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Epidemiological Characteristics of Rodents and Chiggers with Orientia Tsutsugamushi in the Republic of Korea

A survey of rodents and chiggers associated with Orientia tsutsugamushi was conducted in a rural region of the Republic of Korea (Korea) between 2014 and 2018. Overall Apodemus agrarius 15.2% had the highest seropisitive for O. tsutsugamushi, followed by Myodes regulus 11.4%. Monthly risk factors using logistic regression analysis were not associated with O. tsutsugamushi infections in rodents. The overall prevalence rate of O. tsutsugamushi among chiggers was 0.3%. The chigger (Leptotrombidium scutellare) and monthly (October) risk factors were associated with O. tsutsugamushi human infections (P<0.05). Orientia tsutsugamushi infections are endemic in rodents in Korea and people, for example, soldiers who are active outdoors, must employ preventive measures, especially during October (P<0.05). When there are many reports of O. tsutsugamushi infections in Korea. The Boryong strain 85.7% (2/14) was the most common strain detected in chiggers, followed by the Shimokoshi 7.1% (1/14) and Karp 7.1% strains.


INTRODUCTION
Orientia tsutsugamushi, the causative agent of scrub typhus, is transmitted by chiggers (larval trombiculid mites), and can result in death if untreated. Additionally, chigger bites cancause severe itching in people spending time outdoors, especially during fall [1]. Scrub typhus is distributed throughout much of East and Western Central Asia to northern Australia; cases have also been reported in Africa and Europe [2]. Seasonal outbreaks of scrub typhus in Asia are more frequently reported in the fall and early winter in Korea [3]. Studies on scrub typhus prevalence during fall, the primary period of transmission, have primarily focused on chiggers as vectors of O. tsutsugamushi and their distribution [4][5][6][7].
The geographic distribution of scrub typhus depends on the population density and vector competence of local chigger vectors [6]. Statistical studies aimed at predicting and preventing the occurrence of scrub typhus infections have been con-ducted both inside and outside Korea with respect to human outdoor activities and to the time spent on activities such as preventive measures as well as the duration of exposure in chigger habitats such as grasses [8,9]. A statistical model was recently presented for scrub typhus occurrence and included climatic factors such as precipitation and humidity, and the occurrence of scrub typhus cases [9]. It is thus necessary to understand the epidemiological parameters for the seasonal prevalence of O. tsutsugamushi infections, based on vector distributions and population densities to predict the occurrence of exposure-associated infections [17]; however, there have been few such studies in Korea.
Therefore, this study examined the seasonal trends of scrub typhus and established the appropriate preventive measures. It also includes a cross-sectional study to determine the risk factors associated with the relationship between the occurrence of O. tsutsugamushi infections in rodents and the associated chigger vectors from 2014 to 2018 using a statistical program (SPSS 20, IBM, Armonk, New York, USA).

Ethics statement
All procedures were conducted under an animal use proto-col approved by the Chosun University Animal Ethics Committee (CIACUC 2016-S0003).

Rodent and chigger collections
Rodents were collected monthly from a suburban area (35°13'N126°54'E) with uncut grasses using Sherman live traps (3× 3.5× 9 inches) (BioQuip, Rancho Dominguez, California, USA). The study area was located in a densely populated area in the southwestern part of Korea, where scrub typhus patients are reported frequently. The information for the human infection rate was obtained from Korea Centers for Disease Control and Prevention(K-CDC) Disease Web Statistics System [3]. In total, 100 traps baited using crackers covered with peanut butter were set monthly from 10:00 to 14:00 hr the following day. Traps positive for rodents were numbered sequentially, placed in a secure container, and then transported to a central laboratory [4]. The rodents were euthanized using chloroform (Merck, New Jersey, USA) soaked in cotton (1× 1 cm), and blood was collected by cardiac puncture using a 10-cc syringe. The collected blood was centrifuged at 3,000 rpm for 20 min, serum was separated, and then stored at 4˚C. An indirect immunofluorescence assay was performed for antibody detection at 24 hr after the rodents were euthanized. After autopsy, the rodents were hung upside down over a dish containing distilled water for 24 hr. Chiggers were then collected from the water surface using a fine brush.

Indirect immunofluorescence assay for Orientia tsutsugamushi in wild rodents
In total, 10 µl of serum from each rodent was used. Antigen spot slides for O. tsutsugamushi were provided by the Korea Centers for Disease Control and Prevention (KCDC), and indirect immunofluorescence assays were conducted as described previously [14]. Nested polymerase chain reaction (nPCR) was performed as described previously for O. tsutsugamushi detection in chigger mites [11]. A total of 30 chiggers/rodent were examined and placed individually on glass slides with 20 µl of PBS. The abdomen of each chigger was gently pressed using 2 fine needles until the internal contents were expelled and then observed after suspension in 30 µl of PBS under a stereomicroscope (Carl Zeiss, Oberkochen, Germany). The chiggers were mount-ed with polyvinyl alcohol medium (BioQuip) and identified using a morphological key [12]. DNA was extracted from 20 µl of chigger material using a G-spin Total DNA Extraction Kit (iNtRON Biotechnology, Daejeon, Korea). and then analyzed by PCR for the O. tsutsugamushi 56-kDa TSA gene as described previously [13,14].

Risk factors for O. tsutsugamushiin rodent hosts
This study was conducted to identify the relationship between the risk factors (month, species) and prevalence of O. tsutsugamushi infection in rodents and chiggers. The month associated with the mean distribution of disease was evaluated as a risk factor in rodents by logistic regression analysis using SPSS version 20 software (IBM).The 95% confidence interval (CI), and the chi-square analysis was performed for the rodent species and proportion of infection by month as the risk factors in chiggers (P < 0.05).

Sequence and phylogenetic analysis
Amplified PCR products were submitted to Cosmogenetech (Daejeon, Korea) for sequencing using an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, California, USA). For phylogenetic tree construction, the twelve sequences (KX363949-KX363951, KX363952-KX363953, KY266824-KY266830) examined by Park in previous studies [13,14] and 2 sequences (MK451703 and MK451704) that were newly identified in 2017 were compared with O. Tsutsugamushi sequences downloaded from the National Center for Biotechnology Information database, and phylogenetic analyses were conducted using MEGA X. A neighbor-joining tree with 1,000 bootstrap replicates was constructed using the Kimura 2-parameter model.  (Fig. 1).

DISCUSSION
O. tsutsugamushi is endemic in Korea throughout the year, and personal protection should be reported to prevent chigger bites, especially a month before the onset of increased numbers of human infections.
Although O. Tsutsugamushi was present in rodents throughout the year, analysis of O. tsutsugamushi and the monthly factors in rodents did not demonstrate any association (Table 1). However, this study showed a lower prevalence of infection in rodents than that observed in other studies [15][16][17][18][19][20]. Because previous studies have only been conducted seasonally (spring, fall), they do not reflect the monthly O. tsutsugamushi antibody prevalence in rodents.
The monthly distribution of chiggers revealed a particularly high prevalence during the fall season (Fig .1), when the incidence of scrub typhus patients was the highest in the population of the country. However, O. tsutsugamushi positive chiggers were unexpectedly found to occur throughout the year, except during summer. Chiggers were first observed in September and a sharp increase in the mean number of chiggers was observed in October, after which the number of chiggers declined to low numbers in December and persisted until July. Therefore, people should be especially aware of the risks associated with outdoor activities in the spring and fall. There have been reports of scrub typhus onset in humans during spring when a large number of chiggers are present; the increased incidence of spring scrub typhus has also raised concerns related to global warming [17].
The prevalence of O. tsutsugamushi in chiggers was most commonly associated with L. scutellare, and is consistent with L. scutellare as the primary vector of O. tsutsugamushi in Korea [4,5,21]. Chigger species as a risk factor for rodents was not as-    [3]. Our results, supported by the incubation period (7-30 days) of scrub typhus in humans [8], which correspond to the time interval from infection to visiting the hospital to receiving treatment after symptom development.
In a study by Park in 2014-2016 [13,14] conducted in the same region, the O. tsutsugamushi genotype for the 56-kDaTSA gene detected in L. scutellare belonged to the Boryong strain, whereas other genotypes (Karp, Shimokoshi) were identified in 2017. Although there wasonly one unique type, the Shimokoshi strain was identified in L. palpale.
In conclusion, these results indicate that people must be aware of the risks of the exposure to chiggers that are vectors of O. tsutsugamushi, especially during October when transmission is the highest. According to our study, soldiers, farmers, and other people that spend time outdoors belong to high-risk group sand should be careful when performing outdoor activities, especially during October. To establish effective preventive measures for O. tsutsugamushi infections, long-term data on meteorological and ecological factors are needed. Studies conducted in additional areas might increase the reliability of disease prediction throughout Korea.

ACKNOWLEDGMENTS
This study was financially supported by the Health and Environment Research institute (HERI) of Gwang-ju, Korea. This was a joint research project between HERI of Gwang-ju and Chonnam National University.