WO2010042970A1

WO2010042970A1 - A method of vaccination pigs

Info

Publication number: WO2010042970A1
Application number: PCT/AU2009/000061
Authority: WO
Inventors: Peter Mckenzie; Virgil Anthony Fahy
Original assignee: Pork Crc Ltd
Priority date: 2008-10-15
Filing date: 2009-01-21
Publication date: 2010-04-22
Also published as: AU2009100018A4; AU2009100018B4

Abstract

The present invention relates to a method of vaccinating pigs to increase their resistance to infection by Actinobacillus pleuropneumoniae. More particularly, the present invention relates to methods of inducing an immune response in a pig against porcine pneumonia caused by Actinobacillus pleuropneumoniae.

Description

A METHOD OF VACCINATING PIGS

Field of the Invention

The present invention relates to a method of vaccinating pigs to increase their resistance to infection by Actinobacillus pleuropneumoniae. More particularly, the present invention relates to methods of inducing an immune response in a pig against porcine pneumonia caused by Actinobacillus pleuropneumoniae.

Background of the Invention

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 7 are reportedly the most common types of Actinobacillus pleuropneumoniae found in the United 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 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 affected 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 still 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 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. This 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 pleuropneumonias

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 non- recited 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 essentially of excludes, for instance, the co-administration or subsequent administration of antibiotics.

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 ActinobacHlus pleuropneumoniae.

In another aspect the present invention is directed to a method of inducing an immune response in a pig to ActinobacHlus pleuropneumoniae consisting of administering to the pig an effective amount of a vaccine preparation consisting of live ActinobacHlus 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 ctinobacHlus pleuropneumoniae.

A further aspect of the invention is directed to a method for protecting a susceptible pig against infection by ActinobacHlus pleuropneumoniae the method comprising the steps of:

(i) identifying the ActinobacHlus pleuropneumoniae serotype present within the pig's locus; and

(ii) administering to a pig an effective amount of live Actinobacillus pleuropneumoniae serotype of step (i) 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.

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:

(i) identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus;

(ii) isolating the Actinobacillus pleuropneumoniae serotype of step (i); (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,

Accordingly in yet another embodiment the invention is directed to a method for protecting a susceptible pig against infection by Actinobacillus pleuropneumoniae the method comprising the steps of:

(ii) isolating the Actinobacillus pleuropneumoniae serotype of step (i);

(iii) culturing the isolated Actinobacillus pleuropneumoniae serotype of step (ii);

(iv) testing the sow herd for the presence of Actinobacillus pleuropneumoniae antibodies; and (v) 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, and optionally testing the antibiotic sensitivity of the Actinobacillus pleuropneumoniae;

Brief Description of the Drawings

Figure 1 is an image of an intranasal vaccination of sucker pigs with live APP. Figure 2 is an image of an intranasal vaccine gun showing delivery nozzle.

Figure 3 is a graphical representation showing mortality rates on two farms (Ink and SP) using live APP delivered by aerosol in 2008. Production on SP was confounded by an outbreak of S. suis in 2008, see Figure 4.

Figure 4 is a graphical representation showing mortality rates at slaughter of batches treated with live APP or the killed autogenous vaccine over 2007 and 2008. Units INK and SP, 2007 and 2008. N=3600 per treatment cycles: 1^st conventional^ first group of pigs given the killed vaccine; 2^nd aerosol=second group of pigs given live APP by aerosol delivery; 3^rd conventional third group of pigs given the killed vaccine; 4^th aerosol^ fourth groups given live APP by aerosol delivery. The data demonstrate the superior performance of pigs vaccinated with live APP compared to the killed vaccine over the high risk 13-16 week age group.

Figure 5 is a graphical representation showing mortality rates (% weekly inventory) of sequential batches treated with live APP or an autogenous killed vaccine (controls). N (treatment) = 3600, N (control) = 7200. Farm three. Controls were designated C-4 for the first four weeks of control treatments before the vaccinated groups were introduced and C+4 for the four weeks of control treatments after the vaccinated pigs were put into the system.

Figure 6 is a graphical representation of cumulative mortality rate of batches in Figure 5. Farm three.

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 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 Actinobacillus 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.

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 ActinobacHlus 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 ActinobacHlus pleuropneumoniae, as opposed to the passive immunity provided by maternal antibodies. The induction of an immune response as referred to herein therefore increases the resistance of the pigs to infection by and/or disease caused by future challenge with ActinobacHlus pleuropneumoniae. Preferably, said immune response is the production of antibodies against ActinobacHlus pleuropneumoniae. Preferably, said immune response is a mucosal immune response. Preferably, said immune response correlates to the induction of long term immunity.

Accordingly, reference to an "effective amount" means an amount of live ActinobacHlus pleuropneumoniae bacteria sufficient to induce an immune response which increases the resistance of a pig to disease caused by ActinobacHlus pleuropneumoniae. The effective amount of live ActinobacHlus pleuropneumoniae administered per unit dose depends, among other things, on the particular serotype of ActinobacHlus 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 10³ to 10¹⁰ cfu of bacteria are administered to a pig. More preferably approximately 10⁵ to 10¹⁰ cfu of bacteria are administered. Even more preferably 10⁷ to 10¹⁰ cfu of bacteria are administered. Preferably the bacteria are administered in solution in a volume of about 0.5mL of a physiologically acceptable carrier. Such physiologically acceptable carriers will be well known to those of skill in the art and include, for example, normal saline. In an embodiment the bacteria is administered in an about 0.9% normal saline solution at a volume of about 0.5mL.

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 response may be achieved without the detection of εinti-Actinobacillus pleuropneumoniae antibodies 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 determined by subsequent challenge with Actinobacillus pleuropneumoniae. The selection of a suitable method of monitoring the response of pigs administered the preparations of the 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, the present inventors have determined that active immunity to the Actinobacillus pleuropneumoniae serovar administered develops by 5 days following vaccination. Accordingly, it is preferable when carrying out the method if the invention that the treatment of pigs with antibiotics is avoided for 5 days after vaccination. Preferably the treatment of pigs with antibiotics is avoided for 5 days before and after vaccination.

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. More preferably, the vaccine preparation comprising live Actinobacillus pleuropneumoniae is administered to pigs which are younger than 4 weeks of age. Even more preferably, the vaccine preparation is administered to pigs aged between 3 days of age and weaning or 28 days 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 non- limiting 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 more 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 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 bacterium 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.

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 15. More preferably one or more of serovars 5 and 15.

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 (e.g. using slow release molecules). As detailed herein before, Actinobacillus pleuropneumoniae causes pleuropneumonia, an infection of the respiratory mucosa of pigs primarily centered in the lungs. The protective threshold is provided by IgA antibodies produced in the respiratory mucosa. Accordingly, to the extent that a mucosal immune response is sought, the vaccine preparation is preferably administered orally or intranasally. In a more preferred embodiment of the invention the vaccine preparation is administered intranasally.

Inocula are typically prepared as a solution in a physiologically acceptable carrier to form an aqueous preparation suitable for veterinary use. The physiologically acceptable carrier 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. A preferred diluent carrier is normal saline.

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 (e.g., 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 (e.g., jet nebulizers, ultrasonic nebulizers, etc.) and atomizers may be used to produce aerosols from solutions (e.g., using a solvent such as ethanol); metered-dose inhalers and dry-powder inhalers may be used to generate small- particle aerosols. The desired aerosol particle size can be obtained by employing any one of a number of methods known in the art, including, without limitation, jet-milling, spray 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 multi- component 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 (CT). Other suitable adjuvants are for example aluminium hydroxide, phosphate or oxide, oil-emulsions (e.g. of Bayol F (R) or Marcol 52 (R), 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. In addition, to ensure that the sow herd is clinically stable prior to administration of the live Actinobacillus pleuropneumoniae preparation to a pig the antibody titre of the sows, of the Actinobacillus pleuropneumoniae to be administered, may be tested.

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:

(i) identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; and

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 (i) and (ii).

Preferably, the vaccine preparation is administered intranasally. Preferably the dose is administered as a single dose the range 10⁷ 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 Actinobacillus pleuropneumoniae may be determined in a number of ways such as by Western 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 binding assays. These assays also include direct binding of a labelled antibody to a target 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 Actinobacillus pleuropneumoniae serovar for administration to a pig.

(ii) isolating the Actinobacillus pleuropneumoniae serotype of step (i); (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,

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

Preferably, the method consists essentially of steps (i) 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.

To assist with future treatment requirements it should also be understood that it may be desirable to test the antibiotic sensitivity of the Actinobacillus pleuropneumoniae to be administered. Therefore, in relation to the embodiment of the invention described above it would be expected that any one or more of the following steps could be employed in combination with, or in addition to, steps (i) to (iv).

As described herein above the antibiotic sensitivity of the isolate to be administered may be tested. Suitable methods for testing antibiotic sensitivity will be well known to those of skill in the art.

In addition, as also described herein, to ensure that the sow herd to which the live Actinobacillus pleuropneumoniae vaccine is to be administered is clinically stable, the antibody titre of the sows, of the Actinobacillus pleuropneumoniae serovar to be administered, may be tested.

Accordingly, in yet another embodiment the invention is directed to a method for protecting a susceptible pig against infection by Actinobacillus pleuropneumoniae the method comprising the steps of:

(i) identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; (ii) isolating the Actinobacillus pleuropneumoniae serotype of step (i);

(iii) culturing the isolated Actinobacillus pleuropneumoniae serotype of step (ii); (iv) testing the sow herd for the presence of Actinobacillus pleuropneumoniae antibodies; and (v) 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, and optionally testing the antibiotic sensitivity of the Actinobacillus pleuropneumoniae;

Preferably said pig is between 3 days of age and weaning or 28 days of age.

Preferably the bacteria is administered in of about 0.9% normal saline solution at a volume of about 0.5ml.

Preferably administration is intranasally.

Preferably said pigs are not treated with antibiotics for 5 days before or after vaccination.

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 "LB", 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 30° C. and 42° C, 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% CO₂ with 80% to 95% air; 5% to 20% CO₂ with 80% to 95% N₂; or 5% to 10% O₂ with 10% to 20% CO₂ with 70% to 85% N₂; and optionally in the presence of a divalent cation chelator, such as, but not limited to 0 to 100 μM, preferably 25 μM, of BAPTA/AM (2'(ethylenedioxy)dianilinen,n,n',n'-tetraacetic acid/acetoxymethyl ester; Molecular Probes, Eugene, OR), 0 to 10 mM of EGTA (ethylenebis(oxyethylenenitrilo)-tetraacetic acid; Sigma Chemical Co., St. Louis, Mo.), 0 to 100 μM of EGTA/ AM (ethylenebis(oxyethylenenitrilo)-tetraacetic acid/acetoxymethyl ester; Molecular Probes, Eugene, OR).

It should be understood that the Actinobacillus pleuropneumoniae bacteria may be stored for 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 -80° 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 37° 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. Preferably cultures in broth are stored at -80°C or less.

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.

The invention will now be described with reference to the following examples which are intended 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.

Examples

Example 1

* Results confounded by S.suis outbreak in weaners and growers.

None of the combined mortality rates were statistically different. Results of the individual site data are shown in Figures 3 and 4.

The postweaning mortality results in Figures 3 to 6 demonstrate the efficacy of administration of the live vaccine in the prevention of mortality in the face of endemic APP infection.

The weaner accommodation on farm three is all in all out, so that at no time during the weaner phase of the trial did trial replicates share a common airspace. However, from 10 weeks onwards at farm three, all housing is continuous flow. So from 10 to 18 weeks, the Control -4 replicates shared their airspace mostly with non-vaccinates suffering endemic APP infection, and accordingly Control -4 mortality was relatively high. Only the later Control -4 replicates shared airspace with vaccinates.

Vaccinates shared a common airspace firstly with Control -4 pigs, and lastly with Control +4 pigs. The Control +4 pigs shared airspace firstly with vaccinates, and lastly with non- vaccinates.

The postweaning mortality results are consistent with these variations in the sharing of airspace. In the weaner accommodation, where no airspace was shared between groups, the difference between the vaccinated group and each of the control groups was of similar magnitude, in favour of the vaccinates.

In the grower and finisher accommodation there was a clear mortality advantage for the vaccinated group over the Control -4 group. However, there was little difference in mortality between vaccinate and Control +4 groups.

The slaughter check results (Table 2) show a significantly lower rate of pleurisy in vaccinates compared to Control -4 pigs. However Control +4 pigs had pleurisy rates scarcely higher than vaccinates. Table 2: Slaughter check results for controls and pigs vaccinated with APP-alive

Slaughter checks #1

Check # Mean

Control~4 N 30 30 30

Pleurisy% 72.7 68.4 86.4 75.2

Vaccinates N 30 30 30

Pleurisy% 48.1 45.5 42.1 46.1

Difference (absolute) 24.6 23.0 44.3 29.1

Uncorrected 0.04 0.07 0.001 Chi-square

Slaughter checks #2

Check # Mean

Control-4 N 30 30 30

Pleurisy% 42.3 41.7 42.2 42.1

Vaccinates N 30 30 30

PleuήsyVo 41.2 41.7 40.0 40.9

Difference (absolute) 1.1 0.0 2.2 1.1 Example 2

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 0.5 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 5.

Unvaccinated pigs had significantly more Actinobacillus pleuropneumoniae lung lesions (PO.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.

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 3 Trial Log

Table 4 Treatment groups for each challenge

Challenge

At seventeen weeks of age all pigs were commingled and intranasally challenged with the homologous Actinobacillus pleuropneumoniae serovar 5

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 4.

Table 5 Demeanour score

Score Demeanour

0 Normal

1.0 Quiet/ reluctant to move

1.5 Depressed/ reluctant to move even when encouraged

2.0 Very depressed/moribund

Breathing score

Each pig was observed daily, more frequently during the first days after challenge for evidence of respiratory distress. Table 6 Breathing score

Score Breathing condition

0 Normal

1.0 Shallow uneven

1.5 Uneven breathing and coughing

2.0 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.

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 7).

Similarly the control pigs had consistently significantly impaired breathing from day 4, compared to pigs that were intranasally vaccinated (Table 8).

Table 7 Daily frequency of demeanour scores for vaccinated and Non- vaccinated pigs

Day Control Intranasal vaccination P values

Frequency x (score in Frequency x (score in Control v Intranasal brackets) brackets)

__

1 15(0),l 16(0)

2 14(0),2(l) 16(0) 0.24

3 14(0),2(l) 16(0) 0.24

4 l l(0),4(l), 1(3) 16(0) 0.02

5 10(0VKl), 1(3) 16(0) 0.02

6 10(0),5(l), 1(3) 16(0) 0.01

8 H(OVKi), 1(3) 15(0) 0.03

9 H(OVKi), 1(3) 15(0) 0.03

10 8(0),7(l), 1(3) 15(0) 0.002

11 9(0),6(l), 1(3) 15(0) 0.004

pig euthanised for App was given score 3 for remainder of trial Table 8 Daily frequency of breathing scores for vaccinated and Non-vaccinated pigs

Day Control Intranasal Vaccination P values

Frequency x (score in brackets) Frequency x (score in Control > brackets) Intranasal

1 l l(0)₌5(l) 16(0) 0.02

2 14(0),2(l) 16(0) 0.24

3 15(O)₉I 16(0) 0.50

4 10(0)₅5(l), 1(3) 16(0) 0.01

5 7(0),7(l), 1(3) 16(0) 0.001

6 6(0),9(l), 1(3) 16(0) <0.001

7 4(O)₅I l(I), 1(3) 15(0) O.001

8 4(0),10(l),l(2), 1(3) 15(0) <0.001

9 4(O)₅I l(I)₅ 1(3) 15(0) O.001

10 4(0),10(l),l(2), 1(3) 15(0) O.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 9).

Table 9 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 There were significantly more pigs with lung abscesses in the control pigs compared with pigs that had been Intranasally vaccinated (Table 10).

Table 10 Frequency distribution for Abscesses by treatment, and significances of treatment differences.

Lung Abscesses present P values

Treatment No

Control 4 12

Oralvacc 14 2

_____

There were no significant treatment effects on pleurisy scores (Table 11).

Table 11 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 Post Mortem results Table 12

Animal ID Treatment group Post mortem findings

Fluid filled right cardiac lobe. Multiple

WlOl 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 Wl 03 Control abscess right lobe Wl 04 Control Two 10cm diameter abscesses

Multiple abscesses 8cm diameter, 4 cm

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 Control abscesses; 12cm and 8cm diameter Table 13

Animal ID Treatment group Post mortem findings

B451 Intranasal vaccination normal

B452 Intranasal vaccination Oedema of left lobe of lung and a 4* 1.5cm abscess in the left apical lobe grade 1 pleurisy affecting 50% of left B453 Intranasal vaccination apical lobe

Intranasal vaccination

B454 normal

Intranasal vaccination B455 normal

Intranasal vaccination B456 normal

Intranasal vaccination B457 normal

Intranasal vaccination B458 normal

Intranasal vaccination B459 normal

Intranasal vaccination B460 normal

Intranasal vaccination B461 normal

Intranasal vaccination

B462 normal

10% of Left cardiac lobe affected with B463 Intranasal vaccination enzootic pneumonia

Intranasal vaccination

B464 normal

Enzootic pneumonia affecting 20% of left diaphragmatic lobe. 4cm diameter abscess B465 Intranasal vaccination present and grade 2 pleurisy over the lesion

Intranasal vaccination

B466 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 and 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.

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.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. 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 Actinobacillus pleuropneumoniae.

2. 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.

3. A method for protecting susceptible pigs against infection by Actinobacillus pleuropneumoniae the method comprising the steps of:

4. A method for protecting susceptible pigs against infection by Actinobacillus pleuropneumoniae the method comprising the steps of:

(iii) culturing the isolated Actinobacillus pleuropneumoniae serotype of step (ii); and

(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 acceptable for veterinary use,

5. A method for protecting a susceptible pig against infection by Actinobacillus pleuropneumoniae the method comprising the steps of: (i) identifying the Actinobacillus pleuropneumoniae serotype present within the pig's locus; (ii) isolating the Actinobacillus pleuropneumoniae serotype of step (i);

(iii) culturing the isolated Actinobacillus pleuropneumoniae serotype of step (ii); (iv) testing the sow herd for the presence of Actinobacillus pleuropneumoniae antibodies; and

(v) 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, and optionally testing the antibiotic sensitivity of the Actinobacillus pleuropneumoniae;

6. A method according to any one of claims 1 to 5 wherein the pig having a protective level of maternal antibodies to Actinobacillus pleuropneurnoniae is a suckling pig.

7. A method according to any one of claims 1 to 6 wherein the vaccine preparation administered orally or intranasally.

8. A method according to claim 7 wherein the vaccine preparation is administered intranasally.

9. A method according to any one of claims 1 to 8 wherein approximately 10³ to 10¹⁰ cfu of bacteria are administered.

10. A method according to claim 9 wherein approximately 10⁵ to 10¹⁰ cfu of bacteria are administered.

11. A method according claim 10 wherein 10⁷ to 10¹⁰ cfu of bacteria are administered.

12. A method according to any one of claims 1 to 11 wherein the vaccine preparation is administered as a single dose.

13. A method according to any one of claims 1 to 12 wherein the vaccine preparation is administered in an about 0.9% normal saline solution.

14. A method according to claim 13 wherein the volume of the vaccine preparation is about 0.5 mL.

15. The method according to any one of claims 1 to 11 wherein the vaccine preparation is administered as more than one dose.

16. A method according to any one of claims 1 to 15 wherein said immune response is the production of antibodies against Actinobacillus pleuropneumoniae.

17. A method according to any one of claims 1 to 16 wherein said immune response is a mucosal immune response.

18. A method according to any one of claims 1 to 17 wherein administration of an effective amount of the vaccine preparation or Actinobacillus pleuropneumoniae produces an elevation of anti-bacterial antibody titer to at least two times the antibody titer prior to vaccination.

19. A method according to any one of claims 1 to 18 the vaccine preparation is administered to pigs which are younger than 12 weeks of age.

20. A method according claim 19 wherein the vaccine preparation comprising live Actinobacillus pleuropneumoniae is administered to pigs which are younger than 4 weeks of age.

21. A method according to claim 20 wherein the vaccine preparation is administered to pigs between 3 days of age and weaning or 28 days of age.

22. A method according to any one of claims 1 to 21 wherein the Actinobacillus pleuropneumoniae is to be administered un-attenuated.

23. A method according to any one of claims 1 to 22 wherein the Actinobacillus pleuropneumoniae is to be administered attenuated.

24. A method according to any one of claims 1 to 23 wherein the Actinobacillus pleuropneumoniae is one or more of serovars 1, 5, 7 and 15.

25. A method according to any one of claims 1 to 23 wherein the Actinobacϊllus pleuropneumoniae is serovar 5.

26. A method according to any one of claims 1 to 23 wherein the Actinobacillus pleuropneumoniae is serovar 15.

27. A method according to any one of claims 1 to 24 wherein the vaccine preparation further comprises additional active compounds such as antigens or immune stimulating compounds.

28. A method according to any one of claims 1 to 27 wherein the vaccine preparation further comprises an adjuvant.

29. A method according to any one of claims 1 to 27 wherein the vaccine preparation is freeze-dried and reconstituted before use.

30. A method according to any one of claims 1 to 29 wherein the pigs are not treated with antibiotics for 5 days before or after vaccination.

31. A method according to any one of claims 1 to 30 which comprises the further step of testing the antibiotic sensitivity of the Actinobacillus pleuropneumoniae to be administered.