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.

Two chymotrypsin‐like proteases were purified from the secretory and excretory material of first‐instar larvae of Lucilia cuprina. The hydrolysis of N‐succinyl‐L‐phenylalanine‐nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor‐Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease {Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis‐causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.

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.

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.

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.

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<0.05) than other groups. Seven adjuvants mediated an immune response which caused significant inhibition of larval growth (P<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<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.

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.