JPH04182433A - Preventive/therapeutic agent for avian coccidiosis - Google Patents

Abstract

PURPOSE:To provide the title preventive/therapeutic agent containing bacterial cell-spalled product or cell wall components obtained by fractionation of said spalled product. CONSTITUTION:Cells with high immunoactivation effect for cell-spalled product (e.g. Brevibacterium lactofermesitum ATCC 13869, Bacillus satillus ATCC 13952) are aerobically incubated in an enriched medium of pH 4.0-9.5 at 2-40 deg.C for 12hr to 5 days. The resulting microbial cells are either mechanically spalled using a French press or ultrasonic spalling machine or put to cell wall degradation by the addition of cell wall digesting enzyme (e.g. lysozyme, protease) to obtain a bacterial cell-spalled product. This product is then fractionated by e.g. centrifugation to obtain a cell wall component-containing product. It is appropriate that 0.01-2 (pref. 0.05-1.0)wt.%, on a dry basis, of said cell-spalled product or cell wall component-contg. product be added to a feed and administered.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a prophylactic/therapeutic agent for chicken coccidiosis.

(Conventional technology and problems) Chicken coccidiosis is caused by the sporozoan Eimeria.
It is a disease of chickens caused by oral infection of several protozoa belonging to the genus a), and is one of the major diseases that causes great economic losses to the poultry industry, especially the broiler industry.

Monenshishi to prevent and treat chicken coccidiosis,
Polyether antibiotics such as salinomycin and lasalocid, or sulfa drugs such as sulfadimethoxy are used, but frequent use of these drugs can lead to the emergence of drug-resistant protozoa, and there is also the problem of the drugs themselves remaining in the body or in chicken egg white. Since their use is severely restricted, the use of these drugs is not effective or safe for the prevention or treatment of chicken coccidiosis. In recent years, progress has been made in the development of live vaccines, but there is still no one that is completely effective.

Therefore, poultry farmers are looking for new drugs that are safe and highly effective, rather than drugs such as rutame antibiotics, to prevent and treat chicken coccidiosis.

On the other hand, bacterial cell wall components have long been known as immunostimulants (adjuvants), and those prepared from the cell wall of the bacterium Mycobacterium bovis have been investigated for their immune-enhancing activity and effectiveness as a cancer immunotherapeutic agent. There is.

(Cancer, Vol. 65, pp. 493-505, 1974) Also, targeting livestock diseases, the use of a drug prepared from the cell wall of the bacterium Bifidobacterium thermophilum as a preventive and therapeutic drug for livestock diarrhea has been reported. has been done (
Jpn.

J, Vet, Sci, Vol. 49, pp. 235-243, 1987, JP-A-62-265231). However, the preventive and therapeutic effects of these immunostimulants against protozoan infectious diseases such as chicken coccidiosis are currently unknown.

(Means for Solving the Problems) As a result of extensive research in view of the above-mentioned circumstances, the present inventors have found a bacterial cell fragment obtained by mechanically crushing cultured bacterial cells and/or enzymatically decomposing them. The present inventors have also discovered that a cell wall component-containing product obtained by fractionating the cell fragments has a preventive and therapeutic effect on chicken coccidiosis, and has completed the present invention.

That is, the present invention provides at least one of a bacterial cell fragment obtained by mechanically crushing and/or enzymatically decomposing a cultured bacterial cell, and a cell wall component-containing material obtained by fractionating the cell fragment. The present invention relates to preventive and therapeutic agents for chicken coccidia and feed containing them.

As the bacteria used in the prophylactic/therapeutic agent of the present invention, any bacteria can be used as long as its cell fragments have a high immunostimulatory effect, but Brevibacterium lactofermentum (Brevibacterium lactofermentum)
fermentum) 8TCC13869, Corynebacterium daltamicum
m glutamicum) ATCC13060, Bacillus subtilis (Bacillus 5ubtilis)
) ATCC13952 is an example.

Furthermore, for culturing bacteria, any nutrient source that can be used by these bacteria can generally be used. For example, carbohydrates such as glucose and sucrose, alcohols such as ethanol and glycerol, organic acids such as acetic acid and propionic acid,
Carbon sources such as soybean oil or mixtures thereof, enzyme extracts,
Peptone, meat extract, cornstarch liquor, ammonium sulfate,
A conventional culture medium containing a nitrogen-containing inorganic organic nutrient source such as ammonia, an inorganic nutrient source such as phosphate, magnesium, iron, manganese, and potassium, and vitamins such as biotin and thiamine is used. The cultivation method is to keep the pH of the nutrient medium in the range of 4.0 to 9.5 at 20°C to 40°C for 12
What is necessary is just to culture|cultivate aerobically for 5 hours to 5 days.

The method for disrupting the bacterial cells obtained by culture may be either a mechanical method or a method using enzymes. As a mechanical method, for example, using a French press etc.
Cells may be crushed under a pressure of 00 to 2000 kg/C11l, or cells may be crushed using an ultrasonic crusher or the like. When bacterial cells are disrupted using enzymes, cultured bacterial cells or mechanically disrupted cultured bacterial cells are suspended in physiological saline or the like, and a cell wall lytic enzyme is added thereto to decompose the cell walls of the bacterial cells. The enzyme used in this case may be any enzyme as long as it has the ability to decompose cell walls, and representative examples include lysozyme and protease. Enzyme treatment conditions may be according to known methods. In both the mechanical method and the enzymatic method, the cell crushing rate should be about 30% or more (preferably about 60% or more) as measured by the absorbance (wavelength 560 nm) fI & low when suspended in water. By using mechanical methods and enzymatic methods in combination to increase the rate, a cell wall component-containing product with high immunostimulatory effects can be prepared.

Cell wall component-containing substances can also be fractionated from the bacterial cell fragments by centrifugation or the like and used.

The prophylactic/therapeutic agent of the present invention can be used by giving crushed bacterial cells orally to poultry in the form of a liquid, or by drying it if necessary and adding it to feed, feed, etc. in the form of a powder. You may. Although the timing of administration does not matter, it is preferable to give the drug from the time of infusion to prevent coccidiosis.

The dosage is about 1 to 5 g per day as a dry substance.

In addition, the feed contains 0.01 to 2% as dry matter, preferably 0.
．． It is recommended to add 0.05 to 1.0%. The feed can be used by appropriately mixing general feed materials used for chickens and the like. Hereinafter, it will be explained in detail using examples.

Example-1 Preparation of cell fragments and cell wall component-containing products (1) Preparation of bacterial cells Glucose 1.0 g/di, Yeast extract 1.0 g/d
1, peptone], Og/a-(NHa)zSO-0,
5g/d1, K2HPO.

0.3gl, KHzPOa O, 1g/dfl and M
Medium containing gSO4・7H, OO, 05g/min (pH 7
) in a 500111 volume flask and heated at 115°
Sterilize at C for 15 minutes. Add this to bouillon agar medium for 30
Brevibacterium lactofermentum ATCC 13869, Corynebacterium daltamicum ATCC 13060 and Bacillus subtilus A cultured for 1 day at
A loopful of TCC13952 was inoculated and cultured with shaking at 30°C for 24 hours.

After the cultivation was completed, each nutrient solution was centrifuged to collect the bacterial cells. Each bacterial cell was suspended in the same amount of physiological saline as the culture solution, heated at 100° C. for 10 minutes, and the bacterial cells were collected again by centrifugation.

(2) Each bacterial cell (wet bacterial cell) prepared in mechanical crushing treatment (1) was placed in a 25 m
The suspension was suspended in M phosphate buffer (pH 7.0) to a concentration of 10%. This bacterial cell suspension was poured into a stainless steel bottle (5
0Id volume) and ultrasonic crusher (UR-200P type,
(manufactured by Tomy Seiko Co., Ltd.) with an oscillation frequency of 20 kHz, 200
It was treated with a power of W for 15 minutes. After the treatment, the cell wall fraction content was further fractionated by centrifugation.

(3) Each bacterial cell (wet bacterial cell) prepared in (1) by enzymatic decomposition treatment of cultured bacterial cells was dissolved in 25+++H phosphate buffer containing 10% of IE as a solid substance (
pH 7.0) and egg white lysozyme (manufactured by Sigma) 0.01
Weight% and actinase (manufactured by Kaken Pharmaceutical, 70,000 units)
0.02% by weight was added and treated at 37°C for 12 hours.

Thereafter, the enzyme was inactivated by heat treatment at 100°C for 2 minutes.

(4) Egg white lysozyme (
0.1% by weight (manufactured by Sigma) and 0.2% by weight of actinase (70000 units manufactured by Kaken Pharmaceutical Co., Ltd.) were added, and
Time processed. Thereafter, the enzyme was inactivated by heat treatment at 100°C for 2 minutes.

(5) Immunostimulatory effect RPM11 using mouse lung cells
10% non-working fetal calf serum in 640 medium, 5 x 10-5M
2-mercaptoethanol was added and filter sterilized. Lung floating cells prepared from the lungs of a 10 week old female DBA/2 mouse were suspended in this at a concentration of 2.5 x 106 cells/d. Also (1), (2) (3) and (4)
The bacterial cells and enzymatically decomposed products prepared in step 1 were added to each concentration and cultured for 4 days at 37°C in a 5% CO2 incubator. After culturing, the culture supernatant was diluted 1000 times with 0.01M Tris-HCl buffer (pH 8.1) containing 1% bovine serum albumin, and then subjected to ELIS.
The immunostimulatory effect of each additive preparation was determined by measuring the total amount of mouse IgM produced in the culture medium by Method A. In addition, as a standard substance for testing the immunostimulatory effect, Lipopoly1! derived from pathogenic Escherichia coli is used. (LPS)
was used.

The results are shown in Figure 1.

FIG. 1 shows the relationship between sterilized bacterial cells of each genus and mouse IgM/antibody production of their enzymatically decomposed products. In the figure, the horizontal axis shows the added concentration (μg/d), and the vertical axis shows the concentration (μg/d) of total mouse IgM produced in the culture solution of cultured lung cells.

The broken line with black circles indicates the case of the sterilized bacterial cells prepared according to Example (1), the broken line with white circles indicates the case of the mechanically disrupted cells prepared according to Example (2), and the solid line with black circles indicates the case of the enzymatically crushed cells prepared according to Example (3). In the case of decomposition treatment, the solid white circles indicate the cases in which the bacterial cells prepared according to Example (4) were subjected to both mechanical crushing treatment and enzymatic decomposition treatment.

As is clear from FIG. 1, the disrupted cultured cells of Examples (2), (3), and (4) have a higher ability to enhance antibody production than the sterilized cells of Example (1), and are more effective than LPS. It shows high activity exceeding the maximum value of antibody production (150μg/-),
It can be seen that it has an excellent immunostimulatory effect.

Example 2 Preparation and Effects of Feed Containing Crushed Cells and Materials Containing Cell Wall Components (1) Effect of various crushed bacterial cells on chicken coccidiosis Seventy-six commercially available broiler chicks were divided into four groups of 19 each.

One area is the control (non-administration group), and the other three areas are (4
) Brevibacterium ATCC138 prepared according to
69, Corynebacterium glutamicum ATCC13
060 and Bacillus subtilis ATCC 13952, the dry powder of the enzymatically decomposed bacteria was used as an antibiotic-free broiler early matush feed (64% cornmeal,
0.3% was added to 24% vegetable oil cake, 8% fishmeal, and 4% other ash, etc.) and fed.

104 Eimeriatenela oocysts in all districts at 7 days old
The bird was orally administered per bird to forcefully infect the bird.

Seven days after infection, 5 birds in each group were necropsied and lesions were observed based on macroscopic necropsy findings. The severity of the lesions was scored from 0 to 4 (4 is severe) and the average value was calculated, using standard methods for evaluation. Furthermore, the cecum was sampled and the number of oocysts per 1 g was counted. Then, the obtained data are tested for mean difference (D
UNCAN'S MULTIPLE RANGE TES
Statistical processing was performed using T). The results are shown in Table 1.

Table 1: Effect of various bacterial cell fragments on chicken coccidiosis As is clear from Table 1, there were no cases of death after infection in the treated groups, the average lesion value was low, and the number of oocysts in the cecum was significantly reduced. The effect on chicken coccidiosis is clear.

(2) Effect of various treatments on chicken coccidiosis of shredded products 114 commercially available broiler chicks were divided into 6 groups of 19 each. One group was a control (non-administration group), and the other five groups were prepared according to Examples (1), (2), (3), and (4).

Brevibacterium lactofermentum ATCC1
3869 cells and dried powder of the processed product were added to the feed.
The mice were administered at a concentration of 7%, or an aqueous solution containing 10 g/a of dry powder was directly orally administered once daily for 2 days. Early broiler Matushu feed, which does not contain antibiotics, was used for breeding in all plots. A chicken coccidia forced infection test was conducted in the same manner as in section (1) below. The results are shown in Table 2.

A: There is a significant difference at a 1% risk rate between C, D, E, and F. Mechanically disrupted bacterial cells, enzymatically decomposed bacterial cells, or a combination of both have higher anti-coccidial effects than sterilized bacterial cells. . It is also effective whether added to feed or administered directly.

[Brief explanation of the drawing]

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

Claims (1)

[Scope of Claims] 1. Bacterial cell fragments obtained by mechanically crushing cultured bacterial cells and/or enzymatically decomposing them, and cell wall component-containing products obtained by fractionating the cell fragments. Preventive and therapeutic agent for chicken coccidiosis 2 comprising at least one of these, a feed containing the preventive and therapeutic agent for chicken coccidiosis according to claim 1.