Dinesh Mittal

Brucellosis, a contagious reproductive disease of livestock, has a significant economic impact in terms of abortions and stillbirths and has zoonotic importance. A study was conducted to estimate the seroprevalence of brucellosis in a bovine population in Haryana state, India. This study was carried out on 4325 bovine serum samples (Cattle: 2151, Buffalo: 2174) using the Rose Bengal Plate Test (RBPT) and indirect enzyme-linked immunosorbent assay (i-ELISA). The seroprevalence, estimated individually by the RBPT and i-ELISA, was found to be 6.86% (95% CI: 6.11–7.62) and 6.05% (95% CI: 5.34–6.76), respectively. In total, 258 out of 4325 (5.96%; 95% CI: 5.25–6.67) samples were found to be positive by both assays. The prevalence was found to be significantly higher in the cattle population (7.58%) as compared to buffalo (4.37%) (Chi-square = 19, p < 0.001). Seroprevalence was highest in the agroclimatic zone I (8.73%), followed by zone II (7.33%) and zone III (1.45%) (Chi-square = 76.27, p < 0.001).

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.

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis, which causes significant economic losses to the poultry industry. The growing resistance of bacteria to antibiotics is a major global public health concern. However, there is limited data on the efficacy of phage therapy in effectively controlling and treating APEC infections. In this study, a novel lytic Escherichia phage, vB_EcoS_PJ16, was isolated from poultry farm wastewater and characterized in both in vitro and in vivo conditions. Transmission electron microscopy analysis revealed the presence of an icosahedral head and a long non-contractile tail, classifying the phage under the Caudoviricetes class. Host range determination showed that Escherichia phage vB_EcoS_PJ16 exhibited lytic activity against multiple strains of pathogenic E. coli, while no significant signs of lysis for Klebsiella pneumoniae, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus. Biophysical characterization revealed that the isolated phage was sturdy, as it remained viable for up to 300 days at temperatures of 30 °C, 37 °C, and 42 °C and for up to 24 h at pH 5 to 11, with only minor changes in titer. Kinetic analysis at multiplicity of infection (MOI) 0.1 showed a latency period of about 20 min and a burst size of 26.5 phage particles per infected cell for phage vB_EcoS_PJ16. Whole genome sequencing unveiled that the phage vB_EcoS_PJ16 genome consists of a double-stranded linear DNA molecule with 57,756 bp and a GC content of 43.58%. The Escherichia phage vB_EcoS_PJ16 genome consisted of 98 predicted putative ORFs, with no transfer RNA identified in the genome. Among these 98 genes, 34 genes were predicted to have known functions. A significant reduction in APEC viability was observed at MOI 100 during in vitro bacterial challenge tests conducted at different MOIs (0.01, 1, and 100). In vivo oral evaluation of the isolated phage to limit E. coli infections in day-old chicks indicated a decrease in mortality within both the therapeutic (20%) and prophylactic (30%) groups, when compared to the control group. The findings of this study contribute to our current knowledge of Escherichia phages and suggest a potentially effective role of phages in the therapeutic and prophylactic control of antibiotic-resistant APEC strains.

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.

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.