Differential Expression Analysis Of Transcriptome And MicroRNA In Sarcoptes Scabiei-host And Functional Research Of Four Sarcoptes Scabiei Genes

Scabies is a contagious skin disease that affects millions of humans,wild and domestic mammals worldwide,most frequently in socioeconomically disadvantaged populations in tropical regions.The mites burrow into the skin and obtain nutrients from serum and epidermal cells,causing severe itching,scabs and skin thickening,which potentially result in appetite loss,and secondary bacterial infections.Thus,searching for an antigenic component of scabies with high diagnostic sensitivity and specificity is a crucial task.Research exploring the immunopathogenesis and allergens of the scabies mite,and host immunological protective mechanisms is warranted as these genes are potential targets for vaccine development or are potential markers for diagnostic tests.1 Differential expression analysis of transcriptome and microRNA in Sarcoptes scabiei and hostThe goal of this study was to examine the immune interaction mechanism of both Sarcoptes scabiei and infected hosts.mRNA-seq and microRNA-seq were conducted on the S.scabiei mite and on infected and uninfected hosts.We focused on differential expression of unigenes and microRNAs,as well as the real targets of unigenes in enriched immune signaling pathways.S.scabiei enhanced host immune function and decreased metabolism after infection,while the immune response of the host inhibited S.scabiei proliferation and metabolism signaling pathways.Differentially expressed unigenes of S.scabiei were enriched in the JAK-STAT signaling pathway and the Toll-like receptor signaling pathway.The differential expression analysis indicated that microRNAs of S.scabiei and hosts have major roles in regulating immune interactions between parasites and hosts.2 Preliminary functional analysis and serological diagnostic potential of calmodulin from Sarcoptes scabieiCalmodulin(CaM)is an important calcium sensor that participates in various critical physiological processes.In this study,the CaM of Sarcoptes scabiei(SsCaM)was cloned and expressed,and sequence analyses were performed using bioinformatics tools.Recombinant SsCaM(rSsCaM)was used to detect antigenicity using immunoblottingassays,and the serodiagnostic potential of rSsCaM was assessed by indirect ELISA.The calcium binding properties and 8-anilinonaphthalene-1-sulfonic acid(ANS)fluorescence of rSsCaM were also measured.The results indicated that SsCaM contains a 450-bp open reading frame that encodes for a polypeptide with 149 amino acids,and SsCaM was expressed as a soluble protein.Multiple sequence alignment and phylogenetic analyses indicated similarity and genetic distance between SsCaM and other species.The calcium binding properties and ANS fluorescence of rSsCaM indicated typical calcium binding characteristics.Immunolocalizaton assay showed that SsCaM was widespread in S.scabiei.SsCaM-based ELISA exhibited a sensitivity of 87.5%(28/32)and a specificity of 22.5%(9/40)for detecting anti-CaM antibodies in the sera of naturally infected rabbits.The findings of this study provide a comprehensive molecular characterization of SsCaM and suggest that rSsCaM is inappropriate for detecing S.scabiei.3 Serological diagnostic potential of glyceraldehyde 3-phosphate dehydrogenase from Sarcoptes scabieiGlyceraldehyde 3-phosphate dehydrogenase(GAPDH)is a classical metabolic enzyme of the glycolytic pathway,and it has many other nuclear functions.In this study,we cloned and expressed the GAPDH of Sarcoptes scabiei(SsGAPDH);the sequence analysis was performed using bioinformatic tools.The antigenicity of recombinant SsGAPDH(rSsGAPDH)were evaluated using western blot.Indirect enzyme-linked immunosorbent assay(ELISA)was used to assess the serodiagnostic potential of rSsGAPDH.The results indicate that SsGAPDH contains a 918-bp open reading frame that encodes a polypeptide with 305 amino acids.Multiple sequence alignment and phylogenetic analyses indicated the similarity and genetic distance between SsGAPDH and other species.The western blot analysis suggested the strong reactivity and good antigenicity of rSsGAPDH.Indirect ELISA revealed a sensitivity of 81.3% and specificity of 22.2% for detecting anti-GAPDH antibodies in the serum of naturally infected rabbits.The antigenic cross-reactivity indicated that rSsGAPDH was unsuitable for detecting S.scabiei.4 Serodiagnostic potential and allergenicity potential of chitinase-like protein from Sarcoptes scabieiChitinase-like protein(CLP)belongs to glycoside hydrolase family 18,CLPs participated in a variety of biological functions,including inflammatory reactions and infections.Here,we expressed,identified and located the chitinase-like protein of S.scabiei(SsCLP),and evaluated its potential as an early-stage diagnostic antigen for rabbitscabies.Indirect ELISA using recombinant SsCLP(rSsCLP)exhibited diagnostic sensitivity of 94.4%(17/18)and specificity of 86.7%(26/30).Early diagnostic test after artificial infection of rabbits with S.scabiei for 1 week showed a positive detection rate of96.7%(29/30).Immunolocalization assays showed that fluorescence signals were localized on the surface of mites and,in infected rabbits,were observed in keratinized skin and embedded mites.Intradermal skin tests of rabbits by injecting rSsCLP showed a wheal,flare and erythema reaction.5 Serodiagnostic potential and allergenicity potential of triosephosphate isomerase from Sarcoptes scabieiTriosephosphate isomerase(TIM)is an enzyme that catalyzes the reversible interconversion of the triose phosphates glyceraldehyde 3-phosphate and dihydroxyacetone phosphate,which occurs at a non-linear step in the catabolic process to enhance the efficiency of glycolysis without producing pyruvate.Herein,we identified,cloned and recombinantly expressed triosephosphate isomerase from S.scabiei.Immunolocalization assays showed that fluorescence signals were localized to the chewing mouthparts,legs,and in keratinized skin and embedded mites in infected rabbit skin.Intradermal skin tests of rabbits injected with rSs-TIM revealed a wheal,flare and erythema reaction.