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.

Comparative analysis of the anthelmintic efficacy of European heather extracts on Teladorsagia circumcincta and Trichostrongylus colubriformis egg hatching and larval motility.

BACKGROUND: Gastrointestinal nematode (GIN) control is traditionally achieved with the use of anthelmintic drugs, however due to regulations in organic farming and the rise in anthelmintic resistance, alternatives are sought after. A promising alternative is the use of bioactive plant feeding due to the presence of plant secondary metabolites (PSMs) such as proanthocyanidins (PAs). This study focussed on the perennial shrub heather (Ericaceae family), a plant rich in PAs, highly abundant across Europe and with previously demonstrated anthelmintic potential. METHODS: In vitro assays were used to investigate heather's anthelmintic efficacy against egg hatching and larval motility. Heather samples were collected from five European countries across two seasons, and extracts were tested against two GIN species: Teladorsagia circumcincta and Trichostrongylus colubriformis. Polyphenol group-specific ultraperformance liquid chromatography-tandem mass spectrometry analysis was performed to identify relevant polyphenol subgroups present, including the PA concentration and size and ratio of the subunits. Partial least squares analysis was performed to associate efficacy with variation in PSM composition. RESULTS: Heather extracts reduced egg hatching of both GIN species in a dose-dependent manner by up to 100%, while three extracts at the highest concentration (10 mg/ml) reduced larval motility to levels that were not significantly different from dead larvae controls. PAs, particularly the procyanidin type, and flavonol derivatives were associated with anthelmintic activity, and the particular subgroup of polyphenols associated with the efficacy was dependent on the GIN species and life stage. CONCLUSIONS: Our results provide in vitro evidence that heather, a widely available plant often managed as a weed in grazing systems, has anthelmintic properties attributed to various groups of PSMs and could contribute to sustainable GIN control in ruminant production systems across Europe.

Parasitic strongyle nemabiome communities in wild ruminants in Sweden.

BACKGROUND: Wildlife hosts may serve as reservoirs for strongyles, which can be transmitted to domestic livestock. Therefore, studies evaluating nemabiome compositions in wildlife ruminants are of great use in assessing the possibility of transmission of important nematode pathogens to domestic sheep in Sweden. METHODS: First, fecal samples were collected from roe deer (n = 125), fallow deer (n = 106), red deer (n = 18) and mouflon (n = 13) in south central Sweden during the hunting season in 2019. Second, after fecal examination samples were cultured and the larvae were harvested, followed by DNA extractions. Third, all samples were barcoded and processed for sequence analysis on the PacBio platform. Finally, bioinformatic sequence analysis was conducted with DADA2, while species diversity and richness, as well as interactions between the different hosts, were calculated and analyzed in R. RESULTS: Nematode ITS2 sequences were found in 225 of 262 (86%) samples. In total, 31 taxa were identified, among which 26 (86%) to the species level. These were found in different combinations, among which 24 (77%) occurred in roe deer, 19 (61%) in fallow deer, 20 (65%) in red deer and 10 (32%) in mouflon. Five of the species found are known to be associated with livestock (Chabertia ovina, Haemonchus contortus, Oesophagostomum venulosum, Teladorsagia circumcincta and Trichostrongylus axei). However, in the present study the relative abundance and prevalence of most of these species were low. The most striking exception was T. axei, which was relatively abundant in all wildlife hosts. Mostly a wide range of wildlife specific nematodes such as Ostertagia leptospicularis and Spiculopteragia spp. were identified including the invasive nematode Spiculopteragia houdemeri, which was found for the first time in red deer, fallow deer, and mouflon in Sweden. The difference in the number of shared species between mouflon and all cervids (n = 6) was less than among all three cervids (n = 8). CONCLUSION: In this study, we investigated the community structure of parasitic intestinal nematodes in four wildlife hosts, and we found that the majority of the parasite species identified were wildlife specific. We also found a new, potentially invasive species not reported before. After comparing the nemabiome of the wildlife hosts in this study with a previous study in sheep from the same geographical region, we conclude that the horizontal transmission potential appears to be relatively low. Still, cross-infections of nematodes between game and sheep cannot be completely ignored.

Naturally acquired ovine-adapted nematode infections in young cattle and their treatment with eprinomectin 5% w/v extended-release injection.

The objective of this controlled study was to determine the anthelmintic efficacy of eprinomectin 5% w/v extended-release injection (Eprinomectin ERI; LONGRANGE®, Boehringer Ingelheim Animal Health) against primarily ovine-adapted gastrointestinal strongylid nematode (GIN) parasites in naturally infected young cattle. Eighteen calves which grazed on a sheep-dominated mixed sheep-cattle farm were randomly allocated into two equal groups (saline [control] or Eprinomectin ERI, administered each at 1 mL/50 kg body weight once subcutaneously), treated and euthanized 14 days thereafter for a differential GIN count. Ten species of strongylid nematodes were recovered from the control calves (ovine-adapted Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Cooperia curticei, Nematodirus battus, Chabertia ovina; bovine-adapted Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus; 'generalist' Trichostrongylus axei). Adult GIN counts ranged from 1,540 to 5,244 for the control calves and from zero to 110 for the Eprinomectin ERI-treated calves. Accepting the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products anthelmintic guidelines criteria on adequacy of infections to be demonstrated in the control animals, this study showed that Ch. ovina, C. curticei, H. contortus, N. battus, T. axei, and T. colubriformis were reduced significantly (p < 0.0001) by ≥ 98.7% in the animals treated with Eprinomectin ERI. In conclusion, Eprinomectin ERI treatment was efficacious against a range of ovine-adapted nematode parasites in naturally infected young cattle.

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.

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.

Ostertagia ostertagi macrophage migration inhibitory factor is present in all developmental stages and may cross-regulate host functions through interaction with the host receptor.

Macrophage migration inhibitory factor (MIF) of Ostertagia ostertagi, an abomasal parasite of cattle, was characterised in the present study. Phylogenetic analysis identified at least three O. ostertagi MIFs (Oos-MIFs), each encoded by a distinct transcript: Oos-MIF-1.1, Oos-MIF-1.2 and Oos-MIF-2. Oos-MIF-2 is only distantly related to Oos-MIF-1s, but has higher sequence similarity with the Caenorhabditis elegans MIF2. Oos-MIF-1.1 and Oos-MIF-1.2 are similar (93%) and thus collectively referred to as Oos-MIF-1 when characterised with immunoassays. Recombinant Oos-MIF-1.1 (rOos-MIF-1.1) is catalytically active as a tautomerase. A mutation (rOos-MIF-1.1P1G) or duplication of Pro1 residue (rOos-MIF-1.1P1+P) resulted in reduced oligomerisation and loss of tautomerase activity. The tautomerase activity of rOos-MIF-1.1 was only partially inhibited by ISO-1 but was abrogated by a rOos-MIF-1.1-specific antibody. Oos-MIF-1 was detected in all developmental stages of O. ostertagi, with higher levels in the adult stage; it was also detected in adult worm excretory/secretory product. Oos-MIF-1 was localised to the hypodermis/muscle, reproductive tract and intestine, but not to the cuticle. rOos-MIF-1.1, but not rOos-MIF-1.1P1G, was able to specifically bind to human CD74, a MIF cell surface receptor, with an affinity comparable with human MIF. Immunostaining indicated that macrophages were able to internalise rOos-MIF-1.1, further supporting receptor-mediated transportation. Herein we also show that rOos-MIF-1.1 inhibited migration of bovine macrophages and restored glucocorticoid-suppressed, lipopolysaccharide-induced TNF-α and IL-8 in human and/or bovine macrophages. Given its dual role in self-regulation and molecular mimicry, this secreted parasite protein warrants investigation as a vaccine candidate against O. ostertagi infections in cattle.

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.

Structure of Ostertagia ostertagi ASP-1: insights into disulfide-mediated cyclization and dimerization.

The cysteine-rich secretory/antigen 5/pathogenesis-related 1 (CAP) protein superfamily is composed of a functionally diverse group of members that are found in both eukaryotes and prokaryotes. The excretome/secretome of numerous helminths (parasitic nematodes) contains abundant amounts of CAP members termed activation-associated secreted proteins (ASPs). Although ASPs are necessary for the parasitic life cycle in the host, the current lack of structural and functional information limits both understanding of their actual role in host-parasite interactions and the development of new routes in controlling parasitic infections and diseases. Alleviating this knowledge gap, a 1.85 Å resolution structure of recombinantly produced Oo-ASP-1 from Ostertagia ostertagi, which is one of the most prevalent gastrointestinal parasites in cattle worldwide, was solved. Overall, Oo-ASP-1 displays the common hallmark architecture shared by all CAP-superfamily members, including the N-terminal CAP and C-terminal cysteine-rich domains, but it also reveals a number of highly peculiar features. In agreement with studies of the natively produced protein, the crystal structure shows that Oo-ASP-1 forms a stable dimer that has been found to be primarily maintained via an intermolecular disulfide bridge, hence the small interaction surface of only 306.8 Å(2). Moreover, unlike any other ASP described to date, an additional intramolecular disulfide bridge links the N- and C-termini of each monomer, thereby yielding a quasi-cyclic molecule. Taken together, the insights presented here form an initial step towards a better understanding of the actual biological role(s) that this ASP plays in host-parasite interactions. The structure is also essential to help to define the key regions of the protein suitable for development of ASP-based vaccines, which would enable the current issues surrounding anthelmintic resistance in the treatment of parasitic infections and diseases to be circumvented.

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.

The kinetic properties of the glutamate dehydrogenase of Teladorsagia circumcincta and their significance for the lifestyle of the parasite.

Like other nematodes, both L(3) and adult Teladosagia circumcincta secrete or excrete NH(3)/NH(4)(+), but the reactions involved in the production are unclear. Glutamate dehydrogenase is a significant source NH(3)/NH(4)(+) in some species, but previous reports indicate that the enzyme is absent from L(3)Haemonchus contortus. We show that glutamate dehydrogenase was active in both L(3) and adult T. circumcincta. The apparent K(m)s of the L(3) enzyme differed from those of the adult enzyme, the most significant of these being the increase in the K(m) for NH(4)(+) from 18mM in L(3) to 49mM in adults. The apparent V(max) of the oxidative deamination reaction was greater than that of the reductive reaction in L(3), but this was reversed in adults. The activity of the oxidative reaction of the L(3) enzyme was not affected by adenine nucleotides, but that of the reductive reaction was stimulated significantly by either ADP or ATP. The L(3) enzyme was more active with NAD(+) than it was with NADP(+), although the activities supported by NADH and NADPH were similar at saturating concentrations. While the activity of the oxidative reaction was sufficient to account for the NH(3)/NH(4)(+) efflux we have previously reported, the reductive amination reaction was likely to be more active.

A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

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 complexity of the secreted NPA and FAR lipid-binding protein families of Haemonchus contortus revealed by an iterative proteomics-bioinformatics approach.

Two different classes of small nematode specific lipid-binding proteins, the nematode polyprotein allergens/antigens (NPAs) and the fatty acid- and retinol-binding (FAR) proteins, are secreted by helminth parasites. Until now, there was no evidence of the expression or secretion of these two families of proteins in Haemonchus contortus. In this study, we applied proteomic and bioinformatic tools in an iterative manner to reveal the expression and complexity of these proteins in the excretory/secretory products (ESP) of adult H. contortus at the protein and gene levels. Initial examination of the mass spectra of ESP fractions against standard databases returned nine peptides mapping to Ostertagia ostertagi NPA and FAR sequences. Searches of the H. contortus EST and genomic contig databases with the O. ostertagi and Caenorhabditis elegans homologues retrieved diverse sequences encoding H. contortus NPA and FAR proteins. H. contortus sequences were then integrated into a customized database and a new search of the mass spectra achieved a 10-fold improvement in coverage of the predicted H. contortus NPAs. The final analyses of the mass spectra achieved 49-60% coverage of H. contortus NPAs and 7-47% coverage of H. contortus FARs. Moreover, the diversity in structures of the encoding genes was revealed by assembling the genomic sequence data with predicted protein sequences confirmed by the peptide evidence. We predict there are at least one Hc-NPA gene and six Hc-FAR genes in H. contortus, and life stage gene expression of Hc-FAR-1 to -6 revealed unique transcription patterns for each of these genes.

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.

Analysis of the translationally controlled tumour protein in the nematodes Ostertagia ostertagi and Caenorhabditis elegans suggests a pivotal role in egg production.

The translationally controlled tumour protein (TCTP) is a conserved protein which has been described for a wide range of eukaryotic organisms including protozoa, yeasts, plants, nematodes and mammals. Several parasitic organisms have been shown to actively secrete TCTP during host infection as part of their immuno-evasive strategy. In this study, we have studied TCTP in Ostertagia ostertagi, a parasitic nematode of cattle, and in the free-living nematode Caenorhabditis elegans. An analysis of the transcription and expression patterns showed that TCTP was present in the eggs of both species. This localisation is consistent for some other Strongylida such as Teladorsagia circumcincta, Cooperia oncophora and Haemonchus contortus. TCTP was also detected at low levels in excretory-secretory material from adult O. ostertagi worms. The role of TCTP in nematode biology was also investigated by RNA interference in C. elegans. Knock-down of C. elegans tctp (tct-1) transcription reduced the numbers of eggs laid by the hermaphrodite in the F(0) and F(1) generations by 90% and 72%, respectively, indicating a pivotal role of TCTP in reproduction.

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.

Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi--comparison with other strongylid nematodes and Caenorhabditis elegans.

The transthyretin-like (ttl) gene family is one of the largest conserved nematode-specific gene families, coding for a group of proteins with significant sequence similarity to transthyretins (TTR) and transthyretin-related proteins (TRP). In the present study, we investigated the ttl family in Ostertagia ostertagi (a nematode of the abomasum of cattle). Mining of expressed sequence tag (EST) databases revealed the presence of at least 18 ttl genes in O. ostertagi (Oo-ttl), most of which are constitutively transcribed from the free-living, third larval stage onwards. The full-length cDNA of one of these genes (Oo-ttl-1) was amplified and cloned for recombinant expression. Western blot analysis using a specific antiserum showed that the native protein Oo-TTL-1 was highly present in the excretory-secretory (ES) products of adults of O. ostertagi. The protein was immunolocalized to the pseudocoelomic fluid of adult worms. A phylogenetic-bioinformatic analysis of all amino acid sequence data for TTL proteins from a range of strongylid nematodes showed that they could be divided into at least five different classes. This classification was based on conserved amino acids in the first TTL signature domain and the number and location of cysteine residues. The biological role(s) of the TTLs in nematode biology is still unclear. A theoretical three-dimensional model of Oo-TTL-1 indicated that it had a similar structure to TTRs (i.e., containing ß-sheets, arranged in a ß-sandwich). In contrast to TTRs, competitive binding studies using recombinant Oo-TTL-1 indicated that the protein was devoid of any hydrophobic ligand- or thyroid hormone-binding properties. Finally, combinatorial analysis by double-stranded RNA interference of five ttl genes in the free-living nematode Caenorhabditis elegans did not reveal any visible phenotypes. More information on the transcription profile and tissue distribution of TTLs in nematodes is needed to provide new insights into the biological role of this gene family.