Partial Protection of Goats against Haemonchus contortus Achieved with ADP-Ribosylation Factor 1 Encapsulated in PLGA Nanoparticles.

BACKGROUND: Haemonchus contortus (H. contortus), a nematode with global prevalence, poses a major threat to the gastrointestinal health of sheep and goats. In an effort to combat this parasite, a nanovaccine was created using a recombinant ADP-ribosylation factor 1 (ARF1) antigen encapsulated within poly lactic-co-glycolic acid (PLGA). This study aimed to assess the effectiveness of this nanovaccine in providing protection against H. contortus infection. METHODS: Fifteen goats were randomly divided into three groups. The experimental group received two doses of the PLGA encapsulated rHcARF1 (rHcARF1-PLGA) nanovaccine on days 0 and 14. Fourteen days after the second immunization, both the experimental and positive control groups were challenged with 8000 infective larvae (L3) of H. contortus, while the negative control group remained unvaccinated and unchallenged. At the end of the experiment on the 63rd day, all animals were humanly euthanized. RESULTS: The results showed that the experimental group had significantly higher levels of sera IgG, IgA, and IgE antibodies, as well as increased concentrations of cytokines, such as IL-4, IL-9, IL-17, and TGF-ß, compared to the negative control group after immunization. Following the L3 challenge, the experimental group exhibited a 47.5% reduction in mean eggs per gram of feces (EPG) and a 55.7% reduction in worm burden as compared to the positive control group. CONCLUSIONS: These findings indicate that the nanovaccine expressing rHcARF1 offers significant protective efficacy against H. contortus infection in goats. The results also suggest the need for more precise optimization of the antigen dose or a reassessment of the vaccination regimen. Additionally, the small sample size limits the statistical rigor and the broader applicability of the findings.

PLGA-Encapsulated Haemonchus contortus Antigen ES-15 Augments Immune Responses in a Murine Model.

Haemonchus contortus is a gastrointestinal parasite that adversely impacts small ruminants, resulting in a notable reduction in animal productivity. In the current investigation, we developed a nanovaccine by encapsulating the recombinant protein rHcES-15, sourced from the excretory/secretory products of H. contortus, within biodegradable poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). The development of this nanovaccine involved the formulation of PLGA NPs using a modified double emulsion solvent evaporation technique. Scanning electron microscopy (SEM)verified the successful encapsulation of rHcES-15 within PLGA NPs, exhibiting a size range of 350-400 nm. The encapsulation efficiency (EE) of the antigen in the nanovaccine was determined to be 72%. A total of forty experimental mice were allocated into five groups, with the nanovaccine administered on day 0 and the mice euthanized at the end of the 14-day trial. The stimulation index (SI) from the mice subjected to the nanovaccine indicated heightened lymphocyte proliferation (*** p < 0.001) and a noteworthy increase in anti-inflammatory cytokines (IL-4, IL-10, and IL-17). Additionally, the percentages of T-cells (CD4+, CD8+) and dendritic cell phenotypes (CD83+, CD86+) were significantly elevated (** p < 0.01, *** p < 0.001) in mice inoculated with the nanovaccine compared to control groups and the rHcES-15 group. Correspondingly, higher levels of antigen-specific serum immunoglobulins (IgG1, IgG2a, IgM) were observed in response to the nanovaccine in comparison to both the antigenic (rHcES-15) and control groups (* p < 0.05, ** p < 0.01). In conclusion, the data strongly supports the proposal that the encapsulation of rHcES-15 within PLGA NPs effectively triggers immune cells in vivo, ultimately enhancing the antigen-specific adaptive immune responses against H. contortus. This finding underscores the promising potential of the nanovaccine, justifying further investigations to definitively ascertain its efficacy.

Eimeria tenella 14-kDa phosphohistidine phosphatase stimulates maturation of chicken dendritic cells and mediates DC-induced T cell priming in a Th1 cytokine interface.

Given the central role of dendritic cells (DCs) in directing cell-mediated immunity, this study investigated the capability of Eimeria tenella 14-kDa phosphohistidine phosphatase (EtPHP14) to mature chicken DCs and initiate DC-induced T cell immunity. With the aim of identifying novel protective Eimeria antigen, EtPHP14 gene was successfully cloned and EtPHP14 recombinant protein (rEtPHP14) was expressed in Escherichia coli expression system. rEtPHP14 binding was identified on the surface of chicken DCs by Immunofluorescence assay. DC phenotypes were evaluated by flow cytometry and results indicated that MHCII, CD80, CD86, CD1.1 and CD11c were up-modulated in DCs following rEtPHP14 treatment. RT-qPCR showed increased transcript levels of DC maturation markers CCL5, CCR7 and CD83 in rEtPHP14-treated DCs. Moreover, transcript profile of genes associated with intracellular signaling pathways that characterize the immunogenic (TLR signaling) or tolerogenic (Wnt signaling) state of DCs revealed that TLR signaling was stimulated and Wnt signaling was inhibited in rEtPHP14-treated DCs. Furthermore, proliferation of T cells and differentiation of CD4+ cells were promoted when rEtPHP14-treated DCs were co-cultured with autologous T cells. DCs incubated with rEtPHP14 alone expressed increased IL-12 and IFN-γ levels while IL-10 and TGF-ß levels remained unaffected. Likewise, similar trend of IFN-γ expression was noted in rEtPHP14 treated DC-T cell coculture, whereas IL-4 expression remained unchanged. These findings indicate that EtPHP14 is an important molecule that can upregulate host immune response, particularly Th1, during host-parasite interaction, suggesting its importance as a novel candidate for coccidiosis vaccine.

Em14-3-3 delivered by PLGA and chitosan nanoparticles conferred improved protection in chicken against Eimeria maxima.

Eimeria maxima (E. maxima) are an intracellular apicomplexan protozoan that causes intestinal coccidiosis in chickens. The purpose of this research was to develop a novel delivery approach for recombinant E. maxima (rEm) 14-3-3 antigen to elicit enhanced immunogenic protection using poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NPs) against E. maxima challenge. The morphologies of prepared antigen-loaded NPs (PLGA/CS-rEm14-3-3 NPs) were visualized by a scanning electron microscope. The rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs-immunized chicken-induced changes of serum cytokines, IgY-antibody level, and T-lymphocyte subsets and protective efficacies against E. maxima challenge were evaluated. The results revealed that encapsulated rEm14-3-3 in PLGA and CS NPs presented spherical morphology with a smooth surface. The chickens immunized with only rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs elicited a significant (p<0.05) higher level of IFN-γ cytokine, stimulated the proportions of CD4+/CD3+, CD8+/CD3+ T-cells, and provoked sera IgY-antibody immune response compared to control groups (PBS, pET-32a, PLGA, and CS). Whereas, PLGA-rEm14-3-3 NP-immunized chicken provoked a higher level of IFN- γ production and IgY-antibody response rather than CS-rEm14-3-3 and bare antigen, relatively. The animal experiment results ratified that PLGA-rEm14-3-3 NP-immunized chicken significantly alleviated the relative body weight gain (%), decreased lesion score, and enhanced oocyst decrease ratio compared to CS-rEm14-3-3 NPs and only rEm14-3-3. The anti-coccidial index of the chicken vaccinated with the PLGA-rEm14-3-3 NPs was (180.1) higher than that of the Cs-rEm14-3-3 NPs (167.4) and bare antigen (165.9). Collectively, our statistics approved that PLGA NPs might be an efficient antigen carrier system (Em14-3-3) to act as a nanosubunit vaccine that can improve protective efficacies in chicken against E. maxima challenge.

Nanoparticles of Chitosan/Poly(D,L-Lactide-Co-Glycolide) Enhanced the Immune Responses of Haemonchus contortus HCA59 Antigen in Model Mice.

BACKGROUND: Hepatocellular carcinoma-associated antigen 59 (HCA59) from excretory/secretory products of Haemonchus contortus is known to have the ability to modulate the functions of host cells. However, its immunogenicities using different nanoparticles adjuvants remain poorly understood. PURPOSE: The study aimed to select an efficient nanoparticle antigen delivery system, which could enhance the immune responses of Haemonchus contortus HCA59 in mice. METHODS: Here, the immune responses induced by the recombinant protein of HCA59 (rHCA59) with poly-D,L-lactide-co-glycolide (PLGA) nanoparticles, Chitosan nanoparticles, mixture of PLGA and Chitosan nanoparticles (rHCA59-Chitosan-PLGA), and Freund's complete adjuvant were observed, respectively, in mice. Cytokine and antibody levels induced by different groups were detected by ELISA assay. The effects of lymphocyte proliferations on different groups were examined using CCK-8 kit. Phenotypes of T cells and dendritic cells were analyzed by flow cytometry. RESULTS: On day 14 post vaccination, levels of IgM, IgG1, IgG2a, IFN-γ, IL-4, and IL-17 were significantly increased in the groups immunized with rHCA59 encapsulated with nanoparticles. After mice were vaccinated with rHCA59 loaded with Chitosan/PLGA nanoparticles, lymphocytes proliferated significantly. Additionally, the percentages of CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+ CD8+), and dendritic cells (CD11c+ CD83+, CD11c+ CD86+) were obviously up-regulated in the mice immunized with nanoparticles, especially in the rHCA59-Chitosan-PLGA antigen delivery system group. CONCLUSION: The findings of this research demonstrated that rHCA59-Chitosan-PLGA antigen delivery system could induce higher immune responses in mice model and indicated that rHCA59 might be a good candidate molecule to develop nanovaccines against Haemonchus contortus in future study.

The immunogenic maturation of goat monocyte-derived dendritic cells and upregulation of toll-like receptors by five antigens of Haemonchus contortus in-vitro.

Previously, it was found that several proteins of Haemonchus contortus were involved in the stimulation of the host immune system. However, the information about the selection of superlative antigens with immunogenic efficacies on host DCs is lacking. In the current study, the stimulatory effects of five recombinant proteins (elongation factor-1α, arginine kinase, ES-15, ES-24, and ADP-ribosylation factor 1) of H. contortus on the maturation of goat monocyte-derived dendritic cells (md-DCs) were reported. Recombinant proteins were purified separately in E. coli expression and incubated with isolated goat peripheral blood mononuclear cells (PBMC). Immunofluorescence assay (IFA) results confirmed the binding of these molecules to the md-DC's surface as compared to control groups. In the flow cytometry analysis, recombinant proteins induced md-DC stimulation via the up-regulation of the expression of the costimulatory molecule (CD80) and MHC-II. Quantitative RT-PCR data showed a significant increase in the expression of specific genes of the WNT and toll-like receptor (TLR) signaling pathways. The result of ELISA indicated the higher levels of cytokine (IL-10, IL-12, IFN-γ, and TNF-α) secretion in the md-DC compared to the negative (pET-32a His-Tag) and blank (PBS) control groups. The data gives valuable support in the selection of potential antigens for future studies on the immunomodulation of the host against the infection of H. contortus.

Actin-depolymerizing factor from Eimeria tenella promotes immunogenic function of chicken dendritic cells.

Dendritic cells play a crucial role in inducing antigen-specific immunity to pathogens. During host-parasite interaction, host immune response to the parasite molecules is considered essential for recognizing novel antigens for control strategies. Therefore, in the present study, chicken dendritic cells (DCs) (ChDCs), derived from spleens were used to evaluate their capacity to proliferate and differentiate autologous T lymphocytes in response to actin-depolymerizing factor from Eimeria tenella (EtADF). Immunoblot analysis showed that recombinant EtADF protein (rEtADF) was able to interact with rat anti-rEtADF antibodies. The immunofluorescence test confirmed rEtADF binding on ChDCs surface. Flow cytometric analysis revealed that phenotypes for MHCII, CD1.1, CD11c, CD80, and CD86 were increased in ChDCs after rEtADF treatment. qRT-PCR results indicated that ChDCs triggered TLR signaling in response to rEtADF, and suppressed Wnt signaling. Transcript levels of CD83, CCL5, and CCR7 in ChDCs were improved following rEtADF treatment. In addition, rEtADF promoted DC-directed T cell proliferation and differentiation of naïve T cells into CD3+/CD4+ T cells in DC/T cell co-incubation system. Cytokine analysis of rEtADF-pulsed ChDCs showed increased levels of IL-12 and IFN-γ, while IL-10 and TGF-ß remained unchanged. Moreover, rEtADF-treated ChDCs enhanced production of IFN-γ when incubated with T cells, and IL-4 secretion remained unchanged. Our findings indicted that rEtADF could facilitate the polarization of Th1 immune cells by triggering both host DCs and T cells. Our findings provide useful insights into future work aimed at anticoccidial vaccine strategies.

Nanoparticles (PLGA and Chitosan)-Entrapped ADP-Ribosylation Factor 1 of Haemonchus contortus Enhances the Immune Responses in ICR Mice.

ADP-ribosylation factor 1 (HcARF1) is one of the Haemonchus contortus (H. contortus) excretory/secretory proteins involved in modulating the immune response of goat peripheral blood mononuclear cells (PBMC). Here, we evaluated the immunogenic potential of recombinant HcARF1 (rHcARF1) against H. contortus infection in Institute of Cancer Research (ICR) mice. Briefly, rHcARF1 was entrapped in poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NP) and injected into mice as a vaccine. Fifty-six ICR mice were assigned randomly into seven groups, with eight animals in each group, and they were vaccinated subcutaneously. At the end of the experiment (14th day), the blood and the spleen were collected from euthanized mice to detect lymphocyte proliferation, cytokine analysis, and the production of antigen-specific antibodies. Scanning electron microscope was used to determine the size, morphology, and zeta potential of nanoparticles. Flow cytometry was performed, which presented the increase percentages of CD4+ T cells (CD3e+CD4+), CD8+ T cells (CD3e+CD8+) and dendritic cells (CD11c+CD83+, CD11c+CD86+) in mice vaccinated with rHcARF1+PLGA NP. Immunoassay analysis show raised humoral (Immunoglobulin (Ig)G1, IgG2a, IgM) and cell-mediated immune response (Interleukin (IL)-4, IL-12, and IL-17, and Interferon (IFN)-γ) induced by rHcARF1+PLGA NP. Experimental groups that were treated with the antigen-loaded NP yield higher lymphocyte proliferation than the control groups. Based on these results, we could propose that the rHcARF1 encapsulated in NP could stimulate a strong immune response in mice rather than administering alone against the infection of H. contortus.

Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina modulates the functions of chicken dendritic cells to boost Th1 type immune response and stimulates autologous CD4+ T cells differentiation in-vitro.

Dendritic cells (DCs) play a pivotal role to amplify antigen-specific immune responses. Antigens that sensitize T cells via antigen-presentation by DCs could enhance the capacity of host immunity to fight infections. In this study, we tested the immunogenic profiles of chicken DCs towards Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina (EaGAPDH). Immunoblot analysis showed that recombinant EaGAPDH (rEaGAPDH) protein was successfully recognized by rat sera generated against rEaGAPDH. Interaction and internalisation of rEaGAPDH by chicken splenic-derived DCs (chSPDCs) was confirmed by immunofluorescence analysis. Flow cytometry revealed that chSPDCs upregulated MHCII, CD1.1, CD11c, CD80, and CD86 cell-surface markers. Moreover, mRNA expressions of DC maturation biomarkers (CCL5, CCR7, and CD83) and TLR signalling genes (TLR15 and MyD88) were also upregulated whereas those of Wnt signalling were non-significant compared to negative controls. rEaGAPDH treatment induced IL-12 and IFN-γ secretion in chSPDCs but had no effect on IL-10 and TGF-ß. Likewise, DC-T cell co-culture promoted IFN-γ secretion and the level of IL-4 was unaffected. Proliferation of T cells and their differentiation into CD3+/CD4+ T cells were triggered in chSPDCs-T cells co-culture system. Taken together, rEaGAPDH could promote Th1 polarization by activating both host DCs and T cells and sheds new light on the role of this important molecule which might contribute to the development of new DCs-based immunotherapeutic strategies against coccidiosis.

In vitro effects of 5 recombinant antigens of Eimeria maxima on maturation, differentiation, and immunogenic functions of dendritic cells derived from chicken spleen.

Eimeria maxima possesses integral families of immunogenic constituents that promote differentiation of immune cells during host-parasite interactions. Dendritic cells (DCs) have an irreplaceable role in the modulation of the host immunity. However, the selection of superlative antigen with immune stimulatory efficacies on host DCs is lacking. In this study, 5 recombinant proteins of E. maxima (Em), including Em14-3-3, rhomboid family domain containing proteins (ROM) EmROM1 and EmROM2, microneme protein 2 (EmMIC2), and Em8 were identified to stimulate chicken splenic derived DCs in vitro. The cultured populations were incubated with recombinant proteins, and typical morphologies of stimulated DCs were obtained. DC-associated markers major histocompatibility complex class II, CD86, CD11c, and CD1.1, showed upregulatory expressions by flow cytometry assay. Immunofluorescence assay revealed that recombinant proteins could bind with the surface of chicken splenic derived DCs. Moreover, quantitative real-time PCR results showed that distinct gene expressions of Toll-like receptors and Wnt signaling pathway were upregulated after the coincubation of recombinant proteins with DCs. The ELISA results indicated that the DCs produced a significant higher level of interleukin (IL)-12 and interferon-γ secretions after incubation with recombinant proteins. While transforming growth factor-ß was significantly increased with rEmROM1, rEmROM2, and rEmMIC2 as compared to control groups, and IL-10 did not show significant alteration. Taken together, these results concluded that among 5 potential recombinant antigens, rEm14-3-3 could promote immunogenic functions of chicken splenic derived DCs more efficiently, which might represent an effective molecule for inducing the host Th1-mediated immune response against Eimeria infection.

Immunomodulatory dynamics of excretory and secretory products on Th9 immune response during Haemonchus contortus infection in goat.

CD4+ T cells play critical roles in mediating adaptive immunity to a variety of pathogens. Recently, new subset of CD4+T named as T helper 9 cells that express the prototypical interleukin-9 (IL-9) cytokine have been recognized in human and mice models during different parasitic infections. Haemonchus contortus is a gastrointestinal nematode of small ruminants which cause high mortality in young animals. During infection, Excretory and Secretary Products (ESPs) are released in the host body. No other study has reported yet on immunomodulatory dynamics of H. contortus ESPs on Th9 immune response in vitro or in vivo. In this study, immunomodulatory effects of ESPs (5, 10, 20, 40, 80; µg/mL) incubated with goat PBMCs on Th9 cells, IL-9 immune response and TGF-ß/Smad signaling regulator were evaluated in vitro. Moreover, for in vivo study, goats were infected with different doses (P-800, P-2400, and P-8000) of H. contortus infective larva (L3) and immunomodulatory effects on Th9 cells, IL-9 immune response and TGF-ß/Smad signaling regulator were evaluated at 7, 10, 14, 18, 21, 28 Days Post Infection (DPI). Flow cytometry was performed to evaluate the effects on Th9 cells and quantitative real time polymerase chain reaction was performed to evaluate the IL-9 cytokine transcription level. Additionally, fecal egg counting was also performed in parallel to confirm the infection. All goats were dewormed at 29 DPI and all experiments were also performed at 35 DPI, one week post deworming. The finding indicated that 10, 20, 40, 80 µg/mL concentration of ESPs incubated with goat PBMCs showed significant increase in the production of Th9 cells, signature cytokine IL-9 and expression of TGF-ß/Smad signaling regulator as compared to control group in vitro.All infected groups showed significant increase in production of Th9 cells and IL-9 cytokine and expression of TGF-ß/Smad key genes at 18, 21, and 28 DPI as compared to control group. Likewise, at 14 DPI, P-2400 and P-8000 groups showed significant increase in production of Th9 cells, IL-9 cytokine and expression of TGF-ß/Smad key genes. While at 10 DPI, production of Th9 cells and IL-9 was significantly increased in P-2400 & P-8000 groups, and at 7 DPI only P-8000 showed significantly increase in IL-9 production. No immunomodulatory effects were observed at 0 and 3 DPI. Additionally, significant gradually up-regulated key genes expression of TGF-ß/Smad signaling regulator in all infected groups confirmed the above results. After deworming, production of Th9 cells, associated immune response and expression of signaling regulator in each group were significantly decreased. Based on this study, it is concluded that Th9 immune response was induced during H. contortus infection in goat by up-regulation of TGF-ß/Smad signaling key genes.

Recombinant ubiquitin-conjugating enzyme of Eimeria maxima induces immunogenic maturation in chicken splenic-derived dendritic cells and drives Th1 polarization in-vitro.

Dendritic cells (DCs) are key linkages between innate immunity and acquired immunity. The antigens that promote the functions of DCs might be the effective candidates of novel vaccine. In this research, the ability of ubiquitin-conjugating enzyme (UCE), a recognized common antigens among chicken Eimeria species, to stimulate DCs of chickens were evaluated. We cloned UCE gene from Eimeria maxima (EmUCE), and its protein expression was confirmed by SDS-PAGE and western-blot. Immunofluorescence assay confirmed the binding of rEmUCE on the surface of chicken splenic-derived DCs (ChSP-DCs). Flow cytometric analysis showed that rEmUCE-treated ChSP-DCs increased MHCII, CD1.1, CD11c, CD80, and CD86 phenotypes. qRT-PCR indicated that transcript levels of maturation markers CCL5, CCR7, and CD83 in ChSP-DCs were upregulated in response to rEmUCE. Following rEmUCE treatment, chSP-DCs activated TLR signaling and inhibited Wnt signaling. Moreover, rEmUCE promoted DC-mediated T-cell proliferation in DC/T-cell co-incubation. Interestingly, CD3+/CD4+ T-cells were significantly enhanced when rEmUCE-treated chSP-DCs were co-incubated with T-cells. Cytokine secretion pattern of rEmUCE-stimulated ChSP-DCs revealed that the production of IL-12 and IFN-γ was increased whereas IL-10 and TGF-ß were unchanged. Likewise, the co-incubation of ChSP-DCs with T-cells indicated increased production of IFN-γ but not IL-4. Collectively, rEmUCE could polarize DCs to immunogenic phenotype and shift the immune cells towards Th1 response. Our observations provide valuable insight for future research aimed at vaccine development against avian coccidiosis.

Characterization of Haemonchus contortus Excretory/Secretory Antigen (ES-15) and Its Modulatory Functions on Goat Immune Cells In Vitro.

Small size excretory/secretory (ES) antigens of the Haemonchus contortus parasite have intense interest among researchers for understanding the molecular basis of helminths immune regulation in term of control strategies. Immunomodulatory roles of H. contortus ES-15 kDa (HcES-15) on host immune cells during host-parasite interactions are unknown. In this study, the HcES-15 gene was cloned and expression of recombinant protein (rHcES-15) was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG). Binding activity of rHcES-15 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and immunohistochemical analysis showed that H. contortus 15 kDa protein localized in the outer and inner structure of the adult worm, clearly indicated as the parasite's ES antigen. The immunoregulatory role on cytokines production, cell proliferation, cell migration, nitric oxide (NO) production, apoptosis, and phagocytosis were observed by co-incubation of rHcES-15 with goat PBMCs. The results showed that cytokines IL-4, IL-10, IL-17, the production of nitric oxide (NO), PBMCs apoptosis, and monocytes phagocytosis were all elevated after cells incubated with rHcES-15 at differential protein concentrations. We also found that IFN-γ, TGF-ß1, cells proliferation and migration were significantly suppressed with the interaction of rHcES-15 protein. Our findings indicated that low molecular ES antigens of H. contortus possessed discrete immunoregulatory roles, which will help to understand the mechanisms involved in immune evasion by the parasite during host-parasite interactions.

Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat.

BACKGROUND: Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. 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. RESULTS: Purified rHc-CS exhibited a clear band, with a molecular weight 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 detected at 21, 28 and 35 D.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 against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 µg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit 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 negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. CONCLUSION: These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Hepatocellular carcinoma-associated antigen 59 of Haemonchus contortus modulates the functions of PBMCs and the differentiation and maturation of monocyte-derived dendritic cells of goats in vitro.

BACKGROUND: Hepatocellular carcinoma-associated antigen 59 (HCA59), which is one of the most important excretory/secretory products of Haemonchus contortus (HcESPs), is known to have antigenic functions. However, its immunomodulatory effects on host cells are poorly understood. METHODS: Here, we cloned the HCA59 gene and expressed the recombinant protein of HCA59 (rHCA59). Binding activities of rHCA59 to goat peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were checked by immunofluorescence assay (IFA) and the immunoregulatory effects of rHCA59 on cytokine secretions, cell migration, cell proliferation, nitric oxide production, and changes in expression of genes in related pathways were observed by co-incubation of rHCA59 with goat PBMCs and DCs. Monocyte phagocytosis and characterization of goat blood DC subsets were detected by flow cytometry. RESULTS: The IFA results revealed that rHCA59 could bind to PBMCs and DCs. Treatment of PBMCs with rHCA59 significantly increased cellular proliferation and NO production in a dose-dependent manner, while cell migration was vigorously blocked. Treatment with rHCA59 significantly suppressed monocytes phagocytosis. The quantity of surface marker CD80 on DCs increased significantly after rHCA59 treatment. In addition, the expression of genes included in the WNT pathway was related to the differentiation and maturation of DCs, and the production of IL-10 and IL-17 produced by PBMCs was altered. CONCLUSIONS: Our findings illustrated that rHCA59 could enhance host immune responses by regulating the functions of goat PBMCs and DCs, which would benefit our understanding of HCA59 from parasitic nematodes contributing to the mechanism of parasitic immune evasion.

The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells.

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host-parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-ß1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future.

Effects of Recombinant Toxoplasma gondii Citrate Synthase I on the Cellular Functions of Murine Macrophages In vitro.

Toxoplasmosis, which is one of the most widespread zoonoses worldwide, has a high incidence and infection can result in severe disease in humans and livestock. Citrate synthase (CS) is a component of nearly all living cells that plays a vital role in the citric acid cycle, which is the central metabolic pathway of aerobic organisms. In the present study, the citrate synthase I gene of Toxoplasma gondii (T. gondii) (TgCSI) was cloned and characterized. The TgCSI gene had an open reading frame of 1665 bp nucleotides encoding a 555 amino acid protein with a molecular weight of 60 kDa. Using western blotting assay, the recombinant protein was successfully recognized by the sera of rats experimentally infected with T. gondii, while the native protein in the T. gondii tachyzoites was detected in sera from rats immunized with the recombinant protein of TgCSI. Binding of the protein to murine macrophages was confirmed by immuno fluorescence assay. Following incubation of macrophages with rTgCSI, the rTgCSI protein was found to have a dual function, with low concentrations (5-10 µg/mL) enhancing phagocytosis and high levels (80 µg/mL) inhibiting phagocytosis. Investigation of murine macrophage apoptosis illustrated that 5 µg/mL rTgCSI protein can significantly induce early apoptosis and late stage apoptosis (*p < 0.05), while 10 µg/mL rTgCSI protein significantly induced early apoptosis, but had no effect on late stage of apoptosis (**p < 0.01), and 80 µg/mL rTgCSI protein inhibited late stage apoptosis of macrophages (*p < 0.05). Cytokine detection revealed that the secretion of interleukin-10, interleukin-1ß, transforming growth factor-ß1 and tumor necrosis factor-α of macrophages increased after the cells were incubated with all concentration of rTgCSI, with the exception that 5 µg/mL rTgCSI had no effect on the secretion of interleukin-10 and interleukin-1ß. However, secretion of NO and cell proliferation of the macrophages were substantially reduced. Taken together, these results suggested that TgCSI can affect the immune functions of murine macrophages by binding to the cells in vitro.

Recombinant Haemonchus contortus 24 kDa excretory/secretory protein (rHcES-24) modulate the immune functions of goat PBMCs in vitro.

A 24 kDa protein is one of the important components in Haemonchus contortus (barber pole worm) excretory/secretory products (HcESPs), which was shown to have important antigenic function. However, little is known about the immunomodulatory effects of this proteinon host cell. In the present study gene encoding 24kDa excretory/secretory protein (HcES-24) was cloned. The recombinant protein of HcES-24 (rHcES-24) was expressed in a histidine-tagged fusion protein soluble form in Escherichia coli. Binding activity of rHcES-24 to goat PBMCs was confirmed by immunofluorescence assay (IFA) and its immunomudulatory effect on cytokine secretion, cell proliferation, cell migration and nitric oxide production were observed by co-incubation of rHcES-24. IFA results revealed that rHcES-24 could bind to the PBMCs. The interaction of rHcES-24 increased the production of IL4, IL10, IL17 and cell migration in dose dependent manner. However, rHcES-24 treatment significantly suppressed the production of IFNγ, proliferation of the PBMC and Nitric oxide (NO) production. Our findings showed that the rHcES-24 played important regulatory effects on the goat PBMCs.