Am. J. Trop. Med. Hyg., 68(3), 2003, pp. 299-300
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene
SHORT REPORT: VARIATION IN THE 56-KD TYPE-SPECIFIC ANTIGEN GENE OF ORIENTIA TSUTSUGAMUSHI ISOLATED FROM PATIENTS IN THAILAND
THOMAS M. KOLLARS, JR.,
DHARADIDA BODHIDATTA,
DUANGPORN PHULSUKSOMBATI,
BOURSARAPORN TIPPAYACHAI, AND
RUSSELL E. COLEMAN
Entomological Sciences Program, United States Army Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, Maryland; Department of Biochemistry, and Department of Pathology, Division of Research, Royal Thai Army Component, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Department of Entomology, United States Army Medical Component, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
ABSTRACT
The sequences of a 300-base pair region of the 56-kD type-specific antigen gene from 12 Orientia tsutsugamushi isolates from Thailand were compared with isolates from other regions in Asia. A high degree of heterogeneity was found among the 12 Thai isolates, with the C3 strain most commonly found. The abundance of the C3 strain of O. tsutsugamushi is of particular concern because doxycycline and chloramphenicol resistance have previously been reported in this strain.
Orientia tsutsugamushi (Hyashi), the etiologic agent of scrub typhus, is found throughout Southeast Asia from Australia to Pakistan. Regional variation in phenotype and genotype have been described.1–4 Eight prototype strains of O. tsutsugamushi are found in Thailand: Karp, Kato, Gilliam, TA678, TA686, TA716, TA763, and TH1817.5,6 The vectors of O. tsutsugamushi are larval mites (commonly called chiggers) in the family Trombiculidae. In Thailand, the principal vector species are Leptotrombidium deliense, L. imphalum,7 and the recently described L. chiangraiensis.8 Strain O. tsutsugamushi chiangraiensis was recently isolated from the chigger L. chiangraiensis.9 Antibiotic resistance has been reported in several patient isolates in northern Thailand.10 We compared the similarities of a nucleic acid sequence from a region of the 56-kD type-specific antigen (TSAg) gene from O. tsutsugamushi isolated from patients from northern Thailand with isolates from throughout Asia. The TSAg was selected because strains of O. tsutsugamushi show distinct antigenicity in the 56-kD protein.
The TSAg of 15 strains of O. tsutsugamushi were obtained from GenBank. The 15 strains included Karp, Kato, and Gilliam (Ikeda strain); Thailand strains TA678, TA686, TA716, TA763, and TH1817; strain Sxh951 from China; the Boryong strain from Korea; and strains Kawasaki, Kuroki, LA1, LF1, LX1, and Shimokoshi from Japan. A total of 12 isolates from scrub typhus patients in northern Thailand were included in the study. These isolates included C1, C3, C7, C11, C27, C32, C53, C57, C59, P44, SV193, and SV240.10
Genomic DNA was isolated from cultured O. tsutsugamushi using the Ready Amp kit (Promega, Madison, WI) as specified by the manufacturer’s protocol. Amplification was accomplished by a polymerase chain reaction using two primers: OtP 56.809 5'-GATTTAGAGCAGAGCTAGGGTTTATGTACC-3' and OtM 56.1221 5'-TGGATTTCCAACAGGATTAGGGTTACC-3' of variable domain IV of the TSAg region of the O. tsutsugamushi Karp strain. The four variable regions of the TSAg show the greatest diversity among strains, with this polymorphism probably arising from point mutation and recombination processes.11 Automated DNA sequencing was accomplished using the ABI PRISM 377 System (Perkin Elmer, Wellesley, MA) following the manufacturer’s protocol and will be submitted to GenBank. A 300-base pair (bp) product corresponding to the 818–1,118 bp region of the TSAg of Karp strain was aligned and compared using Cluster Analysis Percent Disagreement using Statistica software (StatSoft, Inc., Tulsa, OK).
The relationship (based upon the percent disagreement of sequences from the TSAg segment) among the various isolates of O. tsutsugamushi is shown in Figure 1
. The clustering of the Thai O. tsutsugamushi isolates based upon the TSAg is similar to that reported by Enatsu and others.12 We found that isolate C27 was most similar to prototype strains TA716 (89% homology) and Kato, whereas isolates C1, C32, and P44 were most closely related to the Japanese strain LA1 (homology = 93%). Isolates C3, C11, C53, C57, and SV240 were identical to one another, with 86% homology to C7. These isolates were closely related to prototype strain TA763. Isolate SV193 was the most divergent, but was most closely related to TH1817 (Figure 1).
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FIGURE 1. Percent disagreement of a 300-base pair sequence of the 56-kD antigen gene of 27 isolates of Orientia tsutsugamushi, including 12 isolates from patients in northern Thailand. Strains C53, C57, C59, and SV240 are included as a C3 genotype.
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Watt and others10 reported that O. tsutsugamushi isolates C1, C3 and C27 were resistant to chloramphenicol, with isolates C1 and C3 also resistant to doxycycline. Forty-two percent (5 of 12) of the O. tsutsugamushi isolates from northern Thailand assessed in this study were the C3 type. To fully elucidate the genetic heterogeneity of O. tsutsugamushi in Thailand and to define the distribution of antibiotic resistance it will clearly be necessary to assess additional isolates.
Received March 4, 2002.
Accepted for publication May 1, 2002.
Financial support: This work was supported by funds from the U.S. Army Medical Research and Material Command and the Royal Thai Army.
Disclaimer: The views of the authors do not purport to represent the position of the Department of the Army or the Department of Defense.
Authors’ addresses: Thomas M. Kollars, Jr., Entomological Sciences Program, United States Army Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, MD 21010. Dharadida Bodhidatta, Department of Biochemistry, Division of Research, Royal Thai Army Component, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand. Duangporn Phulsuksombati, Department of Pathology, Division of Research, Royal Thai Army Component, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand. Boursaraporn Tippayachai and Russell E. Coleman, Department of Entomology, United States Army Medical Component, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand.
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