Characterization And Immunomodulatory Effects Of Five Recombinant Proteins Of Hamonchus Contortus On Goat PBMCs And Their Potential Role In Diagnosis

Haemonchus contortus is one of the most important gastrointestinal nematodes of small ruminants.This parasitic nematode feeds on the blood from capillaries in the abomasum of sheep,goat,cattle,and other ruminants.Infection with this parasite often results in anemia,diarrhea,weight loss,or even death in young animals,which causes large economic losses.Mortality ranging from 30%to 50%has been reported in lambs and kids in acute cases.Ruminants are infected by ingestion of the third-stage larva(L3).The worms develop into adults at about 21 days post-infection(DPI)and begin to shed eggs through feces.Excretory and secretory products(ESPs)are produced and released by parasites during infection.H.contortus ESPs(HcESPs)contain many proteins that can perform various functions including modulating the host immune response.ESPs were reported as diagnostic antigens for prepatent detection of H.contortus infection in sheep.Therefore,ESPs antigens to identify their potential value in host immune regulation and serological diagnosis,so as to provide scientific basis for the prevention and control of nematode infection.Meanwhile,the identified ESPs proteins acting as candidate vaccine and diagnostic antigens may have applications for clinical research against H.contortus infections.1 Immunological characteristic of Haemonchus contortus BLAODA and its potential for diagnosis of goat infectionIn this study,HcBLAODA gene of Haemonchus contortus were cloned,expressed and the recombinant proteins rHcBLAODA was obtained.Localization of rHcBLAODA with in H.contortus adult worm and binding with goat peripheral blood mononuclear cells(PBMCs)were performed using immunofluorescence assay(IFA).Moreover,effects of rHcBLAODA on nitric oxide production and signature cytokine IL-9 expression in goat PBMCs were evaluated.Monocyte phagocytosis,Th9 cells production and Cell apoptosis were detected by flow cytometry.The results showed that rHcBLAODA were successfully cloned,expressed in expression vector and localized in the gut surface of adult H.contortus sections.The rHcBLAODA protein specifically cross-reacted with anti-H.contortus antibodies as shown by IFA analysis.Further functional analysis showed that interaction of rHcBLAODA with host cells significantly enhanced the Th9 cells production,IL-9 expression,apoptosis effciency and nitric oxide production.rHcBLAODA significantly decreased the proliferation and migration of PBMCs cells.Goat monocytes were incubated with rHcBLAODA was signifcantly decreased the Monocyte phagocytosis.In addition,a sandwich enzyme-linked immunosorbent assay(ELISA)was produced based on anti-rHcBLAODA monoclonal antibodies.Sandwich ELISA showed 90.6%sensitivity and 90.6%specificity.Our findings indicated that rHcBLAODA protein engaged in regulatory roles on host immune cells and could enhance protective immunity by inducing Th9 cells secreted IL-9 in vitro.Moreover,anti-rHcBLAODA monoclonal antibody was a potential antibody for the diagnosis of H.contortus infection in goats.2 Immunological characteristics of Haemonchus contortus EPH and activates Th9 cells in vitroIn this study,HcEPH gene of Haemonchus contortus was cloned,expressed and the recombinant proteins rHcEPH was obtained.Localization of rHcEPH with in H.contortus adult worm and binding with goat PBMCs were performed using immunofluorescence assay(IFA).Moreover,effects of rHcEPH on nitric oxide production and signature cytokine IL-9 expression in goat PBMCs were evaluated.Monocyte phagocytosis,Th9 cells production and Cell apoptosis were detected by flow cytometry.The results showed that rHcEPH was successfully cloned,expressed in expression vector and localized in the gut surface of adult H.contortus sections.The rHcEPH protein specifically cross-reacted with anti-H.contortus antibodies as shown by IFA analysis.Further functional analysis showed that interaction of rHcEPH with host cells significantly enhanced the Th9 cells production,IL-9 expression,nitric oxide production,cell migration,apoptosis effciency and monocyte phagocytosis.rHcEPH significantly decreased the proliferation of PBMCs cells.Our findings indicated that rHcEPH protein engaged in regulatory roles on host immune cells and could enhance protective immunity by inducing Th9 cells secreted IL-9 in vitro.3 Characteristics of Haemonchus contortus LIC on immune cell functionsIn this study,HcLIC gene of Haemonchus contortus were cloned,expressed and the recombinant proteins rHcLIC was obtained.Localization of rHcLIC with in H.contortus adult worm and binding with goat PBMCs were performed using immunofluorescence assay(IFA).Monocyte phagocytosis and Cell apoptosis were detected by flow cytometry.The results showed that rHcLIC was successfully cloned,expressed in expression vector and localized in the gut surface of adult H.contortus sections.The rHcLIC protein specifically cross-reacted with anti-H.contortus antibodies as shown by IFA analysis.rHcLIC had no significant effect on cells proliferation and monocytes phagocytosis.In addition,rHcLIC significantly promoted the apoptosis effciency of PBMCs cells.The percentage of migration was signifcantly decreased in cells incubated with rHcLIC.Our findings indicated that rHcLIC protein engaged in regulatory roles on host immune cells.4 Characteristics of Haemonchus contortus ADRM on Th9 cell differentitation and its potential for early diagnosis of goat infectionIn this study,the recombinant Haemonchus contortus adhesion-regulating molecule protein(rHCADRM)was expressed and purified,and its diagnostic potential was evaluated.Serum samples from goats experimentally infected with H.contortus(n=5)were collected at 0(before infection,negative control),7,14,21,35,49,63,85,and 103 days post-infection(DPI).The reactions between rHcADRM and goat serum were tested using Western blot(WB).The results show that rHcADRM can be recognized in the serum as early as 14 DPI,and the antibody against rHcADRM in infected goat could be maintained for over 89 days.Further functional analysis showed that interaction of rHcADRM with host cells significantly enhanced the Th9 cells production and IL-9 expression.No reaction was found between rHcADRM and antibodies against Trichinella spiralis,Fasciola hepatica,or Toxoplasma gondii.An indirect enzyme-linked immune sorbent assay(ELISA)was developed based on rHcADRM.The optimal coating antigen(279 ng of rHcADRM/well)and serum dilutions(1:50)were determined by checkerboard titration.A total of 64 serum samples,including 32 from H.contortus infection goats and 32 from helminth-free goats,were used to determine the positive(0.362)and negative(0.306)cut-off values for the ELISA.The results show this serological diagnosis method is highly sensitive(90.6%)and specific(93.75%).To apply this indirect ELISA during field examination,51 serum samples were randomly collected from goat farms and tested using this method.The result showed that 19.6%(10/51)of goats were infected with H.contortus,which was 100%consistent with the necropsy result,higher than that of fecal examination(15.7%,8/51).These results indicate that the rHcADRM could be a potential antigen for diagnosis of H.contortus infection in goats.5 Development and Potential Application of Ras Domain-Containing Protein from Haemonchus contortus for Diagnosis of Goat InfectionHaemonchus contortus is an important gastrointestinal nematode of small ruminants that causes significant mortality in goats worldwide.Diagnosis of this infection mainly depends on the evaluation of clinical signs and fecal examination.However,limitations often occur in early or mild infections.For this purpose,serological diagnosis seems to be more accurate and reliable.In this study,the recombinant H.contortus Ras domain-containing protein(rHcRas)was expressed and purified and its diagnostic potential was evaluated.Reactions between rHcRas and goat sera were tested using Western blotting(WB).The results showed that rHcRas could be recognized by sera as early as 14 days post infection(DPI),and antibodies against rHcRas in infected goats could be maintained for over 89 days.No reaction was found between rHcRas and antibodies against Trichinella spiralis,Fasciola hepatica,or Toxoplasma gondii.An indirect enzyme-linked immunosorbent assay(ELISA)was produced based on rHcRas.The optimal coating antigen(157 ng of rHcRas/well)and serum dilutions(1:50)were determined via checkerboard titration.Indirect ELISA based on rHcRas showed 87.5%sensitivity and 90.6%specificity.The cut-off values for this experiment were determined to be 0.324(positive)and 0.273(negative),respectively,and the variation coefficient(CV)was less than 15%.The results of the indirect ELISA in-field examination showed that 17.6%(9/51)of the goats were infected with H.contortus,higher than the fecal examination results(15.7%,8/51).When compared the results of the indirect ELISA and necropsy testing,98.0%(50/51)consistency was found.These results indicated that rHcRas was a potential antigen for the diagnosis of H.contortus infection in goats.