ES2292322B1 - NEW LACTOBACILLUS SALIVARIUS CEPAS AND COMPOSITIONS OF THE SAME. - Google Patents

Abstract

New strains of lactobacillus salivarius and their compositions. The present invention relates to a substantially pure biological culture of a bacterial strain of the species Lactobacillus salivarius, deposited with the number CECT4930, and a substantially pure biological culture of a bacterial strain isolated from the previous rifampicin resistant strain, deposited with the CECT number 7063. It also refers to veterinary compositions comprising said strains. It also refers to its use to prepare compositions that eliminate colonization by a pathogenic strain of Salmonella spp. of the gastrointestinal tract in domestic birds. The effectiveness of the strains of the invention turns out to be very high whether it is administered directly or if they are incorporated into the feed with which the birds and / or drinking water are fed.

Description

New strains of Lactobacillus salivarius and their compositions.

Technical field

The present invention relates to new strains of the species Lactobacillus salivarius and compositions that include them to eliminate colonization by Salmonella spp . in domestic birds.

Prior art

The bacteria of the genus Salmonella ( Salmonella spp. ), Including Salmonella enteritidis, Salmonella typhimurium , and Salmonella heidelberg , are pathogenic bacteria that are responsible for 50% of cases of salmonellosis due to ingestion of food in poor conditions.

Among them, Salmonella enteritidis is one of the most worrisome to the world health authorities for its adaptation to birds, and because in addition to colonizing the gastrointestinal tract of birds, it is also transmitted through eggs.

Extensive use of antibiotics on farms of animals with the purpose of promoting growth and increasing the efficiency of feed conversion, and prevent intestinal infections, has led to an imbalance of the beneficial intestinal flora and the appearance of bacteria resistant.

The prohibition of the use of certain antibiotics Frequently included in feed to promote growth, led to the use of probiotics to prevent colonization of Birds by intestinal pathogens.

Since Nurmi et al ., Nature, 1973, 241, 210-211 described the pretreatment of chicks with microbiota isolated from the gastrointestinal tract of salmonella-free adult birds to protect them from Salmonella spp . Various indefinite products were developed, but such products are ineffective as described in Stavric et al ., in the book Colonization control of human bacteria enteropathogens in poultry, Academic Press, USA, 1991, pages 323-330.

Mead et al ., In the book Elimination of pathogenic organisms from meat and poultry, Elsevier, Amsterdam, the Netherlands, 1987, pages 57-77, describes defined mixtures of bacteria for commercial use, in order to prevent the transmission of pathogenic microorganisms when using indefinite compositions.

According to the publications found in the state of the art, the experimental results that are obtained with such combinations of well defined bacteria are contradictory.

Jin et al ., J. Anim. Sci., 1996, 9, 397-404, describes that such crops reduce mortality and improve the weight gain of the chicks, and at the same time prevent coliform colonization.

Hejlicek et al ., Veterinarstvi., 1995, 45,16-18, describes that defined crops protect birds against salmonellosis.

However, Watkins et al ., Poult. Sci., 1983, 62, 2088-2094, describes that probiotic agents have no positive effects on chicks, nor do they improve weight gain; and Stavric et al ., op. cit ., describes that neither reduce the burden of Salmonella spp .

There is therefore a need to have defined bacterial cultures that are capable of colonizing the gastrointestinal tract of birds and eliminating or preventing colonization by pathogenic strains of the genus Salmonella .

\hbox{en el buche y en los  intestinos ciegos de los
pollos.}

Garriga et al ., J. App. Microbiol, 1998, 84,125-1 132, describes a strain of Lactobacillus salivarius called CTC2197, antagonist against enteric pathogens, very adherent to chicken patch epithelial cells and which is able to colonize and predominate over the endogenous microbiota present

 \ hbox {in the crop and in the blind intestines of
chickens.} 

Pascual et al ., Applied and Environmental Microbiology., 1999, 65, 4981-4986, describe that the strain Lactobacillus salivarius called CTC2197 is capable of colonizing and completely eliminating the bacteria of the Salmonella enteritidis species from the gastrointestinal tract of chickens.

The strains of Lactobacillus salivarius described in these two publications are not available to the public, so that the information contained in both articles cannot be reproduced, and consequently they do not constitute sufficient descriptions ("enabling disclosures") for the purposes of patentability.

Object of the invention

The object of the present invention is to provide substantially pure biological cultures of two new strains Lactobacillus salivarius .

A veterinary composition for eliminating colonization by a pathogenic strain of Salmonella spp . of the gastrointestinal tract of domestic birds comprising any of the strains of the invention.

Another object of the invention is the use of said strains to prepare veterinary compositions to eliminate colonization by a pathogenic strain of Salmonella spp . of the gastrointestinal tract of domestic birds.

Detailed description of the invention

The inventors have managed to isolate two strains of Lactobacillus salivarius that colonize the gastrointestinal tract of domestic birds and eliminate colonization of it by pathogenic bacteria.

The strains of the present invention have been isolated from the selection of a set of 296 strains of lactic bacteria from the intestinal contents and the crop of 50 chicks aged between 15 and 42
days.

The inhibitory activity of said bacteria against the indicator strains E. coli and S. enteritidis was tested according to the method of gout in agar, described in Schillinger et al ., Applied and Environmental Microbiology, 1989, 55, 1901-1906.

This trial allowed the selection of 35 strains of  lactic bacteria that had inhibited the growth of the two Indicator strains

Their ability to adhere to chicken epithelial cells was then evaluated according to the in vitro adhesion assay described by Fuller, Journal of Applied Bacteriology, 1973, 36, 131-139.

The results of the mentioned trial allowed  the selection of 14 strains of lactic bacteria that presented a high adhesion to chicken epithelial cells.

Strains belonging to this group were able to inhibit other bacteria of animal origin such as Salmonella thyphimurium , and also bacteria of human origin such as Camphylobacter jejuni .

The strains were identified by biochemical and physicochemical tests such as Lactobacillus salivarius because they had the following characteristics:

- grew at 45 ° C,

- there was no gas production from glucose, and

- there was only acid production L-lactic.

In addition, the fermentation model presented according to the API test procedure (API System 50 CHL, BioMerieux, France) was indicative that it was Lactobacillus salivarius .

To rule out the possible identical strains, the lysis of the strains and the isolation of plasmid DNA were carried out according to the method described by Anderson et al ., Applied and Environmental Microbiology, 1983, 46, 549-552. This trial allowed us to identify eight different strains that underwent an additional coaggregation test between them, according to the method described by Vandevoorde et al ., Journal of Applied Bacteriology, 1992, 72, 214-219. The results of said test were evaluated according to the method described in Handley et al ., Journal of General Microbiology, 1987, 133, 3207-3217.

One of the strains object of the present invention  it was the strain that had the best coaggregation behavior, and a crop of it has been deposited, in accordance with the provisions of the Budapest Treaty on the recognition of deposit of microorganisms for the purpose of the procedure of patents, in the international deposit collection authority Spanish of Type Crops, domiciled at the Faculty of Science  Biological, University of Valencia, 46100 Burjassot (Valencia) Spain, which has been assigned the deposit number CECT4930.

This strain CECT4930 has properties of antagonist against pathogenic bacteria, good adherence to chicken epithelial cells, and a good behavior of coaggregation.

In vivo tests it is convenient to use a strain resistant to rifampicin, because it facilitates its identification between endogenous Lactobacillus salivarius or other Lactobacillus that are present in birds, and the Lactobacillus salivarius of the invention that have been administered in such tests.

To select colonies resistant to rifampicin, strain CECT4930 was grown on supplemented agar with rifampin.

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A colony resistant to rifampicin, which has inhibition properties against pathogenic bacteria, adhesion to epithelial cells of chickens, and coaggregation behavior as good as those of the origin strain, and has the same plasmid profile.

Said strain of rifampin has been also deposited with in the international deposit authority Spanish Type Culture Collection, which has been assigned the number of deposit CECT7063.

As mentioned, the strain CECT7063, resistant to rifampin, is a strain from a culture of strain CECT4930, and it has been proven that both strains have a high degree of genomic similarity by sequencing the 16S subunit of ribosomal RNA and the subsequent comparison with that of the Lactobacillus salivarius subsp type strain. DSM20555 salivarius .

Said sequencing was carried out by amplifying the gene encoding 16S ribosomal RNA by PCR with the universal primers 616V and 630R, resulting in a 1,576 base pair fragment. Said fragment was sequenced using a sequencing kit containing the 7-dGTP fluorescent labeled Thermo Sequenase primer (Amersham Life Science) and subsequently the 97KCy5 reverse primer was added with a fluorescent modification at the 5 'end. A coincidence percentage close to 100% was obtained, when the sequences of the CECT4930 and CECT7063 strains were compared with that of the Lactobacillus salivarius subsp type strain. DSM20555 salivarius , in the area of maximum variation between bases 50 and 350. These results confirm that the CECT4030 and CECT7063 strains belong to the species Lactobacillus salivarius subsp. Salivarius

The present invention aims at a substantially pure biological culture of a strain of Lactobacillus salivarius selected from the group formed by the strain with the deposit number CECT4930 and by the strain with the deposit number CECT7063.

The CCT4930 and CECT7063 strains can be obtained, both in the laboratory and on an industrial scale, through its culture in an appropriate medium, using conventional techniques sufficiently known to the expert.

For example, they can be obtained through strain culture in MRS medium (Difco Laboratories, USA) in anaerobic conditions at temperatures between 20 ° C and 37 ° C for periods of time between 15 and 30 hours. Once the incubation period is over, a Approximate concentration of 10 9 CFU / ml.

If deemed convenient, microorganisms  can be separated from the culture medium by techniques Conventional centrifugation, sedimentation, or filtration, and they can resuspend in other appropriate liquids, such as, for example, culture broth, or saline phosphate solution.

The culture of the bacteria subject to invention can also be preserved, for example, by freezing of the culture broth itself with the addition of vegetables appropriate auxiliaries, or by lyophilization thereof in presence of suitable auxiliary agents.

Freezing is done, for example, adding between 15 and 25% (volume / volume) of glycerin to broth, or can also be done by adding between 5% and 10% (weight / volume) of glucose and between 7% and 13% (weight / volume) of skim milk to the culture broth.

The frozen crop is preserved at temperature of, for example, -80 ° C, and its viability is controlled periodically making a series of dilutions (1:10) in solution saline from frozen crops, later planted in Rogosa agar plates (Difco Laboratories, USA), and incubation anaerobic at 37 ° C for 2 days.

Lyophilization can be carried out, by example, adding 7% to 13% to the broth (weight / volume) of skim milk and between 5% and 10% (weight / volume) of glucose.

Freeze drying conditions are the common in this type of processes, for example, the temperature it can be between -40 ° C and -60 ° C, and the pressure between 0.3 Pa and 0.7 Pa.

The lyophilized culture is usually maintained  at 4 ° C, and its viability is periodically controlled in an analogous way to the frozen crop.

Frozen crops and crops lyophilisates of the strains of the invention show a good stability after long periods of conservation.

It has been observed that, after 18 months a -80 ° C, the viability of frozen crops both in presence of MRS culture medium and glycerin, such as glucose and milk skim, undergoes a reduction that is usually included between 1.0 and 1.5 logarithmic units of viable cells per gram of cultivation

In the case of lyophilized cultures, the viability decreases in a greater proportion after periods conservation lengths For example, after 12 months maintained at 4 ° C, a decrease of less than 2 is usually observed logarithmic units of viable cells per gram of culture.

Another object of the invention is a veterinary composition to eliminate colonization by a pathogenic strain of Salmonella spp . of the gastrointestinal tract of domestic birds comprising a strain of Lactobacillus salivarius selected from the group formed by strain CECT4930 and strain CECT7063, and an auxiliary vehicle.

Preferably the pathogenic strain Salmonella spp . it is selected from the group formed by Salmonella enteritidis, Salmonella typhimurium , and Salmonella heidelberg . More preferably, the pathogenic strain is Salmonella enteritidis .

The composition of the invention preferably comprises between 10 5 and 10 10 CFU / g of a strain of Lactobacillus salivarius selected from the group formed by strain CECT4930 and strain CECT7063. More preferably between 10 7 and 10 8 CFU / g.

Preferably the domestic birds to which  administer compositions comprising one of the strains of the invention, are selected from the group consisting of: chickens, laying hens, and turkeys. More preferably the birds are select from the group consisting of chickens and chickens layers.

The veterinary composition of the invention includes an auxiliary vehicle that can be selected among others, for example, between wetting agents such as surfactants anionic or nonionic; inert fillers such as glucose, lactose, or mannitol; hydroxy compounds such as glycerin, propylene glycol, or polyethylene glycol, anti-caking agents such as calcium stearate; liquid vehicles such as broth, or phosphate solution saline; stabilizing agents such as skim milk; and their combinations

Preferably the auxiliary vehicle is select from the group consisting of glucose, skim milk, glycerin, and mixtures thereof.

As is well known, the formulations that comprise bacterial strains do not include products with activity antibiotic that would affect their viability, and by consequently the effectiveness in eliminating the colonization by pathogenic bacteria of the gastrointestinal tract of the birds.

The veterinary compositions of the invention can be administered to birds directly, through the I think, or through drinking water, they are usually administered during the first day of the breeding period, and eventually you can repeat the administration before the end of the period of breeding.

Direct administration refers to the direct dosage of a composition comprising the appropriate amount of a strain of Lactobacillus salivarius of the invention to the proventriculus of the birds, for example, by means of a syringe having a coupled cannula of sufficient length.

Administration of the composition of the invention by direct route is carried out during the first day of the breeding period to domestic birds and is usually administered between 10 5 and 10 8 CFU of a Lactobacillus strain salívarius selected from the group formed by strain CECT4930 and strain CECT7063.

The incorporation of the compositions of the invention into the feed with which the birds are fed, can be carried out using powdery compositions, or liquid suspensions comprising the Lactobacillus salivarius strains of the invention.

An appropriate powder form is the one that results from lyophilization of the culture broth of the strains of the invention in the presence of, for example, skim milk and glucose.

The incorporation of the strains of the invention into  I think it should be done after the granulation process of same, since the temperatures used during this process affect the viability of the strains.

In order to incorporate the compositions of the invention into the drinking water, powdery compositions can be used, for example, lyophilized as already mentioned, or compositions comprising the strains of the invention in suspension form in a solution, for example, of broth of culture or phosphate solution
saline.

The feed and / or drinking water that is administered to the birds comprises an amount of a composition that includes a strain of Lactobacillus salivarius selected from the group formed by strain CECT4930 and strain CECT7063, so that the feed and / or The drinking water includes between 10 5 and 10 8 CFU per gram of feed or between 10 5 and 10 8 CFU per ml of drinking water.

The Lactobacillus salivarius strains of the invention have good stability when lyophilized or in the form of a liquid culture are incorporated into feed formulations for birds.

For example, a lyophilized culture containing 9.89 log 10 CFU / g (colony forming units per gram) is mixed with different proportions of a commercial feed based on corn and soybeans to obtain a concentration of Lactobacillus salivarius of 10 ^ {5}, 10 <6> and 10 <8> CFU per gram of feed.

After keeping these mixtures at temperature environment for a week, it is observed that the viability of crop is reduced by approximately 2.5 logarithmic units of viable cells per gram of feed.

The strains of the invention also have a good stability when mixed with feed containing acidifying substances such as formic acid, propionic acid, acid lactic acid, phosphoric acid, citric acid, fumaric acid, or their mixtures

Compositions comprising a strain of Lactobacillus salivarius selected from the group formed by strains CECT4930 and CECT7063 can be used effectively to eliminate colonization by Salmonella spp . of the gastrointestinal tract of domestic birds, such as chickens, turkeys or laying hens, as is clearly shown in different in vivo trials discussed below.

When 10 8 CFU of a Lactobacillus salivarius strain of the invention is administered directly to the proventriculus of 1-day-old chickens, it is observed that at 14 days 50% of the chickens are still colonized by Salmonella spp ., But that at 21 days all inoculated chickens are protected against colonization by said
microorganism.

Even when Salmonella enteritidis is administered simultaneously with the strain of the invention, Salmonella enteritidis is completely eliminated from the gastrointestinal tract of the birds.

For example, when administering 10 8 CFU of the strain Lactobacillus salivarius CECT4930 simultaneously with 10 6 CFU of Salmonella enteritidis C-114 directly to the proventriculus of 1-day-old chickens, it is observed that at 14 days 100% of the chickens are colonized by Salmonella enteritidis , but that at 21 days all the chickens are exempt from said bacteria.

Salmonella enteritidis C-114 is a non-invasive strain isolated from chickens, which was obtained from the IRTA-Laboratory for Animal Health strains collection (Spain). The strain is resistant to nalidixic acid (200 mg / ml), and mercuric chloride (12 mg / ml).

The results of these tests indicate that a single administration of the strain of the invention in the bird proventriculus is sufficient to completely eliminate colonization by Salmonella enteritidis at 21 days.

In another trial, three groups of 1-day-old chicks are inoculated directly into the proventriculus with different bacteria. The first group is inoculated with 10 6 CFU of S. enteritidis C-114; the second group with 10 8 CFU of Lactobacillus salivarius CECT7063; and the third group with 10 8 CFU of Lactobacillus salivarius CECT7063 and 10 6 CFU of S. enteritidis C-114. During the first day, the chicks have access to feed and drinking water containing a concentration of Lactobacillus salivarius CECT7063 of 10 8 CFU / g of feed and 10 7 CFU / ml of drinking water.
drink.

The group that is inoculated only with Salmonella represents the control group.

At 21 days, all chicks that have been inoculated with the strain of the invention are exempt from colonization by Salmonella enteritidis .

When three groups of 1-day-old chickens are fed on the first day with a feed containing a concentration of the strain of the invention CECT7063 between 10 5 and 10 8 CFU per gram of feed, After 28 days, all chickens have Lactobacillus salivarius bacteria of the strain of the invention in their gastrointestinal tract. There were no significant differences in the count of Lactobacillus salivarius CECT7063 between chickens that had received different concentrations of inoculum in the feed.

To ensure the maintenance of an appropriate concentration of Lactobacillus salivarius of the invention in the gastrointestinal tract of domestic birds until the end of the breeding period, the birds are usually fed with a second dose of feed comprising between 10 5 and 10 8 CFU per gram of feed and / or with drinking water comprising between 10 5 and 10 8 CFU per ml of drinking water.

As mentioned above, the advantage of the rifampicin-resistant strain is that it allows the presence of the strains of the invention to be easily identified in domestic birds to which they have been
administered.

It has been found that the rifampicin resistant Lactobacillus salivarius strains isolated from the birds to which the strains of the invention had been administered had a plasmid profile that corresponded in all cases with that of the administered strain.

Another object of the present invention is the use of a strain of Lactobacillus salivarius selected from the group formed by the strain with the deposit number CECT4930 and by the strain with the deposit number CECT7063 to prepare veterinary compositions to eliminate colonization by a strain pathogen of Salmonella spp . of the gastrointestinal tract of domestic birds.

Preferably the pathogenic strain Salmonella spp . it is selected from the group formed by Salmonella enteritidis, Salmonella typhimurium , and Salmonella heidelberg . More preferably, the pathogenic strain is Salmonella enteritidis .

Surprisingly it is found that the strains of the invention are suitable for colonizing the gastrointestinal tract of domestic birds and for eliminating colonization thereof by Salmonella spp .

This represents an important advance in terms of food safety since the use of the strains of the invention minimizes the risk of food poisoning by Salmonella . (salmonellosis).

The examples set out below are they must interpret as an auxiliary means for a better understanding of the invention, and not as limitations to the object thereof.

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Examples

Example one

Preparation of the strains of the invention

The Lactobacillus salivarius CECT4930 strain was grown in MRS broth (DIFCO Laboratories, USA) for 18 hours at 37 ° C under anaerobic conditions, reaching an approximate concentration of 10 9 CFU / ml.

Following the same procedure was prepared also strain CECT-7063.

To preserve strains by freezing, prepared compositions thereof from the broth of MRS culture, which comprised 20% by volume / volume of glycerin, and kept frozen at a temperature of -80 ° C

Frozen compositions at the same temperature were also preserved, compositions comprising the same MRS culture medium to which 10% by weight / volume of skim milk was added, and 7.5% by weight / volume of
glucose.

Powder compositions of the  strains of the invention by lyophilization of the culture broth, at that 10% by weight / volume of skim milk was added, and 7.5% by weight / volume of glucose.

The composition was lyophilized in a lyophilizer 1-4 Christ Alpha with a controller LDC-1M (Braun-Biotech, Germany) a a temperature of -50 ° C, for a period of 24 h at a pressure 0.5 Pa. A powdery composition containing 9.89 was obtained log_ {10} CFU / g.

The freeze-dried powdery composition for each of the strains was stored at 4 ° C.

The viability of the strains present in the frozen and lyophilized compositions was determined periodically by making a series of dilutions (1:10) in saline solution from the frozen and lyophilized cultures, and subsequently seeded in Rogosa agar plates (Difco Laboratories, EE. .UU.), And anaerobic incubation at 37 ° C for 2
days.

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Example 2

Stability of the strains of the invention in commercial mixtures of feed

The stability of the strains of the invention in I think it was determined using the compositions of strain CECT7063 prepared in Example 1, corresponding to a composition lyophilized powder and a liquid composition comprising Frozen broth.

This strain was used because it is resistant to rifampicin, which facilitated the task of identifying strains

The lyophilized culture of strain CECT7063 It contained 9.89 log 10 CFU / g and mixed with amounts of one commercial mixture of a feed containing corn and soybeans until obtain compositions containing the strains of the invention with concentrations of 10 5, 10 6 and 10 8 CFU / g of I think.

The liquid culture was centrifuged at 4,950 g for 10 minutes, the sediment obtained was resuspended in a volume of saline phosphate solution 20 less than the volume of original culture, diluted with different amounts of solution saline to obtain the desired concentrations, and finally mixed with the commercial feed mixture in a 1:20 ratio.

The mixtures were kept at temperature Environment for several days.

The number of viable strains of strain CECT7063 in commercial feed formulations, it was determined periodically at 2, 3, 6 and 7 days. For this, 3 g were mixed feed with saline solution in a 1:10 ratio, were performed serial dilutions, and were seeded on Rogosa agar plates and in Rogosa agar plates with rifampin (100 µg / ml). The plates are incubated anaerobically at 37 ° C for 2 days, and the number of viable strains in duplicate and expressed as log_ {10} CFU / g.

The results are shown in Table I obtained with the lyophilized culture and with the liquid culture:

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TABLE I

one

It can be seen that the number of viable strains  in feed of strain CECT7063 when incorporated into it in liquid culture form, is comparable to when it is in the form of lyophilized culture.

After a week at room temperature, it  you can check that the stability of the mixed strain CECT7063 With commercial feed it is good.

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Example 3

In vivo trials of elimination of colonization by Salmonella in the gastrointestinal tract of domestic birds

For in vivo tests cultures of the strains of the invention were prepared in a solution of saline phosphate.

CECT4930 strain was grown in culture broth MRS for 18 hours under anaerobic conditions at a temperature of 37 ° C. It was then centrifuged at 4,950 g for 10 minutes, and the sediment obtained was resuspended in a phosphate solution saline (PBS, pH 6; 136.89 mM NaCl, 2.50 mM KH 2 PO 4, K 2 HPO 4, 6.95 mM) in a 1:10 ratio.

Following an analogous procedure, a  CECT7063 strain culture in a saline phosphate solution.

In vivo assays a culture of Salmonella enteritidis C-114 was used. This bacterium is a non-invasive strain isolated from chickens, and was obtained from the IRTA-Laboratory of Animal Health (Spain) strain collection. The strain is resistant to nalidixic acid (200 mg / ml), and mercuric chloride (12 mg / ml). It was maintained at -70 ° C in vials for bacteria storage (Protect, Great Britain).

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Salmonella enteritidis C-114 was grown in a tryptic soy agar (Difco Laboratories, USA) supplemented with nalidixic acid (200 mg / ml) and mercuric chloride (12 mg / ml) for 18 h at 37 ° C. The cells were suspended in a polyvalent medium (General Purpose Medium Plus, BioMérieux, France) until an optical density of 0.15 to 450 nm was obtained, corresponding to 10 8 CFU / ml. The suspension was diluted 1: 100 with polyvalent medium.

In all trials, the birds were placed randomly in Petersime boxes with six levels and four compartments per level, and each treatment occupied a level.

The birds of the different treatments were orally administered Salmonella enteritidis C-114 and / or Lactobacillus salivarius CECT7063, as indicated below, and had free access to water and food.

At the time of sampling, several chickens from each  group were sacrificed for cervical dislocation. He picked up the content of one blind for each chicken, was homogenized in solution of saline phosphate in a 1:10 ratio, and dilutions were made serials before proceeding to seeded in Rogosa agar and / or agar  Rogosa supplemented with rifampicin to account for lactobacilli and lactobacilli resistant to rifampicin. The Plates were grown anaerobically at 37 ° C for 2 days.

The predominance of the inoculated strain was determined by comparing the plasmid profiles of the rifampin-resistant colonies with the plasmid profile of the Lactobacillus salivarius strain CECT7063.

Isolation of the Salmonella enteritidis D C-114 strain from the other blind was carried out using the procedure described in Saco et al ., In Franchini, A, and Mulder, RWA (Eds), Prevention and control of potentially pathogenic microorganisms in poultry and poultry meat processing, Flair / Cost Meeting, Bologna, Italy, October 14-16, 1993, pages 33-37; and in Saco et al ., Salmonella, 1995, 1 (4), 44-51.

The impedimetric procedure was carried out in a Bactometer system (BioMérieux Vitek). Wells impedimetrics were subcultured in bright green modified agar (Oxoid, Great Britain), and XLT4 (Difco).

Isolated colonies were selected alestoriamente and confirmed by culture in tryptic soy agar (Difco) supplemented with nalidixic acid (200 mg / ml), and chloride mercuric (12 mg / ml).

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a) Trial 1

128 day old Leghorn chickens divided into four groups of 32 birds each, corresponding to The following treatments:

-

A: inoculation with 10 6 CFU of Salmonella enteritidis C-114 strain

-

B: inoculation with 10 8 CFU of the strain Lactobacillus salivarius CECT7063

-

C: inoculation with 10 8 CFU of the strain Lactobacillus salivarius CECT7063 and with 10 6 CFU of the strain Salmonella enteritidis C-114.

A group of chickens was not subjected to any treatment.

Both strains of bacteria were inoculated at proventriculus using a syringe that had a built-in semi-rigid cannula 12 cm long and 3 mm in diameter.

In each sampling at 14 and 21 days after oral inoculation, 4 chickens of each were sacrificed group and the blind were collected as already mentioned.

Table II shows the percentage of chickens colonized by Salmonella corresponding to the day on which the sampling was carried out:

TABLE II

2

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It can be seen that at 21 days, 100% of the chickens that have been inoculated with the Lactobacillus salivarius strain CECT7063 are not colonized by Salmonella .

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b) Trial 2

96 1-day-old Leghorn chickens were divided  in three groups of 32 birds each, corresponding to the treatments:

-

D: inoculation with 10 6 CFU of Salmonella enteritidis C-114 strain

-

E: inoculation with 10 8 CFU of the strain Lactobacillus salivarius CECT7063

-

F: inoculation with 10 8 CFU of the strain Lactobacillus salivarius CECT7063 and with 10 6 CFU of the strain Salmonella enteritidis C-114.

Bacterial strains were inoculated directly into the proventriculus during the first day, as described above, and the Lactobacillus salivarius strain CECT7063 was also administered, during the first day, mixed with the feed at a concentration of 10 8 CFU / g, and with drinking water at a concentration of 10 7
CFU / ml.

The feed and drinking water that contained the strain of the invention prepared by mixing them with the appropriate amount of a lyophilized culture of strain CECT7063 containing 9.89 log_10 CFU / g.

In each sampling at 14 and 21 days after oral inoculation, six chickens of each were sacrificed group and the blind were collected as already mentioned.

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Table III shows the percentage of chickens colonized by Salmonella corresponding to the day on which the sampling was carried out:

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TABLE III

3

It can be verified that at 21 days, 100% of the chickens that have been inoculated with the Lactobacillus salivarius strain CECT7063 are not colonized by Salmonella enteritidis .

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c) Trial 3

90 day old Leghorn chickens They divided into 4 groups of 30 chickens each.

A commercial feed additive was prepared with a lyophilized culture of Lactobacillus salivarius CECT-7063 containing 9.89 log 10 CFU / g, so that the chickens were fed on the first day of the test with a feed containing 10 ^ 5 CFU / g, 10 7 UFG / g, and 10 8 CFU / g.

In each sampling at 7, 14, 21, and 28 days after feed administration, six were sacrificed chickens from each group and the blind were collected as already mentioned.

The presence of the strain Lactobacillus salivarius CECT7063 was detected in all chickens subjected to the test. Throughout the study, no significant differences were observed in the count of Lactobacillus salivarius CECT7063 among the chickens that had received different concentration of inoculum in the feed.

Claims (12)

1. Substantially pure biological culture of a strain of Lactobacillus salivarius selected from the group formed by the strain deposited in the international deposit authority Spanish Collection of Type Crops with the deposit number CECT4930, and by the strain deposited in the international deposit authority Spanish Type Crop Collection with deposit number CECT7063. 2. Biological culture according to claim 1, characterized in that it has the deposit number CECT4930. 3. Biological culture according to claim 1, characterized in that it has the deposit number CECT7063. 4. Veterinary composition to eliminate colonization by a pathogenic strain of Salmonella spp . of the gastrointestinal tract of domestic birds characterized in that it comprises a strain of Lactobacillus salivarius of claims 1 to 3, and an auxiliary vehicle. 5. Composition according to claim 4, characterized in that the pathogenic strain Salmonella spp . it is selected from the group formed by Salmonella enteritidis , Salmonella typhimurium , and Salmonella heidelberg . 6. Composition according to claim 5, characterized in that the pathogenic strain Salmonella spp . It's Salmonella enteritidis . Composition according to claims 4 to 6, characterized in that it comprises between 10 5 and 10 10 CFU / g of a strain of Lactobacillus salivarius of claims 1 to 3. 8. Composition according to claims 4 to 7, characterized in that the domestic birds are selected from the group consisting of chickens, laying hens, and turkeys. 9. Composition according to claims 4 to 8, characterized in that it comprises an auxiliary vehicle selected from the group consisting of glucose, skim milk, glycerin, and mixtures thereof. 10. Use of a strain of Lactobacillus salivarius selected from the group formed by the strain with the deposit number CECT4930, and by the strain with the deposit number CECT7063 to prepare veterinary compositions to eliminate colonization by a pathogenic strain of Salmonella spp . of the gastrointestinal tract of domestic birds. 11. Use according to claim 10, characterized in that the pathogenic strain Salmonella spp . it is selected from the group formed by Salmonella enteritidis, Salmonella typhimurium , and Salmonella heidelberg . 12. Use according to claim 11, characterized in that the pathogenic strain is Salmonella enteritidis .