Classification of Tick Species and Detection of Tick-borne Pathogens in Yanbian, China


 BackgroundYanbian is located at the junction between China, Russia, and North Korea. We aimed to determine the species distribution and pathogens carried by ticks in Yanbian.MethodsA total of 2673 unattached ticks were collected from eight counties and cities in Yanbian and classified morphologically. Candidatus Rickettsia tarasevichiae (CRT), spotted fever group Rickettsia (SFGR), severe fever thrombocytopenia syndrome virus (SFTSV), Theileria, and other pathogens were detected using polymerase chain reaction (PCR) and real-time quantitative polymerase chain reaction followed by phylogenetic and genotypic analyses.ResultsAccording to the morphological classification, the main tick species in Yanbian were Haemaphysalis longicornis, Ixodes persulcatus, Dermacentor silvarum, Haemaphysalis japonica, and Haemaphysalis concinna. Candidatus Rickettsia tarasevichiae, spotted fever group Rickettsia, severe fever thrombocytopenia syndrome virus, and Theileria orientalis were detected in H. longicornis, Candidatus Rickettsia tarasevichiae, spotted fever group Rickettsia, and severe fever thrombocytopenia syndrome virus were detected in I. persulcatus, H. japonica, and D. silvarum, but only severe fever thrombocytopenia syndrome virus was detected in H. concinna. Mixed infection with Candidatus Rickettsia tarasevichiae and severe fever thrombocytopenia syndrome virus was found in I. persulcatus and H. japonica. The gene sequences of all tested pathogens exhibited 95.7%–100% homology with sequences registered in GenBank. Phylogenetic analysis showed that different spotted fever group Rickettsia and severe fever thrombocytopenia syndrome virus genotypes were closely related to the Korean strains. We provide the first evidence for the presence of the spotted fever group Rickettsia genotypes of Candidatus Rickettsia longicornii, ompA, ompB, sca4, and rrs, in Haemaphysalis longicornis in Yanbian. Conclusions﻿These results provide epidemiological data to support the prevention and control of ticks and tick-borne diseases in the border areas of China, North Korea, and Russia.


Pathogen gene detection
Nucleic acids were extracted using a nucleic acid extraction kit (Suzhou Tianlong Science and Technology Co., Ltd., Suzhou, China). The Candidatus Rickettsia tarasevichiae (CRT) ompA gene and 17-kDa genes were detected as described by Jia et al. [11]. The spotted fever group Rickettsia (SFGR) Candidatus Rickettsia longicornii ompA, ompB, sca4, rrs genes were detected as described by Jiang et al. [12], the severe fever thrombocytopenia syndrome virus (SFTSV) Small, Medium, and Large gene segments were detected as described by Liu et al. [13], the Theileria orientalisMPSP gene was detected as described by Ota et al. [14], and the Theileria sinensisMPSP gene was detected as described by Liu et al. [15]. The primers for these genes are listed in Table 1.

Homology and phylogenetic analyses
The PCR products of positive samples were sent to Shanghai Shenggong Co., Ltd. for sequencing. Correct gene sequences were analyzed via DNAStar and GenBank, and phylogenetic trees were constructed using the neighbor-joining method with a Kimura 2-parameter model using MEGA7 software.

Statistical analysis
Data were processed using Excel 2007 and statistical analysis was carried out using SAS8.2 software. Numerical data were expressed as a constituent ratio (%) and positive rate (%), where the constituent ratio (%) = number of each tick species in same location / total number of all tick species in same location × 100%, and the positive rate (%) = number of positive samples detected for pathogens / total number of tested samples of the same species (n) × 100%.   (Table 3).  (Fig. 2).

Tick species survey results
The SFTSV Large (MT517309), Medium (MT517308), and Small (MT517307) gene sequences showed 98%-99% homology with the SFTSV gene sequences identi ed in China and South Korea according to homology analysis. Phylogenetic analysis showed that the SFTSV Small gene sequence from ticks in Yanbian was in the same clade as the SFTSV gene sequence (KT890282.1) of Jilin ticks in China. The Medium gene sequence was located in the same branch as the Chinese JS2014-18 strain (KR230781.1), and the Small sequence was located in the same branch as the Chinese JS2014-18 strain (KR230761.1) and was closely related to SFTSV isolated from Zhejiang and South Korea (Fig. 3).
There was 99.4% homology between the T. orientalis MPSP gene (MT517304) and the published GenBank entry number (MG664537.1). Phylogenetic analysis showed that the sequence of the T. orientalis MPSP (MT517304) gene in the ticks was located in the same branch as MPSP (MG664537.1) in the Chinese Chongqing strain (Fig. 4).

Discussion
Yanbian is located at the junction of China, North Korea, and Russia, and has a long border. Strengthening ecological and environmental protection in China means that the species distribution along the border has gradually diversi ed; the numbers and species of ticks are thus constantly changing, and their activity is increasing. Ticks and other vectors in the border zone can migrate to each other using various methods, increasing the risk of tick-borne diseases. In this study, 2673 ticks collected from eight counties and cities in Yanbian were classi ed and analyzed. H. longicornis and I. persulcatus were the dominant tick species in Yanbian. H. longicornis has strong reproductive ability and environmental adaptability, and is widely distributed throughout Asia and the Paci c, including China, Russia, South Korea, Japan, Australia, New Zealand, and the South Paci c islands. It is often parasitic in medium and large wild and domestic animals, whereas humans are accidental hosts. H. longicornis spreads a variety of pathogens that can affect wild animals and livestock, as well as human health.
Ticks can be infected with viruses, bacteria, including Rickettsia and spirochetes, and other pathogens. In addition, ticks can act as both vectors and hosts in the process of infectious disease transmission. The main research into tick-borne pathogen co-infections is currently focused on Borrelia burgdofferi, Babesia microti, Ehrlichia, and Anaplasma phagocytophilum [16]. Previous studies con rmed that one-third of patients with a CRT infection had neurological symptoms that differed from other SFGR infections [17], and were associated with a higher case-fatality rate when co-infected with SFTSV [18]. More attention should thus be paid to SFTSV transmission through both tick bites and close contact with infected cases [19]. In this study, we con rmed the existence of CRT/SFTSV co-infection in I. persulcatus (n = 13), H. japonica (n = 5), and D. silvarum (n = 1) in Yanbian. I. persulcatus is a common dominant tick species in Yanbian, and is especially widely distributed in Hunchun, Yanji, Helong, and other regions, resulting in a high risk of CRT and SFTSV infection via tick bites in these regions.
SFGR forms a long-lasting infection cycle between ticks and mammals and can also be transmittedvertically through tick eggs, making ticks the main host and vector of SFGR. Liu et al. [20]  SFTS is a novel infectious disease that was rst discovered in China. Its main clinical manifestations consist of acute fever, thrombocytopenia, and leukopenia. The pathogen was isolated from a patient's serum and termed SFTSV [21]. At present, most ticks detected and isolated from cases of SFTS were H. longicornis. The rst report of the disease occurred when SFTSV was detected in H. longicornis on the skin of sheep in Henan Province, China [22], and SFTSV was isolated from H. longicornis on sheep in the SFTS epidemic area in Shandong Province, China [23]. SFTSV was rst isolated from H. longicornis in Korea [24]. In the current study, the SFTSV Small, Medium, and Large gene sequences were obtained by gene ampli cation, and homology analysis indicated 98%-99% homology with the SFTSV gene sequence found in South KoreaPhylogenetic analysis showed that the SFTSV Small, Medium, and Large gene sequences were in the same clade as isolates from Jilin and Jiangsu, respectively, and were closely related to SFTSV isolated from Zhejiang and South Korea. This may be related to the parasitism of migratory birds by ticks in the east or SFTSV transmission by migratory birds themselves during cross-sea migration. Korean researchers suggested that migratory birds may play an important role in the spread of SFTSV [25]. The above results suggest that the border area of China, North Korea, and Russia is a key region for preventing tick-borne SFTSV, and should thus be considered in the prevention and control of imported infectious diseases in the border region.
T. orientalis is a protozoon that infects cattle and buffalo, and which is generally transmitted by ticks of the genus Haemaphysalis [26]. T. sinensis was originally isolated from naturally infected cattle by Bai Qi and others in Gansu Province of China. Chinese researchers investigated the taxonomic status of this undetermined species by comparing it with other bovineTheileria worms via morphological comparison, and inoculation transmission and host speci city tests. It was nally termed T. sinensis [27]. T. orientalis is transmitted by H. longicornis, H. concinna, and H. japonica. However, the vector tick species differ between regions; for example, H. japonica is the main vector of Oriental Taylor disease in Russia, followed by H. concinna, while H. concinna is also the main vector in Korea, whereas H. longicornis is the main vector in China and Japan. In this investigation, we detected T. orientalis in H. longicornis but failed to detect T. sinensis in any ticks. This may be related to the collection area and small numbers of its vector, H. japonica.

Conclusions
H. longicornis and I. persulcatus are the dominant tick species in Yanbian, China. Four pathogens (CRT, SFGR, SFTSV and T. orientalis) were detected in the tick species collected in this study, and CRT/SFTSV co-infection was also identi ed in I. persulcatus and H. japonica. Moreover, this study provides the rst evidence of the SFGR genotypes Candidatus Rickettsia longicorniiompA, ompB, sca4, and rrs in H. longicornis in Yanbian, China. Moreover, T. orientalis was detected in H. longicornis. These ndings provide epidemiological data to support the prevention and control of ticks and tick-borne diseases in the border region of China, North Korea, and Russia.