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The genus Leishmania includes approximately 53 species, 20 of which cause human leishmaniais; a significant albeit neglected tropical disease. Leishmaniasis has afflicted humans for millennia, but how ancient is Leishmania and where did it arise? These questions have been hotly debated for decades and several theories have been proposed. One theory suggests Leishmania originated in the Palearctic, and dispersed to the New World via the Bering land bridge. Others propose that Leishmania evolved in the Neotropics. The Multiple Origins theory suggests that separation of certain Old World and New World species occurred due to the opening of the Atlantic Ocean. Some suggest that the ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia evolved on Gondwana between 90 and 140 million years ago. In the present study a detailed molecular and morphological characterisation was performed on a novel Australian trypanosomatid following its isolation in Australia’s tropics from the native black fly, Simulium (Morops) dycei Colbo, 1976. Phylogenetic analyses were conducted and confirmed this parasite as a sibling to Zelonia costaricensis, a close relative of Leishmania previously isolated from a reduviid bug in Costa Rica. Consequently, this parasite was assigned the name Zelonia australiensis sp. nov. Assuming Z. costaricensis and Z. australiensis diverged when Australia and South America became completely separated, their divergence occurred between 36 and 41 million years ago at least. Using this vicariance event as a calibration point for a phylogenetic time tree, the common ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia appeared in Gondwana approximately 91 million years ago. Ultimately, this study contributes to our understanding of trypanosomatid diversity, and of Leishmania origins by providing support for a Gondwanan origin of dixenous parasitism in the Leishmaniinae.

The cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae) (Bouché), is the most common flea species found on cats and dogs worldwide. We investigated the genetic identity of the cosmopolitan subspecies C. felis felis and evaluated diversity of cat fleas from Australia, Fiji, Thailand and Seychelles using mtDNA sequences from cytochrome c oxidase subunit I (cox1) and II (cox2) genes. Both cox1 and cox2 confirmed the high phylogenetic diversity and paraphyletic origin of C. felis felis. The African subspecies C. felis strongylus (Jordan) is nested within the paraphyletic C. felis felis. The south East Asian subspecies C. felis orientis (Jordan) is monophyletic and is supported by morphology. We confirm that Australian cat fleas belong to C. felis felis and show that in Australia they form two distinct phylogenetic clades, one common with fleas from Fiji. Using a barcoding approach, we recognize two putative species within C. felis (C. felis and C. orientis). Nucleotide diversity was higher in cox1 but COX2 outperformed COX1 in amino acid diversity. COX2 amino acid sequences resolve all phylogenetic clades and provide an additional phylogenetic signal. Both cox1 and cox2 resolved identical phylogeny and are suitable for population structure studies of Ctenocephalides species.

Fleas (Siphonaptera) are ubiquitous blood-sucking pests of animals worldwide and are vectors of zoonotic bacteria such as Rickettsia and Bartonella. We performed Ion Torrent PGM amplicon sequencing for the bacterial 16S rRNA gene to compare the microbiome of the ubiquitous cat flea (Ctenocephalides f. felis) and the host-specific echidna stickfast flea (Echidnophaga a. ambulans) and evaluated potential bias produced during common genomic DNA-isolation methods. We demonstrated significant differences in the bacterial community diversity between the two flea species but not between protocols combining surface sterilisation with whole flea homogenisation or exoskeleton retention. Both flea species were dominated by obligate intracellular endosymbiont Wolbachia, and the echidna stickfast fleas possessed the endosymbiont Cardinium. Cat fleas that were not surface sterilised showed presence of Candidatus 'Rickettsia senegalensis' DNA, the first report of its presence in Australia. In the case of Rickettsia, we show that sequencing depth of 50 000 was required for comparable sensitivity with Rickettsia qPCR. Low-abundance bacterial genera are suggested to reflect host ecology. The deep-sequencing approach demonstrates feasibility of pathogen detection with simultaneous quantitative analysis and evaluation of the inter-relationship of microbes within vectors.

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BackgroundFleas of the genus Ctenocephalides serve as vectors for a number of rickettsial zoonoses, including Rickettsia felis. There are currently no published reports of the presence and distribution of R. felis in India, however, the ubiquitous distribution of its vector Ctenocephalides felis, makes it possible that the pathogen is endemic to the region. This study investigates the occurrence of Rickettsia spp. infection in various subspecies of C. felis infesting dogs from urban areas of Mumbai, Delhi and Rajasthan in India.MethodsIndividual fleas collected off 77 stray dogs from Mumbai, Delhi and Rajasthan were screened for Rickettsia spp. by a conventional PCR targeting the ompB gene. Further genetic characterisation of Rickettsia-positive fleas was carried out using nested PCR and phylogenetic analysis of partial DNA sequences of the gltA and ompA genes. Ctenocephalides spp. were morphologically and genetically identified by PCR targeting a fragment of cox1 gene.ResultsOverall, 56/77 fleas (72.7%), including 22/24 (91.7%) from Delhi, 32/44 (72.7%) from Mumbai and 2/9 (22.2%) from Rajasthan were positive for Rickettsia DNA at the ompB gene. Sequences of gltA fragments confirmed the amplification of Rickettsia sp. genotype RF2125. The ompA gene of Rickettsia sp. genotype RF2125 was characterised for the first time and shown 96% identical to R. felis. Three species of Ctenocephalides were identified, with the Ctenocephalides felis orientis being the dominant flea species (69/77; 89.6%) in India, followed by Ctenocephalides felis felis (8/77; 10.4%).ConclusionsHigh occurrence of Rickettsia sp. genotype RF2125 in C. felis orientis and the absence of R. felis suggests a specific vector-endosymbiont adaptation and coevolution of the Rickettsia felis-like sp. within subspecies of C. felis.

Australian mosquito species significantly impact human health through nuisance biting and the transmission of endemic and exotic pathogens. Surveillance programmes designed to provide an early warning of mosquito-borne disease risk require reliable identification of mosquitoes. This study aimed to investigate the viability of Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a rapid and inexpensive approach to the identification of Australian mosquitoes and was validated using a three-step taxonomic approach. A total of 300 mosquitoes representing 21 species were collected from south-eastern New South Wales and morphologically identified. The legs from the mosquitoes were removed and subjected to MALDI-TOF MS analysis. Fifty-eight mosquitoes were sequenced at the cytochrome c oxidase subunit I (cox1) gene region and genetic relationships were analysed. We create the first MALDI-TOF MS spectra database of Australian mosquito species including 19 species. We clearly demonstrate the accuracy of MALDI-TOF MS for identification of Australian mosquitoes. It is especially useful for assessing gaps in the effectiveness of DNA barcoding by differentiating closely related taxa. Indeed, cox1 DNA barcoding was not able to differentiate members of the Culex pipiens group, Cx. quinquefasciatus and Cx. pipiens molestus, but these specimens were correctly identified using MALDI-TOF MS.