Ticks can transmit bacterial, parasitic, and viral pathogens (often zoonotically) and often harbor more than one agent simultaneously [37]. Thus, obtaining broader information about pathogens in ticks is important from the perspective of proper diagnosis and treatment of these diseases [38]. In this study, we demonstrated that the recently released NGS instrument, iSeq100, is useful for screening of bacteria in ticks. NGS approaches have the ability to identify a wide range of known or unknown pathogens or discover new organisms from a single test [21] without the need to design specific primers for each pathogen. This method makes it possible to identify pathogens immediately, not only in ticks, but also in arthropods that serve as vectors and reservoirs for pathogens, such as mosquitoes, tsetse flies, and sand flies.
Considering this advantage, in this study, we screened the tick-borne bacteria using iSeq100 and found that 18 of the 31 ticks harbored pathogens of the R. rickettsii species. For an accurate taxonomic classification of the species of the detected Rickettsia spp., we used conventional PCR to amplify gltA and ompA sequences of the Rickettsia spp. and compared them with sequences deposited in GenBank using BLAST. In the phylogenetic analysis performed using MEGA-X software, the sequence similarity to sequences of Ca. R. jingxinensis isolate Xian-Hl-79, Ca. R. jingxinensis isolate F18, and Ca. R. longicornii isolate ROK-HL727 was 100 % for gltA, indicating a close relationship between rickettsial isolates from H. longicornis from Korea with those from other East Asian countries. The close clustering of the Chinese and Korean strains of Rickettsia spp. may indicate a close epidemiological link between these strains.
In this study, we used iSeq100 and PCR with specific primers for screening tick-borne bacteria, and more Rickettsia-positive samples were detected with the iSeq100 method. In iSeq100 analysis, 18 samples showed positive results, while in the pathogen-specific PCR method, 14 samples were positive. However, sequencing using conventional PCR remains essential for identifying the specific strain of the pathogen species.
Ixodid ticks (e.g. H. longicornis, H. flava, Ixodes persulcatus, and I. nipponensis) in Asia have the potential to be primary vectors/repositories of rickettsiae of medical and veterinary importance [16]. In 2006, the first case in Korea of R. japonica was isolated from a spotted fever patient [14]. Recent studies show a high prevalence of the emerging pathogen, Rickettsia raoultii, in canine ticks [43].
In our study, the sequences of gltA and ompA are identical or highly homologous to those of Ca. R. jingxinensis isolate Xian-Hl-79, Ca. R. jingxinensis isolate F18, and Ca. R. longicornii isolate ROK-HL727. Ca. R. jingxinensis, a novel Rickettsia species in Rhipicephalus microplus and H. longicomis ticks, was first discovered in China (Shenyang and Wuhan) [44–46] and subsequently reported in Korea (Chungnam, Jeonbuk, and Gwangju) [47, 48]. Many associated Ca. R. jingxinensis sequences have been deposited in GenBank. Of these, a gltA sequence (KU853023) was recovered from a patient, suggesting its potential pathogenicity to humans [49]. The pathogenicity of Ca. R. longicornii is yet to be determined. However, 99.6% identity was detected with the Ca. R. longicornii ompA sequence of an isolate from rodent spleen tissue obtained in Korea, and 99.9% identity was detected with the Ca. R. longicornii gltA sequence of an isolate from a human blood sample obtained in China [19, 49]. These results suggest that Ca. R. longicornii has the potential to infect mammalian hosts, including humans [50].
Although not detected in our samples, Borrelia burgdorferi, B. afzelii, and B. garinii, which are implicated in Lyme disease, have been isolated from Ixodes persulcatus, I. nipponensis, I. granulatus, which are distributed in parts of Korea [51, 52]. In addition, several Lyme disease (borreliosis) cases have been reported in Korea [53–56].
Coxiella AB001519, known as a Coxiella-like symbiont of H. longicornis, was detected in all 39 samples [57]. A bacterium belonging to group Coxiella was reported as the primary endosymbiont of Amblyomma americanum ticks and was found to improve the reproductive health of the ticks [58–60]. Coxiella AB001519 was first identified in Japan in a phylogenetic association with the Coxiella-like endosymbiont of H. longicornis, and the Coxiella-like endosymbiont presenting more than 99% homology with Coxiella AB001519 in Thailand, China, and Korea was found in H. longicornis [61–63].
In the present study, the abundance of Actinomycetospora chiangmaiensis found to be higher in Rickettsia spp.-positive samples than in Rickettsia spp.-negative samples, whereas most other bacterial species showed lower abundances in Rickettsia spp.-positive samples. A. chiangmaiensis, a Gram-positive bacterium, was isolated from the soil of a tropical rainforest in northern Thailand in 2008 [64]. Its presence in ticks has been related to the characteristics of the environment, and it can also potentially infect the ticks [65].