WO2003037367A2

WO2003037367A2 - New bacterium causing poultry disease and vaccine derived thereof

Info

Publication number: WO2003037367A2
Application number: PCT/EP2002/011899
Authority: WO
Inventors: Maria Elena Vazquez; Raul Campogarrido; Carlos Gonzales-Hernandez; Vaithianathan Sivanandan
Original assignee: Boehringer Ingelheim Vetmedica S.A. De C.V.
Priority date: 2001-10-26
Filing date: 2002-10-24
Publication date: 2003-05-08
Also published as: AR067732A2; HRP20040366B1; HU228692B1; JP2005511032A; MXPA04003941A; MY138203A; NZ532805A; DE10152307A1; RU2481393C2; MA26293A1; RU2007148219A; RU2430967C2; RU2334792C2; PT1442114E; RU2004116134A; ES2304466T3; AU2002363173B2; RU2007129099A; US7794735B2; WO2003037367A3

Abstract

The invention belongs to the field of animal health and in particular the causative agent of a new bacterial poultry disease, Pasteurella trehalosi and/or Mannheimia haemolytica. The invention provides said Pasteurella trehalosi and/or Mannheimia haemolytica bacteria, vaccine comprising inactivated Pasteurella trehalosi and/or Mannheimia haemolytica, and a method of immunizing to prevent the disease in chicken.

Description

Novel bacterial poultry disease

Field of the invention

The invention belongs to the field of animal health and in particular the causative agents of a new bacterial poultry disease, Pasteurella trehalosi and/or Mannheimia haemolytica. The invention provides said Pasteurella trehalosi and/or Mannheimia haemolytica bacteria, a vaccine comprising inactivated Pasteurella trehalosi and/or Mannheimia haemolytica, and a method of immunizing chicken to prevent said disease in chicken.

Background of the invention

During the last decade, intensive poultry farming methods to increase productivity, has resulted in an increase of disease manifestation throughout all major poultry producing countries. This has caused an increasing need for new and better vaccines and vaccination programs to control these diseases. Nowadays, most animals are immunized against a number of diseases of viral and bacterial origin. Examples of viral diseases in poultry are Newcastle Disease, Infectious Bronchitis, Avian Pneumovirus, Fowlpox, Infectious Bursal Disease etc. Examples of bacterial diseases are Avian Coryza caused by Haemophilus paragallinarum (upper respiratory tract), Bordetella avium (upper respiratory tract), Ornithobacterium rhinotracheale (lower respiratory tract), Salmonella infections (digestive tract), Pasteurella multocida, which is the causative agent of fowl cholera (septicemic), and E. coli infections.

Therefore, the technical problem underlying this invention was to identify a new bacterial poultry disease, to provide the causative agent of said disease and a to provide a vaccine to prevent said disease.

Figure legends

A) From field outbreaks

FIG. 1) Broilers: Nasal discharge and swollen areas around the eye. FIG. 2) Broilers: Haemorrhage in heart and coronary fat.

FIG. 3) Broilers: Conjuctivitis and inflammation around the eye.

FIG. 4) Layers: Nasal discharge and displaced comb with cyanosis.

FIG. 5) Layers: Inflammation and haemorrhage around the eye.

FIG. 6) Layers: Haemorrhage in the dermal tissue behind entrance to auditory orifice.

FIG. 7) Layers: Inflammation of kidneys.

FIG. 8) Layers: Haemorrhages in oviduct.

FIG. 9) Layers: Deformed ovarian follicles.

FIG. 10) Layers: Haemorrhage in the junction between proventriculus and gizzard.

FIG. 11 ) Layers: Congestion and haemorrhage in oviduct.

B) Experimental Infection

FIG. 12) Layers: Inflammation and haemorrhage in kidney.

FIG. 13) SPF: Prostration.

FIG. 14) Layers: Haemorrhage in joint and muscle.

FIG. 15) Layers: Nasal discharge and pale comb.

FIG. 16) Layers: Haemorrhage in muscle.

FIG. 17) SPF: Haemorrhage in heart and coronary fat.

FIG. 18) Layers: Healthy bird on the left and sick bird on the right with ruffled feathers.

FIG. 19) Layers: Greenish diarrhea.

FIG. 20) SPF: Haemorrhage in muscle.

FIG. 21 ) SPF: Prostration (locomotive problems) and greenish diarrhea.

FIG. 22) Layers: Enlarged liver with haemorrhage.

Disclosure of the invention

Definitions of terms used in the description:

Before the embodiments of the present invention it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a Pasteurella trehalosF' includes a plurality of such Pasteurella trehalosi bacteria reference to the "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. It is irrelevant whether a word is capitalized or not, therefore both "Arabinose" and "arabinose" have the same meaning, unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies as reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Surprisingly, a new bacterial poultry disease has been observed by the present inventors, which occurs primarily in layers and less frequent in broilers. The disease was seen in chicken that had been vaccinated against the bacterium Haemophilus paragallinarum (causative agent of avian Coryza), and Pasteurella multocida (causative agent of fowl cholera). The symptoms of this new disease differ from the specific symptoms of Coryza. Given the fact that the newly discovered disease clearly shows the clinical signs of a upper respiratory tract infection as described below, H. paragallinarum could be ruled out as the causative agent.

The present invention relates in a first embodiment to Gram-negative, facultative anaerobic, pleomorphic rod-shaped bacteria causing a novel disease of the upper respiratory and of the reproductive tract of poultry, wherein said bacteria are selected from the group of Pasteurella trehalosi and/or Mannheimia haemolytica.

Said bacteria according to the invention may be isolated from infected trachea, palatine cleft, ovary, liver, heart, kidney and gonads (broilers). They can be identified as Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention based on the tests listed below :

Beta haemolysis + Gram stain Oxidase + Catalase +

Urease

Nitrate +

Indole

The bacterial isolates may be purified and biotyped according to the method described by Jaworski et al. (1 ). This method is also exemplified in the examples. Important method to classify bacteria are DNA-DNA hybridization, REA (restriction enzyme analysis see e.g. J. Clinical Microbiol, 1993, 31 : 831-835) and ribotyping. Said methods may be applied by the artisan to find out whether bacteria are within the scope of the present invention. A challenge model to validate Koch's postulates is also exemplified in the examples.

Thus, an important embodiment of the present invention are Pasteurella trehalosi and/or Mannheimia haemolytica, wherein said Pasteurella and/or Mannheimia are beta(β)-haemolysis-positive, Gram-negative, oxidase-positive, catalase-positive, urease-negative, nitrate-positive and indole-negative. Preferably, said Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention are also MacConkey-positive. Even more preferred, said Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention are additionally Glucose-positive, Sucrose-positive, Mannitol-positive, Arabinose-negative, Celobiose-negative, Xylose- positive, Salicin-negative, Omithine-negative, Esculin-negative, alpha-Fucosidase- negative, beta-Galactosidase-positive. Most preferred are Pasteurella trehalosi according to the invention, wherein said Pasteurella are also Arabinose-negative and Trehalose-positive. Preferably also, said Pasteurella trehalosi according to the invention are also beta(β)-Glucosidase-negative or -positive, depending on the biotype. Also most preferred are Mannheimia haemolytica according to the invention, wherein said Mannheimia are furthermore Arabinose-negative and Trehalose-negative. Preferably also, said Mannheimia haemolytica according to the invention are also beta- Glucosidase negative.

These characteristic properties of the bacteria according to the invention renders the bacteria according to the invention novel over other known bacterial poultry pathogens (Diseases of Poultry, Tenth Edition, Edited by B.W. Calnek, Iowa State University Press, Iowa, U.S.A. 1997). Another preferred embodiment of the present invention are Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention, wherein said poultry is selected from the group of chicken, turkey, duck, goose, dove, pigeon and quail.

The invention provides a novel type of Gram-negative, facultative anaerobic, pleomorphic rod-shaped bacteria, said novel type of bacteria being characterized by the bacteria deposited at the American Type Culture Collection (ATCC), 1081 , University Boulevard, Manassas, VA 20110 - 2209, USA, under the following deposit numbers:

- Pasteurella trehalosi ATCC No. PTA-3667 (internal designation BIV-4985);

- Pasteurella trehalosi ATCC No. PTA-3668 (internal designation BIV-AVICOR);

- Mannheimia haemolytica ATCC No. PTA-3669 (internal designation BIV-07990). The date of deposit was August 22, 2001.

Thus, a most preferred embodiment of the present invention are Pasteurella trehalosi as deposited at the under accession number ATCC No. PTA-3667. These bacteria are further exemplified in table 3 of example 1.

Another most preferred embodiment of the present invention are Pasteurella trehalosi as deposited at the under accession number ATCC No. PTA-3668. These bacteria are further exemplified in table 2 of example 1.

Another most preferred embodiment of the present invention are Mannheimia haemolytica as deposited at the under accession number ATCC No. PTA-3669. These bacteria are further exemplified in table 1 of example 1.

The results of tests from sections A and B in the examples (Tables 1 , 2 and 3) confirms the bacteria BIV- 4895; ATCC No. PTA-3667 and BIV-AVICOR; ATCC No. PTA-3668 to belong to the family Pasteurellaceae (Pasteurella trehalosi, which are Trehalose positive and arabinose negative), while the bacteria BIV-07990; ATCC No. PTA-3669 belong to the family Mannheimia (Mannheimia haemolytica, which are Trehalose negative and arabinose negative).

The invention also relates to microbiological culture comprising bacteria according to the invention as disclosed above. The culture may be made by growing said bacteria at a temperature of between 35° and 37°C. The bacteria may be grown under normal atmospheric oxygen pressure. The bacteria can be grown in a variety of different general-purpose bacterial growth promoting media known to the skilled person, e.g. Tryptose Broth (TB), Soy Trypticasein Broth or Brain Heart Infusion broth or any enriched media. The bacteria may also be grown on sheep blood agar incubated at 37°C for 24 hours.

Various physical and chemical methods of bacterial inactivation are known in the art. Examples of physical inactivation are UV- radiation, X-ray radiation, gamma-radiation and heating. Examples of inactivating chemicals are beta-propiolactone, glutaraldehyde, beta-ethyleneimine and formaldehyde.

Preferably the bacteria according to the invention are inactivated with formaldehyde. Surprisingly, the use of formaldehyde at a final concentration of 0.2% is an excellent method to inactivate the bacteria according to the invention.

Thus, in another important aspect, the invention relates to a method for inactivation of a Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention comprising the use of formaldehyde at a final concentration of 0.2%.

Said bacteria according to the invention which are inactivated by the methods disclosed supra and by other methods of inactivating the bacteria known to the skilled person are embodied in the present invention. Therefore, another important aspect are inactivated Pasteurella trehalosi and/or Mannheimia haemolytica obtainable by a method according to the invention or by a method known in the art. Preferably, said inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention are selected from the group of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA-3668 and/or Mannheimia haemolytica ATCC No. PTA-3669.

Therefore, another important aspect are live attenuated Pasteurella trehalosi and/or Mannheimia haemolytica obtainable by a method known in the art. Preferably, said live attenuated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention are selected from the group of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA-3668 and/or Mannheimia haemolytica ATCC No. PTA-3669. Said Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention are attenuated by multiple passages in appropriate culture media or by any other method known in the art. Inactivated as understood herein means, that the Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention are killed without possible replication to cause clinical disease.

Attenuated as understood herein means, that the Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention are live bacteria with possible replication but will not cause clinical disease.

Yet another important aspect are fractions or fragments of Pasteurella trehalosi and/or Mannheimia haemolytica obtainable by a method known in the art. Said fragments may be prepared by detergent solubilization of Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention or by any other method known in the art. Preferably, said fractions or fragments are purified antigens of said Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention. Preferably, said fractions/fragments are outer membrane proteins of Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention.

A ..fragment" according to the invention is any immunogenic subunit of a said Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention, i.e. any polypeptide subset.

Thus, the invention relates to fragments containing at least one antigen of Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention. Most preferably, said fragments are containing at least one antigen of bacteria selected from the group of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA- 3668 and/or Mannheimia haemolytica ATCC No. PTA-3669. Said fragment may comprise whole bacterial cells of said strain(s), bacterial extracts, Outer Membrane Fractions, bacterial exo- and/or endotoxins, and purified proteins. Antigenic polypeptides or fragments thereof may for example be obtained from purified bacterial proteins or by expression of the corresponding genetic material in some prokaryotic or eukaryotic expression system or by organo-chemical synthesis. Said methods are known to the skilled person.

The invention further relates to live, and/or live attenuated, and/or inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica for use in a vaccine.

The invention further provides a vaccine derived from the newly identified bacteria disclosed above. Thus, the invention further relates to a vaccine composition comprising a live, and/or live attenuated, and/or inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica.

The term "vaccine" as understood herein is a vaccine for veterinary use comprising antigenic substances and is administered for the purpose of inducing a specific and active or passive immunity against a disease provoked by said Pasteurella trehalosi and/or Mannheimia haemolytica. The live or live attenuated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention confer active immunity that may be transferred passively via maternal antibodies against the immunogens it contains and sometimes also against antigenically related organisms. The inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica confer passive immunity. Additional components to enhance the immune response are constituents commonly referred to as adjuvants, like e.g. aluminium hydroxide, mineral or other oils or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins or growth factors.

In a preferred embodiment, said vaccine comprises inactivated bacteria.

Preferably, a vaccine of the invention refers to a vaccine as defined above, wherein one immunologically active component is a live Pasteurella trehalosi and/or Mannheimia haemolytica. The term "live vaccine" refers to a vaccine comprising a particle capable of division/multiplication.

Preferably also, a vaccine according to the invention comprises attenuated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and a pharmaceutically acceptable carrier or excipient. Said vaccine may also be administered as a combined vaccine comprising two or more strains of said live, and/or live attenuated, and/or inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fractions of two or more strains of said Pasteurella trehalosi and/or Mannheimia haemolytica. Most preferably said live, and/or live attenuated, and/or inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica in the vaccine are selected from the group of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA-3668 and/or Mannheimia haemolytica ATCC No. PTA-3669.

Preferably also, a vaccine according to the invention comprises inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and a pharmaceutically acceptable carrier or excipient. Said vaccine may also be administered as a combined vaccine comprising two or more strains of said inactivated Pasteurella trehalosi and/or Mannheimia haemolytica.

Furthermore, fractions of whole cells may also be used as the relevant immunogen in the vaccine according to the invention. Therefore, preferably a vaccine according to the invention comprises fractions of Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and a pharmaceutically acceptable carrier or excipient. Said vaccine may also be administered as a combined vaccine comprising two or more strains of said inactivated Pasteurella trehalosi and/or Mannheimia haemolytica. In particular, the invention relates to vaccines comprising fragments which contain at least one antigen of Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention. Most preferably, the invention relates to vaccines comprising fragments which contain at least one antigen of bacteria selected from the group of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA-3668 and/or Mannheimia haemolytica ATCC No. PTA-3669. Said fragment may comprise whole bacterial cells, bacterial extracts, Outer Membrane Fractions, bacterial exo- and/or endotoxins, and purified proteins. Antigenic polypeptides or fragments thereof may for example be obtained from purified bacterial proteins or by expression of the corresponding genetic material in some prokaryotic or eukaryotic expression system or by organo-chemical synthesis. Said methods are known to the skilled person. Preferably, the vaccine according to the invention also comprises an adjuvant. Therefore, the invention further relates to a vaccine composition according to the invention, further comprising one or more suitable adjuvant(s) and/or excipient(s) and/or carrier(s). Adjuvants as used herein comprise substances that boost the immune response of the injected animal. A number of different adjuvants are known in the art. Adjuvants as used herein include Freund's Complete and Incomplete Adjuvant, vitamin E, non-ionic block polymers, muramyldipeptides, Quil A, mineral and non-mineral oil, vegetable oil, and Carbopol (a homopolymer). In a preferred embodiment, the vaccine according to the invention bacterin comprises a water-in-oil emulsion adjuvant. Said vaccine is also called a bacterin comprising inactivated (killed) bacteria according to the invention and a water-in-oil emulsion adjuvant. Other ways of adjuvating the bacteria known to the skilled person are also embodied in the present invention.

Also preferably, the vaccine according to the invention may comprise one or more suitable emulsifiers, e.g. Span or Tween.

Preferably also, said live, and/or live attenuated, and/or inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica in the vaccine are selected from the group o Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA-3668 and/or Mannheimia haemolytica ATCC No. PTA-3669.

Preferably, the vaccine in the present invention comprises at least one antigen of bacteria selected from the group of Pasteurella trehalosi ATCC No. PTA- 3667,

Pasteurella trehalosi ATCC No. PTA- 3668 and/or Mannheimia haemolytica ATCC No.

PTA- 3669. Said vaccine may comprise whole bacterial cells of said strain(s), bacterial extracts, Outer Membrane Fractions, bacterial exo- and/or endotoxins, and purified proteins. Antigenic polypeptides or fragments thereof may for example be obtained from purified bacterial proteins or by expression of the corresponding genetic material in some prokaryotic or eukaryotic expression system or by organo-chemical synthesis.

Said methods are known to the skilled person. Most preferably, the invention further relates to a vaccine composition according to the invention, further comprising at least one other antigen from a virus or microorganism pathogenic to poultry. Preferably, said antigen is in the form of live, attenuated or inactivated viruses or microorganisms or fragments thereof. Said fragment may comprise whole bacterial cells or viral particles, bacterial extracts, viral antigens, viral subunits, Outer Membrane Fractions, bacterial exo- and/or endotoxins, and purified proteins. Antigenic polypeptides or fragments thereof may for example be obtained from purified bacterial proteins or by expression of the corresponding genetic material in some prokaryotic or eukaryotic expression system or by organo-chemical synthesis. Said methods are known to the skilled person.

Most preferably, the invention further relates to a vaccine composition according to the invention, further comprising at least one other antigen from a virus or microorganism pathogenic to poultry, wherein said virus or microorganism is selected from, but not restricted to, the group consisting of Infectious Bronchitis Virus, Newcastle Disease Virus, Infectious Bursal Disease Virus (disease: Gumboro), Chicken Anaemia agent, Avian Reovirus, Mycoplasma gallisepticum, Avian Pneumovirus, Haemophilus paragallinarum (disease: Coryza), Chicken Poxvirus, Avian Encephalomyelitis virus, Pasteurella multocida and E. coli.

A "pharmaceutical composition" essentially consists of one or more ingredients capable of modifying physiological e.g. immunological functions of the organism it is administered to, or of organisms living in or on the organism. The term includes, but is not restricted to antibiotics or antiparasitics, as well as other constituents commonly used to achieve certain other objectives like, but not limited to, processing traits, sterility, stability, feasibility to administer the composition via enteral or parenteral routes such as oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal or other suitable route, tolerance after administration, controlled release properties. Thus, in another important aspect of the invention the invention relates to a pharmaceutical composition comprising a live, and/or live attenuated, and/or inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica. The invention relates to a method of treating a Pasteurella trehalosi and/or Mannheimia haemolytica-^'mfected animal (e.g. the live bacteria as described above) belonging to the group of poultry wherein said live, attenuated, inactivated Pasteurella trehalosi and/or Mannheimia haemolytica and/or fractions and/or fragments thereof according to the invention as described supra, are administered to the poultry animal in need thereof at a suitable doses as known to the skilled person and the reduction of symptoms caused by said Pasteurella trehalosi and/or Mannheimia haemolytica infection is monitored. Said treatment preferably may be repeated.

Yet another important embodiment is a method of immunizing poultry against the disease of the respiratory and reproductive tract caused by a Pasteurella trehalosi and/or Mannheimia haemolytica (e.g. the live bacteria as described above) comprising administration of an immunologically effective amount of a vaccine according to the invention and the reduction of symptoms caused by said Pasteurella trehalosi and/or Mannheimia haemolytica infection is monitored.

Another important embodiment is the use of an inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or live Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or live attenuated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fragments or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention in the manufacture of a vaccine for the prophylaxis of Pasteurella trehalosi and/or Mannheimia haemolytica infections.

The invention also relates to a method of diagnosis of a disease caused by comprising the steps of obtaining a sample from poultry, wherein said sample is selected from the group of blood, serum, plasma, tissue scrapings, washings, swabs, tissue, analysing said sample for the presence of Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention.

In a preferred embodiment the presence of Pasteurella trehalosi and/or Mannheimia haemolytica is determined by an immune test. An immune test uses monoclonal antibodies or polyclonal antisera specific to Pasteurella trehalosi and/or Mannheimia haemolytica. The generation of monoclonal antibodies is known in the art (3, 4). Immune tests include the methods of detection known in the art such as the ELISA test (enzyme-linked immuno-sorbent assay) or the so-called sandwich-ELISA test, dot blots, immunoblots, radioimmuno tests (radioimmunoassay RIA), diffusion-based Ouchterlony test or rocket immunofluorescent assays) or agglutination tests (rapid plate or micro- plate agglutination tests). Another immune test is the so-called Western blot (also known as Western transfer procedure or Western blotting). The purpose of Western blot is to transfer proteins or polypeptides separated by polyacrylamide gel electrophoresis onto a nitrocellulose filter or other suitable carrier and at the same time retain the relative positions of the proteins or polypeptides obtained from the gel electrophoresis. The Western blot is then incubated with an antibody which specifically binds to the protein or polypeptide under consideration. These methods of detection can be used by the average skilled person to perform the invention described herein. Literature references in which the skilled person can find the above-mentioned methods and other detection methods are listed as follows: An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

In another, most particular embodiment, the sample as disclosed supra is incubated with antibodies which are specific to Pasteurella trehalosi and/or Mannheimia haemolytica and the antigen/antibody complex thereby formed is determined.

In a particularly preferred embodiment of the method according to the invention, the presence of Pasteurella trehalosi and/or Mannheimia haemolytica in a sample as disclosed supra is determined by molecular biology methods. Molecular biology methods as used herein means detection methods which include, for example, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR) or may be Northern or Southern blots which the skilled person can find in the standard reference books (e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and Bertram, S. and Gassen, H.G. Gentechnische Methoden, G. Fischer Verlag, Stuttgart, New York, 1991 ). The invention also includes a diagnostic test kit according to the invention which is characterised in that it contains all the necessary elements for detecting Pasteurella trehalosi and/or Mannheimia haemolytica.

A diagnostic test kit is a collection of all the components for carrying out a method of diagnosis according to the invention. Some examples (not an exhaustive list) of other elements for performing a method according to the invention include containers such as 96-well plates or microtitre plates, test tubes, other suitable containers, surfaces and substrates, membranes such as nitrocellulose filter, washing reagents and buffers. A diagnostic test kit may also contain reagents which may detect bound antibodies, such as for example labelled secondary antibodies, chromophores, enzymes (e.g. conjugated with antibodies) and the substrates thereof or other substances which are capable of binding antibodies.

The invention further relates to a diagnostic test kit according to the invention which is characterized in that it contains all the necessary elements for carrying out a PCR or RT-PCR to detect Pasteurella trehalosi- and/or Mannheimia

DNA or RNA. Said kit may contain, but is not limited to in addition to test tubes or 96-well plates or microtitre plates, other suitable containers, surfaces and substrates, membranes such as nitrocellulose filters, washing reagents and reaction buffers (which may vary in pH and magnesium concentrations), sterile water, mineral oil, BSA (bovine serum albumin), MgCI₂, (NH )₂S0₄, DMSO (dimethylsulphoxide), mercaptoethanol, nucleotides (dNTPs), enzymes such as Taqf-polymerase and reverse transcriptase and, as the DNA matrix, the DNA or cDNA specific for Pasteurella trehalosi and/or Mannheimia haemolytica, oligonucleotides specific for a Pasteurella trehalosi and/or Mannheimia haemolytica DNA or RNA, control template, DEPC-water, DNAse, RNAse and further compounds known to the skilled artisan. Oligonucleotides according to the invention are short nucleic acid molecules from about 15 to about 100 nucleotides long, which bind under stringent conditions to the nucleic acid sequence which is complementary to a Pasteurella trehalosi and/or Mannheimia haemolytica protein. By stringent conditions the skilled person means conditions which select for more than 85%, preferably more than 90% homology (cf. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and Bertram, S. and Gassen, H.G. Gentechnische Methoden, G. Fischer Verlag, Stuttgart, New York, 1991 ). The following examples serve to further illustrate the present invention; but the same should not be construed as limiting the scope of the invention disclosed herein.

Example 1

Field disease outbreaks associated with Pasteurella trehalosi and Mannheimia haemolytica

Clinical signs

From field observations

Gross Lesions

Ovarian atrophy with haemorrhages Haemorrhages in gonads and regression

Ovarian follicles deformed Upper part of trachea with haemorrhages

Enlarged liver Enlarged liver with haemorrhages

Inflammation of kidneys Airsacculitis

Haemorrhages in abdominal fat Enlarged spleen with haemorrhages

Haemorrhages in thoracic cavity Haemorrhages in muscle

Haemorrhages in oviduct Haemorrhages in the heart and hydropericardium

Haemorrhages of coronary fat Haemorrhages in the thoracic cavity

Three strains of a novel type of Gram-negative, facultative anaerobic, pleomorphic rod- shaped bacteria, were deposited at the American Type Culture Collection (ATCC), 1081 , University Boulevard, Manassas, VA 20110 - 2209, USA, under deposit number : ATCC No. PTA - 3667 for BIV-4985 Pasteurella trehalosi; ATCC No. PTA-3668 for BIV-AVICOR Pasteurella trehalosi and ATCC No. PTA-3669 for BIV-07990, Mannheimia haemolytica. The date of deposit being August 22, 2001.

The deposited bacteria were typed according to standard determination methods, using Bergey's Manual of Systematic Bacteriology Volume I (1984. Williams and Wilkins, 428 East Preston Street. Baltimore, USA.)

TABLE 1

Mannheimia haemolytica : BIV - 07990 (Biotype 1); ATCC No. PTA-3669

Macroscopic morphology

Colonies grown on Sheep Blood Agar for 24 hours, range from 1.0 to 1.5 mm in diameter, bright translucent, low convex, smooth and creamy, β hemolysis. Microscopic morphology Gram-negative , non-motile, pleomorphic rods, often exhibit bipolar staining.

Biochemical and other tests

Section A

Section B

TABLE 2

Pasteurella trehalosi BIV-AVICOR (Biotype 2); ATCC No. PTA-3668

Macroscopic morphology

Biochemical and other tests

Section A

Section B

80% are positive

TABLE 3

Pasteurella trehalosi : BIV - 4895 (Biotype 4); ATCC No. PTA-3667

Macroscopic morphology

Colonies grown on Sheep Blood Agar for 24 hours, range from 1.0 to 1.5 mm in diameter, bright translucent, low convex, particulate and dry, β hemolysis. Microscopic morphology Gram-negative , non-motile, pleomorphic rods, often exhibit bipolar staining.

Biochemical and other tests

Section A

Section B

The results of tests from Section A and B (Tables 1 , 2 and 3) confirms the bacteria BIV- 4895; ATCC No. PTA-3667 and BIV- AVICOR; ATCC No. PTA- 3668 belong to the family Pasteurellaceae, (Pasteurella trehalosi, which are Trehalose positive and arabinose negative), while the bacteria BIV- 07990; ATCC No. PTA- 3669 belong to the family Mannheimia (Mannheimia haemolytica, which are Trehalose negative and arabinose negative).

Identification of the causative agent

Bacteria were isolated form infected trachea, palatine cleft, ovary, liver, heart, kidney and gonads (broilers). They were identified as Pasteurella trehalosi and Mannheimia haemolytica based on the tests listed below :

Beta haemolysis + Gram stain Oxidase +

Catalase +

Mac Conkey + Urease Nitrate Indole

No isolations of virus or any other bacteria were made.

Biotyping:

Bacterial isolates were purified and biotyped according to the method described by Jaworski et al.(1 ). Three different biotypes (4, 2, 1 ) were identified. Briefly, from the purified isolates, a single colony was inoculated into tubes containing 3 ml of Tryptose Broth and incubated at 37°C for 8 hours. A loop of inoculum (20 μl) from the tube was then transferred into another tube containing 3 ml of 1% sugar to be tested and incubated for 7 days at 37°C before results were recorded

Challenge Model:

Following purification of the bacteria, isolates were grown in tryptose media to obtain large quantities of pure pathogens. In order to validate Koch postulates, 3 different groups (20 birds per group) of specific pathogen free (SPF) chicken 13 weeks of age were infected with each biotype (0.2 ml / bird; 3x10⁸ CFU / ml) by intravenous route. The birds were observed daily for 3 days for morbidity and mortality. At the end of the 3^rd day, all birds were sacrificed, post-mortem lesions recorded and organ samples (liver and gonads) were collected for re-isolation. Post-mortem lesions of birds that died were also recorded.

Results

Clinical signs: Prostration, Lameness, Displaced comb, Ruffled feathers, Cyanosis at the tip of the comb.

Lesions:

EXAMPLE II

Growth of the bacteria according to the invention, preparation of the vaccine and vaccination of SPF birds.

Strains were grown on Tryptose Broth (TB). Harvest was done at logarithmic growth phase around 6-8 hours post-inoculation depending on the strain. Plate count was made in sheep blood agar for titration. Colony forming units per mililiter (CFU/ml) was determined using 1 :10 dilutions of the harvest. Cells were killed by adding formaldehyde to a final concentration of 0.2%. Following a sterility check of this suspension, a minimal titer of 10⁸ CFU/ml was added to the final vaccine. The vaccine was prepared by mixing the two strains (BIV-4895, ATCC No. PTA-3667 and BIV-AVICOR, ATCC No. PTA - 3668) and oil-adjuvant (a water-in-oil emulsion on the basis of a mineral oil with a ratio of 60% oil / 40% water) to a minimal concentration of 10^{7 0} CFU/strain/ml.

Specific pathogen free (SPF) chicken were vaccinated at 2, 5 and 9 weeks of age by injection of 0.5 ml of the vaccine subcutaneously halfway down the neck.

EXAMPLE III

Preparation of challenge strains and challenge of vaccinated and control groups.

Bacterial strains BIV-4895, ATCC No. PTA-3667 and BIV-AVICOR, ATCC No. PTA - 3668, were grown on sheep blood agar for 24 hrs. at 37°C. The cells were harvested in Tryptose Broth (TB) until a suspension with an Optical Density of 2.0 was obtained, using a spectrophotometer at wavelength of 540 nm. For challenge, preparations were made that contain the following number of cells in the final challenge-volume :

3 x 10⁹ CFU/ml BIV - AVICOR; ATCC No. PTA-3668 1.45 x 10¹⁰ CFU/ml BIV-4895; ATCC No. PTA-3667

At 13 weeks of age, 20 vaccinated and 20 non-vaccinated birds were challenged by intravenous route of 0.2 ml of the inoculum (at least 10⁸⁰ CFU/bird). Birds were observed for 3 days for morbidity and mortality. After three days of observation all the remaining birds were sacrificed and re-isolation of the bacteria from liver and gonads were made from each bird. Post-mortem lesions of birds that died were also recorded. RESULTS

Group of birds Challenge Mortality + Protection inoculum Reisolation %

Control Negative N/A 0 N/A

Control Positive ATCC No.PTA-3667 77 23

Control Positive ATCC No. PTA-3668 54 46

Vaccinated ATCC No.PTA-3667 0 100

Vaccinated ATCC No. PTA-3668 5 95

EXAMPLE IV

Strains were grown on Tryptose Broth (TB). Harvest was done at logarithmic growth phase around 6-8 hours post-inoculation depending on the strain. Plate count was made in sheep blood agar for titration. Colony forming units per mililiter (CFU/ml) was determined using 1 :10 dilutions of the harvest. Cells were killed by adding formaldehyde to a final concentration of 0.2%. Following a sterility check of this suspension, a minimal titer of 10⁸ CFU/ml was added to the final vaccine.

The vaccine was prepared by mixing the three strains (BIV-4895, ATCC No. PTA-3667; BIV-AVICOR, ATCC No. PTA - 3668 and BIV-07990, ATCC No. PTA-3669) and oil- adjuvant (a water-in-oil emulsion on the basis of a mineral oil with a ratio of 60% oil / 40% water) to a minimal concentration of 10^7'° CFU/strain/ml.

EXAMPLE V

Bacterial strains BIV-4895, ATCC No. PTA-3667; BIV-AVICOR, ATCC No. PTA - 3668 and BIV-07990, ATCC No. PTA-3669, were grown on sheep blood agar for 24 hrs. at 37°C. The cells were harvested in Tryptose Broth (TB) until a suspension with an Optical Density of 2.0 was obtained, using a spectrophotometer at wavelength of 540 nm. For challenge, preparations were made that contain the following number of cells in the final challenge-volume :

8.3 x 10⁹ CFU/ml BIV - AVICOR; ATCC No. PTA-3668 2.2 x 10⁹ CFU/ml BIV-4895; ATCC No. PTA-3667 1.0 x 10¹⁰ CFU/ml BIV-07990; ATCC No. PTA-3669

Group of Birds Challenge Mortality + Protection Inoculum Reisolation (%)

Control Negative N/A 0 N/A

Vaccinated

Control Negative Non- N/A 0 N/A vaccinated

Vaccinated ATCC No.PTA- 27.3 72.7 3669

Vaccinated ATCC No. PTA- 20.9 79.1 3668

Vaccinated ATCC No.PTA- 16.7 83.7 3667

Control Positive ATCC No. PTA- 53.3 46.7 3669

Control Positive ATCC No.PTA- 53.3 46.7 3668

Control Positive ATCC No.PTA- 64.3 35.7 3667

Seroloqical test

Hyperimmune sera were produced in rabbits with isolate representing each biotype, according to the method of Biberstein et. al. (2)

The isolates were grown on blood agar overnight, then harvested in saline containing 0.3% formalin. The cells were washed once and adjusted to 10% transmittance at 575 nm for injection. The injections were by IV route according to the following schedule : 0.5 ml, 1.0, 2.0, 3.0, 3.0, 3.0 at 4 day intervals and all rabbits were bled 4 days after the final injection.

The hyperimmune serum was tested for their specificity using the 3 biotype strains and were reacted with homologous and heterologous rabbit antiserum (2 fold dilutions) by rapid plate agglutination.

Antiserum of each biotype was diluted until the end point was reached to determine the highest dilution that was positive.

The biotype specific hyperimmune sera was then used as positive control in micro-plate serum agglutination test.

EXAMPLE VI

Preparation of challenge strains and challenge of vaccinated and control groups. Bacterial strains BIV-4895, ATCC No. PTA-3667, BIV-AVICOR, ATCC No. PTA - 3668 and BIV - 07990, ATCC No. PTA-3669, were grown on sheep blood agar for 24 hrs. at 37°C. The cells were harvested in Tryptose Broth (TB) until a suspension with an Optical Density of 2.0 was obtained, using a spectrophotometer at wavelength of 540 nm. For challenge, preparations were made that contain the following number of cells in the final challenge-volume :

1.5 x 10¹⁰ CFU/ml BIV - AVICOR; ATCC No. PTA-3668 1.7 x 10¹⁰ CFU/ml BIV-4895; ATCC No. PTA-3667

1.6 x 10¹⁰ CFU/ml BIV-07990; ATCC No. PTA-3669

At 13 weeks of age, 20 vaccinated and 20 non-vaccinated birds were challenged by intravenous route of 0.2 ml of the inoculum (at least 10⁸⁰ CFU/bird). Birds were observed for 3 days for morbidity and mortality. After three days of observation all the remaining birds were sacrificed, post mortem lesions were recorded and re-isolation of the bacteria from liver, heart and gonads were made from each bird.

RESULTS

Table 1. Evaluation on the effect of vaccine based on mortality and re-isolation.

Group of birds inocu .lu ^*m _D Re ■iso ■la ϊt-ion Protection %

Control negative non-

N/A N/A N/A vaccinated

Control Positive BIV - 4895 70 30

Control Positive BIV - AVICOR 80 20

Control Positive BIV - 07990 88.8 11.2

Vaccinated BIV - 4895 10 90

Vaccinated BIV - AVICOR 10 90

Vaccinated BIV - 07990 15 85 Table 2. Evaluation on the effect of vaccine based on gross lesions following challenge.

Challenge

Group of birds % of Lesions Protection % inoculum

Control negative

N/A N/A N/A non-vaccinated

Control Positive BIV - 4895 74.4 25.6 Control Positive BIV - AVICOR 27.0 73.0 Control Positive BIV - 07990 7.0 93.0

Vaccinated BIV - 4895 4.0 96.0 Vaccinated BIV - AVICOR 1.1 99.0

Vaccinated BIV - 07990 2.4 98.0

References

1 ) Jaworski M.D., D. L. Hunter, A. C. S. Ward. Biovariants of isolates of Pasteurella from domestic and wild ruminants. J. Vet. Invest. 1988, 10: 49-55.

2) Biberstein EL. , Meyer M.E. , and Kenedy P.C. Colonial variation of Pasteurella haemolytica isolated from sheep. J. Bact. 1958, 76: 445-452.

3) Kearney, J.F., Radbruch A., Liesegang B., Rajewski K. A new mouse myeloma cell line that has lost imunoglobulin expression but permits construction of arrf/body-secreting hybrid cell lines. J. Immunol. 1979, 123: 1548-1550.

4) Kohler, G., Milstein, C. Continous culture of fused cells secreting antibody of predefined specifity. Nature 1975, 265: 495-497.

1/2

PCT 1-1265-PCT

Original (for SUBMISSION) - printed on 23.10.2002 09:05:55 AM

0-1 Form - PCT/RO/134 (EASY) Indications Relating to Deposited Microorganism(s) or Other Biological Material (PCT Rule 13bis)

0-1-1 Prepared using PCT-EASY Version 2 . 92 (updated 01 . 10.2002)

0-2 International Application No. PCT/E? 0 2 / 1 1 8 9 9

0-3 Applicant's or agent's file reference 1-1265-PCT

1 The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on:

1-1 page 18 1-2 line 5

1-3 Identification of Deposit

1-3-1 Name of depositary institution American Type Culture Collection 1-3-2 Address of depositary institution 10801 University Blvd. , Manassas ,

Virginia 20110-2209United States of

America

1-3-3 Date of deposit 22 August 2001 (22 . 08 .2001 ) 1-3-4 Accession Number ATCC PTA-3667

1-4 Additional Indications BIV-4895 Avian Pasteurella trehalosi

1-5 Designated States for Which all designated States Indications are Made

1-6 Separate Furnishing of Indications NONE

These indications will be submitted to the International Bureau later

2 The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on:

2-1 page 17 2-2 line 7

2-3 Identification of Deposit

2-3-1 Name of depositary institution American Type Culture Collection 2-3-2 Address of depositary institution 10801 University Blvd. , Manassas ,

Virginia 20110-2209United States of

America

2-3-3 Date of deposit 22 August 2001 (22 . 08 .2001 ) 2-3-4 Accession Number ATCC PTA-3668

24 Additional Indications BIV-AVICOR Avian Pasteurella trehalosi

2-5 Designated States for Which all designated States Indications are Made

2-6 Separate Furnishing of Indications NONE

These indications will be submitted to the International Bureau later 2/2

PCT 1-1265-PCT

Original (for SUBMISSION) - printed on 23.10.2002 09:05:55 AM

3 The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on:

3-1 page 16

3-2 line 12

3-3 Identification of Deposit

3-3-1 Name of depositary institution American Type Culture Collection

3-3-2 Address of depositary institution 10801 University Blvd. , Manassas , Virginia 20110-2209United States of America -3-3 Date of deposit 22 August 2001 (22 .08 .2001) -3-4 Accession Number ATCC PTA-3669

3-4 Additional Indications BIV-07990 Avian Mannheimia haemolytica -5 Designated States for Which all designated States Indications are Made -6 Separate Furnishing of Indications NONE

These indications will be submitted to the International Bureau later

FOR RECEIVING OFFICE USE ONLY

-5 This form was received by the international Bureau on: -5-1 Authorized officer

SEP-13-2ΘΘ1 08: 58 ATCC 703 365 2745 P.01

AMERICAN TYPE CULTURE COLLECTION

10801 University Blvd.

Manassas, VA 20110-2209

Telephone: 703-365-2700

Fax: 703-365-2745

FACSIMILE

Date: September 12, 2001

To: Abelardo Aquilar

Fax Number. 52 3 668 80 80

From: ATCC Patent Depository Number of pages: 1 (Including this page)_^

REFERENCE: Patent Deposit

Avian Pasteurella trehalosi; BIV-4895 assigned ATCC No. PTA-3667, . Avian Pasteurella trehalosi: BIV-AVICOR assigned ATCC No. PTA-3668 and Avian Mannheimia haemolytica: BIV-07990 assigned ATCC No. PTA-3669.

Date of Deposit: August 22, 2001. Paperwork will be forwarded to you in a few days. An invoice will be sent under separate cover:

Standard storage/informing $ 3,450.00

Viability Test 480.00

Total amount to PTA-3667 to PTA-3669 $ 3,930.00

Marie Harr s, Patent Specialist ATCC Patent Depository

* Please note: Patent deposit fees have increased effective January I, 2001. Deposit forms were revised February 2000. Current deposit forms and fees can be obtained from our web site: www.atcc.org.

The information contained in this facsimile is intmded only for the use of the individual or entity named above. If the reader of this message is oot the intended recipient, you are notified that any dissemination or distribution, except to the intended recipient of this communication, is prohibited. If you h_-vc received this cotrunuπitfution in error, please call us immediately at the telephone number listed above. Budapest Treaty Deposits

American Type Culture Collection

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Phone (703) 365-2700; fax (703) 365-2745; e-mail applied-sci@atcc.org

TO DEPOSIT OR TO CONVERT A DEPOSIT TO MEET THE REQUIREMENTS OF THE BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

ALL QUESTIONS MUST BE COMPLETED IN ENGLISH. PLEASE USE ONE FORM FOR EACH STRAIN DEPOSITED.

1. Name of deposit. If microorganism, give complete scientific name including genus and species and source of material; If virus, give name, whether plant or animal, and source including geographic location; if cell line, give tissue and species, geographical source of isolation, and any known hazards associated (HIV, EBV, etc.); if genetic materials, give name of organism from which vector, clone or library is derived, source of the DNA insert identified by species (e.g. human, mouse) or scientific name, and give name of gene and identity of the host organism; if consortia or mixed culture, each component of the mixture must be identified; If seeds, embryos, insect eggs, etc., give common name, scientific name, and geographical source.

Avian Mannheimia haemoly ica formerly known as Past nr-ella haemol yf i r

2. Strain designation (i.e., number, symbols, etc.) BliZ — ~ — Q7990

The strain designation must correspond with the vial labels. 3. Is this an original deposit under the Budapest Treaty? Yes

4. Is this a rreeqquueesstt ffoorr aa ccoonnvveerrssiioonn ooff aa ddeeppoossiitt aallrreeaaddyy aatt AAlTCC to meet the requirements of the Budapest Treaty? If so please indicate ! A ι TCC designation. Not applicable

5. Is this deposit a mixture of microorganisms or cells? ™_

Provide details necessary to cultivate, test for viability, and store deposit. If mixture, provide description of components and a method to check presence. If plasmid, provide name of host and antibiotic resistance. See annex 3

7. Provide sufficient description so that ATCC may confirm deposit properties (e.g., Gram negative rod). See annex 3

a. If deposit is a cell culture, is it being cultured in the presence of antibiotics? If so, please list the antibiotics.

Not applicable b. If deposit is hybridoma, what is the isotype of antibody produced? Not applicable

8. Is this strain hazardous to humans? No Animals? Y ? Plants? No If yes, what is the recommended blosafety level for working with this strain? ^ (Refer to Biosafety in Microbiological and Biomedical Laboratories, 4th ed. U.S. Dept. of Health and Human Services at www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm].

9. Availability: Prior to the issuance of a U.S. Patent, ATCC will only make a culture available as instructed by the depositor or relevant patent office. Samples must be provided to a specific investigator if a pertinent patent office under the Budapest Treaty Instructs ATCC to do so. The following questions must be answered: a) As of date of deposit or conversion to meet the requirements of the Budapest Treaty, do you wish the deposit to be made available to anyone who requests a culture? If yes there are no restrictions on distribution. Answering no will ensure the deposit Is not available until the patent has issued. Yes No X b) As of date of deposit or conversion to meet the requirements of the Budapest Treaty, do you wish the deposit to be made available to requestors which satisfy patent offices in countries not signatory to the Budapest Treaty? Yes No X

If yes state which countries.

Please note that if you are converting your deposit to meet the requirements of the Budapest Treaty and your deposit has already been released far distribution due to the issuance of a U.S. patent, you cannot restrict it from further distribution.

Credit Card number. Please indicate MasterCard VISA, or AE.

Exp. Date. Nfa e shown on card:

(Please type or pnm dearly \

Signature of card holder

PAYMENT- A^TCC MUST HAVE A BILLING ADDRESS, CONTACT PERSON, PHONE AND FAX NUMBER FOR ALL DEPOSITS: on ar-t- ^prcm- . — fthel ardo ΔqLi lS_kV-

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Phone: f ? ? . fifiR anm Fax. - (52 3 ) 668 80 80

12. Name, address, telephone and facsimile number of your attorney of record.

. (Ref: Docket or Case No. . , ,)

13. MUST BE COMPLETED. Deposited on behalf of (Vdπfy with your management who owns the deposit The owner a usuall «, company or institute and not an individual.) ^■

Bo^βhringer ingelheim Vetmedica/ S.A. de C.V. ₍

I undereitand and agree that the deposit may not be Withdrawn by me for a period specified in Rule

Phone: < ? ^ *«R an nπ Fax- ( 52 '3 ) 668 80 8QE-mall: suchi l@gua.boehringer-ιrι_telhei .

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ADDRESS SHIPMENTS AND FORM TO THE ATTENTION OF Patent Depository

American Type Culture Collection

10801 University Blvd. Manessas, VA 20110-2209 U S A longer, ,ιs

^*

ATCC USE ONLY: ATCC DESIGNATION _. RFcvn _ . V.T RESULT

' 1

Name of Deposit ,, Strain Designation:

' • form BP/1 'lev. 2100

25 glass vials containing freeze 'dried Avian Mannheimia haemolytica formerly known as Pasteurella haemtplytica. Identified as BIV-07990

8 QUANTITY. FREQUENCY OF IMPORTATION, AND EXPECTED COM^PLETION DATEfesttnβte,)

7. PROPOSED USE OF MATERIALAND DERIVATIVES (Also, forlni al pathogens or vectors describe faciliSβstblosafβty procedures)

See annex 6 V

Φ Boehringer Ingelheim

A NEX 3

Bu apest Treaty Deposits

Answer to question No. 6*.

Culture.' ι I^s This strain could be grown in Brain Heart Infusion broth (Difco) or Tryptose Soy broth. The log phase growth is around six to eight hours of incubation at 37^βC.

In Blood Agar peak of growth is observed after twenty four hours at 37^βC, an advantage of this method is that hemolysis could be observed.

Preservation by lyophilizatlon. Bacteria is grown in Brain Heart Infusion broth (Difco) for six to eight hours. Use a 10% sterile solution of Bacto Sk_ Milk (Difco) as stabilizer at a ratio of 1 : 1 (v / v)

Viability test. Take a freeze dried vial and reconstitute with one ml of sterile PBS IX, pH 7.2.

Make serial ten fold dilutions with stenle PJJS, pH 7.2. Seed 100 μl of each dilution on Blood Agar plates.

Incubate for 24 hours at 37^βC . Check viab^ty by colony counting methodology.

Y

Answer to question No. 7^<B.

to 1.5 mm in diameter, bright

staining.

Φ Boehringer Ingelheim

ANNEX 6

•, 'Importation Permit-

Answer to question No. 6^,h (Treatment of xna'-erial prior to importeiion into the U. S.)

BIV -07990 strain

1. Isolation made from layer oviduct

2. Organ maceration

3. Inoculate in nutrient brotia (20 ml of BHI from Difco)

4. Incubation 24 hours at 37°C

5. Seed on sheep blood agar plates

6. Isolate a β haeαiolytic colony

7 Seed on sheep blood agar plates

8. Identification (Biochemical and other tests)

9. Seed on BHI broth from Difco

10. Grow the bacteπa in Brain Heart Infusion broth (Difco) for six to eight hours

11. Add 10% sterile solution of Bacto Skim Milk (Difco) as stabilizer at a ratio of 1 : 1 (v / v)

12. Samples freeze dried

13. Viability, purity and biochemical tests.

Budapest Treaty Deposits

American Type Culture Collection

10801 University Blvd., Manassas, VA 20110-2209

Phone (703) 365-2700; fax (703) 365-2745; e-mail applied-sci@atcc.org

Avian Pasteurella trehalosi, formerly known as Pasteurella haemolytica

2. Strain designation (i.e., number, symbols, etc.) BIV - AVICOR

The strain designation must correspond with the vial labels. 3. Is this an original deposit under the Budapest Treaty? Y^βS

4. Is this a request for a conversion of a deposit already at ATCC to meet the requirements of the Budapest Treaty? If so please indicate ATCC designation. Not applicable

5. Is this deposit a mixture of microorganisms or cells? No i 6. Provide details necessary to cultivate, test for viability, and store deposit. If mixture, provide description of components and a method to check presence. If plasmid, provide name of host and antibiotic resistance.

See annex 2

7. Provide sufficient description so that ATCC may confirm deposit properties (e.g., Gram negative rod).

See annex 2

8. Is this strain hazardous to humans? Jto_ Animals? V^pg Plants? Mn . If ves. what is the recommended biosafety level for working with this strain? ± . (Refer to Biosafety in Microbiological and Biomedical Laboratories, 4th ed. U.S. Dept. of Health and Human Services at www.cdc.gov/od/ohs/biosfly/bmbl4/bmbl4toc.htm].

9. Availability: Prior to the issuance of a U.S. Patent, ATCC will only make a culture available as instructed by the depositor or relevant patent office. Samples must be provided to a specific investigator if a pertinent patent office under the Budapest Treaty instructs ATCC to do so. The following questions must be answered: a) As of date of deposit or conversion to meet the requirements of the Budapest Treaty, do you wish the deposit to be made available to anyone who requests a culture? If yes there are no restrictions on distribution. Answering no will ensure the deposit la net available until the patent hββ iββuo . Yes No "" b) As of date of deposit or conversion to meet the requirements of the Budapest Treaty, do you wish the deposit to be made available to requestors which satisfy patent offices in countries not signatory to the Budapest Treaty' Yes No X

If yes state which countries

Please note that if you are converting your deposit to meet the requirements of the Budapest Treaty and your deposit has already been released for distribution due to the issuance of a U.S. patent, you cannot restrict it from further distribution.

25 glass vials containing freeze dried Avian Pasteurella trehalosi formerly known as Pasteurella haemolytica. Identified as BIV-AVICOR

i

6. QUANTITY, FREQUENCY OF IMPORTATION, AND EXPECTED COMPLETION A^esV ete)

7. PROPOSED USE OF MATER.ALAND DERIVATIVES (Also, far animal pathogens αr vectors describe facllltleslbiosefat protβdums)

f). IF FOR USE IN ANIMALS.≤EEςiQ. THE ANIMAL SPECIES

9 TREATMENT OF MATERlALPRIQp TO IMPORTATION INTO THE U S (Pmcessinglpyuiβcabon methods, including time et specific temperatures, pH, oitier tnβtmenta, disease safeguard etc)

See annex 5 i

ιo. MEΓΓHOO OF FINAL DISPOSITION OF IMPORTED MATERIAL AND DERIVATIVES

r CERTIFY AS AUTHORIZED BY THE COMPANY/INSTITUTION THAT I REPRESENT, THAT THIWATERIAL WILL BE USED IN ACCORDANCE WITH ALL RESTRICTIONS AND PRECAUTIONS AS MAY BE SPECIFIED IN THE 3ERMIT.

11. SIGNATURfc OF APPLICANT 12 TYPED NAME AND TITLE

13. DATE 14. APHIS USER FEE CREDIT ACCOUNT NO. OR METHOD OF USER FEE PAYMENT (for VISA or Mastercard Include number end .xplratk date )

VS FORM 1β-3 (NOV 99) Φ Boehringer Ingelheim

ANNEX 2

Budapest Treaty Deposits

Answer to question No. 6*

Culture. This strain could be grown in Brain Heart Infusion broth (Difco) or Tryptose Soy broth.

The log phase growth is around six to eight hours of incubation at 37°C.

In Blood Agar peak of growth is observed after twenty four hours at 37°C, an advantage of this method is that hemolysis could be observed.

Preservation by lyophilizatlon. Bacteria is grown in Brain Heart Infusion broth (Difco) for six to eight hours. Use a 10% sterile solution of Bacto -Skim Milk (Difco) as stabilizer at a ratio of 1 : 1 (v / v)

A

ViabiMty test. Take a freeze dried vial' and reconstitute with one ml of sterile PBS IX, pH 7.2.

Make serial ten fold dilutions with sterile PBS, pH 7.2. Seed 100 μl of each dilution on Blood Agar plates.

Incubate for 24 hours at 37^βC . Check viability by colony counting methodology.

Answer to question No. 7*.

7.2. BIV- AVICOR

Macroscopic morphology

Colonies grown on Sheep Blood Λ '^g. ar for 24 hours, range from 1.0 to 1.5 mm in diameter, bright traslucent, low convex, smooth and reamy, β hemolysis.

Microscopic morphology A

Gram negative , πon motile, pleomorphic rods, often exhibit bipolar staining.

,ri '

Biochemical and other tests

80% are positive

Φ Boehringer Ingelheim

ANNEX 5

Answer to question No.6^ (Treatment of material prior to importation the U. S,)

BIV -AVICOR strain

1. Isolation made from heart of layer hen

2. Organ maceration •

3. Inoculate in nutrient broth (20 ml of BHI from Difco) ϊ'frf

4. Incubation 24 hours at 37°C

5. Seed on sheep blood agar plates

6. Isolate a β haemolytic colony

7. Seed on sheep blood agar plates

8. Identification (Biochemical and other tests) 9- Seed on BHI broth from Difco ^,ζ;'J

1 . Grow the bacteria in Brain Heaπ Infusion broth (Difco) for six to eight hours

11. Add 10% sterile solution of Bacto Skiπ ilk (Difco) as stabilizer at a ratio of 1 : 1 (v / v)

12. Samples freeze dried

13. Viability, purity and biochemical tests.

Budapest Treaty Deposits

American Type Culture Collection

10801 University Blvd., Manassas, VA 20110-2209

Phone (703) 365-2700; fax (703) 365-2745; e-mail applied-sci@atcc.org

■r — ^ ,

Avian Pasteurella t-r hal ήsf ^:. . formerly kno rf s Pa^pnrpl l a hapm l ÷ i a

2. Strain designation (i.e., number, symbols, etc.) ^BJ- ~ ^°^

The strain designation must correspond with the vial labels.

3. Is this an original deposit under the Budapest Treaty? ^1C°

4. Is this a request for a conversion of a deposit already at ATCC to meet the requirements of the Budapest Treaty? If so please indicate ATCC designation. M ><- ap l i cable _______

5. Is this deposit a mixture of microorganisms or cells? fcfo

Provide details necessary to cultivate, test for viability, and store deposit. If mixture, provide description of components and a method to check presence. If plasmid, provide name of host and antibiotic resistance.

See annex 1

7. Provide sufficient description so that ATCC may confirm deposit properties (e.g., Gram negative rod). See annex 1

a. If deposit is a ceil culture, is it being cultured in the presence of antibiotics? If so, please list the antibiotics. Not applicable b. If deposit is hybridoma, what is the isotype of antibody produced? — Not applicable

8. Is this strain hazardous to humans? No Animals? Yes Plants? No If yes, what is the recommended biosafety level for working with this strain? ) (Refer to Biosafety in Microbiological and Biomedical Laboratories, 4th ed. U.S. Dept. of Health and Human Services at www.cdc.gov/od/ohs/biosfty bmbl4/bmbl4toc.htm].

9. Availability: Prior to the issuance of a U.S. Patent, ATCC will only make a culture available as instructed by the depositor or relevant patent office. Samples must be provided to a specific investigator if a pertinent patent office under the Budapest Treaty instructs ATCC to do so. The following questions must be answered: a) As of date of deposit or conversion to meet the requirements of the Budapest Treaty, do you wish the deposit to be made available to anyone who requests a culture? If yes there are no restrictions on distribution. Answering no will ensure the deposit is not available until the patent has issued. Yes No % b) As of date of deposit or conversion to meet the requirements of the Budapest Treaty, do you wish the deposit to be made available to requestors which satisfy patent offices in countries not signatory to the Budapest Treaty? Yes No X

If yβe ctato u/hioh oouπlrlθS.

12. Name, address, telephone and facsimile number of your attorney of record.

, (Ref: Docket or Case No..

13. MUST BE COMPLETED. Deposited on behalf of (Ver/fy ith your management who owns the deposit The owner is usually a n co-immnpaanπyu n orr I Innssttiittuutete s aπnrdl n noott a ann I Innddiivviidduuaall \ . "ι

Boehxing^r Ingel eim Vetmedi a, S.A. de C.V.

Address- Cal l * 30 Nn, 761 4 ?,nna Jnrlns r.i fll ___Q — ftnarfelaj— "a , Jaluaro. Mav- co

_Pnonβ (52 3 ) 668 80 00 _Fax (52,3 ) 668 80 80 _E._mai jsuc l 1— -qua . boehr . noer-inαel e im

ADDRESS SHIPMENTS AND FORM TO THE ATTENTION OF Patent Depository

,)_f American Type Culture Collec ion

'-^•& 10801 University Blvd.

> Manassas, VA 20110-2209 U.S.A

, - ^•

STORAGE ; Cultures are stored for 30 years from date of dϋposii or 5 years after the last request for a sample, whichever a longer, as required under the rules of patent offices in most countries.^ ^

FEES For current fees, check our Web site at www atcc drgf request a fee sheet by e-mail to applied--.ciQeius.org, or call (703) 365-2700 ext.320. All fees are subject to change jf

ATCC USE ONLY: ATCC DESIGNATION. J. REC'D V.T RESULT. i

Name of Deposit . -^ Strain Designation;

Φ Boehringer Ingel eim

" ANNEX :

Answer to question No. 7^th. 7.1, BIV- 4895

Macroscopic morphology »"β

Colonies grown on Sheep Blood Agar for 24 hours, range from 1.0 to 1.5 mm in diameter, bright traslucent, low convex, low solubility and dry, β hemolysis

Microscopic morphology

Gram negative , non motile, pleomorphic rods, often exhibit bipolar staining.

Biochemical and other tests

Φ Boehringer Ingelheim

ANNEX4 Importation Permit

Answer to question No. δ* (Treatment of material prior to importation into the U. S.)

1

BIV - 4895 strain ,

A

1. Isolation made from palatine cleft of layer hen

2. Inoculation on nutrient broth (20 ml of BHI from Difco)

3 Incubation 24 hours at 37°C , |

4 Seed on sheep blood agar plates ,' '

' .

5. Isolate a β haemo /tic colony ϊ

6. Seed on sheep blood agar plates ' !

7. Identification (Biochemical and other tests)

8. Seed on BHI broth from Difco * .

9. Grow the bacteria in Brain Heart Infusion broth (Difco) for sw to eight hours

10 Add 10% sterile solution of Bacto SkinϊMttk (Difco) as stabilizer at a ratio of 1 : 1 (v / v)

11. Samples freeze dried

12. Viability, purity and biochemical tests.

Claims

1. Gram-negative, facultative anaerobic, pleomorphic rod-shaped bacteria causing a novel disease of the upper respiratory and of the reproductive tract of poultry, wherein said bacteria are selected from the group of Pasteurella trehalosi and/or Mannheimia haemolytica.

2. Pasteurella trehalosi and/or Mannheimia haemolytica, wherein said Pasteurella and/or Mannheimia are beta haemolysis-positive, Gram-negative, oxidase- positive, catalase-positive, urease-negative, nitrate-positive and indole-negative.

3. Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 2, wherein said Pasteurella and/or Mannheimia are MacConkey positive.

4. Pasteurella trehalosi and/or Mannheimia haemolytica according to any one of claims 2 or 3, wherein said Pasteurella and/or Mannheimia are Glucose-positive, Sucrose-positive, Mannitol-positive, Arabinose-negative, Celobiose-negative, Xylose-positive, Salicin-negative, Omithine-negative, Esculin-negative, alpha- Fucosidase-negative, beta-Galactosidase-positive.

5. Pasteurella trehalosi according to any one of claims 2 to 4, wherein said Pasteurella are Arabinose-negative and Trehalose-positive.

6. Mannheimia haemolytica according to any one of claims 2 to 4, wherein said Mannheimia are Arabinose-negative and Trehalose-negative.

7. Pasteurella trehalosi and/or Mannheimia haemolytica according to any one of claims 2 to 6, wherein said poultry is selected from the group of chicken, turkey, duck, goose, dove, pigeon and quail.

8. Pasteurella trehalosi as deposited at the under accession number ATCC No. PTA-3667.

9. Pasteurella trehalosi as deposited at the under accession number ATCC No. PTA-3668.

10. Mannheimia haemolytica as deposited at the under accession number ATCC No. PTA-3669.

11. Method for inactivation of a Pasteurella trehalosi and/or Mannheimia haemolytica according to any one of claims 1 to 10 comprising the use of formaldehyde to a final concentration of 0.2%.

12. Inactivated Pasteurella trehalosi and/or Mannheimia haemolytica obtainable by a method according to claim 11 or by a method known in the art.

13. Inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 12, wherein said Pasteurella trehalosi and/or Mannheimia haemolytica are selected from the group of Pasteurella trehalosi ATCC No. PTA- 3667, Pasteurella trehalosi ATCC No. PTA- 3668 and/or Mannheimia haemolytica ATCC No. PTA- 3669.

14. Live attenuated Pasteurella trehalosi and/or Mannheimia haemolytica wherein the Pasteurella trehalosi and/or Mannheimia haemolytica according to any one of claims 1 to 10 are attenuated.

15. Live attenuated Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 14, wherein said Pasteurella trehalosi and/or Mannheimia haemolytica are selected from the group of Pasteurella trehalosi ATCC No. PTA- 3667, Pasteurella trehalosi ATCC No. PTA- 3668 and/or Mannheimia haemolytica ATCC No. PTA- 3669.

16. Fragments or fractions containing at least one antigen of Pasteurella trehalosi and/or Mannheimia haemolytica according to any one of claims 1 to 10.

17. Fragments or fractions according to claim 16, wherein said fragments are containing at least one antigen of bacteria selected from the group of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA-3668 and/or Mannheimia haemolytica ATCC No. PTA-3669.

18. Vaccine composition comprising an inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 12 or 13 and/or live Pasteurella trehalosi and/or Mannheimia haemolytica according to any one of claims 1 to 10 and/or live attenuated Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 14 or 15 and/or fragments or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 16 to 17.

19. Vaccine composition according to claim 18, further comprising one or more suitable adjuvant(s) and/or excipient(s) and/or carrier(s).

20. Vaccine composition according to claim 18 or 19, comprising inactivated Pasteurella trehalosi and/or Mannheimia haemolytica, wherein said Pasteurella trehalosi and/or Mannheimia haemolytica are selected from the group of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA- 3668 and/or Mannheimia haemolytica ATCC No. PTA-3669.

21. Vaccine composition according to any one of claims 18 to 20, wherein said vaccine comprises two or more strains of live, and/or live attenuated, and/or inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to claims 1 to 10 and 12 to 15 and/or fractions of two or more strains of said Pasteurella trehalosi and/or Mannheimia haemolytica according to claims 16 or 17.

22. Vaccine composition according to any one of claims 18 to 21 , wherein said live, and/or live attenuated, and/or inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to the invention and/or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica in the vaccine are selected from the group of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA-3668 and/or Mannheimia haemolytica ATCC No. PTA- 3669.

23. Vaccine composition according to any one of claims 18 to 22, further comprising at least one other antigen from a virus or microorganism pathogenic to poultry.

24. Vaccine composition according to any one of claims 18 to 23, wherein said virus or microorganism is selected from the group consisting of Infectious Bronchitis Virus, Newcastle Disease Virus, Infectious Bursal Disease Virus, Chicken Anaemia agent, Avian Reovirus, Mycoplasma gallisepticum, Avian Pneumovirus, Haemophilus paragallinarum, Chicken Poxvirus, Avian Encephalomyelitis virus, Pasteurella multocida and E. coli.

25. Use of an inactivated Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 12 or 13 and/or live Pasteurella trehalosi and/or Mannheimia haemolytica according to any one of claims 1 to 10 and/or live attenuated Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 14 or 15 and/or fragments or fractions of said Pasteurella trehalosi and/or Mannheimia haemolytica according to claim 16 to 17 in the manufacture of a vaccine for the prophylaxis of Pasteurella trehalosi and/or Mannheimia haemolytica infections.

26. Method of immunizing poultry against the disease of the respiratory and reproductive tract caused by a Pasteurella trehalosi and/or Mannheimia haemolytica according to claims 1 to 10, comprising an immunologically effective amount of a vaccine according to any one of claims 18 to 24 and the reduction of symptoms caused by said Pasteurella trehalosi and/or Mannheimia haemolytica infection is monitored.

27. Method of diagnosis of a disease caused by comprising the steps of obtaining a sample from poultry, wherein said sample is selected from the group of blood, serum, plasma, tissue scrapings, washings, swabs, tissue, analysing said sample for the presence of Pasteurella trehalosi and/or Mannheimia haemolytica according to any one of claims 1 to 10.

28. Method of diagnosis according to claim 27, wherein said presence of Pasteurella trehalosi and/or Mannheimia haemolytica is determined by an immune test.

29. Method of diagnosis according to claim 27, wherein said presence of Pasteurella trehalosi and/or Mannheimia haemolytica is determined by an molecular biology methods.

30. Diagnostic test kit comprising all the components necessary for carrying out a method according to any one of claims 27 to 29.