Search

Variation in the intensity and duration of infections is often driven by variation in the network and strength of host immune responses. While many of the immune mechanisms and components are known for parasitic helminths, how these relationships change from single to multiple infections and impact helminth dynamics remains largely unclear. Here, we used laboratory data from a rabbit-helminth system and developed a within-host model of infection to investigate different scenarios of immune regulation in rabbits infected with one or two helminth species. Model selection suggests that the immunological pathways activated against Trichostrongylus retortaeformis and Graphidium strigosum are similar. However, differences in the strength of these immune signals lead to the contrasting dynamics of infections, where the first parasite is rapidly cleared and the latter persists with high intensities. In addition to the reactions identified in single infections, rabbits with both helminths also activate new pathways that asymmetrically affect the dynamics of the two species. These new signals alter the intensities but not the general trend of the infections. The type of interactions described can be expected in many other host-helminth systems. Our immune framework is flexible enough to capture different mechanisms and their complexity, and provides essential insights to the understanding of multi-helminth infections., Competing Interests: The authors have declared that no competing interests exist.

Background and Objectives: The possibility of manipulating the immune response in lambs to the gastrointestinal nematode Trichostrongylus colubriformis to reduce production losses associated with infection was investigated. In a series of four experiments, attempts to immunize sheep via the mucosal route to modify the immune response and induce mucosal tolerance are outlined. Initially, a proof of concept study was conducted with lambs being injected with multiple doses of a somatic T colubriformis antigen without an adjuvant in the rectal submucosa and subsequently challenged with T colubriformis L3 larvae. This was followed by a dose-response study comparing different antigen doses to identify the optimum dose of the nematode antigen for successful induction of mucosal tolerance. The final two studies were conducted to determine the larval stage specificity of the parasite antigen and the most suitable site of delivery required to stimulate mucosal tolerance., Methods: In the proof of concept study, lambs either received repeated injections in the rectal submucosa at 3 × weekly intervals with 15 µg of L3, 11 µg of L4 and 21 µg of immature adult (L5) somatic T colubriformis antigens (ANT) or not (INF) prior to infection with T colubriformis. In the dose-rate study, antigen dose rates of 100%, 50%, 10%, 1% or 0% of the antigen concentration used in the proof of concept study were compared while the larval stage study compared antigen from either L3, L4, L5 stages or combination of all (COMB) and the route of administration study compared antigen delivery into either the rectal submucosa (RE) or sub-cutaneous injection (SC)., Results: During infection, lamb growth was improved by antigen treatment between days 21 and 42 in the proof of concept study (P = .009), for groups 10%, 50% and 100% in the dose-rate study (P < .05 for all) and in RE in the route of administration study with no improvement observed in the larval stage study. No differences in faecal egg counts were observed (P > .05 for all). Parasite-specific IgA and IgE showed a dose-response (the dose-rate study), were not affected by larval stage (the larval stage study) and were greater in RE than SC (the route of administration study). IL-4 production following lymphocyte stimulation was greatest in COMB (the larval stage study) and RE (the route of administration study)., Conclusions: Although antigen treatment improved performance, this was inconsistent and appeared to stimulate immunity rather than induce tolerance. Combined larval stages were more efficient than individual stages, and intra-rectal administration was more effective than sub-cutaneous., (© 2020 John Wiley & Sons Ltd.)

Aims: Most breeds of goat are more susceptible to nematode infection than sheep, and this appears to be a consequence of less effective immune responses. Several papers have considered the effectiveness of eosinophils and immunoglobulin A (IgA) in goats but differences in the induction of responses have not been studied in the same detail. The aim of this study was to look at the induction of eosinophil and IgA responses in Boer goats reared indoors under intensive conditions., Methods and Results: The goats were experimentally infected with a low dose of 2400 Haemonchus contortus, Trichostrongylus spp. and Oesophagostomum spp. at a 6:1:1 ratio. Faecal egg counts (FEC), packed cell volume (PCV), IgA activity against third-stage larvae and peripheral eosinophilia were measured twice a week for eight weeks. The infection generated an IgA response but did not significantly increase peripheral eosinophilia in the 25 infected kids compared with the 4 control animals. FEC was not associated with IgA activity or eosinophilia., Conclusion: A detailed analysis of IgA and eosinophil responses to deliberate nematode infection in Boer goats showed that there was an increase in nematode-specific IgA activity but no detectable eosinophil response. In addition, there was no association between increased IgA activity or eosinophilia with egg counts and worm burdens. These suggest that IgA and eosinophils do not act to control nematode infection in goats., (© 2020 John Wiley & Sons Ltd.)

It is normal for hosts to be co-infected by parasites. Interactions among co-infecting species can have profound consequences, including changing parasite transmission dynamics, altering disease severity and confounding attempts at parasite control. Despite the importance of co-infection, there is currently no way to predict how different parasite species may interact with one another, nor the consequences of those interactions. Here, we demonstrate a method that enables such prediction by identifying two nematode parasite groups based on taxonomy and characteristics of the parasitological niche. From an understanding of the interactions between the two defined groups in one host system (wild rabbits), we predict how two different nematode species, from the same defined groups, will interact in co-infections in a different host system (sheep), and then we test this experimentally. We show that, as predicted, in co-infections, the blood-feeding nematode Haemonchus contortus suppresses aspects of the sheep immune response, thereby facilitating the establishment and/or survival of the nematode Trichostrongylus colubriformis ; and that the T. colubriformis -induced immune response negatively affects H. contortus This work is, to our knowledge, the first to use empirical data from one host system to successfully predict the specific outcome of a different co-infection in a second host species. The study therefore takes the first step in defining a practical framework for predicting interspecific parasite interactions in other animal systems., (© 2018 The Authors.)

Immunological and performance characteristics were explored in Romney sheep from lines selected for either resistance or resilience to parasite infection. At a mean 78 days-of-age, twin lambs from a line selected for resistance (RT) and lambs from a line selected for resilience (RL) were infected with the intestinal nematode Trichostrongylus colubriformis for 100 days (I) while their twin remained as an uninfected control (C). Compared with RL, RT animals had lower levels of circulating CD4 + T-cells (P = 0.003) but a greater proportion of these were activated (CD4 + CD25 + ) in response to infection (P = 0.007). Differences between the lines in humoral immune responses to nematode infection varied with higher levels of T. colubriformis specific immunoglobulin (Ig) E in RT-I than RL-I (P = 0.002) but similar levels of both IgG (P = 0.926) and IgA (P = 0.321) responses. Temporal differences in the immune response also existed between the lines with RT-I animals displaying an earlier peak and more rapid reduction in FEC and an earlier peak in T. colubriformis specific IgA. In addition, compared with their RT-C and RL-C counterparts, infection caused a 22% reduction in feed intake from day 56 (P = 0.001) with total feed intake reduced by 15% and 9% for RT-I and RL-I, respectively. Cumulative liveweight gain was greatest for RL animals (P = 0.026) and relative to RT-C and RL-C was reduced by 5.8 kg and 4.9 kg for RT-I and RL-I, respectively. Overall, the selection lines appear to have differences in immunological characteristics that are both dependent on, and independent of parasite infection. Further, the difference in growth in the uninfected animals coupled with the similar cost of infection suggests the lower liveweight gain of RT-I compared with RL-I may be due to inherent differences between the lines in their growth potential, rather than a greater cost of infection in animals selected for resistance., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species., Competing Interests: The authors wish to declare no competing interests. This study was funded by the New Zealand Ministry for Business, Innovation and Employment. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The hypothesis tested in this experiment was that Trichostrongylus colubriformis infection would reduce growth rates of grazing meat-breed lambs; however production loss would be reduced by suppression of the host immune response. The experiment had a 3×2 factorial design using 6-7 month old meat-breed lambs which remained uninfected or infected (IFY) with 2000 or 4000 T. colubriformis L3/week for 12 weeks and were immunosuppressed (SUPY) using methylprednisolone acetate once weekly or remained non-immunosuppressed (SUPN). Immunosuppression increased worm egg counts (WEC) of infected lambs (SUPY 2421 eggs per gram (epg), SUPN 1154 epg on day 84, p<0.05) and T. colubriformis burdens (p<0.05-0.10) and reduced circulating eosinophils (p<0.05 on days 11, 42, 56 and 84) and intestinal total antibody titres (p<0.02). There was a significant (p<0.05) interaction between the main effects of infection and immunosuppression with infection having a larger negative effect on the liveweight of non-immunosuppressed lambs. The immunological response of the host to T. colubriformis infection accounted for 75% of the overall cost of infection (3.1kg) with the majority of this cost occurring during the first 35 days of infection. In contrast, most of the cost associated with the direct effect of infection occurred after day 35. These results confirm in grazing meat-breed lambs that the host's immunological response to T. colubriformis infection is the major component of production loss., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Gastrointestinal nematodes pose a major risk to the farming of small ruminants worldwide. Infections are typically controlled by anthelmintics, however as resistance to anthelmintics increases, it is necessary that the mechanism of host responses are understood in order to develop alternative control options. It is hypothesised that basophils are involved in the initiation of an anti-parasite immune response, independent of IgE. In this study, the in vitro activation states of CD203c(+) basophil-like KU812 cells were determined in the presence of Trichostrongylus colubriformis parasitised HT29 epithelial cells with or without mucin. Cell surface expression of CD164, CD107a and CD13 antigens on gated CD203(+) cells were determined and qRT-PCR was used to examine gene expression changes of IL33 (a Th2 cytokine) and the high affinity IgE receptor (FcɛRIα) within the co-culture. When KU812 basophils encountered T. colubriformis and/or mucin in a parasitised epithelium, the basophils increased cell surface expression of CD13 and CD164 antigens, independent of IgE. T. colubriformis also increased the number of CD203c(+) KU812 cells that expressed CD13 and CD164 antigens. These data support the in vivo observations of T. colubriformis primary infections in guinea pigs and sheep., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Given their global distribution and abilities to persist in the host, helminths can play a crucial role in affecting risk of infections by increasing individual variation in infection. Helminth co-infections are of particular interest because by altering host immune responses, they can modify host susceptibility and thus intensity and transmission of other parasites/pathogens. The dynamics of co-infection were examined using two helminths of the European rabbit. Individuals were simultaneously challenged with a primary dose of both parasites, and changes in intensity were examined in relation to local and systemic immune responses. Both helminths persisted in co-infected rabbits; however, contrasting dynamics and immune responses were observed. Graphidium strigosum intensity was high throughout the co-infection, while Trichostrongylus retortaeformis intensity decreased but was not completely cleared. A Th2 response was observed against G. strigosum, while a mixed Th1/Th2 profile was found to T. retortaeformis. A comparison with our previous work on single infections showed that G. strigosum intensity was higher in co-infected than single infected hosts, while T. retortaeformis showed no significant changes. Differences were also observed in the cytokine profiles, blood cell concentrations and antibody trends. Overall, host variability during helminth co-infections can be generated by significant differences in immune responses and/or parasite dynamics., (© 2013 John Wiley & Sons Ltd.)

This study aimed to identify regions of the genome affecting resistance to gastrointestinal nematodes in a Creole goat population naturally exposed to a mixed nematode infection (Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum) by grazing on irrigated pasture. A genome-wide quantitative trait loci (QTL) scan was performed on 383 offspring from 12 half-sib families. A total of 101 microsatellite markers were genotyped. Traits analysed were faecal egg count (FEC), packed cell volume (PCV), eosinophil count and bodyweight (BW) at 7 and 11 months of age. Levels of activity of immunoglobulin A (IgA) and activity of immunoglobulin E (IgE) anti-Haemonchus contortus L3 crude extracts and adult excretion/secretion products (ESPs) were also analysed. Using interval mapping, this study identified 13 QTL for parasite resistance. Two QTL linked with FEC were found on chromosomes 22 and 26. Three QTL were detected on chromosomes 7, 8 and 14 for eosinophil counts. Three QTL linked with PCV were identified on chromosomes 5, 9 and 21. A QTL for BW at 7 months of age was found on chromosome 6. Lastly, two QTL detected on chromosomes 3 and 10 were associated with IgE anti-L3, and IgE anti-ESP was linked with two QTL on chromosomes 1 and 26. This study is the first to have identified regions of the genome linked with nematode resistance in a goat population using a genome scan. These results provide useful tools for the understanding of parasite resistance in small ruminants., (© 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.)

This study was carried out to evaluate the immune response in young Ile de France (IF) and Santa Ines (SI) sheep naturally infected by Oestrus ovis and gastrointestinal nematodes (GIN). Mast cells, eosinophils and globule leucocytes were enumerated in the upper respiratory tract (septum, middle meatus and ventral nasal conchae) and in the mucosa of abomasum and small intestine. Immunoglobulin G (IgG) levels in serum samples and immunoglobulin A (IgA) levels in mucus from the nasal, abomasum and small intestinal mucosae were determined against O. ovis, Haemonchus contortus and Trichostrongylus colubriformis antigens. Significant positive correlation coefficients were observed in both breeds between the number of O. ovis larvae×IgG against Oestrus crude extract (IF: r=0.58; SI: r=0.66; P<0.05), and between O. ovis larvae x IgG against Oestrus excretory and secretory products (IF: r=0.59; SI: r=0.63; P<0.05). Apparently, the presence of antibodies in the serum or nasal mucus, as well as inflammatory cells, was not able to efficiently protect against O. ovis infestation. With regard to GIN, the levels of immunoglobulins and the inflammatory cell numbers in the gastrointestinal mucosa presented a significant inverse relationship with H. contortus worm burden in SI animals and this may have contributed to the fact that these animals presented the lowest FEC and worm burden compared to IF. In conclusion, the immune responses against O. ovis and GIN are very similar and involve the recruitment of inflammatory cells and production of immunoglobulins against the parasites., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A variety of single-chain variable fragments (scFv) that had been previously developed to the surface epitopes of infective Trichostrongylus colubriformis L3 pathogenic gut nematodes of sheep were fused to a trimeric version of polyoleosin (three head-to-tail repeats of oleosin) and expressed in planta under the control of an Arabidopsis oleosin promoter. The fusion products were found to accumulate in oil bodies (OBs) at the range of 0.25-0.9% of the total seed protein which is comparable with the main 18 kDa isoform of Arabidopsis seed oleosin. Immunofluorescence microscopy and immuno-binding were used to demonstrate that it is possible to both purify the recombinant protein via enrichment for OBs as well as use the OBs emulsion to deliver functional recombinant scFv. This work presents a novel fusion strategy platform to boost the productivity and simplify the delivery of recombinant single chain antibodies and other like proteins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A pen experiment was conducted to investigate the interaction of early-weaning and nutrient supply on the periparturient relaxation of immunity to the gastrointestinal nematode (GIN) Trichostrongylus colubriformis in Merino ewes. Mixed-age pregnant and non-pregnant (dry) ewes were infected with 8,000 T. colubriformis L(3)/week, and fed either a high or low quality diet. Following parturition, lambs were either removed from their mothers at 2 days of age or allowed to continue suckling. Systemic immunity began to wane during late pregnancy with circulating eosinophils and plasma total antibody (Ab) levels declining from day -37 (relative to the midpoint of lambing) and day -24, respectively. Pregnant ewes fed the low quality diet exhibited an increasing faecal worm egg count (WEC) from day -24 and had higher intestinal worm burdens on day 13, whereas ewes fed the high quality diet had a delayed transient rise in WEC of lower magnitude. Dry and early-weaned ewes remained highly resistant to T. colubriformis at all times. In the post-lambing/lactation period, ewes fed the high quality diet had higher levels of local total Ab and numbers of goblet cells (GC) in the small intestine on days 13 and 41. Lactating/suckled ewes had a lower anti-parasite local immune response as indicated by reduced titres of total Ab, IgG(1), IgM and IgA and lower numbers of mucosal mast cells (MMC), globule leukocytes (GL) and GC in small intestinal tissue compared to their dry and early-weaned counterparts. Early-weaning resulted in rapid recovery of blood eosinophils and total Ab. On day 13 post-lambing, titres of total Ab, IgG(1), IgM, IgA and IgE, and numbers of MMC and GL were greater than those measured in dry and suckled ewes. When fed the high quality diet, ewes had a higher dry matter (DM) intake, maternal weight, fat score, greater fat depth and eye muscle depth, birthed heavier lambs that had higher growth rates, and produced more milk. The physiological status of pregnancy resulted in a higher DM intake but lower measures of fat depth and eye muscle depth, and suckling led to an increase in DM intake but a reduction in body weight and fat score through mobilisation of fat and muscle reserves. Despite the marked effect of diet quality on production traits, some inconsistencies were observed between body composition and apparent parasite resistance, measured by WEC and worm counts, suggesting that the nutritional influence was not necessarily always mediated through changes in body composition. Although reproductive status affected blood leptin levels, diet had no effect within suckled ewes and therefore it was concluded that leptin has no causative role in maintaining the periparturient relaxation of immunity to T. colubriformis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The glycolipid CarLA (carbohydrate larval antigen) is present on the epicuticle of the infective-stage larvae of gastrointestinal nematode parasites infecting livestock. The molecule is lost from the surface of the larvae in the few days post-ingestion by a host animal, and the resulting anti-CarLA antibody response has been demonstrated to be protective in vivo. Both the anti-CarLA response, and anti-parasite immunity in general, are slow to develop, and several months of natural exposure to ingested larvae is required. The current study was designed to provide information on how the anti-CarLA response develops, and focuses on the initial recognition of the molecule by human monocyte derived dendritic cells (mdDC) in vitro. Immunofluorescence and flow cytometry demonstrated that mdDC recognise and internalise both the purified and the native form of CarLA, in the case of the latter once it is shed from the larval surface. However, the recognition of CarLA did not result in classical maturation of DC, while there was only transient or minor up-regulation of CD86, CD83, HLA-DR and CD40. Exposure of mdDC to purified CarLA resulted in the increased production of the pro-inflammatory cytokines IL-6 and to a lesser extent of IL-8 and TNF-α, and a reduced production of the anti-inflammatory cytokine IL-1RA. CarLA therefore has little ability to mature and functionally alter monocyte derived dendritic cell function., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Gastrointestinal nematodes represent a major production problem for ruminant livestock. Enhancing immunity to gastrointestinal nematodes through vaccination is desirable but mechanistic understanding of initial host responses that facilitate gastrointestinal nematode protective immunity is limited. We hypothesise that gastrointestinal nematode invasion induces mucosal epithelium damage and alarmin (e.g. IL33) release, thereby contributing to initiation of protective gastrointestinal nematode immunity. To test this, an in vitro air-liquid interface human HT-29 epithelial cell-Trichostrongylus colubriformis co-culture system was developed. Exsheathed L3 T. colubriformis exhibited both sinusoidal and burrowing motions in the co-culture system. Burrowing parasites, but not ivermectin-paralysed larvae, induced necrotic death of epithelial cells (annexin V(+)/propidium iodide(+)/caspase 3/7(-)). Microscopy confirmed that larvae consumed labelled necrotic epithelial cell contents. Trichostrongylus colubriformis larvae and their post-exsheathment antigens (excretory/secretory products) significantly induced IL33 mRNA expression in the epithelial cells. Immunoblot confirmed that IL33 was released from epithelial cells due to the damage caused by motile larvae. Exposure of HT-29 cells to alum or Sigma proprietary adjuvants induced significant epithelial cell IL33 mRNA expression without inducing cellular necrosis. Hence, the intracellular contents were not released externally where they might exert alarmin activity and this may limit their ability to trigger a protective anti-gastrointestinal nematode response. We conclude that T. colubriformis motion at the infection site induces intestinal epithelial cell necrosis which facilitates the release of intracellular contents, including IL33, and may be fundamental to the initiation of an appropriate host response to gastrointestinal nematodes. Our co-culture model is useful for studying initial epithelial cell-parasite interactions without conducting expensive animal trials., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Background: Gastrointestinal nematode (GIN) infections are the predominant cause of economic losses in sheep. Infections are controlled almost exclusively by the use of anthelmintics which has lead to the selection of drug resistant nematode strains. An alternative control approach would be the induction of protective immunity to these parasites. This study exploits an ovine microarray biased towards immune genes, an artificially induced immunity model and the use of pseudo-afferent lymphatic cannulation to sample immune cells draining from the intestine, to investigate possible mechanisms involved in the development of immunity., Results: During the development of immunity to, and a subsequent challenge infection with Trichostrongylus colubriformis, the transcript levels of 2603 genes of cells trafficking in afferent intestinal lymph were significantly modulated (P < 0.05). Of these, 188 genes were modulated more than 1.3-fold and involved in immune function. Overall, there was a clear trend for down-regulation of many genes involved in immune functions including antigen presentation, caveolar-mediated endocytosis and protein ubiquitination. The transcript levels of TNF receptor associated factor 5 (TRAF5), hemopexin (HPX), cysteine dioxygenase (CDO1), the major histocompatability complex Class II protein (HLA-DMA), interleukin-18 binding protein (IL-18BP), ephrin A1 (EFNA1) and selenoprotein S (SELS) were modulated to the greatest degree., Conclusions: This report describes gene expression profiles of afferent lymph cells in sheep developing immunity to nematode infection. Results presented show a global down-regulation of the expression of immune genes which may be reflective of the natural temporal response to nematode infections in livestock.

Resistance to an acute gastrointestinal nematode (GIN) infection is dependent on the ability of the host to recognise the parasite and mount a protective Th2 response. It is hypothesised that lambs which are genetically susceptible to GIN will differentially up-regulate Th1-type genes and therefore remain susceptible to chronic parasitism compared with genetically resistant lambs which will differentially up-regulate Th2-type genes and clear the parasite infection. Two selection flocks, in which lines of Merino sheep produced lambs genetically resistant or susceptible to GIN, were acutely challenged once or thrice with either Haemonchus contortus or Trichostrongylus colubriformis. Faecal-egg counts (FECs), and plasma and tissue anti-parasite (H. contortus or T. colubriformis) antibody isotype responses showed that resistant animals challenged three times with T. colubriformis established a protective Th2 response (negligible FEC, IgG1 and IgE) whereas susceptible animals required multiple challenges to establish a significant IgG1 response despite FECs remaining high. Trichostrongylus colubriformis elicited a more pronounced host response than H. contortus. RNA extracted from tissues at the site of each parasite infection and associated lymph nodes were interrogated by microarray and quantitative PCR analyses to correlate host gene expression to FECs and antibody responses. The IFN-gamma inducible gene cxcl10 was up-regulated in the susceptible line of the Trichostrongylus selection flock sheep after a tertiary challenge with the parasites H. contortus and T. colubriformis. However, a uniform pattern of genes was not up-regulated in resistant animals from both selection flocks during both parasite infections, suggesting that the mode of host resistance to these parasites is different, although some similarities in host susceptibility were apparent., ((c) 2009. Published by Elsevier Ltd. All rights reserved.)

To characterize the role of a range of oxidant, antioxidant and mucous-related genes in the primary response to gastrointestinal nematodes, groups of genetically resistant sheep were challenged with either Haemonchus contortus or Trichostrongylus colubriformis and necropsied for retrieval of tissue at days 0, 3, 7, 14 and 21. To determine if the response was localized to the site of parasite infection, four different gut tissues were sampled: the abomasum, proximal and distal jejunum and ileum. Basal expression patterns of all candidate genes were determined using the day 0 (pre-challenge) samples. A conserved innate response involving elevated expression of dual oxidase, glutathione peroxidase and trefoil factor was initiated within 3 days of challenge and extended out to 21 days. An increase in host gene expression levels at the preferred site of infection (the abomasum for H. contortus and the proximal jejunum for T. colubriformis) was also common to both nematodes. However, these increases were concomitant with reduced expression in other areas of the gut suggesting a compartmentalized response. Other aspects of the response were parasite-specific, with T. colubriformis challenge inducing expression peaks at times corresponding to nematode life-stage transitions.

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

Aim: To evaluate and compare the effects of a variety of diets on the resistance of young, lightweight Merino lambs to repeated low-dose infections of Trichostrongylus colubriformis., Methods: Ninety-six 12-week-old lambs were fed balanced or unbalanced diets, and given primary (at 19 weeks of age) or both primary (at 12 weeks) and secondary (at 19 weeks) infections of T. colubriformis. Suboptimal diets were low in total intake, rumen undegradable protein, total protein, readily available carbohydrate or minerals, or supplemented with cod liver oil. Liveweight was monitored fortnightly, protection was assessed from worm counts at the end of the study (25 weeks) and faecal nematode egg counts (FEC) conducted weekly, and mast-cell response assessed at the conclusion of the study from concentration of mast-cell protease in the jejunum. Concentrations of glucose in plasma were measured at 21 weeks., Results: Diets unbalanced for total protein, readily available carbohydrate, Mo or cod liver oil each increased the worm counts after 6 weeks of secondary infection (at 25 weeks of age) and FEC during secondary infection. Low intake of a high-nutrient diet restricted liveweight gain but did not affect the development of resistance, as indicated by worm counts and FEC. Deficiency of available carbohydrate resulted in reduced liveweight gain and reduced concentration of glucose in plasma. There was a reduction in jejunal mast-cell protease concentration in sheep fed unbalanced rations., Conclusions: The parasitology results and reduction in the concentration of jejunal mast-cell protease observed in sheep fed unbalanced rations suggest that the nutrient deficiencies and the fish-oil supplement may have acted via inhibition or retardation of the host's acquired immune response. Diets unbalanced for a range of components adversely affected the acquired protective response to T. colubriformis to a similar extent and appeared to involve a similar mechanism as each other. The quality of the diet was more important than quantity for the development of resistance, and soluble carbohydrate was an essential component.

This paper describes a series of five pilot trials to test the feasibility of inducing a protective mucosal immune response against a non-blood-feeding intestinal nematode by delivery of antigens across the mucosal epithelium. A number of antigen preparations from Trichostrongylus colubriformis (viable larvae, larval homogenate and recombinant 17kDa excretory-secretory protein) were delivered to the luminal surface of the mucosal epithelium overlying jejunal or rectal lymphoid tissue in cellulose or chitosan formulations. Significant protection was induced following delivery of viable larvae, larval homogenate or recombinant protein to the epithelium overlying rectal Peyer's patches, and recombinant protein to the epithelium overlying jejunal Peyer's patches. Viable larvae were associated with a jejunal IgE/IgG1 response, while the 17kDa antigen was associated with a jejunal IgA response. The results demonstrate that delivery of Trichostrongylus native and recombinant antigens across the epithelium overlying rectal lymphoid patches can result in significant protective immunity even in the absence of adjuvant. They warrant the further investigation of appropriate mucosal delivery methods and adjuvants for induction of protective mucosal responses to stages and species of gastrointestinal helminths which do not ingest serum antibodies.

This study investigated the hypothesis that dietary deficiency of readily available carbohydrate (raCHO) modifies the immune response of lambs to the gastrointestinal nematode Trichostrongylus colubriformis. Sixty helminthologically naive Merino lambs were fed throughout the experiment diets containing three levels of raCHO that provided adequate, moderate or low intakes according to recommended standards and were given primary or both primary and secondary infections of T. colubriformis. A further 20 uninfected lambs received the low diet for 9 weeks, after which they were returned to the standard diet. Immune status was assessed by the measurement of plasma and jejunal antibody concentrations and blood and jejunal cell numbers and function. Diets low in carbohydrate resulted in a failure of the lambs to gain weight and decreases in plasma glucose concentration, blood lymphocytes expressing CD8 or Tcrgammadelta, monocytes, eosinophils, platelets and red blood cells, jejunal and plasma antibody concentrations, lymphocyte proliferation to worm antigen and numbers of jejunal CD8(+) and Tcrdeltagamma(+) lymphocytes, eosinophils and CD1b(+) dendritic cells. Thus, a low dietary concentration of raCHOs impaired the constitutive availability of lymphocytes and antigen-presenting cells, and the cellular and humoral immunological responses. A hypothesis is suggested for the mechanism and for the possible wider implications.

Gastrointestinal nematode infections of livestock animals are prevalent and costly problems worldwide. Currently, infections are controlled by anthelmintic chemicals but increasing drug resistance has prompted research interest to shift towards alternative methods of control such as vaccine development and selection of worm-resistant animals. The present study analyses proteins from Trichostrongylus colubriformis infective L3s that are recognised by IgG of immune sheep. Following protein separation via two-dimensional electrophoresis and Western blot probing with plasma from sheep resistant to T. colubriformis, mass spectrometry-based proteomic analyses were used to identify immuno-reactive protein spots. We were able to identify 28 immune targets, including aspartyl protease inhibitor, enolase, chaperone proteins, galectin, glycolytic enzymes, kinase, phosphatase and structural muscle proteins such as myosin, paramyosin, calponin and DIM-1. The data suggest that immune responses to T. colubriformis are dispersed over a relatively large number of parasite antigens, including several cytoplasmically expressed proteins. The results have new implications for understanding the molecular mechanisms that underpin host-parasite interaction during gastrointestinal nematode infections.

The expression of interleukin (IL)-4, IL-5, IL-10, IL-13, TNF-alpha and IFN-gamma genes, and parasite-specific IgM, IgG1, IgG2, IgA and total IgE levels, were monitored daily in intestinal lymph of sheep infected repeatedly with the nematode parasite Trichostrongylus colubriformis. Host genotype had a significant influence on IL-13 gene activity, with resistant-line (R) sheep consistently expressing higher levels of mRNA than susceptible-line (S) sheep. Mean gene expression of IL-13, IL-4 and IFN-gamma did not differ significantly between the first and second nematode challenge. Field-primed R and S as well as field-primed R and naïve S sheep had lower mean gene expression of IL-5 and IL-10, respectively, during the second when compared to primary challenge. Genes for IL-13 and IL-5 were transiently and strongly up-regulated after nematode infection, particularly in animals with previous exposure to nematodes. Genes for TNF-alpha and IFN-gamma were also transiently up-regulated, but to a lesser extent and more typically after primary challenge. Naïve sheep of both genotypes produced relatively little antibody response after primary challenge. A second nematode challenge resulted in large increases in the lymphatic levels of all antibody sub-classes which were significant for adult antigen-specific IgA and larval antigen-specific IgG1. In naïve S line sheep, the larval-specific IgA and IgG2 response appeared delayed when compared to the R line animals. Field-primed R and S line sheep had relatively high lymphatic IgG1 levels prior to experimental infection and these did not change significantly afterwards. These results demonstrate that during nematode infections, the intestinal micro-environment of sheep is transiently skewed towards Th2 cytokine dominance, although IFN-gamma gene expression continues. This response is accompanied by increases of nematode-specific IgG1, IgA, IgG2 and IgM, as well as of total IgE in lymph plasma.

The transference of immunoglobulins from six New Zealand Romney ewes to their lambs was examined. Immunoglobulin levels were determined in ewe plasma, colostrum and lamb plasma shortly after birth and before the lambs fed, in lamb plasma 2 days after birth, and lamb plasma, ewe plasma and milk 30 days after parturition. Levels of total IgE, and IgE, IgG1, IgG2, IgM, and IgA with specificity for Trichostronglus colubriformis third stage larval secretory/excretory products (TcL3E/S) were determined. Mean levels of total IgE were three times higher in colostrum than in parturient ewe plasma while only trace amounts were detected in milk at 30 days after birth (107.7, 34.3, and 0.2U ml(-1), respectively, differences between means P< or =0.01). Mean total IgE in lamb plasma rose from being undetectable before suckling to levels comparable to those of the ewes by 2 days after birth (21.7U ml(-1)) and then declined to low levels by 30 days (0.4U ml(-1)). Total IgE levels in lamb plasma were significantly correlated with levels in ewe plasma and colostrum (r=0.91, P< or =0.01; r=0.96, P< or =0.003, respectively). The transference of TcL3E/S-specific IgE, IgG1 and IgA was substantial with mean levels of these antibodies in lamb plasma at 2 days comparable to that in parturient ewe plasma (absorbance levels in lamb plasma of 0.283, 0.537, and 0.334, respectively). Proportionally less maternal IgM and IgG2 appeared to be transferred to the lambs (absorbance of 0.112 and 0.081, respectively). Levels of TcL3E/S-specific IgE and IgG1 in lamb plasma at 2 days were significantly correlated with levels in parturient ewe plasma and colostrum (r=0.89 and 0.82, 0.85 and 0.96; all P< or =0.05, respectively). These results indicate that IgE is concentrated in ewe colostrum and that substantial amounts of maternal IgE are transferred to lambs via colostrum. Further, the results suggest that humoral immunity against gastro-intestinal nematode parasites and potentially other parasites in colostrum-fed lambs may approximate that of the ewe. The implications of the transference of humoral immunity through colostrum in ruminants for the passive protection and the development of active immunity against parasites remains to be fully explored.

Cytokine gene expression in cells migrating in afferent and efferent intestinal lymph was monitored for extended time periods in individual sheep experimentally infected with the nematode Trichostrongylus colubriformis. Animals from stable selection lines with increased levels of either genetic resistance (R) or susceptibility (S) to nematode infection were used. Genes for interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha (TNF-alpha), but not for IL-4, IL-10, or gamma interferon (IFN-gamma), were consistently expressed at higher levels in both afferent and efferent lymph cells of R sheep than in S sheep. However, only minor differences were observed in the surface phenotypes and antigenic and mitogenic responsiveness of cells in intestinal lymph between animals from the two selection lines. The IL-4 and IL-10 genes were expressed at higher levels in afferent lymph cells than in efferent lymph cells throughout the course of the nematode infection in animals of both genotypes, while the proinflammatory TNF-alpha gene was relatively highly expressed in both lymph types. These relationships notwithstanding, expression of the IL-10 and TNF-alpha genes declined significantly in afferent lymph cells but not in efferent lymph cells during infection. Collectively, the results showed that R-line sheep developed a strong polarization toward a Th2-type cytokine profile in immune cells migrating in lymph from sites where the immune response to nematodes was initiated, although the IFN-gamma gene was also expressed at moderate levels. Genes or alleles that predispose an animal to develop this type of response appear to have segregated with the R selection line and may contribute to the increased resistance of these animals.

Total and antigen-specific IgE responses in afferent (AIL) and efferent (EIL) intestinal lymph of sheep with a nematode resistant (R) or susceptible (S) genotype during challenge infection with the intestinal nematode parasite Trichostrongylus colubriformis were examined. Within each sheep line, lambs with a nematode naive or nematode field-primed pre-challenge status were used. Total IgE level in AIL and EIL was dependent on nematode infection and was further influenced by genotype or the immune phenotype (nematode immune mean FEC+/-SDM=77+/-179 or non-immune mean FEC+/-SDM=4016+/-4318) of the animal. During T. colubriformis challenge immune animals had higher levels of total IgE in lymph than non-immune sheep, R line sheep had higher concentrations of total IgE than S line sheep, and field-primed animals had higher total IgE levels than nematode naive animals. Concentrations of total IgE were consistently higher in AIL than EIL or serum and were higher in lymph draining the proximal than the distal jejunum demonstrating that polyclonal IgE in AIL was largely derived from the intestinal mucosa of the anatomical compartment where the nematodes reside. The consistently higher concentration of total IgE in AIL was dependent on phenotype or genotype and in S genotype sheep also on the pre-challenge status. Concentrations of nematode specific IgE were significantly higher in EIL than AIL indicating a preference for the production of IgE reacting with excretory secretory products of the infective T. colubriformis larvae in the regional lymph node.

To compare the protection acquired by infection with Haemonchus contortus or Trichostrongylus colubriformis, successive cross infections with the two species were conducted in two genetically similar groups of 150 ewe lambs from 30 sires of INRA 401 breed. At 6 and 7.5 months of age, each group received two doses of 10000 L3 separated by a treatment of one of the species, followed by a similar infection with the second species at 9 and 11.5 months. Faecal egg counts (FECs) were done 28 and 35 days after each dose. There were very high genetic correlation within and between species suggesting that the environmental conditions were very similar. The heritability of FEC of H. contortus ranged from 0.39 to 0.48 and the genetic correlations between FEC after the first and second infections with the same or different species were all near 1. Selection against this species could be done on the response to only one dose. For T. colubriformis, only the response to challenge infection had a similar heritability (0.47); the genetic correlations within and among species were also near one. These results will assist the selection of sheep resistant to nematode parasites.

The J558L cell line, previously transfected with the ovine Cepsilon gene, was induced to secrete a chimeric IgE protein composed of the ovine heavy chain and a mouse light chain with MW of approximately 80 and 26 kDa, respectively. After purification, the chimeric protein was used to immunise BALB-c mice and monoclonal antibodies (mAbs) were generated. The mAb 2F1, which had greatest anti-IgE activity in preliminary screens, was chosen for further characterisation and an examination of systemic and local IgE responses to the intestinal nematode, Trichostrongylus colubriformis. The chimeric IgE protein was not recognised in enzyme linked immunosorbent assay (ELISA) by mAbs raised against ovine IgG1, IgG2, IgA or IgM. However, 2F1 was highly specific to the chimeric IgE protein, and did not cross-react with ovine IgG1, IgG2 or IgA. Western blot analysis also showed that 2F1 and secretory IgA (sIgA) did not cross-react, and that 2F1 and the anti-IgA mAb identified different MW bands from colostrum (approximately 200 and 400 kDa, respectively). 2F1 bound to mucosal mast cells (MMC) isolated from the intestines of lambs infected with T. colubriformis, but cultured bone marrow-derived mast cells (BMMC) required prior incubation with the chimeric IgE protein for this binding to occur. Distinctive staining of plasma cells and putative mast cells were observed using 2F1 on immunohistological sections of mesenteric lymph node and jejunum.ELISA incorporating 2F1 was able to detect >0.4 ng chimeric protein. Total IgE in ovine colostrum and intestinal homogenates was quantified using a capture ELISA, with known amounts of chimeric protein used to produce a standard curve. Colostrum from outbred Merino ewes had 0.55-11.05 ng ml(-1) total IgE, and their lambs, at necropsy after infection with a total of 18,000 T. colubriformis infective larvae over a 9-week period, had 45-620 ng g(-1) total IgE in intestinal tissue. Compared to genetically susceptible lambs, antigen-specific levels of IgE were significantly higher in genetically resistant lambs after infection with 4500 T. colubriformis infective larvae (TcL3) per week for 9 weeks (161.4 versus 44.8 geometric mean titres; P=0.043). In western blots, distinctive bands (19-21 and 27 kDa) from T. colubriformis larval antigen were differentially recognised by IgE, as identified by 2F1, in intestinal homogenates from genetically resistant animals. These results have demonstrated the value of 2F1 for quantification of IgE responses in samples derived from ovine fluids and tissues using ELISA, western blots and immunohistology. In this respect, it recognises native ovine IgE and does not require pre-treatment of the sample with denaturing agents or ammonium sulphate.

Monoclonal antibodies (MAbs) were developed against recombinant ovine interleukin-5 (IL-5) produced in the baculovirus expression vector system. One MAb, D11 (isotype IgG1), neutralised the activity of both recombinant and native sources of IL-5 in a biological assay (Baf cell assay) but was only weakly reactive in immunocytochemistry. Conversely, a second MAb, A8 (isotype IgA), successfully detected IL-5 in immunocytochemistry but did not display neutralising activity. The development of these MAbs will enable the assay of ovine IL-5 in vitro and permit studies into the role of hypersensitivity reactions in sheep by neutralisation of IL-5 in vivo.

We investigated whether the generation of protective memory humoral immunity in Cooperia oncophora infected calves occurs in a dose-dependent way and whether it depends on the animal responder types. To this end, serum and mucus antibody responses were measured in animals primary-infected with 30000 or 100000 L3, treated with anthelmintics and subsequently challenged with 100000 L3. A detailed phenotypic and functional analysis of B cells was done in animals infected once or twice with 100,000 L3. Based on the similarity in parasitological variables of animals primed with 30000 or 100000 L3, we concluded that with these doses priming conferred protection in a dose-independent way. Upon challenge significant increases in Cooperia-specific serum and mucus IgG1 and IgA and total serum IgE titres were induced in primed animals in a dose-independent way. In contrast, intermediate and low responders differed in the onset of the production of Cooperia-specific serum IgG1. Furthermore, not only the onset but also the level of total serum IgE significantly differed between intermediate and low responders. Phenotypic and functional analysis of B lymphocytes revealed that (i). priming induced the generation of memory B cells which upon challenge readily differentiated into antibody secreting cells; (ii). sensitised B cells were more efficiently recruited to the intestinal effector sites; (iii). based on the expression of CD62L and CD86 two distinct B cell subpopulation could be differentiated. CD62L(+)CD86(-) B cells that were likely lymphocytes not yet activated and with an enhanced recirculation capacity, and CD62L(-)CD86(+) B cells that were activated B cells with a reduced recirculation ability; and finally (iv). the increased expression of CD86 and subsequent correlations with parameters of the T helper 2 immune response induced by C. oncophora, suggested that CD86- interactions are involved in the generation of protective immunity against Cooperia.

Cooperia oncophora is the most prevalent intestinal nematode of cattle occurring in Western Europe. Primary infection with 100000 third stage infective larvae (L3) induces acquired immunity in a high proportion of the animals but there is little information on immunity against re-infection. In the current experiment, the contribution of the T-cell mediated immunity in protection against re-infection with C. oncophora was investigated in detail. Priming elicited long-lasting protective immunity that was evidenced by a significantly decreased worm burden and egg excretion in primed animals compared to challenge control animals. Lymphocyte proliferation tests with excretory/secretory products (ESP) of C. oncophora and with three distinct ESP fractions indicated an enhanced reactivity in primed animals and suggested that by fractionating of ESP we selected for proteins involved in protective immunity against re-infection with C. oncophora. Phenotypic analysis of T cell subsets at diverse anatomical locations revealed that the enhanced reactivity of lymphocytes from peripheral blood and lymph nodes of the infected animals coincided with a significantly increased frequency of CD4(+) cells at these locations but a deceased frequency of CD4(+) cells in the lamina propria. These findings were independent of the immune status of the animals but more pronounced in the primed animals than in the challenge control animals. In addition we demonstrated that primary and secondary infections with C. oncophora were associated with two waves of eosinophils and that the kinetics of this cell population differed as a result of priming. Based on the observed correlations we propose that the early increase of eosinophils is T cell independent and merely a consequence of inflammation in the parasitised gut. In contrast, the second wave of eosinophils depends upon CD4(+) cells and correlations with parasitological parameters at this time point support a role of eosinophils as effector cells against adult stages of C. oncophora.

Allergens were identified from the gastrointestinal nematode of sheep, Trichostrongylus colubriformis, by probing Western blots of infective larvae (third stage) somatic antigen with IgE purified from the serum of sheep grazed on worm contaminated pasture. A 31 kDa allergen was frequently recognised by sera from immune sheep, particularly those deriving from a line that has been genetically selected over 23 years for parasite resistance. Using a proteomic approach, the 31 kDa allergen was identified as an aspartyl protease inhibitor homologue. The entire coding sequence of T. colubriformis aspartyl protease inhibitor (Tco-api-1) was obtained and the mature protein expressed in Escherichia coli. Anti-Tco-API-1 antibodies revealed that a commonly observed 21 kDa T. colubriformis allergen species is a truncated form of Tco-API-1. Specific IgE responses to T. colubriformis aspartyl protease inhibitor were significantly correlated with the degree of resistance to nematode infection as measured by faecal egg count in sheep. Surprisingly, IgE responses to Tco-API-1 were not correlated with breech soiling (dag score), which is thought to be caused, in part, by allergic hypersensitivity to worms. Therefore, a specific IgE response to this allergen may be a suitable marker for identifying lambs at an early age that will develop strong immunity to gastrointestinal nematodes.

The response of the mucosal immune system of 4-6-week old lambs to viable Trichostrongylus colubriformis larvae was compared in two groups of animals, one exposed to T. colubriformis antigen and the other to saline while in utero. Exposure to larval antigen two-thirds of the way through gestation resulted in significant reduction in the frequency of jejunal goblet cells and of ileal eosinophils, CD 1b(+) antigen-presenting cells and CD4(+), CD5(+) and CD8(+) cells. However, it resulted in a significant increase in the jejunal CD8(+) response to postnatal challenge. The expression of the cytokines TNF-alpha and IL-1 beta in the ileum, and of jejunal NSE, was significantly reduced by in utero exposure, whereas those of jejunal TNF-alpha and ileal TGF-beta were increased. The observed changes in cellular and cytokine responses to challenge with viable larvae, in those lambs previously exposed in utero, indicated that the intestinal mucosal immune system remains susceptible to down-regulation until considerably later in foetal development than is the case for other components of the immune system.

In an accompanying paper we show that antibodies in intestinal mucus that recognize a 35-kDa antigen from the surface of the L3 stage of the sheep intestinal nematode parasite, Trichostrongylus colubriformis, are strongly associated with immune rejection of L3 in a truncated infection model of immunity in sheep. Monoclonal antibody PAB-1 was used to immunopurify this antigen from T. colubriformis L3. The antigen is resistant to digestion with a range of proteases including proteinase K and does not stain on gels or blots treated with protein-detecting reagents but does stain with carbohydrate-detecting reagents. The antigen is also resistant to degradation by the action of lipases and is not soluble in organic solvents, suggesting that lipid components are not present or not accessible. Treatment with glycosidases does not affect the antigen, indicating either that sialic acid and N-linked or O-linked sugars are not present or that they are not accessible to enzyme attack. The antigen is not destroyed by harsh alkaline degradation with up to 8 m NaOH with or without borohydride reducing agent, or by extensive hydrazinolysis. Strong acid hydrolysis with trifluoroacetic acid or boiling in hydrochloric acid for 20 min does destroy the antigen. The antigen migrates as a poorly defined high molecular weight complex on native electrophoresis gels, but is detected as a major band at 35 kDa on SDS PAGE either by carbohydrate staining or by immunoblotting with antibody from immune sheep intestinal mucus and with mAb PAB-1. Proteinase K digestion and alkaline degradation of extracts from L3 of 10 other parasitic nematode species revealed that L3 of each species contained a carbohydrate staining molecule which can be detected by mAb PAB-1 and by mucus antibody from immune sheep. Because antibodies in intestinal mucus are directed against these antigens and may be responsible for protective immunity, carbohydrate larval antigens (CarLA) could represent a new family of molecules with potential as targets for stimulating host immunity.

Sheep that have been immunized by multiple truncated infections with the parasitic nematode Trichostrongylus colubriformis contain anti-larval activity in their intestinal mucus and high-speed mucus supernatants. This activity induces T. colubriformis L3 to clump in vitro and causes a significant reduction in larval establishment in naive sheep after infusion of larvae and mucus into the intestinal lumen via a duodenal cannula. In this report, we provide evidence that one factor contributing to the anti-larval activity of immune mucus is antibody against a 35-kDa L3-specific cuticular antigen. The anti-larval activity in mucus is > 100 kDa by membrane filtration, is heat labile and sensitive to either protease digestion or reduction with DTT. Immunoblotting showed that mucus and supernatants of ultracentrifuged mucus from immune sheep contained IgG1 and IgA antibodies that recognized predominantly a larval antigen with an estimated molecular weight of 35 kDa on SDS-PAGE. Antibodies eluted from the surface of washed larvae that had been incubated in immune mucus also reacted specifically with the 35 kDa antigen on blots of larval homogenate. Immunofluorescence and immunogold electron microscopy showed that the 35 kDa antigen is present on the epicuticle of L3 and is shed during the moult to L4. The antigen is not present in eggs, L1, L2, L4 or adult worms and is found only in extracts of sheaths and L3 before infection and up to 4 days after infection. We hypothesize that the binding of antibody to the larval surface prevents larvae from establishing at their preferred site, causing them to be eliminated from the intestine. Monoclonal antibody PAB-1 recognizes the 35 kDa T. colubriformis larval antigen and also cross-reacts with antigens of similar molecular weight on blots of L3 extracts of the parasitic nematodes Haemonchus contortus and Ostertagia circumcincta; and with a 22-kDa antigen on blots of L3 extracts from Cooperia curticei and Nematodirus spathiger. This indicates that an antigenically related surface antigen with immunizing potential is present on several nematode species and can be identified by mAb PAB-1. The 35 kDa T. colubriformis larval antigen and related molecules in other nematodes are potential novel targets for stimulating host-protective immunity against nematode infections.

The effect of infection of sheep with the gastrointestinal nematode parasite Trichostrongylus colubriformis on expression of adhesion molecules CD11a, CD11b, CD11c, CD18, CD44, CD49d and CD62L by peripheral blood eosinophils was examined by flow cytometry. Initially, to establish the sensitivity of adhesion molecules to inflammatory signals, eosinophil-rich exudates were elicited in non-lactating mammary glands of immune sheep by infusion of 50 microg of soluble antigen extract from T. colubriformis third stage larvae. Eosinophils comprised 40.8% of mammary leucocytes and 4.5% of peripheral blood leucocytes. In comparison with blood, the percentage of eosinophils expressing CD18 increased and the percentage expressing CD62L decreased in exudates and the mean fluorescent intensity, an indicator of receptor number per cell, for CD11a and CD49d also decreased on exudate eosinophils. Peripheral blood eosinophils were examined over 8 weeks during trickle infection of immune sheep with infective or irradiated third stage larvae of T. colubriformis. During the last 3 weeks of infection, CD11a staining decreased in infected sheep and CD44 staining decreased in sheep receiving either infective or irradiated larvae. Other surface markers did not change. The results indicate that systemic changes in expression of adhesion molecules by eosinophils occur during T. colubriformis infection in sheep.

Haemonchus contortus is known as a prolific parasite, producing high numbers of eggs. It could therefore be used as a cheap source of larvae for immunisation of lambs. The value of immunisation would be improved if the immunity produced gave protection against not only homologous but also heterologous infections. Because antibody cross-reactivity between Haemonchus and Ostertagia has been well established, we wanted to know whether drug-abbreviated infections of H. contortus would stimulate production of antibodies that would cross-react with Trichostrongylus colubriformis. The results obtained from these trials indicated that H. contortus drug-abbreviated infections produced significant immunity against not only Haemonchus but also O. circumcincta. Immunisation increased the level of immunoglobulin E (IgE) and IgE-specific antibodies against T. colubriformis, but the differences between experimental and control animals were not statistically significant. Significantly higher levels of IgG-specific antibodies against T. colubriformis were observed.

Jirds (Meriones unguiculatus) were infected with 700 infective larvae (L3) of Trichostrongylus colubriformis. The fecal egg counts (epg) were followed regularly until 340 days postinoculation, and worm numbers were determined on necropsy at 315 and 350 days postinoculation. The epg increased to a peak of 2,600 on day 24 postinoculation. Thereafter, the epg showed continuous decrease from day 31 until day 318 postinoculation. The rates of inoculum recovery were 0.29-1.43% on day 315 and 0.142-0.428% on day 350 postinoculation. On day 315 postinoculation, 12 male worms were recovered from 2 of 3 animals necropsied. On day 350 postinoculation, all 6 animals were infected, and 6 female and 4 male worms were recovered. The systemic IgM, IgG, and intestinal mucous IgA responses were significantly higher in infected groups compared with negative control groups necropsied on 315 and 350 days postinoculation (P < 0.001). These results confirm that the T. colubriformis-jird system is a useful model for studies on the various host-parasite relationships of chronic trichostrongylosis.

Proteinases are known to be capable of prolonging the survival of endoparasites in a host. We were therefore interested in knowing whether immunization of lambs against a proteasome (multisubunit proteinases) preparation obtained from Trichostrongylus colubriformis infective third-stage larvae (L3) would have any effect on the immune response to a single challenge infection with the same organism. A total of 21 penned lambs aged 8 months were divided into 3 equal groups. Group 1 was immunized on three occasions with increasing amounts of a proteasome-enriched fraction obtained from infective L3. Group 2 was given a similar amount of protein from the initial supernatant of homogenized larvae. Group 3 (controls) received adjuvant plus saline solution only. All groups were challenged with 60,000 infective T. colubriformis larvae at 28 days after the last immunization. Significant protection was obtained only when the initial supernatant extract was used to immunize lambs. The proteasome preparation seemed to have immunosuppressive effects through the stimulation of nonspecific IgE production. Significantly lower levels of specific IgE were observed in lambs immunized with the proteasome-enriched fraction, and levels of specific IgG antibodies were increased. We suggest that proteasome fractions of T. colubriformis may serve as useful preparations for the study of mechanisms of IgE production in parasitized sheep.

In vitro leucocyte proliferative responses to parasite antigens and to mitogens as well as lymphocyte sub-types were compared in guinea pigs with genetically determined differences in their ability to express protective immunity against Trichostronylus colubriformis infection. Proliferative responses to parasite antigens were greatest in high-responder (HR) animals, but cells from low-responder (LR) animals were generally more responsive to mitogens. However, HR circulating leucocytes were more responsive to the T-cell-dependent B-cell mitogen pokeweed mitogen (PWM), and the response of HR, but not LR, cells increased during primary infection with T. colubriformis. Flow cytometry revealed significantly greater numbers of circulating B-cells in HR animals and, as observed for responsiveness to PWM, the number of circulating B-cells increased in HR, but not LR, animals during primary infection with this parasite. These findings suggest a larger and more labile population of B-cells in HR guinea pigs.

Numerous studies have examined the interactions between protein nutrition and the response to nematode parasitism in sheep, but very few in goats. Compared with other ruminants, goats are less resistant to nematode infection. In addition, in dairy goats, high producing animals have been shown to be less resistant and less resilient to infection compared to low producing ones. The objective of the present study was to examine the consequences of protein supplementation on both resistance and resilience of dairy goats to nematode trickle infection, taking into account the initial level of milk production of the animals. During a 14-week period, 40 milking goats received a high protein (HP) diet supplying 130% of the protein requirements, and 38 goats were fed a intermediate protein (IP) diet (120% of the protein requirements). In addition, half of each group was given a weekly trickle infection with Trichostrongylus colubriformis larvae, the other part of the flock remained non-infected. Faecal egg counts (FEC), eosinophil counts and pathophysiological data (urea, albumin and inorganic phosphate concentrations in the serum) were measured twice a month. Milk production data (milk yield, protein and fat contents) were also recorded every 15 days. The results showed that FECs were lower (p < 0.05) and eosinophil counts higher (p < 0.05) in the animals receiving the HP diet suggesting that resistance was enhanced by protein supplementation. Meanwhile, milk parameters (related to resilience) were not affected by the level of protein in the diet when considering the whole groups. In contrast, in the high producing goats, the milk production and milk composition parameters were improved with the HP diet. To conclude, we have seen that the expression of both resistance and resilience did not appear when the coverage of the protein requirements was insufficient. Because the milk production is dependent on the protein supply, we suggest that there is a competition in the use of the protein between the development of resistance and the milk production.

The aim of this research was to determine whether metabolisable protein supply during the early period of infection with Trichostrongylus colubriformis influenced resilience and the later stages in the development and magnitude of host resistance in previously nematode-naïve lambs. Eighty TexelxGreyface lambs were fed pelleted feeds calculated to provide grossly different amounts of metabolisable protein. Sixty of the lambs received a trickle infection of T. colubriformis and 20 lambs were kept as uninfected controls. There were four initial groups, namely infected or uninfected and fed either a moderate or a high protein feed. After 5weeks of infection, a further four groups were established by changing the feed of half of the animals fed the moderate protein feed to the high protein feed and of half of the animals fed high protein to the moderate protein feed. Live weight gain and feed conversion ratio were greatest for lambs fed the high protein feed and were reduced by infection. Faecal egg counts, worm burdens and per capita fecundity of adult female nematodes were unaffected by changes to metabolisable protein supply. Decreasing metabolisable protein supply following 5weeks of infection reduced live weight gain without any effect on resistance to T. colubriformis. Haematological variables, indicative of improved resistance, were also largely unaffected by metabolisable protein supply. It is concluded that the requirements of immune function probably had priority over those of growth and that the metabolisable protein supply provided by the moderate protein feed was sufficient to account for the requirements for the expression of immunity. It is probable that the potential for metabolisable protein supply to enhance resistance to infection from T. colubriformis is dependent on the levels and magnitude (i.e. in relation to maintenance requirements) of metabolisable protein supply being compared and the demand of other competing physiological functions.

Background and Objective: A hypersensitive inflammatory response, associated with the ingestion of trichostrongylid larvae, is a major cause of diarrhoea and soiling of the breech with faeces in adult Merino sheep ('hypersensitivity scours'). This response is characterised by an infiltration of eosinophils and altered lymphocyte populations in the gut of affected sheep. The objective of this study was to investigate the numbers of Ostertagia circumcincta and Trichostrongylus vitrinus infective larvae that were needed to induce diarrhoea in adult Merino sheep grazing improved pastures., Method: Sheep from three farms were categorised as either being susceptible, or not susceptible, to hypersensitivity scours, then transported to an experimental farm and grazed on pastures with low numbers of trichostrongylid infective larvae. They were ranked by farm of origin and previous worm egg count, then systematically allocated to one of five dose-rate groups and infected with 0, 2, 4, 10 or 20 x 10(3) trichostrongylid infective larvae per week for 13 weeks., Results: In sheep selected as being susceptible to hypersensitivity scours, the lowest dose rate of infective larvae (2000 per week) was as effective in inducing diarrhoea as the highest dose rate (20,000 per week). In contrast, even the highest larval dose did not induce diarrhoea in sheep selected as not susceptible to hypersensitivity scours. Sheep selected with an increased susceptibility to hypersensitivity scours also had a significantly higher amount of moisture in their faeces at all times except during summer., Conclusions: The apparent lack of a threshold dose of trichostrongylid infective larvae needed to induce hypersensitivity scours suggests that even improved worm control programs, which substantially decrease the ingestion of larvae, may not be able to reduce the prevalence of hypersensitivity scours or the severity of breech soiling. Controlled-release anthelmintic capsules will control breech soiling but are often not a cost-effective strategy. Thus, genetic selection of less susceptible sheep is probably the best long-term option for the control of hypersensitivity scours, and the assessment of faecal moisture may be a convenient marker of susceptibility to this syndrome.

Phenotypic variation in faecal worm-egg count (FEC) was studied in 100 Angora goats between 6 months and 2 years old as part of a wider investigation into the potential use of FEC as a selection trait in breeding goats for improved natural resistance to nematode infection. Shortly after weaning at 4-5 months old the goats were subjected to a procedure involving successive natural and experimental challenges, which was continued at 6-monthly intervals until the goats were 2 years old. Experimental challenges consisted of Trichostrongylus colubriformis infective larvae administered orally at a dose rate of 1000 larvae/kg liveweight. FECs resulting from both natural and experimental infections varied considerably between individual goats as well as between different sampling times. Mixed model analysis of variance of natural infections confirmed that a significant component of this variation was attributable to differences between individual goats (P < 0.01) as opposed to other potential sources of variability, indicating that although generally deemed to be more susceptible than sheep, Angora goats do nevertheless vary in their ability to resist nematode infection. Rank correlations between pairs of FECs derived from the same infection were relatively high for both natural and experimental infections (r = 0.63-0.70 and 0.54-0.79, respectively; all P < 0.01). However, those between mean FECs measured on successive 6-monthly sampling occasions were considerably lower and more variable (r = 0.01-0.41 and 0.19-0.62 for natural and experimental infections, respectively). Nevertheless, FECs resulting from natural infections showed moderate correlations with those from corresponding experimental infections (P < 0.01) on all except the first sampling occasion (r = 0.23-0.61). Correlations were marginally higher when FECs from experimental infections were adjusted for infecting dose size (r = 0.32-0.63). FECs resulting from experimental infections were significantly higher on the first sampling occasion (at 6 months) than on subsequent occasions (P < 0.01) suggesting that some degree of resistance to nematode establishment had developed in the goats by 12 months old. On the basis of unadjusted FECs there appeared to be no marked increase in resistance beyond that age, although analyses based on FECs adjusted for infecting dose size suggested an increase in acquired resistance up to 18 months of age. FECs following natural challenge in goats which had also received experimental infections were not significantly different from those exposed only to natural challenge, indicating that the drench-abbreviated experimental infections had had no immunizing effect. Does that had given birth and were lactating at 24 months had significantly higher FECs than 'dry' does in the flock (P < 0.01), suggesting that like sheep, goats exhibit a post-parturient relaxation of immunity. The use of FECs as potential indicators of resistance status in goats and as a criterion for the selection of breeding animals is discussed.

The premise that any bias of immune reactivity in neonatal lambs towards T-helper (TH)2 responses could benefit the induction of protection against gastrointestinal nematodes was investigated. In two trials, lambs were either trickle-immunised with 2000 infective larvae of Trichostrongylus colubriformis (TcL3), 3 times weekly from the day of birth for 6 weeks or inoculated with a recombinant T. colubriformis 17 kDa antigen in incomplete Freund's adjuvant (IFA). In trial 1, trickle immunised and control neonates challenged at 7 weeks of age had similar worm counts 10 days after challenge, but from 25 days, significant reductions (P<0.01) in mean faecal egg count and worm count in excess of 75% were displayed by the immunised lambs. The results of a second, similar trial, gave 85-91% reductions in parasitism in trickle immunised neonates (P<0.001) and around 50% protection in neonates vaccinated with recombinant 17 kDa antigen. Parasitism in immunised neonates in Trial 2 was significantly reduced (P<0.001) compared to that in 4-month-old animals. Antibody responses in trickle-immunised (protected) and challenge control (infected) neonates were almost exclusively of the IgG1 isotype compared to vaccinated animals which exhibited increased levels of anti-17kD IgG2. Trichostrongylus colubriformis infection, but not specific vaccination, induced interleukin-5 production by mesenteric lymph node cells. The results offer the tantalising prospect of generating protective immunity to gastrointestinal parasites prior to weaning in sheep; this was most effectively generated by viable parasites in this investigation.

Weaner sheep that had been hand-fed on diets containing increasing concentrations of protein for a 9-week period (when uninfected, or infected with Haemonchus contortus) were studied during the next 69 weeks when put on to pasture as a single, unsupplemented flock. During the 9-week period, groups of 12 sheep (six infected, six uninfected) were offered one of five iso-energetic (9.0 MJ kg(-1)) diets containing 10, 13, 16, 19 or 22% crude protein. All sheep were treated with anthelmintic at the end of the 9 weeks and then put out to pasture for 69 weeks, where they were all subject to the same environmental variables including nematode larval challenge. During the grazing period, animals that had previously received the higher protein diets consistently had higher live-weight gain and wool production, higher antibody responses to both H. contortus and Trichostrongylus colubriformis antigenic challenge in vitro, and lower faecal nematode egg counts than did the lambs previously offered the lower protein diets. Faecal egg counts of the grazing sheep that had been artificially infected with H. contortus while being hand-fed were similar to those of the uninfected sheep and there was no interaction between previous infection and dietary protein concentration. We conclude that short periods of enhanced post-weaning nutrition can have long-term and perhaps life-long effects on production.

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration of challenge infection and collection of intestinal fluids. Sheep were slaughtered at various times after challenge and the distribution of larvae along the small intestine was determined. Results showed that immune sheep had significantly fewer larvae in their intestines and that some sheep could expel the challenge infection within 2 h. Mucus samples from immune sheep contained increased parasite-specific antibody, histamine and anti-parasite activity as measured by larval migration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids of immune sheep after challenge. Immunisation of sheep by truncated infections stimulated serum IgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections before challenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers of mucosal mast cells and globule leucocytes after challenge, compared with naive sheep. When challenge larvae were mixed with mucus from immune sheep and infused back into naive recipient sheep, there was a distinct displacement of the larval population towards the distal part of the intestine, compared with the profile of larval establishment after infusion with mucus from naive sheep. These results are further evidence for an immediate hypersensitivity reaction in the intestine of immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larval properties of mucus. The cannulated-sheep challenge model described here will be a useful tool to unravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.

Antibody levels were compared in guinea pigs with genetically determined differences in their ability to generate protective immunity against the small-intestine nematode parasite Trichostrongylus colubriformis. Animals with the most effective immune response (high responders) developed significantly higher anti-T. colubriformis IgG1 antibody titres than low-responder animals. However, there were no significant differences between their IgG1 antibody responses to a systemically administered protein antigen (ovalbumin). High-titre anti-T. colubriformis serum from high-responder animals did not transfer significant passive protective immunity to low-responder recipients. It is suggested that anti-T. colubriformis IgG1 antibodies mediate the release of mast-cell and basophil products at the site of infection and thus contribute to the more effective immunity expressed by high-responder animals.

Two groups of second grazing season cattle, which had been treated with an ivermectin-sustained-release bolus (ISRB) in their first grazing season, were monitored during consecutive years (1995 and 1996) on the same second grazing season pasture. In the preceding year (1994), this pasture had been grazed by yearlings that had not received chemoprophylaxis in their first grazing season. The objectives of the study were, firstly, to investigate whether the chemoprophylactic-treated yearlings were less resistant to gastrointestinal nematodes upon subsequent exposure, and hence excreted more strongyle eggs compared to the control yearlings; secondly, whether an increased susceptibility of the previously treated animals resulted in a yearly increase of the pasture infestation on the second grazing season pasture; and finally, whether this affected the second year weight gain of the animals. In 1996, the yearlings that had been chemoprophylactic-treated in 1995 excreted higher numbers of nematode eggs, compared to the previously 'untreated' yearlings. In addition, the proportion of Cooperia larvae was markedly higher in the faecal cultures from the chemoprophylactic treated-animals, suggesting a negative effect of preventive treatment with an ISRB on the acquired resistance of the animals. However, there was no evidence that the slightly higher egg output in the previously treated yearlings had an effect on the larval contamination of the second grazing season pasture. A significant yearly decrease in the second season average daily weight gains was observed, but it could not be inferred from the results of the parasitological parameters that the differences in second year growth were caused by different levels of resilience between chemoprophylactic-treated and -untreated animals. As the study covered three consecutive second grazing seasons, an effect of differences between years (e.g. in weather conditions or grass growth) on the results cannot be excluded.