JPS6259214A - Antimicrobial agent against bacteria caused by animal infectious disease - Google Patents

Abstract

PURPOSE:An antimicrobial agent, consisting of bile acid, a material containing the bile acid or/and a plant containing curcumin or an extract thereof, having almost no fear of side effect, resistance and environmental pollution, capable of suppressing growth of bacteria caused by animal infectious diseases and treating the infectious diseases of cultured fishes, domestic animals, etc. CONSTITUTION:An antimicrobial agent, containing bile acid or a material containing the bile acid, preferably dried powder of cattle or pig bile or/and a plant containing curcumin, e.g. Curcuma aromatica rhizome of Curcuma aromatica or Curcuma longa, or an extract thereof and useful for bacteria caused by animal infectious diseases, e.g. streptococci or bacteria belonging to the genus Pateurella. This agent is capable of suppressing the growth of causative bacteria and treating the infectious diseases by administering to pseudotuberculosis caused by streptococcal infection or pasteurellosis in culture farms of young yellowtail preventing the spread of the infectious diseases. The plant containing the curcumin has hemostatic action on hemorrhage caused by infection. There is no problem such as side effect and resistance as in administration of antibiotic substances.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an antibacterial agent against infectious disease-causing bacteria that occurs when aquatic or terrestrial animals are raised.

(Prior Art) Collective cultivation and rearing of aquatic or terrestrial animals is widely practiced in the fields of fisheries and livestock, but bacterial infections often occur and cause serious damage.

As a countermeasure, antibiotics and the like are being administered.

(Problems to be Solved by the Invention) However, administration of antibiotics generally causes the development of resistant bacteria, and there is also a risk that residual antibiotics may have an adverse effect on humans if they ingest the meat of animals to which they have been administered.

(Means for Solving the Problems) The present inventor has conducted various studies in an attempt to prevent the above-mentioned infectious diseases using natural products, and as a result, has established the present invention.

The present invention is an antibacterial agent against bacteria causing animal infectious diseases, which is made of a bile acid or a substance containing it, or/and a curcumin-containing plant or an extract thereof.

Bile acids are components of animal bile, and their structure and content in bile may vary depending on the type of animal. Includes taurotesoxycholic acid, glycodesoxycholic acid, ursotesoxycholic acid, taurourtesoxycholic acid, chenotesoxycholic acid, and the like.

Examples of bile acid-containing substances include bile and its dried products. Dried bile powder is preferred from the standpoint of storage and cost.

Furthermore, bile acids, bile, etc. may be used in the form of a composition mixed with other drugs, diluents, carriers, excipients, etc., if desired, and such compositions are included in the bile acid-containing substances. .

From the viewpoint of availability, preferred animals as sources of bile include cows and pigs.

Curcumin-containing plants include Curcuma aromatica, which is the rhizome of Curcuma aromatica, and Curcuma aromatica.
Examples include turmeric, which is the rhizome of the plant (S. onga).

It is convenient to use curcumin-containing plants either as a dry powder or as an aqueous extract.

The antibacterial agent of the present invention can inhibit the growth of bacteria that cause animal infectious diseases, such as bacteria belonging to Streptococcus and Pastorella.

Bacteria belonging to Streptococcus and Pastorella cause infectious diseases in breeding aquatic or terrestrial animals. For example, in yellowtail farms, streptococcal disease and pass!
- Massive outbreaks of nodularity caused by Lera may occur.

The antibacterial agent of the present invention can be administered to animals infected with causative bacteria, suppress the growth of the bacteria in the body, and restore health.

Administration can be carried out orally. In the case of bile acids or their contents, the dosage is generally 1 to 100 μl for bile acids.
/kq (body weight), preferably 5 to 50 D/kty, and for bile-dried rice, generally 10 to 160 D/kty, preferably 20 to 80 D/kty. In the case of curcumin-containing plants or their extracts, herbal medicines such as turmeric and kyooh are generally used in the dry rice range of 20 to 20 seconds, o o
oda/kq, preferably 100~t, oooMl
/kq.

The antibacterial agent of the invention is conveniently administered in a mixture with feed or feed. It goes without saying that materials suitable for the animal to be cultured or reared are selected as feed or feed.

Animals to which the antibacterial agent of the present invention is administered include, for example, aquatic animals such as fish and crustaceans, and terrestrial animals such as cows, horses, pigs, sheep, and chickens.

(Effect) In the present invention, the bile acid or its content and/or the curcumin-containing plant or its extract act antibacterially on infectious disease-causing bacteria in the body of the animal to which it is administered, suppressing their growth and suppressing symptoms caused by infection. Go into remission.

In addition, curcumin-containing plants or extracts thereof also have a hemostatic effect on bleeding caused by infection.

The present invention will be further described below with reference to Reference Examples and Examples.

Reference Example 1 Investigation of Fish Intestinal Bacteria Like terrestrial animals, bacteria exist in the intestines of fish, and the type and amount of bacteria is determined by the species of fish, the water area they live in, whether and when they feed, and the amount of food they eat. It varies depending on the condition and physiological state. Enterobacteriaceae in the narrow sense are Enterobacteriaceae (Enterobacteriaceae).
e) include the genus E., Escherichia, Salmonella, and Proteus; however, in this study, we focused on bacterial genus in a broad sense, including the bacterial groups that invade and exist in the intestine through symbiosis, contamination, and contamination. . Some of these include potentially pathogenic bacteria and others that are currently harmless.

(1) Intestinal viable bacterial materials and methods for clinically healthy yellowtail hamachi Three yellowtails (600 to 1.000 da) that are considered to be clinically healthy and sampled at each fishing ground in the Nagasaki and Wakayama fishing areas were collected on the spot. Dissect the intestinal tract aseptically for 5 to 10 minutes.
(7) Cut and immerse in sterilized 50% glycerin saline solution, or aseptically collect the intestinal tract from fresh fish directly frozen on dry ice and send it to us.
Weigh out 10 g, add 9 volumes of sterile physiological saline, dilute 10 times, homogenize, and then add 1 volume of sterile physiological saline.
00 times, ], OO dilution steps up to 0 times were made.

Each dilution was applied to two plates of 2% salt-added plain heart infusion (BI(I) agar medium with a Conrage stick in an amount of 0.1 ml, and after culturing at 25°C for 5 days,
Those with a colony count of ~300 were selected, and the average value of the two sheets was determined to determine the number of viable bacteria.

In addition, a sterility test was also performed on the culture medium, physiological saline, etc., and the kidney, brain, and heart were also stamped to check for the presence of bacteria.

The results are shown in the table below.

Table 1 No bacteria were isolated from the kidney, brain, or heart of each sample fish.

As mentioned above, the bacterial populations in Nagasaki Prefecture and the Wakayama region were completely different. This is thought to be due to the relationship between the habitat and food sources, and therefore, it appears that common symbiotic bacteria do not exist in yellowtail.

(2) Intestinal bacteria of clinical northern yellowtail diseases, especially streptococcal disease Materials and methods Yellowtail (800 to 1.5001) sampled at fishing grounds in Kochi, Miyazaki, Ehime, and Nagasaki prefectures (2 each) The tail was examined for viable bacteria using the same method as in (1) above.

Bacteria isolation from the kidneys was also performed at the same time.

The results are as shown in the table below.

Table 2 in the margin below *10 Mild ← Moderate + High concentration ** S, Nis I leptochosoccus *** G, N, Nigerum negative bacilli As is clear from the above, the intestinal bacteria in fish affected by streptococcal disease are mostly Only streptococcus.

The number of bacteria isolated from the kidney is proportional to the number of bacteria isolated from the intestinal tract, which shows how proliferation in the intestinal tract influences the severity of the disease.

Example 1 Test method for inhibiting streptococci from yellowtail using bile and its components Paper Tisfu method Test drug (5) Beef bile dry powder ■) Urtudesoxycholic acid (C) Yellowtail bile stock sample test strain Kochi Prefectural Hamachi Streptococcus (5trep) isolated from
tococcus sp, ) 2nd generation strain culture medium and device-sensitive disk culture [manufactured by Eiken Kagaku 1] Sterile shallow P petri dish [manufactured by Nissui Seiyaku Aji] diameter 9a filter paper (manufactured by Toyo Seisaku Shin) diameter 8
The experiments were conducted after confirming that the culture medium, equipment, and diluted drug solution were sterile through various preliminary tests.

Add the above test drug (4) to 1%, 2%, (B
) diluted to 1%, 2%, (C) 1%, 20%,
Paper disks were prepared for each 0.02 ml and stored dry.

The content of the test drug in the paper disk is (4) 200μ
m1, 400μg CB) 200μg, 400μg (0200μg, 4~).

According to the Showa disk method, one platinum loop amount (platinum wire thickness 0.6 mm, ear inner diameter 1 mm) of the test bacterial strain was added to sterile physiological saline.
．． 1 vtl, one platinum loop of the same was applied to the entire surface of a susceptible disk medium, each sample paper disk was placed on top of it, and after culturing at 25°C for 24 hours, judgment was made, and the results shown in the following table were obtained. .

Table 3: Table 3 As shown above, bile powder (5) was found to be most effective in inhibiting 14 to 23 cases. Urtudesoxycholic acid CB) was found to inhibit 17 ph at 400 μf.
Inhibition of bile (0) between 4 and 10 was also observed.

When the test was repeated twice using different strains of bacteria, almost the same results were obtained, although there was some variation in the inhibition diameter.

Therefore, bile powder and bile components such as urtudesoxycholic acid clearly have an inhibitory effect on streptococci.

11-Example 2 Inhibition test of Durum-negative bacteria derived from red sea bream The results shown in the following table were obtained in the same manner as in Example 1, except that Edwardsiella tarda isolated from red sea bream was used as the test bacterial strain. .

Table 4 As shown above, bile powder and bile components such as urtudesoxycholic acid also exhibit an inhibitory effect on Durham-negative bacteria such as Edwardsierra spp.

Example 3 Streptococcus inhibition test by dilution method Using the same test agent, bacterial strain, and petri dish as in Example 1, the medium was Brae 12 = In Heart Infusion (BHI) agar medium to which 2% salt was added. It was carried out as follows using

The culture medium, equipment, and diluted drug solutions were all preliminarily tested to ensure that they were sterile.

Test drug (4) is 2%, 03) is 2%, and (O is 20%).
The sample was diluted 0 times, and the maximum dilution factor was 10-2.

Sterile physiological saline alone was used as a control. Add 0.01 tel of a solution of 1 platinum loop of the test bacterial strain suspended in 0.1 ml of sterile physiological saline to each diluted drug solution, and after culturing at 25°C for 24 hours, transfer the solution to BHI agar medium supplemented with 2% sodium chloride. ,01
yttl was applied to the entire surface and cultured again at 25°C for 24 hours, and the number of bacteria was determined visually, and the results shown in the following table were obtained.

Table 5 * The control was set as +5, and the case where no bacteria grew was set as 0.

**Although expressed as +5, the growth layer was thinner than the control, no clear colonies were formed, and growth was poor.

Example 4 Comparison of the suppressive effects of bile powder and antibiotics on streptococcus. Streptococcus sp. Bile powder 200μ
y1 Erythromycin 20μg, Spiramycin 20μg
The following results were obtained in the same manner as in Example 1 except that paper disks each containing g were used.

As shown in Table 6 above, bile powder was used at 10 times the amount of each antibiotic, but showed the same inhibitory power as spiramycin.

Example 5 As a test bacterium for suppression of a bacterium that causes tuberculosis of yellowtail, Pa5teurella pisci, a bacterium that causes tuberculosis of yellowtail, was used as a test bacterium.
cida) 2 as a test drug using a foreshadowed substitute strain (4)
The procedure of Example 1 was followed except that ox bile powder, CB) yellowtail bile stock solution was used, and a paper disk containing 200 μp of each (A) and 411 W of (f3) was used, and the results shown in the following table were obtained.

Table 7 As shown above, bile (terminal) suppresses the bacteria that cause yellowtail tuberculosis.

Example 6 Vibrio anguillarum (Vib
rio amguillarum), and using (4) ox bile powder and ■) yellowtail bile stock solution as test drugs, paper discs (A) 200 ttl and 400 ttl each were used.
μg, (B) 201zp.

The procedure of Example 1 was followed except that one containing 4■ was used, and the results shown in the following table were obtained.

Table 8 * Thin inhibition circle As mentioned above, bile (end) suppresses the bacteria that cause Vibrio disease in sweetfish.

Example 7 Suppression test of bacteria causing streptococcus disease and nodularity in yellowtail by turmeric Two strains of streptococcus (5 treptococcus sp.) isolated from yellowtail from Mie prefecture and a yellowtail from Ehime prefecture were used as test bacterial strains. Pastorella
Pa5teurella piscicid
The second foreshadowing stock of a) was used. Turmeric powder was used as a test drug, but it is difficult to dissolve in water as it is, so 200 g J of purified water was added to 10 f of turmeric powder, boiled until it had a resistance of 100, and then filtered through sterile gauze, and then
r, p, m,, centrifuged for 5 minutes, and 0.05 of the supernatant
ml was dropped onto a paper tissue (diameter: 8) and air-dried in a dark place before use. Other than that, the test was carried out in the same manner as in Example 1, and the results were that the diameter was gmm for Streptorella bulbus, and the diameter was 12 for Pastorella bisicida.
My sabotage circle has been approved.

Therefore, it has been revealed that turmeric suppresses streptococcal and nodularis-causing bacteria in yellowtail.

Example 8 Field test on farmed yellowtail Test period: August 1st to November 80th Test location: Nagasaki-based yellowtail farm A Sample fish: This yellowtail was caught in early June and weighed around 230 g after a preliminary period. Divided into 3 test groups for 3,000 fish: 1 group each accommodated in 3 nylon fishing net Aba (float) type rectangular Ikes [8m x 8m x 6n7 (depth)].

Feed: During the fry stage, sand eel was mainly used, and during the test period, semi-thawed and minced sardines, chub mackerel, etc. were used.

Test drugs: 20% ox bile powder, 10% kyowo, 10% turmeric, 30% alpha gluten, 1 alpha starch
Granules consisting of 0% and 20% of guar gum.

Dosing method: A spreading agent was added to 0.5 g of the test drug per 1 kq of fish weight, and this was mixed with the feed and administered.

Feeding was performed 2 to 3 times a day, and medication was performed once a day.

Medication program: A, No-medication area B, Area 1 (1 ql administration area) From the first day of the month, 0.5g/9 (fish body weight) was administered once a day for 5 consecutive days C, H Ward (2-kil administration area) 5 days from the 1st day of the month (1st course) and 1st course
Divide into 5 days (2nd course) from the 5th, once a day 0.5
91kljc fish weight) was administered.

Test method: The collected Mojako were divided into 15,000 fish units and raised from the beginning of June to July, during which time the results were recorded for one section, which was used as a preliminary test section.

During the mojako period (4-28011, nodular disease and ascites occur frequently, and the mortality rate is high. Therefore, in this study, we avoided this period to keep the survival rate as stable as possible and to ensure that food intake is vigorous due to high water temperature. A comparison test was conducted by selecting the period when the greatest increase in meat could be expected.

Select the size from one unit area of 15,000 fish to reduce the deviation value, 8. The test was started by dividing the OOO tails into three areas and housing them in the small compartments mentioned above.

The body weight was measured at 1 i at the end of each month by randomly selecting 100 fish per section. For dead fish, we observed them every day, picked them up each time they occurred, recorded the number of fish, and investigated the cause of death. Table 9.1.0.11.14 χ Also, at the end of the feeding test period, randomly select several fish, take a piece of their muscle and liver in 10% polymeric solution, embed it in paraffin, and then Tissue specimens were prepared and examined by staining (Section 4).
Figure 5.6).

On the other hand, heart blood was collected from several fish in the same way, serum was separated, and RA
BA-AC! A liver function test was conducted using E (manufactured by Chugai Pharmaceutical Aji) (Table 13).

As is clear from these tables and figures, a significant difference was clearly observed when comparing the non-administration area and the medicated area.

In the test, a relatively high margin was allowed for the feeding density, but in general, it is normal for the feeding density to be higher, so it is thought that there would be a greater difference in production yield.

Streptococcus disease was overwhelmingly the most common cause of death, and in this respect it appears that the medication was especially effective (Table 14).

Growth rates were above standard for this region in both Wards 1 and H (Figure 1).

The visual and taste sensory tests were conducted by five third-party people selected at the end of the feeding test period, and all of them yielded similar opinions (Table 12).

Histological examination of muscle and liver showed results supporting the above sensory test (Figure 4.5.6, Table 12)
.

In other words, Figure 4a shows muscle tissue images (X200) in the non-administration area and the medicated area, but in the non-administration area muscle fibers are poorly developed and there is a lot of intercostal fat, whereas in the medicated area The muscle fibers are well developed and there is little fat.

Figure 5a is a liver histological image of a non-administered area and a medicated area (X4
00), however, in the non-administration area, many lipid droplets were seen around the cells and a slight back view was observed, whereas in the administration area, the cell nucleus was clear and there were few sebaceous droplets.

Figure 6a is a histological image of the small intestine in the non-administered area and the medicated area (X2
00), in the non-administered area, the hairs, which are important for the absorption of nutrients, are coarse and poorly developed, whereas in the treated area, the hairs are well-developed and the muscle layer is dense.

Liver function tests revealed some significant differences in total cholesterol and triglycerides, which were also found to be high in serum (Table 13).

23 spaces below = Table 11 *Aggregation of the results of a questionnaire survey of 5 people about 10 fish each**Relationship between body length and circumference* The cause of death was determined based on clinical findings, autopsy findings, and some pathological assessments. 1 piece.

(Effects of the Invention) According to the antibacterial agent of the present invention, it is possible to suppress the growth of bacteria that cause animal infectious diseases, thereby treating infectious diseases in farmed or bred animals, and preventing the spread of such diseases. Also, there are no tolerance issues,
There is almost no risk of environmental pollution.

[Brief explanation of the drawing]

Figures 1, 2, and 3 are graphs showing changes in growth rate, mortality rate, and water temperature in each plot in the field test of Example 8, and Figures 4, 5, and 6 are graphs, respectively. Micrographs of muscle tissue, liver tissue, and small intestine tissue of fish collected at the end of the feeding test in Example 8 (200x magnification each)
(400x and 200x) In each figure, a shows the non-administration area, and b shows the medication chart. Figure 1 ('/day) 8/I B/:al 9/30
IQ/31 1p Color 0 Figure 2 Look at original key let comparison Figure 3 Own room clam jaw 4mi W day. , bi - Change/Ihi Figure 4a Figure 4b Procedural amendment (spontaneous) October 4, 1985 2, Title of invention Antibacterial agent caused by animal infectious disease 3, Relationship with the case of the person making the amendment Patent applicant address: 801 Nishihama, Wakayama City, Wakayama Prefecture Name: Kiyowa Pharmaceutical Co., Ltd. Representative: Tomihisa Kawaguchi 4, Agent address: 46 Mansei Building, Kitahama 4, Higashi-ku, Osaka City Name: Patent attorney (6249) Jurisdiction: Taku)'1'e (06)
202-5858 ``'5, date of amendment order
(Voluntary) 6. Subject of amendment Detailed explanation column 7 of the specification, Contents of the amendment As shown in the attached sheet, Contents of the amendment Page 271 of the specification, Table 9, Test period 1, above the section ■ 3rd row, (row of total weight in the starting period) f7>
1199.30 J to r 699.30 J to YI positive L/su. that's all

Claims (1)

[Scope of Claims] 1. An antibacterial agent for animal infectious disease-causing bacteria, which is made of bile acid or its contents or/and curcumin-containing plants or extracts thereof. 2. The antibacterial agent according to claim 1, wherein the bile acid-containing substance is bile powder. 3. Claim 1, wherein the curcumin-containing plant is the rhizome of Curcuma aromatica or Curcuma longa.
Antibacterial agents listed in section. 4. The antibacterial agent according to claim 1, wherein the animal infectious disease-causing bacterium is streptococcus. 5. The antibacterial agent according to claim 1, wherein the animal infection-causing bacterium is a Pastorella bacterium.