Haemonchus Contortus:Immunomodulatory Effects Of Two Recombinant Proteins On Goat PBMCs And Prepatent Diagnostic Potential Of Three Recombinant Proteins In Goat

Haemonchosis is one of the most important and highly pathogenic diseases of small ruminant caused by H.contortus.Heavily infected animal can lose over 250 mL of blood per day,leading to decreased wool production,reduced carcass quality,anemia and death.In the present studies immunomodulatory effects of two recombinant proteins of H.contortus on immune functions of goatPeripheral Blood Mononuclear cells(PBMCs)and T helper cells and early diagnostic potential of three recombinant proteins were evaluated as follows.1-Galectin Domain containing protein from Haemonchus contortus modulates the immune functions of goat PBMCs and regulates CD4+T-Helper cells in vitroGalectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses.CD4+T-helper cells differentiate into effector subsets in response to cytokines.T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied.In this study,galectin domain containing protein from Haemonchus contortus(Hc-GDC)was cloned,expressed in pET32a,and immunoblotting was performed.Localization of recombinant(r)HcGDC on outer and inner surface of H.contortusworm and binding with goat Peripheral BloodMononuclear cells(PBMCs)were performed using immunofluorescence assay.Moreover,effects of rHc-GDC on proliferation,apoptosis,cell migration,and the nitric oxide production in goat PBMCs were evaluated.Furthermore,modulatory effects of rHc-GDC on production of Th1,Th2,and Th9 cells were evaluated by flowcytometiy and on interferon gamma,interleukin(IL)-4 and IL-9 were evaluated by quantitative real-time polymerase chain reaction.The results demonstrated that rHc-GDC was successfully cloned,expressed in expression vector as well as in the gut surface of adult H.contortus worm and successful binding with PBMCs surface were observed.Immunoblotting results revealed that rHc-GDC is an important active protein of H.contortus excretory and secretoiy products.Moreover,the interaction of rHc-GDC with host cells increased the production of Th2,Th9 cells,IL4,IL-9,PBMC proliferation,nitric oxide,and cell migration.No effects of rHc-GDC were observed on PMBC apoptosis,production of Th1 cells,and secretions of IFN-? and IL-10 cytokines.These findings indicate that recombinant GDC protein from H.contortus modulates the immune functions of goat PBMCs and has the potential to enhance protective immunity by inducing T helper-9-derived IL-9 in vitro.2-Immunodynamics of novelTRIC domain containing protein(DUF714)from Haemonchus contortus on goat PBMCs derived-T helper and regulatory T cellsHaemonchus contortus is blood ingesting nematode of small ruminant that is responsible for huge economic losses worldwide.Development of anti-helminthic resistance against this parasite is becoming a challenge in the effective control so alternative measures are urgently needed.Excretory-secretory products are released during H.contortus infection;TRIC domain containing protein(TDC)is one of these products whose functions are still unknown.In this study,TDC gene was cloned,recombinant HcTDC protein was synthesized and its effects on the functions of goat PBMCs and production of T helper cells®ulatory T cells were evaluated.Recombinant Hc-TDC significantly decreased cell proliferation,migration,nitric oxide production,Thl cells,IFNy and increased apoptosis,Th17,regulatory T cells,IL-17,1L-10 at dose dependent manner.Results concluded that recombinant Hc-TDC has potential to suppress the immunity by up regulating the production of Th17,regulatory T cells and associated immune response in vitro.3-Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goatHaemonchus contortus(H.contortus)is one of the most important parasites that causehuge economic losses to small ruminant industryworldwide.Effective prognosis and treatment depend upon theearly diagnosis of H.contortus infection.To date,no widelyapproved methods for the identification of prepatentH.contortus infection are available to identify prepatentH.contortus infection.The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H.contortus protein(rHc-CS)during early and late infections of H.contortus in goat.Purified rHc-CS exhibited a clear band,with a molecularweight about 38 kDa.H.contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection(D.P.I).However,eggs were detectedat 21,28 and 35D.P.I.Hence,results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage(14 D.P.I)and late stage(21-103 D.P.I)of H.contortus infection.Furthermore,no cross reactivity was observed againstTrichinella spiralis,Fasciola hepatica and Toxoplasma gondiior uninfected goats.Among several evaluated rHc-CS indirect-ELISA format variables,favorable antigen coating concentration was found 0.28?g/well at 37? 1h and overnight at 4?.Moreover,optimum dilution ratio of serum andrabbit anti-goat IgG was recorded as 1:100 and 1:4000,respectively.The best blocking buffer was 5%Bovine Serum Albumin(BSA)while the best time for blocking,serum incubation and TMB reaction were recorded as 60,120 and 10 min,respectively.The cut-off value for positive and negativeinterpretation was determined as 0.352(OD450).The diagnostic specificity and sensitivity of the rHc-CS,bothwere recorded as 100%.These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H.contortusinfections in goat.4-Immunodiagnostic potential of recombinant Tropomyosin during prepatent Haemonchus contortus infection in goatExcretory and secretory products(ESPs)are released by the parasites during Haemonchus contortus(H.contortus)infection.In this study,Tropomyosin(TpMy),one of these ESPs was used to develop western blotting and optimized Enzyme Linked immunosorbent assay(ELISA)for detection of H.contortusduring early infection in goat.Microscopic examination was performed parallel for comparison.Recombinant tropomyosin protein was purified successfully.Western blotting results revealed that antirecombinant H.contortusTropomyosin(rHc-TpMy)antibodies could recognize the natural protein in Sprague Dawleyrats and rHc-TpMy antigen did not show any cross-reaction with goat anti-sera of Fasciola hepatica,Trichinella spiralis,and Toxoplasma gondii.Moreover,initial antibodies were detected by both western blotting and indirect ELISA at 14 days post infection(DPI)and persisted till 30 DPI but fecal eggs count couldn't detect the eggs in feces at early stage(7 and 14 DPI).The optimized antigen coating concentration was calculated as 10?g/mL(P/N Optimum Density450?4.165)with optimized dilution of serum(1:50)and secondary antibody(1:2500).Positive and negative cutoff value of the indirectELISA assay was calculated as 0.392 and 0.344,respectively.Receiver operating characteristic curve analysis validated the cutoff value(0.392)based on a high specificity and sensitivity.Indirect ELISA showed 90%diagnostic sensitivity and 100%diagnostic specificity.In comparison of serological and conventional method,rHc-TpMy based indirect ELISA showed more positive results(30%;9/30)than microscopic examination(20%;6/30).These results demonstrated that rHc-TpMy is a potential immunodiagnostic antigen to detect specific antibodies at early stage of infection in goat and serological methods are more reliable as compared to microscopic examination.5-Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in GoatHaemonchus contortus is recognized as one of the important health problems in small ruminants,leading to reduced production and economic loss for farmers worldwide.Prepatent diagnosis ofH.contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia,edema,diarrhea,and ultimately death in young animals.In this study,one of the excretory and secretory products,rHC-HCA59,was purified and used as antigen to detect specific antibodies in H.contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay(ELISA)as screening test.All goats(n=38)were housed indoor,experimentally infected with 8000 infective larvae(L3)of H.contortus,and serum samples were collected prior to infection and at 14th day of infection.Immunoblotting was performed to confirm the results of indirect ELISA,evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting helminthes and rectify the false negative samples.Furthermore,three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA.No eggs were detected in feces of all goats collected at 7th and 14th day of infection but,H.contortus eggs were detected at 21 days post infection in the feces.Indirect ELISA performed in this study showed 87%sensitivity and 100%specificity.The western blot analysis confirmed immuno-reactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA.Furthermore,all false negative sera(n=5)that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot.Moreover,no crossreactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii,Fasciola hepatica,and Trichinella spiralis.The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H.contortus infection during prepatent period in goats.