Antibody degradation in wound exudates from blowfly infections on sheep.

Sheep were immunised with ovalbumin and then infected with the sheep blowfly, Lucilia cuprina in order to study immunoglobulin and specific antibody degradation at the wound site. Serum and wound exudates were collected over the infection period and the dry weight and protein content of the exudates were determined. Exudates were analysed by SDS-PAGE and immunoblotting for IgG degradation. Levels of IgG and specific anti-ovalbumin antibodies in the exudates were measured by ELISA. The total weight of exudates increased over the whole period of the infection, while protein content increased in the first 24 h and then remained relatively constant. Immunoglobulin was present 6 h after infection and levels increased with protein content. However, the levels of IgG measured were quite different depending on the secondary antibody used in the ELISA. A monoclonal antibody measured mainly intact IgG while a polyclonal anti-IgG measured intact and degraded IgG. This allowed an estimation that approximately 60% of the IgG in exudates was degraded from 6 h after infection. Assays in vitro showed that L. cuprina larval enzymes degraded sheep antibody. However, measurement of specific anti-ovalbumin levels in exudates suggested that although high levels of antibody were degraded this did not necessarily decrease the level of antigen binding. As a result, IgG degradation may assist and not hinder vaccine development by allowing antibody fragments to penetrate the peritrophic membrane and access gut cell antigens.

A comparison of inflammatory exudates released from myiasis wounds on sheep bred for resistance or susceptibility to Lucilia cuprina.

Sheep bred for resistance (R) or susceptibility (S) to fleece rot and myiasis (blowfly strike) were experimentally infected with L. cuprina larvae. Exudates released from the wound site were collected during the infection at 6, 12, 18 and 24 h. The exudates were separated using two-dimensional gel electrophoresis, and proteins were silver stained and identified by immunoblotting with specific antibody and by their isoelectric points and molecular weights. Comparisons of exudate composition were made over time and between R and S sheep. Between 6 and 12 h post larval implantation the exudate was rich in IgG and fibrinogen, which is before extensive tissue damage and suggests that the exudate is not simply tissue haemorrhage but the result of an inflammatory response by the sheep to L. cuprina. The exudate grew in complexity between 12 and 18 h and contained a maximum of 74 distinct peptide spots by 24 h. Exudate from wounds on resistant sheep contained many more peptides in the first 12 h of infection, suggesting a more rapid inflammatory response. The source of proteins from the exudate remains speculative; it appears to be composed of many acute-phase proteins, large amounts of immunoglobulin G and proportionally low levels of serum albumin. Exudate composition is likely to be influenced by the local synthesis of acute-phase proteins and perhaps immunoglobulins, selective transport to the infection site and also enzymic degradation by L. cuprina larval enzymes. The more rapid exudation of acute-phase and serum proteins at infection sites on R sheep may allow the inhibition of the establishment of fleece rot bacteria or L. cuprina larvae under natural challenge.

The production and characterisation of monoclonal antibodies to Lucilia cuprina larval antigens.

A panel of murine monoclonal antibodies was produced against three Lucilia cuprina larval preparations that are unlikely to be exposed to the sheep's immune system during a normal infection. Antibodies were successfully produced against a crude third instar midgut homogenate preparation (MG), and Sodium dodecyl sulphate (SDS) and Triton X-114 (TX) detergent extracts of first instar larvae. Characterisation of the relevant antigens was performed using 1- and 2D gel electrophoresis and immunoblotting, immunoperoxidase histological studies and in vitro larval growth cultures. All the mAbs were of the IgM isotype. Common recognition of bands at 40, 50 and 80 kDA was evident on 1D blots of larval organ preparations by most mAbs while recognition of antigens in the 2D blots appeared to be more specific. Immunohistological studies suggested that a number of the antibodies specifically bound to intracellular structures within the midgut epithelium. However, antibodies derived from one clone also recognised the epithelium of Malpighian tubules, oenocytes and muscle fibres. None of the antibodies raised against TX extracts were observed to bind to larval structures. Results of larval cultures suggested that certain antibodies could significantly inhibit larval survival and growth in vitro.

The sheep antibody response to repeated infection with Lucilia cuprina.

The specific serum antibody responses of sheep exposed to 10 consecutive infections of L. cuprina have been analysed by enzyme-linked immuno-sorbent assay and immunoblotting using monoclonal antibodies specific for sheep immunoglobulin isotypes. Recognition of a number of larval excretory-secretory products by IgM antibodies appeared to be non-specific. IgG1 was the major antibody class stimulated by the infection protocol and marked increases in antibody to specific excretory-secretory antigens were observed. Three molecules of 35, 30 and 25 kDa were particularly recognized although the extent of recognition of these molecules varied considerably between individual sheep serum. A pooled serum composed of sera collected after five to seven infections significantly inhibited larval growth in in vitro cultures when compared to a sera pool consisting of sera collected both prior to infection and after infections 1 and 2. The degree of inhibition was greater when serum with high specific antibody titre was used.

Development of immunity to incoming radiolabelled larvae in lambs continuously infected with Ostertagia circumcincta.

Infective larvae of Ostertagia circumcincta were radiolabelled with 75selenium by a method which did not affect their viability. Three groups of five-month-old lambs were infected daily with 1000 unlabelled infective larvae for four, eight and 12 weeks, respectively. After each period one of these groups and a group of worm-free controls were challenged with three consecutive daily doses of 1000 radiolabelled third stage larvae. The lambs were killed 13 days after the first dose of challenge larvae and their worm burdens examined. The first indication of immunity was retardation of developing worms observed at four weeks. Resistance to the establishment of incoming worms developed between four and eight weeks and a brief period of population turnover probably took place at this time. Simultaneously a greater inhibition of worm development occurred resulting in an increase in the number of parasites recovered as early fourth stage larvae. By 12 weeks the animals were almost completely immune to incoming worms. The development of resistance to incoming worms correlated with a rise in serum antibody titre and an increase in the number of intraepithelial globule leucocytes in the gastric mucosa.

Development of immunity to incoming radiolabelled larvae in lambs continuously infected with Trichostrongylus vitrinus.

Infective third stage larvae (L3) of Trichostrongylus vitrinus were radiolabelled with 75 selenium by a method which did not affect their viability. Three groups of six-month-old lambs were infected daily with 1000 L3 for four, eight and 12 weeks, respectively. After each period, one of those groups (n = 5) and a group (n = 4) of worm-free controls were challenged with three consecutive daily doses of 1000 radiolabelled L3, killed 10 days after the first dose, and their worm burdens examined. After four weeks of continuous infection partial immunity to the establishment of challenge L3 was apparent, and by eight and 12 weeks, with the exception of one sheep, there was almost total resistance to incoming worms. Immunity was also expressed as an inhibition of the development of established worms.

The effect of method of infection on the pathway of juvenile Strongyloides ratti in the host.

Subcutaneous injection of the larvae is the almost universally adopted means of initiating experimental infections of skin-invading roundworms but, so far, the possibility that this procedure introduces artefacts of one kind or another has not been critically studied. Experiments described in this paper were used to compare the effect of (a) injection and (b) skin application, of a small, precisely counted ('exact') dose of larvae. Results with two strains of S. ratti showed that the same proportion of the dose developed to adults in the intestines of rats irrespective of the method. With the same exact dose technique it has been shown that milk-borne infection of the pups of lactating rats is not an artefact produced by injection. Large doses (mean 4000) of larvae of the homogonic strain of S. ratti carrying a radioactive label of 75Se were tracked in their migration to the mammary gland following injection or skin application at two different sites on the right-hand side of nursing mother rats. The broad conclusion of earlier work in this laboratory using injection, that larvae move by a local route and not a systemic one, was supported by the results. The detailed distribution of the label and of unlabelled worms of the heterogonic strain in families was, however, different for the two methods, indicating that subtle variations in pathway can be brought about by the use of injection. If migration involves the lymphatic system, then the interpretation of immunological experiments in terms of lymphatic anatomy must take account of such procedural effects. The extent to which these results contribute to theories of migration in Strongyloides ratti is discussed.

Cytokine production by NZB, C58, and NZB X C58 recombinant inbred mice.

Humans with autoimmune disease and autoimmune mouse strains such as NZB have been shown to produce reduced levels of the cytokines interleukin 1 and interleukin 2. The NZB X C58 recombinant inbred (N X 8 RI) strains exhibit certain of the autoimmune characteristics of the NZB strain. Their abilities to produce interleukin 1 and interleukin 2 have been tested. Deficiencies in the production of one or both of these cytokines were observed in the N X 8 RI strains. Decreased interleukin 2 production was not due to inability to respond to concanavalin A or allogeneic stimulation, nor to altered kinetics of the response. No correlation was observed between cytokine deficiencies and the inherited autoimmune characteristics previously studied in these strains. One especially interesting strain was N X 8 RI 16, which made high amounts of interleukin 2 but no detectable interleukin 1.