| Ixodes is a kind of arthropod that relies on the blood of the host.There are many species of Ixodes in Xinjiang,about 45 species.Xinjiang has the highest number of horses in China,and related industries are relatively developed.Ixodes are more frequent and easier to attach with horses due to mixed grazing of herbivorous livestock,and this situation caused livestock more susceptible for Babesiosis that restricts the economic development of Xinjiang animal husbandry.In addition,Babesiosis is one of the protozoan global epidemic diseases transmitted by ticks.Acute clinical case display the death of horses,while the chronic and subacute cases are lifelong infection.At present,there is no ideal deworming agent or vaccine for the prevention and treatment of Babesiosis,and there are some problems including unclear species and biological characteristics of tick in our region,lack of monitoring and control of spreading Babesiosis and research data of relevant vaccine candidate antigen proteins.In order to solve those problems,using the methods of morphology,molecular biology and statistics for detecting the pathogens of tick and horse blood samples which were collected from 5 test sites in northern and southern Xinjiang in this study.In addition,the HSP70 gene of B.caballi was cloned and expressed to identified its effect on Peripheral blood mononuclear cell(PBMC),which support further development of related vaccines and the invasion mechanism of B.caballi.This present study also laid a foundation for the comprehensive prevention and control of ticks-Babesiosis in our region.(Ⅰ)In this test,504 horse blood samples and 620 Ixodes were collected from 5 tested areas.The Ixodes species were identified using morphology and molecular biology.And the dominant speciesD.marginatus "for the rabbit feeding tick" experiments to observe the life cycle.The morphology and ultrastructure of each age were observed by anatomical microscope and ESEM.The PCR method was used to amplify the Bc48 gene target fragment of B.caballi in samples of D.marginatus and horse blood.The results showed that 599 out of 620 Ixodes were identified as D.marginatus;Under laboratory conditions(temperature:25±1℃,humidity:75±5%),the life cycle of D.marginatus was about 93 days.After observation,ultrastructure of D.marginatus was identified in different at different ages(the larvae had 3 pairs of podomere).It does not have spiracular plate and genital aperture,and the dental formula was 2|2.Nymphs can be distinguished by vaives with 3 pairs of setae;none of genital aperture;4 pairs of podomere;the cornua begins to appears;dental formula 3-2|2-3.Male of adult ticks have spiracular plate and genital aperture;there are 5.5 pairs of setae on surface of anal vaives;3 odontoid processes on ventral idiosoma of podomere Ⅳ(femur,tibia and metatarsus),its dental formula is 3|3.Female of adult ticks can be also distinguished by basis capituli with a thin and shallow transverse sulure,dorsal of basis capitulum,each side of theporosa area has one setae,5 pairs of setaes on the anal vaives,3 small odontoid processes on ventral idiosoma of podomere Ⅳ.Top of hypostome have small denticle,Its dental formula is 4-3|3-4.The 451 bp target bands of Bc48 gene were amplified in both haematopoietic and tick-derived samples,which were consistent with the results of gene sequencing.The results of statistical analysis showed that the total positive rate of 504 samples of horse blood was 10.71%.The positive rate of the tested areas in northern part was higher than southern part in Xinjiang.Horses older than 3 years old were susceptible,with significant differences between different test sites and no differences among all age groups.The carrying rate of 599 D.marginatus is 36.91%,of which,the carrier rate of the engorged female ticks was the highest,reaching 79.17%.The total positive rate of female ticks(43.62%)was higher than that of male ticks.There was a significant difference between the them.In addition,the B.cabalii has been detected in the eggs of D.marginatus,which is the first time that D.marginatus and its eggs have been found in China.This will provide data reference for the study of the morghology and biological characteristics of "Transovarial transmission" of D.marginatus and the comprehensive control of Babesiosis.(Ⅱ)This study based on the screening of functional genes and protective candidate antigens of B.caballi,PCR amplification of obtaining the HSP70 truncated gene fragment of B.caballi(Xinjiang),and the contruction of HSP70-pET 32a recombinant plasmid.Sequencing was performed and HSP70 was sequence analyzed using NCBI,ProtParam and other software software.Bioinformatics data of the protein was obtained.It was induced by inducible expression and identified by SDS-PAGE and Western Blot.The results showed the target fragment(429 bp)was successfully cloned and encoded 143 amino acids.The genetic distance between the gene and the corresponding Babesia HSP70 gene in GenBank was 0.Bioinformatics data presented the recombinant protein was a hydrophilic secretory protein with good stability and 7 antigen epitopes.It was proved that the recombinant protein was an inclusion body protein with a size of 33 kDa.After purification,the purity is 1.8,the concentration is 0.63 mg/mL,has a good immunogenicity,the polyclonal antibody titer can reach 1:1 024 000.(Ⅲ)Focusing on the invasion mechanism of B.caballi,and healthy horse PBMC were isolated,with CCK8 kit,IFA and flow cytometry detection analysis reorganization HSP70 protein on PBMC,binding,proliferation,and apoptosis.Recombinant HSP70 protein(10 μg/mL)was co-incubated with horse PBMC,DAPI(stained the nucleus),and the recombinant protein was labeled with Goat Anti-Horse IgG H&L(FITC).Under a fluorescence inverted microscope,compared with the control group,it was found that the DAPI stained nuclei in the test group emitted blue fluorescence,the recombinant protein also labeled with Goat anti-horse IgG H&L(FITC)at the same position showed green fluorescence.After adjusting the excitation light,indicating that the recombinant protein could bind to PBMC.Furthermore,the PBMC was stimulated with 10 μg/mL,20 μg/mL,and 40μg/mL recombinant HSP70 protein,and recombinant HSP70 protein of 40 μg/mL was significantly promoted PBMC proliferation.The promotion effect was not significant at 10 μg/mL and 20 μg/mL.Recombinant HSP70 protein(10 μg/mL)could significantly promoted the phagocytosis of PBMC,and at 20 μg/mL and 40μg/mL was not significant.Detection of flow cytometry during FITC Annexin V and PI staining revealed that different concentrations of recombinant HSP70 protein had different effects on PBMC apoptosis,the effect was more obvious in early PBMC apoptosis during early and late apoptosis. |
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