Enhancement of progesterone biosynthesis via kisspeptin stimulation: Upregulation of steroidogenic transcripts and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in the buffalo luteal cells.

The presence of Kisspeptin (Kp) and its receptors in the corpus luteum (CL) of buffalo has recently been demonstrated. In this study, we investigated the role of Kp in the modulation of progesterone (P4) synthesis in vitro. The primary culture of bubaline luteal cells (LCs) was treated with 10, 50, and 100 nM of Kp and Kp antagonist (KpA) alongside a vehicle control. The combined effect of Kp and KpA was assessed at 100 nM concentration. Intracellular response to Kp treatment in the LCs was assessed by examining transcript profiles (LHR, STAR, CYP11A1, HSD3B1, and ERK1/2) using quantitative polymerase chain reaction (qPCR). In addition, the immunolocalization of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the LCs was studied using immunocytochemistry. Accumulation of P4 from the culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). The results indicated that LCs had a greater p-ERK1/2 expression in the Kp treatment groups. A significant increase in the P4 concentration was recorded at 50 nM and 100 nM Kp, while KpA did not affect the basal concentration of P4. However, the addition of KpA to the Kp-treated group at 100 nM concentration suppressed the Kp-induced P4 accumulation into a concentration similar to the control. There was significant upregulation of ERK1/2 and CYP11A1 expressions in the Kp-treated LCs at 100 nM (18.1 and 37fold, respectively, p < 0.01). However, the addition of KpA to Kp-treated LCs modulated ERK1/2, LHR, STAR, CYP11A1, and HSD3B1 at 100 nM concentration. It can be concluded that Kp at 100 nM stimulated P4 production, while the addition of KpA suppressed Kp-induced P4 production in the buffalo LCs culture. Furthermore, an increment in p-ERK1/2 expression in the LCs indicated activation of the Kp signaling pathway was associated with luteal steroidogenesis.

Spatio-temporal dynamics and distributional trend analysis of African swine fever outbreaks (2020-2021) in North-East India.

African swine fever (ASF) is a highly contagious, notifiable, and fatal hemorrhagic viral disease affecting domestic and wild pigs. The disease was reported for the first time in India during 2020, resulted in serious outbreaks and economic loss in North-Eastern (NE) parts, since 47% of the Indian pig population is distributed in the NE region. The present study focused on analyzing the spatial autocorrelation, spatio-temporal patterns, and directional trend of the disease in NE India during 2020-2021. The ASF outbreak data (2020-2021) were collected from the offices of the Department of Animal Husbandry and Veterinary Services in seven NE states of India to identify the potential clusters, spatio-temporal aggregation, temporal distribution, disease spread, density maps, and risk zones. Between 2020 and 2021, a total of 321 ASF outbreaks were recorded, resulting in 59,377 deaths. The spatial pattern analysis of the outbreak data (2020-2021) revealed that ASF outbreaks were clustered in 2020 (z score = 2.20, p < .01) and 2021 (z score = 4.89, p < .01). Spatial autocorrelation and Moran's I value (0.05-0.06 in 2020 and 2021) revealed the spatial clustering and spatial relationship between the outbreaks. The hotspot analysis identified districts of Arunachal Pradesh, Assam and districts of Mizoram, Tripura as significant hotspots in 2020 and 2021, respectively. The spatial-scan statistics with a purely spatial and purely temporal analysis revealed six and one significant clusters, respectively. Retrospective unadjusted, temporal, and spatially adjusted space-time analysis detected five, five, and two statistically significant (p < .01) clusters, respectively. The directional trend analysis identified the direction of disease distribution as northeast-southwest (2020) and north-south (2021), indicate the possibility of ASF introduction to India from China. The high-risk zones and spatio-temporal pattern of ASF outbreaks identified in the present study can be used as a guide for deploying proper prevention, optimizing resource allocation and disease control measures in NE Indian states.

Differential expression of inflammatory cytokines, prostaglandin synthases and secretory leukocyte protease inhibitor in the endometrium and circulation in different graded CEH-pyometra in bitch.

Cystic endometrial hyperplasia (CEH)-pyometra (CEH-P) is one of the most common reproductive disorders in bitches, posing a risk to both future fertility and life. The aims of the current study were to elucidate the differential expression patterns of inflammatory mediators at transcript and protein levels in the endometrium and to assess the concentrations of key inflammatory mediators in the peripheral circulation of bitches with different graded CEH-P. A total of 25 client-owned intact mixed breed bitches of 3-10 years presented to the outpatient department of RVP-TVCC of the institute were considered for the study. Of which, 22 cases suggestive of pyometra and 3 cases of CEH obtained during routine elective ovariohysterectomy were subjected to histopathological examination. Uteri were categorized into CEH (n = 3), moderate CEH-P (mCEH-P, n = 9), severe CEH-P (sCEH-P, n = 6) and atrophic pyometra (AT-P, n = 7). A group of age matched (n = 12) bitches without pyometra served as control. Endometrial transcripts such as IL6, IL8, PTGS2, PGFS, and SLPI were expressed differentially in the CEH and CEH-P bitch. In addition, a strong immunoreactivity (IR) of IL6, IL8, PTGS2, and mPGES1 was recorded in the sCEH-P uterus, while expression of IL10 was noticed in AT-P. In circulation, serum IL6 was the most relevant marker with high sensitivity of 96.2% and specificity of 84.6% at a cut off concentration 8.5 pg/mL followed by SLPI with 95.2% sensitivity, and 84.6% specificity at cut off concentration of 1.3 ng/mL. Serum IL10, PGFM and SLPI concentration in the peripheral circulation were 1.5-2.23 fold higher in mCEH-P, 0.87-2.5 fold higher in sCEH-P and 2.9-3.5 fold higher in AT-P than that of control. It is concluded that monitoring the serum concentration of IL6, IL10 and SLPI would be useful adjunct to the established hematobiochemical parameters in the management of pyometra in the bitch with critical illness.

Occurrence and antimicrobial resistance of Campylobacter isolates from broiler chicken and slaughter house environment in India.

Campylobacteriosis is among the most frequently reported foodborne zoonoses. A total of 848 samples were screened for Campylobacter spp. and occurrence was found to be 8.7%, 2.3% and 1.65% in broiler cecum samples, chicken meat samples and slaughter house environment swabs, respectively. High level of antimicrobial resistance was found against tetracycline (64.1%), doxycycline (54.4%), ampicillin (46.6%), nalidixic acid (42.7%), kanamycin (35.9%), and ciprofloxacin (33.33%). Resistance to co-amoxiclav (19.4%) and erythromycin (21.4%) was less common. The MAR index of the isolates was in the range of 0.11-0.78. Multi-drug resistance was observed in 54.4% of the isolates, with 53.2% C. jejuni and 55.3% C. coli isolates found resistant against three or more classes of antimicrobials. Presence of mutations in gyrA and 23S rRNA genes was investigated, which revealed that all the fluoroquinolone resistant isolates possessed Thr-86-Ile point mutation, whereas only 68% of erythromycin resistant isolates had A2075G mutation. The tetO gene was present in 91.7% tetracycline resistant isolates and blaOXA-61 gene was detected in 97.9% of the ampicillin resistant isolates. The occurrence of antimicrobial resistant Campylobacter spp. in broiler chicken samples and slaughter house settings is a public health risk and calls for judicial use of antimicrobials.

Replication competence of canine distemper virus in cell lines expressing signaling lymphocyte activation molecule (SLAM) of goat, sheep and dog origin.

Cellular receptors play an important role in entry and cell to cell spread of morbillivirus infections. The cells expressing SLAM and Nectin-4 have been used for successful and efficient isolation of canine distemper virus (CDV) in high titre. There are several methods for generation of cells expressing receptor molecules. Here, we have used a comparatively cheaper and easily available method, pcDNA 3.1 (+) for engineering Vero cells to express SLAM gene of goat, sheep and dog origin (Vero/Goat/SLAM (VGS), Vero/Sheep/SLAM (VSS) and Vero/Dog/SLAM (VDS), respectively). The generated cell lines were then compared to test their efficacy to support CDV replication. CDV could be grown in high titre in the cells expressing SLAM and a difference of log two could be recorded in virus titre between VDS and native Vero cells. Also, CDV could be grown in a higher titre in VDS as compared to VGS and VSS. The finding of this study supports the preferential use of SLAM expressing cells over the native Vero cells by CDV. Further, the higher titre of CDV in cells expressing dog-SLAM as compared to the cells expressing SLAM of non-CDV hosts (i.e. goat and sheep) points towards the preferential use of dog SLAM by the CDV and may be a plausible reason for differential susceptibility of small ruminants and Canines to CDV.

Faecal carriage of extended spectrum beta-lactamase (ESBL) and New Delhi metallo beta-lactamase(NDM) producing Escherichia coli between piglets and pig farmworkers.

A cross-sectional study on five organized pig farms was conducted to assess the faecal carriage of ESBL and blaNDM carbapenemase-producing E. coli in piglets and pig farmworkers. Faecal samples from piglets (n = 155) and pig farmworkers (n = 21) were processed for isolation and characterization of E. coli. A total of 124 E. coli isolates from piglets and 21 E. coli isolates pig farmworkers were recovered and screening for ESBL production showed that 44.4 % (55/124) of the isolates from piglets and 42.9 % (9/21) of the isolates from farmworkers were ESBL positive. The ESBL positive isolates from piglets and farmworkers harbored blaCTX-M and also co-harbored other beta-lactams, sulphonamide, quinolone and tetracycline resistance genes. Diarrhoeic (50%, 49/98) and crossbred piglets (52.7%, 39/74) harbored a significantly higher number of ESBL producing isolates than non-diarrhoeic (23.1 %, 6/26) and purebred piglets (32%, 16/50) (p < 0.05). Piglets and pig farmworkers harbored nine and two carbapenem-resistant isolates, respectively. Interestingly, two isolates from piglets and one isolate from farmworkers harbored the blaNDM gene. The blaNDM positive E. coli isolated from piglets and farmworkers of the same farm revealed similar antibacterial resistance patterns, resistant genes, sequence (ST-167) and plasmid type (IncX3). In India, carbapenems are not used in food animal treatment, hence carbapenem resistant E. coli in piglets possibly originated from the human contact or common environment and is of public health importance.

Bluetongue in India: a systematic review and meta-analysis with emphasis on diagnosis and seroprevalence.

Bluetongue (BT) is an infectious viral disease which affects a wide range of ruminants and was first reported in India in 1964. In view of the absence of comprehensive information on the BT status in India, this study presents the seroprevalence on BT in farm animals of India based-on a systematic review and meta-analysis. A systematic review was conducted to identify the published articles (2001-2018) reporting the seroprevalence of BT in sheep, goats, cattle, buffalo, camels, and Mithun (Bos frontalis) from India. From 409 research articles, 71 fulfilled the inclusion criteria and meta-analysis for proportions was carried out targeting the eligible studies. From these, 144 strata level data were extracted with a sample size of 14048 sheep, 14696 goats, 5218 cattle, 2653 buffaloes, 2062 camels, and 222 Mithun. Overall, the analyses showed that the BT seroprevalence of 43% (95% CI: 38-49%) in goats, 39% (95% CI: 33-46%) in sheep, 38% (95% CI: 25-45%) in cattle, 34% (95% CI: 20-51%) in buffaloes, 16% (95% CI: 10-22%) in camels, and 66% (95% CI: 17-95%) in Mithun. Furthermore, the meta-regression analysis suggested that serological tests, geographical region, and sample size were the prime moderators. Meta-analytic study indicates the BT seropositivity in 25.35 million sheep (95% CI: 21.5-29.9), 58 million goats (95% CI: 51.3-66.2), 66.8 million cattle (95% CI: 47.7-86), 37.0 million buffaloes (95% CI: 21.7-55.4), 0.06 million camels (95% CI: 0.04-0.09), and 0.19 million Mithun (95% CI: 0.05-0.28). The findings highlight the variation of BT seropositivity in different geographical regions of India.

Cross-sectional study on rotavirus A (RVA) infection and assessment of risk factors in pre- and post-weaning piglets in India.

Rotavirus A (RVA) infections are known to retard the piglets' growth and minimize the profit to the pig farming community. Between August 2014 and July 2017, in a cross-sectional study, we surveyed 13 organized pig farms located in the eight states of India representing northern, north-eastern and southern regions, to identify the risk factors associated with RVA infection in pre- and post-weaning piglets. Faecal samples (n = 411) comprising of non-diarrhoeic (n = 320) and diarrhoeic (n = 91) were collected and screened for RVA infection using VP6 gene-based RT-PCR. RVA positivity of 52.5% (168/320) in non-diarrhoeic and 59.3% (54/91) in diarrhoeic piglets was noticed. Further, 53.3% (120/225) and 54.8% (102/186) of the samples from pre- and post-weaned samples were positive for RVA, respectively. To note, no statistically significant association was noticed between RVA infection, health and weaning status. Additionally, a questionnaire-based survey was conducted to identify the risk factors for RVA infections in piglets. The analysis revealed that good ventilation (OR 0.2, 95% CI 0.15-0.39), use of deep well water (OR 0.2, 95% CI 0.13-0.43) and feeding of commercial feed (OR 0.3, 95% CI 0.18-0.41) were associated with reduced risk of RVA infection compared with poor ventilation, use of shallow well water and feeding of own milled feed, respectively. Contrarily, mixed farms (OR 2.1, 95% CI 1.26-3.37), use of heater or cooler (OR 5.9, 95% CI 3.74-9.30), sheds in different elevation (OR 2.5, 95% CI 1.20-5.01) and weekly and occasional use of disinfectant for surface cleaning (OR 1.8, 95% CI 1.12-2.96) were associated with higher RVA infection. Mitigating the risk factors might help in better health management of piglets and increase the economic return to pig farming community in the country.

Risk factor analysis, antimicrobial resistance and pathotyping of Escherichia coli associated with pre- and post-weaning piglet diarrhoea in organised farms, India.

A cross-sectional study was conducted from 2014 to 2017 in 13 organised pig farms located in eight states of India (Northern, North-Eastern and Southern regions) to identify the risk factors, pathotype and antimicrobial resistance of Escherichia coli associated with pre- and post-weaning piglet diarrhoea. The data collected through questionnaire survey were used to identify the risk factors by univariable analysis, in which weaning status, season, altitude, ventilation in the shed, use of heater/cooler for temperature control in the sheds, feed type, water source, and use of disinfectant, were the potential risk factors. In logistic regression model, weaning and source of water were the significant risk factors. The piglet diarrhoea prevalence was almost similar across the regions. Of the 909 faecal samples collected (North - 310, North-East - 194 and South - 405) for isolation of E. coli, pathotyping and antibiotic screening, 531 E. coli were isolated in MacConkey agar added with cefotaxime, where 345 isolates were extended spectrum ß-lactamase (ESBL) producers and were positive for blaCTX-M-1 (n = 147), bla TEM (n = 151), qnrA (n = 98), qnrB (n = 116), qnrS (n = 53), tetA (n = 46), tetB (n = 48) and sul1 (n = 54) genes. Multiple antibiotic resistance (MAR) index revealed that 14 (2.64%) isolates had MAR index of 1. On the virulence screening of E. coli, 174 isolates harboured alone or combination of Stx1, Stx2, eaeA, hlyA genes. The isolates from diarrhoeic and post-weaning samples harboured higher number of virulence genes than non-diarrhoeic and pre-weaning. Alleviating the risk factors might reduce the piglet diarrhoea cases. The presence of multidrug-resistant and ESBL-producing pathogenic E. coli in piglets appears a public health concern.

Spatial molecular epidemiology of carbapenem-resistant and New Delhi metallo beta-lactamase (blaNDM)-producing Escherichia coli in the piglets of organized farms in India.

AIM: A cross-sectional study was conducted in 10 government-organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli. METHODS AND RESULTS: In this study, fecal sample (n = 673) from non-diarrheic (n = 501) and diarrheic (n = 172) piglets were processed for isolation of carbapenem resistant E. coli. Of 673, E. coli isolate (n = 112) was genotyped for confirming the carbapenem resistance and associated virulence factors. Of the 112 isolates, 23 were phenotypically resistant to carbapenem and 8 were carrying the New Delhi metallo beta-lactamase (blaNDM) gene. The carbapenem-resistant isolates also produced extended spectrum beta-lactamases and were multidrug resistant. The PCR-based pathotyping revealed the presence of stx1, stx2, eae and hlyA genes. The enterobacterial repetitive intergenic consensus PCR dendrogram analysis of the isolates yielded three distinct clusters. The statistical analysis revealed no association between carriages of carbapenem-resistant E. coli in different breed of piglets however, location, sex, health status of piglets and age showed significant difference. The spatial analysis with SaTScan helped in identification of carbapenem-resistant clusters. CONCLUSIONS: The presence of carbapenem resistant E. coli isolates with virulence genes in the piglet poses a potential public health risk through possible access and spread via the food chain and environment. Efflux pump may also play an important role in carbapenem resistance in piglet E. coli isolates. Furthermore, identification of risk factors in relation to spatial clusters will help in designing preventive strategies for reducing the risk of spread of carbapenem resistant bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: 1. Piglets harbor carbapenem resistant E. coli and have great public health significance. 2. Apart from carbapenemase, efflux pump is also important for carbapenem resistance. 3. This is the first report of blaNDM in the piglets from India.

First Complete Genome Sequence of Genotype III Japanese Encephalitis Virus Isolated from a Stillborn Piglet in India.

We report here the first complete genome of the Japanese encephalitis virus (JEV) genotype III strain JEV/SW/IVRI/395A/2014, isolated from stillborn piglets in India. It shares 99% identity with strain JaOArS982 and a few other strains from Japan.

Apparent seroprevalence, isolation and identification of risk factors for brucellosis among dairy cattle in Goa, India.

Brucellosis is a highly contagious zoonotic infection affecting livestock and human beings. The disease has been reported worldwide except in few countries where it has been eradicated. The prevalence of brucellosis among cattle from 11 farms having a history of abortions was studied. A total of 481 samples comprising of blood, milk, vaginal swabs, vaginal discharges, placental tissues and fetal tissues were collected from 296 animals. Clinical samples were processed for the isolation of Brucella. Serum samples (n=296) were tested by Rose Bengal Plate Test (RBPT) and indirect ELISA. A total of 90 (30.40%) and 123 (41.55%) samples were positive by RBPT and indirect ELISA, respectively. Also 27.02% samples were positive by both the tests. Brucella isolates (n= 8) were recovered from clinical samples using Brucella selective media. All the isolates demonstrated PCR amplification for the bcsp31 and IS711 genes. Amplification of Brucella abortus specific primer was demonstrated by all the isolates in AMOS PCR indicating isolates to be of either B. abortus biotype 1, 2 or 4. Risk factors for transmission of brucellosis among cattle population were studied by field surveys. It was observed that lack of awareness about brucellosis (OR=8.739, P=0.138) and inadequate floor space (OR=0.278, P=0.128) were crucial risk factors for transmission of bovine brucellosis.

Sanitizing effect of salts on experimentally inoculated organisms on pork carcasses.

Pork forequarters procured from freshly slaughtered animals were decontaminated with hot water and inoculated with Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes, Serratia marcescens, Pseudomonas aeruginosa and Proteusvulgaris. The forequarters were individually spray washed with 5% potassium sorbate and a combination of 5% sodium chloride and 2.5% each of sodium acetate, sodium citrate, sodium lactate and potassium sorbate solutions. The total viable count (TVC) of the treated meat samples was reduced by 0.96 and 1.31 log units by spraying with salt and salt combination respectively with marginal changes in colour and odour scores. Inoculated organisms were found to be highly sensitive to salt combination treatment as compared to potassium sorbate alone. Shelf-life of salt and salt combination treated samples was increased to 8 and 11days as against 4days in untreated samples. Carcass washing with salt and salt combination was found to be suitable for extension of shelf-life and improvement in the sensory and microbiological quality of meat.

Effect of food grade organic acids on inoculated S. aureus, L. monocytogenes, E. coli and S. Typhimurium in sheep/goat meat stored at refrigeration temperature.

Sheep/goat forequarters procured from freshly slaughtered animals were decontaminated with hot water and inoculated with Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium. The forequarters were individually spray washed with 2% lactic acid and 1.5% acetic + 1.5% propionic acid combination. Total viable count (TVC) of the treated meat samples was reduced by about 0.52 and 1.16 log units with marginal changes in colour and odour scores. Inoculated organisms were found to be highly sensitive to acid combination treatment as compared to lactic acid alone. Shelf-life of acid and acid combination treated samples was increased to 8 and 11 days as against 3 days in untreated samples. Carcass washing with acid alone or acid combination was found to be suitable for extension of shelf-life and improvement in the sensory and microbiological quality of meat.