JPS59101425A - Control of infectious disease of livestock - Google Patents

Abstract

PURPOSE:To control infectious diseases of livestock, by using a drug containing polyhexamethylene biguanidine or its salt through spraying, scattering, or washing. CONSTITUTION:0.01-10wt%, preferably 0.03-5wt% solution of a compound shown by the formula (n is polymerization degree corresponding to 700-1,300 molecular weight of the compound ) or its salt (e.g., inorganic salt such as hydrohalide, sulfate, etc. or organic salt such as lactate, tartrate, succinate, etc.) or its fine powder is used by spraying, scattering, or washing, to control infectious diseases of livestock (e.g., swine, chicken, cattle, etc.). Salmonella pullorum, Salmonella typhimurium, pathaogenic Escherichia coli, etc. may be cited as a mold of the cause of the infectious diseases. The active ingredient shows a sterilizing action in 2,000-16,000 times dilution degree by 10-15min sensitization, and sterilizing action on Escherichia coli, etc. will not decrease much even in the presence of fowl droppings.

Description

[Detailed description of the invention] The present invention relates to a method for suppressing infectious diseases in livestock animals.

Recently, mycobacteria disease in pigs has been occurring frequently in various places, and it is becoming a public health problem. The causative bacteria of mycobacterial disease in pigs are atypical mycobacteria such as Mycobacterium smegmatis and Mycobacterium intra7rulare. 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 diseases is slow, it is difficult to recognize any abnormalities in general clinical symptoms (slightly poor growth, and most cases are only discovered during slaughterhouse inspections. Lesions are generally localized to lymph nodes in the head and neck or mesentery, and vary from yellowish gray-white caseating lesions several millimeters in diameter to cases in which the entire lymph node is swollen.Effective for prevention and treatment of this disease. There are no known vaccines or drugs for this disease.Therefore, the only countermeasures are to detect infected pigs as soon as possible using the tuberculin skin reaction, isolate them from other pigs, and thoroughly clean and rinse the pigpen with water before disinfecting it.

However, it is said that no matter what disinfectant is used, it cannot be completely sterilized.

The present inventors have discovered that polyhexamethylene biganadine or its salt has excellent bactericidal activity against Mycobacterium smegmatis and Mycobacterium intracellulare, which are the causative bacteria of this disease, and that It was discovered that, unlike disinfectants, it retains strong bactericidal activity even in the presence of organic matter such as chicken manure.

Furthermore, in chickens, for example, chick dysentery, respiratory diseases, etc.
Colibacillosis, etc. occurs frequently in groups.

The causative bacteria of these infectious diseases are Salmonella prorum,
These include Salmonella typhimirium, Haemophilus gallinarum, Mycoplasma zinovini, 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, infections caused by anthrax bacteria in raw livers have also become a problem.

The present inventors also studied these and found that the polyhexamethylene biganadine or its salt exhibits bactericidal activity at a dilution of 2,000 to 16,000 times upon sensitization to the bacteria for 10 to 15 minutes. It was also found that the bactericidal effect against Escherichia coli and the like 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 uses a drug containing polyhexamethylene biganadine or a salt thereof represented by the formula % formula % (wherein n is a number indicating the degree of polymerization corresponding to a molecular weight of 700 to 1600), This is a method for controlling infectious diseases in livestock animals, which is characterized by spraying, dispersing, or washing.

Compounds of formula ■ are disclosed in U.S. Pat.
No. 428,576, and is known to exhibit antibacterial activity against common bacteria. Also, special public service in 1977-
According to Publication No. 42611, it is also known to be effective against Clostridium botulinum. However, acid-fast bacteria such as Mycobacterium smegmatis, Salmonella prorum,
Salmonella teifimirium, Haemophilus carinarum, Mycoplasma zinovini, pathogenic Escherichia coli,
It exhibits excellent bactericidal activity against anthrax bacteria, and even in the presence of various organic substances such as chicken manure and blood, its bactericidal activity against Mycobacterium smegmatis and pathogenic Escherichia coli does not decrease significantly. It is a new finding by the present inventors that scales can effectively suppress infectious diseases in livestock animals by spraying, dispersing or washing.

On the other hand, other disinfectants such as chlorine, iodine compounds, and antibacterial soaps have a significantly reduced effect on Mycobacterium smegmatis and the like due to the presence of organic substances, and are rarely effective. .

When carrying out the present invention, the compound of formula (1) or a salt thereof is sprayed or scattered on poultry houses, equipment, equipment, etc., or cleaned 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 compound of formula (1) has a molecular weight of 700 to igoo.

Preferably it is 900-1100. Inorganic acid salts such as hydrohalides, sulfates, nitrates, etc. or organic acid salts such as lactates, tartrates, conophosphates, maleates, malonates, curconates, etc. of the compound of formula ■ Since it is relatively well soluble in water and 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 (1) or its salt in the solution or fine powder is 0.01 to 10% by weight, preferably 0.03% by weight.
~5% by weight.

The daily usage amount of the compound of formula I or its salt is, for example, 10 to 1000 m9, preferably 60 to 8 m9 per pig.
00 m9, 1 to 100711 g per 11 chickens, preferably 6 to 80 m7, 0.1 to 10 I per cow, preferably 0.3 to 8 I. Spraying or spraying or washing with this spraying is usually carried out 1 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 60 seconds to 60 minutes per time.

Example Mycobacterium smegmatis IFO'3153, a type of atypical mycobacteria (not pathogenic to humans),
/]・Medium (1-e3 is obtained by dissolving 10 g of potassium dihydrogen phosphate and 10 g of sodium glutamate in distilled water and sterilized at 100°C for 60 minutes. To 100 ml of this solution, add 6 ml of glycerin and 6 ml of 2% malachite green solution.
Add 200 ml of whole eggs and mix, filter through gauze, dispense, and prepare a slant in a serum coagulator. On the first day, heat at 85°C for 40 minutes, and on days 2 and 6 at 80°C. (use after sterilizing for 40 minutes), incubate at 67℃ for 5 minutes,
Scrape off the bacteria that has grown on the whites of your eyes with a platinum looper.

The number of bacteria in sterile saline in an agate mortar is Z0 x 107
cells/ml (measured by plating and culturing in the culture medium after preparation). This bacterial solution was used as an inoculum solution.

A predetermined amount of each disinfectant was added to 2 ml of Dubos liquid medium, and 2 drops (approximately 0.013 ml) were added using the above-mentioned bacterial syringe, mixed well, and then cultured at 67°C for 5 days.
The minimum inhibitory concentrations of various disinfectants against Mycobacterium smegmatis were determined.

As a disinfectant, the hydrochloride of the compound of formula (molecular weight 900
~1100), as well as benzalkonium chloride, cresol soap liquid (Japanese Pharmacopoeia), and iodophor (
A formulation containing 1.75% available iodine and 11% nonionic active agent) and a methyldodecylbenzyltrimethylammonium chloride formulation (abbreviated as 1viDBTC formulation) were used.

The test results are shown in Table 1.

Dubos liquid medium: 1.6 g of potassium dihydrogen phosphate, 2.2 g of sodium hydrogen phosphate, 2 g of asparagine, 5.2 g of peptone, 0.1 g of magnesium sulfate, 0.01 g of iron ammonium citrate, 0 °o o o o of zinc sulfate. i
y, copper sulfate 0.0001g, calcium chloride o, oo
osg, malachite green o, o.

2g and 0.5.9g of polyoxyethylene sorbitan monooleate were dissolved in distilled water to make a solution of 900ml.
and sterilize at 121°C for 15 minutes. This includes 5g of glycerin, 7.5g of glucose, and food! , 100 g of a solution of 0.9 g and 5 g of bovine albumin in sterile distilled water.
Add m1 and mix to prepare.

Table 1. The results show that the hydrochloride of the compound of formula I exhibits extremely superior antibacterial activity against Mycobacterium smegmatis compared to other disinfectants.

Examples Mycobacterium smegmatis cultured in Ogawa medium according to the method of Experimental Example 1 was suspended in sterile physiological saline at a bacterial count of 1.9 x 108/ml. The bactericidal effect against this bacterium was measured. As a disinfectant, hydrochloride of the compound of formula (molecular weight 900-11
00), benzalkonium chloride, Fresol soap solution (Japanese Pharmacopoeia), iodopol (preparation containing 1.75% available chlorine and 11% nonionic surfactant), and phenol were used as comparative drugs.

Dilute the disinfectant by 2-fold serial dilution using sterile distilled water to prepare 5 to 6 dilutions, each with 10 m
Each sample was dispensed into sterile test tubes and left in a 20°C water bath for 10 minutes. Each of these drug dilutions was inoculated with 0.1 ml of the bacteria A prepared in advance, stirred well, and then placed in a 200G water bath again. 25 minutes, 5 minutes, 10 minutes, and 15 minutes (each sensitization time) after inoculating the bacterial solution,
0.1 ml of each diluted drug solution for bacterial inoculation was collected with a sterile pipette and dropped onto the surface of Ogawa medium.

The samples were then cultured at 67° 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 compound No. 2, and Table 6 shows the highest dilution factor that inhibited bacterial growth at each sensitization time by comparing various disinfectants.

Table 2 Indicates that the

Table 6 When sensitized with the drug for 15 minutes, the highest dilution that inhibited bacterial growth was 1:8000 for the hydrochloride of the compound of formula I, 1:600 for iodophor, and 4 for penzalkonium chloride.
1[1x, RiL/ Sole soap liquid was 160x1, phenol was 80x. For reference, the phenol coefficient of various disinfectants against Mycobacterium smegmatis is found to be 100 for the hydrochloride of compound No. 1, 17.5 for iodophor, Z5 for benzalkonium chloride, and 6 for cresol soap solution. Ta. Therefore, it is clear that the hydrochloride of the compound of formula I exhibits an excellent bactericidal effect against Mycobacterium smegmatis, an atypical acid-fast bacterium, which is not seen in other disinfectants.

Example Chicken manure (nitrogen content 2.9%, moisture 10°8%) in a sterile test tube
Add 4.9 me of distilled water diluted to 10%,
Add 0.1 ml of the bacterial solution obtained in Experimental Example 1 to this and mix thoroughly to make 5 ml. Separately, each disinfectant used in Experimental Example 2 was diluted to a multiple of 1/2 of each measured dilution factor.
ml is added to the above chicken manure liquid. After 2.5 minutes, 5 minutes, 10 minutes, 15 minutes, and 60 minutes (each sensitization time), 0.1 ml of each solution was collected and cultured in fresh 1% Ogawa medium. After culturing at 67° 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 4 Note: -: No bacterial growth was observed; +: 20 or less bacterial colonies were observed; ox: 20 or more bacterial colonies or bacterial growth was observed close to the entire surface of the medium. 41] indicate that bacterial growth was observed on the entire surface of the medium.

In the presence of 5% chicken manure, the hydrochloride of the compound of formula (■) showed a bactericidal effect when diluted 1000 times and sensitized for more than 5 minutes, but benzalkonium chloride showed a bactericidal effect when diluted 100 times and for more than 60 minutes, and when diluted 50 times. A sensitization time of 10 minutes or more was required. The cresol soap solution required sensitization time of 15 minutes or more when diluted 160 times, and 5 minutes or more when diluted 80 times. Iodophor showed a bactericidal effect when diluted 10 times, but was ineffective when diluted 25 times or more, and the color of iodine had completely disappeared. Phenol showed a complete bactericidal effect when diluted 40 times, but sensitization time of 15 minutes was required when diluted 80 times. For reference, the phenolic coefficients of various disinfectants against Mycobacterium smegmatis in the presence of 5% chicken manure are 25 for the hydrochloride of the compound of formula I, 1.6 for benzalkonium chloride, and 2 for cresol soap. , iodophor is 0.29,
It was confirmed that the fungicide of the present invention has an extremely excellent bactericidal effect even under such conditions.

Example: Each disinfectant was diluted with distilled water to the desired multiple by 2-fold serial dilution (phenol was diluted in 10-fold increments (50 to 100 times))
, each dilution e, 10 m/! 1 m and 1 each of Salmonella prorum and Pseudomonas aeruginosa 647 cultured in non-infusion broth and spore liquid of Bacillus anthracis 64F2 are placed in the solution and mixed. The number of bacteria in each inoculated bacterial solution is 6.9 x 1087 m1.1.3 x 1087 m
1 and 7.5 x 108/ml. Then 0.
After 5 minutes, 2.5 minutes, 5 minutes, 10 minutes and 15 minutes (each sensitization time), one loopful of the fluid in the working test tube is transferred to 10 ml of fresh heart infusion broth. Note that the sensitization temperature was 20°C. After 48 hours of incubation at 37°C, the presence or absence of bacterial growth was determined for Salmonella, Prorum, and Pseudomonas aeruginosa 64 for each disinfectant.
The bactericidal effect on spores of B. 7 and Bacillus anthracis 64F2 was investigated. 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 500
0x, iodophor at 250x, benzalkonium chloride at 5000x, MDBTC formulation at 1000x, cresol soap solution at 100x, and phenol at 80x. For reference, the phenolic coefficients of various disinfectants against Salmonella prolumum are 59 for the hydrochloride of the compound of formula (■), 4.4 for iodophor, 59 for benzalkonium chloride, and 17. for the lA D B T C preparation. 6. The cresol soap solution was 1.2. Therefore, it is clear that the hydrochloride of the compound of formula (1) exhibits excellent bactericidal activity against Salmonella prolumum.

Table 5 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 formula (1), 200 times for iodophor, and 4 times for benzalkonium chloride.
00 CJ times, MDBTC preparation 800 times, cresol soap liquid 100 times, and phenol 80 times. For reference, the phenol coefficients of various disinfectants against Pseudomonas aeruginosa are 100 for the hydrochloride of the compound of formula (■), 3.8 for iodophor, 50 for benzalkonium chloride, and MDBTC.
The rating was 7.5 for the formulation and 1.9 for the cresol soap solution.

Table 6 Table 7 shows the bactericidal effects of various disinfectants on anthrax spores.
Shown in the table. When the drug was sensitized for 15 minutes, the highest dilution that inhibited bacterial growth was 800
0x, iodophor 50x, benzalkonium chloride 1000x, MDBTC formulation 100x, and cresol soap liquid 5x or less. Although it is effective against spores, it is not used for livestock farming because it is a mercury agent.
Bacterial growth was suppressed at 0x.

Table 7 Examples Chicken manure (nitrogen content 2.9%, moisture 10°6%) in a sterile test tube
5 m of distilled sphagnum moss containing 2% and 10% of
1 of pathogenic Escherichia coli isolated in the field and cultured for 24 hours in no-infusion broth.
m6 (bacteria count 1.0 x 1 oQ/fnl) and mix. Separately, a solution is prepared by mixing 5 ml of distilled water instead of the chicken manure solution with 1 rnl of the bacterial solution. Add 5 ml of a solution obtained by diluting the hydrochloride salt of the compound of formula (1) (molecular weight 900 to 1100) to a multiple of 1/2 of each measured dilution factor to the above-mentioned canned solution. Then 2.5 minutes, 5 minutes, 10 minutes, 15 minutes and 60 minutes
After a few minutes of incubation, the bactericidal effect of the hydrochloride of the compound of formula (1) on pathogenic Escherichia coli was examined based on the presence or absence of bacterial growth. The results are shown in Table 8.

The hydrochloride of the compound of formula (■) is 2000 times more effective against pathogenic E. coli after 2.5 minutes of sensitization, 4000 times more effective after 5 minutes, and 8 times more effective after 60 minutes.
A bactericidal effect was observed at dilutions up to 1,000 times. Even if chicken manure is present as an organic substance at 1%, the sensitization time is 10 minutes and 4000 times greater. Even if chicken manure is present at 5%, the sensitization time is 2.5 minutes and 2000 times greater.
A bactericidal effect was observed at a dilution factor of 0 times.

Table 8 Note: + indicates that the bacteria have grown, and - indicates that the bacteria have not grown.

Example 1 A parent pig infected with acid-fast bacteria (positive tuberculin reaction, nodule-like symptoms observed in the head and neck lymph nodes, lymph fluid was collected from the area, cultured in Ogawa medium, and examined for atypical anti-acid bacteria) Four pigs (from which the bacteria were isolated) were obtained, and two pigs were placed in separate pig pens and reared for 7 days. One pig pen was treated with a solution of hydrochloride (molecular weight 900 to 11 oo) o, i weight% of the compound of formula (■) for one day. After spraying 4 times for 5 minutes each time and disinfecting for 6 days, 10 healthy young pigs were
Both heads were placed in a pigpen and raised for six months. During breeding, the above-mentioned disinfection was carried out every day, and the breeder disinfected his hands with the above-mentioned disinfectant every day. The amount of spray liquid is 1.9 koku per day, and the amount of hydrochloride of the compound of formula ■ used per pig is approximately 160 koku per day.
rv1 day. Another pigsty was disinfected as described above for 6 days, and then 10 healthy young pigs were placed in the pigsty together, and thereafter they were raised for 6 months without any disinfection as described above, except for the disinfection of the handlers' hands.

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 observed in the mesenteric lymph nodes in 4 out of 10 adult pigs in pigpens that were not disinfected, whereas there were no lesions at all in adult pigs in pigpens that were disinfected. It was not recognized and showed good growth.

Example 2 200 chickens for meat were kept in separate chicken houses. The chicken house on the i side uses the hydrochloride of the compound of formula (molecular weight 9
00-1100) After cleaning the floor in advance with a 0.1% by weight solution, spray the same solution twice a day for 5 minutes each time,
Disinfected for 6 days. The disinfection described above was carried out daily during breeding, and the caretakers disinfected their hands daily with the disinfectant solution described above.

The amount of spray liquid is 6.0 koku per day, and the amount of hydrochloride of formula ■ compound used per chicken is approximately 60 mL? / day. In another poultry house, the chickens were kept for two months without any disinfection other than the hands of the breeders.

Sixty chickens died in a poultry house where the above disinfection was not performed during rearing. Pathogenic E. coli was isolated from the liver, heart, or spleen of 19 of them. 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 6 Anthrax caused by infection with Bacillus anthracis is a legal infectious disease for livestock, and livestock carrying the disease must be killed immediately upon discovery. Therefore, the hydrochloride of the compound of formula ■ is
In the case of Bacillus anthracis, it is more suitable for disinfecting livestock barns than for livestock.

In order to prevent the anthrax bacteria from dissipating into the natural environment, we conducted the following experiment by temporarily constructing the walls of the livestock barn in an infected room.

A 50 CTL x 5 (11 cm) plywood board was vertically placed in the infection chamber, and a 25 Crn x 25 C1n aluminum foil was attached to it.

A piece of F paper (Toyo p Kamiya 2) cut into a 1cm x 10CrfL rectangle was attached to the center of the aluminum foil with sellotape, and 7.5 x spores of Bacillus anthrax 64F2 were added to it.
0.1 ml of water containing 107/ml! I let it soak in. Anthrax 64F2 on another rectangular sheet of paper a little apart.
0.1 ml of 5% cow dung solution containing 7.5 x 1077 ml of spores was soaked in the tube. A 5000-fold diluted solution of the compound of formula (1) (hydrochloride) was sprayed uniformly over the entire plywood board onto these filter papers. The amount of spray was adjusted to 25 ml.

Similarly, Bordholm, 1vlD BTC preparation, benzalkonium chloride, cresol soap solution (pharmacopoeia) 100 each
The diluted solution was sprayed.

Immediately after spraying, the cells were placed in heart infusion broth and cultured at 67°C for 48 hours.

The results are shown in Table 9.

Table 9 Note: + indicates that anthrax bacteria have proliferated, and - indicates that anthrax bacteria have not proliferated.

It can be seen that the hydrochloride salt of the compound of formula (1) is effective at a dilution factor of 5,000 times, and its efficacy is not affected by the presence of cow dung.

Applicant Ueno Pharmaceutical Co., Ltd. Agent: Patent Attorney Masao Kobayashi

Claims (1)

[Claims] 1. Using a drug containing polyhexamethylene biganadine or a salt thereof represented by the formula (wherein n is a number indicating the degree of polymerization corresponding to a molecular weight of 700 to 1600) A method for controlling infectious diseases in livestock animals, which comprises spraying, dispersing or washing. 2. The method according to claim 1, wherein the livestock animal is a pig, chicken, or cow.