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Mastitis is a multi-etiological complex disease. The cow udder is an ideal environment for microbial growth and under optimum udder conditions, such as temperature, nutrition and freedom from outside influence, pathogenic organisms multiply astronomically and it is this factor that causes udder damage and triggers the response that is recognized as mastitis (Sharma &Vohra, 2011). Previously, the mastitis researchers associated the mastitis with physical factors like cold and mechanical injuries. Frank in the year 1876, transmitted the disease by inoculating inflammatory secretions from diseased quarters into the canals of healthy quarters. He thus, proved the infectious nature of this disease and put forward an entirely new concept in the investigation of mastitis etiology. An association between mastitis and pathogenic micro-organisms was established in 1887. Most major pathogens were identified by the 1940s (Sharma et al., 2011b).
S.aureus is the most prevalent and economically significant pathogen causing inflammatory infections in dairy ruminants (Katsudaet al., 2005). The bacterium is frequently found associated with subclinical mastitis in dairy cattle (Adesiyunet al., 1998).Staphylococci are gram positive, non-motile and non-spore forming bacteria. Pathogenic staphylococci are identified by their ability to produce coagulase thus clot the blood (Harris et al., 2002). S. aureusis one of the most extensively studied bacteria of genus Staphylococci.S. aureusis both commensal and pathogen. It is found as a commensal associated with skin, skin glands and mucous membranes. S. aureusaffects skin, soft tissues, bloodstream and lower respiratory tract. It also causes severe deep-seated infections like endocarditis and osteomyelitis (Schito, 2006). The prevalence of S. aureuscan most likely be attributed to the wide distribution of the organism inside mammary glands and on the skin of teats and udders. S. aureusadapts very well in the udder and establishes chronic and subclinical infections. From there it is shed into the milk, which serves as a source of infection for healthy cows during the milking process.
Coagulase-positive S.aureus is recognized worldwide as a major pathogen causing subclinical intramammary infection in dairy cows (Robensonet al., 1996). S.aureus produces coagulase, an extracellular enzyme that binds to protein to form a complex with thrombin-like activity which converts fibrinogen to fibrin (Konemanet al., 1992). Although this enzyme is also produced by Staphylococcus intermedius and some Staphylococcus hyicus strains (Holt et al., 1994), coagulase production is one of the most reliable criteria for the identification of S.aureus (Sperberet al., 1975).
Methicillin-resistant Staphylococcusaureus(MRSA) has emerged as a significant public health problem both in human and veterinary medicine. MRSA are the strains of S. aureusthat are resistant to all available Penicillins and other ?-lactam antimicrobial drugs (David et al., 2010). Strains of S. aureus resistant to ?-lactam antibiotics are known as MRSA (Kumar et al., 2011). Overuse of antibiotics has been ascribed for MRSA emergence. MRSA in cattle was first reported in 1972. MRSA causes incurable intra-mammary infection and skin diseases in cattle. The S. aureus with MRSA represent pathogens with signi?cant impact on food safety, human and animal health. Milk and its derivates are considered as a major source of S. aureus infection in man (Zecconi&Piccinini, 2000). The bacterium is frequently found associated with subclinical mastitis in dairy cattle (Adesiyunet al., 1998).
Those Staphylococcal infections which are frequently treated with antimicrobials and consequence of which antimicrobial resistance has developed out of which of the MRSA is critical one.MRSA has been increasingly reported as emerging problem in veterinary medicine.MRSA has been isolated from cattle, dogs, cats, pigs, horses and poultry worldwide (Leonard & Markey, 2008).
MRSA is considered as a critically important human pathogen that is also an emerging concern for the veterinary medicine.Staphylococcus infection is one of the bacterial infection routinely found isolated from the domestic animal in veterinary clinical practice. Methicillin-resistant strain of S. aureuswas evolved one year after the development of the methicillin, 1961 which was beneficial to treat the penicillin resistant strain of Staphylococcusspp.The combination of increased bacterial virulence and drug resistance creates a potential for increased risk of morbidity and mortality for animals and humans that some have extrapolated to a catastrophic potential.The first case of MRSA in veterinary species was identified in the milk of a cow with mastitis in 1972 (Khan etal., 2010). Although MRSA was emerged in the form of nosocomial pathogen, it has been causing hospital-associated infections throughout the world. This trend of evolution in the strains of MRSA simultaneously resistance for a large numbers of different antibiotics, including ?-lactams, Vancomycin i.e. the last line of antibacterial defense nowadays.
The most important factor that determines the resistance are genetic factor of the bacteria. MRSA has the mecA gene that is located on a genetically mobile chromosomal determinant termed staphylococcal cassette chromosome mec (SCCmec). The mecA gene codes for the modified penicillin binding protein 2a (PBP2a) that is located in cell wall with low binding affinity to ?-lactams (Kocket al., 2013). The reduction in binding affinity of antibiotics by PBP2a allows the cell wall synthesis to continue. The production of the beta-lactamase enzyme by blaZ gene (Kumar et al., 2011) is responsible for drug inactivation.
Emergence and rapid spread of Antimicrobial resistance (AMR) is a major problem globally because it creates the limitations in the efficacy of the antimicrobial drugs for the treatment of diseased human and varieties of animals. Antimicrobials are the valuable therapeutic agents constantly used in human as well as veterinary medicine. The other major factor in the growth of antibiotic resistance is spread of the resistant strains of bacteria from person to person, or from the non-human sources in the environment. There is a fact that the non-therapeutic use of antimicrobial is the major factor for the development of resistivity by microorganism. In addition, the trend uses of such antimicrobials in therapeutic, other non-therapeutic and prophylactic purposes in veterinary medicine are in increasing order. Inappropriate and overuse of antibiotics for non-bacterial infections such as colds and other viral infections and their inadequate doses may lead problem in selection of alternative drugs.
1.2 Rationaleof Study
In Asia, major mastitis causing organisms are S. aureus, Streptococci, E. coli, Corynebacterium spp. and Klebsiella spp., recent reports indicating the changing trends from S. aureusto Coagulase Negative Staphylococci (CNS) as major mastitis causing organism (Sharma et al., 2012). Subclinical mastitis (SCM) may be of public health significance as SCM causing organisms have been isolated in several milk borne diseases in human beings (Vermaet al., 1978). Subclinical mastitis is a disease of major economic importance to dairy industry causing reduced milk quality and leading to a loss in its production (Yalcin, 2000). It is now a well-known fact that the SCM is more serious and is responsible for much greater loss to the dairy industry (Kader et al., 2002). Dhakal and Thapa (2003) estimated the losses of Rs.4287 or $63 per buffaloes per lactation in the Nepalese context. Decreased milk production accounts for about 70% of the total cost of mastitis (Sapkota, 2012). About 10-26% of total milk loss occurs in quarters with subclinical intra-mammary infection (Dhakal, 2007). If an animal can be screened for SCM and treated, it will prevent economic loss resulting from disease state (Bansel& Singh, 2004).
S. aureus is frequently associated with subclinical mastitis. Most commonly isolated highly contagious pathogen recovered from bovine raw milk and infected mammary glands (Haveri, 2008). The S. aureus with MRSA represent pathogens with signi?cant impact on food safety, human and animal health. MRSA has potential to onset the nosocomial and community associated infections. Nosocomial infection is a major problem in the world today. MRSA strains, usually resistant to several antibiotics, show a particular ability to spread in hospitals and are now present in most of the countries. So, it is an important emerging zoonotic problem.The bacterium is frequently found associated with subclinical mastitis in dairy cattle.
Multiple drug resistance pattern is shown by S. aureus. It has been emerged as a significant public health problem both in human and veterinary medicine. This study helps to determine the prevalence of resistance gene and analyse the pattern of distribution of the gene in different dairy farms in the research site. This paves the way for measures to control the transmission of drug resistant bacteria.
1.3 Limitations of study
? Coverage of wider area and large number of sample is not possible due to the limitation of fund, time and other variable causes.
? This study was conducted in Kathmandu Valley focusing dairy farms. Sample units of farms were selected purposively. The result of this study might underestimate or over estimate of prevalence of S.aureusdue to selection bias.
? Some antibiotics which are not commonly used in Nepal were tested against the S. aureus to understand the horizontal gene transfer.
1.4Objectives
General objectives
? Molecular identification of Methicillin Resistant Staphylococcusaureus(MRSA) isolated from raw cattle milk.
Specific objectives
? To find the prevalence of S. aureus isolated from raw cattle milk in Kathmandu Valley.
? To determine antibiotic sensitivity profile of isolated S. aureus.
? To detect and find out the prevalence of methicillin resistant gene, mecA in S. aureusby using PCR.

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