Diversity, spatiotemporal distribution, and host associations of flea species, with detection of co-infection with Rickettsia asembonensis and Wolbachia spp.

Fleas are ectoparasites of wild and domestic animals and can transmit various pathogens, including Rickettsia species, while also harboring endosymbiotic Wolbachia species. Although some studies have morphologically identified certain fleas along with their hosts, no reports have documented the spatiotemporal occurrence of fleas and/or molecular screening of flea-associated microorganisms in Pakistan. A total of 617 hosts were examined, including dogs (253, 41.0%), goats (282, 45.7%), humans (44, 7.1%), rats (19, 3.1%), and porcupines (19, 3.1%). The collected fleas were morphologically identified as Ctenocephalides canis (347/534, 65.0%), Ctenocephalides felis (120/534, 22.5%), Xenopsylla cheopis (16/534, 3.0%), and Pulex irritans (51/534, 9.6%). Overall, 259 hosts (42.0%) were found to be infested, with the highest prevalence in dogs (152/253, 60.1%), followed by porcupines (8/19, 42.1%), goats (87/282, 30.9%), rats (5/19, 26.3%), and humans (7/44, 15.9%). Ctenocephalides canis and C. felis were collected on both dogs and goats, while X. cheopis was collected from rats, and P. irritans on both humans and porcupines. In the collected samples, more fleas were observed during summer than winter. The obtained Rickettsiales gltA sequence from C. canis showed 99.7% identity with Rickettsia asembonensis previously reported from C. canis and C. felis from Brazil, Colombia, and Peru, whereas amplification of ompA and ompB was unsuccessful. The obtained 16S rDNA sequence of Rickettsiales detected in C. canis and C. felis showed 99.8% and 99.7% identity, respectively, with siphonaptern-associated Wolbachia spp. reported from Malaysia and Brazil. Similarly, the obtained coxA sequence of Rickettsiales from C. canis and C. felis showed 100% and 99.7% identity, respectively, with siphonaptern-associated Wolbachia spp. reported from Malaysia and the United States. Out of 80 screened fleas, 35/80 (43.8%) were positive for Wolbachia spp., and 16/80 (20%) for R. asembonensis, while 7/80 (8.8%) fleas were co-infected with Wolbachia spp. and R. asembonensis. Phylogenetically, gltA sequence of R. asembonensis was clustered with the same species reported from Peru, Brazil, and Colombia. Similarly, 16S rDNA sequences of Wolbachia spp. from C. canis and C. felis clustered with the Wolbachia spp. previously detected in C. canis from China and Turkey and Ctenocephalides orientis from Malaysia, and in C. felis from Brazil and the United States, respectively. The coxA sequences of Wolbachia spp. from C. canis and C. felis clustered with Wolbachia spp. previously detected in C. orientis in Malaysia and in C. felis from United States, respectively. Besides flea diversity, this preliminary study, for the first time in Pakistan, investigated spatiotemporal distribution and host associations of fleas, as well as molecular assessment of flea-associated microorganisms.