Simultaneous serological assessment of four zoonotic rickettsiae among dogs near the United States-Mexico border

Obligately intracellular rickettsiae cause a broad spectrum of disease in humans and animals, ranging from mild illness to life-threatening infections. Multiple species co-circulate along the southern United States of America-northern Mexico border, yet their seroprevalence in susceptible hosts remains incompletely understood. Dogs serve as key amplifying hosts for several of these pathogens, including Rickettsia rickettsii and Rickettsia massiliae, and have been shown to be infected by flea-borne Rickettsia typhi and Rickettsia felis. To better characterize exposure and potential co-infection patterns, we conducted a large binational seroepidemiologic study of 779 dogs from urban households and shelter settings across seven subregions along both sides of the border. Using a custom multiplex micro-immunofluorescence assay, we simultaneously screened for antibodies to R. rickettsii, R. massiliae, R. typhi, and R. felis. Overall, 41.2% of dogs were seroreactive to at least one pathogen, with the highest seroprevalence for R. felis (19.3%), followed by R. massiliae (15.7%), R. typhi (14.5%), and R. rickettsii (9.8%). Co-seroreactivity was common, particularly between R. typhi and R. felis, with 34.2% of R. typhi-seroreactive samples also seroreactive to R. felis, and 16.7% exhibiting high titers to both. In contrast, R. rickettsii and R. massiliae showed limited overlap (15.2% co-seroreactive; 6.4% with high titers), suggesting possible cross-protection or competitive exclusion. Spatial analyses revealed distinct geographic patterns: R. massiliae predominated in western Baja California, R. rickettsii was concentrated in Mexicali and the Rio Grande Valley, and R. felis was widely distributed. Seroreactivity patterns were generally consistent across age and sex but varied modestly between shelter and neighborhood dogs, particularly for R. typhi. These findings highlight the complex ecology of rickettsial pathogens in a binational context, underscore the importance of dogs as sentinels for human risk, and provide a foundation for future studies on vector-host-pathogen interactions, cross-protective immunity, and spatial epidemiological risk.