"A METHOD FOR PREVENTING SPECIFIC BACTERIOPHAGE INACTIVATION OF PROBIOTIC STRAIN MIXTURES USED IN ANIMAL FEEDS"

DESCRIPTION

State of the art

Just as in humans, animals cohabit with microorganisms, the number of which exceeds that of the cells in their bodies. It has been observed experimentally that, if protected, animals can grow and multiply even in the absence of microbes, however, with changes observed in their bodies.

According to the definition by Fuller (R. Fuller 1989. Probiotics in man and animals. J. Appl. Bacteriol, 66 (5):365-378) probiotics are dietary supplements, consisting of living microorganisms, which have a beneficial effect on treated animals, in as much as they have improved intestinal flora.

Through many years of research into the "microbe-animal" and "microbe-man" relationship, it has been observed that the presence of "friendly" microorganisms limits the use of antibiotics for impeding the growth of pathogenic microorganisms, the causative agents for a number of diseases. Of late, both in vitro and in vivo, a reduction in immune-related disorders such as allergies, has been observed, if specific germs regulating the development of the immune system are administered at birth, thus preventing autoimmune disorders in various animal species (EJ. Schiffrin and S. Blum 2002. European Journal of Clinical Nutrition 56 (Suppl. 3) S60-S64). The 1960s saw the production of dietary supplements containing " 'Enter ΌCOCCUS faecalis" strains; but such preparations, after having given good results, with reduced incidence of disease and improved growth, lost their efficacy due to bacteriophages present in the environments which destroyed the cells of the probiotics administered. Although the most recent publications have shown that each animal has its own specific microorganisms, the majority of the probiotic preparations on the market consist of one or two microorganisms, which are administered to all species of animals, including humans. Current knowledge of the intestinal flora has highlighted that over 400 bacterial species populate our bodies, and that each species is represented by a number of genetically diverse strains. It should be pointed out that the Enterococcus and the Lactobacillus represent a small percentage of the intestinal flora. This knowledge

has lead to the development of certain products for controlling salmonellosis in poultry farms.

Notable among such products are:

BROILACT (Orion Corporation, Finland), US patent N° US4689226. This is a formulation of 32 strains isolated from the faeces of a single adult chicken, strains for which the distinguishing characteristics are unknown. The aim of the product is that of establishing the natural flora of the chicken blind intestine capable of specifically preventing the onset of salmonellosis.

PREEMPT (MS BioScience, Inc., USA), uses the same principle as the previous product with the difference being that the numerous strains present have been isolated from the faecal flora of a single adult turkey, and is intended solely for controlling salmonellosis.

AVIGUARD (Bayer), which includes lactobacillus and anaerobes isolated from chickens. The product is intended for the control of avian pathogens in general.

Other probiotic mixtures isolated from the entire digestive tract, instead of just a part of the intestine, are capable of preventing the onset of all the gastroenteric disorders of farmed animals including certain viral diseases, and stimulating animal growth (Mulder, R. W. A. W., R. Havenaar and J.H.J. 1997 Huis in't Veld. Intervention strategies: the use of probiotics and competitive exclusion micro- floras against contamination with pathogens in pigs and poultry. In: Probiotics 2: Application and practical aspects (edited by R. Fuller) Chapman & Hall, pp 187- 207; EJ. Schiffrin and S. Blum 2002. European Journal of Clinical Nutrition 56 (Suppl. 3) S60-S64).

The diseases which can be controlled by means of the administration of mixtures of strains isolated from the entire digestive tracts of the animals of interest include:

gastroenteritis from Bacillus cereus;

staphylococcal poisoning;

beta-haemolytic streptococcus infections

enteritis from Clostridium perfringens

Campylobacter- jejuni enterocolitis

diarrhoea from pathogenic Escherichia coli

salmonellosis

shigellosis

In mixtures with several microorganisms, the problem of inactivation due to phage attack is only delayed over time, as over prolonged periods, all the strains in the mixture are inactivated in any case (Mulder, R.W.A.W., R. Havenaar and J.HJ. 1997 Huis in't Veld. Intervention strategies: the use of probiotics and competitive exclusion micro-floras against contamination with pathogens in pigs and poultry. In: Probiotics 2: Application and practical aspects (edited by R. Fuller) Chapman & Hall, pp 187-207; EJ. Schiffrin and S. Blum 2002. European Journal of Clinical Nutrition 56 (Suppl. 3) S60-S64).

The "microbe eating" phages are the bacterial viruses: a phage which enters into a cell and is then multiplied by the same to give approx. 100 new virus particles, prior to the cell expiring. It is easy to understand the damage phages can cause with a multiplication rate of 1 to 100: starting from a low number, in just a few multiplication cycles it can destroy a culture of billions of bacterial cells. Phages have their origins in the same microbial cells. Indeed, every microbial genome, for which the DNA has been sequenced, contains from 1 to over 10 of them. Once separated from the bacterial chromosome, the phage forces the host cell to multiply it, and by infecting the environment and other host cells, it is multiplied and destroys them. In the fermentation industry, and particularly for probiotics, it is the phages from the environment that are responsible for the damage caused: If the rearing environment is infected with phages specific for the probiotic strain, then its administration to animals only allows the phages to multiply. While in other fields, the problems associated with phages are well understood, and there is active research into limiting the damage caused by them, this problem is completely ignored in relation to probiotics: the numerous failures in the use of probiotic cultures may be easily explained by their presence.

Detailed description of the invention

The subject of the present invention is a method for preventing phage mediated inactivation of probiotic strain mixtures comprising the alternating administration of specific strain mixtures, for each reproductive cycle.

The mixtures are administered in rotation, from the start of each new reproductive cycle, on average, every eight days.

The duration of the beneficial effects is proportional to the number of strains in the mixture and the administration strategy implemented to prevent the onset of phage attack.

Indeed: the presence of phages in microbial populations is strain-specific and arises inevitably after several reproductive cycles; without any intervention, phage accumulation causes the deactivation of the strains and the mixtures; the method proposed is capable of maintaining the number of phage particles present below the threshold level which causes the undesired effects.

This method eliminates phage related problems in environments where probiotics are used, both in humans and in animals. It is based on the principle that the phage is specific for the species and strain of the microorganism. Strains with the same characteristics may have different phage susceptibilities, and be sensitive to one phage and resistant to another and vice versa. The effect is obtained by not introducing the phage-specific cells (strains) into the working environment where the phage may continue to multiply.

The fact that suitable host cells are not present because they are not administered with the new mixture impedes the replication of the phages, which thus disappear. Thus, sensitive strains may be re-used after some time has elapsed. The method of the present invention consists of practicing strain rotation, using a number of strains with the same characteristics, but with different phage susceptibility, so as to maintain the specific phage at such a low level as to not kill all the cells used. Over time, while the strain-specific phages multiply, the sensitive strain is continually replaced with another that is phage resistant. In order to increase the

likelihood of eliminating the phage problem, it is necessary to include two strains for each species, with different phage susceptibilities, in the mixture. Preferred strains for obtaining a mixture, useful for obtaining the described effects are: strains of crop lactobacillus species, strains of lactic ferments isolated from the glandular stomach, strains of blind intestine lactobacillus species, strains of blind intestine coccus species, strains of species of anaerobic microorganisms from the blind intestine, strains of various species isolated from the stomach, strains of various species isolated from the small intestine, strains of species of lactobacillus from the large intestine, strains of large intestine coccus species, strains of species of anaerobic microorganisms from the large intestine.

Most preferred microorganisms for obtaining a useful mixture for achieving the effects described are (MIXTURE 1):

1) Lactobacillus spp.l isolated from the crop, 2) Lactobacillus acidophilus spp.3 isolated from the crop, 3) Lactobacillus fermentum spp. 23 isolated from the crop, 4) Lactobacillus spp. 25 isolated from the ileum, 5) Lactobacillus spp. 41 isolated from the ileum, 6) Lactobacillus delbrueckii spp. 7 isolated from the ileum, 7) Lactobacillus spp.123 isolated from the blind intestine, 8) Lactobacillus spp.350 isolated from the blind intestine, 9) Lactobacillus delbrueckii spp. 32 isolated from the blind intestine, 10) Lactobacillus delbrueckii spp. 250 isolated from the blind intestine, 11) Lactobacillus fermentum spp. 58 isolated from the blind intestine, 12) Lactobacillus crispatus spp. 47 isolated from the blind intestine, 13) Clostridium spp.37 isolated from the blind intestine, 14) Clostridium spp.9 isolated from the blind intestine, 15) Clostridium spp.234 isolated from the blind intestine, 16) Enterococcus faecium spp. 17 isolated from the blind intestine, 17) Enterococcus faecium spp. 75 isolated from the blind intestine, 18) Bacteroides spp. 27 isolated from the blind intestine, 19) Bacteroides spp. 168 isolated from the blind intestine.

Materials and methods

Strain mixture preparation process: 1) Strain isolation.

Isolation of the microbes from the various sections of the digestive apparatus of the animal of interest using the method described in patent US4689226 for isolation from chicken blind intestine, including the isolation of lactobacillus from crop.

2) Definition of the mixture.

Characterisation of the strains according to the procedures and their mixture, using also cultures from the strain collection.

3) Industrial preparation of the basic mixture (MIXTURE 1).

The basic mixture is prepared using the method described in patent US4689226.

4) Use of the basic mixture (MIXTURE 1) in rearing.

In the case of fowl, the mixture is administered from hatching for a period of approx. eight days.

5) Laboratory search for phage-resistant strains.

Again, in the case of fowl, after approx. 8 days, strains from the mixture are isolated from the rearing environment in order to isolate phages specific for the same strains and identify resistant strains in the laboratory.

6) Preparation of a second mixture (MIXTURE 2).

MIXTURE 2 is prepared using the strains resistant to the phages found in the environment and used in rearing.

7) Preparation of other mixtures.

The procedure described in parts 3 to 6 is repeated until a total of at least 4 iso- strain mixtures resistant to the various isolated phages are obtained.

Definition of the phage specificity matrix defined in table 1 (T. Sozzi 1998, Batteriofagi. In: De Simone C. et al, Prospettive Terapeutiche dei Batteri Lattici

Teoria e Applicazioni nel Dismicrobismo Intestinale, Ed. Piccin, pp 107-115).

Table 1 : - resistant 4- sensitive

Procedure for using the mixtures in rearing

The plan of use envisages changing the above defined mixtures with every reproductive cycle of the animals being treated, which can vary in accordance with the following, given by way of example.

Poultry: During the first eight days of the lives of the fowl of cycle 1 , the above defined MIXTURE 1 is used; in the following eight days of the life of a fowl of cycle 2, MIXTURE 2 is used and so on, up to MIXTURE 4.

Then the cycle begins again with MIXTURE 1. Swine: during the first eight days following birth the mixture adapted to swine is used, and the procedure then continues in accordance with the example reported above for poultry.

In the case of reduced growth rate or disease among the animals, or the onset of any pathology, then proceed to the immediate administration of another mixture in the same rearing cycle, possibly checking for any phages present in the environment.

EXAMPLES

From application of the above method, it is possible to derive the following strain mixtures, to be used in rearing, comprising four phage-resistant iso-strain variants as illustrated in the method.

Example 1 Mixture 1 for fowl

Composed of equal parts of: three strains of crop lactobacillus species; three lactic ferment strains isolated from the glandular stomach; three strains of blind intestine lactobacillus species; three strains of blind intestine coccus species; three strains of anaerobic microorganisms from the blind intestine;

15 strains in total

Example 2

Mixture 1 for swine

Composed of equal parts of: three strains of various species isolated from the stomach; two strains of various species isolated from the small intestine; three strains of large intestine lactobacillus species; three strains of large intestine coccus species; three strains of anaerobic microorganisms from the large intestine;

Example 3

Process for administering the mixtures for fowl:

MIXTURE 1 is administered by means of spraying the hatching eggs, the feed and drinking water during the first 8 days of life (approx. 10 ⁵ to 10 ^s microorganisms per gram of feed or cm ³ of water).

MIXTURE 2 is administered at the start of the second reproductive cycle. The process continues in an analogous manner, with the administration of MIXTURES 3 and 4 at the beginning of the subsequent reproductive cycles.

If any changes in the fowl are observed during rearing, replace administration of the mixture in use with that of the alternative phage-resistant mixtures described.

Example 4

Process for administering the mixtures to swine:

The mixture for swine is administered at birth, in the form of a liquid, to be taken orally for 8 days. In feeds during the viable cycle to rearing health determining cycles, alternate the phage-resistant mixtures in administration periods of 4-10 days, with doses of 10 ⁶ to 10 ⁸ microorganisms per gram of feed.