ABSTRACT:Nested‐PCR and loop‐mediated isothermal amplification (LAMP) were applied to identify the Borrelia burg‐dor f eri (B .burgdor f eri) in ticks in this study .A total of 112 adult ixodes were collected from vegetation in a forest area and farm animals in Xunhua County ,Qinghai Province and Xinbin County ,Liaoning Province .The ticks were examined for the presence of B .burgdorferi by nested‐PCR and LAMP .Results showed that 12 in 51 samples were found positive in Xunhua County (23 .53% ) .While positive rate in Xinbin County was 29 .51% with 18 samples positive in 61 samples .In total of 112 tick samples ,the PCR‐positive rate was 17 .86% with 20 positive samples identified ,whereas 15 positive samples were con‐firmed with positive rate of 13 .39 % by LAMP assay .There was no significant difference between the two assays (Х2 =0 .85 , P>0 .05) .Results suggest that both nested‐PCR and LAMP could be used in identifying B .burgdorferi in ticks .Combina‐tion of these two assays could improve the testing results .This is the first report of B .burgdorferi in ticks in Xunhua and Xinbin counties ,and helps to complete the database of the infection rate of B .burgdor f eri in ticks in the widely‐forested area of China .

Objective To clone and express the outer surface protein C ( OspC) from a Chinese Borrelia afzelli FP1 strain and to evaluate the immune protectivity of the recombinant OspC protein ( rOspC) . Methods The gene encoding OspC protein of Borrelia afzelli FP1 strain was amplified by polymerase chain reaction (PCR) and then inserted into pET-30a plasmid to construct the recombinant expression plasmid pET-30a-OspC. The transformed E. coli BL21 strains carrying pET-30a-OspC plasmid were induced by IPTG to express OspC protein. The expressed proteins were purified by Ni-IDA resin chromatography and analyzed by SDS-PAGE and Western blot assay. Indirect immunofluorescence assay ( IFA) was performed to detect anti-rOspC protein antibodies in serum samples from rabbits immunized with rOspC protein. In vitro neutral-ization test was performed for evaluation the immune protectivity of rOspC protein. Results The recombi-nant expression plasmid pET-30a-OspC was successfully constructed and highly expressed in E. coli BL21. A strong antigen-antibody reaction between the rOspC protein and polyclonal antibody against Borrelia afzelli FP1 strain was detected by Western blot assay. The titers of IgG in serum samples from rabbits immunized with rOspC protein were significantly elevated. The in vitro neutralization test indicated that 106/ml of Borre-lia afzelli FP1 strains were neutralized by every anti-OspC protein serum sample from the experiment group. Conclusion The rOspC protein showed a strong immune protectivity against Borrelia afzelli, which could be used in the development of polyvalent subunit vaccine against lyme disease.

Objective To predict the potential geographic distribution of Lyme disease in Qinghai by using Maximum Entropy model (MaxEnt).Methods The sero-diagnosis data of Lyme disease in 6 counties (Huzhu,Zeku,Tongde,Datong,Qilian and Xunhua) and the environmental and anthropogenic data including altitude,human footprint,normalized difference vegetation index (NDVI) and temperature in Qinghai province since 1990 were collected.By using the data of Huzhu Zeku and Tongde,the prediction of potential distribution of Lyme disease in Qinghai was conducted with MaxEnt.The prediction results were compared with the human sero-prevalence of Lyme disease in Datong,Qilian and Xunhua counties in Qinghai.Results Three hot spots of Lyme disease were predicted in Qinghai,which were all in the east forest areas.Furthermore,the NDVI showed the most important role in the model prediction,followed by human footprint.Datong,Qilian and Xunhua counties were all in eastern Qinghai.Xunhua was in hot spot area Ⅱ,Datong was close to the north of hot spot area Ⅲ,while Qilian with lowest sero-prevalence of Lyme disease was not in the hot spot areas.The data were well modeled in MaxEnt (Area Under Curve=0.980).Conclusions The actual distribution of Lyme disease in Qinghai was in consistent with the results of the model prediction.MaxEnt could be used in predicting the potential distribution patterns of Lyme disease.The distribution of vegetation and the range and intensity of human activity might be related with Lyme disease distribution.

Objective:To clone and express the 126-274 aa OspA peptide (OspA-pep) of Chinese Borrelia garinii ( B. garinii) strain PD91 and to preliminarily study its immune protectivity. Methods:The gene encoding the 126-274 aa OspA-pep of B. garinii PD91 was amplified by polymerase chain reaction (PCR) and then cloned into the prokaryotic expression vector pET-30a to construct the recombinant plasmid pET-30a-OspA-pep. Escherichia coli BL21 (DE3) competent cells transfected with the recombinant plasmid were induced by IPTG to express the target protein. The recombinant OspA-pep (rOspA-pep) was purified with Ni-IDA resin chromatography and its immunogenicity was analyzed by Western blot. New Zealand white rabbits were immunized with different doses of rOspA-pep (20, 30, 40, 50, 60, 80 and 100 μg). The titers of specific IgG antibodies in rabbit serum samples before and after immunization were detected by indirect immunofluorescence assay (IFA). The optimal immune dose was determined according to the antibody titer after immunization. In vitro neutralization test was performed to detect the immune protection of rOspA-pep using serum samples of the optimal immunization group. The optimal dose of rOspA-pep was used to immunize New Zealand white rabbits to observe the changes in antibody titer. Results:The recombinant plasmid pET-30a-OspA-pep was successfully constructed and highly expressed in host bacteria. Western blot showed that rOspA-pep had obvious antigen-antibody reaction with polyclonal antibody against B. garinii PD91 strain. IFA results showed the titers of IgG antibody in serum samples of rabbits immunized with rOspA-pep increased significantly (up to 1∶2 480) and 40 μg was the optimal dose. The neutralization rates of antibodies induced by 40 μg of rOspA-pep were 100% against 10 6 strain/ml of representative B. garinii PD91 and Borrelia afzelii ( B. afzelii) FP1 strains, 100% against 10 7 strain/ml of FP1 strain, and 60% against 10 7 strain/ml of PD91 strain. After immunization with 40 μg rOspA-pep on 1 d and 30 d, the titers of specific IgG antibody in rabbit serum samples reached the peak within two months, and maintained at that level for about 3-4 months before a gradual decline. Conclusions:The 126-274 aa OspA peptide fragment of Chinese B. garinii PD91 strain possessed good immunogenicity and induced antibodies with better in vitro neutralizing activity, which suggested that it could be used as a candidate component of the second generation subunit vaccine in China.