Host-pathogen interplay of Orientia tsutsugamushi: pathomolecular epidemiology and phylo-immuno profiling in naturally infected rodents and shrews.

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.