The Study Of Molecular Taxomomic Status About Scabies Mites Isolated From Rabbits And Vaccine In Sarcoptes Scabiei

Sarcoptic mange is a common,widespread,highly contagious,Sarcotes scabiei mite-caused,skin disease in rabbits.Clinical signs of acute sarcoptic mange include intense pruitus,erythematous eruptions,papule formation,seborrhea and alopecia.The prevalence of sarcoptic mange is very high and the transmission of sarcoptic mange is very quick.It is a severe threat to rabbits which often causes the growth and development slowly,degrades the quality of fur,even causes death if it is untreated.The precisely diagnose of etiological agent is the key point of curing and controling the epidemic of the sarcoptic mange.Howerver,a generally accepted consensus has not yet been obtained about the taxonomic status of sarcoptic isolates from different animals,and the immune response mechanisms of scabies antigens in naive hosts and in hosts expressing protective immunity are not understood.So far,the treatment with drugs is generally effective for controling of sarcoptic mange,but the cost is relatively high and these drugs cause side effect,drug-resistence,drug-remaining,even carcinogenic to human.These problems in drugs provides impetus for alternative control strategies such as vaccination.Here we sequenced the cytochrome oxidase subunitⅠ(COI) gene of scabies mite isolated from rabbits and swine,cloned paramyosin gene by reverse transcription-polymerase chain reaction(RT-PCR),analysed the sequence of paramyosin,expressed the paramyosin in Escherichia coli-expressed system and COS-7 cell,observed the humoral and cellular immune responses in BALB/c mice induced by vaccines.The results of research are summarized as following:1.The cytochrome oxidase subunitⅠ(COI) gene was investigated for the first time on 4 isolates obtained from rabbits and swine from China by PCR.The COI gene from these specimens were sequenced and compared with the homologous genes of other fourteen Sarcoptes isolated from other countries deposited in GenBank.The results of sequence analysis indicated that the lengths of COI in the four Sarcoptes were all 1 427 bp.There was no insert or deletion.The base composition was obviously A+T biased,averaging 73% A+T.Homology analysis indicated that the identity levels of nucleotide of COI among the 4 isolates from China ranged from 99.1%to 100.0%,while the gene homology of COI between the Sarcoptes isolated from China and the Sarcoptes isolates from human in Australia and those from other animals abroad ranged from 98.4%to 99.6%.The phylogenetic analysis showed that the relationships among Sarcoptes isolates from China and the Sarcoptes isolates from human in Australia and other animals abroad were close. Homology and phylogenetic analysis indicated that the Sarcoptes isolates from China and the Sarcoptes isolates from human in Australia and other animals abroad are conspecific.2.The full-lenth open reading frame of the paramyosin cDNA was amplified by reverse transcription-polymerase chain reaction(RT-PCR) from total RNA of Sarcoptes mites isolated from rabbits for the first time.After sequencing,we did the bioinformatics analysis of the paramyosin gene.The results of sequence analysis indicated that the lengths of paramyosin was 2 628 bp.The base composition of A+T and G+C were 58.1%and 41.9%, respectively.The sequence of Sarcoptes isolated from rabbits had 99.6%,99.5%,83.0%, 82.6%,82.1%and 82.0%identity with the paramyosin genes of Sarcoptes isolates from swine,red fox,human population,chorioptes isolates from Giant panda and those from Holstein cow,Dermatophagoides pteronyssinum,D.farinae,Blomia tropicalis, respectively.The deduced amino acid had 100.0%,97.6%,97.6%,94.4%,97.1%and 95.2%identity with the expressed paramyosins of Sarcoptes isolates from swine,red fox, human population,chorioptes isolates from Giant panda and those from Holstein cow, Dermatophagoides pteronyssinum,D.farinae,Blomia tropicalis,respectively.The full length of paramyosin sequence coded a 875 amino acid multi-peptide.The Glu,Leu and Gln are the most amino acid composition in multi-peptide.Furthermore,the encoded protein molecular weight and pI are 102.36 kDa,5.59,respectively.There are no signal peptide cleavage site,no transmembrane region,major of hydrophilicity region,four N-glaycosylation sites,96.91%α-helix,0.11%β-turn,2.86%coil in secondary structure.3.The DNA fragments encoding paramyosin from scabies mite isolates from rabbits was subeloned to the pET-32a(+) expression vector and highly expressed in E.coli host BL21(DE3) under 0.2 mmol/L IPTG induction 6 hours at 37℃.The expressed proteins fused by Trx with the molecular mass of 124.36 kDa,was found to be sequestered into inclusion bodies.Recombination S.scabiei paramyosin expressed in Escherichia coli was recognized by sera of rabbits infected S.scabiei.COS-7 cell transfected with the pararnyosin cloned into pVAX1 expression vectors.RT-PCR and indirect immunofluorescence assay revealed that paramyosin was normally transcribed in COS-7 cells.4.The plasmid pVAX1-Pmy encoding antigen paramyosin from scabies mite isolated from rabbits,was used for DNA-mediated innunization of BALB/c mice,employing doses of 100μg,delivered by intramuscular route of three times biweekly.The blood,heart,liver, spleen,lung,kidney,brain,muscle of the injected site were sampled at 24h,7d,14d,21d, 28d,35d,49d,63d,77d and 105d after the first immunization,for distribution and safety study by PCR method.Genome DNA were extracted for PCR analysis.The pVAX1-Pmy DNA vaccine can distribute in different tissues and blood from 24 h to 49 d after the first immunization.On the sixty-three day,the PCR result of brain was negative, and the fragment was amplificated from blood and other tissues,On the seventy-seven day, the fragment was amplificated only from blood,heart and muscle of the injected site,but the plasmid could exist at least 105 d in muscle of the injected site.The safety experiment indicated that each gel-purified genomic DNA was negative for PCR assay.No genomic integration of DNA vaccine was detected in the immunized mice.5.The gene IL-2,IL-4,WN-γwere respectively subcloned into BamHⅠand XhoⅠ,EcoRⅠand XhoⅠ,EcoRⅠand HindⅢsite of eukaryotic expression vector pVAX1, construction of the eukaryotic expression plasmid pVAX1-IL-2,pVAX1-IL-4 and pVAX1-WN-γ.They were identified by restriction endouclease digestion analysis.6.To evaluate the immunogenicity of the plasmid DNA vaccine and protein vaccine from Sarcoptes isolates from rabbits,120 BALB/c mices were allocated into eight groups. Groups of BALB/c mice were inoculated intramuscularly in the tibialis anterior muscle with 100μg plasmid pVAX1-Pmy(group Pmy),pVAX1-Pmy+pVAX1-IL-2(group Pmy+IL-2),pVAX1-Pmy+pVAX1-IL-4(group Pmy+IL-4),pVAX1-Pmy+pVAX1-WN -γ(group Pmy+IFN),pVAX1(group pVAX1) and 100μL PBS(group PBS) as control. Goups of BALB/c mice were inoculated intradermicly in back with 50μg protein purified paramyosin protein(group PmyP),intact soluble mite protein(group ScTP).Three identical doses were given,with a 2-week interval.The mice immunized with paramyosin protein and intact soluble mite protein showed higher IgG and IgG1 antibody responses, compared with the groups injected with DNA vaccination.Co-injection of pVAX1-IL-2, pVAX1-IL-4 and pVAX1-IFN-γshowed higher levels,of IgG responses,compared with vaccination with pVAX1-Pmy alone(from 14d to 24d).However,Co-injection of pVAX1-IL-4 showed significant IgG1 antibody responses(P<0.05),compared with the group inj ected with pVAX1-Pmy alone(exception 14d).Co-injection of pVAX1-IL-2,and pVAX1-IFN-γshowed higher levels of IgG2a responses,compared with vaccination with pVAX1-Pmy alone.However,Co-injection of pVAX1-IL-2 showed significant IgG2a antibody responses(P<0.05),compared with the group injected with pVAX1-Pmy alone (from 14d to 35d).Co-injection of pVAX1-IL-4 showed significant IgE antibody responses(P<0.05),compared with the groups injected with pVAX1-Pmy, pVAX1-Pmy+pVAX1-IL-2,pVAX1-Pmy+pVAX1-IFN(on 14d).The mice immunized with paramyosin protein and intact soluble mite protein showed higher IgM antibody responses,compared with the groups injected with DNA vaccination,these groups showed significant IgM antibody responsess(P<0.05),compared with the group injected with pVAX1-Pmy alone(from 7d to 35d).Co-injection of pVAX1-IL-2 showed significant IgM antibody responses(P<0.05),compared with the groups injected with pVAX1-Pmy,pVAX1-Pmy+pVAX1-IL-4,pVAX1-Pmy+pVAX1-IFN-γ(from 21d to 42d).Two weeks after third immunization,the immunized mice showed significant SI values(P<0.05),compared with the control proup injected with pVAX1.The mice immunized with pVAX1-Pmy,pVAX1-Pmy+pVAX1-IL-2,pVAX1-Pmy+pVAX1-IL-4, pVAX1-Pmy+pVAX1-IFN showed higher SI values,compared with the groups injected with paramyosin protein and intact soluble mite protein.Co-injected of pVAX1-IL-2 and pVAX1-IFN significantly increased the production of IL-2 compared with the other groups(P<0.05),and significantly increased the production of IFN-γ(P<0.05) compared with groups injected with pVAX1-Pmy+pVAX1-IL-4,paramyosin protein and intact soluble mite protein.In contrast,the IL-4 levels were significantly decreased(P<0.05), compared the other groups.Co-injected of pVAX1-IL-4 significantly increased the production of IL-4 and IL-5 compared with the other groups(P<0.05).Two weeks after third immunization,the immunized mice showed significant number of angigen-specific CD4~+,CD8~+ T-lymphocytes ccells(P<0.05),compared with the control proups injected with PBS and pVAX1.The groups injected with paramyosin protein,intact soluble mite protein and pVAX1-Pmy+pVAX1-IL-4 enhances the Th2-dominant immune response, however,the groups injected with pVAX1-Pmy+pVAX1-IL-2 and pVAX1-Pmy+pVAX1 -IFN-γ,enhances the Th1-dominant immune response.The immunized mice showed more number of antigen-specific CD4~+,CD8~+ T-lymphocytes cells,compared with the control groups injected with PBS and pVAX1.The present study provide a sound basis for a feasible vaccine of preventing and curing the Sarcoptic mange in domestic animanl populations.