| WO/2003/061674 | DIETARY SUPPLEMENTS COMPRISING GROWTH MEDIA |
| JP4651814 | Use of Lactobacillus to reduce blood fibrinogen levels |
| WO/2022/239012 | MICROBIAL CONSORTIUM AND USES THEREOF |
| US3139382A | 1964-06-30 | |||
| US4237115A | 1980-12-02 | |||
| US3127318A | 1964-03-31 | |||
| US3992521A | 1976-11-16 |
| 1. | What is claimed is: A composition comprising a mixture of non heat killed bacteria, wherein the bacteria are Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp. |
| 2. | The composition of Claim 1, wherein the bacteria are killed with a chemical selected from the group consisting of formalin, glutaraldehyde, lysine, periodate, paraformaldehyde, and phenol, or mixtures thereof. |
| 3. | A method of providing therapeutic treatment to humans and animals, comprising administering to the human or animal an amount of the composition of Claim 1 effective to achieve a therapeutic treatment. |
| 4. | The method of Claim 3, wherein the therapeutic treatment is reduction of bacterial toxin levels, or reduction of diarrhea, or reduction of bacterial levels, or stimulation of the immune system, or stimulation of growth, or elevation of the protein to fat ratio, or reduction of gastrointestinal bacterial content. |
| 5. | The method of Claim 4, wherein the therapeutic treatment stimulates growth in the human or animal. |
| 6. | The method of Claim 4, wherein the therapeutic treatment reduces levels of bacterial toxins in the human or animal. |
| 7. | The method of Claim 4, wherein the therapeutic treatment reduces bacterial levels of Escherichia sp., Pseudomonas sp., Salmonella sp., or Aerobacter sp. in the human or animal. |
| 8. | The method of Claim 4, wherein the therapeutic treatment reduces diarrhea in the human or animal. |
| 9. | The method of Claim 4, wherein the therapeutic treatment stimulates the immune system of the human or animal. |
| 10. | The method of Claim 4, wherein the therapeutic treatment increases the protein to fat ratio in the human or animal. |
| 11. | The method of Claim 4, wherein the therapeutic treatment decreases the gastrointestinal bacterial content in the human or animal. |
| 12. | A method of stimulating growth of an animal comprising administering the composition of Claim 1 to the animal's mother prior to birth of the animal, in an amount effective to stimulate growth, and to increase the protein to fat ratio of the animal. |
| 13. | A method of stimulating growth of a human comprising administering the composition of Claim 1 to the human's mother prior to conception of the human, in an amount effective to stimulate growth, and to increase the protein to fat ratio of the human. |
| 14. | A composition comprising, serum derived from a hyperimmune animal previously immunized with an amount of the composition of Claim 1, effective to elicit an immune response in the animal immunized. |
| 15. | The composition of Claim 14, wherein the serum is effective to treat or prevent diarrhea when administered to a human or animal. |
| 16. | The composition of Claim 14, wherein the hyperimmune animal is a cow. |
| 17. | A method of treating or preventing diarrhea in a human or animal comprising administering to the human or animal an amount of the composition of Claim 15 effective to treat or prevent diarrhea. |
| 18. | The composition of Claim 1, wherein the bacteria are Escherichia coli, Pseudomona aeruginosa, Salmonella enteritidis and Aerobacter aerogenes. |
BACTERIA
Cross-Reference to Related Applications This application is a continuation-in-part application of U.S. Patent Application Serial No. 08/318,030 filed October 4, 1994, which is currently pending. This application is also a continuation-in-part application of U.S. Patent Application Serial No. 08/318,502 filed October 5, 1994, which is currently pending. This application is also a continuation-in-part application of a U.S. Patent Application entitled, "Therapeutic Treatment of Humans and Animals by Hyperimmunization with Killed Bacteria" filed on October 3, 1995, serial number not assigned as of the time of filing. The present application claims priority to these three applications.
Field of the Invention
The present invention relates to a composition and method for providing a therapeutic treatment for humans and animals by vaccination with enteric non heat-killed bacteria for the treatment of several conditions, including but not limited to the following; intestinal diseases such as diarrhea, bacterial infections, toxic infections, for reducing the bacterial content of the gastrointestinal system, for stimulation of the immune system, for accelerating growth and for increasing the protein to fat ratio.
This invention also provides a composition and method for the treatment of intestinal diseases such as diarrhea and includes
administration of serum from an animal, such as a cow, immunized by means of specific, non heat-killed bacteria that are pathogenic to humans or animals.
Background of the Invention
Composition and Treatment to Affect Growth and Protein Content of Humans and Animals
Increasing growth of animals while increasing protein content and increasing their protein to fat ratio are long¬ standing goals of food producers. Many approaches have been undertaken including treatment of food animals with pituitary extracts and recombinant growth hormone. The acceleration of growth rate has also been a goal of food producers since decreasing the time required for development before slaughter reduces costs dramatically thereby decreasing the cost to the consumer. In addition, food producers have become increasingly aware of the need to produce leaner meat from all kinds of food animals as consumers demand a healthier meat product with a higher protein to fat ratio. Food producers are also increasingly concerned about reducing bacterial contamination of food products during slaughter and processing of animal products in order to increase the shelf life of products and to reduce the risk of dangerous and sometimes lethal bacterial food poisoning of humans. One means to reduce bacterial contamination of food is to decrease the gastrointestinal content of bacteria in an animal before slaughter.
Current methods of producing lean animals and meat utilize hormones to increase the body protein to fat ratio, "Designing Foods: Animal Product Options in the Marketplace,"
Committee on Technological Options to Improve the Nutritional Attributes of Animal Products, Board on Agriculture, National Research Council, National Academy Press, Washington, D.C. 1988; and Mersmann, H.J., Prog. Food Nutr. Sci. 11: 175-201
(1987). Hormones that have been used to regulate the carcass ratio of protein to fat include anabolic steroids and growth hormone, and also neurotransmitter adrenergic agonists.
To avoid the problems associated with exogenous administration of hormones, including anabolic steroids and growth hormone, attempts have been made to regulate animal growth immunologically, by actively immunizing the animal against a specific component or hormone, the lack of which promotes the development of a relatively lean animal. For example, a single injection, into growing rats of antibodies raised against fat cell plasma membranes significantly lowered the levels of body fat and the number of fat cells in the rats for a period of several months. Flint, D.J., et al., Hannah Res. (1985), pp. 123- 127.
Normally, upon exposure to a foreign antigen, e.g., a bacterium, the immune system of the host will produce antibodies that will neutralize the effects of the antigen. Exposure to such foreign antigens can occur either naturally, or deliberately by administration of the antigen in vaccine form. The latter is generally referred to as active immunization of the host species exposed to the antigen. In general, merely inducing an immune state in an animal does not alter the levels of carcass fat or protein. Williamson, E.D., et al., Livestock Prod. Sc. 12:251-264 (1985). Attempts have also been made to increase growth hormone levels in growing animals by immunizing lambs and beef cattle against somatostatin, also known as growth hormone release-inhibiting hormone, the hormone responsible for suppression of growth hormone secretion from the anterior pituitary gland. This method has also been tried on sheep and has been reviewed. Designing Foods, Chapter 6, pp. 115-132; and Schelling, G.T., et al., Designing Foods, supra, pp. 200-207. In theory, by immunizing against somatostatin, the levels of
somatostatin would decrease causing plasma levels of growth hormone to increase. However, this method has produced conflicting results. Spencer, G.S.G., et al., Livestock Prod. Sci. 10:25-37 (1983). Lambs immunized repeatedly against somatostatin grew larger, faster, and were heavier than non- immunized lambs but there was no change in the proportion of muscle or fat. However, repeatedly immunizing with an antiserum against a hormone such as somatostatin is relatively expensive when treating large numbers of herd animals.
It has been known in the prior art to produce a hyperimmune state in an animal wherein that hyperimmune state produced a milk having a variety of therapeutic effects. Beck, for example, has disclosed a milk containing antibody to Staphylococcus mutans that has dental caries-inhibiting effects (Beck, U.S. Patent No. 4,324,784; British Patent 1,505,513).
Beck has also described a milk having anti-arthritic properties, U.S. Patent No. 4,732,757, and has patented a method of treating inflammation using milk from hyperimmunized cows. Beck, U.S. Patent No. 4,284,623. Stolle, et al., have disclosed a method of using milk from a hyperimmunized cow for the treatment of diseases of the vascular and pulmonary systems. U.S. Patent No. 4,636,384.
It had been disclosed in the art that multiple injections of a collection of 26 types of heat-killed bacteria into cows produces hyperimmune serum. Administration of this serum into lambs increased muscle thickness and decreased body fat but did not affect growth rate. Beck, U.S. Pat. No. 5,128,127.
Other authors having noted that therapeutic products can be derived from the milk of animals by specifically immunizing or hyperimmunizing those animals against a known antigen. Heinbach (U.S. Patent No. 3,128,230), Singh (U.S.
Patent No. 3,911,108), Peterson (U.S. Patent No. 3,376,198 and
Canadian Patent No. 587,849), Holm (U.S. Application (published) Serial No. 628,987), and Tannah et al. (British Patent No. 1,442,283).
These references suggest that the repeated injection of expensive reagents, such as somatostatin antisera, is not feasible and cost-effective in large numbers of animals. In addition, some treatments use anabolic steroids, recombinant growth hormone, and genetically engineered animals which raise concerns in food consumers.
These goals of increasing growth, and increasing the ratio of protein to fat in the composition of animals, and decreasing the gastrointestinal content of bacteria are also objectives in the treatment of humans. Similar concerns regarding the use of anabolic steroids for growth promotion and protein synthesis, for example, also relate to the treatment of humans since many undesirable side effects such as sterility and cancer may occur. In addition, treatment of children with recombinant growth hormone or other growth promoting agents is very expensive and the long term implications of such hormonal interventions are not yet understood.
The present invention provides an composition and method for the treatment of developing humans to promote growth. The present invention also provides a composition and method to increase the ratio of protein to fat in the human body. The present invention also provides a composition and method for the reduction of the gasttointestinal content of bacteria in humans.
Composition and Treatment for Bacterial Infections, Intestinal Disease and Diarrhea in Humans and Animals
Bacterial infections annually cause suffering and death in millions of humans and animals, account for great expenditures of human effort and resources, and result in the loss of productivity at work. Intestinal infections, such as diarrheal infections, are a major cause of mortality and illness throughout the world and affect numerous travelers. Many bacterial infections are caused by bacteria such as Escherichia sp., Pseudomonas sp., Salmonella sp. and Aerobacter sp. Diarrhea is a major intestinal disease annually affecting billions of humans and animals. Diarrhea is a major cause of mortality in neonates, infants and the elderly, accounting for about 25% of the annual infant mortality. Diarrhea is also a major cause of illness and mortality in livestock and other farm animals, resulting in a tremendous loss of life, food sources and financial resources. Most treatments involve antibiotics which sometimes have undesirable side effects such as reducing immunological resistance to other diseases and decreasing food absorption due to exfoliation of the intestinal epithelium. In addition, many strains of bacteria that cause intestinal diseases such as diarrhea develop resistance to antibiotics, rendering conventional treatment ineffective.
Enterobacteria is defined generally as any of various gram-negative, rod-shaped bacteria of the family Enterobacteriaceae that includes some pathogens of humans and animals, such as the Escherichia coli and Salmonella sp. The specific nature of the enterobacteria involved in such infections, as well as that of the subjects, should be stressed. Enterobacteria which are not pathogenic to animals, may be pathogenic to man. Furthermore, certain enterobacteria which are pathogenic to infants do not cause intestinal infections in adults.
Vaccination with compositions consisting of killed bacterial may provide protection against bacterial infections and also facilitate the immune response to a bacterial infection. In
addition, such vaccinations may provide a general protective effect through stimulation of the immune system.
It has been disclosed that molecular biological methods can be employed for the production of non-toxic subunits of the heat stable enterotoxin of E. coli and Vibrio cholerae to use as antigens in the production of polyvalent antisera. These antisera are disclosed as being useful for providing passive immunization following oral administration. Clements, U.S. Patent No. 5,308,835. These methods are sophisticated and the technology and reagents are not commonly available.
It has been known in the prior art to produce a serum in horses hyperimmunized by means of cultures of the enterobacteria pathogenic to the infant. Le Minor, U.S. Patent No. 3,992,521, for example, disclosed a horse serum produced against the enterobacteria Escherichia coli and Salmonella. These sera were produced separately against heat-killed Escherichia coli and Salmonella and were administered either separately or in combination through the oral route. However, horse serum can produce undesirable side effects as described by J. Fechner, S. Hirzel "Schutzimfungen bet Haustieren" (Vaccines and
Vaccination) Leipzig. In addition, repeated oral administration is not convenient for large numbers of individuals or farm animals. Treatment of bacteria with heat can also have adverse consequences on their antigenicity.
Thus, there remains a need for a composition and method that treats and prevents diarrhea in humans and animals. This composition should produce fewer side effects than previous compositions and methods. The composition should be capable of being administered through intraperitoneal, intravenous, intramuscular or subcutaneous injection to facilitate treatment of humans and animals, especially herd animals. These modes of administration would be much faster and more economical for
treatment of large numbers of animals than previous methods of oral administration.
Thus, what is needed is a composition and methodology that is relatively inexpensive and applicable to large numbers of humans and animals. This composition and method should also promote growth in humans, especially developing humans, and increase the protein to fat ratio in humans. This composition and method should promote growth and increase the protein to fat ratio in the offspring of pregnant humans that received this composition before becoming pregnant, and in the offspring of pregnant animals that received this composition before or during pregnancy. This composition and treatment should be effective in increasing growth rates of animals and in producing lean animals that are acceptable to consumers. This composition and method should also effectively reduce the intestinal content of bacteria in animals before slaughter, thereby producing meat products with reduced levels of bacterial contamination. This composition should avoid the use of hormones such as anabolic steroids, expensive genetically engineered products, and expensive anti-hormone antisera.
In addition, what is needed is a method and composition which provides a treatment for bacterial infections, intestinal diseases such as diarrhea, and for conditions resulting from affliction with toxins, such as toxic bacterial products. This method and composition should also provide for stimulation of the immune system. This composition should be a serum obtained from animals hyperimmunized with selected bacteria.
Summary of the Invention
The present invention provides a therapeutic treatment for humans and animals comprising administering to the human or animal an amount of a composition comprising a mixture of non heat-killed bacteria, wherein the bacteria are
Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp., effective for therapeutic treatment.
The present invention provides a new vaccine composition and method of administering that vaccine composition to provoke an immune response that increases growth in animals and humans. The increased growth is accompanied by an increase in the protein content and relative decrease in fat content of the animal or human, thereby producing a higher protein to fat ratio.
The present invention provides a vaccine composition comprising a mixture of non heat-killed bacteria wherein the bacteria are selected from the group consisting of Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp. Chemical means are preferred for killing bacteria. Use of formalin is a preferred method of killing the bacteria. Optionally, the vaccine composition of the present invention contains an adjuvant such as an oil, lanolin or liquid Vaseline, although other adjuvants including, but not limited to, aluminum hydroxide or aluminum potassium sulfate could be used. Optionally, the vaccine composition may be administered in the form of microspheres, including but not limited to polylactide microspheres.
In one embodiment of the present invention, the vaccine composition comprising a mixture of four, formalin- killed bacteria from Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp., is administered to humans to provide a therapeutic treatment for a variety of conditions including but not limited to the following; infections, bacterial infections, complications due to toxins, especially bacterial toxins, and poor growth rate. Treatment of animals with this composition is effective in reducing the bacterial content of the gastrointestinal lumen. This treatment of an animal before slaughter decreases the bacterial contamination of animal products such as meat.
The present invention also provides a method of increasing the protein content and ratio of protein to fat in an animal or human by hyperimmunizing the animal by administering to the animal a composition comprising a mixture of non heat-killed bacteria wherein the bacteria are selected from
Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp.
According to the present invention, after administration of the composition to the animal, the animal's growth is increased substantially. In cattle, growth increase has been shown to be 20% or more over non-vaccinated cattie. In another embodiment of the present invention, the vaccine composition can be administered to birds, such as chickens, to substantially increase the size of eggs from the adult female bird.
Accordingly, it is an object of the present invention to provide a therapeutic treatment for humans and animals by administration of a composition comprising a mixture of the following non heat-killed bacteria; Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp.
The present invention also provides a method of treating diarrhea by administering to an animal or human with diarrhea antidiarrheal serum obtained from an animal, desirably a cow, that has been immunized with a composition comprising a mixture of non heat-killed bacteria selected from Escherichia sp,
Pseudomonas sp, Salmonella sp and Aerobacter sp.
The antidiarrheal serum and method of the present invention is especially useful for treating diarrhea in newborns and in the elderly. The antidiarrheal serum of the present invention can be administered by injection. This composition is effective in both treating and preventing diarrhea caused by infection with enteric bacteria. The antidiarrheal serum is highly
effective after only one administration, and rarely requires a second administration.
Accordingly, it is an object of the present invention to provide antidiarrheal serum derived from cows to which has been administered the composition comprised of a combination of the following non heat-killed bacteria; Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis and Aerobacter aerogenes.
Still another object of the present invention is to provide antidiarrheal serum from animals other than cows, including but not limited to goats, sheep, and pigs, to which has been administered the composition described above wherein the selected bacteria are pathogenic to the species receiving the antidiarrheal serum.
Another object of the present invention is to provide a therapeutic treatment for diarrhea in humans and animals by administration of a composition comprising a Combination of the following non heat-killed bacteria; Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis and Aerobacter aerogenes
A further object of the present invention is to provide a method of treating diarrhea in humans especially neonates, infants and the elderly.
Another object of the present invention is to provide a method of treating diarrhea in non-human animals, including, but not limited to, livestock, pigs, sheep, birds, rodents, and rabbits.
A further object of the present invention is to provide a method of preventing diarrhea in humans especially neonates, infants and the elderly.
Another object of the present invention is to provide a method of preventing diarrhea in non-human animals, including but not limited to livestock, pigs, sheep, birds, rodents, and rabbits.
Still another object of the present invention is to increase growth and increase the protein to fat ratio in animals and humans.
Another object of the present invention is to increase growth of developing humans.
Yet another object of the present invention is to preferentially increase the growth of the offspring of pregnant animals that received this composition before or during pregnancy.
Still another object of the present invention is to preferentially increase the growth of the offspring of pregnant humans that received this composition before pregnancy.
A further object of the present invention is to increase growth by administering to animals and humans one injection of a composition that induces an immune response.
Still another object of the present invention is to provide a method to stimulate an immune response in animals and humans by administering the composition.
Another object of the present invention is to preferentially increase the protein content and the protein to fat ratio of animals and humans receiving this composition.
Still another object of the present invention is to preferentially increase the protein content and the protein to fat ratio of the offspring of pregnant animals and pregnant humans that received this composition before becoming pregnant.
Yet another object of the present invention is to preferentially increase the protein content and the protein to fat ratio of the offspring of pregnant animals that received this composition during pregnancy.
Another object of the present invention is to mix the non heat-killed bacteria with an adjuvant for administration to animals and humans.
Yet another object of the present invention is to administer the vaccine composition of the present invention in a single injection.
Yet another object of the present invention is to increase the size of eggs from birds.
Another object of the present invention is to decrease the cost of raising livestock and birds by increasing growth rate.
Still another object of the present invention is to decrease the time required for livestock and birds to grow to a desired size before sacrifice.
Yet another object of the present invention is to provide meat with higher protein content and higher protein to fat ratio from livestock and birds.
Another object of the present invention is to decrease the bacterial content of the gastrointestinal tract of animals.
Still another object of the present invention is to decrease the bacterial contamination of animal products, such as meat, obtained at slaughter of the animal.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the preferred embodiments.
Detailed Description of the Preferred Embodiments
Definitions
The term "administered" or "administration" means oral administration and intramuscular, intravascular, intraperitoneal or subcutaneous injection of an animal or human with the composition of the present invention in a pharmaceutically acceptable vehicle. The term "non heat-killed bacteria" refers to bacteria killed through means other than heat. The term "therapeutic treatment" means administration of the combination of the following non heat-killed bacteria; Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp., optionally combined with one or more adjuvants in order to stimulate growth, reduce bacterial levels, reduce the gastrointestinal bacterial content, decrease diarrhea, reduce bacterial toxin levels, stimulate the immune system, or increase the protein to fat ratio. The term "therapeutic treatment" also means administration of serum obtained from an animal immunized with a combination of these non heat-killed bacteria in order to decrease diarrhea. The term "growth" or "increased growth" means an increase in the length of the axial skeleton, an increase in the length of the long bones such as the femur or humerus, an increase in the muscle mass of the body, or an increase in the girth of the body. The term "diarrhea" means excessive and frequent evacuation of watery feces, usually indicating gastrointestinal distress or disorder or intestinal disease. The term "food animal" means any animal that is consumed in the diet of humans or other animals. Typical food animals include bovine animals, for example, cattle; ovine animals, for example, sheep; swine, for example, pigs; fowl, for example, chickens and turkeys; ratites, for example emus and ostriches; fish; rabbits and the like.
Composition and Treatment to Affect Growth and Protein Content of Humans and Animals: The Vaccine
The present invention encompasses a vaccine composition comprising a mixture of non heat-killed bacteria wherein the bacteria are selected from Escherichia sp. , Pseudomonas sp., Salmonella sp., and Aerobacter sp. and a method for using the composition to increase growth, to increase the protein to fat ratio, to stimulate the immune system, to treat and prevent diarrhea, to reduce bacterial levels, to decrease the bacterial gastrointestinal content, to reduce the level of bacterial toxins, to increase the growth of the offspring of animals receiving this composition during or before pregnancy, and to increase the growth of the offspring of humans receiving this composition before conception. In a desirable embodiment, the bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis and Aerobacter aerogenes. Various means may be used to kill bacteria including chemicals, radiation and other methods. Radiation sources may include the use of x-ray, gamma and ultraviolet irradiation. Chemicals which may be used to kill bacteria include, but are not limited to, formalin, phenol, glutaraldehyde, acrolein, alcohol, paraformaldehyde, sodium periodate, lysine, other cross-linking agents and mixtures thereof. In one embodiment, the bacteria are killed with formalin. The bacteria can be killed by other means such phenol, and the like.
While not wishing to be bound by the following statement, it is believed that chemicals such as formalin and others recited above preserve the antigenicity and also the secondary and tertiary structure of antigenic sites found on and in bacteria, thereby enhancing the immune response of a human or animal to the administration of a mixture of non heat-killed bacteria.
Optionally, the composition of the present invention comprises bacteria that are selected from the geographical area in which the vaccine composition is to be used. Although it has been found that including at least the four species listed above provides
excellent growth increase, it is contemplated that the vaccine composition of the present invention can include additional bacteria.
The amounts of bacteria used in a typical preparation of the present invention are between approximately 10 8 and 10 12 bacteria per ml with the desired concentration of bacteria of between 10 10 to approximately 10 11 bacteria per ml. In one embodiment, the amounts of bacterial species per 10 ml are: 7 x 10 10 for Escherichia sp., and 1 x 10 for each of Pseudomona sp., Salmonella sp., and Aerobacter sp. An adult cow will receive
10 ml of the foregoing combination of non heat-killed bacteria for a total bacterial dose of approximately 10 1 1 . Preferred methods of administration to cows include intramuscular and subcutaneous administration. Dosages for different species are approximately 1.5 to 1.7 x 10 bacteria per pound of body weight. In poultry, a sufficient response can be obtained with half the standard concentration of bacteria and 10% of the above dosage as inoculum. The optimal volume of the vaccine for each administration will vary depending upon the species of animal to be vaccinated. For example, for cattle, pigs, sheep, and goats, the optimal volume of the vaccine for administration is between approximately 1 ml and 15 mis with the desired volume of approximately 10 mis. For chickens the optimal volume of the vaccine for administration to the chick is between approximately 0.05 mis and 2 mis at half the concentration of bacteria.
For humans, the optimal volume of the vaccine for administration to the human is between approximately 0.05 to 0.2 ml per 10 kg of body weight at a desired concentration of non heat-killed bacteria of approximately 10 9 bacteria per ml. The preferred method of administration to humans is intramuscular administration. Neonates, infants, and children under 40 kg body weight are not to be treated with the vaccine. Pregnant women should also not be treated with the vaccine.
It is to be understood that the volume of the vaccine to be administered to the human or animal is at least partially dependent upon the individual being treated, the concentration of the bacteria in the vaccine, on the type of adjuvant being used, the condition being treated and the method of administration. Thus, the desired volume may be outside the stated ranges depending upon the final formulation and method of administration of the vaccine.
Desirably, the composition of the present invention contains an adjuvant to increase the immune response such as an oil or Vaseline, although aluminum hydroxide or aluminum potassium sulfate may be used. In the preferred embodiment, hyperimmunization of food animals is achieved by a single administration of a composition of the present invention with the adjuvant. Many different compositions for the slow release of vaccines have been described which would be applicable to the method of the invention, for example, as described in Sanders, H.J., Chem. & Engineering News, Apr. 1, 1985, pp. 30-48, which is incorporated herein by reference. It is to be understood that the composition of the present invention is desirably injected only once, but can be injected more than once over a period of time. For example, a booster injection may be given several weeks or up to a year after the first injection. Other methods of administration, including but not limited to administration of microspheres, such as polylactide microspheres and polyglycolide microspheres, may be used in the present invention. Microspheres are described in Cleland, J.L., Design and Production of Single-Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems, pp. 439-462 in Vaccine Design: The Subunit and Adjuvant Approach, Powell, M.F. and
Newman, M.J., eds., Plenum Press, New York (1995).
Composition and Treatment to Affect Growth and Protein Content of Humans and Animals:
The Treatment Method
The present invention provides a method for increasing the protein content and increasing the protein to fat ratio in an animal or human by vaccinating the animal or human through administration of a composition comprising non heat- killed bacteria wherein the bacteria are selected from Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp. The vaccine of the present invention can also be administered to birds, such as chickens, turkeys, ducks and the like, in order to increase the size of eggs laid by the birds.
The method of the present invention is also applicable to any animal used as a source of food and especially as a source of meat such as mutton and lamb from sheep; beef and veal from cattle; pork from pigs; poultry meat including meat from chickens and turkeys, and rabbit meat.
In a preferred embodiment, animals and humans are vaccinated while still growing and maturing into the adult state. The first vaccination may be administered as soon as the animal is competent to immunologically respond to the antigen. Animals and humans immunologically competent to respond to the antigen are also capable of being placed in a hyperimmune state. In another embodiment, pregnant animals are injected with the composition. In still another embodiment, pregnant animals are injected with the composition and their offspring are injected as soon as the offspring animal is competent to immunologically respond to the antigen.
An advantage of the present invention is that only a single injection is required to accomplish increased growth and increased ratios of protein to fat. Accordingly, there is usually no need to perform booster injections, resulting in savings in labor and materials, especially in the application to large numbers of farm animals such as cattle, chickens and turkeys. A single injection is also less stressful to the animals and decreases the
chances of infection, thereby increasing the overall livestock survival rate. Additional savings are provided by the nature of the composition itself because all components (bacteria and optionally adjuvant) are inexpensive, especially when compared to prior art methods of increasing growth such as beta receptor agonists, somatostatin antisera, growth hormone and synthetic steroids as taught in the prior art. The financial benefits provided by the present invention also decrease the cost of food production thereby cutting the cost of food to the consumer.
The vaccine composition of the present invention does not involve administration of hormones such as anabolic steroids or growth hormone, neurotransmitter agonists such as beta adrenergic agonists, or genetic alteration of animals, so the consumer concerns regarding the effects of these treatments on lean meat are avoided.
Another aspect of the present invention is the reduction or elimination of a species of microorganism from the gut of an animal before slaughter. For example, if one desires to reduce the population of E. coli from the gut of an animal before slaughter, a variation of the vaccine composition of the present invention could be used to achieve this purpose. Cows destined for the slaughterhouse are vaccinated 4 to 6 months before sacrifice with a variation of the vaccine composition containing higher amounts E. coli at a dose of approximately 10 mis at approximately 10 11 bacteria per ml. The relative proportions and components of the vaccine composition may be selected based on the animal selected or the microorganisms prevalent in a particular geographical area or animal. Additional selection of the amounts of other bacteria may occur to decrease the relative percentage of a particular microorganism in the gastrointestinal tract of a selected animal. The present invention is particularly useful for reducing the gastrointestinal content of microorganisms in livestock, especially cattle, cows, sheep, goats, buffalo and pigs; and birds such as chickens, turkeys, hens, doves, and quail.
To reduce the population of bacteria in the gut of an animal, vaccinations of cows may occur annually starting at 6 months of age. Pigs, sheep, and goats could be injected annually after attaining approximately 9 to 11 kg of body weight. Chickens and turkeys could be injected 3 weeks after birth and twice a year thereafter. It is contemplated that other administration schedules may be used. This treatment would greatly reduce or eliminate a selected species or group of species of microorganisms from the gut of an animal before slaughter, thereby decreasing bacterial contamination of the animal products, especially meat, reducing the costs of producing products low in bacterial contamination and decreasing the risks of food poisoning.
Occasionally it is desirable to reduce or eliminate a species of microorganism from the gastrointestinal tract of humans, especially if imbalances in the relative proportions of enteric bacteria produce distress, upset or infection. For example, if one desires to reduce the population of E. coli in the gastrointestinal tract of a human, a variation of the vaccine composition of the present invention could be used to achieve this purpose. Such a treatment might include a vaccine containing an enrichment of formalin-killed E. coli relative to the other antigenic bacteria.
Composition and Treatment for Bacterial Infections and Diarrhea in Humans and Animals:
The Serum Composition
The present invention provides a method of treating diarrhea by administering to an animal or human, preferably in a single injection, an antidiarrheal serum obtained from cows or other animals previously injected with a composition comprising a mixture of non heat-killed bacteria selected from Escherichia sp, Pseudomonas sp, Salmonella sp and Aerobacter sp. In a desirable
embodiment, the non heat-killed bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis and Aerobacter aerogenes. In one embodiment, the bacteria are killed with formalin. Optimally, the antidiarrheal composition of the present invention comprises bacteria that are selected from the geographical area in which the vaccine composition is to be used. The composition is optionally mixed with an adjuvant and administered to the animal. The adjuvant can be liquid Vaseline, lanolin, and/or vegetable oil although other adjuvants are within the scope of the present invention. Alternatively, the vaccine composition may be administered in the from of microspheres, such as polylactide and polyglycolide microspheres.
The amount of bacteria used in immunizing the animal from which the serum is to be extracted generally is between approximately 10 8 and 10 12 per ml, although higher or lower concentrations may be used. The optional volume of the vaccine for each injection will vary defending upon the species of animal to be vaccinated. For example, for cattie the optimal volume of the vaccine for administration to the cow is between approximately 5 mis and 15 mis of inoculum having 10 8 and 10 12 bacteria per ml. For chickens the optimal volume of the vaccine for administration to the chick is between approximately 0.05 mis and 2 mis of inoculum having 10 8 and 10 12 bacteria per ml. The preferred animal for immunization and subsequent collection of serum is the cow. It has been determined that serum from a cow causes fewer side effects in recipients when administered to other species including humans.
The present invention includes an antidiarrheal composition comprising serum from an animal immunized with formalin-killed bacterial cultures pathogenic to an infant. This serum contains antibodies useful for treating the intestinal infections induced by these bacteria in infants, young children and trn* elderly and is administered by injection. This invention is a m dicament enabling successful treatment of enterobacterial
infection without antibiotics, thus avoiding the cost drawbacks and risks associated with antibiotic administration.
Typically, the animal is immunized with an antigen mixture of non heat-killed bacteria, such as formalin-killed bacteria, selected from Escherichia coli, Pseudomona aeruginosa,
Salmonella enteritidis and Aerobacter aerogenes, combined with an adjuvant containing liquid Vaseline, lanolin, and vegetable oil. The antigen mixture is typically injected 3 times into an adult animal over a 30 day period. The number of injections and the timing of the injections can vary. At approximately 45 days after the third administration of the antigen mixture, blood is collected from the animal and serum is prepared from the blood using standard methods, well known to those of ordinary skill in the art. Typically, the blood is allowed to clot and serum is collected either by compression of the clot or by centrifugation. The serum is collected, extracted, mixed with 0.5% phenol, and stored. Alternatively, the vaccine composition may be administered in the from of microspheres, such as polylactide and polyglycolide microspheres, and blood collection may occur at later dates.
In the antigen mixture that is used to vaccinate the animals, it is advantageous to use the bacteria selected from Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp., desirably Escherichia coli, Pseudomona aeruginosa, Salmonella enteritidis and Aerobacter aerogenes responsible for intestinal infections of infants and young children. These bacteria are described generally in the following references: Kaufmann, F., "The bacteriology of Enterobacteriaceae," publisher Munsksgaard, Copenhagen 1966; Edwards, P.R. and W. H. Ewing, "Identification of Enterobacteriaceae" , 3rd Edition,
Burgess publisher, Minneapolis 1972; and Le Minor, L. "Laboratory diagnostic of Gram negative I bacilli, " Enterobacteria, 4th Edition, published by La Tourelle, St. Mandre 1972.
Thus, according to the invention, the composition is a serum preferably obtained from a cow immunized against a mixture of non heat-killed bacteria Escherichia sp., Pseudomonas sp., Salmonella sp., and Aerobacter sp., preferably formalin- killed Escherichia coli, Pseudomona aeruginosa, Salmonella enteritidis and Aerobacter aerogenes, optionally mixed with an adjuvant. Immunization of a cow can be effected in accordance with the general techniques of immunization well known to those of ordinary skill in the art.
Composition and Treatment for Bacterial Infections and Diarrhea in Humans and Animals:
Treatment with the Serum Composition
The new serum composition of the invention can be administered orally or through a single subcutaneous, intravascular or intramuscular injection. For neonates, a desirable average dose is approximately 0.1-1.0 ml/kg. For infants and the elderly, a desirable average dose is between approximately 0.1-0.2 ml/kg. The doses to administer to neonates and infants, as well as other humans such as adults and the elderly will vary from individual to individual. Consequently, a medical provider may elect to administer a low dosage to a particular patient initially, followed by one or more additional administrations. The dose to be provided to animals through subcutaneous, intramuscular, or intravascular injection may vary depending upon the type of animal, its individual characteristics and its condition of health.
The dose of the serum from hyperimmunized cows to be injected into animals subcutaneously, intravascularly, or intramuscularly is tailored to the animal's species, age and body weight. A single injection is the preferred mode of treatment, although some types of infections and some animals may require additional injections.
Although the use of the serum from cows is the preferred embodiment of the invention, some recipient species may respond better to serum produced in the same species. Accordingly, for these cases, serum is produced in the same species as the recipient animal using a composition of the four bacteria selected for maximal compatibility with the animal to be immunized.
This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.
EXAMPLE I
Preparation of the Antigen
The preparation of the formulation is as follows: 10 Roux flasks, 1,000 ml capacity each, with 1% dextrose nutritive agar are autoclaved at about 121 °C for 30 minutes. Seven test tubes containing nutritive agar are seeded with a selected bacteria from a strain of Escherichia coli, one test tube with Pseudomonas aeruginosa (pyocyanic bacillus), one test tube with Salmonella enteritidis, and one test tube with Aerobacter aerogenes. The tubes are incubated at approximately 37°C for 24 hours. Each test tube of culture is washed with 10 ml of sterile physiological solution until all colonies are washed loose. Each Roux flask is inoculated with the wash of each test tube of E. coli, P. aeruginosa, S. enteritidis, and A. aerogenes. The Roux flasks are incubated at approximately 37°C for 36 hours, with the agar in the internal superior surface of the flasks. The agar surface is washed using the liquid contained in the bottom part of each Roux flask and these mixtures are saved in a sterile container. Next,
each Roux flask is washed with 45 ml of sterile physiological solution until all the colonies are washed loose. These mixtures are collected in the same sterile container. Formalin is added to the sterile container in a proportion of 1% of the total collected volume. Flasks are incubated at approximately 37°C for 24 hours or at room temperature for 72 hours.
EXAMPLE II
Preparation of the Adjuvant
The components of the adjuvant are 400 ml of liquid Vaseline, 400 ml of vegetable oil and 200 gm of anhydrous lanolin (Sigma Chemical Co., St. Louis MO) (ratio of 2:2:1, respectively). The mixture is melted in a water bath, boiled for 30 minutes, and slowly cooled at room temperature. Ratios of adjuvant components may be adjusted for application to different animals.
EXAMPLE m
Preparation of the Vaccine for Injection
Equal parts of the antigen and the adjuvant are admixed so that the bacterial count is approximately 1 x 10 11 per 10 ml. The mixture remains undisturbed at 22 to 26°C for 72 hours. This formulation tends to sediment in layers especially at temperatures lower that 22 to 26 β C. For injection into cattle, 10 mis of the vaccine are injected subcutaneously.
EXAMPLE IV
Antidiarrheal Immune Serum
The antidiarrheal immune serum of the invention is obtained after hyperimmunizing a cow with an antigen and adjuvant. The antigen composition comprises a mixture of non heat-killed, preferably formalin-killed bacteria, selected from Escherichia coli, Pseudomona aeruginosa, Salmonella enteritidis
and Aerobacter aerogenes, combined with an adjuvant containing Vaseline, lanolin, and vegetable oil.
The antigen is administered in 3 injections at 1, 15 and 30 days in 10, 15, and 20 ml, respectively, to adult cows no less than 18 months old. Approximately 45 days after the third administration, 1,000 ml of blood per 100 kg body weight is removed from the jugular vein. Booster injections are performed approximately three times a year with blood removal 35 to 45 days after each injection at 1,000 ml/100 kg body weight. The serum can be obtained following formation of a clot at 4°C - 24°C and compression, or by centrifugation at 1,000 x g. The serum is extracted with techniques familiar to those skilled in the art. The serum may be stored with 0.5% phenol and bottled in 30 ml flasks and stored at -90°C to 4°C.
EXAMPLE V
Antidiarrheic Immune Serum for Treatment of Humans, Especially Neonates, Infants and the Elderly
The serum described in Example 1 is injected intramuscularly, intravenously, or subcutaneously into neonates, infants, and elderly persons with diarrhea based on their body weight and the titer of the serum.
A 70 kg man with diarrhea is injected with 5.0 ml of the serum intramuscularly in the gluteal region and with 9.0 ml of the serum intravenously.
EXAMPLE VI
Method of Treating Diarrhea in Cows
A new born calf was infected at two days after birth with a 5 ml volume of a live bacterial culture containing 10 7 bacteria/ml, where the type and number of bacteria in culture were in the same proportion as was used to prepare the antigen,
27
which in turn was used to make the antidiarrheal serum of the invention. The infected calf subsequently developed diarrhea by day five. Five day-old calves suffering from diarrhea were treated by administering a single injection of 50 mis of the antidiarrheal serum of Example IV. Generally the calves were cured of diarrhea in approximately 6 to 12 hours.
It should be understood, of course, that the foregoing relates only to specific examples of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the invention as set forth in the appended claims.