Comparative assessment of faecal diagnostics for detection and predictive modelling of endemic Fasciola hepatica infection in sheep and cattle on Australian farms.

The diagnosis, monitoring and flukicide efficacy testing of fasciolosis on-farm is reliant on non-terminal methods. The coproantigen ELISA (cELISA) has been recommended for diagnosis of fasciolosis and associated flukicide efficacy testing as an alternative to fluke egg counts for monitoring parasitism. Recently experimental multi-age infections have suggested that the reliability of efficacy results can be improved by a second cELISA testing at 6 weeks post-treatment (wpt) in addition to the generally accepted 1 wpt. A field study was conducted to determine the suitability of faecal fluke egg counts (FFEC) and cELISA as diagnostic, drug efficacy testing and epidemiological tools on Australian sheep and cattle farms. Faecal samples from sheep and/or cattle on three endemic farms were taken at monthly intervals for 12 months and examined by both methods. Normal farm management was maintained during the study period and opportunistic efficacy testing, in line with each farm's normal flukicide management was undertaken. Additionally, the suitability of the Ollerenshaw Index as a predictive model for fasciolosis under Australian conditions was examined. While both diagnostics demonstrated their value in the farm environment, the current data demonstrate a distinct and significant increase in diagnostic sensitivity for epidemiological studies by using the two tests in parallel. The agreement between the two diagnostics was found to be higher in cattle, despite the poor sensitivity of FFEC in this species. Similar levels of agreement between the two tests were demonstrated at both sheep properties, regardless of the marked difference in the intensity of F. hepatica challenge. Linear regression models demonstrated the results of the two diagnostics utilized in parallel were explained substantially (R2 = 0.91) as were series data (R2 = 0.88) when the respective models were fitted. In contrast, the fitted models for FFEC (R2 = 0.54) and cELISA (R2 = 0.58) were poor explanations for test outcomes. The outcomes of these models support previous findings that suggest that the two diagnostic tests are best utilized together, particularly in parallel. The application of the Ollerenshaw Index to Australian conditions requires further investigation.

Comparative assessment of faecal diagnostics for detection and predictive modelling of endemic Fasciola hepatica infection in sheep and cattle on Australian farms.

The diagnosis, monitoring and flukicide efficacy testing of fasciolosis on-farm is reliant on non-terminal methods. The coproantigen ELISA (cELISA) has been recommended for diagnosis of fasciolosis and associated flukicide efficacy testing as an alternative to fluke egg counts for monitoring parasitism. Recently experimental multi-age infections have suggested that the reliability of efficacy results can be improved by a second cELISA testing at 6 weeks post-treatment (wpt) in addition to the generally accepted 1 wpt. A field study was conducted to determine the suitability of faecal fluke egg counts (FFEC) and cELISA as diagnostic, drug efficacy testing and epidemiological tools on Australian sheep and cattle farms. Faecal samples from sheep and/or cattle on three endemic farms were taken at monthly intervals for 12 months and examined by both methods. Normal farm management was maintained during the study period and opportunistic efficacy testing, in line with each farm's normal flukicide management was undertaken. Additionally, the suitability of the Ollerenshaw Index as a predictive model for fasciolosis under Australian conditions was examined. While both diagnostics demonstrated their value in the farm environment, the current data demonstrate a distinct and significant increase in diagnostic sensitivity for epidemiological studies by using the two tests in parallel. The agreement between the two diagnostics was found to be higher in cattle, despite the poor sensitivity of FFEC in this species. Similar levels of agreement between the two tests were demonstrated at both sheep properties, regardless of the marked difference in the intensity of F. hepatica challenge. Linear regression models demonstrated the results of the two diagnostics utilized in parallel were explained substantially (R2 = 0.91) as were series data (R2 = 0.88) when the respective models were fitted. In contrast, the fitted models for FFEC (R2 = 0.54) and cELISA (R2 = 0.58) were poor explanations for test outcomes. The outcomes of these models support previous findings that suggest that the two diagnostic tests are best utilized together, particularly in parallel. The application of the Ollerenshaw Index to Australian conditions requires further investigation.

Trichostrongylus colubriformis and Haemonchus contortus infections in light bodyweight Merino lambs.

UNLABELLED: OBJECTIVE; To show that low bodyweight is a predisposing cause of high Trichostrongylus colubriformis and Haemonchus contortus burdens and egg counts in Merino lambs. DESIGN: A comparison was made, among lambs of different bodyweights, on the effect on immunity of a primary or secondary viable infection with T colubriformis or H contortus larvae. PROCEDURE: Sixty-one Merino lambs, 1 or 6 months of age, were penned indoors and given primary, or both primary and secondary, infection of T colubriformis or H contortus. Faecal egg counts, worm counts and parasite-specific immunoglobulin concentrations were examined for their relationships with bodyweight. RESULTS: Bodyweight at the start of a primary infection was correlated with worm burden, worm fecundity and jejunal IgA antibody concentration. Merino lambs weighing less than 23 kg at the time of first exposure to T colubriformis or H contortus had impaired ability to develop protective mucosal immunity and to resist homologous challenge. CONCLUSION: If the goal is to ensure that lambs develop immunity before weaning, then every endeavour should be made to achieve the combination of critical bodyweight and exposure to moderate levels of nematode infection as soon as possible.

Application of a coproantigen ELISA as an indicator of efficacy against multiple life stages of Fasciola hepatica infections in sheep.

At present diagnosis of true resistance and determination of drug efficacy in Fasciola hepatica infection rely solely on terminal experiments. The coproantigen ELISA (cELISA) has been reported previously as a sensitive and specific tool appropriate to detect treatment failure, and potentially drug resistance. Two studies were conducted to determine whether the cELISA was appropriate for on-farm efficacy and resistance testing in Australian Merino sheep. In Study 1 sheep were infected orally with 50 F. hepatica metacercariae on three occasions, twelve, six and two weeks prior to a single flukicide treatment with triclabendazole, closantel or albendazole. Sheep were sampled weekly for a further seven weeks prior to necropsy. Following effective treatment, no faecal antigen was detected from 1 week. When immature stages (≤6 weeks) survived treatment, coproantigen reappeared from 6 weeks post-treatment. Therefore, cELISA conducted 1-4 weeks after treatment will demonstrate obvious treatment failure against adult F. hepatica, but is not sufficiently sensitive to detect survival of immature fluke until these reach maturity. In study 2, fluke burdens of sheep necropsied 13 weeks post single infection were compared to fecal worm egg counts (FWEC) and cELISA at necropsy. Regression analysis demonstrated that cELISA correlated strongly with fluke burden, whilst FWEC correlated weakly with cELISA. The correlation between FWEC and fluke burden was also weak, although stronger than that of FWEC with cELISA. The cELISA is an appropriate tool for monitoring effectiveness of treatments against Fasciola hepatica if an adult infection is present, however when immature stages of the parasite are present it is not as reliable. Where immature parasites are present it is recommended that initial cELISA be followed with a secondary cELISA at least 6 weeks after treatment to ensure resistance to immature stages is detected. Further testing is justified for monitoring the effectiveness of control programs by detecting adult populations that have survived a treatment regime.

Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot.

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.

Bilateral growth-related protein expression suggests a transient increase in regenerative potential following brain trauma.

The potential of mature central nervous system (CNS) neurons to regenerate after injury represents a fundamental issue in neurobiology. The regional expression of proteins associated with axonal elongation, such as microtubule-associated protein 1B (MAP1B), its phosphorylated isoform (MAP1B-P), growth-associated protein 43 (GAP-43), and polysialylated neural cell-adhesion molecule (PSA-NCAM), was examined using immunohistochemistry from 24 hours to 2 months following lateral fluid percussion brain injury of moderate severity (2.4-2.6 atmospheres) in anesthetized rats. Uninjured (control) rats were subjected to anesthesia and surgery without injury or were subjected to anesthesia alone. Within the site of maximal injury, only increases in MAP1B and MAP1B-P were observed. Increased immunoreactivity was observed bilaterally for all growth-related proteins that were evaluated. By 24 hours postinjury, MAP1B and MAP1B-P increased within the cortex (P < 0.01) and the hippocampus (P < 0.001), whereas MAP1B-P also was elevated in the thalamus (P < 0.05). Within the dentate gyrus, increased immunoreactivity was observed for all proteins examined. By 48 hours postinjury, GAP-43 was elevated bilaterally within the inner molecular layers of the dentate gyrus (P < 0.005) and within the stratum lacunosum moleculare (P < 0.01), the stratum radiatum (P < 0. 005), and the stratum oriens (P < 0.05) of the hippocampus. Increased numbers of PSA-NCAM-labeled neurons were observed in the granule cell layers of the dentate gyrus from 48 hours through 2 weeks postinjury (P < 0.0005). The bilateral nature of increased expression of growth-related proteins differs from unilateral patterns of neuronal degeneration previously characterized for the lateral fluid-percussion model of brain injury. Taken together, these results suggest the existence of a temporary posttraumatic state in which the CNS may have increased regenerative potential. Enhancement of such a response may be one therapeutic strategy in treating CNS injury.

Demonstration of a range of inflammatory mediators released in trichostrongylosis of sheep.

The levels of inflammatory mediators in the intestinal contents of sheep immunized with Trichostrongylus colubriformis larvae increased in the first 6 days after challenge. These mediators were histamine, leukotriene C4, 6-keto-prostaglandin F1 alpha (from prostacyclin) and thromboxane B2. Leukotriene C4 was released in the greatest quantities. Leukotriene B4 was present but its concentration remained unchanged after challenge. The presence of these particular mediators in the intestinal contents after challenge is consistent with antigen-induced mediator release from the mucosal mast cells found in immune sheep undergoing challenge infection. This is the first sequential analysis of mediator release in sheep that also demonstrates the release of prostacyclin and thromboxane into the intestine during expulsion of a nematode infection.

Studies on the inhibition of rejection of Trichostrongylus colubriformis larvae from immune sheep.

A total of 8 drugs were examined for their ability to suppress the rejection of Trichostrongylus colubriformis infective larvae (L3) from immune sheep. Specific antagonists of leukotrienes (piroxicam), prostaglandin (indomethacin) and the immunosuppressant cyclosporin A were given orally, while injectable preparations of dexamethasone, chlorpheniramine, BN 52051, WEB 2086 (anti-platelet-activating factor) and theophylline were administered as directed. The drugs were given for 5 days prior to and 4 days after challenge with 20,000 L3, when worm counts were done. Corticosteroids inhibited rejection by around 70% in two experiments, and none of the remaining compounds were effective. In the third study, six of the drugs were given to susceptible sheep and did not affect the establishment of a primary infection with T. colubriformis.

The use of electroblotted antigens of Trichostrongylus colubriformis to induce proliferative responses in sensitized lymphocytes from sheep.

Merino sheep were immunized against the intestinal nematode, T. colubriformis, by repeated infections, and proliferative responses of their peripheral blood lymphocytes (PBL) against parasite extracts and excretory-secretory (ES) antigens were monitored over 130 days. Maximal responses occurred 7-14 days after challenge. The ability of soluble proteins and parasite antigens to induce proliferation was compared with that of antigen-bearing particles obtained after antigen was adsorbed onto nitrocellulose. Blank particles increased c.p.m. two- to three-fold above that obtained in medium alone, and to elicit proliferative responses of comparable magnitude between 10 and 100 times more antigen was required when antigen-bearing particles were used instead of soluble extracts or defined proteins. Blood leucocytes as well as T-cell lines established by stimulation with parasite antigens in vitro reacted to moieties of from 5000 to 38,000 mol. wt in ES antigens on nitrocellulose particles. Direct comparisons of T-lymphocyte responses with antibody responses as assessed by immunoblots revealed different profiles of immunogenicity among ES proteins within individual sheep, but the 10,000, 30,000 and 75,000-90,000 mol. wt proteins were immunodominant. These proteins were also those consistently recognized by T-lymphocytes and sera from sheep immunized with ES proteins in adjuvant. Thus, this technique can be applied to identify parasite material which is immunogenic for T-lymphocytes, but the sensitivity of the procedure in sheep is less than reported in human studies.

The effect of specific immunization or infection with Trichostrongylus colubriformis on production of eosinophil differentiation factors in guinea pigs.

The cultivation of bone marrow was used to quantitate the levels of eosinophil differentiation factors (EDF) produced in conditioned medium (CM) by incubation of mesenteric lymph node cells (MLNC) with mitogens or specific antigens from the intestinal nematode parasite, Trichostrongylus colubriformis. In liquid cultures with 20 units ml-1 recombinant murine interleukin-5 (IL-5), bone marrow cells (BMC) from either normal or infected donors contained less than 5% eosinophils and differentiated to greater than 50% eosinophils over 2-3 weeks. Conditioned medium from 3-4 week infected donors produced between 20 and 50% eosinophils when donor MLNC were stimulated with the specific antigen preparation SP3, but macrophages predominated when using CM from MLNC incubated with Concanavalin A (ConA). CM from MLNC of challenged donors incubated with SP3 produced 30-70% eosinophils in BMC assays, with highest levels induced by CM from high responder (HR) donors. Marrow from parasitized or normal donors gave rise to comparable proportions of eosinophils. CM was also produced from LNC of donors given protein or parasite antigens in adjuvant where between 28 and 35% eosinophils were produced in culture. There were no differences between activities attributable to the antigen, but Freund's complete adjuvant induced earlier differentiation of BMC than alum-induced CM. The results confirm that high levels of EDF activity are specifically induced by parasitic infection, and can also be produced by intraperitoneal and subcutaneous inoculation of adjuvanted antigens. Consistent with the greater eosinophilia exhibited by HR guinea pigs to infection with T.colubriformis L3, their MLNC also produced the highest levels of EDF activity.

The sensitization of mucosal mast cells during infections with Trichostrongylus colubriformis or Haemonchus contortus in sheep.

Responses of isolated mucosal mast cells (MMC) during infections with either Trichostrongylus colubriformis or Haemonchus contortus were examined by measuring the release of sheep mast cell protease (SMCP) in a degranulation assay. MMC from sheep immune to T. colubriformis released maximal amounts of SMCP and histamine within 0.5h of incubation with larval antigen whereas maximum secretion of leukotrienes occurred 3h after addition of antigen. It was only after 8 weeks of a primary T. colubriformis infection, that MMC released significantly elevated levels of SMCP (23%); this occurred when the worm burden was being rejected. In contrast, the SMCP release from MMC of immune sheep was significantly higher at 40%, and occurred within 1-4 days after challenge (DAC). The SMCP release peaked at 6-8 DAC at 51%, and declined after 56 DAC to < 25%. MMC isolated from the duodenum and mid-small intestine of immune sheep released 2-3 times higher proportion of SMCP than did cells recovered from the terminal ileum. Mast cell numbers were similar in the 3 regions but the quantity of globule leucocytes (GL) was 2.5 times higher in the duodenum. During infections with H. contortus in the abomasum, MMC isolated from the small intestine released greater levels of SMCP when incubated with larval antigens than did abomasal MMC. There was no increased release during the first 12 weeks of a primary infection although the SMCP release (23%) from immune MMC at 7-10 DAC was significantly enhanced. Once again the release from MMC isolated from the three intestinal regions of sheep immune to H. contortus was lowest in the terminal ileum.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Isolation and degranulation of mucosal mast cells from the small intestine of parasitized sheep.

The isolation of mucosal mast cells and globule leucocytes from the small intestine of sheep immunized with Trichostrongylus colubriformis is described. Sheep mast cell protease was released from these cells in a dose-dependent fashion after incubation with soluble protein from T. colubriformis larvae. Release also occurred with other T. colubriformis antigens whereas non-parasite antigens at comparable protein concentrations evinced only a minimal response. Mucosal mast cells prepared from worm-free sheep also produced a similar minimal response. This is the first report describing the release of sheep mast cell protease from isolated sheep intestinal mucosal mast cells after addition of specific parasite antigens.

Induction of protective immunity to Trichostrongylus colubriformis in neonatal merino lambs.

The premise that any bias of immune reactivity in neonatal lambs towards T-helper (TH)2 responses could benefit the induction of protection against gastrointestinal nematodes was investigated. In two trials, lambs were either trickle-immunised with 2000 infective larvae of Trichostrongylus colubriformis (TcL3), 3 times weekly from the day of birth for 6 weeks or inoculated with a recombinant T. colubriformis 17 kDa antigen in incomplete Freund's adjuvant (IFA). In trial 1, trickle immunised and control neonates challenged at 7 weeks of age had similar worm counts 10 days after challenge, but from 25 days, significant reductions (P<0.01) in mean faecal egg count and worm count in excess of 75% were displayed by the immunised lambs. The results of a second, similar trial, gave 85-91% reductions in parasitism in trickle immunised neonates (P<0.001) and around 50% protection in neonates vaccinated with recombinant 17 kDa antigen. Parasitism in immunised neonates in Trial 2 was significantly reduced (P<0.001) compared to that in 4-month-old animals. Antibody responses in trickle-immunised (protected) and challenge control (infected) neonates were almost exclusively of the IgG1 isotype compared to vaccinated animals which exhibited increased levels of anti-17kD IgG2. Trichostrongylus colubriformis infection, but not specific vaccination, induced interleukin-5 production by mesenteric lymph node cells. The results offer the tantalising prospect of generating protective immunity to gastrointestinal parasites prior to weaning in sheep; this was most effectively generated by viable parasites in this investigation.

Studies on the host-parasite relationship between Trichostrongylus colubriformis and susceptible and resistant sheep.

This study examined whether infective larvae (L3) of Trichostrongylus colubriformis could establish throughout the small intestine and were not restricted to the anterior duodenum in susceptible and resistant sheep. The location of worms was similar in susceptible animals given doses of T. colubriformis between 10,000 and 80,00 T. colubriformis larvae, with 90% of worms located in the proximal 3 m of the small intestine. Those worms recovered from resistant sheep were also found in the first 9 m of the intestine. However, worms recovered from immune sheep were significantly (P = 0.0074) relocated posteriorly from the first 3 m into the next 6 m of the intestine. By the surgical introduction of worms, it was found that T. colubriformis could establish at any site in the small intestine and to some extent in the caecum. Immunity was generated principally in the site of predilection in the anterior 3 m of the small intestine and effectively expelled challenges given at distal sites and caecum, indicating dissemination of immunity throughout the gastrointestinal tract. Moreover, the rejection episode had removed worms from the entire small intestine within 2 h of introduction through the pylorus.

Attempts to generate immunity against Trichostrongylus colubriformis and Haemonchus contortus in young lambs by vaccination with viable parasites.

The ability of young Merino lambs to achieve protective immunity following vaccination via viable nematode infections was assessed. Lambs were infected from 1 month of age by repeated continuous low dose (trickle) administration of Trichostrongylus colubriformis or Haemonchus contortus infective larvae (L3), or by truncated infections with high doses of viable T. colubriformis L3. After 7 weeks all groups were drenched with anthelmintic and at 3 months of age they were re-infected with the homologous species. Protection was assessed by faecal egg counts at 3, 4, 5, 6 and 7 weeks after challenge, and worm count at 7 weeks after challenge. Young lambs were partially protected by 3 months of age against Trichostrongylus by trickle infection. This protection correlated with local mast cell and T-cell priming, increased numbers of local antigen-presenting cells and T-cells and increased worm-specific antibody titres in the intestine. However, there was no evidence that young lambs were capable of immunologically recognising H. contortus antigens following trickle infection, nor did trickle infection significantly protect young lambs against Haemonchus challenge.

A new simplified assay for larval migration inhibition.

A simple method is described for the in vitro detection of substances that impair the motility of third-stage larvae of gastro-intestinal nematodes. The test is based on the ability of larvae to freely migrate through selected mesh sizes of nylon sieves and the reduced ability of larvae to migrate after preincubation with, and in the presence of, substances that inhibit or reduce larval motility.

Changes in inflammatory mediators and larval inhibitory activity in intestinal contents and mucus during primary and challenge infections of sheep with Trichostrongylus colubriformis.

Helminthologically naive merino sheep were given either a single infection of 30,000 or a trickle infection of 6000 Trichostrongylus colubriformis infective larvae (TcL3) per week. Faecal egg counts started to fall after 8 weeks in the single infection and after 11 weeks in the trickle infection. Small intestinal contents were collected from indwelling intestinal fistulae over the next 14 weeks. Concentrations of sheep mast cell protease (SMCP) in these contents increased to highest levels 9-11 weeks and 6-10 weeks after the single infection and from the start of the trickle infection, respectively. Similarly, peptidyl leukotriene (PLT) concentrations were highest at 6 weeks and at 6-9 and 13 weeks, respectively. Histamine concentrations increased slightly after both infections to peak values at 7 weeks and 9 weeks, respectively. Inhibition of migration of larvae in vitro was increased in contents sampled at 8 weeks after the single infection and after 6-10 weeks of the trickle infection. Another 2 groups of sheep were immunised by repeated infections with TcL3. Gut contents from 1 group sampled immediately before and after challenge with 30,000 TcL3 at 0 and 18 days had increased levels of larval migration inhibitory (LMI) activity throughout the 35 day period, especially 7-14 days after challenge (DAC). The mediators SMCP increased significantly 5-7 DAC while PLT increased 7-14 DAC. In the second group of immunised sheep, levels of SMCP and PLT increased rapidly within 1 DAC and further increased 3-14 DAC.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Freund's adjuvants on guinea pigs infected with, or vaccinated against, Trichostrongylus colubriformis.

Soluble antigens that protected guinea pigs against experimental challenge with Trichostrongylus colubriformis were found to be less effective if injected as emulsions in Freund's adjuvants. This occurred despite the production of higher antibody titres in guinea pigs given emulsified antigen. Investigations of this phenomenon showed that an intraperitoneal injection of Freund's complete adjuvant (FCA) delayed rejection of primary infections and partially abrogated resistance to challenge infection. Administration of FCA/antigen emulsion to infected guinea pigs resulted in a prolonged blood eosinophilia which paralleled the increased longevity of the parasitosis. These findings suggest the need for caution in the selection of adjuvants for vaccination against gastrointestinal nematodes.

Antigen delivery strategies for mucosal vaccines.

Progress towards effective vaccines to control internal parasites, especially those affecting mucosal compartments, has been inhibited by the combined problems of the antigenic complexity of parasites and the lack of understanding of the host response. However, the accumulation of information regarding regulation of mucosal immunity has enabled a reappraisal of vaccination options to provide appropriate mucosal effector responses. The pivotal role of T cell influences, and in particular the contribution of cytokine signals, has been clearly established from in vitro studies, but data emerging from our laboratories provide evidence for these effects in vivo. We have demonstrated the role of T cells in determining the outcome of an intestinal response and propose a role for local Th2 cytokine production in this regard. To support this proposition, the distribution of cytokine mRNA has been determined by in situ hybridisation techniques in normal and parasitised animals. Further, we have shown that in the absence of Th2 cytokines (using gene knockout animals) mucosal responses are grossly deficient; we have also shown that this defect can be overcome by vector-directed gene therapy. These studies have indicated that new mucosal immunisation opportunities exist by combining traditional immunisation approaches with strategies to upregulate local cytokine production. However, the success of these new strategies will depend on selection of highly immunogenic subunit antigens, coupled with techniques for cytokine manipulation and delivery with appropriate adjuvant/vehicle formulations. This paper reviews delivery technologies available to chaperone labile antigenic and genetic material to appropriate sites for mucosal stimulation after systemic or oral administration.