대한민국 제주도와 전라북도 지역의 야생쥐에서 확보한 털진드기에서 리케치아 종들의 분자적 검출

Abstract

Background

Due to global warming and climate variations, there is an increment in both vector population and pathogen growth rate. Surviving conditions of vectors and vector-borne pathogens are favorable due to warmer winter. As a result of atypical fluctuations in climate, the incidence of tick-borne diseases such as scrub typhus, anaplasmosis, ehrlichiosis, and rickettsiosis showed an alarming increase. It is very important to investigate the tick-borne diseases and its pathogenic distribution in wild animals and ticks. Therefore, in this study, genetic tests were performed to investigate and identify the infections of Orientia tsutsugamushi, Ricekttsia genus, Anaplasma phagocytophilum, and Ehrlichia species in mites collected and obtained from wild rodents from Jeju Island and Jeollabuk-do.

Methods

The wild rodents were captured using Sherman trap between April and November 2018 in Seogwipo-si,Jeju Island, and from April to September 2019 in Jinan-gun, Jeollabuk-do. The collected mites, which were attached to the wild rodents were pooled and homogenized for further study. Genomic DNA was extracted from the homogenized mite lysate. Nested PCR (N-PCR) was performed to detect the presence of each pathogen using target specific gene primers i.e. Sca1 and rompA for Rickettsia strains, 56 kDa for O. tsutsugamushi, ankA and 16s rRNA for A. phagocytophilum. Real time PCR (RT-PCR) was performed using sodB gene for Ehrlichia species. In addition, nucleotide sequencing and phylogenetic analysis was performed for PCR amplicons to determine the presence of the pathogens.

Results

From April to November 2018, 56 wild rodents were captured from Jeju Island, all were identified as Apodemus agrarius, and 684 mites were collected and sampled into 43 pools. From April to September 2019, 44 wild rodents were captured from Jeollabuk-do, with 40 Apodemus agrarius and 4 Clethrionomys rufocanus, and 839 mites were collected and sampled into 43 pools. Of the 1,513 mites, nested PCR targeting Rickettsia genus specific sca1 and rompA genes were performed, both genes were not detected. In the case of scrub typhus, nested PCR was performed targeting 56 kDa gene, amplicons were identified in 0.72 %( 11/1513). The sequencing analysis revealed three cases of O. tsutsugamushi Boryong strain and one case of O. tsutsugamushi Gilliam strain . Anaplasma was subjected to N-PCR targeting ankA and 16s rRNA genes and confirmed as negative in all samples. RT- PCR was performed for Ehrlichia species targeting the sodB gene and confirmed as negative in all samples.

Conclusions

This study was performed for the detection of various Rickettsia species, including O. tsutsugamushi, Anaplasma and Ehrlichia, from the mites collected from wild rodents in Korea. O. tsutsugamushi was detected in 11 samples of 1,513 mites which were collected from the rodents captured from Jeju in 2018 and Jeollabuk-do in 2019. O. tsutsugamushi Boryong strain was identified in three pools whereas Gilliam strain in one pool. However, Rickettsia, Anaplasma, and Ehrlichia were negative in all the mite samples. In conclusion this study identified the infection of O. tsutsugamushi in the mites obtained from rodents in Jeju and Jeollabuk-do areas. Due to the global climate change and increment in outdoor activities, the incidence of tick-bone infection is increasing not only in Korea but also around the world, with more diversified infectious conditions. Therefore, regular monitoring and conducting research on various infectious diseases, specially scrub typhus and rickettsiosis is very important. Systematic investigations of mites and ticks will assist in identifying and monitoring the presence of pathogens in forthcoming infectious diseases, subsequently it can be a serious threat to human health in the future.

Keywords: nested PCR, real time PCR, O. tsutsugamushi, A. phagocytophilum, Ehrlichia, Rickettsia