A. Pernthaner

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

ABSTRACT 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-α), but not for IL-4, IL-10, or gamma interferon (IFN-γ), 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-α gene was relatively highly expressed in both lymph types. These relationships notwithstanding, expression of the IL-10 and TNF-α 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-γ 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.

Antibodies to surface epitopes of the carbohydrate larval antigen CarLA are associated with passive protection in strongylid nematode challenge infections

Sheep were immunized by multiple truncated infections with the gastrointestinal nematodes Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. Three infections with T. colubriformis of 14 days plus five booster doses of L3 stimulated highly effective protection against challenge (99%). Three infections of 14 days plus three booster doses with H. contortus also resulted in significant protection against challenge infection (87%), but the same procedure was not effective for T. circumcincta. Antibodies derived from gastrointestinal mucus of these immunized sheep were tested for their ability to reduce worm burden following injection of antibody‐coated exsheathed larvae into the abomasum (H. contortus and T. circumcincta) or duodenum (T. colubriformis) of nematode‐naïve sheep in a passive immunity test. The IgG fraction from the mucus of immunized sheep reduced worm burdens by 62%, 76% and 91% in three tests with T. colubriformis but was not effective for either of the abomasal dwelling nematodes H. contortus and T. circumcincta. Antibodies in immune mucus predominantly recognized two larval surface antigens on immunoblots of L3 extract, a high MW surface glycoprotein and the carbohydrate larval antigen (CarLA). Antibodies raised against purified T. colubriformis glycoprotein Tc‐120 and CarLA were tested in the passive immunity model and it was found that only the antibody against CarLA resulted in a significant reduction of infection (87%). The protective anti‐CarLA antibodies strongly recognized the surface of living T. colubriformis L3. Antibodies from abomasal mucus of sheep immunized by H. contortus and T. circumcincta infections reacted weakly with CarLA and the larval surface and did not reduce worm counts in a passive immunity test. The results provide further evidence that the larval surface carbohydrate antigen CarLA has potential as a mucosal immunogen for a strongylid nematode vaccine.

Immune mechanisms of resistance to gastrointestinal nematode infections in sheep.

Infections with gastrointestinal nematode parasites are a major problem for the sheep industry in Australia and New Zealand and have been the subject of intensive research to define mechanisms of resistance. The ability to take continuous biopsy samples of infected organs and cannulate both afferent and efferent lymphatics of draining lymph nodes has been particularly useful in illuminating the kinetics of immune responses at the site of infection. Distinct localized immune responses were shown to occur within and between sheep breeds at different sensitization regimes, as well as at different developmental stages of the parasite within the host. Using localized antibodies derived from mucus and lymph nodes, two major antigens have been identified on the infective L3 stage, which may be responsible for inducing protection and have potential as vaccine targets. Recent advances in sheep genomics also offer the potential of gaining further insight into the underlying genetics of resistance to nematode infections.

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

BackgroundGastrointestinal 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.ResultsDuring 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.ConclusionsThis 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.

Immunization of sheep against parasitic nematodes leads to elevated levels of globule leukocytes in the small intestine lumen

In sheep that had been given three immunizing infections with Trichostrongylus colubriformis and Ostertagia circumcincta infective (L3) larvae, drenched after the last infection and challenged with larvae of the same species, there was a significant increase in numbers of small intestine mucosal tissue globule leukocytes (TGLs) and lumenal globule leukocytes (LuGLs) compared with sheep that had only been drenched and challenged. There was a positive correlation between the numbers of LuGLs and TGLs in the small intestine but the ratio of these two cell types was lower in non-immunized than immunized sheep. In immunized sheep positive correlations were observed between LuGLs and levels of arylsulphatase and peroxidase in the intestinal mucus and between arylsulphatase and larval migration inhibition (LMI) activity in mucus. Lumen eosinophils correlated with blood eosinophils, serum antibody against T. colubriformis correlated with peroxidase in the mucus and blood eosinophils correlated with nematode specific IgM levels in the intestinal mucus. In the abomasum, TGLs were present but not LuGLs. Sheep repeatedly infected with T. axei also had significantly more LuGLs in the small intestine than control animals. Two sheep that had a surgically prepared isolated small intestinal loop, after oral infection with T. colubriformis had TGLs and LuGLs in the intact intestine, but not in the isolated loop. Significantly more LuGLs were produced in sheep by allowing repeated T. colubriformis L3 infections to develop to adult stages compared to sheep treated with the same number of larvae, but where the infections were terminated by drenching at various intervals.

Phenotypic diversity of antigen-presenting cells in ovine-afferent intestinal lymph.

The phenotypic and functional repertoire of antigen-presenting cells (APCs) remains incompletely characterized, particularly during the migratory phase of their life history, when these cells leave peripheral tissues and travel via afferent lymphatic vessels to regional lymph nodes. Lymphatic cannulation procedures were used to collect ovine APCs as they migrated from the mucosa of the small intestine to regional lymph nodes. A panel of 19 new monoclonal antibodies (mAbs) was produced to characterize surface molecules expressed on APCs by means of two-color flow cytometry and microscopy. Two broad patterns of mAb reactivity were evident. Twelve mAbs reacted almost exclusively with cells in the APC-gated region, because all these mAbs stained < 3% of cells in the lymphocyte-gated region. Within this group, some mAbs identified distinct subsets of dendritic cells (DCs). The second group of seven mAbs displayed high-intensity staining on cells in the APC-gated region but also reacted with variable numbers (4-26%) of cells in the lymphocyte-gated region. This indicates that molecules recognized by these mAbs are highly expressed on APCs but also occur on other lineages. When new mAbs were analyzed by two-color flow cytometry of cells in afferent intestinal lymph, a wide range of differences in reactivity were observed, especially on CD11b(+), CD11c(+), CD4(+), MHCII(+), and gammasigmaTCR(+) cells. Although molecular specificities of mAbs reported here remain undefined, marked heterogeneity of staining patterns indicates considerable phenotypic, and probably functional, diversity within APC population in afferent intestinal lymph. MAbs reported here will provide useful tools to explore these features further.

Drug-abbreviated infections and development of immunity against Trichostrongylus colubriformis in sheep

A protective immune response without liveweight loss can be induced in sheep against T. colubriformis but results depend on the anthelmintic used and duration of immunizing infections. More than 90% protection was achieved in sheep immunized by three 15- or 7-day oxfendazole abbreviated infections or three 21-day nonabbreviated infections. Only 41% protection was induced by 3-day oxfendazole abbreviated infections. Significantly higher worm burden and faecal egg counts were present after challenge in sheep immunized by 7-day levamizole abbreviated infections compared to 7-day oxfendazole abbreviated infection. Liveweight gains of sheep immunized by 15- and 7-day abbreviated infections were not significantly different than non infected controls. Liveweight loss seemed to be associated with high activity of mucus peroxidase and high numbers of eosinophils in the intestinal lumen. High parasite numbers seemed to be associated with low activity of alkaline phosphatase in mucus. Mucus peroxidase, arylsulphatase, larval migration inhibition of mucus, mucus or serum antibody against L3 excretory/secretory antigen or somatic L3, L4 and adult antigen were not associated with protection.

The immune responsiveness of Romney sheep selected for resistance or susceptibility to gastrointestinal nematodes: Lymphocyte blastogenic activity, eosinophilia and total white blood cell counts

Blastogenic activity, eosinophil and total white blood cell counts (TWBC) were examined over a period of 14 weeks in Romney lambs, genetically resistant or susceptible to gastrointestinal nematodes. The lambs were infected with 5000 infective Trichostrongylus colubriformis larvae twice weekly. Compared to preinfection levels, the blastogenic activity of unstimulated lymphocytes in lambs of both lines peaked at week 3, and was significantly higher in resistant than in susceptible lambs. These changes may have been due to in vivo polyclonal activation. Lymphocytes from susceptible sheep responded more strongly to Con A, PHA and PWM than cells from resistant sheep. Counts per minute (c.p.m) for Con A- and PHA-stimulated lymphocytes increased in both lines of sheep from week 2 to week 7 and then returned to initial levels. An increase in c.p.m. in PWM-stimulated cell cultures was observed from weeks 3 to 5 in both groups. The blastogenic activity for LPS-stimulated cultures was significantly higher for resistant than susceptible sheep at weeks 3 and 4. No significant correlations between the decline in faecal egg counts (FEC) and the blastogenic activity was observed. Eosinophil counts in peripheral blood began to increase one week earlier in resistant than in susceptible sheep. No significant correlation between FEC and eosinophil counts was observed in resistant lambs, whereas in susceptible lambs a significant correlation was found between FEC and eosinophil counts at some sampling times. TWBC in resistant lambs steadily increased with infections whereas susceptible lambs showed a decrease until week 5 and then steadily increased. There was no significant correlation between the decline in FEC and TWBC.(ABSTRACT TRUNCATED AT 250 WORDS)

Digested and Fermented Green Kiwifruit Increases Human β-Defensin 1 and 2 Production In vitro

The intestinal mucosa is constantly exposed to a variety of microbial species including commensals and pathogens, the latter leaving the host susceptible to infection. Antimicrobial peptides (AMP) are an important part of the first line of defense at mucosal surfaces. Human β-defensins (HBD) are AMP expressed by colonic epithelial cells, which act as broad spectrum antimicrobials. This study explored the direct and indirect effects of green kiwifruit (KF) on human β-defensin 1 and 2 (HBD-1 and 2) production by epithelial cells. In vitro digestion of KF pulp consisted of a simulated gastric and duodenal digestion, followed by colonic microbial fermentation using nine human faecal donors. Fermenta from individual donors was sterile filtered and independently added to epithelial cells prior to analysis of HBD protein production. KF products obtained from the gastric and duodenal digestion had no effect on the production of HBD-1 or 2 by epithelial cells, demonstrating that KF does not contain substances that directly modulate defensin production. However, when the digested KF products were further subjected to in vitro colonic fermentation, the fermentation products significantly up-regulated HBD-1 and 2 production by the same epithelial cells. We propose that this effect was predominantly mediated by the presence of short-chain fatty acids (SCFA) in the fermenta. Exposure of cells to purified SCFA confirmed this and HBD-1 and 2 production was up-regulated with acetate, propionate and butyrate. In conclusion, in vitro colonic fermentation of green kiwifruit digest appears to prime defense mechanisms in gut cells by enhancing the production of antimicrobial defensins.

Condensed tannins from Botswanan forage plants are effective priming agents of γδ T cells in ruminants.

The potential impact of extracts from forage plants on γδ T cell activity in ruminants was evaluated using an in vitro immunoassay. This study investigated whether plant extracts could prime γδ T cells via up-regulation of CD25 (interleukin-2 receptor alpha). Purified Sephadex LH-20 fractions, isolated from Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius and Grewia flava, were screened against γδ T cells on kid, lamb and calf peripheral blood lymphocytes. Condensed tannins (CT) from G. flava significantly primed γδ T cells in kids up to 64.75% at 10 μg/mL, which was statistically significant relative to the negative control at 22.66% (p=0.004). CT from T. oleifolius also induced priming of γδ T cells in kids, while fractions from V. rotundifolium and V. verrucosum induced minimal priming of γδ T cells. In contrast, there was no significant priming of γδ T cells from lambs and calves for any of the tested fractions (p>0.05). These findings suggest that CT from a selected range of Botswanan forage plants can stimulate the immune system in vivo in selected ruminant species and may participate in enhancing host innate immune responses.