AU664054B2 - Flystrike antigen and vaccine and method for preparation - Google Patents

Description

641

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name of Applicant: Actual Inventor: Address for Service: Invention Title: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION ROSS L. TELLAM CRAIG H. EISEMANN CHRIS ELVIN IAIN EAST CULLEN CO., Patent Trade Mark Attorneys, 240 Queen Street, Brisbane, Qld. 4000, Australia.

FLYSTRIKE ANTIGEN AND VACCINE AND METHOD FOR PREPARATION *o 0 00a 0 00 0000s 0 0 oo a o 0 *a 6 o S a 0* 0 0 a0 0 0 0 Ks Details of Associated Provisional Applications: Nos. PK9743 The following statement is a full description of this invention, including the best method of performing it known to us:

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(11) AU-B-29716/92 -2- 664054 18. A native antigen obtained from extraction of peritrophic membrane having a molecular weight (Mr) of 44,000 determined by SDS-PAGE under reducing conditions.

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L-,¢i I- This invention is described in the following statement: THIS INVENTION relates to a vaccine which when administered to sheep results in the production of an immune response which is capable of retarding the growth of blowfly larvae feeding on the vaccinated sheep thereby restricting or limitig the effects of blowfly strike. The invention also relates to the identification of antigens for use in the vaccine and methods for the preparation of the antigens.

BACKGROUND

Blowfly strike in sheep is caused by fly maggots feeding on the tissue and tissue fluids of the sheep. The problem is of significant economic importance to the Australian sheep industry (Brideoke, 1979; Arundel and Sutherland, 1988; full details of these references as with all other references are provided in the LIST OF REFERENCES hereinafter). It has been i estimated that up to 3 million sheep per annum or t approximately 2% of the Australian flock are killed by blowfly strike even with existing control practices (Brideoake, 1979).

The major species of blowfly which initiates 80-90% of all .o primary strikes in Australia is Lucilia cuprina. This fly has a widespread distribution throughout Australia (Arundel and Sutherland, 1988).

CCC' Adult female flies deposit eggs on susceptible animals.

The eggs hatch in 8-24 hours and the resulting 'maggots (larvae) release substances which cause inflammation of the skin and the loss of outer skin layers. The maggot then feeds on the weeping wound. At the end of third instar, the maggot f '7 t

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head has penetrated deeply into the sheep tissue and is feeding on tissue fluid and blood. This wound then makes the sheep susceptible to additional secondary fly strikes. The maggots remain on the sheep for about 3 days after which they fall off onto the soil where they can remain viable for long periods. These larvae pupate and adults emerge 6-8 days later thereby allowing the life cycle to continue (Arundel and Sutherland, 1988). Susceptible sheep are those that have wet wool due to sweat, rain or urine or have bacterial infections such as fleece rot or wounds. Blowfly strike in sheep can express itself in 6 different forms depending where on the sheep the strike develops i.e. crutch or breech strike, tail strike, body strike, head or poll strike, pizzle strike and wound strike.

The damage to the sheep caused by the feeding maggots results in significant losses in wool production and quality, reduced body weight, decreased fertility, death and increased costs associated with the control of the problem. Sheep can die from toxaemia associated with heavy infestations of blowfly larvae (Broadmeadow et al., 1984).

Curren management practices include mulsing and crutching (surgical procedures), jetting (pesticide use) and flock selection (culling) and breeding. Mulsing and crutching only prevent blowfly strike in particularly vulnerable areas of the sheep but do not prevent other forms of strike such as body strike. Further, these surgical procedures result in significant sheep losses due to infections such as tetanus.

They are also labour intensive and costly. Substantial problems exist with resistance developing to the 4 organophosphate pesticides (Oakeshott et al., 1990).

Australia (Whitten et al., 1980). Further, there are increasing consumer and grower concerns about the presence of trace levels of chemical residues present in sheep products.

Selection against animals with susceptibility to fleece rot, blowfly strike and excess skin wrinkle is also being used in current management practices.

There is little evidence for effective naturally acquired immunity gained in sheep against the blowfly larvae even after repeated infestations. However, there are some reports of weak acquired resistance in older sheep after repeated infestations (Watts et al., 1979; O'Donnell et al., 1980; Sandeman et al., 1985; Sandeman et al., 1986). There is some evidence, from largely in vitro feeding experiments, which demonstrated a reduction in the weight or number of larvae feeding"on sera from animals repeatedly infested with blowfly S larvae (Eisemann et al., 1990). Further, it was demonstrated that this effect could be mediated by isolated immunoglobulin from the serum of sheep repeatedly infested with blowfly larvae (Eisemann et al., 1990). These studies showed significant immunological cross-reactivity between sera from S these repeatedly infested sheep and proteins from the larval excretory and secretory material (Sandeman et al., 1986).

Skelly and Howells (1987) demonstrated that sera from infested sheep react with a range of proteins from different larval tissues. They showed immunological interactions between this sera and proteins from larval salivary glands, midguts and larval excretory and secretory material. Notably, a cuticle I it extract, which presumably may have contained crude peritrophic membrane material reacted very poorly with the sera from infested sheep. The effect on feeding larvae of sera from animals vaccinated with larval or adult L. cuprina cuticular components was not assessed. This widespread immunoreactivity of sera from infested sheep with different larval tissues may be questionable in view of the possibility of the development of antisera with carbohydrate cross-reactivity which could react with a number of unrelated proteins (Willadsen and McKenna, 1991).

In general, the principal larval molecules directly in contact with the ovine immune system are those in the excretory and secretory material from the larvae. This material is thought to promote inflammation and wound formation. Some component of the larval excretory/ secretory material may be responsible for the weak acquired immunity which largely expresses itself as hypersensitivity (Sandeman, I 1990). Bowles et al (1987) have exposed sheep in a variety of ways to the second instar larval excretory and secretory products. They demonstrated a significant reduction in feeding larval numbers when this crude material was exposed to sheep as an intranasal aerosol. Seaton et al (1991) have j concluded that this approach to vaccination depends on the specific immunization procedure.

Sn,' Analysis of this excretory and secretory material has demonstrated the presence of a number of active proteases which may interact with the ovine coagulation and inflammation cascades (Bowles et al., 1988; Bowles et al., 1990; Sandeman et al., 1990; Sandeman et al., 1991).

United Kingdom patent specification 2029222 (1980) describes a method for the prevention or reduction of fly strike in sheep. This patent describes the isolation of crude excretory secretory material from blowfly larvae cultures and the vaccination of sheep with this material. The individual effective components in this material were not identified. The material tested in vaccination trials was the soluble component. There was no assessment of the protective effect of the insoluble component.

An early attempt to vaccinate sheep against blowfly was referred to briefly by Mackerras (1936), who provided no details of the procedure used, but indicated that no success was achieved. O'Donnell et al (1981) demonstrated that sera from sheep injected with crude soluble components of homogenates from blowfly third instar larvae inhibited first oo instar larval growth in in vitro feeding experiments. There oo was no 4effect of the vaccination against implant challenge with first instar larvae. Recently, Eisemann et al (1991) and Johnston et al (1991) demonstrated a larval weight retarding activity in the sera of sheep vaccinated with extracts from o o° whole first or second instar larvae and from the guts of ao a second instar larvae. They showed that this activity resided S o in both the supernatant (or soluble component) as well as the 0 0 pellet or (insoluble component) of extracts from both the o 0whole larvae and from the guts of the larvae. The sera from S° sheep vaccinated with insoluble material derived from larvae or their guts have no effect on the growth of larvae on the sheep itself in an in vivo assay). Campbell and Howells (1991) showed inhibition of blowfly larvae feeding on sera p 7 from rabbits vaccinated with extracts from the midgut of first/second. instar larvae. However, there was no effect on feeding larvae in an in viva assay system.

IFry et al (1991) have produced a number of monoclonal antibodies against larval antigens from various extracts of the blowfly, L. cuprina. Many of these monoclonal antibodies showed cross-reaction with several larval antigens suggesting the presence of cross-reacting carbohydrate epitopes and not specific protein epitopes. The monoclonal antibodies were raised against midguts and detergent extracts from whole first instar larvae. Some of these monoclonal antibodies inhibit the growth of larvae in in vitro assays. However, the specificities of these antibodies have not been determined.

In addition to this pragmatic approach, specific proteins have been targeted for purification as they may have potential immuno-protective activity when injected into sheep. These proteins include dopa-decarboxylase and phenyloxidase (Barrett and Trevalle, 1989; Barrett, 1987; Campbell and Howells, 1991). However, in both cases there was no effect on larvae when they were fed on sera from sheep vaccinated with these enzymes.

In this latter approach, it is assumed that these potential protein targets are accessible to ingested ovine Santibody. Both of these enzymes are relatively inaccessible being in the haemolymph, brain or cuticle. There is a considerable barrier, called the peritrophic membrane, between the gut lumen of blowfly larvae and the underlying digestive cells and haemolymph. One study has shown that the peritrophic membrane of L. cuprina has pores of sufficient

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size to allow passage of immunoglobulin molecules into the haemolymph (Campbell and Howells, 1991). Evidence from other studies suggests that the pores of the peritrophic membrane are too small to allow substantial access of intact antibody molecules to the gut membrane. For exampe, dextrans larger than 32,000 Mr could not penetrate the peritrophic membrane of a variety of insects (Peters and Wiese, 1986). In addition, Eisemann et al (1992) have shown that only gold particles of size less than approximately 10 nm diameter pass through the peritrophic membrane of L. cuprina. Immunoglobulin molecules are approximately this size or larger and thus antibody access to the gut epithelial cells is likely to be severely restricted. Eisemann et al (1992) have measured the specific Ig concentration in the haemolymph of 3rd instar larvae. They demonstrated a marked decrease in this concentration compared with that present in an artificial agar-based medium containing 75% serum (on average a 10,000 fold reduction).

Thus, it is unlikely that sufficient Ig can penetrate into the haemolymph to target proteins in this or similar environments.

This study further demonstrates that the highest concentrations of immunoglobulin ingested by the larvae are found in the gut. This information suggests that the most feasible targets in the larvae for immunological attack are in 0 the gut.

There have been several attempts to vaccinate against insects, particularly mosquitos, using extracts derived directly from these insects (Hatfield, 1988; Ramasamy et al., 1988; Alger and Cabrera, 1972; Schlein and Lewis, 1976; Kaaya and Alemu, 1982; Ben-Yakir and Mumcuoglu, 1988; Sutherland and i 9 Ewen, 1974). In general, these vaccinations show only minimal effects in in vitro assay systems and have not been developed further. In addition, none of these studies identified the active component in the crude insect extracts. Ramasamy et al (1988) used mosquito head/thorax, midgut and abdomen to vaccinate rabbits. They showed a reduction in the fecundity of mosquitos feeding on these vaccinated rabbits. It has also been reported that mortality in Anopheles stephensi Liston increased when they were fed on rabbits immunized with mosquito midgut (Alger and Cabrera, 1972; Hatfield, 1988) and the fecundity of Aedes aegypti decreased when fed on rabbits immunized with whole fly extracts (Sutherland and Ewen, 1974).

Apart from vaccinating against mosquitos using mosquito extracts, there have also been attempts to vaccinate against various flies using tissue from these flies. 'chlein and Lewis (-1976) demonstrated the development of lesions and increased mortality in the haematophagous fly, Stomoxys calcitrans when these flies fed on rabbits immunized with tissue from this fly. In a similar manner, Khan et al (1960) reported that the injection of an extract of the first instar larvae of Hypoderma into infested warbled calves could stimulate a host immune reaction which killed the larvae. In both cases the individual protective agents in these crude extracts were not identified. Maget and Boulard (1970) demonstrated that a partially purified, soluble serine protease (hypoderma C) from Hypoderma, when injected into cattle, protected those cattle from Hypoderma infestations.

Recently, 3 serine proteases (hypoderma A, B and C) from tt 41

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r 1 Hypoderma lineatum have been identified, purified, cloned and expressed (Pruett et al., 1989). Two of these proteases have been shown to inactivate bovine complement C 3 and are postulated to facilitate the escape of Hypoderma infestations from the bovine host immune defence system. These soluble proteins are the essential components in a vaccine being developed to protect cattle against Hypoderma (Pruett et al., 1989).

The object of the present invention is to provide a vaccine which may be used to retard the growth of blowfly larvae when they feed on vaccinated sheep.

It is a further object of the invention to provide antigens for use in the abovementioned vaccine as well as methods for the production of the antigens.

The antigens in relation to the present invention are peritrophic membrane derived from Lucilia cuprina and related blowflies as described hereinafter or extracts derived therefrom which may be obtained from adult blowflies or more preferably blowfly larvae.

In this regard the gut of the blowfly larvae is bathed in ingested ovine immunoglobulin. The cells lining the gut are protected from abrasive action and bacterial attack by S °the tough cuticular membrane called the peritrophic membrane.

There are pores in this membrane which allow the passage of small molecules from the gut to the ectoperitrophic membrane space where gut epithelial cells take up digested material.

11 The peritrophic membrane has been found to possess the following characteristics: Composition: mainly chitin (poly N-acetyl glucosamine) and proteins.

Thickness: ranges from approximately 50 to 100 nm.

Structural characteristics: Only a single peritrophic membrane is present in larvae of L. cuprina although 3 may be found in adult flies. The peritrophic membrane extends from the cardia at the anterior end of the midgut through the midand hind guts, often protruding through the anus. Peritrophic membranes consist of a skeleton of chitin-containing microfibres embedded in a matrix of other material. These fibres may be arranged regularly or in a random "feltwork".

In transmission electron micrographs, 2 electron-dense layers, separated by a very narrow zone of less dense material, occur at the face in contact with the gut lumen contents; the inner electron-dense layer is the more strongly marked. A more diffuse and irregular electron-dense layer is usually apparent on the opposite face of the peritrophic membrane, nearest the gut cells. In first instar larvae, the relative width of the 2 electron-dense layers on the gut lumen side and their separating electron-lucent layer is greater (30-40% of the *;whole) than in later instars (10-15%).

SThe peritrophic membrane if desired may be obtained from blowflies or blowfly larvae by dissection methods but this is very labour intensive and may not be a commercial process. It is therefore preferred that the peritrophic membrane be obtained by in vitro production in larval culture. This lI I I pr; r: 12A inhibiting flystrike in sheep by vaccination of sheep with whole intact peritrophic membrane or parts thereof or extracts obtained therefrom which may retard the growth of larvae feeding on the vaccinated sheep. The vaccine would contain in addition to peritrophic membrane a suitable o 0 0 040, *000 00 00 0* *a 0 0 o e S o* 0 4 1 0044 O 1 4 ~fi 12 method therefore comprises a FIRST ASPECT OF THIS INVENTION which includes the following steps: obtaining blowfly larvae from blowfly eggs which are preferably maintained in vitro; (ii) propagating the larvae in vitro; and (iii)obtaining peritrophic membrane from the propagated larvae as it is continually synthesised and shed as it passes out of the anus.

In step blowflies may be obtained from flystruck sheep and eggs collected from the blowflies as they are maintained in artificial culture. The eggs may be incubated for a suitable period before being placed in suitable laboratory growth media which may contain serum and preparations derived from yeast. After addition of an o appropriate protease inhibitor, the resulting larvae produced from the eggs are then sieved or filtered in step (ii) to 0 S" produce the peritrophic membrane which may subsequently be subjected to further purification such as centrifugation.

In another alternative method peritrophic membrane may be obtained from blowfly tissue or cells derived from blowfly tissue, said method including the step of growing Sblowfly tissue or cardia in artificial culture and obtaining the peritrophic membrane from the resulting tissue or cells by filtration or centrifugation.

's' t In a SECOND ASPECT OF THE INVENTION the peritrophic membrane may be injected into sheep which may provide relatively high levels of circulating antibody. Thus, it is within the scope of the invention to provide a method of It 13 r.

Ii commercially acceptable adjuvant and could be administered by intamuscular. injections, subcutaneous injections, intraperitoneal injections or infusion techniques.

It has also been found that isolated immunoglobulin from the sera of sheep vaccinated with peritrophic membran'e (or extracts derived therefrom) also retards the growth of feeding larvae thereby showing that such retardation is mediated by antibody.

In a THIRD ASPECT OF THE INVENTION peritrophic membrane may be dissolved in suitable solubilising agents so as to solubilize components of the peritrophic membrane which may be responsible for retardation of larval growth. Suitable solubilizing agents include detergents or strong disassociation agents.

Thus, the invention also includes within its scope, vaccination with extracts of peritrophic membrane which also have been found to produce relatively high levels of circulating antibody causing retardation of the growth of larvae feeding on vaccinated sheep.

In this aspect of the invention the peritrophic membrane may be extracted with strong ionic detergents which suitably include (but are not limited to) CHAPS, CHAPSO, CTAB, sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, Zwittergent 3-08, Zwittergent 3-10, Zwittergent 2-12, Zwittergent 3-14 and Zwittergent 3-16. The useful concentration of these detergents will typically vary between 0.1 and However, concentrations outside this range also may be effective.

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14 It also will be appreciated that the peritrophic membrane may be extrapted with disassociation agents which suitably are such that while effective in the solubilization of peritrophic membrane-bound proteins, do not chemically alter the structure *of these proteins, nor render such proteins in a useless state in a state which does not cause the production of antisera in vaccinated sheep capable of retarding the growth of larvae). Generally, these agents disrupt protein water structure, hydrogen bonds or ionic interactions which bind proteins to the peritrophic membrane. Examples of this class of agents include, but are not limited by, high concentrations of urea and guanidine hydrochloride. These dissociation agents can be used alone or in conjunction with detergents, reducing agents, heat or alkaline and acid conditions. These examples represent general procedures for the extraction of peritrophic membrane-bound proteins but are in no way limiting.

Examples of powerful disassociation agents which may be a used for the extraction of 3 useful antigens of the invention (referred to hereinafter as PM44, PM90 and PM95) include 4 M o urea or 4 M guanidine hydrochloride.

In accordance with the invention, initial extraction of S the peritrophic membrane with a strong ionic detergent Sfollowed by extraction with a strong disassociation agent is not absolutely necessary for the solubilization of the effective antigenic fraction. Whole unextracted peritrophic membrane and peritrophic membrane directly extracted with urea both yield solubilized fractions which are effective in vaccination trials. The advantage of an initial detergent extraction is the removal of some proteins thereby resulting in a much simpler pattern of proteins in the subsequent urea extract. To this end, even non-ionic detergents (which are weaker in their solubilization abilities) may at least partially fulfil this role although the protein pattern extracted subsequently from the peritrophic membrane with urea would be more complex.

In a FOURTH ASPECT OF THE INVENTION individual antigens can be isolated and purified from peritrophic membrane. These antigens can then be injected into sheep either individually or in combination and thereby induce an immune response which retards the growth of feeding blowfly larvae. The type of adjuvant and route of administration to sheep are described in the Second Aspect of the Invention.

In a FIFTH ASPECT OF THE INVENTION a method is provided for the artificial production of the effective antigens as recombinant proteins produced in bacteria or other cellular expression systems. Both the glycosylated and nonglycosylated forms of these effective antigens may be used as immunogens.

Homologues of the effective antigens either from the same or different insect species may also be used. The recombinant proteins can be produced in bacterial, fungal or eucaryotic expression systems. Suitable adjuvants and routes for administration into sheep are described in the Second Aspect of the Invention. In addition, it is possible to deliver these effective antigens by live viral delivery systems.

In a SIXTH ASPECT OF THE INVENTION synthetic peptide vaccines based on the regions within the effective antigens or carbohydrate antigens from whatever source may be used as o GG Ilt Ii G t o Of

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rrrrr)ir S I t I 14 tIi i I L 4 4 t a 1 i \i i rt Ea 16 immunogens in a vaccine. These antigens will induce an immune response upon injection into a sheep or other animal which is capable of recognising the effective antigens.

In a SEVENTH ASPECT OF THE INVENTION the effective components in the vaccine could comprise at least one antiidiotypic antibody capable of inducing a protective immune response by mimicking the effective antigens.

The blowflies to which the present invention is applicable include not only Lucilia cuprina but also related flies causing myasis in their hosts. These flies are characterised by the ability of their larval stages to parasitise their hosts (typically cattle or sheep) by feeding on host tissue or tissue fluid. These flies are generally related and belong to the family Calliphoridae and subfamilies Calliphorinae and Chrysominae. While not exclusive the following list represents the range of flies applicable to this invention: Lucilia curpina, Lucilia sericata, Calliphora auger, Calliphora stygia, Calliphora nociva, Calliphora albifrontalis (also called Calliphora australis or Calliphora maryfulleri), Calliphora hilli, Calliphora vicinia (also called Calliphora erythrocephala), Chrysomya rufifacies, Chrysomya varipes, Chrysomya bezziana, Chrysomya albiceps and Cochliomyia hominivorax. However, it is recognised that the vaccine or variations thereof based on the same or similar effective antigens, may have general application for the control of Dipteran insects or their larvae which have a similar peritrophic membrane structure to L. cuprina and which feed on the tissue or tissue fluids of vertebrate hosts.

17 The invention is further described in the following examples which are illustrative of the invention but in no way limiting on its scope.

TRADE NAMES Zwittergent 3-14 Tris Centricon Aquapore RP-300 C-8 Rota vac Superose 12 SUPPLI ER Calbiochem sigma Ini con Waters Savant Pharmacia 00140C 4 4 4444 *994 0* 00 C 40 .4 04 is C Ca *4 C 4 40 C ''Cc.

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ABBREVIATIONS

AMP

BSA

CHAPS.

CHAP SO

CTAB

DIG

dNTP

DTT

EDTA

Endo Lys C Endo Glu C Amp icil1lin Bovine serum albumin (3-cholamidopropyl) dimethylaimmonio]-l -propane-sulfonate (3-cholamidopropyl) dimethylanunonio] -2 -hydroxy-l-propane-sulfonate Cetyltrimethylammonium. bromide Digoxigenin Deoxynucleotide triphosphate Dithiothreitol Ethylenediamine tetra acetic acid Endoproteinase Lys C Endoproteinase Glu C k 18 Elisa Enzyme-linked immunoabsorbtion assay HPLC High performance liquid chromatography IPTG Isopropylthiogalactoside

PBS

PCR

PTH-amino acid

SDS

SDS-PAGE

Phosphate-buffered saline Polymerase chain reaction Phenylthiohydantoin Sodium dodecylsulfate Sodium dodecylsulfate polyacrylamide gel electrophoresis Tris (hydroxymethyl) aminomethane Tris-buffered saline 5-bromo-4-chloro-3-indolyl-B-D-galactoside Tris

TBS

X-Gal I o o 0* 0 o0 S o o 0o o f oe 1$ 0 0 0 r

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*rr 0 0000 00* 0 01 MATERIALS AND GENERAL METHODS Unless otherwise mentioned, laboratory chemicals were of analytical grade and purchased from the Sigma Chemical Company (St. Louis, Acids and solvents were analytical 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 Boehringer-Mannheim (North Ryde, Australia).

Biotinylated lectins were obtained from Vector Laboratories Inc (Burlingham, CA, USA).

Sodium Dodecyl Sulphate Gel Electrophoresis (SDS-PAGE): All protein samples were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis on 6-18% gradient gels.

All gels included molecular weight standards (Pharmacia).

'4 i Fi 3.9 Gels were silver-stained using the method of Morrissey (1981).

Glycoproteins were detected using biotinylated lectins to probe proteins separated by SDS-PAGE and transferred by electroblotting onto nitrocellulose. Briefly, proteins were separated by SDS-PAGE and then electroblotted onto nitrocellulose using an LKB semi-dry NOVA blotter (used according to the manufacturer's instructions). The nitrocellulose was blocked for 1 h at 37 0 C in TBS containing gelatin, incubated with a 1/500 dilution of the appropriate biotinylated lectin for 1 h at 37 0 C, washed (x3) in TBS containing 0.1% Tween 20, incubated with a 1/1000 dilution of streptavidin-peroxidase (Amersham) for 30 minutes at room temperature and then developed with chloronapthol/H 2 0 2 substrate according to the manufacturer's instructions.

S. Immunofluorescence: Post-vaccination sera from sheep vaccinated with the purified peritrophic membrane proteins o PM44, PM90 or PM95 and from control sheep were diluted 1:2000 in phoshate-buffered saline, pH 7.3 (PBS). Pieces of peritrophic membrane collected from larval culture as o described below (Example 1) and stored at -1000C and lengths of peritrophic membrane freshly dissected from the midguts of S'3rd instar larvae of L. cuprina fed on larval rearing medium ,(Singh and Jerram, 1976) were incubated overnight at 70C in each of the diluted sera. After 4 washes in PBS, the peritrophic membrane was incubated in a 1:25 dilution of fluoroscein isothiocyanate-labelled rabbit anti-sheep Ig serum in PBS for 2 h at 24°C. After 2 further washes in PBS, the L'i samples of peritrophic membrane were mounted on slides, examined in a fluorescence microscope and photographed.

Sheep: Experimental animals were 6-12 months 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.

Vaccination: In general, each sheep was injected with the material extracted from 2.5 g of peritrophic membrane.

The peritrophic membrane extracts were each homogenised with an equal volume of Freund's coiiplete adjuvant. The first injection was intramuscular, given half into each rear leg.

The second injection used Freund's incomplete adjuvant and was intramuscular, given in the neck 28 days later. All animals were bled from the jugular vein prior to each injection. 14 o days after the second injection, the effect of vaccination was assessed by in vitro or in vivo larval growth assays (Eisemann 0 et al., 1989; Eisemann et al., 1990). The in vitro assay consisted of allowing 1st instar larvae to feed on an agarbased medium containing 75% serum from vaccinated animals.

S The in vivo assay consisted of feeding larvae directly on a St.. restricted area* on the back of sheep. In both assays the number of surviving larvae and their weights were measured.

There were 3-6 animals in each group. Antibody titres were assessed by Elisa as described by Eisemann et al (1990).

Protein concentration determinations were made using the Pierce BCA kit with BSA as a standard.

r -a i 21 Establishment of a L. cuprina colony: Laboratory populations of L. cuprina, which had originated from flystruck sheep, were maintained on an artificial medium (Singh and 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. cuprina for 4-5 h. The eggs were then incub .,ed overnight at 16 0 C and. 100% relative humidity before surface sterilisation.

Molecular biology Many of the standard procedures used for the cloning, sequencing and expression of PM44 are described by Sambrook et al (1989). Other methods are described in the text.

EXAMPLES

Preferred procedure for the isolation of protective antigens from the larvae of Lucilia cuprina Example 1: In vitro production of peritrophic membrane in Slarval culture An L. cuprina eggs were washed in 1% sodium hypochlorite for minutes at room temperature to separate and surface sterilise the eggs. During this time, the egg clumps were gently dispersed. Approximately 7000 eggs were placed into a flask containing 250 ml of PBS containing 40% newborn calf serum, 0.2 mg/ml gentamicin and 2% yeastolate. The growth medium was absorbed into standard kitchen sponges placed in iu Fr I i i I 22 the flask. 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 a 850 pm sieve. The larvae were returned to the flask and 30 ml of PBS containing 2.5.mM benzamidine and 5 mM EDTA (protease inhibitor buffer) were added. The latter two reagents were added to inhibit proteases secreted by the larvae. The flasks were then incubated for a further 24 h at 37 0 C. During this time, the peritrophic membrane in each larva is continually synthesised and shed as it passes out the anus. The flask contents were then sieved to remove the larvae and the filtrate centrifuged at 10,000xg for minutes at 4 0 C. The pellet was resuspended in inhibitorbuffer and recentrifuged. The final pellet was stored at 100 0 C. This technique for producing peritrophic membrane yielded a mean of approximately 40 mg of peritrophic membrane per 1000 larvae. Examination by light microscopy showed that the peritrophic membrane was the major component with a minor S" quantity of cast-off larval cuticle.

The in vitro culture of whole larvae of L. cuprina allows the collection of gram quantities of peritrophic membrane with minimal manipulation (production of greater than 10 grams per week). Previously, it has been possible to grow small amounts of peritrophic membrane from adults of the blowfly, Calliphora erythrocephala by organ culture of isolated cardiae (Becker et 1975). The procedure described above for the production of peritrophic membrane from L. cuprina is the first report of 23 production of sufficient material to allow vaccination trials to be carried out in sheep.

In immunofluorescence assays, the antisera produced by vaccination with peritrophic membrane shed from cultured larvae reacted equally well with peritrophic membrane collected from cultures or freshly dissected peritrophic membrane. (There was little or no immunofluorescence associated with peritrophic membrane incubated with control sera.) This, and the effect of vaccination on live larvae strongly suggests that the two sources of peritrophic membrane are antigenically equivalent.

Example 2: Demonstration of retardation of growth of L.

cuprina larvae feeding on sera from sheep vaccinated with whole peritrophic membrane Peritrophic membrane was cultured as described in Example 1. The' peritrophic membrane was washed in PBS and the insoluble peritrophic membrane material was injected into sheep as described above. The immunization resulted in high Slevels of circulating antibodies (as measured by Elisa).

Larvae were either fed directly on these vaccinated animals for 20 h or 50 h or were fed for 20 h on sera derived from S these vaccinated animals. In addition to the sera from the control animals, the in vitro assay was also performed with prevaccination sera as another control. There were no differences between the mean larval weights determined by :'.*feeding larvae on the sera from control sheep or prevaccination sera. The average weights of the larvae for 4 independent experiments are shown in Table 1. Overall, there 24 was an average decrease in larval weight compared to controls of 30±8% for the 20 h in vivo assay, 18±9% for the 50 h in vivo assay and 32±3% for the 20 h in vitro assay. For the in vivo assay the effect at 50h was always less than at 20 h.

Generally, the in vitro assay gave a greater percentage decrease in larval weight than the in vivo assays. The conclusion from this experiment is that vaccination of sheep with whole peritrophic membrane induces an immunological response in the sheep which is capable of retarding the growth of feeding larvae.

Example 3: Enhanced retardation of the growth of larvae of L.

cuprina when feeding on sheep serum with increased concentrations of Ig from immunised animals.

Immunoglobulin (Ig) was isolated using method E of SMostratos and Beswick (1969), from post-vaccination sera of two sheep which had been vaccinated with whole peritrophic membrane (and which responded strongly to vaccination as measured by reduction in larval growth in the in vitro assay) Sand also from pooled sera of 12 control sheep which had been injected with adjuvant only. The percentage yields of Ig obtained from the sera were estimated using Elisa. With the S' anti-peritrophic membrane sera, this was performed on a range of dilutions of both the original sera and of the isolated Ig using procedures described previously (Eisemann et al., 1990).

An antigen capture Elisa was performed for the. control sera and the isolated Ig. 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 Ln 1 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 Ig samples were concentrated by vacuum dialysis against PBS. Aliquots of each Ig solution were added to 4 ml of pooled control 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 in the in vitro assays described above. The results of an in vitro experiment using these antibody-enriched sera are shown in figure 1. The results of this experiment show a greater :2 retardation of larval growth when larvae feed on immune sera enriched with Ig from animals vaccinated with peritrophic membrane. For example, the retardation for sheep #2 changed S from 71% of controls at IxIg to 92% of controls at 4xIg. The retardation in the growth of larvae feeding on these vaccinated animals is therefore mediated by antibody and the S: extent of the effect depends on the concentration of relevant S antibody in the serum.

Example 4: Vaccination of sheep with detergent extracts from the peritrophic membrane.

Peritrophic membrane was extracted with two strong ionic detergents, 4% SDS (also containing 10% 2-mercaptoethanol) and 26 CTAB in TBS) in an attempt to solubilize the components of the peritrophic membrane responsible for the retardation of larval growth described in Example 2. Immunisation with peritrophic membrane detergent extracts resulted in high levels of circulating antibodies in sheep (as shown by Elisa).

The results of the vaccination trial are summarized in Table 2. Both detergent soluble extracts of the peritrophic membrane caused inhibition of larval growth in the in vitro and in vivo assays. These results indicate that both of these detergents solubilized at least some fraction of the effective components from the peritrophic membrane. However, a greater retardation in larval growth was generally observed with the detergent-insoluble fraction. For example the CTAB-insoluble fraction caused a 56% reduction in larval growth after hours feeding of larvae on sheep. These detergent insoluble peritrophic membrane fractions had strong effects in both the in vivo'and in vitro assays.

Even after extraction with these ionic detergents, some of the most effective antigens were still located in the insoluble material. It was clear that strong ionic detergents were not capable of full solubilization of all of the effective antigens from the peritrophic membrane. Strong disassociation agents were therefore required for the full extraction of effective antigens.

Example 5: Demonstration of the further solubilization of effective antigens from the peritrophic membrane: successive extraction of peritrophic membrane proteins .4 ,3 27 The peritrophic membrane was subjected to a series of successively more severe extractions which were then injected individually into sheep. Each extraction step involved suspending the peritrophic membrane in a specific extraction solution (2 ml per gram of peritrophic membrane), homogenizing with an Ultra-turrax type 18\10 blender (Janke and Kunkel, Germany) and allowing the homogenate to stand for 2-16 h at 4°C. The homogenate was then centrifuged (50,000 x g, 40C, min.) and the supernatant (or extracted component) retained while the pellet was further processed. The peritrophic membrane was initially washed in distilled water and then extracted in 0.1 M Tris/HC1 pH 7.5 containing 150 mM NaCl, mM EDTA (TBS) and 5 mM benzamidine for 1 h and then the remaining pellet was successively extracted with TBS containing 2% Zwittergent 3-14 (2 TBS containing 4 M Urea (6 TBS containing 6 M Guanidine HCl (12 h) and Sfinally- with 0.1 M HCl (12 Each extract was dialysed H against PBS (3 x 3 litres). This dialysis was necessary to S.remove disassociation agents before the samples were injected S into sheep.

Each of these extraction solutions solubilized a different group of proteins from the peritrophic membrane as was shown in a silver-stained SDS-PAGE gradient gel.

Immunization of sheep with these fractions resulted in high levels of circulating antibodies with all groups having broadly similar titres (as measured by Elisa). Immunization with the fractions extracted with Zwittergent 3-14, urea or guanidine HCL were effective in inhibiting larval growth (Table The final pellet and the HCl-soluble material had Lu i .1 28 no significant effect when assessed in the bioassays (data not shown). This result indicated that the effective antigens from the peritrophic membrane had been either completely extracted by prior procedures or that it had been inactivated by the extraction process. The 50 h in vivo results showed strongest activities with the detergent and urea extracts.

When larvae were grown on sera in vitro, the antisera from sheep vaccinated with the urea-soluble fraction reduced larval growth by 50% (p<0.01) and the sera from sheep vaccinated with the Zwittergent 3-14-soluble fraction reduced growth by 43% (p<0.05).

Two further experiments using an identical protocol confirmed that the material extracted from peritrophic membrane by 4 M urea or 2% Zwittergent 3-14 induced immune responses in sheep that caused reduced growth of larvae grown in vitro and in vivo (results not shown).

Example 6: Demonstration that sera from sheep vaccinated with a purified 44,000 Mr protein "(PM44) from the peritrophic membrane of L. cuprina retard the growth of larvae feeding on that serum The 4 M urea extract of peritrophic membrane described in Example 5 was further fractionated by gel permeation chromatography. A Superose 12 column (1.6 cm diameter, 50 cm length) was equilibrated with 50 mM Tris-HCl, pH containing 6 M urea. The 4 M urea extract from the peritrophic membrane was concentrated on a Centricon concentrating cell (Amicon) and then applied to this column running at a flow rate of 0.5 ml/min at room temperature.

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I I 1*4 K I *1A AI K c- rra~--n~aLI-Foeah 29 Fractions were collected and pooled into 5 groups (Table 4).

The relative-molecular weight of the proteins in each of these pools was analysed by SDS-PAGE under reducing conditions and the results are also listed in Table 4. Pool D contained a single protein of Mr=44,000. This protein has been called PM44.

Each of these pools was used to immunize sheep. Larvae were then fed on sera from these vaccinated sheep (in vitro assay). There was a strong antibody response as measured by Elisa to each of these pools except pool E which contained little or no protein. Table 5 shows the reduction in larval weight when the larvae were fed on the sera from these vaccinated animals for two independent vaccination trials.

There was significant larval growth retardation when larvae fed on sera from animals vaccinated with all of the pools except pool E. Pool D, in both vaccination trials, gave approximately 40% retardation in larval growth compared with controls. This pool contains only one protein, PM44 which was a also found in pool C.

44 Example 7: Demonstration that sera from sheep vaccinated S with the purified proteins of Mr 90,000 (PM90) or 95,000 (PM95) from the peritrophic membrane of L. cuprina retard the growth of larvae feeding on that sera The 4 M urea extract of peritrophic membrane described in Example 5 was concentrated in a Centricon 30K cell (Amicon) and fractionated by preparative SDS-PAGE 'Model 491 Prep Cell; Biorad) on a 10% acrylamide gel. This procedure was carried out according to the manufacturer's instructions. Fractions Ii ci collected from this procedure were subjected to analytical SDS-PAGE and proteins stained with silver. Fractions containing proteins of Mr=90,000 (PM90) and Mr=95,000 were independently pooled, concentrated in a Centricon cell and then used to vaccinate sheep as described for PM44 (see Example The weights of larvae feeding on the sera from these vaccinated and control sheep (in vitro assay) were measured and are shown in Table 6. Both PM90 and PM95 induced a strong immune response in sheep as measured by Elisa. The sera from sheep vaccinated with either of these proteins was effective in retarding the growth of feeding larvae i.e. there were means of 61.6% and 57.5% larval weight reductions for and PM95 respectively.

It is recognised that the proteins PM44, PM90 and could be purified from peritrophic membrane by a number of biochemical methods including, but not limited by, preparative SDS-PAGE, preparative isoelectric focussing, gel filtration, e" ion exchange chromatography or affinity chromatography on chitin.

Example 8: Demonstration that many of the proteins isolated from the peritrophic membrane and in particular PM44, PM90 and 04 4" PM95 are glycoproteins.

The presence and type of glycosylation present on proteins solubilized from the peritrophic membrane by 4 M urea in TBS were assessed by probing the proteins with biotinylated lectins. Purified PM44 and other proteins solubilized from peritrophic membrane were separated by non-reducing SDS-PAGE and electroblotted onto nitrocellulose according to standard 31 procedures. 10 jg/lane of PM44 and 80 /Ig/lane of the ureaextracted proteins were loaded onto each lane of the polyacrylamide gel. The nitrocellulose was then incubated with one of the biotinylated lectins and reacting proteins visualised with streptavidin-peroxidase and a chloronaphthol/H 2 0 2 substrate. Table 7 lists the relative molecular weights of the reacting glycoproteins.

Most of the proteins from the urea extract of peritrophic membrane react with biotinylated lens culinaris lectin. In particular, purified PM44 and PM90 and PM95 have specific reactivities with this lectin. This reactivity was demonstrated to be specific by showing a complete lack of reactivity of this biotinylated lectin with PM44 when the lectin was preincubated with 100 mg/ml of methylmannose (results not shown). The individual glycoprotein relative molecular weights shown in Table 7 do not correspond exactly S, with particular proteins identified in Table 4. This result is a reflection of the use of reducing conditions for the separation of proteins in the experiment described in Table 4 and non-reducing conditions for the protein separations reported in Table 7. There are also minor differences in the i relative molecular weights of individual proteins measured on .silver-stained gels and cn the nitrocellulose.

Example 9: Localization of PM44, PM90 and PM95 in L. cuprina Antisera from sheep vaccinated with either PM44, PM90 or PM95 were used to localize these proteins to peritrophic membrane by immunofluorescence techniques. Peritrophic membrane was obtained by both larval culture (Example 1) or by 32 fresh dissection of third instar L. cuprina larvae. Prevaccination .serum did not react with either of these peritrophic membrane preparations (Fig. Sera from animals vaccinated with either PM44 or PM90 or PM95 gave essentially the same result i.e. strong fluorescence labelling of both dissected and cultured peritrophic membrane. These results are exemplified by the localization of PM44 to freshly dissected peritrophic membrane as shown in figure It is concluded from these results that all three of these proteins PM90 and PM44) are present on peritrophic membrane obtained by either larval culture or by fresh dissection of larvae.

Antiserum to PM44 also reacted with the peritrophic membrane from Haematobia exigua irritans suggesting the presence of a similar immunoreactive antigen in this blood feeding fly.

Example 10: The production, purification and amino acid i a sequences of peptides from PM44.

PM44 and other identified proteinr such as PM90 and can be obtained from peritrophic membrane by larval culture.

a However, this method of production of peritrophic membrane and a. its effective antigens may not be commercially viable.

S Sufficient quantities of these effective antigens can be produced artificially in bacteria. This can be achieved by the expression of the genes coding for these peritrophic membrane proteins in bacteria or other suitable cell expression systems, using recombinant DNA technology. To this end, peptides from PM44 have been isolated and their 33 amino acid sequences determined as a first step in the process for making .recombinant antigens. The general methods described below for the cloning of the gene coding for PM44 and its expression as a recombinant protein are also an applicable strategy for the production of recombinant PM90 and proteins given relevant peptide amino acid sequences from each of these proteins. PM44 (300 Ag), purified as described in Example 6, was mixed with 100 p1 of 0.1 M Tris/HCl, pH 8.3 containing 20 mM dithiothreitol and 2% SDS and then incubated for 30 minutes at 56 0 C. The solution was cooled to room temperature and sodium iodoacetate added to a final concentration of 0.14 M. After 45 minutes in the dark, cold methanol was added in a ratio of 9:1 methan6l/sample The sample was stored at 20 0 C overnight, centrifuged, the supernatant removed and the pellet dried.

The pellet was dissolved in 76 pl of 0.1 M Tris-chloride buffer containing 4 M urea, pH 8.5 and then 4 pl of Endo Lys C (6 units/ml) or 4 Al of Endo Glu C (6 units/ml) were added.

After 2 hr at 37°C, another 4 Al of the appropriate protease Swas added and the digestion continued for a further 17 hrs.

The digest of PM44 was applied directly to an Aquapore S. RP-300 C-8 column in 0.1% trifluoroacetic acid and peptides Swere eluted in a linear gradient from 0-60% (v/v) acetonitrile/water in 0.1% trifluoroacetic acid by HPLC. If necessary, peptides were rechromatographed in the same solvent system using an Aquapore C-18 column. Peptides were collected, concentrated to 50-100 Al by rotary desiccation in a Rota Vac. The amino acid sequences cf the peptides were determined using an Applied Biosystems amino acid sequencer.

j The following peptide amino acid sequences were obtained for PM44 (Table The one letter code for amino acids has been used.

Some of the glutamate residue assignments in the peptides may be carboxy-methyl cysteine residues because of the coelution: of each of these PTH-derivitised amino acids in the HPLC system being used for the separation of the PTH amino acids. Peptides PM215, PM1702 and PM1501 were sequenced at very low levels (<20 pmoles) and therefore there is some doubt about the confidence of the sequences of these peptides. The amino acid sequences of each of these peptides have been used to search the NBRF and Swiss amino acid sequence databases.

No significant homologies were found.

Example 11: Molecular cloning of the gene coding for PM44 Oligonucleotide Synthesis The amino acid sequences described in Example 10 (Table 8) were used to design suitable oligonucleotide probes that could be used in the polymerase chain reaction (Saiki et al., 1988) to amplify DNA fragments of the PM44 gene. Degenerate oligonucleotide primers were designed and synthesised on a Pharmacia LKB Gene Assembler Plus oligonucleotide synthesiser. The following primers were synthesised for use in the PCR reaction sense primer (based on peptide PM30022) a G C G C T G C CCAGATGGATTTATCGCAGATCC 3' T T A T C C C PM2704) G G G G G G CCACCATAATTATAAGCCATACC 3' ST T T C C C Preparation of genomic DNA from L. cuprina Kr Genomic DNA was prepared from first instar larvae of L.

Scuprina essentially as described by Henry et al (1990). The starting material was 2 g of first instar larvae which was i ground to a fine powder using a freezer mill .'pex j Industries). Nuclei were not isolated. Instead SDS wps added directly to the lysed cell suspension. The lysis buffer volumes) consisted of 0.2 M EDTA (pH 100 pg/ml proteinase K and 0.5% sarkosyl. The resulting suspension was heated to 55°C for 3 h with gentle mixing every 15 minutes after which it was centrifuged it 6000xg for 10 minutes (4 0

C)

to remove insoluble material. The supernatant was extracted with an equal volume of phenol/chloroform (1:1 The organic phases were separated by centrifugation (6000xg) for 10 minutes at 4 0 C. The upper aqueous phase containing the DNA i i i was precipitated by ethanol (2 volumes). The genomic DNA was then isolated from the solution and washed (x3) in 70% ethanol (centrifugation conditions 20,000xg, 30 min., The pellet was dried and then resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to a concentration of 1 mg/ml. The DNA was treated with DNAase-free RNAase (100 Ag/ml) at 37°C for 3 h. The DNA was then extracted with phenol/chloroform and dialysed extensively against TE buffer.

Preparation of a L. cuprina genomic library.

High molecular weight genomic DNA was digested with Sau 3a in order -to produce DNA fragments ranging from 15-25 kb in si:ze. This size range of fragments was purified by centrifugation through a 5-25% NaCl gradient using standard protocols (Sambrook et al., 1989). Purified Sau 3a fragments (15-25 kb) were ligated to lambda gem-11 arms (digested with Bamhl and treated with calf intestinal phosphatase; Promega) and packaged in vitro using standard procedures (Sambrook, et al., 1989). Packaged phage were then titred using E. coli LE392 as host.

Preparation of first instar cDNA g of first instar larvae were frozer at -80°C and pulverized at -170 0 C to a fine powder using a freezer mill.

Total RNA was isolated by the guanidinium thiocyanate method of Chomczynski and Sacchi (1987). PolyA RNA was isolated according to standard procedures using oligo dT-cellulose chromatography (Sambrook et al., 1989). cDNA was prepared from PolyA+ RNA using a commercially available kit (Amersham cDNA synthesis system plus). 3 jg of cDNA was synthesised from 10 jg of poly A+ RNA.

Preparation of a probe for screening the genomic library A PM44-specific double stranded DNA probe was prepared using the polymerase chain reaction (PCR). The procedure was based on that describe by Saiki et al (1988) and used a recombinant form of Taq DNA polymerase obtained from Perkin Elmer Cetus (Amplitaq). The PCR was performed on genomic DNA that had been purified as described in section above. The reaction mixture contained 1 yg genomic DNA or 10 ng cDNA, 1 cc 003 *00 4944 4 .4 4, 4 0 .~a 040 0 :Ci I L- l-:i u a rrC

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3 7 I M of each of the oligonucleotide primers listed in section 500 LM .of each dNTP, 2.5 mM MgCl 2 0.5 L1 of Taq DNA polymerase and 1 l of perfect match enhancer (Stratagene; 1 unic/reaction) in 100 kl. Amplification took place over cy les, each of which consisted of denaturation for 150 s at 94 0 C, annealing for 150 s at 59 0 C and extension for 150 s at 72°C. Appropriate controls, as described by Saiki et al (1988), were also included.

Samples of the PCR reaction were analysed on 1% agarose gels at the end of the reactions. A unique band of 860 bp was seen when either genomic DNA or cDNA was used (Fig. 3) suggesting the absence of introns in this fragment of the genomic DNA. A band also of 860 bp was obtained in the PCR reaction with DNA isolated from the screw worm fly, Chrysomya bezzianna suggesting the presence of PM44 in the larvae of this fly and the potential for its control using a vaccine based on homologous antigens to those isolated from L.

cuprina. The L. cuprina PCR product (500 ng) was isolated I from 3.0% low melting agarose according to the method described by Ericson (1990) and treated with T4-DNA polymerase min. at 37 C; 6 units T4-DNA polymerase in 50 mM Tris-HCl, S"pH 8.3, 50 mM NaCl, 10 mM MgC12, 10 mM DTT, 0.1 mg/ml BSA, I 0.25 mM of each dNTP. The blunt-ended PCR fragment (500 ng) was precipitated with ethanol, resuspended in 7 pl H20 and ligated in 10 il total volume with 100 ng pSK II plasmid vector (Stratagene pBluescript II) which had been linearized with the restriction enzyme Eco R1. The ligation reaction was carried out overnight at 14 0 C. The buffer used in this 38 ligation reaction was a ligation buffer supplied by Boehringer.

Competent E. coli SURE strain (Stratagene) cells were produced using the procedure described by Inove et al (1990) and were transformed with 5 g1 of ligation mix and AMPr colonies were selected on X-gal/IPTG/AMP plates using standard procedures (Sambrook et al., 1989). White colonies were isolated from these selection plates and plasmid DNA isolated by a standard "miniprep" method (Sambrook et al., 1989). DNA sequence analysis was carried out using a commercially available kit (USB Sequenase version Sequencing primers T 3 and T 7 homologous to sequences in the vector, were used in sequencing reactions according.. to the manufacturer's instructions. The remaining sequence of the original PCR DNA fragment was obtained using "internal primers" #2045 (sense) and #2046 (reverse sense) positioned within the original PCR DNA fragment. These "internal primers" were based on the DNA sequences obtained from analysis using the T 3 or T 7 primers.

t primer# 2045 5' ACC CAT ACA AGT TTG GCC ATC AGG 3' primer# 2046 5' TTG CTA TTG TTC TAA TAA AAA AGC 3' The DNA and deduced amino acid sequence of the PCR amplified S: o <DNA is shown in figure 4.

I There was only one open reading frame throughout this sequence which coded for 286 amino acids. There was no indication that the 3' end of coding region had been sequenced (lack of a stop codon or a poly A tail). Likewise, it is i 1 unlikely that the 5' end of the coding region had been sequenced because of the lack of an initiating methionine residue or a predicted signal sequence. The deduced amino r 39 acid sequence did confirm regions of sequences of peptides PM30022 and PM2704 which were determined by direct amino acid sequencing of the protein and which were not used in the design of the original PCR oligonucleotide primers. Further, the deduced amino acid sequence identified the position of peptides PM4401, PM1001, PM4408, PM4404 and possibly PM1501.

The latter sequence was determined at very low levels and therefore the sequence is in considerable doubt. There are 2 consensus sequences Gavel and von Heijne, 1990) for N-linked glycosylation. The most striking feature of the deduced amino sequence is the abundance of cysteine residues.

Proteins homologous to this sequence were searched for in the NBRF (National Biomedical Research Foundation) and Genbank databases using the ANGIS computer system (Australian National Genomic Information Service; Sydney, Australia). No highly significant homologous sequences with identified. However, a Snumber "of proteins which also contained an abundance of cysteine residues showed low degrees of matching.

Isolation and sequence of a genomic clone coding for PM44 Genomic DNA plaques were transferred to Hybond N nylon filter discs (Amersham) and prehybridized for 3 h at 65 0 C in co 0.1% sarkosyl, 0.02% SDS, 1% blocking agent (Boehringer), 1 mM pyrophosphate, 75 gg/ml herring sperm DNA, 5xSSPE (Sambrook et al., 1989). After 60 s in contact with the LB (Luria Broth) S plate containing recombinant plaques, filters were placed f (DNA-side up) on a pad of 3mm chromatography paper soaked in M NaOH and 1.5 M NaC1 for 10 min. followed by contact with a neutralizing solution of 0.5 M Tris/Cl and 1.5 M NaC-l Filters were then placed on a pad soaked in 0.4 N NaOH for min., followed by a brief rinse in 10xSSPE (Sambrook et al., 1989). The PM44 PCR fragment was labelled with DIG (Boehringer Mannheim; according to the manufacturer's instructions) and used as probe to screen 120,000 recombinant plaques.

Following hybridization 60 0 membranes were rinsed in 2xSSC (Sambrook et al., 1989) at room temperature followed by 2x20 minute washes in 2xSSC at 60 0 C. "Positive" plaques were identified by a colour detection method relying on the alkaline phosphatase activity associated with an anti- DIG antibody (Boehringer Mannheim) used according to the manufacturer's directions. Positive plaques were then identified using the method described by West et al (1990).

Six positive plaques were obtained. Plaques A, B, C and D appeared more slowly than plaques E and F. Plaque E was chosen for sequencing since it hybridized strongly to the PM44 PCR fragment.

The PM44 clone E was digested with a number of S restriction enzymes and the DNA fragments transferred to Hybond N+ membrane. The DIG-labelled PM44 PCR fragment was used to probe the fragments derived from clone E. A 4.0 kbp Dra I fragment which hybridized strongly to the PCR fragment was subcloned into the Sma I site of plasmid pGEM 7zf The kbp insert was sequenced using a commercially available kit (USB: Sequenase version Unidirectional deletions S into the insert sequence were produced using the Erase-a-base system (Promega), according to the manufacturer's instructions. The procedure produced a series of deletion

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clones, each differing by approximately 200 bp into the PM44 sequence.

The nucleotide and deduced amino acid sequences of this PM44 genomic DNA are shown in figure 5. There is a single open reading frame coding for 323 amino acids. The presence of a stop codon followed by a poly A sequence indicated that the 3' end of the gene had been sequenced. However, there was no indication that the 5' end of the gene had been sequenced.

On the basis of the size of the protein, it is estimated that 20-30% of the structural gene sequence (at the 5' end) has not been obtained in this clone. Comparison of the deduced amino acid sequence of the PCR fragment with the equivalent region in the genomic sequence reveals 20 (out of 969) nucleotide differences which results in 10 amino acid differences. The reasons for these small differences between the PCR and genomic sequences are not clear but-could be the result of one or more of the following: errors introduced by the Tag polymerase during the PCR reaction; the presence of more than one strain of Lucilia cuprina (with minor variants of PM44) being represented in the DNA used for PCR amplification or in the genomic library or; allelic variants of PM44 present in a single larva of Lucilia cuprina. The amino acid sequence deduced from the genomic nucleotide sequence shows the presence of 2 potential N-linked glycosylation sites however one of these is at a slightly different position to that present in the PCR frugent.

From the foregoing it will be appreciated that there may be homologues of PM44 which may be equally as effective in the vaccine. Thus, the invention includes within its scope these

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i 42 homologues produced as either native proteins or as recombinant proteins. These recombinant peritrophic membrane proteins can be produced in bacteria, yeast, insect cells or other suitable expression systems. In a preferred option the vaccine will consist of one or more of these recombinant proteins as well as a suitable adjuvant such as (but not limited to) Montanide/Marcol, saponin, Quill A, ISCOMS, alum, aluminium phosphate, poly anions (eg. dextran sulfate) and chitin. Routes of administration, dosage and frequency of injections are all factors which can be optimised using ordinary skills in the art.

From the foregoing therefore it will be appreciated that the invention also includes within its scope native antigens corresponding to PM44, PM90 and PM95 as well as DNA sequences or recomrbinant antigens corresponding to the sequences shown in FIG 4 or FIG 5 as well as hybrids or antigenic fragments derived from these sequences. There also will be provided structural homologues of these sequences having at least 50% identity of the DNA sequences shown in FIG 4 or FIG 5 and structural homologues of the amino acid sequences shown in FIG 4 or FIG 5 having at least 70% homology of the amino acid sequences shown in FIG 4 or FIG I pr 43 LIST OF REFERENCES Alger, N.W.E. and Cabrera, E.J. (1972) An increase in death rate of Anopheles stephensi fed on rabbits immunised with mosquito antigen. Journal of Economic Entomology 65: 165-168.

Arundel, J,H. and Sutherland, A.K. (1988) Blowflies of Sheep in: Animal Health in Australia Vol. 10, Ectoparasitic diseases of sheep, cattle, goats and horses. (ed. Arundel J. H. and Sutherland, A. Aust. Govt. Publishing Service, Canberra.

pp 35-60.

Barrett, F.M. (1987) Phenoloxidases from larval cuticle of the sheep blowfly, Lucilia cuprina: Characterization, developmental changes and inhibition by antiphenoloxidase antibodies. Arch. Insect Biochem. 5: 99-118.

Barrett, M. and Trevalle, W. (1989) The immune response of the sheep popliteal lymph node to a purified phenoloxidase from larval cuticle of the sheep ectoparasite, Lucilia cuprina, J.

Parasitology. 75: 70-75.

1 Becker, Peters, W. and Zimmermann, U. (1975) Investigations on the transport function and structure of peritrophic membranes VI. In vitro synthesis of peritrophic Smembranes of the blowfly, Calliphora erythrocephala. Journal of Insect Physiology. 21: 1463-1470.

1 c- -un- i~iaiarpa*i 44 Ben-Yakir, D. and Mumcuoglu, Y.K. (1988) Host resistance to the human body louse (Pediculus humanus) induced by immunisation with louse extracts. Proceedings of the XVIII International Congress of Entomology, Vancouver. pp. 282.

Bowles, Carnegie, P.R. and Sandeman, R.M. (1987) Immunisation of sheep against infection with larvae of the blowfly Lucilia cuprina. International Journal of Parasitology. 17: 753-758.

Bowles, Carnegie, P.R. and Sandeman, R.M. (1988) Characterization of proteolytic and collagenolytic enzymes from the larvae of Lucilia cuprina, the sheep blowfly.

Australian J. Biol. Res. 41: 269-278.

Bowles, Feehan, J.P. and Sandeman, R.M. (1990) Sheep plasma protease inhibitors influencing protease activity and growth of Lucilia cuprina larvae in vitro. International S Journal for Parasitology. 20: 169-174.

Brideoke, B.R. (1979) The estimated cost of blowfly control in S* the Australian sheep industry: 1969-70 to 1975-76. in: National, Symposium on Sheep Blowfly and Flystrike in Sheep.

(Edited by Roadsma, pp. 7-21. New South Wales Department of Agriculture, Sydney.

Broadmeadow, Gibson, Dimmock, Thomas, R.J. and O'Sullivan, B.M. (1984) The pathogenisis of fly-strike in sheep. Wool Technology and Sheep Breeding. 32: 28-32.

Campbell, PiM. and Howells, A.J. (1991) Rabbit antisera against DOPA decarboxylase and other larval antigens from Lucilia cuprina: Limited effects on larval growth. in Immunological Control of Blowfly Strike. pp 66. Editors R.M.

Sandeman, J.H. Arundel and E.A. Scheurmann. Australian Wool Corporation, Melbourne.

Chomczynski, P. and Sacchi, N. (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162: 156-159.

Eisemann, Johnston, L.A.Y. and Kerr, J.D. (1989) New techniques for measuring the growth and survival of larvae of Lucilia cuprina on sheep. Australian Veterinary Journal. 66: 187-189.

o Eisemann, Johnston, Broadmeadow, O'Sullivan, S Donaldson, Pearson, Vuocolo, T. and Kerr, on J.D. (1990) Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro. International Journal S. for Parasitology. 20: 299-305 Eisemann, Pearson, Donaldson, Cadogan, L.C.

and Vuocolo, T. (1992) Uptake and fate of specific antibody in ,o feeding larvae of the sheep blowfly, Lucilia cuprina. Medical and Veterinary Entomology (in press).

46 Eisemann, Standfast, Tellam, R.L. and East, I.J.

(1991) Vaccination of sheep against larvae of Lucilia cuprina using novel antigens. in: Immunological Control of Blowfly Strike. pp 76. Editors R.M. Sandeman, J.H. Arundel and E.A.

Scheurmann. Australian Wool Corporation, Melbourne.

Ericson, M.L. (1990) Quick DNA recovery from agarose gels by an ultracentrifuge run. Trends in Genetics. 6: 278.

Fry, Farrell, J.M. and Sandeman, R.M. (1991) Characterization of potential vaccine antigens in blowfly larvae using monoclonal antibodies. in: Immunological Control of Blowfly Strike. pp 82. Editors R.M. Sandeman, J.H. Arundel and E.A. Scheurmann. Australian Wool Corporation.

Gavel, Y. and von Heijne, G. (1990) Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng. 3, 433-442.

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.

Henry, Raina, A K. and Ridgway, R.L. (1990) Isolation of high molecular weight DNA from insects. Anal. Biochem. 185: C 147-150.

47 Inoue, Nojima, H. and Okayama, H. (1990) High efficiency transformation of E. coli with plasmids. Gene 96: 23-28.

Kaaya, G.P. and Alemu, P. (1982) Fecundity and survival of Tsetse maintained on immunised rabbits. Insect Science and its' Applications. 3: 237-241.

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I c~---~rlcp~ 48 Mostratos, A. and Beswick, T.S.L. (1969) Comparison of some simple methods of preparing gamma-globulin and anti-globulin sera for use in the indirect immunofluorescence technique.

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.I "I 9 t.

52 LIST OF TABLES AND FIGURES Table 1: Effect of sera from sheep vaccinated with peritrophic membrane on the weight of feeding larvae.

Table 2: Vaccination of sheep with detergent extracts from peritrophic membrane.

Table 3: Vaccination of sheep with extracts from peritrophic membrane.

Table 4: Description of pools obtained from Superose 12 chromatography.

Table 5: Effect on larval weight caused by the vaccination of sheep with pools of proteins obtained by gel filtration.

Table 6: Effect on larval weight caused by vaccination of sheep with proteins purified by preparative SDS-PAGE.

Table 7: Glycosylated proteins extracted from L. cuprina peritrophic membrane.

Table 8: PM44 peptide amino acid sequences.

o Figure 1: The effect of sera -and various concentrations of isolated anti-peritrophic membrane immunoglobulin-on growth of o o L. cuprina larvae in vitro.

Figure 2: Indirect immunofluorescence localization of PM44 on o, peritrophic membrane.

S Figure 3: DNA amplified using the polymerase chain reaction and PM44 oligonucleotide primers.

j Figure 4: Nucleotide and deduced amino acid sequence of a fragment of DNA obtained by PCR which codes for a fragment of SPM44.

a 4 Figure 5: Nucleotide and deduced amino acid sequence of the PM44 genomic clone E.

.4 I 53 Table 1: Effect of sera from sheep vaccinated with peritrophic membrane on the weight of feeding larvae Experiment Assay Control 2

PM

1 Mean decrease in weight 89/30 in vivo 20h 1.9±0.2 1.4±0.2 20.9 in vivo 50h 25.4±5.1 24.1±1.2 5.1 in vitro 20h 2.8±0.4 2.0±0.3 28.3 89/31 in vivo 20h 1.6±0.4 1.1±0.1 31.0 in vivo 50h 20.2±7.0 16.7±2.4 17.1 in vitro 20h 3.2±0.1 2.1±0.3 33.0.

89/38 in vivo 20h 1.4±0.4 1.0±0.1 26.5 in vivo 50h 13.3±4.0 10.5±2.8 20.8 in vitro 20h 3.0±0.3 1.9±0.2 36.2 89/30 in vivo 20h 1.9±0.2 1.1±0.2 41.1 in vivo 50h 23.6±9.0 17.0±6.5 28.2 in vitro 20h 2.7±0.1 1.9±0.1 30.3 1pM, Peritrophic membrane; 2 Each control and vaccinate group contained 3 sheep; For each "assay the average larval weight in milligrams *o was measured (and the standard deviation in the measurements) and is listed in the Table; 4 Percentage decrease compared to controls.

Ip*t Table 2: Vaccination of sheep with detergent extracts peritrophic membrane from

S

s

I:

14~

I

Experiment Mean larval weights (mg) 81/44 Assay control 3 CTAB sol. PM 4 CTAB insol. PM in vivo 20h 1.9±0.31 1.0±0.1 (47.3) 2 0.8±0.2 (55.9) in vivo 50h 22.8±5.2 18.7±1.1 (18.3) 15.5±4.7 (32.1) in vitro 20h 2.8±0.2 1.8±0.6 (33.5) 1.0±0.2 (63.6) Experiment Mean larval weights (mg) 81/44 Assay control SDS sol. PM SDS insol. PM in vivo 20h 1.6±0.2 1.3±0.1 (18.6) 1.0±0.1 (35.4) in vivo 50h 18.4±4.0 13.4±1.7 (27.2) 13.7±2.3 (25.6) in vitro 20h 2.9±0.1 2.5±0.2 (14.3) 1.9±0.2 (33.6) t r I Standard error of mean; 2 The percentage reduction compared with controls is shown in the brackets; 3Each group contained 6 animals; 4 pM, peritrophic membrane.

4444 *0

S.'

Table 3: Vaccination of sheep with extracts from peritrophic membrane Assay Mean Larval Weights (mg) control 2 Z'3 144 urea G-HC1 in vivo 2.4±0.41 1.1±0.1(55.5)3 1.4±0.3(38.8) 1.4±0.4(42.2) in vivo 24.5±3.1 19.1±0.6(22.0) 15.6±5.9(36.4) 21.8±7.2(11.2) in vitro 3 4 2.1±1.0(43.5) 1.9±0.5(48.9) 2.4±1.1 (35.2) 1 Standard error of mean; 2ec group contained 4 sheep; 3 the percentage reduction in larval weight compared to controls is shown in brackets; 4 Z-314, Zwittergent 3-14; 5 G-HC1, guanidine HCl.

4 1 t ii U~ 56 Table 4: Description of pools obtained from Superose 12 chromatography Pool Fraction 2 Relative Mr I of Proteins in Pool A 8-11 187000, 110000, 95000, 90000, 80000 B 12-13 110000, 95000, 90000, 80000, 61000 C 14-17 110000, 95000, 90000, 80000, 61000, 44000 D 18-22 44000 E 23-30 no detectable proteins 1Mr, relative molecular weight of proteins after reducing SDS-PAGE: the proteins were present in different relative abundances; 2 fractions from Superose 12 column.

Ct t

I

rr r u a a ar r« e a 6o 8

I,

It II 57 Table 5: Effect on larval weight caused by the vaccination of sheep with pools of protein obtained by gel filtration Experiment Dose 1 Mean larval weight 4 Reduction compared 91/76 (Gg) (mg) to control pool A 150 2.6±0.42 21 B 50 2.4±1.1 27 C 200 1.9±0.5 42 D 375 1.9±0.6 42 E 162 3.1±0.5 6 control 3.3±0.2 Experiment Dose Mean larval weight Percentage reduction 91/79 (Ag) (mg) compared to control pool A 812 2.2±0.6 33 B 212 2.2±0.6 33 C 425 2.2±1.2 33 D 512 2.0±0.5 39 E n.d.

3 control 3.3±0.2 o 0 a o 0 00 4 t 1 The dose is gg of pr'tein per injection per sheep; there were-.4 sheep in each group 2 standard error of mean; 3n.d., not determined; 4in vitro assay.

O

*000 0 0o~

*COC

00 4i 0 11___1 58 Table 6: Effect on larval weight caused by vaccination of sheep with proteins purified by preparative SDS-PAGE

PM

1 Protein Dose 2 Mean larval weight Reduction compared (mg) to controls 158 1.43±0.703 61.6 225 1.58±0.60 57.5 Control 3.72±0.60 1 PM, peritrophic membrane; 2 gg protein per animal per injection; there were 4 sheep in each group; 3 standard error of the mean.

0 0 0 0.I H 0 o 0 0 ou~ a a 0 o a o o

OB

01I rtrt o

.P.

Table 7: Glycosylated proteins extracted from the peritrophic membrane of L. cuprina Lectin 'Mr of PM proteins Reactivity with detected by lectin purified PM44 (kD) 2 Sculinarls 201, 193, 173, 162, 151 Strong 125, 95, 90, 44, 36, 24 Wheat germ 93, 62, 42 Not detected Soybean 42, 62 Not detected 'Mr, relative molecular weight; 2 kD, kilodaltons; PM, peritrophic. membrane #0 0 000000 o *000 .0 0 400000 0000 0 a 4* *4 00 a i I twwwrnww Table 8: PM44 peptide amino acid sequences Peptide Sequence PM215 1 I G T L M P S M I S Q Q D Y Y PM1702 G I Q L G N L V Y D T K PM601 Q G A N T V F D K PM1001 N G P G I X G X 2 PM1501 X Q S H Y Y X C E Q PM30022 T Q E Y T P D G F I A D P N S Q Q S Y G Y Q K PM0701 Y L K Q T T D V E PM3106 F G K P Q L M D Q P P N T Y F T Y Y F Q Q Q T G QDN FIPAPTQ E PM2704 T S Q G M A Y N Y G G Y I G L P Q T D K N Y P F F N E PM4404 N G P G I W G K PM4401 N N Q L V G T G K PM4408 N G P G I W G K X P K G L H 1The lysine or glutamate residues bracketed at the beginning of each of these peptides are understood to be there as a consequence of the specificity of the proteases Endo Lys C or Endo Glu C respectively used for the digestion of this protein. 2 represents an unidentified amino acid. Residues with a question mark below them are uncertain. The one letter code for amino acids has been used.

090 *4 0t *9 000900 000*

Claims (29)

1. A method of obtaining peritrophic membrane from blowflies, said method including the steps of: obtaining blowfly larvae from blowfly eggs; (ii) propagating zhe larvae in vitro; and (iii) obtaining peritrophic membrane from the propagated larvae as it is continually synthesised and shed as it passes out of the anus.

2. A method as claimed in claim 1 wherein the blowfly eggs are maintained in vitro.

3. A method as claimed in claim 1, wherein the larvae are filtered to produce the peritrcphic membrane. S 15 4. A method as claimed in claim 3, wherein the peritrophic membrane is subjected to further purification 4 after filtering such as centrifugation. oo 5. A method of inhibiting flystrike in sheep which includes the step of vaccinating sheep with whole intact peritrophic membrane or parts thereof.

6. A method of inhibiting flystrike in sheep which includes the step of vaccinating sheep with a composition containing peritrophic membrane of blowfly or antigenic fragments thereof. oQ 25 7. A vaccine for inhibiting flystrike in sheep including whole intact peritrophic membrane of blowfly or antigenic fragments thereof. o8

8. A method of inhibiting flystrike in sheep 1: including the step of vaccinating sheep with extracts of peritrophic membrane.

9. A vaccine for inhibiting flystrike in sheep including an extract of peritrophic membrane in combination with an adjuvant. A vaccine as claimed in claim 9, wherein the extract of peritrophic membrane contains strong ionic detergent.

11. A vaccine as claimed in claim 10, wherein the Sconcentration of detergent is from 0.1 to p., .I 00 0 *0 0 oo a 00 0 0 0 62

12. A vaccine as claimed in claim 10 or 11, wherein the extract of peritrophic membrane contains a disassociation agent.

13. A vaccine as claimed in claim 12 wherein the disassociation agent is selected from 4M urea or 4M guanidine hydrochloride.

14. A method of extraction of antigens for vaccination of sheep to inhibit flystrike including the steps of initially extracting peritrophic membrane with a strong ionic detergent; (ii) dissolving the mixture obtained in step with a disassociation agent; and (iii) isolating protective antigens from the mixture obtained in step or (ii). A method as claimed in claim 14, wherein the concentration of detergent used in step is from 0.1 to

16. A method as claimed in claim 14 or 15, wherein 20 the disassociation agent is selected from 4M urea or 4M guanidine hydrochloride.

17. Antigens when obtained from the method of any one of claims 14 to 16.

18. A native antigen obtained from extraction of peritrophic membrane having a molecular weight (Mr) of 44,000 determined by SaS-PAGE under reducing conditions.

19. A native antigen obtained from extraction of peritrophic membrane having a molecular weight (Mr) of 90,000 determined by SDS-PAGE under reducing conditions.

20. A native antigen obtained from extraction of peritrophic membrane having a molecular weight of (Mr) 95,000 determined by SDS-PAGE under reducing conditions.

21. An isolated DNA molecule comprising a nucleotide sequence encoding PM44 or antigenic fragments thereof.

22. An isolated DNA molecule comprising a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in FIG 4 or FIG 5, wherein said a;~i~ 63 sequence having at a least 70% homology encodes a protein having the antigenic properties of PM44.

23. A recombinant antigen corresponding to the PM44 amino acid sequence shown in FIG 4 or FIG 5 or an antigenic fragment thereof, or a structural homologue of said recombinant antigen having at least 70% homology with the amino acid sequences shown in FIG 4 or FIG wherein said structural homologue has the antigenic properties of PM44.

24. Peptide PM215, as hereinbefore defined. Peptide PM1702, as hereinbefore defined.

26. Peptide PM601, as hereinbefore defined.

27. Peptide PM1001, as hereinbefore defined.

28. Peptide PM1501, as hereinbefore defined.

29. Peptide PM30022, as hereinbefore defined. 5 30. Peptide PM0701, as hereinbefore defined.

31. Peptide PM3106, as hereinbefore defined.

32. Peptide PM2704, as he sinbefore defined.

33. Peptide PM4404, as hereinbefore defined.

34. Peptide PM4401, as hereinbefore defined. Peptide PM44Q,0 as hereinbefore defined. S36. An isolated DNA molecule comprising a nucleotide sequence encoding PM215, PM1702, PM601, SPM1001, PM1501, PM30022, PM0701, PM3106, PM2704, PM4404, PM4401 or PM4408, as hereinbefore defined, or antigenic fragments thereof. K 37. A vaccine comprising an antigen according to any one of claims 18 to 20 or 23, or a peptide according 0 'to any one of claims 24 to 36, or mixtures thereof N 30 38. A vaccine as claimed in claim 37 which further Scomprises an adjuvant.

39. A method of obtaining peritrophic membrane from blowflies, which method is substantially as hereinbefore described with reference to Example 1.

40. A method of extraction of antigens for vaccination of sheep to inhibit flystrike, which method is substantially as hereinbefore described with reference to Example 4 or Example c r 64 Dated this 14th day of July 1995. COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION by Their Patent Attorneys CULLEN CO mi i i i i c I i i ABSTRACT Specific methods of obtaining peritrophic membrane from blowflies as well as methods of inhibiting fly strike in sheep which includes the step of vaccinating sheep with a composition containing peritrophic membrane of blowfly or S antigenic fragments thereof as well as extracts of peritrophic I membrane. There is also provided a method of extraction of peritrophic membrane from blowfly using a disassociation agent suitably in combination with a strong ionic detergent. There is also provided native antigens obtained from extraction of Speritrophic membrane as well as a recombinant antigen. There is also provided vaccines containing the abovementioned antigens suitably in combination with an adjuvant. 4, 1~