Title of Invention

"VACCINE COMPOSITION COMPRISING GRAM-NEGATIVE, PLEOMORPHIC ROD-SHAPED BACTERIUM"

Abstract The invention belongs to the field of animal health and in particular the causative agent of a new bacterial poultry disease, Pasteurella irehalosi and/or Mannheimia haemolytica. The invention provides said Pasteurella trehalosi and/otMannheimia haemolytica bacteria, vaccine comprising inactivated Pasteurella trehalosi and/or Mannheimia haemolytica, and a method of immu nizing to prevent the disease in chicken.
Full Text 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 Haernophilus paragallinarum (upper respiratory tract), Bordetella avium (upper respiratory tract), Omithobacteriurn rhinotracheale (lower respiratory tract), Salmonella infections (digestive tract), Pasteurella multocida, which is the causative agent of fowl cholera (septicemic), and E,co// 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(p)-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, Ornithine-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(p)-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, interieukins 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 of 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. coll.
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 fcaemo/yf/ca-infected 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, radioimrnuno 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 a/., 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, 2nd 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 haemolytica-spec\f\c 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), MgCIa, (NH4)2SO4, DMSO (dimethylsulphoxide), mercaptoethanol, nucleotides (dNTPs), enzymes such as rag-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, 2nd 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 haemolvtica
Clinical signs
From field observations
(Table Remove)
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 Sergey'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, p hemolysis. Microscopic morphology Gram-negative , non-motile, pleomorphic rods, often exhibit bipolar staining.
Biochemical and other tests(Table Remove)
TABLE 2
Pasteurella trehalosi BIV-AVICOR (Biotype 2); ATCC No. PTA-366S
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, p hemolysis. Microscopic morphology Gram-negative , non-motile, pleomorphic rods, often exhibit bipolar staining. (Table Remove)
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, p hemolysis. Microscopic morphology Gram-negative , non-motile, pleomorphic rods, often exhibit bipolar staining.
Biochemical and other tests
Section A
Section B
(Table Remove)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 jal) 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; 3x1 Ofl CPU / ml) by intravenous route. The birds were observed daily for 3 days for morbidity and mortality. At the end of the
3rd 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:
(Table Remove)
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
of1070CFU/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 1.45x1010 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 10*° 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(Table Remove)

EXAMPLE IV
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 liter 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 1070 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 V
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:
8.3 x 109 CFU/ml BIV - AVICOR; ATCC No. PTA-3668 2.2 x109 CFU/ml BIV-4895; ATCC No. PTA-3667 1.0 x1010 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 10"° 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(Table Remove)

Seroloaical 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.
Dilution (log ) (Table Remove)

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 :
1.5x 1010 CFU/ml BIV-AVICOR; ATCC No. PTA-3668 1.7 x1010 CFU/ml BIV-4895; ATCC No. PTA-3667 1.6 x1010 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.
Oroupo, birds » Protec-on,.
(Table Remove)
References
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.
Biberstein EL., Meyer M.E. , and Kenedy P.C. Colonial variation of Pasteurella
haemolytica isolated from sheep. J. Bact. 1958, 76: 445-452.
Kearney, J.F., Radbruch A., Liesegang B., Rajewski K. A new mouse myeloma
cell line that has lost imunoglobulin expression but permits construction of
anf/body-secreting hybrid cell lines. J. Immunol. 1979,123: 1548-1550.
Kohler, G., Milstein, C. Continous culture of fused cells secreting antibody of
predefined specifity. Nature 1975,265: 495-497.


1. Gram-negative, facultative anaerobic, pleomorphic rod-shaped bacterium
causing a disease of the upper respiratory and of the reproductive tract of
poultry, wherein said bacterium is from the family Pasteurellaceae and wherein
said bacterium is beta haemolysis-positive, oxidase-positive, catalase-positive,
urease-negative, nitrate-positive and indole-negative.
2. The bacterium according to claim 1, wherein said bacterium is MacConkey
positive.
The bacterium according to claim 2, wherein said bacterium is Glucose-
positive, Sucrose-positive, Mannitol-positive, Arabinose-negative, Celobiose-
negative, Xylose-positive, Salicin-negative, Ornithine-negative, Esculin-
negative, alpha-Fucosidase-negative, beta-Galactosidase-positive.
The bacterium according to claim 3, wherein said bacterium is Arabinose-
negative and Trehalose-positive.
The bacterium according to claim 4, wherein said bacterium is Arabinose-
negative and Trehalose-negative.
The bacterium according to claim 4, wherein said bacterium is deposited under
accession number ATCC No. PTA-3667.
The bacteria according to claim 4, wherein said bacterium is deposited under
accession number ATCC No. PTA-3668.
The bacterium according to claim 5, wherein said bacterium is deposited under
accession number ATCC No. PTA-3669.
Method for inactivation of a bacterium according to any one of claims 1 to 8
comprising the use of formaldehyde to a final concentration of 0.2%.
The bacterium according to any one of claims 1 to 8, wherein said bacterium
belonging to the family Pasteurellaceae is inactivated by formaldehyde.
A live attenuated bacterium strain of the bacterium according to any one of
claims 1 to 8.
Live attenuated bacterium strain according to claim 11, wherein the live
attenuated bacterium strain is derived from the bacterium deposited under the
accession number ATCC No. PTA-3667, ATCC No. PTA- 3668 or ATCC No.
PTA- 3669.
Preparation comprising fragments or fractions containing at least one antigen
of the bacterium according to any one of claims 1 to 12.
A vaccine for immunizing against a disease of the upper respiratory and of the
reproductive tract in poultry, wherein said disease is caused by bacterium
belonging to the family Pasteurellaceae and said bacterium is beta
haemolysis-positive, oxidase-positive, catalase-positive, urease-negative,
nitrate-positive and indole-negative.
The vaccine according to claim 14, wherein said bacterium is MacConkey
positive.
The vaccine according to claim 15, wherein said bacterium is Glucose-
positive, Sucrose-positive, Mannitol-positive, Arabinose-negative, Celobiose-
negative, Xylose-positive, Salicin-negative, Ornithine-negative, Esculin-
negative, alpha-Fucosidase-negative, beta-Galactosidase-positive.
The vaccine according to claim 16, wherein said bacterium is deposited under
accession number ATCC No. PTA-3667, ATCC No. PTA-3668 or ATCC No.
PTA-3669.
A vaccine comprises a bacterium according to any one of claims 1 to 8.
A vaccine comprises the inactivated bacterium according to claim 10.
A vaccine comprises the live attenuated bacterium according to claim 11 or 12.
A vaccine comprises the preparation according to claim 13.
1f 22. The vaccine according to any one of claims 18 to 21, wherein said vaccine further comprises one or more suitable adjuvant(s) and/or excipient(s) and/or carrier(s).
A vaccine according to any one of claims 18 to 20, wherein said vaccine
comprises bacteria according to claims 1 to 8 of two or more different bacteria
strains, inactivated bacteria according to claim 10 of two or more different
bacteria strains and/or live attenuated bacteria according to claim 11 or 12 of
two or more different bacteria strains.
The vaccine according to claim 23, wherein said vaccine comprises two or
more live, and/or live attenuated, and/or inactivated bacteria of the bacteria
deposited under accession numbers ATCC No. PTA-3667, ATCC No. PTA-
3668 and ATCC No. PTA-3669.
The vaccine according to any one of claims 18 to 24, further comprising at
least one other antigen from a virus or microorganism pathogenic to poultry.
The vaccine according to claim 25, 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.
A method of immunizing poultry against a disease of the respiratory and
reproductive tract caused by a bacterium belonging to the family of
Pasteurellaceae comprising the step, administer a poultry the vaccine
according to claims 18 to 26.
Method of diagnosis of a disease in poultry caused by a bacterium belonging
to the family of Pasteurellaceae comprising the steps:

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 a bacterium according to claims
1 to 8
The method of diagnosis according to claim 28, wherein said presence of said
bacterium is determined by an immune test.
The method of diagnosis according to claim 29, wherein said presence of said
bacterium is determined by an molecular biology methods.
A diagnostic test kit comprising the components necessary for carrying out the
method according to any one of claims 28 to 30.

Documents:

00856-delnp-2004-abstract.pdf

00856-delnp-2004-claims.pdf

00856-delnp-2004-correspondence-others.pdf

00856-delnp-2004-description (complete).pdf

00856-delnp-2004-drawings.pdf

00856-delnp-2004-form-1.pdf

00856-delnp-2004-form-13.pdf

00856-delnp-2004-form-18.pdf

00856-delnp-2004-form-2.pdf

00856-delnp-2004-form-3.pdf

00856-delnp-2004-form-5.pdf

00856-delnp-2004-gpa.pdf

00856-delnp-2004-pct-134.pdf

00856-delnp-2004-pct-304.pdf

00856-delnp-2004-pct-306.pdf

00856-delnp-2004-pct-308.pdf

00856-delnp-2004-pct-332.pdf

00856-delnp-2004-pct-401.pdf

00856-delnp-2004-pct-402.pdf

00856-delnp-2004-pct-409.pdf

00856-delnp-2004-pct-416.pdf

00856-delnp-2004-pct-request form.pdf

00856-delnp-2004-pct-search report.pdf

856-delnp-2004-abstract-(01-03-2008).pdf

856-DELNP-2004-Abstract-(26-02-2008).pdf

856-delnp-2004-claims-(01-03-2008).pdf

856-DELNP-2004-Claims-(26-02-2008).pdf

856-DELNP-2004-Correspondence-Others-(26-02-2008).pdf

856-DELNP-2004-Description (Complete)-(26-02-2008).pdf

856-DELNP-2004-Drawings-(26-02-2008).pdf

856-delnp-2004-form-1-(01-03-2008).pdf

856-DELNP-2004-Form-1-(26-02-2008).pdf

856-delnp-2004-form-2-(01-03-2008).pdf

856-DELNP-2004-Form-2-(26-02-2008).pdf

856-DELNP-2004-Form-3-(26-02-2008).pdf

856-DELNP-2004-Form-5-(26-02-2008).pdf

856-DELNP-2004-GPA-(26-02-2008).pdf


Patent Number 216563
Indian Patent Application Number 00856/DELNP/2004
PG Journal Number 13/2008
Publication Date 28-Mar-2008
Grant Date 17-Mar-2008
Date of Filing 02-Apr-2004
Name of Patentee BOEHRINGER INGELHEIM VETMEDICA S.A. DE C.V.
Applicant Address CALLE 30 NO. 2614, ZONA INDUSTRIAL, GUADALAJARA, JALISCO 44940, MEXICO,
Inventors:
# Inventor's Name Inventor's Address
1 MARIA ELENA VAZQUEZ RIO AUTLAN NO.2034, COLONIA ATLAS, GUADALAJARA, JALISCO, MEXICO;
2 RAUL CAMPOGARRIDO SAN ERNESTO NO. 194, COLONIA JARDINES DE SAN IGNACIO, ZAPOPAN, JALISCO, MEXICO;
3 CARLOS GONZALES-HERNANDEZ PRIVADA COMONFORT NO. 74, HDA. DEL SUR, 45645 C.P. JALISCO, MEXICO
4 VAITHIANATHAN SIVANANDAN 213 TURF COVE, AUSTIN, TX 78748, USA,
PCT International Classification Number A61K 39/00
PCT International Application Number PCT/EP02/11899
PCT International Filing date 2002-10-24
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 101 52 307.6 2001-10-26 Germany