Characterisation of protective vaccine antigens from the thiol-containing components of excretory/secretory material of Ostertagia ostertagi.

Previous vaccination trials have demonstrated that thiol proteins affinity purified from Ostertagia ostertagi excretory-secretory products (O. ostertagi ES-thiol) are protective against homologous challenge. Here we have shown that protection induced by this vaccine was consistent across four independent vaccine-challenge experiments. Protection is associated with reduced cumulative faecal egg counts across the duration of the trials, relative to control animals. To better understand the diversity of antigens in O. ostertagi ES-thiol we used high-resolution shotgun proteomics to identify 490 unique proteins in the vaccine preparation. The most numerous ES-thiol proteins, with 91 proteins identified, belong to the sperm-coating protein/Tpx/antigen 5/pathogenesis-related protein 1 (SCP/TAPS) family. This family includes previously identified O. ostertagi vaccine antigens O. ostertagi ASP-1 and ASP-2. The ES-thiol fraction also has numerous proteinases, representing three distinct classes, including: metallo-; aspartyl- and cysteine proteinases. In terms of number of family members, the M12 astacin-like metalloproteinases, with 33 proteins, are the most abundant proteinase family in O. ostertagi ES-thiol. The O. ostertagi ES-thiol proteome provides a comprehensive database of proteins present in this vaccine preparation and will guide future vaccine antigen discovery projects.

Characterization of IL-10-producing neutrophils in cattle infected with Ostertagia ostertagi.

IL-10 is a master regulator of immune responses, but its cellular source and function in cattle during the initial phase of immune priming have not been well established. Despite a massive B cell response in the abomasal draining lymph nodes in Ostertagia ostertagi (OO)-infected cattle, protective immunity is slow to develop, and partial protection requires years of repeated exposure. In addressing this problem, our initial hypothesis was that B cells produce IL-10 that downregulates the host protective immune response. However, our results showed that neutrophils made up the majority of IL-10-producing cells in circulation and in secondary lymphoid tissues, particularly the spleen (80%). Conversely, IL-10-producing B cells were rare. In addition, approximately 10% to 20% of the neutrophils in the blood and spleen expressed MHC II and were IL-10 negative, suggesting that neutrophils could also participate in antigen presentation. In vitro investigation of bovine neutrophils revealed that exposure thereof to OO extract increased IL-10 and MHC II expression in these cells in a dose-dependent manner, consistent with IL-10+/MHC II+ neutrophils detected in cattle shortly after experimental OO infection. Co-culture of untreated neutrophils with anti-CD3 antibody (Ab)-stimulated CD4+ T cells led to enhanced T cell activation; also, IL-10 depletion with neutralizing Ab enhanced the stimulatory function of neutrophils. OO extract depressed neutrophil stimulation of CD4+ T cells in the presence of IL-10-neutralizing Ab, suggesting that OO utilizes both IL-10-dependent and independent mechanisms to manipulate the bovine immune response. Finally, contact and viability were required for T cell-stimulatory neutrophil function. This report, to the best of our knowledge, is the first to demonstrate that neutrophil-derived IL-10 is directly involved in T cell regulation in cattle. Our data suggest that neutrophils and neutrophil-derived IL-10 are co-opted by nematode parasites and other pathogens to attenuate host immune responses and facilitate pathogen survival.

Analysis of the mucosal immune responses induced by single and trickle infections with the bovine abomasal nematode Ostertagia ostertagi.

The purpose of this study was to provide more information on the kinetics of the immunological changes occurring in the abomasal mucosa after single and trickle infections with the bovine parasite Ostertagia ostertagi. The time course analysis of gene expression revealed that the major changes coincided with the emergence of adult worms from the gastric glands. These changes consisted of a simultaneous upregulation of Th1- and Th2-type cytokines. In addition, a single O. ostertagi infection elicited an upregulation of the epithelial-derived cytokine IL33, while TSLP expression levels were not impacted. Apart from the massive increase in inflammatory cytokines IL6, IL17 and IL21, O. ostertagi infection also elicited an upregulation of the immunosuppressors TGFB, IL10 and ARG1, as well as NK and γδ-T cell markers. Furthermore, the cytotoxic factors granulysin, perforin and granzyme B were upregulated following an O. ostertagi infection. Analysis of cytokine transcript levels in animals receiving trickle infections for 60 days showed a similar trend as observed following a single infection except for IL33, IL6, GATA-3, TBX21 and NCR1, which were no longer upregulated after trickle infections. Finally, the long trickle infections were associated with mucosal eosinophilia and mastocytosis.

Galectin-11 induction in the gastrointestinal tract of cattle following nematode and protozoan infections.

Galectin-11 (LGALS11) has been suggested to play an important role in protective immunity against gastrointestinal nematodes in ruminants. However, in cattle, this molecule has not been characterized in detail. In the current study, it was shown that transcription of LGALS11 was highly inducible in the bovine abomasal mucosa after an Ostertagia ostertagi infection. LGALS11 protein expression was also increased in the abomasal mucosa following O. ostertagi infection and localized to the nucleus and cytoplasm of epithelial cells and the mucus. Using in vitro abomasal epithelial cell cultures, it was shown that LGALS11 induction was associated with the proliferative and dedifferentiated status of cells. However, LGALS11 was not induced following stimulation with O. ostertagi excretory-secretory products. These results suggest that LGALS11 induction in vivo may be an indirect rather than a direct effect of the parasite on the epithelium. In addition, LGALS11 transcript was also detected in the abomasal lymph nodes where it was shown to be transcribed in MHCII+ cells; however, transcription levels in the lymph nodes were not altered after O. ostertagi infection. In addition, LGALS11 was also induced in the small intestine by different types of parasites, including the nematode Cooperia oncophora and the protozoan parasite Giardia duodenalis.

Effects of Teladorsagia (Ostertagia) circumcincta infection on lambs selected for high fleece weight.

The physiological processes leading to the expression of the resilient phenotype, which allow animals to maintain a relatively higher production level during infection, have been investigated in lambs from a closed flock selected for 40 generations for high fleece weight (HFW), but with higher FEC and worm burdens than their unselected control (C) flock run in parallel. After recovery from surgery to implant abomasal cannulae, eight parasite-naive lambs from each flock were infected intraruminally at 4.5 months-of-age with 50,000 Teladorsagia circumcincta L3. Blood, abomasal fluid and faecal samples were collected daily for measurement of serum gastrin and pepsinogen concentrations, blood eosinophils, abomasal pH and FEC. Four lambs from each flock were euthanased on Day 8 post-infection and the other four on Day 28 post-infection. At necropsy, abomasal contents and tissues were collected for worm counts, abomasal lymph nodes and fundic tissue for cytokine gene expression and fundic tissue for histopathology. Expression of resilience appeared to be age-dependent as there were no significant differences in either FEC or worm burden between lambs from the two flocks, unlike older HFW lambs in a previous study. Abomasal secretion did not differ between flocks. Histopathological changes were typical of parasitism: inflammatory cells, mainly eosinophils and lymphocytes, were numerous in nodular areas and there were fewer TGF-alpha positive parietal cells, many of which were vacuolated. By Day 28 p.i., globule leucocytes were present. Mucosal thickness was significantly greater on Day 8 than Day 28 p.i. (p=0.000) and in C than HFW lambs. There were fewer parietal cells on Day 28 than on Day 8 p.i. (p=0.003) for pooled data. Circulating eosinophil counts increased moderately in both groups, significantly less in the HFW lambs. Fewer tissue and blood eosinophils in the HFW than C group on Day 8 p.i. were consistent with cytokine gene expression patterns, particularly lower IL-5 levels. Worm count decreased by 90% by Day 28 p.i., along with declining tissue eosinophil counts and IL-13 gene expression and increasing IL-10 and IL-4 gene expression. Food intake was depressed less in the HFW lambs, suggesting that maintenance of appetite could be an important aspect of the physiological basis for resilience. Although the resilient phenotype was not apparent at the younger age, lesser effects on food intake, differences in ALN cytokine profiles and lower blood and tissue eosinophil numbers in the HFW lambs may lead to the expression of resilience when older.

The effect of corticosteroid treatment on local immune responses, intake and performance in lambs infected with Teladorsagia circumcincta.

The nutritional cost of, and the sequential cellular changes associated with the developing immune response to the abomasal parasite Teladorsagia circumcincta were investigated using corticosteroid-induced immune-suppression. Six-month-old lambs with minimal nematode experience were either infected with 4000 L3 T. circumcincta per day (group IF), similarly infected and concurrently immune-suppressed with methylprednisolone acetate (group ISIF), immune-suppressed only (group IS) or remained as controls (group C). Food intake, faecal egg count (FEC) and antibody titres in plasma were recorded weekly, worm burden at necropsy on day 63 p.i. and body composition by X-ray computed tomography on days -2 and 62 p.i. Furthermore, sequential immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from additional animals that were fitted with an abomasal cannula and either infected with the same regime as IF animals above (group CnIF) or concurrently infected and immune-suppressed as above (group CnISIF). Corticosteroid treatment resulted in greater FECs (P<0.01) and worm burdens (P<0.01) in both ISIF and CnISIF compared with IF and CnIF sheep, respectively. Infection reduced feed intake by 17% between 14 and 28 days p.i. (P<0.05) and efficiency of energy utilisation by 20% (P=0.07) in IF animals but not in ISIF animals. Mast cells, globule leukocytes and IgA in tissue biopsy samples were elevated in CnIF from 42 days p.i., all of which were abrogated by corticosteroid treatment. The ability to regulate the worm population appeared to be associated with a rise in tissue IgA concentration and numbers of globule leucocytes (GL). The results support the hypothesis that a majority of the production losses that occur during infection of lambs with T. circumcincta in lambs are a consequence of the host immune response. These findings may have implications for regimes that promote the development of a strong host immune reaction to gastrointestinal parasites in lambs.

Vaccination against Ostertagia ostertagi with subfractions of the protective ES-thiol fraction.

Previous vaccination trials against Ostertagia ostertagi in cattle have demonstrated the protective capacity of a protein fraction termed ES-thiol, which is enriched for activation-associated secreted proteins (ASPs) and cysteine proteases. In this study, ES-thiol was subfractionated through Q-Sepharose anion exchange chromatography to determine whether the ASPs and/or the cysteine proteases are responsible for the induced protection. Calves (seven/group) were immunized three times intramuscularly with 100 microg of ES-thiol or equivalent amounts of an ASP-enriched fraction, a cysteine protease-enriched fraction or a rest fraction, with QuilA adjuvant. A negative control group only received QuilA. After the final immunization the animals were challenged with a trickle infection of 25,000 infectious L3 larvae (1000 L3/day; 5 days/week). During a 2-month period the geometric mean cumulative faecal egg count (FEC) of the ES-thiol group was reduced by 62% compared to the QuilA control group (P<0.05). Groups injected with the ASP-enriched, the cysteine protease-enriched and the rest fraction demonstrated a reduction in cumulative FEC of 74, 80 and 70%, respectively (P<0.01). Although no significant reductions in worm burdens were observed, adult male and female worms were significantly smaller in all vaccinated groups (P<0.05), except for male worms from the ES-thiol group. These results suggest the protective capacity of ASPs and the presence of other protective antigens in the ES-thiol fraction.

Validation of the protective Ostertagia ostertagi ES-thiol antigens with different adjuvantia.

Intramuscular immunization of calves with an excretory-secretory antigen fraction enriched for cysteine proteinase activity (ES-thiol) and QuilA as adjuvant induces a protective immune response against the abomasal nematode Ostertagia ostertagi. The objectives of the present study were to confirm the protective capacity of ES-thiol in combination with QuilA, to test Al(OH)(3) as adjuvant for vaccination against O. ostertagi and to look for correlations between protection and immunological effector responses. Calves(seven animals/group) were vaccinated three times intramuscularly with 100 micro g antigen and/or adjuvant (ES-thiol with QuilA, ES-thiol with Al(OH)(3), QuilA alone and Al(OH)(3) alone) and subsequently challenged with a trickled oral infection of 25 000 infective larvae in total over 25 days. Faecal egg counts in the ES-thiol QuilA group were reduced by 56% during the two-month period of the trial compared to the QuilA control group (P < 0.002). Calves immunized with ES-thiol QuilA had significantly smaller adult worms (P < 0.002) and less eggs/female worm (P < 0.05) compared to the QuilA control group. No differences in egg output, worm counts or parameters of worm fitness were observed in the ES-thiol Al(OH)(3) group compared to the Al(OH)(3) control group. Although the protective immune mechanism against O. ostertagi remains unknown, protection in the ES-thiol QuilA group was associated with high levels of parasite-specific antibodies in the abomasal mucosa.

Vaccination with an Ostertagia ostertagi polyprotein allergen protects calves against homologous challenge infection.

As an alternative to antihelminthic drugs, we are exploiting vaccination to control infections with the abomasal nematode Ostertagia ostertagi in cattle. Our focus for vaccine targets is excretory-secretory (ES) products of this parasite. One of the most abundant antigens in larval and adult Ostertagia ES products is a protein homologous to nematode polyprotein allergens. We found that the Ostertagia polyprotein allergen (OPA) is encoded by a single-copy gene. OPA comprises three or more repeated units, and only the 15-kDa subunits are found in ES products. The native antigen is localized in the intestinal cells of third-stage larvae and in the hypodermis and cuticle of fourth-stage larvae and adult parasites. Vaccination of cattle with native OPA (nOPA) in combination with QuilA resulted in protection against Ostertagia challenge infections. The geometric mean cumulative fecal egg counts in the nOPA-vaccinated animals were reduced by 60% compared to the counts in the control group during the 2-month course of the experiment. Both male and female adult worms in nOPA-vaccinated animals were significantly shorter than the worms in the control animals. In the abomasal mucus of vaccinated animals the nOPA-specific immunoglobulin G1 (IgG1) and IgG2 levels were significantly elevated compared to the levels in the control animals. Reductions in the Ostertagia egg output and the length of the adult parasites were significantly correlated with IgG1 levels. IgG2 titers were only negatively associated with adult worm length. Protected animals showed no accumulation of effector cells (mast cells, globular leukocytes, and eosinophils) in the mucosa. In contrast to the native antigen, recombinant OPA expressed in Escherichia coli did not stimulate any protection.

Effects of gastrointestinal nematode infection on the ruminant immune system.

Gastrointestinal (GI) nematodes of ruminants evoke a wide variety of immune responses in their hosts. In terms of specific immune responses directed against parasite antigens, the resulting immune responses may vary from those that give strong protection from reinfection after a relatively light exposure (e.g. Oesophagostomum radiatum) to responses that are very weak and delayed in their onset (e.g. Ostertagia ostertagi). The nature of these protective immune responses has been covered in another section of the workshop and the purpose of this section will be to explore the nature of changes that occur in the immune system of infected animals and to discuss the effect of GI nematode infections upon the overall immunoresponsiveness of the host. The discussion will focus primarily on Ostertagia ostertagi because this parasite has received the most attention in published studies. The interaction of Ostertagia and the host immune system presents what appears to be an interesting contradiction. Protective immunity directed against the parasite is slow to arise and when compared to some of the other GI nematodes, is relatively weak. Although responses that reduce egg output in the feces or increase the number of larvae undergoing inhibition may occur after a relatively brief exposure (3-4 months), immune responses which reduce the number of parasites that can establish in the host are not evident until the animal's second year. Additionally, even older animals that have spent several seasons on infected pastures will have low numbers of Ostertagia in their abomasa, indicating that sterilizing immune responses against the parasite are uncommon. In spite of this apparent lack of specific protective immune responses, infections with Ostertagia induce profound changes in the host immune system. These changes include a tremendous expansion of both the number of lymphocytes in the local lymph nodes and the number of lymphoid cells in the mucosa of the abomasum. This expansion in cell numbers involves a shift away from a predominant classic T cell population (CD2 and CD3 positive), to a population where T cell percentages are decreased and B cells (immunoglobulin-bearing) and gamma-delta cells are increased. At the same time the expression of messenger RNAs for T cell cytokines (IL2, IL4, IL10 and gamma-interferon) is changed to that of increased expression of IL4 and IL10 and decreased expression of IL2 and perhaps of gamma-interferon. The reasons for these changes remain to be elucidated, but it is evident that the lack of protective immune responses is not the result of a poor exposure of the host to parasite products, or to the stomach being an immunoprivileged site. In fact, a superficial look at the responses elicited indicates that Ostertagia induces responses (the so-called TH2 mediated responses) that are widely considered to be the type of responses necessary for protection against GI nematodes. There are many factors that could lead to this apparent lack of immunity in the face of a strong stimulation of immune responses including: (1) the elicitation of suboptimal responses; (2) the failure of the abomasum to function as an efficient effector organ; (3) active evasion of the functional immune response by the parasite; and (4) that these classic responses are not protective in this particular ruminant-parasite system and that novel protective mechanisms may be required. The strong stimulation of the host gut immune system by Ostertagia and perhaps by other GI nematode infections, raises questions about the potential effects of such infections on the overall well-being of the host. A number of authors have indicated that Ostertagia infections may diminish the host's ability to mount subsequent immune responses to antigenic challenges such as vaccination against other infectious organisms. In addition, recent studies have indicated that infections with GI nematodes may result in increased circulatory levels of stress-related hormo

Surface and excretory/secretory antigens of fourth-stage larvae and adult Ostertagia circumcincta.

With the use of an enzyme-linked immunosorbent assay (ELISA), it was revealed that surface antigens of both adult and fourth-stage larvae (L4) of Ostertagia circumcincta induced high levels of serum IgG antibodies, while serum IgA antibody levels were low but increased significantly (P < 0.01) after infection. Immunofluorescence studies on the surface of viable L4 and adult nematodes showed that the IgG response was stage-specific only in animals vaccinated with adult surface extracts. The results of Western blot analysis using these antibodies suggested that at least eight polypeptides were shed from the L4 surface to the environment and that infection induced (or boosted) IgG antibody against a further four polypeptides. A comparison of reactivity of pre- and post-infection sera of sheep vaccinated with adult nematode surface antigens suggested that only one of the antigens stripped from the nematode surface was immunogenic and/or present in a concentration sufficient to induce an IgG response following parenteral vaccination. Infection boosted the IgG antibodies to a further four polypeptides. Only one polypeptide of 63 kDa seems to be shed in vivo from the adult nematode surface. Ten to eleven antigens were recognised in adult excretory/secretory products by serum IgG of multiple-infected sheep.

Local eosinophil- and mast cell-related responses in abomasal nematode infections of lambs.

Eosinophil numbers in peripheral blood and eosinophil potentiating activity (EPA) and sheep mast cell protease (SMCP) in efferent gastric lymph were monitored in lambs during infections with Ostertagia circumcincta. Worm burdens, eosinophil numbers in bone marrow, abomasal mucosa and gastric lymph node, as well as mast cell numbers and SMCP concentrations in mucosa and mucus, were determined in post mortem samples. In naive lambs, high and relatively uniform worm burdens were present 10 days after primary infection and these were associated with only mild blood and tissue eosinophilia. By day 21 worm burdens were markedly lower and more variable. There was more evidence of eosinophil and mast cell accumulation in mucosa, and numbers in bone marrow were also higher than on day 10. However, neither EPA nor SMCP were detectable in lymph. By contrast, EPA and SMCP were present in substantial amounts in draining lymph within 48 h of challenge (secondary) infection of previously exposed lambs. EPA was inversely related to worm burdens recovered on day 10, as were abomasal mucosal and mucus SMCP concentrations. Elevated eosinophil numbers were also consistently detected in blood, bone marrow, mucosa and gastric lymph node. The results suggest that host immune defence against secondary, but not primary, exposure to O. circumcincta involves a rapidly mobilised local inflammatory component.

Characterisation of ovine mast cells derived from in vitro culture of haemopoietic tissue.

Ovine mast cells generated in vitro from bone marrow (BMMC) were compared with mucosal mast cells (MMC) isolated from parasitised abomasum. Ultrastructurally, the granules of BMMC were partially developed and immature. Both cells types contained beta-hexosaminidase, arylsulfatase, histamine, dopamine and sheep mast cell proteinase (SMCP). Greater amounts of beta-hexosaminidase, but less SMCP, histamine and arylsulfatase were present in BMMC. Stimulation with calcium ionophore A23187 caused the secretion of granule constituents and generation of leukotriene C4 by BMMC in a dose-dependent manner. An additional [3H]diisopropylfluorophosphate-binding 31,500 mol. wt. serine esterase, antigenically related to SMCP (27,000 mol. wt.) was present in cultures of BMMC but was not detected in isolated MMC. Both enzymes were detected in BMMC by Day 7 of culture and were secreted concomitantly following stimulation of BMMC with ionophore.

Celiac trunk cannulation for obtaining abomasal lymph from cattle.

Cannulation of the celiac trunk was surgically performed in 26 Holstein steers. The procedure was successful in 23 (88.5%) of the steers. Twenty-two of the steers were infected either naturally or experimentally with the abomasal nematode, Ostertagia ostertagi and/or other gastrointestinal parasites. The remaining 4 steers were not infected. Lymph obtained after surgery was used in various immunologic and biochemical assays. Daily lymph flow rate and total and differential WBC counts were determined after surgery in 4 of the infected and 3 of the noninfected steers. Steers were euthanatized for tissue specimen collection 7 days after surgery. At the time of euthanasia, lymph was still flowing from the cannula of 13 (56.5%) of the steers in which surgery was successful. This surgical procedure represents a valuable technique for studying at the local level, immunologic and physiologic responses of cattle to infection with O ostertagi.

Relationship between cytologic changes in bronchoalveolar lavage fluid and weight gain in calves with gastrointestinal nematodes and lungworms.

Two of three groups of 10 calves each were infected with either 100,000 infective larvae (L3) of Ostertagia spp. and 100,000 L3 of Cooperia spp. or with 4000 L3 of Dictyocaulus viviparus, respectively, at the age of 14 weeks. The third group was not infected. After treatment with an anthelminthic five calves from each group were challenged with either 100,000 L3 of Ostertagia spp. and 100,000 L3 of Cooperia spp. or 4000 L3 of Dictyocaulus at the age of 20 weeks. The calves were 25 weeks old when slaughtered. Total and differential cell counts were determined in bronchoalveolar lavage fluid and showed that neutrophils were the most frequent and eosinophils the least frequent cell present. There was a significant negative relationship between eosinophil levels and weight gain of the calves.

Transfer of immunity to Ostertagia circumcincta and IgA memory between identical sheep by lymphocytes collected from gastric lymph.

Sheep rendered immune to Ostertagia circumcincta were challenged with 50,000 larvae and lymphocytes were collected from the gastric lymph up to eight days after challenge. The cells were transferred intravenously to genetically identical worm-free sheep which, together with controls, were challenged with 50,000 larvae and killed nine days later. Cells obtained during the donors lymphoblast response to challenge transferred partial immunity, measured either as stunting or loss of worms. Significantly less immunity was transferred by cells collected either before or after this response. Thus the responding cells can mediate protective immunity to O circumcincta. On the other hand the donor sheep remained immune to their challenge infection despite being depleted of these functional cells, showing that their presence was not essential for immunity to be maintained. Comparison of the immunoglobulin A (IgA) concentrations in the gastric lymph of recipient and control sheep showed that a local IgA response had also been transferred. Enumeration of mucosal mast cells suggested that a mastocytosis had been transferred to the two recipients which were most immune to challenge.