Sample collection and species identification
In May 2018, a total of 149 ticks were collected from the body surface of cattle and goats in the Yakeshi County-Level City of Hulunbuir City, Inner Mongolia (49.17°N, 120.40°E). Based on morphological observation, three species were identified: 99 Ixodes persulcatus, 24 Haemaphysalis concinna, and 26 Dermacentor silvarum (Fig. 1). For further confirmation, the COI sequences of all three tick species have nucleotide similarities higher than 99% with reference sequences.
Detection and analysis of the Rickettsia spp.
Based on the PCR results and sequencing of 16S rRNA sequences, a total of three Rickettsia species including four genotypes were identified. Candidatus Rickettsia tarasevichiae was detected in all I. persulcatus (99/99, 100%), 7 of 24 H. concinna (29.17%), and 2 of 26 D. silvarum (7.69%). All the 16S sequences are 100% identical to Ca. Rickettsia tarasevichiae isolate Dog-145, Ca. Rickettsia tarasevichiae isolate Bayan-68, and Ca. Rickettsia tarasevichiae isolate Mulan-11, which were all detected in the Heilongjiang Province of Northeast China. Similarly, the gltA (1007 bp) sequences show highest 99.90% nucleotide similarity to these strains, and the groEL (1060 bp) sequences have 99.65–99.88% (coverage 76–80%) to previously uploaded sequences.
Two genotypes of R. raoultii were detected only in D. silvarum, with positive rates of 50.00% (13/26) and 26.92% (7/26), respectively. All three genes of type I (strains N78, N83, and N95) were 100% identical to Rickettsia conorii subsp. raoultii strain IM16. In contrast, the 16S sequences of type II were 100% identical to Rickettsia conorii subsp. raoultii isolate Tomsk, R. conorii strain Malish_7, and R. massiliae MTU5, while their gltA sequences were 100% to that of R. raoultii isolate Binxian-91. As shown in Fig. 2, these strains clearly divided into two distinct clades.
Four R. heilongjiangensis strains were detected only in H. concinna ticks. All their 16S rRNA and gltA sequences show 100% and 99.90% identities to both R. heilongjiangensis CH8-1 and R. japonica strain YH_M. For the groEL sequences, they are 100% identical to those of R. heilongjiangensis CH8-1 and R. heilongjiangensis HCN-13, but only 99.53% to R. japonica strains. These results confirmed that these strains should be classified as R. heilongjiangensis.
Detection and analysis of the Anaplasma spp.
Based on analysis of the 16S sequences, a total of three Anaplasma species were detected: A. phagocytophilum, A. bovis, and an unclassified Anaplasma sp., showing 100%, 100%, and 99.87% to A. phagocytophilum str. JM, A. bovis clone Am-Hc60, and A. centrale isolate LP10, respectively. Notably, based on gltA and groEL sequences, the A. phagocytophilum strains divided into two types in the phylogenetic trees: A. phagocytophilum N3 represent type I while strains N54, N55, and N136 belong to type II (Fig. 3). The gltA sequences of type II are 100% identical to Anaplasma sp. KhabIx detected in the Russian Far East, and only 82.73–88.11% to A. phagocytophilum strains. Similarly, the groEL sequence of type I (strain N3) was 100% identical to A. phagocytophilum isolate Ip11, but sequences of type II have 98.59–100% similarities to A. phagocytophilum strains identified in Tomsk and Omsk in Russia. These results showed the genetic diversity of A. phagocytophilum in this area. Out of our expectation, the gltA sequence of Anaplasma sp. N127 was 100% identical to Anaplasma sp. BL126-13, but its groEL sequence has a long genetic distance to that of Anaplasma sp. BL126-13 (Fig. 3). In contrast, it was 100% identical to the groEL sequence of Anaplasma sp. clone B251. The 23S sequence of Anaplasma sp. N127 was also obtained, showing highest 96.79% identity to A. ovis str. Haibei and 94.81% to A. marginale str. Florida (Fig. S1). We propose it as a novel species, namely “Candidatus Anaplasma mongolica”.
Detection and analysis of the Ehrlichia sp. and Lariskella sp.
Seven Ehrlichia strains were detected in I. persulcatus, and all of them were identified as Ehrlichia muris. In addition to the 16S rRNA (456 bp) sequences which show 100% to E. muris strains, the gltA (986 bp), and groEL (1121 bp) sequences were also successfully obtained. The gltA sequences have highest 99.47–99.80% identities to E. muris strains in rodents (isolate Khab-85_Mruf, AS145) and I. persulcatus ticks (isolate Omsk-563_Ip) from Japan and Russia. The groEL sequences are also highly homologous to E. muris strains from Japan and Russia, with nucleotide identities of 99.29–100% (Fig. 4). To date, there is only one E. muris sequence in the GenBank Database from mainland China. This study may provide more data on the genetic diversity of E. muris in China.
Unexpectedly, Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae, order Rickettsiales, was detected in as many as 47 of the 99 Ixodes persulcatus ticks (47.47%) using the primers screening Anaplasmataceae. All the 16S rRNA sequences are 100% identical to each other and have 98.83–100% identity to Candidatus Lariskella arthropodarum, 98.82–99.06% to Candidatus Lariskella guizhouensis we previously reported. Interestingly, their gltA sequences (400 bp) are only highly homologous to Ca. Lariskella guizhouensis, with similarities of 99.50-99.73% (Fig. 5). Due to the absence of gltA sequences of previously reported Ca. Lariskella arthropodarum or other Lariskella strains in the GenBank, it is hard to determine whether the detected strains should be classified as Ca. Lariskella arthropodarum or Ca. Lariskella guizhouensis. Actually, it is also quite possible that these two species be the same species.
Detection and analysis of the Borrelia sp. and Borreliella spp.
One Borrelia sp. (Borrelia miyamotoi) and two Borrelialla spp. (Borreliella afzelii and Borreliella garinii) were detected. As shown in Table 1, all of them were detected in I. persulcatus, with positive rates of 3.03% (3/99), 3.03% (3/99), and 8.08% (8/99), respectively. The flaB and 16S sequences of B. miyamotoi strains were all closely related to B. miyamotoi strain Yekat-31 from Russia, with nucleotide similarities of 98.72% and 100%, respectively. Notably, phylogenetic analysis indicated that both the flaB and 16S gene sequences of B. garinii showed remarkable genetic diversity (shown in Fig. 6).
Table 1
Prevalence of Rickettsia spp., Anaplasma spp., Borreliella spp., Borrelia miyamotoi, Ehrlichia muris, and Candidatus Lariskella sp. in different tick species from Hulunbuir City of Inner Mongolia.
| Ixodes persulcatus | Haemaphysalis concinna | Dermacentor silvarum | Total |
Candidatus Rickettsia tarasevichiae | 99/99 (100%)a | 7/24 (29.17%) | 2/26 (7.69%) | 108/149 (72.48%) |
Rickettsia raoultii type I | 0/99 (0.00%) | 0/24 (0.00%) | 13/26 (50.00%) | 13/149 (8.72%) |
Rickettsia raoultii type II | 0/99 (0.00%) | 0/24 (0.00%) | 7/26 (26.92%) | 7/149 (4.70%) |
Rickettsia heilongjiangensis | 0/99 (0.00%) | 4/24 (16.67%) | 0/26 (0.00%) | 4/149 (2.68%) |
Anaplasma phagocytophilum type I | 1/99 (1.01%) | 0/24 (0.00%) | 0/26 (0.00%) | 1/149 (0.67%) |
Anaplasma phagocytophilum type II | 3/99 (3.03%) | 0/24 (0.00%) | 0/26 (0.00%) | 3/149 (2.01%) |
Anaplasma bovis | 0/99 (0.00%) | 1/24 (4.17%) | 0/26 (0.00%) | 1/149 (0.67%) |
Candidatus Anaplasma mongolica | 1/99 (1.01%) | 0/24 (0.00%) | 0/26 (0.00%) | 1/149 (0.67%) |
Ehrlichia muris | 7/99 (7.07%) | 0/24 (0.00%) | 0/26 (0.00%) | 7/149 (4.70%) |
Candidatus Lariskella sp. | 47/99 (47.47%) | 0/24 (0.00%) | 0/26 (0.00%) | 47/149 (31.54%) |
Borrelia miyamotoi | 3/99 (3.03%) | 0/24 (0.00%) | 0/26 (0.00%) | 3/149 (2.01%) |
Borreliella afzelii | 3/99 (3.03%) | 0/24 (0.00%) | 0/26 (0.00%) | 3/149 (2.01%) |
Borreliella garinii | 8/99 (8.08%) | 0/24 (0.00%) | 0/26 (0.00%) | 8/149 (5.37%) |
a positive samples/total samples. |