D.L. Emery

A serial study of rejection of Trichostrongylus colubriformis by immune sheep

Host responses and the rejection of worms were measured at intervals following challenge of immune and susceptible sheep with T. colubriformis infective larvae. Immune sheep rejected most of their larvae within the first day after infection. This early rejection was associated with local appearance of globule leucocytes and increased concentration of T. colubriformis-specific IgG1 and IgG2 in intestinal mucus. Rejection of the remaining worms occurred between 3 and 14 days after infection and was associated with increased T. colubriformis-specific IgA and IgG2 in intestinal mucus, local T cell infiltration, activation, differentiation and epithelial necrosis. Local T cell changes included expansion of the T19- gamma delta+ populations in the villous lamina propria and epithelium.

Influence of antigens and adjuvants on the production of gamma-interferon and antibody by ovine lymphocytes

The production of gamma‐interferon (γ‐IFN) and antigen‐specific immunoglobulin isotypes was monitored in sheep given primary and secondary inocula of protein and polysaccharide antigens with or without adjuvants. In efferent lymph from prefemoral nodes draining the site of inoculation, γ‐IFN levels increased within 24 h after injection of adjuvants Quill A, dextran sulfate (DXS) and 6 days after oil adjuvants in saline. The increased synthesis of γ‐IFN was prolonged by 1–2 days by the presence of adjuvanted antigen, but the major stimulus to γ‐IFN production was provided by the adjuvant. Alhydrogel (AH) did not initiate γ‐IFN synthesis. Following a second inocula of antigen in saline, increased levels of γ‐IFN were detected in efferent lymph after 3–4 h, and persisted for at least 2 days. Following cultivation of leucocytes from immunized sheep with specific antigen in vitro, strong positive correlations between levels of interleukin (IL) IL‐2, ‐IFN, proliferative responses and antibody titres were observed. However, after analysis of the antigen‐specific isotypes of immunoglobulin (Ig) there was no correlation between the production of γ‐IFN and particular Ig isotypes. Together with findings that AH did not preferentially induce IgG1 or IgG2, these results suggest that the specific subpopulations of helper T lymphocytes which regulate the production of antibody isotypes by differential secretion of γ‐IFN or IL‐4, are not as clearly defined in sheep as in mice.

Production of vaccines against gastrointestinal nematodes of livestock

Three international collaborations involving Australian research scientists are currently developing vaccines against Haemonchus, Trichostrongylus and Ostertagia parasites using recombinant DNA technology. The variety of protective antigens identified can be classified as ‘conventional’ (stimulate naturally acquired immunity) or ‘novel/’covert‘/’concealed’ (protective once immunity is induced by vaccination). To date, the most gratifying progress has resulted in 60–90% protection against Haemonchus and other blood‐sucking parasites (e.g. ticks) using novel antigens, where high titres of serum antibody ingested by feeding worms leads to their demise. A great deal of research effort is unravelling the complexity of naturally acquired immunity so that conventional antigens, which may be the principal means of removing ‘mucosal‐browsing’ parasites, may be formulated and delivered to achieve optimal efficacy. This work reveals that to remove early stages of parasites before they take up residence, deliberate induction of hypersensitivity responses akin to asthma, may be a desirable goal for vaccines and that the two models have much in common.

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 Freunds 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.

An analysis of cellular proliferation, and synthesis of lymphokines and specific antibody in vitro by leucocytes from immunized cattle.

The relationships between the production of lymphokines, cellular proliferation and antibody synthesis by immune bovine PBL in vitro was examined to identify the cellular reactions responsible for differences in the titres of serum antibodies in calves from selected sire lines and MHC Class I phenotypes. Leucocytes from 22 calves immunized with ovalbumin and DNP-BSA proliferated specifically in vitro in the presence of 1-10 micrograms/ml ovalbumin 7-28 days after the second vaccination. Significant correlations between the production of IL-2, IFN-gamma and maximum proliferation were observed for the total group. The quantity of specific antibody produced when PBL were incubated alone or with 10(-1)-10(-2) micrograms/ml ovalbumin was also correlated significantly with the maximum proliferation and the serum antibody titre between 7 and 14 days. Anti-ovalbumin IgG was also synthesized in MLRs where the quantity of antibody was significantly correlated with the magnitude of proliferation. The responses in vitro to DNP-BSA were too low to provide meaningful comparisons. The results indicate that at intervals during the period of increasing serum titres, events in the bovine antibody response in vivo can be replicated in vitro. In addition, assays for proliferation, IL-2 or gamma-IFN, or specific antibody can be used to assess the magnitude of the immune response in vivo in experimental groups of cattle. Significant sire line differences in the serological responses to ovalbumin were observed but DNP-BSA was a poorer antigen and differences in the responses to this antigen were not significant. However, the sire line differences in vivo were not reflected in vitro in proliferative and lymphokine assays; only the production of antibody in vitro was significantly correlated with the in vivo serum titre.

Immunological Reactivity Between Chimeric Cattle Twins: I. Homograft Reaction

SUMMARY The response of chimeric calves to grafts of cotwin skin was examined both by direct observation of the fate of the graft and by collection of lymph from the lymph node which drained the grafted area. Whilst grafts of body skin exchanged between 16 pairs of twins survived for longer periods than did grafts transferred between unrelated calves, all of the former had been rejected after 10 weeks. Secondary grafts of cotwin body skin were rejected significantly faster. In contrast, grafts of auricular skin exchanged between cotwins survived indefinitely in some instances despite the rejection of simultaneously transferred body skin. The magnitude of the blast cell response observed in efferent lymph in the case of grafts exchanged between cotwins was generally intermediate between that observed in responses to allografts and control autografts. However, an early peak of blast cell response which occurred some 50 hr after placement of allogeneic grafts was not observed in response to cotwin grafts.

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.

Studies on the purification of the leucocidin of Fusobacterium necrophorum and its neutralization by specific antisera.

Leucocidin from several strains of Fusobacterium necrophorum was partially purified by gel filtration on Fractogel HW55 (F), the majority of the activity being present in the 50 ml of filtrate collected after 1.1 void volumes had passed through the column (termed Fraction 1, or #1). The material also contained lipopolysaccharide in 12.5% SDS-PAGE gels run under reducing conditions, but the protein did not migrate into 7.5% PAGE gels run under non-reducing conditions. Rabbit and bovine antisera to the leucocidin possessed antibodies against antigens in concentrated, washed culture supernates from toxigenic F. necrophorum and neutralized the leucocidal activity of such supernates. Absorption of the antisera with homologous, washed F. necrophorum cells reduced ELISA antibody titres by greater than 50%, but decreased neutralization titres by 15%. Absorbed rabbit IgG anti-#1 precipitated a single rocket in crossed immunoelectrophoresis and identified two proteins, of molecular weights (M.W.) 14 000 and 13 000, and 1 protein of M.W. 13 500 in immunoblots from toxigenic and non-toxigenic strains, respectively. An additional protein of M.E. 103 000 was present after SDS-PAGE separation of supernates from toxigenic but not non-toxigenic F. necrophorum and was not present in whole cell components. It was considered that the leucocidin may be present in a dimeric form in culture supernates from toxigenic strains. Antisera to leucocidins from several strains of F. necrophorum exhibited variable neutralization titres against leucocidins from heterologous bacteria.

Immunological reactivity between chimeric cattle twins. III. Mixed leukocyte reaction.

SUMMARY Leukocytes from chimeric calves invariably failed to mount a proliferative response in excess of control responses when cultured with cotwin cells as stimulators. The levels of cellular proliferation in control cultures of leukocytes from chimeric donors were so low as to exclude the occurrence of any autostimulation, and it was clear that the absence of excess proliferation in mixed cultures could not be attributed to elevated background levels. Attempts to restore reactivity in mixed leukocyte culture by removal of cell subpopulations from chimeric calf leukocytes and by modification of cell surfaces were unsuccessful. It was inferred that the unreactivity between chimeric cotwins manifest in the normal lymphocyte transfer and mixed leukocyte reactions was unlikely to be produced by humoral or cellular blocking mechanisms.

Immunological reactivity between chimeric cattle twins. II. Normal lymphocyte transfer.

SUMMARY The response of normal single-born calves to the s.c. injection of allogeneic lymphocytes and that of chimeric calves to allogeneic and cotwin cells were studied. Whereas allogeneic cells produced local dermal swelling along with an efflux of blast cells and specific cytolytic antibody in the regional lymph, the injection of cotwin lymphocytes into chimeric calves failed to produce responses in excess of those of controls. There was little indication of specific reduction in responsiveness of the lymphocytes of normal calves following challenge with allogeneic cells.