Total and nematode-specific IgE responses in intestinal lymph of genetically resistant and susceptible sheep during infection with Trichostrongylus colubriformis.

Total and antigen-specific IgE responses in afferent (AIL) and efferent (EIL) intestinal lymph of sheep with a nematode resistant (R) or susceptible (S) genotype during challenge infection with the intestinal nematode parasite Trichostrongylus colubriformis were examined. Within each sheep line, lambs with a nematode naive or nematode field-primed pre-challenge status were used. Total IgE level in AIL and EIL was dependent on nematode infection and was further influenced by genotype or the immune phenotype (nematode immune mean FEC+/-SDM=77+/-179 or non-immune mean FEC+/-SDM=4016+/-4318) of the animal. During T. colubriformis challenge immune animals had higher levels of total IgE in lymph than non-immune sheep, R line sheep had higher concentrations of total IgE than S line sheep, and field-primed animals had higher total IgE levels than nematode naive animals. Concentrations of total IgE were consistently higher in AIL than EIL or serum and were higher in lymph draining the proximal than the distal jejunum demonstrating that polyclonal IgE in AIL was largely derived from the intestinal mucosa of the anatomical compartment where the nematodes reside. The consistently higher concentration of total IgE in AIL was dependent on phenotype or genotype and in S genotype sheep also on the pre-challenge status. Concentrations of nematode specific IgE were significantly higher in EIL than AIL indicating a preference for the production of IgE reacting with excretory secretory products of the infective T. colubriformis larvae in the regional lymph node.

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.