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

Inwmrrtiomd Journrrlfor Printed in Greol Brimin

Parusilology

Vol. 23, No. 2,pp.

221-229,

1993 0

LUCILIA CUPRINA: IMMUNIZATION

002&7519/93 56.00 + 0.00 Pergamon Press Lrd Society for Parasitology

1993 Ausrralian

INHIBITION OF LARVAL GROWTH INDUCED BY OF HOST SHEEP WITH EXTRACTS OF LARVAL PERITROPHIC MEMBRANE

I. J. EAST,* C. J. FITZGERALD, R. D. PEARSON, R. A. DONALDSON, T. VUOCOLO, L. C. CADOGAN, R. L. TELLAM and C. H. EISEMANN CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3, Indooroopilly, Queensland 4068, Australia (Received 14 May 1992; accepted 20 October 1992)

I. J., FITZGERALD C. J., PEARSONR. D., DONALDSON R. A., VUOCOLOT., CADOGANL. C., TELLAMR. L. and EISEMANNC. H. 1993. Luciliu cuprina: Inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane. International Journal for Parasitology 23: 221-229. 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. Abstract-E.wr

INDEX KEY WORDS: Lucilia cuprina; peritrophic membrane; immunization; sheep; reduced larval growth.

INTRODUCTION THE blowfly, Lucilia cuprina, causes losses in product-

ivity to the Australian sheep industry which exceed one hundred million dollars per annum (Arundel & Sutherland, 1988). Traditional methods of control include both radical surgical procedures and repeated application of insecticides. Resistance to insecticides is now a major problem in the industry (Arundel & Sutherland, 1988) and surgery is becoming less acceptable. Clearly, new strategies for the control of blowfly strike must be developed. Vaccination of sheep to control the blowfly is one alternative. O’Donnell, Green, Connell & Hopkins (1981) demonstrated impairment of larval development in vitro when L. cuprina larvae were fed on serum from sheep vaccinated with a soluble extract of third instar larvae. Bowles, Carnegie & Sandeman (1987) have also had some success in vaccinating sheep with

* To whom all correspondence should be addressed.

blowfly larval secretory products. The effects, however, were only partial and transient. An alternative strategy may be to direct a vaccine to components of the digestive tract. Recently, Willadsen, Riding, McKenna, Kemp, Tellam, Nielsen, Lahnstein, Cobon & Cough (1989) described the isolation and characterization of a glycoprotein from the midgut epithelial cells of the cattle tick, Boophilus microplus. This protein, when used to vaccinate cattle, reduced tick numbers on vaccinated cattle by more than 90%. Antibodies ingested in the blood meal of the tick caused severe damage to the gut epithelial cells. The gut of L. cuprina, however, has a substantially different structure and function. Unlike the tick, where digestion occurs intracellularly by endocytosis of ingested blood, digestion in the fly takes place within the lumen of the gut. In addition, the gut of L. cuprina is lined with a peritrophic membrane (PM). Evidence 221

222

1. J. FASTet af.

from various studies suggests that the pores of the PM are too small to allow substantial access of intact antibody molecules to the underlying gut digestive cells. For example, dextrans larger than 32,000 M, could not penetrate the PM of a variety of insects (Peters & Wiese, 1986). In addition, Eisemann, Kemp & Vuocolo (manuscript submitted) have shown that only gold particles smaller than 10 nm in diameter will pass though the PM of L. cuprina. Immunoglobulin molecules are approximately this size and therefore antibody access to the gut epithelial cells is likely to be severely restricted. However Fab, fragments of immunoglobuiin are produced in the insect gut and these, whilst smaller than immunoglobulin, still have the ability to inhibit larval growth (Eisemann, unpublished results). The PM protects the underlying digestive epitheIia1 cells from attack by bacteria and mechanical damage from ingested particles. The PM also aids digestion by the selective partitioning of specific proteases and the provision of a structure which promotes recycling of digestive enzymes by allowing anterior flow of fluid in the ecto-peritrophic space (Terra, 1990). In addition, a number of non-structural proteins are associated with the PM, e.g. lectins and aminopeptidases (Walker, Geer & Williamson, 1980; Peters, Kolb & Kolb-Bachofen, 1983). This range of functions for the PM suggests that it is essential for maintaining viabiliy of the insect. Disruption of any of these functions may cause adverse effects in the larvae. The accessibility of the PM to ingested immunoglobulins makes the PM an attractive target for an anti-blowfly vaccine. Here we describe a novel culture method for the production of PM and the use of the PM as a source of vaccine antigen for the control of blowfly strike. MATERIALS AND METHODS ~ufer~als. Unless otherwise mentioned. laboratory chemicals were of AR grade and purchased from the Sigma Chemical Company (St. Louis, U.S.A.). Acids and solvents were of AR grade and purchased from Ajax Chemical Company (Auburn, Australia). Newborn calf serum, Freund’s complete adjuvant and gentamicin were purchased from Commonwealth Serum Laboratories (Parkville, Australia). Zwittergent 3-14 was purchased from BoehringerMannheim (North Ryde, Australia). Blotvfly colony. Laboratory populations of L. cuprina, which had originated from Aystruck sheep, were maintained on an artificial medium (Singh & Jerram, 1976) for up to 10 generations. Eggs were collected by placing small trays of minced liver covered with fine nylon gauze inside cages of adult L. cup&a for 4-5 h. The eggs were then incubated overnight at 16°C and 100% relative humidity before surface sterilization. Produefion of PM in larval culture. L. cuprina eggs were washed in I% sodium ~ypoch~orite for 5 min at room temperature to separate and surface sterilize theeggs. During

this time, the egg clumps were gently dispersed. Approximately 7000 eggs were placed into a flask containing 250 ml of 0.012 M-phosphate buffered saline (pH 7.3) containing 40% newborn calf serum, 0.2 mg ml-’ gentamicin and 2% yeastolate. The growth medium was absorbed into standard kitchen sponges placed in the llask. The flask was vented with an aquarium pump and incubated at 28-30°C for 72 h. The flask was opened, the sponges shaken to dislodge adhering larvae and removed from the flask. The larvae were washed with sterile distilled water and drained in an 850 pm sieve. The larvae were returned to the flask and 30 ml of PBS containing 2.5 mM- benzamidine and 5 mM-disodium N,N’ethylenediamine tetraacetate (EDTA) (inhibitor buffer) was added. The later two reagents were added to inhibit proteases secreted by the larvae. The flasks were then incubated for a further 24 h at 28-30X. During this time, the PM in each larva is continually synthesized and shed as it passes out the anus. The flask contents were then sieved to remove the larvae and the filtrate centrifuged at 10,~~ for 30 mm at 4°C. The peilet was resuspended in inhibjtor-buffer and recentrifuged. The final pellet was stored at - 100°C. Exfraction of the peritrophic membrane. Each extraction step involved suspending the PM in 2 ml per gram of a specific extraction solution, homogenizing with an Ultraturrax type 18jlO blender (Janke & Kunkel, Germany) and allowing the homogenate to stand for 4-16 h at 4°C. The homogenate was then centrifuged {50.~0 g, 4X, 30 min) and the supernatant retained while the pellet was further processed. In experiment 1, the PM was extracted in 0. I Fn-Tris/HCl pH 7.5 containing 150 mM-NaCl, 5 mM-EDTA (TBS) and S rn~-~nzamidine. In experiment 2, the PM was extracted in TBS and then the insoluble pellet was further extracted with either 4% sodium dodecyl sulphate and 10% &mercaptoethanol or 5% cetyitrimethylammonium bromide. In experiment 3, the PM was extracted sequentially with distilled water, TBS, TBS containing 2% (w/v) Zwittergent 3-14,4 Murea, 6 M-guanidine HCl and 0.1 M-HCI. Each extract was dialysed against PBS (3 x 3 I). This dialysis, which was necessary to remove denaturants, resulted in substantial precipitation of protein. The precipitate and dialysed solution were stored together until used for vaccination. Sheep. Experimental animals were 6-12 month old Merino ewes. These animals had not previously suffered flystrike and were maintained in pens on a diet of lucerne pellets. Animals were randomly assigned to various treatment groups. Animals were maintains under veterinary supervision and all manipulations were in accord with the Australian code of practice for the care and use of animals for experimental purposes (Anonymous, 1990). Vuccinarion. Each animal received the material extracted from 2.5 g of PM at each injection. The extracts were each homogenizd with an equal volume of Freund’s complete adjuvant (FCA). The first injection was intramuscular, given half into each rear leg. The second injection was intramuscular, given in the neck 28 days later. Ail animals were bled from the jugular vein prior to each injection. Fourteen days after the second injection the effect of vaccination was assessed by both in viva and in vitro larval growth assays (Eisemann, Johnston & Kerr, 1989; Eisemann, Johnston, Broadmeadow, O’Sullivan, Donaldson, Pearson, Vuocolo &

Immunization

of sheep against

Kerr, 1990). There were three to six animals in each group. Antibody titres were assessed by ELISA as described by Eisemann et al. (1990). Immunoglobulin isolation and concentration. Using method E of Mostratos & Beswick (1969), immunoglobulin (Ig) was isolated from post-vaccination sera of two sheep which had been vaccinated with extracts of PM and also from pooled serum of 12 control sheep which had been injected with FCA only. The percentage yields of Ig obtained from the sera were estimated by ELISA. With the anti-PM sera, this was determined by comparison of a range of dilutions of both the original sera and of the isolated Ig. For the control sera and Ig, an antigen capture ELISA was used. Wells of microtitre plates were coated with rabbit anti-sheep Ig serum and a range of dilutions of the original serum or isolated Ig were then added. Subsequent steps were essentially those described by Eisemann et al. (1990). Relative concentrations of Ig in the original sera and in isolation were estimated in each case by comparing curves obtained by plotting log ELISA optical densities against log dilution. The samples were concentrated by vacuum dialysis against PBS. Aliquots of each Ig solution were added to 4 ml of pooled serum to give Ig concentrations equal to twice and four times those in the original sera. The total volume was adjusted to 5 ml with PBS. It was necessary to supplement the Ig preparations with the control serum to provide sufficient nutrition for satisfactory larval growth. The 5 ml preparations so obtained were formulated into diets and larvae grown on them as described above. SDS-PAGE. All samples were analysed by SDS-PAGE on 618% gradient gels, using the method ofLaemmli (1970). All gels included mol. wt standards (Bio-rad) and were silverstained using the method of Morrissey (1981). Glycoproteins were detected by electrotransfer of gel contents to nitrocellulose and staining with a 1 in 500 dilution of biotin-labelled

223

blowfly strike

lectins (Vector Laboratories, Burlingame, CA, U.S.A.) followed by a 1 in 800 dilution of streptavidin-horseradish peroxidase conjugate (Amersham, U.K.). The detailed procedure followed the immunoblot method of Kerlin & Allingham (1992). Immunofluorescence. Post-vaccination sera from sheep vaccinated with Zwittergent 3-14-solubilized or guanidine HCl-solubilized material of PM and from control sheep which had received injections of FCA only were diluted 1:2000 in 12 m&PBS, pH 7.3. Pieces of PM collected from culture as described above and stored at - 1OOC and lengths of PM freshly dissected from the midguts of third instar larvae of L. cuprina fed on larval rearing medium (Singh & Jerram, 1976) were incubated overnight at 7’C in each of the diluted sera. After four washes in PBS, the PM was incubated

in a 1:25 dilution of fluoroscein isothiocyanate-labelled rabbit anti-sheep Ig serum in PBS for 2 hat 24°C. After three further washes in PBS, the samples of PM were mounted on slides and examined and photographed in microscopes using fluorescence and Nomarski differential interference contrast techniques. RESULTS

The new technique for producing PM yielded a mean of appoximately 40 mg of PM per 1000 larvae. Examination by light microscopy showed that the preparation consisted mainly of PM but contained small amounts of larval exuviae and sponge (results not shown). The cultured PM was homogenized in TBS and the soluble and insoluble fractions were used to immunize separate groups of six sheep. The immunization resulted in relatively high levels of circulating antibodies in both groups (Fig. la). Larvae were grown on

b

i1

caadcdubb Pdet

contml SosPebl ClBPdd SDssahue

Contml Zv&rg~U Guaniine

cTBso&b

ItiUe

lk

HCI

Treatment FIG. 1. (a) The antibody titres of sheep immunized with insoluble extracts of peritrophic membrane. (b) The antibody with detergent extracts of peritrophic membrane. (c) The immunized with various extracts of peritrophic

buffer-soluble and buffertitres of sheep immunized antibody titres of sheep membrane.

I. J. EASTet al.

224

TABLE 2-T~E~FFE~OFIMMUNI~TIONOFSHEEPWITH ANDIN~Lu6LEE~=~~sOFPERITROPHICMEMBRANEON=H~

DETERGENT-SOLUBLEAND-INSOLUBLEFRA~IONSOFPERITROPHIC

GROWTHOFBLOWFLYLARVAEFEEDINGON~ESHEEPANDON

ME~BR~NEONTKEGROW~OFBLOWFLYLARVAEFEEDINGONTHE

THEIRSERA

SHEEP ANDON

Larval weight(mg)* Group

n

Control 6 Soluble 6 Insoluble 6

THEIR SERA

Larval weight (mg)* Group

n

20 h

50 h

20 h

20 h

50 h

20 h

in vivo

in vivo

in vitro

in vivo

in viva

in vitro

1.7*0.1”

22.lrt2.5”

3.0fO.l”

1.6kO.l” 1.3f0.1b

20.6f 1.5” 2.4f0.2” 20.4f 1.8” 2.1 fO.lb

1.6+0.2” 1.3*0.1= I.O*O.lb

18.4+3.9* 2.9~kO.i” 13.4f 1.7” 2.510.2 13.7i2.3” I.9~kO.2~

2.0f0.1” 1.4 + 0.2b l.O+O.tb

25.1zk22.0” 2.7iO.l” 23.5f2Yb 20i02b . , I 16.6f I.gb 1.3kO.2’

Within each column, figures with the same superscript are not sjgni~can~y different (P > 0.05). *Values are group means rt S.E.M.

these sheep and control sheep to assess the level of protective immunity induced (Table 1). Larvae grown on sheep immunized with the insoluble material were 26% smaller than those on control sheep after 20 h (P < 0.05); however, after 50 h there was no significant difference in larval size. When sera from these groups of sheep were used to grow larvae ipl vitro, the larvae grown on sera from both the group vaccinated with soluble antigen and the group vaccinated with insoluble antigen produced smaller larvae (P < 0.05) than control sera but the effect was greater with sera from the group vaccinated with insoluble antigen (69% of controls) than with sera from the group vaccinated with soluble antigen (82% of controls) (Table 1). The most effective vaccine fraction was the material that was insoluble after extraction with buffer. SampIes of this insoluble material were therefore treated with two different detergents in an attempt to solubilize the effective antigens. The buffer-insoluble material was treated with either 4% SDS/IO% 2-mercaptoethan01 or 5% ~etyl-triethylammonium bromide. Both of these ionized detergents are considered to be strong solubilizing agents and each solubilized a proportion of the buffer-insoluble material. The resulting detergent-soluble fractions from each treatment were used to immunize sheep. As with the previous experiment, immunization with PM extracts resulted in relatively high levels of circulating antibodies (Fig. 1b). With both detergents, immunization with either soluble or insoluble fractions resulted in larvae in the challenge infection being of lower weight than those grown on control sheep (Table 2). When larvae were grown in vivo, the animals immunized with either the SDS- or the

Experiment A

Control SDS solublef SDS insoluble

6 6 6

ExperimentB Control 6 CTAB solublet 6 CTAB insoluble 6

For each experiment within each column, figures with the same superscript are not significantly different (P > 0.05). *Values are group means f s E.M. WDS-sodium dodecyl sulphate.

$CTAB-cetyltrimethylammonium bromide. CTAB-insoluble fractions reduced lava1 weights to 65 and 52%. respectively, of control weights (P < 0.05) after 20 h. Further, after 50 h infestation, a reduction in weight (74 and 66% of control weight, respectively) was still observed although the reduction with sera from the animals vaccinated with the SDS-insoluble material was not statistically significant. Even after extraction with these ionic detergents, the most effective protective antigens were still located in the insoluble material. It was clear that strong ionic detergents were not capable of solubilization of the protective activity associated with the peritrophic membrane. Stronger solubili~tion agents were therefore required fo the extraction of antigens responsible for the protective activity. A batch of peritrophic membrane was subjected to exhaustive extraction by sequential treatment with TBS, 2% Zwittergent 3-14 in TBS 4 M-urea, 6 M-guanidine HCl and 0.1 M-HCl. Each of these agents extracted a different group of proteins from the peritrophic membrane and these can be seen in the silver-stained SDS-PAGE gradient gel shown in Fig. 2. The major band extracted with both distilled water and TBS had a relative molecular weight of approximately 60,000 and probably corresponded to residual bovine serum albumin from the culture medium. The other four reagents each extracted a wide range of proteins with various molecular weights. In particular, the 4 M-Urea

Immunization of sheep against blowfly strike

225

ABCDEFG

FIG. 2. Silver stain of a SDS-PAGE gel of various extracts of peritrophic membrane. (A) Molecular weight standards, (B) distilled water extract, (C) buffer extract, (D) Zwittergent 3-14 extract, (E) urea extract, (F) guanidine HCl extract, (G) HCl extract.

TABLE

3-THEAPPARENTMOL.WTOFPROTEINSFROMTHEUREA EXTRACTOFPERITROPHICMEMBRANE

Mannose-containing glycoproteins 224,000 195,000 158,000 123,000 105,000 98,000 79,000 69,000 48,000 38,000 31,000 28,000 25,000

Major proteins identified by silver stain

158,000 120,000 105,000 83,000 79,000 60,000 48,000 42,000 32,000 28,000 25,000

solution was the most effective solubilizing agent and the molecular weights of the major proteins extracted by 4 M-Urea are listed in Table 3. The most abundant proteins in this fraction were of sizes 60, 48, 42 and 32 kDa. The material extracted from PM by 4 M-urea was subjected to further analysis by lectin blotting of SDS-PAGE gels electrotransferred to nitrocellulose. Most of the proteins that were identified by the silver stain reacted strongly with Lens culinaris lectin. This indicated a high mannose content typical of many insect glycoproteins (Kuroda, Geyer, Geyer, Doerfler & Klenk, 1990). The molecular weights of these glycoproteins are listed in Table 3. The same proteins reacted weakly with Phaseolus vulgaris lectin which binds to fucose and Ulex europaeus lectin which binds to various oligosaccharides. Several high molecular weight proteins reacted with wheat germ lectin which binds to N-acetyl glucosamine (Fig. 3). Immunization of sheep with these fractions resulted in relatively high levels of circulating antibodies with all groups having broadly similar titres

I. J. EASTet al.

226

E

F

FIG. 3. Identification. of gl~~prote~ns in the urea extract of per~trop~~c membrane. @,D,F,H) Prestained molecular weight standards; (A,C,E,G) urea extract of ~ri~opbjc membrane, stained with (A) wheat germ lectin, (C) i/%x europaeus leetin, (E) P~~eo~u~ ~~~ari~ &tin and G Lens cuharis iectin. Each lectin was labelled with biotin. TARLE~-THEEFFECTOPIMMUNIZAT~ONOFSHEEPWITHVARIOUS EXTRACTS OF BLOWFLY

PERITROPHICMEMBRANE ON THE GROWTH OF

LARVAE FEEDING irtV&r0

Group

Control L Zwittergent 3-14 Urea Guanidine HCI Hydrochloric acid Residual pellet

n

4 4 4 4 4 4

ON

SERUM.BASED

MEDIA

Larval weight (mg)* 20 h in vim 3.72rtO.W 2.1OitO.50”’ 1.9fktO.26~ 2.41 &0.56”~b 3.15 f 0.2oa,’ 3.52f0.17”

Figures with the same superscript are not significantly different (P > 0.05).

*Values are group means f s2.M. (Fig. 16). Only immu~i~tion with the fractions extracted with Z~tter~ent 3-14 or urea was effective in inhibiting larval growth (Table 4). When larvae were grown on media in vitro, the antisera from sheep vaccinated with the urea-soluble fraction reduced

larval growth by 50% (P -=z0.01) and the sera from sheep vaccinated with the Zwittergent 3-14 soluble fraction reduced growth by 43% (P < 0.05) (Table 4). A further ex~~ment using an identical protocol confirmed that the material extracted from PM by 4 Murea induced a protective immune response in sheep that caused reduced growth of sheep lavae in vim (results not shown). Strong uniform fluorescence was seen in both cuttured and freshly dissected PM samples after incubation with anti-Pi sera and a ~ITC-labelled anti-sheep IgG (Fig. 4a), but no fluorescence was observed with PM samples incubated in control sera (Fig. 4b). The vaccination had thus produced antibodies which reacted with the PM. The effect of increased concentrations of specific antibody on larval growth was assessed by isolating and concentrating antibodies and using them in vitro assays. Concentated Ig solutions from anti-PM sera produced substantial reductions in larval weight at all concentrations tested (Fig. 5) and the observed reduction in growth increased with increasing Ig concentration. In addition, the percentage of larvae recovered

Immunization of sheep against blowfly strike

227

FIG. 4. Indirect immunofluorescence staining of isolated peritrophic membranes with (a) antiperitrophic membrane antiserum, (b) control serum. The bar repesents a distance of 30 pm. from 1xeparations containing anti-PM sera tended to be lov ver than that from the corresponding control prepai rations and was lower at higher Ig concentrations. This was partly due to the difficulty in finding smalle :r sized larvae in the growth medium. Dead larvae were found in all concentrations of anti-PM Ig but or 11yin the four-fold concentration of control Ig. DISCUSSION

The in vitro culture of whole larvae of L. cuprina allows : the collection of gram quantities of PM with

minimal manipulation. Previously, other work ers have been able to grow small amounts of PM frcom adults of the blowfly, Calliphora erythrocephala by organ culture of isolated cardiac (Becker, Peters & Zimmermann, 1975). This, however, is the first rep ort of production of sufficient material to allow vaccil nation trials. We do not know whether the PM is damaged or partly digested during its passage throw tgh the gut but importantly it retains sufficient immui sogenicity to induce a protective immune response. In immunofluorescence assays, the antisera produced hy

228

I. .I. EASTef al.

I%, 5. The effect of serum and various concentrations of isolated anti-PM i~uno~ob~in on growth of L. cuprina larvae in vitro. 1 x , 2 x and 4 x are estimated ratios of Ig concentrations in these preparations compared to those in the original sera. ) Serum from unvaccinated sheep; ($$//$ serum from sheep 1171 vaccinated with the urea extract of perltrophic membrane; M) serum from sheep

1180 vaccinated with the urea extract of peritrophic membrane. vaccination with PM shed from cultured larvae reacted equally well with PM collected from cultures or freshly dissected PM. This, and the effect of vaccination on live larvae strongly suggest that the two sources of PM are antigen&ally equivalent. Immunization of sheep with extracts of PM results in an immune response that causes reduced growth of blowfIy larvae feeding on the sheep. Although mortality of larvae was rarely observed, slower development may expose the larvae to the possibility of dehydration as the fleece dries, and thus increase larval mortality. Vaccination with midgut-associated antigens has previously been attempted with several insect species including Anop~eles s~~~hensi(see Alger & Cabrera, 1972), Stomoxys calcitrans (see Schlein & Lewis, 1976), ~~5s~~~ ~ortj~a~ (see Kaaya L Alemu, 1982), Andes uegj?pti (see Hatfield, 1988; Ramasamy, Ramasamy, Kay & Kidson, IQ%), Pediculus hmmzis (see Ben-Yakir, D. & Mumcuoglu, Y.K., 1988, Abstract in Proceedings of the XIIIth International Congress of Entomoiogy, Vancouver, p. 282) and L. cuprina (see Johnston, Ei~mann, Donaldson, Pearson & Vuocolo, 1992). However, it is not known whether the protective antigens were associated with the PM or the midgut proper. Vaccination with cultured PM has two major advantages over whole extracts or dissected guts; first the relatively large yield and second the comparative biochemical simplicity. SDS-PAGE gels of the urea extract of PM suggest that this fraction contains seven to I 1 major proteins compared to tens of thousands in a whole larval extract. One further reason why the PM may be a good source of antigen for vaccination is the possibility of a

synergistic interaction with antigens from other sources. When larvae were fed on media containing sera from various va~inat~ sheep, we observed, on several occasions, a synergistic enhan~ment of the growth inhibitory effect when an anti-PM antiserum was combined with an anti-whole first instar larval extract antiserum (results not shown). The mechanism of action of the anti-PM antisera is being investigated. The small larvae from effectively vaccinated sheep show no gross abno~alities of the digestive tract (results not shown). Therefore it is unlikely that the immune response is destroying the PM. An alte~ative expl~ation is that the antibody molecules adhering to the PM may sterically hinder the passage of digestive enzymes and nutrient substances through the PM or inhibit the functions of crucial proteins associated with the PM. The protective antigen fractions identi~ed are not readily solubjli2~ except with strong denatu~ng agents. When these agents are used, however, the antigens retain sufficient structural integrity to generate an immune response which is capable of reducing the weight of larvae feeding on the vaccinated sheep. These antigens therefore must produce antibodies which react with the native antigens. It is highly likely that proteins such as these that retain intact protective epitopes in denaturing solvents will retain activity and integrity through bioche~cal ma~pulation. This makes them ideai for further pu~fi~tion and characterization to produce a vaccine for the control of flystrike in sheep. Acknowledgements-The

authors wish to acknowledge the substantial financial contributions of the late Mr. L. W. Bett to this work.

ALGERN. W. E. & CABRERA E. I. 1972. An increase in death rate of Anopheies stephensi fed on rabbits immunised with mosquito antigen. JaumaI of Economic Enio~o~ogy 6% i65-168. AF*‘ONYMOUS 1990. Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Australian

~ovemment

Publ~shiug Service, Canberra.

ARWNDEL J. H. & SUTHERLAND A. K. 1988. Blowflies of sheep. In: Animal Health in Australia, Vol. 10, Ectoparasitic Diseases of Sheep, Cattle, Goats and Horses (Edited by ARUNDELJ. J. &SUTHERLANDA. K.), pp. 35-60. Australian

Government Publishing Service, Canberra. BECKERB., PETERSW. & ZIMMERMANN U. 1975. Investiga-

tions on the tr~spo~ function and structure of peritrophic membranes--VI. In vitro synthesis of peritropbic membranes of the blowfly, Cailiphora erythra~ep~ala. Journal of Insect Physia~ogy 21: 146%1470. BOWLES V. M., CARNEGIEP. R. & SA~DK~A~ R. M. 1987. Immunisation of sheep against infection with larvae of the

Immunization

of sheep against

blowfly Lucilia cuprina. International Journal for Parasitology 17: 153-758. EISEMANN C. H., JOHNSTONL. A. Y. & KERR J. D. 1989. New techniques for measuring the growth and suvival of larvae of Lucilia cuprina on sheep. Australian Veterinary Journal 66: 187-189. EISEMANNC. H., JOHNSTON L. A. Y., BROADMEAD~W M., O’SULLIVANB. M., DONALDSON R. A., PEARSON R. D., VUOCOLO T. & KERR J. D. 1990. Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro. International Journalfor Parasitology 20: 299-305. HATFIELD P. R. 1988. Anti-mosquito antibodies and their effects on feeding, fecundity and mortality of Aedes aegypti. Medical and Veterinary Entomology 2: 331-338. JOHNSTON L. A. Y., EISEMANN C. H., DONALDSON R. A., PEARSON R. D. & VUOCOLOT. 1992. Retarded growth of Lucilia cuprina larvae on sheep and their sera following production of an immune response. International Journal for Parasitology 22: 187-193. KAAYA G. P. & ALEMU P. 1982. Fecundity and survival of tsetse maintained on immunised rabbits. Insect Science and ils Applications 3: 231-241. KERLIN R. L. & ALLINCHAM P. G. 1992. Acquired immune response of cattle exposed to buffalo fly (Haematobia irritans exigua). Veterinary Parasitology 43: 115- 129. KURODA K., GEYER H., GEYER R., DOERFLERW. & KLENK H.D. 1990. The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector. Virology 174: 418429. LAEMMLIU. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 68&685. MORRISSEYJ. H. 198 1. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analytical Biochemistry 117: 307-310.

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