Outer membrane vesicles as nanovaccine candidates against pathogenic Leptospira in experimental Guinea pig model.

Leptospira interrogans serovar Hardjo is a long slender bacterium of size 0.1-0.3 µm × 5-50 µm. It is one of the major causes of bovine leptospirosis and is of economical importance because of the reproductive failure, still birth, abortion, and reduced productivity in cattle. It is also a zoonotic disease-causing infection in humans characterized by headaches, fever, chills, sweats and myalgia, lethargy, aching joints, pulmonary haemorrhages, and death in severe cases. Control of the disease involves antibiotic therapy, management and vaccination, of which immunization is the cheapest and effective means of disease prevention. The present study was developed to isolate and characterize the outer membrane vesicles of Leptospira interrogans serovar Hardjo and to evaluate their vaccine potential in guinea pig model. The OMVs were isolated from the culture by sonication and ultracentrifugation. In transmission electron microscopy, the isolated OMVs appeared as small spherical structures of 50-200 nm size. In Western blot and indirect ELISA, antibodies specific to OMVs were observed as indicative of a good humoral immune response elicited by L. interrogans serovar Hardjo OMV. The OMV-based Leptospira vaccine was able to prevent kidney lesions and renal colonization compared to the control and bacterin vaccinated group as proven by histopathology and PCR.

Development and validation of multiplex PCR based molecular serotyping of Salmonella serovars associated with poultry in India.

Salmonella species are Gram-negative bacteria with more than 2600 serovars. Among these serovars, many are associated with various diseases in livestock and humans. White Kauffman Le-Minor (WKL) serotyping scheme applies specific serum to determine the serovars of Salmonella. Recent studies have applied molecular methods for serovar predictions. These methods include PCR, hybridization and sequence data to detect/predict serovar-specific genetic elements. Among these, PCR is a robust method if the unique genetic element is already known. Within this context, also involving novel primers, two multiplex PCR assays were standardized to detect six important Salmonella serovars viz. Typhimurium, Enteritidis, Kentucky, Infantis, Virchow and Gallinarum associated with poultry in India. The developed PCR assays showed targeted serovar specificity. Serial dilution experiments of both kit-based and crude lysate DNA preparations indicated similar applicability of both methods for testing from pure cultures. Further the developed assays were validated with 25 recent field isolates to confirm the applicability in routine diagnosis. The PCR assay could predict all the targeted serovars (17/25) with 100% specificity (CI-95%; 0.63-1). Molecular serotyping can reduce the number of serum used in comparison to the conventional serotyping which involves more random application of serum.

Foremost report of the whole genome of Spirabiliibacterium mucosae from India and comparative genomics of the novel genus Spirabiliibacterium.

Several Pasteurella like organisms isolated from various avian species were recently reclassified into new genus based on whole genome sequence analysis. One such Pasteurella like organism, Bisgaard taxon 14 was classified as Spirabiliibacterium mucosae. In the present study, a Gram-negative organism was isolated from ailing pigeons with respiratory infection from a farm in Tamil Nadu, India and the organism was misidentified as Burkholderia mallei by Vitek 2 compact system based on biochemical characterization. Since, B. mallei is highly pathogenic and zoonotic, to further confirm, 16S rDNA sequencing and analysis was carried out which revealed that the strain belonged to Bisgaard taxon 14 (Spirabiliibacterium mucosae). To further confirm the findings, whole genome sequencing of the isolate was performed. Whole genome phylogeny and average nucleotide identity (ANI) analysis showed that the genome was closely matching with Spirabiliibacterium mucosae type strain 20,609 /3. Hence, the strain from pigeon was named as Spirabiliibacterium mucosae TN_CUL_2021 and the genome was submitted in NCBI SRA database. The genome of S. mucosase TN_CUL_2021 is only the second genome available worldwide in the NCBI database. Comparative genome analysis of 26 Pasteurellaceae family strains revealed 1101 genes specific for Spirabiliibacterium mucosae. Similarly, luxS virulence gene was found only in S. mucosae and Bisgaardia hudsonensis strains. Since there are only 2 genomes available in the NCBI genome database, further studies on isolation of S. mucosae needs to be carried out to identify its epidemiology and pathogenesis so as to develop better diagnostic assays and vaccines.

Comparative phylogenomics of Trueperella pyogenes reveals host-based distinction of strains.

Trueperella pyogenes, an opportunistic pathogen causes various ailments in different animals. Different strains from different animals have distinct characters phenotypically and genotypically. Hence understanding the strains in a particular geographical location helps in framing the preventive measures. Comparative genomics of all the available T. pyogenes genome in the NCBI was conducted to understand the relatedness among strains. Whole genome phylogeny showed host associated clustering of strains recovered from swine lungs. Core genome phylogeny also showed host associated clustering mimicking whole genome phylogeny results. MLST analysis showed that there was higher diversity among cattle strains. Multidimensional scaling revealed five swine clusters, two cattle and buffalo clusters. Pangenome analysis also showed that T. pyogenes had an open genome with 57.09% accessory genome. Host specific genes were identified by pangenome analysis, and (R)-citramalate synthase was specific for swine strains of Asian origin. Host specifc genes identified by pangenome analysis can be exploited for developing a molecular assay to specifically identify the strains. The study shows that MLST having higher discriminatory power can be used as an epidemiological tool for strain discrimination of T. pyogenes.

Whole genome sequencing and comparative genomics of Mycobacterium orygis isolated from different animal hosts to identify specific diagnostic markers.

Introduction: Mycobacterium orygis, a member of MTBC has been identified in higher numbers in the recent years from animals of South Asia. Comparative genomics of this important zoonotic pathogen is not available which can provide data on the molecular difference between other MTBC members. Hence, the present study was carried out to isolate, whole genome sequence M. orygis from different animal species (cattle, buffalo and deer) and to identify molecular marker for the differentiation of M. orygis from other MTBC members. Methods: Isolation and whole genome sequencing of M. orygis was carried out for 9 samples (4 cattle, 4 deer and 1 buffalo) died due to tuberculosis. Comparative genomics employing 53 genomes (44 from database and 9 newly sequenced) was performed to identify SNPs, spoligotype, pangenome structure, and region of difference. Results: M. orygis was isolated from water buffalo and sambar deer which is the first of its kind report worldwide. Comparative pangenomics of all M. orygis strains worldwide (n= 53) showed a closed pangenome structure which is also reported for the first time. Pairwise SNP between TANUVAS_2, TANUVAS_4, TANUVAS_5, TANUVAS_7 and NIRTAH144 was less than 15 indicating that the same M. orygis strain may be the cause for infection. Region of difference prediction showed absence of RD7, RD8, RD9, RD10, RD12, RD301, RD315 in all the M. orygis analyzed. SNPs in virulence gene, PE35 was found to be unique to M. orygis which can be used as marker for identification. Conclusion: The present study is yet another supportive evidence that M. orygis is more prevalent among animals in South Asia and the zoonotic potential of this organism needs to be evaluated.

First report of the whole genome of Moraxella bovoculi genotype 1 from India and comparative genomics of Moraxella bovoculi to identify genotype-specific markers.

Moraxella bovoculi has been isolated frequently from cattle with Infectious bovine keratoconjunctivitis (IBK). Two diverse genotypes of M. bovoculi, 1 and 2 were identified based on whole genome sequence analysis. It is essential to discriminate between the two genotypes to frame prevention and control measures. The whole genome of M. bovoculi TN7 was sequenced and compared to other M. bovoculi strains available in the NCBI database. M. bovoculi TN7 was found to be genotype 1, had an RTX toxin operon and pilA gene that are the known virulence factors in related Moraxella sp., but lacked antimicrobial resistance genes. M. bovoculi was found to have an open pangenome with 4051 (75.31%) accessory genes, and the addition of each new genome adds 18 genes to the pangenome. Comparison of pilin protein amino acid sequences revealed three new sequence types. Furthermore, the presence of linx, nagL, swrC and mdtA genes was found to be genotype 1 specific, whereas hyaD, garR, gbsA, yhdG, gabT, iclR, higB2, hmuU, hmuT and hemS were found only in genotype 2. Polymerase Chain Reaction (PCR) primers were designed and evaluated on strain TN7 plus seven additional strains accessible to us that had not been whole genome sequenced. This initial evaluation of the designed primers for the linX and hyaD genes produced the expected banding patterns on PCR gels for genotypes 1 and 2, respectively, among the 8 strains. The genotype-specific genes identified in this study can be used as markers for accurate diagnosis of genotype 1 isolates and this can aid in the development of autogenous or other molecular vaccines for treatment of infectious bovine keratoconjunctivitis (IBK) in resource-limited research settings.

Comparative genomics of Trueperella pyogenes available in the genome database reveals multidrug resistance genomic islands.

OBJECTIVE: An opportunistic pathogen, Trueperella pyogenes can infect cattle, buffalo, pig, goat, cat, dog, forest musk deer, etc., affecting various organs. The aim of this study was to identify the multidrug resistance genomic islands of T. pyogenes genomes available in NCBI database and also in the recently isolated strain TN_CUL_2020. METHODS: The strain TN_CUL_2020 isolated from swine lung abscess was sequenced by Illumina platform, and all the available T. pyogenes genome in NCBI database was retrieved for the comparative analysis. The ABRicate searches was used to identify antimicrobial resistance genes, and genomic islands (GIs) were predicted using IslandViewer 4. RESULTS: The strains SH01, SH02, and TP1 were predicted with maximum number of drug resistance genes. Genomic islands identified had multidrug resistance genes along with the class I integron and/or IS6100 elements in SH01, SH02, TP1. Composite transposons of IS6100 were noted in T2849, T4479, and TP3 intercalating tet(33) resistance genes. Several strains were predicted with phage elements, type IV secretion system, the toxin-antitoxin system in the GIs. CONCLUSION: Swine strains SH01, SH02 were predicted with multidrug resistance genes along with class I integrons. The presence of class I integrons, insertional elements, type IV secretion system, toxin-antitoxin system, and phage elements may aid in the horizontal transfer of antimicrobial resistance genes.

Genome Sequencing and Comparative Genomics of Indian Isolates of Brucella melitensis.

Brucella melitensis causes small ruminant brucellosis and a zoonotic pathogen prevalent worldwide. Whole genome phylogeny of all available B. melitensis genomes (n = 355) revealed that all Indian isolates (n = 16) clustered in the East Mediterranean lineage except the ADMAS-GI strain. Pangenome analysis indicated the presence of limited accessory genomes with few clades showing specific gene presence/absence pattern. A total of 43 virulence genes were predicted in all the Indian strains of B. melitensis except 2007BM-1 (ricA and wbkA are absent). Multilocus sequence typing (MLST) analysis indicated all except one Indian strain (ADMAS-GI) falling into sequence type (ST 8). In comparison with MLST, core genome phylogeny indicated two major clusters (>70% bootstrap support values) among Indian strains. Clusters with <70% bootstrap support values represent strains with diverse evolutionary origins present among animal and human hosts. Genetic relatedness among animal (sheep and goats) and human strains with 100% bootstrap values shows its zoonotic transfer potentiality. SNP-based analysis indicated similar clustering to that of core genome phylogeny. Among the Indian strains, the highest number of unique SNPs (112 SNPs) were shared by a node that involved three strains from Tamil Nadu. The node SNPs involved several peptidase genes like U32, M16 inactive domain protein, clp protease family protein, and M23 family protein and mostly represented non-synonymous (NS) substitutions. Vaccination has been followed in several parts of the world to prevent small ruminant brucellosis but not in India. Comparison of Indian strains with vaccine strains showed that M5 is genetically closer to most of the Indian strains than Rev.1 strain. The presence of most of the virulence genes among all Indian strains and conserved core genome compositions suggest the use of any circulating strain/genotypes for the development of a vaccine candidate for small ruminant brucellosis in India.

A comprehensive review on avian chlamydiosis: a neglected zoonotic disease.

Avian chlamydiosis is one of the important neglected diseases with critical zoonotic potential. Chlamydia psittaci, the causative agent, affects most categories of birds, livestock, companion animals, and humans. It has many obscured characters and epidemiological dimensions, which makes it unique among other bacterial agents. Recent reports on transmission from equine to humans alarmed the public health authorities, and it necessitates the importance of routine screening of this infectious disease. High prevalence of spill-over infection in equines was associated with reproductive losses. Newer avian chlamydial species are being reported in the recent years. It is a potential biological warfare agent and the disease is an occupational hazard mainly to custom officers handling exotic birds. Prevalence of the disease in wild birds, pet birds, and poultry causes economic losses to the poultry industry and the pet bird trade. Interestingly, there are speculations on the 'legal' and 'illegal' bird trade that may be the global source of some of the most virulent strains of this pathogen. The mortality rate generally ranges from 5 to 40% in untreated cases, but it can sometimes be higher in co-infection. The intracellular lifestyle of this pathogen makes the diagnosis more complicated and there is also lack of accurate diagnostics. Resistance to antibiotics is reported only in some pathogens of the Chlamydiaceae family, but routine screening may assess the actual situation in all pathogens. Due to the diverse nature of the pathogen, the organism necessitates the One Health partnerships to have complete understanding. The present review focuses on the zoonotic aspects of avian chlamydiosis with its new insights into the pathogenesis, transmission, treatment, prevention, and control strategies. The review also briefs on the basic understandings and complex epidemiology of avian chlamydiosis, highlighting the need for research on emerging one health perspectives.