Sequence analysis method.

<div><p>Arthropod-borne rickettsioses comprise a wide variety of subtypes that are endemic in Cambodia, but there remains very little data on the geographic distribution of the pathogens or their vectors. Surveys were conducted in Koh Kong and Preah Sihanouk Provinces between September 2017 and June 2018 to collect ectoparasites from peridomestic animals and the environment using dragging and flagging methods. Collected ectoparasites were sorted and identified morphologically, then pooled by species, host, and location for molecular detection using <i>Rickettsia</i> genus- and species-specific qPCR and/or multilocus sequence typing (MLST) assays. A total of 14,254 ectoparasites were collected including seven new locality records. <i>Rickettsia</i> species were detected in 35.5% (174/505) of the pools screened representing 3,149 randomly selected ectoparasites from the total collected. <i>Rickettsia asembonensis</i> was detected in 89.6% (147/164) of <i>Rickettsia</i>-positive flea pools and 3.6% (6/164) of the flea pools were positive for both <i>R</i>. <i>asembonensis</i> and <i>Rickettsia felis</i>. <i>Candidatus</i> Rickettsia senegalensis from <i>Ctenocephalides orientis</i> fleas and <i>Rickettsia</i> sp. close to <i>Rickettsia japonica</i> and <i>Rickettsia heilongjiangensis</i> from <i>Haemaphysalis</i> ticks were identified by MLST. This appears to be the first report of these new ectoparasite records and rickettsial species in southern Cambodia, suggesting a potential health risk to military and civilians in this region.</p></div>