A Longitudinal Molecular Surveillance of Genetic heterogeneity of Orientia tsutsugamushi in humans, reservoir animals, and vectors in Puducherry, India

Krishan Kumar Sihag^1,2Waseema A³Srikanth Srirama^1*Anand Kumar Chandrasekaran⁴Vinod Raveendran⁵Asayas Bosco Chandrakumar⁶Anand Kasirajan⁷Sivagamy Alias Punitha Thavaraj⁸Lakshmy Srinivasan¹Anoop C Choolayil¹Mathivanan Ashokkumar⁹Amala Ramasamy¹⁰Panneer Devaraju^1,2*

• ¹Unit of One Health, ICMR- Vector Control Research Centre (ICMR-VCRC), Indira Nagar, Puducherry, India
• ²ICMR- Vector Control Research Centre (ICMR-VCRC), affiliated to Pondicherry University, Kalapet, Puducherry, India
• ³Unit One Health, ICMR- Vector Control Research Centre (ICMR-VCRC), Indira Nagar, Puducherry, India
• ⁴Indira Gandhi Medical College and Research Institute, Puducherry, India
• ⁵Sri Venkateshwaraa Medical College Hospital and Research Centre, Ariyur, Puducherry, India
• ⁶Sri Lakshmi Narayana Institute of Medical Sciences, Puducherry, India
• ⁷Mahatma Gandhi Medical Advance Research Institute (MGMARI), Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, India
• ⁸ICMR- Vector Control Research Centre (ICMR-VCRC), Puducherry, Indira Nagar, Puducherry, India
• ⁹Unit of Microbiology and Immunology, ICMR- Vector Control Research Centre (ICMR-VCRC), Puducherry, Indira Nagar, Puducherry, India
• ¹⁰Unit of Biostatistics and VBD Modeling, ICMR- Vector Control Research Centre (ICMR-VCRC), Puducherry, Indira Nagar, Puducherry, India

Background: Scrub typhus (ST), is a vector borne zoonotic disease, transmitted by the larva of the trombiculid mites. The enzootic cycle of the pathogen involves rodents/shrews as the animal reservoirs and humans are the accidental dead-end host. A transposon-like activity in its major antigen 56 kDa, has led to the evolution of several serotypes/strains, and more than 40 serotypes are reported globally. Puducherry, India, is endemic to scrub, but limited data exist on local serotype distribution across hosts. and vectors. Methodology: A longitudinal molecular surveillance was conducted in Puducherry to investigate the genetic diversity of O. tsutsugamushi among humans, animal reservoirs, and vectors. Samples from febrile patients, trapped rodents/shrews, and their infesting mites were screened using real-time and nested PCR. Serotype analysis was performed by partial amplification and sequencing of the 56 kDa gene, followed by phylogenetic, pairwise genetic distance and amino acid analysis. Result: ST infection was detected in 4.37% (95% CI: 3.05%-5.71%) of human, 11.52% (95% CI: 8.6%-14.4%) of rodent/shrew, and 2.36% (95% CI: 0.95%-4.87%) of mite samples. Karp-like (51.72%) and Gilliam-like (41.38%) strains were predominant in both humans and animal hosts, with pairwise genetic distance (<0.1) and amino acid identity (>85%) analysis revealing a close relationship between the strains identified across the region. Notably, the only mite pool that tested positive for the 56 kDa gene, along with a shrew, was identified to belong to the TA678-like serotype (6.90%), which has not been previously reported from Puducherry.This study provides molecular evidence of the enzootic maintenance and active human transmission of O. tsutsugamushi in Puducherry, with multiple co-circulating serotypes.The first detection of the TA678-like strain in the region suggests the possible introduction of new strains and underscores the need to monitor for strain-specific clinical manifestations in future studies