Evaluation of the Role of Galectins in Parasite Immunity.

Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are ruminant specific galectins, first reported in sheep. Although their roles in parasite immunity are still being elucidated, it appears that they influence protection against parasites. In gastrointestinal infections with the nematode Haemonchus contortus, both galectin-11 and galectin-14 appear to be protective. However, in a chronic infection of liver fluke, Fasciola hepatica, these galectins may aid parasite survival. To unravel the structural, functional, and ligand profile of galectin-11 and galectin-14, recombinant production of these proteins is vital. Here we present the recombinant production of soluble galectin-11 and galectin-14 from domestic sheep for in vitro and structural biology studies. These methods include parasite cultivation and infection, galectin staining of host and parasite tissue, surface staining of parasites with recombinant galectins, pull-down assays to identify endogenous galectin binding proteins, and in vitro assays to monitor the effect of galectins on parasite development.

Ovine CD14- an Immune Response Gene Has a Role Against Gastrointestinal Nematode Haemonchus contortus-A Novel Report.

CD14 (also known as the monocyte differentiation antigen) is an important immune response gene known to be primarily responsible for innate immunity against bacterial pathogens, and as a pattern recognition receptor (PRR), binds with LPS (endotoxin), lipoproteins, and lipotechoic acid of bacteria. So far very limited work has been conducted in parasitic immunology. In the current study, we reported the role of CD14 in parasitic immunology in livestock species (sheep) for the first time. Ovine CD14 is characterized as a horse-shoe shaped bent solenoid with a hydrophobic amino-terminal pocket for CD14 along with domains. High mutation frequency was observed, out of total 41 mutations identified, 23 mutations were observed to be thermodynamically unstable and 11 mutations were deleterious in nature, causing major functional alteration of important domains of CD14, an indication of variations in individual susceptibility for sheep against Haemonchus contortus infestations. In silico studies with molecular docking reveal a role of immune response against Haemonchus contortus in sheep, which is later confirmed with experimental evidence through differential mRNA expression analysis for sheep, which revealed better expression of CD14 in Haemonchus contortus infected sheep compared to that of non-infected sheep. We confirmed the above findings with supportive evidence through haematological and biochemical analyses. Phylogenetic analysis was conducted to assess the evolutionary relationship with respect to humans and it was observed that sheep may well be used as model organisms due to better genetic closeness compared to that of mice.

Ovine ß-globin gene: A new qPCR for rapid haplotype identification and association with susceptibility to Haemonchus contortus infection.

Two ß-globin allelic haplotypes (A and B) were identified in domestic sheep, wherein animals which are homozygous for ßB allele (BB haplotype) have a deletion of pre-adult ßC-globin and consequently are less tolerant to anemia and hypoxia. Since Haemonchus contortus infection, is associated with severe anemia, studies performed from 1960s to 1990s investigated the association between ß-globin haplotype and resistance against this parasite. However, the findings were controversial, pointing out from increased resistance in animals harboring the ßA allele to inexistence of association. Thus, our study aimed to develop a qPCR for ß-globin haplotype identification, and to evaluate the association between ß-globin haplotype and resistance against H. contortus in a group of sheep submitted to artificial infection with this parasite. A total of 286 lambs of Morada Nova breed were experimentally challenged with 4000 H. contortus L3 and monitored for 112 days from weaning. Significantly improved (p < 0.05) phenotypic profiles (lower fecal egg counts, higher packed cell volume and birthweight) were observed for AA haplotype animals, especially when compared to BB animals, while AB animals were similar to BB. This is the first report of a qPCR assay for ovine ß-globin haplotype identification. In view of significant differences of phenotypic profiles between haplotype groups, the developed qPCR may constitute an important tool for sheep producers to improve genetic selection of parasite resistant animals.

Haemonchus contortus infection induces a variable immune response in resistant and susceptible Pelibuey sheep.

The immune response and phenotypic characteristics of Pelibuey lambs were analysed after the induction of a Haemonchus contortus trickle infection. Male lambs (n = 29; 20 kg live weight) were infected with 100 H. contortus infective larvae per kg of live weight on day 3, 5 and 7 of the experiment. The number of eggs per gram (epg), seven haematological parameters and the immunoglobulin A (IgA) level were analysed for 56 experimental days. In addition, histopathological samples from the fundic abomasal region and the relative expression of 10 immune-related genes from 15 infected and three non-infected lambs were analysed at day 0 and 49 of the experiment. The epg count and some haematological parameters (leucocytes, red blood cells, haemoglobin and total protein) with statistically significant differences (P < 0.01) were used to identify nine resistant and 20 susceptible lambs (1166 ±â€¯1071 and 3171 ±â€¯1463 epg, respectively). Moreover, acute infiltration of immune cells and parasitic granuloma formation were observed in susceptible lambs; the resistant group had moderate inflammatory cell infiltration. With respect to relative gene expression, resistant lambs showed upregulation (P < 0.001) of 10 genes, from 2.2 to 15.99 fold. Moreover, there was a strong indirect correlation (P < 0.05) between the epg count and interleukin 5 (IL5) gene expression. By contrast, there was an average 0.34 fold downregulation in nine of the immune-related genes (P ≤ 0.05) in susceptible lambs (the only exception was Fc fragment of IgE receptor Ia [FCER1A] upregulation). In addition, there was a direct correlation (P ≤ 0.05) between the epg count and the expression of IL8, which encodes an inflammatory chemokine. In conclusion, this study showed differential IL5 and IL8 gene expression during haemonchosis in resistant and susceptible Pelibuey lambs, respectively, together with a variable immune response based on histopathological and haematological parameters.

The GT1-TPS Structural Domain Protein From Haemonchus contortus Could Be Suppressive Antigen of Goat PBMCs.

Trehalose phosphate synthase (TPS), a key enzyme in trehalose synthesis, is not present in mammals but critical to the viability of a wide range of lower organisms. However, almost nothing is known about the function of Hc-TPS (GT1-TPS structural domain protein from Haemonchus contortus). In this study, Hc-TPS gene was cloned and the recombinant protein (rHc-TPS) was expressed and purified. The quantitative real-time PCR (qPCR) results showed that Hc-TPS was transcribed at different stages of H. contortus, with higher levels of transcription at the molting and embryo stages. Immunofluorescence analysis showed that Hc-TPS was widely distributed in adults, but the expression was mainly localized on the mucosal surface of the intestine as well as in the embryos of female worms. The impacts of rHc-TPS on peripheral blood mononuclear cell (PBMC) proliferation, nitric oxide (NO) generation, transcriptional expression of cytokines, and related pathways were examined by co-incubating rHc-TPS with goat PBMCs. The results showed that rHc-TPS significantly inhibited PBMC proliferation and NO secretion in a dose-dependent manner. We also found that rHc-TPS activated the interleukin (IL)-10/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (IL-10/STAT3/SOCS3) axis and significantly promoted SOCS3 expression, while inhibiting interferon-gamma (INF-γ), IL-4, IL-9, and IL-2 pathways. Our findings may contribute to understanding the immune evasion mechanism for the parasite during host-parasite interactions and also help to provide ideas for discovering new drug targets.

Parasitological and immunological response to Haemonchus contortus infection: Comparison between resistant Garole and susceptible Sahabadi sheep.

Parasitological and immunological responses to the experimentally induced Haemonchus contortus infection were compared between Garole and Sahabadi breeds of sheep. The experiment was conducted in a 2 (breed) × 2 (infection status) factorial arrangement with a completely randomised design. Two breeds of sheep were divided into infected (n = 10) and control (n = 6) groups, and the infected groups were orally infected with H. contortus (500 stage 3 larvae per kilogram of body weight). Faecal egg counts (FEC) were determined from 18 days post infection (DPI) at 3-day intervals until 42 DPI. Average daily body weight gain, packed cell volume (PCV), concentrations of serum immunoglobulin (Ig) G1, IgG2, IgE and peripheral eosinophil count were measured at 14-day intervals from 0 to 42 DPI. Lymphocyte proliferation in response to somatic antigen of H. contortus was determined by in vitro lymphoproliferation assay, and concentrations of interferon gama (IFN-γ) and interleukin 4 (IL-4) in lymphocyte culture supernatant were measured at 14-day intervals until 42 DPI. Variables were analysed using the repeated measures mixed model procedure over DPI. Faecal egg count was significantly (p < 0.01) lower in Garole sheep than Sahabadi sheep and no faecal eggs were detected in the infected Garole sheep on 30 DPI. Infected Garole sheep had significantly (p < 0.05) higher body weight gain and PCV% than the infected Sahabadi sheep. In the infected Garole sheep, serum Ig except IgE increased significantly (p < 0.05) compared to infected Sahabadi sheep. On 28 DPI, peripheral eosinophil number, in vitro lymphoproliferation as well as concentrations of IFN-γ and IL-4 in culture supernatant were significantly (p < 0.05) higher in the infected Garole sheep than in the infected Sahabadi sheep. Parasitological observations indicated that Garole sheep were resistant to H. contortus and they exhibited greater cellular as well as humoral immune responses compared to Sahabadi sheep.

Modulatory functions of recombinant electron transfer flavoprotein α subunit protein from Haemonchus contortus on goat immune cells in vitro.

Suppression and modulation of the host immune response to parasitic nematodes have been extensively studied. In the present study, we cloned and produced recombinant electron transfer flavoprotein α subunit (ETFα) protein from Haemonchus contortus (rHCETFα), a parasitic nematode of small ruminants, and studied the effect of this protein on modulating the immune response of goat peripheral blood mononuclear cells (PBMCs) and monocytes. Immunohistochemical tests verified that the HCETFα protein was localized mainly in the intestinal wall and on the body surface of worms. Immunoblot analysis revealed that rHCETFα was recognized by the serum of goats artificially infected with H. contortus. Immunofluorescence analysis indicated that rHCETFα bound to the surface of PBMCs. rHCETFα was co-incubated with goat PBMCs to observe the immunomodulatory effects exerted by HCETFα on proliferation, apoptosis, cytokine secretion and nitric oxide (NO) production. The results showed that rHCETFα suppressed the proliferation of goat PBMCs stimulated by concanavalin A and induced apoptosis in goat PBMCs. After rHCETFα exposure, IL-2, IL-4, IL-17A and TNF-α expression was markedly reduced, whereas secretion of TGF-ß1 was significantly elevated, in goat PBMCs. Moreover, rHCETFα up-regulated NO production in a dose-dependent manner. FITC-dextran internalization assays showed that rHCETFα inhibited phagocytosis of goat monocytes. These results elucidate the interaction between parasites and hosts at the molecular level, suggest a possible immunomodulatory target and contribute to the search for innovative proteins that may be candidate targets for drugs and vaccines.

Characterization of a rhodanese homologue from Haemonchus contortus and its immune-modulatory effects on goat immune cells in vitro.

BACKGROUND: Modulation of the host immune response by nematode parasites has been widely reported. Rhodaneses (thiosulfate: cyanide sulfurtransferases) are present in a wide range of organisms, such as archaea, bacteria, fungi, plants and animals. Previously, it was reported that a rhodanese homologue could be bound by goat peripheral blood mononuclear cells (PBMCs) in vivo. METHODS: In the present study, we cloned and produced a recombinant rhodanese protein originating from Haemonchus contortus (rHCRD), a parasitic nematode of small ruminants. rHCRD was co-incubated with goat PBMCs to assess its immunomodulatory effects on proliferation, apoptosis and cytokine secretion. RESULTS: We verified that the natural HCRD protein localized predominantly to the bowel wall and body surface of the parasite. We further demonstrated that serum produced by goats artificially infected with H. contortus successfully recognized rHCRD, which bound to goat PBMCs. rHCRD suppressed proliferation of goat PBMCs stimulated by concanavalin A but did not induce apoptosis in goat PBMCs. The production of TNF-α and IFN-γ decreased significantly, whereas secretion of IL-10 and TGF-ß1 increased, in goat PBMCs after exposure to rHCRD. rHCRD also inhibited phagocytosis by goat monocytes. Moreover, rHCRD downregulated the expression of major histocompatibility complex (MHC)-II on goat monocytes in a dose-dependent manner, but did not alter MHC-I expression. CONCLUSIONS: These results propose a possible immunomodulatory target that may help illuminate the interactions between parasites and their hosts at the molecular level and reveal innovative protein species as candidate drug and vaccine targets.

Local and systemic immune mediators of Morada Nova lambs with divergent Haemonchus contortus resistance phenotypes.

AIMS: Local and systemic immune mediators of Morada Nova lambs with divergent Haemonchus contortus resistance phenotypes were evaluated. METHODS AND RESULTS: Lambs were ranked through faecal egg counts (FEC) after two parasitic challenges with 4,000 H.contortus L3 . After the second challenge, the lambs underwent a third artificial infection and were euthanized 7 days later. Immune-related genes were quantified locally in abomasal mucosa and lymph nodes (CD4, IFNγ, IL4, IL5, IL13, IL2RA and MS4A2) and systemically in the whole blood (IL4 and IL13). Anti-H. contortus IgG and IgA antibodies and eosinophils and mast cells counts were also investigated. Resistant animals presented higher systemic IgG and IgA titres, both negatively correlated with FEC. Susceptible animals had higher blood levels of IL4 transcripts. At the local level, resistant lambs had higher eosinophils counts and superior MS4A2 levels in abomasal fundic mucosa, besides higher IgA levels in abomasal mucus, while susceptible lamb had superior IL4 expression in abomasal lymph nodes. CONCLUSION: These data indicate that resistant lambs had an immune response mediated by antibody-mediated cytotoxicity. Also, the systemic humoral profile, particularly IgA isotype, seems to be a good resistance marker for Morada Nova sheep, as we found differences between groups even when FEC did not differ.

Immune response related to Pelibuey sheep naturally infected with gastrointestinal nematodes in a tropical region of Mexico.

We analysed the immune response involved in sheep naturally infected with gastrointestinal (GI) nematodes. Fifteen Pelibuey lambs were grazed in paddocks contaminated with GI nematodes for 13 weeks. To assess the infection, the number of eggs per gram (epg) and the percentage of packed cell volume (pcv) were evaluated. Blood and abomasal tissue samples were collected at week 8 post-infection to analyse the expression levels of IL-4, IL-5, IL-6, IL-8, IL-13, TGF-ß and FCεR1A genes. The nematode Haemonchus contortus was the main species identified. In addition, two groups of lambs were classified based on the x ± SE of epg and pcv values: G-1, with 151 ± 28 and 29 ± 0.33%, respectively, and G-2, with 475 ± 59.5 and 26 ± 0.38%, respectively. For G-1, upregulation of IL-4, IL-8, IL-13, TGF-ß and FCεR1A genes from 2.42- to 14.99-fold was observed in blood and abomasal tissue samples (p > .05), and IL-5, IL-8 and TGF-ß genes had significant gene expression levels in blood (p < .05). For G-2, moderate gene expression levels, ranging from 1.22- to 3.45-fold, were observed in abomasal tissue (p > .05), and the IL-5 gene presented significant gene expression in blood (p < .05). Strong positively correlated values (r) between pcv and IL-4, IL-8 and TGF-ß genes were observed in G-1. In contrast, significant negative correlations between epg and IL-4, IL-5 and FCεR1A genes indicate acute infection for G-2. Our results suggest that IL-4, IL-5, IL-13, TGF-ß and FCεR1A genes are important modulators of GI nematode infections of Pelibuey lambs.

Artificial Haemonchus contortus infection as a strategy to induce protective immune response to natural infection in Pelibuey lambs.

The objective of this study was to evaluate the reduction in nematode faecal egg count (FEC) in Pelibuey lambs segregated as resistant (RES), susceptible (SUS) and intermediate (INT) to gastrointestinal nematodes. Twenty-nine weaned Pelibuey lambs, aged five months old, free of nematode infection, were used. Nine lambs were RES, six were SUS and 14 were INT lambs. The study consisted of two phases: in Phase 1 the lambs were infected experimentally with Haemonchus contortus. In Phase 2, the lambs were naturally infected by grazing. Faecal and blood samples were taken every week. The packed cell volume and total protein were quantified. The FEC value (FECmax) per lamb was recorded together with a natural reduction in FEC in the two phases. The data were analysed with a model of measures repeated over time. During Phase 1, the RES lambs showed the lowest FEC (1061 ± 1053) compared to the other groups (INT: 2385 ± 1794 eggs per gram of faeces (EPG); and SUS: 3958 ± 3037 EPG). However, in Phase 2 no significant differences (p > 0.05) were observed between the groups of lambs (RES: 275 ± 498 EPG; SUS: 504 ± 1036 EPG; and INT: 603 ± 1061 EPG). At the end of Phase 1, the FEC of RES lambs was naturally reduced by 75.5% in respect to FECmax (p < 0.05), and at the end of Phase 2 the reduction in FEC was 90% in respect to FECmax (p > 0.05); the same behaviour was observed in RES and SUS lambs. It is concluded that the artificial infection in the lambs induced a more rapid immune response in RES than SUS lambs, and all lambs developed high acquired resistance by continuous infection.

Unveiling the immunomodulatory properties of Haemonchus contortus adhesion regulating molecule 1 interacting with goat T cells.

BACKGROUND: Gastrointestinal nematodes could release excretory-secretory (ES) proteins into the host environment to ensure their survival. These ES proteins act as immunomodulators to suppress or subvert the host immune response via the impairment of immune cell functions, especially in chronic infections. In our preliminary study, Haemonchus contortus adhesion-regulating molecule 1 (HcADRM1) was identified from H. contortus ES proteins (HcESPs) that interacted with host T cells via liquid chromatography-tandem mass spectrometry analysis. However, little is known about HcADRM1 as an ES protein which may play a pivotal role at the parasite-host interface. METHODS: Based on bioinformatics approaches, multiple amino acid sequence alignment was conducted and the evolutionary relationship of HcADRM1 with ADRM1 orthologues was extrapolated. Employing RT-qPCR and immunohistochemistry assays, temporal transcriptional and spatial expression profiles of HcADRM1 were investigated. Using immunostaining approaches integrated with immunological bioassays, the immunomodulatory potentials of HcADRM1 on goat T cells were assessed. RESULTS: We hereby demonstrated that HcADRM1 with immunodiagnostic utility was a mammalian ADRM1 orthologue abundantly expressed at all developmental stages of H. contortus. Given the implications of ADRM1 proteins in cell growth, survival and development, we further investigated the immunomodulatory property of HcADRM1 as an individual ES protein acting at the parasite-host interface. The rHcADRM1 stimuli notably suppressed T cell viability, promoted intrinsic and extrinsic T cell apoptosis, inhibited T cell proliferation and induced cell cycle arrest at G1 phase. Simultaneously, rHcADRM1 stimuli exerted critical controls on T cell cytokine secretion profiles, predominantly by restraining the secretions of interleukin (IL)-4, IL-10 and interferon-gamma. CONCLUSIONS: Importantly, HcADRM1 protein may have prophylactic potential for anti-H. contortus vaccine development. Together, these findings may contribute to the clarification of molecular and immunomodulatory traits of ES proteins, as well as improvement of our understanding of parasite immune evasion mechanism in H. contortus-host biology.

Characterization of ovine monocyte activity when cultured with Haemonchus contortus larvae in vitro.

AIMS: The objective of this study was to identify and characterize cell populations within ovine peripheral blood mononuclear cells (PBMCs) associated with Haemonchus contortus (Hc) larval morbidity and impairment in vitro. METHODS AND RESULTS: Monocytes and lymphocytes were separated from PBMC from parasite-resistant St. Croix (STC) sheep and parasite-susceptible Suffolk (SUF) sheep. Cells were cultured with Hc third stage larvae (L3) for 9 h. Larval morbidity was assessed using ATP concentration. Activation status was determined through gene expression analysis and enzyme inhibition. Enzymes arginase-1 (Arg1) and inducible nitric oxide synthase (iNOS) were inhibited using BEC (S-(2-boronoethyl)-I-cysteine) and 1400W (N-(3-(aminomethyl)benzyl)acetamidine), respectively. Larval ATP was lower when cultured with STC-derived monocytes (0.015 µmol/L ATP) compared to SUF-derived monocytes (0.067 µmol/L ATP) (P < .001), or lymphocytes from either breed (STC: 0.085 µmol/L, SUF: 0.112 µmol/L ATP) (P < .001). SUF-derived monocytes displayed higher expression of M1 genes, whereas STC-derived monocytes displayed M2 genes continuously. Inhibition of Arg1 decreased monocyte function in both breeds, whereas iNOS inhibition restored SUF-derived monocyte function. CONCLUSIONS: Together, these data indicate STC-derived monocytes favour M2 phenotype when exposed to L3, where SUF-derived monocyte function resembled M1 phenotype and described potential for improving Suffolk sheep through modulating inflammatory responses.

A Novel α/ß Hydrolase Domain Protein Derived From Haemonchus contortus Acts at the Parasite-Host Interface.

The α/ß-hydrolase domain (ABHD) proteins belonging to α/ß-hydrolase (ABH) superfamily are ubiquitously distributed throughout all the organisms, and their functional roles have been implicated in energy metabolism, cell signaling, growth and development. In our preliminary work, we identified a novel ABHD protein derived from Haemonchus contortus excretory-secretory (ES) proteins (HcESPs) that interacted with host T cells. Here, we demonstrated that H. contortus ABHD (HcABHD) protein, expressed in all life-cycle stages of H. contortus, is a mammalian ABHD17 homolog with immunodiagnostic utility and lipase activity. Given its catalytic activities and immunomodulatory potentials, we further investigated the functional diversity of HcABHD as an individual ES protein in parasite-host interactions. HcABHD protein may serve as depalmitoylase or thioesterase to suppress cell viability, inhibit cell proliferation, induce intrinsic and extrinsic T cell apoptosis, and cause cell cycle arrested at G1 phase. Moreover, recombinant HcABHD stimuli exerted critical controls on T cell cytokine production profiles, predominantly by inhibiting the secretions of interleukin (IL)-4, interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-ß) 1, and promoting IL-10 production. As the immunomodulator acting at the parasite-host interface, HcABHD protein may have potential applications for the vaccine development of therapeutic intervention. Together, these findings may help illuminate the molecular and particularly immunomodulatory aspects of ES proteins and contribute to an enhanced understanding of parasite immune evasion in H. contortus-host biology.

Interleukin-13 induces paralysis of Haemonchus contortus larvae in vitro.

AIMS: Interleukin-13 (IL-13) is a Th2-associated cytokine that typically induces gut contractility and mucus secretion to eliminate helminth parasites from the digestive tract. Little evidence exists of IL-13's direct effect on Haemonchus contortus larvae (L3) and thus was the objective of this study. METHODS: To test effects of IL-13 on H contortus, L3 were treated with ovine recombinant (r) IL-13 (1 µg/mL); motility and morbidity were assessed. Monocytes isolated from H contortus-resistant St. Croix (STC) and susceptible Suffolk (SUF) sheep were treated with anti-IL-13 blocking antibody to elucidate differences in host immune response. RESULTS: rIL-13 treatment reduced L3 speed (27 µm/s) and distance (7.5 µm) compared to untreated L3 (speed: 94 µm/s; distance: 27 µm) (P < .001). Comparison of larval speed to known paralytic levamisole (LEV) revealed no difference between treatments (rIL13: 23 µm/s; LEV 27 µm/s). Additionally, rIL-13 had no effect on larval morbidity. Blocking IL-13 reduced monocyte-driven larval morbidity (0.13 µmol/L ATP) and increased larval motility (88 µm/s; 27 µm) compared to larvae treated with STC-monocytes alone (0.07 µM ATP; 34 µm/s; 8 µm) (P < .05). CONCLUSIONS: These data indicate IL-13 has a dual capability paralysing L3 and contributing to monocyte-driven larval morbidity, and also indicate breed differences.

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.

Kinetics of IgA and eosinophils following a low-dose, predominantly Haemonchus contortus infection of Boer goats.

AIMS: Most breeds of goat are more susceptible to nematode infection than sheep, and this appears to be a consequence of less effective immune responses. Several papers have considered the effectiveness of eosinophils and immunoglobulin A (IgA) in goats but differences in the induction of responses have not been studied in the same detail. The aim of this study was to look at the induction of eosinophil and IgA responses in Boer goats reared indoors under intensive conditions. METHODS AND RESULTS: The goats were experimentally infected with a low dose of 2400 Haemonchus contortus, Trichostrongylus spp. and Oesophagostomum spp. at a 6:1:1 ratio. Faecal egg counts (FEC), packed cell volume (PCV), IgA activity against third-stage larvae and peripheral eosinophilia were measured twice a week for eight weeks. The infection generated an IgA response but did not significantly increase peripheral eosinophilia in the 25 infected kids compared with the 4 control animals. FEC was not associated with IgA activity or eosinophilia. CONCLUSION: A detailed analysis of IgA and eosinophil responses to deliberate nematode infection in Boer goats showed that there was an increase in nematode-specific IgA activity but no detectable eosinophil response. In addition, there was no association between increased IgA activity or eosinophilia with egg counts and worm burdens. These suggest that IgA and eosinophils do not act to control nematode infection in goats.

Recombinant elongation factor 1 alpha of Haemonchus contortus affects the functions of goat PBMCs.

Excretory/secretory proteins of Haemonchus contortus (HcESPs) intermingle comprehensively with host immune cells and modulate host immune responses. In this study, H contortus ES antigen named as elongation factor 1 alpha (HcEF-1α) was cloned and expressed. The influences of recombinant HcEF-1α on multiple functions of goat peripheral blood mononuclear cells (PBMCs) were observed in vitro. Immunoblot analysis revealed that rHcEF-1α was recognized by the serum of goat infected with H contortus. Immunofluorescence analysis indicated that rHcEF-1α was bound on surface of PBMCs. Moreover, the productions of IL-4, TGF-ß1, IFN-γ and IL-17 of cells were significantly modulated by the incubation with rHcEF-1α. The production of interleukin IL-10 was decreased. Cell migration, cell proliferation and cell apoptosis were significantly increased; however, nitric oxide production (NO) was significantly decreased. The MHC II molecule expression of cells incubated with rHcEF-1α was increased significantly, whereas MHC-I was not changed as compared to the control groups (PBS control and pET32a). These findings indicated that rHcEF-1α protein might play essential roles in functional regulations of HcESPs on goat PBMC and mediate the immune responses of the host during host-parasite relationship.

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.

RNA-Sequencing of ovine PBMC after exposure to Haemonchus contortus larval antigen.

Mechanisms of immune activation in effector cells during Haemonchus contortus infection in sheep are currently unknown. Microarray experiments have been performed on tissues of H contortus infected sheep of varying parasite resistance during early and late points of infection, but not in immune effector cells. The purpose of this study was to compare early gene activation in peripheral blood mononuclear cells (PBMC) from primed parasite susceptible (Suffolk) and resistant (St. Croix) sheep in response to H contortus larval antigen (HcLA). Peripheral blood mononuclear cells were cultured for 6 hours with HcLA, and RNA-sequencing was performed. St. Croix PBMC upregulated 499 unique genes in response to HcLA while Suffolk PBMC upregulated 130 unique genes and 25 genes were shared between the two breeds. St. Croix PBMC had increased expression of genes associated with immune function, signal transduction, response to stress and others. In addition, while mechanisms of innate recognition of H contortus are unknown, multiple pattern recognition receptors were found to be upregulated in St. Croix PBMC cultured with HcLA and none were found to be upregulated in Suffolk PBMC. These patterns of immune gene activation may contribute to St. Croix's rapid response and ability to resist H contortus infection.