Evaluation Of Diagnostic Values And Immune Efficacy Of Parasitic Stage-specific,highly Expressed Antigens TcHB/TcSP Of Toxocara Canis

Toxocara canis is a common intestinal parasitic nematode found in canids and felids and can also infect humans and other animals,causing serious public health problems because of its role as a neglected zoonotic parasite worldwide.Therefore,researches about its diagnosis and immune control are of great significance in veterinary and public health.Serodiagnosis and vaccination are regarded as ideal strategy candidates for prevention and control of toxocariasis because of their early detection,continuous protection,and effectiveness against migrating larvae in comparison to the traditional fecal egg detection and deworming control.Although currently,parasitologists have tested the potential of T.canis excretory/secretory(TES)proteins as antigens for serodiagnosis and immune protection of toxocariasis,it is still true that these TES proteins are very limited and not easy to be preserved;what’s more,their components are complex and therefore possibly have diagnostic defects and biosafety issues.Herein,based on the developmental transcriptome and secretome datasets of T.canis and experimental screenings of immune molecules finished in our laboratory,this study was designed to investigate the biological properties of two parasitic stage-specific and highly expressed antigens TcHB/TcSP of T.canis by using cloning,prokaryotic expression,immunofluorescence histochemistry and profiling of expression levels of Tchb and Tcsp.Then,the serodiagnostic and vaccine potentials of the recombinant TcHB(rTcHB)and TcSP(rTcSP)proteins were evaluated with the "T.canis-mouse/dog" infection and the "T.canis-mouse" challenging models,respectively.The main results are as follows:Both Tchb and Tcsp genes were amplified from cDNA of the third-stage(L3)larvae of T.canis.The lengths of the complete open reading frames(ORFs)of Tchb and Tcsp genes were 441 bp and 1,416 bp,respectively.Through prokaryotic expression and SDS-PAGE analysis,the molecular weights(MW)of rTcHB and rTcSP were 34 kDa and 50 kDa,respectively.Western-blotting analysis showed that both recombinant proteins could be recognized by T.canis-infected positive sera of mice,dogs and humans,suggesting their good reactivity with T.canis-specific antibodies.Analysis of protein characteristics showed that TcHB and TcSP were widely distributed in the cuticle,muscle,intestine,ovarian wall,uterine wall,and testis of T.canis,while TcSP was also present in the uterine eggs of females.Gene expression analysis showed that the Tchb gene was expressed across all stages of T.canis except for embryonated eggs(L0),while the Tcsp gene was only expressed in four stages of the T.canis,including L0,first-stage(L1),second-stage(L2)and third-stage(L3).Interestingly,a consistently high expression level of Tchb and Tcsp genes was found in the infected second-stage(L2)larva of T.canis.The rTcHB and rTcSP were respectively coated as antigens to establish indirect enzymelinked immunosorbent assays(iELISAs)using T.canis-infected positive sera collected from the "T.canis-mouse/dog" infection models.The results showed that the optimal concentration of the rTcHB-based iELISA(rTcHB-iELISA)was 80 μg/mL,the serum dilution was 1:40 and the cut-off value was 0.115 with the sensitivity and specificity of 96.7%and 100%,respectively.The optimal concentration of the rTcSP-based iELISA(rTcSP-iELISA)was 12 μg/mL,the serum dilution was 1:640 and the cut-off value was 0.131 with the sensitivity and specificity of 16.7%and 100%,respectively.These two iELISAs were further used for the detection of T.canis in clinical dogs and the results showed that the detection rates of rTcHB-iELISA and rTcSP-iELISA were 70.8%and 20.8%,respectively.Therefore,it is reasonable that compared with rTcSP,rTcHB was more suitable as a diagnostic antigen for T.canis detection in dogs.In addition,we also evaluated the immunoprotection effects of the rTcHB and rTcSP by using the "T.canis-mouse" infection model,and the results showed that both recombinant proteins had good immunogenicity which cannot only effectively stimulate vaccinated mice to produce high levels of serum IgG,IgGl and IgG2a and cytokines TNF-α,IL-6,IL-1β and IFNγ but also induced a mixed Th1/Th2 immune response,with respective 52.8%and 25.5%reductions of T.canis larvae in vaccinated mice in comparison with the controls.Therefore,rTcHB can be also used as a potential antigen for development of the vaccine against T.canis.