Brucella abortus S19 Delta per, a vaccine candidate for bovine brucellosis, was developed by deleting the wbkB gene from the currently used Brucella abortus vaccine S19 strain. The mutant, B. abortus S19 Delta per was found to be safe and efficacious, and yielded high biomass. As survival under different stressors is also an integral part of the attributes of a vaccine, we evaluated the viability of B. abortus S19 Delta per under different physical, chemical, oxidative, and membrane stressors in vitro, in comparison with the parent strain. The results revealed that the survival rate of the mutant to the stressors was comparable with that of the parent strain (P>0.05), indicating that deletion of the wbkB gene did not affect the response to the stressors. Collectively, these findings add strength to the claim that the S19 Delta per mutant is a good replacement for the parent, B. abortus S19 strain.

Japanese encephalitis (JE) is a re-emerging viral zoonotic disease transmitted primarily by the bite of infected Culex mosquitoes with ardeid birds as reservoir host for JE virus (JEV). Swine being the amplifier host have been studied extensively, however the role of domestic poultry in JEV epidemiology remains unexplored. Therefore, the present study was undertaken with the aim of quantifying JE viral load and to identify the circulating genotype of JEV in domestic chicken and duck population of India. A total of 206 chicken and 76 duck samples collected across five JE endemic states of India representing three geographical zones were screened using TaqMan real time RT-PCR targeting JEV envelope gene. Positive samples were further amplified using nested RT-PCR targeting the C/PrM gene of JEV and confirmed by sequencing followed by phylogenetic analysis of the amplified products to infer their genetic relationship with other JEV isolates. A total of 53 chicken samples and 9 duck samples were positive for JEV RNA with viral load ranging from 9 to 1.33 × 10⁵ copies/reaction for chickens and 32 to 1.23 × 10⁴ copies/reaction for ducks. Phylogenetic analysis of the partial C/PrM gene sequences (n = 11) revealed dominance of Genotype III in domestic birds. Two novel amino acid insertions, Serine at C87 and Arginine at C94 positions, were detected in one sequence. The findings of this study suggests that domestic poultry might act as reservoir or amplifier host for the virus, however further studies are required to elucidate their role in JEV transmission to humans.

BACKGROUND OBJECTIVES:Japanese encephalitis (JE) is a re-emerging vector-borne zoonotic disease with a significant public health impact in India. JE is endemic in several Indian states, including Assam, which consistently reports high burden of human JE cases in the country. Pigs, as amplifier hosts for the JE virus, plays a critical role in JE transmission cycle and serves as suitable sentinels for predicting human JE outbreaks. Although several studies have assessed JE prevalence in pigs across different districts of Assam, there is no prior report of occurrence of JE in pigs from southern region of Assam. The present study was undertaken to determine the occurrence of JEV infection in pigs from Cachar, a southern district of Assam through serological and molecular testing. METHODS:A total of 100 pig serum samples collected from backyard farms across eight villages in the Cachar district were screened using indirect ELISA(s) to detect JEV-specific IgM and IgG antibodies. Samples were further subjected to nested RT-PCR for viral RNA detection followed by phylogenetic analysis to identify the circulating JEV genotype in the region. RESULTS:Of the 100 pig serum samples tested, 59% were positive for JEV specific IgM antibodies whereas 53% samples were positive for IgG antibodies. The overall seropositivity of JEV among pigs from Cachar district was 66%. JEV RNA was detected in 12 samples and phylogenetic analysis confirmed the presence of genotype III of JEV circulating among pigs from Cachar district of Assam. INTERPRETATION CONCLUSION:This is the first study to report the presence of JEV infection in pigs from Cachar district of Assam, India underscoring the need to initiate JEV surveillance in the pig population of the region.

Salmonella is a worldwide foodborne pathogen and chicken is considered to be the main reservoir for this zoonotic pathogen. In this study, three bacteriophages were isolated from sewage samples that exhibited a broad host range against Salmonella enterica serovar Typhimurium (rpSTIz1), Salmonella enterica serovar Enteritidis (rpSEIz2) and Salmonella enterica serovar Gallinarum (rpSGIz3) were characterized by restriction fragment length polymorphism and random amplification polymorphic DNA. Restriction analysis was performed using AluI, ApaI, AvaI, BamHI, BglI, SacI, BspI, Bsp119I (BstBI), EcoRI, Eco311, EcoRV, HindIII, HindfI, KpnI, NcoI, NotI, PstI, SacI, SalI, SmaI, XbaI and XhoI endonucleases and dendrogram were constructed to analyze the genetic diversity among the three phages. RAPD was performed with 3 randomly chosen 10-base random primers URP-6, OPS-11 and OPP-16 and the data obtained from amplification products by primers were used to estimate genetic similarity among different phages on the basis of shared amplification products. SDS-PAGE of three bacteriophage lysate was carried out. Three phages (rpSTIz1, rpSEIz2 and rpSGIz3) DNA dendrograms showed a distinctive RFLP when digested with restriction endonucleases NcoI, PstI, SalI and XhoI. The SDS-PAGE profile of the three bacteriophages differed in band patterns of major and minor proteins. The present study describes molecular and protein profiling of three phages of Salmonella rpSTIz1, rpSEIz2 and rpSGIz3 using RAPD, RFLP and SDS-PAGE which revealed that all three phages have a common ancestor with genetic differences.

Staphylococci are important mastitis pathogens in dairy animals and serve as reservoirs of antimicrobial resistance genes (ARGs) having zoonotic potential. Genomic characterization of resistant isolates is essential to understand their diversity, resistance mechanisms, and One Health implications. A total of 363 cow and buffalo milk samples—including 108 from animals with mastitis—were screened, yielding 98 staphylococcal isolates, comprising 20 Staphylococcus aureus and 78 coagulase-negative staphylococci (CoNS). Antimicrobial susceptibility testing revealed resistance to cefoxitin (CoNS: 21.7

Scrub typhus is a significant public health concern in India. Despite numerous studies on its prevalence across hosts and vectors, country-specific data remain unavailable. This systematic review and meta-analysis provide comprehensive insights into the prevalence of scrub typhus across humans, animal hosts, and vectors to aid disease control programs. The PECOS framework was used to identify eligible studies. A systematic search across six databases (PubMed, Scopus, Web of Science, Google Scholar, Krishikosh, and Shodhganga) was conducted in March 2024. Data were classified by diagnostics and disease distribution across hosts and vectors for narrative synthesis. Study bias was assessed using a prevalence study checklist. A random- or common-effects meta-analysis model estimated overall and subgroup pooled prevalence across population types and diagnostic tests, with heterogeneity measured by the I2 index. A total of 83 eligible studies published between 2003 and 2024 covered different regions, hosts, and vectors. The pooled prevalence (95

Scrub typhus is a notable zoonosis, yet natural host-associated pathogen prevalence and genotypic diversity remain underexplored. A total of 261 small mammals comprising rodents and shrews captured in Uttar Pradesh, India, were screened for Orientia tsutsugamushi (OT) using nested PCR. The sample included Rattus rattus (n = 28), Rattus norvegicus (n = 42), Rattus tanezumi (n = 42), Bandicota bengalensis (n = 10), Mus musculus (n = 15), and Suncus murinus (n = 124). The overall molecular prevalence was 11.9% (95% CI: 8.2-16.4) with detection rates of 13.1% in rodents and 12.1% in shrews. Prevalence was significantly higher in rural (15.2%) than urban (1.6%) settings, and during monsoon (29.3%) and post-monsoon (11.9%) periods. Phylogenetic analysis of the partial tsa56 gene identified 4 OT strains circulating in rodent and shrew hosts, including Gilliam and TA678, along with novel JJOtsu5 and JJOtsu7. Population genetic analysis revealed substantial tsa56 nucleotide and haplotype diversity under purifying selection, while recombination analysis detected recombination in a subset of sequences, also contributing to genetic diversity. Deduced amino acid sequence analysis of the contiguous partial tsa56 gene revealed distinct mutations and structural variation in Gilliam-like and TA678-like strains, while JJOtsu5 and JJOtsu7 showed no variation relative to reference strains. In silico immunoinformatic analysis predicted 8 CD8 and 55 CD4 T-cell epitopes (TCEs) eliciting a human immune response, predominantly located within the spacer region S-VDIII/IV. The CD8 epitope AQLYKDLVKL was conserved across Gilliam, TA678-like (variant-27S), and JJOtsu7 strains; while the CD4 epitope PVKVLSDKITQIYSD was shared among Gilliam, JJOtsu5 and JJOtsu7 strains with a single S290R substitution. These strains exhibited distinct histopathological responses in OT PCR-positive animal tissues. Gilliam infections caused moderate lesions in liver, spleen, and lungs; TA678 infections exhibited mild pulmonary and renal changes; JJOtsu7 induced severe pulmonary and myocardial changes; and JJOtsu5 caused mild-to-moderate inflammation in liver, spleen, and kidneys. These findings highlight substantial genetic diversity, differential immunogenic potential and variable pathogenicity among rodent and shrew associated OT strains, underscoring the need for extended surveillance and strain-specific virulence characterization for improved control strategies.

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in the Asia-Pacific region. Amplification of JEV in pigs is a potent driver for spillover of the infection to humans, and hence monitoring of virus dynamics in pigs can provide insights into JEV ecology. To study the dynamics of natural JEV infection in a tropical region, two groups of immunologically naïve pigs consisting of six animals per group were kept as sentinels on two different farms in the district of Thanjavur, Tamil Nadu, India. In a longitudinal study conducted from May 2022 to October 2023, nested RT-PCR and indirect ELISA were used to track the dynamics of JEV and the humoral response in pigs. Synchronous and asynchronous seroconversion in pigs was recorded on two different farms with different management practices. Repeated infections with JEV were recorded in all of the sentinel animals throughout the study period, irrespective of the season. Phylogenetic analysis revealed the presence of JEV genotype III in the region. It was observed that the IgG response to natural JEV infection did not last long, which might have been the reason for repeated infections in the sentinel animals. The longest period during which IgG was present at detectable levels in this study was two months, after which the pigs could once again amplify the virus. A significant positive correlation was found between wind speed and JEV incidence in sentinel animals. Our results offer a different perspective on the relationship between JEV and its amplifying host that contradicts the assumption that pre-immune pigs are resistant to JEV amplification. Our findings could have a major impact on our understanding of the ecology of JEV in tropical regions, where there is a high burden of JE despite coordinated prevention efforts that have relied on achieving a long-lasting immune response.

Brucellosis is among the most widespread zoonotic diseases globally, affecting multiple domestic animal species. We report the first isolation of Brucella suis from a vaginal swab collected from an aborted cow in India. The isolate (VS1) was confirmed as B. suis bv. 1 by biochemical assays and species-specific PCR. Whole genome sequencing analysis of the VS1 isolate revealed a 32,81,903 bp genome with a guanosine and cytosine (GC) content of 57.29

Scrub typhus, a vector-borne zoonosis prevalent in the Asia-Pacific region, poses diagnostic challenges due to the pathogen's complex genome and diverse rodent and shrew hosts. The scarcity of reliable diagnostic tests hinders effective sentinel surveillance. This study aims to identify novel diagnostic gene targets using a bioinformatics approach to develop a highly sensitive and specific PCR assay for scrub typhus detection. Genome sequences of Orientia tsutsugamushi, the causative agent, were analyzed, leading to the selection of 11 potential diagnostic biomarkers. In-house conventional PCR assays targeting these biomarkers and published nested PCR assays, were tested on blood and tissue (spleen) samples of 150 field rodent and shrew. Among the tested genes, the tsa56 gene consistently demonstrated the highest detection rate in both conventional (55.6 %, n = 15) and nested (74 %, n = 20) assays, indicating it to be the most reliable diagnostic marker for scrub typhus. A novel nested PCR was designed targeting a unique tsa56 gene segment, which showed an analytical sensitivity of 4.7 (95 % CI: 2-7.4) copies/μL, and was able to detect O. tsutsugamushi in multiple hosts including human and mite samples. Moreover, fewer primer-template mismatches and no mismatches at the critical 3' terminus with O. tsutsugamushi strains were observed. The assay did not show any cross-reaction with available non-target organisms. Thus, the developed nested PCR assay demonstrates enhanced sensitivity, specificity, and broader strain inclusivity. Overall, the study presents a promising tool for scrub typhus detection, which will aid in improved disease surveillance, outbreak prediction, and timely implementation of control measures.

Multidrug resistant non-typhoidal Salmonella infections are a threat to food safety impacting human health in the form of hospitalizations and in severe cases, fatalities. Epidemiological investigations of an outbreak require accurate identification of the infection source and the transmission route for effective implementation of preventive measures against microbial food-borne pathogens. Therefore, the present study investigates the repetitive sequence-based PCR (rep-PCR) fingerprinting techniques for genotyping of multidrug-resistant isolates of Salmonella Typhimurium. ERIC, REP, BOX and GTG5-PCR fingerprinting generated reproducible band patterns ranging from 3-10, 1-5, 6-14 and 2-10 separate bands, respectively. Cluster analysis revealed 9 types from ERIC-PCR, 7 types from REP-PCR, 16 types from BOXPCR and 9 types from GTG5-PCR. Hundred percent typeability was obtained with all genotyping techniques except REP-PCR. The isolates’ sharing similar band patterns is suggestive of genetic relatedness pointing towards the multifactorial involvement in the transmission of Salmonella. Faecal isolates and meat swab isolates sharing identical band patterns and grouped into a single cluster is indicative of a likely cross contamination. No unique pattern was observed in penta resistant (ACSSuT) profile isolates by our typing techniques. Discrimination index (DI) value of BOX-PCR was highest (0.940) followed by GTG5-PCR (0.862), REP-PCR (0.846) and ERIC- PCR (0.843). On comparing the techniques, the BOX-PCR exhibited good DI value, typeability and complex band pattern on gel in differentiating the isolates. Conclusively, the BOX-PCR fingerprinting technique was found useful in the genotyping of isolates thus suitable enough to find its application in an epidemiological investigation during an outbreak.

Brucella melitensis is a highly infectious zoonotic pathogen responsible for brucellosis, which significantly affects both human and livestock health worldwide. This study employed whole-genome sequencing (WGS) to analyze the genetic diversity of 24 B. melitensis isolates from India. Pangenome analysis revealed a highly conserved nature with the involved strains having very limited accessory genes. Multilocus Sequence Typing (MLST) identified sequence type ST8 as predominant among Indian strains. Analysis of virulence genes revealed a total of 43 virulence-related genes in all strains, emphasizing their critical role in the pathogenicity of B. melitensis. Unique gene profiles and distinct phylogenetic clusters suggest regional adaptations and evolutionary pressures. The comprehensive genomic insights from this study help to elucidate the geographic distribution and interspecies transmission of Indian strains, highlighting the importance of targeted brucellosis control measures in India. Additionally, the identification of conserved virulence genes involved in immune evasion and intracellular survival highlights their importance in the bacterium's pathogenicity. This research contributes to the global understanding of B. melitensis genomic diversity, providing valuable insights for broader epidemiological studies and brucellosis management strategies worldwide.IMPORTANCEB. melitensis is a significant cause of illness in both humans and animals, particularly in India, where the disease remains a major concern. This study highlights that only a few genetic types of the bacteria are circulating in the region, which means control efforts can be better focused on these specific types. By understanding the unique characteristics of Indian strains, and how these strains spread and adapt, this research offers valuable guidance for improving brucellosis prevention strategies. These insights can help in developing more effective diagnostic tools, enhancing vaccination efforts, and strengthening disease control programs to reduce the impact of brucellosis on public health and livestock industries.

India’s livestock sector is a major reservoir for bacterial pathogens and a potential source of antimicrobial resistance. We investigated the prevalence and antimicrobial resistance mechanisms in Staphylococcus spp. and Escherichia coli from livestock and poultry isolates from Sambhal district, Uttar Pradesh. A total of 173 samples (cow and buffalo milk and rectal, goat rectal, and poultry cloacal swabs) were collected and cultured. Staphylococci were isolated from 55 of 65 milk samples (84.6%), including 12 S. aureus (21.8% of Staphylococci). Fifteen of 55 isolates (27.3%) were methicillin-resistant. Penicillin (43.6%) and erythromycin (23.6%) resistance predominated, while all isolates remained linezolid susceptible. E. coli prevalence was 71.1% (123/173). High resistance was observed to ampicillin (59.3%), ceftriaxone (39.0%), and cefotaxime (29.3%). ESBL and AmpC phenotypes were detected in 20 (16.3%) and 11 (8.9%) isolates, respectively; carbapenem resistance was rare and mCIM-negative. Multivariate clustering revealed sample-type and host-associated resistance patterns with considerable overlap between bovine and poultry isolates.

We present the draft genome sequences of 23 Brucella melitensis isolates derived from human and animal sources across India with genome size predominantly at 3.207 M and uniform GC content (57.24%) across isolates. The accession numbers and detailed sequencing data enhance the utility of this resource for further genomic studies.