New genotypes of Orientia tsutsugamushi isolated from humans in Eastern Taiwan

Abstract

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.

Figure 1. The tsutsugamushi triangle and geographical relationship of Taiwan with other countries in east Asia.

Hualien and Taitung Countries are in eastern Taiwan and Chungkung Harbour is in Taitung Country. Blood samples were collected from 2002 to 2008 from patients with suspected scrub typhus in Hualien and Taitung and submitted to the laboratory for this study.

Figure 2. Phylogenetic tree of 125 O. tsutsugamushi isolates based on 56-kDa TSA gene sequences.

The tree was constructed using the neighbour-joining method and MEGA software. One thousand bootstrap replicates were used to evaluate the tree reliability. Bootstrap support values greater than 75 are shown. Taiwan-H is shown in bold italics, and Taiwan Gilliam-variant cluster is enlarged in the figure S1. JP-1, Japan-Karp 1; JP-2, Japan-Karp 2; JG, Japanese Gilliam; TG-v, Taiwan Gilliam-variant; Kt, Kato; Kt-v, Kato-variant; Thai, Thailand; IND, Republic of India; KOR, Korea; JPN, Japan; PRC, People's Republic of China; ROC, Republic of China; TSA, type-specific antigen.

Figure 3. BootScan analysis of 56-kDa TSA genes of Taiwan Gilliam-variant (Taitung-5) strain and other reference O. tsutsugamushi strains.

TSA, type-specific antigen. RFLP: tsu-a and tsu-b primer position, 263–430 (Taitung-5 nucleotides 263 to nucleotide 430); variable domain I (VD-I) position, 313–396 (Taitung-5 nucleotides 313 to nucleotide 396); VD-II position, 451–529 (Taitung-5 nucleotides 451 to nucleotide 516); VD-III position, 611–705 (Taitung-5 nucleotides 592 to nucleotide 675); VD-IV position, 1196–1321 (Taitung-5 nucleotides 1153 to nucleotide 1260).

Figure 4. BootScan analysis of 56-kDa TSA genes of Taiwan-P (Hualien-14) strain and other reference O. tsutsugamushi strains.

TSA, type-specific antigen. RFLP: tsu-a and tsu-b primer position, 263–430 (Hualien-14 nucleotides 263 to nucleotide 430); variable domain I (VD-I) position, 313–396 (Hualien-14 nucleotides 313 to nucleotide 396); VD-II position, 451–538 (Hualien-14 nucleotides 451to nucleotide 522); VD-III position, 598–684 (Hualien-14 nucleotides 617 to nucleotide 719); VD-IV position, 1206–1340 (Hualien-14 nucleotides 1165 to nucleotide 1257).