AU2009100018A4 - A Method of vaccination - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR AN INNOVATION PATENT Name of Applicant: Actual Inventor: Address for Service: Invention Title: Pork CRC Ltd, of Level 1, Eastick Building University of Adelaide Roseworthy Campus South Australia 5371 Australia Peter McKenzie Virgil Anthony Fahy DAVIES COLLISON CAVE, Patent Attorneys, of 1 Nicholson Street, Melbourne, Victoria 3000, Australia "A Method of vaccination" The following statement is a full description of this invention, including the best method of performing it known to us: QA\OPER\MAI2OO0\JANUARY\30718229 1401DOC -14/1/09 PAOPERWMAL\Inwvlion NicnIk~u I 1R229 Por CRC Lid de 14A1,m21X)9 0 -1- A METHOD OF VACCINATION 0 Field of the Invention SThe present invention relates to a method of vaccinating pigs to increase their resistance to infection by Actinobacillus pleuropneumoniae. More particularly, the present invention Srelates to methods of inducing an immune response in a pig against porcine pneumonia caused by Actinobacillus pleuropneumoniae.

Background of the Invention The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Actinobacillus pleuropneumoniae (App) is the causative agent of porcine pleuropneumoniae, a severe respiratory disease of pigs that is of economic importance wherever pigs are raised. Actinobacillus pleuropneumoniae severely damages the lungs of growing pigs and can linger on as a chronic infection serving as a source of future outbreaks. It has a significant economic impact on pig production by slowing growth rates and in worst cases causing death. In the Australian pig industry the chronic form of the disease has been estimated to cost $64/sow/year without medication costs.

Actinobacillus pleuropneumoniae is carried in the tonsils and upper respiratory tract and is highly contagious. To date, two different biovars of Actinobacillus pleuropneumoniae are recognised within the species. Within biovar 1, there are thirteen recognised serovars, these being serovars 1-12 and 15, and within biovar 2 two recognised serovars, 13 and 14.

Serovars differ in the way they exert their pathogenic effects on the pig and the severity of infection that they induce. Serovar 1 of biovar 1 is reported to be the most virulent type, and biovar 1 strains usually cause higher morbidity than biovar 2 strains. Serovars 1, 5 and POPERV1ALUnmr-atin PRc,=00718229 Pork CRC Ltd dow-14AJ I/2J9I -2- 7 are reportedly the most common types of Actinobacillus pleuropneumoniae found in the SUnited States and 1, 7 and 15, the most common types in Australian pigs. However, any serovar of Actinobacillus pleuropneumoniae can cause significant damage to swine 0 0 respiratory systems, especially if a secondary bacterial or viral disease exists.

Actinobacillus pleuropneumoniae can affect all age groups, but most frequently affects pigs between weaning and six months of age and growing pigs are most likely to be Saffected when they are 12-16 weeks of age. The stress of moving and mixing animals often causes the disease to break out among the subclinical pigs, and then the bacteria is passed directly from pig to pig or via aerosol over very short distances. Some vertical transmission from sow to piglet can occur, but it is not nearly as common as the direct contact from incoming infected animals.

The current major control mechanism of Actinobacillus pleuropneumoniae outbreaks is management. For example, the adoption of practices including 'all in-all out' weaner accommodation, segregated early weaning and multi-site production. The use of therapeutic levels of antibiotics, either in the water or in the feed, (pulse treatment) at times of high risk has also been shown to be beneficial. In-feed antibiotics are used to reduce the likelihood of Actinobacillus pleuropneumoniae infection, while antibiotic injections are used to treat animals displaying symptoms of Actinobacillus pleuropneumoniae. However, the wholesale use of antibiotics faces strict regulations due to problems concerning resistance, as well as problems associated with the presence of antibiotic residues in pig meat. Prevention by vaccination is a preferred alternative to antibiotic treatment.

However, only a small number of commercial vaccines exist.

Bacterins containing antigens to the prevalent serotypes of Actinobacillus pleuropneumoniae (APP) have been commercially available for several years. These products consist of chemically-inactivated, oil- or aluminum-adjuvanted, whole-cell preparations. However, it has been repeatedly demonstrated both experimentally and with field usage that although vaccination with these products may reduce the clinical symptoms, pneumonia, and mortality associated with acute infection, vaccinated pigs may P:\OPER\MALVnw saion ftt=00371H229 Pork CRC Ud doc-14/0I lt2Xi9 -3still become subclinically or chronically infected. Furthermore, the use of these products can be associated with systemic or local untoward reactions and many of the available vaccines cause side effects such as infection site swelling and abscesses, pyrexia and 00 lethargy. EP-A-420,743 proposes a vaccine containing inactivated toxin of serotype 1 and optionally an inactivated toxin of another serotype of Actinobacillus pleuropneumoniae.

SThis vaccine provides protection against serotype 1 and only partial protection against other serotypes. The lack of a safe effective vaccine for immunisation of pigs against pleuropneumonia has engendered considerable research, but the problems involved have been found to be immunologically very complex.

Accordingly, there exists an ongoing need to develop new safe and effective methods for the immunisation of pigs against Actinobacillus pleuropneumoniae.

Summary of the Invention Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of', and variations such as "consists essentially of' will be understood to indicate that the recited element(s) is/are essential i.e.

necessary elements of the invention. The phrase allows for the presence of other nonrecited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined. It will be appreciated by those skilled in the art that one of the advantages of the method of the present invention is that it is not necessary to administer the vaccine preparation in conjunction with antibiotic treatment. Accordingly, in the context of the present invention one would understand that the phrase "consists P:\OPERWALU-n 1-o n PrUIn307 11229 Pod1 CRC Lid docl-4l/1201 -4essentially of' excludes, for instance, the co-administration or subsequent administration of Santibiotics.

0 O One aspect of the present invention is directed to a method of inducing an immune response in a pig to Actinobacillus pleuropneumoniae comprising administering to the pig an effective amount of a vaccine preparation comprising live Actinobacillus pleuropneumoniae, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to A ctinobacillus pleuropneumoniae.

In another aspect the present invention is directed to a method of inducing an immune response in a pig to Actinobacillus pleuropneumoniae consisting of administering to the pig an effective amount of a vaccine preparation consisting of live Actinobacillus pleuropneumoniae, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae.

A further aspect of the invention is directed to a method for protecting a susceptible pig against infection by Actinobacillus pleuropneumoniae the method comprising the steps of: identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; and (ii) administering to a pig an effective amount of live Actinobacillus pleuropneumoniae serotype of step in the form of a vaccine preparation, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae.

P IOPERW1ALIn- ron ftc 0017I229 Pork CRC Lid doc-I4l A1/2/19 c. Yet another aspect of the invention is directed to a method for protecting a susceptible pig against infection by Actinobacillus pleuropneumoniae the method comprising the steps of: 00 identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; S(ii) isolating the Actinobacillus pleuropneumoniae serotype of step (iii) culturing the isolated Actinobacillus pleuropneumoniae serotype of step (ii); and (iv) administering to the pig an effective amount of the cultured Actinobacillus pleuropneumoniae of step (iii) in the form of a vaccine preparation, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae.

Detailed Description of the Invention Sow herds with Actinobacillus pleuropneumoniae are typically seropositive and the sows pass high concentrations of protective maternal antibodies via colostrum and milk to their offspring. Therefore, vaccination against Actinobacillus pleuropneumoniae is typically carried out after weaning since maternal antibodies have been shown to interfere with the development of active immunity in neonatal pigs. The present invention is predicated on the discovery that administration of live Actinobacillus pleuropneumoniae to pigs which are still protected by maternal antibodies induces an immune response which increases the resistance of the pigs to future disease caused by Actinobacillus pleuropneumoniae. More particularly, in an embodiment, it has been determined that administration of a single dose of unattenuated live Actinobacillus pleuropneumoniae to suckling pigs protects the pigs against subsequent challenge with Actinobacillus pleuropneumoniae.

Accordingly, one aspect of the present invention is directed to a method of inducing an P \PERVMALhm iosn PaiUU0716229 Pork CRC LiddoC.IJIWTZR)9 -6immune response in a pig to Actinobacillus pleuropneumoniae comprising administering Sto the pig an effective amount of a vaccine preparation comprising live Actinobacillus pleuropneumoniae, optionally in combination with one or more carriers and/or diluents 00 acceptable for veterinary use, said pig having a protective level of maternal antibodies to S 5 A ctinobacillus pleuropneumoniae.

In another aspect the present invention is directed to a method of inducing an immune I response in a pig to Actinobacillus pleuropneumoniae consisting of administering to the pig an effective amount of a vaccine preparation consisting of live Actinobacillus pleuropneumoniae, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to A ctinobacillus pleuropneumoniae.

As detailed herein before maternal antibodies present in colostrum and milk provide young pigs with passive immunity against Actinobacillus pleuropneumoniae. Accordingly, reference to a protective level of maternal antibodies is reference to a level of antibodies acquired by passive transfer from a sow to a pig which is sufficient to prevent or reduce disease caused by Actinobacillus pleuropneumoniae.

Weaning of pigs typically occurs or begins when the pigs are between 10 and 29 days of age. However, circulating maternal antibodies may still be present up until 12 weeks of age. Accordingly, it should be understood that reference to a "pig" as used herein is reference to a pig having a protective level of maternal antibodies. This includes both pigs which are still receiving milk from a sow, i.e. a suckling pig, as well as pigs which are also receiving alternative forms of nutrition. In a preferred embodiment of the invention the pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae is a suckling pig.

Reference to the induction of an immune response as used herein and in the claims refers to the ability to induce an active immune response within the pig whereby the pig produces its own immune response against Actinobacillus pleuropneumoniae, as opposed to the P)OPERW4ALfnnovaln Palcnai7 19229 Pork CRC Lid doc-I 112(019 -7- (t Spassive immunity provided by maternal antibodies. The induction of an immune response Sas referred to herein therefore increases the resistance of the pigs to infection by and/or disease caused by future challenge with Actinobacillus pleuropneumoniae. Preferably, 0 0 said immune response is the production of antibodies against Actinobacillus S 5 pleuropneumoniae. Preferably, said immune response is a mucosal immune response.

SPreferably, said immune response correlates to the induction of long term immunity.

SAccordingly, reference to an "effective amount" means an amount of live Actinobacillus pleuropneumoniae bacteria sufficient to induce an immune response which increases the resistance of a pig to disease caused by Actinobacillus pleuropneumoniae. The effective amount of live Actinobacillus pleuropneumoniae administered per unit dose depends, among other things, the particular serotype of Actinobacillus pleuropneumoniae chosen, the body weight of the pig, and the chosen inoculation regimen. In preferred, non-limiting embodiments of the invention, an effective amount of vaccine produces an elevation of anti-bacterial antibody titer to at least two times the antibody titer prior to vaccination.

Preferably approximately 10 3 to 1010 cfu of bacteria are administered to a pig. More preferably approximately 105 to 1010 cfu of bacteria are administered. Even more preferably 10 7 to 1010 cfu of bacteria are administered.

Without limiting the present invention to any one theory or mode of action, to monitor the response of individual pigs administered the preparations of the invention, anti- Actinobacillus pleuropneumoniae antibody levels may be determined. In many instances it will be sufficient to assess the antibody level in serum or plasma obtained from a vaccinated pig. Decisions as to whether to administer another dose or to change the amount of the preparation administered to the pig may be at least partially based on the antibody levels. However, based on the present methodology a single dose is generally sufficient to provide a protective immune response in a pig. Methods for the detection of antibodies or said bacteria in a pig include immunoassays. Such immunoassays are known in the art and include, but are not limited to radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), fluorescent immunoassays, and fluorescence polarization immunoassays (FPIA). However, it should be understood that a protective immune P\OPER\MAL\4n,,o-uion pfit,,I3OIR229 Pok CRC Uddoc-14111209 -8c.s response may be achieved without the detection of anti-Actinobacillus pleuropneumoniae Santibodies within the pig due to interference by maternal antibodies. In this instance, without limiting the invention in any way the response of individual pigs may be 0 o determined by subsequent challenge with Actinobacillus pleuropneumoniae. The selection S 5 of a suitable method of monitoring the response of pigs administered the preparations of Sthe invention will be well within the capabilities of the person skilled in the art.

Without limiting the present invention to any one theory or mode of action, as stated herein before, passive immunity conferred by maternal antibodies has been typically shown to start to disappear around 4 weeks of age and disappear almost entirely by 12 weeks of age.

Accordingly, in a preferred embodiment of the invention the vaccine preparation comprising live Actinobacillus pleuropneumoniae is administered to pigs which are younger than 12 weeks of age. Even more preferably, the vaccine preparation comprising live Actinobacillus pleuropneumoniae is administered to pigs which are younger than 4 weeks of age.

The use of live bacteria to induce an immune response to itself or to a carried vaccine component is an attractive vaccine strategy. Advantages of live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. As detailed hereinbefore, the inventors have determined that administration of live unattenuated Actinobacillus pleuropneumoniae bacteria to pigs has been demonstrated to induce a protective immune response against disease caused by future challenge with Actinobacillus pleuropneumoniae. Accordingly, in a preferred nonlimiting embodiment of the invention the Actinobacillus pleuropneumoniae is unattenuated.

However, it should be understood that the administration of live attenuated bacteria is also contemplated. Reference to attenuated bacteria should be understood as reference to a live bacteria which has reduced virulence as compared to unattenuated bacteria. Without limiting the present invention to any one theory or mode of action, an attenuated bacterium can be obtained in several ways. One possibility is to introduce a mutation into one or M:\PERVAULn 00718229 Pork CRC Lid dow-TJIT If2(IJ9 -9more genes encoding functional proteins of the bacterium, preferably by utilising recombinant DNA techniques. A mutation is understood to be a change of the genetic information in the region with respect to the genetic information present in this region of o 0 the genome of the wild-type bacterium. The mutation is, for example, a nucleic acid substitution, deletion, insertion or inversion, or a combination thereof resulting in a Sbacterium which fails to infect and/or colonize. Methods to introduce the mutations into the specific genomic regions are well-known and will be apparent to the skilled person.

i For instance, the whole gene to be mutated or a fragment is cloned into a vector and modified in order to abolish its expression and/or its biological activity. The modified DNA fragment is reintroduced into the bacterial genome by genetic recombination, preferably by homologous recombination between the bacterial chromosome and the vector. An attenuated bacterium can also be obtained by culturing under conditions that disable their virulent properties. It would fall within the capability of the person of ordinary skill in the art to determine the most suitable attenuation protocol.

As detailed herein before there are 15 known serovars of Actinobacillus pleuropneumoniae. Accordingly, reference to Actinobacillus pleuropneumoniae is reference to any serovar of Actinobacillus pleuropneumoniae. In a preferred embodiment of the invention the serovar is one or more of serovars 1, 5, 7 and The method of the present invention is predicated on the determination that the administration of live Actinobacillus pleuropneumoniae to pigs as a single dose increases the resistance of said pig to future challenge with Actinobacillus pleuropneumoniae.

Accordingly in a preferred embodiment of the invention the dose is administered once.

However, it should be understood that subsequent doses may be administered.

The vaccine preparation may be administered in any convenient manner such as by the oral, intravenous (where water soluble), intranasal, intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting using slow release molecules). As detailed herein before, Actinobacillus pleuropneumoniae causes pleuropneumonia, an infection of the respiratory mucosa of pigs primarily centered in the P.AOPERWMALtnmovation PaRlw1)7I8229 Pork CRC Lid dm- 14lh/21X)9 c. lungs. The protective threshold is provided by IgA antibodies produced in the respiratory Smucosa. Accordingly, to the extent that a mucosal immune response is sought, the vaccine preparation is preferably administered orally or intranasally. In a more preferred 0 0 embodiment of the invention the vaccine preparation is administered intranasally.

SInocula are typically prepared as a solution in a physiologically acceptable carrier to form an aqueous preparation suitable for veterinary use. The physiologically acceptable carrier N is one that does not cause an adverse physical reaction upon administration and one in which the bacteria retain their activity to deliver a pharmaceutically or therapeutically effective amount to the pig to which it is administered. Carriers and/or diluents suitable for veterinary use include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the preparation is contemplated.

For oral administration, the formulation of the vaccine preparation may be presented as capsules, tablets, powders, granules, or as a suspension. The preparation may have conventional additives, such as lactose, mannitol, corn starch, or potato starch. The preparation also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins. Additionally, the preparation may be presented with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose.

The preparation may be further presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. The preparation may be presented with lubricants, such as talc or magnesium stearate.

For intranasal administration nasal sprays) and/or pulmonary administration (administration by inhalation), formulations of the vaccine preparation, including aerosol formulations, may be prepared in accordance with procedures well known to persons of skill in the art. Aerosol formulations may comprise either solid particles or solutions (aqueous or non-aqueous). Nebulizers jet nebulizers, ultrasonic nebulizers, etc.) and P OPER\MALUnnmolion Puenl 071o8229 Pork CRC Ltd doc.4l ll/2l09 -11c. atomizers may be used to produce aerosols from solutions using a solvent such as ethanol); metered-dose inhalers and dry-powder inhalers may be used to generate smallparticle aerosols. The desired aerosol particle size can be obtained by employing any one 0 0 of a number of methods known in the art, including, without limitation, jet-milling, spray S 5 drying, and critical-point condensation.

Supplementary active ingredients can also be incorporated into the vaccine preparations.

The latter is particularly contemplated as far as the present invention extends to multicomponent vaccines. Accordingly, in another embodiment, the vaccine preparations of the present invention may comprise in addition to Actinobacillus pleuropneumoniae, one or more other active compounds such as antigens and or immune stimulating compounds.

Optionally, one or more compounds having adjuvant activity may be added to the vaccine preparation. Adjuvantia are non-specific stimulators of the immune system. They enhance the immune response of the host to the invading pathogen. Examples of adjuvantia known in the art are Freunds Complete and Incomplete adjuvans, vitamin E, non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulating complexes, cf. for instance European Patent ER 109942), Saponins, mineral oil, vegetable oil, and Carbopol (a homopolymer). Adjuvantia, specially suitable for mucosal application are e.g. the E. coli heat-labile toxin (LT) or Cholera toxin Other suitable adjuvants are for example aluminium hydroxide, phosphate or oxide, oil-emulsions of Bayol F or Marcol 52 saponins or vitamin-E solubilisate.

Therefore, in another embodiment, the vaccine preparation according to the present invention comprises an adjuvant.

Without limiting the present invention to any one theory or mode of action to the extent that there is limited cross-reactivity between known serovars of Actinobacillus pleuropneumoniae it will be advantageous to vaccinate pigs with the same Actinobacillus pleuropneumoniae serovar to which the sows from which the pigs have been receiving milk have been exposed.

P 'OPERjAL oon -0-12 I29 Pok CRC Ltd do14MI/(X4 Y -12t- According to this embodiment the invention is directed to a method for protecting a susceptible pig against infection by Actinobacillus pleuropneumoniae the method oO comprising the steps of: identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; and (ii) administering to a pig an effective amount of live Actinobacillus pleuropneumoniae serotype of step in the form of a vaccine preparation, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae.

Preferably the pig is a suckling pig.

Preferably, the method consists essentially of steps and (ii).

Preferably, the vaccine preparation is administered intranasally. Preferably the dose is administered as a single dose the range 10 7 and 1010 cfu.

Reference to a "susceptible pig" is reference to pigs which still have protective levels of maternal antibodies but which would be susceptible to infection by and/or disease caused by Actinobacillus pleuropneumoniae once the protective levels of maternal antibodies are no longer present.

Methods for identifying Actinobacillus pleuropneumoniae strains will be well known to those skilled in the art. Without limiting the present invention in any one, to the extent that antibody based methods of identification are used, the presence of a serovar of P %OPERNAALJ,,,oa:on Paimt\(I71922 Pork CRC Liddoc-1I49)UM O -13- C. Actinobacillus pleuropneumoniae may be determined in a number of ways such as by SWestern blotting, or ELISA. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive 00 binding assays. These assays also include direct binding of a labelled antibody to a target S 5 and other immunoassays as herein before described. Examples of other assays that may be used include, indirect haemagglutination assays (IHA), agar plate fermentation techniques, the ring precipitation test and DNA based identification methods. It should be understood that selection of a suitable identification technique will be well within the capabilities of the person skilled in the art.

Without limiting the present invention to any one theory or mode of action, the method of the invention may also include the additional steps of isolating and culturing the Actinobacilluspleuropneumoniae serovar for administration to a pig.

According to this embodiment the invention is directed to a method for protecting a susceptible pig against infection by Actinobacillus pleuropneumoniae the method comprising the steps of: identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; (ii) isolating the Actinobacillus pleuropneumoniae serotype of step (iii) culturing the isolated Actinobacillus pleuropneumoniae serotype of step (ii); and (iv) administering to the pig an effective amount of the cultured Actinobacillus pleuropneumoniae of step (iii) in the form of a vaccine preparation, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae.

P \OPERVAALkIino,-aion Palnm13)07R1229 Por CRC Uld doc.I!IA/12UN)9 -14- SPreferably the pig is a suckling pig.

Preferably, the vaccine preparation is administered intranasally. Preferably the dose is 00 administered as a single dose the range 107 and 1010 cfu.

SPreferably, the method consists essentially of steps to (iv).

Reference to the locus of the pig should be understood as reference to where it is located.

Without limiting the present invention in any way, a pig's locus may include a farm or research facility.

Isolation techniques will also be well known to those skilled in the art. As an example the isolation of an Actinobacillus pleuropneumoniae serovar may be performed by inoculation of a biological sample such as, for example, blood, fluid removed from lesions and swabs of the respiratory tract of an animal presenting with disease, into culture media or onto agar plates and the bacteria grown under suitable growth conditions. It should be understood that selection of a suitable isolation technique will be well within the capabilities of the person skilled in the art.

Methods used for culturing Actinobacillus pleuropneumoniae bacteria will also be well known to those skilled in the art and include growing bacteria in a suitable basal essential culture medium, such as but not limited to commercially available brain heart infusion broth "BHI", Luria broth sheep blood agar "SBA", Brucella broth, Meuller-Hinton broth, proteose peptone beef extract broth, etc., with various conditions and components including but not limited to 0.05% to 3% bile or 0.025% to 0.6% of one or more bile acids or salts thereof or biological precursors thereof such as cholesterol, at a temperature between 300 C. and 420 until a growth phase at about early log phase, between early log and stationary phases, or at about stationary phase, in air or under microaerophillic conditions, such as 5% to 20% CO 2 with 80% to 95% air; 5% to 20% CO 2 with 80% to 95% N 2 or 5% to 10% 02 with 10% to 20% CO 2 with 70% to 85% N 2 and optionally in the presence of a divalent cation chelator, such as, but not limited to 0 to 100 M, PA0PERMAL\Innosion PhI,,T3U0I7J229 Pork CRC Ltd doc-Jl I4/2W9 0 h preferably 25 M, of BAPTA/AM (2'(ethylenedioxy)dianilinen,n,n',n'-tetraacetic Sacid/acetoxymethyl ester; Molecular Probes, Eugene, OR), 0 to 10 mM of EGTA (ethylenebis(oxyethylenenitrilo)-tetraacetic acid; Sigma Chemical Co., St. Louis, 0 00 to 100 iM of EGTA/AM (ethylenebis(oxyethylenenitrilo)-tetraacetic acid/acetoxymethyl S 5 ester; Molecular Probes, Eugene, OR).

It should be understood that the Actinobacillus pleuropneumoniae bacteria may be stored Sfor future use. There are several ways to store live organisms. Storage in a refrigerator is e.g. a well-known method. Also often used is storage at -800 C in a buffer containing glycerol. Bacteria can be harvested for freezing by any known method, for instance by swabbing the culture and resuspending in BHI containing 30% glycerol. Cultures for analytical experiments or for production fermentations can be prepared by any generally known methods, such as by growing the organism on BHI with 1.5% agar at 370 C under MC or atmospheric conditions and then transferring a single colony to broth and culturing.

Bacteria can be harvested for use by any method generally known to those skilled in the art, such as by centrifugation.

Bacteria can also be kept in liquid nitrogen. Freeze-drying is another way of conservation.

Freeze-dried bacteria can be stored and kept viable for many years. Storage temperatures for freeze-dried bacteria may well be above zero degrees, without being detrimental to the viability. Freeze-drying is equally applicable for subunits. Freeze-drying can be done according to all well-known standard freeze-drying procedures. Optional beneficial additives, such as e.g. skimmed milk, trehalose, gelatin or bovine serum albumin can be added in the freeze-drying process.

Therefore, in another embodiment, the vaccine preparation according to the present invention is in a freeze-dried form. According to this preferred embodiment it would be understood that the vaccine preparation may be reconstituted in a suitable medium before use. The choice of a suitable medium would be well within the capabilities of the person skilled in the art.

POPERWMALsmno- son Patinis,1071129 Pork CRC Lid doc-4AII 2N)9 -16- C-s The invention will now be described with reference to the following examples which are Sintended only for the purpose of illustrating certain embodiments of the invention and are not to be taken as limiting the generality of the invention previously described.

00 Examples Example 1 Evaluation of an intranasal vaccine formulation to control A.pleuropneumoniae (App) infection using pen challenge studies.

The purpose of this study was to determine the efficacy of a live Actinobacillus pleuropneumoniae (App) 5 vaccine given intranasally in promoting immunity against Actinobacillus pleuropneumoniae infection. One group of piglets was given the intranasal vaccine at 17-18 days of age. A second group from the same farm separated by about 500 metres remained unvaccinated. All pigs were treated on day of transport to Bendigo with ml of Draxin providing 14 days of antibiotic cover against Actinobacillus pleuropneumoniae. The vaccinated and non vaccinated were raised in isolation until 18 weeks of age then challenged with a homologous strain of Actinobacillus pleuropneumoniae Unvaccinated pigs had significantly more Actinobacillus pleuropneumoniae lung lesions (P<0.001) than pigs that had been vaccinated. Unvaccinated pigs also had significantly worse demeanour and breathing scores from day 4 after challenge (P>0.02).

The results indicate that the intranasal live vaccine proved highly efficacious in protecting vaccinated pigs against a challenge with the homologous strain of Actinobacillus pleuropneumoniae.

P \OPERWMALVnnion Palw\0l718229 Por CRC Lid dom4)II20O9 17- Methods Sixteen piglets (Group 1) were intranasally vaccinated with 1 ml of live Actinobacillus pleuropneumoniae vaccine at 18 days of age whilst still in their farrowing crates. Sixteen matched pigs from the same farm, but held in a different shed to ensure no aerosol spread from the vaccinated piglets, remained unvaccinated.

Six days after vaccination trial pigs were transported to Bendigo. Pigs vaccinated with the live vaccine were transported and held separately from the unvaccinated pigs until the time of challenge.

Table 1 Trial Log Date Event 16/2/08 Intranasal vaccine group piglets, at 18 days of age, given 1 ml live vaccine intranasally. Control groups of pigs from same farm kept separate from vaccinated pigs 22/2/08 All pigs treated with Draxxin to minimise risk of Glassers disease.

Pigs at 24 days of age collected and transported to Bendigo.

Unvaccinated pigs housed separately from vaccinated pigs at this stage.

8/5/08 Pigs treated with Lincomycin for diarrhoea 16/5/08 All pigs treated with Linco-spectin for looseness 2/6/08 All pigs commingled and challenged intranasally with App serovar 6/6/08 Control pig euthanized (W135) due to severe respiratory distress due to App infection 9/6/08 Intranasally vaccinated pig euthanized (B461) due to severe, debilitating lameness affecting the right shoulder Strep spp.

isolated on autopsy. No App lesions seen at autopsy.

P XOPERMALIn,,- ton PaIcrnoml718229 Pork CRC Ltd doc-4t )112(M)9 18- Date Event 12/6/08 Trial terminated and all surviving pigs euthanized and post mortem inspections performed.

Table 2 Treatment groups for each challenge Number Volume Age at primary Age at booster Group of pigs (mL) vaccination vaccination Live Intranasal One mL intra 16 18 days No booster App vaccine intranasally Control pigs 16 No vaccination Challenge At seventeen weeks of age all pigs were commingled and intranasally challenged with the homologous Actinobacillus pleuropneumoniae serovar Data collected Demeanour Score Each pig was observed daily, (more frequently during the first days after the challenge), for evidence of well being. Demeanour was scored according to Table 2.

Table 3 Demeanour score Score Demeanour 0 Normal Quiet/ reluctant to move Depressed/ reluctant to move even when encouraged Very depressed/moribund PAOPIRF MALInal0on Patmi\30 7IXN229 Pok CRC Ltd CI.14AI /I(M9 -19- Breathing score Each pig was observed daily, more frequently during the first days after challenge for evidence of respiratory distress.

Table 4 Breathing score Score Breathing condition 0 Normal Shallow uneven Uneven breathing and coughing Thumping of flanks and/or gasping Animals with a score of 2.0 were euthanised on welfare grounds.

Lung lesions Post mortem examinations were performed on all pigs. Gross lung pathology was recorded. For analysis lungs were categorised as positive or negative for lung lesions caused by Actinobacillus pleuropneumoniae Statistical Analysis Response variables were: 1. Lung lesions 2. Demeanour 3. Breathing problems Statistical analyses were done using StatExact, Cytel Statistical Software, Cytel Software Corporation, MA, USA.

P AOPERMAL\InAoution PtRcn[01 7 19229 Pork CRC Ltddoc-.4flu/2W9 Results One pig from each group was euthanised during the trial. A post mortem examination of control pig W135 revealed lung lesions consistent with Actinobacillus pleuropneumoniae infection. Pig B461 from the vaccination group was euthanized because of severe lameness. The post mortem examination found the animal had polyarthritis due to streptococcus infection. No lung lesions were evident at necropsy.

From day 4 after challenge, the control pig's demeanour was significantly more impaired than the pigs from the Intranasalvaccination group (Table Similarly the control pigs had consistently significantly impaired breathing from day 4, compared to pigs that were intranasally vaccinated (Table 6).

Table 5 Daily frequency of demeanour scores for vaccinated and Nonvaccinated pigs Day Control Intranasal vaccination P values Frequency x (score in Frequency x (score in Control v Intranasal brackets) brackets) 1 15(0),1 16(0) 2 14(0),2(1) 16(0) 0.24 3 14(0),2(1) 16(0) 0.24 4 1(3) 16(0) 0.02 1(3) 16(0) 0.02 6 1(3) 16(0) 0.01 8 1(3) 15(0) 0.03 9 1(3) 15(0) 0.03 1(3) 15(0) 0.002 11 1(3) 15(0) 0.004 pig euthanised for App was given score 3 for remainder of trial P:'OPERWALInmovaulon P-IcnIVI 7 I X229 Pork CRC Ltd doc.14'i I/2119 -21 Daily frequency of breathing scores for vaccinated and Non-vaccinated pigs Table 6 Day Control Intranasal Vaccination P values Frequency x (score in brackets) Frequency x (score in Control brackets) Intranasal 1 11(0),5(1) 16(0) 0.02 2 14(0),2(1) 16(0) 0.24 3 15(0),1 16(0) 0.50 4 1(3) 16(0) 0.01 1(3) 16(0) 0.001 6 1(3) 16(0) <0.001 7 1(3) 15(0) <0.001 8 1(3) 15(0) <0.001 9 1(3) 15(0) <0.001 1(3) 15(0) <0.001 pig euthanised for App was given score 3 for remainder of trial There were significantly more pigs with Actinobacillus pleuropneumoniae lung lesions in the control pigs compared with pigs that had been intranasally vaccinated (Table 7).

Table 7 Frequency distribution for App lung lesions by treatment, and significances of treatment differences.

Lung lesions present P values Treatment No Yes Control 4 12 Intranasalvacc 14 2 <0.001 POPERMALXInoswion PoRjnU 377 I 229 Pork CRC LUddoc.14)1R1/219 -22- There were significantly more pigs with lung abscesses in the control pigs compared with pigs that had been Intranasally vaccinated (Table 8).

Table 8 Frequency distribution for Abscesses by treatment, and significances of treatment differences.

Lung Abscesses present P values Treatment No Yes Control 4 12 Oralvacc 14 2 <0.001 There were no significant treatment effects on pleurisy scores (Table 9).

Table 9 Frequency distribution for Pleurisy scores by treatment, and significances of treatment differences.

Pleurisy score P values Treatment 0 1 2 3 Control 14 1 1 Intranasalvacc 14 1 1 0.43 PI\OPERWALOnIoaton Palc=,1V718229 Pork CRC Ud docI4lr Il2(X)9 -23 Post Mortem results Table 00 Animal ID Treatment group Post mortem findings 0 Fluid filled right cardiac lobe. Multiple

W

10 1 Control abscesses; diameters 4cm, 2cm, 7 8cm. Left side abscess 7*3cm W102 Control Multiple abscess 9cm, 10*12 cm and 18*9cm Golf ball abscess left diaphragmatic lobe. 4 cm W103 Control abscess right lobe W104 Control Two 10cm diameter abscesses Multiple abscesses 8cm diameter, 4 cm W126 Control W126 Control diameter, and two lesions 7*4 cm W127 Control multiple abscesses W128 Control lesions W129 Control 8cm abscess right lobe W130 Control 8 cm abscess and 40% pneumonia right lobe W131 Control normal W132 Control normal W133 Control normal W134 Control normal W135 Control 100% consolidation, abscess, grade 2 pleurisy W136 Control 8 cm diameter abscess pericarditis, grade 3 pleurisy and multiple W137 abscesses; 12cm and 8cm diameter P kOPERMA\Inor-(on Pateni\10718229 Pork CRC liddw .199)1/21X)9 -24- Table 11 Animal ID B451 B452 B453 B454 B455 B456 B457 B458 B459 B460 B461 B462 B463 B464 B465 B466 Treatment group Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Intranasal vaccination Post mortem findings normal Oedema of left lobe of lung and a 4* 1.5cm abscess in the left apical lobe grade 1 pleurisy affecting 50% of left apical lobe normal normal normal normal normal normal normal normal normal of Left cardiac lobe affected with enzootic pneumonia normal Enzootic pneumonia affecting 20% of left diaphragmatic lobe. 4cm diameter abscess present and grade 2 pleurisy over the lesion normal Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit POPERWMAL\kn[o iron cnI\"0 I22 9 Por CRC Liddoc. II'1/2119 Sand scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims (4)

1. A method of inducing an immune response in a pig to Actinobacillus 00 pleuropneumoniae comprising administering to the pig an effective amount of a vaccine preparation comprising live Actinobacillus pleuropneumoniae, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to Actinobacillus Spleuropneumoniae.

2. A method for protecting susceptible pigs against infection by Actinobacillus pleuropneumoniae the method comprising the steps of: identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; and (ii) administering to a pig an effective amount of live Actinobacillus pleuropneumoniae serotype of step in the form of a vaccine preparation, optionally in combination with one or more carriers and/or diluents acceptable for veterinary use, said pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae.

3. A method for protecting susceptible pigs against infection by Actinobacillus pleuropneumoniae the method comprising the steps of: identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; (ii) isolating the Actinobacillus pleuropneumoniae serotype of step (iii) culturing the isolated Actinobacillus pleuropneumoniae serotype of step (ii); and PXOPERMAL\ncosIion PolcIlVl718229 Pork CRC Lid dcc 14/i/20(X -27- c. (iv) administering to a pig an effective amount of the cultured Actinobacillus pleuropneumoniae of step (iii) in the form of a vaccine preparation, optionally in combination with one or more carriers and/or diluents 00 acceptable for veterinary use, said pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae.

4. A method according to any one of claims 1 to 3 wherein the pig having a protective level of maternal antibodies to Actinobacillus pleuropneumoniae is a suckling pig. A method according to any one of claims 1 to 4 wherein the vaccine preparation administered orally or intranasally.