The effect of immune and inflammatory mediators on growth of Lucilia cuprina larvae in vitro.

Immune and inflammatory responses occurring during dermal infestation by larvae of Lucilia cuprina can retard larval growth and development. This study examined the effect of 4 classes of humoral inflammatory mediators on larval growth in an in vitro assay. Mediators of plasma leakage (histamine, bradykinin, platelet-activating factor and serotonin), leucocyte chemotactic agonists (activated complement, leukotriene B4 and interleukin-8), effector molecules of immune responses (interleukin-1 beta, tumour necrosis factor-alpha and interferon-gamma) and endotoxin all failed to inhibit larval growth. In contrast, immunoglobulins isolated from immune serum caused marked retardation of larval growth. The results suggest that humoral mediators of inflammatory and immune responses do not play a role in immune defence against Lucilia cuprina.

The peritrophic membrane: its formation, structure, chemical composition and permeability in relation to vaccination against ectoparasitic arthropods.

Peritrophic membrane (PM) lines the gut of many arthropods and other animals, and thus separates ingested food from the gut epithelium. Its main functions are connected with its partitioning of the gut lumen into regions between which the transfer of large macromolecules and other particles is limited by its permeability properties. In the context of vaccinating mammalian hosts against parasitic arthropods. PM may either restrict penetration of ingested immune effector components within the parasite, or serve as a target for immunological attack. The properties of PM that are relevant to these potential roles--its site and mode of formation, structure, chemical composition and permeability--are reviewed with reference to ectoparasitic insects. Recent experiments, in which sheep were vaccinated with extracts of PM from larvae of the sheep blowfly, Lucilia cuprina, are outlined. Antibodies ingested from vaccinated sheep slowed the growth of L. cuprina larvae. These antibodies bound specifically to the PM, reducing its permeability and thereby perhaps hampering utilization of food by larvae. The potential for vaccination against parasitic arthropods using antigens from their PMs is discussed.

Inhibition of growth of Lucilia cuprina larvae using serum from sheep vaccinated with first-instar larval antigens.

Whole first-instar Lucilia cuprina larvae were homogenised and sequentially extracted with a series of buffers of progressively more severe solubilising power. The final extract, using a buffer containing 6 M-urea, was fractionated by preparative isoelectric focussing. At each step in this process, protein fractions were tested in sheep vaccination trials for their ability to induce immune responses affecting the growth of L. cuprina larvae which fed on the sera from vaccinated sheep. One isoelectric focussing fraction (pH 5.9-6.7) containing a number of larval proteins induced an immune response which inhibited the growth of larvae by a mean of 84 +/- 7% in an in vitro feeding bioassay. The recovery of larvae after feeding on sera from sheep vaccinated with this fraction was significantly reduced by 35 +/- 13%. This antilarval effect was shown to be mediated by ingested ovine antibodies. Immunofluorescence and immunogold localisations showed that the immune response was directed at proteins from the larval peritrophic membrane, larval cuticle and, to lesser extent, basement membranes and microvilli of digestive epithelial cells. Electron microscopic examination of larvae feeding on sera from sheep vaccinated with this fraction showed that the normally semi-permeable peritrophic membrane was blocked on the luminal side by an electron-lucent layer of undefined composition. It is postulated that this layer prevents nutrients from moving from the gut to the underlying digestive epithelial cells, thereby starving the larvae. The sera from sheep vaccinated with another isoelectric focussing fraction (pH 3.4-5.5) reduced the mean larval weight by 56 +/- 13% without significant effects on larval survival.

The major excretory/secretory protease from Lucilia cuprina larvae is also a gut digestive protease.

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.

Vaccination of sheep with purified serine proteases from the secretory and excretory material of Lucilia cuprina larvae.

Sheep were vaccinated with two purified serine proteases, LCT25a and LCT25b, isolated from the secretory and excretory material from first instar larvae of Lucilia cuprina. The immunization produced a strong antibody response to LCT25b and a weaker response to LCT25a as measured by ELISA. However, neither protease induced an ovine immune response which affected the development of first instar larvae growing on sera derived from these sheep. Further, direct in vivo bioassays of larval growth on the backs of vaccinated sheep also indicated a lack of induction of an immune response which prevented establishment of the larvae. Sera from unvaccinated sheep which had previous experience of blowfly strike, in general, strongly recognised the serine protease LCT25b. It was concluded from all of these results that serine proteases from the secretory and excretory material of L. cuprina are unlikely to be effective antigens in a vaccine designed to protect sheep from blowfly strike.

Role of oligosaccharides in the immune response of sheep vaccinated with Lucilia cuprina larval glycoprotein, peritrophin-95.

The larvae of the fly Lucilia cuprina cause a cutaneous myiasis in mammalian hosts, particularly sheep. The glycoprotein, peritrophin-95, isolated from Lucilia cuprina larval peritrophic matrix, is a candidate vaccine antigen. This protein induced an immune response in vaccinated sheep that inhibited larval growth. Recombinant forms of peritrophin-95 were produced in bacteria and baculovirus-infected insect cells. The bacterial protein was not glycosylated and incorrectly folded whereas the insect cell-expressed protein was glycosylated and probably correctly folded. Sheep immunised with purified native peritrophin-95 generated strong larval growth inhibitory activity in their sera, whereas sheep immunised with either recombinant form of peritrophin-95 generated only relatively weak inhibitory activity. Ingested ovine antibodies to native peritrophin-95 mediated the anti-larval growth activity and this was independent of the presence of ovine complement. The activity was associated with IgG(1) and IgG(2) but not IgM. There were strong antibody responses to both the correctly folded native peritrophin-95 polypeptide and the oligosaccharides present on this glycoprotein. Immuno-affinity isolation of antibody to the peritrophin-95 polypeptide and antibody to peritrophin-95 oligosaccharides demonstrated that the larval growth inhibitory activity resided with both antibodies. Lectin blots and ELISA data showed substantial differences between the oligosaccharides attached to native peritrophin-95 and insect cell-expressed recombinant peritrophin-95. It was concluded that the oligosaccharides attached to native peritrophin-95 and its unique polypeptide structure are essential for the induction of larval growth inhibitory activity in the sera of sheep vaccinated with this antigen.

Ingestion of host antibodies by Bovicola ovis on sheep.

Sheep body lice, Bovicola ovis, collected from moderately heavy infestations on Merino wethers, were assayed by ELISA for their content of host-derived specific immunoglobulin (Ig). Relative concentrations of anti-horse myoglobin antibodies in the lice and in sera from their hosts were used to estimate the total quantities of functional Ig (that which remained capable of binding specifically to its antigen) present, giving a mean of 0.21 +/- 0.20 mg/g of lice. An attempt to demonstrate the presence of antibodies against B. ovis antigens in naturally-infested host sheep using ELISA produced inconclusive results. The implications of the quantities of Ig ingested by feeding B. ovis are discussed in relation to the feasibility of immunological control of this species on sheep.

Retarded growth of Lucilia cuprina larvae on sheep and their sera following production of an immune response.

Attempts were made to immunize sheep against larvae of the sheep blowfly Lucilia cuprina using supernatant and pellet prepared by centrifuging (100,000 g max) homogenates from whole second instar larvae of L. cuprina or their excised guts. Injection of supernatant from whole larvae and from two fractions of this supernatant, prepared by ammonium sulphate precipitation, significantly reduced (by 24-58%) the final weight of larvae grown in vivo (i.e. on immunized sheep) for 20 or 44 h. Serum from animals vaccinated with supernatant from whole larvae reduced larval weights by 12% after growth for 20 h in vitro (i.e. on diet containing serum from treated animals). Pellet material from whole larvae or guts, when injected into sheep, stimulated an immune response which reduced the weight of larvae by 20-23% after 20 or 48 h in vitro. Larvae grown on these animals were not reduced in weight. Immunoglobulin (Ig) isolated from serum of a sheep vaccinated with gut material strongly retarded growth of larvae in vitro, the effect increasing with Ig concentration. These results indicate that an immune response in sheep, induced by injecting extracts of L. cuprina larvae, substantially reduces growth of this parasite.

Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro.

The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro. One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant. The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media. These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.

Growth of Lucilia cuprina larvae following treatment of sheep divergently selected for fleece rot and fly strike with monoclonal antibodies to T lymphocyte subsets and interferon gamma.

Intensive lymphocytic infiltration of the underlying dermis occurs during cutaneous myiasis caused by larvae of the blow fly, Lucilia cuprina. To determine the effect of this infiltrate on larval growth, monoclonal antibodies (mAb) to CD4, CD8 or WC1 lymphocyte subset determinants were injected intravenously before and during experimental infection of sheep with larvae. The effect of intravenous injection of mAb to ovine interferon (IFN) gamma was also examined. The experiments were performed in 18-month-old maiden ewes with genetic resistance or susceptibility to the disease complex, bacterial dermatitis/cutaneous myiasis. mAbs induced profound depletion of CD8+ and WC1+ subpopulations from blood and skin at sites of larval growth. mAb to CD4+ gave only a moderate reduction in lymphocytes from blood or skin. mAb treatments did not modify larval growth or survival at 20 or 50 h after infection. Larval growth rates did not differ between resistant and susceptible genotypes. No evidence was found for a role of T lymphocyte subpopulations or the cytokine IFN, in modifying larval growth during the first 50 h of infection. It seems unlikely that T lymphocyte-dependent immunological effector mechanisms contribute to the lower prevalence of fly strike seen in the resistant genotype in the field.

Variation in immune responsiveness of sheep to the antigens of intestinal nematodes and blowfly larvae.

The total and IgG1 antibody responses to the intestinal nematode parasites Haemonchus contortus and Trichostrongylus colubriformis were measured in the serum of 160 lambs, 4 months of age. These antibodies had developed as the result of natural exposure to the parasites on pasture. Three sires were examined and strong sire effects on half-sib progeny were found. Plotting of ELISA antibody results in two dimensions revealed clustering of responses within sire groups. Bimodal antibody distributions were also observed within sire groups and the whole population for T. colubriformis. A bimodal distribution of antibodies to H. contortus was found for one sire group but not for the whole population. The injection of blowfly larvae (Lucilia cuprina) extract into 42/160 lambs at a later age (12 months) was followed by increased antibodies to L. cuprina and an apparent increase in antibodies to T. colubriformis. A bimodal distribution for antibodies to L. cuprina was found in one sire group and in the whole population. These bimodal distributions of antibodies to L. cuprina did not coincide with the distribution of antibodies to T. colubriformis or H. contortus, measured on the same serum samples. It was concluded that high and low responder sire groups could be differentiated in lamb populations for all three parasites. These effects persisted during lamb maturation and appeared to be genetic effects. Finally, cross-reacting antibodies between L. cuprina and T. colubriformis appear to be stimulated by injection of L. cuprina antigens.

Vaccines against blowfly strike: the effect of adjuvant type on vaccine effectiveness.

Vaccination of sheep with a partially purified extract of Lucilia cuprina larvae in some cases resulted in marked reduction of growth in larvae which fed on the sheep. Twelve adjuvants were assessed, in vitro and in vivo, to determine which induced the largest inhibitory effect on larval growth. The Freund's complete adjuvant and Quil A groups produced ELISA antibody levels significantly higher (P less than 0.05) than other groups. Seven adjuvants mediated an immune response which caused significant inhibition of larval growth (P less than 0.05). When the sheep were assessed by in vivo larval culture, only larvae feeding on sheep vaccinated with the antigen presented in Freund's complete adjuvant or dextran sulphate or a dextran sulphate/Freund's incomplete adjuvant mixture weighed significantly less (P less than 0.05) than larvae feeding on control sheep. The effect on larvae was monitored in vitro for 70 days after vaccination, by which time significant reduction in larval weight was no longer observed. The loss of larval growth inhibition was not associated with a corresponding reduction in overall antibody levels.

Lucilia cuprina: inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane.

A culture system has been established to produce gram amounts of peritrophic membrane from larvae of the sheep blowfly, Lucilia cuprina. Peritrophic membrane obtained from this culture has been used to immunize sheep. The immunization produced an immune response which resulted in the average weight of larvae on immunized sheep being only 50% of that of larvae grown on control sheep (P < 0.05). Fractionation of the components of the peritrophic membrane followed by immunization trials showed that the protective antigen fraction comprised material that could only be solubilized by harsh agents such as 4 M-urea. Even after solubilization by 4 M-urea, the protective antigens were able to produce a protective immune response which reduced growth of larvae on immunized sheep to 55% of larvae grown on control sheep (P < 0.05). This immune response which reduced growth of the larvae did not cause gross morphological damage to the larvae.

Identification and molecular characterisation of a peritrophin gene, peritrophin-48, from the myiasis fly Chrysomya bezziana.

The peritrophic matrix lines the midgut of most insects and has important roles in digestion, protection of the midgut from mechanical damage and invasion by micro-organisms. Although a few intrinsic peritrophic matrix proteins have been characterised, no direct homologues of any of these proteins have been found in other insect species, even closely related species, suggesting that the peritrophic matrix proteins show considerable sequence divergence. We now report the identification of the cDNA and genomic DNA sequences of a Chrysomya bezziana homologue of the Lucilia cuprina intrinsic peritrophic matrix protein, peritrophin-48. The gene for C. bezziana peritrophin-48 spans 1315 bp and consists of three exons (65, 560 and 690 bp, respectively) separated by introns of 566 and 72 bp. The transcriptional start site, identified by a consensus of cDNA clones and primer extension analysis, is probably located 58 bp upstream from the start codon. However, there may be multiple start sites for transcription. Two potential TATA boxes and a consensus arthropod transcription initiator are located within 134 bp of sequence upstream of the putative transcriptional start site suggesting that this region contains the gene promoter. Immuno-fluorescence localization demonstrated that C. bezziana peritrophin-48 was localised to the larval peritrophic matrix. Protein fold recognition analysis indicated structural similarities between peritrophin-48 and wheatgerm lectin. As wheatgerm lectin binds chitin, this result suggested that C. bezziana peritrophin-48 may also bind chitin, a constituent of the peritrophic matrix. Chitin binding studies with a recombinant peritrophin-48 protein confirmed that it binds chitin. A Drosophila melanogaster homologue of peritrophin-48 encoded in an EST and a genomic sequence was also identified. The pairwise percentage identities of the deduced amino acid sequences for the peritrophin-48 homologues from the three higher Dipteran species were relatively low, ranging between 32 and 42%. Despite this sequence variability, the predicted structure of these proteins, dictated by five domains, each containing a characteristic distribution of six cysteines, was strictly conserved. It is concluded that considerable sequence variation can be tolerated in this protein because of the constraints imposed on the structure of the protein by an extensive disulphide bonded framework.

Production of antibodies to recombinant antigens from Lucilia cuprina following cutaneous immunisation of sheep.

Immunological control of cutaneous myiasis of sheep caused by Lucilia cuprina larvae has been an elusive goal. Antibody to antigens derived from the peritrophic membrane can stunt or kill larvae in a dose dependent fashion. Thus efficacy of vaccines employing these antigens may be limited by the amount of antibody in skin available for ingestion by larvae. The potential for elevating antibody concentrations in skin by intradermal immunisation with the recombinant peritrophic membrane antigens peritrophin-44, peritrophin-48 and peritrophin-95 was therefore examined. Using within-animal comparisons, specific antibody was significantly higher in skin transudates from locally immunised sites than from adjacent adjuvant control sites. It was concluded that cutaneous immunisation may assist immunological control of blowfly larvae.

Reduced growth of Lucilia cuprina larvae fed serum from sheep treated with anthelmintics.

The effect of three commonly used anthelmintics, levamisole hydrochloride, ivermectin and closantel, on the development of the sheep blowfly, Lucilia cuprina, was determined. Sheep were treated with each anthelmintic using the manufacturers' recommended dose for helminth control. Both ivermectin and closantel significantly (p < 0.05) reduced the growth rate of larvae of L cuprina cultured in vitro on serum from these sheep. Levamisole hydrochloride had no effect. Ivermectin was effective for less than 6 days after treatment, whereas closantel significantly reduced larval growth 21 days after treatment. Dose-response experiments showed that lower concentrations of both ivermectin and closantel were not as effective in reducing larval growth.

Vaccination against Lucilia cuprina: the causative agent of sheep blowfly strike.

The sheep blowfly, Lucilia cuprina, is responsible for over 80% of cases of blowfly strike in Australia and the losses in production and sheep deaths due to flystrike exceed $200 million per annum. Traditional methods of control are becoming less effective because of the blowfly's resistance to insecticides and thus other methods of control are necessary. In general, sheep develop very little immunity to flystrike even after repeated infestation, however vaccination against L. cuprina has shown considerable potential for controlling flystrike. The most successful sources of antigens have been the larvae's secreted proteases and several extracts from gut or peritrophic membrane. Immunization with these antigens results in retardation of larval growth and in some cases larval mortality. On sheep immunized with peritrophic membrane extracts, the growth retardation appears to be caused by a blocking of the peritrophic membrane which results in the larvae being starved of nutrients. The prospects for vaccine development and the remaining barriers to be overcome are discussed.

'Concealed' antigens: expanding the range of immunological targets.

For many parasites, the interaction between the immunogenicity of the parasite and the immunological response of the host is a dynamic equilibrium that allows both to survive, albeit often with severe consequences for the host. Vaccines, if intended as a means of parasite control, are unlikely to be generally successful if they do no more than mimic an immunological equilibrium that would be reached after natural exposure to the parasites. The situation must be tipped in favour of the host. It has been difficult to find ways around this impasse. One approach has been receiving practical attention over recent years, an approach that Peter Willadsen, Craig Eisemann and Ross Tellam have called vaccination against 'concealed' antigens.

Isolation of a trypsin-like serine protease gene family from the sheep blowfly Lucilia cuprina.

Various protease inhibitors active against both trypsin- and chymotrypsin-like serine proteases were used to characterize gut proteases from Lucilia cuprina by in vitro feeding assays. Significant larval growth retardation was observed on feeding first-instar larvae with trypsin inhibitors, particularly soybean trypsin inhibitor. Feeding of chymostatin, a specific chymotrypsin inhibitor, resulted in no significant growth retardation. This information suggests that trypsin-like serine proteases are probably the major gut digestive enzymes. A DNA fragment obtained by PCR which coded for part of a putative trypsin gene from L. cuprina was used to isolate a four-member multigene family of trypsins. The full nucleotide sequence of one of the genes and partial sequence from the other three genes were determined. Transcription of at least one of the genes has been confirmed. All four of the genes appear to have arisen by two separate gene duplication events.