Molecular epidemiology, antibiogram profile and risk factor analysis of pathogenic Escherichia coli associated with pre-weaning diarrhoea in piglets from Haryana, India.

BACKGROUND: Piglet diarrhoea is a multifactorial disease with serious implications for the swine industry worldwide, including India. The Escherichia coli (E. coli) pathotypes, i.e., enterotoxigenic E. coli (ETEC) and Shiga toxin-producing E. coli (STEC) are among the major bacterial agents attributed as causative agent for piglet diarrhoea, but studies related to genetic diversity, antibiogram profile and their correlation with risk factors of these pathogens are sparse. MATERIAL AND METHODS: A total of 104 faecal swab samples were collected from 32 different piggery units of Haryana, India and confirmed as E. coli by standard microbiological methods. The identified E. coli were characterized as ETEC and/or STEC using PCR assays and were studied for their genetic diversity by phylogenetic analysis of the sequences. All the isolates were subjected to antimicrobial susceptibility testing. Further, the correlation of variables with presence or absence of ETEC and/or STEC was also investigated by using Fisher's exact test. RESULTS: Microbiological isolation led to identification of 208 E. coli isolates. A total of 17.3% (31/208) isolates were characterized as ETEC and 4.8% (10/208) isolates as STEC, whereas 2.4% (5/208) isolates exhibited both ETEC and STEC pathotype. Of the total studied piggery units (n = 32), ETEC were isolated from fourteen and both ETEC and STEC from eight farms. The phylogenetic analysis of Stx2 gene revealed 100% homology with Stx2eA variant from Germany, while analysis of STII gene revealed a distinct nucleotide and amino acid substitution when compared with standard strains. The antibiotic susceptibility testing revealed maximum resistance to moxifloxacin (71.9%) followed by tetracycline (58.1%) and amoxicillin with a total of 41.8% (87/208) E. coli isolates designated as multi-drug resistant (MDR). The multiple antibiotic resistance index varied from 0.05 to 0.75. The statistical analysis suggested three factors viz., number of farm worker(s), frequency of using disinfectant for floor cleaning and use of antibiotic in feed as risk factors significantly associated (p < 0.05) with ETEC associated diarrhoea at piggeries under study. CONCLUSION: Current study warrants a need for systematic studies on the ETEC/STEC associated diarrhea and antibiotic resistance among these isolates to understand the mechanisms of origin and dissemination of drug resistant pathogens and to design suitable prevention and control measures to curb emergence of antibiotic resistance in the farm settings.

First Molecular Confirmed Outbreak of Malignant Ovine Theileriosis in Sheep from North India.

CONTEXT: Malignant ovine theileriosis is a tick-borne disease of sheep and goats, caused by protozoan Theileria lestoquardi. The disease has serious economic implications for small ruminant production around the world. METHODS: An outbreak of malignant ovine theileriosis in a sheep flock was investigated from Hisar district of Haryana, India, in March 2022. The etiological agent was identified using polymerase chain reaction assay with genus specific primers targeting 18S rRNA gene and subsequently confirmed by sequencing. RESULTS: The morbidity, mortality and case fatality rate reported in the outbreak were 22.2, 18.8 and 85%, respectively. The phylogenetic analysis clustered the present study T. lestoquardi isolate in the same clade with T. lestoquardi from Iraq, Iran and Pakistan with maximum nucleotide identity of 99.37% with strains from Iraq. The tick vector Hyalomma anatolicum recovered from dead animals was implicated in the disease's transmission. CONCLUSIONS: Malignant ovine theileriosis resulted in high case fatality rate. This study presents the first molecularly confirmed outbreak of malignant ovine theileriosis outbreak in the North Indian region, with characteristic post-mortem findings.

uspA gene-based phylogenetic analysis and antigenic epitope prediction for Escherichia coli strains of avian origin.

Pathogenic Escherichia coli (E. coli) is responsible for various local and systemic infections in animal and human populations. Conventional methods for the detection and identification of E. coli are time-consuming and less reliable for atypical strains. The uspA gene has been widely used as a target for the detection of E. coli. The present study was aimed at phylogenetic analysis of the uspA gene sequences to determine the evolutionary relationships between the strains and other members of the Enterobacteriaceae family. In addition, the unique differences in the sequences of the current study with Salmonella and Shigella species were tested using Tajima's molecular clock test. Antigenic epitope prediction was performed to locate the B-cell epitope region of the UspA protein. Two E. coli isolates of avian origin and strains from the National Center for Biotechnology Information (NCBI) database were used for prediction. The Immune Epitope Database (IEDB) server, Bepitope, ABCpred, SVMTrip, and ElliPro server were used to identify B-cell epitopes. The 3D structure was predicted using SWISS-MODEL. Phylogenetic analysis of the isolates from the current study revealed that both OM837340 and OM837341 sequences from the current study had maximum nucleotide homology (nt) of 99.87%-100% with E. coli isolates and minimum nt homology of 84.08% with Salmonella enteritidis and S. Hissar. The isolates in the current study had a homology of 98.87%, while the homology with Shigella species was 99.25%. Seven silent mutations were observed in the coding region of the UspA protein of ECO9LTBW (current study). Modeling of the UspA protein revealed a maximum homology of 67.86% with the Protein Data Bank in Europe (PDBe), also validated by the Ramachandran plot. No significant differences were found in the coding regions of uspA of Salmonella, Shigella, and E. coli with Tajima's test. For the E. coli isolates, a total of 24 linear B-cell and seven discontinuous epitopes were predicted using in-silico analysis. When the results of the predicted peptides were compared, two peptides, namely ARPYNA and YSDLYTGLIDVNLGDMQKRISEE, were found suitable candidates. In conclusion, the uspA gene appears to be conserved among E. coli isolates and can be used for molecular detection.

Identification and assessment of stress and associated stressors among veterinary students in India using a cross-sectional questionnaire survey.

Background: Veterinary education, is a rigorous professional training program, which exposes students to significant academic and non-academic pressures. The identification of stressors and stress levels among veterinary students mighty help the designing and implementation of coping strategies to protect the students' mental health. Methods: A 44-item based cross-sectional questionnaire survey was prepared and disseminated among veterinary students in India to identify the stressors responsible, measure the amount of stress, and relate stress to characteristics like gender, degree year, and family income. A total of n = 611 veterinary students across 14 states including 27 colleges/universities participated in the study. The collected data was evaluated for sampling adequacy, construct validity, and reliability using a set of statistical tests. Results: The analysis revealed high sampling adequacy with a KMO value of 0.957 and a highly significant anti-image correlation (p < 0.001). The principal component analysis generated six factors or subscales which effectively explained 51.98% of the variance in the data, depicting high construct validity. The Cronbach's alpha value of 0.957 revealed high internal consistency for the questionnaire. Analysis revealed more than 94% of pupils under stress, with levels ranging from moderate to severe. Academic-related stressor (95.58%) was the leading cause of overall stress in the present study followed by inter- and intrapersonal and career related-stressors (93.12%) and exams and evaluation-related stressor (90.99%). In comparison to male students, female students reported significantly higher levels of overall stress, academic stress, and intrapersonal and interpersonal stress (p < 0.001) using Chi-square. The students from lower-income families experienced significantly higher overall stress as well as stress due to family responsibilities (p < 0.001). The first-year undergraduate students reported significantly higher (p < 0.001) stress due to family responsibilities-related stressors whereas second-year students due to social activities-related stressors. The hierarchal regression model predicted that gender, family income, academic-related stressors, inter- and intrapersonal and career-related stressors, and social activities-related stressors can be employed to evaluate overall stress among students, as they ensured the maximum variance in the data (p < 0.001). Conclusions: To the best of our knowledge, this is the first Indian study to identify stressors, quantify associated stress and predict major attributes to be targeted in future studies for veterinary students.

Attitude, Opinions, and Working Preferences Survey among Pet Practitioners Relating to Antimicrobials in India.

The indiscriminate usage and overuse of antimicrobials in pets or companion animals are underlying causes of antimicrobial resistance (AMR). Despite the multi-faceted global challenge presented by antimicrobial resistance, very few studies have appraised pet practitioners' factors, such as written policy on antimicrobials, dose rate prescribed, use of critically important antimicrobials, and antimicrobial prescription in clean surgical procedures, which can contribute to AMR. In the present study, an online cross-sectional survey among randomly selected pet practitioners (n = 104) of various Indian provinces and union territories was conducted using a questionnaire comprising 33 closed-ended questions on different parameters, viz., the dosage regimen and level of compliance towards guidelines of the World Health Organization (WHO), other relevant veterinary associations, and their opinion while prescribing antimicrobials. Almost every practitioner of the 104 respondents had revealed the difficulties with owner compliance; i.e., incomplete course of the antibiotics, inappropriate follow-ups, and improper care of the sick animals. The majority of practitioners (95%) reported self-prescription of antimicrobials by the owner before presenting the pet(s) to the veterinary clinic, whereas more than half of the respondents (64%) revealed unavailability of antibiogram facilities. Furthermore, a large number (76%) of practitioners stated empirical treatment based on their experience as the main criteria for antimicrobial choice in the absence of timely results from the laboratory. Although non-necessitated use of antimicrobials in clean surgical procedures has been claimed, surprisingly, the majority of pet practitioners (97%) reported their use to reduce the post-operative complications. The use of the highest priority, critically important antimicrobials (HPCIA) listed by the WHO for humans, particularly quinolones and third-generation cephalosporin, also has been reported for different infections. The treatment durations were nearly as per the recommended guidelines issued by the Danish Small Animal Veterinary Association (DSAVA) for different ailments. Analysis using chi-square tests exhibited a significant correlation between less experienced veterinarians (less than 5 years) and prescription of antimicrobials restricted for critically important infections in human medicine. However, there seems to be no association between the experience of the practitioner and the further studied parameters, namely, antimicrobial regimen prescription, weighing the animals before prescription, dose rate calculation, and antimicrobial selection and use after clean surgical operations. The findings suggest periodic awareness campaigns among practitioners regarding the implementation of the official guidelines, the need for systematic surveillance of AMR, awareness among pet owners about antimicrobial resistance, and the importance of rational use of antimicrobials on their pets.

Epidemiology, clinical features, and molecular detection of orf virus in Haryana (India) and its adjoining areas.

Orf is an acute, highly contagious, and economically important viral disease of small ruminants. In this study, six orf suspected outbreaks among goats and sheep were investigated from Haryana state and adjoining areas of Rajasthan state during the year 2021. The disease was diagnosed on the basis of clinical signs and molecular identification. The causative agent of the disease, orf virus (ORFV), was confirmed using polymerase chain reaction (PCR) targeting immunodominant envelope antigen (B2L) gene and confirmed by sequencing. The morbidity in goats ranged from 8.75 to 100%, whereas in sheep, it ranged from 0 to 8%. The higher mortality was observed among flocks with mixed infections of orf and peste des petits (PPR) or orf and haemonchosis as compared to other outbreaks. The phylogenetic analysis of sequenced PCR products clustered the current study strains in the same clad with Indian as well as strains from other countries with nucleotide identity more than 99%, signifying a close genetic relationship. The study highlighted the circulation of strains of a single cluster among sheep and goats in Haryana and adjoining areas. Prompt diagnosis of the disease is highly important for facilitating the implementation of control measures to minimize the losses suffered by small and marginal farmers in this region. Further detailed studies are required to delineate the molecular details of ORFV for better understanding the dynamics and molecular epidemiology of strains circulating in the country and for designing the effective vaccines against the disease which are currently lacking in the country.

Characterization and antimicrobial susceptibility of biofilm-producing Avian Pathogenic Escherichia coli from broiler chickens and their environment in India.

Avian pathogenic Escherichia coli (APEC) is responsible for colibacillosis in poultry. APEC remains a constant problem for the poultry industry, despite the use of antimicrobials and disinfectants in farms. The endemicity of APEC in poultry farms is associated with its biofilm-forming ability, which is further aggravated by various virulence factors and resistance to multiple drugs that help bacteria to thrive under different environmental conditions. To characterize APEC from affected broiler chickens and their environments, samples (n=114) from dead birds (heart, liver, lungs, and cloacal swab) and surrounding environments such as feeder, drinker, litter, PVC pipe, water tank wall, feed, and water were collected. The collected samples were subjected to microbial isolation using MacConkey Lactose agar (MLA) and Eosin Methylene Blue agar (EMB), which led to the isolation of 62 E. coli isolates. This was confirmed by uspA gene amplification and Vitek 2 Compact. These isolates were characterized using a set of five virulence genes (hlyF, ompT, iroN, iss, iutA), which yielded 47 (75.80%) isolates as APEC and the remaining as non-APEC. Furthermore, all the 62 isolates were subjected to microtiter plate assay for biofilm detection and the result showed that 36 (58.06%) isolates were able to form moderate to strong biofilms in Trypticase soy broth (TSB) at 72h of incubation. Of the 36 biofilm-producing isolates, 30 were APEC. Biofilm-related genes (crl, csgA, fimH, luxS, and papC) were also detected with higher prevalence among APEC isolates. Antimicrobial susceptibility test using Vitek 2 Compact revealed 43 (91.48%) of 47 APEC isolates as multiple drug resistant (MDR) and 8 (17.02%) as ESBL positive. This study reveals that APEC with biofilm formation ability is present in poultry farms. Further studies are needed to understand the role of biofilms in the pathogenesis and antimicrobial resistance of APEC.