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.

Clostridium chauvoei is a spore-forming bacterium responsible for black quarter (BQ) in cattle, a disease leading to high mortality. Despite its economic significance in India, genomic characterization of C. chauvoei strains from the country remains scarce. This study reports the genome of a C. chauvoei strain, 23CCJK, associated with a black quarter outbreak in Leh, India. The genome, approximately 2.7 Mb, encodes 2557 protein-coding genes including important known virulence determinants such as CctA, NanA, hyaluronidase, and collagenase. Species pangenome analysis involving 85 global strains revealed 2357 core and 381 accessory genes, indicative of limited gene acquisition. CRISPR analysis revealed conserved repeat patterns among Indian strains, supporting regional relatedness. Phylogenetic analysis indicated three major lineages (L1, L2, and L3), with Indian strains clustering among L3. The major lineage (L3; n=80) represented mainland strains, whereas L1 (n=2) and L2 (n=3) represented strains mainly from Australia and New Zealand. Notably, several novel genes were identified in this analysis with hypermutations, possibly involved in vital metabolism, host-pathogen interaction and virulence. This included discoidin domain-containing proteins, Glycerophosphoryl diester phosphodiesterase, ABC transporters, and histidine kinases, suggesting possible roles in faster adaptation during infection. These findings enrich our understanding of C. chauvoei evolution, population structure, and virulence, while highlighting key genetic markers that may be associated with pathogen adaptation during infections.

This study investigated the antimicrobial resistance (AMR) profile of Salmonella enterica (n = 446) of poultry origin (n = 1020) collected from West Bengal, India. Salmonella Typhimurium (n = 328) were the most frequently isolated serovar and found resistant to tetracycline (72

Salmonella can cause significant infections in poultry, including fowl typhoid and pullorum disease, while Non-Typhoidal Salmonella (NTS) causes mild to severe diarrheal diseases and is potentially zoonotic. NTS, a significant cause of gastroenteritis in humans, is often underestimated in developing countries, where contaminated chicken or poultry products are the main drivers of its spread. Various whole-cell killed, live-attenuated, or subunit vaccines are available for poultry salmonellosis. Poultry producers prefer killed vaccines owing to their safety and minimal public health risks. However, killed vaccines do not eliminate the carrier status and require parenteral administration. Live-attenuated vaccines are supposed to elicit strong mucosal and cell-mediated immune responses, which may enhance protection; however, their use requires additional biosafety considerations. In addition, certain subunit vaccines, including OMV-based vaccines and nanoparticle vaccines, can also elicit strong mucosal, humoral, and cell-mediated immune responses. Vaccine platforms, including mRNA vaccines and multi-epitope-based vaccines, have not been extensively studied against poultry salmonellosis, though they can be evaluated to develop effective vaccine formulations. Challenges, including spatiotemporal variation in serovar distribution, operational difficulties in implementing vaccination programs, and limited awareness among poultry producers and the public, need to be addressed alongside considerations of vaccine efficacy and longevity. This review provides a comprehensive overview of the available Salmonella vaccines for poultry, including their merits and demerits, recent advancements in vaccine development, and the challenges associated with implementing immunization programs, particularly in developing countries.

Brucellosis, a zoonotic disease caused by Brucella species, continues to pose major health challenges for humans and livestock, along with significant economic losses. Traditional serological tests are widely used but are limited, as they cannot reliably distinguish between active infection and past exposure. Molecular tools such as PCR and real-time PCR offer high sensitivity and specificity; however, their dependence on sophisticated equipment and trained personnel restricts their use outside well-equipped laboratories. More recently, isothermal techniques such as LAMP and RPA have emerged as faster, simpler alternatives, and their integration with CRISPR-based platforms has further enhanced detection capabilities. Nevertheless, these methods still face challenges, including the requirement for pre-amplification and the risk of contamination, which limit their practical application in field conditions. In this study, we developed an amplification-free CRISPR-Cas12a assay—HiTECT (High-copy Target Enhanced CRISPR Test)—targeting the high-copy IS711 insertion element for thermocycler free rapid detection of Brucella spp. Using a multiplexed crRNA strategy, HiTECT enabled robust visual detection without nucleic acid amplification. Under optimal conditions (100 nM LbCas12a with a 1:1 Cas12a–crRNA ratio) and employing three crRNAs, fluorescence was significantly enhanced. The assay demonstrated an analytical sensitivity of 461.71 ag/µL of genomic DNA (0.92 fg/ reaction) and approximately 6*10⁴ CFU/mL for Brucella suis 1330. HiTECT was found very specific as it could detect all 16 field isolates and four reference Brucella strains, with no cross-reactivity to any of the non Brucella bacterial species, and showed substantial concordance with real-time PCR (κ = 0.65). HiTECT provides a robust, amplification-free, field-deployable platform for rapid and sensitive detection of Brucella spp., offering clear advantages for resource-limited settings.

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.

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.

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

Clostridium perfringens, a ubiquitous organism that has zoonotic potential, is associated with enteric diseases in humans and various animals, largely due to its potent toxin production and increasing antimicrobial resistance (AMR). The present study involved whole genome sequencing and comparative genomic analysis of four C. perfringens isolates (from pig, dog, and cattle) collected in Tamil Nadu, India, along with 133 global strains of animal origin. The phylogenomic analysis revealed six major clusters and no correlation associated with geographic origin or host were observed. Multilocus sequence typing (MLST) defined sequence types (STs) of two Indian isolates (ST126 and ST200) whereas the remaining two isolates were novel., Pangenome analysis indicated an open genome structure, with 18.28 % core genes and 81.72 % accessory genes. Virulent gene profiling identified widespread presence of plc, pfoA, and cpe, especially among dog isolates, suggesting potential zoonotic risks. wgMLST and MST analysis showed clustering of toxinotype F isolates from dogs and higher genetic diversity for other toxintypess. AMR gene analysis revealed tetracycline resistance as the most prevalent, with several strains harbouring multidrug resistance genes. This study underscores the genetic diversity, adaptive evolution, and One Health significance of C. perfringens in animals and highlights the importance of genomics in understanding host-specific virulence and resistance patterns for effective disease surveillance and control.

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.

Salmonella Typhimurium is one of the leading causes of foodborne illness in humans. Poultry products are the main source of infection for humans. Here we report the whole-genome sequences of Salmonella Typhimurium strain E-5591 (a field poultry isolate) and its mutant strains lacking the various methionine sulfoxide reductase genes.

Mycotic infections in neonatal calves are often undiagnosed but pose significant risks, especially in immunocompromised neonates. In this study, we report a rare case of co-infection with systemic candidiasis and diphtheritic aspergillosis in a 13-day-old female Murrah buffalo calf presented for necropsy at ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly. Gross examination revealed pneumonic changes with a well-demarcated nodule in the right caudal pulmonary lobe. Adherent mycotic pseudo membranes over the tracheal mucosa, and type I b haemorrhagic ulcers in the abomasum were also detected. Histopathological examination revealed fungal pseudo-hyphae, budding yeasts within pulmonary microabscesses, bronchiolar epithelium, and vessels, along with angioinvasion. The abomasum exhibited transmural necrotizing mycotic abomasitis with fungal hyphae invading submucosal vessels. Aseptically collected heart blood and lung tissue samples cultured on Sabouraud Dextrose Agar (SDA) yielded C. tropicalis. Tracheal swabs inoculated on SDA revealed growth of Aspergillus fumigatus and Aspergillus flavus. This study highlights that in calves with immature immune systems, opportunistic fungal infections are more likely, and the risk is further heightened by injudicious alteration of antibiotic regimens and corticosteroid therapy; such practices should be avoided to prevent mixed infections.

The zoophilic Microsporum canis is mainly associated with skin disease in dogs and cats, though increased occurrence in humans has been reported recently. Genome-based comparison of isolates from different host origins shall provide deeper insights into disease epidemiology and zoonotic transmission. However, whole genome sequence data of M. canis isolates, especially of animal origin, are scarce. Considering these facts, whole genome sequencing (WGS) of M. canis isolates of canine (B12-45A) and feline (B12-36A) origins from India was carried out. Both the strains revealed a genome size of similar to 22.8 Mb and GC content of similar to 47.4 %. A total of 158 protein-coding genes were predicted as carbohydrate-active enzymes (CAZymes). Further, comparative genome analysis envisaged the identification of average nucleotide identity (ANI), mash distance, and core SNP-based analysis involving 8 Microsporum isolates. Based on ANI and mash distance, the M. canis strains of the human (n = 4) and animal (n = 2) origins clustered together, distinct from the other two species. Within the M. canis cluster, the two animal-origin strains exhibited close relatedness (ANI value > 99.9) to two human strains (CBS_113480 and BMU_10802). The other two human strains were represented as outliers with lower ANI values. Similarly, lower pairwise SNP distance between animal and some human invasive strains indicates the possibility of diverse genotypes within M. canis, of which some may have high zoonotic potential. Further, the genomic and phylogenetic insights derived from the study may be applied in further epidemiological studies and for devising strategies for the development of effective therapeutics targeting their secretomes, especially the CAZymes.

AbstractBrucellosis is a significant zoonotic disease with major economic implications, particularly in livestock. Traditional diagnostic methods, such as bacterial culture and serology, are often time-consuming and less sensitive. In this study, a PCR assay targeting the per gene was developed. The assay demonstrated 100% sensitivity in detecting Brucella DNAacross various isolates and showed no cross-reactivity with non-Brucella species. It was capable of detecting as little as 38.3 pg of Brucella genomic DNA and 4.8 × 10² CFU/mL in spiked milk samples, suggesting its high sensitivity and specificity. The developed assay offered a rapid, reliable method for Brucella detection, particularly useful for outbreak investigations and zoonotic risk assessment. Future research should focus on optimizing sample preparation and expanding testing across diverse sample types to further enhance the assay’s applicability in field conditions.

BACKGROUND:Trichophyton indotineae (earlier Trichophyton mentagrophytes ITS genotype VIII) is a newly defined dermatophyte species frequently reported from India. The strains representing T. indotineae cause dermatophytosis in humans, and some strains exhibit resistance to terbinafine. OBJECTIVE:This study aims to characterize the animal strains of T. indotineae at the genomic level and compare them with human strains and other Trichophyton species. METHODS:In this study, we report the generation of de novo WGS data for two strains of T. indotineae isolated from canines of northern India. The genome sequence was subjected to functional and structural annotation, followed by comparative genomics and phylogeny. RESULTS:Genome assembly and annotation revealed a 22 Mb genome in both strains, comprising approximately 6800 protein-coding genes, and a 24 Kb contig representing the mitochondrion. Functional annotations revealed the presence of around 200 CAZymes and secretomes. The identified proteins include various proteolytic enzymes, such as keratinases, lyases, fungalysins, subtilisins, and chitinases. Orthologous cluster comparison among T. indotineae and T. rubrum identified a LysM effector virulence factor associated with the sequestration of chitin oligosaccharides unique to T. indotineae. Taxonomic inferences involving 69 species representing the Genus Trichophyton revealed similar clustering in genome ANI, mash distance, and core-genome phylogeny. Within all approaches, a total of 16 Indian strains represented T. indotineae. The SNP difference between any two T. indiotinae strains ranged from 8 to 257, indicating limited strain variation within the species. The core-genome phylogeny revealed two major clusters, with no cluster indicating a specific host association. Correlating their phenotypic susceptibility to the anti-fungal drug terbinafine (TRB), the coding sequences (CDS) maintained no amino acid substitutions at key positions 393 and 397. The comparative analysis of the squalene epoxidase gene across the 16 strains revealed a high frequency of SNPs at the 397th position in the squalene epoxidase genes in terbinafine-resistant strains. CONCLUSION:To conclude, the study indicates similar genome composition in strains of T. indiotineae from canine and human origin, irrespective of the TRB resistance phenotype. The pathogen and genotypes circulating among humans and animals need to be continuously monitored to determine the exact role of animals in the transmission and endemicity of T. indotineae in India.

Bovine tuberculosis (bTB) is a severe infectious disease that affects both domesticated and wild animals and has a negative impact on both public health and the global economy. The causative organism of the disease is Mycobacterium bovis and, sporadically, other pathogenic mycobacteria. The issue of bTB is made more challenging when the infection is linked to multi-drug resistant M. bovis. In the current investigation, immunoinformatics approach has been applied to design a multiepitope peptide-based vaccine, MBOVAC1.0. Based on an initial screening of 3805 proteins, we selected three vital proteins viz., PPE family protein, ESAT-6 and Serine Threonine Protein Kinase for designing the multi-epitope vaccine candidate. The candidate vaccine elicited strong expression in Escherichia coli following codon optimization and in silico cloning. Immunological simulation result indicated release of immune molecules by CD4 + and CD8 + T-lymphocytes and antibodies by B-lymphocytes ensuring in high level of both cell mediated and humoral immunity against the pathogen. The proposed vaccine candidate was found to yield encouraging results in terms of safety, stability, antigenicity and immunogenicity; so, it should be validated by wet lab experiments to assess its efficacy in neutralising bTB.