NZ795066A - Oral vaccine against ruminant respiratory disease comprising polyvinylpyrrolidone - Google Patents

Abstract

The present invention relates to an oral vaccine against respiratory disease in ruminants, comprising live attenuated Mannheimia haemolytica bacteria and a Polyvinylpyrrolidone (PVP). The addition of PVP significantly improves the protective effect of vaccination on lung scores after a challenge infection. Further the PVP allows vaccination by a method of mass administration, such as by oral route via a drink. In addition the invention relates to methods for the preparation of such a vaccine, to methods for the vaccination of ruminants employing such a vaccine, and to medical uses of a composition comprising M. haemolytica bacteria. ection. Further the PVP allows vaccination by a method of mass administration, such as by oral route via a drink. In addition the invention relates to methods for the preparation of such a vaccine, to methods for the vaccination of ruminants employing such a vaccine, and to medical uses of a composition comprising M. haemolytica bacteria.

Description

The present invention relates to an oral vaccine against respiratory disease in ruminants, comprising live attenuated Mannheimia haemolytica bacteria and a Polyvinylpyrrolidone (PVP).

The addition of PVP significantly improves the protective effect of vaccination on lung scores after a challenge infection. Further the PVP allows vaccination by a method of mass administration, such as by oral route via a drink. In addition the ion s to methods for the ation of such a vaccine, to methods for the vaccination of ruminants ing such a vaccine, and to medical uses of a composition comprising M. haemolytica bacteria.

NZ 795066 ORAL VACCINE AGAINST NT RESPIRATORY DISEASE COMPRISING POLYVINYLPYRROLIDONE This application is a onal of New Zealand patent application 754108, which is the national phase entry of PCT international application , filed 11 er 2017and published as The present invention relates to the field of veterinary vaccinology; specifically the invention relates to an oral vaccine against respiratory disease in ruminants, comprising live attenuated imia haemolytica bacteria. In on the invention relates to methods for the preparation of such a vaccine, to methods for the vaccination of ruminants employing such a vaccine, and to medical uses of a composition sing M. haemolytica bacteria.

In the animal husbandry of ruminants, one of the main veterinary problems is respiratory disease. This is a x syndrome of affections with serious negative effects on animal welfare and on economy of operation. Several causes are ered to be relevant in nt respiratory disease (RRD): at the basis is infection by one or more bacteria and viruses, which is facilitated and aggravated by nmental stress factors. Such stress can resort from housing conditions such as heat, thirst, crowding, poor ventilation (dust and a); from social s such as weaning, and co-mingling into new groups; as well as from handling and transportation. This is why RRD is often called ‘shipping fever pneumonia’.

The pathogens involved are bacteria, among others: Mannheimia ytica, Pasteurella multocida, Mycoplasma bovis, and Histophilus somni; and s, amongst others: bovine virus (BCV), parainfluenza-3 (PI3) virus, bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea virus (BVDV) and bovine herpes virus 1 (BHV1), which causes infectious bovine rhinotracheitis (IBR). Some s on the respiratory disease complex in bovines are: D. Griffin (2010, Vet. Clin. Food Animals, vol. 26, p. 57 - 71), and Grissett et al. (2015, J. Vet. Intern. Med., vol. 29, p. 770-780).

Several veterinary treatments are available to prevent or mitigate RRD, often combinations are being used of antibiotics and of vaccines for the viral- and bacterial pathogens.

Of the commercial vaccines for bacterial pathogens involved in RRD, most are inactivatedadjuvated vaccines with antigens of M. haemolytica and P. multocida, in the form of bacterins (inactivated bacteria) or s (de-toxified toxins). Such vaccines are intended for parenteral injection by intramuscular or subcutaneous route. For example: Bovipast™ and Ovipast™ (Merck Animal Health), Rispoval™ (Zoetis), Presponse™ (Boehringer Ingelheim), and Respishield™ l). Only few vaccines against bacterial RRD comprise live attenuated bacteria. Reason is that such live attenuated bacterial vaccines cannot easily be combined with the preventive use of antibiotics. Examples are: Onset™, ONCE™, Vista™, and Respavir™ (all from Merck Animal Health). These live attenuated es are administered either by subcutaneous- or intranasal route. Both these routes are quite stressful to the animal, because the intranasal route lly requires the physical restraint of the , and especially its head; either by a human or by mechanical means.

Further, the intranasal administration of a liquid is apparently very unpleasant. Also, vaccine applied intranasally can be sneezed out again, so that the animal did not receive a full dose of vaccine.

Consequently there is an urgent need in this field for a live attenuated bacterial vaccine against RRD that can be administered in a more convenient and less stressful way.

Of the bacterial pathogens involved in RRD, M. haemolytica is the major cause of disease. While being a sal of the upper respiratory tract in ruminants, it can become the principal bacterial pathogen associated with pneumonic Pasteurellosis in RRD if an opportunity arises. Typical clinical symptoms of disease are fever, depression, and increase of respiration frequency. Histopathological signs are characteristic lung lesions such as necrosis, thrombosis, and exudation.

M. haemolytica is a member of the Pasteurellaceae family, and was previously called Pasteurella ytica (Angen et al., 1999, Int. J. Syst. Bacteriol., vol. 49, p. 67-86).

The Pasteurellaceae are Gram-negative, rod-shaped, non-motile, and facultative anaerobes.

Pasteurellaceae are commonly sub-divided into serotypes based on their capsular antigen. The most prominent pes of M. haemolytica in RRD are A1 and A6. The main virulence factors are the capsular polysaccharide and the oxin. Live attenuated strains of M. haemolytica have been described, comprising different attenuating mutations, for instance in: Chengappa & Carter (1979, Am. J.

Vet. Res., vol. 40, p. 449-450), WO 15670, and A Polyvinylpyrrolidone (PVP) is a synthetic polymer which has been known since the 1930’s and is widely used for a y of purposes: in foodstuffs as a stabiliser ); in technical products, e.g. in paint or glue; and in cosmetics and pharmaceuticals as a binder, thickener, emulsifier or disintegrant.

WO 94/20070 is titled ‘Polymeric mucoadhesives in the ry of immunogens at mucosal surfaces’. It presents a list of compounds ‘considered to act as mucoadhesives’, among which is PVP. ‘070 prefers the use of ymethylcellulose and an adjuvant. The only antigen tested is vated H3N2 influenza virus, which is administered to mice by oral- or intragastric route. Not all experiments showed protection, and the samples that did show seroconversion were later found to be bacterially contaminated. This was then ed to be a ‘bacterial adjuvant’. ‘070 concludes that the viral antigen, with mucoadhesive but without a bacterial nt was unable to provide a significant immune se by oral immunisation (‘070, page 24, 9th - 3rd line from the bottom).

WO 78 is titled ‘Use of bioadhesives and adjuvants for the mucosal delivery of antigens’, and aims to develop an intranasal vaccine against nza for humans. ‘078 lists PVP and y-propyl-methyl cellulose (HPMC) as bioadhesives. The bioadhesive is preferred to be a microsphere with the antigen adsorbed on, or entrapped within the spheres. The only experiment described applies intranasal administration of Influenza HA antigen to rabbits, using a bacterial toxin as adjuvant, and polycarbophil, 40 carbopol, or HPMC as bioadhesive. vaccine administered via the feed or drinking water. ‘330 describes immunisation of turkeys by intra- muscular injection, and of calves by aneous route, or by oral route via the feed. No use of or need for any other excipient is described or suggested.

Bühler (in: ‘Polyvinylpyrrolidone excipients for pharmaceuticals’, ISBN 323412-8, Springer , 2005) bes the suitability of PVP as bioadhesive for the delivery of pharmaceutical compounds to dermal and mucosal surfaces (Bühler, supra: p. 120, n 2.4.9.2). However the suitability of PVP for use in mouthwashes etc. is described to derive from its reduction of the adherence of oral bacteria to tooth , hence its use as microbial anti-adherent agent (Bühler, supra: p. 124, section 2.4.9.9).

WO 93/16680 also describes the microbial anti-adherence effect of PVP in dentifrices.

It is an object of the present ion to overcome a antage in the prior art, and to accommodate to a need in the field by providing a live attenuated bacterial vaccine against ruminant respiratory disease that can be administered by a more convenient and less stressful route of administration, while still inducing an effective immune-protection.

One common method of mass application of vaccines is by oral route. However when the inventors ted the straightforward oral application of existing live attenuated bacterial vaccines prescribed for administration by intranasal route, this was not successful. While effective for P. multocida bacteria, but singly the oral route was no success for the closely related M. haemolytica. This even when doses of the bacterium were applied that were near the maximum levels that can be produced (above 10^9 bacteria per animal dose), even with rich culture media and state of the art industrial systems for bacteriological production. The ors then attempted to concentrate an M. haemolytica culture, but this only d the final titre of live bacteria. The inventors had no indications from the prior art on how to me this problem.

Surprisingly it was found that the object can be met, and consequently one or more disadvantages of the prior art can be overcome, by providing an effective vaccine for RRD comprising live attenuated Mannheimia haemolitica ia, that can be administered by oral route. This was reached by the addition of a Polyvinylpyrrolidone to the vaccine.

The advantageous effect of the novel vaccine is that its administration by oral route, especially when applied as a drink or with the feed, allows the cheap and effective mass vaccination of animals. This no longer requires an additional handling of the animals and so reduces unnecessary stress to the animal, and in on saves considerably on expenses for labour and veterinary services. Because stress is such an ant factor in RRD, therefore the reduction of stress to the animal by vaccination using a method of mass administration helps to reduce onset of RRD. 40 PVP, in the concentration range in which it was found to be effective, was found to be non-toxic to the live attenuated M. haemolytica bacteria.

It is tly not known why the addition of a PVP s the administration of a live attenuated M. haemolytica by oral route. Although the inventors do not want to be bound by any theory or model that might explain these observations, they speculate that a PVP in some way or other associates with the bacteria in the vaccine, which allows these bacteria to more effectively establish a sation of the upper atory tract and/or results in a better exposure of these ia to the immune system.

Therefore in one aspect the invention relates to an oral vaccine against respiratory disease in ruminants, the vaccine sing live attenuated Mannheimia haemolitica bacteria and a pharmaceutically acceptable carrier, wherein the vaccine also comprises Polyvinylpyrrolidone (PVP).

For the invention “oral” refers to a route of administration to a target ruminant via the oral cavity of the animal, typically: via the mouth, also known as: per os. Inter alia this includes routes of stration that are termed: buccal, (sub)lingual, (sub)labial, laryngeal, oro-pharyngeal, tonsilar, or oro-mucosal. Further this also refers to an indirect administration to the oral cavity, such as by administration to the muzzle area of a ruminant; the animal’s natural tendency to clean itself by g, or even by licking another ruminant, will then cause the um to reach the oral cavity.

In ce oral refers to ion of vaccine by some way of eating or ng, but may also comprise spray or nebulisation of a liquid or a powder.

A “vaccine” is well known to be a composition that has a medical effect. A vaccine ses an immunologically active component, and a pharmaceutically acceptable carrier. The ‘immunologically active component’, is one or more antigenic molecule(s), here: live M. haemolytica bacteria, that are recognised by the immune system of a target, and induce a protective immunological response. The response may originate from the targets’ innate- and/or from the acquired immune system, and may be of the cellular- and/or of the humoral type.

A vaccine generally is efficacious in reducing the level or the extent of an infection by the target pathogen, for e by reducing the load or ning the duration of the replication of pathogenic M. haemolytica bacteria in a host ruminant.

Also, or ly as a results thereof, a vaccine generally is effective in reducing or ameliorating the (clinical) symptoms of e that may be caused by such infection or replication, or by the animal’s response to that infection or replication.

The effect of an oral vaccine according to the invention is the prevention or reduction in ruminants of an infection by a pathogenic M. haemolytica, and/or of one or more signs of respiratory disease that are associated with such infection or replication. Such (clinical) signs are: fever, increased respiration rate, nasal discharge, and several types of inflammatory affections to the lungs causing typical lesions.

Consequently, the oral vaccine according to the invention is an aid in the reduction of respiratory disease caused by M. haemolytica, as is e.g. able in reduction of the number and/or the severity of lung 40 lesions caused by M. haemolytica.

Such an M. haemolytica vaccine may colloquially also be referred to as a vaccine ‘against’ M. haemolytica, a vaccine ‘against’ pneumonic Pasteurellosis, or as an ‘M. haemolytica vaccine’.

Embodiments and s of an oral vaccine according to the invention, its production, and its uses will be described herein below.

“Respiratory e” for the invention refers to any disease of a ruminants’ respiratory tract. Typically this is a uence of infection with a pathogenic M. ytica bacteria, commonly in combination with infection by one or more bacteria or s. For a description see veterinary handbooks such as: "The Merck veterinary manual" (10th ed., 2010, C.M. Kahn edt., ISBN: 091191093X). Examples of such a disease are: shipping fever, or pneumonic Pasteurellosis.

A ant” for the invention is an animal assigned to the suborder Ruminantia, and/or an animal applying the process of tion to digest its feed.

The terms “live” and “attenuated” for the invention refer to ia that are alive, meaning replicative, and that have a reduced capacity to induce ion or e in a particular host, as compared to unattenuated , more pathogenic strains of the bacterium. A synonym is: modified-live. The effect of the attenuation is that the bacterium can still replicate in a target animal and so display relevant ns to the targets’ immune system, but without itself causing (serious) disease to the target. This ively ts the target against (the consequences of) an infection with an enuated version of the bacterium.

The use of live attenuated bacteria in vaccination is known since the 1880’s. Stably ated bacteria will carry a genetic mutation that induces a loss in replicative or infective capacity, such as a mutation to their external organelles, their coat or capsule, to the expression of a virulence factor, or to their internal organisation. An attenuated ium can be generated in vitro in a wide variety of ways, such as through a method of induced mutation, either directed or a-specific. Examples are subsequent passageing (in vivo or in vitro); use of mutagens such as als or ionising radiation; or recombinant DNA technology. Also a bacterium can be considered to be attenuated upon infection of a particular target species, while being fully pathogenic when infecting its natural host species.

“M. haemolytica” are ia from the Pasteurellaceae family. They display the characterising features of their taxonomic group-members such as the morphologic, genomic, and biochemical characteristics, as well as the biological characteristics such as logic, immunologic, or pathologic behaviour. As is known in the field, the classification of micro- organisms is based on a combination of such characterising features. The scope of the invention therefore also includes M. haemolytica bacteria that are subclassified therefrom in any way, for instance as a subspecies, strain, isolate, genotype, variant, subtype, serotype, or subgroup and the like.

It will be apparent to a skilled person that while a particular M. haemolytica bacterium for the present 40 invention may currently be classified in a specific species and genus, such a taxonomic classification can change in time as new insights may lead to reclassification into a new or different taxonomic group. This applies in particular to M. haemolytica which was previously classified as Pasteurella haemolytica. Such reclassification does not change the organism itself, its genetic or antigenic repertoire, or the level of genetic relatedness to other bacteria, but only its scientific name or classification. Therefore such sified ia remain within the scope of the invention.

A “pharmaceutically acceptable carrier” is for example a liquid such as water, physiological salt solution, or phosphate buffered saline solutions. In a more complex form the carrier can e.g. be a buffer comprising further additives, such as stabilisers or vatives.

The term "comprises" (as well as variations such as "comprise", “comprising", and "comprised") as used herein, intends to refer to all elements, and in any possible combination conceivable for the invention, that are covered by or included in the text section, paragraph, claim, etc., in which this term is used, even if such elements or combinations are not explicitly recited; and not to the exclusion of any of such element(s) or combinations.

Therefore any such text section, paragraph, claim, etc., can therefore also relate to one or more embodiment(s) wherein the term "comprises" (or its variants) is replaced by terms such as "consist of", "consisting of”, or st essentially of”.

A “Polyvinylpyrrolidone” is a synthetic polymer of 1-vinylpyrrolidinone units. The compound has CAS number 90038, is iated as PVP, and has l synonyms, such as: Polyvidone, and Povidone. PVP is widely available commercially, in ent levels of quality and purity. Well-known brands are for example: Kollidon™ and Luvitec™, both available from BASF or Aldrich; Luviskol™, BASF; Periston™, BCM; and Plasdone™, from Ashland.

PVP is available in polymer sizes differing between 2.500 and 2 million Dalton in weight-averaged molecular weight of the polymer. Alternatively a PVP product can be characterised by a Fikentscher K value. This is an empirical value (referenced in DIN EN ISO 1628-1) representing the ity of a dilute solution of the polymer relative to that of a solvent, as a measure of the r’s molecular weight.

Some indications of the on between K value and the corresponding range of averaged lar weights for PVP are: K 12: 2.000-3.000; K 17: 7.000-11.000; K 25: 28.000-34.000; K 30: 44.000-54.000; K 60: 350.000-550.000; and K 90: 1.000.000-1.500.000. (V. Bühler: Polyvinylpyrrolidone excipients for pharmaceuticals, ISBN 323412-8, Springer Berlin, 2005; p. 25, Table 17).

PVP has been known since the 1930’s and is widely used for a y of es: in foodstuffs as a stabiliser (E1201); in technical products, e.g. in paint or glue; and in cosmetics and pharmaceuticals as a binder, ner, emulsifier or disintegrant. It is registered in Pharmacopoeias, e.g. monograph 685 in PhEur 8.

Different embodiments of an oral vaccine according to the invention, regarding for instance its composition, volume, titre, and physical form are disclosed herein below.

During the preparation and use of the oral vaccine ing to the invention several intermediary 40 compositions will be made and used, such as premixes, more or less concentrated working solutions, rough harvests, etc. These will differ in their composition and concentrations from the complete vaccine as it is ed by a manufacturer for sale and commercial use. Also the vaccine as released may require a further preparation before it is ready for administration, depending on the form in which the e was marketed. In practice this means that when the vaccine is released by a manufacturer in liquid form, the vaccine may be ready for direct administration, or may require dilution, when marketed in a concentrated form. Alternatively, when the vaccine is released by the manufacturer in freeze-dried form, the vaccine may be applied as a granulate or powder. More often the freeze-dried cake will first be reconstituted with the recommended volume of an appropriate diluent, before it is ready for administration.

Therefore the embodiments and details described herein of the oral vaccine according to the invention refer to the final n of the e which is ready for administration to a ruminant target.

This notwithstanding the fact that even the ‘final version of the vaccine which is ready for administration to a ruminant’ of the oral vaccine according to the invention, may be used for a r preparation, for instance by taking up into feed or drink to allow mass administration. As a result, those further dilutions, admixtures, etc., may bring (some elements of) the vaccine outside of the rred) embodiments as bed herein. All this is within the scope of the invention.

The live attenuated M. haemolytica ia for use in the oral vaccine according to the invention can be obtained in different ways, either as a natural isolate, or as result from manipulation and selection in vitro, to achieve ation in any way that provides for their safe but efficacious use in the invention. The preferred method of attenuation is one that has a stable genetic basis in the bacterial genome; such stable mutation will not change to more or less attenuation upon the many rounds of replication that a vaccine strain of ia must undergo. Examples of such rounds of replication are the generation of master- and working stock supplies, next the replication during production runs, and finally the rounds of replication in the target animal to establish an effective and immunising infection. Typically the most stable mutations are the ones that have been introduced by ed recombinant DNA techniques, and that incorporate more than a single or a few tides of change.

To facilitate the marketing authorisation of the resulting vaccine in many countries of the world, such mutant bacteria should preferably not contain any foreign DNA, but in particular not contain any gene of which the expression would provide resistance to an antibiotic agent.

Preferred live attenuated ia of M. haemolytica for use in the invention are bacteria as described in Chengappa & Carter (1979, Am. J. Vet. Res., vol. 40, p. 449-450), comprise a mutation in the oxin operon.

More preferred are live attenuated M. haemolytica bacteria that express an avirulent form of the leukotoxin A n.

In this respect: the avirulence of the M. haemolytica leukotoxin A protein may derive from the size of the expressed leukotoxin A n being shorter than in a wildtype protein, or may derive from the protein not achieving post-translational tion. Either way, the bacteria expressing such avirulent oxin A 40 protein remain viable and replicative, and the expressed form of the leukotoxin A protein can still stimulate an immune response, but causes significantly less pathology than a wildtype version of the leukotoxin A. Such mutants are for example described in Therefore in an embodiment of the oral vaccine according to the invention, the live attenuated M. haemolytica bacteria express an ent form of the leukotoxin A protein.

Even more preferred are live attenuated M. haemolytica bacteria as described in se a deletion in the leukotoxin A gene of the nucleotides that would otherwise encode amino acids numbers 34 - 378 of the wildtype Leukotoxin A protein. Most preferred live attenuated M. haemolytica is the mutant strain of M. haemolytica serotype 1, as described in ΔlktA.

In a preferred embodiment, a “ruminant” for the invention relates to any ruminant of relevance to veterinary science or to commercial farming operations. Preferably this refers to bovine, caprine, ovine or cervine animals.

While there is commercial g of deer, and goats, and in particular of sheep, the economic relevance of bovine-, and in ular of cattle farming has the largest global nce.

Therefore in an embodiment of the oral vaccine according to the invention, the ruminant is a bovine animal.

In a preferred embodiment the bovine animal is e cattle (Bos taurus), zebu cattle (Bos indicus), buffalo, bison, yak, or wisent.

The bovine can be of any type: dairy or beef, or al stock for dairy- or beef type.

The inventors observed that the range n PVP is effective in the oral vaccine according to the invention is quite broad. Only practical limitation is that at higher concentrations of PVP, or when using PVP of types with high average molecular weight ranges, it can take more time to completely dissolve the PVP and achieve complete mixing with the other constituents.

An effective oral vaccine according to the ion can be obtained by sing a concentration of between about 0.01 and about 10 % w/v PVP in the e. As described above, this refers to the final version of the oral vaccine according to the invention which is ready for administration to a nt.

Preferably the concentration of PVP in the oral vaccine according to the invention is between about 0.05 and about 7 % w/v PVP; 0.1 and 5; 0.2 and 4; or even between about 0.3 and 3 % w/v PVP, in that order of preference.

Therefore in an ment of the oral vaccine according to the invention, the concentration of PVP is between about 0.3 and about 3 % w/v. 40 For the invention, a number indicated with the term "about" means that number can vary between ± 25 % around the indicated value; ably: about means ± 20 % around the indicated value, more preferably: about means ± 15, 12, 10, 8, 6, 5, 4, 3, 2 % around the indicated value, or even: about means ± 1 % around the indicated value, in that order of preference.

In a more preferred embodiment of the oral vaccine according to the invention, the concentration of PVP is about 1.3 % w/v.

In an embodiment, the oral vaccine according to the invention comprises a suitable preservative, such as thimerosal, merthiolate, or benzoic compounds, in an amount that is effective but is also tolerated by the live vaccine micro-organisms.

In one ment, the oral vaccine according to the ion is in liquid form. The liquid may be generally aqueous, meaning: like water, but can also be a less fluid semi-solid, e.g. as in a syrup, gel, dispersion, on or paste.

When released in liquid form, the vaccine formulation will comprise a stabiliser to allow prolonged storage of the live attenuated bacteria. For example: when the liquid vaccine is intended to be stored frozen at a temperature below 0 ºC, the stabiliser will be a otectant, for example glycerol, to allow storage at temperatures of -20 °C or less for extended s. Alternatively, when storing the liquid vaccine at temperatures above 0 ºC, a suitable liquid stabiliser may be selected, for example as described in In one embodiment, the oral vaccine according to the invention is in freeze-dried form, also known as: in lyophilised form. This form has a number of advantages over a vaccine in liquid form, as it is lighter and therefore more economical to transport. Further, a freeze-dried vaccine will usually not require to be kept frozen, but can be stored at a more economical 2-8 ºC.

Procedures for -drying are well-known to persons skilled in the art, and equipment for freeze-drying at a variety of scales is available commercially. ore in an embodiment of the oral vaccine according to the ion, the vaccine is in freeze-dried form.

The oral vaccine in freeze-dried form according to the invention can be administered as such to a ruminant, for example as a fast-melt dosing-form, or as a ground powder from freeze-dried cake. More common is that the freeze-dried cake is first tituted in a diluent and then is ready for administration to a ruminant.

The “freeze-dried form” will be a freeze dried cake which itself can be in any form, for example as a layer in a bottle, as a tablet, or as a cal object, for example a lyosphere as described in EP 3. lly such freeze-dried cakes will be stored in moisture-proof ing, such as under vacuum or under nitrous gas in a sealed glass bottle, or packaged in sheet-metal or metal-laminated 40 packaging.

Typically freeze-dried vaccines will contain a freeze-drying stabiliser that will protect the live-attenuated micro-organisms in the vaccine from decay over time in e, but also during the g- and heating cycles of the freeze-drying process itself. Well-known freeze drying stabilisers n a bulking agent such as an amino acid, e.g. glycine or arginine; aspecific protein such as bovine serum albumin or a ysate e.g. NZ amine; and/or polymers such as ne or gelatine.

In an embodiment the oral vaccine in freeze-dried form according to the invention comprises sucrose as a freeze-drying stabiliser. Not only does this provide good stabilisation, but an additional advantage is that its sweet taste makes the ingestion of the oral vaccine more pleasant to the ruminant, which helps to further reduce the stress of the vaccination for the ruminant animal.

Therefore in an embodiment of the oral e in freeze-dried form according to the invention, the vaccine comprises sucrose.

The sucrose is t in the oral vaccine in freeze-dried form according to the invention in a concentration of between about 1 and about 20 % w/v of the vaccine ready to use; preferably between about 2 and about 15 %, between about 3 and about 10, or even n about 4 and about 8 % w/v of the vaccine ready to use, in that order of preference.

More preferred, the oral vaccine in freeze-dried form according to the invention comprises sucrose in a concentration of about 6 % w/v.

In addition, the oral vaccine in -dried form according to the invention may comprise r excipients, for example carry-over compounds from the culture medium of the organism(s) in the vaccine. For the M. haemolytica of the invention, that may be es of the bacterial culture medium comprising yeast extract, dextrose, peptone, etc. Such compounds may even be helpful in the stabilisation, as additional bulking agent.

To reconstitute a freeze-dried vaccine, it is common to re-suspend the freeze-dried cake in a physiologically acceptable diluent. This is commonly done shortly before administration to the target, to ascertain the best quality of the vaccine. The diluent is lly aqueous, and can e.g. be sterile water, or a physiological salt on.

The diluent may se further excipients, such as a stabiliser, or an adjuvant.

The diluent for the oral vaccine in freeze-dried form can be supplied in a separate container, either with- or separate from the freeze-dried e. When provided er, the freeze-dried vaccine and the diluent (each in their own container) form a kit of parts that embodies the oral vaccine according to the invention.

Therefore in an embodiment of the oral vaccine in freeze-dried form according to the invention, 40 the vaccine is a kit of parts with at least two containers, one container comprising the freeze-dried vaccine, and one container comprising a diluent.

In an embodiment, the oral vaccine according to the invention ses a colorant. This can facilitate the administration, by serving as an optical indication of which ruminants have already been vaccinated. Such a colorant will of course need to be pharmaceutically acceptable, such as e.g. a food-colorant. Also the colorant needs to be xic for the live attenuated M. haemolytica bacteria, and for any other microorganisms , antigens, or biologically active molecules that may be included.

In an embodiment of the oral vaccine according to the ion, the vaccine comprises a blue colorant, e.g. FD&C Blue No. 1 (E133).

The colorant for the invention can be comprised in the vaccine that is released by a manufacturer, either when in , semi-solid, or when in freeze-dried form.

In an embodiment of the oral e in freeze-dried form according to the invention, the colorant is comprised in the diluent that is recommended by the manufacturer for the reconstitution of the freezedried cake, and which may be provided together with or separate from the freeze-dried vaccine, e.g. in a kit of parts.

The oral vaccine ing to the invention comprises an amount of live attenuated M. haemolytica bacteria that is immunologically effective.

For the ion “immunologically ive” means an amount of live attenuated M. haemolytica bacteria that is capable of inducing in the ruminant target a protective immune response that is e of reducing the consequences of an infection with a pathogenic M. haemolytica ium; in particular, that is an aid in the reduction of respiratory disease caused by Mannheimia haemolytica. A skilled person in the field of the ion will be more than capable of determining the iveness of an oral vaccine according to the invention, e.g. by monitoring the immunological response following vaccination or after a challenge infection, e.g. by monitoring the targets’ signs of disease, clinical scores, or by re-isolation of the pathogen, and comparing these results to a vaccination-challenge response seen in mock-vaccinated animals.

Ways to determine the number of bacteria in a sample are well-known in the art. For ia that can form colonies on agar plates, the preferred method is by plating-out serial dilutions of the sample on suitable agar plates, incubating those under suitable conditions (e.g. regarding time, temperature, humidity, presence of oxigen, etc.), and ng of the es that have formed. When required a (counter-)staining may be applied to increase visibility. From this the titre of the bacteria in the original sample can be calculated. Consequently such a titre is expressed in: colony g units (CFU).

M. haemolytica can for instance be counted by streaking on agar plates of tryptic soy broth (TSB), and by incubation for 16 - 24 hours at about 36 °C.

The inventors were surprised to learn that the use of PVP in an oral vaccine according to the invention, makes that the titre of M. haemolytica bacteria per animal dose that is required for effective protection by oral route, is now in the range of about 10^8 CFU/dose or less; this for the first time makes the economic 40 production of an oral vaccine of M. haemolytica feasible.

Therefore in an ment, the oral vaccine according to the invention comprises at least about 1x10^7 CFU of live attenuated M. haemolytica bacteria as bed for the invention, per animal dose of the final version of the vaccine which is ready for administration to a ruminant target.

Preferably the oral vaccine ses at least about 3x10^7 CFU of live attenuated M. haemolytica bacteria per animal dose. More preferably at least about , 6x10^7, 7x10^7, 8x10^7, 9x10^7, 1x10^8, 2x10^8, 3x10^8, 4x10^8, 4x10^8, 5x10^8, 6x10^8, or even at least about 7x10^8 CFU of live attenuated M. haemolytica ia as described for the invention, per animal dose, in that order of preference.

More preferred, the oral vaccine according to the invention comprises between about 1x10^8 and about 7x10^8 CFU of live attenuated M. haemolytica bacteria as described for the invention, per animal dose. Such a dose range of M. haemolytica bacteria is immunologically effective, can be economically produced, and provides sufficient margin for losses in titre during tion, formulation and storage.

The volume of one animal dose of the oral vaccine according to the invention is a volume that is practicable from the viewpoint of the cturer and of the user, such as a veterinarian or animal caretaker. In addition the volume of what constitutes one animal dose can be dependent on the type and the age of the ruminant target that is to be vaccinated. As bed, the volume of one animal dose refers to the final version of the oral vaccine which is ready for administration to a ruminant . Also the volume of a dose refers to the vaccine when in liquid form, or to the freeze-dried cake resulting from such a volume.

In an embodiment of the oral vaccine according to the ion, the volume of one animal dose is between about 0.1 and about 10 ml.

Preferably the volume of one animal dose is between about 0.2 and about 8 ml; more preferably n about 0.5 and about 6 ml; between about 0.7 and about 4 ml; or even between about 1 and about 3 ml, in that order of preference.

More preferred, the volume of one animal dose is about 2 ml.

Although it is possible to e commercial ruminant vaccines in packaging for a single animal, that is not very cost efficient, nor is it practicable for use on large number of animals. Therefore commercial forms of packaging of ruminant vaccines can be in containers that comprise the animal doses for 2, 5, 10, , 25, 50, 100, 200, 250, 500, or even 1000 animals. For example, a container of oral e according to the invention for 50 animal doses, may contain a freeze-dried pellet of an original volume of about 30 ml of vaccine formulation. This can be dissolved in 100 ml diluent to provide 50 doses of 2 ml each.

The inventors have found that different types of PVP can be used to achieve the advantageous effect of the invention, for example they have used PVP of types such as K 12 and K 60. The K value being an indication of the weight-averaged molecular weight of the polymer, determined by viscosity measurement, as described above. Also combinations of types of PVP have been tested, such as the ation of K 40 12 and K 60 types, and found to be effective. Consequently, the PVP to be used for the invention can be made up of a single type of PVP, but can also be a combination of types of PVP, e.g. of two, three, or even more types.

Therefore in an embodiment of the oral vaccine according to the invention, the PVP is of a combination of types of PVP.

For the invention the “type” of PVP refers to the size class of the weight-averaged lar weight of the PVP r that is being used. Such a size class can be indicated by a certain size-averaged molecular weight or -weight-range, or by the K value.

In an embodiment of the oral vaccine ing to the invention, the type of PVP is one or more ed from the group consisting of: K 12, K 17, K 24, K 25, K 30, K 60, K 70, K 80, K 90 and K 120.

These different types of PVP are commercially available from a variety of suppliers. They are also available in a variety of qualities and purities. The use of PVP of a pharmaceutical grade is preferred.

The type of PVP as indicated by its K value may be written in different layouts, such as e.g. K 60, K60, K-60, etc. All these are within the scope of the invention.

Surprisingly it was found that the use of PVP provided advantages also in the formulation phase of the oral e according to the ion; specifically in the freeze-drying process. For example: PVP K 12 was found to provide for improved survival of M. ytica bacteria in the freeze-drying process.

Further: PVP K 60 was found to e the ency of the freeze-drying process, such that less time was needed to complete a full freeze-drying cycle In addition, PVP K 60 was found to improve the shelf-life stability of M. haemolytica bacteria in the oral vaccine in freeze-dried form according to the invention.

Therefore in an embodiment of the oral vaccine according to the invention, the type of PVP is K 12, or the type of PVP is K 60, or the type of PVP is a combination of K 12 and K 60.

An example of a way to combine more than one type of PVP in the oral vaccine according to the ion, is by incorporating one or more types of PVP into the culture medium during the production stage, and adding one or more further types at the stage of final formulation, or via a diluent. The PVP in the culture medium is then carried-over with the ia and the medium into the vaccine, when these bacteria are harvested for the subsequent formulation. In the preparation of the oral vaccine according to the invention, significant amounts of the culture medium can be taken up into the vaccine, e.g. 20 % or more of the culture volume.

The oral vaccine according to the invention can advantageously be combined with one or more other ns, micro-organisms or biologically active molecules, into a combination vaccine. However the 40 combination needs to be made with care to safeguard the viability of the replicative vaccine components, and the ity and efficacy of the l combination vaccine. Such choices are within the routine capabilities of the skilled person.

Therefore, in an ment the oral vaccine ing to the invention comprises at least one additional active component.

An “additional immunoactive component” may be an antigen, micro-organisms or a part f, a biologically active molecule, an immune enhancing substance, and/or a vaccine, either of which may comprise an adjuvant. The additional immunoactive component when in the form of an antigen may consist of any antigenic ent of veterinary importance. Preferably the additional immunoactive component is based upon, or d from, a further micro-organism that is pathogenic to a nt animal. It may for instance comprise a biological or synthetic molecule such as a protein, a carbohydrate, a lipopolysaccharide, a nucleic acid encoding a proteinaceous antigen. Also a host cell comprising such a nucleic acid, or a live recombinant carrier micro-organism containing such a nucleic acid, may be a way to deliver or express the nucleic acid or an additional immunoactive component. Alternatively the additional immunoactive component may comprise a fractionated or killed micro-organism such as a parasite, bacterium or virus, or a part thereof, such as an extract, fraction, or sonicate.

The additional immunoactive component(s) may also be an -enhancing substance e.g. a chemokine, or an immunostimulatory nucleic acid. Alternatively, the e according to the ion, may itself be added to a vaccine, while assuring viability and efficacy.

Preferred additional immunoactive components are based on, or derived from, micro-organisms that are pathogenic to ruminants. Examples of such micro-organisms are: For : Neospora spec., caulus spec., Cryptosporidium spec., Ostertagia spec., bovine rotavirus, bovine viral diarrhoea virus, bovine coronavirus, infectious bovine rhinotracheitis virus (bovine herpes virus 1), bovine paramyxo virus, bovine parainfluenza virus, bovine respiratory syncytial virus, rabies virus, bluetongue virus, E. coli, ella spec., Staphylococcus spec., cterium spec., Brucella spec., Clostridia spec., Pasteurella spec., Mannheimia spec., Haemophilus spec., Leptospira spec., and Fusobacterium spec..

For sheep and goats: Toxoplasma gondii, peste des petit ruminant virus, bluetongue virus, Schmallenberg virus, Mycobacterium spec., Brucella spec., Clostridia spec., Coxiella spec., E. coli, Chlamydia spec., Clostridia spec., Pasteurella spec., and Mannheimia spec..

For cervines: Epizootic haemorrhagic disease virus, bluetongue virus, papilloma virus, Borrelia burgdorferi, Mycobacterium bovis, and Trueperella pyogenes.

Preferred micro-organisms pathogenic to ruminants are one or more selected from the group consisting 40 of: Pasteurella multocida, Mycoplasma bovis, Histophilus somni, bovine coronavirus, parainfluenza-3 virus, bovine respiratory syncytial virus, bovine viral diarrhoea virus, and bovine herpes virus 1.

More preferred micro-organisms pathogenic to ruminants are live attenuated Pasteurella ida bacteria.

Therefore in an ment of the oral vaccine according to the invention, the at least one additional immunoactive ent are live attenuated Pasteurella multocida bacteria.

Preferred live attenuated P. multocida bacteria for the invention, are those that are lar, meaning that the bacterium cannot express its normal capsule of hyaluronic acid.

The acapsular phenotype may result from any mutation in the P. multocida genomic locus for capsule biosynthesis. For example in the region encoding one of the transporters of polysaccharide to the e; in the region encoding one of the hya genes; or in the region encoding proteins involved in phospholipid substitution; see: Chung et al. (1998, FEMS Microbiol. Letters, vol. 166, p. 289-296). Either way, the acapsular P. multocida bacteria remain viable and replicative, and can still stimulate an immune response, but cause significantly less pathology than capsular P. multocida can.

Therefore in an embodiment of the oral vaccine sing live attenuated P. multocida bacteria as onal immunoactive component according to the invention, the live attenuated P. multocida bacteria are acapsular.

Even more preferred are live attenuated P. ida as described in deletion of all or part of the hyaE gene.

Most red live attenuated P. multocida is the mutant strain of P. multocida serotype A3, as described in An oral vaccine according to the invention can be used either as a prophylactic- or as a therapeutic treatment, or both, as it interferes both with the establishment and with the progression of an infection by a pathogenic M. haemolytica.

Further or additional ments of the oral e according to the invention are conceivable, and are perfectly achievable for a skilled person. Also these further embodiments may be applied in one or more combination(s) to the embodiments already described.

Therefore in an ment of an oral vaccine according to the ion, one, more, or all of the conditions apply, selected from the group consisting of: - the live attenuated M. haemolytica bacteria express an avirulent form of the leukotoxin A protein, - the live attenuated M. haemolytica is the mutant strain of M. haemolytica serotype 1, as bed in - the ruminant is bovine, caprine, ovine or cervine, 40 - the ruminant is a bovine animal, - the bovine animal is taurine cattle (Bos taurus), zebu cattle (Bos s), buffalo, bison, yak, or wisent, - the oral vaccine comprises a colorant, - the concentration of PVP is between about 0.3 and about 3 % w/v, - the concentration of PVP is about 1.3 % w/v, - the PVP is of a combination of types, - the type of PVP is one or more selected from the group consisting of: K 12, K 17, K 24, K 25, K , K 60, K 70, K 80, K 90 and K 120, - the type of PVP is K 12, or the type of PVP is K 60, or the type of PVP is a combination of K 12 and K 60, - the oral vaccine ses at least about 1x10^7 CFU of live attenuated M. haemolytica bacteria per animal dose, - the oral vaccine comprises between about 1x10^8 CFU about 5x10^8 CFU of live attenuated M. haemolytica bacteria per animal dose, - the volume of one animal dose is between about 0.1 and about 10 ml, - the volume of one animal dose is about 2 ml, - the oral vaccine also comprises at least one additional immunoactive component, - the additional immunoactive component is based on, or derived from, micro-organisms that are pathogenic to ruminants, - the micro-organisms that are pathogenic to ruminants are live attenuated Pasteurella multocida bacteria, - the live attenuated P. multocida bacteria are acapsular, - the live attenuated P. ida is the mutant strain of P. multocida serotype A3, as described in - the oral vaccine is in freeze-dried form, - the oral vaccine in freeze-dried form comprises sucrose, - the oral vaccine in freeze-dried form ses sucrose in a concentration of about 6 % w/v, - the oral vaccine in freeze-dried form is a kit of parts with at least two containers, one container sing the freeze-dried vaccine, and one container comprising a diluent, and - the oral vaccine in freeze-dried form comprises a colorant comprised in the diluent.

In an embodiment of the oral e according to the ion, the live attenuated M. haemolytica is the mutant strain of M. ytica pe 1, as described in ΔlktA; the nt is cattle; the concentration of PVP is about 1.3 % w/v; the type of PVP is a combination of K 12 and K 60; the oral vaccine comprises between about 1x10^8 and about 7x10^8 CFU of live attenuated M. haemolytica bacteria per animal dose; the oral e additionally comprises a live attenuated P. multocida which is the mutant strain of P. multocida serotype A3, as described in WO 2005/003330, named: 1062 ΔhyaE; the oral vaccine is in freeze-dried form; and comprises sucrose in a concentration of about 6 % w/v.

The oral vaccine according to the invention can be prepared from live attenuated M. ytica bacteria, by methods well known in the art, and within the routine capabilities of a person skilled in the art. For example: M. haemolytica is ed in fermenters using standard culture medium, e.g. TSB, with monitoring of temperature and use of variable stirrer speed and oxygen level. The complete culture is harvested at an appropriate time, such as upon reaching a specified culture density, measurable e.g. by optical density. The bacteria are then harvested for example by centrifugation, and are taken up into a pharmaceutically acceptable carrier such as water for ion combined with the necessary stabilisers.

Next an amount of PVP is added, which is gently stirred and given sufficient time to fully mix. This may take significant time; for example in experiments employing 1.3 % PVP K 60 from a 45 % liquid stock, it could take up to 24 hours of stirring at room temperature to have the PVP fully mixed into the other liquids.

Conveniently, the PVP can be added as a sterilised stock solution. Next the vaccine product is apportioned into appropriate sized containers, and can be further formulated such as by freeze-drying, or the product can be released on the market in liquid or semi-solid form.

The various stages of the manufacturing process are monitored by adequate tests, for instance by iological and logical tests for the quality and quantity of the bacteria or any r antigens; by tests for absence of extraneous agents; and ultimately by in vitro or in vivo experiments to determine vaccine efficacy and -safety. All these are well known to a d person, and are prescribed in Governmental regulations such as the Pharmacopoeia, and in handbooks such as: “Remington: the e and practice of pharmacy” (2000, Lippincot, USA, ISBN: 683306472), and: “Veterinary vaccinology” (P. et et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

Therefore in a further aspect the invention relates to a method for the preparation of an oral vaccine ing to the invention, comprising the step of admixing live attenuated M. haemolytica bacteria and a pharmaceutically acceptable carrier, with PVP.

The admixing with PVP can be done in different ways, and at different times, to se tion efficiency or vaccine characteristics.

One option is to add the PVP (either of one or of more types) to the e of the M. haemolytica bacteria during the production stage, as described.

When the oral vaccine according to the invention is provided in freeze-dried form, further embodiments are possible; the PVP can be sed in a diluent for the reconstitution of the freezedried cake. This diluent with PVP may be provided er with, or separate from, the freeze-dried vaccine.

However, the PVP K 60 is preferably added to the vaccine according to the invention before the freeze-drying process. This takes full advantage of the favourable effect of PVP K 60 on the efficiency increase of the freeze-drying cycle, and on the stabilisation of the M. haemolytica ia during the life. 40 At different points in this method, onal steps may be added, for example for additional treatments such as for purification or storage.

Next, the method for the preparation can involve the admixing with further pharmaceutically acceptable excipients such as stabilisers, carriers, adjuvants, diluents, ons, and the like.

As described , an oral vaccine according to the invention can be produced in different forms, for e as a liquid, or semi-solid, and can be either a concentrate, or ready to use for administration.

Alternatively, the e can be formulated in a freeze-dried form. These variations, and optionally many more, can be incorporated as a further step at an appropriate point in the method for preparation according to the invention.

Therefore the method for the preparation according to the invention can comprise any of the embodiments (preferred or not) as described herein for the oral vaccine according to the ion, or any combination of two or more of those embodiments of the oral vaccine according to the invention.

The oral vaccine according to the invention, which can be made by a method for the preparation according to the invention, can be administered to a target ruminant in different ways and at a different time point in its life, as long as the efficacy and the safety are preserved. For example, as will be t to a skilled person, it is preferred that the target ruminant did not receive around the time of vaccination e.g. via feed or as injectable, any significant amount of antibiotics to which the vaccine bacteria are sensitive.

When appropriate the ruminant target may be given a booster ation later in life, but preferably the oral vaccine according to the invention is stered only once per ruminant target, i.e. it is a single dose vaccine.

The oral e according to the invention, in the final version of the vaccine which is ready for administration to a nt target, can conveniently be administered to a ruminant by administering the ed volume of one animal dose, directly into the animal’s mouth. Such oral administration of a fluid to an animal is commonly called a drench. Alternatively, when in semi-solid form, the oral e can be orally administered as e.g. a paste or a gel. A wide variety of tools for the convenient dosing and oral administration are available commercially. Typically this will be an applicator of some sort such as a syringe or injector, with a nozzle that can be placed in the animal’s mouth. Such ators are also available for repeated administration, when treating large number of animals.

This route of stration will commonly not require the animal to be restrained, or not to the same extent as required for intranasal administration, making the oral vaccination less stressful than intranasal. Also there is no danger of the vaccine being sneezed out again. Further, in case the vaccine comprises sucrose as described, the pleasant taste facilitates the oral vaccination.

Therefore in a further , the invention s to a method for the vaccination of ruminants against respiratory disease, the method comprising the step of administering an oral vaccine according to the invention to said ruminants by oral route.

The administration regime for a method for the administration according to the invention, to a target ruminant can be in single or in multiple doses, in a manner compatible with the formulation of the vaccine and with cal aspects of the animal husbandry.

Preferably, the regime for the method for the administration according to the ion is ated into existing vaccination schedules of other vaccines that the target ruminant may require, in order to r reduce stress to the animals and to reduce labour costs. These other vaccines can be administered in a simultaneous, concurrent, or sequential fashion, in a manner compatible with their registered use.

Therefore in an embodiment of the method for the vaccination of ruminants according to the ion, the vaccine is administered in a combination with another ruminant vaccine.

One of the advantages of the oral vaccine according to the invention is that the oral route enables the use of methods of mass administration. Such s are without stress to the animal and are very cheap to apply.

Most prominent among those methods of mass-administration is the administration as a drink or with the feed.

Therefore in an embodiment of the method for the vaccination of ruminants according to the invention, the vaccine is administered to a target ruminant as a drink and/or with the feed.

Administration with the feed preferably regards so called top-dressing of feed, which is the addition of the vaccine to feed directly before feeding. This is advantageous to the vaccine’s stability as compared to use already mixed into the feed. Alternative administrations by feeding are also: as a bait, treat, chew, or lick.

Preferred method of mass-administration of the oral vaccine according to the ion, is administration as a drink, e.g. with drinking water.

Administration via drinking water can conveniently be accomplished by using the water installations present on the farm, such as a ystem for drinking water distribution, with drinkers which are adapted to the target animals. Essentially this would mean the dilution of the oral e according to the invention in drinking water, and ng that the animals to be vaccinated ingest the right amount of this vaccine-dilution.

To apply mass-administration of the oral vaccine according to the invention by drinking water, several practical issues need to be ered, which are all within the routine capabilities of the skilled person, such as: - the water needs to be of ient quality to sustain the viability of the vaccine for a sufficient amount of time, e.g. 1 - 2 hours until all s have ingested their allotted amount of the vaccine-in-water dilution. In this respect, the water quality is determined both by the source of the water, or by the use 40 of a water sanitizer.

- When of insufficient quality, it may be required to rinse the water lines before adding of the vaccine, or to add a stabiliser of some sort to the water, such as skimmed milk powder at e.g. 2 grams per litre. - the dilution of the e in the drinking water needs to be in a dilution that assures that each of the target animals ingests the appropriate amount of drinking water to receive (on e) a full dose of the vaccine. - the ation of the vaccine and the drinking water can be done in different ways, e.g. using a medication tank or a dosing pump; a medication tank typically contains all of the medicated water ed for the treatment, and the water flow from this tank will then replace the external water flow during the time of vaccination. The use of a dosing pump implies the injection of a pre-dilution of the vaccine into the water lines, which then mixes naturally and so forms the vaccine dilution. In either format the (pre-)dilution of the vaccine needs to be carefully calculated to achieve that each animal will receive (on average) of full dose of e. - to monitor vaccine distribution through the water-lines, and the uptake by the animals, a colorant can be added, in on to what may already be t in the vaccine. ore in an embodiment of the method for the vaccination of ruminants according to the invention, the vaccine is administered to a target ruminant in drinking water.

One preferred on for administering the oral vaccine according to the invention is in the preparation of nts for transport, for example to a - or er farm. Such transport and ling is quite stressful to the ruminants, and is often followed by outbreaks of RRD in the weeks after. The timing of such vaccination can be optimised to take place at about 1 - 2 weeks before a planned transportation, e.g. before weaning or before transport to a feedlot farm.

Therefore, in an embodiment of the method for the vaccination of ruminants according to the invention, the vaccine is administered to a target ruminant 1 - 2 weeks before a planned transportation of the ruminant. Preferably such vaccination is administered via drinking.

The age, weight, sex, immunological status, etc. of the target ruminant for a vaccination according to the invention, are not critical although it is favourable to vaccinate healthy targets, and to vaccinate as early as possible to prevent (the consequences of) an early infection by a pathogenic M. haemolytica.

Therefore, in an embodiment of the method for the vaccination of ruminants according to the invention, the oral vaccine according to the invention is administered to young ruminants.

The term “young” refers to the period in the life of a ruminant up to its weaning. This period differs for various species of ruminants; for cattle weaning is typically at about 6 - 8 weeks of age, for lambs weaning is at about 4 - 6 weeks of age. Preferably “young” refers to 0 - 8 weeks of age, more preferably 40 to 0 - 6 weeks of age.

Preferred method of mass-administration of the oral vaccine according to the invention, is, e.g. with drinking water.

One advantageous method for the vaccination of ruminants by the administration as a drink, according to the invention, is to administer the oral vaccine to ruminants by admixing the vaccine with milk, and feeding this mixture to the ruminants. Ruminants of all ages like to drink milk, therefore such stration is totally -free for the animals. This can conveniently be done by feeding an appropriate amount of vaccine-in-milk dilution to the ruminant using a drinking trough or a bucket, or for young ruminants a bottle with a suction nipple. The milk drink with the vaccine dilution can conveniently be prepared and administered to a large number of ruminants at a time.

A further advantage is that the M. haemolytica bacteria are quite stable in milk.

Therefore, in an embodiment of the method for the vaccination of nts according to the invention by administration as a drink, the oral vaccine according to the invention is d with milk and fed to ruminants.

The “milk” to be used for admixing with the oral vaccine according to the ion, can be whole milk, and is preferably from the same species as the target. Alternatively the milk can be prepared from powdered milk, such as a milk replacer. Commercial milk replacer is available in a variety of types, both for general cross-species use, or for species-specific use. The milk evidently needs to be of good quality, and the dilution of the vaccine in the milk is preferably prepared shortly before administration by feeding.

The feeding of the vaccine dilution in milk can be incorporated into the normal milk feeding program of the young ruminant target, the timing and the quantities of which will be ent of its species and age.

While the dilution applied for the vaccine in milk will probably not be a great as for a drinking water administration, this still can be between about 10 and 1000 times. For example one animal dose of the oral vaccine ing to the invention can conveniently be administered to a bovine calf in 1 litre of milk, representing e.g. a 1:500 uent dilution to a prescribed animal dose volume of 2 ml per animal.

The dilution of the vaccine in milk or in ng water can be prepared either from the oral vaccine ing to the ion itself, or from an intermediate on. For example the vaccine when in dried form and already containing the PVP, can be dissolved in a small volume of water or milk and subsequently in a larger volume of water or milk. Alternatively, when the vaccine in freeze dried form did not yet contain PVP, it should first be ved in diluent-containing PVP, and then in water or milk.

Alternative wording can be used to describe the embodiments of the oral vaccine and of the method for the vaccination of ruminants, both according to the invention: 40 In a further aspect the invention relates to an oral vaccine according to the ion, for administration to a ruminant as a drink or with the feed.

In a preferred embodiment, the drink is a dilution of the vaccine in milk, for administration by feeding to young ruminants.

The “milk drink” is composed and prepared as described above.

In a further aspect the invention s to a milk drink for the ation by feeding of young ruminants against respiratory disease, the drink comprising a dilution of an oral vaccine according to the invention.

In a further aspect the invention relates to the use of an oral vaccine according to the invention for the manufacture of a milk drink for the vaccination by feeding of young ruminants against respiratory disease.

In a further aspect the invention relates to a method for the reduction of an infection with M. haemolytica or of associated signs of disease in ruminants, characterised in that the method comprises the administration to said ruminants of an oral vaccine according to the invention.

In a preferred embodiment of the method for the reduction of an infection according to the invention, the e is stered to a target ruminant as a drink and/or with the feed.

The invention will now be further bed by the following, non-limiting, examples.

Examples Example 1: Efficacy study of oral e against M. haemolytica comprising PVP 1.1. Summary This experiment trated the efficacy of an oral vaccine comprising a relatively low dose of live attenuated M. haemolytica in PVP, to protect bovine calves of 2 to 3 weeks of age against respiratory disease caused by a severe challenge infection with M. haemolytica.

Forty colostrum-deprived calves, approximately 2 weeks of age were randomized into two treatment groups. Twenty calves in group A were orally administered the test vaccine in milk replacer, ning per dose: the M. haemolytica strain NADC D153 ΔlktA, at about 2x10^8 CFU, and a live attenuated P. multocida antigen, strain 1062 ΔhyaE, at about 1x10^9 CFU. The second group of 20 calves in group B rly received a control vaccine, containing only the live attenuated P. multocida n. Four weeks after vaccination, all animals were challenged intra-tracheally with a culture of virulent M. haemolytica. After a seven day observation period, which included clinical monitoring for respiratory rate per minute and rectal temperature, the animals were euthanized, sied and the lungs were scored for nic lesions.

The data was analysed statistically to determine the differences in lung lesion scores (LLS) between the two treatment groups. The animals in vaccine group A had significantly lower LLS (mean LLS of 11.5) than those in control group B (mean LLS of 28.4); this as a result of the oral administration of the vaccine with at titre of M. haemolytica of 1.8x10^8 CFU/dose, demonstrating the efficacy of the PVP enhanced oral vaccine. 1.2. Vaccine The vaccine for group A contained the M. haemolytica and the P. multocida bacteria at passage +5 from the master seed. Vaccine was reconstituted from freeze dried formulation with sterile water, to 2 ml per animal dose.

The vaccine for group B, (control group to e group A) contained only the P. multocida bacteria. The composition of the e B was the same as that for vaccine A, except that the volume of M. haemolytica ia in water/sucrose/PVP was replaced by TSB.

The M. haemolytica bacteria were cultured using 1 % PVP K 12 in their growth medium. the whole culture was harvested, and the formulated vaccine contained 80 % of its volume from bacteria and culture medium, providing 0.8 % w/v of PVP K 12 to the vaccine. This was then mixed with a stabiliser containing sucrose and PVP K 60, to a final concentration of 6 % sucrose and 1.3 % w/v total PVP (0.8 % K 12 and 0.5 % K 60). This was -dried according to a standard protocol, and the freeze-dried product was stored at 2 - 7 °C. PVP K 60 was obtained from Ashland lty Chemicals (New Jersey, USA) as 45 % w/v solution in water. 1.3. Study design: 1.3.1. Experimental animals: groups A and B each contained 20 calves, all from the same source, of Holstein breed and of mixed sex.

The calves had been raised colostrum-deprived, and were individually marked. The calves were healthy at the time of ation with no prior history of vaccination against M. ytica or P. multocida.

Calves were randomly assigned to one of the ent groups, and were allowed to acclimatise for about 14 days before the vaccination. Calves were fed 2 and later 3 litres of milk replacer twice a day initially from a bottle and later from a bucket. Water was ble at m, and age-appropriate feed was provided.

Daily nary monitoring for general health began when the calves arrived on site and ued throughout the study. Pre-vaccination rectal temperatures and respiratory rates were recorded for all animals on days -1 and 0, and blood s were taken. These readings all served as confirmation on the proper performance of the trial.

Prior to challenge, the calves were co-mingled and randomly divided over pens for challenge infection. The personnel administering the challenge infection, and those performing the lung lesion scoring were blinded from the grouping codes. 1.3.2. Vaccination: Ampules of vaccines were reconstituted in sterile water, and pooled. A dose of 2 ml per animal of the respective vaccine was mixed with about 3 litres of milk replacer shorty before feeding the milk to the calve. Bacterial titrations were done in 5 fold on the pool vaccines to confirm the average titre per animal dose.

Post-vaccination animals were observed daily; one death in control group B at 20 days post ation was of a cause unrelated to the experiment. 1.3.3. Challenge: At -2, -1 and 0 days prior to challenge, rectal temperatures and respiratory rates were recorded for all calves to establish baseline ters. On day -1 blood samples were taken.

The challenge um was an active culture of virulent M. ytica, strain OSU, that had been grown in TSB with moderate agitation at 37 °C, and harvested at an OD of about 0.77. Prior to challenge, the culture was diluted in e TSB to the approximate target dose.

All the calves were challenged on day 28 post-vaccination, by intratracheal inoculation with 30 ml of TSB ning 3x10^8 CFU virulent M. haemolytica bacteria. The titre in CFU was determined in 5 fold, by streaking serial dilutions on standard blood-agar plates.

Post challenge the calves were ed daily at approximately the same time of day. Rectal temperatures and respiratory rates were ed for seven days.

During post-challenge days 1 - 4 several calves died, or were euthanized based on the attending veterinarian’s recommendation. Necropsy was conducted on those calves, and in all cases the observation was: fibrinopurulent bronchopneumonia.

On hallenge day 7, blood samples were taken from survivors. Next all surviving calves were euthanized, and necropsy was conducted with no identification of treatment groups. Lungs were harvested from the calves and the percentage of pneumonic versus normal lung tissue was determined, ing to the procedure described by Jericho & Langford (1982, Can. J. Comp. Med., vol. 46, p. 287- 292). In short: of isolated lungs, the lung-lobe areas affected (visible consolidation) are identified and noted down using a grid pattern, for both lungs, and for both the ventral and the dorsal side. Next the extent of affection of lobes is d from the number of grids, which is then multiplied by the proportion of the total lung normally represented by that lobe. All calculated values are added, maximal score is 100 Because lung lesion scoring is quite difficult, and the lesions observed are quite variable, the scoring was conducted independently by two observers and the two scores were averaged. Tissue samples of ed lung lobes were collected for bacterial re-isolation. 1.4. Results: 1.4.1. Confirmation of vaccine dose: Titration results indicated that the M. haemolytica titre in vaccine A was 1.81x10^8 CFU/2 ml. The P. multocida titre was 2x10^9 CFU/2 ml dose. 1.4.2. Lung lesion Scores: The analysis of the lung lesion data from all the calves showed a mean LLS of 11.46 for the vaccine group A, and a mean LLS of 28.35 for the control group B. As the ratio of these LLS (vaccine/control) is 0.4, which is below 0.5, this indicates (as described in Example 3) that the challenge-protection was efficacious. 1.4.3. Clinical observations: With respect to the rectal temperatures of all the calves, there were 13 of the 19 animals from the control group B and 15 of the 20 s from the vaccinated group A with a temperature > 40 °C on at least one post-challenge day.

With respect to the respiration rates of all the calves, there were 14 of the 19 animals from the control and 19 of the 20 animals from the ated group with a respiratory rate > 40/min on at least one post-challenge day.

Both these readings confirm the proper execution of this vaccination-challenge trial. 1.4.4. ity is: During the post-challenge period, 10 of the 19 animals from the control group B, and 5 of the 20 animals from the vaccine group A died, indicating that the challenge was severe. Clearly, icantly fewer calves died in the vaccinated group A, compared to the l group B. 1.4.5. ial isolation: Out of the 39 lung tissue samples from which isolation was attempted, growth was observed from 37 samples. Eighteen positive isolations were from the e group A and 19 from the control group B. All positive samples were identified as M. haemolytica. 1.5. Conclusions: - Cattle vaccinated orally at 2 weeks of age with a live attenuated M. haemolytica vaccine with PVP, and subjected to a severe challenge with pathogenic M. haemolytica 4 weeks later, had significantly reduced lung lesion scores, as ed to calves that received a control vaccination before challenge.

- The oral vaccine comprised M. haemolytica at 1.81x10^8 CFU and 1.3 % w/v total PVP per animal dose of 2 ml, and was administered in 3 litres of milk-replacer.

- The data proves that the vaccine complies with the indication: “For the oral vaccination of healthy cattle, 2 weeks of age or older, as an aid in the reduction of atory disease caused by Mannheimia haemolytica.” Example 2: Collected results of studies on oral vaccines against M. haemolytica with/without PVP Prior to the experiments described in Examples 1 and 2, several earlier vaccination-challenge s had been performed in . These tested several oral vaccines, comprising different amounts of live- attenuated M. haemolytica, but without PVP. Those experiments were generally med as described in Example 1.

The common result was that only when very high doses of a live attenuated strain of M. ytica were applied, then effective protection against challenge could be achieved, and lower doses were not protective.

It was only after the introduction of PVP into the vaccine, that lower doses of M. haemolytica also became effective.

The combined results of a representative set of these experiments are presented in Table 1 below, which s on the relative reduction of lung-lesions, as the most ant parameter of effective protection against a challenge with pathogenic M. haemolytica.

Table 1: Combined results of M. ytica vaccination-challenge experiments in calves.

Oral e Average lung lesion scores Chall.-prot. exp. nr. M. haem dose PVP used Vaccinates (%) Controls (%) Ratio V/C to M. haem. 029 1,5 x 10^10 No 8,0 17,6 0,45 Yes 031A 1,1 x 10^8 No 7,1 9,0 0,79 No 034B 7,4 x 10^7 No 3,5 3,9 0,90 No 031B 5,1 x 10^7 No 6,8 9,0 0,76 No Expl. 1 1,8 x 10^8 1.3 % 11,5 28,4 0,40 Yes Indications used in Table 1: ‘exp.nr’ = experiment number; ‘M. haem dose’ = dose/animal of live attenuated M. ytica bacteria in the oral vaccine; ‘Chall.-prot. to M. haem.’ = protection against M. haemolitica challenge; ‘Expl. 1’ refers to the experiments described in Example 1.

To facilitate the interpretation of the relative reduction of lung-lesions, the ratio is indicated of the lung lesion scores of the ates over that of the controls (‘Ratio V/C’). This is a simplification of the advanced statistical analysis that was applied in these experiments. Nevertheless this ratio gives a quick indication of tion: when this ratio is 0.5 or less, the test animals can be considered protected against a severe challenge with pathogenic M. haemolytica.

Table 1 y illustrates the advantageous effects of the use of different concentrations of PVP, in an oral vaccine for ruminants against enic M. haemolytica.

Also a further comparison was made between groups from the experiments described herein in Examples 1 and 2, that did or did not receive PVP added to the vaccine. Specifically: a group receiving vaccine with PVP from Example 1 was compared to a group without PVP from the studies listed in Table 1, both for a vaccine dose of 1x10^8 CFU/dose. Other circumstances were the same: vaccine was administered orally in 1 litre of milk replacer, and nge dose was 2x10^8 CFU/ml at 30 ml challenge dose.

The effect of vaccination on challenge-induced lung scores was compared to unvaccinated controls for both these groups. The comparison demonstrates an impressive improvement is reached upon the addition of PVP, namely: without PVP, a vaccination improves lung scores by 21 % as compared to unvaccinated controls; whereas with 1.3 % PVP added, vaccination (with the same dose of bacteria) improves lung scores by 62 %, relative to controls. Consequently, the on of PVP improves the protective effect of vaccination on lung scores by 3 fold ! Example 3: Duration of immunity trial This experiment is currently in progress and will confirm the duration of the immunity induced by the oral vaccine according to the invention.

The layout of this experiment is largely the same as that described in Example 1: At least 40 colostrum deprived calves, two to three weeks of age, will be ized into two treatment groups of 20 each. One group (group A) will be orally administered a 2 ml dose of the oral vaccine mixed with whole milk or milk replacer (about 3 ) containing the live attenuated M. haemolytica at a titre of about 1.8x10^8 CFU/dose, and P. multocida bacteria at about ^8 CFU/dose. The vaccine comprises 1.3 % w/v total PVP (K 60 and K 12), and a blue colorant, and was freeze-dried with 6 % sucrose. The control group B will receive the same vaccine, , in milk, but will comprise only P. multocida bacteria.

Four months after vaccination, the animals will be gled and challenged intra-tracheally with a culture of virulent of M. haemolytica. After a seven day observation period, the animals will be euthanized, necropsied and scored for pneumonic lesions. The data will be analysed statistically to determine the differences in lung lesion scores between the two treatment groups.

This study will demonstrate that a 4 month duration of immunity can be ed with the oral vaccine according to the invention.

Example 4: Duration of ty trial, as performed 4.1. Introduction: This study is the completion of the experiment already described as Example 3 above. The purpose of this study was to demonstrate the sustained efficacy of the M. haemolytica e according to the invention, at four months after oral administration to 2 week old calves, against respiratory disease caused by M. haemolytica. The vaccine used was a modified live vaccine containing the M. haemolytica strain NADC D153 ΔlktA, which was ated with 1.3 % w/v total PVP (K 60 and K 12), as described above in Examples 1 and 3. The e for group A contained live attenuated bacteria of both M. haemolytica and of P. multocida; the control e for group B only contained live attenuated P. multocida .

The calves were received in two shipments, combined, and randomised over the two treatment groups: 21 in group A (vaccinates) and 20 in group B (controls). 4 months after ation the calves were given a challenge infection, and one week after challenge the calves were euthanized and the lungs were evaluated and scored for pneumonic lesions. 4.2. Materials and methods 4.2.1. Animals and housing The calves were received in two shipments of 24 and 20 calves, Within each shipment, calves were randomly assigned to the two treatment groups using the RAND function in Excell™. All calves were from the same source and no blocking factors were used. The calves in the two ent groups were housed separately in individual huts. After two months they were moved to a different building, and housed separately in pens based on the ent groups, whereby comparable numbers of vaccinates and controls were in each building. The calves were gled prior to challenge, and remained commingled until the end of the study.

The personnel who administered the challenge, performed lung lesion g, and bacterial isolation from lung tissues, were blinded to the study grouping codes.

The calves were of both sexes, and were colostrum deprived Holstein race. Identification was by ear tags, and all animals were healthy at the time of vaccination with no prior history of vaccination against M. haemolytica.

The calves were bottle-fed at least 2 liters of milk er twice a day during the first week of life.

From the second week onwards, the calves were bucket- fed at least 2.5 liters of milk replacer twice a day till they were imately 8 weeks old. Water was provided ad libitum. Post-weaning, they received standard feed for animals of this age.

All calves were allowed to acclimate for at least 13 days prior to vaccination. All animals were under daily observation by a veterinarian and animal caretakers. 4.2.2. Vaccine The vaccine for group A was a lyophilized sample containing the M. haemolytica NADC D153 ΔlktA . Also vaccine A contained an acapsular mutant strain of Pasteurella multocida, strain 1062 with ΔhyaE mutation. One dose of vaccine A contained about 1.8 x 10^8 CFU per 2 mL dose of M. haemolytica, and about 1x10^9 CFU per 2 mL dose of P. multocida. Both bacteria were at the 5th passage level from their master seed.

The vaccine for l group B did not contain the M. haemolytica, but contained the P. multocida, and was otherwise prepared similarly to vaccine A. For both es the Blushadow™ diluent was used for rehydrating the lyophilized vaccines.

For each target, a 2 ml sample was taken from pools of vaccine A or vaccine B, dependent on the planned treatment. This was mixed with approximately 2.5 liters of milk replacer, which was fed completely to the calf, as a single dose. The vaccine pools were back-titrated to verify the dose actually Animals were observed daily for general health and ations recorded. During the postvaccination period, some animals died due to causes unrelated to the experiment. During the experiment and surrounding ent steps, blood samples were taken, and rectal temperatures and respiratory rates were noted down. 4.2.3. Challenge The challenge material was an active e of virulent M. haemolytica (strain OSU), which was grown in Tryptic soy broth with moderate ion at 37°C. Prior to challenge, the culture was diluted in sterile TSB to the imate target dose, which was based on a prior established correlation between the OD value and CFU counts.

The challenge was administered on day 123 post-vaccination. Each calf in the study was inoculated once intra-tracheally with 40 mL of TSB containing at least 1x10^8 virulent M. ytica bacteria.

This challenge dose was determined by standard bacterial titration in 5-fold of the challenge material used.

After challenge the calves were observed daily at approximately the same time of day. Rectal temperatures and respiratory rates (per minute) were recorded daily for seven days after the challenge (post-challenge days 1 through 7). ng, if observed and abnormal respiratory patterns, were recorded. Calves were also observed for l health and signs of any disease.

Following challenge 5 calves in group B died from ‘pneumonia’, that was found to be resulting from the challenge infection.

On day 7 hallenge, all surviving calves were euthanized. Necropsy was ted and lungs were harvested from the calves. The percentage of pneumonic lung tissue was evaluated and percent (score) 40 of lung lesions was calculated according to the procedure described by Jericho and Langford (Can. J.

Comp. Med, 1982, vol. 46, p. 287-292). Lung lesion scoring was conducted independently by two observers and the two scores were averaged.

Titration of bacteria in vaccine pools was done on tryptic soy agar plates. Serial tenfold dilutions of Vaccines A and B were made in sterile TSB. Each dilution was streaked on five plates. Plates were incubated at 36 °C for 16-24 hours. Colonies of M. haemolytica could be counted directly; plates of P. multocida ed a further incubation at room temperature for an additional 16-24 hours. The plates for counting M. haemolytica contained 0.001% Nafcillin to inhibit the growth of P. multocida; the plates for counting P. multocida contained 0.0015% Potassium tellurite to inhibit the growth of M. haemolytica.

Titration of the M. haemolytica challenge material was on standard blood-agar plates. 4.3. Results 4.3.1. Confirmation of vaccine dose Titration results indicated that vaccine A contained 1.64 x 10^8 M. haemolytica per 2 mL dose. 4.3.2. Confirmation of challenge dose Titration s indicated that each animal in the study received approximately 4.06 x 10^8 M. haemolytica organisms in the 40 mL dose administered by the intra-tracheal route. 4.3.3. Mortality analysis: The percent death caused by the M. ytica nge was evaluated by the prevented on (PF) method and 95% ence interval for the PF. The PF and associated confidence intervals were calculated with SAS™ 9.3 using the procedure BINOMIAL from StatXact 10 Procs for SAS.

During the post-challenge period, 5 of the 15 animals from the control group died. There were no deaths in the vaccinated group after challenge. The ted fraction was 1.00, with a lower 95% highest density confidence bound of 0.5. 4.3.4. Lung Lesion Scores: The percent of lung tissue with lesions caused by M. haemolytica infection was evaluated by the mitigated fraction (MF) method with associated 95% highest density confidence interval. The MFs and associated ence intervals were calculated with SAS™ 9.3 using the ure PROC_R with R module.

The analysis of the data showed that the mean LLS for the control group B was 24.32 and the mean LLS for the vaccinated group A was 1.02. The mitigated fraction was 0.74 with a lower 95% highest density confidence bound of 0.5. 4.3.5. Clinical ations: With respect to the rectal temperatures of all the calves, there were 5 of the 15 s from the l 40 group B and 3 of the 18 animals from the ated group A with a temperature of over 40 °C on at least one post-challenge day.

With respect to the respiration rates of all the calves, there were 10 of the 15 s from the control group B, and 4 of the 18 animals from the vaccinated group A with a respiratory rate of over 40/minute, on at least one post-challenge day. 4.4. Conclusions During the post-challenge period, 5 of the 15 animals from the control group B died and there were no deaths in the vaccinated group A. The mortality rate was significant in the control group (p= 0.0092).

Also, icant differences of lung lesion scores were ed between the two treatment groups, where control group B animals scored LLS of 24.32 and ates from group A only scored LLS of 1.02 (p =0.0003). There was also a significant difference between the control and vaccinated groups with respect to the maximum respiratory rate (p= 0.0063).

Consequently, the oral M. haemolytica vaccine according to the invention was demonstrated to be protective at a dose of 1.64x10^8 CFU / 2 mL dose, given by oral route, against a severe challenge infection. The results of this experiment demonstrate convincingly the efficacy of a vaccine according to the invention in protecting calves against respiratory disease caused by Mannheimia ytica infection, even up to 4 months after vaccination. After challenge, calves in the vaccinated group A had significantly less ion and disease as indicated by their much lower scores of mortality and LLS.

Further, the difference found in the tion induced by groups A and B also convincingly demonstrates that protection against M. haemolytica challenge could only be provided by vaccination with M. haemolytica, and not by P. multocida vaccination.

In this specification where reference has been made to patent specifications, other external nts, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated ise, reference to such external documents, or such sources of information, is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common l knowledge in the art.

Claims (15)

Claims

1. Oral vaccine against respiratory disease in ruminants, the vaccine sing live attenuated Mannheimia haemolitica bacteria and a pharmaceutically able carrier, wherein the vaccine also comprises Polyvinylpyrrolidone (PVP).

2. An oral vaccine according to claim 1, wherein the live ated M. haemolytica bacteria are unable to express a virulent leukotoxin A protein.

3. An oral vaccine according to claims 1 or 2, wherein the ruminant is a bovine animal.

4. An oral vaccine according to anyone of claims 1 - 3, wherein the concentration of PVP is between about 0.3 and about 3 % w/v.

5. An oral vaccine according to anyone of claims 1 - 4, wherein the vaccine is in freeze-dried form.

6. An oral vaccine according to anyone of claims 1 - 5, n the PVP is of a combination of types of

7. An oral e according to anyone of claims 1 - 6, wherein the oral vaccine also comprises at least one additional active component.

8. An oral vaccine according to claim 7, wherein the at least one additional immunoactive component are live attenuated Pasteurella multocida bacteria.

9. Method for the preparation of an oral vaccine according to any one of claims 1 - 8, sing the step of admixing live attenuated M. haemolytica bacteria and a pharmaceutically acceptable carrier, with PVP.

10. Method for the vaccination of ruminants against respiratory disease, the method comprising the step of administering an oral vaccine according to any one of claims 1 - 8 to said ruminants by oral route.

11. Method according to claim 10, n the oral vaccine is d with milk and fed to ruminants.

12. An oral vaccine according to anyone of claims 1 - 8, for administration to a ruminant as a drink or with the feed.

13. An oral vaccine according to claim 12, wherein the drink is a dilution of the e in milk, for administration by feeding to young ruminants.

14. Milk drink for the vaccination by feeding of young ruminants against respiratory e, the drink comprising a dilution of an oral vaccine according to any one of claims 1 - 8.

15. Use of an oral vaccine according to any one of claims 1 - 8 for the manufacture of a milk drink for the vaccination by feeding of young ruminants against respiratory disease.