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.     

Expression of cell surface adhesion molecules by peripheral blood eosinophils during Trichostrongylus colubriformis infection in sheep

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.     

Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis

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 distribution and localization of the stage-specific GP31 antigen from infective Ostertagia circumcincta larvae.

The 31,000 mol. wt glycoprotein (GP31) antigen of infective third-stage (L3) Ostertagia circumcincta larvae was shown, by surface labelling experiments and immunofluorescent antibody staining of whole larvae and larval sections, to be distributed internally. When transverse sections of L3 O. circumcincta, taken from the anterior pharyngeal region, were further examined by electron microscopy, after immunogold staining with rabbit anti-GP31 antiserum, the GP31 antigen was found to be specifically located in 'secretory organelles' within the cells of the oesophageal glands. By in vitro culturing L3 O. circumcincta in medium supplemented with 35S-methionine and then analysing the excretory-secretory material released by the larvae, it was found that the GP31 molecule was one of the major components of the excretory-secretory complex. The purified GP31 molecule had no detectable proteolytic activity in protein degradation assays. On examination of Triton X-100 extracts of infective larvae from other nematode parasite species, a predominant antigen similar to GP31 was found in Trichostrongylus colubriformis and Haemonchus contortus, but in Toxocara canis a minor component corresponding in mol. wt to GP31 was also detected. Based on these results the possible role of GP31 as a candidate antigen for a broad spectrum molecular vaccine against gastrointestinal nematode parasites in sheep is discussed.     

Antibody production in guinea pigs with genetically determined high and low responsiveness to Trichostrongylus colubriformis

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.     

Identification of immuno-reactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using two-dimensional electrophoresis and mass spectrometry

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.     

A molecular diagnostic tool to replace larval culture in conventional faecal egg count reduction testing in sheep

The accurate diagnosis of parasitic nematode infections in livestock (including sheep and goats) is central to their effective control and the detection of the anthelmintic resistance. Traditionally, the faecal egg count reduction test (FECRT), combined with the technique of larval culture (LC), has been used widely to assess drug-susceptibility/resistance in strongylid nematodes. However, this approach suffers from a lack of specificity, sensitivity and reliability, and is time-consuming and costly to conduct. Here, we critically assessed a specific PCR assay to support FECRT, in a well-controlled experiment on sheep with naturally acquired strongylid infections known to be resistant to benzimidazoles. We showed that the PCR results were in close agreement with those of total worm count (TWC), but not of LC. Importantly, albendazole resistance detected by PCR-coupled FECRT was unequivocally linked to Teladorsagia circumcincta and, to lesser extent, Trichostrongylus colubriformis, a result that was not achievable by LC. The key findings from this study demonstrate that our PCR-coupled FECRT approach has major merit for supporting anthelmintic resistance in nematode populations. The findings also show clearly that our PCR assay can be used as an alternative to LC, and is more time-efficient and less laborious, which has important practical implications for the effective management and control strongylid nematodes of sheep.     

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.     

Cross-immunity between Haemonchus contortus and Trichostrongylus colubriformis in sheep

Cross-protective immunity between the nematode parasites, Haemonchus contortus and Trichostrongylus colubriformis, was examined in sheep vaccinated with irradiated larvae of either species. Secondary immunological responsiveness stimulated in this manner protected only against challenge infection with the species used for vaccination. Significant cross-protective immunity was not observed. Titres of serum antibody to an extract of adult but not infective larval T. colubriformis reflected the specificity for protective immunity. Immediate hypersensitivity skin reactions to nematode extracts did not reflect the antigen-specificity for protective immunity.     

The effects of immunization of sheep with Trichostrongylus colubriformis larvae on worm burdens acquired during grazing

The effects of immunization of sheep with Trichostrongylus colubriformis larvae on worm burdens acquired during grazing. International Journal for Parasitology 19: 177-181. Romney sheep, reared helminth-free in pens to 5 months of age, were immunized against Trichostrongylus colubriformis by giving two doses of 200,000 T. colubriformis infective larvae at 15 day intervals to assess protection from natural challenge during grazing. Five immunized sheep and five unimmunized sheep were grazed on infested pasture for 4 weeks, and were then returned to the pens for 4 weeks before slaughter. Worm burdens, gastrointestinal histology and mucus antiparasite activity were examined at slaughter. Faecal egg counts and haematological examinations were carried out at regular intervals throughout the trial. Significant protection (P less than 0.05) was afforded immunized sheep against adult T. colubriformis (87%), T. axei (67%), Nematodirus spathiger (91%) and Ostertagia spp. (42%). Greater numbers of immature Nematodirus spp. and Ostertagia spp. were present in immunized sheep Overall, a significant (P less than 0.05) 42% reduction in total nematode burdens was afforded by immunization of the sheep with T. colubriformis larvae. Immunized sheep had significantly (P less than 0.05) more globule leukocytes, mast cells and eosinophils in gastrointestinal tissue and significantly (P less than 0.05) higher levels of mucous antiparasite activity than unimmunized sheep. Haematological observations showed some sheep had transient eosinophilia during immunization or grazing. Both immunized and unimmunized sheep showed depressed (P less than 0.05) total leukocyte counts during grazing which returned to pre-grazing levels within 1 week of return of the sheep to the pens. Overall, haematological parameters reflected parasite challenge and were unrelated to worm burdens acquired.     

Gastrointestinal nematode challenge induces some conserved gene expression changes in the gut mucosa of genetically resistant sheep

Sheep have a varying ability to resist infection with gastrointestinal nematodes. This ability is due in part to genetic differences that exist between individuals. In order to define these differences we have used real-time PCR to quantify gene expression responses in the gut mucosal surface of genetically resistant and susceptible sheep, following a nematode challenge. Expression profiles were determined in response to two different nematode species, Haemonchus contortus and Trichostrongylus colubriformis, and in divergent sheep originating from two different genetic backgrounds. Results show that the response generated differs between resistant and susceptible animals and is further impacted by the origin of the sheep and nematode species used for challenge. However, some conserved features of a response mounted by a resistant or a susceptible animal were identified. Genes found to be more abundantly expressed in resistant animals include markers of an early inflammatory response, several Toll-like receptors (TLR2, 4, 9) and free radical producing genes (DUOX1 and NOS2A). Conversely, genes differentiating susceptible animals indicate a prolonged response and development of a chronic inflammatory state, characterised by elevated expression of members of the NF-kappabeta signalling pathway (IKBKB and NFKBIA) together with delayed expression of regulatory markers such as IL2RA (CD25), IL10 and TGFbeta2. While multiple nematode response pathways were identified, the identification of conserved aspects of the response which associate with resistance provides evidence that alternative nematode control strategies, such as breeding for resistant animals, may be feasible.     

Cellular changes in the intestinal lymph of sheep infected with the enteric nematode, Trichostrongylus colubriformis

Some changes produced in the cell populations of intestinal lymph by infection with the enteric nematode, Trichostrongylus colubriformis, were studied in sheep regularly re-infused with all discharged lymph. Lymphocyte traffic through the intestinal lymphatic duct was reduced until day 35 of primary infection, mainly due to the absence of lymphocytes with smaller cell volumes, but was increased two-fold after day 70 and in immune sheep. Antigen-reactive lymphocytes in blood and lymph were assayed by the uptake of 3H-thymidine in cell culture stimulated by extracts from the larvae of T. colubriformis. Cells from the blood and lymph of immune sheep were highly reactive to worm antigen. A relatively smaller reactivity was present in the blood of worm-free sheep and was abolished during the first 12 days of primary infection. Antigen reactive cells were not detected in intestinal lymph until 12 days after primary infection, and in vitro antigen reactivity in intestinal lymph of immune sheep was increased after challenge with infective larvae. Responses to the mitogens, concanvalin A and phytohaemagglutinin, in cultures of cells from both intestinal lymph and blood were depressed on days 7 and 12 of primary infection. It is proposed that the diminished traffic of lymphocytes in intestinal lymph and the reduced numbers of mitogen and nematode antigen-reactive lymphocytes in both blood and intestinal lymph during the early stages of infection with T. colubriformis is closely related to the slow development of protective immunity to this parasite.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.     

Isolation and degranulation of mucosal mast cells from the small intestine of parasitized sheep.

The isolation of mucosal mast cells and globule leucocytes from the small intestine of sheep immunized with Trichostrongylus colubriformis is described. Sheep mast cell protease was released from these cells in a dose-dependent fashion after incubation with soluble protein from T. colubriformis larvae. Release also occurred with other T. colubriformis antigens whereas non-parasite antigens at comparable protein concentrations evinced only a minimal response. Mucosal mast cells prepared from worm-free sheep also produced a similar minimal response. This is the first report describing the release of sheep mast cell protease from isolated sheep intestinal mucosal mast cells after addition of specific parasite antigens.     

Local and systemic antibody responses in gerbils following vaccination with irradiated or non-irradiated Trichostrongylus colubriformis larvae

Groups of 10 Mongolian gerbils, Meriones unguiculatus, were vaccinated with 1,500 gamma-irradiated Trichostrongylus colubriformis infective larvae (L3) or with non-irradiated larvae. 25 days later five gerbils from each group were necropsied and the remaining gerbils challenged with 1,500 non-irradiated T. colubriformis infective larvae. Systemic, local intestinal and coproantibody levels were compared in each group of gerbils 25 days after vaccination and 26 days after challenge. Strong local intestinal and faecal antibody responses were detected. Coproantibodies reflected antibody levels in the intestinal contents and in mucosal extracts. The results gave further support to the view that coproantibody measurements provide a sensitive index of immunity at mucosal surfaces to intestinal parasites.     

Demonstration of a range of inflammatory mediators released in trichostrongylosis of sheep.

The levels of inflammatory mediators in the intestinal contents of sheep immunized with Trichostrongylus colubriformis larvae increased in the first 6 days after challenge. These mediators were histamine, leukotriene C4, 6-keto-prostaglandin F1 alpha (from prostacyclin) and thromboxane B2. Leukotriene C4 was released in the greatest quantities. Leukotriene B4 was present but its concentration remained unchanged after challenge. The presence of these particular mediators in the intestinal contents after challenge is consistent with antigen-induced mediator release from the mucosal mast cells found in immune sheep undergoing challenge infection. This is the first sequential analysis of mediator release in sheep that also demonstrates the release of prostacyclin and thromboxane into the intestine during expulsion of a nematode infection.     

Basophil leucocytes in cutaneous hypersensitivity reactions in nematode-infected guinea-pigs.

Cutaneous hypersensitivity reactions were elicited in guinea-pigs infected with the parasitic nematode Trichostrongylus colubriformis by injecting them with a crude extract of T. colubriformis fourth-stage larvae. The reaction was characterized by early oedema and pronounced cellular infiltration, initially with neutrophils but later with mononuclear cells, basophils and variable numbers of eosinophils. Because basophils have been implicated as effector cells in the protective immune response of guinea-pigs to this nematode, the capacity to elicit a basophil-rich cellular infiltrate in infected animals might be a useful assay for T. colubriformis protective antigen(s).     

Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

The efficiency of RNA interference (RNAi) delivery to L1 through L3 stage worms of the sheep parasitic nematode Trichostrongylus colubriformis was investigated using several techniques. These were: (i) feeding of Escherichia coli expressing double stranded RNA (dsRNA); (ii) soaking of short interfering (synthetic) RNA oligonucleotides (siRNA) or in vitro transcribed dsRNA molecules; and (iii) electroporation of siRNA or in vitro transcribed dsRNA molecules. Ubiquitin and tropomyosin were used as a target gene because they are well conserved genes whose DNA sequences are available for several nematode parasite species. Ubiquitin siRNA or dsRNA delivered by soaking or electroporation inhibited development in T. colubriformis but with feeding as a delivery method, RNAi of ubiquitin was not successful. Feeding was, however, successful with tropomyosin as a target, suggesting that mode of delivery is an important parameter of RNAi. Electroporation is a particularly efficient means of inducing RNA in nematodes with either short dsRNA oligonucleotides or with long in vitro transcribed dsRNA molecules. These methods permit routine delivery of dsRNA for RNAi in T. colubriformis larval stage parasites and should be applicable to moderate to high-throughput screening.     

Dietary modulation of the mucosal immune response to a parasite

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.     

Characterization of the major immunogen in the excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis

The excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis conferred some protection to guinea pigs against homologous challenge. A glycoprotein with an apparent molecular mass of approximately 94 kDa was the dominant immunogen in post-exsheathment products. Immunoblots revealed IgG antibodies to this glycoprotein in sera from multiply-infected guinea pigs and some sheep, and in sera of guinea pigs after three truncated infections which had been restricted by anthelmintic treatments to development of the third parasitic stage. IgA antibodies to this protein were also found in intestinal lymph of a naturally infected sheep. Fluorescent antibody studies indicated that this 94 kDa component was associated with cells in the central body cavity of third-stage larvae, but was absent from fourth-stage larvae or adult worms. Fractionation and protection assays in guinea pigs revealed that while the native and aggregated 94 kDa protein conferred some host protection, it was not the only protective component of the excretory-secretory products of exsheathed third-stage larvae of T. colubriformis.