Establishment Of The ELISA Method To Identify Routes Of Infection In Toxoplasma Gondii

Toxoplasma gondii(T.gondii)is a specialized intracellular parasite that has a wide range of hosts and can infect almost all warm-blooded animals,posing a serious threat.Sheep is an important source of meat and protein for the Chinese people.Adult sheep infected with T.gondii generally do not show clinical symptoms and show a hidden infection.Pregnant ewes infected during pregnancy may experience symptoms such as miscarriage,stillbirth,and mummified fetuses.Lambs with weaker immune systems may experience significant clinical symptoms after infection,such as fever,depression,and decreased appetite.Therefore,sheep infected with T.gondii not only poses a serious threat to public food safety and hygiene,but also seriously affects the economic development of animal husbandry.T.gondii has a complex life history,and multiple stages of its body are infectious.There are two main ways for sheep to be infected with T.gondii: cyst infection or oocyst infection.However,currently,most of the sheep T.gondii ELISA kits on the market can only detect whether sheep are infected,and cannot distinguish their infection methods.Establishing diagnostic methods to distinguish the way sheep are infected with T.gondii can provide technical tools for epidemiological investigations.In addition,based on the diagnostic results,the main sources of T.gondii infection in different regions can be analyzed to effectively trace the source.Furthermore,corresponding prevention and control measures can be taken to determine the main sources of T.gondii infection in different regions,and targeted measures can be taken to reduce the harm of T.gondii and provide support for the prevention of Toxoplasmosis.Therefore,it is very important to establish a detection method that can distinguish infection pathways.Some proteins expressed during the oocyst stage of T.gondii have low expression levels in other stages,making them the most suitable antigen for differential diagnosis.Therefore,this study screened and expressed three specific expression proteins SRS42,Tg LEA850,and Tg LEA870 during the oocyst stage of T.gondii.The three proteins were used as antigens,and the T.gondii oocyst and cyst standard positive serum(prepared in this study)were used as primary antibodies.Western blot experiments were conducted,and it was found that Tg LEA870 protein can react with oocyst positive serum to produce bands and cannot react with cyst positive serum to produce bands,Prove that Tg LEA870 protein is indeed a highly expressed specific protein during the oocyst stage,and has the potential to serve as a differential diagnostic antigen.At the same time,three proteins were subjected to matrix titration experiments to determine the dominant antigen Tg LEA870.The Tg LEA870 protein was used as an antigen coating to establish an indirect ELISA diagnostic method and detect the concentration of Ig G antibodies.Optimize various conditions to determine the optimal coating concentration of Tg LEA870 protein as 8 μg/m L,the optimal blocking time is45 minutes,the optimal blocking concentration is 1% BSA,the optimal dilution ratio of the serum to be tested is 1:50,the optimal incubation time for the second antibody is45 minutes,the optimal incubation concentration for the second antibody is 1:3000,and the optimal color development time is 15 minutes.The sensitivity of this protein was determined to be greater than 1:200 through sensitivity experiments.Using this method to detect the positive serum of sheep Haemorhabditis spiralis,no cross reaction was found.By detecting 20 sheep negative serum samples validated by MAT,the critical value was determined to be 3.62(S/N),and a S/N value greater than 3.62 can be determined as positive.The Tg LEA870-i ELISA method established in this study was combined with GRA1-i ELISA method to detect 40 goat serum samples stored in our laboratory,including 12 serum samples from follicular infection and 3 serum samples from cyst infection.In summary,the Tg LEA870-i ELISA method has good sensitivity and can be used for the diagnosis of sheep toxoplasmosis.This method can specifically identify sheep serum infected with T.gondii oocysts,and when combined with the diagnostic method established in our laboratory that can detect antibodies against T.gondii throughout the entire period,it can identify the pathways of sheep infection with T.gondii.