Scrub typhus, an acute febrile illness, is caused by the obligate intracellular bacterium Orientia tsutsugamushi. In our study, O. tsutsugamushi was rapidly detected and typed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the 56-kDa type-specific antigen (TSA) gene. To investigate the genotypes of clinical variants of O. tsutsugamushi, we collected 3223 blood samples from eastern Taiwanese patients with suspected scrub typhus from 2002 to 2008. In total, 505 samples were found to be positive for scrub typhus infection by PCR, and bacteria were isolated from 282 of them. Four prototype genotype strains (Karp, Kato, Kawasaki and Gilliam) and eleven different Taiwanese genotype isolates (Taiwan-A, -B, -C, -D, -E, -G, -H, -J, -N, -O and -P) were identified by RPLF analysis. Taiwan-H, the major genotype in eastern Taiwan, exhibited prevalence and isolation rates of 47.3% (239/505) and 42.6% (120/282), respectively. We also assessed the genetic relatedness of the 56-kDa TSA gene among eight Taiwan-H isolates, thirteen other Taiwanese isolates and 104 DNA sequences deposited in the GenBank database using MEGA version 5.0 and PHYLIP version 3.66. We found that the Taiwan-H isolates formed into a new cluster, which was designated the Taiwan Gilliam-variant (TG-v) cluster to distinguish it from the Japanese Gilliam-variant (JG-v) cluster. According to Simplot analysis, TG-v is a new recombinant strain among Gilliam, Ikeda and Kato. Moreover, the Gilliam-Kawasaki cluster had the highest percentage of RFLP cases and was the most frequently isolated type in eastern Taiwan (50.1%, 253/505; 44.0%, 124/282). These findings shed light on the genetic evolution of O. tsutsugamushi into different strains and may be useful in vaccine development and epidemic disease control in the future.
References
[1]
Tamura A, Ohashi N, Urakami H, Miyamura S (1995) Classification of Rickettsia tsutsugamushi in a new genus, Orientia gen. nov., as Orientia tsutsugamushi comb. nov. Int J Syst Bacteriol 45: 589–591.
[2]
Fournier PE, Siritantikorn S, Rolain JM, Suputtamongkol Y, Hoontrakul S, et al. (2008) Detection of new genotypes of Orientia tsutsugamushi infecting humans in Thailand. Clin Microbiol Infect 14: 168–173.
[3]
Lu HY, Tsai KH, Yu SK, Cheng CH, Yang JS, et al. (2010) Phylogenetic analysis of 56-kDa type-specific antigen gene of Orientia tsutsugamushi isolates in Taiwan. Am J Trop Med Hyg 83: 658–663.
[4]
Watt G, Strickman D (1994) Life-threatening scrub typhus in a traveler returning from Thailand. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 18: 624–626.
[5]
Ni YS, Chan TC, Chao CC, Richards AL, Dasch GA, et al. (2005) Protection against scrub typhus by a plasmid vaccine encoding the 56-KD outer membrane protein antigen gene. Am J Trop Med Hyg 73: 936–941.
[6]
Kelly DJ, Fuerst PA, Ching WM, Richards AL (2009) Scrub typhus: the geographic distribution of phenotypic and genotypic variants of Orientia tsutsugamushi. Clin Infect Dis 48 Suppl 3: S203–230.
[7]
Yu Y, Wen B, Niu D, Chen M, Qiu L (2005) Induction of protective immunity against scrub typhus with a 56-kilodalton recombinant antigen fused with a 47-kilodalton antigen of Orientia tsutsugamushi Karp. Am J Trop Med Hyg 72: 458–464.
[8]
Shishido A, Hikita M, Sato T, Kohno S (1969) Particulate and soluble antigens of Rickettsia tsutsugamushi in the complement fixation test. J Immunol 103: 480–490.
[9]
Ohashi N, Koyama Y, Urakami H, Fukuhara M, Tamura A, et al. (1996) Demonstration of antigenic and genotypic variation in Orientia tsutsugamushi which were isolated in Japan, and their classification into type and subtype. Microbiol Immunol 40: 627–638.
[10]
Ohashi N, Tamura A, Sakurai H, Yamamoto S (1990) Characterization of a new antigenic type, Kuroki, of Rickettsia tsutsugamushi isolated from a patient in Japan. J Clin Microbiol 28: 2111–2113.
[11]
Tamura A, Takahashi K, Tsuruhara T, Urakami H, Miyamura S, et al. (1984) Isolation of Rickettsia tsutsugamushi antigenically different from Kato, Karp, and Gilliam strains from patients. Microbiol Immunol 28: 873–882.
[12]
Tamura A, Ohashi N, Koyama Y, Fukuhara M, Kawamori F, et al. (1997) Characterization of Orientia tsutsugamushi isolated in Taiwan by immunofluorescence and restriction fragment length polymorphism analyses. FEMS Microbiol Lett 150: 225–231.
[13]
Tamura A, Yamamoto N, Koyama S, Makisaka Y, Takahashi M, et al. (2001) Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama Prefecture, Japan, and discovery of a new type. Microbiol Immunol 45: 439–446.
[14]
Enatsu T, Urakami H, Tamura A (1999) Phylogenetic analysis of Orientia tsutsugamushi strains based on the sequence homologies of 56-kDa type-specific antigen genes. FEMS Microbiol Lett 180: 163–169.
[15]
Qiang Y, Tamura A, Urakami H, Makisaka Y, Koyama S, et al. (2003) Phylogenetic characterization of Orientia tsutsugamushi isolated in Taiwan according to the sequence homologies of 56-kDa type-specific antigen genes. Microbiol Immunol 47: 577–583.
[16]
Chen HL, Shieh GJ, Chen HY, Horng CB (1995) [Isolation of Rickettsia tsutsugamushi from the blood samples of patients in Taiwan]. J Formos Med Assoc 94 Suppl 2: S112–119.
[17]
Kawamori F, Akiyama M, Sugieda M, Kanda T, Akahane S, et al. (1993) Two-step polymerase chain reaction for diagnosis of scrub typhus and identification of antigenic variants of Rickettsia tsutsugamushi. J Vet Med Sci 55: 749–755.
[18]
Stover CK, Marana DP, Carter JM, Roe BA, Mardis E, et al. (1990) The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope. Infect Immun 58: 2076–2084.
[19]
Ohashi N, Tamura A, Ohta M, Hayashi K (1989) Purification and partial characterization of a type-specific antigen of Rickettsia tsutsugamushi. Infect Immun 57: 1427–1431.
[20]
Ohashi N, Nashimoto H, Ikeda H, Tamura A (1992) Diversity of immunodominant 56-kDa type-specific antigen (TSA) of Rickettsia tsutsugamushi. Sequence and comparative analyses of the genes encoding TSA homologues from four antigenic variants. The Journal of biological chemistry 267: 12728–12735.
[21]
Chen HL, Chiu SC, Chen HY, Wang GR (1999) [Molecular typing of Taiwanese Orientia tsutsugamushi isolates by restriction fragment profile]. Journal of microbiology, immunology, and infection 32: 68–72.
[22]
Furuya Y, Yoshida Y, Katayama T, Yamamoto S, Kawamura A Jr (1993) Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase chain reaction. J Clin Microbiol 31: 1637–1640.
[23]
Felsenstein J (2006) Accuracy of coalescent likelihood estimates: do we need more sites, more sequences, or more loci? Molecular biology and evolution 23: 691–700.
[24]
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular biology and evolution 24: 1596–1599.
[25]
Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, et al. (1999) Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. Journal of virology 73: 152–160.
[26]
Zoll J, Galama JM, van Kuppeveld FJ (2009) Identification of potential recombination breakpoints in human parechoviruses. Journal of virology 83: 3379–3383.
[27]
Sanders-Buell E, Saad MD, Abed AM, Bose M, Todd CS, et al. (2007) A nascent HIV type 1 epidemic among injecting drug users in Kabul, Afghanistan is dominated by complex AD recombinant strain, CRF35_AD. AIDS research and human retroviruses 23: 834–839.
[28]
Lukashev AN (2005) Evidence for recombination in Crimean-Congo hemorrhagic fever virus. The Journal of general virology 86: 2333–2338.
[29]
Sonthayanon P, Peacock SJ, Chierakul W, Wuthiekanun V, Blacksell SD, et al. (2010) High rates of homologous recombination in the mite endosymbiont and opportunistic human pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis 4: e752.
[30]
Feil EJ, Maiden MC, Achtman M, Spratt BG (1999) The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis. Molecular biology and evolution 16: 1496–1502.
[31]
Brown CT, Fishwick LK, Chokshi BM, Cuff MA, Jackson JMt, et al. (2011) Whole-genome sequencing and phenotypic analysis of Bacillus subtilis mutants following evolution under conditions of relaxed selection for sporulation. Applied and environmental microbiology 77: 6867–6877.
[32]
Sun M, Gao L, Liu Y, Zhao Y, Wang X, et al. (2012) Whole genome sequencing and evolutionary analysis of human papillomavirus type 16 in central China. PLoS One 7: e36577.
[33]
Groves MG, Kelly DJ (1989) Characterization of factors determining Rickettsia tsutsugamushi pathogenicity for mice. Infect Immun 57: 1476–1482.
[34]
Blacksell SD, Luksameetanasan R, Kalambaheti T, Aukkanit N, Paris DH, et al. (2008) Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand. FEMS Immunol Med Microbiol 52: 335–342.
