WO2004052379A1

WO2004052379A1 - Antidiarrheal composition

Info

Publication number: WO2004052379A1
Application number: PCT/JP2003/015540
Authority: WO
Inventors: Yoshikatsu Kodama; Hideaki Yokoyama
Original assignee: Ghen Corporation
Priority date: 2002-12-11
Filing date: 2003-12-04
Publication date: 2004-06-24
Also published as: JP2004189672A; TW200423950A; AU2003289181A1

Abstract

It is intended to provide a composition efficacious in preventing and treating diarrhea which can prevent the onset of diarrhea and relieve symptoms characteristic to diarrhea such as loose bowels, vomiting and body weight loss while causing no problem of the appearance of tolerant bacteria. Namely, an antidiarrheal composition which contains an avian egg laid by a bird having been immunized with a diarrheal pathogen or an antigen originating in a diarrheal pathogen or a processed product thereof and a viable bacterial preparation.

Description

Description Antidiarrhea Composition Technical Field

The present invention relates to an antidiarrheal composition comprising an egg produced by birds or a processed product thereof and a viable agent, an antidiarrheal pharmaceutical composition containing the composition, food and feed, and an avian product. The present invention relates to a method for preventing and treating diarrhea in livestock and the like by administering an egg or a processed product thereof and a viable agent. Background art

Diarrhea is one of the most common diseases in humans and livestock, but it often causes dehydration and other complications in young individuals, especially in the suckling period, often leading to serious situations. Causes of diarrhea can be divided into those that are infectious due to viruses and bacteria, and those that are non-infectious due to the environment, stress, food, etc. It often causes great damage.

Viruses and bacteria are the major pathogens of infectious diarrhea. It develops when these pathogens enter the intestinal tract with food or beverages orally and colonize and grow in the intestinal mucosa. In addition, it is not uncommon for two or more pathogens to be combined and make symptoms more serious.

As pathogens of viral diarrhea, rotavirus, parvovirus, coronavirus, norwalk virus, force ricivirus, adenoinoles, and astrovirus are known. The pathogens of bacterial diarrhea include Shigella, Salmonella, pathogenic Escherichia coli, Toxigenic Escherichia coli, Vibrio parahaemolyticus, Vibrio parahaemolyticus, Yersinia, Staphylococcus aureus, Pseudomonas aeruginosa, and Campylobacter, Bacteria belonging to the genus Lycopacter and Clostridium are known.

Various diarrhea caused by the above pathogens are diseases that cause severe vomiting and diarrhea in each animal. Enteritis, in particular, may cause vomiting at first, followed by diarrhea within a few days, and sometimes bloody stools, with loss of appetite, loss of energy, rapid dehydration, and increased body temperature. Some of these diarrhea In contrast, vaccines are now in widespread use.

However, such diarrhea vaccines have had problems with their protective effects. It is important how to produce IgA antibodies to protect against intestinal infections, but inactivated vaccines currently in use produce IgG antibodies when injected intramuscularly. Production of IgA antibodies in the intestine Since the amount is small, it is difficult to obtain the desired effect. In contrast, when a live vaccine is administered orally, the production of IgA antibodies is high and a protective effect is also observed, so the live vaccine is mainly used against viral diarrhea, but the effect is sufficient. That's not true. In the case of bacteria, few live vaccines are recognized due to the problem of reversion to pathogenicity. On the other hand, antibiotics are also used as a treatment for diarrhea, but in the case of bacterial diarrhea, there is a problem that resistant bacteria are generated by the use of antibiotics. In addition, antibiotics added especially to livestock feed to promote growth and prevent diarrhea are also problematic in terms of environmental pollution.

On the other hand, unlike a vaccine, an egg egg antibody is known as an antibody suitable for oral administration. The pharmaceutical composition using hen egg antibody includes the 987P, K88 and K99 antigens of enterotoxigenic Escherichia coli causing porcine colibacillosis, and the enterotoxigenic Escherichia coli causing causative colibacillosis. Any of the K99 antigens, including a polyclonal antibody specific to the antigen, obtained by immunizing chickens with one or more antigens and recovering antibodies from at least the yolk-containing portion of the eggs produced by these immunized chickens Oral prophylactic and therapeutic agents for colibacillosis are known (Patent No. 2034005). Also known are food-poisoning-bacteria-inhibiting materials that contain whole eggs, egg yolk or antibody-containing fractions of eggs produced after obtaining immunity by chickens previously inoculated with food-poisoning bacteria, and that contain antibodies specific to the food-poisoning bacteria. (Patent No. 2615673).

Further, as a pharmaceutical composition for canine parvovirus infection, an anti-parvovirus infection composition containing an antibody obtained from the egg yolk of chicken immunized with canine parvovirus is known (Japanese Patent Laid-Open No. 8-259462). ) In addition, as a pharmaceutical composition for swine epidemic diarrhea virus infection, milk collected from cows immunized with swine epidemic diarrhea virus or milk components thereof and chicken egg yolk or chicken egg antibody collected from chickens immunized with the virus There is known a pharmaceutical composition containing the above (JP-A-10-265393). In addition, it is a feed composition effective against non-infectious diarrhea and infectious diarrhea in domestic animals, poultry and pets, and is a specific antibody against tannins and infectious microorganisms or toxins produced by these. When A combination of these is known (Japanese Patent Laid-Open No. 7-066754).

On the other hand, there are many literatures on the usefulness of live bacteria, and reports that live bacteria are effective in treating simple diarrhea in animals. In addition, immunization of chickens with enterohemorrhagic Escherichia coli cells or toxylated verotoxin as an antigen, and an antibody obtained from the eggs produced by the chickens, and one or more live bacteria agents, contain as active ingredients, A prophylactic and therapeutic agent for enterohemorrhagic E. coli infection is known (Japanese Patent Laid-Open No. 10-298105).

However, it has excellent preventive effects against various diarrhea caused by bacteria and viruses, and has excellent therapeutic effects by reducing the symptoms of diarrhea such as diarrhea, vomiting and weight loss. Things were not known. Disclosure of the invention

The object of the present invention is to prevent the onset of diarrhea and to improve the symptoms such as diarrhea, vomiting and weight loss that are characteristic of diarrhea, and to prevent and treat diarrhea, which is free from the occurrence of resistant bacteria. It is to provide a composition.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have administered an animal a composition containing an egg obtained by immunizing birds with a diarrhea pathogen as an antigen, or a processed product thereof and a viable agent. As a result, the inventors have found that the above problems can be solved, and have completed the present invention. Antibodies contained in eggs or processed products obtained by immunizing birds with diarrhea pathogens as antigens are thought to eliminate their pathogenicity by attaching to the pathogens and neutralizing the pathogens. Alternatively, the pathogenicity is thought to be eliminated by adhering to the cell adhesion factor of the pathogen and preventing the pathogen from attaching to the intestinal cells. Furthermore, administration of a live bacterial agent improves the intestinal microflora, and at the same time, the above-mentioned antibody acts more effectively and exhibits an excellent effect in preventing and treating diarrhea. In addition, by mixing and administering the viable agent and the anti-diarrhea antibody of the present invention, an excellent synergistic effect can be achieved with a small amount of antibody administration.

The present invention relates to an anti-diarrhea composition comprising an egg produced by birds immunized with a diarrhea pathogen or an antigen derived from the diarrhea pathogen or a processed product thereof, and a viable agent.

The present invention also relates to the above composition wherein the diarrhea pathogen is selected from the group consisting of viruses, bacteria and protozoa.

The present invention also relates to the composition according to any one of the above, wherein the viable agent is a lactic acid bacterium. The present invention also relates to an antidiarrheal pharmaceutical composition comprising any of the compositions described above.

The present invention also relates to a feed comprising any of the compositions described above.

The present invention also relates to a food product comprising any of the compositions described above.

The present invention also prevents or treats diarrhea by administering an egg produced by birds immunized with a diarrhea pathogen or an antigen derived from a diarrhea pathogen or a processed product thereof and a viable agent to an animal. Regarding the method.

The present invention also relates to the above-mentioned treatment method, wherein the animal is an animal other than human.

In the present invention, a bird immunized with a diarrhea pathogen or an antigen derived from a diarrhea pathogen is not particularly limited, and examples thereof include chickens and pupae. From the viewpoint of mass production of the antibody, it is preferable to use chickens, particularly spawning species.

In the present invention, diarrhea has a meaning usually used in the art, that is, a symptom in which liquid or semi-solid stool is abnormally repeatedly excreted from the intestine. In the present invention, the diarrhea pathogen means microorganisms such as bacteria, fungi, protozoa and viruses that cause diarrhea in animals. Diarrhea pathogens include, but are not limited to, rotavirus, reovirus, parvowi / res, coronawinoles, enterowinoles, norwalkwinores, force ricinowinoles, adenowinoles, astrowinores , Viruses such as Hismonas vinores and influenza virus; Shigella (such as Shigella), Salmonella (such as Salmonella dublin, Salmonella typhimurium), Escherichia (such as pathogenic Escherichia coli and Toxigenic Escherichia coli), Vibrio (Such as Vibrio cholerae and Vibrio parahaemolyticus), Yersinia (such as Yersinia enterocol itica), Staphylococcus (such as Staphylococcus aureus), Syudomonas (such as Pseudomonas aeruginosa), Campylobacter (such as Campylobacter fetus), Helicopter Connector genus, black stridium genus (Clost It belongs to the genus Serpul ina hyodysenteriae, such as ridium perfringens, Sermoninus genus (Aeromonas hydrophila), Fremonas genus (such as Plesiomonas shigelloides), and Bacillus genus (such as Baci llus cereus). Bacteria; Giardia genus, Eimeria scabra, Cryptosporidium genus, Toxoplasma genus, Trichomonas genus, Penta tricomonas This includes protozoa such as the genus (Pentatrichomonas), the genus Isospora, the genus Entamoeba, the genus Balantidium and the coccidium.

In the present invention, an antigen derived from a diarrhea pathogen means a peptide composed of a part of the components of the diarrhea pathogen as described above, for example, a surface antigen of the diarrhea pathogen, an outer membrane protein, Includes adhesion factors, invasion factors, toxins, metabolites and virulence factors, including those consisting of parts of them. In the present invention, it is preferable to use an antigen derived from such a diarrheal pathogen. Diarrhea pathogens and antigens derived therefrom are contained in cell lysates and the like, and both purified and unpurified ones can be used, but it is preferable to use purified ones. For purification of the antigen, conventional methods such as ammonium sulfate precipitation, column chromatography, and electrophoresis can be used singly or in combination of two or more. In column chromatography, one type of column may be used, or two or more types of columns may be combined. For normal column chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, affinity column chromatography, etc. are used.

More specifically, diarrhea that can be prevented and treated by the composition of the present invention, and pathogens that cause such diarrhea are not limited to these, but include the following: Can be mentioned. In cattle, viral diarrhea, mucosal disease virus (BVDV), bovine viral diarrhea, mucosal disease, bovine coronavirus, bovine coronavirus disease, bovine rotavirus, bovine rotavirus disease, bovine parvovirus, bovine parvovirus disease, Bovine enterovirus disease caused by bovine enterovirus, bovine enteropathy by Clostridium perfringens, Campylobacter fetus i, cattle campinolytic disease, Salmonella dubl in, S. typhi murium, S. enteritidis Bovine salmonellosis caused by protozoa, calf coliform diarrhea caused by protozoal protozoa, and bovine cryptosporidiosis caused by Cryptosporidium parvum. In sheep, there are sheep dysentery and sheep clostriosis due to Clostridium perfringens. In horses, there is equine rotavirus disease due to equine rotavirus. In swine, infectious gastroenteritis due to infectious gastroenteritis virus, swine epidemic diarrhea due to swine epidemic diarrhea virus, reovirus disease due to reovirus, swine colitis due to E. coli Salmonella disease due to Salmonella choleraesuis S. typhimurium ^ S. typhisuis S. derby, knee dysentery due to Serpul ina hyodysenteriae, Kelena mouth toki due to Clostridium perf ringens, 5. Yersinia enterocolitica Y. pseudotuberculosi s ί! ¾ Enorenji / ¾E _N Campylobacter mucosalis C. hyointestinal is caused by intestinal adenoma syndrome, and Eiraeria scabra E. debl iecki caused swine coccidium disease. In dogs and cats, canine parvovirus due to canine parvovirus, canine coronavirus due to canine coronavirus, feline panleukopenia due to feline panleukopenia virus (parvovirus), feline leukemia due to feline leukemia virus, Campylobacter jejuni C. col i dog campylobacterosis, Isospora cams I. ohioensi s. Iel is. Rivolta, Sarcocyst is Eimeria, dogs caused by Toxoplasma 'cat coccyzym disease, dogs caused by Toxoplasma gondi i' cat toxoplasmosis, Giardia canis, G Dogs by cati · Cat giardiasis, Pentatrichoraonas homini s Dogs by cat · Trichomonas cats)) Dogs by Cryptosporidium parvum · Dogs by cat dipspomyosomiasis, dogs by Entamoeba histolyti ca · Cat amoebosis, Balantidium col i have dog balantidium disease. In birds, duck virus enterit is virus duck virus decubitus enteritis, turkey coronavirus turkey coronavirus enteritis, mouth rotavirus avian rotavirus disease, hemorrhagic enteritis virus turkey hemorrhagic enteritis, chicken parvovirus chicken fowl Viral disease, turkey-ost-mouth virus disease caused by turkey-fast mouth virus, birds caused by Yersinia pseudotuberculosi s individual tuberculosis, chick-white dance by Salmonella pol lorum! ], By Salmonella ga 丄丄 lnarum | § Chicken Paratyphoid and Chicken Sanoremonella by Tefs, salmonella typhimurium _N S. enterit idis, S. sofia, S. thompson, S. b 丄 ockley, S. heidelberg _N S. infanti s , Turkey arizonosis due to Salmonel la arizonae, avian campylobacteriosis due to Campylobacter jejuni, Clostridium col inum C. perfringens _N C. septicum C. botul inum 禽 'fowl cross lysium, Histomonas meleagridi s Examples include stomonas disease and chicken cryptosporidiosis caused by Cryptosporidium meleagridi s, C. bai leyi. In humans, rotavirosis caused by rotavirus, sanoremonosis caused by Salmonella typhimurium S. enteritidi s S. typhi S. paratyphi, red fox caused by Shigella sonnei, Vibrio parahaemolyticus ^ V. raimicus V. Vibriosis caused by fluvial is, V. hol li sae, cholera caused by Vibrio cholerae, Aeromonas hydrophi lai Ayona Monas ¾E, Plesiomonas shigel loides i Plesiomonas;) Positive, Uenores in Clostridium perfringens;) Positive, Baci l Celebosis due to lus cereus, Staphylococcus aureus due to Staphylococcus aureus, rotavirus due to rotavirus, enterovirus due to enterovirus, adenovirus due to adenovirus, influenza due to influenza virus, small sphere due to norwalk virus Viral disease, Campylobacter jejuni, etc., E. coli-induced E. coli disease, Yers inia enterocol itica, Y. pseudotuberculosis ί enoreshini / £ Ε, candy ~ ζ and so on.

When immunizing birds with diarrhea pathogens as described above, adjuvants such as Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) can be used as necessary. Immunization is mainly performed by injecting intravenously, subcutaneously, intramuscularly, or intraperitoneally, but can also be performed by oral administration, instillation, instillation, and the like. The interval between immunizations is not particularly limited, and immunization is performed 1 to 10 times at intervals of several days to several weeks. Usually, antibodies that react specifically with the administered antigen are obtained in eggs, especially yolk, within a few weeks after the first immunization. The amount of antigen to be inoculated is the amount of protein, and 0.01 ~:! Omg is preferably used.

Antibody titers in eggs can be measured using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, agglutination antibody method, neutralization reaction, etc. The change in antibody titer can be traced by measuring. Usually, a high antibody titer can be obtained over about 4 months. If a decrease in the antibody titer is observed after immunization, the antibody titer can be kept high by performing additional immunizations at appropriate intervals. In the present invention, an anti-diarrhea composition, a pharmaceutical composition, a livestock feed, a food, and the like are produced using the chicken eggs immunized as described above and processed products thereof. In the present invention, the processed egg is not particularly limited as long as it contains an antibody against the diarrheal pathogen used for immunization of birds as an antigen. For example, whole eggs of immunized eggs, egg yolk and egg white, these Examples include egg liquids and solutions, and extracts obtained by extracting egg liquids with propanol or chloroform. From the viewpoint of the amount of antibody contained, it is preferable to contain an egg yolk component. Also included are those powdered by spray drying or freeze drying. Also, the egg yolk lipid component is removed from the egg yolk by a method using an organic solvent such as hydroxypropinolemethyl senorelose phthalate, polyethylene glycol, dextran sulfate, propanol, ethanol and hexane, and then pulverized. included. Such powders in paste form or liquid form are also included. Further, in the present invention, the processed egg product is purified from the egg by a known method such as ammonium sulfate salting out, sodium sulfate salting out, low temperature ethanol precipitation, ion exchange chromatography, gel filtration, affinity chromatography, etc. It also means the antibody itself. In the present invention, the antibody thus prepared is referred to as a chicken egg antibody. In order to enhance the preservability of the processed product, it is preferable to sterilize the sterilized whole egg yolk or egg yolk liquor by spray drying or freeze drying.

In the present invention, when an animal suffering from diarrhea is given a viable preparation together with the egg and egg processed product obtained as described above, symptoms such as diarrhea, vomiting and a decrease in body weight associated therewith, etc. It was found that the symptoms were effectively reduced.

In the present invention, the viable agent means living bacteria, yeast and fungi, and can be produced by a usual method in this technical field. Bacteria as viable agents preferably used in the present invention are not particularly limited. For example, lactic acid bacteria such as bacteria belonging to the genus Lactobacillus and Streptococcus, genus Clostridium, and Bifidobacterium Useful enteric bacteria such as bacteria, and other bacteria belonging to the genus Enterococcus, Lactococcus and Bacillus. Yeasts as viable bacteria include yeasts belonging to the genus Candida and Pichia. Examples of the fungus as the viable agent include, but are not limited to, fungi belonging to the genus Aspergillus and Saccharomyces. In the present invention, in particular, a bacterium belonging to the genus Lactobacillus, Streptococcus, Lactococcus, Bifidobacterium, Clostridium, Bacillus, Enterococcus, yeast belonging to Candida, Aspergillus, Saccharomyces is used. Is preferred. These live bacteria agents may be used alone or in combination of two or more.

More preferred viable agents for use in the present invention include, but are not limited to, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus. Salinocris, Lactobacillus gasseri, Lactobacillus fermentum, Lactono Chinoles helveticas, lac tono chills' yugurti, streptococca S. Thermophilus, Lactococcus Lactis, Bifidopacterium bifidum, Bifui debactericum 'Breve, Bifui dpacterium' Infan Taste, Bifid debacterum longum, Bifudodacterium gum Bifidobacterium thermofilms, Clostridium 'Buttilium', Bacillus cereus, Enterococcus fuecaris, Enterococcus fuecium, Candida 'Albicans, Aspergillus' Niger, Aspergillus oryzae and Saccharomyces. For example, Lactobacillus acidophilus and Bifidobacterium bifidum and Enterococcus' Fuecarris, Ratatobacillus. Sarivarius and Bifidobacterium thermofilm, Fu, Streptococcus thermofinoles and Bifidobacterium cyudulongum It is preferred to use. For diarrhea caused by viruses, especially rotavirus and parvovirus, it is preferable to use useful enterobacteria, particularly bacteria belonging to the genus Bifidopacteria, as diarrhea caused by bacteria. For this, it is preferable to use a bacterium belonging to the genus Ratatobacillus as a viable agent. In particular, for diarrhea caused by bacteria belonging to the genus Escherichia, it is preferable to use bacteria belonging to the genus Bacillus as viable agents, and for diarrhea caused by Salmonella bacteria as infectious agents It is preferable to use endophytic bacteria, especially Bifidopacteria bacteria, and for diarrhea caused by Clostridium bacteria, it is preferable to use lactic acid bacteria, especially Lactobacillus bacteria, as a viable agent. Yes. Furthermore, for diarrhea caused by protozoa, particularly protozoa of the genus Cryptosporidium, it is preferable to use lactic acid bacteria, particularly bacteria of the genus Lactobacillus as a viable agent.

In the anti-diarrhea composition of the present invention, the mixing ratio of eggs produced by birds immunized with diarrhea pathogens or processed products thereof and viable agents is not particularly limited. Usually, the antibody lg contained in eggs It is used in a ratio of 10 to ¹¹ living bacteria, preferably 10 ⁴ to 10 ⁹ living bacteria for the antibody lg.

The thus obtained anti-diarrhea composition is usually 0.001 to 100% by weight, preferably 0.01 to 10% by weight of a mixture of the above-mentioned egg or a processed product thereof and a viable agent. It can be in the form of a solution, powder, granule, tablet or paste. And since this anti-diarrhea composition has the effect of preventing and treating diarrhea in animals, It can be used as an antidiarrheal pharmaceutical composition. In addition, by containing an anti-diarrhea composition in feed and food for domestic animals, it has a preventive effect on diarrhea even in animals not suffering from diarrhea. The antidiarrheal composition can be added to the pharmaceutical composition, livestock feed and food in a proportion of usually 0.001 to 100% by weight, preferably 0.01 to 10% by weight.

When a pharmaceutical composition containing the anti-diarrhea composition of the present invention is produced, it can be administered orally in the form of tablets, granules, powders, force capsules, liquids, etc., as is or together with conventional additives, by a conventional formulation method. Preparation. Examples of additives include excipients, binders, disintegrants, lubricants, antioxidants, coloring agents, and corrigents, which are used as necessary. In order to prevent digestion and degradation in the stomach and to release slowly so that it can act for a long time at the small intestine site, it can be coated with a known retarder or the like. Excipients include, for example, strength sodium oxymethyl cellulose, agar, light anhydrous caustic acid, gelatin, crystalline cellulose, sorbitol, talc, dextrin, starch, lactose, birch sugar, mannitol, aluminate metasilicate Magnesium, calcium hydrogen phosphate, etc. can be used. Binders include, for example, gum arabic, sodium alginate, ethanol, ethylsenole mouthpiece, sodium caseinate, canoleboxymethinole cellulose sodium, agar, purified water, gelatin, starch, tragacanth, lactose, hydroxy Examples include senorelose, hydroxymethylenorescenellose, hydroxypropenorescenellose, and polybylpyrrolidone. Examples of the disintegrant include carboxymethylenoresenololose, canolepoxymethinoresenorelose sodium, canolepoxymethinoresenorelose calcium, crystalline senorelose, starch, hydroxypropyl starch and the like. Examples of the lubricant include stearic acid, calcium stearate, magnesium stearate, talc, hydrogenated oil, sucrose fatty acid ester, waxes and the like. Examples of the antioxidant include tocofurol, ester gallate, dibutylhydroxyltoluene (BHT), butylhydroxydisole (BHA), and ascorbic acid. In addition, other additives and drugs as required, such as antacids (sodium bicarbonate, magnesium carbonate, precipitated calcium carbonate, synthetic hydrosite, etc.), gastric mucosa protective agents (synthetic aluminum silicate, sucralfate, copper) Chlorophyllin sodium etc.) may be added.

An antidiarrheal composition of the present invention, and a pharmaceutical composition containing the composition, food and feed are provided. The target animal is not particularly limited as long as it can cause diarrhea due to the above-mentioned diarrhea pathogen, and examples thereof include mammals and birds. The anti-diarrheal composition of the present invention is suitably used for mammals such as humans, lions, pigs, horses, hidges and goats, and birds such as chickens and rabbits.

This specification includes the contents described in the specification of Japanese Patent Application No. 2 0 2-3 5 9 6 3 2 which is the basis of the priority of the present application. BEST MODE FOR CARRYING OUT THE INVENTION

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated still in detail, this invention is not limited to this Example at all.

[Example 1]

Ratatobacillus acidophilus strain JCM-1132 as a viable agent was cultivated in MRS medium, collected, and lyophilized to prepare 1 X 1C / g powder. Bifidobacterium thermofilm JCM-1207 as a viable cell agent was cultured in MRS medium, collected, and lyophilized to produce 1 X 10 ^1Q / g powder. Bifidobacterium-cyudron gum JCM-1205 strain as a viable fungus was cultured in MRS medium, collected and freeze-dried to prepare 1 × 10 ^1Q / g powder. Bacillus coagulans strain JCM-2257 as a viable cell agent was cultured on a normal agar medium, collected, and lyophilized. IX 10 ¹ A piece / g powder was prepared.

[Example 2]

Bovine rotavirus Shimane strains were cultured in red-haired monkey kidney cells (MA-104), and virus particles were collected by ultracentrifugation. This virus 10 ⁹ TCID ₅ . The first immunization was carried out by emulsifying a solution containing Freund's adjuvant and Freund's adjuvant and injecting lml into left and right pectoral muscles of a 12-week-old hen. Similarly, a second immunization was performed 6 weeks later. Two weeks after the second immunization, the antibody titer (Arch. Virol., 69: 49-60, 1981) measured by neutralizing the anti-rotavirus antibody in the blood of this chicken was 6400 times. . The antibody titer of eggs laid by this chicken was 6400 times. This antibody titer lasted for 4 months. The eggs were collected. Whole egg powder was prepared by spray drying. The antibody titer of this powder was 6400 times.

Using the feed mixed with this anti-bovine rotavirus antibody and a viable agent, the effect on experimental bovine rotavirus infection (Archives Virology 138: 143-148, 1994) was examined. Serving The test animals should be 1-day-old newborn cows, milk containing 3200-fold antibody titer and milk containing Bifidobacterium thermofilm JCM-1207 strain IX 10 ⁶ separately or mixed. For 10 days. In addition, a mixture of 1 × 10 ⁶ viable bacterial agents and the above 1/10 or 1/100 amount of antibody was administered in the same manner. Infection of bovine rotavirus Shimane strain is IX 10 ^1Q TCID ₅ . And assigned to 6 groups with 4 animals in each group. That is, antibody alone administration group ⁽³ 200 times), probiotic alone administration group ⁽¹⁰⁶ Zg), 3200 times the antibody Namakinzai ¹⁰⁶ Zg, 320-fold, group administered by mixing with 32 times Were tested. A control group in which nothing was administered was also tested. The observation period after infection was 10 days. Antibodies are thought to protect the intestinal mucosa and prevent rotavirus from adhering to the mucosa. Probiotics are thought to improve the gut flora and improve diarrhea symptoms. As tests, daily clinical observations, virus excretion periods and weight gain measurements were performed. The results are shown in Table 1. Significant improvement was observed in the group that received a mixture of antibody and live bacteria in reducing diarrhea and vomiting, viral excretion, and weight gain. In addition, the group administered with the mixture of the viable agent and 1/100 amount of antibody showed the same or better effect than the group administered with the antibody alone. Table 1 Anti-bovine rotavirus antibodies and viable bacteria

*: P 0. 05, **: P 0.

[Example 3]

Canine parvovirus strain Cp83016 was cultured in feline kidney cells (CRFK) and virus particles were collected by ultracentrifugation. This virus 10 ⁹ TCID ₅ . The first immunization was carried out by emulsifying the solution containing Freund's and Freund's adjuvant and injecting 1 ml each into the left and right pectoral muscles of a 12-week-old hen. Similarly, a second immunization was performed 6 weeks later. Second immunization 2 weeks later, the antibody titer (J. Vet. Med. Sci., 60: 973-974, 1998) measured by neutralization of anti-parvovirus antibody in the chicken blood was 50,000 times. It was. The antibody titer of eggs laid by this chicken was 50,000 times. This antibody titer lasted for 4 months. The eggs were collected and whole egg powder was made by spray drying. The antibody titer of this powder was 51,200 times.

Using this feed mixed with anti-canine parvovirus antibody and live bacteria, the effect on experimental infection with canine parvovirus (J. Virology, 68: 4506-4513, 1994) was examined. The test animals are 3 week old dogs, milk containing 25,600 times antibody titer and milk containing Bifidobacterium siudron gum JCM-1205 strain IX 10 ⁶ as a viable agent, separately or mixed For 14 days. Further, a mixture of ⁶ viable agents IX 10 and the above 1/10 or 1/100 amount of antibody was administered in the same manner. The infection of the dog Parvoirs strain Cp83016 is 6 x 10 ⁸ TCID ₅ . And assigned to 6 groups of 3 in each group. That is, the antibody alone administration group (25600 times), probiotic alone administration group ⁽¹⁰⁶ Zg), Namakinzai 10 ⁶ / g 25600 times antibody, 2560-fold, was administered by mixing with 256-fold Groups were tested. A control group in which nothing was given was also tested. The observation period after infection was 14 days. Antibodies are thought to protect the intestinal mucosa and prevent parvovirus from adhering to the mucosa. Probiotics are thought to improve the gut flora and improve diarrhea symptoms. As examinations, daily clinical observations, virus excretion periods and weight gain measurements were performed. The results are shown in Table 2. An improvement effect was observed in the group administered with a mixture of antibody and live bacteria in reducing diarrhea and vomiting, viral excretion and weight gain. In addition, the group administered with the mixture of the viable agent and 1/100 amount of antibody showed the same or better effect than the group administered with the antibody alone.

Table 2 Anti-canine parvovirus antibodies and viable agents

Test group Control group Antibody Viable agent Antibody + Viable agent Antibody + Viable agent Antibody + Viable agent

25600 times 10 ⁶ 25600 times +10 ⁶ 1 2560 times +10 ⁶ 256 times +10 ⁶ Diarrhea incidence (%) 100 67 100 0 0 33 Diarrhea onset period 3 2 2 0 # 氺 0 ** 1 (曰)

Total clinical score 7 3 5 0 ** 0 ** 2 *

Infection 14 days-0. 1 0. 2 ** 0. 1 0. 3 ** 0. 2 ** 0. 2 ** Weight gain (kg)

Virus excretion 12 7 ** 10 6 ** 6 ** 7 ** Duration (days) *: P-p. 05, **: P-p.

[Example 4]

E. coli swine-derived E. coli strain 8199 (with F18-attached pili) was cultured in MINCA medium, and the cells were collected by centrifugation, and F18-attached pili were extracted by heat extraction at 60 ° C (Vet. Microbiol., 45: 281-295). 1995). The first immunization was performed by emulsifying a solution containing 0.5 mg / ml of F18-attached pili and Freund's adjuvant and injecting lml into the left and right pectoral muscles of a 12-week-old hen. . Similarly, a second immunization was performed 6 weeks later. Two weeks after the second immunization, the antibody titer measured by the agglutination antibody method of anti-F18 pilus antibody in the blood of this chicken was 2560 times. The antibody titer of eggs laid by this chicken was 2560 times. This antibody titer lasted for 4 months. The eggs were collected and whole egg powder was made by spray drying. The antibody titer of this powder was 2560 times.

Using this diet mixed with anti-F18 pilus antibody and viable agent, we investigated the effect on experimental infection of swine E. coli (J. Vet. Med. Sci., 59: 917-921, 1997). The test animals are 4-week-old weanling pigs, and 10 times the antibody titer and Bacillus coagulans JCM-1132 1X10 ⁴ / g as live bacteria are mixed separately or together in the feed. For 10 days. In addition, a mixture of 1 × 10 ⁴ live bacteria agent / g and the above 1/10 or 1/100 amount of antibody was administered in the same manner. Infection of pig derived from E. coli 8199 strain and IX 10 ¹¹ pieces, was allocation set for each group 10 animals Dzu' six groups. That is, antibody alone administration group (10-fold), probiotic alone administration group (10 ⁴ Zg), 10 times the antibody to 10 ⁴ probiotic, 1-fold, group and administered in admixture with 1-fold 0.1 Were tested. A control group in which nothing was administered was also tested. The observation period after infection was 10 days. The antibody is thought to protect the intestinal mucosa and prevent F18 fimbriae E. coli from adhering to the mucosa. Probiotics are thought to improve the gut flora and improve diarrhea symptoms. As examinations, daily clinical observations, measurement of attacking E. coli isolation rate and weight gain were performed. The results are shown in Table 3. Significant improvement was observed in the group that received the antibody and the live bacteria agent in reducing diarrhea and vomiting, reducing the isolation rate of attacking E. coli, and weight gain. In addition, the group administered with the mixture of the viable agent and 1/100 amount of antibody showed the same or better effect than the group administered with the antibody alone. Anti-pig-derived Escherichia coli antibodies and live bacteria

氺: P く 0. 05, **: P 0 0. 01

[Example 5]

Salmonel la dubl in 256 strain was cultured in Luria broth medium, and the cells were collected by centrifugation. Was first immunization by injecting each lml of a solution and the Freund adjuvant punt containing the cells 10 ^1Q pieces to the right and left pectoral muscle emulsified 12 weeks old female chickens. Similarly, a second immunization was performed 6 weeks later. Two weeks after the second immunization, the antibody titer measured by the agglutination antibody method of anti-Salmonella antibody in the blood of this chicken was 2560 times. The antibody titer of eggs laid by this chicken was 2560 times. This antibody titer lasted for 4 months. The eggs were collected and whole egg powder was made by spray drying. The antibody titer of this powder was 2560 times.

Using this diet mixed with anti-Salmonella antibody and live bacteria, the effect on bovine Salmonella experimental infection (Am. J. Vet. Res., 59: 81-85, 1998) was examined. The test animals are 1-day-old newborn cows, milk containing 2560-fold antibody titer and milk containing Bifidobacterium thermofilm JCM-1207 strain 1 X 10 ⁶ separately or The mixture was administered for 10 days. In addition, a mixture of ⁶ viable agents IX 10 and the above 1/10 or 1/100 antibody was administered in the same manner. Infection of cattle from Salmonella dubl in 256 shares and 1 X 10 ¹¹ pieces, was assigned set to 5 dogs Dzu' six groups each group. That is, antibody alone administration group (2560 times), probiotic alone administration group ⁽¹⁰⁶ Zg), 2560 times the antibody Namakinzai ¹⁰⁶ Zg, for 256 times, the group administered by mixing with 26 times Tested. A control group in which nothing was administered was also tested. The observation period after infection was 10 days. Antibodies are thought to protect the intestinal mucosa and prevent Salmonella from adhering to the mucosa. Viable bacteria Is thought to improve intestinal flora and improve diarrhea symptoms. Daily clinical observations were performed as tests. The results are shown in Table 4. In the reduction of diarrhea and vomiting, significant improvement was observed in the group that received the antibody and the live bacteria. In addition, the group administered with the mixture of the viable agent and 1/100 amount of the antibody showed the same or better effect than the group administered with the antibody alone. Anti-Salmonella antibodies and viable agents

*: P <0.05, **: P <0. [Example 6]

Clostridium perfringens strain PB6 was cultured in Brucella broth medium and the cells were collected by centrifugation. This cell IX 10 ¹ . The first immunization was performed by emulsifying the solution containing Fred / ml and Freund's adjuvant and injecting lnil into the left and right pectoral muscles of a 12-week-old hen. Similarly, a second immunization was performed 6 weeks later. Two weeks after the second immunization, the antibody titer measured by the agglutination antibody method of the antibody in the chicken blood was 640 times. The antibody titer of eggs laid by this chicken was 640 times. This antibody titer lasted for 4 months. The eggs were collected and whole egg powder was made by spray drying. The antibody titer of this powder was 640 times.

Using this anti-clostridium antibody and a viable agent, the effect on mouse clostridium experimental infection was examined. The test animals were 4-week-old BALB mice, and the antibodies of 32-fold antibody titer and Lactobacillus acidophilus JCM-1132 strain 1 X 10 ⁵ were administered separately or mixed for 3 days. . A mixture of ⁵ IX 10 viable agents and 1/10 or 1/100 amount of the antibody was administered in the same manner. Clostridium perfringens PB6 strain was infected at 1 X 10 ⁷ and assigned to 6 groups of 10 in each group. That is, antibody alone administration group (32 times), probiotic alone administration group (10 ⁵ Zg), 32 times the antibody Namakinzai 10 ⁵ / / g, three times, were mixed with 0.3 times The administered group was tested. A control group in which nothing is administered Were also tested. The observation period after infection was 14 days. Antibodies are thought to protect the gut mucosa and prevent clostridial adhesion to the mucosa. Probiotics are thought to improve the gut flora and improve diarrhea symptoms. A daily clinical observation was performed as a test. The results are shown in Table 5. In the reduction of mortality, a significant improvement was observed in the group that received the antibody and the bacterial agent. In addition, the group administered with the mixture of the viable agent and 1/100 amount of the antibody showed the same or better effect than the group administered with the antibody alone. Table 5 Anti-clostridium antibodies and viable agents

*: P 0 0. 05, **: P 0 0. 01

[Example 7]

SCID mice were orally infected with oocysts of Cryptosporidium parvum Mito strain, feces and intestinal contents were collected, and merozoid was purified. A first immunization was carried out by emulsifying a solution containing 0.5 mg / ml of this merozoid and Freund's adjuvant and injecting 1 ml each into the left and right pectoral muscles of a 12-week-old hen. Similarly, a second immunization was performed 6 weeks later. Two weeks after the second immunization, the antibody titer measured by the fluorescent antibody method of the antibody in the chicken blood was 6400 times. The antibody titer of eggs laid by this chicken was 6400 times. This antibody titer lasted for 4 months. These eggs were collected and whole egg powder was prepared by spray drying. The antibody titer of this powder was 6400 times.

Using this anti-krytosporidium antibody mixed with a viable agent, the effect on experimental infection with mouse cryptosporidium was examined. Test animals are 4-week-old SCID mice, and 20-fold antibody titer and ratatobacillus acidophilus JCM-1132 strain 1 X 10 ⁵ / g as a viable agent are administered separately or mixed for 27 days. did. Further, a mixture of the viable agent IX 10 ⁵ / g and the above 1/10 or 1/100 antibody was administered in the same manner.

The infection of Cryptosporidium parvum Mito was 1 X 10 ⁷ and was assigned to 6 groups of 6 animals in each group. That is, antibody alone administration group (20 times), live bacteria agent alone administration group (10 ⁵ Zg), live bacteria agent 10 ⁵ pieces Zg were mixed and administered 20 times, 2 times, 0.2 times About the group And tested. A control group in which nothing was administered was also tested. The observation period after infection was 27 days. Antibodies are thought to protect the intestinal mucosa and prevent Cryptosporidium from adhering to the mucous membrane. Probiotics are thought to improve the gut flora and improve diarrhea symptoms. As a test, daily clinical observations and measurement of the amount of attack bacteria excretion were conducted. The results are shown in Table 6. In the reduction of the excretion amount of the attacking bacteria, a marked improvement effect was observed in the group administered with the antibody and the live bacteria. In addition, the group administered with the mixture of the live bacterial agent and 1/100 amount of antibody showed the same or better effect than the group administered with the antibody alone or the live bacterial agent alone. Table 6 Anti-Kributospolydium antibodies and viable agents

*: Poku 0.05, **: Pku 0 The anti-diarrhea composition containing the antibody of the present invention and a viable agent was mixed with feed, food or pharmaceuticals and fed to experimentally infected animals with diarrhea pathogens. However, the group of animals administered with a mixture of live bacteria was significantly reduced in onset and weight gain than the group of animals administered with the antibody alone. In addition, even when a small amount of antibody was administered to the viable bacterial agent, synergistic effects were observed to reduce symptoms and increase body weight.

All publications, patents and patent applications used for Bow I in this specification are incorporated herein by reference in their entirety. Industrial applicability

Since the anti-diarrheal composition containing the anti-diarrhea antibody and the viable agent in the present invention acts specifically on diarrhea pathogens, it is extremely superior to vaccines or antibiotics against animal diarrhea. In addition to exerting a preventive effect, it reduces symptoms such as diarrhea, vomiting and weight loss that are characteristic of diarrhea, and can be used as an extremely effective diarrhea preventive and therapeutic agent. Moreover, there is no problem that resistant bacteria like antibiotics are generated.

Claims

The scope of the claims

1. An anti-diarrhea composition comprising an egg produced by birds immunized with a diarrhea pathogen or an antigen derived from a diarrhea pathogen or a processed product thereof, and a viable agent.

2. The composition of claim 1 wherein the diarrhea pathogen is selected from the group consisting of viruses, bacteria and protozoa.

3. The composition according to claim 1, wherein the viable agent is a lactic acid bacterium.

4. The composition according to claim 2, wherein the viable agent is a lactic acid bacterium.

5. An antidiarrheal pharmaceutical composition comprising the composition according to claim 1.

6. A feed comprising the composition according to claim 1.

7. A food comprising the composition according to claim 1.

8. A method for preventing or treating diarrhea by administering an egg produced by a bird immunized with a diarrhea pathogen or an antigen derived from the diarrhea pathogen or a processed product thereof and a viable agent to an animal.

9. The method according to claim 8, wherein the animal is an animal other than human.