Objective To evaluate the application of 5S-23S rRNA and outer ospA genes in PCR?based detection of Borrelia burgdorferi and to investigate the infection with B. burgdorferi in rodents of Zhejiang province, China. Methods PCR was used to amplify 5S-23S rRNA and ospA gene fragments from 100 mice collected from different areas of Pan’an county. PCR products were purified and sequenced. Results Of the 100 mice investigated, 3 were positive for 5S-23S rRNA gene of B. burgdorferi and 5 for ospA gene of B. burgdorferi. The sequences of PCR products were found with high homology to the reference genes. Conclusion B. burgdorferi can be detected from rodent samples by PCR amplification of 5S-23S rRNA and ospA genes.

Objective To analyze the distribution and variation of Leptospira in humans and host animals after leptospirosis outbreak and to provide a scientific basis for prevention of leptospirosis. Methods Sera of clinically diagnosed patients and host animals were collected and examined by microscopic agglutination test (MAT). The kidney samples of rodent, frog, pig, and teal as well as the midstream urine samples of cattle were collected; pathogens were isolated from the samples and then cultured, and the strains were identified by MAT. Results Five cases of leptospirosis were reported in Pan'an county from 2009 to 2012. A total of 40 duck sera were examined, and 8 were positive for antibodies against Leptospira, including 6 cases for antibody against strain 56601 and 2 cases for antibody against strain 56608. A total of 439 small mammals were examined, and 14 strains of Leptospira, all of which were strain 56601, were isolated. No Leptospira was detected in the kidney samples collected from 80 duck, 390 frog, 244 pig, and 160 cattle midstream urine samples. Conclusion Leptospira is prevalent in host animals in Pan'an county, and the epidemic strains in foci may change. Surveillance should be enhanced to prevent leptospirosis.

Objective To detect the infection of Babesia protozoa in rodents in Chun’an county, Zhejiang province. Methods The polymerase chain reaction (PCR) was employed to detect the liver and spleen samples of 106 rodents from Chun’an county and the positive ones were sequenced, followed by phylogenetic analysis. Results It was found that there were three positive samples, 2 of which were obtained from Rattus tanezumi and one of which from R. norvegcus. The sequences of the positive samples were of high similarity with those from Babesia, with the rates of similarity being both 99% . Conclusion DNA segments of Babesia can be detected from rodents in some mountain areas of Zhejiang province, indicating that it is likely that there exists infection of rodents with Babesia in the area.

Objective To identify Borrelia burgdorferi sensu lato infection of rodents in Pan’an county, Zhejiang province. Methods The ospA gene specific fragments from 128 mouse liver and spleen samples were detected using the PCR method and the positive samples were sequenced, followed by phylogenetic analysis. Results It was found that there were four ospA gene positive fragments out of the 128 samples, of which 3 fragments were obtained from Rattus losea and the other one was from Apodemus peninsulae. The 4 positive fragments shared high similarity with each other in terms of the sequences and shared high identities with B. valaisiana. Conclusion B. burgdorferi sensu lato can be detected from samples using the ospA gene. There exists B. burgdorfer sensu lato infection in rodents in some mountain areas of Zhejiang province, and B. valaisiana is found to be predominant.

Objective To discuss the feasibility of lipL32-PCR for detecting Leptospira interrogans. Methods The specificity and sensitivity of primers based on the lipL32 gene were compared with the recommended standard G1/G2 primers. Using these gene-based primers, a PCR assay was conducted for detection of L. interrogans in kidney samples from frogs and rats in Changshan county. PCR identification was performed on isolates taken from Zhejiang in 2008. Results The lipL32-PCR was specific and sensitive to L. interrogans. The PCR identification result for 2008 isolates was consistent with serological testing. A consistency of 95.0% was noted between lipL32-PCR and the assay based on the standard G1/G2 primer. The positive rate was 10.0% for lipL32-PCR and 5.0% for G1/G2-PCR. No significant difference between the two positive rates was shown by Fisher's exact probability test (P＝0.25). Conclusion The lipL32-PCR was specific and sensitive in the detection of L. interrogans and can effectively reflect the carrier rate in animals, thus, providing evidence for the control of leptospirosis.

Objective To determine the prevalence of Anaplasma phagocytophilum in goats and oxen in Zhejiang province. Methods Five goats and five oxen were randomly surveyed in each househould based on registration information. Venous blood samples of 5 ml were collected from each subject, and the separated serum was tested for IgG antibodies against A. phagocytophilum using immunofluorescence assay (mIFA) as recommended by the WHO. The blood cells were used for DNA extraction, which was then amplified for the 16S rRNA genes of A. phagocytophilum. The PCR products were sequenced and analyzed for homology and genetic variation. Results A high carrying rate (39.42%) was found in the local livestock, particularly in Tiantai county. Considerable variation in the 16S rRNA genes was found in the prevalent strains. In addition to 15 independent variations in the nucleic acid sequences, at least five strains with variation in the dominant sequences were found. Conclusion Most domestic animals in Zhejiang province carry A. phagocytophilum warranting the survey and molecular epidemiological investigation of this pathogen in humans.

Objective To get an insight into the carriage status and epidemic characteristics of Bartonella in rodents in Zhejiang province for providing baseline data for the development of measures for the control and prevention of Bartonella infection. Methods DNA was extracted from the livers and spleens of mice and the beta-subunit-encoding gene (rpoB) of Bartonella was selected for PCR amplification, the positive products of which were sequenced and phylogeneticaly analyzed. Results One of 101 rodents captured in Panan county of Jinhua city in Zhejiang was positive for the rpoB gene of Bartonella with the isolation rate of Bartonella in Rattus tanezumi being 4.3％. Phylogenetic analysis showed that the isolated strain was of the same evolutionary branch as B. grahamii (GenBank accession No. AB426694.1) with a bootstrap value of 96%. Conclusion There exists Bartonella infection in rodents in Zhejiang province with R. tanezumi as its possible host. The isolated strain belongs to B. grahamii.

Objective To identify the prevalence of ticks infected with spotted fever group rickettsiae in mountain areas of Zhejiang province, and to explore the feasibility of the PCR approach using rickettsial outer membrane protein A (rOmpA) and citrate synthase (gltA) gene as the target genes. Methods The rOmpA and gltA gene specific fragments were assessed using the PCR method from 46 tick specimens in Tiantai Zuoxi county and Lin’an Xitianmu region. The positive samples were cloned and sequenced followed by cluster analysis. Results The rOmpA and gltA gene specific fragments of spotted fever group rickettsiae were detected from 2 Haemaphysalis longicornis of the 46 specimens, with similar nucleic acid sequences. However, the evolutionary positions of the spotted fever group rickettsia species were presumably different between the two. Conclusion The ticks infected with spotted fever group rickettsiae were found in some mountain areas of Zhejiang province. Spotted fever group rickettsiae were detected from the

【Abstract】 Objective To approach the feasibility of FlaB?PCR used for the detection of leptospira. Methods The proper  primer  was  synthesized  according  to  FlaB  gene and its sensitivity and specificity was evaluated compared to the primer G1/G2 recommended by public health industry standard. It was applied to leptospira detection of frog and rats which were from Longyou and Changshan. And it was identified by PCR. Results FlaB?PCR was highly sensitive and specific, and pathogenic leptospira DNA could be specifically amplified with the selected primer. The PCR results on leptospira isolated from Zhejiang province  in  2007 were in accordance with the serology results. The positive rate of the kidneys of rats and frogs was 20.94% by FlaB?PCR. Compared with the primer G1/G2 recommended by public health industry, the coincidence of two methods was 90.54%. Conclusion FlaB?PCR can detect the pathogenicity leptospira specifically and quickly, which can reflect the carrying rate of leptospira in wild animals accurately and efficiently and provide the evidence for leptospira control.

Objective To investigate the natural infection situation of Borrelia burgdorferi sensu lato in rat-shape animals from Zhejiang province.Methods The segment between 5S rRNA and 23S rRNA of B.burgdorferi sensu lato in the rat-shape animals was amplified with the method of nest-PCR,and the positive PCR products were sequenced and analyzed.Results 128 rat-shape animals were captured in Jindong district of Jinhua city.12 positive samples were detected in Rattus,Apodemus and Shrew,and the positive rate were 9.68%,14.28% and 25.00%,respectively.The sequence analysis results showed the amplified segments from two positive samples were homologous to that of B.valaisiana and their gene serial numbers were AB091447BvalaiOS115 and DQ188933BvalaiQX13P25.The comparability reached 100% between them.Conclusion It suggested that there was B.burgdorferi sensu lato in Zhejiang province,and the rice field mouse,Apodemus and shrew could be the hosts of B.valaisiana.But it still needs be further studied.

Objective To study the infection of Spotted fever Group Ricketfsiase in wild rodent and tick in Zhejiang province. Methods Polymerase chain reaction was used to detect Spotted fever Group Ricketfsiase DNA in tick and rodent's spleen. Results From Jindong district,Jinhua city,2 Rattus confucianus,10 Rattus lossea and 4 Apodemus agrarius were captured. All of them were negative by PCR. 8 specimens of the 49 Lxodes sinensis were positive.The positive rate was 16.33.Conclusion It showed preliminarily that the pathogen of Spotted fever Group Ricketfsiase occurred in Zhejiang,which should be further investigated.

Objective The rapid laboratory detection technology of Dengue fever was applied to provide important evidence in efficient diagnosis and epidemics control of Dengue. Methods Indirect immunofluorescent assay(IFA) was developed to detect the sera from suspicious Dengue patients in 2003-2004. Results By the IFA assay,three Dengue fever epidemics was confirmed including one outbreak. Conclusion The imported Dengue epidemics occurred in Zhejiang in 2003-2004.

Objective To study the epidemic pattern of Leptospirosis in Zhejiang province,analyze the problems in the prevention and contol of Leplospirosis. Methods With MAT to test the serum antibody in patients. Check the leptospira in kidney of duck,pig,forg and rodents,and urine of cattle. Results In 2002 year,the total patients in whole province were 59. The average incidence was 0.13 per 100 000,one of them died. Most of patients were in Quzhou,Lishui and Taizhou city. Of 299 mouse's kidney,620 forg's kidney,80 pig's kidney,28 duck kidney and 54 cattle's urine samples,culturepositive rates of leptospira were 2.01%,1.61%,1.25%,0,0,respectively. In 2003 year,total patients in whole province were 20. The avevage incidence was 0.04 per 100 000 and patients were in Jinhua,Quzhou,Lishui and Taizhou city. Of 577 mouse's kidney,1 070 frog's kidney,127 pig's kidney,40 duck's kidney and 110 cuttle's urine samples,culture posistive rates of leptospira were 0,4.67%,0,0,0. Conclusion The prevention and control of Leptospirosis can not be relaxed. It should be guarded against,especially after flood.

Objective:To study the epidemic pattern of Leptospirosis in Zhejing province,analyze the problems in the provention and control of Leplospirosis.Methods:With MAT to test the serum antibody in patients,ducks and frogs.Culture the Leptospira with the serum of patients,ducks and frogs,pig's kidney,frog's kidney,duck's kidney,cattle's urine,mouse's kidney.Results:In 2000 year,the total patients in whole province are 77.The average incidence is 0.17 per 100000,3 of them were died.Most of patients were in Quzhou,Lishui and Taizhou city. The positive rate of serum antibody in patients,frogs and ducks was 65.22%,13.64% and 50% respectively. The culture positive rate of patient's blood,mouse's kidney and frog's kidney are 0,2.08% and 0.73% respectively.Conclusion:The provention and control of Leptospirosis can not be relared. It should be on guard against its outbreak after flood.