| Objective: Through the identification of Culicoides species and PCR identification of their abdominal blood powder,the source of gastric blood of Culicoides was judged,the blood-sucking preferences of different species of Culicoides were analyzed,their bloodsucking habits were understood,and the possible feeding range of Culicoides and the species groups transmitted by pathogens were determined,so as to provide a reference basis for Culicoides control.Culicoides extract was used to construct Culicoides-mouse allergy model,and to study the immune response model of mice sensitized by Culicoides extract,so as to provide a model tool for further study on the mechanism of Culicoides bite allergy and other allergic diseases.at the same time,it provides a basis for the screening and development of allergic drugs for blood-sucking insect bites.Methods: 1.Culicoides from different habitats in Qijiang Area of Chongqing were collected by lamp trapping method,and the morphological classification and identification of Culicoides were carried out.The results of morphological identification were checked by molecular identification and development tree construction,and the distribution of Culicoides in different habitats was analyzed.2.Based on the CO I gene sequence of species,species-specific primers were designed to extract DNA from the ventral blood powder of Culicoides.The specific primers were used for PCR,and the host species were determined according to the amplification results.3.Mice were sensitized with Culicoides extract in two steps: mice were sensitized by intraperitoneal injection(IP)with Culicoides extract plus alum adjuvant on days 0,7,14 and21,and intradermal injection(ID)of Culicoides extract on days 28,31 and 35,while those in the control group were injected with PBS under the same conditions.4.The behavior of the sensitized mice was observed and the morphology of the sensitized site of Culicoides extract was detected;the expression of specific antibody Ig E and Ig G subclass was detected by ELISA every 14 days;after 42 days of sensitization,the spleen cells of mice were extracted,and the effect of Culicoides extract on the proliferation of spleen cells was determined by MTT method.At the same time,the expression of cytokines in spleen cells of mice treated with Culicoides extract was detected by q PCR.Results: 1.Seven species of Culicoides were collected from four different coops(pig house,sheep house,chicken house and mixed house of chickens and ducks)in Chongqing,and seven species of Culicoides CO I genes were amplified.The species of Culicoides were successfully matched by sequencing and sequence alignment.NJ molecular tree and ML molecular tree were constructed with CO I gene sequence.The results of molecular identification were consistent with those of morphological identification,and all kinds of Culicoides formed a single branch,and there was no species crossing.2.The Culicoides populations distributed in different barns were different.The dominant species of sheep house,chicken house and chicken-duck mixed house were C.arakawai,accounting for 75.26%,93.36% and 94.96% of the total captured,respectively,while the dominant species of pig house was C.kinabaluensis,accounting for 70.77% of the total captured.By random sampling,PCR was detected in gastric blood of 6 species of Culicoides.The results showed that all 6 species of Culicoides had the phenomenon of facultative blood feeding,which could feed on the blood of 4 to 6 species of animals.Among them,C.arakawai,C.kinabaluensis,C.actoni and C.lingshuiensis all feed on chicken blood,C.kinabaluensis also likes sheep blood and pig blood,C.oxystoma and C.punctatus prefer pig blood and sheep blood.In addition,C.actoni and C.arakawai also sucked human blood,and the proportion of both sucking human blood was 56.52% and 1.39% respectively.3.Preparation of Culicoides extract,by intraperitoneal injection(IP)and intradermal injection(ID)of Culicoides extract to construct Culicoides-mouse allergy model.After being sensitized by Culicoides extract,the mice in the sensitized group scratched the sensitized site more significantly than the control group(P<0.001),and the skin of the sensitized site showed tissue edema and lymphocyte infiltration.In addition,the serum levels of Ig E(P<0.05)and Ig G1(P<0.05)antibodies in the sensitized group were significantly higher than those in the control group.In vitro stimulation with Culicoides extract caused significant proliferation of spleen cells in sensitized mice(P<0.001),and the cytokines produced by inducing the immune response of type 2 helper T lymphocytes(Th2)included IL-4,IL-5,The m RNA expressions of IL-10 and IL-13 were significantly up-regulated compared with the control group(P<0.05).In conclusion,the Culicoides-mouse allergic model was successfully constructed.Conclusion: 1.Preliminary clarification of the blood-sucking habits of 6 species of Culicoides in Chongqing.All six species of Culicoides are facultative blood-sucking and have the characteristics of blood-sucking diversity.At the same time,different species of Culicoides have different blood-sucking habits.2.The Culicoides-mouse allergy model was successfully established by using Culicoides extract through two-step sensitization method,which provides a model tool for further study on the mechanism of Culicoides bite allergy and other allergic diseases.at the same time,it provides a basis for the screening and development of drugs for the treatment of blood-sucking insect bites. |
