Molecular evidence for infections with the scrub typhus agent (Orientia tsutsugamushi) in bird-associated chiggers and ground birds

Background Orientia tsutsugamushi is an obligate intracellular bacterium responsible for scrub typhus. It exists as a symbiont within trombiculid mites, of which only the larval stage (or “chigger”) is parasitic. While the role of small mammals in maintaining this vector–pathogen cycle is well-documented, the involvement of birds as hosts for chiggers and/or O. tsutsugamushi remains understudied. Methods Here, we screened chigger mites collected from various bird species in a scrub typhus-endemic country (Malaysia) for O. tsutsugamushi infection, together with organ and tissue samples from red junglefowl ( Gallus gallus ), domestic chickens ( Gallus gallus domesticus ) and house crows ( Corvus splendens ). Chiggers were pooled by species per bird host and assayed using a nested polymerase chain reaction (nPCR) targeting the htrA gene, with a subset of pools subjected to Illumina metagenomic sequencing. Leptotrombidium chiangraiensis chiggers from an O. tsutsugamushi -positive colony maintained in Thailand were used to validate the sequencing strategy. Bird tissue samples were also assayed by htrA -targeted nPCR. Results Illumina sequencing of pools of L . chiangraiensis from an infected colony showed good coverage across almost the entire O. tsutsugamushi genome. However, O. tsutsugamushi was not detected in pooled DNA samples from bird-associated chiggers by nPCR and neither did metagenomic analysis identify O. tsutsugamushi sequences, except in a Blankaartia acuscutellaris pool from a wild slaty-breasted rail ( Lewinia striata ). Multiple contigs related to Rickettsiales amplified genetic elements of Orientia origin were observed in this sample, but no core genes were assembled that could be used for strain typing. Among galliform birds and house crows, O. tsutsugamushi DNA was detected in one red junglefowl and one domestic chicken sample by nPCR, and phylogenetic analysis clustered these sequences with Karp isolates. Conclusions Although Orientia infections in birds and associated chiggers appear rare, our findings suggest that these hosts may contribute to pathogen ecology and dispersal, highlighting the need for broader avian surveillance.