Galectins 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)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear 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 flowcytometry 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 secretory 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-γ 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.

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Haemonchus 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 of H. 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 parasites 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 immunoreactivity 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 cross-reactivity 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.

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-β/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-β/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-β/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-β/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-β/Smad key genes. While at 10 DPI, production of Th9 cells and IL-9 was significantly

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