JP2008044945A - Probiotics as alternative drug confronting infectious disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective alternative treating method acting on both of the humoral and cellular immunity of birds, fishes and mammalian animals infected with pathogenic microorganisms, viruses, parasites, protozoan, etc., such as bird coccidiosis, infectious diseases such as by parvovirus, etc. <P>SOLUTION: This mixed probiotics is provided by mixing Pediococcus acidilactici as a lactobacillus and Saccharomyces cerevisiae boulardii as an yeast, made as powdery states. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

Probiotics, pediococcus, pediococcus acidilactici, saccharomyces boulardii, eimeria, chickens, birds, digestive disorders, dogs, parvovirus, parvovirus

Background field

U.S. Pat. No. 5,968,569 shows a pet food with gelled starch cell stroma containing probiotics, and specifically discloses the genus Saccharomyces and the genus Pediococcus acidilactici. U.S. Pat. US Pat. No. 6,551,633 discloses a powder based on pet milk containing lactase and lactose. U.S. Pat. 5,968,569 also relates to probiotics. U.S. Pat. No. 6,780,447 discloses that it is an animal food containing sorbic acid and microorganisms (live or dead). A very wide variety of microorganisms are published, including Pediococcus acidilactici. U.S. Pat. 6,827,957 discloses a method for producing animal foods that are soft on the inside and hard shells containing probiotics on the outside, and are published on the genus Saccharomyces. U.S. Pat. 6,835,397 is Saccharomyces. boulardi and Pediococcus. An encapsulated yeast containing a wide variety of probiotics including acidic is published. US Published Patent Application No. 2003/0049240 discloses a method for treating Helicobacter infection using the genera Lactobacillus and Bifidobacterium. U.S. Published Patent Application No. 2004/0109025 discloses a method for producing particles containing live coated bacteria. US Published Patent Application No. 2004/0197352 discloses a prebiotic component that contains numerous species of microorganisms and that reduces creatine and urea nitrogen in the blood. US Published Patent Application No. 2003/0165472 publishes information on the storage and transport of microorganisms. US Published Patent Application No. 2006/0008511 discloses a process for wrapping probiotics in xanthan and chitosan mixture capsules or gelatin capsules. The above-mentioned examples that are related to the above and related methods are given as examples, suggesting that they are not always the only methods. And other methods and limits of a statement will become clear from the literature and research.

A method of making probiotic powder by mixing it with animal food. A method for treating infectious gastrointestinal symptoms in birds and mammals by using powdered probiotics or probiotics contained in animal food or animal snack foods (snack) is shown. The probiotic contained in powdered probiotics or animal foods or animal snack foods (snack) will show how to enhance the immune response to antigens in birds and mammals.

Although techniques and methods are used to explain the content of the claims in an easy-to-understand manner, the scope is not limited. While the above claims clearly state that one or more of the problems described above have been reduced or eliminated, other claims indicate that there has been an improvement over other problems.

In addition to the above explanation, the claims are further clarified by reference documents, drawings, and the following explanation.

Problems to be solved by the invention

Avian coccidiosis is a serious infectious disease that causes chicken fat, malabsorption (underutilized feed), reduced growth rate, and reduced egg production in hens (Lellehoj et al., 2004). The most significant problem of avian coccidiosis is a growth disorder such as a decrease in the rate of weight gain or, in severe cases, weight loss, causing economic damage to the poultry industry (Dalloul and Lillehoj, 2006). Drugs and live vaccines have traditionally been a method of suppressing symptoms, but alternative therapies are needed due to growing concerns about the prophylactic use of drugs and the high cost of vaccines (Dalloul and Lillehoj). , 2004). Recent probiotic studies have demonstrated that live bacteria can affect the liquid immunity of the host against rotavirus, E. coli, and Salmonella causing gastrointestinal diseases (Isolauri, E. 2003; Majamaa, et al 1995; Perdigon, et al 2001). In order for probiotics to be used as an effective alternative medicine for broilers infected with Eimeria, it is necessary to prove that they act on both liquid immunity and cellular immunity.

Dogs infected with parvoirus have severe digestive symptoms such as vomiting and bloody stool. If not treated properly, death can occur in a matter of hours. There are no antibiotics in the US to treat dogs infected with parvovirus, and the only way to destroy and recover from the virus is the dog's autoimmune function. We therefore determined that dogs infected with parvovirus are good research subjects to give probiotics.

Means for Solving the Invention

To demonstrate that Pediococcus acidilactici is effective in broilers infected with Eimeria, we The chickens were divided into chickens with and without acidilactici. The oocysts that formed tellena spores were infected. P. to broilers infected with Eimeria. In order to determine the effectiveness of acidilactici, The number of oocysts of feces collected from Eimeria-infected chickens and pre-sivots given acidilactici and the body weight of the chickens were measured.

In order to evaluate the antibody response to the Eimeria antigen EtMIC2, the gene of the Eimeria microprotein revealed and replicated at the molecular level was used (Dalloul et al, 2006). An ELISA method was used to determine antibody production from sera collected from chickens.

To determine the cellular immune response to Eimeria infection, Eimeria fed with acidilactici and Saccharomyces bouularidii was tried to grow T cells in infected chickens. Splenocytes were collected and cultured for 24 hours in the presence of ConA. The increase in cells was measured by [3H] -thymidine assay.

P. acidilactici and S. In order to prove the effectiveness of boulardiii for dogs suffering from gastrointestinal disorders, acidilactici and S. An evaluation test giving bouulardi was performed. The evaluation study was conducted jointly by four veterinarians at three animal hospitals in Sao Paulo, Brazil (Table 2). The dog's weight ranged from 2KG to 26KG and the age ranged from 1 to 15 years. Each dog suffered from a different level of gastrointestinal illness and was given one or two probiotic capsules daily, depending on body weight.

P. acidilactici and S. In order to prove the effectiveness of boulardiii for dogs suffering from digestive diseases caused by infection with pathogenic bacteria, P. acidilactici and S. gave a Booleanardi.

While typical aspects and embodiments have been discussed above, there are revisions and additions to technical methods. In the appended claims, which are set forth below, revisions, modifications, and additions are intended to be included within the scope of the essence of each article.

The invention's effect

Probiotics as an alternative to broiler parasitic infections

Probiotic toxicity testing in broilers infected with Eimeria

One to fifteen broilers one day after birth were placed in each chick as a group. Each group will receive only food as a pre-sivo, and P. Acidilactici was mixed and given. P. Acidilici was fed to the feed at concentrations of 0%, 0.01%, 0.05%, 0.1% and 0.4%. On the 10th day, 5,000 Eimeria acervulina oocysts were infected by caliber administration except for the presivo (no P. acidilactici given, no infection). The body weight was measured on days 0, 6, and 9 (dpi), and an increase in body weight was observed. To measure the number of oocysts, stool was collected on days 4, 6, and 9 after infection.

Pilot treatment is available from GraphPad Software, Inc., San Diego, California. The difference analysis was performed by GRAPHPAD INSTAT, a statistical software owned by the company. A difference was allowed if the probability was P <0.05. Average values were compared by Dunnett comparison test.

It is important how to avoid side effects such as toxicity before conducting drug discovery experiments to determine whether the drug is effective. P. Acidilactici is a natural microorganism that originally lives in the gastrointestinal tract of humans and animals, and no literature has been published that has been reported to be toxic. In broilers, P.I. To investigate whether the acidilactici is toxic, broiler or E. coli. A. acervulina infected chickens were treated with 0.01% to 0.04% P. aeruginosa. Acidilici was given. P. broiler fed with acidilactici, also P. E. acidilici given No weight loss or death was observed in any of the acervulina-infected chickens. Also, 1% to 100% P.I. acidilactici and S. et al. Although there were a group given a diet mixed with boulardii at a concentration of 0.01% and a group given a concentration of 1.0%, the EA-infected group and the ET-infected group showed considerable weight gain even during infection ( Table 1A). During the experiment, after giving probiotics, no abnormalities were observed in the main organs of the chicken (liver, heart, kidney, spleen). A 40-fold concentration range (0.01-0.4% range) for broilers infected with Eimeria, acidilactici S. et al. From the results, it can be seen that bouulardii was mixed and given in a 100-fold concentration range (0.01-1.0% range), but no systematic change was observed.

P. To demonstrate that acidilactici is effective in broilers infected with Eimeria, we The chickens were divided into chickens with and without acidilactici. The oocysts that formed tellena spores were infected.
P. In broilers given acidilactici, oocysts were reduced by 20% -40%. Similarly, E.I. A broiler infected with P. acervulina acidilactici and S. et al. 30% -50%, E. coli, given the bacterial mixture of boulardiii broilers infected with tellena acidilactici and S. et al. There was a 10% -20% reduction when given the bacterial mixture of bouulardii. These indicate that probiotics have the effect of reducing pathogens and parasitic infections.

Probiotics given to broilers infected with Eimeria stimulate a liquid immune response.

FIG. 1 shows that when no probiotics were given to each broiler, acidilactici and S. The antibody response to the EtMIC2 antigen is shown when the bacterial mixture of boulardi is mixed with 0.1% food for 21 days or mixed with 0.2% (NOR, MG0.1, MG0. 2). Birds are not infected, 5,000 E. coli at 12 days after hatching. Some are infected with tellena oocysts, and serum is sampled 10 days after infection. Bars are mean ± standard deviation (N = 3) (P <0.05).

Antibody-induced responses to ET infection were expressed in all infection groups.
Furthermore, P.I. In the birds given acidiliteri, antibodies specific to Eimeria were detected in the serum (P <0.05) much higher than in the birds not given probiotics (FIG. 1). P. This experiment demonstrates that acidilactici can induce liquid immunity against certain parasitic infections of broilers.

Tissue cell immune response: lymphocyte proliferation

FIG. 2 shows the value of concanavalin A (ConA) indicating the proliferation of chicken spleen cells in cases where normal bait only and provaotics were given 0.01%, 0.1% and 1.0%, respectively. Is shown. M is P.M. acidilactici and S. et al. It is a bacterial mixture of bouulardii. Infected with 5,000 EA (E. acervulina) or ET (E. tenella) oocysts 14 days after hatching. Splenocytes were collected and cultured for 24 hours in the presence of ConA. The increase in cells was measured by [3H] -thymidine assay. Bars are mean ± standard deviation (N = 3). (P <0.05).

The cellular immune response to Eimeria infection was determined by the proliferative response of splenocytes induced by concanavalin A (ConA) in birds fed probiotics and birds fed only normal diet.
The spleen cells of birds given 0,1% in the EA infection group had a very high growth rate compared to the group fed only with normal food and the group with different doses of probiotics (P <0 .05). Birds given 0.01% 0.1% in the ET infection group had a very high splenocyte proliferation response that stimulated ConA (P <0.05), with a growth rate of 0% at 0% It was higher than the group that received .01% (FIG. 2).

FIG. The number of oocysts in the feces of birds infected with acervulina (EA) is shown. P. at concentrations of normal bait only (REG), 0.1% (MG0.1), 0.2% (MG0.2). The chicken dung when fed with acidilactici was fed with 5,000 E.D. in units of 6-9 dpi. It is the result of measuring the number of oocysts when tenella was collected. Bars are mean ± standard deviation (N = 5 cage).

FIG. 4 shows 5,000 E. coli at 12 days after hatching. acervulina (EA) oocysts or E. coli. Chickens infected with tenella (ET) oocysts were treated with normal food only (REG) and probiotics of 0.01% (M00.1), 0.1% (M0.1), 1.0% ( M1.0) This is a result obtained by mixing with food and collecting feces after 6 to 10 days and measuring the number of oocysts in the feces. M is P.M. acidilactici and S. et al. The bacterial mixture of bouulardii is shown. Bars are mean ± standard deviation (N = 5 cages).

Probiotics as alternative medicines for dogs with gastrointestinal disorders or dogs infected with viruses

Application of probiotics to dogs with digestive disorders

A broiler infected with Eimeria acidilactici and S. et al. Due to the successful results of giving voulardi's fungus mixture probiotic MITOMAX, we next decided to conduct an evaluation study in dogs with digestive disorders. MITOMAX is a trademark of Imagiline Technology, Inc., located in Potomac, Massachusetts. A mixture of acidilactici and Saccharomyces cerevisiae boulardi (S. boulardi) is wrapped in a gelatin capsule. The evaluation study was conducted jointly by four veterinarians at three animal hospitals in Sao Paulo, Brazil (Table 2). The dog's weight ranged from 2KG to 26KG and the age ranged from 1 to 15 years. Each dog suffered from a different level of gastrointestinal illness and was given one or two probiotic capsules daily, depending on body weight. Within 14 days of giving probiotics, the dog's gastrointestinal illness recovered and there was a significant recovery in symptoms. These results indicate that probiotics are effective against dogs with digestive disorders.

Probiotics as an alternative medicine to stop blood stool in dogs infected with barbovirus

Four dogs diagnosed with parvovirus infection were dosed with Normosol®, Reglan, Cefazolin, Metronazole, Ampicillin, +/− Famotideone, but blood stool did not stop. Therefore, P.P. acidilactici and S. Boulardii fungal mix was given to 4 dogs for 2-3 days. All four dogs not only stopped bloody stool, but also stiffened stool. As a result of continuing probiotic treatment after discharge, bloody stool did not recur even after 2 weeks (Table 3).

Whether probiotics are effective for animals or humans depends on the survival status of the probiotics. Encapsulated P.I. acidilactici and S. The boulardi mixture probiotics were confirmed to have approximately the same number of viable bacteria as before storage even after 2 years storage at room temperature or 4 ° C. Broilers infected with Eimeria have 0.01% -0.4% P.I. Even when acidilactici was given, there was no particular change in functional form or body weight. E. acervulina or E. coli. 0.1% -1% of P. aeruginosa broilers infected with telena. The same result was obtained when acidilactici was given.

For broilers infected with Eimeria acidilactici and S. The effects of giving boulardi's mixed probiotics were judged from the following aspects. 1) Decrease in the number of oocysts separated from feces and measurement, 2) Promotion of antibody production, 3) Promotion of increase in splenocytes. P. acidilactici and S. et al. Evaluation tests have shown that dogs with digestive disorders such as diarrhea, vomiting, loss of appetite, and body odor can be improved by giving boulardi's mixed probiotics. Even dogs suffering from bloody stool due to parvovirus infection may have p. acidilactici and S. et al. Symptom recovery was seen after 2-3 days of mixture probiotics of boulardi.

Suitable probiotics are yeast and lactic acid bacteria. Suitable microbial probiotics are the genera Pediococcus, Lactobacillus, Bifidobacterium, Bacillus, Streptococcus, Enterococcus. A suitable yeast is Saccharomyces cerevisiae bouulardi. Powdered probiotics are effective against gastrointestinal diseases caused by pathogenic microorganisms (bacteria), viruses, fungi, parasites and protozoa. Powdered probiotics are effective against helminths, roundworms, whipworms and tapeworms. Powdered probiotics are effective against parasites such as Giardia.

Probiotics are effective for human infectious gastrointestinal diseases if they are taken in appropriate amounts. Probiotics are effective for infectious gastrointestinal diseases in fish when taken in appropriate amounts. Dry powder probiotics have the same characteristics as capsule-encapsulated probiotics and are effective.

  FIG. 1 is a graphical representation of the effect of probiotics on antibody responses.   FIG. 2 graphically illustrates the effect of probiotics on cellular immune responses.   FIG. FIG. 3 is a graph showing oocysts of feces of birds infected with acervulina. FIG.   FIG. acervulina or E. coli. The oocyst of the stool of the bird infected with tenella is shown by the graph.

Claims (16)

  As a probiotic, a method of making at least one probiotic into powder form.   A manufacturing method in which lactic acid bacteria and yeast are powdered as probiotics.   A method for producing yeasts of the genus Pediococcus genus, Lactobacillus genus, Bifidobacterium genus, Bacillus genus, Streptococcus genus, Enterococcus genus lactic acid bacteria and Saccharomyces genus as probiotics.   A method of making Pediococcus acidilactici and Saccharomyces cerevisiae bouulardi into powder.   Probiotics claimed in claim 1 are one of the means for adjusting and treating immune responses to antigens in living organisms of infected birds, fish and mammals.   The probiotic as a means claimed in claim 5 is contained in animal food, animal snacks, animal seasonings, fermented milk products, or yogurt.   Birds that are subject to the means claimed in claim 5 include chickens, parrots, parakeets, pigeons, turkeys, duck, geese.   Mammals that are the subject of the means claimed in claim 5 include dogs, cats, guinea pigs, hamsters, mice, rabbits, monkeys, chimpanzees, pigs, and humans.   The infectious disease claimed in claim 5 is caused by pathogenic microorganisms, viruses, fungi, parasites, and protozoa.   The viruses claimed in claim 9 are parvovirus, rotavirus, and influenza virus.   Parasites claimed in claim 9 are helminths, roundworms, whipworms, and tapeworms.   The protozoa claimed in claim 9 are Eimeria, Giardia, Cryptosporidium, Toxoplasma, Plasmodium, Leishmania, and cyclospolar.   Modulation of the immune response claimed in claim 5 indicates increasing the antibody or promoting the proliferation of T cells or helper T cells.   Providing the probiotics claimed in claim 1 is one of the means of enhancing the immune response to antigens of birds, fish and mammals.   Modulation of the immune response claimed in claim 14 refers to increasing the ability to produce antibodies or increasing the ability to proliferate T cells.   Probiotics as means claimed in claim 14 are included in animal food, animal snacks, animal seasonings, fermented milk products, or yogurt.

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