Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Kisspeptin stimulates oestradiol biosynthesis by upregulating steroidogenic transcripts and proliferation markers in the bubaline granulosa cells in vitro.

Kisspeptin (Kp), an upstream regulator of GnRH release, is essential for the development and function of reproductive axis. Previously, we demonstrated the localization of Kp and its receptor (Kiss1r) in the active follicle in the bubaline ovary. Present study aimed to determine the effect of Kp on granulosa cell (GCs) functions, especially oestradiol (E2 ) and progesterone (P4 ) production, and differential expression of genes regulating the proliferation, apoptosis and steroidogenesis in the buffalo. The ovaries with 6-10 mm size follicles obtained from the cyclic buffaloes after slaughtering were used for isolation of GCs for in vitro study. The primary GCs culture was treated with Kp (0, 10, 50 and 100 nM) and incubated for 48 h. Production of E2 and P4 was estimated in the culture supernatant by ELISA. The expression of gonadotropin receptors (FSHR and LHR), steroidogenic genes (STAR, 3ß-HSD, CYP19A1), proliferation marker (PCNA), apoptotic factors (CASP3 and BCL2) and Kp signalling molecule (extracellular signal-regulated kinase 1/2, ERK1/2 and p-ERK1/2) was studied in the GCs by qPCR. Significant E2 production was found in the Kp 50 and 100 nM groups (p < .05), whereas P4 production was reduced in Kp 100 nM group (p < .05). There was concomitant upregulation of FSHR, ERK1/2, STAR and CYP19A1 in the Kp 100 nM treated GCs. In addition, Kp at 100 nM stimulated the proliferation of GCs by upregulating the expression of BCL2 (5.0 fold) and PCNA (94.9 fold). Further, high immunoreactivity of p-ERK1/2 was observed in the Kp-treated GCs. It was concluded that Kp at 100 nM concentration stimulated E2 production by upregulating the steroidogenic pathway through ERK1/2, STAR and CYP19A1 and modulating PCNA and BCL2 expressions in the GCs. Further experiments are warranted using Kp antagonist in different combinations to establish the signalling pathway in Kp-mediated steroidogenesis in the GCs for developing strategies to control ovarian functions.

Serum concentrations of secretory leukocyte protease inhibitor and IL6 can predict the onset of sepsis in pyometra bitches.

As onset of sepsis adversely affects the prognosis of canine pyometra, finding biomarkers that would distinguish sepsis status would be useful in the clinical management. Accordingly, we hypothesized that differential expression of endometrial transcripts and circulating concentration of certain inflammatory mediators would discriminate pyometra-led sepsis (P-sepsis+) from those of pyometra without sepsis (P-sepsis-). Bitches with pyometra (n = 52) were classified into P-sepsis+ (n = 28) and P-sepsis- (n = 24) based on vital clinical score and total leukocyte count. A group of non-pyometra bitches (n = 12) served as control. The relative fold changes in the transcripts of IL6, IL8, TNFα, IL10, PTGS2, mPGES1 and PGFS, SLPI, S100A8, S100A12 and eNOS were determined by quantitative polymerase chain reaction. Furthermore, the serum concentrations of IL6, IL8, IL10, SLPI and prostaglandin F2α metabolite (PGFM) were assayed by ELISA. The relative fold changes in S100A12 and SLPI and mean concentrations of IL6 and SLPI were significantly (p < .05) higher in P-sepsis+ than that of P-sepsis- group. Receiver operating characteristic analysis revealed that serum IL6 had a diagnostic sensitivity of 78.6% and a positive likelihood ratio (LR+) of 2.09, at a cut-off value of 15.7 pg/mL to diagnose P-sepsis+ cases. Similarly, serum SLPI had a sensitivity of 84.6% and an LR+ of 2.23, at a cut-off value of 2.0 pg/mL. It was concluded that SLPI and IL6 would serve as putative biomarkers for pyometra-led sepsis in bitches. Monitoring SLPI and IL6 would be a useful adjunct to the established haemato-biochemical parameters in customizing the treatment strategies and arriving at the decision for management of pyometra bitches with critical illness.

Analysis of bluetongue disease epizootics in sheep of Andhra Pradesh, India using spatial and temporal autocorrelation.

Bluetongue (BT) disease poses a constant risk to the livestock population around the world. A better understanding of the risk factors will enable a more accurate prediction of the place and time of high-risk events. Mapping the disease epizootics over a period in a particular geographic area will identify the spatial distribution of disease occurrence. A Geographical Information System (GIS) based methodology to analyze the relationship between bluetongue epizootics and spatial-temporal patterns was used for the years 2000 to 2015 in sheep of Andhra Pradesh, India. Autocorrelation (ACF), partial autocorrelation (PACF), and cross-correlation (CCF) analyses were carried out to find the self-dependency between BT epizootics and their dependencies on environmental factors and livestock population. The association with climatic or remote sensing variables at different months lag, including wind speed, temperature, rainfall, relative humidity, normalized difference vegetation index (NDVI), normalized difference water index (NDWI), land surface temperature (LST), was also examined. The ACF & PACF of BT epizootics with its lag showed a significant positive autocorrelation with a month's lag (r = 0.41). Cross-correlations between the environmental variables and BT epizootics indicated the significant positive correlations at 0, 1, and 2 month's lag of rainfall, relative humidity, normalized difference water index (NDWI), and normalized difference vegetation index (NDVI). Spatial autocorrelation analysis estimated the univariate global Moran's I value of 0.21. Meanwhile, the local Moran's I value for the year 2000 (r = 0.32) showed a high degree of spatial autocorrelation. The spatial autocorrelation analysis revealed that the BT epizootics in sheep are having considerable spatial association among the outbreaks in nearby districts, and have to be taken care of while making any forecasting or disease prediction with other risk factors.

Seasonal pattern in occurrence of rotavirus infection (RV) in diarrheic children, calves and piglets from Bareilly, India.

Rapid and reliable diagnosis for diarrhoeal disease is critically important for the differentiation of etiological agents and subsequent suitable treatment modalities. The objective of the study is to reveal the seasonal pattern in the occurrence of rotavirus in diarrheic children, calves and piglets from Bareilly, Uttar Pradesh, India. A total of 115 diarrhoeal samples were collected, out of which 51 were collected during post-monsoon/autumn (September 2018-November 2018) and 64 during the winter season (December 2018-February 2019). The samples were collected from children <5 years (n = 50), piglets <3 months (n = 35) and calves <6 months of age (n = 30). These samples were screened by ribonucleic acid-polyacrylamide gel electrophoresis (RNA-PAGE) and reverse transcriptase-polymerase chain reaction (RT-PCR) by targeting the VP6 gene of rotavirus A (RVA) and the two were compared. In RNA-PAGE 29.4% (5/17), 6.3% (1/16) and 0% (0/18) samples collected from children, calves and piglets, respectively were rotavirus positive during the autumn season while 45.5% (15/33), 21.4% (3/14) and 17.7% (3/17) samples in the winter season. In RT-PCR, 41.2% (7/17), 12.5% (2/16) and 0% (0/18) samples were rotavirus positive in the autumn season while 51.5% (17/33), 28.6% (4/14) and 29.4% (5/17) samples in winter season collected from children, calves and piglets, respectively. On statistical analysis, no significant difference between the season and number of positives in children and calves (p > 0.05) was observed, however in piglets significantly higher number of RVA positives were detected in the winter season than autumn (p < 0.01). The diagnostic test comparison of RNA-PAGE and RT-PCR showed no statistically significant difference in detecting the RVA positives (p > 0.05). Overall the percent positivity showed a seasonal pattern with higher positivity in winter as compared to autumn season.

Molecular characterization of porcine rotavirus A from India revealing zooanthroponotic transmission.

Rotaviruses A (RVA) are leading causes of diarrhea and dehydration in piglets and imply great economic loss to the pig farming community. In this study, the porcine RVA genotypes circulating in western and northern parts of India were determined by screening 214 fecal samples from diarrheic (n = 144) and non-diarrheic (n = 70) pigs. Subsequently, the structural (VP4 and VP7) and nonstructural (NSP3, and NSP4) genes were amplified, sequenced, and genetically characterized. The RVA positivity percentage was 7.94% (17/214) by RNA-PAGE and 10.28% (22/214) by RT-PCR. Higher RVA positivity was observed in samples from Uttar Pradesh (24.07%) followed by Maharashtra (6.77%) and Goa (2.38%). The sequence and automated genotyping software analysis confirmed the circulation of G4P[6] and G9P[13] RVA strains in porcine population. To note, the sequence similarity of the VP7 gene of Porcine/INDIA/RVA/PK-13 IVRI/Maharashtra/G4 and Porcine/INDIA/RVA/P-8/IVRI/U.P./G9 strain showed a relationship of 96.83 and 98.89% at the nucleotide level with human RVA strains indicating inter-species transmission. Additionally, the NSP3 (T1) and NSP4 (E1) genes (genotypes) also showed genetic relatedness with human RVA strains. Overall, the nucleotide sequences of VP7, NSP3, and NSP4 genes of porcine RVA indicate zooanthroponotic transmission. Further, we report the detection of G9P[13] RVA strain in porcine for the first time from India.HIGHLIGHTSRVA positivity was 7.94% (17/214) by RNA-PAGE and 10.28% (22/214) by RT-PCRThe RVA strain G9P[13] reported for the first time in Indian pigletsVP7, NSP3 and NSP4 genes analysis of porcine RVA showed genetic relatedness with human strains indicating evidence of zooanthroponotic transmission.

Responses to COVID-19 in South Asian Association for Regional Cooperation (SAARC) countries in 2020, a data analysis during a world of crises.

Coronavirus disease (COVID-19) caused by SARS-CoV-2 was notified from Wuhan city, Hubei province, China in the mid of December 2019. The disease is showing dynamic change in the pattern of confirmed cases and death toll in these low and middle-income countries (LMICs). In this study, exponential growth (EG) method was used to calculate the real-time reproductive number (Rt) for initial and later stage of epidemic in South Asian Association for Regional Cooperation (SAARC) member countries (April 2020 - December 2020). Time dependent (TD) method was used to calculate the weekly real -time reproduction number (Rt). We also presented the observations on COVID-19 epidemiology in relation with the health expenditure, poverty, BCG vaccination, literacy population density and Rt for understanding the current scenario, trends, and expected outcome of the disease in SAARC countries. A significant positive correlation was noticed between COVID-19 deaths and health expenditure (% GDP) (r = 0.58, P < 0.05). The other factors such as population density/sq km, literacy %, adult population %, and poverty % were not significantly correlated with number of COVID-19 cases and deaths. Among SAARC countries, the highest Rt was observed in India (Rt = 2.10; 95% CI 2.04-2.17) followed by Bangladesh (Rt = 1.62; 95% CI 1.59-1.64) in initial state of epidemic. A continuous monitoring is necessitated in all countries looking at the medical facilities, available infrastructure and healthcare manpower, constraints which may appear with increased number of critically ill patients if the situation persists longer.

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.

Genotypic determination of human group A rotaviruses from Goa and Meghalaya states, India.

INTRODUCTION: Rotavirus is the leading cause of diarrhoea in young children in India, responsible for an estimated 21357 mean numbers of deaths in 2010. Various genotypes of rotaviruses evolved due to mutational changes have been recognized. In this study, we determined the genotypes of rotaviruses involved in diarrhea in Goa and Meghalaya states of India. METHODS: The dsRNA of rotaviruses was extracted from stool samples and detected by Ribonucleic Acid-Polyacrylamide gel electrophoresis (RNA PAGE) and Reverse transcription-polymerase Chain Reaction (RT-PCR) targeting the partial VP7 gene. The full length VP7 and partial VP4 genes of rotavirus strains were amplified by RT-PCR followed by nucleotide sequencing. The RotaC classification tool was used to determine the genotypes. RESULTS: The positivity of rotavirus by PAGE and RT-PCR was observed to be 43.10% and 39.65% in Goa and 38% and 36% in Meghalaya, respectively. Though long electrophoretic profile was appeared to be the most predominant rotavirus type in circulation in these two states, 96% of long and 84.61% short electropherotype profiles could be detected by RT-PCR. The dsRNA of rotavirus extracted from 36 samples could be transcribed and amplified by beg9end9 primers for G genotyping, while, 41 by con3con2 primers for P genotyping. G1P[8] and G1P[6] genotypes were commonly circulated in Goa and G1P[8] and G1P[4] genotypes in Meghalaya. On nucleotide analysis, 6 samples from Goa showed G1 genotype specificity, while, 3 showed P[8] specificity indicating the G1P[8] rotavirus circulating in Goa. In Meghalaya state, 3 strains showed P[8] and 2 showed P[4] genotype specificity. The majority of the G and P genotypes were closely related to each other and G1 genotypes appeared in two separate clusters, while, P[8] and P[4] appeared in the respective clusters. CONCLUSION: The circulation of G1P[8], G1P[6] genotypes in Goa and the presence of G1P[8] and G1P[4] genotypes in Meghalaya was observed.

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.

Japanese encephalitis virus-induced neuropathology in mouse model infected through the conjunctival route.

Background & objectives: Mouse is a preferred animal model for studying pathogenesis of Japanese encephalitis virus (JEV) infections, and different routes of inoculation have been tried. Some neurotropic viruses can reach the brain following infection through ocular route. This study was undertaken to establish JEV-induced clinical disease in mouse model through conjunctival route and document the neuropathological effects. Methods: Ten two-week old Swiss albino mice were inoculated with 5 µl Vero cell cultured virus containing 104.7 TCID50 JEV through conjunctival route. Clinical signs of mice were observed twice daily. After necropsy examination, different organs including eyes and olfactory bulbs were collected for histopathological examination, quantification of viral copy number and antigen by real-time TaqMan assay and immunohistochemistry, respectively. Results: Infected mice showed characteristic clinical signs of JE by 4 days post-infection (dpi). Histopathological lesions in brain included perivascular cuffing by mononuclear cells, focal gliosis, necrosis of neurons and neuronophagia and astrocytosis in the cerebrum, cerebellum and the brainstem. JEV viral load was highest in the brain followed by intestine, heart, liver, spleen, lung and kidney. JEV antigen was detected in the bipolar and ganglion cells of the retina and in the mitral cells and periglomerular cells of olfactory bulb and other parts of the brain. Interpretation & conclusions: JEV infection in mice through conjunctival route produced characteristic clinical signs of the disease and neuropathological lesions. Demonstration of JEV antigen in association with neuropathological lesions in the central nervous system and neuronal cells of the eye showed that conjunctival route could be an effective alternate route for virus invasion into the brain. These findings have biosafety implications for researchers, veterinary practitioners and pig farmers.

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.

Human brucellosis among pyrexia of unknown origin cases and occupationally exposed individuals in Goa Region, India.

BACKGROUND: Brucellosis is a widespread zoonotic infection. This disease is endemic in many parts of Asia, including India. Brucellosis is a major cause of pyrexia of unknown origin (PUO). Persons exposed to infected animals or contaminated animal products are at high risk. Seropositivity among animal handlers, veterinarians and dairy workers has been documented in India. Thus, the present study was aimed to determine prevalence of brucellosis among PUO cases and occupationally exposed individuals. METHODS: In this study, serum samples (n=282) from cases of pyrexia of unknown origin (PUO) (n=243), and occupationally exposed individuals (n=39) were collected and tested for brucellosis by Rose Bengal plate test (RBPT), serum agglutination test (SAT), indirect ELISA, IgG and IgM ELISA. Blood culture for isolation of Brucella was performed for 10 serologically positive patients using BACTEC 9050 automated blood culture system. Biochemical tests and PCR techniques were used for confirmation of the isolates. RESULTS: Of the samples tested, 4.25%, 3.54%, 6.02% and 4.96% samples were positive by RBPT, SAT, indirect ELISA and IgG ELISA, respectively. None of the sample was positive for IgM ELISA. Of the 10 blood samples cultured bacteriologically, one Brucella isolate was recovered. The isolate was confirmed as Brucella abortus. Amplification of the bcsp31 and IS711 genes was also observed. CONCLUSION: Seropositivity for brucellosis was observed among PUO cases, animal handlers and dairy workers in Goa, India. The serological tests showed variable results. One Brucella isolate was obtained by performing blood culture. Confirmation of the case was done rapidly using molecular tools. General awareness about clinical symptoms should be increased which will improve proper diagnosis within short time frame.

Evaluation of recombinant outer membrane protein based vaccine against Salmonella Typhimurium in birds.

Food-borne diseases caused by Salmonella enterica from poultry sources represent an important public health problem and no reliable control by vaccination has proved effective despite research. The aim of the present study was to evaluate the use of recombinant OmpC protein for immunization of birds to elucidate its protection against virulent Salmonella Typhimurium. The recombinant OmpC protein was prepared after cloning and expressing ompC gene and was characterized by SDS-PAGE and Western blot analyses. The protein preparations were tested as vaccine candidate in layer birds by comparing the immune response, protection and organ clearance against crude lysate and control. The biologically functional recombinant 43 kDa truncated OmpC protein proved to be a good immunogen which induced a significantly high humoral immune response than control. At the same time, it primed a stable cell-mediated immune response. A protective index (based on faecal shedding of organism) of rOmpC based preparations ranged between 50 and 75% as observed for 3 weeks after challenge. Therefore, the protein preparations conferred satisfactory protection against challenge infections with virulent strains of S. Typhimurium as evidenced by limited faecal shedding and minimal detection of Salmonella from edible tissues and eggs. These findings suggest the possibility to explore the use of S. enterica OMP protein for the production of novel vaccine.

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.