| Psoroptes ovis is a major non-burrowing ectoparasite with infestation in various domestic and wild animals.P.ovis is the most epizootic ectoparasite specie in rabbits,sheep,buffalo,and causes severe psoroptic mange characterized by intense cutaneous inflammation,pruritus and scabs,etc.This parasite leads to feed efficiency reduction,wool loss,and in severe cases,even death,which induces severe economic losses in the domestic breeding industries.Diagnosing all infected animals as early as possible is the prerequisite for the prevention and control of psoroptic mange,but the existing etiological diagnostic method is very easy to misdiagnosis in the early and sub-clinical infections.Therefore,the more efficient diagnosis for the early and sub-clinical infections of P.ovis is of great significance.Serine protease inhibitor(Serpin)is involved in a variety of fundamental physiological functions including anticoagulation,inhibition of melanization,regulation the innate immune response and reproductive development,etc.Moreover,serpin has been proven as a promising diagnostic antigen for helminthiasis.Our previous omics study revealed that serpin has been reported to exist in P.ovis,but no further study were conducted.Thus,in this study,two serpin(PsoSP1 and PsoSP2)genes were amplified and expressed using a prokaryotic expression vector.Moreover,the relative m RNA expression levels and localization of these two proteins at different development stages of P.ovis.were analyzed.Finally,establishment the indirect ELISAs using these two recombinant proteins evaluated their early diagnostic potential to P.ovis var.cuniculi infestation in rabbits.1.Prokaryotic expression,transcription levels and localization analysis of PsoSP1 and PsoSP2In this study,PsoSP1 and PsoSP2 of P.ovis were amplified and sequenced with the fulllength of 1572 bp and 723 bp,respectively.The recombinant proteins rPsoSP1 and rPsoSP2 were obtained in prokaryotic expression system and the molecular weight were approximately 75 k Da and 46.29 k Da,respectively.The rPsoSP1 and rPsoSP2 were both recognized by P.ovis-positive sera,revealing their favourable reactivity.Quantitative realtime PCR results showed that PsoSP1 and PsoSP2 both appeared expression across all lifecycle stages but significantly higher of expression in adult female than in any other stage(P< 0.01),and the expression level of PsoSP1 in adults and larvae was significantly higher than that of PsoSP2.Immunofluorescence histochemistry results showed that PsoSP1 was located in the cloaca of female mites,and PsoSP2 was located in the mouthparts of female mites.The results of present study laid the foundation for clarifying the functions of PsoSP in P.ovis physiological activities and further establishment of indirect ELISAs using these two proteins.2.Evaluation of the serodiagnostic potential of rPsoSP1 and rPsoSP2 for psoroptic mange in rabbitsIn this study,the indirect ELISA(iELISA)methods were established using the rPsoSP1 and rPsoSP2 obtained in the preceding chapter,and furtherly were evaluated the potential diagnosis in early infestation of P.ovis in rabbits.The results showed that the optimal working conditions for iELISA were 46 μg/m L of rPsoSP1 and 64.5 μg/m L of rPsoSP2 for the coated antigen,and the cut-off values for rPsoSP1 and rPsoSP2 were 0.633 and 0.490,respectively.In addition,the sensitivity and specificity for the establishment of iELISA were96.0% and 90.91% for rPsoSP1 and 90.00% and 78.18% for rPsoSP2,respectively.Consequently,the AUC of rPsoSP1-iELISA(0.983)was closer to 1 than that of rPsoSP2-iELISA(0.956),indicating that the rPsoSP1-iELISA was a better diagnostic test to detect specific antibodies against P.ovis var.cuniculi than rPsoSP2-iELISA.Thus,rPsoSP1-iELISA was used to evaluate the potential diagnosis in early infestation of P.ovis in rabbits,and revealed that rPsoSP1-iELISA can detected the P.ovis-postive antibodies in 80 % rabbits after 2 weeks p.i.which appared no obvious clinical symptoms,and the serum positive rate increased 100% 4 weeks p.i..Based on this results,the rPsoSP1-iELISA is a potential early diagnosis method forP.ovis infestation in rabbits.Finally,rPsoSP1-iELISA was used to detect 158 rabbit serum samples from a farm with a history of psoroptic mange,and the results showed a 52.53%(83/158)sero-positive rate,which was higher than positive rate of the traditional etiological detection method(11.39%,18/158).In summary,rPsoSP1-iELISA appear a high value in early P.ovis infestation in rabbits,and this method is more efficient than the the etiological detection method in diagnosis the P.ovis subclinical infestation of rabbits.Our study would provide the technical supports for the further diagnosis and control of psoroptic mange in rabbits. |
