Development Of The PCR Method For Detection The Pathogen Of Psoroptic Mange In Rabbits And Its Comparison To Microscopic Examination And IELISA

Psoroptic mange is the most common parasitic disease that harms rabbit husbandry in China,and this disease is caused by non-burrowing Psoroptes ovis.The clinic signs of this disease presents wasting,pruritus and crusted skin lesions.It is easy to cause large-scale spread of the population without timely prevention and control,resulting in severe economic losses in commercial rabbit husbandry.Thus,timely diagnosis of P.ovis infestation in rabbits are of paramount importance for effective disease prevention and control of psoriotic mange.However,the current diagnosis of psoroptic mange mainly relies on microscopic examination of skin scrapings,and although the cost of this method is low,it requires an expertise having professional knowledge to distinguish P.ovis,and this method is extremely time-consuming and inefficient,especially for low densities of mites and sub-clinical infestations in rabbits.Polymerase chain reaction(PCR)has been used in the diagnosis of a variety of parasitic diseases for its advantages of the high sensitivity and simplicity.Unfortunately,PCR diagnostic method for psoroptic mange has not been reported so far.Thus,this study intends to establish a PCR method for diagnosis of psoroptic mange,and compares the diagnostic effects of this developed PCR method with microscopy and serological diagnostic methods.Our study is expected to provide the best method for the clinical diagnosis of psoroptic mange in rabbits,and finally provide technical support for its prevention and control.1 Establishment of PCR method for diagnosis of psoroptic mangeIn this study,the P.ovis DNA was used as template to conduct PCR amplification with designed 12S rDNA,16S rDNA,COX1 and ITS2 gene primers,it found that COX1 primers appeared non-specific bands and were excluded in the following study.Specificity of 12S rDNA,16S rDNA,and ITS2 primers were furtherly confirmed by amplification from P.ovis DNA,Sarcoptes scabiei DNA,and rabbit skin tissue DNA,the results showed that 16S rDNA primers were eliminated for their non-specific bands from rabbit skin tissue DNA.Then,the PCR program was optimized,and results showed that the optimal cycle times,annealing temperature and primer volume were 35,55℃,1 μL for 12S rDNA and25,55℃,1 μL for ITS2,respectively.Finally,the sensitivity of 12S rDNA and ITS2 primers were compared,and it found that the sensitivity of ITS2 primers(40.3 pg/μL)were significantly higher than that of 12S rDNA(403 pg/μL),thus ITS2 gene was determined to be the best molecular target for PCR assay,and this PCR assay had good repeatability.In conclusion,ITS2 was the best molecular target among the four genes,and ITS2-PCR has strong specificity and high sensitivity.2 Comparison of PCR,microscopy and iELISA in diagnosis of psoroptic mange in rabbitsIn this study,New Zealand rabbits were infested with P.ovis,and dry swabs,skin scrapings as well as serum samples were collected during the infection and treatment periods,then these samples were examined by ITS2-PCR established in the preceding chapter,microscopic examination and rPsoSP3-iELISA,previously.The results showed that the positive rates of dry swab ITS2-PCR and serum rPsoSP3-iELISA were comparable at 7 days post-infection(dpi)(ITS2-PCR: 88.9%;rPsoSP3-iELISA: 77.7%),and their detection rate were at least two times higher than that of microscopic examination(33.3%).while at 14,28 and 42 dpi all three methods yielded the same positive rate at 100%.After treatment,microscopy and ITS2-PCR positivity rates rapidly decreased at 7 dpi(ITS2-PCR:0%,microscopy: 0%),but the positive rate of ITS2-PCR returned to 11.1% at 11 dpt,whereas rPsoSP3-iELISA remained 100% positive from 3 to 11 dpt.In summary,this PCR test had advantages over microscopic examination in detection low-level mite infection and serological assay in monitoring treatment outcome.3 Field investiagation using PCR and microscopy in a rabbit farmIn view of the above comparison,iELISA method could not distinguish between past and current infection.Therefore,we compared the effect of the PCR and the traditional microscopy for the detection of clinical samples in a rabbit farm in Chengdu city with epidemic history of psoroptic mange,results showed that the detection rate of ITS2-PCR(7/36;19.4%)was higher than that of microscopic examination(4/36;11.1%),and the samples that were positive by microscopic examination could be detected by ITS2-PCR.In summary,the detection effect of ITS2-PCR was better than microscopy.