Geographical Distribution and Molecular Identification of Larval. Trombiculid Mites in Korea (2005∼2007)

A surveillance of trombiculid mites was conducted by collecting wild small mammals twice a year (spring and autumn seasons) at 24 localities in Korea from 2005 to 2007 to clarify the nationwide distribution of scrub typhus vectors. Among 783 trapped rodents, Apodemus agrarius accounted for 87.4% and the trapping rate at riverside was recorded the highest. A total of 67,325 mites representing 4 genera and 14 species were collected and their chigger index (C. I.) was 86.0. The predominant species were Leptotrombidium pallidum (52.6%), L. scutellare (27.1%), L. palpale (8.2%), L. orientale (5.6%), and Neotrombicula tamiyai (1.7%). However, the portion of L. scutellare in southern area including endemic provinces such as Jeollabuk‐Do (34.3%), Jeollanam‐Do (49.0%), and Gyeongsangnam‐Do (88%) were relatively higher than middle areas where L. pallidum was predominant in. Additionally, in autumn season, the density of L. scutellare was increased up to 42.0% while that of L. pallidum was decrease to 37.6%. Now we are performing a periodic surveillance again (2011～2013) and trying to develop a method to identify these trombiculid mites using molecular markers.     

Seroepidemiological Survey of Zoonotic Diseases in Small Mammals with PCR Detection of Orientia tsutsugamushi in Chiggers,Gwangju, Korea

Serosurveillance for zoonotic diseases in small mammals and detection of chiggers, the vector of Orientia tsutsugamushi, were conducted from September 2014 to August 2015 in Gwangju Metropolitan Area. Apodemus agrarius was the most commonly collected small mammals (158; 91.8%), followed by Myodes regulus (8; 4.6%), and Crocidura lasiura (6; 3.5%). The highest seroprevalence of small mammals for O. tsutsugamushi (41; 26.3%) was followed by hantaviruses (24; 15.4%), Rickettsia spp. (22; 14.1%), and Leptospira (2; 1.3%). A total of 3,194 chiggers were collected from small mammals, and 1,236 of 3,194 chiggers were identified with 7 species of 3 genera: Leptotrombidium scutellare was the most commonly collected species (585; 47.3%), followed by L. orientale (422; 34.1%), Euchoengastia koreaensis (99; 8.0%), L. palpale (58; 4.7%), L. pallidum (36; 2.9%), Neotrombicula gardellai (28; 2.3%), and L. zetum (8; 0.6%). L. scutellare was the predominant species. Three of 1,236 chigger mites were positive for O. tsutsugamushi by PCR. As a result of phylogenetic analysis, the O. tsutsugamushi strain of chigger mites had sequence homology of 90.1-98.2% with Boryong. This study provides baseline data on the distribution of zoonotic diseases and potential vectors for the development of prevention strategies of vector borne diseases in Gwangju metropolitan area.     

Occurrence rates of wild rodent hosts and chigger mites and the infection rate of Orientia tsutsugamushi in the central region of South Korea from 2015 to 2018

Outbreaks of vector‐borne diseases are dramatically increasing because of climate change, consequently increasing the importance of surveillance of endemic disease vectors. In this study, we surveyed chigger mites, vectors for Orientia tsutsugamushi—the bacteria that causes Tsutsugamushi disease—, and their rodent hosts in Gimcheon, central South Korea, in 2015–2018. A total of 225 rodents were collected, with trap rate and percentage of rodents infected by chigger mites of 9.8 and 72.4%, respectively. Six species of rodents from five genera were collected, the most common rodent being Apodemus agrarius (n = 153, infection rate = 90.8%). The highest number of rodents was collected in spring (trap rate = 10.3), but the rate of infected rodents was higher in fall (81.5%) than in spring (61.3%). Trap rate was highest for bank near waterway (17.9), but the chigger index (CI) was highest in hill (224.1). A total of 20,534 (CI 126.0) chigger mite individuals from 10 species and three genera were found on the collected rodents. The most common species was Leptotrombidium pallidum (n = 7,982, 83.6%, CI 49.0), followed by L. palpale and L. scutellare. Chigger mites were most frequent at banks near waterway (n = 11,093, CI 152.0) and hill (n = 2,017, CI 224.1). To detect O. tsutsugamushi in chigger mites, 450 pools of chigger mites (n = 10,991) were analyzed; 24 pools were positive—23 of A. agrarius, the most frequently collected species in South Korea, 1 of Micromys minutus—and the minimum positive rate (MPR) was 0.22. The detected strain types included Boryong (dominant in all years, seasons, and habitats), Jecheon, 07–489, and IIOC1202.     

Three‐year surveillance (2016–2018) of chigger mites vector for tsutsugamushi disease in the Hwaseong‐Si area of Gyeonggi‐Do,Republic of Korea

Owing to climate change, the global resurgence of vector‐borne infectious diseases has emerged as a critical public health issue. Orientia tsutsugamushi is the etiological agent of tsutsugamushi disease (scrub typhus) a mite‐borne acute febrile disease occurring in the Asia‐Pacific region. We investigated the prevalence of tsutsugamushi disease transmitted by chigger mite vectors living on rodents. Using sticky‐type chigger traps for three months during 2016–2018, 1,057 chigger mites were collected (chigger mite index, 1.31) from four locations in the Hwaseong‐si area of Gyeonggi‐do, Republic of Korea. Five species distributed among three genera were identified. In addition, 94 rodents were captured (collection rate: 7.83%) using Sherman live traps over the course of three months (April, October, and November) during 2016–2017. Three rodent species were captured and identified and the striped field mouse (Apodemus agrarius) was the dominant rodent host species in the surveyed area. A total of 10,469 ectoparasitic chigger mites were recovered from the 94 rodents, from which 13 species distributed among four genera were identified. Of the 5,250 chigger mites examined, Leptotrombidium pallidum was most abundant (n = 2,558), followed by L. orientale, L. scutellare, L. zetum, Euschoengastia koreaensis, L. subintermedium, and Neotrombicula tamiyai. Of the examined chigger mites, no groups recovered from rodent hosts tested positive for O. tsutsugamushi. This study provides fundamental regional information on vector‐borne disease data collection in the Hwaseong‐si area, Gyeonggi‐do, and will further contribute to formulating disease control and prevention strategies.     

Scrub typhus caused by Shimokoshi type Orientia tsutsugamushi showing variant 56‐kDa type‐specific antigen gene sequence in Tohoku region,Japan

In 2018, a patient was diagnosed with Shimokoshi type scrub typhus in Yamagata Prefecture, Japan. The causative pathogen was likely a variant type because 43 (8.3%) of 521 deduced amino acid sequences of the 56‐kDa type‐specific antigen (TSA) were different from those of the Shimokoshi prototype strain. The patient's paired sera showed low antibody titers against the Shimokoshi prototype strain. Two cases of scrub typhus reported in the Tohoku region during 2011–2012 also involved the same 56‐kDa TSA gene sequence. These findings suggest the presence of diversity in Shimokoshi type Orientia tsutsugamushi, which may impede the laboratory diagnosis of scrub typhus.     

Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan,and Their Classification into Type and Subtype

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.     

Transovarial Transmission of Orientia tsutsugamushi in Leptotrombidium palpale (Acari: Trombiculidae)

Transovarial transmission of Orientia tsutsugamushi in colonies of Leptotrombidium palpale was studied in the parent and F₁ and F2 generations. Both transovarial transmission and filial infection rates were 100% in the parent and F₁ generations of Leptotrombidium palpale. The filial infection rate in the F₁ generation was 100%, but it declined to 94.3% in the F₂ progeny. The sex ratio of the F₁ generation from infected L. palpale was 1∶0.8 (male:female) and the proportion of males was relatively high. This study is the first to report on the transovarial transmission of O. tsutsugamushi in L. palpale. High transovarial transmission rates in L. palpale suggest that this species might be one of the major vectors of tsutsugamushi disease in Korea.     

Monitoring Chigger Mites for Orientia tsutsugamushi in Field Small Mammals in Hwaseong-si,Gyeonggi-do,Korea, 2019-2020

Incidence of tsutsugamushi disease (scrub typhus) caused by Orientia tsutsugamushi, is steadily increasing. It is a mite-borne disease transmitted by chigger mites. In this study, the chigger mites were collected from field small mammals in Hwaseong-si (city), Gyeonggi-do (province), Korea, 2019 and 2020. The field small mammals captured were 56 Apodemus agrarius (94.9%) and 3 Crocidura lasiura (5.1%). A total of 7,531 chigger mites were collected from the captured small mammals. Using PCR test, 153 chigger mite pools were examined and 17 pools were reported positive for O. tsutsugamushi. The O. tsutsugamushi were identified to 5 strains; Jecheon strain was most prevalent, followed by Boryong strain. The other strains were OI011, Taguchi, and Shimokoshi. Collectively, these results provide essential regional information on mite-borne tsutsugamushi disease in the Hwaseong-si, and further contribute to bring awareness and rapid diagnosis for the tsutsugamushi disease.     

Coincidence between Geographical Distribution of Leptotrombidium scutellare and Scrub Typhus Incidence in South Korea

To clarify the geographical distribution of scrub typhus vectors in Korea, a survey of larval trombiculid mites was conducted from 2005 to 2007 by collecting wild small mammals twice a year (spring and autumn) at 24 sites nationwide. A total of 67,325 mites representing 4 genera and 14 species were collected from 783 trapped rodents, corresponding to a chigger index (number of chigger mites per rodent) of 86.0. The predominant mite species were Leptotrombidium pallidum (52.6%), Leptotrombiduim scutellare (27.1%), Leptotrombidium palpale (8.2%), Leptotrombidium orientale (5.6%), and Neotrombicula tamiyai (1.7%). However, the proportions of L. scutellare in southern areas, including endemic provinces such as Jeollabuk-Do (34.3%), Jeollanam-Do (49.0%), and Gyeongsangnam-Do (88%), were relatively higher than in central Korean regions where L. pallidum was predominant. In autumn, the ratio of L. scutellare increased to 42% while the ratio of L. pallidum decreased. The geographical distribution map of the L. scutellare chigger index was identical to the incidence pattern of scrub typhus, whereas those of overall mites and L. pallidum showed no relationship with case incidence patterns. Distribution mapping analysis shows an identical geographical distribution of L. scutellare and epidemic incidence of scrub typhus in South Korea. L. pallidum could be another vector at all other parts of the Korean peninsula, including the eastern and northern regions that have a low level of scrub typhus incidence.     

Larval Chigger Mites Collected from Small Mammals in 3 Provinces,Korea

A total of 9,281 larval chigger mites were collected from small mammals captured at Hwaseong-gun, Gyeonggi-do (Province) (2,754 mites from 30 small mammals), Asan city, Chungcheongnam-do (3,358 mites from 48 mammals), and Jangseong-gun, Jeollanam-do (3,169 for 62 mammals) from April-November 2009 in the Republic of Korea (= Korea) and were identified to species. Leptotrombidium pallidum was the predominant species in Hwaseong (95.8%) and Asan (61.2%), while Leptotrombidium scutellare was the predominant species collected from Jangseong (80.1%). Overall, larval chigger mite indices decreased from April (27.3) to June (4.9), then increased in September (95.2) and to a high level in November (169.3). These data suggest that L. pallidum and L. scutellare are the primary vectors of scrub typhus throughout their range in Korea. While other species of larval chigger mites were also collected with some implications in the transmission of Orientia tsutsugamushi, they only accounted for 11.2% of all larval chigger mites collected from small mammals.     

Epidemiological characteristics of tsutsugamushi disease in Oita Prefecture, Japan: yearly and monthly occurrences of its infections and serotypes of its causative agent, Orientia tsutsugamushi, during 1984--2005.

Using indirect immunofluorescence assay, we examined the sera of 561 patients from November 1984 to February 2005 to determine the incidence of tsutsugamushi disease (scrub typhus) in Oita Prefecture, Japan. The results obtained were positive in 384 individuals (68.4%). Municipalities where patients were presumed to have been infected with Orientia tsutsugamushi were Taketa City (41.7%), Oyama Town (13.5%), and Ogi Town (8.3%). Infections occurred most often in October, November, and December. A small number of cases occurred from January to May. The serotypes Kuroki (47.5%), Kawasaki (42.5%), and Karp (10.0%) were detected by genetic analysis of O. tsutsugamushi DNA extracted from the blood of 120 patients. The gene sequences of the Kuroki type were highly homologous to that of the Nishino strain. The gene sequences of the Kawasaki type were identical to that of the Kawasaki strain. The gene sequence of the Karp type was highly homologous to that of the JP-2 type. To determine the distribution of vector mites, 558 wild rodents were captured and 72010 mites attached to these rodents were collected from 1982 to 1998. Six genera and 16 species of trombiculid mites were collected. Leptotrombidium pallidum and L. scutellare, which are known to be mite vectors for tsutsugamushi disease, accounted for 20.5% and 5.9%, respectively, of all trombiculid mites collected. The geographical distribution of cases roughly coincided with the distribution of L. scutellare. In Oita Prefecture, L. scutellare is presumed to primarily transmit tsutsugamushi disease. In addition, our results also suggest that L. pallidum transmits the Karp type of the causative rickettsia in some municipalities.     

High Rates of Homologous Recombination in the Mite Endosymbiont and Opportunistic Human Pathogen Orientia tsutsugamushi

Orientia tsutsugamushi is an intracellular α-proteobacterium which resides in trombiculid mites, and is the causative agent of scrub typhus in East Asia. The genome sequence of this species has revealed an unprecedented number of repeat sequences, most notably of the genes encoding the conjugative properties of a type IV secretion system (T4SS). Although this observation is consistent with frequent intragenomic recombination, the extent of homologous recombination (gene conversion) in this species is unknown. To address this question, and to provide a protocol for the epidemiological surveillance of this important pathogen, we have developed a multilocus sequence typing (MLST) scheme based on 7 housekeeping genes (gpsA, mdh, nrdB, nuoF, ppdK, sucD, sucB). We applied this scheme to the two published genomes, and to DNA extracted from blood taken from 84 Thai scrub typhus patients, from 20 cultured Thai patient isolates, 1 Australian patient sample, and from 3 cultured type strains. These data demonstrated that the O. tsutsugamushi population was both highly diverse [Simpson''s index (95% CI) = 0.95 (0.92–0.98)], and highly recombinogenic. These results are surprising given the intracellular life-style of this species, but are broadly consistent with results obtained for Wolbachia, which is an α-proteobacterial reproductive parasite of arthropods. We also compared the MLST data with ompA sequence data and noted low levels of consistency and much higher discrimination by MLST. Finally, twenty-five percent of patients in this study were simultaneously infected with multiple sequence types, suggesting multiple infection caused by either multiple mite bites, or multiple strains co-existing within individual mites.     

Epidemiological characteristics of tsutsugamushi disease in Oita Prefecture, Japan: Yearly and monthly occurrences of its infections and serotypes of its causative agent, Orientia tsutsugamushi, during 1984–2005

Using indirect immunofluorescence assay, we examined the sera of 561 patients from November 1984 to February 2005 to determine the incidence of tsutsugamushi disease (scrub typhus) in Oita Prefecture, Japan. The results obtained were positive in 384 individuals (68.4%). Municipalities where patients were presumed to have been infected with Orientia tsutsugamushi were Taketa City (41.7%), Oyama Town (13.5%), and Ogi Town (8.3%). Infections occurred most often in October, November, and December. A small number of cases occurred from January to May. The serotypes Kuroki (47.5%), Kawasaki (42.5%), and Karp (10.0%) were detected by genetic analysis of O. tsutsugamushi DNA extracted from the blood of 120 patients. The gene sequences of the Kuroki type were highly homologous to that of the Nishino strain. The gene sequences of the Kawasaki type were identical to that of the Kawasaki strain. The gene sequence of the Karp type was highly homologous to that of the JP-2 type. To determine the distribution of vector mites, 558 wild rodents were captured and 72 010 mites attached to these rodents were collected from 1982 to 1998. Six genera and 16 species of trombiculid mites were collected. Leptotrombidium pallidum and L. scutellare , which are known to be mite vectors for tsutsugamushi disease, accounted for 20.5% and 5.9%, respectively, of all trombiculid mites collected. The geographical distribution of cases roughly coincided with the distribution of L. scutellare . In Oita Prefecture, L. scutellare is presumed to primarily transmit tsutsugamushi disease. In addition, our results also suggest that L. pallidum transmits the Karp type of the causative rickettsia in some municipalities.     

DETECTION OF RICKETTSIA TSUTSUGAMUSHI FROM VECTOR TROMBICULID MITE BY DNA AMPLIFICATION USING POLYMERASE CHAIN REACTION TECHNIQUE*

Abstract With reference to the Sta 58 major antigen gene of Rickettsia tsutsugamushi (Karp strain), we had designed and synthesized a pair of DNA primers; and a kilobase fragment of Sta 58 is amplified for using in polymerase chain reaction (PCR) with the primers. This is the first report on detection of R. tsutsugamushi in trombiculids by using PCR technique. The experimental results indicated that the time for which R. tsutsugamushi could exist in the adult of Leptotromhidium deliense when inoculated into its abdomenal cavity was 360 days at least and R. tsutsugamushi could be transovarially transmitted to the offsprings for 4 generations; and the time for which R. tsutsugamushi could exist in L. deliense after biting the infective mouse was 270 days at least and R. tsutsugamushi could be transmitted transovarially to the offsprings for 2 generations. The results also showed that the PCK technique was highly specific and sensitive for detection of R. tsutsugamushi; and the amplification of gene outside body to detect the rickettsiae in the body of trombiculid and mouse can be used as a new method for investigation of scrub typhus epidemiology.     

Geographical distribution of Orientia tsutsugamushi strains in chiggers from three provinces in Korea

Chiggers were collected from the central and southern parts of South Korea between April and November, 2009 with the aim of investigating the seasonal and geographical distribution of Or. A total of 1136 chiggers were identified. They included eight species belonging to four genera, as follows: Leptotrombidium scutellare (27.2%, n = 309), L. pallidum (54.6%, n = 621), L. orientale (6.25%, n = 71), L. palpale (1.59%, n = 18), L. zetum (2.0%, n = 23), Euschoengastia koreaensis (1.5%, n = 17), Cheladonta ikaoensis (0.08%, n = 1) and Neotrombicula japonica (1.05%, n = 12). The density of L. pallidum was high from April to May, whereas L. scutallare was not found in spring, being observed from October. Serotype‐specific nested PCR targeting the 56 kDa protein gene and sequencing analysis identified that the strains of 1136 O. tsutsugamushi in the chiggers as Boryong (6.8%), Kanda (0.4%), Oishi (0.3%), Jecheon (0.1%), Youngworl (0.1%) and Wonju (0.1%). Our findings indicate that L. pallidum and L. scutellare are dominant species in Korea and have geographical and seasonal variations.

     

Consistency of the Key Genotypes of Orientia tsutsugamushi in Scrub Typhus Patients, Rodents, and Chiggers from a New Endemic Focus of Northern China

Scrub typhus is one of the most common infectious diseases of rural south and southeastern Asia and the western Pacific. It emerged in Shandong Province in northern China from autumn to winter of 1986. Since then, the “autumn–winter type scrub typhus” has been found in many areas of northern China. However, the principle genotypes of Orientia tsutsugamushi still remain unknown. This study was undertaken to identify the genotypes of O. tsutsugamushi obtained from scrub typhus patients, chigger mites and rodents from the focal point of the problem in Shandong Province. Forty-four isolates from patients, rodents, and chiggers, 47 blood clots from patients during the acute phase, 10 eschars from patients during the convalescence phase and 16 pools of larval chiggers were tested for the scrub typhus antigen 56-kD protein (Sta56) gene by nested PCR methodology and additional sequence analysis including DNA sequence alignment and phylogenetic analysis. Based on nested PCR, ninety-five initial PCR-positive samples produced amplicons using Kawasaki strain-specific primers, while the other two (the FXS4 and LHGM2 strains) produced amplicons using Karp strain-specific primers. The partial Sta56 gene sequence analysis indicated that the sequence homologies of 3 selected isolates (the B16, FXS2, and XDM2 strains) and 7 eschars out of the 95 samples, which were nested PCR-positive using Kawasaki strain-specific primers, were 94–98 % to that of Kawasaki strain. The sequence homology of the FXS4 and LHGM2 strains to that of the Karp strain was respectively 83 and 96 %. These findings implied that the key genotypes of O. tsutsugamushi in patients, rodents, and chiggers in Shandong Province were identical and similar to Kawasaki strains.     

Serosurveillance of Scrub Typhus in Small Mammals Collected from Military Training Sites near the DMZ, Northern Gyeonggi-do, Korea, and Analysis of the Relative Abundance of Chiggers from Mammals Examined

Comprehensive quarterly serosurveillance on scrub typhus in small mammals collected from military training sites located near the Demilitarized Zone (DMZ), northern Gyeonggi-do (Province), ROK was conducted to determine the potential rodent-borne and associated ectoparasite disease risks to military personnel. A total of 1,196 rodents and insectivores representing 8 species, Apodemus agrarius (87.3%, n = 1,044), Mus musculus (5.4%, n = 65), Crocidura lasiura (3.3%, n = 40), Microtus fortis (2.6%, n = 31), Micromys minutus (0.3%, n = 4), Tscherskia triton (0.3%, n = 4), Rattus norvegicus (0.3%, n = 4), and Myodes regulus (0.3%, n = 4) were assayed for the presence of antibodies to Orientia tsutsugamushi. O. tsutsugamushi antibodies were detected in 6 of 8 species and seroprevalence determined; A. agrarius (45.6%), M. musculus (23.1%), M. fortis (48.4%), M. minutus (50.0%), T. triton (50.0%), and R. norvegicus (25.0%). A total of 31,184 chigger mites collected from 508 rodents and insectivores were slide-mounted and 10 species belonging to 4 genera were identified. Leptotrombidium pallidum (53.4%) was the most frequently collected, followed by L. palpale (15.7%), Neotrombicula tamiyai (14.3%), L. orientale (10.7%), L. zetum (3.1%), Walchia fragilis (2.1%), and L. gemiticulum (0.8%), while the remaining 3 species, L. subintermedium, N. gardellai, and Euschoengastia koreaensis were rarely observed (prevalence < 10%). In contrast to previous surveys, higher chigger indices of the primary scrub typhus vectors, L. pallidum (165.4), L. orientale (45.0), and L. palpale (21.4), were observed during the spring season.     

Rickettsial Agents from Parasitic Dermanyssoidea (Acari: Mesostigmata)

Mites are often overlooked as vectors of pathogens, but have been shown to harbor and transmit rickettsial agents such as Rickettsia akari and Orientia tsutsugamushi. We screened DNA extracts from 27 mites representing 25 species of dermanyssoids for rickettsial agents such as Anaplasma, Bartonella, Rickettsia, and Wolbachia by PCR amplification and sequencing. DNA from Anaplasma spp., a novel Bartonella sp., Spiroplasma sp., Wolbachia sp., and an unclassified Rickettsiales were detected in mites. These could represent mite-borne bacterial agents, bacterial DNA from blood meals, or novel endosymbionts of mites.     

Obligate intracellular bacteria diversity in unfed Leptotrombidium scutellare larvae highlights novel bacterial endosymbionts of mites

It is well known that the mite Leptotrombidium scutellare carries the pathogen of scrub typhus, Orientia tsutsugamushi. However, our understanding of other bacterial endosymbionts of mites is limited. This study investigated the diversity of the obligate intracellular bacteria carried by L. scutellare using 16S rRNA gene amplicon analysis with next-generation sequencing. The results showed that the detected bacteria were classified into the genera Rickettsia, Wolbachia, and Rickettsiella and an unknown genus of the order Rickettsiales. For further classification of the detected bacteria, a representative read that was most closely related to the assigned taxonomic classification was subjected to homology search and phylogenic analysis. The results showed that some bacteria of the genus Rickettsia were identical or very close to the human pathogens Rickettsia akari, Rickettsia aeschlimannii, Rickettsia felis, and Rickettsia australis. The genetic distance between the genus Wolbachia bacteria in the present study and in previous reports is highly indicative that the bacteria in the present study can be classified as a new taxon of Wolbachia. This study detected obligate intracellular bacteria from unfed mites; thus, the mites did not acquire bacteria from infected animals or any other infectious sources. Finally, the present study demonstrated that various and novel bacterial endosymbionts of mites, in addition to O. tsutsugamushi, might uniquely evolve with the host mites throughout overlapping generations of the mite life cycle. The roles of the bacteria in mites and their pathogenicity should be further examined in studies based on bacterial isolation.     

Dual exposure of Rickettsia typhi and Orientia tsutsugamushi in the field‐collected Rattus rodents from Thailand

Field‐collected rodents and fleas from ten provinces covering four regions of Thailand were investigated for possible rickettsial pathogen infections. The 257 trapped‐rodents belonged to 12 species. Five species of Genus Rattus accounted for 93% of the total capture, of which Rattus exulans and Rattus norvegicus were the two major species caught. All flea specimens, removed from trapped rodents, were identified as Xenopsylla cheopis. The PCR technique was performed on ectoparasite specimens to detect the presence of murine typhus pathogen (Rickettsia typhi) and scrub typhus pathogen (Orientia tsutsugamushi). Thirteen flea specimens (2.6 %) were found to be positive for R. typhi but none for O. tsutsugamushi. An ELISA technique was used to detect the rodent's antibodies against R. typhi and O. tsutsugamushi. Sixty‐one rodent serum samples (23.7%) were positive for R. typhi specific IgM, IgG, or both, while 47 of the samples (18.3%) were positive for O. tsutsugamushi. Twenty serum samples from R. norvegicus (7.8%) had detectable antibodies against both R. typhi and O. tsutsugamushi. Our findings revealed the existence of the dual infection of rickettsial pathogens in the same natural hosts.