JPH06128163A - Agent for preventing or treating fish streptococcosis with mixed ingredients - Google Patents

Abstract

PURPOSE:To provide a preventing or therapeutic agent containing a macrolide antibiotic or lincomycin antibiotic and a pleuromutilin derivative and effective for fish (e.g. yellowtail) streptococcosis exhibiting resistance against both the agents. CONSTITUTION:The fish streptococcosis-preventing or treating agent comprises one member selected from the group consisting of a macrolide antibiotic and a lincomycin antibiotic and further a pleuromutilin derivative as active ingredients.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to one kind selected from the group consisting of macrolide antibiotics and lincomycin antibiotics and pleuromutilin (Pleuromutil).
in) derivative as an active ingredient, and to a preventive or therapeutic agent for fish streptococcal infection.

[0002]

2. Description of the Related Art In recent years, as aquaculture of seawater fish and freshwater fish has become popular due to the development of fish culture technology, various diseases have occurred and cause great damage. Streptococcal disease in fish occurs in saltwater fish such as yellowtail and flounder, and freshwater fish such as rainbow trout and ayu. Among them, the most damage is caused by streptococcal disease of yellowtail.

The yellow-tail streptococcal disease first occurred in Japan in 1974 and is now the most seriously ill disease in the yellow-tail aquaculture industry. Attempts have been made to identify the causative bacterium of this streptococcus brii (Bulletin of
the JapanSociety of Sc
authentic Fisheries, 1976, 1
345-1352), this bacterium is Streptococcus faecal.
is) and Streptococcus faecium (St
reptococcus faecium) has similar properties to both species, but is a distinctly different bacterium,
Streptococcus Espi
cus sp. ) Was reported as.

Since then, further research has been conducted (fish disease research,
1982, 6, 17-26), but the species name has not been clarified yet. More recently, the causative agent is Enterococcus ser.
(iolicida) report (Internatio)
nal Journal of Systematic
Bacteriology, 1991, Vol. Four
1, No. 3, 406-409), but the state of enterococcus (Ente) of tiamulin fumarate
MIC for rococcus) is 100 μg / ml
["Tiaceptin (Tiamulin Fumarate) Field Trial Manual". 1976, 6 (Sankyo Co., Ltd.)],
It is said that it has almost no antibacterial activity.

Usually, for the causative bacteria of streptococcal infection,
Ampicillin and oxytetracycline hydrochloride have strong antibacterial activity in In Vitro, but it is known that administration of ampicillin to artificially infected yellowtail with this bacterium has no effect (see Rigaku IV Antibiotic 3 ", P71, Midori Shobo) has the drawback that the effect cannot be confirmed unless actually administered.

Therefore, according to the clinical results in the field, macrolide antibiotics such as erythromycin and spiramycin, lincomycin and the like have been used as prophylactic or therapeutic agents for Streptococcus bris. Initially, the antibacterial substance that was effective against streptococcal disease has become ineffective in the past few years, and the cause is the acquisition of resistance to the antibacterial substance by streptococcus, and In contrast, it is greatly affected by the medium of water, its propagation of resistance is accelerated, and its resistance is multidrug resistant.

[0007]

The present invention is effective against streptococcus fish due to the emergence of multidrug-resistant strains against commonly used antibacterial agents such as macrolide antibiotics and lincomycin antibiotics. There is no cure. In addition, in order to confirm what kind of drug is effective for the streptococcal disease that has occurred, it is necessary to actually administer medication,
Not only does it take a long time to determine the effect of the drug, but inflicting a drug that is not effective causes a great deal of damage.

Therefore, the above streptococcus, for example,
Not only against B. streptococci showing drug sensitivity, but also showing an effective action against B. streptococcus showing multidrug resistance, even if any resistant pattern strain appears in the field clinical, There has been a demand for the development of a preventive / therapeutic agent for effectively acting B. streptococcus.

[0009]

Regarding the effectiveness of a pleuromutilin derivative against Streptococcus which is a causative bacterium of Streptococcus bris, Japanese Patent Application Laid-Open Nos. 3-264526, 4-1131, and 4-11 Further, as a result of examining many field isolates, it was revealed that Streptococcus naturally having resistance to tiamulin fumarate, although partially known, is known. .

Under these circumstances, as a result of intensive studies, the present inventors have found that one of the pleuromutilin derivatives selected from the group consisting of macrolide antibiotics and lincomycin antibiotics. The addition of the compound effectively acts not only against strains showing sensitivity to both macrolide antibiotics, lincomycin antibiotics, and pleuromutilin derivatives, but also against resistant strains. That is, the new fact that a synergistic effect is recognized was found, and the present invention was completed.

The present invention has been made on the basis of the above findings, and uses as an active ingredient a pleuromutilin derivative and one selected from the group consisting of macrolide antibiotics and lincomycin antibiotics. It is a preventive or therapeutic agent for fish streptococcal infections. The present invention also provides a method for preventing or treating streptococcal fish infection, which comprises administering a pleuromutilin derivative and one selected from the group consisting of macrolide antibiotics and lincomycin antibiotics.

The macrolide antibiotics in the present invention include, for example, erythromycin, oleandomycin, kitasamycin, josamycin, spiramycin, milosamycin, carbomycin, midecamycin or malidomycin or their non-toxic salts, for example,
Examples thereof include tartaric acid and citric acid. Examples of the lincomycin antibiotics include lincomycin, clindamycin, and nontoxic salts thereof, such as hydrochloride and sulfate. The pleuromutilin derivative is preferably thiamulin (R in the following general formula
₁ is vinyl, X is sulfur, n is 2, and R ₂ and R ₃ are thiamulin when R ₃ is ethyl).

[0013]

[Chemical 1] (In the formula, R ₁ is an ethyl or vinyl group, and X is a sulfur or group.

[0014]

[Chemical 2] Where each of Y and Z is sulfur, or one of Y and Z is oxygen and the other is sulfur, n is an integer from 2 to 5, and R ₂ and R ₃ are each An alkyl group, or R ₂ and R ₃ together with a nitrogen atom are one nitrogen atom or one nitrogen atom and further sulfur,
Form a heterocyclic ring containing a heterologous members indicated by the oxygen or the group = N-R _4, provided that R ₄ JP-B-57-7147 represented by a is) lower alkyl or lower hydroxyalkyl group Mention may be made of those mentioned and their derivatives, or their non-toxic salts, such as the fumarate, hydrochloride, maleate, embonate or quaternary ammonium salts.

The compounding ratio of the prophylactic or therapeutic agent containing, as an active ingredient, a pleuromutilin derivative and one selected from the group consisting of macrolide antibiotics and lincomycin antibiotics is 1:10 to 1 /. A range of 10 (titer ratio) is preferred. Formulation with these formulations is appropriately performed according to a conventional method in view of the administration method. Further, in order to improve the palatability of the drug, the drug may be coated with a water-soluble polymer substance such as gelatin or methylcellulose, and the amount of the water-soluble polymer substance is usually 20% based on the weight of each drug. It should be processed according to the degree.

The means for administering the prophylactic or therapeutic agent of the present invention is
The method is not particularly limited, and various methods such as oral, injection, application, drug bath (dissolved in seawater or fresh water) can be mentioned, but oral administration is particularly preferable from the form of farmed fish breeding, and the timing of this administration is for preventive purposes in advance. As the above, it may be administered before infection, or immediately after some death due to infection is observed, preferably about 3 to 7 days, or may be continued as appropriate for more than 3 days. The method of oral administration is also not particularly limited, and a method of administering a raw feed or a mixed feed containing a drug is convenient and preferable.

The dose of the prophylactic or therapeutic agent containing, as an active ingredient, a pleuromutilin derivative and one selected from the group consisting of macrolide antibiotics and lincomycin antibiotics is variously used conventionally. Similar to the drug, it will vary considerably depending on the type of fish, the degree of physical exhaustion of sick fish, environmental conditions such as water temperature, etc. However, in the case of oral administration, if the total amount is 5 mg (titer) / Kg fish weight or more, Good, 5 to 1 from the viewpoint of therapeutic effect, economy and palatability
00 (titer) / Kg fish weight, especially 20-80 (titer) /
Kg fish weight is preferred. Regarding actual medication in the field, it may be necessary to take into consideration the amount taken, because the necessary amount may not be taken into the fish body due to factors such as the outflow of the drug.

Further, in the preventive or therapeutic agent for fish streptococcal infection of the present invention, when orally administered, an adhering agent such as sodium alginate or guar gum may be used in combination with moist pellets. Is an additive commonly used for fortifying foods, such as vitamin A, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , vitamin B ₁₂ , vitamin C, vitamin D ₃ , vitamin E, vitamin K _3. Vitamins such as choline, pantothenic acid, biotin, nicotinic acid, inositol and folic acid, calcium, phosphorus, sodium, potassium, magnesium, iron,
Zinc, manganese, copper, cobalt, selenium, molybdenum,
Minerals having elements such as chlorine, iodine and sulfur, amino acids such as methionine, lysine, tryptophan, glutamic acid, glycine and alanine, enzymes such as protease, cellulase, amylase and pectinase, It can be appropriately blended with other antibiotics, vaccines, anthelmintics and the like.

[0019]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. Example 1 Antibacterial Activity of Macrolide Antibiotics, Lincomycin and Tiamulin Fumarate (Fumaric Acid: Tiamulin = 1: 1 Salt) Against Streptococcus pyogenes Macro Macrophyll against Streptococcus pyogenes isolated from aquaculture farms in Kyushu and Shikoku The antibacterial activities of the ride-type antibiotics [erythromycin (EM), spiramycin (SPM), kitasamycin (KTM)], lincomycin (LCM) and tiamulin fumarate (TML) were determined from the minimum inhibitory concentration (MIC). The MIC was determined by the drug plate dilution method according to the standard method of the Japanese Society of Chemotherapy. That is, Table 1
The 11 test strains listed in the above are MUELLER HINTON BROTH (DIFC
O) to the post-logarithmic growth phase and diluted to 1/100 in the same medium, and the test reagents were diluted 2-fold in serial concentrations (100 μg / ml
From 0.0125 μg / ml) to MUELLER HINTON A
GER (DIFCO) was inoculated with 0.025 ml using NI continuous inoculator (Rika Ikeda), cultured at 25 ° C for 24 hours, and MI
The C value was determined. The results are shown in Table 1.

[0020]

[Table 1]

As shown in Table 1 above, there are many strains showing multidrug resistance to macrolide antibiotics and lincomycin, and these strains show sensitivity to tiamulin fumarate. There were also strains that were resistant to tiamulin fumarate. In addition, the test strain No. As shown in 7, 9, and 10, some strains showed spontaneous resistance to thiamulin fumarate alone.

Example 2 Streptococcus aureus (multidrug resistant strain)
Infection test of erythromycin and thiamulin fumarate mixture against cultivated yellowtail fry 13 with an average body weight of 53 g that had been preliminarily kept under running water at a water temperature of 19.6 to 20.9 ° C. (water volume of 500 l)
Streptococcus derived from yellowtail (No. 5 in Table 1) showing multidrug resistance against 0 macrolides (13 plots in each group, 10 in each group) against macrolide antibiotics, lincomycin and tiamulin fumarate. Strain) was intramuscularly infected with 10 ⁷ CFU per rat by intramuscular injection, and 2, 24, 48, 72, 96 after inoculation of the strain.
The following preparation was administered by oral gavage at the time.

These preparations were prepared by mixing erythromycin alone, thiamulin fumarate alone, and a combination of erythromycin and thiamulin fumarate in a fish-culture mixed feed (Moist mash for Marunaka hamachi, trade name "Hamachi Jump" Chubu Feed Co., Ltd.). The amount of each reagent to be added was adjusted so that the compounding ratio was 1: 1), and the drug mixture made into a paste by adding water was added to the stomach of the test fish using a simple sonde. Total amount in cavity 5, 10, 20, 40, and 80 mg
A certain amount of the drug mixture was orally administered by gavage so that (titer) / Kg · BW was obtained. The observation period is 10 days after infection,
The mortality of the sample fish was observed. The results are shown in Table 2.

[0024]

[Table 2]

As shown in Table 2 above, erythromycin was 5 to 80 mg / Kg.
With a death rate of 0%, 80 mg in the tiamulin fumarate single-agent administration group as well as in the erythromycin single-agent administration group
The mortality rate was 100% even after administration of / Kg · BW. On the other hand, regarding the effect of administration of a mixture of erythromycin and thiamulin fumarate (mixing ratio = 1: 1), despite the use of strains resistant to macrolide antibiotics and tiamulin fumarate, 20 mg / Kg
・ The mortality rate is 20% or less at doses of BW and above, and the mortality rate is 10% or less at doses of 40 mg / Kg and BW and above, and synergism by the combination of erythromycin and thiamulin fumarate. The effect was recognized and the effectiveness was suggested.

Example 3 Streptococcus aureus (multidrug resistant strain)
Mixture ratio of erythromycin and thiamulin fumarate with respect to cultivated yellowtail juvenile 6 with an average weight of 287 g that had been preliminarily kept under running water at a water temperature of 19.3 to 21.6 ° C. (water volume of 500 l)
0 fish (test group 6 wards, each group 10 fish) with respect to, macrolide antibiotics, yellowtail from Streptococcus one fish per 10 ⁷ CFU dorsal muscles showing the multidrug resistant against lincomycin and fumaric acid Tiamulin After injecting by internal injection and inoculating the strain in the same manner as in Example 2, the total amount of the combination of erythromycin and thiamulin fumarate having different compounding ratios was 40 mg (titer) / Kg.
-The patient was administered by oral gavage to obtain BW and an infection treatment test was conducted. The results are shown in Table 3.

[0027]

[Table 3]

As shown in Table 3 above, the effectiveness of the erythromycin and tiamulin fumarate combination on the streptococcus resistant to macrolide antibiotics, lincomycin and tiamulin fumarate is determined by the combination of the combination. Although it is affected by the ratio, it is effective in the range of 1:10 to 1/10, and when the ratio is 1: 1, the death rate is 1
It was 0%, which was particularly effective.

Example 4 Streptococcus aureus (multidrug resistant strain)
Infection treatment test of each drug and thiamulin fumarate mixture for the above-mentioned cultured yellow fry 3 having an average weight of 345 g which had been preliminarily kept under running water at a water temperature of 22.5 to 23.7 ° C. (water amount of 500 l).
10 ⁷ CFU dorsal muscles of yellowtail-derived streptococci showing multidrug resistance to macrolide antibiotics, lincomycin and tiamulin fumarate for 0 (3 plots in each test group, 10 in each group) After injecting by injection and inoculating the strain in the same manner as in Example 2, the total amount of the combination of spiramycin and thiamulin fumarate and the combination of lincomycin and tiamulin fumarate were 40 mg (titer) / Kg · BW. Gavage was administered and an infection treatment test was conducted. The results are shown in Table 4.

[0030]

[Table 4]

As shown in Table 4 above, the mortality rate of the non-medication control group was 100%, whereas the combination of spiramycin and tiamulin fumarate and the combination of lincomycin and tiamulin fumarate were: The death rate is 20%, and the target drug for the combination drug with tiamulin fumarate is 1
Not to mention 4-membered macrolide antibiotics, 16
The member-ring macrolide antibiotics and lincomycin-type antibiotics were effective.

Example 5 Field trial of administration of erythromycin and thiamulin fumarate combination to streptococcal infectious disease yellowtail erythromycin single agent, erythromycin and fumar, using three cultured cages in which death due to streptococcal disease was observed A therapeutic test was carried out by administration of the acid tiamulin combination drug. Three yellowtail fish with an average weight of 450 g each were placed in the test cages.
000 fish are bred, and erythromycin alone and erythromycin and thiamulin fumarate mixture (mixture ratio 2: 1) are added to these yellowtail culture groups so that the total amount is 60 mg (potency) / Kg · BW. Mixed in feed. Observation 1
From the day to the 7th day of observation (to confirm infection), the food containing no drug was given, and the food containing the drug was administered for 5 days from the 8th day to the 12th day of observation, and then the food containing no drug was administered. The number of dead tails was counted from the start of observation to the 25th day.

The water temperature during the test was 21.4-25.
The temperature of the feed used for the test was 2 ° C. Frozen sardines were chopped with a granulator (peretta) equipped with a chopper, and then mixed feed for fish farming (moisture mash for marnaka hamachi, trade name "Hamachi Jump" Chubu feed) Co., Ltd.) was mixed at a ratio of frozen sardines: mixed feed = 6: 4, and 1% of a nutritional supplement (“Paramix A” Eisai Co., Ltd.) was used. A predetermined amount of each drug was added, pelletized by the above granulator, and fed. The results are shown in Table 5.

[0034]

[Table 5]

As shown in Table 5 above, in the field, the non-medication control group had an average daily mortality rate of 0.47% (average from observation day 13 to observation day 25), and in the erythromycin single agent administration group. Similarly, the average daily mortality rate was 0.45%, and no effect was observed. On the other hand, the average daily death rate was 0.03% in the combined administration group of tiamulin fumarate and erythromycin, which was remarkably effective for streptococcal infection. A strain showing multidrug resistance to macrolide antibiotics, lincomycin and tiamulin fumarate was isolated from the dead fish after the administration.

[0036]

INDUSTRIAL APPLICABILITY The preventive or therapeutic agent for fish streptococcal infection of the present invention is more effective for preventing streptococcal infection than the conventionally used macrolide antibiotics or lincomycin antibiotics alone. Or treatment can be given.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location A61K 31/22 AFF 9283-4C (72) Inventor Mamoru Suzuki 1-30-2 Matsushiro Tsukuba, Ibaraki Prefecture

Claims (5)

[Claims] 1. A preventive or therapeutic agent for Streptococcus infectious disease of fish, which comprises as an active ingredient a pleuromutilin derivative and one selected from the group consisting of macrolide antibiotics and lincomycin antibiotics. 2. The compounding ratio of one kind selected from the group consisting of macrolide antibiotics and lincomycin antibiotics and the pleuromutilin derivative is 1:10 to 1/1.
The preventive or therapeutic agent for fish streptococcal infection according to claim 1, wherein the range is 0. 3. The total of one kind selected from the group consisting of macrolide antibiotics and lincomycin antibiotics and the pleuromutilin derivative is at least 5 mg (titer).
3. A dose of 1 / kg fish body weight or more.
The preventive or therapeutic agent for the fish streptococcal infection described. 4. The macrolide antibiotic is erythromycin, oleandomycin, kitasamycin, josamycin, spiramycin, mirosamycin, carbomycin, midecamycin or maridomycin or a non-toxic salt thereof, and the lincomycin antibiotic is lincomycin or The clindamycin or a nontoxic salt thereof, and the pleuromutilin derivative is tiamulin or a nontoxic salt thereof, the preventive or therapeutic agent for fish streptococcal infection according to claim 1. 5. A method for preventing or treating streptococcal fish infection, which comprises administering a pleuromutilin derivative and one species selected from the group consisting of macrolide antibiotics and lincomycin antibiotics.