Metagenomic analysis of Rhipicephalus microplus from minority autonomous prefectures in Guizhou province, China

doi:10.11853/j.issn.1003.8280.2023.03.007

Abstract

Abstract: Objective To investigate the microbiota of the dominant tick species Rhipicephalus microplus in the minority autonomous prefectures of Guizhou province, China, and to provide a scientific basis for the prevention and control of tick-borne disease.Methods Rh. microplus ticks were collected from the body surface of cattle and sheep at 18 sampling sites located in Qiandongnan Miao and Dong Autonomous Prefecture, Qiannan Buyi and Miao Autonomous Prefecture, and Qianxinan Buyi and Miao Autonomous Prefecture in April and July of 2019 and 2020. The ticks were grouped by region, growth stage, and sex, with three parallel samples in each group, followed by metagenomic sequencing. The sequencing results were processed with quality control and assembly procedures before homology alignment with the non-redundant protein database of National Center for Biotechnology Information to obtain taxonomic annotation information. R (v. 3.6.3) and GraPhlAn (v. 1.1.3) were used for visual analysis, including species composition analysis, non-metric multidimensional scaling (NMDS) analysis, and analysis of similarities (ANOSIM).Results The dominant bacterial phylum for Rh. microplus was Firmicutes (60.70%), followed by Proteobacteria (33.76%) and Actinobacteria (4.53%). Staphylococcus aureus (38.29%) was the dominant bacterial species, followed by Klebsiella pneumoniae (7.79%) and Streptococcus pneumoniae (5.26%). Several tick-borne pathogens were detected, including various genotypes of spotted fever group Rickettsia and Anaplasma phagocytophilum (1.38%). R. fournieri (2.91%) was an emerging rickettsiae detected from ticks for the first time in China, and R. japonica (1.76%) and R. monacensis (0.04%) were common tick-borne Rickettsia genotypes in China. R. fournieri and R. japonica were mainly carried by male ticks. The NMDS analysis showed that the microbiota composition of Rh. microplus differed by growth stage and sex. The ANOSIM analysis indicated reasonable grouping, with greater intergroup differences than intragroup differences (R=0.147, P=0.014).Conclusions In the minority autonomous prefectures of Guizhou province, Rh. microplus ticks have a diverse microbiota composition, carrying pathogens that may cause spotted fever and human granulocytic anaplasmosis. Therefore, vector surveillance and pathogen detection should be strengthened, and effective control measures should be taken to reduce the risk of tick-borne diseases.

Key words: Rhipicephalus microplus, Metagenomic analysis, Bacteria, Spotted fever group Rickettsia, Anaplasma phagocytophilum, Guizhou province

摘要： 目的调查贵州省少数民族自治州的优势寄生蜱种微小扇头蜱的微生物菌群,为蜱传疾病防控提供科学依据。方法 2019－2020年4和7月在黔东南苗族侗族自治州、黔南布依族苗族自治州和黔西南布依族苗族自治州的18个采样点捕捉牛、羊体表的微小扇头蜱。将捕获的微小扇头蜱按地区、生长阶段和性别分组,每组3个平行样,进行宏基因组测序。测序结果经质控、拼接组装等操作后与美国国立生物技术信息中心（NCBI）非冗余蛋白库进行同源性比对,获得物种分类注释信息。然后通过R（v. 3.6.3）和GraPhlAn（v. 1.1.3）软件进行可视化分析,包括物种组成分析、非度量多维尺度（NMDS）分析和组间相似性分析（ANOSIM）。结果微小扇头蜱的优势菌门为厚壁菌门（60.70%）,其次为变形菌门（33.76%）和放线菌门（4.53%）。优势菌种为金黄色葡萄球菌（38.29%）,其次是肺炎克雷伯菌（7.79%）和肺炎链球菌（5.26%）。检出多种蜱传病原体,包括斑点热群立克次体的多种基因型和嗜吞噬细胞无形体（1.38%）。Rickettsia fournieri（2.91%）是中国首次从蜱中检出的新发立克次体,日本立克次体（1.76%）和R. monacensis（0.04%）是中国常见的蜱传立克次体基因型。R. fournieri和日本立克次体主要由雄蜱携带。NMDS分析表明,不同生长阶段、不同性别微小扇头蜱的微生物菌群组成不同。ANOSIM分析结果显示分组合理,组间差异大于组内差异（R=0.147,P=0.014）。结论贵州省少数民族自治州的微小扇头蜱微生物菌群组成丰富,携带的病原体可能会导致斑点热和人粒细胞无形体病,应加强相关媒介监测和病原体检测,采取相关防控措施,降低蜱传疾病发病风险。

关键词: 微小扇头蜱, 宏基因组分析, 细菌, 斑点热群立克次体, 嗜吞噬细胞无形体, 贵州省

CLC Number:

R384.4
R372

XIANG Yu-long, ZHOU Jing-zhu, ZHANG Yan, HU Yong, LIANG Wen-qin. Metagenomic analysis of Rhipicephalus microplus from minority autonomous prefectures in Guizhou province, China[J]. Chinese Journal of Vector Biology and Control, 2023, 34(3): 319-325.

向昱龙, 周敬祝, 张燕, 胡勇, 梁文琴. 贵州省少数民族自治州微小扇头蜱的宏基因组分析[J]. 中国媒介生物学及控制杂志, 2023, 34(3): 319-325.

References

[1] Wikel SK. Ticks and tick-borne infections:Complex ecology,agents,and host interactions[J]. Vet Sci,2018,5(2):60. DOI:10.3390/vetsci5020060.
[2] Xiang LL,Poźniak B,Cheng TY. Bacteriological analysis of saliva from partially or fully engorged female adult Rhipicephalus microplus by next-generation sequencing[J]. Antonie Van Leeuwenhoek,2017,110(1):105-113. DOI:10.1007/s10482-016-0780-8.
[3] Gómez GF,Isaza JP,Segura JA,et al. Metatranscriptomic virome assessment of Rhipicephalus microplus from Colombia[J]. Ticks Tick-borne Dis,2020,11(5):101426. DOI:10.1016/j.ttbdis. 2020.101426.
[4] Segura JA,Isaza JP,Botero LE,et al. Assessment of bacterial diversity of Rhipicephalus microplus ticks from two livestock agroecosystems in Antioquia,Colombia[J]. PLoS One,2020,15(7):e0234005. DOI:10.1371/journal.pone.0234005.
[5] Anderson JF,Magnarelli LA. Biology of ticks[J]. Infect Dis Clin North Am,2008,22(2):195-215. DOI:10.1016/j.idc.2007. 12.006.
[6] Galay RL,Llaneta CR,Monreal MKFB,et al. Molecular prevalence of Anaplasma marginale and Ehrlichia in domestic large ruminants and Rhipicephalus (Boophilus) microplus ticks from Southern Luzon,Philippines[J]. Front Vet Sci,2021,8. DOI:10.3389/fvets.2021.746705.
[7] 杨茂生,吴位珩,杨莉,等. 贵州微小牛蜱的检测与防制研究[J]. 贵州农业科学,2009,37(11):157-159. DOI:10.3969/j.issn.1001-3601.2009.11.049.Yang MS,Wu WH,Yang L,et al. Study on detection and control of Boophilus microplus in Guizhou[J]. Guizhou Agric Sci,2009,37(11):157-159. DOI:10.3969/j.issn.1001-3601.2009.11.049.(in Chinese)
[8] Maldonado-Ruiz LP,Neupane S,Park Y,et al. The bacterial community of the lone star tick (Amblyomma americanum)[J]. Parasit Vector,2021,14(1):49. DOI:10.1186/s13071-020-04550-z.
[9] 杨小娜,张琳,侯学霞,等. 16S rDNA全长高通量测序在蜱媒病原生物多样性研究中的应用[J]. 中国媒介生物学及控制杂志,2021,32(4):404-411. DOI:10.11853/j.issn.1003.8280. 2021.04.004.Yang XN,Zhang L,Hou XX,et al. Application of 16S rDNA full-length high-throughput sequencing in the study of tick-borne pathogen biodiversity[J]. Chin J Vector Biol Control,2021,32(4):404-411. DOI:10.11853/j.issn.1003.8280.2021.04.004.(in Chinese)
[10] 黄邵军,张一,罗学辉,等. 基于高通量测序技术分析浙江省余姚市蜱中携带细菌菌群多样性[J]. 疾病监测,2020,35(7):642-645. DOI:10.3784/j.issn.1003-9961.2020.07.019.Huang SJ,Zhang Y,Luo XH,et al. High-throughput sequencing based analysis on diversity of pathogens carried by ticks in Yuyao,Zhejiang[J]. Dis Surveill,2020,35(7):642-645. DOI:10.3784/j.issn.1003-9961.2020.07.019.(in Chinese)
[11] 向昱龙,周敬祝,刘英,等. 贵州省部分地区蜱及其携带细菌调查[J]. 中国媒介生物学及控制杂志,2022,33(1):148-152. DOI:10.11853/j.issn.1003.8280.2022.01.027.Xiang YL,Zhou JZ,Liu Y,et al. An investigation of ticks and tick-borne bacteria in some areas of Guizhou province,China[J]. Chin J Vector Biol Control,2022,33(1):148-152. DOI:10.11853/j.issn.1003.8280.2022.01.027.(in Chinese)
[12] Matei IA,Estrada-Peña A,Cutler SJ,et al. A review on the eco-epidemiology and clinical management of human granulocytic anaplasmosis and its agent in Europe[J]. Parasite Vector,2019,12(1):599. DOI:10.1186/s13071-019-3852-6.
[13] Diop A,Barker SC,Eberhard M,et al. Rickettsia fournieri sp. nov.,a novel spotted fever group rickettsia from Argas lagenoplastis ticks in Australia[J]. Int J Syst Evol Microbiol,2018,68(12):3781-3784. DOI:10.1099/ijsem.0.003057.
[14] Li H,Li XM,Du J,et al. Candidatus Rickettsia xinyangensis as cause of spotted fever group rickettsiosis,Xinyang,China,2015[J]. Emerg Infect Dis,2020,26(5):985-988. DOI:10.3201/eid2605.170294.
[15] 韩婧,贺真,邵中军. 常见蜱传立克次体的研究进展[J]. 中华卫生杀虫药械,2022,28(1):86-89. DOI:10.19821/j.1671-2781.2022.01.024.Han J,He Z,Shao ZJ. The research progress of common tick-borne rickettsia[J]. Chin J Hyg Insect Equip,2022,28(1):86-89. DOI:10.19821/j.1671-2781.2022.01.024.(in Chinese)
[16] Li JB,Hu W,Wu T,et al. Japanese spotted fever in Eastern China,2013[J]. Emerg Infect Dis,2018,24(11):2107-2109. DOI:10.3201/eid2411.170264.
[17] Qin XR,Han HJ,Han FJ,et al. Rickettsia japonica and novel Rickettsia species in ticks,China[J]. Emerg Infect Dis,2019,25(5):992-995. DOI:10.3201/eid2505.171745.
[18] Narra HP,Sahni A,Alsing J,et al. Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells[J]. BMC Genomics,2020,21(1):665. DOI:10.1186/s12864-020-07077-w.
[19] Sentausa E,El Karkouri K,Robert C,et al. Sequence and annotation of Rickettsia sibirica sibirica Genome[J]. J Bacteriol,2012,194(9):2377. DOI:10.1128/JB.00150-12.
[20] Li H,Fu XY,Jiang JF,et al. Severe illness caused by Rickettsia sibirica subspecies sibirica BJ-90 infection,China[J]. Emerg Microbes Infect,2017,6(1):1-3. DOI:10.1038/emi.2017.95.
[21] Jia N,Jiang JF,Huo QB,et al. Rickettsia sibirica subspecies sibirica BJ-90 as a cause of human disease[J]. N Engl J Med,2013,369(12):1176-1178. DOI:10.1056/NEJMc1303625.
[22] Ye XD,Sun Y,Ju WD,et al. Vector competence of the tick Ixodes sinensis (Acari:Ixodidae) for Rickettsia monacensis[J]. Parasite Vector,2014,7:512. DOI:10.1186/s13071-014-0512-8.
[23] Li W,Liu L,Jiang X,et al. Molecular identification of spotted fever group rickettsiae in ticks collected in central China[J]. Clin Microbiol Infect,2009,15:279-280. DOI:10.1111/j.1469-0691.2008.02235.x.
[24] 周琦,贺真,邵中军. 嗜吞噬细胞无形体的流行特征及临床诊断进展[J]. 中华卫生杀虫药械,2022,28(2):184-187. DOI:10.19821/j.1671-2781.2022.02.022.Zhou Q,He Z,Shao ZJ. Epidemiological characteristics and progress in clinical diagnosis of Anaplasma phagocytophilum[J]. Chin J Hyg Insect Equip,2022,28(2):184-187. DOI:10.19821/j.1671-2781.2022.02.022.(in Chinese)
[25] 刘增加,郑龙,张爱勤,等. 人粒细胞无形体病临床流行病学与防治研究现状[J]. 中华卫生杀虫药械,2018,24(5):417-422. DOI:10.19821/j.1671-2781.2018.05.001.Liu ZJ,Zheng L,Zhang AQ,et al. Research progress of clinical epidemiology,prevention and treatment on human granulocytic anaplasmosis[J]. Chin J Hyg Insect Equip,2018,24(5):417-422. DOI:10.19821/j.1671-2781.2018.05.001.(in Chinese)
[26] Wu XB,Na RH,Wei SS,et al. Distribution of tick-borne diseases in China[J]. Parasit Vectors,2013,6:119. DOI:10.1186/1756-3305-6-119.