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.

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)

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.

Increased expression of interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha genes in intestinal lymph cells of sheep selected for enhanced resistance to nematodes during infection with Trichostrongylus colubriformis.

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.

Intestinal exposure to a parasite antigen in utero depresses cellular and cytokine responses of the mucosal immune system.

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.

Immunomodulation of lambs following treatment with a proteasome preparation from infective larvae of Trichostrongylus colubriformis.

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.

Quantification of total sheep IgE concentration using anti-ovine IgE monoclonal antibodies in an enzyme immunoassay.

Monoclonal antibodies (mAbs) which recognize separate epitopes on ovine immunoglobulin E (IgE) have been used to develop a non-competitive antibody sandwich enzyme immunoassay (EIA) for quantitating ovine IgE. Purified anti-IgE mAb (YD3) coated onto polystyrene microtitre plates was used to capture IgE in serum samples. Biotinylated anti-IgE mAb (XB6) followed by streptavidin conjugated with horseradish peroxidase were used to detect captured IgE. Tetramethylbenzidine and H2O2 were used as enzyme substrate. A reference serum was prepared by pooling sheep sera containing elevated IgE levels. This reference serum was assigned a value of 100 units ml-1 and used to prepare standard curves for the EIA. The linear region of log-log transformed standard curve data covered a range of 0.05-0.8 units ml-1. The equation of a linear regression line fitted to this curve was used to determine sample concentrations. Using purified IgE, 1 unit of reference serum was equivalent to 0.86 micrograms ml-1 IgE. Maximum intra- and inter-assay coefficients of variation for the EIA were 4.6% and 9.7%, respectively. Subjecting serum samples to 15 freeze/thaw cycles, storage at room temperature for 16 days or incubation at 37 degrees C for 8 h resulted in minimal loss of IgE detection. Incubation of serum at 56 degrees C resulted in rapid reduction in detection of IgE by the EIA. The assay was used to determine IgE levels in adult sheep monospecifically infected with weekly doses of the nematode Trichostrongylus axei. Serum IgE levels increased from 9 to 16 days following first infection and reached maximum levels by days 35-58. Serum IgE responses closely followed IgE positive cell responses in the abomasal mucosa.

Sequential cellular and humoral responses in the abomasal mucosa and blood of Romney sheep dosed with Trichostrongylus axei.

Abomasal cannulae were surgically placed in 7 2-year-old New Zealand Romney sheep which had been maintained parasite-free from birth. Four of these sheep were randomly selected and dosed orally with 10,000 infective Trichostrongylus axei larvae per week for 8 weeks, while the remaining 3 sheep served as uninfected controls. Abomasal biopsy, blood and faecal samples were obtained from all sheep at regular intervals from 5 days before and until 58 days after the first infection. The sheep were then killed, worm burdens assessed and abomasal and small intestinal samples collected Faecal egg counts of all 4 dosed sheep were low and only one (No. 701) had a substantial worm burden (8400) post mortem. Overall, levels of mucosal mast cells/globule leukocytes, eosinophils, T19+ cells and larval migration inhibitory activity increased significantly in the abomasal mucosa of the dosed sheep compared to the controls. The CD4+:CD8+ cell ratio in the abomasal mucosa of the dosed sheep also increased compared to that of the controls (P = 0.06). In blood, T. axei-specific antibody (total and IgG1) and eosinophil numbers increased significantly in the dosed sheep. Mucosal cells staining for IgE (IgE+), and blood and mucosal eosinophils showed the earliest substantive increases in number followed by increases in specific serum antibody levels, numbers of mucosal cells fluorescing under UV light (UVf) and T19+ cells. The difference in the IgE+ and UVf cell responses indicated that expansion of globule leukocyte numbers lagged behind that of mucosal mast cells. The results supported the concept of CD4+ T cell help in the abomasal mucosa and defined the sequential expression of components of the immunological responses potentially mediating resistance to T. axei. In sheep No. 701, persistence of adult worms was associated with lower mucosal IgE+ cell and eosinophil responses compared with the other dosed sheep.

Rejection of heterologous nematodes by sheep immunized with larval or adult Trichostrongylus colubriformis.

Sheep immunized by truncated larval infections or by the adoptive transfer of adult Trichostrongylus colubriformis were subsequently challenged with single infections of T. colubriformis, Nematodirus spathiger, Haemonchus contortus or Ostertagia circumcincta or combinations of the parasites. Sheep vaccinated with larval infections were > 90% protected by 4 days after challenge (DAC) against T. colubriformis L3 given in a single or combined infection, whereas no significant protection was exhibited against a single-species infection with the unrelated nematodes N. spathiger or O. circumcincta. Sheep challenged with T. colubriformis together with N. spathiger or O. circumcincta were equally protected against both intestinal nematodes, but O. circumcincta was not affected. Sheep immunized with adult T. colubriformis and challenged with a combined infection of T. colubriformis, N. spathiger and H. contortus expelled around 40 and 80% of the intestinal parasites by 4 and 11 DAC, respectively, but showed no protection against the abomasal parasite, H. contortus. The results confirm the previous findings on 'self-cure' and the non-specific rejection of unrelated parasites living in the same or downstream niches in the gut when the nematode used to induce immunity is included in the challenge infection. The results also indicate that even though L3 antigens effectively elicited the non-specific rejection, antigens produced by L4 and later stages could also induce rejection of unrelated worms in the second week after infection.

The isolation, characterization and cloning of a globin-like, host-protective antigen from the excretory-secretory products of Trichostrongylus colubriformis.

An 18-kDa component from the excretory-secretory (ES) products of adults of Trichostrongylus colubriformis was isolated and characterized, and was shown to induce 60-84% protection of guinea pigs from challenge infection following a single intraperitoneal injection. Amino-terminal sequence analysis of gel-purified protein enabled oligonucleotides to be synthesized and used to screen a lambda gt10 cDNA library made from young adult worm mRNA, and to synthesize full-length clones from cDNA using the polymerase chain reaction (PCR). The full-length clones coded for a 20-kDa precursor protein of 173 amino acids which had a strongly hydrophobic leader sequence of 15 residues. The mature protein sequence of 158 amino acid residues was rich in charged amino acids (32%), including 8 oppositely charged pairs of amino acids. The protein sequence contained no half-cystine residues and no potential N-glycosylation sites. Unlike 2 other fully characterized ES components which are expressed only in the parasitic stages, mRNA coding for the 20-kDa component was present in both the parasitic and free-living stages of T. colubriformis. The parasite protein had approximately 20% identity with globins from human and from the larvae of the insect Chironomus thummi thummi. The homology included the invariant distal histidine and phenylalanine, and a number of other residues highly conserved in globins.

Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis.

The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep. We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite. This glycoprotein is secreted by the L4 and adult parasitic stages of the worm. The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond. The clones also indicate the existence of sequence variability in this antigen. Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.