Mitigation of production losses associated with ruminant gastrointestinal nematode infections by induction of mucosal tolerance : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University 

Abstract

This thesis describes a series of studies designed to further develop an antigen treatment regime involving mucosal immunisation with somatic nematode antigens to modify the immune response and reduce production losses associated with Trichostrongylus colubriformis infections in sheep. The aim of the first study (Chapter 3) was to determine the larval stage specifity of T. colubriformis antigen to induce successful tolerance. Thirty-six five-month-old Poll Dorset x East Friesian lambs were born and raised indoors and allocated hierarchically by live weight (mean 30.50 ± 0.67 kg) into one of six treatment groups (n=6). The antigens investigated for successful immunisation were evaluated using single somatic and purified antigens that were prepared from each larval stage of T. colubriformis (L3, L4 and L5) and one combination antigen consisting of all three larval stages. Antigen doses containing either 7.5 µg L3, 5.5 µg L4 and 10.5 µg L5 or a combination of all three were contained in 200 µl PBS and injected into the rectal submucosa once per week for three weeks. The ability to induce mucosal tolerance was determined by measuring liveweight gains of lambs trickle infected with 2,000 T. colubriformis day⁻¹ and fed ad libitum of a pelleted complete ruminant diet containing 12.15 MJME kgDM⁻¹ and 173 g CP kgDM⁻¹. Apart from liveweight gains antibodies and cytokines in serum, faecal egg counts (FEC), worm burden, worm length, carcass composition and serum levels of phosphorous, urea, total protein and albumin were measured. The antigen treatment reduced worm establishment and worm length in all groups with the greatest effect in the lambs injected with the antigen combination. Furthermore, it boosted a Th2 immune response reflected as elevated IL-4 cytokines and induced immunity rather than tolerance indicated by elevated antibodies and reduced FEC. The objective of the second study (Chapter 4) was to identify the optimal delivery route of T. colubriformis antigen to stimulate mucosal tolerance by investigating the intra-rectal and subcutaneous routes in four-month-old lambs. Thirty-five Coopworth lambs were born on pasture, allocated hierarchically by sex and live weight (mean 35.73 ± 1.32 kg) into one of five treatment groups (n=7) and housed indoors for the trial. The most efficient antigen from Chapter 3, i.e. the larval combination antigen, was used. Two groups were injected with antigen into the rectal submucosa or subcutaneously in the neck area using the treatment protocol from Chapter 3. One group was immunosuppressed with weekly intramuscular injections of methylprednisolone acetate 40 mg ml⁻¹ at a dose 1 ml 30 kg live weight⁻¹ for six weeks. The lambs were trickle infected with 3,000 T. colubriformis day-1 and fed ad libitum lucerne pellets containing 10.26 MJME kgDM⁻¹ and 214 g CP kgDM⁻¹. The ability to induce mucosal tolerance or immunity was determined by measuring the same parameters as in Chapter 3. Treatment reduced the worm establishment and female worm length in both antigen treatment groups with the greatest effect in the lambs injected intra-rectally. The antigen treatment induced a Th2 immune response with increased serum antibody responses and elevated IL-4 cytokine concentrations. Reductions in serum protein and serum albumin concentrations were alleviated in the antigen treated groups. Moreover, the intra-rectally injected group had greater performance during the last 21 days of the study compared with the untreated infected group. The final study (Chapter 5) was conducted as a field study with 72 sets of crossbred mixed-sex twin lambs that were born on low contaminated pasture and sequentially allocated into one of six treatment groups (n=24) based on birth order, and balanced for sex. The lambs were injected with a somatic antigen either at birth or at weaning (three months of age) to explore the importance of age and previous nematode exposure on the ability to induce mucosal tolerance or immunity to gastrointestinal nematodes. Two groups were injected with the T. colubriformis larval combination used in Chapters 3 and 4 at the intra-rectal submucosa three times a week apart. One group was injected subcutaneously with 1 mg kg live weight-1 of the long-acting anthelmintic Cydectin and used for positive growth control. One of the twins from each set was used as a control for the respective treatment and three of the groups were injected with PBS and acted as control for the level of pathogenicity of infection. The lambs were trickle infected from four months of age with 2,000 T. colubriformis day⁻¹. The results provided no evidence that the antigen treatment induced mucosal tolerance; however it induced immunity indicated by increased antibody production and reductions in FEC. Treatment did not mitigate the costs of immunity on growth. In summary, these studies provided evidence that injection of lambs with a somatic T. colubriformis antigen is associated with a Th2 immune response and that the combination larval antigen was more efficient than individual larval antigens. The intra-rectal route was the most sufficient delivery route for inducing immunity and reducing the establishment of parasites. Extended protection through tolerance was not able to be achieved by administration of larval antigen to neonates.