普氏野马分布区域亚洲璃眼蜱的宏基因组分析与病原体评估

Metagenomic analysis and potential assessment of Hyalomma asiaticum in the distribution area of Przewalski’s horses

目的评估亚洲璃眼蜱在普氏野马分布区域传播疾病的潜在风险,研究雌、雄蜱在宏基因组水平的特征并进行病原体分析。方法2022年4月,在新疆卡拉麦里山有蹄类自然保护区使用“守株待蜱”法采集硬蜱,利用体视镜进行形态学鉴定,提取48只蜱(雌、雄各24只)DNA,PCR扩增线粒体细胞色素C氧化酶第1亚基(COⅠ)序列进行分子生物学鉴定。将雌、雄亚洲璃眼蜱分组进行宏基因组测序,非冗余基因集与非冗余蛋白(NR)数据库进行比对,分析蜱携带的微生物群落组成;与京都基因与基因组百科全书(KEGG)数据库和抗生素抗性基因数据库(ARDB)进行比对,获取蜱及蜱传病原体的基因功能注释结果以及抗生素抗性功能注释结果。数据的比较采用t检验分析。结果共采集硬蜱124只,经形态学鉴定亚洲璃眼蜱成虫119只。硬蜱DNA扩增出约700 bp的阳性条带,经测序与亚洲璃眼蜱(GenBank:MH459386.1)的序列一致性为99%~100%。宏基因组测序经质控过滤共获得469327812条序列读长,开放阅读框预测得到836843~1094994条序列。NR数据库比对结果显示,亚洲璃眼蜱携带的细菌菌落丰度占注释到的总群落丰度的99.13%,共注释到细菌32门2040种,变形菌门和放线菌门为优势菌门,分别占细菌群落丰度的51.52%和44.35%;嗜吞噬细胞无形体为优势菌种,占细菌群落丰度的16.35%。病毒菌落丰度占注释到的总群落丰度的0.004%,共注释到病毒5门21种。雌蜱和雄蜱携带的细菌群落和病毒群落的丰富度及多样性差异均无统计学意义(t=-1.180、-1.729,均P>0.05)。KEGG基因功能注释到亚洲璃眼蜱新陈代谢基因占比最高(54.38%),主要的功能条目为氨基酸代谢、碳水化合物代谢、膜转运等;共识别出11352种蛋白通路,其中154种蛋白通路在雌蜱和雄蜱间的差异有统计学意义(t=-2.348,P<0.05)。ARDB注释到亚洲璃眼蜱携带154种抗生素抗性基因,包括49种抗性基因类型,主要抗性基因类型为Baca(占62.53%),抗生素类型为肝菌肽。大部分注释到的抗性基因与多重耐药相关,如Mexf、Mexb、Emrd、Mexw等。雌蜱和雄蜱在抗生素抗性基因类型的差异性对比结果显示,仅雌蜱的Emrd基因抗性高于雄蜱(t=-7.558,P<0.05)。结论本研究在宏基因组水平上揭示了亚洲璃眼蜱携带的主要细菌、病毒群落及多重耐药相关的抗生素抗性基因,雌蜱及其携带的病原体具有较高的物种丰度和基因丰度。

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 ste‑reomicroscope,and DNA of 48 ticks(24 males and 24 females)was extracted for molecular identification by PCR am‑plification of the mitochondrial cytochrome C oxidase subunit 1(COⅠ)sequence.Metagenomic sequencing of H.asiai⁃cum 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.Addi‑tionally,comparisons were made with the Kyoto encyclopedia of genes and genomes(KEGG)and the antibiotic resis‑tance 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 469327812 sequence reads were ob‑tained from metagenomic sequencing,and open reading frame prediction yielded 836843 to 1094994 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 2040 species identified.The predominant phyla are Proteobacteria and Actinobacteria,accounting for 51.52%and 44.35%of the bacterial community abundance,respec‑tively.The dominant species is Anaplasma phagocytophilum,accounting for 16.35%of the bacterial community abun‑dance.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 11352 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 dif‑ferent 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.