Within-host mechanisms of immune regulation explain the contrasting dynamics of two helminth species in both single and dual infections.

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.

Attempts to induce tolerance to Trichostrongylus colubriformis infection in sheep.

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.

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

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

Predicting the effects of parasite co-infection across species boundaries.

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.

Immune development and performance characteristics of Romney sheep selected for either resistance or resilience to gastrointestinal nematodes.

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.

Identification of Peptide Mimics of a Glycan Epitope on the Surface of Parasitic Nematode Larvae.

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.

Immune-mediated responses account for the majority of production loss for grazing meat-breed lambs during Trichostrongylus colubriformis infection.

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.

Trichostrongylus colubriformis induces IgE-independent CD13, CD164 and CD203c mediated activation of basophils in an in vitro intestinal epithelial cell co-culture model.

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.

A co-infection with two gastrointestinal nematodes alters host immune responses and only partially parasite dynamics.

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.

Recovery of Trichostrongylus colubriformis infective larvae from three grass species contaminated in the autumn / Recuperação de larvas infectantes de Trichostrongylus colubriformis em três espécies de gramíneas contaminadas no outono

This experiment aimed to assess the recovery of infective larvae (L3) of Trichostrongylus colubriformis from Brachiaria decumbens cv. Australiana, Cynodon dactylon cv. Coast-cross and Panicum maximum cv. Aruana. The experimental module comprised six plots, with two plots per herbage species. Larval survival was assessed from autumn to winter, under the effect of two herbage-paring heights (5 and 30 cm). TThe paring was carried out immediately before contamination with faces containing T. colubriformis eggs. The feces and herbage were collected at one, two, four, eight, 12 and 16 weeks after feces had been deposited in the experimental plots. In general, larvae were recovered from both herbage and feces until the 16th week. The longer persistence of these larvae in the environment was probably due to warmer temperatures. The number of L3 recovered from the pasture was not influenced by the height of plants, except for Brachiaria and Aruana herbage in the fourth week. Regarding the concentrations of larvae per kg of dry matter (L3/kg DM), recovery was higher from low pasture in all three herbage species. During the autumn, the development and survival of the T. colubriformis free-living stages were not affected by the different herbage species.(AU)

O experimento teve como objetivo avaliar a recuperação de larvas infectantes (L3) de Trichostrongylus colubriformis em Brachiaria decumbens cv. Australiana, Cynodon dactylon cv. Coast-cross e Panicum maximum cv. Aruana. Foram utilizados módulos experimentais constituídos por seis canteiros, perfazendo dois canteiros por espécie forrageira. A sobrevivência larval foi avaliada do outono até o inverno, sob o efeito de duas alturas de poda (5 e 30 cm). A poda foi realizada imediatamente antes da deposição das fezes contaminadas com ovos de T. colubriformis. A colheita das fezes e da forragem foi realizada uma, duas, quatro, oito, 12 e 16 semanas após a deposição das fezes nos canteiros experimentais. De modo geral, foram recuperadas larvas das forragens e das fezes até a 16ª semana. Essas larvas persistiram por mais tempo no ambiente, provavelmente em razão das temperaturas mais amenas. O número de L3 recuperadas nas pastagens não foi influenciado pela altura das plantas, exceto nos capins braquiária e aruana na quarta semana. Já em relação às concentrações de larvas (L3/kg MS) recuperadas das três forrageiras, houve maior concentração nas pastagens baixas. Durante o outono, o desenvolvimento e a sobrevivência de estádios de vida livre de T. colubriformis não foram afetados pelos diferentes tipos de espécies de forrageiras.(AU)

Production of active single-chain antibodies in seeds using trimeric polyoleosin fusion.

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.

The response of monocyte derived dendritic cells following exposure to a nematode larval carbohydrate antigen.

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.

Immune responses in sheep naturally infected with Oestrus ovis (Diptera: Oestridae) and gastrointestinal nematodes.

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.

Detection of quantitative trait loci for resistance to gastrointestinal nematode infections in Creole goats.

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.

The influence of reproductive physiology and nutrient supply on the periparturient relaxation of immunity to the gastrointestinal nematode Trichostrongylus colubriformis in Merino ewes.

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.

Trichostrongylus colubriformis larvae induce necrosis and release of IL33 from intestinal epithelial cells in vitro: implications for gastrointestinal nematode vaccine design.

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.

The gastrointestinal nematode Trichostrongylus colubriformis down-regulates immune gene expression in migratory cells in afferent lymph.

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.

Nematode challenge induces differential expression of oxidant, antioxidant and mucous genes down the longitudinal axis of the sheep gut.

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.

Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus.

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.

Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

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.