NZ214858A - Vaccines against flystrike - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £14858 214358 NO DRAWINGS >■ Fr;c.-:v Da". ~l C:. p!ft£) Spec.ficunori Filed: .'Ti .".V 8*£' Cm: : Publication PO. NJ: .

No.: Date: NEW ZEALAND PATENTS ACT. 1953 COMPLETE SPECIFICATION "IMPROVED VACCINE' We. COMMONWEALTH SCIENTIFIC & INDUSTRIAL RESEARCH ORGANISATION, a company established under the Science and Industry Research Act, 1949 of Australia, of 14 Limestone Avenue, Campbell, Australian Capital Territory, Australia, and BIOTECHNOLOGY AUSTRALIA PTY. LTD., a company incorporated under the laws of the State of New South Wales, of 28 Barcoo Street, Roseville, New South Wales, Commonwealth o£ Australia, hereby declare the invention for whichtX/ we pray that a patent may be granted totsft/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - - 1 - * " rir.c >>& J. 214858 The present invention relates to a method for the control of fleece rot and fly strike to the body of sheep and to a vaccine for use in such control.

Fly strike is a major problem in sheep management and is 5 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 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 131 weight/volume, i.e. 13g/100ml. The gravid female flies therefore search for such 15 areas to lay their eggs. The flies are attracted to 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 suitable area they can find.

On sheep, there are many such areas. One such area is 20 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 moved to rich pasture. Grazier management practices involve removing this faecal contaminated material by clipping the wool - a process known 25 as crutching. A second area of the sheep which is attractive to flies and provides the necessary nutrients is the area 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 30 strikes are not amenable to treatment by this invention.

By far the major predisposing conditions to strike 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, 35 pizzle and breech of sheep. In general terms, the bacteria 214858 - 3 proliferate following constant wetting of the sheep by rain \ i 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 5 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 across the dermis. This 10 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 volatile compounds and is stimulated to lay eggs (or 15 oviposit) in the region of the lesions. The serum associated with the lesions provides the nutrients required for the survival of the first instar larvae. Once the larvae have survived beyond the first instar stage, they develop abrasive mouthparts and begin to feed on the flesh of the sheep. This 20 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 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 vaccination will prevent the bacteria from causing the dermatitic lesion in the first place. This, in turn, will reduce the incidence of 30 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 to such an extent that the incidence vof fly strikes developing will be reduced.

The means by which the dermatitis may be reduced by 35 vaccination are broadly twofold. In the first instance. 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, 5 damage to the skin may be reduced to such an extent tfiat serum seepage across the dermis will not occur and the nutrients for the further proliferation of the bacteria and for the survival of the larvae will not be available. Thus a strike will not develop.

In the second mechanism, antibodies raised as a result of vaccination will cross the intact skin ar\d prevent the bacteria from growing at the level of the skin. Thus the potentially dermonerotic exoproducts of the bacteria will not be present on the skin and therefore not available to act on 15 the dermal and epidermal layers to cause a severe dermatitis.

The means by which vaccination to prevent the dermatitis is effective may be either of these two mechanisms or by both working in concert. The scope of the invention is not restricted to either mechanism. Evidence has been presented 20 in Patent application No. 34030/84 to suggest 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 the body as well, is extremely complex. It is often extremely difficult to 25 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 found that Pseudomonas species, particularly Pseudomonas aeruginosa, are the major pathogenic organisms which are 30 responsible for predisposition to fly strike of the body of sheep.„ Pseudomonas aeruginosa is the organism responsible for the classical discolouration of fleece associated with a ""condition known as fleece rot. Fleece rot lesions are particularly attractive to oviposition by flies and provide 35 the nutrients for survival of first instar larvae of Lucilia S* 214858 cuprina. Pseudomonas aeruginosa can often be recovered in virtually pure culture from classical pigmented fleecie' xot lesions and has been shown to be pathogenic by a number of criter ia.

One of the present inventors has previously discovered that the predisposition of sheep to fly strike may at least be reduced by preventing or reducing the intensity of the dermatitic lesions caused by the exoproducts of virulent Pseudomonas aeruginosa by vaccination of sheep parenterally 10 with the supernatant of a culture of virulent strain of Pseudomonas aeruginosa or a suitable purified antigen derived therefrom together with a suitable adjuvant (Australian Patent Application No. 34030/84).

The novelty of the current invention lies in the 15 discovery that certain other Pseudomonas and Pseudomonas-like species are capable of causing similar pathogenic processes of the skin and fleece (also referred to as fleece rot) which predispose to body strike of sheep.

The vaccine according to this invention is therefore 20 comprised of antigens from the bacteria Pseudomonas maltophilia, together with a suitable adjuvant. This vaccine may also be prepared in combination with a Pseudomonas aeruginosa vaccine for broad spectrum control of body strike.

The bacterial species from which the antigen comprising 25 this vaccine are drawn were identified as causal organisms of fleece rot and predisposition to body strike by bacteriological surveys on several flocks of sheep during the period October 1983 to October 1985.

The present invention further consists in a method for 30 reducing the predisposition of sheep to fly strike comprising vaccinating the sheep with a vaccine according to the present • "• invention.

The primary bacterial species against which the antigens included within the vaccine according to the present 35 invention is effective is: Pseudomonas maltophilia, however 214858 Pseudomonas putida, and Pseudomonas stutzeri are also * * • implicated to a lesser extent.

Example 1 By way of example# Table 1 indicates the bacteria most 5 commonly recovered from fleece rot and body strike lesions on three properties. The data demonstrates that Pseudomonas ma1tophilia, Pseudomonas putida and Pseudomonas stutzeri occur as frequently, if not more frequently during development of these conditions than Pseudomonas aeruginosa, 10 on these properties.

TABLE I. Bacteria recovered from fleece rot and body strike lesions.

Total Number of samples Type of No. of yielding each species Property lesion samples PA PM PP PS Body strike 17 Fleece rot 28 7 4 13 13 7 1 17 Body strike 16 Fleece rot 23 4 7 12 7 10 4 3 3 Body strike 22 7 4 10 21 Fleece rot 44 20 12 30 41 PA = Pseudomonas aeruginosa PM = Pseudomonas maltophilia PP = Pseudomonas putida PS = Pseudomonas stutzeri Example 2 -v Higher recovery rates of P. putida (97%) and P. stutzeri (45%) from 43 normal fleece samples compared with 35 P. maltophilia (16%) suggest a passive involvement of these • "I ■ni I: 18FEB 1986 h 58 two species in fleece rot lesions. In field studies during the summers of 1982, *83 and '84, 286 strains ot ' *. P. maltophilia were isolated from 721 fleece rot and flystrike samples obtained in three different areas of N.S.N. (a recovery rate of 40%). • By way of example the following experimental results demonstrate the potential for an active role by P. maltophilia in the development of fleece rot. The biochemical and enzymic profiles of ten 10 representative isolates of P. maltophilia were found to be similar to those of a human type strain NCTC 10257 thus indicating the potential for virulence in ovine isolates. Of _ the Pseudomonas species, P. maltophilia is regarded as being second in importance to P. aeruginosa in human infections. 15 The irritant action of proteolytic enzymes on the skin of sheep is thought to be an important step in the development of fleece rot. Sheep isolates of P. maltophilia were shown to produce at least two different proteases by the zymogram technique in which culture supernatant is subjected 20 to electrophoretic separation in agar and then overlain with specific substrates. Like P. aeruginosa, one is elastolytic and both are collagenolytic and gelatinolytic. P. putida and P. stutzeri isolates from sheep showed neglible production of protease further suggesting a less important role than 25 P. maltophilia in development of fleece rot.

Histochemical examination of skin biopsies revealed that experimental exudative dermatitis could be induced after four days of continuous application to the skin of one sheep of a wool pad saturated with P. maltophilia. The simultaneous 30 application of P. putida and P. stutzeri cultures at separate sites on the same sheep did not produce dermatitis.

The following results of immunisation experiments using ' p. maltophilia isolates from sheep show that ovine strains are less serologically diverse than medical strains and that 35 a vaccine prepared from one isolate may afford protection • j 18FEfc!9867,'/ 214858 8 - against practically all ovine strains according to the > . • bacteriostatic mechanism herein described.

One field isolate MCPM001 was grown on horse blood agar for 48 hours at 32°C. The cells were scraped off the plate 5 into sterile 0.851 saline and killed by adding 40% formaldehyde to a final concentration of 1*. For immunisation, a suspension of cells at a concentration of 1.4 Q X 10 /ml in 0.85% saline containing 0.5% formalin was mixed with an equal volume of alhydrogel adjuvant. Two 10 subcutaneous doses, each of 2 ml, were administered to a sheep at a one month interval. Serum was ^collected before the first vaccination and two weeks after the second vaccination, and tested for slide agglutination with 30 P. maltophilia strains obtained from different flocks of 15 sheep. Twenty nine out of thirty of the ovine isolates of P. maltophilia were slide-agglutinated by the antiserum prepared against the one isolate.

While it is recognised that all of the species previously listed other than P. aeruginosa and P. maltophilia 20 may be responsible for a small proportion of fleece rot lesions and flystrike, it is proposed that a vaccine based on the combination of antigens from one or several strains of P. aeruginosa and one strain of P. maltophilia will afford broadspectrum protection against fleece rot and associated 25 body strike in the majority of outbreaks.

It is believed that the products elaborated by Pseudomonas species which may be implicated in the dermal pathology of fleece rot include the enzymes elastase, protease, lecithinase, phospholipase (a haemolysin) and an 30 -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. It is believed, though the inventors are not bound to this 35.. suggestion, that one or more of the above compounds may be 16- 214858 - 9 among the antigenic compounds referred to above as those which, when inactivated by antibodies raised against*then, lead to the marked reduction in the ability of virulent organisms to create a dermatitic lesion.

It is believed that the immunisation of the sheep with the above antigens 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 10 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 15 sheep blowfly and provide the nutrition adequate for development of the first instar larvae.

The strains of Pseudomonas which are cultured for use in the vaccine are preferably virulent isolates. In preferred embodiments of the invention the antigen(s) derived from the 20 supernatant having a molecular weight above about 10,000 daltons and more particularly those having subunit molecular weights in excess of about 55,000 daltons.

The vaccine according to this aspect of the invention is preferably administered together with or otherwise in 25 association with a suitable adjuvant. The adjuvant of preference is an aluminium hydroxide gel, however, other suitable adjuvants including oil based adjuvants could be used.

The vaccination preferably takes place at least twice 30 prior to a period of the year in which the animals are expected to be susceptible to fly strike, thouqh 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 35 varied widely to cope with the widely varying management,- r~ - . i "7 . . « O V 'iT 18 FEB 1986

Claims (11)

21 a ■: c - fUk J. *;- 10 -;climatic, nutritional and environmental conditions which can apply. * The supernatant used as the vaccine, or from which the principal antigens are extracted, may be the product of 5 culturing the particular Pseudomonas strain in any bne of the. 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) J. Bacteriology 88, 1421-1427. The bacterial cells are preferably grown 10 aerobically in fermenters at pH 7.5 from 25 to 48 hours at 37°C. The vaccine is most suitably prepared by removing the cells by centrifligation. The supernatant so obtained may be dialysed against a suitable buffer or filtered through Millipore filters (0.2jim) though these steps are not 15 essential. 0.5% Formalin may then be added to sterilise the supernatant and then mixed with a suitable aluminium hydroxide gel or oil based adjuvant. The vaccines according to this invention contain antigens which are found not only in the supernatant but are 20 also associated with the surface of the bacterial cells. For protection via a bacteriostatic mechanism, a vaccine based on the lipopolysaccharide antigens of the bacteria may be preferred. -I 18FEB1986 y "9 - c ^ c e i \i WHAT l/WE CLAIM IS: 11 214858 The claims defining the invention are as follows:-

1. A vaccine comprised of antigens from the bacteria Pseudomonas maltophilia with a suitable adjuvant.

2. A vaccine as claimed in claim 1 in which the vaccine also includes antigens from Pseudomonas aeruginosa.

3. A vaccine as claimed in claim 1 or claim 2 in which the vaccine also includes antigens from species of bacteria selected from the group comprising Pseudomonas putida, P. stutzeri and mixtures thereof.

4. A vaccine as claimed in claim 3 in which the species of bacteria is Pseudomonas putida.

5. A vaccine as claimed in claim 3 in which the species of bacteria is Pseudomonas stutzeri.

6. A vaccine as claimed in any one of claims 1 to 5 in which the antigens are derived from the supernatant of a culture containing the said bacteria.

7. A vaccine as claimed in any one of claims 1 to 5 in which the antigens are derived from or comprise whole cells.

8. A vaccine as claimed in any one of claims 1 to 7 in which the adjuvant is an aluminium hydroxide gel.

9. A method for reducing the predisposition of sheep to fly strike comprising vacinating the sheep parentally with a vaccine according to any one of claims 1 to 8.

10. A method as claimed in claim 9 in which the sheep are vaccinated twice at intervals of from 1 to 6 weeks.

11. A vaccine substantially as hereinbefore described with reference to the accompanying examples. ^^ By His/Their Authorised Agents. A. J. PARK & SON / / %f FEc ^.