Identification of Vietnamese Flea Species and Their Associated Microorganisms Using Morphological, Molecular, and Protein Profiling

Fleas are obligatory blood-sucking ectoparasites of medical and veterinary importance. The identification of fleas and associated flea-borne microorganisms, therefore, plays an important role in controlling and managing these vectors. Recently, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been reported as an innovative and effective approach to the identification of arthropods, including fleas. This study aims to use this technology to identify ethanol-preserved fleas collected in Vietnam and to use molecular biology to search for microorganisms associated with these fleas. A total of 502 fleas were collected from wild and domestic animals in four provinces in Vietnam. Morphological identification led to the recognition of five flea species, namely Xenopsylla cheopis, Xenopsylla astia, Pulex irritans, Ctenocephalides canis, and Ctenocephalides felis. The cephalothoraxes of 300 individual, randomly selected fleas were tested using MALDI-TOF MS and molecular analysis for the identification and detection of microorganisms. A total of 257/300 (85.7%) of the obtained spectra from the cephalothoraxes of each species were of good enough quality to be used for our analyses. Our laboratory MALDI-TOF MS reference database was upgraded with spectra achieved from five randomly selected fleas for every species of Ctenocephalides canis and Ctenocephalides felis. The remaining spectra were then queried against the upgraded MALDI-TOF MS database, which showed 100% correspondence between morphology and MALDI-TOF MS identification for two flea species (Ctenocephalides canis and Ctenocephalides felis). The MS spectra of the remaining species (three P. irritans, five X. astia, and two X. cheopis) were visually generated low-intensity MS profiles with high background noise that could not be used to update our database. Bartonella and Wolbachia spp. were detected in 300 fleas from Vietnam using PCR and sequencing with primers derived from the gltA gene for Bartonella and the 16S rRNA gene for Wolbachia, including 3 Bartonella clarridgeiae (1%), 3 Bartonella rochalimae (1%), 1 Bartonella coopersplainsensis (0.3%), and 174 Wolbachia spp. endosymbionts (58%).