GB2270562A - Determination of immune and infection status against Aeromonas salmonicida - Google Patents

Abstract

A method is described for the determination of immune or infection status of an animal, with respect to Aeromonas salmonicida, said method employing iron regulated outer membrane proteins and/or external lipopolysaccharides and/or antibodies raised to either or both of these as binding agents together with test kits for use in the method.

Description

METHOD AND KITS FOR DETERMINING IMMUNE AND INFECTION STATUS.

The present invention relates to a method for the determination of immune or infection status of an animal, particularly immune or infection status with respect to organisms of genus Aeromonas, specifically Aeromonas salmonicida (A.salmonicida). The invention further provides test kits for use with the method of the invention.

A.salmonicida is a pathogen of some significance, particularly in activities such as fish farming, due to its causation of the systemic disease furunculosis (Herman,R.L. (1968) Fish furunculosis 1952-66.

Trans. Amer. Fish. Soc. 97:221-230). Acute forms of this disease are associated with rapid growth of the organism in the major body organs which produces a terminal septicemia frequently accompanied by severe tissue necrosis. The organism is particularly known for its effects on the salmonidae but is capable of infecting a wide range of fish species. Further significant economic effects of the organism include its ability to induce erythrodermatitis disease in carp.

Intramuscular injection of as few as 100 virulent cells can produce death within 9 hours and as asymptomatic carriers can die of fulminant furunculosis if subjected to environmental stress it is desirable to vaccinate fish as opposed to attempting curative treatments.

It is known that fish produce antibodies to both virulent and non-virulent A. salmonicida cells or their extracellular products (ECP) but it is found that direct production of antibodies by administration of these antigens to coho salmon provides less effective protection than administration of rabbit raised antisera.

It appears that rabbits are more efficient than fish in responding to protective antigens (Olivier et al, (1985) J.Fish Dis.,8:43-55) but in any case the nature of these antigens (immunogens) has not as yet been determined.

In the applicant's copending patent application PCT/GB92/01016 there is described the use of two components of A. salmonicida cultures as protective agents for use in vaccines against that organism.

Furthermore there are therein described antibodies and antisera raised to these components, some of which are lethal to the infecting organisms. These antibodies and antisera may be derived from a variety of animals and have application in treatment of animals, particularly fish, already infected with the organism.

A first one of the particular culture components of these vaccines is A. salmonicida Extracellular Lipopolysaccharide (ELPS), herein defined as that lipopolysaccharide obtainable from the extracellular fluids derived from cell free supernatant of an A. salmonicida 24 to 48 hour culture, that supernatant being capable of evoking a protective immune response in salmon when administered without adjuvant in an amount of O.lml/fish. This ELPS component is different from the cell wall lipopolysaccharide of A. salmonicida in haemolytic activity, fatty acid methyl ester and sugar composition, as illustrated in Table I in Example 1 below.

A second culture component provided by the invention of PCT/GB92/01016 is capable of evoking production of antibodies that actually kill A.salmonicida bacteria and comprises Iron Restricted Outer Membrane Protein (IROMPs) produced by A.salmonicida cultured under iron limiting conditions.

Iron limiting culture conditions are those in which the amount of iron available to the bacteria in the culture is less than that which is required for normal growth and can be brought about by not including sufficient in the media or by including therein chelating agents which reduce its availability. The sub-culture of organisms several times in such media before production conveniently depletes iron stores.

It is established that bacteria have an absolute requirement for iron and that A.salmonicida is typical in this respect. It has further been shown that A.salmonicida is induced to produce such IROMPs in iron limiting conditions (Chart and Trust, (1983). J. Bacteriol., 156:758-764). These iron regulated OMPs have been characterised using electrophoresis and may be isolated by various standard techniques such as eg. electrophoresis and/or centrifugation (Aoki and Holland, (1985). FEMS Microbiol. Lett. 27:299-305).

Despite their existence and isolation having been reported, neither the suitability of these or related OMPs as vaccine active components or their respective antibodies as therapeutic, bactericidal agents for treating fish for furunculosis infection has been suggested. Where iron regulated OMPs have been prepared for vaccine screening purposes they have not been proved effective (Evenburg et al, (1988) J. Fish Diseases, Vol 11, pp337-349). In this regard it is to be noted that Evenburg's OMPs are apparently not those identified in PCT/GB92/01016 as the culture in which they were prepared contained what has now been found to be insufficient iron-chelating compound (lOpM 2,2'-dipyridyl) to evoke IROMPs production.

The present invention provides a method for assessing the immune or infection status of an animal with respect to organisms of Aeromonas salmonicida comprising use of ELPS and/or IROMPs (as defined herein) obtainable from a culture of Aeromonas salmonicida, and/or antibodies raised thereto, to specifically bind complementary antibodies or antigens specific to Aeromonas salmonicida immunity or infection present in one or more of the animals biological fluids, determining the presence of any fluid derived antibodies or antigens that have been bound, and relating that to the immune or infection status of the animal.

Presence of antibodies to ELPS or IROMPs in the biological fluids is associated with a degree of immunity while the presence of antigens which agglutinate with them is indicative of infection. The biological fluids may be any of the internal fluids obtainable from the animal, in fish that conveniently being blood, serum or plasma.

The binding of the antibodies or antigens specific to infection with A. salmonicida may be carried out using any of the well established binding assays available; these being well described in the art.

In its simplest form the method of the present invention mixes a biological fluid under investigation with one or both of the binding agents described above and the amount of agglutination taking place is used to assess the amount of complementary component present in the fluid. In this form of assay the binding agent may be used in isolated form or in complex form, ie. such as antisera for antibodies or as a culture or its component fractions for ELPS and/or IROMPs. To enhance such method the binding agent may be immobilised onto a solid surface such as a microtitre well plate or latex beads against which the agglutination product will more readily be visualised.

In a further form of the invention, a competitive assay, two forms of the binding agent may be used. One form of the binding agent is immobilised and used to bind to the fluid derived component, while a binding agent complementary to this but in labelled forum, eg.

labelled with gold, an enzyme or radioactivity, is used to compete with antibody or infection component (ie. antigen) present in the sample for binding to an immobilised component complementary to them both. In this manner a maximum amount of label will be bound when there is no competing material present in the sample and thus immune or infection status may be assigned as zero or insignificant. A minimum amount of label will be bound where a maximal amount of competing material is present in the sample and thus immune status or infection may be assessed as maximal or significant.

In a still further form of the invention the binding agents are used in what is termed a capture assay format. In this format a further binding agent is utilised with the binding agents of the invention whereby the antibody or infection component is first captured by the further binding agent and then its identity is confirmed by exposure and thus additional binding to the binding agent of the method of the invention or vice versa. Typical of such capture assay is one in which an antibody raised against the subject animal's IgA, IgG or IgM class antibodies in general is immobilised upon latex or a microtitre well plate. In an example of such assay the immobilised antibody is exposed to a sample under investigation whereupon IgA, IgG or IgM in the sample is selectively bound with it.The immobilised agent is rinsed and then exposed to a further binding agent specific for the target antibody rather than its class, ie. in the present case for IROMPs or ELPS, whereupon any immobilised target antibodies bind the specific binding agent. In this case it is preferred to assess the presence of captured antibody, and thus assess the immune status, by labelling the IROMPs and/or ELPS such that once the final immobilised complex is rinsed, eg. in water or buffer, the amount of captured antibody can be determined by the amount of label present on the solid's surface. Thus typically the IROMPs and/or ELPS will be labelled using a radioactive, chemical (eg. gold) or biological label such as an enzyme, and its presence determined by conventional means.

The IROMPs component may be used in isolated or semi-isolated form for all applications; such semi-isolated form being eg. that obtainable as sodium N-lauryl sarcosinate insoluble inner membrane of IROMPs cells. Alternatively, the whole membrane form such as in formalinised whole membrane or while outer membrane fractions derived from an iron-restricted culture or, more conveniently, formalinised whole cells from such culture, may be used as immobilised components or for production of antibodies.

The iron restriction culture medium comprises any medium known to be suitable for growth or support of A.salmonicida organisms in which the iron component has been omitted or reduced to below levels necessary for normal growth or to which iron limiting agents have been added.

Suitable iron limiting agents are chelating agents such as eg. EDTA, EDDA and dipyridyl and use of these is preferred due to their ability to deplete internal iron stores in the organisms and thus stimulate iron seeking mechanisms associated with the IROMPs components. In this regard it should be noted that 2,2'-dipyridyl levels of lOgM are insufficient. Preferably lOOmg/ml EDDA or 100PM 2,2'-dipyridyl is included.

A typical IROMPs culture is carried out for 24 hours to 72 hours before carrying out inactivation of cells with formalin or other bactericidal treatment or isolation of the IROMPs component. Cultures are conveniently carried out at approximately ambient temperature and are preferably fermented aerobically at constant pH.

In a preferred procedure a sub-culture of A.salmonicida is first grown in an iron-chelating agent containing medium for about 24 hours at ambient temperature (eg.220C), preferably with shaking, after which it is used to inoculate a fermentation vessel containing the same growth medium in which the bacterium is then grown for approximately 48 hours (2 days), again at ambient temperature, under aerobic conditions and at constant pH (eg. pH 7.0). The culture is inactivated after the 48 hour period and a suitable concentration of cells produced as determined by measurement of the amount of cells expressing IROMPS, Cells are sedimented from the medium then added back to achieve the desired numbers. This preferred procedure is particularly convenient as it provides both IROMPs and ELPS components in one culture.A particularly advantageous feature of this procedure is that the ELPS component in the supernatant is not reduced when the sedimented cells are re-added. Thus where both components are provided in the same culture in this way one can ensure that both reach an effective concentration for raising antibodies before the culture is stopped.

The culture so provided can be separated into component parts to provide the IROMPs and ELPS in a form isolated from each other and these or the culture may be used as a vaccine for raising antibodies for use as binding agents. It is found that use of 0.1 ml of the 1x107 cell/ml supernatant produced as above is sufficient to protect fish when injected intraperitoneally (i.p.) although other volume /dilution combinations giving sufficient cells/fish will be suitable, and 0.1 ml of 1x108 cell/ml or more is preferred. In this regard it is found that i.p. administration of 2Opg/fish of isolated IROMPs component as described herein is sufficient to produce bactericidal antisera.However these vaccines have been determined as active when administered in other ways, for example, by immersion of fish in vaccine or by administration in oral delivery forms, eg in capsules.

Where the binding agents are to be used in immobilised form the immobilisation may be carried out by any of the means known in the art of immunoassay. For example, the immobilising material may be in plate, rod or bead form and may for example comprise man-made plastics, cellulose nitrate or latex. Examples of immobilised binding agent assays are given in EP 0158746 wherein immobilising materials or 'matrices' are listed and a method for enhancing such assays provided.

For assays using immobilised antigen or antibody, the selected binding agents, ie. IROMPs, ELPS or their respective antibodies, are conveniently attached to an immobilising surface by passive technique.

For example, when using latex beads as immobilising material it is possible to merely shake the beads together with a suspension or solution of the selected binding agent using protocols such as are described in WO 92/06197. Conveniently such shaking may be carried out with semi-pure binding agent such as antisera or IROMPs and/or ELPS containing fractions, but purified fractions eg. containing affinity purified antibody or IROMPs and/or ELPS as prepared as in the examples below are preferred. It will further be realised that where monoclonal antibodies have been raised that ascites may be used to directly prepare latex bound antibody as in WO 92/06197.

The present invention further provides test kits for use in carrying out the method of the invention. These kits comprise IROMPs and/or ELPS and/or antibodies to either in a form suitable for use in a binding assay. Thus such kits may comprise pure or semi-pure IROMPs, ELPS and/or said antibodies for use in all manner of binding assays and IROMPs comprising membranes or whole cells for use with labelled capture assay or competitive assay components in so far as these can be separated from soluble components, eg. by filtration or sedimentation. Immobilised IROMPs, ELPS and/or antibodies will be included for use with all those assays requiring immobilised binding agents, ie. capture, competitive and latex bead assays.

Thus a simple agglutination assay test kit will comprise IROMPs and/or ELPS and/or their respective antibodies in isolation, suspension or in a form that is readily solubilised or suspended in water or buffer.

Competitive assay test kits will comprise IROMPs and/or ELPS and/or one or both of their antibodies in immobilised form together with labelled IROMPs, ELPS and/or one or both of their respective antibodies in solution or in a form that is readily solubilised.

Alternatively IROMPs may be provided as membranes or whole cells in place of other immobilised forms.

Capture assay test kits will comprise IROMPs and/or ELPS and/or one or both of their respective antibodies in immobilised form together with labelled IROMPs, ELPS or secondary antibodies targeted at these or labelled anti-fish IgA, anti-fish IgG or anti-fish IgM. Thus any target bound to the immobilised agent will further bind the secondary antibody or antigen as discussed. Further test kits based upon the binding agents of the invention will occur to the person skilled in the art.

The production of the IROMPs, ELPS and antibody/antisera components and their immobilised forms for use in the present invention will now be illustrated by way of the following non-limiting Examples. Further embodiments of the present invention will occur to those skilled in the art in the light of these. Figure 1 associated with Example 1 illustrates correlation of the method with infection status.

EXAMPLE 1: PURIFIED ELPS - COMPARISON TO CELL WALL LPS (CWLPS).

Purified ELPS was prepared from cell free extracellular products of a culture of a strain of A.salmonicida, using the method of Westphal and Jann (Methods in carbohydrate chemistry (1965) 5 p83-91), and its chemistry compared to that of LPS isolated from A. salmonicida cell walls (TABLE I). The %weight values given are approximate and are provided in order to aid the skilled worker in identifying the ELPS component in culture supernatants. To avoid increase in cell wall LPS (CWLPS) relatively young cultures should be used, eg. less than 96 hours old, preferably from 24 to 48 hours old.

TABLE I.

ELPS CWLPS Sugar %wt(anhydrous) Sugar %wt(anhydrous) Mannose 13.97 Fucose 7.56 Galactose 2.57 Ribose 7.58 Glucose 1.59 Xylose 0.18 Mannose 0.55 Galactose 0.85 Glucose 6.44 These figures correspond to the ELPS sugar component comprising 77% mannose with CWLPS having a sugar component composition of 32.6% fucose, 32.7% ribose and 27.8% glucose; all in % anhydrous weight.

Lipid determination of both fractions gave approximate figures of 14.8% weight lipid for ELPS and 49.5% weight lipid for CWLPS.

ComDarison of immunogenic activitv of ELPS with CWLPS.

Salmon parr were vaccinated intraperitoneally with O.lml of vaccine comprising 2mg/ml of either ELPS or CWLPS in phosphate buffered saline. Control fish received O.lml of Dulbecco's phosphate buffered saline. Fish were maintained at 140C for 6 weeks post vaccination, a subsample was bled and the serum assayed for antibodies to ELPS or CWLPS. After 6 weeks fish were challenged with a more virulent heterologous strain of A. salmonicida and the mortality of each group monitored; relative percent survival (RPS) was then calculated. A different and superior effect of ELPS in producing a protective response in vaccinated fish thus is clearly demonstrated Cross absorption studies on the antisera produced on vaccination with each of the two LPSs evidences a difference in epitopic structure of these antigens.

EXAMPLE 2: IROMPs OF A. SALMONICIDA.

IROMPs were prepared by harvesting cells from a 500 ml Tryptone Soy Broth (TSB)/100pM 2,2'-dipyridyl A. salmonicida culture by centrifugation (8000g for 30 minutes at 40C), washed with 20mM Tris(hydroxymethyl)methylamine (TrisHCl, pH7.2) resuspended in 20mM Tris-HCl, lOmM EDTA (pH7.2) containing O.lpg DNase and O.lpg RNase per ml and phenylmethylsulphonyl fluoride (PMSF;SOpgml-l) and sonicated on ice (6 x 30 s) . Whole cells and debris were removed by centrifugation (20000g for 20 minutes at 40C). Inner membranes were solubilised by addition of Sarkosyl (sodium N-lauryl sarcosinate) to 1.5% (v/v) and incubated for 30 minutes at 220C.Sarkosyl-insoluble IROMPs were collected by centrifugation at 100,000 g for 1 hour at 40C, washed and suspended in 20mM Tris-HCl and stored at -200C.

The IROMPs containing fractions produced were analysed by the method of Bradford (Analytical Biochem. (1976) 72, 248-254) using the phenol-sulphuric acid method/glucose standard of Dubois (Analytical Chem. (1956) 28 350-356) for carbohydrate determination, several LPS tests and the thiobarbiturate 2-keto-3-deoxyoctonate (KDO) test. All confirmed their status as OMPs. At the concentration of 200pg/ml IROMPs high levels of LPS and carbohydrate were detected although A-layer proficient (virulent) strains had less due to the additional protein. Protein concentrations were standardised in each case prior to calculation of other variables.

TABLE III.

Protein LPS LAL KDOpg/ml Carbohydrate OMP Antigen pg/ml pg/ml EUml-I LPS/mgprotein mg/ml Avirulent 200 230 > 268 5.5 4.3 Iron-replete Avirulent 200 285 > 268 5.0 9.0 IROMPs Virulent 200 52.2 > 268 0.2 0.38 IROMPs Comparison of IROMPS from various strains of A. SALMONICIDA.

To determine effect of iron-restriction on the OMPs of A. salmonicida in general, 18 typical strains and 2 atypical strains were grown on TSB containing EDDA (lOOmg/ml) and the OMPs produced were compared by SDS-PAGE with those from organisms grown in TSB alone and TSB/EDDA plus FeCl3 (0.54 mM). Growth under iron restricted conditions resulted in expression of four novel OMPs (IROMPs) of apparent molecular weight 82, 77, 72 and 70kDa which were not present in cultures of those strains grown under iron-replete conditions (TSB or TSB+EDDA+FeCl3).

Culture of A. salmonicida was carried out under varying conditions to check that disadvantageous temperatures or oxygenation levels does not induce IROMPS production under iron replete conditions. Use of 150C, 220C and anaerobic conditions did not in themselves result in expression of the IROMPs. Use of 2,2'-dipyridyl (lOO M) in place of EDDA however produced identical IROMPs but in quantatively greater amounts, thus indicating this chelator to be preferred. Studies on IROMPs cross-reactivity with salmon antisera and monospecific rabbit anti-IROMP antiserum raised to IROMPs of a single strain and all showed staining using Western blotting techniques.

SDS-PAGE analysis of OMP vaccines indicates that the only difference between iron-replete and iron-restricted OMPs fraction is the presence of the 82, 77, 72and 70 kDa IROMPs.

EXAMPLE R: IROMPS ANTIBODIES.

A salmon antiserum raised against a mixture of IROMPs from two typical A. salmonicida strains grown under iron restricted conditions and shown to contain antibodies directed against the IROMPs by use of Western blotting techniques was used to passively immunise Atlantic salmon against bath challenge exposure to a virulent strain whereon an RPS of 77% was produced.

To ensure that IROMPs were responsible for the aforementioned RPS monospecific anti-IROMP antiserum prepared in rabbit against the gel-isolated IROMPs of the typical strain was also evaluated for its ability to passively immunise against a heterologous bath challenge whereon 55% RPS was achieved. Affinity purified IgG from the rabbit antiserum further gave an RPS of 83%. Such purified IgG is particularly suitable for assay use with regard to presence of antigens.

EXAMPLE 4. IMMOBILISATION OF ANTIBODIES OR ANTIGENS ON LATEX.

Latex beads (Sigma) of approximately 3pm diameter were coated with either ELPS or IROMPs or antibodies to these, all as prepared in Examples 1 to 3, antibodies being affinity purified against the respective antigen (ELPS or IROMPs) using affinity columns. Coating was carried out in accordance with bead manufacturers instructions, conveniently by shaking a solution or suspension of the antigen or antibody as obtained above with beads for 6 to 14 hours then drying by conventional means.

EXAMPLE 5. ASSAY OF IMMUNE STATUS OF FISH, Salmon sera under investigation was mixed with latex beads coated with either ELPS or IROMPs fractions obtained by the methods of Examples 1 and 2 in a ratio of 1:1 vol:vol fraction:suspension in buffer. Known fully positive fish produced agglutination of antibodies with both types of beads while fish that had only developed one antibody response produced agglutination with only one of the types of beads.

Non-responders gave no agglutination with either bead type. Figure 1 shows the correlation of assay using ELPS/IROMPs immobilised on latex beads with slide agglutination tests from A. salmonicida infected fish.

Claims (23)

CLAIMS.

1. A method for assessing immune or infection status of an animal with respect to organisms of Aeromonas salmonicida comprising use of Extracellular Lipopolysaccharide (ELPS) and/or Iron Regulated Outer Membrane Protein (IROMPs) obtainable from a culture of Aeromonas salmonicida, and/or antibodies raised thereto, as binding agents to specifically bind complementary antibodies or antigens specific to Aeromonas salmonicida immunity or infection present in one or more of the animals biological fluids, determining the presence and/or amount of any fluid derived antibodies or antigens that have been bound, and relating that to the immune or infection status of the animal.

2. A method as claimed in claim 1 wherein the biological fluid or fluids are selected from the group comprising whole blood, serum or plasma.

3. A method as claimed in claim 1 or 2 wherein a biological fluid under investigation is mixed with one or more of the binding agents and the amount of agglutination taking place is used to assess the amount of specific antibodies and/or antigens present in the fluid.

4. A method as claimed in claim 3 wherein the binding agent is an antibody and is used in the form of whole antisera.

5. A method as claimed in any one of claims 1 to 3 wherein the binding agent comprises IROMPs in the form of membranes or whole cells.

6. A method as claimed in any one of claims 1 to 3 wherein the binding agent comprises IROMPs in the form of a fraction obtainable as sodium N-lauryl sarcosinate insoluble inner membrane of iron restricted A.salmonicida cells.

7. A method as claimed in any one of claims 1 to 3 wherein the binding agent comprises IROMPs in the form of one or more of the IROMPs obtainable by SDS-PAGE separation of the fraction obtainable as sodium N-lauryl sarcosinate insoluble inner membrane of iron restricted A.salmonicida cells.

8. A method as claimed in any one of claims 1 to 3 wherein the binding agent comprises ELPS in the form of cell free extracellular lipopolysaccharide obtained from a 24 hour to 96 hour culture of A.

salmonicida.

9. A method as claimed in claim 8 wherein the culture is a 24 to 48 hour culture.

10. A method as claimed in any one of the claims 1 to 9 wherein the binding agent is used in immobilised form.

11. A method as claimed in claim 10 wherein the binding agent is immobilised on a plastics, nitrocellulose or latex material.

12. A method as claimed in claim 7 wherein the binding agent is immobilised on beads, rods or on a plate.

13. A method as claimed in claim 8 wherein the plate is a microtitre well plate.

14. An immune or infection status test kit comprising A. salmonicida IROMPs and/or ELPS and/or antibodies to either in a form suitable for use in a binding assay.

15. A test kit as claimed in claim 14 wherein the IROMPs, ELPS and/or antibodies to either are in isolated or semi-pure form.

16. A test kit as claimed in claim 15 wherein the IROMPs are in the form of cell membranes or whole cells comprising IROMPs.

17. A test kit as claimed in claim 14 comprising IROMPs and/or ELPS and/or their respective antibodies in solution or suspension or in a form that is readily solubilised or resuspended in water or buffer.

18. A test kit as claimed in claim 14 comprising A. salmonicida IROMPs. ELPS or antibodies to either, in immobilised form.

19. A test kit as claimed in claim 18 further comprising labelled IROMPs, ELPS and/or their respective antibodies in solution or suspension or in a form that is readily solubilised or resuspended in water or buffer.

20. A test kit as claimed in claim 18 further comprising labelled secondary antibodies targeted at IROMPs, ELPS or labelled anti-fish IgA, anti-fish IgG or anti-fish IgM.

21. A test kit as claimed in any one of claims 18 to 20 wherein the IROMPs, ELPS and/or antibodies are immobilised on a plastics, nitrocellulose or latex material.

22. A test kit as claimed in claim 20 or 21 wherein the binding agent is immobilised on beads, rods or on a plate.

23. A test kit as claimed in claim 22 wherein the plate is a microtitre well plate.