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.

Plant-based production of a protective vaccine antigen against the bovine parasitic nematode Ostertagia ostertagi.

The development of effective recombinant vaccines against parasitic nematodes has been challenging and so far mostly unsuccessful. This has also been the case for Ostertagia ostertagi, an economically important abomasal nematode in cattle, applying recombinant versions of the protective native activation-associated secreted proteins (ASP). To gain insight in key elements required to trigger a protective immune response, the protein structure and N-glycosylation of the native ASP and a non-protective Pichia pastoris recombinant ASP were compared. Both antigens had a highly comparable protein structure, but different N-glycan composition. After mimicking the native ASP N-glycosylation via the expression in Nicotiana benthamiana plants, immunisation of calves with these plant-produced recombinants resulted in a significant reduction of 39% in parasite egg output, comparable to the protective efficacy of the native antigen. This study provides a valuable workflow for the development of recombinant vaccines against other parasitic nematodes.

Ostertagia ostertagi Mediates Early Host Immune Responses via Macrophage and Toll-Like Receptor Pathways.

Ostertagia ostertagi is an abomasal parasite with significant economic impact on the cattle industry. Early host immune responses are poorly understood. Here, we examined time course expression of Toll-like receptors (TLRs) in peripheral blood mononuclear cells (PBMC) during infection where PBMC macrophages (Mϕ) generated both pro- and anti-inflammatory responses when incubated with excretory/secretory products (ESP) from fourth-stage larvae (OoESP-L4) or adult worms (OoESP-Ad). First, changes in cell morphology clearly showed that both OoESP-L4 and OoESP-Ad activated PBMC-Mϕ in vitro, resulting in suppressed CD40 and increased CD80 expression. Expression of mRNAs for TLR1, -4, -5, and -7 peaked 7 days postinfection (dpi) (early L4), decreased by 19 dpi (postemergent L4 and adults) and then increased at 27 dpi (late adults). The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) (transcript and protein) increased in the presence of OoESP-Ad, and the anti-inflammatory cytokine interleukin 10 (IL-10) (protein) decreased in the presence of OoESP-L4 or OoESP-Ad; however, IL-10 mRNA was upregulated, and IL-6 (protein) was downregulated by OoESP-L4. When PBMC-Mϕ were treated with ligands for TLR4 or TLR5 in combination with OoESP-Ad, the transcripts for TNF-α, IL-1, IL-6, and IL-10 were significantly downregulated relative to treatment with TLR4 and TLR5 ligands only. However, the effects of TLR2 ligand and OoESP-Ad were additive, but only at the lower concentration. We propose that O. ostertagi L4 and adult worms utilize competing strategies via TLRs and Mϕ to confuse the immune system, which allows the worm to evade the host innate responses.

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.

Pathophysiology in Teladorsagia (Ostertagia) circumcincta-infected sheep selected for high fleece weight.

Resilience to parasitism is considered to be the maintenance of growth and production during infection, probably associated with an immune response with lesser detrimental effects on the host relative to adverse effects on the parasite. Resilience to infection with Teladorsagia circumcincta was investigated in lambs from a flock selected for forty 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, four parasite-naïve lambs from each flock were infected intraruminally at 6.5 months-of-age with 50,000 T. circumcincta L3, then from Day 35 to 70 post infection with 10,000 larvae at weekly intervals. Blood, abomasal fluid and faecal samples were collected daily to Day 35 and thence twice weekly for measurement of serum gastrin and pepsinogen concentrations, blood eosinophils, abomasal pH and FEC. Abomasal worm counts were made after necropsy on Day 94. Skin biopsies were collected weekly for estimation of the percentage of wool follicles containing paracortical cells. Total serum immunoglobulin and IgG1, IgG2, IgA and IgM titres specific for T. circumcincta antigens were estimated twice weekly to Day 42 p.i., then weekly. After the primary challenge, FEC were higher in the HFW lambs, whereas neither group shed many eggs during the 5-week trickle infection; worm burdens were small at post mortem. Resilient HFW lambs showed a lesser inflammatory response, but relatively small differences in abomasal secretion. Circulating eosinophil counts increased moderately in both groups, less in the HFW lambs, during the primary infection and more markedly during the subsequent trickle infection, when the increase in the C lambs became significantly greater. All measured serum antibody titres were low in both groups throughout. Selection for HFW altered the wool characteristics of parasite-naïve lambs (fewer follicles containing paracortical cells). There was a slower increase in the percentage of follicles containing these cells after primary infection. Abomasal function was similar in the two groups, both exhibiting typical increases in abomasal pH and serum gastrin and pepsinogen concentrations. The most marked differences in the HFW lambs were a greater rise in serum pepsinogen during the primary infection and the 2-day delay in onset of hypoacidity. Resilience to parasitism in this flock is consistent with maintenance of wool quality and small differences in abomasal secretion resulting from an attenuated immune response causing fewer detrimental effects on host tissues.

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.

Vagal and splanchnic afferent nerves are not essential for anorexia associated with abomasal parasitism in sheep.

Heavy burdens of the abomasal nematode, Ostertagia (Telodorsagia) circumcincta, in growing lambs result in a reduction in liveweight gain due largely to a drop in voluntary feed intake. The present study investigated: (1) the role of subdiaphragmatic vagal and non-vagal visceral afferent nerves in mediating a reduction in voluntary feed intake, using subdiaphragmatic vagal deafferentation (vagotomy) either alone or in combination with coeliac-superior mesenteric ganglionectomy (vagotomy and sympathectomy); and (2) the association between appetite, abomasal pH, selected blood values (amidated gastrin (G-17-amide), glycine-extended gastrin (G-17-Gly), pepsinogen and leptin) and worm burden, in sheep experimentally infected with 100,000 O. circumcincta infective larvae per os. Neither vagotomy alone nor vagotomy and sympathectomy in combination adversely affected the establishment or course of development of the parasite burden, when compared with a control group subject to sham surgery. Furthermore, neither surgical procedure prevented the drop in appetite seen 5-10 days post-infection, although combined vagotomy and sympathectomy did reduce voluntary feed intake prior to the start of the study. Ostertagia infection resulted in a significant increase in abomasal pH in all three groups, which was accompanied by an increase in blood G-17-amide and in G-17-Gly, the latter reported for the first time in parasitized ruminants. There were no significant differences in blood leptin, also reported for the first time in parasitized sheep, either between groups or in comparison with pre-infection levels, though weak negative correlations were established between blood leptin and appetite from day 5 to the end of the study in all three groups and a positive correlation with blood G-17-amide in the control group over the same period. These data suggest that neither intact subdiaphragmatic vagal afferent nerves or coeliac-superior mesenteric ganglion fibres, nor changes in circulating gastrin and leptin concentrations play a major role in mediating the hypophagic effects of O. circumcincta in parasitized sheep.

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.

Evidence for a parasite-mediated inhibition of abomasal acid secretion in sheep infected with Ostertagia leptospicularis.

The acid secretory capacity of the abomasal mucosa was studied in sheep experimentally infected with Ostertagia leptospicularis. The acidity of the abomasal contents, permanently recorded by a pH probe located inside the abomasum, decreased markedly to mean levels between pH 5 and 6. Subcutaneous administration of histamine or carbachol successfully stimulated acid secretion (pH 3.4). The results indicate that the abomasal mucosa harboured a population of functional parietal cells which were also identified immunohistochemically (H(+)/K(+)-ATPase). Ultrastructural investigation before stimulation revealed that the majority of these cells was in a resting state. Despite high serum gastrin levels, the acid secretion was blocked either at the level of the parietal cell or the enterochromaffin-like cell by an unknown factor, possibly mediated by the parasites. This is the first report of a parietal cell dysfunction associated with a nematode infection in the abomasum. It is suggested that the parasites induce changes in their environment which favour their survival and/or increase their reproduction.

A sequential study of the pathology associated with the infection of sheep with adult and larval Ostertagia circumcincta.

Disturbances in the physiology of the abomasa of sheep infected with either adult Ostertagia circumcincta given via abomasal cannulae, or larvae (L3) given intraruminally were matched by pathological changes in tissues collected by repeated mucosal biopsy. Within 2-3 days of the transplant of adult worms, abomasal pH had increased markedly in five out of six animals, and there also had been rapid increases in serum gastrin and pepsinogen concentrations in all animals. Reductions in parietal cell number were recorded as early as 1 day after the transplant of adults and were associated with the rapid accumulation of many neutrophils and eosinophils. Mucosal hyperplasia, with increased numbers of cells closer in appearance to mucous/mucous neck cells, was a relatively late development, being most pronounced in the latter part of the infection. In sheep given larvae, changes in secretory physiology were again matched by a concurrent fall in parietal cell number and by the accumulation of inflammatory cells. Changes became maximal when most worms could be expected to be present as adults, confirming the role of adults in the natural disease. Some abnormalities were detected in biopsies collected from animals maintained free of parasites and, although milder in degree, there were similarities to those observed in parasitised tissues, there being fewer parietal cells, a modest degree of mucous cell hyperplasia and inflammatory infiltrates of predominantly neutrophils. These changes were the likely result of trauma to the tissues in the immediate vicinity of the cannula, due either to the presence of the cannula itself or to the frequent collection of biopsy material from areas close to it.

Proteinases released in vitro by the parasitic stages of the bovine abomasal nematode Ostertagia ostertagi.

Host tissue penetration, feeding and immune evasion by helminth parasites may be mediated by both mechanical processes and histolytic products released by the parasite. The aim of this study was to investigate potential histolytic products released during in vitro maintenance of exsheathed third (L3) and 4th larval stage (L4) and adult Ostertagia ostertagi. Therefore, the pH optima, substrate specificity, molecular size and inhibitor sensitivity of in vitro released (IVR) proteinases were analysed by spectrophotometry and substrate gel electrophoresis. It was shown that L3, L4 and adult IVR proteinases degrade a variety of protein substrates in a pH-dependent and stage-specific manner. At alkaline pH, gelatin, casein and fibrinogen were degraded by metallo- and serine proteinases. In contrast, mucin, fibrinogen, albumin and haemoglobin were degraded at acidic pH by aspartyl protease- and cathepsin L-like activity. At pH 3, the heavy chain of bovine IgG was completely degraded by an aspartyl proteinase secreted by all 3 parasitic stages. The specificity of the L3, L4 and adult Ostertagia ostertagi proteinases against the different substrates indicates variable functional requirements.

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

[Ostertagiasis in bulls used for bullfights]. / Ostertagiosis en 'Toro de lidia'.

In this paper, an outbreak of ostertagiosis in fighting-bull calves is described. Of 95 calves, 12 were affected and 10 died. Grossly, the abomasal mucosa was aedematous and showed multiple whitish tiny nodules. Differential diagnoses at a macroscopic level included parasitization by E. gilruthi. However, histologically the presence of Ostertagia spp. larvae in the lumen of the gastric glands was confirmed. The inflammatory reaction observed in the abomasal mucosa, together with the clinical picture, correspond to type-II ostertagiosis, with metaplasia, hyperplasia, dilatation, and parasitic forms in the gastric glands.

Indirect gene diagnoses for complex (multifactorial) diseases--a review.

The analysis of multifactorial diseases requires the efficient investigation of large numbers of (gene) loci and patient (family) samples. Since simple repetitive DNA markers are dispersed all over the chromosomes, molecular techniques employing these tools render most conventional screening procedures obsolete. Examples of tumors, autoimmune diseases and infections are presented to validate concepts of indirect gene diagnoses via simple, tandemly arranged, repetitive DNA sequences. The salient advantages of microsatellite technologies vs. those of multilocus DNA fingerprinting are weighed.

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.