MXPA00001497A - Control of enterohemorrhagic e. coli - Google Patents

Abstract

Dominant probiotic bacterial strains have been isolated. The isolated bacterial strains fed to a ruminant animal are able to prevent the establishment of the pathogen i(E. coli) O157:h7 in the animal. The strains can be re-isolated from the gastrointestinal tract contents of inoculated animals at about 28 days post-inoculation. Animals previously infected by i(E. coli) O157:h7 can be treated to reduce or eliminate the pathogen by administering any of the dominant probiotic bacteria of the invention.

Description

CONTROL OF E. COLI 0157.H7 ENTEROHEMORRAGICO IN LIVESTOCK FOR PROBIOTIC BACTERIA BACKGROUND OF THE INVENTION E. coli 0157: H7, a major human pathogen that causes hemorrhagic colitis and haemolytic uraemic syndrome, has been reported frequently increased during the past decade as a cause of human disease [for reviews, see Bell. P.B. and collaborators (1994) JAMA 272: 13 9-1353; _Griffin, P.M. and collaborators (1991) Epi demi ol. .Rev. 1_3: 60-98; Padhye, N..Y. and collaborators (1992) J. Food Pro t. 55: 555-565]. Cattle, especially young animals, have been implicated as a major reservoir of E. coli 0157: H7, with undercooked ground meat that is a major vehicle of outbreaks of disease caused by food. Also, the number of outbreaks related to fruits, fruit juices, vegetables (lettuce), and water (including recreational lakes) has increased dramatically in recent years. A recent national surveillance conducted by the National Animal Health Monitoring System of the United States revealed that 1.6% of REF. : 32508 feedlot scattered fecally E. col i 0157: H7 and 0.4% scattered E. col i 0157: NM [Dagatz, D. (1995) USDA: APHIS: VS, Centers for epidemiology and animal health. Fort Collins, CO (personal communication)]. A larger study of calves in dairy farms revealed that 1.5% that were between the weaning age and 4 months spread E. col i 0157: H7 in their stools [Zhao, T. et al. (1995) Appl. They sent. My crobi ol. 61: 1290-1293], Experimental infection of calves and adult cattle with E. coli 0157: H7 varies widely among animals of the same age group, but persists more in calves than in adults, and previous infection does not prevent reinfection by the same strain of E. coli 0157: H7 [Cray, C. and collaborators (1995) Appl. They sent. My crobi ol. 61: 1585-1590]. Other animals, such as chickens, deer and sheep have also been determined to have the ability to carry E. coli 0157: H7 for a prolonged period of time. Many public health interests have emerged regarding the contamination of food by E. coli 0157: H7. Such interests have been emphasized by the unique acid tolerance of E. coli 0157: H7. Proper cooking is an effective method to kill E. col i 0157: H7 in foods.

However, unsanitary practices in food preparation often result in food-borne illness, and therefore in reducing or eliminating the burden of E. coli 0157: H7 in cattle, it is necessary to reduce public exposure to the pathogen in food and the environment [ang G. et al., (1996) Appl. They sent. My crobi ol. 62_: 2567-2570]. Vaccination has been the traditional procedure to protect livestock from carrying dangerous bacteria. However, E. col i 0157: H7 does not adhere to or infect livestock. The main location sites of E. coli 0157: H7 in calves, are the rumen and the colon. The rumen seems to be the most important site for the long-term carrying of E. coli 0157: H7 and can serve as the source of bacteria found in the colon [Brown, C., et al., (1995) Ve t. Pa th ol. 32: 587], Histological examination of colon tissue revealed no evidence of E. col i 0157: H7 to colon tissue. Therefore the presence of E. coll 0157: H7 in the colon appears to be a transient state through which the bacteria is passing through, rather than colonizing the colon. The vaccines are not likely to be effective in reducing the amount of E. coli 0157: H7 carried and spread by livestock. The amount of E. coli 0157: H7 carried by calves can be affected by nutrition and management practices. Rasmussen et al [(1993) FEMS Microbiol. Lett. 114: 79-84] determined that E. coli 0157: H7 developed unrestricted in the rumen fluid collected from fasted cattle. Some strains of E. coli can produce colicins that inhibit in vitro diarrheogenic E. coli strains, including strains of serotype 0157: H7 [Bradley D.E., et al., (1991) Can J. Microbiol. 3_7: 97-104; Murinda S.E. and collaborators, (1996) App. Environ Microbio! . 62: 3196-3302]. Murinda et al. Tested forty-eight colicin-producing strains of E. coli, and determined that all strains of E. coli 0157: H7 evaluated were sensitive to Col El to E8, K and N on agar containing itomycin C, and also to Col G, Col H and MccB17 on Luria agar. The patterns of sensitivity to colicin and resistance have been used for the identification of the strains. The biological control of a bacterial strain based on its bacteriocin sensitivity has not been achieved.

However, Doyle et al., Patent North American No. 5,302,388, described the prevention or inhibition of colonization of Campyl oba c t er j ej uni of the poultry by administering a select bacterial strain capable of competing with C. j ej uni to colonize sites in the caecum of poultry and to inhibit the development of C. j ej uni.

BRIEF DESCRIPTION OF THE INVENTION The present invention includes the specific strains of E. probiotic coli, its isolation, characteristics and methods of use to prevent or treat the carrying of E. col i 0157: H7 by a ruminant animal. By "probiotic" are meant the bacteria that have the property of preventing the establishment of E. col i 0157: H7 in a ruminant animal previously administered with an effective dose of said probiotic bacteria. For example, calves first administered with an effective amount of a strain of probiotic bacteria, and then subsequently administered with E. coli 0157: H7 do not become carriers of E. col i 0157: H7, while calves administered only with E. coli 0157: H7 continue to carry the strains for weeks, spreading the bacteria to the environment in the feces. Of eighteen probiotic strains isolated in the present study, four were identified as 'dominant', which means that they could be re-isolated from the contents of the gastrointestinal tract of animals inoculated at approximately 28 days after inoculation. the dominant probiotic bacteria are capable of reducing or eliminating E. coli 0157: H7 from calves previously inoculated with E. coli 0157: H7 and carrying the pathogen.The invention therefore provides a method to prevent the carrying of E. coli 0157: H7 by a ruminant, through the step of administering an effective amount of a strain or combination of strains of probiotic bacteria to the ruminant prior to exposure to E. coli 0157: H7.The method is especially useful for treating young ruminants , such as bovine calves, at an early age, before exposure to E. coli 0157: H7, which may be present in the environment. The method is also useful to prevent animals on a feedlot being contaminated at the feedlot. The invention further provides a method for reducing or eliminating E. coli 0157: H7 of a ruminant, to administer an effective amount of a strain or combination of strains of dominant probiotic bacteria. The method is useful to keep herds of cattle free of E. coli 0157: H7 and reduce the portability of E. col i 0157: H7 before slaughtering. The administration of probiotic bacteria is carried out by the administration of a food supplement or additive comprising an effective amount of probiotic bacteria, or by supplying an additive or inoculum of treatment in water to the drinking water of the animals. The invention therefore provides a food supplement composition comprising probiotic bacteria, and a water additive comprising probiotic bacteria. The invention further includes a method for isolating probiotic bacteria, which includes the selection of the supernatant for development of the isolating strains of the stool or intestine, for the ability to inhibit the development of E. coli 0157: H7 on agar. A method for identifying a dominant probiotic bacterium includes the step of inoculating a ruminant animal with a mixture of strains of probiotic bacteria, then isolating the strains of the animal after a defined period of time, for example, of about 4 weeks. Reisolated strains are those that have persisted successfully in the animal, hence the term dominant probiotic bacteria. The reisolation and identification of dominant probiotic strains can be facilitated by the use of marker characteristics, whether endogenous, selected or manipulated by genetic engineering which allow each strain to be identified and / or selected from a mixture. The methods for the isolation of probiotic bacteria, for the isolation of dominant probiotic bacteria, to prevent the contribution of E. col i 0157: H7 and to treat an animal to reduce or eliminate E. coli 0157: H7 of the animal, are all applicable to other animals besides cattle, especially other ruminants, which have been observed to carry E. coli 0157: H7. To date, the cattle are the most numerous carriers of E. coli 0157: H7. The methods of isolation and use of the dominant probiotic strains are achieved by studies carried out with livestock. A large number of strains are available from natural sources, which can meet the criteria of probiotic and dominant probiotic bacteria. The repeats of the isolation process described herein may yield the same or different strains as those described herein. Such strains fall within the general categories of probiotic and dominant probiotic bacteria, as described herein.

DESCRIPTION OF THE DRAWINGS Figure 1 is a graph - showing the destination of E. coli 0157: H7 in the calf rumen fluid to which only E was administered. col i 0157: H7. The arrow indicates the detection of E. col i 0157: H7 only by an enrichment procedure. Figure 2 is a graph showing the destination of E. col i 0157: H7 in feces of calves to which only E was administered. coli 0157: H7. The arrow indicates the detection of E. col i 0157.H7 only through an enrichment procedure. Figure 3 is a graph showing the destination of E. coli 0157: H7 in the rumen fluid of calves given probiotic bacteria and two days subsequently E. col i 0157: H7. The arrow indicates the detection of E. coli 0157: H7 only by an enrichment procedure. Figure 4 is a graph showing the destination of E. coli 0157: H7 in calf feces that were given probiotic bacteria and two days subsequently E. coli 0157: H7. The arrow indicates the detection of E. col i 0157: H7 only by an enrichment procedure. Figure 5 is a graph of the destination of E. col i 0157: H7 in the rumen fluid of calves to which E was administered. coli 0157: H7 only. The arrow indicates the detection of E. coli 0157: H7 only by an enrichment procedure. Figure 6 is a graph of the destination of E. col i 0157: H7 in feces of calves to which E was administered. coli 0157: H7 only. The arrow indicates the detection of E. coli 0157: H7 only by an enrichment method. Figure 7 is a graph of the destination of E. col i 0157: H7 in the rumen fluid of calves treated with dominant probiotic bacteria, 1 to 3 days after administration of E. col i 0157: H7. The arrow indicates the detection of E. col i 0157: H7 only by an enrichment procedure. Figure 8 is a graph of the destination of E. coli 0157: H7 in feces of calves treated with the dominant probiotic bacteria, 1 to 3 days after administration of E. col i 0157: H7. The arrow indicates the detection of E. col i 0157: H7 only by an enrichment procedure. Figure 9 describes the digestions with Xbal of the genomic DNA of the dominant probiotic bacteria by pulsed-field gel electrophoresis. The left and right bands are the stairs? (48.5 kb). Bands 1 to 8 are strain 271. Figure 10 describes the Xbal digestions of the genomic DNA of the dominant probiotic bacteria by pulsed-field gel electrophoresis. The left and right bands are the stairs? (48.5 kb). Band 1 is strain 786, and bands 2 through 6 are strain 797.

DETAILED DESCRIPTION OF THE INVENTION Histological examination of colonic tissue has not revealed evidence of E adhesion. col i 0157: H7 to bovine colonic tissue. The rumen seems to be the most important site for the long-term carrying of E. col i 0157: H7. The presence of the 0157-H7 bacteria in the colon is considered to be a transient state, with the bacteria passing through from the source of the rumen and being scattered in the feces. Animals can be initially infected by ingestion of contaminated grass, feed or water. The results of the present study show that E. coli 0157: H7 persists in the regimens of untreated calves up to 30 days after a single inoculant dose, and scattered in the feces throughout the same period of time. The 057: H7 bacteria are therefore carried by the cattle along with any other microbial strains that inhabit the digestive tract of the animals. For purposes of this invention, cattle and other animals in whose tissues or feces can be detected E. col i 0157: H7 is said to carry E. col i 0157: H7. The amount of E. col i 0157: H7 carried by each animal, is measurable in different ways, including taking samples from various tissues. More conveniently, the presence of E. coli 0157: H7 can be measured in stool. Such measurement is of practical importance, since fecal contamination is the apparent source of contamination of the meat and also of the reintroduction and infection of other animals. As shown herein, the amount of S. coli 0157: H7 scattered in the stool is reflected in the amount measurable in the rumen. Therefore, the amount of E. col i 0157: H7 in feces is a measure of the amount carried by the animal. The quantitative measurement of E. coli 0157: H7 is expressed as colony forming units (CFU) per gram of faeces or per milliliter of rumen. * Probiotic "is used in the present as an adjective to describe bacteria isolated from a natural source and which has the property of inhibiting the development of E. coli 0157: H7.The test of an inhibition used in the present was a test in vi On a solid medium in which the culture supernatants of the isolated candidate bacteria were observed for their property of inhibiting the development of E. coli 0157: H7 when applied to the surface of the solid medium, typically an impregnated paper disc with the culture supernatant of a candidate strain was placed on the surface of an agar plate seeded with E. coli 0157: H7.The probiotic bacterial supernatants caused a ring of clear agar or of reduced growth density, indicating the inhibition of E. coli. coli 0157: H7 in the vicinity of the disc There are other tests for inhibition that are available or could be considered, including competency tests and direct growth, vi n vi o vi n vi, which can generate a panel of probiotic bacteria similar to those described herein. The bacterial strains identified by such a test are within the category of probiotic bacteria, as the term is used herein. The term "dominant probiotic" is applied in probiotic bacteria that persist in, and are reaisable from, an animal to which the bacteria have been administered.The criterion used in the work described here was reisolation 26 days after inoculation The bovine calves were fed with a mixture of 18 probiotic strains, then, from a variety of tissues, samples of digestive contents and feces were taken 26 days after inoculation Four strains were retrievable, designated probiotic strains Other criteria may be employed, including shorter or longer periods of time between inoculation and sampling.It is advisable to choose a sufficiently long period of time, so that the persistence of the dominant probiotic strains can provide useful reduction of the amount of E. coli 0157: H7 carried by the animal The isolation of the bacteria Dominant probiotics can be carried out by those of ordinary skill in the art, following the principles and procedures described herein. Of 1200 isolated colonies of feces and cattle tissues, 18 were probiotic and 4 were dominant probiotics. Therefore, testing similar numbers of independent isolates is likely to reasonably and successfully produce probiotic dominant bacteria. The administration of dominant probiotic bacteria can be carried out by any method that probably introduces organisms into the digestive tract. Bacteria can be mixed with a carrier and applied to liquid or solid food or drinking water. The carrier material must be non-toxic to bacteria and to the animal. Preferably, the carrier contains an ingredient that promotes the viability of the bacteria during storage. The bacteria can also be formulated as an inoculant paste to be directly injected into the animal's mouth. The formulation may include added ingredients to improve palatability., improve shelf life, impart nutritional benefits, and the like. If a reproducible and measured dose is desired, the bacteria can be administered by a rumen cannula, as described herein. The amount of dominant probiotic bacteria that will be administered is governed by factors that affect efficacy. In the present study, 1010 CFU were administered in a single dose. Lower doses may be effective. When it is administered in food or drinking water, the dose can be spread over a period of days or even weeks. The cumulative effect of lower doses administered in several days may be greater than a single dose of 1010 CFU. By periodically checking the numbers of E. col i 0157: H7 in faeces before, during and after administration of the dominant probiotic bacteria, those skilled in the art can easily assess the level of dose necessary to reduce the amount of E. coli 0157: H7 carried by the animals. One or more strains of dominant probiotic bacteria can be co-administered. A combination of strains may be advantageous, because the individual animals may differ in the strain that is more persistent in a given individual. The dominant probiotic bacteria can be administered as a preventative, to prevent animals that do not currently carry E. coli 0157: H7 acquire the strain by exposure to other animals or to environments where E is present. col i 0157: H7. Young calves and mature animals coming to be transferred to a new site, such as a feedlot, are attractive candidates for preventive management. The treatment of animals that carry E. col i 0157: H7 can be carried out to reduce or eliminate the amount of E. coli 0157: H7 carried by the animals, by administering a dominant probiotic bacteria to animals infected with E. col i 0157: H7. The animals that are known are dispersing E. col i 0157: H7 in the stool, or those produced where it is known that E. coli 0157: H7 exists, are suitable candidates for treatment with dominant probiotic bacteria. The methods for administering dominant probiotic bacteria are essentially the same, either for prevention or treatment. Therefore, the need to determine first if E. coli 0157: H7 is being carried by the animals, it is eliminated. By routinely administering an effective dose to all animals in a herd, the risk of contamination by E. col i 0157: H7 can be substantially reduced or eliminated by a combination of prevention and treatment.

EXAMPLES Example 1. Isolation of Probiotic Bacteria Probiotic bacteria were isolated from cattle feces or from gastrointestinal tissue of cattle (intestine and colon). Faecal samples were collected from the cattle that were confirmed as negative for E by faecal test. col i 0157: H7. Fecal samples were serially diluted (1:10) in 0.1 M phosphate buffer, pH 7.2 (PBS) and 0.1 ml of each dilution was placed in plates, on sorbitol-MacConkey agar plates (SMA). The plates were incubated for 16 hours at 37 ° C and up to 10 colonies were randomly selected and each transferred to a test tube containing 10 ml of chicken broth Tripticase (TBS) [(BBL, Becton Dickinson, Cockeysville, MD). The cultures were incubated for 16 hours at 37 ° C. The tissue samples (1 g each) were homogenized (Ultra-Turrax Homogenizer T25, Janke &Kundel IKA-labortechnik, Germany) at 9,500 rpm for 1 minute and then 0.1 ml portions were plated on the surface of the plates. of SMA. The plates were incubated for 16 hours at 37 ° C. Up to 10 colonies were each transferred to test tubes containing 10 ml of TSB and incubated for 16 hours at 37 ° C. The supernatant fluid from each culture was sterilized by filtration (0.2 μm cellulose acetate membrane, Nalgene Co., Rochester, NY).

Example 2. Crop Selection for Anti-i Properties ?, coli 0157.H7 A mixture of 5 strains of E. coli 0157: H7, including strains 932 (human isolate), C7927 (human isolate), E009 (meat isolate), E0018 (cattle isolate) and E0122 (cattle isolate) was used to select culture supernatants for anti-J? metabolites. coli 0151: The.

Approximately 107 E. coli 0157: H7 from approximately equal populations of each strain in 0.1 ml were plated onto surface in SMA and TSA in duplicate. A disc (12 mm in diameter) was placed on the surface of each SMA and TSA plate and 0.1 ml of sterile supernatant was filtered by a simple culture and applied to the disc surface. In addition, a disc with 70% ethanol (positive control) and a disc with PBS (negative control) were applied to each plate. The cultures were incubated for 18 hours at 37 ° C and observed for the zones of inhibition. A clear area of more than 1 mm was considered a positive response.

Example 3. Preparation of Col cultures i 0157: H7 The same mixture of 5 strains described above was used. To facilitate the counting of these bacterial isolates, the strains were induced for resistance to nalidixic acid (50 μg / ml). Each strain of E. coli 0157: H7 resistant to nalidixic acid was transferred to 10 ml of tryptic soy broth (TSB) containing nalidixic acid (50 μg / ml) and incubated for 16-18 hours at 37 ° C with shaking (150 rpm). A suspension of 2 ml of each isolate was transferred to 300 ml of TSB. After incubation at 37 ° C for 16 to 18 hours, the bacteria were pelleted by centrifugation (4,000 x g for 20 minutes) and washed 3 times in PBS. The PBS was added to the sedimented bacteria in an amount necessary to obtain an optical density (O.D.) of 0.5 to 630_ nm (108 CFU / ml). A mixture of the 5 isolates (2 x 109 CFU of each strain) of E. col i 0157.H7 was mixed in 250 ml of 2% sterilized skim milk just before the oral inoculation of the calves. Bacterial populations were confirmed by counting on TSA and SMA plates containing nalidixic acid (50 μg / ml, SMA-NA).

Example 4: Preparation of Probiotic Bacteria All probiotic bacterial isolates were selected for resistance to nalidixic acid (50 μg / ml) for ease of counting in feces. The bacteria were individually developed in 10 ml of TSB containing nalidixic acid (50 μg / ml). A 1 ml portion of each isolate was transferred to 100 ml of TSB. After incubation at 37 ° C for 16 to 18 hours, the bacteria were sedimented by centrifugation, washed, and adjusted to an optical density at 630 nm of 0.5, using the method described above. The 18 strains of probiotic bacteria (1010 CFU) were mixed in 250 ml of sterilized skim milk, 2% just before the oral inoculation of the calves. The bacterial population was confirmed by counting the serial dilutions on TSA and SMA-NA plates in duplicate.

Example 5 Preparation of the Calves Fifteen male milk calves from a single source were bred with a milk replacer and weaned at 6 weeks of age before transferring them to the University of Georgia. The calves were housed individually in rooms of concrete BL-2 climate controlled. Each room had a single floor drain and were cleaned once a day. The calves were fed a mixture of alfalfa pellets and sweet food twice a day and had free access to water. During a 2-week conditioning period, fecal samples from all calves were taken and tested as negative, via fluorescent antibody staining, for bovine virus diarrhea, coronavirus, rotavirus, pilus E antigens. col i, and Crypt ospori di a. Fecal flotation for intestinal parasites and bacterial culture for Salmonel l and E. coli 0157: H7, was performed and the fecal pH was determined. After a preconditioning period of 2 weeks, the calves were surgically equipped with cannulas in the rumen (flexible cannula for rumen). They were allowed at least 10 days to recover from the surgery and were given the necessary care before the experiment began.

Example 6 Cannulation of the Rumen The food was removed from the calves for 12 hours. The left paralumbar fossa was sheared and cleaned for standard surgical preparation. The fossa was anesthetized by using a paravertebral nerve block, the inverted L block, Lidocaine, slightly smaller than the local anesthetic, a circular incision was made, slightly smaller than the diameter of the cannula, and the circular piece of skin and the underlying external abdominal and cuticular oblique muscles were removed.The vessels were ligated as necessary and the oblique transverse abdominal muscles, internal, and peritoneum were separated and retracted leaving them blunt to create an opening to expose the wall The rumen wall was clamped with two large towel clamps, for traction, to exteriorize the rumen.The rumen wall was then sutured to the skin using cat # 3 or vetafil gut incorporating the muscle layers with a skin pattern. continuous suture The incision was made in the wall of the rumen and a circular piece of the rumen was removed and the cannula was inserted . Calves were treated for 5 days after surgery procaine penicillin G intramuscularly. The area between the cannula and the rumen wall was gently cleaned daily with betadine solution.

Example 7 Inoculation of Calves After 12 hours of fasting, the calves were inoculated via cannulation of the rumen with 250 ml of skimmed milk containing probiotic bacteria. After 48 hours, the mixture of 5 strains of E. coli 0157: H7 was inoculated via the same route. The control calves were challenged with the mixture of 5 strains of E. coli 0157: H7 only. After the challenge, the calves were examined daily for clinical signs including depression, pyrexia, diarrhea and anorexia. The rectal stools or samples collected from the fiscular tube were determined for pH, and the count of E. coli 0157: H7 and probiotic bacteria. Isolation and Counting of Inocula Bacteria: A 10 g sample of stool or rumen content was collected through cannulation of rectum or rumen recovery daily after inoculation of E. coli 0157: H7. The samples were placed in a tube containing 15 ml of Cary-Blair transport medium, kept at 5 ° C, and transported to the Center for Food Safety and Quality Improvement (Center for Food Safety and Quality Enhancement) for analysis . A volume containing 1 g of faeces was serially diluted (1:10) in 0.85% sodium chloride to 10"6, and 0.1 ml of each dilution was placed in duplicate on SMN-NA. complete gastrointestinal, collected at necropsy were contained at 5 ° C until analysis. The content of each tissue from each segment was separated and weighed, and the tissue was rinsed with 100 ml of PBS. The rinsed tissue was d to 9 ml of PBS and homogenized for 1 minute at 9,500 rpm with an Ultra-Turrax tissue homogenizer. A sample of 0.1 ml of tissue or suspension of tissue content was inoculated onto SMA-NA plates in quadruplicate and incubated at 37 ° C for 24 hours for the E count. coli 0157: H7 or probiotic bacteria. If these bacteria were not detected by the direct plating method, a selective enrichment method (17) (modified TSB containing 50 μg of nalidixic acid / ml) was performed. The samples were each placed in 100 ml of selective enrichment medium and incubated at 37 ° C for 24 hours with shaking at 150 rpm. The dilutions of the cultures were plated on SMA-NA and the isolates were selected and subsequently tested. The typical colonies of E. coli 0157: H7 (negative sorbitol) were seeded again on plates on SMN-NA and confirmed as E. coli by biochemical methods, and as 0157 and H7 by serological methods. The probiotic bacteria were confirmed by DNA fingerprinting by pulsed-field gel electrophoresis (PFGE).

Example 8. Genomic Fingerprint of Bacterial Isolates The PFGE procedures similar to those previously described were used (14) The bacteria were developed in 10 ml of TSB at 37 ° C for 24 hours with agitation at 200 rpm. The bacteria were sedimented by centrifugation (4000 X g, 20 minutes), washed three times in 75 mM NaCl containing 25 mM EDTA at pH 7.4 (SE), and resuspended in 0.5 ml of SE. The bacterial suspension was mixed with 0.5 ml of 2% (w / v) low melting point agarose in buffer consisting of 10 mM Tris, 10 mM magnesium chloride and 1 mM EDTA (ME), pH 7.5. This mixture was supplied in sample molds and the agarose plugs were digested with proteinase K (2 mg proteinase K, 50 mM Tris, 50 mM EDTA, 1% N-lauroyl sarcosine / ml, pH 8.0) at 5 ° C the night. The samples were washed in 10 mM Tris, 5 mM EDTA, pH 7.5 (TE), and digested with 50 U of Xbal. After incubation at 37 ° C overnight, the reaction was stopped by the addition of 20 μl of 0.5 M EDTA. The DNA samples were subjected to electrophoresis on a 1.2% agarose gel in 0.5 X TBE buffer for a homogeneous electric field device, with clamp on the contour (CHEF MAPPER, BioRAd). After electrophoresis for 24 hours at 200 V with pulse times of 5-50 seconds and linear inclination and an electric field angle of 120 ° C to 14 ° C, the gels were stained with ethidium bromide and the bands were visualized and photographed with UV transillumination. The results for 3 dominant probiotic strains, 271, 786 and 797 are shown in Figures 9 and 10.

Example 9 Carcass Necropsy The calves were sacrificed with intravenous sodium pentobarbital. The gastrointestinal tract was pinched in the esophagus and rectum and completely removed. Stretches of four to six cm of the duodenum, proximal, intermediate, middle, and distal jejunum, proximal and distal ileum, proximal and distal caecal bow, proximal curl of the ascending colon, centripetal and centrifugal spin of the spiral colon, transverse colon, and colon descending were double bound to allow sampling of all sections for the count of E. coli 0157: H7 and the probiotic bacteria in the tissue, and their content with minimal cross-contamination. Sections and contents of the rumen, reticulum, omasum and abomasum, and sections of kidney, spleen, liver, gallbladder, lymphoid jejunal lymph node, lymph node, cecal lymph node and tonsil were also collected for culture and counting of E coli 0157: H7 and / or probiotic bacteria. Sections from all of these sites, as well as sections of the prescapular lymph node, skeletal muscle, skin, tonsil, thyroid, thymus, esophagus, heart, pancreas, navel, adrenal, urinary vegija, and testicles were also placed in formalin 10 % buffered for histological examination.

Example 10. Histopathology and Immunohistochemistry of Tissues Fixed tissues were embedded in paraffin by standard methods, sectioned at 5 μm and stained with hematoxylin and eosin. The selected sections were stained with Gram. Sections showing large numbers of surface or luminal bacteria histologically were selected and treated by an alkaline phosphatase immunostaining procedure to identify E. coli 0157: H7. The tissues were deparaffinized in xylene for 10 minutes, rehydrated through gradient alcohols, and rinsed in PBS. The sections were covered with the specific antibody of E. col i 0157: H7 (Kirkegaard &Perry Laboratories, Inc., Gai Thersburg, MD) and incubated in a humidity chamber for 30 minutes at room temperature. After a 10 minute rinse in PBS, the slides were covered with a biotin-labeled second antibody for 20 minutes in a humid chamber at room temperature. The slides were rinsed for 10 minutes in PBS, returned to the humidity chamber, and the tissue sections were covered with conjugated streptavidin alkaline phosphatase. After incubation at room temperature for 20 minutes, the slides were rinsed in PBS for 10 minutes, placed in the humidity chamber, and bathed in a substrate solution for 15 minutes. The slides were rinsed in PBS for 10 minutes, counterstained for 3 minutes with Mayer's hematoxylin, mounted with aqueous medium followed by the non-aqueous mounting medium, and examined microscopically.

Example 11 Quantities of coli 0157: H7 carried in the absence and presence of probiotic bacteria Selection in vitro of potential probiotic bacteria that secrete inhibitory metabolism (s) to E. coli 0157: H7. Eighteen of 1,200 colonies isolated from cattle tissue and feces inhibited E. coli 0157: H7 i n vi tro. Among them, five colonies were isolated from faeces, five from the small intestine, and eight from the colon. Seventeen of the 18 colonies were identified as E. coli and the others as Pro t eus mi rabili s. All were evaluated for Shiga toxin production and none produced Shiga toxin. The fingerprint of the genomic DNA of PFGE revealed 13 different profiles among the 18 isolates.

Colonization of calves by probiotic bacteria. One calf was initially fed a strain of probiotic bacteria (E. coli with 1010 CFU). The calf appeared to be normal, and this E. col i was recovered by the enrichment procedure from only the ileum and the caecum at the termination of the experiment (12 days). Two calves were then fed with the 18 complete strains (approximately equal concentrations, 5 x 108 CFU each) of probiotic bacteria (1010 CFU / per calf) as a mixture. The feces of the calves were of normal consistency and the bacteria colonized the gastrointestinal tract for up to 27 days (at the end of the study the counts were 50 to 200 CFU / g of feces).

Determination of dominant probiotic bacteria strains by PFGE. Twenty-one colonies isolated from the tonsil, omasum, reticulum, proximal ileum, distal caecal bowel, proximal curl of the ascending colon, transverse colon, and stool at 26 days after inoculation were analyzed by PFGE. The isolates with four DNA profiles were dominant and all were JE7. coli Among the twenty-one colonies, nine were from strain # 797, seven were from strain # 786, three were from strain # 721, and two were from strain # 1019.

Pathological changes in calves by probiotic bacteria. Although some strains of the inoculated bacteria were recovered at necropsy from tissue specimens from different parts of the gastrointestinal tract, there were no pathological changes in any of the tissue samples evaluated.

Efficiency of probiotic bacteria to reduce the carrying of E. coli 0157: H7 in calves.

Of the 9 calves to which they were administered only E. coli 0157: H7, all remained healthy without evidence of fever or diarrhea. E. coli 0157: H7 was intermittently isolated from the rumen fluid of all animals during 3 weeks after inoculation (Figure 1). The dispersion of E. coli 0157: H7 in stools at various levels was continuous throughout the experiment (average 28 days) (Figure 2). At the necropsy, E. col i 0157: H7 was isolated from the rumen contents of eight out of ten, and colon from 10 out of 10 calves. No pathological changes of any of the tissue samples examined were observed. All calves given probiotic bacteria 2 days before treatment with E. coli 0157: H7 remained healthy without evidence of fever or diarrhea. E. coli 0157: H7 was detected in rumen samples collected through the fistula tube, up to 9 days in two animals, 16 days in an animal, 17 days in two animals, and 29 days in an animal (Figure 3). E. coli 0157: H7 was detected in feces for up to 11, 15, 17, 18, 19, and 29 days (at the end of the experiment) in each of the animals (Figure 4). At necropsy (average 30 days), E. coli 0157: H7 was not recovered from the rumen samples of any of these six animals; however, these bacteria were recovered from the colon of one of the six animals. The animal positive to E. col i 0157: H7 was twice fasted for periods of 2 days (day 16, 17 and day 23, 24) after inoculation during the study. Four of the six animals treated with probiotic bacteria were fasted according to this protocol.

Example 12. Efficacy of Dominant Probiotic Bacteria as a Treatment to Reduce / Eliminate E. col i 0157: H7 in Cattle Preparation of Dominant Probiotic Bacteria. Four strains of E. coli (271, 797, 786 and 1019) previously determined as inhibitors of E. coli 0157: H7, were selected for resistance to nalidixic acid (50 μg / ml) for ease of counting in feces. Approximately equal populations of each of the four stages were mixed (total 10 10 CFU) in 50 ml of skimmed milk sterilized at 2% for administration to calves. The populations of E. coli were confirmed by duplicate counting of TSA and SMA-NA plates.

Inoculation of calves. A total of 16 calves were used. After a period of 24 hours of fasting, each was administered a mixture of 5 strains of E. col i 0157: H7 in 50 ml of skimmed milk 2% sterile (1010 CFU) through a cannula tube. Two calves were treated with probiotic bacteria (1010 CFU) through a cannula tube 1 day after the administration of E. coli 0157: H7. Two additional calves were treated with probiotic bacteria (1010 GFU) 3 days after the administration of E. coli 0157: H7. The rumen samples collected through the tube of the cannula and rectal stools were counted daily for E. coli 0157: H7 and the probiotic bacteria.

Efficiency of the treatment of probiotic bacteria in the reduction / elimination of the carrying of E. coli 0157: H7 in calves. Of the 12 calves to which they were administered only E. col i 0157: H7 as positive controls, 0157 was isolated intermittently from the rumen for up to 2 weeks from 1 animal, for 3 weeks from 3 animals, and for 4 weeks from 8 animals (Figure 5). The continuous dispersion of E. coli 0157: H7 at various levels in the feces of 11 of 12 calves occurred throughout the study (Figure 6). At necropsy, E was isolated. col i 0157: H7 of the contents of the rumen of 9 of 12 calves and of the colon of 10 of 12 calves. Four calves were treated with a mixture of four strains of probiotic bacteria (strains 271, 786, 797, and 1019) 1 to 3 days after administration of E. col i 0157: H7. E. coli 0157: H7 was detected in rumen samples up to 6 days in 1 animal, 8 days in two, and 9 days in one (Figure 7). E. col i 0157: H7 was detected in feces for up to 10 days in two animals, 11 days in one, and days in one (Figure 8). At a necropsy (two at 22 days and two at 27 days), E. coli 0157: H7 was not recovered from the rumen or colon samples (tissue or contents) of any of these four animals. Only strains 271, 786 and 797 of the probiotic bacteria were recovered from the four animals at the end of the study. Strain 1019 was not detected in any of the animals at the time of the necropsy. The examples above illustrate the principles and practices for preventing and treating the carrying of E. col i 0157: H7 by administering probiotic or dominant probiotic bacteria to an animal carrying E. coli 0157: H7. Strains 271, 786 and 797 of E. coli were isolated and showed to be dominant probiotic bacteria. From the ease of isolation and the number of strains obtained, it is evident that other dominant probiotic strains can be isolated and used to inoculate cattle or other animals to prevent or treat the carrying of E. coli 0157: H7. Different strains can be advantageous for different applications depending on the animal species, breeding, age, diet, housing environment and management practices. All bacterial strains of this type, isolated and / or used as generally described herein to effectively reduce or eliminate the amount of E. coli 0157: H7 carried by an animal, are within the scope of the invention. Administration methods capable of providing an effective dose of probiotic or probiotic dominant bacteria to an animal, include a variety of feeding, drinking, and other administration methods known in the art, using a variety of formulations containing the bacteria, all within the scope of the invention. All such variations and modifications, based or derived from the teachings and descriptions herein, are considered within the scope of the appended claims. The following strains are deposited with the American Collection of Species (American Type Culture Collection) in accordance with 37 CFR 1.801-1.809: E. col i 271 ATCC Access No. 202020, August 13, 1997 E. coli 786 ATCC Access No. 202018, 13 August 1997 E. coli 797 ATCC Access No. 202019, August 13, 1997 It is noted that in relation to this date, the best method known to the applicant to carry out said invention is that which is clear from the present description of the invention. invention

Claims (13)

CLAIMS ~ - Having described the invention as above, the content of the following claims is claimed as property:

1. A microorganism selected from the group of dominant probiotic bacteria, characterized in that they consist of E. col i 271, E. col i 786, E. col i 797 and E. coli 1019.

2. A method for selecting dominant probiotic bacteria, characterized in that it comprises the steps of: the isolation of bacterial strains of natural origin from the fluids of ruminant animal tissues, digestive contents or feces, and the obtaining of isolated strains: the cultivation of strains isolated in liquid or solid media; the individual test for the ability of each isolated strain to inhibit E growth. col i 0157: H7 in vi tro, thereby strains that have the ability to inhibit the growth of E. coli 0157: H7 in vi tro are identified as probiotic bacteria, the subculture of probiotic bacteria, and the administration of one or more strains of probiotic bacteria to a ruminant animal, and isolating again the probiotic bacteria reaisables from the digestive contents or the faeces of said animal, after a predetermined time, whereby the rearabilable probiotic bacteria are identified as dominant probiotic bacteria.

3. The method according to claim 2, characterized in that the probiotic bacteria are provided with a selectable marker trait.

4. The method according to claim 2, characterized in that the probiotic bacteria are distinguishable from one another by digestion patterns of restriction fragments. DNA

5. The method according to claim 2, characterized in that the animal is a bovine.

6. A method to prevent or treat the carrying of E. coli 0157: H7 by a ruminant animal, characterized in that it comprises administering an effective amount of one or more strains of the dominant probiotic bacteria selected according to the method according to claim 3, to the digestive tract of said animal.

7. The method according to claim 6, characterized in that the dominant probiotic bacterium is selected from the group of E. coli 271, E. coli 18 6, or E. coli 797.

8. The method according to claim 6, characterized in that the dominant probiotic bacterium is administered in a series of doses combined with the drinking water.

9. The method according to claim 6, characterized in that the dominant probiotic bacteria is administered in a series of doses combined with the food.

10. The method according to claim 6, characterized in that the animal is a bovine.

11. An "igniter" composition, characterized in that it comprises a carrier and one or more strains of dominant probiotic bacteria selected from a group consisting of E. coli 271 ATCC 202020, E col i 786 ATCC 20218 and E. coli ATCC 20219.

12. The composition according to claim 11, characterized in that the carrier comprises water.

13. The composition according to claim 11, characterized in that the carrier comprises a substance edible by livestock.