Abstract:

Objective To evaluate the potential risk of disease transmission by Hyalomma asiaticum in the distribution area of Przewalski’s horses, investigate the metagenomic characteristics and conduct pathogen analysis of male and female ticks. Methods In April 2022, tick samples were collected using the “waiting for ticks” method in the Kalamaili Mountain Ungulate Nature Reserve in Xinjiang. The ticks were morphologically identified under a stereomicroscope, and DNA of 48 ticks (24 males and 24 females) was extracted for molecular identification by PCR amplification of the mitochondrial cytochrome C oxidase subunit 1 (COⅠ) sequence. Metagenomic sequencing of H. asiaicum was conducted by grouping the ticks according to sex. The non-redundant sequences were compared to the non-redundant protein (NR) database to analyze the composition of the microbial communities carried by the ticks. Additionally, comparisons were made with the Kyoto encyclopedia of genes and genomes (KEGG) and the antibiotic resistance genes database (ARDB) to obtain the annotations for gene function and the antibiotic resistance functions in ticks and tick-borne pathogens. The data were analyzed using t-test. Results A total of 124 ticks were collected and morphological identification revealed that 119 were H. asiaicum adult. The PCR amplification result showed that the positive production with a length of 700 bp were amplified from tick DNA and the sequences were 99%-100% identical to H. asiaicum (Genbank: MH459386.1). After quality control filtering, a total of 469 327 812 sequence reads were obtained from metagenomic sequencing, and open reading frame prediction yielded 836 843 to 1 094 994 sequences. The NR database comparison revealed that the bacterial community abundance carried by H. asiaicum accounted for 99.13% of the total community abundance, with bacteria from 32 phyla and 2 040 species identified. The predominant phyla are Proteobacteria and Actinobacteria, accounting for 51.52% and 44.35% of the bacterial community abundance, respectively. The dominant species is Anaplasma phagocytophilum, accounting for 16.35% of the bacterial community abundance. The viral community abundance accounts for 0.004% of the total community abundance, with viruses from 5 phyla and 21 species identified. There were no significant differences in the richness and diversity of bacterial and viral communities between female and male ticks (t = -1.180、-1.729, both P > 0.05). KEGG gene function analysis revealed that the highest proportion of genes in H. asiaticum were involved in metabolism (54.38%), with the primary functional categories include amino acid metabolism, carbohydrate metabolism, and membrane transport. A total of 11 352 pathways were identified, 154 of which exhibited statistically significant differences between female and male ticks (t = -2.348, P < 0.05). ARDB analysis revealed that H. asiaticum carried 154 antibiotic resistance genes, comprising 49 different types. The major type of resistance gene was Baca (62.53%), and the main class of antibiotics was glycopeptides. The majority of resistance genes were associated with multi-drug resistance, including Mexf, Mexb, Emrd, Mexw and others. The resistance of Emrd gene in female ticks were higher than male (t = -7.558, P < 0.05). Conclusion This study revealed the major bacterial and viral communities carried by the H. asiaticum at the metagenomic level, along with multidrug resistance-related antibiotic resistance genes. Female ticks and their associated pathogens exhibited higher species richness and gene abundance.

Key words: Hyalomma asiaticum, Tick-borne disease, Metagenomics, Gender difference, Przewalski’s horses