JP3353063B2 - Inhibitory agent for enterohemorrhagic Escherichia coli O157 carrier cow - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a microorganism belonging to Streptococcus bovis or Lactobacillus galinarum and having an antibacterial inhibiting effect on enterohemorrhagic Escherichia coli O157 (hereinafter referred to as "Escherichia coli O157") carrier cows as an active ingredient. The present invention relates to an antibacterial agent for Escherichia coli O157-bearing cattle characterized by containing

[0002]

[Prior Art] Veterinary drug database published by the Animal Drug Laboratory (http://www.nval.go.jp/yakkou/Y
According to AK056.html), lactic acid bacteria (Lactobacillus) and lactic acid-producing spores (Clostridium) for the purpose of preventing and treating simple diarrhea in domestic animals, treating ruminal abnormal fermentation, and obtaining intestinal effects. Live bacterial preparations for livestock using Bacillus, Enterococcus (Streptococcus), yeast, and the like are commercially available. Also, by Fuller (1992), Prob
iotics, Chapman & Hall (Cambridge), Tomohari Mitsuoka (1990), Enterobacteriaceae, Asakura Shoten, and the like also describe live bacterial preparations using various microorganisms.

[0003] However, the above publications and databases contain Streptococcus bovis for the purpose of preventing the elimination of cattle carrying Escherichia coli O157.
vis) and Lactobacillus g
allinarum) has not been reported.

[0004]

If human E. coli O157, which is a direct public or indirect source of food poisoning of humans due to Escherichia coli O157, which is a major public health problem, can be suppressed or prevented from eradicating Escherichia coli O157, humans can be controlled. It is possible to reduce the risk of infection. The present invention has been made under such a technical background, and Escherichia coli O1
57 It is intended to provide a means for suppressing the elimination of bacteria from a carrier cow.

[0005]

[Means to solve the problem] According to a domestic survey, the carriage rate of Escherichia coli O157 in cattle bred outdoors is 0.62%,
The carriage rate of cattle brought into slaughterhouses is 1.4% and the rate of meat contamination is 0.3% (1996, Ministry of Health and Agriculture survey). A survey in the United States reported that the carriage of cattle was 2.8%, and that the carriage of adults was significantly lower than that of calves [Faith et al. (1996);
pplied and Environmental Microbiology, 1519-1525
Page, Hancock et al. (1994), Epidemiology and Infectio
n, pp. 199-207, Wells et al. (1991), Journal of Clinica
l Microbiology, 985-989]. Also, Cray et al. (199
5), Applied and Environmental Microbiology, 1586
According to ~ 1590, cattle were divided into calves and adult cattle, and E. coli O157
Was experimentally infected and its eradication process was investigated.
It is said that the calf continued to eradicate for a long time while the adult calf often stopped evacuation relatively early.

The present inventors speculated that such a difference in evacuation between calves and adult cows was due to the presence of useful bacteria having a function of inhibiting colonization of Escherichia coli O157 present in the intestinal tract of adult cows. As a result of selecting bacteria derived from bovine rectal stool from such a viewpoint, a strain having a bacterial elimination inhibitory effect was successfully isolated, and the present invention was completed. That is, the present invention relates to an Escherichia coli O157-bearing cattle eradication inhibitor comprising, as an active ingredient, a microorganism belonging to Streptococcus bovis or Lactobacillus gallinarum and having an antibacterial effect against Escherichia coli O157-bearing cattle. is there.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The antibacterial agent of the present invention belongs to Streptococcus bovis or Lactobacillus galinarum and is characterized by containing, as an active ingredient, a microorganism having an antibacterial effect against Escherichia coli O157-bearing cows. Here, "E. coli
The term "microorganism having an effect of inhibiting the excretion of O157-bearing cattle" means a microorganism that has an effect of reducing the excretion of the cow by administration to Escherichia coli O157-bearing cattle.

[0008] The microorganism used in the present invention is not particularly limited as long as it belongs to Streptococcus bovis or Lactobacillus gallinarum and has a bacterial elimination inhibitory effect on E. coli O157-bearing cows. It is preferable to use LCB6 strain) or Lactobacillus galinalum LCB12 strain (hereinafter simply referred to as "LCB12 strain").

The properties of the LCB6 strain are as follows. (1) General characteristics: Gram-positive bacilli, aerobic growth positive, 15
℃ growth negative, 45 ℃ growth positive, lactic acid type L, gas production negative, β hemolytic negative (the above are the results obtained by general bacteriological techniques) (2) Other biochemical properties: acetoin Production positive, hippuric acid hydrolysis negative, esculin positive, pyrrolidonyl allyl amidase negative, α-galactosidase positive, β-glucuronidase negative, β-galactosidase positive, alkaline phosphatase negative, leucine allyl amidase positive, arginine dihydrolase negative, β-hemolytic negative, sugar assimilation; D-ribose negative, L-arabinose negative, D-mannitol negative, D-sorbitol negative, lactose positive, D-trehalose negative, inulin negative, D-raffinose positive, starch positive, Glycogen-positive (The above was obtained by Apistrep 20 from BioMérieux Biotech Japan) Grade)

(3) Comparison of 16S rRNA nucleotide sequences: Mori et al., Int.
ernational Journal of Systematic Bacteriology, 54
And primers for amplifying the 16S rDNA of the bacterium according to the method of 5757 (1997).
The nucleotide sequence of the position region was determined. When a homology search was performed by the FASTA program from the EMBL / GenBank database, 99.413 was found between Streptococcus bovis and
% Similarity was observed (Reference: S. Macedonix)
(S. macedonicus) 94.721%, S.capr.
inus) 94.910%, S. gallolyticus
us) 91.202%, S. dysagalae
ctiae) 90.909%).

The properties of the LCB12 strain are as follows. (1) General properties: Gram-positive bacilli, aerobic negative, 15
℃ growth negative, 45 ℃ growth negative, lactic acid type DL, gas production negative (the above are the results obtained by general bacteriological techniques) (2) Sugar assimilation: glycerol negative, erythritol negative, D -Arabinose negative, L-arabinose negative, ribose negative, D-xylose negative, L-xylose negative, Adonitol negative, 8-methyl-D-xyloside negative, galactose negative, glucose positive, fructose positive, mannose negative, sorbose negative, Rhamnose negative, dulcitol negative, inositol negative, mannitol negative, sorbitol negative, α-methyl-D-mannoside negative, α-methyl-D-glucoside negative, N-acetylglucosamine positive, amygdalin negative, arbutin negative, esculin positive, salicin Negative, cellobiose positive, maltose positive, lactose positive, melibi Over the scan negative, sucrose-positive, trehalose-positive, inulin negative, melezitose negative, raffinose negative, starch negative, glycogen-negative, xylitol negative, gentiobiose negative, D- turanose negative, D- lyxose negative, D- tagatose negative,
D-fucose-negative, L-arabitol-negative, L-arabitol-negative, gluconate-negative, 2-ketogluconate-negative, 5-ketogluconate-negative (The above results were obtained with Api 50 CH and Api 50 CHL manufactured by BioMérieux Biotech Japan) )

(3) Comparison of 16S rRNA nucleotide sequences: Mori et al. (199
7), International Journal of Systematic Bacteriol
ogy, a primer for amplifying the 16S rDNA of the bacterium was prepared according to the method of pages 54 to 57, PCR was performed, and 3
The nucleotide sequence of the region at position 00 was determined. When a homology search was performed using the FASTA program from the EMBL / GenBank database, 99.39 was found between Lactobacillus galinalum.
0% similarity was observed (Reference: El Crispatis)
(L. crispatus) 94.340%, L. acidophilus (L. a.
cidophilus) 91.892%, L. helveticas (L. helvet)
icus) 90.189%, L-acetotolerance (L. acetotol
erans) 89.888%).

The bacterial elimination inhibitor of the present invention may be used in the form of a cultured microbial cell as it is, but it is preferable to add a suitable excipient or stabilizer to prepare a viable bacterial preparation. The microorganisms used are
It can be collected from feces of adult cattle, but LCB6 strain and LC
The B12 strain has been deposited at the National Institute of Bioscience and Biotechnology, Accession No. FERM P-17313 and FERM P-17314, respectively (deposit date: March 16, 1999). Cultivation of microorganisms can be performed according to a conventional method, for example, GAM
Culture can be performed at a temperature of 37 ° C using a commercially available medium such as broth.

The amount of cells contained in the bacterial elimination inhibitor can be appropriately changed depending on the form of the preparation, but when used as a viable preparation, it is preferably 10 ¹⁰ cfu / g.
The dose to Escherichia coli O157-bearing cows can be appropriately selected depending on the concentration of the bacterial cells in the eradication inhibitor, but it is preferable that the dose per cow be 10 ¹¹ cfu. The cow to which the antibacterial agent is administered may be either a calf or an adult cow,
Further, it may be either male or female.

[0015]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. [Example 1] Separation and identification of LCB6 strain and LCB12 strain The bacterial LCB6 strain and LCB12 strain used in the method of the present invention can be separated and identified as follows, for example.

(1) Isolation of 6 strains of LCB and 12 strains of LCB: 3 adult calves about 3 years old (all ♂) and 3 calves about 4 months old
(# 2, # 1) rectal stool was collected, and diluent (A) (monopotassium phosphate) described in Intestinal Infection Study Group (1973), How to Set Up an Infection Model, pages 171-173 (Modern Publishing) 4.5 g, disodium phosphate 6.0 g, L-cysteine hydrochloride 0.5 g
, Tween 80 0.5 g, Agar 1.0 g and purified water 1,000
ml) and serially diluted with BL agar medium (Eiken Chemical)
Was inoculated. After cultivation under anaerobic conditions at 37 ° C. for 3 days, the grown colonies were roughly classified according to their size, color tone and discoloration of the medium due to acid production, and then isolated, and arranged for adult cattle and calves. Among them, (i) the bacterial count is high and predominant in the rectum of adult cattle, but the bacterial count is low or undetectable in the rectum of calves, and (ii) the growth in the artificial medium is good. And (iii) vigorous acid production, and two types of bacteria isolated from the stool of adult cattle were obtained, which were designated as LCB6 strain and LCB12 strain, respectively.

(2) Identification of LCB6 strain and LCB12 strain A commonly used bacteriological technique and a commercially available identification kit, Apistrep 20, Api 50 CH and Api 50
Using CHL (Nippon BioMeryu Biotech Co., Ltd.), the morphological and biochemical properties of the above LCB6 and LCB12 strains were examined, and 16S rRNA which is considered to be useful for identifying bacterial species at the gene level And the homology comparison of the base sequences was performed. As a result, the morphological properties and the like of the LCB6 strain and the LCB12 strain were as described above.

Based on the above-mentioned morphological properties of the LCB6 strain,
Apistrep 20 Profile Index (BioMérieux Vitec Japan) and Colman (1990), Topl
ey &Wilson's Principles of Bacteriology, Virology
and Immunity volume 2 Systematic Bacteriology, 120
~ 159 pages (Edward Arnold, London)
This strain was identified as Streptococcus bovis.

Further, based on the morphological properties and the like of the aforementioned LCB12 strain, Fujisawa et al. (1992), International Journal
of Systematic Bacteriology, pp. 487-491 and API 50
This strain was identified as Lactobacillus galinalum by referring to the CHL profile index (BioMerieux Biotech Japan).

Example 2 Bactericidal Inhibition Test by Oral Administration of Fresh Culture Bacterial Cultures of LCB6 and LCB12 Strains (1) Preparation of Escherichia coli O157 Challenge Strain Resistance to both nalidixic acid and rifampicin drugs from Escherichia coli O157 MN157 The strain was selectively induced and used as an orally infected challenge strain (hereinafter, this challenge strain is simply referred to as "Escherichia coli MN157NR").

(2) Experimental Infection One 210-day-old Japanese black cattle (黒, cattle No. 1) and one 180-day-old Japanese black cattle (♀, cattle No. 2) were used as test cows. . 10 ^10.62 cfu for cow No.1, 10 for cow No.2
Experimental infection was performed by orally administering ^10.49 cfu of a fresh culture of Escherichia coli MN157NR. In cow No.1 and No.2,
In both cases, evacuation of Escherichia coli MN157NR was observed from the first day of the experimental infection, and evacuation continued thereafter.

(3) Measurement of Bacterial Elimination The measurement of the bacterial evacuation was carried out by the following method. [Measurement Method of Bacterial Elimination] After collecting rectal feces of test cows, diluent (A) (4.5 g of monopotassium phosphate, disodium phosphate)
6.0 g, L-cysteine hydrochloride 0.5 g, Tween 80 0.5
g, agar 1.0 g and purified water 1000 ml) to prepare a 10-fold serial dilution series. Nalidixic acid and rifampicin the added sorbitol MacConkey agar to a final concentration of 20 [mu] g / ml respectively (Eiken Chemical, hereinafter referred to as selective medium plate) subjected to quantitative cultured in E. coli MN157NR 10 ^-5, 1
Select 0 ^-3 and 10 ^-1 dilutions for each 0.1 ml, 1
0 ^-1 dilution of each 2 ml was inoculated into five selection plate medium. The detection sensitivity by this method is 1 cfu / g of feces.

When the amount of bacteria is less than 1 cfu per 1 g of feces, the MPN (Most Probable Number) method, 5 methods [Kurata et al. (1977), Microorganism Testing Method for Pharmaceuticals and Cosmetics, 300-305 (Kodansha)] ] Was applied. That is, 10 g, 1 g of feces,
0.1 g each of 100 ml, 10 ml and 10 ml of novobiocin-added mEC
5 sets each of which was added to the medium (Eiken Chemical),
After enrichment culture in novobiocin-supplemented mEC medium at 37 ° C for 18 hours, 0.1 ml of the resulting culture was inoculated into a selective plate medium, and MPN was determined based on the number of novobiocin-supplemented mEC medium in which the growth of E. coli
The values were converted. The detection sensitivity of E. coli MN157NR in this MPN method is
2 cfu / 100 g of feces.

(4) Preparation of fresh cultures of LCB6 and LCB12 strains Both LCB6 and LCB12 strains were cultured in GAM broth (Nissui Pharmaceutical) at 37 ° C. for 18 hours with shaking at 10 ⁸ cfu / ml each. A bacterial solution of a certain degree was prepared. In order to compare the eradication inhibitory effect of the test cattle in the short and long term after the start of E. coli MN157NR eradication,
The following tests were performed.

(5) Escherichia coli MN157NR Bactericidal Inhibition Test Cattle No. 1 contains LCB6 strain and LCB12 prepared in (4) above.
Each 250 ml (10 ¹¹ cfu each) of a fresh culture of the strain was mixed and orally administered once daily for 3 days from the sixth day after the experimental infection using an oral catheter for cattle. On the first day after the start of the administration (7 days after the experimental infection), the amount of E. coli MN157NR shed significantly decreased,
From day 3 onward, eradication was negative (that is, not detected).

In the same manner as cattle No. 1, 2.5 ml (10 ⁹ cfu each) of freshly cultured bacterial liquids of LCB6 strain and LCB12 strain were mixed with bovine No. 2, and for 3 days from 28 days after the experimental infection, It was orally administered once daily using a bovine oral catheter, but E. coli MN157NR was used.
The evacuation volume only decreased temporarily during the administration period. Therefore, the fresh culture fluid of 3 days also LCB6 strain and LCB12 strains 35 days after experimental infection subsequently 10 ¹¹
When cfu was orally administered once a day, the amount of Escherichia coli MN157NR was greatly reduced on the first day after the start of the administration, and was negative on the third and subsequent days.

From these results, fresh cultures of the LCB6 strain and the LCB12 strain were added to each 2.5 ml (10 ⁹ c
fu) administration, the eradication only temporarily decreases, but about 250 ml (10 ¹¹ cfu) per cow
Escherichia coli MN157NR by administering LCB6 strain and LCB12 strain
Was found to be completely prevented. FIG. 1 shows the results of the bacterial elimination test of Example 2.

Example 3 Bactericidal Inhibition Test by Oral Administration of LCB6 and LCB12 Strains as Viable Bacterial Preparations Based on the results of Example 2, the LCB6 strain and LCB12 having the effect of inhibiting E. coli O157 elimination were confirmed. For the purpose of easier administration of the strain to cattle, a bacterial elimination test was carried out using live bacterial preparations in which both bacteria were powdered. As a method for measuring the amount of bacteria to be removed, three MPN methods (detection sensitivity: 3 cfu / 100 g)
The same method as in Example 2 was used except that the method was changed to.

(1) Preparation of Viable Bacterial Preparation Preparation of viable bacterial preparations of LCB6 strain and LCB12 strain was requested to Calpis Co., Ltd. The outline of the preparation method is as follows. The LCB6 strain and the LCB12 strain were each used in MRS broth (pancreatin digest of gelatin 10.0 g, beef extract 8.0 g, yeast extract 4.0 g, glucose 20.0 g, potassium monohydrogen phosphate)
2.0 g, polysorbate 80 1.0 g, sodium acetate 5.0
g, ammonium citrate 2.0 g, magnesium sulfate 0.
2 g, manganese sulfate 0.05 g and purified water 1,000 ml)
After culturing at 37 ° C. for 2 days, the mixture was concentrated by centrifugation at 5,000 g. This was freeze-dried and powdered, and dextrin was added as a shaping / stabilizing agent to obtain a viable bacterial preparation. The amount of bacteria in the viable cell preparation was adjusted to 10 ¹⁰ cfu per 1 g of the preparation powder for both LCB6 and LCB12. Store this drug at 4 ° C or less until immediately before use.

(2) Escherichia coli MN157NR Bactericidal Inhibition Test As test cows, eight Holstein breeds of about 4 months (Cow N
o.1-8: No.1,6,7,8 used ♀, No.2,3,4,5 used ♂). Each test cow was experimentally infected with E. coli MN157NR, which was cultured and prepared in the same manner as in Example 2 (1), by orally administering 10 ^10.11 cfu per cow.

In all test cows, E. coli
Eradication of MN157NR was observed, and evacuation continued thereafter.
Four out of eight test animals (cattle Nos. 5 to 8) became negative for evacuation on the 7th day after the experimental infection, but the other four test cows (cattle Nos. 1 to 4) showed a negative infection. Since evacuation of 10 ² to 10 ⁵ cfu / g was still observed on the 7th day, these four animals were prepared in the above Example 2 (1) for 7 days from the 7th to 13th days after the experimental infection. L
CB6 and LCB12 live bacterial preparations are each 10 g per cow
(10 ¹¹ cfu each) was orally fed twice a day in the morning and evening together with the concentrate.

Rectal stool was collected periodically to examine the effect of the live bacterial preparation on eradication. Escherichia coli after administration of viable bacterial preparation
On the 14th day after the experimental infection, three out of four cows (Cow Nos. 1, 3, and 4) became negative for evacuation of MN157NR.
However, the remaining one cow (Cow No. 2) had 43 to 500 cows / 100 g feces.
Since a very small amount of bacterial excretion was continued, a live bacterial preparation was again administered to all four cattle (Cow Nos. 1 to 4) for 7 days from the 17th to 23rd day after the experimental infection. In the eyes, all four feces were negative for evacuation. Thereafter, the measurement was continued for 3 weeks, but no re-elimination was observed.

Based on these results, the LCB6 strain and LCB12
The same E. coli MN15 as in Example 2 was also obtained by continuously administering the strain as a viable bacterial preparation to cattle together with feed.
The antibacterial effect of 7NR was confirmed. FIG. 2 shows the results of the bacteria elimination test of Example 3. From the results of Example 2 and Example 3, the LCB6 strain and LCB12 strain derived from adult cattle feces were
It was clarified that continuous administration of 10 ¹¹ cfu orally per cow carrying Escherichia coli O157 completely prevented the elimination of Escherichia coli O157.

[0034]

According to the present invention, the elimination of E. coli O157 from a carrier cow, which is a direct or indirect source of human food poisoning, can be suppressed or prevented.

[Brief description of the drawings]

FIG. 1 shows the outline of the eradication-inhibiting effect obtained in Example 2.

FIG. 2 shows the outline of the eradication inhibition effect obtained in Example 3.

Claims (2)

(57) [Claims] [Claim 1] An agent for inhibiting the elimination of enterohemorrhagic Escherichia coli O157-bearing cattle, comprising as an active ingredient Streptococcus bovis LCB6 strain or Lactobacillus galinarum LCB12 strain. 2. The bacterial elimination inhibitor according to claim 1, wherein the preparation is a viable bacterial preparation.