Study On Life Cycle And Activity Of Antibacterial Peptides Of Hyalomma Anatolicum Anatolicum And Development Of A Two-temperature PCR Method For Detecting Theileria Annulata Infection In Cattle

In this study,I was completed life cycle of Hyalomma anatolicum anatolicum in under the conditions of the laboratory,and after isolation of antibacterial peptides from hemolymph of Hyalomma anatolicum anatolicum,purified by AU-PAGE.The blood samples were collected from epidemic area for detecting Theileria annulata infection in cattle,to develop a two-temperature PCR method,which was useful to study prevention of ticks,and control of Theileria annulata infection in cattle.Hyalomma anatolicum anatolicum,a kind of three-host tick,which mainly distributed in desert and grassland of Xinjiang in China,was one of the vector of Theileria annulata,which resulted in a lot of economic loss in livestock.Biological parameters of the tick's life cycle were studied in this experimental study.It showed that the tick needed 138d to completed generation at 22-28℃,60-85% relative humidity,by feeding on the rabbit body.Adult ticks spent 8.5,10.5,27.5 and 31.5d on blood-sucking period,pre-spawning period,spawning period,eggs incubation,respectively.The hatching rate was 80%.The larvae was 3d pre-blood-sucking period,5.5d blood-sucking period,and 18.5d exuviation period,respectively.The nymph was 4d pre-blood-sucking period,10d blood-sucking period,and 19d exuviation period respectively.This study suggested that Hyalomma anatolicum anatolicum can be propagated under laboratory conditions and also be stored at 4℃,which provided a large number of ticks and low-cost experimental materials for the study of tick-borne diseases.To study the extraction method of antimicrobial peptide and activity of cationic antibacterial peptides from haemolymph of Hyalomma anatolicum anatolicum,and the haemolymph was collected from adult female tick,and extracted with 5% acetic acid.The components of antimicrobial peptides were analyzed by AU-PAGE.The antibacterial peptides of samples were screened with polyacrylamide gel electrophoresis dispersion methods.The result showed that antibacterial peptides had strong antibacterial activities to Escherichia coli and Staphylococcus aureus in vitro,and a case of faster-moving than lysozyme was found.In conclusions,there were antibacterial peptides which differ from lysozyme in Hyalomma anatolicum anatolicum haemolymph.AU-PAGE was a convenient method for purification of antibacterial peptides. A specific,sensitive and rapid two-temperature PCR assay for detecting Theileria annulata infection in cattle was established with aseptic blood collected from endemic area.And the primers were designed according to T.annulata merozoite surface antigen(Tams1),and a gene fragment of 154 bp was amplified by the two-temperature PCR.Analysis of cloning and sequencing showed that sequence of PCR products had 96% similarity to the Tams1.Fifty blood samples collected from cattle in Theileria annulata epidemic area of Xinjiang were detected.Out of the 88% samples was positive by two-temperature PCR assay,while the positive rate was only 58% detected from same carrier cattle by blood smears examination.The results indicated that this assay could be widely used to clinical diagnosis or suppressive infection and epidemiological investigation for Theileria annulata infection in cattle.