JP2621588B2 - Prevention and treatment of white and diarrhea in livestock - Google Patents

Description

The present invention relates to a novel agent for preventing and treating white and diarrhea in livestock, poultry, and pets.

[Conventional technology]

At present, for example, white and diarrhea during the suckling period is one of the biggest problems for breeding pig farmers. That is, about 45% to 70% of
% Suffer from white diarrhea and diarrhea, which is a significant economic loss for pig farmers and a major problem in the pig farming industry. This trend is similar in developed countries. Antibiotics such as sulfur drugs are generally used to prevent and treat this disease. However, in recent years, the use of antibiotics has been limited due to the emergence of resistant bacteria and the problem of persistence of drugs in the body.

Live bacteria have been developed as agents for preventing and treating white and diarrhea in livestock and poultry other than antibiotics. Probiotics are administered directly by live, useful bacteria, which either colonize the gut of livestock or antagonize harmful bacteria in the gut, such as pathogenic Escherichia coli, as they pass through the gut. It eliminates and improves the intestinal microflora to prevent and treat livestock diarrhea / white diarrhea. Therefore, it is necessary that the cells are alive for the expression of the effect, and as a result,
In order to enhance physicochemical stability as a probiotic agent in feed and in vivo, spore-forming bacteria may be used (Veterinary and Livestock Shimpo N
o.695 343 pages, 1979
In addition, there have been reports of cases in which multidrug-resistant bacteria are used in consideration of combination with feed additive antibiotics (Bifidoba)
cteria Micrcflora, Vol. 4, p. 15, 1985).

However, there are still few bioactive agents having excellent stability, and their effect is slow because their mechanism of action is to eliminate harmful bacteria by antagonizing harmful bacteria.

On the other hand, Bifidobacterium thermophilum (Bifidobacteriuntherm
A prophylactic / therapeutic agent for vomiting and diarrhea in livestock and poultry has been proposed which comprises an enzyme digest of the cell wall of mophilum as an active ingredient (see JP-A-56-108717). It has also been reported that peptidoglycan is an active ingredient in the cell wall degradant of this genus Bifidobacterium, and its mechanism of action is thought to activate the immune system of livestock and enhance the ability of livestock to protect against infection. It has a completely different mechanism of action from the conventional probiotic (see Japanese Patent Application Laid-Open No. 62-265231). These inventions cultivate useful bacteria originally inhabiting the intestine of livestock, take out only the active ingredient, and return it to the host.Bifidobacterium used here is, Cultivation of bacterial cells that are obligate anaerobic bacteria and are the raw material requires anaerobic operation, and the difference is complicated.Moreover, expensive raw materials such as vitamins are required for growth, and the yield of cells is low and cost is high. Disadvantages.

[Problems to be solved by the invention]

Prevention and treatment of diarrhea and diarrhea due to immunostimulation in livestock is different from antibiotics without the emergence of resistant bacteria and the problem of drug persistence in the body. Those using the bifidobacterium, which is a bacterium, are not practical because of the difficulty in handling the bacteria and the low yield of the cells.

Therefore, an object of the present invention is to develop a preventive and therapeutic agent for white and diarrhea in livestock, poultry, and pets, which is easy to prepare in large quantities, is easy to handle, and has an excellent effect by immunostimulation.

[Means for solving the problem]

The present inventors have conducted intensive studies in view of the above-mentioned circumstances, and as a result, it has been found that Bacillus sp., Brevibacterium sp., Corynebacterium sp., Esthesia sp. Genus, Lactobacillus,
The present inventors have found that a cell wall component-containing substance of a bacterium belonging to the genus Streptococcus or Streptomyces has a preventive and therapeutic effect on white and diarrhea in livestock, poultry, and pets, thereby completing the present invention.

That is, the present invention is not a gut bacterium derived from the gut of a domestic animal, and is capable of growing under aerobic conditions Bacillus, Brevibacterium, Corynebacterium, Eselicia,
Lactobacillus, Streptococcus, or Streptomyces bacterium, a bactericidal product of a bacterium belonging to the genus Streptomyces, a crushed cell obtained by subjecting the bacterium to mechanical disruption or enzymatic degradation, and the crushed cell Which contains at least one of livestock, poultry, and companion animals that have immunostimulatory ability.・ This is related to the prevention and treatment of diarrhea.

Examples of the bacterium used for the preventive or therapeutic agent of the present invention include Bacillus bacterium such as Bacillus subtilis ATCC13952, Brevibacterium bacterium such as Brevibacterium lactofermentum ATCC13869, and Corynebacterium. Bacterium glutamicum (Corynedacterium glutamicum) ATCC13060
Escherichia bacteria such as Corynebacterium bacteria such as Escherichia coli ATCC8739;
Lactobacillus acidophilus (Lacto-bacillus ac
Bacteria belonging to the genus Lactobacillus such as idophilus ATCC4356, Streptococcus ther
mophilus) Streptococcus bacteria such as ATCC19987,
Streptomyces t
anashiensis) ATCC15238 and the like.

In culturing these bacteria, any nutrient source that these bacteria can usually utilize can be used. For example, glucose, carbohydrates such as sucrose, ethanol, alcohols such as glycerol, acetic acid, organic acids such as propionic acid, carbon sources of soybean oil and the like or a mixture thereof, yeast extract, peptone, meat extract, corn steep liquor,
A normal medium appropriately containing an inorganic nutrient such as ammonium sulfate and ammonium, an inorganic nutrient such as phosphate, magnesium, iron, manganese and potassium, and a vitamin such as biotin and thiamine is used. As a culture method,
The pH of the nutrient medium is in the range of 4.0 to 9.5 at 20 to 40 ° C for 12 hours to 5 hours.
The culture may be aerobic for days.

Cells obtained by culturing can be used as they are, separated from the culture and sterilized by heat treatment or the like. However, it is preferable that the cells have been subjected to a crushing treatment. The cells to be crushed may be live cells or sterilized cells. The crushing method may be either a mechanical method or a method using an enzyme. As a mechanical method, for example, using a French press or the like, about 800 to 2000 kg /
The cells may be disrupted at a pressure of cm ² , or the cells may be disrupted using an ultrasonic disrupter or the like. When bacterial cells are disrupted using an enzyme, the cultured cells or a mechanically disrupted product of the cultured cells are suspended in a physiological saline solution or the like, and a cell lysing enzyme is added thereto to decompose the cell walls of the cells. The enzyme used at this time may be any enzyme as long as it has the ability to degrade the cell wall, and lysozyme and protease are typical examples. Enzyme treatment conditions may be in accordance with a known method. In both the mechanical method and the enzymatic method, the cell crushing rate is preferably about 20% or more, and more preferably about 60% or more. The crushing rate can be measured by the turbidity reduction rate of the suspension at a wavelength of 660 nm. In addition, it is preferable to use both a mechanical method and an enzymatic method in order to increase the crushing rate.

It is also possible to separate and use the cell wall component-containing substance by fractionating the cell crushed product. The fractionation method may be simply centrifugation to remove insolubles, and a known method for fractionating proteins or the like by molecular weight, for example, a method such as ultrafiltration or gel filtration can be used.

As a method of using the preventive or therapeutic agent of the present invention, the prepared bacterial cells, the crushed product thereof, or the cell wall component-containing material may be orally given to livestock, poultry or companion animals as a liquid, or if necessary. It may be dried and added as a powder to livestock, poultry, pet feed, feed and the like. The administration period is not limited, but a high effect can be obtained for the preventive treatment of leukemia and diarrhea in the suckling period by continuously administering for 1 to 2 weeks from birth. The dose is 10mg to 50mg / day as dry matter.
g, preferably about 100 mg to 2 g. In addition, in the weaning period or poultry, 0.001 to 5
%, Preferably 0.05 to 1%.

In the present invention, livestock are pigs, cows, horses, goats, sheep, etc., poultry is birds such as chickens, pets are dogs,
Cats and the like.

〔Example〕

Example 1 (1) Preparation of bacterial cells Glucose 1.0 g / dl was obtained by adding yeast extract 1.0 g / dl and peptone.
_{1.0g / dl, (NH 4)} 2 SO 4 0.5g / dl, K 2 HPO 4 0.3g / dl, KH 2 PO 4
A medium (pH 7) containing 0.1 g / dl and MgSO ₄ .7H ₂ O 0.05 g / dl was added to 5
500 ml was placed in a 00 ml flask and sterilized at 115 ° C. for 15 minutes.
Bacillus subtilis ATCC 13952, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 13060, Escherichia coli ATCC 8739, Lactobacillus acidophilus ATCC 4356, Streptococcus thermophilus ATCC 19987 cultured in bouillon agar medium at 30 ° C for 1 day. One platinum loop of Myces tanaciensis ATCC15238 was inoculated, and cultured with shaking at 30 ° C. for 24 hours. After completion of the culture, each culture was centrifuged to collect the cells. Each of the cells was suspended in the same amount of physiological saline as the culture solution, heated at 100 ° C. for 10 minutes, and collected again by centrifugation.

As a control, the obligate anaerobic bacterium Bifidobacterium
Thermophilum ATCC25525 (derived from pig intestinal tract) and Clostridium butyricum (derived from human intestinal tract) were used. glucose
1.0 g / dl, yeast extract 1.0 g / dl, peptone 1.0 g / dl, MgSO ₄
・ 7H ₂ O0.02g / dl, Tween80 0.05g / dl, ammonium acetate
280 ml of a medium (pH 7) containing 0.2 g / dl, 10% of tomato juice filtrate, and 1.0 g / dl of CaCO _{3 is} placed in a 300 ml volumetric flask at 115 ° C.
Sterilized for 15 minutes. This was inoculated with a control bacterium punctured on the same agar medium, and cultured at 37 ° C. for 2 days under anaerobic conditions. After completion of the culture, each culture was centrifuged to collect the cells. Suspend each cell in the same amount of physiological saline as
Heat treatment was performed at 0 ° C. for 10 minutes, and the cells were collected again by centrifugation.

Table 1 shows the weight of the wet cells obtained per 100 ml of the medium after the culture.

(2) Each of the cells (wet cells) prepared in (1)
The suspension was adjusted to 10% by weight of a 25 mM phosphate buffer (pH 7.0). This cell suspension is placed in a stainless steel bottle (50 ml
And treated with an ultrasonic crusher (UR-200P, manufactured by Tommy Seiko Co., Ltd.) at an output of 200 W and an oscillation frequency of 20 kHz for 15 minutes. After the treatment, the contents contained in the cell wall fraction were further fractionated by centrifugation.

(3) Enzyme-decomposed product 0.01% by weight of egg white lysozyme (manufactured by Sigma) was added to 25 mM phosphate buffer (pH 7.0) containing 10% by weight of the mechanically processed product of the cell prepared in (2) as a fixed substance. 0.02% by weight of actinase (7000 units, manufactured by Kaken Pharmaceutical Co., Ltd.) was added, and the mixture was treated at 37 ° C. for 12 hours. Thereafter, the enzyme was inactivated by heat treatment at 100 ° C. for 2 minutes.

(4) Immunostimulatory effect using mouse spleen cells 10% fetal calf-inactivated serum in PRMI1640 medium, 5 × 10 ⁻⁵ M2
Mercaptoethanol was added and sterilized by filtration. to this
Spleen prepared from spleen of 10-week-old female DBA / 2 mouse
The suspended cells were suspended at 2.5 × 10 ⁶ cells / ml. In addition, the bacterial cells prepared in (1) and (3) and Kluyvera citophilia IF081 prepared by enzymatic degradation or in the same manner as in (1) and (3)
93, Beijerinckia ind
ica) ATCC9037 cells and enzymatically degraded products were added to each concentration, and cultured in a 5% CO ₂ incubator at 37 ° C. for 4 days. After the culture, the culture supernatant was diluted with 0.01 M Tris-HCl buffer (pH 8.1) containing 1% bovine serum albumin for 10 minutes.
After dilution by a factor of 00, the immunostimulatory effect of each of the added preparations was measured by measuring the amount of 1 gM of the total mouse produced in the culture solution by ELISA. In addition, lipopolysaccharide (LPS) derived from pathogenic Escherichia coli was used as a standard substance for assaying the immunostimulatory effect.

 The results are shown in FIG.

FIG. 1 shows the relationship between sterilized cells of each genus in FIG. 1 and mouse IgM / antibody production of the enzymatically degraded products. In the figure, the abscissa indicates the added concentration (μg / ml), and the ordinate indicates the concentration (μg / ml) of total mouse IgM produced in the culture of spleen cultured cells. The dashed line indicates the case where the sterilized cells were added, and actually the case where the enzyme-decomposed product was added.

As is clear from FIG. 1, Bacillus subtilis ATCC13952, Brevibacterium lactofermentum ATCC13869, Corynebacterium glutamicum A of the present invention.
TCC13060, Escherichia coli ATCC8739, Lactobacillus acidophilus ATCC4356, Streptococcus thermophilus ATCC19987, Streptomyces tanaciensis ATCC15238 are sterilized bacterial cells, and the antibody production ability of both enzyme-treated products and the enzyme-treated product is a control anaerobic anaerobic anaerobic. It is higher than the bacteria Bifidobacterium thermophilum and Clostridium butyricum, and the aerobic bacteria Kluyvera sitophilia and Beijerinkia indica.

In addition, the antibody-producing ability of the enzyme-treated product of the present invention tends to be higher than that of the bactericidal cells, and each shows a high activity exceeding the maximum value of antibody production in LPS (150 μg / ml), and is excellent. It can be seen that it has an immunostimulatory effect. In contrast, no activity exceeding LPS was observed in the control group, and as can be seen from FIG. 1, in these control groups, enhancement of antibody production could not be expected even if the added concentration was increased.

(5) Effect on suckling swine diarrhea and diarrhea Sixty suckling pigs were divided into nine groups of seven each. One section was used as a control (non-administration group), and the other 8 sections were suspended in a dry powder of enzymatically digested bacteria prepared according to (4) at a rate of 200 mg once a day in 3 ml of water. Oral administration was performed for 7 days from the beginning.

The state of white diarrhea / diarrhea was observed for 20 days from the 8th day to the 27th day after birth. As shown in Table 2, the administration group to which the enzyme-decomposed product of the bacterium according to the present invention was administered was in each case less susceptible to white and diarrhea than the control and the group to which the enzyme-decomposed product of Bifidobacterium thermophilum was administered. The incidence was low and the average weight gain was high.

(6) Twenty-eight suckling pigs were divided into four groups by head casting.

One section was used as a control (non-administration group), and the other three sections (administration group) were heat-treated products of the bacteria of Brevibacterium lactofermentum ATCC13869 prepared in (1), (2) and (3) and a machine. Crushed product, enzymatic digestion product, and fractionated powder after enzymatic digestion process were each 1
Once a day, 200 mg was suspended in 3 ml of water and orally administered for 7 days from birth.

The state of white diarrhea / diarrhea was observed for 20 days from the 8th day to the 27th day after birth. As shown in Table 3, in each case, the incidence of white and diarrhea was lower in the administration group than in the control, and the average weight gain was higher.

In each administration group, the cells treated with mechanical disruption or enzymatic digestion were more likely to undergo leukemia and
The effect of preventing diarrhea was high.

(7) Breeding test of calves by adding feed One 12-week-old Holstein bull was divided into three sections, one section was used as a control (non-administered group), and the other two sections were Brevi prepared according to (4). A dry feed of 0.1% and 1% of a dry powder of an enzymatically degraded product of the bacterium lactofermentum ATCC13869 was prepared, and a feed mixture for weaning calves to which 1% was added was fed (administration group).
After nursing for a week, the incidence of diarrhea, loose stool and weight gain in each section were measured.

As shown in Table 4, the incidence of diarrhea and loose stool was lower and the weight gain was better in the administration group than in the control.
In the administration group, the 1% administration group showed slightly better results than the 0.1% administration group.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between sterilized cells of each genus and mouse IgM / antibody production of the enzymatically degraded products.

Claims (1)

(57) [Claims] 1. A genus of Bacillus, Brevibacterium, Corynebacterium, Eselicia, Lactobacillus,
A sterilized product of cells of a bacterium belonging to the genus Streptococcus or Streptomyces, a cell crushed product obtained by subjecting the cells of the bacterium to mechanical crushing treatment or oxygen decomposition treatment, and fractionating the cell crushed material. Livestock, poultry, pet animal white diarrhea, which contains at least one of the livestock, poultry, and companion animals having immunopotentiating ability.
Prevention and treatment of diarrhea