Prevalence of Staphylococcus aureus Isolated from Mastitic Milk, Udder Surfaces and Milkers’ Hands from Different Farms in Bikaner, Rajasthan

Staphylococcus aureus is recognized worldwide as one of the most important contagious mastitis pathogen and is frequently isolated from mastitic milk and extramammary sites. The present study was undertaken to study prevalence of S. aureus strains isolated from mastitic milk, udder surfaces and milkers’ hands from organized (n=5) and unorganized dairy farms (n=2). For this, a total of 197 samples (80 mastitic milk samples, 66 udder swabs and 51 swabs of milkers’ hands) were collected from different places. A total of 107 isolates including 51 from mastitic milk samples, 35 from udder and 21 from milkers’ hands, were presumptively identified as S. aureus on the basis of cultural and biochemical properties and then genotypically confirmed using 23S rRNA ribotyping followed by PCR amplification of nuc gene. An overall recovery rate of S. aureus was 54.3% with highest (63.8%) recovery from mastitic milk samples followed by udder (53.0%) and milkers’ hands (41.2%). The unorganized dairy farm showed highest prevalence (65.4%) of S. aureus than that in other farms.

Mastitis is one of the most complex and multifactorial disease affecting dairy animals. Staphylococcus aureus is one of the chief causative agent of clinical mastitis in bovines in India (Padhy et al., 2014;Kutar et al., 2015;Bhagat et al., 2017;Choudhary et al., 2018) and worldwide (Abdel-Tawab et al., 2016;Hanon, 2017). The milk from infected mammary glands serves as the primary reservoir of the bacterium which may be transferred to other animals in the herd during milking (Capurro et al., 2010). In addition human handlers, milking equipment, environment, udder and teat skin of infected animals are possible sources of infection to the animals. In dairy workers, the hands may be considered the most important site of S. aureus because the hands are in intimate contact with the udder.
Although various PCR based detection systems have been developed so far for identification of S. aureus but not found sufficiently reliable to detect all strains of S. aureus. A PCR system developed by Straub et al. (1999) based on amplification of species-specific 23S rRNA allows specific detection of all strains of species. Thus this method is extensively used throughout the world for genotypic identification and confirmation of S. aureus from various clinical and subclinical infections (Salasia et al., 2004;Upadhyay et al., 2010;Khichar et al., 2014;Yadav et al., 2015).
Staphlyococcus aureus is a contagious mastitis pathogen and its transmission is thought to occur from animal to animal principally through the mastitic milk, udder surfaces, milkers' hands, milking utensils, or milking machine (Radostits et al., 2006). However, its epidemiological behaviour is not definite with strains signifying infectious and/or environmental transmission patterns (Fernandez et al., 2013). Hence the present study was undertaken to study the molecular epidemiology of S. aureus strains in and around Bikaner, Rajasthan, India by 23S rRNA ribotyping and amplification of nuc gene.

Animals and sample collection
In the present study, a total of 197 samples were collected from seven different locations (five organized and one unorganized dairy farm in and around Bikaner, Rajasthan, India and one unorganized dairy farm in Bhiwani, Haryana). Animals of selected locations were diagnosed with clinical mastitis by clinical symptoms. From each location, three types of samples were collected which comprised of milk samples from cows with clinical mastitis, swabs from udder of infected cows and swabs of milkers' hands who were working in that farm or location. A total of 80 mastitic milk samples, 66 udder swabs and 51 swabs of milkers' hands were collected in the morning hours and were immediately transported on ice to the laboratory for further processing as per standard procedures (Quinn et al., 1994). All the procedures have been carried out in accordance with the guidelines laid down by the Institutional Ethics Committee and in accordance with local laws and regulations.

Isolation and identification of bacteria
The samples were inoculated in nutrient broth over night and then swabbed on nutrient agar followed by overnight incubation at 37 ºC. Bacterial colonies were closely observed for their morphology, color and consistency. Gram's staining was used as primary identification test and suspected colonies were streaked on mannitol salt agar and incubated for 24 h at 37 o C under aerobic conditions.

RESULTS AND DISCUSSION
Details of samples and recovery of S. aureus isolates in samples collected from different locations of Bikaner have been depicted in Table 1. On the basis of cultural and biochemical properties, out of 197 samples, 107 S. aureus isolates were presumptively identified of which 51 isolates were from mastitic milk samples, 35 from udder and 21 from milkers' hands.
In the present investigation, all the 107 isolates produced an amplicon of 1250 bp following 23S rRNA based ribotyping (Fig. 1) and an amplicon of 270 bp upon PCR amplification of nuc gene confirming them to be S. aureus (Fig. 2). The overall recovery rate was 54.3%. Highest prevalence of S. aureus was observed in mastitic milk samples being 63.8% followed by 53% and 41.2% prevalence in udder and milkers' hands, respectively. Among the seven groups under study, the overall highest prevalence of S. aureus was found in group no. 5 (unorganized dairy farm) being 65.4% followed by group no. 7 (60.0%), group no. 3 (58.3%), group no. 2 (55.3%), group no. 4 (55.0%), group no. 1 (40.0%) and group no. 6 (35.7%). Further, all the groups showed higher occurrence of S. aureus in mastitic milk than in extramammary sites (Table 1).
Genotypic confirmation by 23S rRNA ribotyping using similar primers has been reported by Sanjiv et al.   Kateete et al. (2010) considered identification of S. aureus using PCR amplification of the nuc gene as a gold standard method. Detection of nuc gene for the identification of S. aureus strains isolated from intramammary infections has been adopted by many workers in India and from other parts of the world. Similar prevalence of 100% nuc gene carrying S. aureus isolates has been reported in previous studies on intramammary infections in cattle by Kalorey et al. (2007), Sarkar et al. (2014), Xu et al. (2015), Nazir et al. (2017) and Javid et al. (2018) in different parts of the world.
The overall occurrence of S. aureus (54.3%) from all the locations in present study is similar to that reported by Souza et al. (2012) who collected 446 samples from several sites in dairy farms and recovered 244 (54.7%) S. aureus isolates. Sarkar et al. (2014) also reported 73.6% prevalence of S. aureus in the farm from mastitic milk samples and nasal swabs of farm workers similar to present study. Nathiya et al. (2018) reported recovery of 47.45% S. aureus isolates from milk of cows with clinical mastitis from same area of work as in present study. Similarly, Kutar et al. (2015) detected higher (56%) incidence of S. aureus in clinical mastitis cases in Uttar Pradesh, India. Likewise, Parth et al. (2016) found that 54.29% of clinical mastitic milk samples yielded S. aureus (cows 61.90% and buffaloes 42.85%) in Gujrat, India. A high prevalence of 63.8% of S. aureus isolates from mastitic milk samples observed in present study is similar to the findings of Sori et al. (2011) who isolated 86 (52.4%) S. aureus strains from 164 high CMT score milk samples in Ethopia. Hanon, (2017) reported incidence of 47.69% S. aureus from bovine mastitis in Iraq and Baloch et al. (2018) isolated 46.2% S. aureus strains from bovine mastitic milk in China.
In the present study, the occurrence of S. aureus in mastitic milk, udder skin and milkers' hands was found to be different in all groups owing to the difference in management practices of maintaining animal health as well hygiene of the farm and farm workers.
Similar to the present research work of isolating S. aureus from mastitic milk samples and extramammary sites, studies have been conducted in other parts of India (Sarkar et al., 2014) and world reporting the prevalence of S. aureus in milk, body sites and environment in varying degrees (Capurro et al., 2010;Souza et al., 2012;Mekuria et al., 2013;Schmidt et al., 2017). When compared to the prevalence rates detected by other workers worldwide, it was found that alike the prevalence rate of S. aureus in milk, the prevalence rate of S. aureus on the udder skin and in the hands of animal worker population varies depending upon the size, geographical area and management practices of the herd under study.
In conclusion, the results of the present study demonstrate that genotyping methods involving 23S RNA ribotyping and nuc gene amplication provides easy identification of S. aureus isolates in bovine mastitis. Furthermore this study also substantiates the previous reports of S. aureus being most prevalent pathogen causing clinical mastitis in dairy animals revealing higher prevalence in samples of bovine origin as compared to human samples. The isolation of S. aureus from milkers hands in fairly good numbers indicate transmission of this pathogen between animals and man on a farm. Hence it may be suggested from the present study that stringent management practices should be adopted to prevent the spread of these bacteria and of mastitis in a herd of animals.