Study On Detoxification Pathway Of Orf And Mother To Fetal Transmission Pathway Of ORFV

Orf is an infectious disease caused by orf virus（ORFV）in sheep and goat.The disease is particularly harmful to lambs and kids less than 30 days old,and once the disease breaks out,the morbidity and culling rate of kids is above 90%.The long-held view of ORFV infection was that goat infected the disease through direct contact with infected animals or ORFV-contaminated utensils,through puncture of the mucous membranes of the mouth or scratches on the skin caused by foraging on thorny plants or harder hay.However,there is a lack of scientific research evidence for this view so far,and most of the infected kids are less than 10days old,and they have little access to thorny hay during this period of lactation.More importantly,this view is difficult to give a reasonable explanation for the outbreak of Orf in kids within 5 to 15 days age in multiple sheep farms.In view of the fact was proved previously by our laboratory that 30%-98%of the blood samples of parturient goat were ORFV positive,the hypothesis of possible mother-to-fetus transmission of ORFV was proposed.In addition,in many goat farms,there is no record of the incidence of Orf,but there are still many cases of Orf in newborn kids.Through clinical investigation,our laboratory detected ORFV in the milk and saliva of lactating dairy goat,so proposed that ORFV-carrying goat may excrete ORFV through milk and saliva,leading to infection of lactating lambs and other goat.Based on the above hypothesis,this study used the placental cotyledon,umbilical cord,amniotic fluid,saliva and blood of newborn lambs as materials,and the blood,saliva and milk of dairy goats as materials respectively.PCR,Real-time PCR,phylogenetic analysis and amino acid homology analysis were used to detect ORFV in samples.ORFV pathogenicity in ORFV positive samples was detected by in vitro cell infection test and animal infection test,providing experimental evidence to support the two hypotheses proposed above.The results are as follows:1.The total detection rates of ORFV in peripheral blood,saliva and milk samples of dairy goats were 32.4%,53%and 44.7%respectively.In the three farms,the detection rates of ORFV in peripheral blood samples were 52.8%,95%and 7.5%,the ORFV detection rates in saliva samples were 46.7%,100%and 23.8%,the ORFV detection rates of milk samples were82.6%,94.4%and 16.1%respectively.The Pearson correlation coefficients between the detection rates of the three samples were 0.97,0.93 and 0.82,showing strong positive correlation;2.The conserved region B2L gene and variable region ORF128 gene of ORFV in the saliva or milk of dairy goats detected in this study were most closely related to the published ORFV Hub13 strain from Fujian Province of China and ORFV D1701 strain isolated from Germany respectively.The similarity of amino acid levels with other published ORFV strains was 94.5-100.0%and 94.9-99.9%respectively;3.The results of in vitro infection experiment showed that the primary goat cells inoculated with ORFV in saliva and milk showed some typical cytopathic features,such as condensation and round,loss of adhesion,and a small number of cell lysis and shedding.The results of animal infection showed that the wounds infected with saliva ORFV and milk ORFV showed typical Orf symptoms,such as papules,pustules and scabs.;4.The ORFV detection rates in blood of kids were 70%,90.9%and 87.5%,and the ORFV detection rates in blood of adults were 23.1%,48.5%and 52.8%respectively.The ORFV detection rates of adults and kids were 0.96 by Spearman correlation analysis,showing a strong positive correlation;5.The positive rates of maternal goats,amniotic fluid,umbilical cord,placental cotyledon,saliva and blood samples of newborn lamb not fed milk were 57.1%,68.0%,54.5%,58.3%,70.4%and 50.5%respectively.The ORFV-positive placenta and umbilical cord tissues showed a lot of specific green fluorescence staining;6.The copy numbers of ORFV in 27 blood samples of maternal goats ranged from 10^5.34to 10^7.07 copies/m L,with an average of 10^6.14 copies/m L;the copy numbers of ORFV in 11blood samples of lambs ranged from 10^5.77 to 10^6.35 copies/m L,with an average of 10^6.11copies/m L;the copy numbers of ORFV in 14 placental tissues ranged from 10^3.38 to 10^5.76copies/mg,with an average of 10^4.25 copies/mg;the copy numbers of ORFV in 12 umbilical tissue samples ranged from 10^2.28 to 10^4.68 copies/mg,with an average of 10^3.50 copies/mg;7.After ORFV isolated from placental tissue was infected with goat primary cells,the cells became partially round 24 h later,almost all the cells became round 48 h later,and cell clusters appeared in some places.A large number of cells were broken 72 h later,and a small number of clustered cells stuck to the bottom,and the specific green fluorescence of ORFV gradually increased with the extension of infection time.Conclusion:1.The ORFV-carrying dairy goats were able to excrete infectious ORFV through saliva and milk,thus forming an ORFV-contaminated biological environment;2.ORFV can infect the kid fetus through the maternal-fetal route,resulting in newborn kids already carrying ORFV,which provides an experimental basis for the outbreak of Orf in newborn kids and lambs under stress.