Rickettsial agents from fleas infesting stray dogs and cats in rural and urban communities in Malaysia

Abstract

Background: Rickettsial infections are rarely diagnosed in Malaysia, due to the lack of awareness and useful diagnostic tools in the clinical setting. Seroprevalence studies among the indigenous people of Malaysia have shown evidence of exposure to rickettsial infections; however, not much is known about the causative agents. More studies are necessary to generate the resources, including genetic information and pathogen isolates, for development of more effective diagnostics. Stray dogs and cats are commonly found in rural and urban areas in Malaysia. Fleas infesting these animals have shown to harbour rickettsial agents which could be transmitted to humans. This study aims to identify and isolate the rickettsial agents in fleas recovered from stray animals found in two study sites in Malaysia. Methods & Materials: The study sites include a rural community of indigenous people in Perak and a mixed residential-commercial area within Kuala Lumpur, the capital city of Malaysia. Fleas were recovered from stray animals near residences or shop-lots. Fleas were pooled according to species, gender and animal hosts, and were used for DNA extraction. Polymerase chain reaction (PCR) amplification of rickettsial partial 16s rRNA gene sequence was performed to determine the presence of rickettsial DNA. For isolation, flea tissues were inoculated into tick cell cultures and monitored periodically for signs of cytopathic effects, and the presence of bacteria monitored by Giemsa-staining and PCR. Results: The fleas recovered were morphologically identified as Ctenocephalides felis and Ctenocephalides orientis. 47/54 flea pools (total individuals = 277) were tested positive in the PCR. The amplicon sequences were closely related to Rickettsia asembonensis (identity: 99–100%), a Rickettsia felis-like organism (RFLO). Sequences closely related to Bartonella clarridgeiae (identity: 98–100%) and Bartonella henselae (identity: 100%) were also detected. Attempts to isolate these bacteria in cell cultures are currently underway. On-going efforts include the whole genome sequencing of these flea-associated bacteria for further genetic characterization. Conclusion: These findings suggest that RFLO and Bartonella spp. are commonly present in fleas infesting stray dogs and cats in rural and urban communities in Malaysia. Further investigation is necessary to determine if the RFLO contribute to the rickettsial infections evidenced by past seroprevalence studies.