NZ209793A - Vaccination against fly strike: antigenic preparation derived from pseudomonas aeruginosa - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £09793 2 09793 PATENTS ACT, 1953 No..

Date: COMPLETE SPECIFICATION "VACCINE" fc/We. BIOTECHNOLOGY AUSTRALIA PTY LTD, an Australian company incorporated under the laws of the State of New South Wales, of 28 Barcoo Street, Roseville, New South Wales,Australia, and COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANIZATION, a body corporate established under the Science Industry Research Act 1941, as amended, carrying on Scientific and Industrial Research, of Limestone Avenue, Campbell, Australian Capital Territory, Australia, hereby declare the invention for which * / we pray that a patent maybe granted to xwr/us, and the method by which it is to be performed, to be particularly described in and by the following statement:- / . /.' .4,,, r.. , • • ^....,.1 , ;■ /, 209793 The present invention relates to a method for the treatment of fly strike to the body or breech area of sheep and to a vaccine for use in such control.

Fly strike is a major problem in sheep management 5 and is caused by the blowfly Lucilia cuprina. This species of fly can be considered as a parasite of sheep. The flies must lay their eggs in an environment containing soluble proteins which provide the nutrients for the fly larvae. The first instar larvae have no 10 abrasive mouthparts and therefore, the first instar larvae are totally dependent upon a source of soluble protein for survival. it has been estimated that the concentration of protein required for survival of the first instar larvae is of the order of 13% w/v, i.e. 15 13g/100ml. The gravid female flies therefore search for such areas to lay their eggs. The flies are attracted co odours on sheep in regions where conditions are suitable for survival of the larvae and, as the gravid female flies must oviposit, they do so in the most 20 suitable area they can find.

On the sheep, there are many such areas. One such area is in wool stained by faecal material, particularly on sheep which have diahorrea as a result of an intestinal infection, or sheep which have been recently 25 moved to rich pasture. Grazier management practices involve removing this faecal contaminated material by clipping the wool - a process known as crutching. A second area of the sheep which is attractive to flies and provides the necessary nutrients is the area 30 surrounding a wound where blood and serum are present.

This type of lesion has been estimated to be responsible for approximately 4% of body strikes which occur. These types of strikes are not amenable to treatment by this invention.

By far the major predisposing conditions to strike 209793 are those caused by microbial activity in the fleece and on the skin of the sheep. Such microbiological predisposition is responsible for strikes which occur on the body, head, tail, pizzle and breech of the sheep.

In general terms, the bacteria proliferate following constant wetting of the sheep by rain or urine. This proliferation is particularly severe where the moisture collects in folds of the skin such as are found in the breech area of non-Mulesed ewes and between the 10 shoulders of sheep. When the bacteria proliferate to the skin level, pathogenic strains secrete products, mainly proteins with enzymatic or haemolytic activities which damage the dermal and epidermal layers. The dermatitic lesions which ensue result in serum seeping 15 across the dermis. This serum provides nutrients for the further proliferation of bacteria and an extensive lesion results.

Volatile compounds emanate from these lesions. The blowfly is attracted to the site of the lesion by the 20 volatile compounds and is stimulated to lay eggs (or oviposit) in the region of the lesions. The serum associated with the lesions provide the nutrients required for the survival of the first instar larvae. Once the larvae have survived beyond the first instar 25 stage, they develop abrasive mouthparts and begin to feed on the flesh of the sheep. This results in the lesion becoming more severe and, in time more attractive to further oviposition by the flies and a severe strike develops. Unless treated at an early stage, these 30 strikes will lead to the death of the sheep within two to three days.

The basic premise of the present invention is that by vaccinating sheep with components of pathogenic bacteria, antibodies produced as a result of the 35 vaccination will prevent the bacteria from causing the 209793 dermatitic lesion in the first place. This, in turn, will reduce the incidence of oviposition by flies and, even if flies do deposit eggs, soluble protein will not be present so the survival of the larvae will be reduced 5 to such an extent that the incidence of fly strikes developing will be reduced.

The present invention consists in a method for reducing the predisposition of sheep to fly strike comprising vaccinating the sheep parenterally with an 10 antigen having a molecular weight greater than 10 kilodaltons which is derived from cells of a virulent strain of Pseudomonas aeruginosa or from the whole or a fraction of a supernatant of a culture of that organism, together with a suitable adjuvant. 15 In a further aspect the present invention consists in an antigenic preparation effective in reducing the predisposition of sheep to breech strike or other fly strike comprising an antigen having a molecular weight greater than 10 kilodaltons which is derived from cells 20 of a virulent strain of Pseudomonas aeruginosa or from the whole or a fraction of a supernatant of a culture of than organism, together with a suitable adjuvant.

The means by which the dermatitis may be reduced by vaccination are broadly twofold. In the first instance, 25 antibodies generated as a result of vaccination may cross the intact skin and inactivate the bacterial proteins or enzymes which are responsible for the damage to the dermis and epidermis. If the activity of the enzymes is inhibited, damage to the skin may be reduced 30 to such an extent that serum seepage across the dermis will not occur and the nutrients for the further pr<j?L iteration of the bacteria and for the survival of the larvae will not be available. Thus a strike will ^\not develop.

A 0i' In the second mechanism, antibodies raised as a •' e \ sl 20979 3 - 5 result of vaccination will cross the intact skin and prevent the bacteria from growing at the level of the skin. Thus the potentially dermonecrotic exoproducts of the bacteria will not be present on the skin and 5 therefore not available to act on the dermal and epidermal layers to cause a severe dermatitis.

The means by which vaccination to prevent the dermatitis is effective may be by either of these two mechanisms or by both working in concert. The scope of 10 the invention is not restricted to either mechanism.

Evidence suggests that either mechanism can predominate and an efficacious vaccine result.

The bacteriology of fly strike lesions, particularly of the breech, pizzle, head and tail but of 15 the body as well, is extremely complex. It is often extremely difficult to identify the pathogenic organism(s) and to distinguish these from opportunistic or non pathogenic organisms. The most extensively studied form of strike is body strike. It has been 20 found that Pseudomonas species, particularly Pseudomonas aeruginosa, are the major pathogenic organisms which are responsible for predisposition to fly strike of the body of sheep. Pseudomonas aeruginosa is the organism responsible for the classical discolouration of fleece 25 associated with a condition known as fleece rot. Fleece rot lesions are particularly attractive to oviposition by flies ana provide the nutrients for survival of first instar larvae of Lucilia cuprina. Pseudomonas aeruginosa can often be recovered in virtually pure 30 culture from these lesions and has been shown to be pathogenic by a number of criteria.

The vaccine covered in this patent is preferably comprised of antigens derived from pathogenic ovine isolates of Pseudomonas aeruginosa. A biochemically or 35 seralogically similar isolate of Pseudomonas aeruginosa 209793 from a different animal species may also be used for the production of the antigens. If desired the vaccine may comprise a mixture of antigens derived from a variety of different strains of Pseudomonas aeruginosa.

It has been found that the antigens of particular relevance are those contained in a high molecular weight fraction of the supernatant. This fraction of the supernatant substantially comprises material having a molecular weight of about 10 kilodaltons and above. 10 Most prefereably antigens of subunit molecular weight in excess of 55 kilodaltons will be protective as analysed by 10% acrylamide SDS gel electrophoresis and compared with molecular weight standards in daltons: phosphorylase B (MW 92.5 x 10^, bovine serum albumin (67 x 10^), 15 ovalbumin ( 43 x 103), carbonic anhydrase (31 x 10"*), soybean trypsin inhibitor (21.5 x 10^) and lysozyme (14.4 x 103). Antigens derived from whole cells or an extract of whole cells may also be used.

Some of these protective antigens in native form 20 are found as part of a larger complex with a molecular weight greater that 150 kilodaltons as the supernatant components which pass through a column of Ultrogel AcA44 in the void volume are capable of leading to protection from the development of dermatitis when used in a 25 vaccine.

It is believed that the products elaborated by Pseudomonas aeruginosa which may be implicated in the dermal pathology of fleece rot include the enzymes elastase, protease, lecithinase, phospholipase (a 30 haemolysin) and an exotoxin. In addition other bacterial components such as surface slime lipopolysaccharides, deoxyribonuclease and intracellular toxins may be important in their contribution to pathogenesis of the dermatitis exhibited by the sheep. 35 is believed, though the inventors are not bound to 0|j 11 SEP 1987 j! 209793 this suggestion, that one or more of the above compounds may be among the antigenic compounds referred to above as those which, when inactivated by antibodies raised against them, lead to the marked reduction in the ability of virulent organisms to create a dermatitic lesion.

In addition, somatic surface antigens such as lipopolysaccharides may be important in that antibodies against these antigens may prevent the growth of the virulent organisms on the surface of the skin and thereby reduce the severity of incidence of dermatitic lesions by a bacteriostatic effect.

It is believed that the immunisation of the sheep with the above antigens to provide substantial immunity to at least experimental dermatitis induced by Pseudomonas aeruginosa is considered to involve an immune mechanism consisting of an antibody mediated reaction neutralising the action of those products of the bacteria which are pathogenic to the skin and/or a bacteriostatic reaction preventing the elaboration of these products. Reduction in the degree of dermatitis by vaccination renders potential fleece rot less productive of the proteinaceous exudate that would otherwise act as a substrate for the production of volatile attractants for oviposition by the sheep blowfly and provide the nutrition adequate for development of the first instar larvae.

It has most surprisingly been found that the antigenic composition according to the present invention not only reduces the incidence of body strike in sheep but also reduces the incidence of breech strike. This is most surprising as Pseudomonas aeruginosa does not appear to be the principal bacterium implicated in breech strike. At the breech of sheep the soluble protein needed for development of the first instar 209793 larvae of the Lucilia cuprina can come from faeces collected on the wool but most commonl'' from a seropurulent exudate resulting from the ulcerative dermatitis caused by a proliferation of microorganisms in the breech area, of ewes in particular, as a result of constant wetting of the skin with urine. Management procedures such as crutching, mulesing, and pizzle dropping result respectively in removing the wool from the breech area, removing the folds of skin in which urine collects and the microorganism proliferate, and reducing the degree of urine staining at the skin level.

The surprising aspect of this latter discovery is that the bacteria principally responsible for the dermatitis in the breech, unlike that on the body, are not Pseudomonas aeruginosa. The bacteria present in the breech folds, and elsewhere in the breech area, do however appear to share important virulence factors or somatic antigens with the Pseudomonas isolates found in fleece rot and implicated, as a precursor, in body strike. Antibodies against these virulence factors or somatic antigens apparently provide a bacteriostatic action which prevent the bacteria proliferating to such an extent that a severe dermatitis develops.

The strains of Pseudomonas which are cultured for use in the vaccine are preferably virulent isolates of Pseudomonas aeruginosa. In preferred embodiments of the invention the antigen(s) derived from the supernatant are contained in those fractions of the supernatant having a molecular weight above 10 kilodaltons and more particularly those having subunit molecular weights in excess of about 55 kilodaltons.

The vaccine according to this aspect of the invention is preferably administered together with or otherwise in association with a suitable adjuvant. The adjuvant of preference is an aluminium hydroxide gel, ' • * ^ »■ „4 , ■ 209793 however, other suitable adjuvants including oil based adjuvants could be used.

The vaccination preferably takes place at least twice prior to a period of the year in which the animals 5 are expected to be susceptible to fly strike, though a single vaccination may be effective. The vaccinations preferably take place at intervals of from 1 to 6 weeks, preferably 3 to 4 weeks. The actual vaccination regime may, however, be varied widely to cope with the widely 10 varying managment, climatic, nutritional and environmental conditions.

The supernatant used as the vaccine, or from which the principal antigens are extracted, may be the product of culturing the Pseudomonas strain in any one of the 15 media in which the strain is known to grow. Preferred media are however, the media described by Morihara (1964) Journal of Bacteriology, 88, 745-757 or Liu (1964) Journal of Bacteriology 88, 1421-1427. The bacterial cells are preferably grown aerobically in 20 fermenters at pH 7.5 from 25 to 48 hours at 37°C. The vaccine is most suitably prepared by removing the cells by centrifugation. The supernatant so obtained may be dialysed against a suitable buffer or filtered through Millipore filters (0.2 Mm) though these steps are not 25 essential. 0.5% formalin or another sterilising preparation may then be added to sterilise the supernatant. The sterile mixture is then mixed with a suitable aluminium hydroxide gel or oil based adjuvant. The vaccines according to this invention have been 30 found to contain antigens which are found not only in the supernatant but are also associated with the surface of-the Pseudomonas cells and are common to all isolates of Pseudomonas aeruginosa obtained from fleece rot l^esions. The isolates which have been tested include 35 ri|ppresentatives of all of the 17 serogroups 11 SEP 1987 7/ / ? ,3 it t'v. *■-. 2097 - distinguished by the Difco sero grouping scheme. For protection via a bacteriostatic mechanism, a vaccine based on the lipopolysaccharide antigens of a number of Pseudomonas aeruginosa strains may be preferred.

Hereinafter given by way of example only are examples illustrating the present invention.

EXAMPLE 1 A virulent isolate of Pseudomonas aeruginosa (isolate 88 of the C.S.I.R.O. McMaster Laboratory 10 collection in Sydney which corresponds to Bacto Antisera Set serotype 6) was cultured for 48 hours in the glucose, yeast extract, salts media described by Morihara (1964) Journal of Bacteriology 88, 745-757. The cells were removed by centrifugation at 5,500 g for 15 10 minutes. The supernatant was dialysed against two changes of 100 volumes of lOmM tris buffer pH 7.5 to remove the small molecular weight media components and salts. The dialysate was then made 0.5% with respect to formalin and incubated at 4°C for 24 hours. Virulent 20 bacteria were estimated to be less than 1 per ml by plating on nutrient agar plates. The supernatant so obtained was mixed with an equal volume of alhydrogel ad juvant.

EXAMPLE 2 The supernatant from a culture of virulent isolate of Pseudomonas aeruginosa (isolate 88 of the C.S.I.R.O. McMaster collection in Sydney which corresponds to Bacto Antisero Set serotype 6) was prepared as described in Example 1 . 2 ml of the mixture was used to vaccinate 30 each of three sheep on two occasions at a three week interval, control sheep received adjuvant alone. Three weeks after the second vaccination, areas of the backs of each sheep were shorn and wool pads saturated with Pseudomonas aeruginosa isolate 88 were placed over the 35 shorn area and held in position with a plastic support. ^09793 -J: V Twice each day, 20 ml of sterile media was injected into the wool pad beneath the support. On days 4 and 7 following challenge, skin biopsies were taken, one from each of the two challenge sites, histochemically stained 5 and examined microscopically for integrity of the dermal and epidermal layers. The results of this experiment clearly demonstrate tha*". control sheep developed severe dermatitic lesions as a result of this intensive challenge whereas the dermal and epidermal layers of the 10 vaccinated sheep were intact. This result demonstrates that protection from dermatitis induced by a homologous strain could be obtained by vaccination.

EXAMPLE 3 Sheep were vaccinated with the supernatant from a 15 culture of Pseudomonas aeruginosa isolate 42 of the McMaster collection (serotype 1) as described in Example 2 except in that the synthetic dialysable medium of Lui (1964) was used. The wool pad challenge was set up on three separate areas of the backs of the vaccinated 20 sheep using isolate number, 38, 88 and 21 of the McMaster collection (serotypes 3 (10), 1 and 7 respectively) in the challenges. 4 and 7 days after the challenge was initiated, skin biopsies were taken and examined for the integrity of the dermal and epidermal 25 layers. The results clearly showed that sheep vaccinated with isolate 42 were protected from the dermatitis induced in control sheep by isolates 38, 88 and 21. Thus the vaccination is capable of providing cross serotype protection from dermatitis. 30 EXAMPLE 4 The supernatant from a culture of Pseudomonas aeruginosa isolate 88 was fractionated by ion exhange chromatography and Ultrogel AcA44 chromatography to give fractions which were purified with respect to the major 35 proteins located in the supernatant. These fractions 1 - / / 1 • V, / ^ V v - || », im ' 9*' / 209793 were characterised by a series of techniques including SDS polyacrylamide gel electrophoresis, immunoblots, isoelectric focusing, specific enzyme assays, mouse toxicity and rabbit skin necrotising activities. Each 5 of seven different fractions was mixed with alhydrogel adjuvant and used to vaccinate sheep. Three weeks after the second vaccination, the sheep were challenged with wool pads saturated with isolate 88. Skin biopsies were taken and examined as above. The results indicated that 10 animals vaccinated with fractions which were excluded from the Ultrogel column, i.e. those fractions having a molecular weight above 55 kilodaltons, whether purified or in conjunction with other supernatant components, were protected from the dermatitis induced by the 15 challenge with Pseudomonas aeruginosa.

EXAMPLE 5 Forty-five 3-4 month old sheep which had not been shorn, Mulesed or in contact with insecticides were selected. Twenty-five were vaccinated with two doses at 20 three weekly intervals of 2 ml of a mixture of a supernatant of a culture of Pseudomonas aeruginosa isolate 88 and alhydrogel adjuvant. Three weeks following the second vaccination, the sheep were housed in a fly proof facility and sprayed daily with two 25 applications of water equivalent to approximately 6 mm of rain per day. Following the above treatment for one week, 3000 gravid female Lucilia cuprina blowflies were released into the facility each day. The animals were thoroughly inspected at regular intervals for 30 ciscolouration of the fleece characteristic of fleece rot and for deposition of eggs and larvae. The results (Table 1) clearly demonstrate that vaccinated animals had a much lower incidence of body strike than did the controls. The Pseudomonas aeruginosa recovered from the 35 discoloured fleece and strike lesions in this experiment 209793 were serotype 7 on the Difco serotyping scheme indicating that cross serotype protection from fly strike was afforded by vaccination.

TABLE 1 No. of sheep Fleece discolouration (surface) Fleece discolouration (to skin) Body Strikes EXAMPLE 6 Vaccinated Group 25 (100%) 13 (50%) 1 ( 4%) Non-Vaccinated Group 25 (100%) 10 (50%) 6 (30%) Three separate groups of sheep identified with numbered ear tags were vaccinated with 2 ml of a mixture of the supernatant from a culture of Pseudomonas aeruginosa isolate 88 together with an equal volume of alhydrogel adjuvant. The vaccinations took place on two occasions at a 4 week interval. The sheep were allowed to run with control sheep and normal managerial practices were used with the exception that insecticides were not employed on the group of sheep. The animals were regularly inspected over the following three months and strikes recorded. The results (Table 2) clearly demonstrate that the incidence of strikes in the vaccinates was less than in the controls.

TABLE 2 Vaccinates Controls Property No.of Sheep No. % Struck No.of Sheep No. % Struck in Group Struck in Group Struck 1-Hoggets 100 6 6.0 100 26* 26.0 2-Lambs 40 1 2.5 40 6 15.0 3-Lambs 76 3 3.9 76 11 13.2 * Unknown number struck in range 26-32; 6 dead controls in which there was no record kept of whether body strike or tail strike occurred. The data were analysed conservatively by assuming the number struck to be 26.

- I ■■> ■■■•• Hit in i in itii ./ -%-u.. . ■*'" 209793 These results are extremely statistically significant on all three properties. Thus it has been demonstrated that vaccination which leads to protection from dermatitis caused by Pseudomonas aeruginosa will 5 lead to a reduction in the incidence of body strike in the field.

The mechanism of protection in these experiments is at least partly due to the antibodies raised as a result of vaccination inactivating the potentially 10 dermonecrotic exoproducts of the virulent Pseudomonas challenge organisms. However, in some cases, it has been found that the serum from vaccinated sheep also agglutinates and inhibits the growth on plates of the challenge organisms particularly when the challenge 15 organisms are of the same serotype as that used in the vaccine. Therefore, in these cases the mechanism of protection may also be in part or entirely due to a bacteriostatic effect of the antibodies raised as a result of vaccination which prevent or reduce the growth 20 of the pathogenic bacteria on the skin.

EXAMPLE 7 Forty ewes which had not been Mulesed were vaccinated with 2 ml of a vaccine comprised of supernatant from a culture of Pseudomonas aeruginosa 25 isolate 88 mixed with an equal volume of alhydrogel adjuvant. The vaccination was repeated three weeks after and the animals were regularly inspected. None of the 40 vaccinated animals developed ulcerative lesions in the breech and none was struck. Of the 39 control 30 animals, 9 developed ulcerative lesions and 6 of those 9 were struck. In a separate experiment under conditions more favourable for strike, one out of 12 vaccinated ewes developed an ulcerative lesion but none was struck whilst 9 of 11 control ewes developed lesions and 8 of 35 those 9 were struck in the breech region. 209793 This result is verv surprising as Pseudomonas aeruginosa has never been reported to be associated with breech strike and, as will be demonstrated in the example to follow, is not isolated from breech strike 5 lesions or from ulcerative dermatitic lesions of the breech of ewes.

EXAMPLE 8 On five different properties, groups of ewe lambs which had not been Mulesed were vaccinated with a 10 vaccine prepared as above. The animals were allowed to run with a similarly sized group of control ewes and each animal identified with a numbered ear tag. Over the next three months, the animals were regularly inspected and strikes and restrikes of the breech of the 15 animals recorded. The date are summarised in Table 3.

TABLE 3 Vaccinates Controls Test of indepencer.ce Property No., in No. 'i Struck No. in No. ^Struck of incidence & Group Struck Group Struck treatment X Significance 1 60 24 40.0 60 37 61.7 .63 p=0.02 d. 60 IS .0 60 39 65.0 14.74 p=0.0001 *5 40 17 42.5 40 62.5 3.21 p=0.07 •-t 76 19 .0 76 37 48.7 9.16 p=0.002 40 3 7.5 40 .0 4.50 p=0.02

Claims (8)

209793 - 16 - These results indicate that significant protection from breech strike can be afforded by vaccination with antigens derived from cultures of Pseudomonas aeruginosa. This is highly surprising as this organism 5 or other Pseumononads have not been reported to be associated with breech strikes from the breech of ewes. During the course of these experiments, swabs were taken for microbiological examination from the breech of ewes both before and after the development of ulcerative 10 lesions and both before and after strikes. Analysis of at least 80 independant samples over the summers of 1982-83 and 1983-84 has failed to yield any Pseudomonas aeruginosa from these lesions. The bacteria which are responsible for predisposing the sheep to breech strike 15 are therefore very different from those used in the vaccine. It is believed that the vaccines according to the present invention may also be effective in the treatment of other pathogenic effects of Pseudomonas infections. 20 The incorporation of the abovementioned antigens into a suitable vaccine are believed to provide protection against Pseudomonas associated afflictions such as haemorrhagic pneumonia in mink, mammitis in cattle and septicaemia in man. 209793 - 17 - WHAT^E CLAIM IS:

1. A method for reducing the predisposition of sheep to fly strike comprising vaccinating the sheep parenterally with an antigen having a molecular weight greater than 10 kilodaltons which is derived from cells of a virulent strain of Pseudomonas aeruginosa or from the whole or a fraction of a supernatant of a culture of that organism, together with a suitable adjuvant.

2. A method as claimed in claim 1 wherein the fly strike is breech strike.

3. A method as claimed in claim 1 or claim 2 in which the virulent strain of Pseudomonas aeruginosa is a pathogenic ovine isolate of that organism.

4. A method as claimed in any one of claims 1 to 3 in which the fraction of the supernatant is a fraction containing or consisting of antigens having a molecular weight greater than 55 kilodaltons.

5. A method as claimed in any one of claims 1 to 4 in which the sheep are each vaccinated twice prior to a period of the year in which the animals are expected to be susceptible to fly strike.

6. A method as claimed in claim 5 in which the vaccinations are spaced apart by an interval of from 1 to 6 weeks.

7. An antigenic preparation effective in reducing the predisposition of sheep to fly strike comprising an antigen having a molecular weight greater than 10 kilodaltons which is derived from cells of a virulent strain of Pseudomonas aeruginosa or from the whole or a fraction of a supernatant of a culture of that organism,together with a suitable -adjuvant.

8. An antigenic preparation as claimed in claim 7 in which the fly strike is breech strike. An antigenic preparation as claimed in claim 7 or 8 t 1 SEP1987 '•H wh^c1*1 the fraction contains or consists of antigens .. .v,- ^ V f 209793 18 having a molecular weight greater than 55 kilodaltons. i >1 ■ An antigenic preparation as claimed in any one of claims 7 to 9 in which the antigenic preparation is rendered sterile by the addition of a sterilising preparation.

11. An antigenic prepartion as claimed in any one of claims 7 to 10 in which the adjuvant is selected from the group comprising aluminium hydroxide adjuvants and oil based adjuvants.

12. An antigenic preparation as claimed in any one of claims 7 to 11 in which the antigenic preparation contains antigens from a plurality of strains of Pseudomonas aeruginosa.

13. A method £or reducing the predisposition of sheep to fly strike substantially as hereinbefore described with reference to any one of examples 2 to 8.

14. An antigenic preparation effective in reducing the predisposition of sheep to fly strike substantially as hereinbefore described with reference to any one of examples 2 to 8. At* fc+wvA is- l-tol r'y>*rAuthorise! Agent#, A.'J. PAH.< & SON 5 OCT 1984