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.

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.

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 IgG1 and IgG2 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.

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 peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

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 deteraminants were injected intravenously before and during experimental infection of sheep with larvae. The effect of intravenous injection of mAb to ovine interferon (IFN) γ was also examined. The experiments were perfermed 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 grwoth rates did not differ between resistant and susceptible genotypes. No evidence was found for a role of T lymphocyte subpopulations or the cytokine IFNγ 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.

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsin (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.

Gravid females of Lucilia cuprina (Wiedeman) in a laboratory cage orientated positively to samples of fleece. Newly-wetted fleece was significantly more attractive than dry fleece, an effect resulting from the action of water on the fleece and not just addition of water vapour to the volatile fleece kairomones. Fleece contaminated with serous exudate, resulting from myiasis by L. cuprina, was much more attractive than wet, uncontaminated fleece from the same sheep. Kairomones from wetted fleece consistently augmented the attractive effects of 2-mercapto-ethanol and indole in separate experiments, and of hydrogen sulphide (released from saturated aqueous sodium sulphide solution) in one trial out of three, but not overall. It is suggested that volatile fleece kairomones play a part in eliciting orientation to sheep by gravid L. cuprina. Fleece kairomones may augment the efficacy of kairomones released by putrefactive conditions in the fleece, which are known to predispose sheep to fly strike. They may also provide an input which helps to retain L. cuprina populations in the sheep's peridomestic precinct.

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.

Medical and Veterinary EntomologyVolume 9, Issue 4 p. 448-450 Digestion of ovine immunoglobulin G in larvae of the sheep blowfly Lucilia cuprina C. H. EISEMANN, Corresponding Author C. H. EISEMANN CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Indooroopilly, Queensland, Australia2 CSIRO Division of Tropics Animal Production, Long Pocket Laboratories, Private Bag No. 3, P.O. Indooroopilly, Queensland 4068, Australia.Search for more papers by this authorR. A. DONALDSON, R. A. DONALDSON CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Indooroopilly, Queensland, AustraliaSearch for more papers by this authorL. C. CADOGAN, L. C. CADOGAN CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Indooroopilly, Queensland, AustraliaSearch for more papers by this author C. H. EISEMANN, Corresponding Author C. H. EISEMANN CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Indooroopilly, Queensland, Australia2 CSIRO Division of Tropics Animal Production, Long Pocket Laboratories, Private Bag No. 3, P.O. Indooroopilly, Queensland 4068, Australia.Search for more papers by this authorR. A. DONALDSON, R. A. DONALDSON CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Indooroopilly, Queensland, AustraliaSearch for more papers by this authorL. C. CADOGAN, L. C. CADOGAN CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Indooroopilly, Queensland, AustraliaSearch for more papers by this author First published: October 1995 https://doi.org/10.1111/j.1365-2915.1995.tb00025.xCitations: 10AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References Allingham, P.G., Kerlin, R.L., Tellam, R.L., Briscoe, S.J. & Standfast, H.A. (1992) Passage of host immunoglobulin across the mid-gut epithelium into haemolymph of blood-fed buffalo fly Haematobia irritans exigua. Journal of Insect Physiology, 38, 9–17. East, I.J., Fitzgerald, C.J., Pearson, R.D., Donaldson, R.A., Vuocolo, T., Cadogan, L.C., Tellam, R.L. & Eisemann, C.H. (1993) Lucilia cuprina: inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane. International Journal for Parasitology, 23, 221–229. Eisemann, C.H. & Binnington, K.C. (1994) The peritrophic membrane: its formation, structure, chemical composition and permeability in relation to vaccination against ectoparasitic arthropods. International Journal for Parasitology, 24, 15–26. Eisemann, C.H., Pearson, R.D., Donaldson, R.A., Cadogan, L.C. & Vuocolo, T. (1993) Uptake and fate of specific antibody in feeding larvae of the sheep blowfly, Lucilia cuprina. Medical and Veterinary Entomology, 7, 177–185. Lemos, F.J.A. & Terra, W.R. (1991) Properties and intracellular distribution of a cathepsin D-like proteinase active at the acid region of Musca domestics midgut. Insect Biochemistry, 21, 457–465. Pendola, S. & Greenberg, B. (1975) Substrate-specific analysis of proteolytic enzymes in the larval midgut of Calliphora vicina. Annals of the Entomological Society of America, 68, 341–345. Waterhouse, D.F. (1940) Studies of the physiology and toxicology of blowflies. 5. The hydrogen ion concentration in the alimentary canal. CSIR Pamphlet No. 102, pp. 7–27. Willadsen, P., Eisemann, C.H. & Tellam, R.L. (1993) ‘Concealed’ antigens: expanding the range of immunological targets. Parasitology Today, 9, 132–134. Citing Literature Volume9, Issue4October 1995Pages 448-450 ReferencesRelatedInformation

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.

Larvae of the blowfly Lucilia cuprina (Wied.) (Diptera: Calliphoridae) were grown in vitro on a serum-free medium in the presence of a number of lectins. Lectins with specificities for beta-(1,4)-N-acetylglucosamine (wheat germ lectin) and alpha-D-mannopyranosyl and (alpha-D-glucopyranosyl residues (lentil lectin and Con A) caused strong concentration-dependent inhibition of the growth of the larvae and substantial mortality. Wheat germ lectin had the strongest effects, showing 50% inhibition of larval growth at a concentration of 2 mu M and 100% mortality at 25 mu M. Other lectins with different sugar specificities had much less effect. The mechanism of the larvicidal action(s) of wheat germ lectin, lentil lectin and Con A was investigated. There were at least three effects of these lectins on L. cuprina larvae. First, these lectins bound to and reduced the permeability of the peritrophic membrane of the larvae. Second, they reduced ingestion of diet medium by larvae. Third, the lectins also bound to the apical membranes of larval gut epithelial cells although there were no obvious signs of damage to these cells. It is concluded that the combination of these effects probably results in the starvation of the larvae. The implications of these results in terms of possible control strategies for L. cuprina are discussed.

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.

A large and diverse family of serine protease genes was identified in first‐instar larval cDNA of the sheep blowfly (Lucilia cuprina). This complex repertoire of genes was identified via a PCR approach using highly degenerate primers based on structurally conserved regions which surround the active site His and Ser residues found in all serine proteases. PCR products from entire first‐instar larval cDNA, or from third‐instar larval salivary glands or cardia, generated using a microscale RT‐PCR method, were cloned into a plas‐mid vector. Comparison of the restriction fragment patterns of PCR products generated from the three different sources suggests a highly diverse tissue‐specific pattern of serine protease expression in this organism. Detailed analysis of the restriction fragment patterns of sixty‐nine randomly selected clones from entire first‐instar larvae revealed forty‐nine different classes of PCR product. Maximum likelihood analysis of these data indicate that between 125 and 220 different serine protease genes are expressed in first‐instar larvae of L. cuprina. DNA sequence analysis of ten randomly‐selected clones, derived from the three tissue sources, indicated that all ten encoded serine protease gene fragments. A frequently occurring PCR product, generated from both first‐instar total cDNA and third‐instar cardia cDNA, showed 73% amino acid identity to a digestive protease expressed in Droso‐phila melanogaster larval gut cells.

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.

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.

Abstract. The quantity of specific antibody ingested by larvae of Lucilia cuprina and its fate after ingestion were studied in larvae grown on sheep and on an artificial diet. Larvae grown to late first or early second instar on sheep vaccinated with horse myoglobin contained 66% less specific antibody detected by enzyme linked immunosorbent assay than larvae grown to a similar stage on an artificial diet containing 75% serum from the same sheep. A similar result was obtained when larvae were grown to mid‐third instar. Larvae grown on sheep to first or second instar contained approximately the same quantity of specific antibody per unit weight of larvae as those grown to third instar. Larvae grown on diet to third instar contained 22% less specific antibody per unit weight than those grown to first or second instar. In larvae grown on diet to late third instar, ingested diet retained 91 ± 12% of its original specific antibody activity in the crop, 50 ± 11% in the anterior midgut, 8 ± 2% in the posterior midgut and 13 ± 6% in the hindgut. The mean concentration of total immunoglobulin detectable in the haemolymph of individual third instar larvae grown on diet was 1.7 ± 2.8 ug/ml. Assays of specific antibody in the haemolymph of similarly reared larvae indicated that all or most of this immunoglobulin remained functional. The implications of the quantities and distribution of ingested functional antibody found in feeding larvae of L.cuprina are discussed in relation to the possibility of vaccinating sheep against these larvae and the selection of likely internal targets as sources of potential protective antigens.

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 protecive 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.

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.