关键词: 蜱, 立克次体, 无形体, 埃立克体, 疏螺旋体, PCR检测

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