Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2–5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.

Chigger mites are vectors of the bacterial disease scrub typhus, caused by Orientia spp. The bacterium is vertically transmitted in the vector and horizontally transmitted to terrestrial vertebrates (primarily wild small mammals), with humans as incidental hosts. Previous studies have shown that the size of the chigger populations is correlated with the density of small mammals in scrub typhus-endemic regions. Here, we explore interactions between the small mammals and chiggers in two oil palm plantations located in the Perak and Johor states of Peninsular Malaysia. The location in Perak also contained an aboriginal (Orang Asli) settlement. A ~5% sub-sample from 40,736 chigger specimens was identified from five species of small mammals (n = 217), revealing 14 chigger species, including two new records for Malaysia. The abundance and species richness of chiggers were significantly affected by habitat type (highest in forest border), state (highest in Perak), and season (highest in dry). The overall prevalence of Orientia tsutsugamushi DNA in small-mammal tissues was 11.7% and was not significantly affected by host or habitat characteristics, but in Johor, was positively associated with infestation by Leptotrombidium arenicola. These findings highlight the risk of contracting scrub typhus in oil palm plantations and associated human settlements.

No abstract

While fleas are often perceived simply as a biting nuisance and a cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat (Ctenocephalides felis) and dog (Ctenocephalides canis) fleas, as well as Ctenocephalides orientis (restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused by Bartonella spp.) and Rickettsia spp., including Rickettsia felis (agent of flea-borne spotted fever) and Rickettsia asembonensis , a suspected pathogen. These Rickettsia spp. are members of a phylogenetic clade known as the ‘transitional group’, which includes both human pathogens and arthropod-specific endosymbionts. The relatively depauperate flea microbiome can also contain other endosymbionts, including a diverse range of Wolbachia strains. Here, we present circularized genome assemblies for two C. orientis-derived pathogens ( Bartonella clarridgeiae and R. asembonensis ) from Malaysia, a novel Wolbachia strain (wCori), and the C. orientis mitochondrion; all were obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated two Wolbachia strains from Malaysian C. felis into tick cell culture and recovered circularized genome assemblies for both, one of which (wCfeF) is newly sequenced. We demonstrate that the three Wolbachia strains are representatives of different major clades (‘supergroups’), two of which appear to be flea-specific. These Wolbachia genomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors and the biotin operon of obligate intracellular microbes. The first circularized assembly for R. asembonensis includes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the USA. Analysis of loci under positive selection in the transitional group revealed genes involved in host–pathogen interactions that may facilitate host switching. Finally, the first B. clarridgeiae genome from Asia exhibited large-scale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity of Ctenocephalides-associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.