• View in gallery

    Small mammals collection sites at A (35°13′51.7″ N, 126°54′23.8″ E) and B(35°09′19.2″ N, 126°45′05.4″ E) in the southwest area of Korea, from September 2014 to December 2016. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Monthly chigger indices by species collected from small mammals captured in the southwest area of Korea, from September 2014 to December 2016. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Phylogenetic tree of Orientia tsutsugamushi based on the 56-kDa type specific gene sequence detected in chiggers; closed rectangle ■ (KX363952, KX363953, KY266824-KY266830), rodent (KX363954), human; open circle ○ (KY946003, KY946031, KY946061, KY946107, KY946108, KY946128, KY946124-KY946127). Phylogenetic tree was constructed by neighbor-joining method with the Kimura’s two-parameter model (bootstrap 1,000) using MEGA 6.0. Genbank accession numbers of O. tsutsugamushi are indicated for each sequence.

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Molecular Epidemiology of an Orientia tsutsugamushi Gene Encoding a 56-kDa Type-Specific Antigen in Chiggers, Small Mammals, and Patients from the Southwest Region of Korea

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  • 1 Division of Infectious disease investigation, Health and Environment Research Institute of Gwangju City, Gwangju, Korea;
  • 2 Clinical Pathology, Gwangju Health University, Gwangju, Korea;
  • 3 Departments of Internal Medicine, School of Medicine, Chosun University, Gwangju, Korea;
  • 4 Premedical Science, College of Medicine, Chosun University, Gwangju, Republic of Korea;
  • 5 Division of Zoonoses, Korea Centers for Disease Control and Prevention, Cheongju, Korea

A phylogenetic analysis of Orientia tsutsugamushi was performed to elucidate its antigenic diversity in chiggers, small mammals, and patients. Between September 2014 and December 2016, a total of 3,816 chiggers were identified within nine species of four genera in the southwest region of Korea: Leptotrombidium scutellare (49.9%; 1,907/3,816), Leptotrombidium orientale (21.1%; 804/3,816), Leptotrombidium pallidum (12.4%; 474/3,816), Euchoengastia koreaensis (7.2%; 273/3,816), Leptotrombidium palpale (6.7%; 256/3,816), Neotrombicular gardellai (1.3%; 50/3,816), Leptotrombidium zetum (0.8%; 32/3,816), Walchia fragilis (0.5%; 18/3,816), and Neotrombicular japonica (> 0.1%; 2/3,816). Twelve chiggers (11 L. scutellare and one L. palpale) tested positive for O. tsutsugamushi by polymerase chain reaction and, except for 1 chigger (KY266830), were part of the Boryong strain cluster. Of the 413 small mammals that were analyzed for O. tsutsugamushi, Apodemus agrarius was the most common rodent species (89.5%; 370/413), followed by Crocidura lasiura (6.8%; 28/413) and Myodes regulus (3.6%; 15/413). The sequence identity of an O. tsutsugamushi sample obtained from the A. agrarius sample population belonged to the Saitama strain cluster. Furthermore, a phylogenetic analysis in 125 patients revealed four clusters (Boryong cluster: 82.4% [103/125], Karp: 13.6% [17/125], Kawasaki: 3.2% [4/125], and Saitama: 0.8% [1/125]). This study clarified the phylogenetic relationship for O. tsutsugamushi in chiggers, small mammals, and patients. The Boryong strain was the most common strain in chiggers and patients. In addition, various strains were identified, except for the Boryong strain, in the southwest region of Korea. Overall, the data presented here will be helpful for the establishment of prevention strategies for scrub typhus.

INTRODUCTION

Orientia tsutsugamushi is a rod-shaped, gram-negative, intracellular bacterium that belongs to the order Rickettsiales and is the causative agent of scrub typhus or Tsutsugamushi disease.1,2 Unlike other bacteria, the cell walls of O. tsutsugamushi lack a peptidoglycan and lipopolysaccharide layer. Scrub typhus has been recognized as a major cause of acute febrile disease that is encountered in rural regions such as bushes and abandoned grain field.3 This disease is endemic to the Western Pacific region, northern Australia, and Central Asia. Clinical characteristics of scrub typhus include eschar, fever, rash, meningitis, intracellular coagulation, lymphadenopathy, and multiple organ failure. Moving forward, if patients are not treated, the fatality rate will continue to increase.4

Furthermore, O. tsutsugamushi can be transmitted by the larval trombiculid mite (chigger) while feeding, which then can propagate to its progeny through transovarian transmission. The Leptotrombidium species are known to act as the primary vector for O. tsutsugamushi,5,6 where the major species, Leptotrombidium pallidum and Leptotrombidium scutellare, can be found in the central and southern regions of Korea, respectively.7,8

Wild rodents, such as Apodemus agrarius, are also natural hosts of scrub typhus as well as for chiggers.9,10 At the same regions where this study was performed, a serosurveillance for scrub typhus in small mammals was performed in 2014–2015 by Park, and a seropositive rate of 25% (37/145) in A. agrarius was observed.11

The 56-kDa type-specific antigen (TSA) is an immunodominant protein located on the surface of O. tsutsugamushi that is not expressed in other bacteria. Therefore, phylogenetic analysis of the 56-kDa TSA gene sequence is useful to clarify variations of scrub typhus.1215

Worldwide, more than 20 variations of scrub typhus are caused by various prototype strains of O. tsutsugamushi. The Boryong strain is predominant in Korea. The Giliam, Karp, Kato, TA678, TA686, TA716, TA763, and TH1817 strains have been reported in Southeast Asia, Australia, and Taiwan. In addition, the Gilliam, Karp, Kato, Kawasaki, Kuroki, and Shimokoshi strains have been found in Japan.1 Furthermore, differences in virulence according to genotype variations of O. tsutsugamushi have been identified.16

Recently, climate change may have affected the life cycle of wild rodent and chiggers.17 Therefore, clarifying the relationship between the vector and environment may help predict future cases of scrub typhus.1823 In northern China, a phylogenetic analysis for scrub typhus in chiggers, rodents, and patients was conducted, and consistency of circulating strains in the hosts was demonstrated.24

According to the Korea Centers for Disease Control and Prevention, 10,000 cases of scrub typhus were reported annually between 2013 and 2015, as well as 6,000 cases in 2005. The distribution of chiggers has also expanded from the southern region to the central region for the past decade.25 Therefore, scrub typhus is an increasingly important disease in Korea.2629

For the development of strategies to control this disease, a better understanding of the vector’s life cycle is required as well as knowledge of the O. tsutsugamushi strain in the host. We surveyed a distribution of chiggers and performed a phylogenetic analysis of the 56-kDa TSA gene sequence to identify the genetic characteristics of O. tsutsugamushi in chiggers, small mammals, and patients in the southwest region of Korea.

MATERIALS AND METHODS

Collection of small mammals and chiggers.

All procedures were conducted under an animal use protocol approved by the Chosun University Animal Ethics Committee. From September 2014 to December 2016, small mammals were trapped monthly using Sherman live traps (3 in × 3.5 in × 9 in, USA) baited with peanut butter–covered biscuits in two southwest regions of Korea (Figure 1). After wild rodents were euthanized, their organs (spleen, kidney) were collected for detection of O. tsutsugamushi. Chiggers collected from the rodents were stored in 70% ethyl alcohol until used to acquire sample fluids for DNA extraction.

Figure 1.
Figure 1.

Small mammals collection sites at A (35°13′51.7″ N, 126°54′23.8″ E) and B(35°09′19.2″ N, 126°45′05.4″ E) in the southwest area of Korea, from September 2014 to December 2016. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 98, 2; 10.4269/ajtmh.17-0070

Patients’ blood sample.

From 2014 to 2015, blood samples were collected from patients who had visited Chosun University Hospital with suspected scrub typhus in the southwest region of Korea. Blood sample analysis was blinded. All experiments were conducted under approval of the Chosun University Medical College (IRB number: 2013-10-001).

Acquisition of fluids from chiggers and species identification.

The fluids from the engorged chiggers were acquired as previously described.30 The chigger’s internal contents (organ, fluid, and hemolymph) were squeezed out with two fine pins under a stereomicroscope (Carl Zeiss, Oberkochen, Germany). The chigger exoskeleton was identified by Ree’s fauna key.31

DNA extraction from chigger samples.

DNA from the chigger samples were purified using the G-spin Total DNA Extraction Kit (Intron Biotechnology, Seoul, Korea). The DNA from the rodent’s organs as well as the patients’ blood was purified using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA was stored at −20°C until use.

Nested polymerase chain reaction (PCR) amplification for the 56-kDa TSA gene.

PCR was performed via the INNOPLEX TSUTSU detection kit (catalogue number: IPC10040; Intron Biotechnology). The kit was designed using primer sets (First F: 5ʹ-GCA ATA TTG CTA GTG CAA TGT CTG C-3ʹ, First R: 5ʹ-ATG CAT GCA TGR CGC TKC AAT TTA-3ʹ; Second F: 5ʹ-ATA GGC CTA TAA GTA TWG CKG ATC G-3ʹ, Second R: 5ʹ-CAT CTA GAY GCA CTA TTA GGC AAA-3ʹ) to detect the 56-kDa TSA gene of O. tsutsugamushi. The target was a 426–862-bp region that includes variable domain 2, 3 in the total TSA 56 gene (1,566 bp). The first PCR was amplified at 94°C for 5 minutes, followed by 40 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 40 seconds, and a final elongation at 72°C for 5 minutes. The second PCR was amplified at 94°C for 5 minutes, followed by 30 cycles at 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 40 seconds, and a final elongation at 72°C for 5 minutes using the GeneAmp 9700 Biosystem (ABI, California). The final 475-bp PCR products were evaluated by 1.5% agarose gel electrophoresis and visualized with ethidium bromide and an ultraviolet transilluminator.

Sequence and phylogenetic analysis.

The amplified PCR products were sent to Cosmogenetech (Daejeon, Korea) for sequencing using the ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). The nucleotide sequences were aligned using ClustalW to a reference sequence downloaded from the National Center for Biotechnology Information database, and phylogenetic analyses were conducted via the MEGA6 program. A neighbor-joining tree with 1,000 bootstrap replicates was constructed using the Kimura’s two-parameter model. Sequences obtained from chiggers, rodents, and human samples were submitted to GenBank (accession number: KY266824-KY266830, KX363954, and KY946003-KY946128, respectively).

RESULT

Small mammal collection and chigger infestation rate.

During the study period, 413 small mammals, including two species of rodents and one species of shrew, were captured. Apodemus agrarius were the most common small mammals (370; 89.5%), followed by Crocidura lasiura (28; 6.8%) and Myodes regulus (15; 3.6%). Furthermore, A. agrarius were captured year-round. The infestation rate of chiggers on the small mammals was 51.8% (214/413), A. agrarius 53.5% (198/370), M. regulus 100% (15/15), and C. lasiura 3.5% (1/28). Crocidura lasiura were infested with fewer chiggers than other small mammals. The infestation rate for M. regulus was 100% during the study period (Table 1).

Table 1

Chigger infestation rates (%) on small mammals (number of chigger infested small mammals/number of captured small mammals) in the southwest area of Korea, from September 2014 to December 2016

MonthRodentsShrewTotal (chigger infested/captured small mammals)
Apodemus agrarusMyodes regulusCrocidura lasiura
September 20149/109/10
October12/1212/12
November18/220/118/23
December0/51/10/11/7
January 20158/143/30/111/18
February3/71/14/8
March5/101/16/11
April3/91/14/10
May11/221/10/312/26
June2/162/16
July0/180/18
August4/134/13
September8/100/18/11
October15/151/116/16
November17/171/118/18
December14/1514/15
January 20164/61/14/7
February7/87/8
March12/131/10/113/15
April9/100/29/12
May6/251/10/17/27
June0/220/22
July0/130/10/14
August0/70/7
September6/121/10/17/14
October5/51/56/10
November12/120/412/16
December8/252/20/510/32
Total198/370 (53.5)15/15 (100)1/28 (3.5)214/413 (51.8)

Monthly species distribution of chiggers.

A total of 3,816 chiggers were collected from 214 small mammals. Leptotrombidium scutellare was the most commonly collected species (1,907; 49.9%), followed by Leptotrombidium orientale (804; 21.1%), L. pallidum (474; 12.4%), Euchoengastia koreaensis (273; 7.2%), Leptotrombidium palpale (256; 6.7%), Neotrombicula gardellai (50; 1.3%), Leptotrombidium zetum (32; 0.8%), Walchia fragilis (18; 0.5%), and Neotrombicular japonica (2, < 0.1%). Regarding distribution, 3,512 (92.0%) chiggers were found on A. agrarius, followed by 301 on M. regulus (7.9%), and 3 (< 0.1%) on C. lasiura. Eight species of chiggers were observed on A. agrarius. Furthermore, W. fragilis was only collected from M. regulus (Table 2).

Table 2

Species of chiggers collected from small mammals captured in the southwest area of Korea, from September 2014 to December 2016

Number of chiggers collected (%)Total
Host speciesLeptotrombidium scutellareLeptotrombidium pallidumLeptotrombidium orientaleLeptotrombidium palpaleLeptotrombidium zetumNeotrombicular gardellaiNeotrombicular japonicaEuchoengastia koreaensisWalchia fragilis
Rodents
 Apodemus agrarius1,866474585237305022683,512 (92.0)
 Myodes regulus38219192518301 (7.9)
Shrews
 Crocidura lasiura33 (> 0.1)
Total1,907 (49.9)474 (12.4)804 (21.1)256 (6.7)32 (0.8)50 (1.3)2 (> 0.1)273 (7.2)18 (0.5)3,816 (100)

Monthly fluctuation of chiggers by host is presented in Table 3 and Figure 2. We observed seasonal differences in chigger species. During the autumn season from September to December, the predominant species was L. scutellare whereas, during the spring season between March and May, the predominant species was L. orientale.

Table 3

Monthly distribution of chigger species (positive chigger for Orientia tsutsugamushi) collected from small mammals captured in the southwest area of Korea

MonthLeptotrombidium pallidumLeptotrombidium scutellareLeptotrombidium palpaleLeptotrombidium orientaleLeptotrombidium zetumNeotrombicular gardellaiNeotrombicular japonicaEuchoengastia koreaensisWalchia fragilisTotal
September 201456212768154
October12257 (2)27278 (2)
November172651570112380
December6112120
January 2015113510531146
February752261
March560166
April62 (1)62
May1168979
June22
July
August8210
September156353095
October41272 (2)41287407
November453781914447
December654084252216
January 2016957573126
February31153411293
March1021098108228
April4424745147
May1071835
June
July
August
September1942215684
October1132 (6)18151
November51258 (1)3419326
December191444342203
Total4741,907 (11)256804 (1)32502273183,816 (12)
Figure 2.
Figure 2.

Monthly chigger indices by species collected from small mammals captured in the southwest area of Korea, from September 2014 to December 2016. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 98, 2; 10.4269/ajtmh.17-0070

Prevalence of O. tsutsugamushi in samples.

Individual nested PCRs were used to screen 3,816 chiggers for O. tsutsugamushi. A total of 12 chiggers (0.3%; 12/3,816) were positive for O. tsutsugamushi, and 11 samples of L. scutellare and one L. orientale were confirmed. Furthermore, one A. agrarius specimen from the 413 small mammals tested was confirmed to have O. tsutsugamushi (0.2%; 1/413). Finally, 125/402) of the patients with suspected scrub typhus tested positive (Table 4).

Table 4

Distribution of Orientia tsutsugamushi strains in chigger, small mammals, and humans in the southwest area of Korea

HostTestPositive (%)Cluster
BoryongKawasakiSaitamaKarpEtc.
11442171
Chiggers3,81612 (0.3)111
 Leptotrombidium scutellare1,90711 (0.6)101
 Leptotrombidium pallidum474
 Leptotrombidium orientale2561 (0.4)1
 Leptotrombidium palpale804
 Leptotrombidium zetum32
 Neotrombicular gardellai50
 Neotrombicular japonica2
 Euchoengastia koreaensis273
 Walchia fragilis18
Small mammals4131 (0.2)1
 Apodemus agrarius3701 (0.3)1
 Myodes regulus15
 Crocidura lasiura28
Human402125 (50.5)1034117

Phylogenetic analysis for the 56-kDa TSA gene of O. tsutsugamushi.

The 56 kDa TSA gene sequence identified in 11 chiggers, one rodent, and 125 patients corresponded to four different strains of O. tsutsugamushi. The Boryong strain was the most common in chiggers (11/12; 95.8%) and patients (103/125; 82.4%). Interestingly, the sequence (KX363954) identified from the rodents was 84.3% homologous to the CH0711a strain (GQ342749), which is related to the Saitama strain cluster (Table 5). Peculiarly, there was a difference between the sequence (KY266830) identified from the chigger and Boryong cluster (Similarity percent: 74.0–79.8%) (Table 5). In patient samples, the 17 sequences (KY946107-KY946123) identified (17/125; 13.6%) are related to the Karp cluster. In addition, the four sequences (KY946124-KY946127) that were identified (4/125; 3.2%) are related to the Kawasaki cluster, one sequence (KY946128) that were identified (1/125; 0.8%) are related to the Saitama cluster (Figure 3; Table 6). For composition of phylogenetic tree, sequences obtained from chiggers (KX363952, KX363953, KY266824-KY266830), rodent (KX363954), human (KY946003, KY946031, KY946061, KY946107, KY946108, and KY946128, KY946124-KY946127) were used (Table 6).

Table 5

Identity matrix of Orientia tsutsugamushi strains in chigger, small mammals, and humans in the southwest area of Korea

Percent identity
Divergence12345678910111213141516171819202122232425262728293031323334353637
192.192.276.767.167.067.067.085.687.488.393.773.870.972.470.082.766.171.881.295.587.465.767.968.868.487.787.287.290.389.990.189.472.484.183.282.71KY946003_Human_Boryong
20.099.876.773.874.573.874.983.485.285.686.666.464.165.262.880.558.875.885.488.480.064.167.366.866.480.380.091.094.494.494.093.976.988.487.587.02KY946031_Human_Boryong
30.00.076.574.074.774.074.783.285.085.786.866.664.365.363.080.359.076.085.688.680.163.967.566.666.280.580.191.294.694.694.294.076.988.687.787.23KY946061_Human_Boryong
48.48.98.959.659.458.759.782.380.981.671.157.257.057.955.184.151.860.576.472.977.471.555.461.761.475.578.967.971.171.170.870.657.965.364.463.94KY946128_Human_Pajoo
534.734.734.637.599.398.498.964.666.267.761.781.682.991.268.462.677.670.667.963.562.864.393.392.191.761.962.368.269.369.369.569.158.563.763.563.05KY946124_Human_Kawasaki
634.234.334.237.00.299.199.664.466.167.561.681.482.390.667.762.577.171.368.663.462.664.192.891.991.561.762.169.070.070.070.269.959.264.464.363.76KY946125_Human_Kawasaki
733.934.334.136.90.20.098.963.765.367.561.681.482.390.467.561.777.171.369.163.462.663.492.691.090.661.762.169.970.870.471.170.459.965.264.864.37KY946126_human_kawasaki
833.933.933.936.50.50.20.064.866.467.561.681.482.390.367.362.877.171.368.463.462.664.492.491.991.561.762.168.869.969.970.069.759.264.364.163.58KY946127_human_kawasaki
98.69.29.25.536.536.136.035.798.284.882.968.864.167.364.496.062.164.479.484.786.868.860.571.771.591.787.274.577.877.877.477.364.171.870.970.49KY946107_Human_Karp
108.49.09.05.535.935.435.435.10.086.584.570.665.769.066.194.463.566.181.086.388.367.362.171.871.593.388.676.479.679.679.279.165.573.672.772.210KY946108_Human_Karp
119.59.59.40.334.834.334.134.16.16.284.871.369.768.667.382.564.472.089.986.891.961.470.464.864.488.491.782.383.883.684.783.069.979.177.877.611AF430142_Human_Pajoo
121.00.00.08.734.734.233.933.910.19.79.275.371.169.573.680.970.966.675.596.687.561.967.566.666.686.885.683.284.784.785.684.368.183.481.981.812M63380_Human_Kuroki
1325.728.027.931.511.110.610.410.427.526.826.724.591.289.778.266.489.265.062.675.173.863.986.385.985.673.371.165.365.365.066.465.054.961.661.061.413DQ485289_Gilliam
1431.333.933.833.210.010.19.89.832.131.630.630.97.788.177.461.794.865.561.269.369.161.289.283.883.467.566.263.063.062.664.162.653.160.559.759.914AF173033_lkeda
1531.934.734.634.60.20.20.20.534.233.732.531.910.69.376.565.985.965.762.571.371.166.192.495.595.170.270.263.563.963.564.663.453.258.558.357.815M63383_Human_Kawasaki
1630.232.632.533.525.926.526.627.034.133.631.629.423.523.923.763.076.266.659.471.370.862.574.572.472.068.267.361.661.761.462.561.052.759.458.158.316AY836148_Kato
178.99.59.55.536.035.535.435.11.31.15.59.127.432.232.532.461.762.377.183.886.170.858.570.470.890.886.871.774.974.974.574.461.269.068.167.517AY956315_Human_Karp
1831.233.933.833.210.010.19.89.833.132.330.629.87.50.09.423.331.160.356.069.969.158.183.982.382.768.166.857.857.857.458.857.447.857.957.457.418U19903_Human_Yonchon
1931.231.531.433.835.635.134.934.935.835.232.030.832.032.134.828.235.632.171.868.467.159.667.963.262.865.566.471.773.372.973.372.962.567.366.666.119U19904_Rattus._rattus_TA6.7.8
2014.712.212.12.837.437.037.537.19.19.25.115.235.640.040.539.88.640.037.077.383.256.964.660.560.180.084.384.787.986.886.887.274.081.481.080.720KX363954_Apod.e.mus_agrarius
211.00.00.08.734.734.233.933.910.19.79.00.224.630.631.929.49.129.330.815.290.663.766.168.468.890.388.885.086.586.387.586.169.981.880.580.321JQ898368_Human_Boryong
2210.210.510.53.434.834.334.134.18.68.43.410.125.529.731.029.27.328.832.08.09.862.865.568.168.492.894.676.978.778.379.678.064.873.572.472.622AF430141_Human_Young-worl
2327.028.328.329.730.329.829.729.328.228.228.627.028.631.528.225.127.830.931.433.327.027.459.969.969.562.162.157.859.459.059.058.851.353.152.352.223M63381_Shimokoshi
2433.334.734.636.20.20.20.20.535.534.932.632.610.79.40.024.134.99.434.840.532.932.629.787.987.563.562.867.066.266.167.765.956.564.163.563.424AF173039_Kanda
2532.535.035.034.10.50.00.00.234.033.933.532.310.49.80.225.331.99.835.937.832.331.727.80.299.667.167.560.861.961.962.161.751.456.356.155.625AF173038_Taguchi
2632.535.035.034.10.50.00.00.234.233.933.532.210.49.80.225.331.79.835.937.832.131.527.80.20.067.567.960.561.661.661.761.451.156.055.855.226AF.173037_Oishi
279.39.59.45.835.935.535.235.21.91.75.99.224.230.232.031.40.929.534.512.18.96.728.234.032.732.693.576.478.578.379.277.864.473.172.071.727AF430143_Human_Jecheon
289.510.010.00.835.234.834.534.57.67.50.69.526.330.831.531.25.930.132.75.69.43.328.233.632.131.96.275.377.877.478.276.963.472.271.570.928GQ332749_CH0711a_TAIWAN
295.64.14.114.535.935.535.235.614.013.715.96.031.938.036.637.014.538.036.217.16.016.832.737.236.336.315.515.692.493.093.992.679.890.488.889.029KY266825_L.scutellare
304.12.12.112.027.236.836.936.911.811.514.44.832.838.939.537.512.238.936.013.34.814.730.639.737.637.614.414.57.396.095.896.479.290.188.688.630KY266826_L.scutellare
31391.91.911.636.936.536.736.611.511.314.54.633.239.439.338.011.939.435.914.74.815.031.139.837.337.314.514.36.84.496.297.779.491.090.389.431KY266827_L.scutellare
323.01.61.611.435.535.134.935.211.311.112.63.230.136.036.035.511.736.034.314.03.012.930.036.135.935.912.412.55.43.93.796.079.191.591.090.832KY266828_L.scutellare
334.81.91.911.736.335.936.436.011.611.315.04.832.939.140.038.412.039.136.114.94.815.330.539.936.736.715.015.47.13.71.83.279.490.189.489.433KY266829_L.scutellare
3425.922.823.133.857.056.456.656.130.429.735.426.953.460.059.655.131.660.054.732.326.935.849.660.656.856.833.934.224.624.724.625.024.475.573.674.034KY266830_L.scutellare
353.92.12.111.836.836.436.636.511.911.714.26.534.541.938.140.812.343.935.914.34.615.331.940.637.237.213.813.63.94.63.73.24.224.092.192.435KY266824_L.scutellare
366.04.34.214.638.437.938.538.114.314.016.68.936.044.139.644.014.845.738.415.36.717.435.042.338.838.816.415.76.17.55.13.95.626.48.596.036KX363952_L.scutellare
376.34.54.515.038.938.439.038.614.714.316.99.135.343.640.043.515.245.738.915.96.917.134.742.739.339.316.416.06.37.06.34.65.626.48.44.337KX363953_L.scutellare
12345678910111213141516171819202122232425262728293031323334353637
Figure 3.
Figure 3.

Phylogenetic tree of Orientia tsutsugamushi based on the 56-kDa type specific gene sequence detected in chiggers; closed rectangle ■ (KX363952, KX363953, KY266824-KY266830), rodent (KX363954), human; open circle ○ (KY946003, KY946031, KY946061, KY946107, KY946108, KY946128, KY946124-KY946127). Phylogenetic tree was constructed by neighbor-joining method with the Kimura’s two-parameter model (bootstrap 1,000) using MEGA 6.0. Genbank accession numbers of O. tsutsugamushi are indicated for each sequence.

Citation: The American Journal of Tropical Medicine and Hygiene 98, 2; 10.4269/ajtmh.17-0070

Table 6

Strain of Orientia tsutsugamushi obtained in rodent, chigger, and humans from September 2014 to December 2016 in the southwest area of Korea

SourceGenbank accession numberCollection dateClusterReference
Chigger (Leptotrombidium scutellare)KX363949October 2014Boryong11
Chigger (L. scutellare)KX363950October 2014Boryong11
Chigger (Leptotrombidium orientale)KX363951May 2015Boryong11
Chigger (L. scutellare)KX363952October 2015Boryong
Chigger (L. scutellare)KX363953October 2015Boryong
Rodent (Apodemus agrarius)KX363954December 2015Saitama
Chigger (L. scutellare)KY266824October 2016Boryong
Chigger (L. scutellare)KY266825October 2016Boryong
Chigger (L. scutellare)KY266826October 2016Boryong
Chigger (L. scutellare)KY266827October 2016Boryong
Chigger (L. scutellare)KY266828October 2016Boryong
Chigger (L. scutellare)KY266829October 2016Boryong
Chigger (L. scutellare)*KY266830November 2016
HumanKY9460032014Boryong
HumanKY9460312015Boryong
HumanKY9460612015Boryong
HumanKY9461072014Karp
HumanKY9461082014Karp
HumanKY9461282014Saitama
HumanKY9461242015Kawasaki
HumanKY9461252015Kawasaki
HumanKY9461262015Kawasaki
HumanKY9461272015Kawasaki

Different strain from other chiggers.

DISCUSSION

Scrub typhus is a common endemic in the southwest region of Korea. The purpose of this study was to confirm the strains of O. tsutsugamushi for the 56-kDa TSA gene in chiggers, small mammals, and humans with chigger surveillance.

Seasonal chigger infestation rate for small mammals can be a criterion for the prevalence of scrub typhus. Furthermore, the abundance of chiggers influences the number of cases of scrub typhus. The degree of chigger infestation in small mammals varies from species to species. We found a high infestation rate in A. agrarius, which is the main host for scrub typhus in the southwest region of Korea,9 and which is the most abundant rodent species captured in this study.

Leptotrombidium scutellare, the predominant chigger species in the southern area of Korea, begins to appear in September and reaches a peak in autumn from October to November.31 In this study, L. scutellare was also the predominant chigger species identified during this period and was the primary contributor to the increased chigger infestation rate. However, during spring (March to May), L. orientale was the predominant species. Furthermore, the W. fragilis strain was identified from M. regulus. However, W. fragilis has never been reported in chigger species in the southwest region of Korea, but has been previously reported in M. regulus in the central region of Korea.32

Here, we analyzed for the 56-kDa TSA gene sequence of O. tsutsugamushi, and identified the Boryong strain to predominate in chiggers and patients. The Boryong strain is primarily distributed in the southern region of Korea where L. scutellare is found.15 In the present study, the results suggest that the Boryong strain can be transmitted through L. scutellare in the southwest region, and that L. orientale can act as a vector for scrub typhus in the spring. However, strains of O. tsutsugamushi carried by L. scutellare can differ between East Asian nations. For instance, in China, L. scutellare is considered a vector of the Karp strain,24 whereas in Japan, L. scutellare is considered the main vector of the Kawasaki strain.14

Unlike the Boryong strain, the Karp- and Kawasaki-related strains had different genotypes in patients than what was predicted. In the central and southwest regions of Korea, the Karp and Kawasaki strain had already been identified in patients.33,34 The Karp strain was classified into a higher virulence group, and the Boryong and Kawasaki strains were classified into a lower virulence group.16

Furthermore, a Saitama-related strain was found in a single A. agrarius, but was not found in chiggers. The Saitama strain is closely related to the Karp strain based on the 56-kDa TSA gene sequence. This strain was first reported in Japan in 1993 by Tamura et al.35 by identifying it in wild rodents, which tend to carry various strains in East Asia. In addition, Wu et al. (2015) confirmed the Kato, Gilliam, Karp, and TA763 strain in the Guangdong region of China.36,37 These strains were also reported in Taiwan.38 Finally, the CH0711a strain was reported in a patient in 2010 in Taiwan.39

Our study had several strengths. First, this study was conducted on a monthly basis. Secondly, this is the first study to compare the O. tsutsugamushi strains between chiggers, rodents, and patients in the southwest region. Despite a low infection rate, the data presented here provide a foundation for evidence of other strains in chiggers and small mammals. Unlike the strains identified in chiggers and patients, a Saitama-related strain was demonstrated in rodents.

In conclusion, the 56-kDa TSA gene showed variations in O. tsutsugamushi strains from different hosts. We confirmed that the Boryong strain is the most common strain in chiggers and patients, whereas wild rodents harbored a Saitama-related strain. In patients, Kawasaki-related strains prevalent in Japan were observed. To identify strains that are circulating in a host at any given point in time, a survey should be conducted continuously. The results provided here will be helpful for minimizing future incidences of scrub typhus by understanding disease transmission vectors.

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 Chosun University.

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Author Notes

Address correspondence to Jae Keun Chung, Division of Infectious Disease Investigation, Health and Environment Research Institute of Gwangju City, Gwangju 61986, Korea. E-mail: jkchung@korea.kr

Authors’ addresses: Jung Wook Park, Sun Hee Kim, Duck Woong Park, So Hyang Jung, Hye Jung Park, Mi Hee Seo, Dong Ryong Ha, Eun Sun Kim, and Jae Keun Chung, Division of Infectious Disease Investigation, Health and Environment Research Institute of Gwangju City, Gwangju, Korea, E-mails: jwpvet@korea.kr, sunny1989@korea.kr, vetpo2005@korea.kr, mist324@korea.kr, chj0103@korea.kr, mihee0105@korea.kr, dongryongha@korea.kr, keunsun@korea.kr, and jkchung@korea.kr. Hyeon Je Song and Jung Yoon Lee, Clinical Pathology, Gwangju Health University, Gwangju, Korea, E-mails: songha1@ghc.ac.kr and kumho_ljr@hanmail.net. Dong Min Kim, Departments of Internal Medicine, School of Medicine, Chosun University, Gwangju, Korea, E-mail: drongkim@hanmail.net. Choon-Mee Kim, Premedical Science, College of Medicine, Chosun University, Gwangju, Republic of Korea, E-mail: choonmee@hanmail.net. Byong Chul Gill, Hang Jin Jeong, and Jeong Min Lee, Division of Zoonoses, Korea Centers for Disease Control and Prevention, Cheongju, Korea, E-mails: gilri@korea.kr, jhjin9035@korea.kr, and jeongminlee@korea.kr.

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