It is my pleasure to bring this annual technical report for the fiscal year 2071/72( 2014-
. This annual technical report is a routine publication and presents an overview
of the programs and activities of Regional Veterinary Laboratory, Pokhara. This
report includes progress and achievements, laboratory findings and investigation
works carried out during the period. Western development region (WDR) has
tremendous scopes for livestock production. Most of the districts in the region have
good infrastructures and access to markets, growing tourism and changing food habits
of the people favor livestock production to become commercialization.
In Nepal, infectious diseases and parasites are the major constraint for the improvement in livestock production and productivity. Many infectious diseases are also zoonotic, which increase the significance of the disease. A number of exotic diseases such as infectious bursal disease and leechi heart disease in poultry and Peste des Petits Ruminants (PPR) in goats have been introduced into the country with the import of live animals or animal products. WDR experienced several outbreaks of highly pathogenic avian influenza and foot and mouth disease (FMD) during this period. These diseases are responsible to cause a considerable amount of economic loss to livestock industry of the country, compelling the need of a massive disease control program. Correct diagnosis and epidemiological information of animal diseases and parasites are required to formulate an effective disease control program. For perfect diagnosis of the diseases, laboratory services are essential components. So strengthening diagnostic facilities not only in central level but also in regional and district level is indispensable. Recently with the assistance of Zoonotic Control Project diagnostic capabilities of Regional Veterinary Laboratory(RVL) pokhara have been upgraded to some extent and capacity building efforts are continuing. On behalf of Regional Veterinary Laboratory, Pokhara I would like to express my sincere thanks to Central Veterinary Laboratory , Veterinary Epidemiological Center and all DLSOs of this region for continuous support. I also like to extend my gratitude to all my staffs for their contribution in preparing this report. I always welcome your comments and suggestions regarding this publication. Dr. Kedar Raj Pande
Senior Veterinary Officer
Regional Veterinary Laboratory
Ramghat, Pokhara, Nepal
Introduction RVL
Mission of RVL
Objective RVL
Major Laboratory Test Facilities
Annual work program and summary of achievement of RVL FY069/70
Laboratory Services
Parasitological Services Microbiological Services Pathological Services Serological Services Hematological Services Biochemical Services REGIONAL VETERINARY LABORATORY
Western Development Region (WDR) is situated between 82º 30' to 85º 15' east longitude and from 27º 15' to 29º 30' north latitude. It occupies about 20% (29355 Sq. Km.) of total areas of Nepal. The region shares boundaries with Uttar Pradesh of India in the south and Tibet of China in the north. The region is bulging between Central and Mid-western development regions of Nepal in the east and west respectively. Geographically, WDR is divided into the following three main domains: Himalayan region

Himalayan region is located in the northern part of the WDR, covering Mustang, Manang and upper belt of Gorkha districts. Yak/Nak, sheep, alpine goats (Chyangra) and mule rearing formed the way of life of the people in this region. Hilly region

Hilly region lies in between the Himalayan and Terai regions. This region comprised of Arghakhanchi, Gulmi, Palpa, Shyanga, Kaski, Tanahu, Lamjung, and lower belt of Gorkha, Parwat, Baglung and Myagdi districts. People of divergent ethnic groups, casts and cultures share their common way of living. Agro-based livestock industry in this region is the main source of income of the people. Poultry farming, goat rearing and dairy industries are becoming familiar near the cities/towns and in the areas where market is Terai region

Terai region covers Nawalparasi, Rupandehi and Kapilbastu districts. This plain extends from east to west of the region and stretched from 15 to 40 Kilometers in width. Sediments and silt are main constituents of soil deposited by rivers making it more fertile and this belt supplies the food and fibers to other regions of the country. Compared to mountains and hilly regions, this region has relatively better infrastructure and market accessibility. People of this region are motivated to adopt livestock farming in commercial scale. The population of livestock in the region is very high as compared to their production. Though many factors are contributory, the health of animal plays a vital role to increase the production and productivity of animal. Every year, several diseases and parasitic problems attribute a considerable amount of economic loss to livestock rearing farmers of the country warranting switching on a massive disease control program. Major economically important diseases of cattle and buffaloes are Foot and mouth disease (FMD), Haemorrhagic septicemia (HS), Helminthiosis, infertility, mastitis and blood protozoan diseases whereas PPR, Gastrointestinal nematodosis and Clostridial diseases in sheep and goats. Swine fever and FMD in pigs and Avian Influenza, New Castle disease (ND), Infectious bursal disease (IBD), Coccidiosis, Hemorrhegic Enteritis and Mycotoxicosis in chickens are major disease problems. 2. Mission of the Regional Veterinary Laboratory, Pokhara

The mission of the Regional Veterinary Laboratory, Pokhara is to promote the health of livestock, poultry and companion animals and to insure safe animal products for the consumer by assisting District Livestock Development Offices, veterinarians, clients, and others responsible for animal health in the detection and prevention of disease by conducting responsible investigation on animal diseases and providing accessible, accountable, timely, and accurate diagnostic services. 3. Objectives of Regional Veterinary Laboratory
 To provide accessible, timely and accurate diagnostic services to the livestock and poultry farmers and to veterinarians, veterinary technicians and their owners in the  To conduct diagnostic examinations, record results, report information, and assist in the interpretation of results to submitting DLSOs, Veterinarians, and veterinary  To investigate the animal disease epidemics in the region and assist, advice and support DLSOs to control them.  To prepare epidemiological profile of livestock and poultry diseases and maintain and disseminate the regional epidemiological information database on animal health in the regional as well as in the national networks.  To investigate relatively important livestock diseases in the region and formulate control measures for the same with wider consultation to the experts.  To monitor and report the incidence and threat of animal diseases, as well as diseases that are transmissible form animal to humans.  To supervise and assist in diagnostic services to basic and primary laboratories based at DLSO's of the region.  To conduct and support the laboratory and animal health related training programs for the Para vets in the region.  To coordinate national disease control and eradication programs in the region. These objectives are accomplished by the application of different diagnostic assays, interpretation of diagnostic procedures, consultation with animal health professionals of the Animal health directorate and Department of Livestock Services and training and continuing education of persons responsible for delivering animal health care services. 4. Major Laboratory Tests Facilities of Regional Veterinary

Laboratory, Pokhara
Regional Veterinary Laboratory, Pokhara, located at Ramghat, the centre of Pokhara city, provides diversified veterinary Laboratory test facilities for the farmers, private veterinary practitioners and district Livestock Service Office of this region. It mainly tests the following categories of the samples: a. Parasitological Unit:
Parasitological unit tests for external parasites the parasitology unit performs microscopic examination of skin scrappings for the identification of mange mite species. It conducts blood parasite test using blood smear examination and for the blood filaria examination using Knot's method. b. Microbiological Unit:
Microbiology unit tests diversified samples like milk, tissues, blood, aspirated fluids and tissues etc. Both aerobic and anaerobic culture facilities are available. It also perform identification of the Bacterial and fungal organisms using various biochemical tests, staining, morphology etc. The laboratory is capable of handling Mycobacterium and Mycoplasma species for culture. The microbiology unit also performs antibiotic susceptibility test and advice for the appropriate antibiotic for the treatments. In virology the laboratory is capable for the isolation of NewCastle Disease and Infectious Bronchitis virus using egg inoculation method. c. Pathology Unit:
Pathology unit mainly perform Post mortem examination on various species of animals and collect appropriate samples for the histopathological examination and dispatched to the histopathology unit of Central Veterinary Laboratory for the examination. The unit perform annual necropsy of about 194 animals and birds mostly poultry birds. It performs various types of cytological studies for the disease diagnosis. d. Serology Unit:
Serology unit of Regional Veterinary Laboratory, Pokhara mainly perform Brucellosis test using RBPT antigen, Mycoplasmosis and Pullorum disease of poultry using Mycoplasma gallisepticum and Mycoplasma synovae antigen by Plate agglutination test. e. Biochemistry Unit:
Biochemistry unit analyze mainly serum for the estimation of Calcium, Phosphorus, Magnesium and total proteins as well as different biochemical profile of different animals. It is performing the Urine tests by estimating Albumine, Bilirubin, and Urobilinogen using dipstik test kit. f. Hematology Unit:
The Hematology Unit of Regional Veterinary Laboratory is providing routine hematological parameters of all the animals and Poultry. Table 1: Annual work Program and summary of achievements of Regional
Veterinary Laboratory, Pokhara (for fiscal year 2069/70 & Budget No:312125-34)

Programmes and Activities Capital Expenditure
Construction of Remaining Compound Wall Purchase of Fridge Running Expenditure
Laboratory Services
Parasitological Examinations EPG Count and Larva Culture Examination of Blood Parasites Pathological Examinations Clinical Hematological Tests Calcium, Phosphorus, T. Protein, Glucose, CU,CO,Iron Postmortum Examination Microbiological Examinations Isolation and Identification of Bacteria Isolation and Identification of Fungus Virological Tests (Ranikhet,Gumboro,AIV) PPR testing using penside test Serological Tests Mycoplasma tests in Poultry using PAT Salmonella tests in Poultry using PAT Disease Diagnosis using ELISA Tests ND diagnosis using HA HI Molecular diagnostic test Sample collection and dispatch Disease Investigation and Surveillance Program
Antibiotic resistant bacteria surveillance and sharing Finding out the Basic production and Fertility Profile in Cattle Investigation of Parasitic Diseases in Migratory Sheep Management of Regional EDIT Investigation of Epidemic Laboratory Diagnosis of Zoonotic Diseases
Brucella Diagnosis using RBPT Rabies Rapid Test Tuberculosis Testing using Tuberculin Laboratory pogramme under food security
Campylobacter and Salmonella isolation in chicken Monitoring and Supervision
Follow up tour of district Laboratories Workshops and seminars
Animal Disease Investigation Interaction Workshop Participation in Budget and Programme formulation workshops at Central Level Publication Program
Half Yearly Epidemiologic Bulletin publication Annual Epidemiological Bulletin Publication Material Preparation for Annual Technical Report Purchase of Journal
Laboratory Management
Civil Servent Use in Anti Rabies Vaccine Civil Servent Health Check Lab Animal Management Contract Service
Machinary Tools and Computer Management
Computer Operator,Mali,Swiper,Helper,Driver and Lab Equipments Repair Management etc. P.P.R. Sero-Monitoring F.M.D Sero-Monitoring Swine Fever Sero-Monitoring Ranikhet Sero-Monitoring 5. Laboratory Services
5.1 Parasitological Examination
Parasitological Examination of Blood Protozoa in the fiscal year 2071/072 Blood sample
Staining Blood smears. A total of 106 blood samples from animals were examined for different Animal. 5.2 Microbiological Examinations

Microbiological examinations include the isolation and identification of bacteria and fungi from the pathological samples received in the laboratory. Bacteriological culture,Fungus Culture and antibiotic sensitivity tests were performed of the samples received for microbiological investigation. During 2071/72 a total of 521 samples were examined in microbiology unit of the laboratory. Table 3: Different Bacteria isolates of Mastitis
Name of the isolates Percentage of cases Klebsiella sps Staphylococcus sps. Streptococus sps. Bacillus sps All the organisms shown in the culture were subjected for the antibiotic sensitivity test and gave the following result: 5.3 Pathological Examinations

Pathological examinations mostly consisted of necropsy examination of carcasses presented in the laboratory where poultry dominates all. In the pathology unit, the cause of death of chickens presented was generally drawn on the basis of both the post mortem lesions observed and laboratory investigation of samples collected during necropsy Table 4 Diseases of chickens diagnosed on the FY 2071/72 are summarized in the
following table.
It can be seen during the FY 2071/72, Colibacillosis was the most prevalent poultry disease followed by Mycotoxicity. There was increase in the cases of Colibacillosis,which might be due to the increased number of farming and poor management condition of the Infectious Bursal Disease New Castle Disease Others (Enteritis, hepatitis,etc) Total cases (n) = 2537 Maximum cases are mixed infections farming system. The temporal pattern of major poultry diseases during the FY 2069/70 is as 5.4 Serological examinations
Serological examinations mainly consisted of plate agglutination test of chicken serum to
detect antibody against Mycoplasma gallisepticum and Salmonella pullorum organisms.
Similarly, serum samples from cattle, buffalo, sheep, goats and dogs were tested for
brucella antibodies using Rose Bengal Plate Agglutination Test (RBPT). During the fiscal
year 2071/72, the serum samples tested and their results are presented as follows:
Table 5: Serological Test Result in RVL in FY 2069/70
Table 6 Following is the virological test results of samples of different species
5.5 Haematological Examinations

Hematological unit of the laboratory is well equipped to determine a range of hematological parameters such as Total Erythrocyte Count (TEC) and Total Leukocyte Count (TLC), Differential Leucocytes Counts (DLC), Erythrocyte Sedimentation Rate (ESR), determination of hemoglobin (HB) and Packed Cell Volume (PCV) and staining of blood smears for blood protozoa and bacteria. A total of 106 blood samples from animals were examined for different hematological parameters. 5.6 Biochemical examinations

Biochemical examinations included biochemistry of serum and routine and microscopic examination of urine. Multistick strip was used for routine urine analysis. Microscopic examination of urine was done after centrifugation of the urine samples. 5.7 Sample Collection and Dispatch

During 2071/72, serum samples, brain and tissue samples of different animal species and poultry were collected from the disease investigation sites. A total of 474 various samples were dispatched to Central Veterinary Laboratory, Kathmandu and National FMD and TADs laboratory, Kathmandu for confirmatory disease diagnosis. 6. Avian Influenza Surveillance (Laboratory Surveillance)

The annual avian influenza laboratory surveillance programme was continuing in the
laboratory and it was successful in the detection of flu A cases. During this fiscal year
071/72 all together 17 flu A cases were identified out of 2537 cases registered for the
disease diagnosis. Four out of 17 flu A case were confirmed as H9 by CVL.Monthly
pattern of the cases of avian influenza was shown by the following figure.
Lokraj Joshi1, Ananta Tiwari1, Shiva Prasad Devkota2,Kedar Raj Pande2
Objective: To determine prevalence of Methicillin- resistant Staphylococus aureus (MRSA)
in dairy farms of Pokhara valley. Design: Cross- sectional study
Methodology: Ten commercial cattle dairy farms within Pokhara valley were selected
purposively. From each farms, 10 cattle were selected making sample size of 100 cattle. Milk sample was taken from all the four teats. Thus, 400 milk samples were used for analysis. Staphylococus aureus was isolated from milk samples. MRSA were identified using Cefoxitin disk diffusion method using CLSI (2007) protocol. S. aureus isolates having zone of inhibition <= 21 mm were identified as MRSA. Antibiotic susceptibility test for all the S. aureus isolates was done on Muller Hilton Agar. Results: Out of 400 milk samples, S. aureus was isolated from 119 (29.7%) samples. MRSA
was found in 45 (11.25%) milk samples. No significant difference was found between farm- wise prevalence of MRSA (P>0.05). Ciprofloxacin, Ceftriaxone, Gentamicin and Tetracycline were found highly sensitive against S. aureus and statistically no significant difference was found between the sensitivity of these four antibiotics. However, Cefoxitin and Cotrimoxazole were significantly less effective (P< 0.05) than Ciprofloxacin, Ceftriaxone, Gentamicin and Tetracycline. Conclusion and Recommendations: This study showed 11.25% prevalence of MRSA in
dairy farms of Pokhara. This indicates MRSA as an emerging zoonotic pathogen posing threat to both animal and public health. Rational use of antibiotics and public awareness are needed to control MRSA spread. Further research using greater sample size and employing molecular techniques like Polymerase Chain Reaction (PCR) are needed for precise result. Key words: S. aureus, MRSA, Cefoxitin, Cattle, Pokhara
1 Institute of Agriculture and Animal Science,Rampur,Chitwan
2 Regional Veterinary Laboratory, Pokhara
Background information
Antibiotics are used both for therapeutic and sub-therapeutic purpose in veterinary medicine. Reason for using antibiotics for therapeutic purpose is analogous to human medicine that is to treat the bacterial infections. In addition to treatment, antibiotics are used for sub-therapeutic purpose in veterinary medicine to enhance feed efficiency and promote growth of animals. Veterinary sector uses approximately 50% of total antibiotics used around the globe (Teuber, 2001). There is currently increased public and scientific interest regarding the administration of therapeutic and sub-therapeutic antimicrobials to animals, due to the emergence and dissemination of multiple antibiotic resistant zoonotic bacterial pathogens (Hardy, 2002). Such antibiotic resistant bacteria do not respond to regular antibiotic treatments and prolong the duration of illness. The emergence of Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious public health threat. Strains of S. aureus resistant to β-lactam antibiotics are known as Methicillin-resistant Staphylococcus aureus (MRSA) (Kumar et al., 2011). First described as a cause of nosocomial infection in hospital settings, now MRSA has gained attention as community pathogen (Said-Salim et al, 2003). In recent years, Methicillin-resistant Staphylococcus aureus (MRSA) has been increasingly reported as emerging problem in veterinary medicine. MRSA has been isolated from both healthy and diseased animals of many species. MRSA has been found in cattle, dogs, cats, pigs, horses and poultry worldwide (Leonard, 2008). MRSA colonization in animals possesses an occupational risk to the humans who remain in close contact with them. In cattle, Staphylococcus aureus is an important mastitic pathogen. Beta lactam antibiotics are drug of choice for the treatment of Staphylococcal mastitis in cattle. Heavy use of beta- lactam antibiotics in cattle have led to the emergence of MRSA (Hendriksen et al., 2008). Devrise et al (1972) first reported the prevalence of MRSA in dairy cows in Belgium. Since then, many reports describing the prevalence of MRSA in dairy cows in different parts of the globe have been published (Spohr et al., 2011; Juhasz-Kaszanyitzky et al., 2007; Moon et al., 2007; Kwon et al., 2005). In Nepal, MRSA prevalence in human hospitals has been studied by several human health workers. Rijal et al (2008) have found 31% prevalence of MRSA in Western Regional Hospital of Pokhara. But, no research has been done in Nepal till now to determine the prevalence of MRSA in animals. This research aims to determine the prevalence of MRSA in dairy farms of Pokhara valley for the first time in Nepal. Description of the project area
This research was carried out in Pokhara valley of Kaski district. Pokhara is one of the highest milk producing places of Nepal. Kaski district has got 73,150 cattle and 129,404 buffalo and milk production was 39,479 Metric Ton (MT) in the fiscal year 2009/10 (MOA, 2010). Intensive cattle farming is increasing in Pokhara. The number of commercial dairy farms is rising. Mastitis is one of the common problems faced by farmers in this area. Out of 241 milk samples brought to Regional Veterinary Laboratory, Pokhara, 160 (66.39%) milk samples were found positive for Sodium Lauryl Sulphate Test (SLST) test (RVL, 2011). Due to unregulated use of antibiotics there is severe threat of antibiotic resistant problem in this region. Beta lactam antibiotics like amoxicillin, penicillin and cephalosporin are being used haphazardly. This led us to hypothesize the presence of MRSA in dairy farms of Pokhara All the laboratory works were carried out at Microbiology section of Regional Veterinary Laboratory, Pokhara. Established in 2049 B.S, this is one of the well equipped veterinary laboratories of Nepal. This lab has long experience of antibiotic sensitivity testing. Hundreds of milk samples are brought by farmers every year in this lab for mastitis detection and antibiotic sensitivity test. Justification of study
Commercial dairy farming is increasing in Pokhara. This has not only helped to raise the living standard of the farmers but also playing a vital role in solving youth unemployment problem. Antibiotics have proved themselves as a boon to dairy industry in terms of increasing growth, performance and treating ailments. But, use of antibiotics is not well regulated in Nepal. Antibiotics are administered without undergoing antibiotic sensitivity assays. This has led to the threat of emergence of resistant bacteria like Methicillin-resistant Staphylococcus aureus (MRSA). Mastitis is one of the common problems faced by dairy farmers in Pokhara. According to Rana (2009), the prevalence of sub-clinical mastitis in Pokhara is 38%. He has also showed that major cause (53%) of sub-clinical mastitis is S. aurues. Since, use of beta lactam antibiotics like ampicillin, cloxacillin, penicillin, ceftriaxone is a common practice in this region, some strains of S. aureus may have acquired ressistance to beta-lactam antibiotics leading to emergence of MRSA. Presence of MRSA in cattle poses serious threat to the public health. They may be transmitted to human beings especially to the farmers, milk handlers and veterinarians (Lee, 2003). So, it is necessary to know the prevalence of such pathogenic organisms in the farms. On the other hand, human health workers have found high prevalence of MRSA in human beings in Nepal (Kumari and Mohapatra., 2008; Rijal et al., 2008). Due to close human- livestock interface, chances are higher that human MRSA may have been transmitted to Data regarding prevalence of MRSA in dairy animals are now available in most of the countries. But there has not been single research done in Nepal regarding prevalence of MRSA in animals. So, this research will pave pathway for further research in this area. Objectives of the study
General objective
 To determine the prevalence of Methicillin-resistant Staphylococcus aureus in dairy farms of Pokhara valley Specific objectives
 To know the prevalence of S. aureus in cattle milk in the farms of Pokhara valley  To determine the prevalence of MRSA in cattle milk  To study the antibiogram of S. aureus isolated from bovine milk samples of dairy farms of Pokhara MRSA transmission between cattle and humans
There are various reports published to provide evidence of MRSA transmission between cattle and humans. Lee (2003) isolated 12 MRSAs from milk samples which were genetically similar to human MRSA. Wulf et al (2006) screened vets and vet students attending a livestock conference in Netherland. Out of 179 vets and vet students who had contact with animals, 7 (4.6%) were MRSA positive. But, out of 27 vets and vet students who had no contact with animals, no one was found MRSA positive. Similarly, transmission of MRSA between Hungarian cows with sub-clinical mastitis and farm workers has been reported by Juhasz-Kaszanyitzky et al (2007). According to Nunang and Young (2007) MRSA can be transmitted from animal to humans through direct contact, environmental contamination and through handling of infected animals product MATERIALS AND METHODS
Site of study
The research was focused on commercial dairy farms of Pokhara valley. Lab works were carried out at Regional Veterinary Laboratory, Pokhara. Duration of study
October 2012 to January 2013 Sample size and sampling method
Ten commercial dairy farms within Pokhara valley were selected purposively. From each farm, again 10 cattle were selected purposively making the sample size of 100 cattle. Milk was collected from all the four quarters of each cattle. So, a total of 400 milk samples from 100 cattle of 10 dairy farms were used for analysis. Collection and transportation of sample
All the teats were swabbed with 70% ethyl alcohol and then allowed to dry. First few streaks of milk were discarded. Then milk was collected aseptically in sterile conical tubes and was labeled as fore right, fore left, hind right and hind left. And they were transported to laboratory in ice-packed containers within three hours of collection. Microbiological analysis of sample
Isolation of Staphylococcus aureus
Milk sample was cultured on blood agar soon after they arrive at lab. 10 µl of milk was spread on blood agar and then incubated at 37°C for 24 hours. Then, golden yellow colored colonies showing beta- haemolysis were suspected as Staphylococci. Then, Gram's staining was done. Gram negative samples were discarded. Samples which revealed gram positive cocci were further inoculated on nutrient agar and incubated for 24 hours at 37°C for pure culture isolation. After incubation, colony morphology was observed. Staphylococcus produces whitish to yellow colonies on nutrient agar. Again, Gram's staining was done and sample having Gram positive cocci were used for further analysis. All the gram positive cocci samples were further subjected to catalase test. This test distinguishes Streptococcus from Staphylococcus as Staphylococcus is catalase positive while streptococcus is catalase negative. All the catalase positive samples were identified as Staphylococcus spp. These catalase positive samples were further subjected to coagulase test which distinguishes coagulase positive S. aureus from other Staphylococci. Identification of Methicillin resistant Staphylococcus aureus (MRSA)
MRSA was identified using CLSI (2007) protocol. S. aureus isolated from above procedure was first suspended on peptone water and incubated for 4 hours at 37°C. Turbidity of bacterial suspension was maintained at 0.5 Mc Farland by further incubation if turbidity was lower and addition of normal saline if turbidity was higher. Then, antibiotic sensitivity test was done on Muller Hilton Agar according to Kirby Bauer Disk Diffusion method. Cefoxitin (30 µg) disk was used for identification MRSA. S. aureus isolates resistant to Cefoxitin (having zone of inhibition less ≤ 21 mm) were identified as MRSA. Antibiotic sensitivity of test
Antibiotic sensitivity test was done for S. aureus according to Kirby Bauer disk diffusion method. Briefly, 0.5 Mc Farland of bacterial suspension was inoculated on Muller Hilton Agar and following disk were placed: a) Cefoxitin (30 µg) b) Ceftriaxone (30 µg) c) Ciprofloxacin(30 µg) d) Gentamicin (30 µg) e) Tetracycline(30 µg) f) Cotrimoxazole (25 µg) Then, plates were incubated at 37°C for 18 hours and then zone of inhibition was measured with the help of zone scale. Antibiotics were classified as resistant, intermediate sensitive and sensitive on the basis of zone of inhibition produced by each antibiotic disk. Data analysis
Data entry was done in Ms- Excel 2007. For prevalence determination, bar graphs and pie charts Ms- Excel 2007 was used. Significant differences between different groups were analyzed using Chi Square test on PH Stat 2.0 (Pearsons). Table 1. Biochemical reactions of Staphylococcus aureus (Source: Chakraborty, 2003) Proportion of Grams' positive and Gram's negative bacteria in milk
Milk samples from 400 teats of 100 cattle were analyzed microbiologically. Out of 400 samples, Gram's positive bacteria were seen in 256 samples (64%) and Gram negative bacteria were seen in 132 samples (33%). No growth was seen in remaining 12 (3%) milk samples on blood agar. Types of Gram's positive bacteria in bovine milk
Staphylococcus spp. was most frequently isolated pathogen. Out of 256 Gram's positive samples, Staphylococcus spp. were isolated from 202 milk samples. It was followed by Streptococcus spp, which were isolated from 42 samples. Other Gram's positive organisms like Gram's positive rod, Gram's positive diplococci were seen in remaining 12 samples. Prevalence of Staphylococcus aureus in dairy farms of Pokhara Out of 400 milk samples, S. aureus was isolated from 119 milk samples. Thus prevalence of S. aureus in dairy farms of Pokhara was 29.75% . Prevalence of MRSA in dairy farms of Pokahara valley
Out of 400 samples, 45 samples were tested positive for MRSA . Thus , prevalence of MRSA was 11.25% (Fig. 4). Table 3 shows farm-wise prevalence of MRSA. Using Chi Square Test, no significant difference was found among farm-wise prevalence of MRSA (P> 0.05) Result of antibiotic susceptibility test
All the S. aureus isolates were subjected to AST. Most of the isolates were found sensitive to Ciprofloxacin (94.1%), while least samples were found sensitive to Cotrimoxazole (69.7%) as shown in Figure 6. Number of isolates sensitive to different antibiotics has been shown in Table 5. All the intermediate samples were integrated into sensitive ones on the basis of fact that intermediate samples are susceptible at higher dose of antibiotics. Using Chi square test no significant difference was found in the sensitivity of Ceftriaxone, Ciprofloxacin, Tetracycline and Gentamicin (P>0.05). However, significant difference was found in the sensitivity of Cefoxitin and Cotrimoxazole in comparision of above four antibiotics. Thus, Cefoxitin and Cotrimoxazole were found less sensitive than Ceftriaxone, Ciprofloxacin, Tetracycline and Gentamicin (P< 0.05). DISCUSSION
The prevalence of S. aureus was found to be 29.7% in dairy farms of Pokhara. This indicates high prevalence of S. aureus in udder of the cow, which is responsible for clinical and sub clinical mastitis. This finding has been supported by various authors. Pradhan et al (2011) has found 34.01 % prevalence of S. aureus in cattle milk in India. Simlary, our finding is in agreement with the finding of Abera et al (2012) who found 28.1% S. aureus in Ethopia. Our finding contradicts with the finding of Suddan et al (2005), Shrestha and Bindari (2012) who found 56% and 50% prevalence of S. aureus in India and Bhaktapur (Nepal) respectively. Similarly, Rana (2009) reported 53% S. aureus from Pokhara. This difference may be due to analysis of only those milk samples which were positive for sub clinical mastitis by those authors, while this research analyzed all the milk samples irrespective of presence of sub clinical mastitis. Prevalence of MRSA in Pokhara valley was found to be 11.25%. This is in agreement with the finding of Kumar et al (2011) who found 13.1% prevalence of MRSA in Sahiwal cattle in India. Suleiman et al (2012) found 8% prevalence of MRSA in Ethopia. Our finding contradicts with the finding of many authors who reported lower prevalence of MRSA. Juhasz- Kaszanyitzky et al (2007) reported 4.53% prevalence MRSA, Huber et al (2010) reported 1.4% MRSA in Switzerland. Haran et al (2012) reported 4% herd prevalence of MRSA in Minnesota, USA. Lower prevalence reported by these authors may be due to use of PCR for MRSA detection instead of disk diffusion method we used. Good regulations regarding use of antibiotics in developed countries may be attributed to lower MRSA prevalence in those countries. Higher prevalence in our research may be due to comparatively lower sample size. Indiscriminate use of beta lactam antibiotics for the treatment of mastitis in Nepal may have led to increased emergence of MRSA. Antibiotic susceptibility test reveals higher sensitivity of Ciprofloxacin, Gentamicin, Ceftriaxone and Tetracycine in descending order, while Cefoxitin and Cotrimoxazole has been found less sensitive to S. aureus. This finding has been supported by Sekhan et al (2011) who found Ciprofloxacin (91.97%) most sensitive to S. aureus. Similarly, our finding is in with Jakee et al (2011) who reported high resistance of S. aureus to methicillin a beta and Sulphamethoxazole- trimethoprim. High resistance of S. aureus against Cefoxitin also indicates the emergence of MRSA in Pokhara. CONCLUSION AND RECOMMENDATIONS
There is high prevalence of (29.7%) S. aureus in dairy farms of Pokhara. This high prevalence poses threat of clinical and sub clinical mastitis in farms of Pokhara. Prevalence of MRSA in dairy farms of Pokhara is 11.25%. This is the first report of MRSA prevalence in cattle in Nepal. This prevalence is comparatively higher than those reported in other many countries. Most of the S. aureus isolates are resistant to beta a lactam antibiotic, which indicates rising antibiotic resistant problem in dairy farms of Pokhara. Ciprofloxacin, Ceftriaxone, Gentamicin and Tetracycline are effective against S. aureus. a) Antibiotics sensitivity assay should be performed before prescribing antibiotics b) Emphasis on farm sanitation and personal hygiene to reduce the chance of MRSA c) Research with greater sample size and employing molecular techniques are needed for d) Further researches are needed to study cattle to human transmission of MRSA. REFERENCES
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