Scrub typhus, also known as tsutsugamushi disease, is an acute febrile disease that is characterized by fever, rash, and eschar. The disease is transmitted to humans by the bite of larval trombiculid mites harboring O. tsutsugamushi and is endemic in the Asia-Pacific region . In Japan, scrub typhus is the most common rickettsiosis, more than 300 cases being reported each year . O. tsutsugamushi strains found in humans in Japan are classified into six serotypes: Gilliam, Karp, Kato, Kawasaki, Kuroki and Shimokoshi . Currently, Gilliam, Karp, Kawasaki and Kuroki are the major scrub typhus serotypes associated with human infections [3, 4]. Kato type is rarely reported  and Shimokoshi type was first reported in 1984 , only a few cases having been reported thereafter [6-8]. These serotypes are genetically distinguishable by nested PCRs targeting the O. tsutsugamushi 56 kDa protein gene [6, 9].
It is presumed that human scrub typhus is primarily transmitted by mites of the genus Leptotrombidium . In Japan, L. pallidum, L. scutellare and L. akamushi are believed to transmit Gilliam and Karp types, Kawasaki and Kuroki types and Kato type, respectively . However, the vector of the Shimokoshi type is yet to be identified; this failure acts as a bottleneck in the clarification of the epidemiology of Shimokoshi type scrub typhus in Japan.
The Tohoku region, which includes Yamagata Prefecture, is located in the northern part of Japan's main island of Honshu and is known to be endemic for Gilliam and Karp types . Recent studies have shown that 90% of cases of Karp type scrub typhus are caused by the JP-1 type, which is a Karp genotype [11, 12]. As for the Shimokoshi type, only eight cases have been reported to date [6-8]. In the present study, we used larval trombiculid mites to conduct a survey in Yamagata Prefecture, Japan to investigate potential vectors for Shimokoshi type O. tsutsugamushi.
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- MATERIALS AND METHODS
This is the first study to detect Shimokoshi type O. tsutsugamushi DNA in L. palpale. A previous study reported that, like L. pallidum, L. scutellare and L. akamushi, which have been proved to transmit O. tsutsugamushi to humans, L palpale also bites humans . Furthermore, researchers isolated uncharacterized O. tsutsugamushi-like organisms from L. palpale in Japan between 1960s and 1980s before Shimokoshi type O. tsutsugamushi had been recognized . In this study, we detected Shimokoshi type O. tsutsugamushi DNA from three mites, all of which we identified as L. palpale, whereas other Leptotrombidium species were negative for this type (Table 2). Taken together, these results indicate that it is highly possible that L. palpale is a key reservoir for Shimokoshi type O. tsutsugamushi and thus that it transmits Shimokoshi type scrub typhus to humans. In addition, of nine cases diagnosed with Shimokoshi type scrub typhus in Japan, six occurred in spring and three in autumn [5-8]. L. palpale is mainly found during spring in natural environments [15, 16]. Consistent with this, we collected three L. palpale mites positive for Shimokoshi type O. tsutsugamushi DNA between April and May. The seasonal pattern of activity of these mites also tends to support our contention that L. palpale is a potential vector of Shimokoshi type O. tsutsugamushi.
Karp type O. tsutsugamushi can be divided into two genotypes (JP-1 and JP-2) based on the 56 kDa type-specific antigen protein gene . The vector for JP-2 type is known to be L. pallidum , whereas that for JP-1 type is controversial. In this study, we identified JP-1 type O. tsutsugamushi DNA in L. pallidum (Table 2). Because L. pallidum is the predominant trombiculid mite species in the Tohoku region  and JP-1 type scrub typhus is dominant in this region , it is reasonable to speculate that L. pallidum transmits JP-1 type O. tsutsugamushi to humans. In an earlier study, researchers also isolated JP-1 type O. tsutsugamushi from L. intermedium . However, as this species rarely bites humans , L. pallidum is more likely to play a significant role in disease transmission to humans.
It should be mentioned that this study has the following limitations. First, because we collected larval trombiculid mites from field rodents, we cannot exclude the possibility that O. tsutsugamushi-positive larvae pick rickettsia up from infected rodents and transiently harbor them, but are unable to transmit them to humans. We showed that three of four rodents did not have antibodies against O. tsutsugamushi whereas all of them were negative for O. tsutsugamushi infection on the basis of nested PCR (Table 4). Futhermore, whereas these rodents were parasitized by seven different mite species (Table 3), serotype-specific PCRs showed that all species except L. palpale and L. pallidum were negative for both Shimokoshi and JP-1 types (Tables 2 and 3). These lines of evidence suggest that mites that PCR finds positive for O. tsutsugamushi are infected with rickettsia and did not acquire rickettsia or rickettsial DNA transiently from the rodents.
Second, we developed a real-time PCR assay based on the 16S rRNA gene. Since the primers and probe were designed to anneal to a region well conserved among the different serotypes and even among closely related rickettsial species , it is possible that not only the six known serotypes but also other uncharacterized serotypes of O. tsutsugamushi and/or other related rickettsial species are detected by this real-time PCR. In particular, despite the fact that we found four samples of G. saduski (R01-m044, R16-m090, R16-m102 and R17-m002) and one sample of L. intermedium (R18-m027) to be positive for O. tsutsugamushi by real-time PCR with low Ct values, we found them all to be negative by serotype-specific nested PCR (Table 2), suggesting that these mites may harbor previously uncharacterized O. tsutsugamushi serotypes or related rickettsial species.
Third, serotype-specific nested PCRs were negative in six samples of L. pallidum and one sample of L. palpale(Table 2). Since the assays amplify the gene encoding a 56 kDa outer membrane protein, which is polymorphic , one possibility is that the nucleotide sequences of primer annealing sites were not conserved in those samples. Considering that most of those samples were positive by real-time PCR with high Ct values, it is also possible that serotype-specific nested PCRs were negative due to the difference in sensitivity between nested PCR and real-time PCR.
So far, only a few cases of Shimokoshi type scrub typhus have been reported in Japan [5-8]. This could be because no laboratory diagnostic technique for Shimokoshi type scrub typhus is widely available. Almost all commercial laboratories in Japan only test specimens from patients with suspected scrub typhus for Gilliam, Karp and Kato type antibodies. In addition, the regional institutes for public health, which are in charge of infectious disease diagnosis, seldom test for Shimokoshi type scrub typhus . However, we expect that Shimokoshi type scrub typhus cases may no longer be overlooked because researchers recently reported a nested PCR method for detection of Shimokoshi type O. tsutsugamushi . Based on our results, further investigations, including transmission studies in laboratory animals, are necessary to clarify the role of L. palpale as a vector of Shimokoshi type scrub typhus.