Development And Utilization Of Nested-PCR Diagnostic Method For Detection Of Canis Babesia

Babesia as a tick-borne hemoprotozoan parasite is the causative agent ofBabesiosis in animal and people. This disease causes enormous economis losses. Theclinic manifestations are fever, anemia, icterus, hemoglobinuria and even mortality.With the rapid development of biological technology, various diagnostic methods forBabesiosis detection has been reported, however, each diagnostic method has itsadvantages and disadvantages. A method which is more sensitive, rapid and simplediagnostic for detection of Babesia than before was urgently needed. In this study, aNested-PCR for detection of Babesia in Canis blood samples were developed andutilized.⑴A nested-PCR diagnostic method for detection of canis Babesia was developed.In order to develop the nested-PCR diagnostic method for rapidly detection of canisBabesia, three groups of specific primers were designed according to the publishedgene18s ribosome RNA of Babesia of canis in GenBank.⑵Aepidemiological survey of henan province (Luoyang, Pingdingshan,Sanmenxia) was done by the nested-PCR diagnostic method, which was developed inthe study. A total of2960dogs and813ticks were measured separately by nested-PCR and blood smear. Ticks identification was performed with a stereomicroscope.⑶25samples are randomly selected from115positive samples and sequenced.Blast is one of the most important programs in NCBI BLAST Basic Local AlignmentSearch Tool toolkits. The sequenced result were blasted in GenBank, then the resultwere described with system analysis and phylogenetic tree by DNA Star5andBioEdit software.The results showed that: the best of three specific primers(PCR:GGCTTTCGGTGATTCATA,CCTTCCGTCAATTCCTTT,nPCR:TGGACCATTCAAGTTTCTG,TCGTCTTCGATCCCCTA) was selected. And then the nested-PCR was developed. The dogs were infected by Babesia in west of Henan province, and the prevalence rate was3.89%; Those858bp fragments were obtained and confirmed that was18S rRNA geneof Babesia gibsoni. Compared with published previously (KF878944/KF171474/AB478328/AB478330/JX112784), the sequences similarities were98.8%,98.8%,98.8%,98.8%,100%,respectively; The morphological types for ticks were Rhipicephal-ushaemaphysaloides (52.4%) and Rhipicephalus sanguineus, and the tick parasite ratewas2.33%.Conclusion:⑴A established method of nested-PCR assay was a precise, sensitive and specifictechnique, and could be used to rapidly detect Babesia and conduct and itsepidemiological survey in the small scale.⑵Infection rate of Babesia is low, and the pathogenic strains were Babesiagibsoni; Rhipicephalus haemaphysaloides is of dominant species parasitized on dogs,and the parasite rate of Babesia is low.