Detection And Isolation Of Babesia Spp. In Ticks In Heilongjiang Province Of China

Objectives:The objectives of the study were to investigate the infection status of Babesia in ticks from Heilongjiang province of China, and to characterize the area strains from ticks by molecular techniques, and to obtain isolated strains of Babesia.Methods:In this study,we selected Dashigou forest farm and Heiniubie forest farm from Heilongjiang Province in northeast china as the survey area.18S rRNA gene of Babesia was amplified using PRIO-A/PRIO-B primer to analyze the infection situation of Babesia in ticks in the survey area. The PCR products were sequenced. Babesia sequences were compared with those of known Babesia species in GenBank for maximal homology. The phylogenetic analysis was performed in MEGA5.0software with the neighbor-joining method. It is to investigate the infection status of Babesia spp. in ticks in the survey area and to identify the genotype of Babesia in the survey area.Mice were bited by Ixodes persulcatus collected from Mudanjiang City, Heilongjiang Province in2011from May to July. Then mice were detected by polymerase chain reaction method with PRIO-A/PRIO-B primer and identification of the morphological. Positive mice were observated regularly and subcultured. Identification of the gene aspects:18S rRNA gene was amplified by polymerase chain reaction method with PRIO-A/PRIO-B primer. Identification of the morphological aspects: Including the optical microscope after Giemsa staining and electron microscope. Preservation:Including the continuous passage culture and frozen in liquid nitrogen.The SPSS16.0software was used to process statistical analyses, including χ2-test and Fisher’s exact test.Results:A total number of2841ticks were collected from the survey area during2010～2012, including2380Ixodespersulcatus and461Haemaphysalis conicinna. The overall prevalence of Babesia in ticks was 1.27%(36/2841). The rates of Babesia infection in Haemaphysalis conicinn and Ixodespersulcatus were2.82%(13/461) and0.97%(23/2380). There is statistical difference of the rates of Babesia infection between Haemaphysalis conicinn and Ixodespersulcatus. There is statistical difference of the structure of the speices of ticks between Dashigou forest farm and Heiniubie forest farm.There is no significant difference of the infections of Babesia between the two forest farms. Babesia positive rates in2010,2011and2012were1.08%,1.60%and1.01%, Babesia positive rates were no significant difference in three years. There was no significant difference about Babesia infection rates in the different environmental information. Sequence analysis showed that Babesia in ticks in the survey area exist genotypes diversity, including Babesia sp. EU1、B. microti、B. bigemina、B. divergens、Babesia sp. hlj242-like、Babesia sp. hlj874.By ticks biting mice, B. divergens was successfully isolated. The Successfully isolated B. divergens was confirmed by PCR, optical microscope after Giemsa staining and electron microscope.Conclusion:Through this study, we have obtained the infection status of Babesia in ticks in Heilongjiang forest, Sequence analysis revealed the genotypes diversity of Babesia. B. divergens was successfully isolated from ticks.This study laid a foundation for the further research of Babesia in china.