Shahid Hussain Farooqi

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.

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.

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 nos; 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.

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 milkers hands, unhygienic milking procedures, and higher use of beta lactam of antibiotics were risk factors that were prevalent in increasing order from sub-district Faisalabadu202f>u202fJaranwalau202f>u202fSamundary. 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.

Economic Significance of Tropical Theileriosis on a Holstein Friesian Dairy Farm in Pakistan

Abstract The dairy industry in Pakistan is booming, and investors are anxious to fund dairy farms that are using high-milk-producing (exotic) cattle breeds such as Holstein Friesians that are not native to the country. Unfortunately, the benefits of increased milk production do not provide resistance to pathogens present in regions where the exotic breeds are introduced. Therefore, the current study was conducted to evaluate the economic impact of Theileria annulata on a commercial Holstein Friesian dairy farm in the District of Ranjanpur, in the Province of Punjab, Pakistan. The economic impact of T. annulata infection was calculated for cattle with subclinical and clinical theileriosis. Losses were estimated based on milk production, morbidity, mortality, and tick control costs (organophosphate sprays). Animals were classified into groups after screening for mastitis, teat abnormality, abnormal parturition, intestinal parasites, and hemoparasites (T. annulata, Babesia spp., and Anaplasma spp.). Microscopy was done for hemoparasites and intestinal parasites. PCR was used to confirm microscopic identification of T. annulata. Animals were classified into 3 groups: group A (normal), group B (subclinical theileriosis), and group C (acute theileriosis). Hemoparasites were observed microscopically in 28.7% of cows. Theileria annulata was found in 8%, and the herd incidence (new cases) of T. annulata was 2.8%. Milk production, animal rectal temperature, and body condition scores between group A and groups B and C were significantly different (P < 0.05). But the enlargement of sub-scapular lymph node and interval of body condition score of the 3 groups were not significant (P > 0.05). The total expenditure incurred due to theileriosis was US

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

74.98 per animal and 13.83% of total farm costs. Hence theileriosis caused significant economic loss of US

Molecular epidemiology of bovine anaplasmosis in Khyber Pakhtunkhwa, Pakistan

18,743.76 (0.02 million) on this Holstein Friesian dairy.

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

BACKGROUNDnA widespread epidemic of equine influenza (EI) occurred in nonvaccinated equine population across multiple districts in Khyber Pakhtunkhwa Province of Pakistan during 2015-2016.nnnOBJECTIVES AND METHODSnAn 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.nnnRESULTSnHA 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.nnnCONCLUSIONnEpidemiological 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.

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

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 (cattleu2009=u2009479, buffalou2009=u2009421) 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 ratiou2009>u20091). The 611xa0bp 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

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.