Epidemiological Characteristics And Mechanism Study On The Expansion Of Epidemic Area Of Autumn-winter Scrub Typhus

BackgroundScrub typhus, a natural focal disease caused by Orientia tsutsugamushi, is widely endemic to a13,000,000km2area in the Asia-Pacific region and threatens a population of one billion people. The main animal host of the disease is rodents, and the vector is chigger mites. Scrub typhus is a severe health problem in China. The disease was once endemic only in tropics and subtropics to the south of latitude31°in China, with the epidemic season mainly in summer. However, it emerged in temperate zone of northern China with the first outbreak in Shandong Province in1986, and new epidemic areas formed and expanded rapidly, with the epidemic season mainly in autumn and winter. Genetic mutation of the pathogen, development of eco-tourism, and underestimate of the epidemic intensity and disease burden are new impetetus of the study on scrub typhus. Surveillance of the changes in the epidemic features of scrub typhus, and exploration of the characteristics and influencing factors of the expansion of the epidemic area are essential to enrich the etiology and epidemiology of scrub typhus, and to improve the diagnosis and prevention of the disease.ObjectiveThe present study performed a comparative analysis of the species composition and distribution features of animal hosts and vectors, the genetic evolutionary characteristics of type-specific antigen gene of O. tsutsugamushi, and the epidemic and clinical features of infected population of scrub typhus between old and new epidemi area, to explore the epidemiological characteristics and mechanism of the expansion of epidemic area of scrub typhus, to provide important clues for the prediction of epidemic trends of the disease, and provide scientific evidence for its prevention and diagnosis. Materials and MethodsWe chose Yinan, Yishui from old epidemic area and Xintai, Daiyue, Jimo, and Jiaonan from new epidemic area of scrub typhus in Shandong Province as the study sites. During September2010to March2012, small mammals were captured from the study sites every season. The spleen and liver were dissected aseptically, and the chigger mites were collected from the ears of captured mammals. The mammals and chigger mites were identified into speicies and counted, and infestation rate and chigger index of the mammals were calculated. DNA was extracted from the samples, and screened for O. tsutsugamushi using nested PCR. Partial56kDa type-specific antigen gene was amplified and sequenced. Phylogenetic analysis and homology analysis was performed based on the sequences obtained in the study and reference ones retrived from Genbank. Relationships were determined on the basis of the partial56-kDa type-specific antigen gene of O. tsutsugamushi by the neighbor-joining method with the Kimura2-parameter distance model using Mega Software Version5.0. The homology analysis based on nucleotide sequences and the deduced amino acid sequences was performed using Lasergene Software Version7.1. The molecular evolutionary genetic analysis was performed with Bayesian Markov Chain Monte Carlo (MCMC) algorithm using BEAST Software Version1.7.4, to estimate the evolutionary rate, divergence time, the time to the most common ancestor (TMRC A), and the molecular clock model. The rates of O. tsutsugamushi positivity in captured animals and the minimum positive rate of O. tsutsugamushi in chiggers were calculated. Comparative analysis on the species composition of host animals and vectors, the infection status of O. tsutsugamushi, and the genetic evolutionary characteristics of O. tsutsugamushi TSA gene were performed between old and new epidemic areas.Information of reported cases of scrub typhus during2006yo2013were retrived from the Shandong Disease Report Information System, to describe the epidemic features and seasonal distribution of the disease. From Janurary2010to December2011, anti-coagulated whole blood and naturally desquamated eschars were collected to screen for Ot DNA, type-specific amplification and sequence analysis. A cross-sectional study was performed in the monitoring hospitals of the study sites during2010, to investigate the clinical features of scrub typhus. The database was built using Epidata Software Version3.1, and analyzed using Microsoft Excel and SPSS Software Version16.0.Results1. The composition of animal hosts and the infection status of O. tsutsugamushi in animalsA total of1158small mammals were captured in the new epidemic area of scrub typhus, including Rattus norvegicus (58.4%), Rattus rattus (16.3%), Mus musculus (24.2%), Apodemus agrarius (0.7%), Cricetus triton (0.2%), and shrew (0.3%). Rattus norvegicus is the predominant rodent in every season in the new epidemic area of scrub typhus, followed by Mus musculus and Rattus rattus. A total of736small mammals were captured in the old epidemic area of scrub typhus, including Rattus norvegicus (50.5%), Rattus rattus (0.7%), Mus musculus (19.7%), Apodemus agrarius (14.8%), Cricetulus barabensis (11.4%), Cricetulus migratorius (1.2%), and shrew (1.6%). Rattus norvegicus is the predominant rodent in spring, summer, autumn, and Apodemus agrarius is the predominant rodent in winter in the old epidemic area of scrub typhus.The positive rate of O. tsutsugamushi is2.4%in the animal hosts captured inside residences in the new epidemic area, and that in the Rattus norvegicus and Mus musculus was1.1%and5.2%, respectively. The positive rate in captured animals was2.9%,0,3.3%, and1.1%during spring, summer, autumn, and winter, respectively. O. tsutsugamushi was only detected from Rattus norvegicus captured during autumn in the field. The positive rate of O. tsutsugamushi is0.7%in the animal hosts captured inside residences in the old epidemic area, and that in the Rattus norvegicus and Mus musculus was0.7%and1.1%, respectively. The positive rate in captured animals was0.6%,0,2.5%, and0during spring, summer, autumn, and winter, respectively. O. tsutsugamushi was only detected from Rattus norvegicus and Cricetulus barabensis captured during autumn in the field of old epidemic area, and the positive rate was 2.3%and9.0%, respectively.2. The composition and distribution of vectors and the infection status of O. tsutsugamushiLeptotrombidium intermidium, L. palpale, L. scutellare, L. taishanicum, L. linhuaikongense, Walchia pacifica, and Odontacarus majesticus were found in new epidemic area of scrub typhus, and Odontacarus majesticus and Walchia pacifica were predominant species in summer and L. palpale, L. intermidium, and L. scutellare were predominnat in autumn and winter. Among the3species of animals captured inside residences in new epidemic area, the infestation rate and chigger index were highest in Rattus norvegicus (20.8%and8.72), followed by Mus musculus (12.9%and3.77). The infestation rate and chigger index of wild-captured host animals were7.7%and0.54, respectively.L. tanshanicum, L. palpale, L. scutellare, L. intermedium, L. laxoscutum, Gahrliepia octosetosa, and Walchia pacifica were found in old epidemic area of scrub typhus, and Gahrliepia octosetosa and Walchia pacifica were predominant species in summer and L. palpale and L. scutellare were predominnat in autumn and winter. Among the animals captured inside residences in old epidemic area, the infestation rate and chigger index were highest in Rattus norvegicus (17.1%and2.89), followed by Mus musculus (10.6%and1.00). The infestation rate and chigger index of wild-captured host animals were11.9%and1.53, respectively. Among the5species of wild-captured animals in old epidemic area, the infestation rate and chigger index were highest in Apodemus agrarius (22.1%and3.74), followed by Rattus norvegicus (22.0%and2.17), Cricetulus barabensis (3.2%and0.03), and Mus musculus (2.0%and0.02).Natural infection of O. tsutsugamushi was found in L. intermedium, L. scutellare, L. palpale, L. taishanicum and L. Linhuaikongense in new epidemic area of scrub typhus in Shandong. The minimum positive rate of O. tsutsugamushi in chiggers collected from domestic animals was0.8%. The minimum positive rate was highest in L. taishanicum (6.3%), followed by L. linhuaigongense (3.2%), L. intermedium (1.2%), L. scutellare (0.7%), and L. palpale (0.6%). O. tsutsugamushi was detected from L. scutellare paratisizing on wild animals in new epidemic area, with the minimum positive rate14.3%. Natural infection of O. tsutsugamushi was found in L. intermedium, L. scutellare, L. palpale, and L. taishanicum in old epidemic area. The minimum positive rate of O. tsutsugamushi in chiggers collected from domestic animals was1.7%. The minimum positive rate was highest in L. taishanicum (5.5%), followed by L. scutellare (1.9%), L. palpale (1.8%), and L. intermedium (1.3%). O. tsutsugamushi was detected from L. palpale and L. taishanicum parasitizing on wild animals in old epidemic area, and the minimum positive rate was5.0%and3.8%, respectively.3. Genetic characteristics and distribution of O. tsutsugamushiPhylogenetic analysis based on56kDa TSA gene of O. tsutsugamushi demonstrated a genotypic diversity of O. tsutsugamushi in animal hosts and chiggers inside residences in new epidemic area of Shandong Province. Eight genotypes, including KWS1-4, SKS, FJS, SDM1, and SDM2, were identified. Kawasaki-related genotypes were predominant in animal hosts and chiggers. In new epidemic area, natural infection of K.WS1, KWS2, and KWS4genotypes were found in Rattus norvegicus, KWS1in Cricetulus barabensis, KWS2, KWS3, FJS, SDMI and SDM2in Mus musculus, KWS1in L. scutellare, and KWS2and SKS in L. palpale. Less variaty in O. tsutsugamushi was revealed in animal hosts and chiggers in old epidemic area, with KWS1and KWS2genotypes identified. Natural infection of KWS2genotype was found in Apodemus agrarius, Rattus norvegicus, and Mus musculus, that of KWS1was found in L. palpale, L. intermedium, and L. taishanicum.Homology analysis showed that the nucleotide identities between KWS1-4genotypes and Japanese Kawasaki strain were96.6%,96.5%,96.1%, and95.8%, respectively; and the amino acid identities were92.1%,91.7%,91.2%, and91.2%, respectively. SKS genotype had a nucleotide identity of99.3%and an amino acid identity of99.2%with the Japanese Shimokoshi strain. FJS genotype had a nucleotide identity of96.5%and an amino acid identity of92.5%with the Japanese Fuji strain. The nucleotide identity ranged from69.6%-100%(amino acid identity:53.8%-100%) among O. tsutsugamushi strains prevalent in animal hosts and vectors in new epidemic area of autumn-winter scrub typhus. And the nucleotide identity ranged from99.7%-99.9%(amino acid identity:99.2%-99.6%) among O. tsutsugamushi strains prevalent in animal hosts and vectors in old epidemic area. The divergence of O. tsutsugamushi strains in new epidemic area was significantly higher than those in old epidemic area.Evolutionary genetic analysis by BEAST indicated that the overall evolutionary rate of56kDa TSA gene for O. tsutsugamushi was1.15×10-4s/s/y (nucleotide substitution per site per year,95%HPD:5.75×10-6-2.52×10-4s/s/y). KWS1-4and Japanese Kawasaki strain grouped into one branch, which diverged679years ago and evolved at at a high rate; then the evolutionary rate decreased. The divergence time of KWS1-4genotypes were1934,1968,1990, and1990. The branch of SDM1and SDM2genotypes diverged2552years before, and evolved at a low rate. SDM1and SDM2diverged in1983. FJS genotype grouped with Japanese Fuji strain, and SKS genotype grouped with Japanese Shimokoshi strain. Since the two branches diverged before1061years, they have evolved at a relatively high rate.4. Epidemic and Clinical features of scrub typhusSurveillance data from Shandong Disease Reporting Information System showed that scrub typhus had been reported in Linyi, Tai’an, Rizhao, Qingdao, Laiwu, Weifang, Yantai, Zibo, Weihai, Jinan, Zaozhuang, Binzhou, Jining, and Liaocheng. The annual incidence of scrub typhus increased from0.23per100,000people in2006to0.65per100,000people in2013. Farmers are the most susceptible population to the disease in old and new epidemic areas, and population that between40and69years old accounted for more than65%of the total cases. Scrub typhus cases occurred in every month in old epidemic area, with a significant peak in October and November and a slight peak in May. In new epidemic area, scrub typhus cases were reported to occur in every month except for Janurary, March, and April, with a peak in October and November. A total of12cases were reported in May in new epidemic area. Spring cases and summer cases of scrub typhus were confirmed using rapid immunochromatographic immunoassay in old and new epidemic area, respectively. The main clinical manifestations of scrub typus cases were high fever, eschar or skin ulcer, rash and fatigue. No significant difference was shown in laboratory findings of scrub typhus cases between old and new epidemic area. Difference was statistically significant in the presence of headache (P=0.013), myalgia (P=0.032), liver percussion (P=0.022), and complication (P=0.008) between patients from old and new epidemic area. KWS1and KWS2were predominant genotypes infecting humans, which had an identity>96.5%to the Kawasaki prototype. It was the first report of Sdu-2genotype O. tsutsugamushi infection in humans. And a co-infection of Sdu-2and KWS2genotypes was identified in a scrub typhus patient.Conclusions1. Species composition of animal hosts and vectors were similar between old and new epidemic areas, with some differences. Natural infection of KWS1, KWS2, and KWS4genotypes were found in Rattus norvegicus, KWS2, KWS3, FJS, SDM1and SDM2in Mus musculus, and KWS2and SKS in L. palpale in epidmic areas of Shandong Province. Genetically diverse strains of O. tsutsugamushi had extensive adaption and non-excludability to animal hosts and vector mites. Similar elements of natural focus and the extensive adaption and non-excludability of O. tsutsugamushi to animal hosts and vector mites may contribute to the expansion of epidemic area of autumn-winter scrub typhus.2. Genotypic diversity of O. tsutsugamushi was revealed in animal hosts and chiggers in new epidemic area of Shandong Province. Eight genotypes, including KWS1-4, SKS, FJS, SDM1, and SDM2, were identified. Less variaty in O. tsutsugamushi was revealed in animal hosts and chiggers in old epidemic area, with KWS1and KWS2genotypes identified. Kawasaki-related genotypes were predominant in animal hosts and chiggers in both old and new epidmic areas, and the homologies ranged from70.0%to100%.3. Spring scrub typhus cases were confirmed in epidemic areas in Shandong Province. Natural infection of O. tsutsugamushi was identified in rodents and chiggers in spring in local area, which implies that the requirements for spring scrub typhus epidemic were reached, and transmission chain was formed in spring in Shandong. 4. Natural infection of O. tsutsugamushi were identified in domestic rodents and the parasitizing chiggers. Besides wild rodents, domestic rodents may play an important role in the transmission of scrub typhus in Shandong. The transmission of scrub typhus in old epidmic area was largely related to wild rodents, while that in new epidemic area was largely related to domestic rodents.5. The distribution in population and seasonal distribution were similar between old and new epidemic areas. KWS1and KWS2genotypes of O. tsutsugamushi were predominant among patients in the two types of epidemic areas. A case that co-infected with Sdu-2and KWS2genotypes of O. tsutsugamushi was first reported in the study. Difference was statistically significant in the presence of some symptoms, signs, and complication between patients from old and new epidemic area.