Molecular epidemiology and characterization of Babesia bovis in cattle of Khyber Pakhtunkhwa province, Pakistan.

Babesiosis is a tick-borne disease found globally but most prominent in tropical and subtropical regions. It is responsible for huge mortality and morbidity, especially in developing countries like Pakistan. The current study was designed to determine the molecular epidemiology and characterization of Babesia bovis (B. bovis) infection in cattle populations of districts Mardan, Kohat and Swat of Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 434 tick-infested animals were sampled. Blood samples were collected, processed and then examined initially by microscopy for the presence of Babesia and were later confirmed through PCR by targeting cytochrome b gene, and the PCR products were sequenced. Phylogenetic analysis of sequenced isolates of the current study showed close sequence similarity with the reported strain of China. A non-significant association (p > 0.05) was observed between the prevalence of infections and risk factors. The overall prevalence of infection in all three districts was 10.11%. In district Swat (12.61%), the prevalence was recorded as the highest for B. bovis infection followed by district Mardan (10.60%) and district Kohat (06.90%). The Friesian breed of cattle, females and adult animals were highly susceptible to B. bovis infection. The prevalence of infection was recorded highest during the summer season and lowest during the winter season. This study concludes that B. bovis infection is prevalent in three studied districts of KP province and the sequenced isolates of the current study showed close sequence similarity with the reported strain of China.

Concurrent Trypanosomosis, Babesiosis and Toxocarosis in a Dog: a Case Report.

Background: Among the blood-borne parasitic infections of dogs, trypanosomosis and babesiosis are highly prevalent in the Indian subcontinent and highly pathogenic. Methods: A six-month-old dog was presented with complaint of lethargy, anorexia and loss of body weight over the previous 15 days on October 11, 2021. A full clinical and targeted haematologic and parasitological examination was undertaken. Results: Wet smears were positive for the motile trypanosomes while stained thin blood smears showed large number of extracellular trypanosomes and intra-erythrocytic ring-like inclusion bodies of Babesia spp. A high burden of Toxocara canis was identified on coprological examination. Conclusion: This report describes the clinical manifestation of concurrent infection of haemoparasites and gastrointestinal nematodes in Pakistani dogs, and highlights the importance of prompt veterinary intervention.

First Report on Molecular Characterization of Anaplasmosis in Small Ruminants in Pakistan.

Anaplasmosis is caused by a Gram-negative obligate intracellular bacterium of the genus Anaplasma with the pathogen having a zoonotic impact. The study aimed to estimate the prevalence of anaplasmosis in Pakistan, to unravel the association of potential risk factors, and to investigate the effect on hematological parameters in affected small ruminants. A total of 150 (n = 75 sheep; n = 75 goats) blood samples were initially screened microscopically and then subjected to PCR targeting the amplification of the 16S rRNA gene fragment of Anaplasma. The PCR-based positive samples were then processed for sequencing. Statistical analysis regarding risk factors was performed using R software. The study revealed an overall 29.33% (44/150) prevalence of anaplasmosis in small ruminants. Sheep had higher (P > 0.05) prevalence (32%) as compared to goats (25.30%). The final statistical model resulting from backward elimination showed only tick infestation as a significant predictor of infection status. The phylogenetic analysis of 16S rRNA gene of Anaplasma spp. revealed 9 study isolates clustered together and showed a close resemblance (99%) with Anaplasma ovis isolate (DQ837600) from Hungary. One of the isolates showed (99%) similarity with the isolate of Anaplasma marginale (MH155594) from Iraq. Furthermore, the hematological parameters pack cell volume, red blood cells, hemoglobin, white blood cells, granulocytes, monocytes, lymphocytes, and platelet count were decreased in Anaplasma-positive animals. This is the first study at the molecular level to characterize Anaplasma spp. in small ruminants of Pakistan, and it will be useful in developing control strategies for anaplasmosis.

Spatiotemporal analysis of dengue outbreaks in Samanabad town, Lahore metropolitan area, using geospatial techniques.

Dengue is endemic to Pakistan with its usual peak incidence in the post-monsoon period. In the last decade, dengue outbreaks have occurred in major urban areas particularly Karachi and Lahore, affecting large numbers of people. This study is an attempt to analyze the spatiotemporal variation of dengue fever (DF) in Samanabad town, Lahore metropolitan area. The study is based on secondary data, acquired from concerned government departments. Point level geo-coding is used to transform the relative location to the absolute location using Google Earth, and Global Position System (GPS) is used to validate the geo-coded location. Geographic information system (GIS) has been used to perform spatial analysis. It has been found that temporally DF prevalence varies from month to month and year to year. Major outbreak was observed in the year 2013 with more than 900 confirmed DF cases. Rainfall, temperature, and humidity have played a central role in outbreaks. The land cover pattern and population density further intensified the outbreak. Spatially, the number of DF incidence was high in those localities where the entire land is built-up and with little/no green space areas. Analysis reveals that DF is still a major threat to the area as socioeconomic and geographic conditions favor vector breeding and transfer of disease from one person/place to another. This study presents useful information regarding spatiotemporal patterns of dengue outbreak and may bring the attention of public health departments to formulate dengue-combating strategies. The methodology is general for spatiotemporal analysis and can be applied to other infectious diseases as well.

The first report on serotyping of bluetongue virus in small ruminants of Khyber Pakhtunkhwa province, Pakistan.

Bluetongue virus (BTV), a member of Orbivirus genus (family Reoviridae), is a non-contagious infection of domestic and wild ruminants. The current study was designed to detect various serotypes of BTV in small ruminants of Khyber Pakhtunkhwa (KPK) province of Pakistan, along with their effects on hemato-biochemical parameters. A total of 408 serum samples in four districts (Mansehra, Abbottabad, Swabi, and Kohat) of KPK from small ruminants were screened based on competitive ELISA (cELISA). A total of 204 (50%) samples were found positive for BTV group-specific antibodies. The seropositive samples were processed for the detection of BTV serotypes through real-time polymerase chain reaction (qPCR). Out of 204 cELISA-positive samples, 60 (29.41%) were found positive through qPCR. Three serotypes [6, 8, 9] were detected from Mansehra District and two from Kohat [2, 8] and Abbottabad [6, 8], while only one from Swabi [8]. The serotype "8" was found consistently in all the four study districts. A significant (p < 0.05) increase in the level of blood urea nitrogen (BUN) and alkaline phosphatase (ALP) was recorded in goats, whereas aspartate aminotransferase (AST) in sheep infected with BTV, compared to healthy animals. The hematological parameters showed significantly (p < 0.05) raised total leucocyte count (TLC) in both sheep and goats, whereas only hematocrit (HCT) value was increased significantly (p < 0.05) in infected sheep. This is the first report on serotyping of BTV among small ruminants in Pakistan.

A comprehensive review on past, present and future aspects of canine theileriosis.

Canine theileriosis is a notorious tick borne piroplasmid infection of wild and domestic canines. The causative agent has not yet been accurately classified. PCR studies revealed that causative agent resembles to Theileria genus and thus provisionally named as Theileria annae. The other Theileria species reported in canines is Theileria annulata, Theileria equi and unnamed Theileria specie. This emergent canine infection is considered to be endemic in most of the European countries. However in Asia this disease has not been reported till date. The vectors responsible for transmission of this disease have not been determined. It has been suggested that DNA of Theileria annae has been detected in hard tick Ixodes hexagonus in Northwestern Spain and several other tick species. Clinically canine theileriosis is characterized by severe weakness, fever, hemoglobinuria and anemia. Recently atovaquone or buparvaquone plus azithromycin therapy showed better clinical efficacy. This comprehensive review is intended to summarize the current knowledge on prevalence and epidemiology of canine theileriosis in different countries of the world and associated tick vectors.

RETRACTED: An epidemiological investigation of associated risk factors with equine influenza (H3N8) epidemic 2015-16 in Pakistan.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editors-in-Chief. The article duplicates significant parts of a paper that had already appeared in Preventative Veterinary Medicine, 149, 132-139; https://doi.org/10.1016/j.prevetmed.2017.12.005. One of the conditions of submission of a paper for publication is that authors declare explicitly that the paper has not been previously published and is not under consideration for publication elsewhere. Re-use of any data should be appropriately cited. As such this article represents a misuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

Molecular epidemiology of bovine anaplasmosis in Khyber Pakhtunkhwa, Pakistan.

Bovine anaplasmosis is endemic in Pakistan where it reduces livestock productivity and leads to high mortality, especially in young animals. This study was aimed to identify the potential risk factors responsible for the occurrence and spread of anaplasmosis in cattle and buffaloes for the first time in Pakistan. A total of 900 (cattle = 479, buffalo = 421) blood samples were collected irrespective of age and sex from three distinct zones of Khyber Pakhtunkhhwa (KP) province of Pakistan. Polymerase chain reaction (PCR) technique was used for the molecular detection of anaplasmosis. Data collected on a piloted questionnaire including 11 predicting variables which were analyzed using R-statistical software, and association between the dependent and independent variables was assessed using univariable analysis. Automated and manual approaches were exercised, producing comparable models. Key risk factors identified in all the approaches included species of the animal, breed of animal, sex of animal, tick infestation status, previous tick history, tick control status, and acaricides used (odds ratio > 1). The 611 bp DNA fragment specific for 16S rRNA gene of Anaplasma spp. was produced from 165 samples. The samples were confirmed for anaplasmosis through sequencing and BLAST queries. The findings of the current study conclude that by enhancing the protective measures to control the identified risk factors can reduce the spread of anaplasmosis in Pakistan.

Equine Granulocytic Anaplasmosis 28 years later.

Equine granulocytic anaplasmosis (EGA) is an important tick borne disease of equines that is caused by Anaplasma phagocytophilum (A. phagocytophilum). The etiological agent has veterinary as well as public health importance because of its zoonotic nature. A. phagocytophilum causes an acute illness in equines with loss of appetite, lethargy, hemorrhages and lameness. Clinically, EGA is diagnosed upon examination of morulae within neutrophils especially granulocytes in the blood. The best diagnostic tool for the detection of EGA is Polymerase chain reaction (PCR). Previous studies suggested that EGA is a self-limiting disease and tetracycline therapy is considered as a best treatment regimen. There is no comprehensive summary on the occurrence and distribution of the infection at global level. Therefore, we intended to provide a comprehensive summary on the prevalence and epidemiology of EGA in different areas of the world. It includes mapping the global distribution of EGA in different areas of the world to identify the endemic regions which may be a source of potential disease outbreak. For this purpose, the published data from 1990 to 2018 on EGA was reviewed and collected by electronic literature search of five databases including Google, Google Scholar, Science Direct, PubMed and Web of Science.

Development and evaluation of vaccine against Staphylococcus aureus recovered from naturally occurring mastitis in she-camels.

The predominant Staphylococcus aureus (S. aureus), an etiological agent of camel mastitis is becoming drug resistant that invites prevention and control strategies. Vaccine production would have a valuable impact on public health. Therefore, in present study, inactivated vaccine with different adjuvants was prepared and evaluated against S. aureus. The vaccinal isolate recovered from camel subclinical mastitis was coagulase positive (PCR based), having expressed pseudocapsule, holding alpha-beta hemolysin characteristics, and multiple drug resistant. Inactivated alum precipitated S. aureus vaccine (APSV) and oil adjuvant S. aureus vaccine (OASV) were prepared after confirming its antigenicity in rabbits. Three groups of rabbits were randomly inoculated with APSV, OASV, and placebo (Unvaccinated, UV). Each group was further divided into two groups based on single and booster dose inoculation. Booster dose of vaccines in rabbits at day 15th of primary inoculation was given. Serum samples were taken on 15, 30, 45 and 60 days of primary inoculation from all rabbits. Analysis of variance was applied to compare geometric mean titer (GMT) of three groups, while t-test was applied to estimate the difference between single and booster dose response. The study found 1010 CFU/mL S. aureus as standard bacterial load for vaccines with higher and sustained antigenicity. The vaccines were safe from morbidity and mortality, and proved effective and stable for 7 and 4 months at 25 °C and 37 °C, respectively. The OASV produced significantly (p < 0.05) higher immune response followed by APSV throughout trial. The highest GMT by APSV and OASV vaccines with single dose inoculation was 37.92 and 69.92 at day 45th post primary inoculation, respectively. Similarly, 59.20 and 142.40 GMTs were noted with booster dose in case of APSV and OASV, respectively. The booster dose presented significantly (p < 0.05) higher GMT than that of single dose inoculation of vaccines. The study concluded APSV and OASV safe, effective, and stable with significant immunogenic results in experimental rabbits.

Sero-epidemiology of bluetongue virus (BTV) infection in sheep and goats of Khyber Pakhtunkhwa province of Pakistan.

Bluetongue virus (BTV) infection is an emerging hazard in small ruminants having socio-economic impacts on animals and associated people. The current study was aimed to estimate the sero-prevalence and associated risk factors in sheep and goat from Khyber Pakhtunkhwa (KP) province of Pakistan. Three distinct zones (northern, central and southern) with four districts (Mansehra, Abbottabad, Swabi, and Kohat) with a higher population of small ruminants were selected. A total of n = 408 sera originating from sheep (n = 212) and goats (n = 196) were randomly collected for detection of BTV group specific antibodies through competitive ELISA (c-ELISA). Univariable and multiple logistic regressions were applied to assess the potential risk factors associated with the occurrence of this disease. Results showed an overall prevalence of 50.00% (CI = 44.17-54.83) of BTV in both sheep and goats with a significant difference (p < 0.05) among different districts. The prevalence of BTV in sheep was found higher (56.60%, CI = 49.6-63.4) than goats (42.86%, CI = 35.8-50.1). The risk factors identified based on chi-square test were; 1-2 year of animals, herd size and location in sheep while, milking status, ticks infestation, location and herd size for goats (p < 0.05). On the basis of univariable analysis, 1-2 year of animals, and location for sheep while, ticks infestation and location for goats (OR > 1). Multiple logistic regressions conferred only herd size and location as potential risk factors (OR > 1) for BTV in sheep and goats. The study concluded higher prevalence of BTV in sheep than the goats, the risk factors were significantly associated with the occurrence of disease, and together ascertaining the needs to design appropriate disease management and control strategies in sheep and goats.

Emerging discrepancies in conventional and molecular epidemiology of methicillin resistant Staphylococcus aureus isolated from bovine milk.

Methicillin resistant Staphylococcus aureus (MRSA) is an emerging public health concern from dairy milk, and its diagnosis by phenotypic methodology is experiencing higher discrepancies. The present study was planned to estimate discrepancies in phenotypic identification of MRSA and MSSA (Methicillin sensitive Staphylococcus aureus) in relation to mecA, and prevalent risk factors from various localities. In-vitro oxacilline antibiotic disks were used for phenotypic identification of MRSA, whereas mecA gene was used as MRSA marker in Staph aureus by PCR. Total of 900 bovine milk samples from private and public farms located in district Faisalabad using convinent sampling technique were collected. Potential risk factors for MRSA prevalence identified by non-parametric statistical tests were compared among different subdistricts. Discrepancy in MRSA was calculated as percentage of mecA negative strains while that of MSSA was determined as percentage of mecA positive strains. Molecular identification presented 17.97% (55/306) of discrepancy in MRSA in terms of negative mecA strains from district Faisalabad while sub-district Faisalabad, sub-district Jaranwala, and sub-district Samundary presented 13.98% (13/93), 18.28% (17/93), and 20.83% (25/120) discrepant results, respectively. On the other hand, 29.1% (55/189) of discrepancy in MSSA in terms of mecA positive strains from MSSA isolates was noted. MSSA results were more discrepant than that of MRSA. Hence discrepancy ratio of MSSA over MRSA was noted to be 1.53, 1.50, and 1.21 from tehsil Faislabad, Samundary, and Jaranwala. Tick infestation, lactation stage, frequency of milking, dirty milker's hands, unhygienic milking procedures, and higher use of beta lactam of antibiotics were risk factors that were prevalent in increasing order from sub-district Faisalabad > Jaranwala > Samundary. The study concluded higher prevalence of MRSA in bovine milk samples, and found remarkable discrepancies in phenotypic and genotypic identification which demand immediate attention to tackle exacerbation in resistance patterns.

First molecular evidence of equine granulocytic anaplasmosis in Pakistan.

Anaplasma phagocytophilum (A. phagocytophilum) is an obligate intracellular bacterium that causes equine granulocytic anaplasmosis (EGA) disease in equines. This pathogen has zoonotic potential, which makes it very important to be detected and controlled as early as possible. This study was aimed to assess the molecular prevalence, associated risk factors of EGA along with its effects on various hematological parameters. This study revealed an overall 10.67% prevalence in equine. Horses showed highest prevalence followed by mules and donkeys presenting 11.86, 10.53 and 9.43% prevalence, respectively. The samples were confirmed for anaplasmosis through sequencing. The BLAST queries confirmed very high homology of our isolates with Chinese and Japanese isolates of A. phagocytophilum (Accession no's; KX505303, KY242456 and LC002836). The phylogenetic analysis found the study isolates clustered with each other and this cluster closely resembled Chinese isolate of A. bovis (FJ169957), A. phagocytophilum (HQ872464) and A. phagocytophilum (NR_044762) human isolate from northern Minnesota and Wisconsin. The key risk factors identified for occurrence of EGA in equine species on the basis of univariable analysis were sex of animal, housing type, tick infestation, previous tick history and tick control status, type of acaricides used, rearing system and farm hygiene, respectively. The hematological parameters like Hemoglobin (Hb), Total Leukocyte Count (TLC), Total Erythrocytes Count (TEC), and granulocytes were decreased in diseased animals. The mules showed no typical hematological variations which make sense for its nature as carrier of infection to the susceptible species. This is the first molecular evidence of EGA in Pakistan. The disease needs to be handled seriously as it has zoonotic potential. The animals should be properly attended in disease conditions as leukopenia, neutropenia and lymphopenia can aggravate the condition by making the animal prone to secondary infections.

Investigating the epidemiology of EI epidemic spread in the Province of Khyber Pakhtunkhwa, Pakistan in 2015-16.

EI in non-vaccinated population causes disruption and economic losses. To identify the risk factors associated with the EI epidemics in equids in Pakistan, a 1:1 matched case control study was conducted during 2015-2016. Including a total of 197 laboratory confirmed cases and negative controls, matched on the basis of geography, time of sampling, specie and age. A piloted questionnaire was used to collect data regarding risk factors associated with the occurrence of EI in face to face interviews. Conditional logistic regression was performed to analyze the data. A total of 16 out of 23 variables were found associated as risk factors in Univariable conditional logistic regression analysis. Multivariable conditional logistic-regression analysis was also performed. Monthly removal of manure doubles the risk of EI (EI) compared to its daily removal. Due to lack of vaccination; the spread of disease was favored by high equine density. Investigating the index-case it was recorded that infected cases were imported from Afghanistan. Most of these risk factors related to biosecurity and management were due to low awareness level regarding EI amongst the respondents. These findings are in line with the results of many other studies identifying similar risk factors for EI infection in various countries. Adopting protective practices, vaccination and controlling the risk factors identified in the present study could reduce the spread and future outbreaks of EI in Pakistan.

Sero-epidemiology and hemato-biochemical study of bovine leptospirosis in flood affected zone of Pakistan.

The bovine leptospirosis is an economically important zoonotic disease of flood affected areas worldwide, but scarce information is available about its epidemiology in Pakistan. This is a first study on sero-epidemiology of bovine leptospirosis in Pakistan. The objectives of this study were to investigate the sero-prevalence and associated risk factors of bovine leptospirosis in flood affected zone of Punjab, Pakistan. A total of 385 serum samples were randomly collected from four tehsils of district Muzaffargarh, Pakistan. The serum samples were subjected to indirect ELISA for the detection of anti-leptospira antibodies. The overall sero-prevalence of leptospirosis was 30.39%. The prevalence was significantly higher (p<0.001) in cattle (56.25%) than buffaloes (4.66%). The key risk factors identified based on multivariable logistic regression were; confined system of rearing, flooded area, and lesser to graduate level of education as risk factors for leptospirosis. The values of hematological parameters varied significantly (p<0.05) for Hb, TEC and PCV while there was no significant (p>0.05) difference among TLC values among sero-positive and sero-negative animals. The serum biochemical profile revealed significant differences (p<0.05) in values of ALT, AST and creatinine among the sero-positive and negative animals. Hence, it can be concluded that leptospirosis is an emerging and neglected disease in flood affected zone of Pakistan, and the disease needs to be explored comprehensively in other parts of the country to sort out solid strategies for its control and eradication.

Report: Prevalence and antibiotic trials against Salmonella enterica isolated from diarrheic lambs and kids.

Salmonella enterica (S. enterica) is the major zoonotic threat for small ruminants and humans responsible for huge economic losses and high mortality in Pakistan. Lambs and kids of Lahore district were examined to determine the prevalence, hematology and chemotherapy of S. enterica. A total of 200 diarrheic samples (n=100 lambs; n=100 kids) were collected and examined; 59 (29.50%) were found positive for S. enterica. Lambs had lightly greater prevalence (31%) than kids (29%). The frequency analysis (OR=1.16 [reciprocal =0.87]) showed non-significant difference in both the lambs and kids. The significant decrease (Pℜ0.001) in hemoglobin, pack cell volume and total erythrocyte count was observed in infected lambs and kids. Results of in-vitro antibiotic susceptibility test revealed that S. enteric isolated from both lambs and kids were susceptible to levofloxacin, ciprofloxacin, ofloxacin, gentamicin, azithromycin, tobramycin, amoxicillin, ampicillin and nalidixic acid. Where as the results of in vivo antibiotic trials showed that isolates from both lambs and the kids with diarrhea were susceptible to levofloxacin and ciprofloxacin.

Molecular Epidemiology of a novel re-assorted epidemic strain of equine influenza virus in Pakistan in 2015-16.

BACKGROUND: A widespread epidemic of equine influenza (EI) occurred in nonvaccinated equine population across multiple districts in Khyber Pakhtunkhwa Province of Pakistan during 2015-2016. OBJECTIVES AND METHODS: An epidemiological surveillance study was conducted from Oct 2015 to April 2016 to investigate the outbreak. EI virus strains were isolated in embryonated eggs from suspected equines swab samples and were subjected to genome sequencing using M13 tagged segment specific primers. Phylogenetic analyses of the nucleotide sequences were concluded using Geneious. Haemagglutinin (HA), Neuraminidase (NA), Matrix (M) and nucleoprotein (NP) genes nucleotide and amino acid sequences of the isolated viruses were aligned with those of OIE recommended, FC-1, FC-2, and contemporary isolates of influenza A viruses from other species. RESULTS: HA and NA genes amino acid sequences were very similar to Tennessee/14 and Malaysia/15 of FC-1 and clustered with the contemporary isolates recently reported in the USA. Phylogenetic analysis showed that these viruses were mostly identical (with 99.6% and 97.4% nucleotide homology) to, and were reassortants containing chicken/Pakistan/14 (H7N3) and Canine/Beijing/10 (H3N2) like M and NP genes. Genetic analysis indicated that A/equine/Pakistan/16 viruses were most probably the result of several re-assortments between the co-circulating avian and equine viruses, and were genetically unlike the other equine viruses due to the presence of H7N3 or H3N2 like M and NP genes. CONCLUSION: Epidemiological data analysis indicated the potential chance of mixed, and management such as mixed farming system by keeping equine, canine and backyard poultry together in confined premises as the greater risk factors responsible for the re-assortments. Other factors might have contributed to the spread of the epidemic, including low awareness level, poor control of equine movements, and absence of border control disease strategies.

Antibiotic susceptibilities and prevalence of Methicillin resistant Staphylococcus aureus (MRSA) isolated from bovine milk in Pakistan.

The study was designed to investigate bovine milk for prevalence of an emerging zoonotic pathogen Methicillin resistant Staphylococcus aureus (MRSA), and in-vitro therapeutic response of various antibiotics against MRSA. Nine hundred (900) milk samples were collected (half from cattle and half from buffalo) from private and public farms located in various tehsils of district Faisalabad, using the convenient sampling method. Milk samples were put to biochemical identification of Staphylococcus aureus and later oxacilline disk sensitivity testing for confirmation of MRSA. The MRSA isolates were confirmed by PCR targeting mecA gene in Staphylococcus aureus. The study found 34% prevalence of MRSA in overall bovine milk from district Faisalabad with 30% and 38% prevalence in cattle and buffalo, respectively. Tehsil Samundari presented comparatively higher MRSA prevalence followed by tehsil Jaranwala and tehsil Faisalabad. However, there was non-significant difference of MRSA prevalence between cattle and buffalo, and among different tehsils. All assumed risk factors except specie were significantly associated with mastitis spread. The in-vitro drug trial against MRSA from buffalo milk presented 100% efficacy of Ciprofloxcin, Moxifloxacin, Linezolid, and Trimethoprim plus Sulphamethoxazole combination, followed by Gentamicin and Levofloxacin presenting 90%, and Amikacin becoming 80% efficacious against MRSA from buffalo milk. The MRSA isolates of cattle milk presented similar pattern with some variations of higher susceptibility against Oxytetracycline, and Fusidic acid. The conclusion of the study states uniform prevalence of MRSA in cattle and buffalo milk in study area having assumed risk factors positively associated with disease spread, while Ciprofloxcin, Moxifloxacin, Linezolid, and Trimethoprim plus Sulphamethoxazole drugs showed the highest efficacy to combat this pathogen.