Diversity and genotype analysis of tick-borne pathogens in grasslands in the central and western part of Inner Mongolia

doi:10.12140/j.issn.1000-7423.2021.01.004

Abstract

Abstract:

Objective To investigate the structure of tick populations, the diversity of pathogens they carry, and the genotypes of tick-borne Rickettsia-related pathogens in central and western part of Inner Mongolia of China. Methods Tick samples were collected from body surface of animals in the grasslands in central and western Inner Mongolia in spring and summer between 2016 and 2019. The tick species was identified. The salivary glands of ticks were dissected and genomic DNA was extracted. PCR was performed to amplify citrate synthase A (gltA) in spotted fever Rickettsia, flagellin gene B (flaB) in Borrelia, outer membrane protein 1 (omp1) in Ehrlichia, and major surface protein 2 (msp2) in Anaplasma. The samples positive for Rickettsia gltA were classified by restriction fragment length polymorphism (RFLP), and then 20 to 30 representative samples were selected for each type for each species and region for gene sequencing of gltA and Rickettsia outer membrane protein A (rOmpA). Homology analysis was performed by BLAST, Clustal W and MEGA 7.0 softwares, and the phylogenetic tree was constructed using the adjoining method. Results A total of 3 822 ticks were collected, 3 genera of 2 species were identified by morphological observation and genotyping of the specific 18S rRNA, which were Dermacentor nuttalli, Hyalomma asiaticum and H. marginalis, of which D. nuttalli (55.7%, 2 129/3 822) and H. asiaticum (30.0%, 1 147/3 822) constituted the predominant species. PCR results showed that 1 899 ticks were positive for Rickettsia gltA, with positive rate of 49.7% (1 899/3 822). The gltA-positive samples were divided into two groups according to the RFLP results. The gltA sequence was 581 bp in both groups, and was 100% homologous to R. raoultii (DQ365804) or R. aeschlimanni (KT873466). The rOmpA length of the two groups was 367 bp, which was 100% homologous to R. Raoultii (AH015610) or R. aeschlimanni (U83466), consistent with the results of gltA. The positive rates of R. raoultii and R. aeschlimanni in the 3 822 ticks were 37.2% (1 422/3 822) and 12.5% (477/3 822), respectively; 58.5% (1 245/2 129) and 11.1% (477/2 129) in D. nuttalli, respectively; 15.4% (177/1 147) and 0 in H. asiaticum; and 0 and 44.0% (240/546) in H. marginalis, respectively. A total of 28 ticks (0.7%, 28/3 822) were positive for Borrelia flaB, with positive rate of 0.8% (16/2 129) in D. nuttalli and of 1.0% (12/1 147) in H. asiaticum. A total of 10 sequences of Borrelia flaB were obtained, with 90.6%-100% and 95.6%-100% homology to B. garinii (AB035602) and B. afzelii PKO (NC008277), the main pathogens of Lyme disease. The positive rates of B. garinii and B. afzelii were 0.9% (10/1 147) and 0.2% (2/1 147) in H. asiaticum, respectively, and were both 0.4% (8/2 129) in D. nuttalli. Of the 3 822 ticks examined, 1 tick was positive for Ehrlichia omp1. After TA cloning, 8 clones with the same amino acid sequence and 3 with different amino acid sequences were obtained. The amino acid sequences of the 11 clones had the highest homology with E. muris, although the homology was only 65%-69%. No Anaplasma microbiome was detected in the 3 822 ticks. The phylogenetic tree analysis showed that the Rickettsia that infected the three tick species clustered with R. raoultii and R. aeschlimanni. Among the 10 Borrelia flaB sequences obtained, one sequence from D. nuttalli and H. asiaticum clustered with B. garinii, another sequence from D. nuttalli also clustered with B. afzelii, and the remaining 8 sequences were in different branches from the flaB sequences of B. garinii and B. afzelii. The Ehrlichia infected by D. nuttalli was also far from the known Ehrlichia, forming a separate cluster. Conclusion D. nuttalli, H. asiaticum and H. marginalis are found in grasslands in the central and western part of Inner Mongolia. Infection of spotted fever Rickettsia and Lyme disease spirochete are widespread in the ticks, creating potential natural foci of R. raoultii, R. aeschlimanni, B. garinii, B. afzelii and Ehrlichia infection. It is necessary to strengthen the prevention and control of tick-borne infectious diseases in these areas.

Key words: Tick, Rickettsia, Anaplasma, Ehrlichia, Borrelia, PCR detection

CLC Number:

R384.41

Wulantuya, YIN Xu-hong, CUI Yun-hong, LIU Dan, WANG Ting-fu, MIAO Yu-run, , CAO Min-zhi, ZHAO Zhi-wei, XING Fang-chao, LU Jian-ying, GAO Wa. Diversity and genotype analysis of tick-borne pathogens in grasslands in the central and western part of Inner Mongolia[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2021, 39(1): 27-35.

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病原体Pathogen	基因名称 Gene name	引物名称 Primer name	引物序列（5′→3′） Primer sequence（5′→3′）	片段长度/bp Fragment length/bp	反应温度/℃ Reaction temperature/℃	文献 Reference
斑点热立克次体 Spotted fever group Rickettsia	gltA	gltA-F	CGAACTTACCGCTATTAGAATG	581	55	[11]
		gltA-R	CTTTAAGAGCGATAGCTTCAAG
	rOmpA	rOmpA-F	TGGTGGAGCTCATAAGTTACA	367	58	[11]
		rOmpA-R	AGTTACATTTCCTGCACCTAC
无形体属Anaplasma	msp2	msp2-F	GCTAAGGAGTTAGCTTATGA	521	55	[12]
		msp2-R	AGAAGATCATAACAAGCATTG
埃立克体属Ehrlichia	Omp1	omp1-F	ATYAGTGSAAARTAYRTRCCAA	700	55	[11]
		omp1-R	TTARAARGYAAAYCTKCCTCC
疏螺旋体Borrelia	flaB	flaB-F	GCTGAAGAGCTTGGA ATGCACC	397	50	[13]
		flaB-R	TGATCAGTATCATT CTA ATAGCA

蜱种 Tick species	蜱虫数 No. of ticks	立克次体阳性蜱数（阳性率/%） No. of ticks positive for Rickettsia (Positive rate/%)		埃立克体阳性蜱数（阳性率/%） No. of ticks positive for Ehrlichia sp. (Positive rate/%)	疏螺旋体阳性蜱数（阳性率/%） No. of ticks positive for Borrelia (Positive rate/%)
蜱种 Tick species	蜱虫数 No. of ticks	R. raoultii^a	R. aeschimannii^a		B. garinii^a	B. afzelii^a
草原革蜱 D. nuttalli	2 129	1 245（58.5）	237（11.1）	1（0.0）	8（0.4）	8（0.4）
边缘璃眼蜱 H. marginatum	546	0（0）	240（44.0）	0（0）	0（0）	0（0）
亚东璃眼蜱 H. asiaticum	1 147	177（15.4）	0（0）	0（0）	10（0.9）	2（0.2）
合计Total	3 822	1 422（37.2）	477（12.5）	1（0.0）	18（0.5）	10 （0.3）

地区 Area	蜱种 Tick species	数量 Number	感染蜱数 No. of ticks infected
地区 Area	蜱种 Tick species	数量 Number	立克次体 Rickettsia	无形体 Anaplasma	埃立克体 Ehrlichia	疏螺旋体 Borrelia
锡林郭勒 Xilingol	草原革蜱D. nuttalli	835	545	0	0	4
	亚东璃眼蜱H. asiaticum	381	59	0	0	6
	边缘璃眼蜱H. marginatum	76	0	0	0	0
乌兰察布Ulanqab	草原革蜱D. nuttalli	197	90	0	0	2
	边缘璃眼蜱H. marginatum	202	80	0	0	0
巴彦淖尔Bayannur	亚东璃眼蜱H. asiaticum	134	36	0	0	2
	边缘璃眼蜱H. marginatum	268	160	0	0	0
	草原革蜱D. nuttalli	487	421	0	1	6
鄂尔多斯Erdos	草原革蜱D. nuttalli	430	318	0	0	4
乌海Wuhai	草原革蜱D. nuttalli	180	108	0	0	0
阿拉善Alxa	亚东璃眼蜱H. asiaticum	632	82	0	0	4
合计Total		3 822	1 899	0	1	28