Brucella melitensis: Divergence Among Indian Strains and Genetic Characterization of a Strain Isolated from Cattle.

Brucella melitensis primarily affects sheep, goats and is associated with brucellosis in humans, which is one of the world's most widespread neglected zoonotic disease. The current study attempted the determination of genetic diversity through comparative genome analysis of B. melitensis strains reported from India with other countries. The study also reports the isolation and identification of B. melitensis BMNDDB8664 from a cow with a history of abortion, whole-genome sequencing (WGS), determination of virulence factors, genotyping, and comparative genome analysis. Multilocus sequence typing, Multiple locus variable number of tandem repeats analysis (MLVA), and WGS based phylogeny revealed the predominance of ST-8 and genotypes (116 and II respectively) that clustered to the East Mediterranean lineage. Identification of hitherto unreported genotypes by MLVA also indicated the existence and circulation of West Mediterranean and American lineages in India. Though the AMOS-PCR results suggest the BMNDDB8664 isolate as Brucella abortus, the outcomes from multiplex PCR, ribosomal multilocus sequence typing, and WGS analysis confirmed it as B. melitensis. The analysis revealed the presence of adeF gene (aids conferring resistance to fluoro-quinolone and tetracyclines). The isolate lacked two important T4SS genes virB2 and virB7 genes (roles in infection and rifampicin resistance respectively) and also lacked the Brucella suis mprF gene that aids intracellular survival. Further, BMNDDB8664 lacked some of the genes associated with LPS synthesis (wbkB, wbkC) and transport (wzm, wzt) and hence, is most likely a rough strain. WGS-based phylogenetic analysis revealed close genetic relatedness of this BMNDDB8664 with a sheep isolate and two human isolates. The results prompt systematic, broad-based epidemiological studies on brucella infection at the species level. For effective control of human brucellosis, a concerted One Health approach with studies encircling the identification of aetiology at species, strain level to find their prevalence, spread, and inter-host transmission patterns need to be understood, for better design and implementation of effective control strategies in India and other endemic regions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01081-w.

Molecular detection of Anaplasma, Babesia, Theileria, and Trypanosoma infection in cattle and buffaloes in India.

Tick-borne pathogens pose a significant global threat, causing substantial economic losses to the dairy industry. In India, tropical theileriosis, anaplasmosis, babesiosis, and trypanosomiasis are major hemo-parasitic diseases affecting bovines. A cross-sectional study was conducted to determine the prevalence of hemo-parasites in different farms in India. PCR assays were employed to detect carrier status, using gene targets msp1b, tams1, rap-1, ama1, and ITS1 for A. marginale, T. annulata, B. bovis, B. bigemina, and Trypanosoma species, respectively. Out of the 578 apparently healthy animals screened, 30.45% (95% CI: 26.84-34.32%) were infected with at least one hemo-parasite. Cattle showed an overall positivity of 32.87%, while buffaloes had a prevalence of 15.19%, which was statistically significant (p < 0.001). Interestingly, prevalence was higher in indigenous cattle (47.81%) compared to cross-breeds (25.53%) and exotics (14.62%), with a statistically significant difference (p < 0.001). The prevalence of hemo-parasites varied widely among the farms, ranging from 5.77 to 100%. A. marginale was the most prevalent parasite (23.70% of animals), followed by T. annulata (13.67%), Babesia species (1.90%), and Trypanosoma species (1.56%). Enzootic instability was observed in six of the eight farms, indicating a potential for future outbreaks. Co-infection was detected in 60 out of 176 animals positive for hemo-parasites, with 59 animals co-infected with A. marginale and T. annulata, and only one cross-breed cattle infected with both Anaplasma marginale and Babesia bigemina. The findings highlight the prevalence of hemo-parasites in farms, underscoring the need for whole-herd screening, treatment of infected animals, and improvement in farm management practices to prevent production losses caused by these pathogens. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01673-3.

Development and laboratory validation of duplex real-time PCR for simultaneous detection of Brucella and bovine alphaherpesvirus from clinical specimens.

A duplex real­time PCR was developed and validated for the simultaneous detection of Brucella and bovine alphaherpesvirus­1 (BoHV­1) from bovine clinical specimens. The bcsp31 gene of Brucella and gB gene of BoHV­1 were used as targets in the assay. The limit of detection for BoHV­1 was 0.03 TCID50 of virus and 10 plasmid copies containing the target gene while for Brucella it was 4.1 × 101 CFUs. Intra­assay and inter­assay values showed high repeatability and reproducibility of the assay. The diagnostic sensitivity (dsn) and diagnostic specificity (dsp) of the duplex assay were determined by screening 443 clinical specimens and comparing the results with the respective individual assays. The dsn and dsp for detection of Brucella were found to be 95.24% and 95.65%, respectively whereas for BoHV­1, the dsn (100%) and dsp (99.47%) were slightly higher. The duplex assay had a very good degree of agreement with the respective individual real­time PCR test {kappa value 0.97 for Brucella and 0.95 for BoHV­1}. The results of the current study suggest that the duplex assay would be a cost­effective and time­saving alternative for the individual real­time PCR assay for the detection of Brucella and BoHV­1.