Ticks are second only to mosquitoes as vectors to transmit viral, bacterial, and protozoan agents in humans and animals, some of which pose a threat to human and animal health and are frequently zoonotic. Among tick-borne bacteria, members of the genera Anaplasma and Ehrlichia of the family Anaplasmataceae cause anaplasmoses and ehrlichioses in humans and animals (Ismail et al., 2010). The two most important species include Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME), and Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis (HGA). Less commonly, ehrlichiosis induced by Ehrlichia ewingii was first discovered by Buller et al. (1999). Pritt et al. (2011) identified a third species of Ehrlichia muris in patients who had fever, malaise, headache, and lymphopenia in Wisconsin and Minnesota, the United States, and it has also been found in Europe and Asia (Rar et al., 2010; Tateno et al., 2015). Other species that infect animals include A. marginale, Anaplasma centrale, A. ovis, E. canis, and Ehrlichia minasensis (Wen et al., 2003; Kocan et al., 2015; Cabezas-Cruz et al., 2016). Several species have been described in China, such as A. ovis, Anaplasma bovis, and A. platys in Gansu province (Li et al., 2016), A. centrale and E. canis in Fujian province (Ge et al., 2016; Yu et al., 2016), and E. chaffeensis in Guangxi province (Yang et al., 2015b). In Asia, Ixodes persulcatus is considered the primary vector of A. phagocytophilum and E. muris (Jin et al., 2012; Ivanova et al., 2016).

The tick-borne protozoa of the genus Babesia, the causative pathogen of babesiosis in humans and animals, are considered as emerging diseases worldwide. Approximately 100 Babesia species have been identified to infect a broad range of animals, in which malaria-like disorders are induced (Rozej-Bielicka et al., 2015). Babesiosis has a great effect on the animal production and on companion animals; however, human babesiosis has attracted increased attention (Ord and Lobo, 2015). In immunocompetent individuals, the infection is usually asymptomatic, or shows mild, self-resolving symptoms, but babesiosis can be life-threatening in neonates/infants, elderly persons, asplenic patients, and the immunocompromised populations (Fang et al., 2015; Ord and Lobo, 2015). The three most important species to infect humans are B. microti, B. divergens, and B. venatorum. Other species, such as B. ovis, B. major, B. bovis, B. bigemina, B. ovata, B. orientalis, B. motasi, and B. caballi, cause animal infections (Fang et al., 2015). In China, B. microti has been found in rodents in Fujian, Zhejiang, Henan, and Heilongjiang provinces (Sun et al., 2008; Zhao et al., 2013; Chen et al., 2014); B. divergens has been described in I. persulcatus, Haemaphysalis concinna, and H. japonica and striped field mice in Heilongjiang province, where B. venatorum has also been reported in I. persulcatus (Fang et al., 2015; Jiang et al., 2015). Most ixodid tick species, such as Ixodes scapularis in the United States, Ixodes ricinus in Europe, and I. persulcatus in Asia, can transmit Babesia parasites to their natural hosts (Brasseur and Gorenflot, 1996; Sun et al., 2008; Schulze et al., 2013; Zamoto-Niikura et al., 2016).

Members of Hepatozoon, belonging to Apicomplexa protozoa, parasitize mainly erythrocytes in amphibians, reptiles, and avian hosts, whereas they are found primarily in leukocytes in mammals (Baneth, 2011). In humans, sporadic cases have been reported in Russia (Shuikina et al., 2004). In China, Hepatozoon canis has been reported in dogs from Beijing, Henan, Jiangsu, Shaanxi, and Xingjiang provinces (Xu et al., 2015). Hepatozoon DNA has been detected in I. ricinus, Dermacentor spp., and Haemaphysalis spp., but the vector competence remains to be confirmed (Giannelli et al., 2013; Hamsikova et al., 2016).

The topography of northeastern China includes both plains and mountains, where Changbai Mountains, Da Hinggan Mountains and Xiao Hinggan Mountains are not only the important natural barriers of protecting the ecosystem, but also host to a wide range of natural focal diseases, among which Lyme borreliosis and tick-borne encephalitis are the most common tick-borne diseases (Wu et al., 2013), and emerging tick-borne zoonoses, induced by Rickettsia raoultii, Candidatus Neoehrlichia mikurensis, and B. venatorum, have been reported (Fang et al., 2015). To the best of our knowledge, there is no wide survey of tick-borne pathogens in northeastern China. The objective of this study was to characterize tick-borne bacteria (Anaplasma and Ehrlichia) and protozoa (Babesia and Hepatozoon) in ticks collected from Jilin and Heilongjiang provinces, northeastern China. The data obtained here would contribute to understanding the epidemic and distribution of these tick-borne pathogens, which could be used to design effective control measures for tick-borne diseases in China.

Several species of Anaplasma, including A. phagocytophilum, A. bovis, A. marginale, and A. ovis, have been described in China (Li et al., 2016; Zhang et al., 2016). A. phagocytophilum is considered as an emerging human pathogen of public health importance, which is naturally maintained in tick-mammal cycles, and has been found in sheep, goats, cattle, rabbits, and rodents (Jin et al., 2012). Although, A. phagocytophilum was detected in the genera of Ixodes, Dermacentor, and Haemaphysalis, I. persulcatus may play the most important role in the transmission of the bacterium in northeastern China, due to the high infection rate in the tick species and its high abundance. In Hebei province, A. phagocytophilum was detected in H. longicornis and D. nuttalli, where H. longicornis is the predominant tick species for the transmission of this pathogen (Yaxue et al., 2011). Phylogenetically, A. phagocytophilum detected in the study was more likely to infect ruminants (Yang et al., 2015a).

Anaplasma bovis has a wide host range, whose susceptible species include cattle, goats, dogs, cats, and deer (Kocan et al., 2015). In recent years, molecular detection of A. bovis infection showed an overall prevalence of 10–16% in goats, and 9.7% in sheep in China (Liu et al., 2012; Zhang et al., unpublished). Both H. concinna and H. longicornis ticks were found to be infected with A. bovis, suggesting these two tick species may be responsible for the transmission of A. bovis between ticks and mammals, but this assumption needs further experimental evidence.

Ehrlichia spp. are obligate intracellular bacteria residing within the cytoplasmic vacuoles of monocytes, granulocytes, or platelets of humans and animals, and can cause illnesses with fever, leukopenia, and thrombocytopenia (Dumler and Bakken, 1995). Serological and molecular evidences show a wide distribution of the bacteria infection in ticks, animals, and humans (Yu et al., 2016). In China, E. chaffeensis was detected in Amblyomma testudinarium, H. yeni, and I. persulcatus (Cao et al., 2000; Zhang et al., 2014). E. canis has been identified in Rhipicephalus sanguineus and Rhipicephalus microplus (Wen et al., 2003), and human infection usually presents fever, malaise, thrombocytopenia, and lymphopenia (Johnson et al., 2015). E. muris has been detected in R. microplus in Hunan (Yu et al., 2016). In the present study, we first detected E. muris in I. persulcatus collected from Jilin and Heilongjiang provinces of northeastern China, with a prevalence of 1.9–4.3%, showing that I. persulcatus may be a vector for this bacterium in northeastern China. Further studies are needed to assess the possible emergence of the infections in northeastern China.

Candidatus N. mikurensis is an emerging tick-borne pathogen causing neoehrlichiosis, whose clinical symptoms may include fever, localized pain in muscles and/or joints, vascular and thromboembolic events (Silaghi et al., 2016). In China, the human infection was first reported in Heilongjiang province of northeastern China in Li et al. (2012), and the bacterium may have wide geographic distribution in China (Li et al., 2013). Only I. persulcatus was positive for Candidatus N. mikurensis in this study, implying that I. persulcatus may be a vector for this bacterium in northeastern China.

Babesiosis, the causative pathogens including B. microti, B. venatorum, and B. divergens in humans, is considered an emerging threat in China, where there are approximately 1.3 billion people at risk of infection (Qi et al., 2011; Jiang et al., 2015; Vannier and Krause, 2015). B. microti was detected in I. persulcatus, H. longicornis, and H. concinna in Fujian, Zhejiang, Henan, and Heilongjiang provinces (Saito-Ito et al., 2008; Sun et al., 2008; Zhou et al., 2014). B. venatorum has been reported in I. persulcatus ticks collected from forested areas of northeastern China (Jiang et al., 2015). Other species of Babesia, including B. ovis, B. major, B. ovata, B. orientalis, B. motasi, and B. caballi, have not been shown to infect humans (Fang et al., 2015). In the present study, we found B. divergens, B. microti, and B. venatorum in I. persulcatus, and also first detected B. divergens in H. longicornis, indicating that I. persulcatus may be the main vector for the Babesia species of human babesiosis in northeastern China, since only two H. longicornis pools were positive for this parasite.

Previous studies have shown that B. microti is the predominant species in southeastern and northeastern of China while B. divergens may be the main pathogen in Inner Mongolia, and Xinjiang Uygur Autonomous Region of China (Zhou et al., 2014). In this study, of the 47 Babesia-positive samples, 19 (40.2%) were B. venatorum, followed by 9 (19.1%) B. microti, 3 (6.4%) B. divergens, and 16 (34.3%) sequence variants, suggesting that B. venatorum may be the predominant species responsible for human babesiosis in northeastern China.

Additionally, several sequence variants, which are closely related to the groups of carnivores, cattle and small ruminant Babesia, have also been found in the present study. For example, Babesia sp. Ip-hlj179 was phylogenetically associated with B. gibsoni; Babesia sp. Ip-hlj179 was related to B. motasi; Babesia sp. Ip-hlj238 was related to B. crassa; and Babesia sp. Ip-hlj212 was related to B. major (Figure 3). Thus, it is necessary to monitor these variants infection in domestic animals in northeastern China. Isolation of parasites and identification of transmission vector should also be included. Interestingly, a high genetic variability of Babesia has been described in Russia, which included all variants found in northeastern China (Rar et al., 2014), and four variants were detected in Heilongjiang province while only two variants were found in Jilin province. These data showed the cross-border spread of Babesia in northeastern China may occur.

More than 300 Hepatozoon species have been identified in amphibians, reptiles, birds, marsupials, and mammals (Smith, 1996). Of these, more than 120 species infect snakes, and approximately 50 have been described in mammals. Ticks and other blood-sucking arthropods may serves as definitive hosts for Hepatozoon spp. Unlike, other vector-borne pathogens that are transmitted via the bite of arthropods, the vertebrate host becomes infected by ingestion of the arthropods that contains mature oocysts. Three species H. canis, H. americanum, and H. felis can cause hepatozoonosis in dogs and cats, showing different clinical symptoms. H. canis is primarily found in hemolymphatic tissues, causing fever, lethargy, weight loss, anemia, and hyperglobulinemia in dogs, while H. americanum infects mainly muscular tissues, causing myositis and lameness (Baneth, 2011). There is only one report of infection with a Hepatozoon sp. in a person from Russia; the patient was anemic and icteric, and gamonts were detected in the blood (Shuikina et al., 2004).

In China, H. canis infection in dogs was detected 1.1% in Jiangsu, 1.2% in Xinjiang, 8.9% in Shaanxi, and 4.9% in Henan and Beijing (Xu et al., 2015). A new species, H. chinensis has been found in king rat snakes (Elaphe carinata) from Shanghai (Han et al., 2015). In this study, two Hepatozoon species, the most closely related to the isolates of Japanese martens, were found in ticks in northeastern China, and both of them were detected in Ixodes, Haemaphysalis, and Dermacentor ticks, suggesting that specificity for the final host may be low in Hepatozoon; however, the intermediate hosts and the resulting disease still remain to be determined.

Not only the high infection rates but also the high abundance of I. persulcatus makes it the most important vector tick in the area. Moreover, Candidatus N. mikurensis, B. venatorum, and B. microti were only detected in I. persulcatus. These findings show that I. persulcatus may be an important vector of tick-borne bacteria and protozoa in northeastern China.

In summary, we detected four species of bacteria and three species of protozoa in four tick species in northeastern China, including A. phagocytophilum, A. bovis, E. muris, Candidatus N. mikurensis, B. venatorum, B. microti, and B. divergens, which are associated with emerging diseases in humans and/or animals. Additionally, four Babesia sequence variants, and two Hepatozoon sp. were also found. These findings showed the genetic variability of Anaplasma, Ehrlichia, Babesia, and Hepatozoon spp. circulating in ticks in northeastern China, highlighting the need for further research of these tick-associated pathogens and their role in human and animal diseases. Further studies will be necessary to confirm the vectorial capacity of ticks, to improve understanding of the epidemiology of these tick-borne diseases, and to monitor emerging tick-borne pathogens and factors influencing their prevalence, which will facilitate implementing integrated strategies for controlling ticks and tick-borne pathogens in China.

JQ and QL designed the study in collaboration with FW, MS, and HL. HM conducted the fieldwork with assistance from MS, ZZ, and QL. HL, BW, ZW, and ZL conducted the laboratory work; HL and SW conducted the statistical analysis and drafted the manuscript. FW and MS contributed to the interpretation of the data. All authors contributed to the manuscript editing and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.