Dromedary camels (Camelus dromedarius) are widely distributed in Africa, the Middle East and northern India. In this study, we aimed to detect tick-borne pathogens through investigating prokaryotic and eukaryotic microorganisms in camel blood based on a metagenomic approach and then to characterize potentially pathogenic organisms using traditional molecular techniques. We showed that the bacteria circulating in the blood of camels is dominated by Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. At the genus level, Sediminibacterium, Hydrotalea, Bradyrhizobium and Anaplasma were the most abundant taxa. Eukaryotic profile was dominated by Fungi, Charophyta and Apicomplexa. At the genus level, Theileria was detected in 10 out of 18 samples, while Sarcocystis, Hoplorhynchus and Stylocephalus were detected in one sample each. Our metagenomic approach was successful in the detection of several pathogens or potential pathogens including Anaplasma sp., Theileria ovis, Th. separata, Th. annulate, Th. mutans-like and uncharacterized Theileria sp. For further characterization, we provided the partial sequences of citrate synthase (gltA) and heat-shock protein (groEL) genes of Candidatus Anaplasma camelii. We also detected Trypanosoma evansi type A using polymerase chain reaction (PCR) targeting the internal transcribed spacer 1 (ITS1) region. This combined metagenomic and traditional approach will contribute to a better understanding of the epidemiology of pathogens including tick-borne bacteria and protozoa in animals.

Ticks are the second most important vector capable of transmitting diseases affecting the health of both humans and animals. Amblyomma testudinarium Koch 1844 (Acari: Ixodidae), is a hard tick species having a wide geographic distribution in Asia. In this study, we analyzed the composition of A. testudinarium whole mitogenomes from various geographical regions in Japan and investigated the population structure, demographic patterns, and phylogeographic relationship with other ixodid species. In addition, we characterized a potentially novel tick species closely related to A. testudinarium from Myanmar. Phylogeographic inference and evolutionary dynamics based on the 15 mitochondrial coding genes supported that A. testudinarium population in Japan is resolved into a star‐like haplogroup and suggested a distinct population structure of A. testudinarium from Amami island in Kyushu region. Correlation analysis using Mantel test statistics showed that no significant correlation was observed between the genetic and geographic distances calculated between the A. testudinarium population from different localities in Japan. Finally, demographic analyses, including mismatch analysis and Tajima’s D test, suggested a possibility of recent population expansion occurred within Japanese haplogroup after a bottleneck event. Although A. testudinarium has been considered widespread and common in East and Southeast Asia, the current study suggested that potentially several cryptic Amblyomma spp. closely related to A. testudinarium are present in Asia.

Members of the genus Spiroplasma are Gram-positive bacteria without cell walls. Some Spiroplasma species can cause disease in arthropods such as bees, whereas others provide their host with resistance to pathogens. Ticks also harbour Spiroplasma, but their role has not been elucidated yet. Here, the infection status and genetic diversity of Spiroplasma in ticks were investigated using samples collected from different geographic regions in Japan. A total of 712 ticks were tested for Spiroplasma infection by PCR targeting 16S rDNA, and Spiroplasma species were genetically characterized based on 16S rDNA, ITS, dnaA, and rpoB gene sequences. A total of 109 samples originating from eight tick species were positive for Spiroplasma infection, with infection rates ranging from 0% to 84% depending on the species. A linear mixed model indicated that tick species was the primary factor associated with Spiroplasma infection. Moreover, certain Spiroplasma alleles that are highly adapted to specific tick species may explain the high infection rates in Ixodes ovatus and Haemaphysalis kitaokai. A comparison of the alleles obtained suggests that horizontal transmission between tick species may not be a frequent event. These findings provide clues to understand the transmission cycle of Spiroplasma species in wild tick populations and their roles in host ticks.

Ticks serve as important vectors of a variety of pathogens. Recently, the viral and prokaryotic microbiomes in ticks have been explored using next-generation sequencing to understand the physiology of ticks and their interactions with pathogens. However, analyses of eukaryotic communities in ticks are limited, owing to the lack of suitable methods. In this study, we developed new methods to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, another PCR using non-metazoan primers, referred to as UNonMet-PCR, was performed for comparison. We performed each PCR using tick-derived DNA and sequenced the amplicons using the Illumina MiSeq platform. Almost all sequences obtained by conventional PCR were derived from ticks, whereas the proportion of microeukaryotic reads and alpha diversity increased upon using the newly developed method. Additionally, the PNA- or LNA-based methods were suitable for paneukaryotic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi. The newly described methods enable analyses of the eukaryotic microbiome in ticks. We expect the application of these methods to improve our understanding of the tick microbiome.