on 5% sheep blood agar and MacConkey s lactose agar plates, with the help of a 4 mm diameter platinum loop. The plates were incubated at 37 0 C for 24 to 48 hours. Sub-cultures of the resulting growth were made on blood agar for purification of isolates and identified on the basis of Gram s reaction, morphology and colony characteristics. All the isolates were characterized up to species level following standard bacteriological procedures (Quinn et al. 1994). The SCC in milk samples was performed and the milk smears were stained with Newman-Lampert stain (Schalm et al. 1971). Different strains of various organisms isolated from udder infections were subjected to invitro drug sensitivity testing, using 20 antimicrobials by disc-diffusion method (Bauer et al., 1966). 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 (Bauer et al., 1966). RESULTS AND DISCUSSION Results of cultural examination and SCC in milk samples of healthy cows are presented in Table 1. The overall animal wise and quarter wise prevalence on the basis of cultural examination was 64.21(61/95) and 39.83% (145/364), respectively. Similar prevalence of SCM has also been reported (Nagal et al. (1999), Maiti et al. (2003), Jain (2005) and Sharma et al. 2010). In contrast to the present study, several workers (Saravanan et al. 2000, Saluja et al, 2004 and Chavan et al. 2007) reported very high animal wise and quarterwise prevalence of SCM. These differences in the prevalence rates of SCM are perhaps, due to the difference in 349 managemental and hygienic practices adopted in different dairy herds. The incidence of mastitis varied among farms and the risk increased with increasing parity (Sargeant et al. 1998). Thirunavukarasu and Prabaharan (1998) reported that the incidence of mastitis was significantly associated with animal factors such as breed (Dego and Tareke 2003), milk yield, stage of lactation (Sharma et al. 2007) and udder morphology, besides farm practices and sanitation. The climatic conditions also affect the prevalence of mastitis (Schultze 1985). Thus the higher rate of prevalence as observed in present investigation, might be attributed to intensive management and lack of proper hygienic and control measures. The per cent prevalence of sub-clinical mastitis was found to be lower on the basis of SCC (>5x10 5 /ml) alone (47.36) as compared to cultural examination (64.21). However, the quarter wise per cent prevalence on the basis of SCC (20.00) was lesser as compared to bacteriological examination (47.36). According to the criteria of International Dairy Federation (IDF), per cent quarters of cows were diagnosed as sub-clinically mastitic on account of having SCC more than 5,00,000 per ml of milk and (International Dairy federation,1987). This is closer to the findings of Tuteja (1999) and Saluja et al. (2004) but Jain (2005) disagreed with this who reported a higher rate of infection on the basis of IDF criteria. However, the per cent prevalence of latent mastitis (SCC < 5 x 10 5 /ml and ) as observed in present study (24.45). Serieys (1985) and Roder and Gedek (1986) reported that the SCC could be influenced by the type of infecting organisms and season. Thus, a low cell count does not reflect the true bacteriological status of udder. The TABLE 1: Prevalence of sub-clinical mastitis in cows. Species (No of Animals/ Quarters) COW (95/364) Animals 61 (64.21) Animals show ing lac/m l 45 (47.36) Quarters 145 (39.83) lac/ml 73 (20.00) lac/m l and 56 (15.38) Quarters showing SCC < 5 la c/m l a nd 89 (24.45) lac/m l and negative 17 (4.67) Figures in parentheses indicate percentage.

3 350 INDIAN JOURNAL OF ANIMAL RESEARCH TABLE 2: Frequency distribution of different organisms isolated from milk of cows suffering from sub clinical mastitis. Organisms No. (per c ent) Coagulase staphyloco cci 58 (3 8.67) Coagulase negative staphylococci 44(29.33) Streptococcus agalactiae 10(6.67) Streptococcus dysgalactiae 34(22.67) Streptococcus uberis 4(2.6 7) significance of latent mastitis cannot be undermined, since some of these are likely to convert into subclinical form, followed by clinical mastitis, particularly under unfavorable environmental conditions. Moreover, latent infection also reflects the possibility of teat canal infections serving as a potential source of infection to the milk secretory tissue. Even mammary parenchyma may be damaged due to liberation of bacterial toxins, in the infected teat canal (Nickerson et al. 1986). There were 4.67 per cent quarters having x 10 5 /ml of milk and negative, which were considered as nonspecifically mastitic. Tuteja et al. (1993) and Sindhu et al. (2009) reported 7.7 and 2.19 per cent quarters having non-specific mastitis, respectively. Failure to detect pathogens in such cases might be due to intermittent excretion of the organisms or their disappearance because of spontaneous recovery. Salsberg et al. (1984) observed that SCC increased during summer months from June to August in Holstein cows, than in cooler months. The possibility of mycoplasmal mastitis can not be ruled out in such TABLE 3. Characterization of staphylococci isolated from cows. Coagulase test Organisms Number Coagulase staphylococci (CPS) Staphylococcus aureus 52 Staphylococcus hyicus 3 Staphylococcus intermedius 3 Coagulase negative staphylococci (CNS) Staphylococcus hyicus 15 Staphylococcus epidermidis 4 Staphylococcus hominis subsp. hominis 3 Staphylococcus caprae 3 Staphylococcus pasteuri 2 Staphylococcus arlettae 2 Staphylococcus kloosii 2 Staphylococcus felis 2 Staphylococcus chromogenes 2 Staphylococcus schleiferi coagulans 2 Staphylococcus haemolyticus 1 Staphylococcus saprophyticus subsp. saprophyticus 1 Staphylococcus piscifermantans 1 Staphylococcus sciuri subsp. scuiri 1 Staphylococcus gallinarum 1 Staphylococcus cohnii subsp. urealyticus 1 Staphylococcus condimenti 1 TOTAL 102

4 TABLE 4. Antibiogram of Staphylococci and Streptococci isolated from milk. Antimicrobials P er ce nt se nsitivity Staphylococci Vol. 46, No. 4, 2012 Strepto cocc i Erythromycin P enicillin Streptomycin Tetracycline C hloram phenicol Ampicillin Neomycin Cloxacillin Enrofloxacin Ge ntam icin Am ikacin Amoxycillin Ceftrioxone Cefoperazone Ciprofloxacin Colistin Co-Triamoxazole Nitrofurantion Lincomycin Cephalexin cases, since the organism can not be cultivated on common bacteriological media. Out of 145 quarters of cows, total 150 organisms were recovered (Table 2). Of these, % were coagulase staphylococci (CPS) and % were coagulase negative staphylococci(cns) followed by Streptococcus agalactiae 6.66 %, Streptococcus dysgalactiae 22.66% and Streptococcus uberis 2.66%, and 3.33% quarters revealed mixed infections Staphylococcus spp. and Streptococcus spp. All the CPS and CNS were further characterized to species level. Staphylococcus aureus was found to have the highest prevalence followed by other species. Results indicated substantial differences in the prevalence of pathogens among different herds. In the present study, contagious bacteria like staphylococci and Str. agalactiae caused most of the infections. These infections are usually spread from animal to animal at the time of milking. The mastitis situation can be improved by improving milking practices and hygiene. Rani et al., 2008 reported that the prevalence of mastitis varies with 351 breed, age, lactation and season. Amongst the various mastitogenic bacteria isolated, staphylococci were the most prevalent, accounting for per cent of the infections followed by streptococci (31.98%). Similar findings were also reported in India (Sharma and Kapur,2000; Bulla,2002)..The high prevalence of staphylococci has been reported by several workers in India (Tuteja 1999, Kaya et al and Sharma et al. 2007) and abroad (Hawari and Dabas 2008, Tenhagen et al and Nickerson and Stephen. 2009). Prevalence of pathogen is influenced by parity, type of sample and season (Sharma et al. 2007; Hagnestan et al. 2009). Distribution of pathogens changes over time, therefore, bacteriological examination at herd level must be taken regularly to monitor udder health. In the current study, amongst streptococcal isolates, Str. dysgalactiae were the predominating organisms (22.66%) followed by Str. agalactiae (6.66%) and Str. uberis (2.66%) (Table 3). The higher prevalence of Str. dysgalactiae than Str. agalactiae was reported by Kalra and Dhanda (1964) and Tuteja (1999). In contrast to the present study, higher prevalence of Str. agalactiae than Str. dysgalactiae was reported (Hameed et al. 2007, Chavan et al. 2007, Getahun et al and Ferguson et al. 2008), whereas other workers (Javed and Siddique 1999 and Sampimon et al. 2009) found higher prevalence of Str. uberis than that recorded in the present study. The findings of present study are in close agreement with Petzer et al. (2009) who reported isolation of CNS, Str. agalactiae and Str. uberis. Among staphylococci, Staph. aureus was found to be the most prevalent followed by Staph. hyicus and Staph. Epidermidis (Table3). Many workers have found Staph. aureus to be more prevalent than Staph. epidermidis (Saini et al. 1994, Armenteros et al and Unnerstad et al. 2009).Contrary to this, several workers (Chavan et al. 2007, Ferguson et al. 2008, Petzer et al. 2009, Tenhagen et al and Sampimon et al. 2009) reported high prevalence of CNS. These findings show the increasing importance of CNS which were formally described as a minor pathogen in case of mastitis. Moreover, Staph. epidermidis is supposed to be normal flora of teat skin, its higher prevalence, as observed in present study, might be a

5 352 INDIAN JOURNAL OF ANIMAL RESEARCH consequence of unhygienic milking practice, due to which the organisms gained access into mammary gland, through milkers hands, causing increase in SCC and inflicting pathogenicity in the alveolar tissue. All the isolates from sub-clinical mastitis cases were subjected to antimicrobial sensitivity against 20 antimicrobial agents (Table 4). Results of in-vitro sensitivity are presented in Table 4. The invitro antimicrobial sensitivity patterns of isolates recovered from SCM revealed 100 per cent sensitivity to Cloxacillin, Ceftriaxone and Cefoperazone and a high ( %) sensitivity towards Enrofloxacin, Cephalexin and Lincomycin. In comparison to present study, Ranjan et al also found high sensitivity towards Enrofloxacin (91.67%) where as they observed lower sensitivity towards Ceftriaxone (84.10%). It was interesting to note that Staphylococci isolates revealed 100 per cent sensitivity towards Tetracycline, and Streptococci isolates showed high ( %) sensitivity to ciprofloxacin and penicillin. Studies conducted by several workers (Dhakal et al. 2007, Sharma et al. 2007, Chavan et al. 2007, Roychoudhury and Dutta 2009) have showed increased resistance towards different traditional and newly introduced antibiotics. In contrast to these studies, the antibiogram obtained in current study indicated high sensitivity towards newer and older antibiotics showing rationale use of these antibiotics at farms under study. To institute an effective treatment of mastitis, in vitro antimicrobial sensitivity testing of mastitogenic isolates is suggested. REFERENCES Armenteros, M., Ponce P., Capdevila J., Zaldivar V. and Hernandez R.. (2006). Prevalence of mastitis in first lactation dairy cows and sensitivity pattern of the bacteria isolated in a specialized dairy.revi. de Salud Anim., 28: Bauer, A.W., Kirby W.W.M., Sherris J.C. and Turch. M. (1966). Antibiotic susceptibility testing by a standardized single disc method. American J. Clin. Pathol., 45: Bulla, T.R. (2002). Studies on diagnosis and treatment of sub-clinical mastitis in buffaloes. M.V.Sc thesis submitted to CCS HAU, Hisar, Haryana. Chavan, V.V., Digraskar S.U., Dhonde S.N. and Hase P.B.. (2007). Observation on bubaline subclinical mastitis in and around Parbhani. Indian J. Field Vet., 3:50. Dego, O.K. and Tareke F.. (2003). Bovine mastitis in selected areas of southern Ethiopia. Trop. Anim. Health Prod., 35: Dua, K. (2001). Studies on incidence, etiology and estimated economic losses due to mastitis in Punjab and India -An update. Indian Dairyman, 53: Ferguson, J.D., Azzaro G., Gambina M. and Licitra G..(2008). Prevalence of mastitis pathogens in Ragusa, Sicily, from 2000 to J. Dairy Sci., 90: Getahun, K., Kelay B., Bekana M. and Lobago F.. (2008). Bovine mastitis and antibiotic resistance patterns in Selalle smallholder dairy farms, central Ethiopia. Trop. Anim. Health Prod., 40: Hagnestan, N.C., Emanuelson U., Berglund B. and Strandberg E. (2009). Relationship between somatic cell count and milk yield in different stages of lactation. J. Dairy Sci., 92: Hameed, K., Galal A., Sender G. and Korwin K.A.. (2007). Public health hazard due to mastitis in dairy cows. Anim. Sci. Papers and Reports, 25: Hawari, AD. and Al Dabbas F.. (2008). Prevalence and distribution of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Jordan. American J. Anim. Vet. Sci., 3: International Dairy Federation (1987). Bovine mastitis.definition and guidelines for diagnosis.bulletin of International Dairy Federation No 211, Brussels, Belgium Jain, A.K. (2005). Detection of Staphylococcus spp. by double antibody sandwich enzyme linked immuno-sorbent assay (DAS-ELISA) in bovine sub-clinical mastitis. M.V.Sc. Thesis. CCS Haryana Agricultural University, Hisar, Haryana, India. Javed, I. and Siddique M.. (1999). Some epidemiological aspects of mastitis in cows and the characterization of the isolates. Pakistan Vet. J., 19: Kalra, D.S. and Dhanda M.R..(1964). Incidence of mastitis in cows and buffaloes in North West India. Vet. Record, 76: Kaya, O., Kirkan S., Gulal M. and Unal B.. (2000). Identification and antibiotic susceptibility of microbes causing mastitis in dairy cows. Vet. Bull., 70: 290.

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