Epidemiologic Study On The Autumn-Winter Type Scrub Typhus In Shandong, China

BackgroundScrub typhus,also known as tsutsugamushi disease,is a zoonosis which is epidemic mainly in the Pacific region of Asia.Classified in the genus Rickettsia,its etiologic agent is now identified as O.tsutsugamushi(Ot),a gram-negative obligate intracellular bacterium.Clinical manifestations of the disease range from mild fever with few other symptoms such as enlarged lymph nodes,skin rash,splenomegaly, hepatomegaly to a fatal syndrome characterized by multiple-organ failure.In China, before 1986 scrub typhus was only found in southern Provinces of China(South of the Yangtze River),with the main epidemic season in summer.After 1986,however, scrub typhus was identified in northern China(North of the Yangtze River)including Shandong Province.Scrub typhus is an emerging infectious disease in northern China, which is epidemic primarily in autumn and winter.In Shandong Province,the first scrub typhus outbreak was in Linyi District in 1986.In 1988,the disease appeared in Jinan District.Before 1996,many cases were notified only in Linyi and Jinan Districts,and no case was found in other Districts of Shandong Province.However,there was a scrub typhus epidemic in Jining District in 1996.The disease was found in Yantai District in 1997 and in Weifang District in 2000.In autumn of 2000,65 scrub typhus cases,including two deaths,were diagnosed in Tai'an District.Therefore,the "old" epidemic areas of scrub typhus in Shandong are the areas where scrub typhus cases were firstly diagnosed and notified from 1986 to 1996,while the "new" epidemic areas in Shandong are the areas where scrub typhus cases were firstly identified after 1996.Since this disease was not required to monitor in the whole China after 1990,the epidemiologic status of scrub typhus in the entire Shandong Province was not clearly understood.ObjectivesAlthough epidemiological investigation on the autumn-winter type scrub typhus has been developed in some districts of Shandong Province,many questions still remained to be solved.Several aspects had been developed mainly in the epidemiologic study of the autumn-winter type scrub typhus in Shandong,China. Such as,to clarity the genotypes of Ot prevailing in Shandong,the phyIogenetic relationship of Ot between the new and old epidemic areas of scrub typhus, ultrastructure of the Shandong Ot isolates and the pathological changes occurred on host cells,epidemiologic status and clinical features of scrub typhus in the entire Shandong Province.Materials and Methods1.Molecular epidemiological studies of scrub typhus1.1 Investigation areas and source of specimen Linyi and Tai'an Districts,the representatives of old and new epidemic areas,were selected as study areas. Acute-phase blood specimens from the scrub typhus patients were collected before the antibiotics treatment over the period of September,2004 to December,2006. During the convalescent phase,the spontaneously desquamated eschars were also collected from the patients.In autumn and winter,the epidemic seasons of scrub typhus in Shandong,rodents in the above two study areas were captured in both patient' house and wild field.1.2 Extract DNA DNA was extracted from the peripheral blood and eschar of scrub typhus patients,and the spleen tissue of rodents by the method of phenol/chloroform/isoamyl alcohol.1.3 Nested PCR The primers,including group and type specific primers,were designed based on the nucleotide sequence of scrub typhus antigen 56-kilodalton surface protein gene.PCR amplification was adopted to identify Ot-DNA and clarity the genotype of Ot.1.4 Nucleotide sequence Based the results of genotype identified by nested PCR, the PCR products were purified and sequenced.1.5 Sequence analysis The nucleotide sequences of O.tsutsugamushi isolates in Shandong,China were compared with those of other O.tsutsugamushi obtained from GenBank of National Center for Biotechnology Information(NCBI).Alignment of the nucleotide sequence was carried out using MEGA3.1.Some sequences were trimmed,conserving only the informative regions necessary for compatible analysis. Homologous comparison analysis was performed by the MegAlign program using DNAStar software.Phylogenetic analysis was conducted by the Neighbor-joining method using MEGA3.1.One hundred bootstrap replicates were performed.2.Ultrastructural observation on the low-virulent O.tsutsugamushi2.1 Isolation of Ot.The animal model of immuno-suppression built by injecting cyclophosphamide was used to isolate the low-virulent Ot epidemic in Shandong, China.Ot was examine by light microscopy(×1000).2.2.Genotype identification Genotypes of the Ot isolates were identified by nested PCR.2.3.Ultrastructural observation Mouse spleen tissue infected with the low-virulent Ot was observed by transmission electron microscopy(TEM).3.Investigation on the epidemiologic status of scrub typhus Scrub typhus has been monitored through Shandong Diseases Reporting Information System(SDRIS) in the entire Shandong,China in 2006.And questionnaires about clinical signs and symptoms were applied in some endemic areas where scrub typhus was prevailing heavily. Results1.Molecular epidemiological studies1.1 Detection of O.tsutsugamushi by nested PCR1.1.1 Specimens of patients Among the 16 peripheral bloods of patients from Linyi,12 of them appeared the specific bands as expected,indicating that the blood specimens were O.tsutsugamushi positive.Three of the 4 eschar specimens of patients from Tai'an were infected with O.tsutsugamushi.All the second PCR products were recognized only with the Kawasaki type-specific primer pair,but no DNA fragment was amplified with other primer pairs specific to Gilliam,Karp, Kato,Kuroki and Saitama strains.1.1.2 Specimens of rodents In the epidemic seasons of scrub typhus,a total of 128 rodents were captured in Linyi,including 79 Cricetulus(C.)tyiton(Greater Long-tail Hamster),47 Apodemus(A.)agrarius(Striped Field Mouse)and 2 Rattus (R.)norvegicus(Brown Rat).Among 68 rodents captured in Tai'an,34 were R. norvegicus,24 were A.agrarius and 10 were Mus(M.)musculus(House Mouse).In Linyi,the overall infection rate of O.tsutsugamushi was 13.28%among the captured rodents,and positive rates of O.tsutsugamushi in A.agrarius and C.tyiton were 31.91%and 2.53%,respectively.No infection of O.tsutsugamuhi was detected in R.norvegicus.In Tai'an,the overall O.tsutsugamushi infection rate was 5.88%in rodents.Among those,it was 12.5%in A.agrarius and 10.00%in M. musculus,and no infection of O.tsutsugamushi was identified in R.norvegicus.All isolates from rodents were the Kawasaki strain of Ot.1.2 Nucleotide sequencing All the nested PCR products of eschars and rodents from Tai'an,and patient's blood from Linyi,were purified and sequenced.Some of nested PCR products from rodents in Linyi were selected to purify and sequence, Nucleotide sequences of the two Shandong strains were submitted to GenBank and had been assigned the accession numbers of DQ489310(WG-2)and EF543196 (55P).1.3 Sequence analysis All the sequences of O.tsutsugamushi isolated from Shandong and other published in NCBI were aligned and compared using the MEGA3.1 program.Some sequences were trimmed,while 458bp,the informative regions,was conserved and used for nucleotide sequence analysis.1.3.1 Homologous comparison When comparing the sequences of 16 O.tsutsugamushi isolated from Linyi District with the sequences of 7 O.tsutsugamushi from Tai'an District,there were high identities among these isolates(from 73.9%to 100.0%).The 55P and 56P isolates from rodents in Tai'an, which had a 92.5%identity with each other,were in the same category.However, other O.tsutsugamushi isolated from Linyi and Tai'an Districts formed another category,with 99.3%～100.0%identities among them.In addition,the 55P and 56P from Tai'an showed 73.9%～76.6%identities with other Shandong isolates.1.3.2 Phylogenetic tree The phylogenetic tree based on the 56-kDa gene sequence homologies was divided into two branches.The majority of Shandong O.tsutsugamushi isolates,including the Sdu-1 strain,formed an independent lineage distinct from other O.tsutsugamushi,together with Kawasaki,Kanda, Taguchi and Oishi strains from Japan.There were 95.5%～95.9%identities between Sdu-1 and the four Japan strains.TW461 and Hualien-2 strains isolated from Taiwan,Yonchon from Korea and Sxh951 from Shanxi Province,China were belonging to another clade.The 56P and 55P(Sdu-2)isolated from A.agrarius in Tai'an,formed a separate clade.The first branch was constructed by these clades described above.The second branch was divided into three clades.One clade was built by the UT177 and Karp isolated in Thailand,Boryon from Korea,and Kuroki from Japan.The Kato isolated in Thailand formed another separate clade.The TA763,TA686 and TA716,all isolated in Thailand,were located in the third clade.2.Ultrastructural observation on the low-virulent O.tsutsugamushi2.1 Isolation and genotype of Ot Two low-virulent Ot strains were isolated successfully,and identified as the Kawasaki strain of Ot.2.2 TEM findings of infected spleen tissue and the low-virulent Ot The Ot parasitized in the spleen were different in size,shape and electron density.More detailed pathological changes were observed on organelles of the mouse spleen cells. Swollen perinuclear cisterna was observed in mononuclear cells,and multivesicular body was found in macrophage.In the phagosome of macrophage,many Ot enveloped with an additional membrane were found to push the phagosomal membrane outward from the inside.In addition,the phenomenon of autophagy was observed on the low-virulent Ot frequently.3.Epidemiologie characteristics of scrub typhus in Shandong In 2006,206 patients were diagnosed as scrub typhus and enrolled in the SDRIS.All patients responded well to treatment with antibiotic,and no fatal patient was reported.3.1 Geographical distribution Among the seventeen districts of Shandong Province,scrub typhus was reported in twelve districts which are located in the coast of Shandong or the southern of the Yellow River.The overall annual incidence rate of scrub typhus was 2.21 per million populations in Shandong,China.The incidence rates of scrub typhus were higher in Laiwu District and Rizhao District.In addition, 52.9%scrub typhus patients in Shandong,China were reported in Linyi and Tai'an Districts,74 in Linyi,and 35 in Tai'an.3.2 Seasonal distribution Scrub typhus was epidemic mainly in autumn and winter,and peaked at October and November.In addition,there were a few patients in spring.3.3 Age,gender and occupation distributions The majority of patients were adults with a median age of 51 years old(range,2-88 years).Among the 206 scrub typhus patients,100 were males,and 106 were females.Occupations of patients were farmer,worker,student,retiree,teacher,preschool child and fisherman.One hundred seventy five patients(84.9%)were farmers with 82 males and 93 females.3.4 Clinical features Common clinical signs and symptoms included fever (97.9%),eschar(89.4%),rash(78.7%),and lymphadenopathy(53.2%).25.5% patients of scrub typhus had the symptoms of splenomegaly,19.1%had headache, 17.0%had hepatomegaly,and 4.3%had conjunctival hyperemia.10.6%patients of scrub typhus appeared interstitial pneumonia,a complication of scrub typhus. Conclusion1.The Sdu-1 type was the main genotype of O.tsutsugamushi in Shandong,China. The low-virulent O.tsutsugamushi were successfully isolated from scrub typhus patients in Shandong,China,and the isolates were Kawasaki type identified by nested PCR.2.There was only less genetic variation of O.tsutsugamushi in both new and old epidemic areas in Shandong Province.With 73.9%～100.0%identities,all of these Shandong O.tsutsugamushi isolates were located in the same branch of the phylogenetic tree.3.Transmission electron microscopy observed the morphological characterization of the low-virulent O.tsutsugamushi,and the pathological changes occurred on host cells.O.tsutsugamushi released itself by pushing out the phagosomal membrane from the inside like the budding process of viruses,which might be the escape mechanism of O.tsutsugamushi from the phagosome.In addition,the low-virulent O.tsutsugamushi has the phenomenon of autophagy.4.Scrub typhus was epidemic in 12 of 17 districts in Shandong Province.And the 12 districts are located in the coast of Shandong or the southern of the Yellow River.Scrub typhus was epidemic mainly in autumn and winter in Shandong, China.A few cases were reported in the spring.In addition,majority of patients were farmers.Fever,eschar,rash,and lymphadenopathy were the major clinical characteristics of scrub typhus patients.