Haemonchus contortus HcL6 promoted the Th9 immune response in goat PBMCs by activating the STAT6/PU.1/NF-κB pathway.

Th9 cells play a crucial role in parasite immunity. The development of Th9 cells is facilitated by several cytokines. Key transcription factors, such as STAT6, STAT5, and PU.1, are known to enhance IL-9 expression during the Th9 immune response. NF-κB-mediated transduction pathways participate in the induction of IL-9. In a previous study, we unveiled a unique ribosomal protein derived from Haemonchus contortus excretory-secretory proteins (HcESPs) that interact with host Th9 cells. In the present study, the effects of the Haemonchus contortus ribosomal protein L6 domain DE-containing protein (HcL6) on IL-9 secretion, Th9 differentiation, and IL-9 transcription were assessed by employing ELISA, flow cytometry, and qPCR methodologies. The observations revealed the transcriptional upregulation of several key genes within the Th9 immune response pathway. Moreover, silencing STAT6, PU.1, and NF-κB was found to attenuate the Th9 immune response. In this study, we unveiled the Th9 immune response-inducing capabilities of HcL6 and elucidated some of its underlying mechanisms. These findings suggest that HcL6 is an immunostimulatory antigen capable of inducing the Th9 immune response. These insights could prove instrumental in identifying potential candidate antigens for the development of immunoprophylactic strategies against H. contortus infections.

Immunization With Recombinant Haemonchus contortus Y75B8A.8 Partially Protects Local Crossbred Female Goats From Haemonchus contortus Infection.

Haemonchus contortus Y75B8A.8 (Hc8) derived from H. contortus excretory-secretory (ES) products was identified as a functional inhibitor of goat interleukin 2 (IL-2). It may act as a vaccine candidate for the development of therapeutic strategies against H. contortus infection. In this research, recombinant Hc8 (rHc8) and goat anti-rHc8 polyclonal antibodies were employed to evaluate the protective capacities of Hc8 antigen against H. contortus infections via active and passive immunization trials, respectively. In both trials, local crossbred female goats aged 9-12 months old were randomly divided into three groups, five in each group, respectively. Parasitological examinations, including fecal egg counts (FEC), cumulative FEC (cFEC), and worm burdens, were performed. In addition, antibody levels in mucosal homogenate (MH) samples and hematological and immunological parameters were detected. In the passive trial, goats were intravenously immunized with 5 mg total IgG containing anti-rHc8 goat polyclonal antibodies. After twice immunization, compared with the challenged control group, cFEC was reduced by 39%. In addition, there was a 46% reduction of worm burdens compared with the challenged controls. In the active immunization trials, 500 µg of recombinant Hc8 protein was given subcutaneously twice to 9-12-month-old local crossbred female goats with a 2-week interval, resulting in the generation of high levels of antigen-specific circulating antibodies. Besides, cFEC and abomasal worm burden were reduced by 70 and 55%, respectively, compared with the challenged control group. In addition, immunized goats had higher mucosal homogenate IgA and hemoglobin levels than the challenged controls in both passive and active immunization trials. These preliminary results demonstrated the immunoprophylactic effects of Hc8 antigen and will inform new studies on ES proteins in developing subunit recombinant vaccines against H. contortus.

Immunization of Goats with Recombinant Protein 14-3-3 Isoform 2(rHcftt-2) Induced Moderate Protection against Haemonchus contortus Challenge.

A previous study identified that isoform 2 (Hcftt-2) of the 14-3-3 protein of Haemonchus contortus (H. contortus) could suppress immune functions of goat peripheral blood mononuclear cells (PBMCs) and might be a potential vaccine target, as neutralization of the protein function may enhance anti-parasite immunity. In this research, the recombinant Hcftt-2 was evaluated for its immunoprotective efficacy against H. contortus infection in goats. Five experimental goats were immunized twice with rHcftt-2 along with Freund's adjuvant. The five immunized goats and five nonimmunized goats (adjuvant only) were challenged with 5000 L3-stage H. contortus larvae after 14 days of second immunization. Five nonimmunized and uninfected goats (adjuvant only) were set as the uninfected group. A significant increase in the serum immunoglobin G(IgG) and serum IgA levels were identified in the rHcftt-2 immunized animals. The mean eggs per gram in feces (EPG) and the worm burdens of rHcftt-2 immunized group were reduced by 26.46% (p < 0.05) and 32.33%, respectively. In brief, immunization of goats with rHcftt-2 induced moderate protection against H. contortus challenge.

HcFAR, a functional inhibitor of goat TGF-ß1 identified from excretory and secretory products of Haemonchus contortus.

Haemonchus contortus has developed complexed and multifaceted mechanisms of immune evasion to enable the survival in the host. Generating excretion and secretion products (ESPs) to subvert or suppress the functions of host cytokines is a newly immune regulatory pattern found during recent years. Transforming growth factor-ß (TGF-ß) has critical immune regulatory functions in nematode infections. In this study, co-immunoprecipitation (co-IP) assay was used to identify the goat TGF-ß1 binding proteins from HcESPs. The interaction between TGF-ß1 and nematode fatty acid retinoid binding domain containing protein of H. contortus (HcFAR) was analyzed by glutathione S-transferase (GST)-pull down assay. The suppressive effect of rHcFAR on TGF-ß1-induced immunoglobulin A (IgA) secretion was observed by co-incubation of rHcFAR and TGF-ß1 with goat peripheral blood mononuclear cells (PBMCs). The IgA concentrations were determined using enzyme linked immunosorbent assay (ELISA) kit. Meanwhile, the suppressive effect of rHcFAR on TGF-ß1-induced T helper (Th) 9 differentiation was investigated by co-incubation of rHcFAR, TGF-ß1 and interleukin (IL)-4 with goats PBMCs. In parallel, IL-4 was replaced by IL-6 to determine the effects on the Th17 differentiation. The transcriptions of IL-9 and IL-17 in PBMCs were then evaluated by real-time PCR. Finally, we found that HcFAR from HcESPs could bind to goat TGF-ß1 in vitro. The ELISA results of IgA showed that 40 µg/mL rHcFAR could suppress the IgA secretion of PBMCs induced by TGF-ß1. Additionally, rHcFAR (at 10 µg/mL and 20 µg/mL) could inhibit the mRNA transcription of IL-9 induced by TGF-ß1 and IL-4. Meanwhile, rHcFAR could also downregulate the transcription of IL-17 induced by TGF-ß1 and IL-6 in a dose-dependent manner. These results indicated that HcFAR was a functional inhibitor of goat TGF-ß1 and this information may help contribute to understanding of the relationship between the ESPs and host cytokines.

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.

Haemonchus contortus transthyretin domain - containing protein (HcTTR): A promising vaccine candidate against Haemonchus contortus infection.

Haemonchus contortus transthyretin domain-containing protein (HcTTR) with 136 amino acids belongs to a transthyretin-like (TTL) family member. In our previous study, it was reported that HcTTR was a novel antagonist of the goat cytokine Interleukin 4 (IL-4), and was involved in the regulation of host immune responses, implying that it might be applied for therapeutic strategies or vaccine development against the infection of H. contortus. Thus, the protective capacities of HcTTR against H. contortus infections via active and passive immunization trials were examined. For the passive protection trials, H. contortus-infected goats were intravenously immunized twice with 5 mg of total IgG containing anti-rHcTTR goat polyclonal antibodies. The results showed that the significant rates of reduction in egg shedding and worm burden was 58.12% and 64.61%, respectively, as compared with the positive control group. For the active protection trials, local goats were vaccinated twice with 500 µg of recombinant HcTTR to generate antigen-specific circulating antibodies, resulting in 63.7% reduction in eggs shedding and 66.4% reduction in worm burdens after H. contortus challenge. In both passive and active trials, the immunized goats displayed higher mucosal IgA levels and less anaemic compared to the challenged positive controls. Pen trials indicated that HcTTR generated partial immune protective effects against H. contortus challenge and it could be a promising vaccine candidate for development of effective strategy to control H. contortus.

Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection in Goats.

Haemonchus contortus, a blood-sucking nematode of ruminants, causes large economic losses worldwide. Diagnosis of infection mainly depends on the evaluation of clinical signs and fecal examination. However, this has limitations for the diagnosis of early or light infections, where serological diagnosis seems to be more accurate and reliable. In this study, the recombinant H. 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) analysis. 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. 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%). The coefficient of variation within run and between runs was less than 11%. 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 rHcADRM could be a potential antigen for diagnosis of H. contortus infection in goats.