Ticks and tick-borne Anaplasma spp. and Rickettsia spp. from hilly area in central China

Background Ticks are widely distributed in diverse habitats across the globe. Different tick habitats breed different tick varieties and tick-borne diseases. Pingdingshan City, located in central China, is a hilly area where livestock grazing is an everyday activity. Till now, the prevalence of tick-borne Rickettsiales around Pingdingshan City has not yet been explored. Methods We used PCR and nest-PCR detected the 16S rRNA gene of ticks, 16S rRNA gene of Anaplasma spp. and 17 KDa gene and gltA gene of Rickettsia spp. in the ticks collected from livestock and vegetation in hilly area of central China, Pingdingshan City. unravel its pathogenicity. Besides, other tick-borne disease-causing pathogens in H. longicorni, which pose a signicant threat to humans and livestock, demand further assessment. homology


Introduction
The recent decade has witnessed a steep rise in infectious disease cases across the globe. A majority of human pathogens are transmitted through animals. As livestock are present in close vicinity to humans, they spread pathogens more quickly as compared to wild animals. Usually, in developing countries, people lack awareness about livestock or vector transmitted diseases. Vectors, such as ticks, play a crucial role in wildlife and livestock mediated transmission of pathogens to humans [1].
Ticks (Acari: Ixodidae) are notorious sanguisuge ectoparasites of humans, domestic, and a wide range of wild animals. Till now, more than two hundred tick-borne pathogens have been identi ed, which includes viruses, bacteria, protozoa, and helminths [2]. The distribution of ticks within a speci c habitat depends on several environmental and climatic factors, such as annual rainfall, atmospheric temperature and relative humidity (RH), vegetation cover, altitude, and host availability [3]. In the hilly area of central China, climatic conditions are hot and rainy in summer, dry and cold in winter, clear in spring and autumn. Due to undulating terrains, this area is inapt for crop farming, and so, in this area, livestock grazing is commonly exercised. Hence, in the hilly area of central China, ticks and tick-borne diseases have a speci c pattern, which affects human health.
However, the epidemiological investigations on ticks and tick-borne infectious agents such as Anaplasma spp. and Rickettsia spp. [4] have not been investigated so far in Pingdingshan City, the hilly area in central China. In this study, we collected ticks from livestock and vegetation from six counties of the Pingdingshan City, identi ed the tick species, and investigated the prevalence of Anaplasma spp. and Rickettsia spp. through 16S rRNA gene sequencing for ticks and Anaplasma spp., and 17 kDa and gltA gene sequencing for Rickettsia spp.

Ticks collection
Ticks were collected from the surface of livestock, including goat, sheep, cattle, and vegetation from 11 sampling sites of six counties of Pingdingshan City (see Fig. 1) during the period of June-July, 2018. These ticks were starved for 2-3 days before the morphological examination, as described in a previous study [5,6]. The ndings were validated by ampli cation, sequencing, and analysis of the 16S rRNA partial sequences [7]. The details of this experiment are demonstrated in Table 1. All ticks were stored at -80 °C until DNA extraction. RpCS.1258n ATTGCAAAAAGTACAGTGAACA DNA extraction DNA was extracted from ticks, as described previously [8]. Ticks were washed and disinfected thrice with 75% ethanol and sterile deionized water, 5 min each time. DNA was extracted by using the TIANamp Genomic DNA Kit (TIANGEN Biotech Co., Ltd., Beijing, catalog no: DP304), as per the manufacture's instruction.
Detection of Anaplasma spp. and Rickettsia spp. by nested PCR The molecular identi cation of ticks and Anaplasma spp. was made through the 16S rRNA gene (390 bp) ampli cation by using nested PCR [9] and sequencing. Rickettsia spp. was identi ed by the ampli cation of Rickettsia spp. speci c 17 kDa gene by using nested PCR [10], and gltA gene ampli cation was used to validate the positive samples [11]. The details of these experiments are demonstrated in Table 1.
The PCR products of the 16S rRNA gene of tick and Anaplasma spp., and 17 kDa gene, gltA gene of Rickettsia spp. were visualized by 1.5% agarose gel electrophoresis. Positive bands were cut and sent with the appropriate primers to BGI (The Beijing Genomics Institute) for sequencing by ABI 3730 with the Sanger sequencing method.

Sequence analysis
NCBI BLAST was used to analyze the gene sequences. To understand the relationship between different sequences, Mega 5.0 [12] was used to construct the phylogenetic tree with bootstrap 1000 [13] by the Neighbor-Joining (NJ) method [14].

Collection of ticks
The altitude of small hills ranges from 200-800 meters in Pingdingshan City. The livestock grazing around the hill is widespread from April to October or even later when the bush and grass grow copiously. Around 686 ticks were Detection of Rickettsia spp. and Anaplasma spp.
Rickettsia spp. and Anaplasma spp. are tick-borne pathogens, and nested-PCR analysis showed that the tick samples collected in this study were positive for Rickettsia spp. and Anaplasma spp. We determined the proportion of tick samples that were positive for Rickettsia spp., Anaplasma spp., and the co-infection, which were collected from counties in Pingdingshan City (

Genetic and phylogenetic analysis
To decipher the correlation of the sample sequences with the reference sequences, we constructed a phylogenetic tree based on the tick 16S rRNA sequences (Fig. 2) from Anaplasma spp. (Fig. 3) and 17 kDa and gltA from Rickettsia spp. (Fig. 4).

Discussion
Ticks are widely distributed across the globe, and this distribution depends on several environmental and climatic factors [3]. H. longicornis mostly occurs in temperate secondary forests, mountainous and hilly fringe areas, [15] and transmits the virus, bacteria, spirochetes, rickettsia, blood protozoa, chlamydia, and other disease-causing and emerging infectious pathogens [1,16]. In our research,H. longicornis is the dominant tick species in hilly area in Pingdingshan. Not only in the hilly area we studied, in other hilly areas, such as Shandong Province [17], the hilly area in eastern Liaoning Province [18], the hilly area in Hebei Province [19,20], southwest forest in Beijing [21], Huichun and Tumen in Jilin Province [22], Huaian in Jiangsu Province [23], Anhui Province [24], Jiangxi Province [25] in China, even in Japan (ref) and South Korea (ref), H. longicornis is the dominant tick species. One common feature of these areas is that they are all hilly areas. Therefore, the distribution characteristics of H. longicornis remind us that the hill area is a unique ecological environment (Habitat), in which H. longicornis was bred. In other words, the hilly area and H. longicornis are a natural pair. Meanwhile, H. longicornis transmits different pathogens, and inhabits the body of livestock close to humans, suggesting it may be a substantial threat to healthy human beings in the hilly area.
R. microplus is also widely distributed in China and abroad (ref ?). We found a small amount of R. microplus in the collected ticks, which is consistent with the report of XX (Ref). We believe that the proportion of R. microplus in this area is relatively small, far lower than that of H. longicornis, so that some research teams have not found the existence of R. microplus in the whole Henan Province, including Pingdingshan (Ref. ). In addition, the main hosts we collected were goats and sheep, which were not the main hosts of R. microplus (Ref.), which may be the reason for the low proportion of tick. Therefore, we believe that although R. microplus is not the main tick species in the region, its proportion may be slightly larger than that detected by us. In other words, the tick in the area and the diseases it spreads still cannot be underestimated.
In addition, Anaplasma central (A. central) is a member of the genus Anaplasma. A.central causes a mild form of infectious disease. A. marginale is closely related to A. central, and widely used in immunization and to protect against virulent A. marginale challenge [37]. A. centrale primarily occurs in wild swine, deers [38,39], sheep [40], and vectored by Ixodes persulcatus [41], Rhipicephalus pumilio, Hyalomma asiaticum [40]. In this study, A. centrale was detected for the rst time in H. longicornis in China. These outcomes draw attention to the increased risk of A. centrale infection in the areas where H. longicornis primarily occurs.
Candidatus Rickettsia jingxinensis (Ca. R. jingxinensis) is an uncultured Rickettsia spp. identi ed in H. longicornis from north-eastern China [42], and its gltA sequence (KU853023) was detected in a human subject. Besides, it was detected in H. longicornis from Japan [43], H. longicornis and rodents from South Korea [44], H. longicornis from Hebei Province [45], Liaoning Province [46] and R. microplus from Wuhan Province of Hubei [47] in China; also H. longicornis was found in Shaanxi Province [4], and H. longicornis in Yunnan Province [48] in China. In our research, 17 kDa and gltA gene sequences of Ca. R. jingxinensis were also detected in H. longicornis ticks. These results indicate that Ca. R. jingxinensis is widely distributed in H. longicornis in hilly area in east Asia and suggests potential health risk to human and animals.

Conclusion
In summary, in the current study, we investigated ticks and tick-borne Anaplasma spp. and Rickettsia spp. from livestock and vegetation from the hilly area in central China due to a high prevalence of H. longicornis, high infection rate of Anaplasma spp. and Rickettsia spp., even co-infection of ticks in this hilly area. Further epidemiological studies are required to assess the role of these disease-causing pathogens and other pathogens transmitted by H. longicornis in hilly area and its impact on human health with an emphasis on people related to animal husbandry and eld workers. In addition, in hilly area, the pathogenicity of A. phagocytophilum, A. central, and C. Ra. jingxinensis demands further investigations.

Declarations
Ethics approval and consent to participate All the experiments with animals were approved by the Animal Ethics Committee of Pingdingshan University, China (PDSAEC-2018-001).

Competing interests
The authors declare no con ict of interest.