Epidemiological Investigation Of Toxoplasmosis In Dogs And Cats In Some Areas Of Wuhan And Establishment Of ELISA Method For Cat Oocyst Infection

Toxoplasma gondii(T.gondii)is a zoonotic parasite that is exclusively intracellular and widespread worldwide,and animal-to-human transmission is an important route of infection for human toxoplasmosis.Understanding the prevalence of toxoplasmosis in the most common companion animals-dogs and cats-not only provides insight into their health status,but also helps people to manage the risk of toxoplasmosis infection.Felines,both wild and domestic,are the only end-hosts for T.gondii,and the formation of oocysts in their intestines,which are excreted in faeces after sexual reproduction,is an important link in the transmission of toxoplasmosis.At the same time,cats can be infected by ingesting prey containing tissue envelopes or water or food contaminated with sporulated oocysts.The differential diagnosis of the route of infection of feline toxoplasmosis is important for the tracing of feline toxoplasmosis,the interruption of toxoplasmosis transmission and the prevention and control of toxoplasmosis in humans and other intermediate hosts.To investigate the epidemiology of T.gondii in dogs and cats in some areas of Wuhan,a total of 688 blood samples were collected from dogs and cats,315 from cats and 373 from dogs,at the Animal Hospital of Huazhong Agricultural University.The MIC17A-i ELISA-Ig G method was used to test the cat blood samples and the modified agglutination test(MAT)was used to test the dog samples,and the SPSS software was used to analyse the correlation between the breed,The correlation between sex and age and T.gondii infection.The results showed that 15.01%(56/373)of dogs were seropositive for T.gondii and 46.03%(145/315)for T.gondii in cats.SPSS chi-square test was used to analyze the test results of blood samples.The analysis results showed that the breed of dog had a very significant effect on Toxoplasma infection,while the breed of cat had no significant effect on Toxoplasma infection.There was a significant difference in the serum positive rate of Toxoplasma between castrated/neutered cats and female cats(P < 0.01),but no significant difference in the infection rate between female and male cats.There was no significant difference in the serum positive rate of Toxoplasma among female,male and castrated/neutered dogs.Dogs and cats were divided into five age groups,including 0-2 years old,2-4 years old,4-7 years old,7-10 years old and 10 years older.There were significant differences in the serum positive rates of Toxoplasma in five groups,dog and cat.For dogs,there were significant differences in Toxoplasma infection rate between dogs over 10 years old and dogs 4-7 years old and dogs 0-2years old(P < 0.01),there were significant differences between dogs 7-10 years old and dogs 0-2 years old(P < 0.05),and there were significant differences between dogs 2-4 years old and dogs 0-2 years old and dogs 4-7 years old(P < 0.05).For cats,the positive rate of Toxoplasma was significantly different between 0-2 years old and4-7 years old(P < 0.01),and was significantly different from that of cats over 10 years old(P < 0.05).To establish an ELISA assay for the T.gondii infection pathway in cats,the proteins TGME49＿226230 and Tg LEA880,which were specifically expressed during the oocyst period of T.gondii,were screened as candidate diagnostic targets in this study,and TGME49＿226230 and Tg LEA880 proteins were successfully expressed and purified.WB confirmed that Tg LEA880 could react with positive serum of cats infected with oocysts,but not with positive serum of cats infected with cysts and negative serum.Therefore,Tg LEA880 can be used as an ELISA diagnostic antigen to identify the route of Toxoplasma infection in cats.The reaction conditions of the established ELISA method were optimized,and the optimal encapsulation concentration of Tg LEA880 was 8 μg/m L,the optimal blocking concentration was0.5% BSA,and the optimal blocking time was 45 min.The optimal primary antiserum dilution concentration was 1:100,and the incubation time of the optimal primary antiserum was 45 min.The optimal dilution concentration of secondary antibody was1:5000 and the optimal action time of secondary antibody was 45 min.The best color development time is 20 min.Feline panleukopenia positive sera,feline herpesvirus positive sera,feline infectious peritonitis positive sera and feline coccidia positive sera were not cross-reacted with the ELISA method using optimised reaction conditions.Feline positive sera for feline oocyst infection can still be tested positive at a serum dilution of 1:400 using this method.These results indicate that the ELISA method established in this study has good sensitivity and specificity.The ELISA method established in this study was used to detect the above-mentioned collected cat clinical serum samples,combined with the MIC17A-i ELISA-Ig G method established in our laboratory,and a total of 117 cat blood samples were detected,including 15 cat blood samples with oocyst infection and 39 cat blood samples with cyst infection.These results indicate that the main infection mode of Toxoplasma in companion animals is cystosis infection,and owners can prevent and control it by limiting its hunting or not giving uncooked raw meat.