1. NCRIAH, Russian Academy of Agricultural Sciences, Krasnodar
Bacteriophages for Meat Animal Husbandry: from fundamental genetic research to application in farms
Andrei Zimin
PhD in Biology,
Nikolai Skoblikow
PhD in Medicine,
IBPM, Russian Academy of Sciences, Pushchino
NCRIAH, Russian Academy of Agricultural Sciences, Krasnodar

2. THE CHARACTERISTIC OF PHAGES IN COMPARISON WITH ANTIBIOTICS
Bacteriophages (Phages) are the viruses of bacteria. They may be able for specific effective and securing therapy of bacterial infection diseases as soon humans as animals.
The phages possess a number of significant advantages
in comparison with antibiotics:
they specifically act to concrete species and even strains of bacteria;
they do not target the intestinal normal microflora;
they are indifferent for animal’s eukaryotic cells;
they capable to self-multiplication with subsequent self-elimination.
Side effects
Antibiotics
Toxicity, Selection of resistant strains; Allergies; Dysbioses
Bacteriophages
Transducing (transferring the genes into different bacteria)

3. Modern requirements to phage preparations
Bacteriophages mast be:
Non-lysogenic, virulent
Not able to transduce the genes
With short living circle
Lower immunogenic

4. TRANSDUCING
Now the serious restrictions of phage therapy associated with ability of phages to transduce the various genes, in particular ― the determinants of antibiotic resistance.
By this cause the forming of non-tansducing phages may be considered as a basis for design of successful strategy of reception the perspective phages, accords to requirements to modern medical and veterinary medicines.

5. T4- type bacteriophage is potential optimal candidate for phage therapy
All virulent bacteriophages can be able to transduce the genes of virulent factors, antibiotic resistance
BUT bacteriophage T4 can trancduce genes only after mutation in 6 genes.
The frequency of such mutation lower than (VERY low)
The transducing by T4- type bacteriophages primary discovered in IBPM RAS in 1998.

6. Scheme of isolation of bacteriophages for phage therapy
Collection of strains of phages from various regions of Russia.
Collection of strains of patogenic bacteria from particular region.
Selection of bacteriophages lysing strains of patogenic bacteria.
Selection of T4-type bacteriophages
Проверка бактериофагов на способность к трансдукции

7. FORMING THE COLLECTION OF NON-TRANSDUCING BACTERIOPHAGES
The forming of collection of non-transducing phages, received from different and most microbial diversified sources, may be considered as a basis for design of successful strategy of reception the perspective phages, accords to requirements to modern medical and veterinary medicines.
Isolation of bacteriophages has been made from sampled sewages and natural water ecosystems of Vladivostok, Krasnodar, Moscow, Tula, Astrakhan, Ufa regions of Russia. The bacteriophages characterized by DNA-analysis and electron microscopy. The collection of 378 (at now) bacteriophages formed.

8. Geography of phage sampling
The geographic points of water sampling for forming the biodiversified collection
of non-transducing bacteriophages

9. Swine Escherichiosis as model of investigation of efficiency of non-transducing bacteriophages
One of the general veterinary problem of agriculture are escherichiosis as infection disease induced by pathogenic strains of E. coli, which pathogenic potential in most degree released in animal youth in during the post-weaning period.
Pathogenic E.coli

10. The forming of working collection of pathogenic E.coli from
Krasnodar
region
Azov sea
All of pathogenic E. coli strains analyzed on biochemical and serological properties.
The collection of 13 (at now) pathogenic E.coli strains, isolated from pig farms of Krasnodar region (6 different districts) formed.
Krasnodar
city
Kuban river
Black sea

11. MICROBIOLOGICAL ANALYSIS OF SELECTED PHAGES
The test of sensitivity of pathogenic E. coli strains to selected phages has been executed. In result of it the all bacterial strains divided on two groups. The bacteria of each of these groups characterized by sensitivity for self phages. Only few of isolated phages has been lysed the members of other bacterial group.

12. MOLECULAR-GENETIC ANALYSIS OF SELECTED PHAGESА Б В Г
The collection of phages isolated from pig biomaterial, has been analyzed by PCR with using the primers to 23 and 1 genes of T4-type phages. The gene 23 codes the major phage head protein and is characteristic for this group of viruses. To this sign all isolated phages have been carried to T4-type.

13. MORHOLOGICAL ANALYSIS OF SELECTED PHAGES
Рис. 1. Электронная микроскопия бактериофагов: А и Б – бактериофаг φVZ4, В - φVZ7, на фотографии Б видна агрегация бактериофагов, возникающая при снижении ионной силы водной суспензии, при этом большая часть головок деформирована. A Б В
To morphological signs all isolated phages have been carried to T4-type.

14. MOLECULAR-GENETIC ANALYSIS OF SELECTED PHAGES
The phage DNA hydrolysis by site-specific nucleases

15. Tree of the major capsid protein of bacteriophages 4 and 7
Systematic positioning of isolated bacteriophages
Tree of the major capsid protein of bacteriophages 4 and 7

16. THE TRANSDUCTION TEST

17. TESTING OF EFFICIECY OF SELECTED BACTERIPHAGES in vivo
Constructing the experimental phage veterinary medicines for its application in practical animal husbandry and investigation of its application in experimental farm of NCRIAH, Krasnodar.

18. NEXT steps…
Enlarging the collections of phages and model pathogenic bacteria strains. Selection the bacteriophages, effective on other pathogenic bacteria (Salmonella spp. etc) Investigation of experimental genetically securing phage veterinary medicines on other agricultural animals. Combined using phage medicines with probiotics on the basis of bacteria isolated from animals of local populations. PCR-selection of low immunogenic T4-type bacteriophages

19. Capsid of bacteriophage T4
PCR-selection of low immunogenic T4-type bacteriophages

20. THANKS for your interest !
DANKE SCHÖN !
Andrei Zimin
PhD in Biology,
IBPM, Russian Academy of Sciences, Pushchino
Nikolai Skoblikow
PhD in Medicine,
NCRIAH, Russian Academy of Agricultural Sciences, Krasnodar