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Title:  Control of acidosis
United States Patent: 
7,011,826
Issued: 
March 14, 2006
Inventors: 
Rowe; James Barber (Armidale, AU); Al Jassim; Rafat A M (Gatton, AU)
Assignee: 
The University of New England (New South Wales, AU)
Appl. No.: 
786253
Filed: 
July 3, 2000
PCT Filed: 
July 3, 2000
PCT NO: 
PCT/AU00/00805
371 Date: 
August 15, 2001
102(e) Date: 
August 15, 2001
PCT PUB.NO.: 
WO01/02008
PCT PUB. Date: 
January 11, 2001


 

Pharm Bus Intell & Healthcare Studies


Abstract

The present invention relates to a vaccine for the prevention of lactic acidosis in a vertebrate, said vaccine comprising at least one isolated microorganism, or fragment or fragments thereof, wherein said microorganism is capable of producing lactic acid within the gut of said vertebrate, and wherein said microorganism is selected from the group consisting of: Clostridium-like species, Prevotella-like species, Bacteroides-like species, Enterococcus-like species, Selenomonas species, non-dextran slime producing Streptococcus species and non-slime producing lactic acid bacterial isolates.

BEST MODE OF PERFORMING THE INVENTION

1. Isolation of Microorganisms involved in acidosis

The following method provides a means of isolating microorganisms involved in acidosis. Fluid samples are taken from caecal, colonic, rectal or faecal material from animals or humans consuming a diet containing more than half of the dry matter as sugars, oligosaccharides or starch. This material (one part) may then be mixed well with distilled water (9 parts), prior to straining through 4 layers of cheese cloth and serially diluting in ten-fold steps using anaerobic dilution solution (ADS) (Caldwell and Bryant, 1966) to a final dilution of 10-8.

The material so diluted (10-6, 10-7 and 10-8) is then used to inoculate media roll tubes prepared with modified semi-selective MRS-Agar medium, Oxoid, England, (de Man et al., 1960), together with adjustment of the pH of the medium to 5.5 Three replicate tubes are used for each dilution and they are incubated at 39 C. for three days.

The colonies so prepared are then carefully studied under a low power (4) microscope to identify the most common colonies based on physical appearance and growth characteristics. These colonies are then enumerated at the three dilutions (10-6, 10-7 and 10-8) to confirm a consistent representation. At this stage, samples are taken of at least five colonies that have been counted as being of the same most common characteristics to confirm similarity, and are examined under high power magnification (>40) using gram straining to determine that the cells are similar.

Once this is done, viable colonies representing the dominant copy type are picked and used to inoculate a broth of basal medium 10 containing glucose (0.5%). This process of inoculation is repeated into MRS roll tubes and again examined for uniformity among colonies. At this stage, at least three examples of the most common colonies are examined and if these appear identical, representative colonies are picked and used to inoculate a broth of BM10. The process of roll tube and broth cultures is repeated until it is clear that a purified isolate has been obtained. In some cases, it is possible that two or more bacteria are very closely associated and in this case a crude isolate is maintained as the antigenic unit.

In the case of Streptococcus isolates, dextran (slime) characteristics are examined by centrifugation at 17,000 g for15 minutes. The absence of a bacterial "pellet" following centrifugation indicates a dextran type slime. One of the aims of the present invention was the selection for S. bovis bacterial that did not produce dextran slime.

Finally, characteristics of the isolate can then be determined by measuring the range of substrate utilisation and rate of lactic acid production.

2. Vaccine/Pharmaceutical Composition and Methods for Control of Acidosis

In a process of preparing a vertebrate vaccine of the invention, a typical protocol includes: washing the microbial growth free of nutrient medium, killing, harvesting and suspension of the dead cells of the microorganisms in a pharmaceutically/veterinarily acceptable carrier, diluent, excipient and/or adjuvant.

An alternative typical protocol includes: washing the microbial growth free of nutrient medium, rupturing to form outer membrane and associated proteins, separating whole cells from outer membranes and associated proteins, suspension of the outer membrane and associated proteins in a pharmaceutically/vertinarily acceptable carrier, diluent, excipient and/or adjuvant.

In delivery systems utilising the parenteral route it is preferred that dead cells of the microorganisms and/or outer membrane and associated proteins, are suitably washed, harvested and resuspended in a pharmaceutically/veterinarily acceptable carrier, diluent and/or adjuvant suitable for injection, utilising methods of administration as are well known in the art.

In the administration of therapeutic formulations in accordance with the present invention and herein disclosed, there are preferred non-toxic pharmaceutical carriers, diluents, excipients and/or adjuvants. For administration of the above formulations the microorganism or fragment or fragments thereof of the present invention are admixed with these non-toxic carriers, diluents, excipients and/or adjuvants and may be in the form of capsules, aqueous or oily suspensions, emulsions, micelles or injectable solutions.

Examples of pharmaceutically and veterinarily acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; crrageenan; gum tragcanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides, cytokines and buffering agents.

In general to induce the production of antibodies to the vaccines of the invention, they can be oleogenous or aqueous suspensions formulated in accordance with known methods in the art using suitable dispersing, suspension and/or wetting agents. Examples of suitable dispersing, suspension and wetting agents include Freund's complete/incomplete adjuvant. Montenide Marcol adjuvant and phosphate buffered saline, and mannan.

it will be appreciated that the examples referred to above are illustrative only and other suitable carriers, diluents, excipients and adjuvants known in the art may be employed without departing from the spirit of the invention.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include. Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

Further, a vaccine composition containing a recombinant polypeptide as encoded by at least one of the nucleic acid molecules in accordance with the twelfth or thirteenth embodiment of the invention, may be prepared for use by standard methods, well known to those of ordinary skill in the art.

In one embodiment, the immunogenic polypeptide, glycopeptide or the like, may be produced in a recombinant system by expression of the polynucleotide sequence (or a fragment thereof) in accordance with the twelfth or thirteenth embodiments of the invention, and subsequently isolated. For example, microbial cells containing the nucleic acid molecule of interest may be cultured in large volume bioreactors, then collected by centrifugation and subsequently ruptured, for instance by high-pressure homogenisation. The resulting cell lysate may be resuspended in appropriate diluent such as those described herein, and filtered to obtain an aqueous suspension of the immunogen. The recombinant protein can be administered in crude form, for example, by diluting in a 0.1M phosphate buffer (pH 7.4) to 50-500 μg/ml concentration, and then passing through a sterile 0.22 micron filter.

Alternatively, a vaccine composition containing the recombinant immunogenic polypeptide, glycopeptide or the like, may be prepared in a mammalian expression system, utilising host cells such as Chinese Hamster Ovary (CHO) cells. The recombinant polypeptide, glycopeptide or the like, (or fragment thereof) may be manufactured using batch fermentation with serum free medium. After fermentation the recombinant polypeptide, glycopeptide or the like, (or fragment thereof) may be purified via a multistep procedure incorporating chromatography and viral inactivation/removal steps. For instance, the recombinant polypeptide, glycopeptide or the like, (or fragment thereof) may be first separated by Protein A affinity chromatography and then treated with solvent/detergent to inactivate any lipid enveloped viruses. Further purification, typically by anion and cation exchange chromatography may be used to remove residual proteins, solvents/detergents and nucleic acids. The purified recombinant polypeptide glycopeptide or the like, (or fragment thereof) may be further purified and formulated into 0.9% saline using gel filtration columns. The formulated bulk preparation may then be sterilised and viral filtered and dispensed.

Alternatively, a vaccine composition containing an immunogenic polypeptide, glycopeptide or the like, of the microorganism of the present invention may be prepared by synthesis of a peptide, using standard methods known to those in the art, such as by automated synthesis on, for instance, an Applied Biosystems model 430A. For example, the peptide may comprise selected amino acid regions of the CDR and/or FR of the polypeptide of the invention. The synthetic peptide can be administered, for example, after diluting in a 0.1M phosphate buffer (pH 7.4) to 50-500 μg/ml concentration, and passing through a sterile 0.22 micron filter.

Alternatively, the vaccine may be a DNA based vaccine. In one aspect, the DNA based vaccine may comprise naked DNA comprising a nucleic acid encoding an immunogenic polypeptide of the microorganism of the present invention, or a fragment thereof.

In another aspect, the DNA based vaccine may comprise a nucleic acid molecule encoding an immunogenic polypeptide of the microorganism of the present invention, or a fragment thereof, cloned into an expression vector. Typically, the expression vector is a eucaryotic expression vector and may include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.

A typical vaccination regime is to deliver the vaccine in multiple doses generally one, two or three equal doses.

The vaccines of the invention are typically formulated for administration by parenteral route, by inhalation or topically, The term parenteral as used herein includes intravenous, intradermal, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred.

A vaccine or pharmaceutical composition of the invention may also be administered topically, such as externally to the epidermis, to the buccal cavity and instillation of such an antibody into the ear, eye and nose.

The amount of the vaccine or pharmaceutical composition of the invention required for therapeutic or prophylactic effect will, of course, vary with the vaccine or pharmaceutical composition chosen, the nature and severity of the condition being treated and the animal undergoing treatment, and is ultimately at the direction of the physician or veterinarian. A suitable topical dose of a vaccine or pharmaceutical composition of the invention will generally be within the range of about 1 to about 100 milligrams per kilogram body weight daily; preferably about 0.05 to about 50, more preferably about 0.5 to about 25, even more preferably about 0.5 to about 10 milligrams per kilogram body weight per day.

The parenteral dosage regimens for employing compounds of the invention to prophylactically or therapeutically control lactic acidosis will generally be in the range of about 0.01 to about 100, preferably about 0.01 to about 50, more preferably about 0.05 to about 25, even more preferably about 0.1 to about 2 milligrams per kilogram body weight per day. Alternatively, dosage rates can be determined in relation to metabolic rate or surface area of the body.

A vaccine or pharmaceutical composition of the invention may also be administered by inhalation, that is, intranasal and/or inhalation administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques. The preferred dosage amount of a compound of the invention to be employed is generally within the range of about 0.05 to about 100, preferably about 0.05 to about 50, more preferably about 0.5 to about 25, even more preferably about 0.5 to about 10 milligrams per kilogram body weight per day.

Typically, the dosage rate for immunisation is between 1106 and 11011 bacterial cells per administration.

Typically, the dosage rates are approximately equivalent to between 1108 to 1109 bacterial cells per kg body weight. More typically, the dosage rates are approximately equivalent to between 1108 and 5108 bacterial cells per kg body weight. Even more typically, the dosage rates are approximately equivalent to 2.5108 bacterial cells per kg body weight.

Typically, the dosage rate for immunisation of small animals, such as sheep, is between 1109 and 51010 bacterial cells per injection. More typically, the dosage rate for immunisation of small animals, such as sheep, is approximately 5109 bacterial cells per administration.

Typically, the dosage rate for immunisation of large animals, such as cattle and horses, is between 1109 and 11012 bacterial cells per injection. More typically, the dosage rate for immunisation of large animals, such as cattle and horses, is approximately 11010 bacterial cells per administration.

Typically, the injection volume for sheep is between 1 mL to 3 mL, and 2 to 7 mL for cattle and horses 3 to 5 mL. More typically, the injection volume for sheep is between 1 mL to 2 mL, and 1 to 5 mL for cattle and horses.

In accordance with any one of the third through eleventh embodiments of the invention, the administered dose of the antibiotic can vary and will depend on several factors, such as the condition, age and size of the human or animal patient, as well as the nature of the lactic acid producing bacteria.

Dosages will typically range from between any one of the following: 0.01 and 100 mg per kg of bodyweight; 0.01 and 75 mg per kg of bodyweight; 0.01 and 50 mg per kg of bodyweight; 0.01 and 25 mg kg of bodyweight; 0.01 and 15 mg per kg of bodyweight; 0.01 and 10 mg per kg of bodyweight; and 0.01 and 5 mg per kg of bodyweight. More typically dosages will range from between 0.2 and 2.0 mg per kg of bodyweight. More typically dosages will range from between 0.5 and 1.0 mg per kg of bodyweight. Even more typically dosages will range from between 0.1 and 0.5 mg per kg of bodyweight. Yet even more typically, the antibiotic is administered to the human or animal at a rate of 0.4 mg per kg of bodyweight.

Typically, the antibiotic is administered at a rate of between 1 and 100 mg per kg of dry weight of food. More typically, the antibiotic is administered at a rate of between 1 and 75 mg per kg of dry weight or food. Even more typically, the antibiotic is administered at a rate of between 1 and 50 mg per kg of dry weight of food. Yet even more typically, the antibiotic is administered at a rate of between 5 and 40 mg per kg of dry weight of food.

Typically, antibiotic preparations are selected and/or formulated for delivery to the hind gut and for little or no absorption from the digestive tract. Formulations include encapsulation and/or coating with materials resistant to acid and enzymatic digestion in the stomach and small intestine. Formulation can also include chemical treatment to reduce the solubility of the antibiotic.

As above, the administered dose of the enzyme preparation can vary and will depend on several factors, such as the condition, age and size of the human or animal patient, as well as the nature of the carbohydrate. Dosages will typically range from between 0.01 and 50 g/kg food dry matter. Typically, the enzyme is administered at a rate of between 0.1 and 3 g per kg of dry weight of food. More typically, the enzyme is administered at a rate of between 1 g per kg of dry weight of food.

Similarly, the administered dose of the clay preparation can vary and will depend on several factors, such as the condition, age and size of the human or animal patient, as well as the nature of the carbohydrate. Dosages will typically range from between 0.5 and 100 g/kg food dry matter. Typically, the clay is administered at a rate of between 1 and 50 g per kg of the dry weight of food. More typically, the clay is administered at a rate of between 10 and 20 g per kg of dry weight of food.

Typically, the administered dose of the probiotic preparation can vary between 104 and 1012 bacteria per kg of body weight. More typically, dose of the probiotic preparation can vary between 104 and 1010 per kg of body weight. Even more typically, dose of the probiotic preparation can vary between 104 and 106 per kg of body weight.

Typically, probiotics are formulated in such a way as to deliver viable bacteria and/or other microorganisms to gastrointestinal tracer including the hind gut. These formulation techniques include coatings and encapsulation using materials resistant to gastric and intestinal digestion.

According to another form of the invention, the active agents can be used together.

According to another aspect of the invention, the formulation of the active agent ensures that it is administered in a palatable form to the animal or human and in a form which retains activity and is properly mixed in the appropriate compartment(s) of the gastrointestinal tract.

Generally, the active agent is administered regularly throughout the period the animal or human is subjected to a high carbohydrate diet or to sugars or other fermentable compounds which are not efficiently absorbed prior to reaching the large intestine, colon and caecum.

More typically, the active agent is administered 1-3 times daily. Even more typically, the active agent is administered once daily or can be included in human food and animal feeds. They can be fed as powders or suspended in water, included in pellets as well as being fed in premixes.

More typically the active agent is mixed with the food, or is added to feeds which contain starch or sugars which may produce an acidic pattern of fermentation in the gastrointestinal tract. The active agent can also be added to water included in tablets and the like.

A suitable treatment may include the administration of a single dose or multiple doses. Usually, the treatment will consist of administering one dose daily of the active agent for a period sufficient to control the accumulation of acid by fermentation of the carbohydrate in the gastrointestinal tract. Dosing may continue while sources of carbohydrate known to cause problems of acidic fermentation in the gastrointestinal tract are included in the diet.

More typically the active agent may be administered in a single dose immediately before consuming meals containing sources of carbohydrate which are poorly digested and rapidly fermented.

More typically, the active agent is administered for only day prior to and daily during the consumption of excessive quantities of food stuffs containing readily fermentable carbohydrates.

Typically, the active agent is administered orally.

3. Antibiotics

Antibodies or immunoglobulins are typically composed of four covalently bound peptide chains. For example, an IgG antibody has two light chains and two heavy chains. Each light chain is covalently bound to a heavy chain. In turn each heavy chain is covalently linked to the other to form a "Y" configuration, also known as an immunoglobulin conformation. Fragments of these molecules, or even heavy or light chains alone, may bind antigen.

A normal antibody heavy or light chain has an N-terminal (NH2) variable (V) region, and a C-terminal (COOH) constant (C) region. The heavy chain variable region is referred to as VH (including, for example, Vγ), and the light chain variable region is referred to as VL (including Vκ or Vλ). The variable region is the part of the molecule that binds to the antibody's cognate antigen, while the Fc region (the second and third domains of the C region) on the heavy chain determines the antibody's effector function (eg. complement fixation, opsonization). Full-length immunoglobulin or antibody "light chains" are encoded by a variable region gene at the N-terminus and a κ (kappa) or λ (lambda) constant region gene at the COOH-terminus. Full-length immunoglobulin or antibody "heavy chains", are similarly encoded by a variable region gene and one of the constant region genes, e.g., gamma. Typically, the "VL" will include the portion of the light chain encoded by the VL and JL (J or joining region) gene segments and the "VH" will include the portion of the heavy chain encoded by the VH, and DH (D or diversity region) and JH gene segments.

An immunoglobulin, light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, also called complementarity-determining regions or CDRs. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space. The CDRs are primarily referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus.

The two types of light chains, κ (kappa) and λ (lambda), are referred to as isotopes. Isotypic determinants typically reside in the constant region of the light chain, also referred to as the CL in general, and Cκ or Cλ in particular. Likewise, the constant region of the heavy chain molecule, also known as CH, determines the isotype of the antibody. Antibodies are referred to as IgM, IgD, IgG, IgA, and IgE depending on the heavy chain isotype. The isotopes are encoded in the λ (mu), δ (delta), γ (gamma), α (alpha), and ε (epsilon) segments of the heavy chain constant region, respectively.

The heavy chain isotopes determine different effector functions of the antibody, such as opsonisation or complement fixation. In addition, the heavy chain isotype determines the secreted form of the antibody. Secreted IgG, IgD, and IgE isotypes are typically found in single unit or monomeric form. Secreted IgM isotype is found in pentameric form; secreted IgA can be found in both monomeric and dimeric form.

In a related aspect, the invention features a monoclonal antibody, or an Fab, (Fab)2, scFv (single chain Fv), dAb (single domain antibody), bi-specific antibodies, diabodies and triabodies, or other immunologically active fragment thereof (eg., a CDR-region). Such fragments are useful as immunosuppressive agents. Alternatively, the antibodies of the invention may have attached to it an effector or reporter molecule. For instance, an antibody or fragment thereof of the invention may have a macrocycle, for chelating a heavy metal atom, or a toxin, such as ricin, attached to it by a covalent bridging structure. In addition, the Fc fragment or CH3 domain of a complete antibody molecule may be replaced or conjugated by an enzyme or toxin molecule, such as chelates, toxins, drugs or prodrugs, and a part of the immunoglobulin chain may be bonded with a polypeptide effector or reporter molecule, such as biotin, fluorochromes, phosphatases and peroxidases. Bispecific antibodies may also be produced in accordance with standard procedures well known to those skilled in the art.

The present invention further contemplates genetically modifying the antibody variable and/or constant regions to include effectively homologous variable and constant region amino acid sequences. Generally, changes in the variable region will be made to improve or otherwise modify antigen binding properties of the antibody or fragment thereof. Changes in the constant region will, in general, be made in order to improve or otherwise modify biological properties, such as complement fixation, interaction with membranes, and other effector functions.

Typically, the antibodies in accordance with the sixteenth embodiment of the invention can be comprised of a polyclonal mixture, or may be monoclonal in nature. Further, antibodies can be entire immunoglobulins derived from natural sources, or from recombinant sources. The antibodies of the present invention may exist in a variety of forms, including for example as a whole antibody, or as an antibody fragment, or other immunologically active fragment thereof, such as complementarity determining regions.

Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. For example, spleen cells from an animal immunised with a desired antigen are immortalised, commonly by fusion with a myeloma cell, in a manner as described for example, in Kohler and Milstein, Eur. J. Immunol., 6:511-519 (1976), the disclosure of which is incorporated herein by reference.

Alternative methods of immortalisation include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalised cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Various techniques useful in these arts are discussed, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York, (1988), the disclosure of which is incorporated herein by reference, including: immunisation of animals to produce immunoglobulins; production of monoclonal antibodies; labelling immunoglobulins for use as probes; immunoaffinity purification; and immunoassays.

4. An antibody/nucleic acid based method and kit for detecting acidosis activity

The present invention also encompasses a method of detecting in a sample the presence of microorganisms involved in lactic acidosis and/or the potential acid producing characteristics of the microorganisms, wherein the method comprises:

(a) contacting a sample with the antibody (or fragment thereof) as defined in accordance with the sixteenth embodiment of the invention, and

(b) detecting the presence of the antibody (or fragment thereof) bound to a microorganism or (fragment thereof) involved in lactic acidosis.

Typically, the method of detecting in a sample the presence of microorganisms involved in lactic acidosis, or potential lactic acid producing microorganisms may also comprise:

(c) measuring lactic acid present in the digesta or faecal sample or measuring pH of said sample; or

(d) measuring amount of lactic acid produced when microorganisms ferment carbohydrate.

Conditions for incubating an antibody (or fragment thereof) with a test sample vary widely, depending on the format of detection used in the assay, the detection method, and the type and nature of the antibody used. A person of ordinary skill in the art would readily appreciate that any one of the commonly available immunological assays could be used in performing the method of detection. For example, these assays include: radio immunoassays, enzyme-linked immunosorbent assays, and/or immunoflourescent assays.

A kit for performing the above method of the invention contains all the necessary reagents to carry out the above methods of detection. For example, the kit may comprise the following containers:

(a) a first container containing the antibody (or fragment thereof) in accordance with the sixteenth embodiment of the present invention;

(b) a second container containing a conjugate comprising a binding partner of the antibody (or fragment thereof), together with a detectable label.

Typically, the kit may further comprise one or more other containers, containing other components, such as wash reagents, and other reagents capable of detecting the presence of bound antibodies. More typically, the detection reagents may include: labelled (secondary) antibodies, or where the antibody (or fragment thereof) of the present invention is itself labelled, the components may comprise antibody binding reagents capable of reacting with the labelled antibody (or fragment thereof) of the present invention.

Further, the kit of the present invention, as described above in relation to antibodies, can be readily incorporated, without the expenditure of inventive ingenuity, into a kit for nucleic acid probes. One skilled in the art would select the nucleic acid probe from the polynucleotides of the present invention, according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of vertebrate tissue.

Such a kit comprises at least one container means having disposed therein the above-described nucleic acid probe. The kit may further comprise other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe. Examples of detection reagents include, but are not limited to radiolabelled probes, enzymatic labelled probes (horseradish peroxidase, alkaline phosphatase), and affinity labelled probes (biotin, avidin, or steptavidin).

In detail, a compartmentalised kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and like), and containers which contain the reagent detect the hybridised probe, bound antibody, amide product, or the like.

Furthermore, one skilled in the art would readily recognise that the nucleic acid probes of the present invention can readily be incorporated into one of the established kit formats, which are known in the art.

In terms of measuring lactic acid production, the pH of digesta or faecal material can be measured using colour sensitive reagents or a pH meter. Lactic acid can be detected using colorimetric techniques using enzymes, colour reagents and assays assessed with the naked eye or using spectrometric techniques. Further, identifying potential lactic acid producing bacteria involves incubation of the sample with carbohydrate followed by measurement of lactic acid, pH and lactic acid producing bacteria.
 


Claim 1 of 12 Claims

1. A vaccine comprising at least one isolated microorganism or living or dead cells thereof wherein the microorganism is selected from the group consisting of: (a) Streptococcus bovis strain SbR1 Accession number NM99/04455, (b) Streptococcus equinus strain SER1 Accession number: NM99/04456, (c) Streptococcus equinus strain SER2 Accession number: NM99/04457, (d) Selenomonas ruminantium strain SRR1 Accession number: NM99/04458, (e) Selenomonas ruminantium strain SRR3 Accession number: NM99/04460, (f) Clostridium vitulinus strain LVR3 Accession number: NM99/04461, (g) Clostridium vitulinus strain LVR4 Accession number: NM99/04462, (h) Prevotella isolates LAB01 Accession number: NM00/12630, (i) Prevotella isolate LAB03 Accession number: NM00/12632, (j) Bacteroides isolates LAB07 Accession Number: NM00/12636, (k) Bacteroides isolate LAB05 Accession number: NM00/12634, (l) non-dextran slime producing Streptococcus isolate LAB04 Accession number: NM00/12633, (m) non-slime producing lactic acid bacterial isolates LAB02 Accession number: NM00/12631, (n) non-slime producing lactic acid bacterial isolate LAB06 Accession number: NM00/12635, and (o) non-slime producing lactic acid bacterial isolate LAB08 Accession number: NM00/12637.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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