WO1994005783A1

WO1994005783A1 - Novel haemophilus organism and vaccines containing the same

Info

Publication number: WO1994005783A1
Application number: PCT/AU1993/000457
Authority: WO
Inventors: Patrick Blackall
Original assignee: Bioproperties (Australia) Pty. Limited
Priority date: 1992-09-07
Filing date: 1993-09-06
Publication date: 1994-03-17

Abstract

Essentially pure H.paragallinarum organisms which on mutagenesis with a mutagenising agent give rise to mutant organisms of low or absent pathogenicity when compared to wild type reference strains, and mutants thereby produced are described. Particularly preferred H.paragallinarum organisms are strains HP286, HP19, HP286/10, HP286/15 and HP19/4. Vaccines containing H.paragallinarum organisms are also described.

Description

NOVEL HAEMOPHILUS ORGANISM AND VACCINES CONTAINING THE SAME

The instant invention to novel Haemophilus paragallinarum (hereafter H.paragallinarum) organisms and mutant H.paragallinarum organisms and to components and parts thereof useful in the development of diagnostic assays and vaccines against Haemophilus infection.

The bacterial genus Haemophilus causes a number of diseases in animals, particularly respiratory diseases. In poultry, and in particular chickens, infection with H.paragallinarum causes a disease known as infectious coryza which results in decreased production yield. Infection with

Haemophilus is often associated with mycoplasma infection which may give rise to serious respiratory and other diseases. It is, therefore, desirable to reduce or prevent Haemophilus infection in animals.

H.paragallinarum is a gram-negative coccobacillus, which is oxidase positive, catalase negative, typically dependent upon nicotinamide adenine dinucleotide for growth in vitro, non- motile, produces acid from glucose, sucrose and mannitol, does not produce acid from arabinose, gaiactose, lactose and trehaiose. There are three serovars or groups of H.paragallinarum (determined serologically) known as groups A, B and C. H.paragallinarum is a major poultry pathogen for which urgent control is required.

It has previously been proposed to protect poultry from H.paragallinarum infection by administration of a vaccine comprising killed H.paragallinarum organisms. The killed vaccines only protect against the serovar of H.paragallinarum administered and generally require two or more administrations in order to achieve protection against the particular vaccinating serovar.

Furthermore, killed vaccines generally require coadministration with adjuvants in order to achieve an acceptable immune response in the vaccinated animal. Adjuvant administration may cause local inflammation which causes stress to the animal with associated side effects, as well as the possibility of granuloma formation.

This invention arises from the identification of H.paragallinarum isolates, which on mutagenesis with mutagenising agents give rise to mutant H.paragallinarum organisms of low or absent pathogenicity, with excellent growth characteristics. Such mutant organisms or fragments or antigens thereof may be used in the production of vaccines against Haemophilus infection.

Accordingly, in a first aspect this invention is directed to an essentially pure H.paragallinarum organism which on mutagenesis with a mutagenising agent gives rise to mutant organisms of low or absent pathogenicity when compared to wild type H.paragallinarum reference strains on administration of live organisms to poultry. More particularly said H.paragallinarum organisms are selected from H.paragallinarum isolates HP286 and HP 19.

HP286 and HP 19 are H.paragallinarum isolates which were isolated from chickens in

Australia suffering from infectious coryza. Whilst these strains are pathogenic in their own right and cause infectious coryza in poultry, mutants of these strains produced using conventional mutagenic techniques well known in the field (Lai et al, 1990, Infection and Immunity 58:2289-2296), have a propensity toward being non-pathogenic, yet capable of colonizing birds and establishing a protective immune response to H.paragallinarum challenge regardless of serovar specificity.

Strains HP286 and HP19 were deposited with the Australian Government Analytical Laboratories, a Budapest Treaty Depository, on 3 September 1993, respectively under accession numbers N93/37100 and N93/37103.

In another aspect, this invention relates to mutants of strain HP286 or HP 19 which exhibit low or absent pathogenicity compared to wild type H.paragallinarum reference strains on administrations of live organisms to poultry. Such mutants require no adjuvant when administered as a live vaccine and may protect against challenge with H.paragallinarum organisms of one or more of serovars A, B and C. Preferably the mutant strains protect against challenge with two or more of H.paragallinarum serovars A, B and C. This is of considerable practical significance as there are now a number of wide spread serovars of H.paragallinarum which cause respirator)' diseases such as infectious coryza.

Preferred mutants of strains HP286 are strains HP286/10 and HP286/15. Other mutants of HP286 which exhibit low pathogenicity are strains HP286/2, HP286/4, HP286/5 and HP286/7. A preferred mutant strain of HP 19 is HP 19/4. H.paragallinarum organisms HP286/10. HP286/15 and HP 19/4 have been deposited with the Australian Government Analytical Laboratory and deposit particulars are set out at the end of the description of the invention. All of these organisms have excellent growth characteristics.

It is to be understood that the invention is not restricted to the aforementioned mutants of

HP286 and HP 19, but instead relates to any mutant of these organisms, produced according to standard mutagenic techniques well known in the art, which have low or absent pathogenicity when administered as live organisms to poultry. Pathogenicity of mutant H.paragallinarum organisms is readily and simply determined by administration of organisms (generally from about 1 x 10⁴ to 1 x 10⁹ organisms) to chickens, such as by oral or sinus administration or by instillation into the eye. If birds show standard clinical signs oϊ H.paragallinarum infection (acutely swollen sinuses and nasal discharge) during a seven day test period, the organisms are pathogenic.

It has been found that those mutant organisms which are temperature sensitive (unable to grow under temperature conditions of 40°C), and/or which give rise to small colonies when compared to the parent strain are likely to be of low or absent pathogenicity. These selection procedures are not absolutely determinative but provide a good practical guide for selection of organisms which can then be routinely tested in a standard chicken challenge test as described above, and as set out in more detail in the Examples.

The above mentioned H.paragallinarum organisms may be used as live vaccines without the necessity for killing the organisms, this being due to low or absent pathogenicity as mentioned above. Surprisingly, as discussed in more detail hereafter, live vaccines so produced comprising a single isolate may provide protection against all of the H.paragallinarum serovars.

Mutants of H.paragallinarum strains HP286 and HP 19, may be prepared according to methods well known in the art of microbiology, such as irradiation, chemical mutagenesis (using agents such as nitrosoguanidine), and directed mutagenesis techniques such as transposon mutagenesis. Preferred mutagenic agents are N-methyl-N'-nitro-nitrosoguanidine (NTG) and the ethyl ester of methane sulfonic acid (EMS). Any such mutants are within the scope of this invention with the proviso that such mutants possess low or absent pathogenicity to host animals (compared to wild type pathogenic organisms) as well as good growth characteristics as mentioned above, thus allowing production of large amounts of the organism for formulation into vaccines or other uses.

In a further aspect of this invention there is provided an antigen from a mutant strain of H.paragallinarum HP286 or HP 19 which antigen is capable of eliciting an immune response on administration to poultry which may be protective against H.paragallinarum infection. Preferred organisms from which antigens may be derived are HP286/10, HP286/15 and HP 19/4.

Antigens may be in the form of proteins, peptides or fragments thereof, lipids, carbohydrates, lysed cells, sonicated cells, cell extracts and cell components and fragments of cells, or combinations of one or more of the aforementioned components, produced according to methods well known in the art. Antigens of the aforementioned H.paragallinarum strains may be incorporated into vaccines for the control oϊ Haemophilus infection, in particular H.paragallinarum infection in animals, such as poultry, and particularly chickens. Immune response generated by administration of antigens oϊ H.paragallinarum organisms may not necessarily be protective of infection, but may produce immune serum useful in diagnosis/detection oϊ Haemophilus infection.

In another aspect the invention is directed toward isolated nucleic acids encoding antigens capable of eliciting an immune response whether or not protective, and parts or derivatives thereof, for example, comprising twenty or more nucleotides thereof.

Nucleic acids of H.paragallinarum organisms of the invention may be expressed in appropriate expression vectors either within a host, such as suitable bacterial or prokaryotic cells, or in a cell free system, as are well known in the art (Sambrook et al, Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1989). Expressed antigens may be formulated into vaccines, such as subunit vaccines or multicomponent vaccines in association with suitable adjuvants (such as Freunds Adjuvant, alum, and the like) and pharmaceutically or veterinarily acceptable carriers.

The present invention further extends to oligonucleotides useful as genetic probes or antisense molecules, or as primers for use in Polymerase Chain Reactions (PCRs) for the detection oϊ Haemophilus, particularly H.paragallinarum according to methods well known in the art. Such oligonucleotides are derived from isolated nucleic acids of mutants as herein described. Oligonucleotides generally comprise ten or more nucleotides, preferably from ten to forty nucleotides.

In a further aspect, the invention relates to claim a method for ameliorating the effects of, or protecting animals from Haemophilus infection comprising administering to an animal an effective amount of a live vaccine comprising a H.paragallinarum organism which is a mutant of strain HP286 or HP19 of low or absent pathogenicity compared to wild type reference strains, optionally in association with a pharmaceutically or veterinarily acceptable carrier.

In a particularly preferred aspect, the method of this invention is directed to the treatment oϊ H.paragallinarum infection in birds such as chickens, turkeys, ducks, pigeons, quail and the like. Preferably this aspect is concerned with the treatment oϊ H.paragallinarum in chickens, particularly those used for the commercial production of eggs.

The preferred mutant strains of HP286 or HP 19 which may be used in the method of this invention are strains HP286/10, HP286/15 and HP19/4. Other mutants which may be used in invention include strains HP286/2, HP286/4, HP286/5, HP286/6 and HP286/7. It is to be appreciated that this aspect of the invention is not restricted to any specific mutant strain or HP286 or HP 19 but rather extends to any strain which is a mutant of the aforementioned strains and which is of low or absent pathogenicity compared to wild type reference strains. Suitable mutant strains may be prepared and screened according to methods previously described in this application.

Organisms may be administered to animals by various methods well known in the art, such as by oral administration in animal feed or water, or by installation into the eye, mouth or other orifice. The organisms according to this invention need only be administered once to provide protection against infection with any H.paragallinarum serovar. Having said this, organisms may be administered as many times as are necessary to elicit an immune response. The amount of organisms administered to an animal, preferably poultry (chickens, turkeys, ducks, geese, pigeons and the like) and particularly chickens, is not critical as long as a protective immune response is produced. In general terms, and without limiting the invention, mutant organisms of HP286 or

HP19 of low or absent pathogenicity are administered in an amount of about 10⁴ to 10¹⁰ organisms, preferably about 10⁵ to 10⁷ organisms. The administration of live organisms obviates the need for use of an adjuvant because of the self-adjuvancy of the live organisms. Notwithstanding this, organisms may be administered with an adjuvant, particularly where organisms are administered by injection, such as subcutaneous or intramuscular injection.

In a further aspect, this invention relates to a method for ameliorating the effect of, or protecting animals from, Haemophilus infection which comprises administering to an animal an vaccine effective amount of an antigen derived from a H.paragallinarum organism which is a mutant of strain HP286 or HP 19 of low or absent pathogenicity compared to wild type reference strains, optionally in association with a pharmaceutically or veterinarily acceptable carrier.

Antigens, as previously described, are generally administered parenterally such as by transdermal intramuscular injection. In these circumstances it may be necessary to use adjuvants well known in the art (such as Freunds Adjuvant, alum, polysaccharide and monosaccharide adjuvants, and the like).

The H.paragallinarum organisms as herein described or, fragments or antigens thereof may be combined with pharmaceutically/veterinarily and or other ingredients conventionally used in vaccine preparation. Thus, in accordance with a further aspect of this invention there is provided a vaccine for the treatment/prevention oϊ Haemophilus infection.

In a still further aspect, the invention relates to a live infectious coryza vaccine which comprises a mutant strain of HP286 or HP 19 which exhibits low or absent pathogenicity compared with wild type H.paragallinarum reference strains, optionally in association with a pharmaceutically or veterinarily acceptable carrier. Preferably the vaccine comprises one or more of strains

HP286/10, HP286/15 and/or HP19/4. While the aforementioned strains are preferred, any mutant of HP286 or HP 19 may be used in the invention as long as it has low or absent pathogenicity and elicits a protective immune response in animals, preferably poultry, more preferably chickens.

Any convenient means can be used to administer vaccines. For example, vaccines may be administered by injection (such as subcutaneous or intramuscular injection), in animal feed or water or by instillation into the eye. mouth or other orifice as previously described. With regard to poultry, the preferred means of administration is by instillation to the eye, for example either by direct instillation or using a mist. The isolates of this invention need only be administered once to provide protection against infection with any H.paragallinarum serovar as discussed above.

The invention will now be described with reference to the following non-limiting examples.

EXAMPLE I

ISOLATION OF H.paragallinarum

STRAINS HP286 AND HP19

The strains HP286 and HP 19 were separately isolated from chickens suffering from mild respiratory disease. Strain HP 19 was isolated from a chicken flock in Queensland, Australia and strain HP286 from a chicken flock in South Australia, Australia. Each strain was isolated from a nasal swab of an affected chicken. Swabs were plated onto blood agar that was then cross-streaked with a nurse colony oϊ Staphylococcus aureus. HP286 and HP 19 were identified as members of the H.paragallinarum species by standard microbiological tests (Blackall, P J and Yamamoto R [1990] Infectious coryza. In: A laboratory manual for the Isolation and Identification of Avian Pathogens, 3rd Edition, pages 27-31, American Association of Avian Pathologists).

HP286 and HP 19 were serotype tested using serovar specific antibody reagents. HP286 was shown to belong to serovar C. HP 19 was shown to belong to serovar A. These organisms exhibit all of the characteristics oϊ H.paragallinarum (Blackall, P J and Yamamoto, Supra).

HP286 and HP 19 were tested in a standard test to ascertain whether or not these organisms were useful as live vaccines against H.paragallinarum infection. In these tests, an overnight culture of the organisms, generally 10⁶ organisms, was administered in a volume of 0.2ml directly into the sinus of chickens. Chickens were then observed everyday for seven days for clinical signs of H.paragallinarum infection and pathogenicity, which is characterized by swollen sinuses and nasal discharge. Birds are scored for severity of infection according to according to the following scale: 0 normal; 1+ mild swelling of sinus and/or nasal discharge; 2+ frank swelling of sinus; 3+ gross swelling of the sinus; 4+ gross swelling of the sinus that extends completely around the eye socket. While HP286 was only mildly pathogenic in the animals from which it was isolated, testing as above showed that neither HP286 nor HP 19 were mild pathogens but rather were fully pathogenic causing acutely swollen sinuses and nasal discharge in infected chickens. As a consequence, neither of these strains were suitable as candidate live vaccine strains.

Mutants of HP286 and HP 19 were then produced according to standard mutagenic techniques to assess whether or not such mutants would be suitable as live vaccine candidates. It was suφrisingly found that these strains had a propensity to produce, on mutation, organisms of low or absent pathogenicity when tested in animals, but at the same time capable of colonizing the animals to produce a protective immune response. The production of mutant strains is described in Example 2.

EXAMPLE 2

Mutants of strains HP286 and HP 19 were produced according to conventional procedures (Lai et al, [1990] Biological evaluation of Mycoplasma pulmonis temperature-sensitive mutants for use as possible rodent vaccines. Infection and Immunity 58:2289-2296).

Chemical mutagenesis was carried out as follows. Strain HP286 and HP 19 were first grown in nutrient broth medium for eight hours. One milligram of a lmg/ml solution of N-methyl-N'- nitro-nitroguanidine (NTG) or the ethyl ester of methane sulphonic acid (EMS) was then added to 5ml of the culture suspension and the mixture was incubated with agitation at 37°C for thirty minutes. Bacterial cells were then washed three times in saline and the final resuspension was plated out onto agar plates and incubated at 33°C.

From a myriad of potential selection procedures, such as growth in minimal nutrients, antibiotic selection, growth in presence of specific substrates and the like, a decision was serendipitously made to select mutant organisms on the basis of temperature sensitivity (using an incubation temperature of 40°C) and/or on the basis of colony size, selecting for small colonies compared to the parent strains as determined by visual analysis of bacterial plates. These selection criteria turned out to be the most fortuitous as temperature sensitive mutants and small colonies had a marked statistical propensity (greater than about 40%) of low to absent pathogenicity, as determined by challenging chickens with organisms (about 10⁶ organisms) and determining whether or not pathogenic signs appear, according to the procedure set forth in Example 1. Some organisms of low or absent pathogenicity were also temperature sensitive and small colony mutants. Mutants which were produced by mutation with N-methyl-N'-nitro-nitroguanidine (NTG) include strains HP286/2, HP286/4, HP286/5, HP286/6, HP286/7 and HP286/10. Mutants produced on mutation with the ethyl ester of methane sulphonic acid (EMS) included HP286/15 and HP 19/4. HP286/10 and HP286/15 are temperature sensitive mutants and were selected through failure to grow at 40°C. HP 19/4 is a small colony mutant

Mutants of low or absent pathogenicity are produced by irradiation of strains HP286 and HP 19 as follows. Cells of strain HP286 and HP 19 are washed three times in phosphate buffered saline and then dispensed in shallow (5mm deep) sterile containers. The cultures are then exposed to a UV light for upto 200 seconds. Bacterial cells are washed three times in phosphate buffered saline and the final resuspension plated onto agar plates and incubated at 33°C.

The mutant organisms do not differ from their respective parents except that they show either temperature sensitivity and/or small colony size. Neither of these markers are present on reisolation of organisms administered to birds. All of the mutants show excellent growth characteristics. The following table shows the properties of preferred mutant strains.

TABLE 1: Basic Properties of Parents and Mutants

^A NTG = N-methyl-N'-nitro-nitroguanidine

EMS = ethyl ester of methane sulphonic acid

EXAMPLE 3

A particularly useful feature of H.paragallinarum mutants, and particularly strains HP286/10, HP286/15 and HP19/4 is that all of these strains have been rendered non-pathogenic. 10

but are capable of colonizing birds (for varying time periods) and stimulating a protective immune response.

Chickens were inoculated with H.paragallinarum organisms of Example 2 according to the procedures of Example 1 and observed over a seven day period. Results of this are shown in Table 2 below.

TABLE 2: Pathogenicity Trial Results

Presence at Day 2 of swollen sinus and/or nasal discharge. Figure in brackets is average score per bird. Scoring system as follows: 0 normal; 1 + mild swelling of sinus and/or nasal discharge; 2+ frank swelling of sinus; 3+ gross swelling of the sinus; 4+ gross swelling of the sinus that extends completely around the eye socket.

Presence of mucus in sinus at post mortem. Figure in brackets is average score per bird. Scoring system as follows: 0 normal; 1+ small amount of mucus; 2+ frank presence of mucus; 3+ large amount of mucus present. ^c Presence of organisms in sinuses at post mortem. Figure in brackets is average score per bird. Scoring systems as follows: 0 no haemophili detected; 1+ 1 to 10 haemophili detected; 2+ 10 to 100 haemophili detected; 3+ >100 haemophili but not a caφet of growth; 4+ heavy growth of haemophili that forms a caφet of growth.

Pathogenicity. The pathogenicity data presented in Table 2 have been shown as an average score per bird as well as the total number of birds positive for a particular characteristic. The following sections present an analysis of the results.

Strain HP286 caused clinical signs of infection similar to serious clinical infection associated with wild-type infection. HP286 induced the production of mucus in the sinuses of three out of ten chickens studied one week after challenge. The organism was present in the sinuses of nine out of ten chickens one week after challenge.

All the mutant strains failed to produce any clinical signs when inoculated by the intra- orbital route into groups often chickens with the exception that strain HP286/10 caused one chicken to produce a mild swelling of the sinus. In these same chickens, the mutants all also failed to induce the production of mucus in the sinuses one week after challenge, with the exception that strain HP286/10 and HP286/4 induced the production of mucus in three or two chickens, respectively. In these same chickens, the mutant HP286/5 could not be recovered one week after the original inoculation. For the remaining mutants, the number of chickens that were shown to be colonized were three for HP286/10, three for HP286/7 and one each for HP286/5, HP286/4 and HP286/7. The extent of pathogenicity of the mutant strains was negligible indicating these strains could be used as live vaccines.

The cross protection provided by HP286 and mutant organisms HP286/10, HP286/15 and HP19/4 when administered as live vaccines via eye drop inoculation was then assessed on challenge with wild type H.paragallinarum organisms HP 14 (serovar A) and HP31 (serovar C). Wild type organisms were administered directly to the sinus by infraorbital administration. From 10⁴ to 10⁷ organisms (generally about 10⁶ organisms) were administered in a volume of 0.2ml via the intraorbital route and chickens were then observed for seven days. Results are set out in Tables 3 through 6.

Presence of swollen sinus and/or nasal discharge. Figure in brackets is average score per bird.

Presence of mucus in the sinuses at post mortem. Figure in brackets is average score per bird.

Protection Rate is the number of chickens with no clinical signs and no lesions at post mortem divided by the number of chickens (expressed as a percentage).

^{A B C} As for Table 3.

^{A B C} As for Table 3.

A, B, C As for Table 3.

HP286, HP286/10, HP286/15 and HP19/4 protected against subsequent challenge from a virulent strain of either H.paragallinarum serovar A (as represented by HP 14) or serovar C (as represented by HP31) with the exception that HP286/10 provided poor protection against serotype

A. The score of clinical signs and lesions was low for the mutant organisms (below 0.8) indicating mild symptoms.

MICROORGANISM DETAILS

All strains were deposited on 3 September 1993 with the Australian Government Analytical Laboratory, a Budapest Treaty Depository, of P O Box 385, Pymble, New South Wales, 2073, Australia. 14

MICROORGANISM ACCESSION NUMBER

HP286 N93/37100 HP19 N93/37103 HP286/10 N93/37101 HP286/15 N93/37102 HP 19/4 N93/37104

Claims

1. An essentially pure H.paragallinarum organism which on mutagenesis with a mutagenising agent gives rise to mutant organisms of low or absent pathogenicity when compared to wild type H.paragallinarum reference strains on administration of live organisms to poultry.

2. An essentially pure H.paragallinarum organism according to claim 1 selected from H.paragallinarum strains HP286 or HP 19, or a mutant of said strains.

3. A H.paragallinarum organism which is a mutant of strain HP286 and which exhibits low or absent pathogenicity compared to wild type H.paragallinarum reference strains on administration of live organisms to poultry.

4. A H.paragallinarum organism which is a mutant of strain HP 19 and which exhibits low or absent pathogenicity compared to wild type H.paragallinarum reference strains on administration of live organisms to poultry.

5. A H.paragallinarum organism according to claim 3 selected from HP286/10 or HP286/15.

6. A H.paragallinarum organism according to claim 4 which is HP 19/14.

7. An antigen from a H.paragallinarum organism as defined in any one of claims 3 to 6 which antigen is capable of eliciting an immune response on administration to poultry which may be protective against H.paragallinarum infection.

8. An antigen according to claim 7 which is an antigen of HP286/10 or HP286/15.

9. An antigen according to claim 7 which is an antigen of HP 19/4.

10. An antigen according to any one of claims 7 to 9 which is a protein, peptide or fragment thereof, lipid, carbohydrate, cell extract or cell fragment, or mixture of any of the aforementioned components derived from H.paragallinarum.

11. A method for ameliorating the effects of, or protecting animals from, Haemophilus infection comprising administering to an animal an effective amount of a live vaccine comprising a H.paragallinarum organism which is a mutant of strain HP286 or HP 19 of low or absent pathogenicity compared to wild type reference strains, optionally in association with a pharmaceutically or veterinarily acceptable carrier.

12. A method according to claim 1 1 wherein said H.paragallinarum organism is HP286/10 or HP286/15.

13. A method according to claim 1 1 which said H.paragallinarum mutant organism is strain

19/4.

14. A method according to claim 1 1 wherein said organisms are administered orally or by instillation in the eye.

15. A method according to claim 1 1 wherein said animals are poultry species.

16. A method according to claim 11 wherein amelioration or protection from Haemophilus infection is in respect of one or more oϊ H.paragallinarum serovars A, B, or C.

17. A method according to claim 1 1 which further comprises the administrations of a separate vaccine component for the treatment of disease.

18. A method for ameliorating the effect of, or protecting animals from, Haemophilus infection which comprises administering to an animal a vaccine effective amount of an antigen derived from a H.paragallinarum organism which is a mutant of strain HP286 or HP 19 of low or absent pathogenicity compared to wild type reference strains, optionally in association with a pharmaceutically or veterinarily acceptable carrier.

19. A method according to claim 19 wherein said antigen is an derived from strain HP286/10 or HP286/15.

20. A method according to claim 19 wherein said antigen is an antigen derived from strain 19/4.

21. A method according to any one of claims 19 to 21 wherein said antigen is a protein, peptide or fragment thereof, lipid, carbohydrate, cell extract or cell fragment, or mixture of any of the aforementioned components derived from said H.paragallinarum organisms.

22. A live infectious coryza vaccine which comprises one or more of mutant strains HP286 or HP 19 which exhibit low or absent pathogenicity compared to wild type H.paragallinarum reference strains, optionally in association with a veterinarily acceptable carrier.

23. A vaccine according to claim 23 which comprises one or more of strains HP286/10, HP286/15 and/or HP 19/4.