From September 2014 to August 2015, small mammals were collected using Sherman live traps (3"×3.5"×9", USA) in 2 regions (Gwangsangu and Bukgu) of Gwangju Metropolitan Area (MA), ROK. Collection sites were just within the city perimeter. The 2 regions where traps were installed were in a similar environment. Both included 5 types of locations. The locations were fallow ground, a ridge between rice fields, a boundary between forest and field, around tombs, and around water. Traps were baited with biscuits covered with peanut butter and set up in the late afternoon, and collected from 8-10 a.m, the following morning. Gwangju MA surrounded by Chonnam Province is a densely populated area (Fig. 1).

Traps positive for small mammals were numbered, placed into secure shipping containers, and then transported to the Health and Environment Research Institute of Gwangju MA. Small mammals were euthanized using chloroform (Merck, West Point, Pennsylvania, USA) soaked cotton (1×1 cm). Then, blood was centrifuged at 3,000 rpm for 20 min, and serum was separated and maintained at 4˚C. Indirect immunofluorescence assay test (IFAT) and passive hemagglutination assay (PHA) were performed 24-36 hr after the small mammals were euthanized for antibody detection. The carcasses of the small mammals were hung over a glass bowl containing water to harvest chiggers, and chiggers were collected the following day.

A total of 10 μl of sera from each small mammal was used for serial dilutions of 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1,024, and 1:2,048 in PBS (pH 7.2). Diluted sera were deposited on an antigen spot slide, incubated at 37˚C for 30 min in a humidified chamber, and then washed as in step 2. First, the slide was washed for 3 min with PBS, and second, washed for 3 min with distilled water to remove PBS salt. A total of 25 μl fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma, St. Louis, Missouri, USA) was added to a spot slide, and the slides were incubated at 37˚C for 30 min in a humidified chamber. The slides were washed for 3 min with PBS, distilled water, and then air-dried. After mounting medium (Sigma) was added to a spot slide, and covered with coverslip, the slides were examined for specific spots using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). A cutoff titer of 1:16 was used to identify seropositivity. Antigen spot slides for O. tsutsugamushi, hantaviruses, and Rickettsia spp. were provided by KCDC.

Genedia Lepto PHA (Green Cross, Seoul, Korea) kit reagents were used for detection of leptospirosis by PHA. A total of 25 μl serum from each small mammal was added to a 96-well plate, and diluted 1:80 in a dissolved solution of Genedia Lepto kit. A total of 75 μl of sheep blood containing red blood cells sensitizied by Leptospira spp. was placed on a diluted serum for agglutination assay. Ag-Ab agglutination reaction in 1: 80 tested positive for leptospirosis.

The method used by Ree et al. [8] was applied for the detection of O. tsutsugamushi from individual chiggers. Individual chiggers were placed on a glass slide with PBS 10 μl. The chigger’s internal contents were squeezed out with 2 fine pins and observed after suspension with 50 μl PBS under stereomicroscope (Carl Zeiss). The chigger exoskeleton was mounted with polyvinylalcohol medium (Bioquip, Gardena, California, USA) and identified by Ree’s fauna key [9]. Because it was difficult to perform with all chiggers, 30 chiggers per small mammals were used.

DNA was extracted from 20 μl of chigger suspension using G-spin total DNA extraction kit (cat. no. 17046; Intron Biotechnology, Seoul, Korea). 200 μl of lysis buffer and 10 μl of proteinase K solution (20 mg/ml) was added to 20 μl of chigger suspension. The lysate was incubated at 56˚C on a heating block for 30 min. After lysis, 200 μl of binding buffer was added. Then, the mixture was incubated at 70˚C for 5 min. The mixture was applied to the spin column, and centrifuged at 13,000 rpm for 1 min. After discarding the filtrate, washing buffer A was added to the spin column and centrifuged at 13,000 rpm for 1 min. After discarding the flow-through, the column was placed into a 2.0 ml collection tube. Washing buffer B was added to the spin column and centrifuged at 13,000 rpm for 1 min. After discarding the flow-through, placed column into a new 1.5 ml collection tube, and a total of 50 μl of elution buffer was added directly onto the membrane. After incubating for 1 min at room temperature, it was centrifuged for 1 min at 13,000 rpm to elute. DNA extract was stored at -20˚C until amplification.

PCR was performed as INNOPLEX TSUTSU detection kit (cat. No. IPC10040; Intron Biotechnology). The kit was designed using primer sets to detect the 475 bp fragment gene encoding the 56 kDa antigen of O. tsutsugamushi. The first PCR was performed with 2 μl of DNA extract and 18 μl of distilled water treated with diehyl pyrocarbonate (DEPC; Gendepot, Barker, Texas, USA) in the first PCR premix tube. PCR conditions were as follows: initial denaturation at 94˚C for 5 min; 40 cycle at 94˚C for 30 sec, 58˚C for 30 sec, 72˚C for 40 sec; and final elongation at 72˚C for 5 min using a Geneamp 9700 Biosystem (ABI, Foster City, California, USA). The second PCR was performed with 2 μl of the first PCR product and 18 μl of distilled water with DEPC in the second PCR premix tube. PCR conditions were as follows: initial denaturation at 94˚C for 5 min; 30 cycle at 94˚C for 30 sec, 58˚C for 30 sec, 72˚C for 40 sec, and final elongation at 72˚C for 5 min using a Geneamp 9700 Biosystem (ABI). The final PCR products were evaluated by 1.5% agarose gel electrophoresis containing ethidium bromide.

The nucleotide sequence homology was aligned with sequence of previously published O. tsutsugamushi in Genbank by the BIOEDIT software program. The phylogenetic tree was generated by the neighbor-joining method (MEGA 6.0).

A total of 172 small mammals were collected. Apodemus agrarius was the most frequently collected 158 (91.8%), followed by Myodes regulus 8 (4.6%) and Crocidura lasiura 6 (3.5%). Overall, monthly mean trap rate was 14.3% (range 7-26%) (Table 1). For all periods, the trap rate for A. agrarius was higher for fallow ground (34.2%), followed by around water (29.1%), a boundary between forest and field (17.7%), around a tomb (13.9%), and a ridge between grain fields (5.1%) (Table 2).

Blood samples were obtained from 156/172 (90.7%). A total of 41/156 (26.3%) were seropositive for O. tsutsugamushi followed by small mammals positive for hantaviruses (24/156, 15.4%), Rickettsia spp. (22/156, 16.7%), and Leptospira spp. (2/172, 1.3%). Especially, tested 12 rodents were all positive for O. tsutsugamushi in October 2014. The prevalence for 4 vector-borne (deletion) infectious diseases was confirmed all the year round in Gwangju MA. Total 21 cases of mixed infections were confirmed in A. agrarius (Table 3).

A total of 2,772 chiggers were collected from 158 A. agrarius (Table 4). L. scutellare 579 (54.1%) was the most commonly collected chiggers, followed by L. orientale 281 (1.8%), E. koreaensis 94 (8.8%), L. palpale 46 (4.3%), L. pallidum 36 (3.3%), and L. zetum 7 (0.7%). The majority of L. scutellare were collected during the autumn (September-November, mean 5.6-21.4), while L. orientale was collected in late autumn (October, mean 1.7) through spring (May, mean 3.0). A high number of E. koreaensis was collected in fall (September, 6.8), but infrequently during other months (Fig. 2).

The distribution of chiggers by species of small mammals was 7 chigger species on A. agrarius, 4 species on M. regulus, and no chiggers on C. lasiura (Table 5). A total of 3 chiggers (0.24%) were positive for O. tsutsugamushi (Table 5). Each sequence (1,410 mite-1 L. scutellare, 1,410 mite-2 L. scutellare, 1,504 mite-3 L. orientale) showed homology of 90.1%, 93.2%, and 98.2% with the Boryong strain (accession no. L04956) (Fig. 3). Two L. scutellare collected from A. agrarius (September 2014), and 1 L. orientale from M. regulus (April 2015) were confirmed as positive for O. tsutsugamushi.