Spatio-temporal epidemiology of Japanese encephalitis virus infection in pig populations of eastern Uttar Pradesh, India, 2013-2022.

AIMS: Japanese encephalitis (JE) is endemic in India. Although pigs are considered important hosts and sentinels for JE outbreaks in people, limited information is available on JE virus (JEV) surveillance in pigs. METHODS AND RESULTS: We investigated the spatio-temporal distribution of JEV seroprevalence and its association with climate variables in 4451 samples from pigs in 10 districts of eastern Uttar Pradesh, India, over 10 years from 2013 to 2022. The mean seroprevalence of IgG (2013-2022) and IgM (2017-2022) was 14% (95% CI 12.8-15.2) and 10.98% (95% CI 9.8-12.2), respectively. Throughout the region, higher seroprevalence from 2013 to 2017 was observed and was highly variable with no predictable spatio-temporal pattern between districts. Seroprevalence of up to 60.8% in Sant Kabir Nagar in 2016 and 69.5% in Gorakhpur district in 2017 for IgG and IgM was observed, respectively. IgG seroprevalence did not increase with age. Monthly time-series decomposition of IgG and IgM seroprevalence demonstrated annual cyclicity (3-4 peaks) with seasonality (higher, broader peaks in the summer and monsoon periods). However, most variance was due to the overall trend and the random components of the time series. Autoregressive time-series modelling of pigs sampled from Gorakhpur was insufficiently predictive for forecasting; however, an inverse association between humidity (but not rainfall or temperature) was observed. CONCLUSIONS: Detection patterns confirm seasonal epidemic periods within year-round endemicity in pigs in eastern Uttar Pradesh. Lack of increasing age-associated seroprevalence indicates that JEV might not be immunizing in pigs which needs further investigation because models that inform public health interventions for JEV could be inaccurate if assuming long-term immunity in pigs. Although pigs are considered sentinels for human outbreaks, sufficient timeliness using sero-surveillance in pigs to inform public health interventions to prevent JEV in people will require more nuanced modelling than seroprevalence and broad climate variables alone.

Sero-positivity of Japanese Encephalitis Virus in Equine Population of India Using IgG ELISA: Unraveling the Need for Vaccination.

Japanese encephalitis (JE) is a mosquito borne flaviviral zoonoses, causing fatal disease in equines and humans. JE is endemic in most of the states of India with occurrence of human cases every year. The horses are not vaccinated against JE in India and thus they are at more risk of acquiring the disease. Due to nonavailability of indigenously developed ELISA and high cost of imported kits, regular sero-surveillance is not being carried out to assess the true picture of JE virus in equine population of India. Therefore, a recombinant NS1 protein based indirect IgG ELISA was developed with the objective to assess the sero-positivity of JE virus in equine population of India. The diagnostic sensitivity and specificity of developed ELISA was 84.73% and 86.70%, respectively. The validation studies revealed good reproducibility of ELISA with kappa value ranging from 0.75 to 1 between the results of different laboratories. A total of 2,069 horse serum samples were screened using the developed ELISA and 401 samples were positive for IgG against JEV with an overall sero-positivity of 19.38% in equine population of India. A sero-positivity of 25.90% and 12.22% was recorded in Himachal Pradesh and Jammu-Kashmir, both hill states of North zone of India for the first time, revealing the spread of virus to the nonendemic parts of the country. The high sero-positivity of JE virus recorded in equine population warrants the need for initiation of vaccination of horses in India to prevent the morbidity and mortality.

Extended-spectrum beta-lactamase (ESBL) producing and multidrug-resistant Escherichia coli in street foods: a public health concern.

Antimicrobial resistance (AMR) pattern and virulence genes of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli from foods of animal origin were evaluated. Based on combination disc method and ESBL E test, 42 of the 213 E. coli isolates were confirmed as ESBL producers where a high presence was observed in raw foods (60.62%), environmental samples (46.73%) and ready to eat foods (42.99%) of which 31(26.49%), 3(6.97%) and 7(15.21%) samples harbored ESBL E. coli, respectively. Higher contamination rates were observed in samples collected from meat vendors (54.36%), milk vendors (48.88%) and egg vendors (45.20%) of which 16.1%, 11.11% and 2.05%, respectively were ESBL E. coli. Among the 42 ESBL isolates, 85.71% (36/42) were multidrug-resistant. On polymerase chain reaction (PCR) analysis, expression of beta-lactamase genes viz., blaCTXM was noted in 69.04% (29/42) ESBL isolates, blaTEM in 66.66% (28/42) and blaOXA-1 in 19.04% (8/42) isolates, while blaSHV was not detected in any of the isolates. Other AMR genes viz., blaAmpC, sul1, sul2, tet(A), tet(B), catI, dhfrI, aac(3)-IIa(aacC2), aph(3')-Ia(aphA1), qnrB, qnrS were detected by PCR in 39, 28, 29, 3, 9, 5, 17, 11, 6, 6 and 33 isolates, respectively. None of the isolates harbored chloramphenicol (floR) and plasmid-mediated quinolone resistance (PMQR) (qnrA) genes. However, 21 isolates were positive for class I integron (int1), 5 for EPEC (eae) and 9 for ETEC (lt) while none were carrying bfp or stII genes. All ESBL producing isolates formed a single group when subjected to enterobacterial repetitive intergenic consensus (ERIC PCR) genotyping. The presence of multidrug-resistant ESBL E. coli in street foods of animal origin raises the issues of food safety and public health.

Virulent methicillin resistant Staphylococcus aureus (MRSA) in street vended foods.

Street foods are one of the important sources of foodborne infections and Staphylococcus aureus is an important infectious agent transmitted through various sources including street foods. The methicillin-resistant S. aureus (MRSA) are of public health significance, hence the study was taken to assess the street foods as a source of MRSA, for which 430 street vended foods of animal origin (meat, milk, eggs and their products) and associated environmental samples were processed for isolation and characterization. A total of 52 (12.1%) S. aureus were isolated and resistant was observed to oxacillin (36.5%), cefoxitin (25%) and penicillin G (82.7%) by disc diffusion test. On genotypic screening, mecA and blaZ have detected in 17.3% and 69.2% isolates, respectively. The virulence typing identified nuc, coa, clfA, spA, FnbA and enterotoxin A (sea) genes in 100%, 96.2%, 30.8%, 55.8, 50% and 7.7% isolates, respectively. Genetic diversity among the isolates was observed by enterobacterial repetitive intergenic consensus PCR with a D value of 0.77. The presence of virulent MRSA in street vended foods trigger the public health concern and emphasis to educate the consumers and street food vendors about quality and safety of such foods.

Antimicrobial resistance and typing of Salmonella isolated from street vended foods and associated environment.

The present study was carried out to find out the occurrence and types of Salmonella present in street vended foods and associated environment, and their resistance pattern against various antibiotics. About 1075 street vended food and associated environment samples were processed for isolation and confirmation of different Salmonella spp. by targeting gene specific invA gene and serotype specific Sdf I, Via B and Spy genes by PCR. Selected Salmonella isolates were screened for antibiotic resistance by using Baeur-Kirby disk diffusion test. Out of 1075 samples, only 31 (2.88%) isolates could be amplified the invA gene of which 19 could be recovered from meat vendors; 8 from egg vendors while remaining 4 from milk vendors. Though, majority of Salmonella recovered from raw foods the ready-to-eat food like chicken gravy and rasmalai also showed its presence which pose a serious public health threat. Overall, 19, 6 and 1 isolates of S. Typhimurium, S. Enteritidis and S. Typhi could be detected by PCR while remaining 5 isolates could not be amplified suggesting other type of Salmonella. Selected Salmonella isolates were completely resistance to Oxacillin (100%) followed by Cefoxitin (30.43%) and Ampicillin (26.10%). Thus, it is observed that the street vended foods of animal origin and associated environment play an important role in transmission of food borne pathogens including Salmonella.

Prevalence and genotypic (G and P) determination of porcine group A rotaviruses from different regions of India.

Group A rotaviruses can infect both humans and animals and have been recognized as an important cause of diarrhea in porcine. In this study, we report the prevalence and molecular epidemiology of rotaviruses detected in piglets in different regions of India. A total 275 fecal samples (180 diarrheal and 95 non-diarrheal) from piglets were collected from the western (135), southern (60), northern (20), and North-Eastern Hill (NEH) (60) regions of India and tested for rotaviruses. All the samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reverse transcription-polymerase chain reaction (RT-PCR). Rotaviruses were detected in 10.18 % of samples by SDS-PAGE and/or RT-PCR with a maximum of 30 % from the NEH region followed by 7.4 % from the western region. Samples from the southern and northern regions were found to be negative. Only 10 isolates were subjected to genotypic characterization using amplification of VP7 and VP4 genes followed by two separate multiplex PCR assays for G genotyping and another two for P genotyping using genotype-specific primers. Of these, three isolates could be typed as G4 specificity, one with G9, and three as P[6] leading to identification of an uncommon strain, G4P[6]. One isolate was further confirmed by nucleotide sequencing. The data demonstrate genetic diversity of porcine rotavirus strains and suggest that pig farms may serve as potential reservoirs for human infections.

Use of a phospholipase-C assay, in vivo pathogenicity assays and PCR in assessing the virulence of Listeria spp.

Listeria spp. was isolated from 19.8% of animals with a history of reproductive disorders. A total of 333 faecal, genital swab and blood samples from 111 animals (cattle, buffaloes, sheep and goats) were subjected to PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap) and pathogenicity testing by the phosphatidylinositol phospholipase-C (PI-PLC) assay, and by mouse and chick embryo inoculation. One isolate of Listeria ivanovii recovered from a genital swab from a sheep was found to be pathogenic. Virulence assessment was then carried out on two L. ivanovii and 29 Listeria monocytogenes isolates from various sources using these assays. Haemolytic L. monocytogenes isolates lacking the plcA gene and PI-PLC activity were deemed non-pathogenic when assessed by mouse and chick embryo inoculation tests, in spite of having the hlyA gene. The results suggested that the PI-PLC and PCR assays are reliable in vitro alternatives to in vivo pathogenicity tests for L. monocytogenes.