Establishment And Application Of Detection Method Of O.ostertagi And Function Analysis Of O. Ostertagi Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor （MIF） is a proinflammatory molecule inmammals that, unusually for a cytokine,exhibits tautomerase and oxidoreductaseenzymatic activities.Homologues of this well conserved protein are found within diverse phylaincluding a number of parasitic organisms.The present study is the firstcharacterization of Ostertagia ostertagi macrophage migration inhibitory factor（OoMIF）. BLAST-N analysis of OoMIF revealed, though Teladorsagia circumcinctaMIF （TciMIF） and OoMIF do not have the same nucleotide sequence, which thereare14nucleotide differences between these two MIF, they do have an identicalanimo acid sequence. Recombinant OoMIFs （rOoMIF） lacks oxidoreductase activitybut exhibits tautomerase activity with a specific activity of10⁵μmol/min/mg thatcannot be inhibited completely by the human MIF inhibitor ISO-1while OoMIFmhad neither tautomerase nor oxidoreductase. rOoMIF wasable to compete withrecombinant human MIF for a MIF receptor （CD74）, suggesting that OoMIF doesbind to this MIF receptor. Immunofluorescence staining demonstrated that OoMIFwas localized to all over the nematode. OoMIF was abundant in the L3and L4stageand present in excretory and secretory antigen （ESAg） preparations.We also demonstrate that OoMIF can elicit IL-8and TNFа production frombovine peripheral blood mononuclear cells and U937cells while incubated withPMA, LPS and glucocorticoid. Therefore OoMIF maybe play an immunomodulatoryrole during Ostertagi ostertagia infection in bovine. In this study,to develop a simple, convenient and fast PCR for detecting O.ostertagi, according to the GenBank reported O. ostertagi ITS-2sequence （Accessnumber: AB245023.2）, a pair of specific PCR primers were designed by usingmolecular biology software Primer Primier5.0, and respectively optimizes conditionssuch as the concentration of Mg²⁺,dNTP, primers, Taq and annealing temperature,theresults showed that the optimum reaction conditions:3Mm Mg²⁺,0.25mM dNTP,0.5μM Taq（5U/μL）and the optimum annealing temperature was57℃. Thesequencing result showed the amplified ITS-2is identical with the one reported inGenBank,so we confirmed that the amplified gene definitely was the target gene.A specific fluorescence quantitative PCR was also developed for detecting O.ostertagi by using the same gene ITS-2. according to the GenBank reported O.ostertagi ITS-2sequence （Access number: AB245023.2）, a pair of specific PCRprimers were designed by using molecular biology software Primer Primier5.0,Ostertagia ostertagi ITS-2gene for target genes, and designs a specific fluorescencequantitative PCR primers by molecular biology software PrimerExpress3.0, and thereaction conditions were optimized and the specifity and sensitivity were established.The curvilinear equation is Y=-3.425X+39.79and R²is0.998. the sensitivity of therealtime PCR is1x10³copies/μL, which is100fold more sensitive than theconventional PCR.This research also uses Ostertagia ostertagi ITS-2gene as target genes todesigns6primers of LAMP using online primer design softwareprimerexplore（https://prime rexplorer.jp/lamp3.0.0/index.html）. After respectivelyoptimizing the amplification conditions, we developed the LAMP method fordiagnosing the Ostertagia ostertagi.The above three methods were used to test Ostertagia ostertagi50samples, andthe results showed: PCR method used in this study showed a sensitivity of90%and aspecificity of100%, the detecton rate was94%; the realtime PCR assay has asensitivity100%, specificity of100%, the rate was100%, LAMP method sensitivityof96.6%and a specificity of100%, the rate was98%; therefore the realtime PCR was the superior method compared toPCR and LAMP method of detectingOstertagia ostertagi.