Molecular Epidemiology Of Orientia Tsutsugamushi Infection In Shandong Province

BackgroundOrientia tsutsugamushi, the etiologic agent of scrub typhus, is an obligate intracellular bacterium. The pathogen is transmitted to the rodent host or humans through the bites of infected chiggers (trombiculid mite larvae). Scrub typhus is characterized by high fever, eschar or skin ulcer, enlarged lymph glands and rashes. Some patients may suffer with severe complications, including pneumonia, encephalitis, multi-organ failure or even death. Scrub typhus is widely prevalent in rural area of Asia-Pacific region. It has been estimated that about 1 billion people are at risk for scrub typhus, and almost 1 million scrub typhus cases occur annually. Hitherto, infection of O. tsutsugamushi has been found in at least 29 provinces, municipalities, and autonomous regions of China. Shandong Province is considered as a typical endemic area of scrub typhus in northern China, annual reported cases of scrub typhus have continuously increased in the past few years. Which drive renewed efforts to develop a suitable vaccine.The severity of scrub typhus and clinical presentation may relate to the genotypes of O. tsutsugamushi patients infected with. Evident antigenic diversity exists throughout the region of endemicity. The protective immunity of O. tsutsugamushi generates against homologous strains is high and long-lasting, while the protective immunity it generates against heterologous strains is low and short-lived. Strain identication, as well as establishment of strain prevalence for the different regions and reservoirs, could increase specicity and sensitivity of diagnostic tests in scrub typhus patients, and provide benecial to vaccine development as well.Objectives1. To understand the broad-coverage molecular epidemiology of O. tsutsugamushi and the most prevalent strains in Shandong Province, we conducted the present study to identify the genotypes of O. tsutsugamushi by phylogenetic analysis based on 56-kDa TSA gene, and draft geographic distribution from a sampling of scrub typhus cases and captured rodents covering the major endemic areas of Shandong Province.2. Eyeing a recommendation of potential vaccine candidates for scrub typhus, we conducted the phylogenetic analysis and sequence homologies in our study to identify the genetic characterization of STA-07, a novel genotype of O. tsutsugamushi in Shandong Province. Gene diversity measured at the nucleotide and amino acid level in the alignment of the highly variable protein domains VD I-VD IV.Materials and MethodsThe study was conducted from June 2013 to December 2015. Patients were recruited from 18 hospitals located at the major endemic areas of scrub typhus in Shandong encompassed six districts (Tai’an, Laiwu, Zibo, Zaozhuang, Linyi, and Qingdao). Acute-phase blood specimens and spontaneously desquamated eschars were collected from scrub typhus patients. Rodents in the above study areas were trapped in rural residences and wild in fields. DNA was extracted from each specimen and nested PCR was used to amplify the coding sequences of the 56-kDa TSA. Complete or partial sequences encoding 56-kDa TSA of O. tsutsugamushi reference strains were retrieved from GenBank, Multiple sequence aligned by using ClustalW and trimmed to the appropriate size. Phylogenetic analysis was carried out and the resulting dendrogram was performed by Mega software using neighbor-joining method with 1000 bootstrap replicates. Sequence homologies were calculated using MegAlign of Lasergene software. We also explored the geographic distribution of genotypic variants of O. tsutsugamushi.Genetic characterization of STA-07 compared to reference strains were measured at the nucleotide and amino acid level in the alignment of the highly variable protein domains Variable domains (VDI-VDIV). The bioinformatics techniques has been used to analyze the molecular structure of 56-kDa antigen epitopes.Results1. Detection of O. tsutsugamushi in samples.The overall O. tsutsugamushi infection rate was 0.76%(18/2360) among captured rodents in major endemic areas of Shandong Province, and positive rates of O. tsutsugamushi in Rattus norvegicus, Mus musculus, Apodemus agrarius, and Cricetulus tyiton were 0.45％,1.63％,1.78％和4.00％, respectively. Among the 352 acute whole blood samples and 6 eschar specimens,115 of them were O. sutsugamushi positive.2. Phylogenetic analysis and sequence homologiesThe genotypes of O. tsutsugamushi in Shandong Province were divided into two genotype groups (Kawasaki-related and STA-07). In present study, the majority sequences were related to the Kawasaki strain, with the nucleotide sequence identities ranging from 95.8％to 96.3％ with Japanese Kawasaki strain, and 99.3％to 100％ to each other.8 sequences from this study including 2 sequences from patient eschar、 one from patient blood sample and 5 from rodents clustered in the STA-07 group, which had 99.5～100％ to each other. And 64.2％～76.1％identity with Gilliam、Karp、 Kato、Kuroki、Boryong、TA763、Shimokoshi、Fuji、Ikeda and Kawasaki-related strain.3. O. tsutsugamushi distribution by geographical regionKawasaki-related genotype was distributed in Tai’an, Linyi, Zibo, Zaozhuang, Laiwu and Qingdao. STA-07 was distributed in Tai’an, Linyi, Zibo, Zaozhuang and Qingdao. Fuji-related and Shimokishi-related genotype were found in Tai’an, and Karp-related in Linyi, which were only detected from truncated version of previous reported.4. Genetic characterization of STA-07We sequenced 1562bp in the cloned O. tsutsugamushi DNA of STA-07. The nucleotide sequences consists of 245bp of 5’-non coding region and 1317 bp of open reading frame. The base composition of the sequenced DNA showed a higher A+T contents (58.47%) in coding region. The 354 amino acid sequence have the state of a-helix (36.67%), β-sheet (9.79%), Random coil (37.36%) and Extended strand (16.17%). From comparison of the amino acid sequences in the NH2-terminal region of TSA, the sequences of 22 amino acid residues on the NH2-terminal side was predicted as signal peptides. We analyzed the molecular structure of 56-kDa antigen epitopes of STA-07, and the results indicated that 57-65、122-135、153-169、191-199、 219-230、251-258、339-353aa would be the potential antigenic determinant regions.5. Amino acid sequences of VDI-IV of STA-07In this comparative analysis, substitutions or deletions of one or several contiguous amino acid residues are recognized, but the significant differences in the sequence among strains are seen in the regions indicated VD (variable domain) I-IV. Phylogenetic trees based on nucleotide sequences of each VD show the same results with amino acid analysis, and form a separate branch. The amino acid sequences of the STA-07 and Kawasaki-related strains in VD Ⅰ area are very similar, and the homology is 87.5％(80.0％ in nucleotide sequences), with two amino acid deletions and one substitution(Ala-Thr). The homology between the STA-07 and Kawasaki strains, is also relatively high,83.3％, in the amino acid sequences (77.8％in the nucleotide sequences). However, the similarities among other strains in VDI are less than 60.0％in the amino acid sequences. In VD Ⅱ area, the similarities between STA-07 and others are less than 50.0%. STA-07 show a great degree of amino acid substitution in comparison with Kawasaki-related (D-I-L-A-Q-A-A were replaced by G-N-M-T-R-A-E, Q-Q-L-T-V-E were replaced by Q-D-D-D-V-I). The similarities are 67.6％ in amino acid sequences between STA-07 and Kuroki/Boryong, and 64.7% with Karp strain in VD III area. While the similarities with other strains in this area are less than 60.0%. VD IV is the largest variable domain, the sequences of STA-07 are similar to Kuroki/Boryong with the homologies are 74.3％in amino acid sequences (67.8％in nucleotide sequences),and similarities are 71.4％with Gilliam and Karp strains, with other strains are less than 60.0%.Conclusions1. The infection rate of O. tsutsugamushi among rodents was 0.76%, C.tyiton was the major host of scrub typhus in Shandong, A.agrarius, M.musculus and R.norvegicus might also be reservoirs.2. Kawasaki-related genotypes were dominant, reflecting thus the most widely distributed, or ubiquitous, in Shandong Province. STA-07, the second most frequently detected strain in Shandong, was distributed in Tai’an, Linyi, Zibo, Zaozhuang and Qingdao. Fuji-related and Shimokishi-related genotype were found in Tai’an, and Karp-related in Linyi, which were only detected from truncated version of previous reported. Genotypes of O. tsutsugamushi in Tai’an and Linyi were diverse,with more than three types.3. From comparison of the amino acid sequences in the NH2-terminal region of TSA, the sequences of 22 amino acid residues was predicted as signal peptides. The processing site of the signal peptide is at the bond between the 22nd and 23rd amino acids. The amino acid residues that we sequenced were located in the outside of membrance.4. Potential antigenic determinant regions of 56-kDa of STA-07 may be at residues 57-65、122-135、153-169、191-199、219-230、251-258 and 339-353aa.5. STA-07 genotype showed a high level of genetic diversity of four variable domains of 56-kDa TSA compared to reference strains of O. tsutsugamushi, especially VD Ⅱ.