Experimental Study On The Establishment Of Reference Material For Pathogenic Detection Of Schistosoma Japonicum Using Schistosoma Sinensis As Model

Objective: In view of the similarity between Schistosoma sinensis and Schistosoma japonicum in many aspects,and the convenience of sample preparation for Schistosoma sinensis,which is safe and non-infectious to humans,the feasibility of using Schistosoma sinensis as a model to prepare positive reference and quality control products for Schistosoma japonicum testing is to be explored,providing innovative ideas and laying the foundation for the effective and convenient acquisition of positive reference products for Schistosoma japonicum testing with low biosafety risk and standard application in laboratory testing.Methods:The animal infection experiments for both Schistosoma sinensis and Schistosoma japonicum were established in the laboratory to obtain the necessary sample materials for the study.1.The feasibility of replacing infective Oncomelania Hupensis positive reference with infective Tricula Humida by crushing microscopy method: Following the double-blind principle,30 test personnel with more than 5 years of experience in schistosomiasis detection were blind-tested using the crushing microscopy method with samples of Oncomelania Hupensis infected with S.japonicum and Tricula Humida with S.sinensis,respectively.The collected cercariae were fixed,and those with good morphology were photographed using a microscopic imaging system.Four parameters-body length,body width,tail length,and tail spine length-were measured,and statistical software SPSS was used to conduct independent sample t-tests on the four sets of data.2.Feasibility of using Schistosoma sinensis eggs as a positive reference product for hatching,instead of using Schistosoma japonicum eggs: The nylon stocking method was used to collect eggs from the intestines of wild rodents infected with S.sinensis and feces of laboratory mice infected with S.sinensis.A parallel control group was set up using liver homogenate from mice infected with S.japonicum.Different egg concentrations,p H values,and temperatures were used to compare cercarial hatching between liver homogenates from wild rodents infected with S.sinensis and mice infected with S.japonicum.3.Feasibility of using serum from mice infected with Schistosoma sinensis as a positive reference product for quality control of the indirect hemagglutination assay,instead of using serum from mice infected with Schistosoma japonicum: Serum from mice infected with S.japonicum and S.sinensis were collected to evaluate the feasibility of detecting S.japonicum antibodies using the indirect hemagglutination assay.Results:1.Blind test of the crushing microscopy method: 30 schistosomiasis detection personnel correctly identified the negative results of all nine samples,with three infected Oncomelania hupensis snails and three infected Tricula Humida snails being identified as S.japonicum positive snails.2.Measurement of external morphological indicators of cercariae:independent sample T-tests were performed on the four parameters,with T-values for body length = 0.000,body width = 0.372,tail length = 0.000,and tail spine length = 0.651.The differences in body length and tail length between the two types of cercariae were statistically significant,while there was no statistical significance in the differences in body width and tail spine length.3.Cercarial hatching results: A total of 111 rodents were captured in Yongsheng County,among which 15 had visible liver nodules and Schistosoma sinensis eggs were observed in liver tissue sections under a microscope.2 mice hatched cercariae using nylon stocking egg collection and hatching methods.Schistosoma sinensis eggs were found in the feces of 5 mice using the Kato-Katz method.The liver homogenate from mice infected with S.japonicum in the parallel control group hatched cercariae.The movement patterns of both cercariae were similar.7 mice infected with Schistosoma sinensis had liver nodules,and Schistosoma sinensis eggs were detected in liver sections.However,no cercariae hatched using nylon stocking egg collection and hatching methods,and only two mice showed Schistosoma sinensis eggs in their fecal sediment by smear microscopy.In the parallel control group,cercariae hatched from the feces of mice infected with S.japonicum.The comparison experiment of cercarial hatching showed that cercariae hatched from S.japonicum eggs but not from S.sinensis eggs.4.Cross-experiment of serum antibodies: The antibody titer of S.japonicum mouse serum was 1:10～1:10240,and the antibody titer of S.sinensis mouse serum was negative to 1:10.Conclusion: This study successfully established the animal infection experiments of Schistosoma sinensis and Schistosoma japonicum in a local laboratory,providing a good way to obtain convenient and low biosafety-risk Schistosoma japonicum positive reference materials,it lays a foundation for the establishment and verification of alternative experimental methods.In particular,the possibility of replacing infected snails with infected Schistosoma japonicum snails as positive reference and quality control products for crush microscopy is high,the feasibility of using the eggs of Schistosoma sinensis and the positive sera of infected animals as reference and quality control products for hatching of Schistosoma japonicum eggs and detection of serum antibodies still needs to be further studied.