Intestinal infusion of soluble larval antigen stimulates rejection of heterologous nematode larvae by immune sheep.

Sheep immunised by multiple truncated infections with Trichostronglyus colubriformis were highly resistant to subsequent challenge with homologous exsheathed larvae, administered via a surgically implanted duodenal cannula. The duration of immunity after truncated infections was 12-14 weeks against challenge with T. colubriformis or Cooperia curticei, but there was little cross-protection against challenge with Nematodirus spathiger. When immune sheep were given booster doses of T. colubriformis larvae before challenge with N. spathiger, there were 97% fewer N. spathiger larvae in the first 5 m of small intestine, and an overall 79% reduction of N. spathiger larvae in immunised sheep, compared with naive controls. Boosting T. colubriformis immune sheep with killed T. colubriformis larvae plus soluble T. colubriformis L3 antigen, or with soluble antigen alone, also caused significant displacement of N. spathiger challenge larvae (98% and 100% respectively), indicating a non-specific expulsion process. These results indicate that N. spathiger can be used as an indicator species in T. colubriformis immune sheep, to quantify the effects of stimulating mucosal immunity with specific antigens, which may lead to identification of the antigens required for immunisation against nematodes.

Studies on the role of mucus and mucosal hypersensitivity reactions during rejection of Trichostrongylus colubriformis from the intestine of immune sheep using an experimental challenge model.

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

Serum IgE responses during primary and challenge infections of sheep with Trichostrongylus colubriformis.

Serum IgE responses during primary or challenge infections with Trichostrongylus colubriformis were examined by measuring total IgE and parasite-specific IgE by ELISA. Three- to four-month-old lambs reared nematode-free received a single primary infection of 30,000 T. colubriformis-infective larvae (TcL3). Infections were terminated after 100 days by anthelmintic treatment, at which time faecal egg counts had fallen to low levels. Total serum IgE increased slowly from 20 days p.i. until anthelmintic treatment. The specific IgE response to T. colubriformis adult antigen peaked at 20-27 days p.i. before returning to near baseline levels. A further slight increase occurred between 56 and 100 days p.i. The animals were then divided equally into two groups. A first series of challenge infections consisting of either weekly 10,000 TcL3 infections (Group A) or a single 30,000 TcL3 infection (Group B) produced low faecal egg counts. Total and specific IgE to adult and L3-ES antigens increased rapidly over a 21-day period, then remained elevated irrespective of challenge regime. Termination of primary and the first challenge infections with anthelmintic resulted in a rapid decline in serum IgE responses but not other T. colubriformis-specific Ig responses. A second series of challenge infections consisting of repeated 10,000 (Group A) or 20,000 TcL3 infections (Group B) resulted in all IgE responses peaking within 7-8 days p.i. Marked softening of faeces coincided with peaks of specific and total IgE responses during challenge infections. The results of this study showed that infection of sheep with T. colubriformis leads to elevated levels of total and parasite-specific IgE in serum. IgE responses following challenge suggest involvement of this antibody isotype in protection from T. colubriformis infections.

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.

Pilot field trial of a recombinant Taenia ovis vaccine in lambs exposed to natural infection.

Previous trials of an experimental Taenia ovis vaccine using the recombinant antigen GST--45W(B/X) established that it was possible to achieve >90% protection against a single artificial challenge of T. ovis eggs. This trial was undertaken to assess vaccine efficacy against artificial challenge and natural infection acquired by lambs grazing contaminated pasture. Two hundred Romney lambs were vaccinated at 6 and 12 weeks of age. One hundred control lambs were not vaccinated but were allowed to run with the vaccinated mob. At 15 weeks of age, 10 controls and 18 vaccinated lambs were artificially challenged with 2000 T. ovis eggs. The remaining control and vaccinated lambs were allowed to graze contaminated pasture for 3 weeks and were then moved to clean pasture for 5 months. The artificially challenged lambs plus 24 of the field-infected lambs were slaughtered and the carcasses dissected to obtain cyst counts. The remaining field-infected lambs were slaughtered at a commercial processing plant and the carcasses examined by conventional meat inspection. The results showed that the vaccine provided a high level of protection against artificial challenge (92%) and natural infection (98%) when assessed by carcass dissection. The data from commercial meat inspection showed that vaccination provided 89% efficacy against downgrading or condemnation compared to non-vaccinated control lambs. The average difference in carcass values between vaccinated and non-vaccinated groups was 4.36 dollars, representing a 35% loss in value due to T.ovis infection in non-vaccinated lambs.

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.

Production and characterisation of monoclonal antibodies recognising ovine IgE.

Two monoclonal antibodies (mAbs), XB6 and YD3, which recognise ovine immunoglobulin E (IgE) were produced. Mast cells isolated from ovine intestinal mucosa were used as a source of IgE to immunize mice. Culture supernatants of hybridomas were screened by immunoassays on small-intestine tissue sections, isolated mucosal cells, and dot blots of lysed mast cell homogenate. Two mAbs were chosen for their specific binding to mast cells. Antigen bound by these mAbs was purified by immunoaffinity chromatography using XB6 mAb, and this produced two bands consistent with IgE heavy chain (86,000 Daltons) and immunoglobulin light chain (28,000 Daltons) when run under reducing conditions on SDS-PAGE gels. Purified IgE was shown on dot blots to react weakly with mAb to chimeric ovine IgE and strongly to polyclonal anti-sheep antibodies. The two mAbs induced an immediate hypersensitivity-like reaction when injected into the skin of sheep. The mAbs bound to mast cells and other mononuclear cells, presumably IgE-secreting B-cells in mesenteric lymph node sections. These mAbs proved useful for detecting IgE-bearing cells in various ovine tissues, for purifying mast cells from cell isolates by panning and immunomagnetic bead separation, for purifying serum IgE using immunoaffinity chromatography and for detecting IgE in an ELISA. Competitive binding assays showed that the two mAbs bind to different epitopes on IgE. These mAbs will be useful in research applications and in diagnostic assays.