JPH0237882B2 - CHIKUSANDOBUTSUNODENSENSEISHITSUKANNOYOKUSEIHOHO - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for suppressing infectious diseases in livestock animals. Recently, there have been frequent outbreaks of mycobacterial infections in pigs in various places, and this is becoming a public health problem. The causative bacteria of mycobacterial disease in pigs are atypical mycobacteria such as Mycobacterium smegmatis and Mycobacterium intracellulare. Infection with these bacteria forms lesions similar to tuberculosis, resulting in so-called atypical anti-acid bacteria. Acidobacterosis occurs.
Because the disease of atypical mycobacterial disease is slow, it is difficult to recognize any abnormality in general clinical symptoms, and the only thing that can be seen is slightly poor growth, and in most cases, it is only discovered during slaughterhouse inspection. The lesions of this disease are generally localized to lymph nodes in the head and neck and mesentery, and vary from yellowish gray-white caseating lesions several millimeters in diameter to cases in which the entire lymph node is swollen. There are no known vaccines or drugs that are effective in preventing or treating this disease. Therefore, infected pigs should be detected as soon as possible using the tuberculin test and isolated from other pigs.
The only countermeasure is to thoroughly clean and wash the pigpen with water and then disinfect it. However, it is said that no matter what disinfectant is used, it cannot be completely sterilized. The present inventors have demonstrated that polyhexamethylene biganadine or its salts are effective against Mycobacterium smegmatis and Mycobacterium smegmatis, which are the causative bacteria of this disease.
It has been found that it has an excellent bactericidal effect against intracellulare and, unlike other disinfectants, maintains strong bactericidal activity even in the presence of organic matter such as chicken manure. Furthermore, in chickens, for example, chick dysentery, respiratory diseases, and coliosis occur frequently. The causative bacteria of these infectious diseases include Salmonella prolum, Salmonella teifimirium, Haemophilus gallinarum, Mycoplasma synobiae, and pathogenic Escherichia coli. Furthermore, in cases of mastitis in cows or infectious diseases in pigs and chickens, healing may be delayed or infections may occur due to the colonization of Pseudomonas aeruginosa due to bacterial alternation. In recent years, anthrax infections in cattle and pigs have also become a problem. As a result of the inventors' research on these,
The above-mentioned polyhexamethylene biganadine or its salt is applied to the above-mentioned bacteria by sensitizing it for 10-15 minutes to
It was found that it exhibits bactericidal activity at a dilution of 16,000 times, and that its bactericidal activity against Escherichia coli and other bacteria does not decrease significantly even in the presence of chicken manure. As a result of research into methods of disinfecting poultry houses, equipment, equipment, etc. using this compound, the present invention was achieved. The present invention is based on the formula (In the formula, n is a number indicating the degree of polymerization corresponding to a molecular weight of 700 to 1300) This is a method for controlling infectious diseases in livestock animals, which is characterized by spraying, dispersing, or washing the animals. Compounds of formula are described in U.S. Pat.
It is known from the various specifications of No. 3428576, and is known to exhibit antibacterial activity against general bacteria. According to Japanese Patent Publication No. 55-42611, it is also known to be effective against Clostridium botulinum. However, it exhibits excellent bactericidal activity against acid-fast bacteria such as Mycobacterium smegmatis, Salmonella prolum, Salmonella teifimirium, Haemophilus carinarum, Mycoplasma synobiae, pathogenic Escherichia coli, and Bacillus anthracis. Even in the presence of organic matter such as chicken feces or blood, the bactericidal activity against Mycobacterium smegmatis and pathogenic Escherichia coli does not decrease significantly, and this can be used to eliminate infectious diseases in livestock animals by spraying, spraying, or washing. It is a new finding by the present inventors that this can be effectively suppressed. On the other hand, other disinfectants such as chlorine, iodine compounds, and antibacterial soaps are hardly effective against Mycobacterium smegmatis and the like because their activity is significantly reduced by the presence of organic substances as described above. When carrying out the present invention, the compound of the formula or its salt is sprayed or sprinkled on poultry houses, equipment, equipment, etc., or washed with the same. This makes it possible to sterilize pathogenic bacteria in the house, in manure and on the body surfaces of livestock animals. The molecular weight of the compound of formula is 700-1300, preferably 900-1100. Inorganic acid salts such as hydrohalides, sulfates, nitrates or organic acid salts such as lactates, tartrates, succinates, maleates, malonates, gluconates of compounds of formula Since it is relatively soluble in organic solvents, it can be used in the form of a solution for spraying, spraying, or cleaning.
Moreover, instead of a solution, it may be made into a fine powder and sprayed or dispersed together with a suitable powder diluent. The concentration of the compound of formula or its salt in solution or fine powder is
0.01-10% by weight, preferably 0.03-5% by weight. The daily usage amount of the compound of formula or its salt is, for example, 10 to 1000 mg per pig, preferably 30 mg per pig.
~800mg, 1~100mg per chicken, preferably 3~
80mg, 0.1-10g per cow, preferably 0.3-8
It is g. Spraying or spraying or washing with this is usually done once to 10 times a day, every day during the breeding period of livestock animals. In that case, it is preferable to allow the fine particles (about 0.1 to 60 microns) of the drug to stay in the indoor space for about 30 seconds to 30 minutes each time. Experimental example 1 Mycobacterium, a type of atypical mycobacteria
Smegmatis IFO3153 (not pathogenic to humans)
and Ogawa medium (10 g of potassium dihydrogen phosphate and 10 g of sodium glutamate dissolved in distilled water.
and sterilize at 100℃ for 30 minutes. 100ml of this solution, 6ml of glycerin, 6ml of 2% malachite green solution
ml and 200 ml of whole eggs, mix, pass through gauze, dispense, make a slope in a serum coagulator, and place in the first
85℃ for 40 minutes on the second day and 40 minutes at 80℃ on the second and third days.
After sterilizing for 5 minutes before use), the bacteria were incubated at 37℃ for 5 minutes, and on the 5th day, the proliferated bacteria were scraped off with a platinum loop, and the number of bacteria was 7.0 x 10 ⁷ in sterilized physiological saline in an agate mortar.
cells/ml (measured by plating on the same medium after preparation). This bacterial solution was used as an inoculum solution. Add the specified amount of various disinfectants to 2 ml of Dubos liquid medium, and add 2 drops (approximately 0.013 ml) of the above bacterial solution with a syringe.
The disinfectants were added dropwise, mixed well, and cultured at 37°C for 5 days, and the minimum inhibitory concentration of each disinfectant against Mycobacterium smegmatis was measured. As disinfectants, in addition to the hydrochloride of the compound of the formula (molecular weight 900 to 1100), for comparison, benzalkonium chloride, cresol soap solution (Japanese Pharmacopoeia),
Iodophor (preparation containing 1.75% available iodine and 11% nonionic active agent) and methyldodecylbenzyltrimethylammonium chloride preparation (abbreviated as MDBTC preparation) were used. The test results are shown in Table 1. Dubos liquid medium: 1.3 g of potassium dihydrogen phosphate,
Sodium hydrogen phosphate 2.2g, asparagine 2g,
Dissolve 5.2 g of peptone, 0.1 g of magnesium sulfate, 0.01 g of iron ammonium citrate, 0.0001 g of zinc sulfate, 0.0001 g of copper sulfate, 0.0005 g of calcium chloride, 0.002 g of malachite green, and 0.5 g of polyoxyethylene sorbitan monooleate in distilled water. Take 900 ml of the solution prepared in step 1 and sterilize it at 121℃ for 15 minutes. To this, 100 ml of a solution of 5 g of glycerin, 7.5 g of glucose, 0.9 g of common salt, and 5 g of bovine albumin dissolved in sterile distilled water is added and mixed.

[Table] From these results, it is recognized that the hydrochloride of the compound of the formula exhibits extremely superior antibacterial activity against Mycobacterium smegmatis compared to other disinfectants. Experimental Example 2 A bacterial solution prepared by suspending Mycobacterium smegmatis cultured in Ogawa medium using the method of Experimental Example 1 in sterile physiological saline to a bacterial count of 1.9 x 10 ⁸ /ml was used as the inoculum solution, and various disinfectants were used. The bactericidal effect against this bacterium was measured. As a disinfectant, the hydrochloride of the compound of formula (molecular weight 900-1100), benzalkonium chloride cresol soap solution (Japanese Pharmacopoeia) as a comparative agent, iodophor (available chlorine 1.75%, nonionic surfactant 11%) (containing formulations) and phenols were used. Dilute the disinfectant by 2-fold serial dilution using sterile distilled water to prepare 5 to 6 dilutions, dispense 10 ml of each into sterile test tubes, and leave them in a 20°C water bath for 10 minutes. did. Each of these drug dilutions was inoculated with 0.1 ml of the previously prepared bacterial solution, stirred thoroughly, and then placed in a water bath at 20°C again. 25 minutes, 5 minutes, 10 minutes, and 15 minutes (each sensitization time) after inoculating the bacterial solution, collect 0.1 ml of each diluted drug solution from the bacterial solution inoculation with a sterile pipette, and apply it to the surface of Ogawa medium. dripped. The samples were then cultured at 37°C for 5 days, and the bactericidal effect of each disinfectant against Mycobacterium smegmatis was examined based on the presence or absence of bacterial growth. Table 2 shows the dilution test results for the compound of the formula, and Table 3 shows the highest dilution factor that inhibited bacterial growth at each sensitization time by comparing various disinfectants.

[Table] is shown below.

[Table] Shows dilution factors.
When sensitized with the drug for 15 minutes, the highest dilution that inhibited bacterial growth was 8000 for the hydrochloride of the compound of formula
1,600 times more iodophor, 400 times more benzalkonium chloride, 160 times more cresol soap, and 80 times more phenol. For reference, when calculating the phenol coefficient of various disinfectants against Mycobacterium smegmatis, the hydrochloride of the compound of the formula is
100, iodophor was 17.5, benzalkonium chloride was 7.5, and cresol soap was 3. Therefore, it is clear that the hydrochloride of the compound of the formula exhibits an excellent bactericidal effect against Mycobacterium smegmatis, an atypical acid-fast bacterium, which is not seen in other disinfectants. Experimental example 3 Chicken manure in a sterile test tube (nitrogen content 2.9%, moisture 10.8%)
Add 4.9 ml of distilled water diluted to 10%, add 0.1 ml of the bacterial solution obtained in Experimental Example 1, and mix thoroughly to make 5 ml. Separately, 5 ml of each disinfectant used in Experimental Example 2 was diluted to 1/2 of the dilution ratio measured, and added to the chicken manure solution. Combine both liquids
2.5 minutes, 5 minutes, 10 minutes, 15 minutes and 30 minutes (each sensitization time)
After that, collect 0.1ml from each and add a new one.
% Ogawa medium. After culturing at 37°C for 5 days, the effectiveness of each disinfectant was determined based on the number of Mycobacterium smegmatis colonies that grew.
The results are shown in Table 4.

【table】

[Table] In the presence of 5% chicken manure, the hydrochloride of the compound of formula is
A sensitization time of 5 minutes or more at a 1000-fold dilution showed a bactericidal effect, but benzalkonium chloride required a sensitization time of 30 minutes or more for a 100-fold dilution and 10 minutes or more for a 50-fold dilution. Cresol soap solution is diluted 160 times for 15 minutes or more.
The sensitization time required for 80-fold dilution was more than 5 minutes. Also, iodophor showed bactericidal effect when diluted 10 times, but 25
It was ineffective when diluted more than twice as much, and all the color of iodine disappeared. Phenol showed a complete bactericidal effect when diluted 40 times, but 15 minutes of sensitization time was required when diluted 80 times. For reference, when calculating the phenol coefficient of various disinfectants against Mycobacterium smegmatis in the presence of 5% chicken manure, the hydrochloride of the compound of the formula is 25, benzalkonium chloride is 1.3,
The value was 2 for cresol soap and 0.29 for iodophor, and it was confirmed that the disinfectant of the present invention has an extremely excellent disinfectant effect even under these conditions. Experimental Example 4 Each disinfectant was diluted with distilled water to the required number by 2-fold serial dilution (phenol was diluted in 10-fold increments from 50 to 100 times), and 10 ml of each diluted solution was incubated with Salmonella spp. cultured in heart infusion broth. Bacterial fluids of Program Nakayoshi and Pseudomonas aeruginosa 347, and Bacillus anthracis 34F ₂
Add 1 ml of each spore solution and mix. The number of bacteria in the inoculated bacterial solution was 3.9×10 ⁸ /ml, 1.3× ^{10 8} /ml, and
It was 7.5×10 ⁸ /ml. Then 0.5 minutes, 2.5 minutes, 5
After 1, 10 and 15 minutes (each sensitization time), transfer one loop of the fluid in the working test tube into a fresh 10 ml heart infusion broth. The sensitization temperature was 20°C. After culturing at 37°C for 48 hours, the bactericidal effect of each disinfectant on Salmonella prorum, Pseudomonas aeruginosa 347, and Bacillus anthracis 34F ₂ spores was examined based on the presence or absence of bacterial growth.
Table 5 shows the effective dilution factors for each sensitization time of various disinfectants against Salmonella prolumum. When sensitized with the drug for 10 or 15 minutes, the highest dilution that inhibited bacterial growth was 5000 times for the hydrochloride of the compound of formula, 250 times for iodophor, 5000 times for benzalkonium chloride, and 5000 times for MDBTC preparation. 1000 times,
The cresol soap solution was 100 times more concentrated, and the phenol was 80 times more concentrated. For reference, the phenol coefficients of various disinfectants against Salmonella prorum are 59 for the hydrochloride of the compound of formula, 4.4 for iodophor,
Benzalkonium chloride is 59, MDBTC preparation is
17.6, cresol soap liquid 1.2. It is therefore clear that the hydrochloride of the compound of the formula exhibits excellent bactericidal activity against Salmonella prolumum.

【table】

[Table] Table 6 also shows the bactericidal effects of various disinfectants against Pseudomonas aeruginosa. When sensitized with the drug for 15 minutes, the highest dilution that inhibited bacterial growth was 16,000 times for the hydrochloride of the compound of the formula, 200 times for iodophor, 4,000 times for benzalkonium chloride, and 800 times for the MDBTC preparation.
The cresol soap solution was 100 times more concentrated, and the phenol was 80 times more concentrated. For reference, the phenol coefficients of various disinfectants against Pseudomonas aeruginosa are 100 for the hydrochloride of the compound in the formula, 3.8 for iodophor, 50 for benzalkonium chloride, 7.5 for MDBTC preparations, and 1.9 for cresol soap. Ta.

[Table] Next, Table 7 shows the bactericidal effects of various disinfectants against anthrax spores. If you sensitize the drug for 15 minutes,
The highest dilution that inhibited bacterial growth was 8000 times for the hydrochloride of the compound of formula, 50 times for iodophor, 1000 times for benzalkonium chloride, and 100 times for MDBTC preparation.
The cresol soap solution was 5 times or less. Although it is effective against spores, Shohei, which is not used for livestock production because it is a mercury agent, inhibited the growth of bacteria by a factor of 2,000.

[Table] Experimental example 5 Chicken manure in a sterile test tube (nitrogen content 2.9%, moisture 10.6%)
5 each of distilled water containing 2% and 10% of
1 ml of pathogenic Escherichia coli bacteria isolated in the field and cultured in heart infusion broth for 24 hours (bacterial count: 1.0 x 10 ⁹ /ml) and mixed. Separately, a solution is prepared by mixing 5 ml of distilled water instead of the chicken manure solution with 1 ml of the bacterial solution. Add 5 ml of a solution obtained by diluting the hydrochloride salt of the compound of the formula (molecular weight 900 to 1100) to a multiple of 1/2 of each measurement dilution factor to each of the above solutions. Then 2.5 minutes, 5 minutes, 10 minutes, 15 minutes and 30 minutes
After 1 minute, remove one loopful from each and incubate in 10 ml of fresh heart infusion broth. 37
After culturing at ℃ for 48 hours, the bactericidal effect of the hydrochloride of the compound of formula against pathogenic Escherichia coli was examined based on the presence or absence of bacterial growth. The results are shown in Table 8. The hydrochloride salt of the compound of formula
Sensitization time: 2,000 times in 2.5 minutes, 4,000 times in 5 minutes, 30 minutes
A bactericidal effect was observed at dilutions up to 8000 times.
Even if chicken manure is present as an organic substance at 1%, the sensitization time is 2000 times greater for 10 minutes, and 4000 times greater for 30 minutes; even if chicken manure is present at 5%, the sensitization time is 1000 times greater for 2.5 minutes, and 2000 times greater for 15 minutes or more.
A bactericidal effect was observed at double dilution.

[Table] Indicates that the species did not propagate.
Example 1 Parent pig infected with antibacterial acid (positive tuberculin test, nodule-like symptoms observed in lymph nodes in the head and neck,
Lymph fluid was collected from the area, cultured in Ogawa medium, and atypical acid-fast bacteria were isolated.) Four pigs were obtained, and two pigs were kept in separate pig pens. One pigpen is the hydrochloride of the compound of formula (molecular weight
900-1100) 0.1% by weight solution was sprayed 4 times a day for 5 minutes each time, and after disinfecting for 3 days, healthy young pigs 10
Both heads were placed in a pigpen and raised for 6 months. During breeding, the above-mentioned disinfection was carried out every day, and the caretakers disinfected their hands with the above-mentioned disinfectant every day. The amount of spray liquid is 1.9 per day, and the amount of hydrochloride of the formula compound per pig is approximately 160 mg/day. Another pigpen was disinfected as described above for 3 days, and then 10 healthy young pigs were found.
The heads of the pigs were placed together in a pig pen, and the pigs were raised for 6 months without any disinfection as described above, other than the hand disinfection of the handler. After rearing, all grown pigs were sacrificed and dissected to examine the presence or absence of tuberculosis-like lesions. As a result, tuberculosis-like lesions were found in the mesenteric lymph nodes in 4 out of 10 adult pigs in pigpens that had not been disinfected, whereas there were no lesions in adult pigs in pigpens that had been disinfected. It was not recognized and showed good growth. Example 2 200 meat chickens were kept in separate chicken houses. In one chicken house, the floor was cleaned in advance with a 0.1% by weight solution of hydrochloride of the compound of the formula (molecular weight 900-1100), and then the same solution was sprayed twice a day for 5 minutes each time to disinfect for 3 days. During breeding, the above-mentioned disinfection was carried out daily, and the caretakers disinfected their hands with the above-mentioned disinfectant solution every day. The amount of spray liquid is 6.0 per day.
The amount of the compound hydrochloride used per chicken is approximately 30 mg/day. In another poultry house, the above disinfection was not performed after that, except for the hand disinfection of the breeder.
It was kept for several months. 30 in poultry houses that did not carry out the above disinfection during breeding.
Feather died. Pathogenic E. coli was isolated from the liver, heart, or spleen of 19 of these birds. On the other hand, 17 chickens in the chicken house that had been disinfected as described above died, but no pathogenic E. coli was detected upon autopsy. Example 3 Anthrax caused by Bacillus anthracis infection is a legal infectious disease for domestic animals, and domestic animals carrying the disease must be killed as soon as they are discovered. Therefore, in the case of anthrax, the hydrochloride of the compound of the formula is more suitable for disinfecting livestock barns than for livestock. In order to prevent anthrax from dissipating into the natural environment, the following experiment was conducted in an infected room with the walls of a livestock barn temporarily installed. A 50 cm x 50 cm plywood board was placed vertically in the infection chamber, and a 25 cm x 25 cm piece of aluminum foil was attached to it. Attach a 1 cm x 10 cm strip of paper (Toyo Paper No. 2) to the center of this aluminum foil with sellotape, and add 7.5 x spores of Bacillus anthrax _34F2 to this.
0.1 ml of water containing 10 ⁷ /ml was soaked. A short distance away, another strip of paper was soaked with 0.1 ml of a 5% cow feces solution containing 7.5 x 10 ⁷ /ml of B. anthracis 34F ₂ spores. A 5,000-fold diluted solution of the compound (hydrochloride) of the formula was sprayed onto these papers evenly over the entire plywood board. The amount of spray was adjusted to 25ml. Similarly, iodoform, MDBTC preparations, benzalkonium chloride, cresol soap liquid (pharmacopoeia)
A 100-fold dilution of each was sprayed. Immediately after spraying, the cells were placed in heart infusion broth and cultured at 37°C for 48 hours. The results are shown in Table 9.

【table】

[Table] shows that there was no proliferation.
It can be seen that the hydrochloride salt of the compound of the formula is effective at a dilution factor of 5000 times, and its efficacy is not affected even in the presence of cow dung.

Claims (1)

[Claims] 1 formula (In the formula, n is a number indicating the degree of polymerization corresponding to a molecular weight of 700 to 1300) using a drug containing polyhexamethylene biganadine or its salt, poultry house and/or its equipment, characterized by spraying, dispersing or cleaning equipment;
Methods for controlling infectious diseases in livestock animals. 2. The method according to claim 1, wherein the livestock animal is a pig, chicken, or cow.