BACTERIOPHAGE STRAINS AND THEIR APPLICATIONS

Abstract

New strains of bacteriophages and their applications are revealed, useful especially in fish farming.

Claims

1. A bacteriophage for use in prevention and treatment of infections of farm animals, especially fish, caused by pathogenic bacterial strains sensitive to these bacteriophages, wherein said bacteriophage is intended to be given to endangered animals via immersion, favorably at 24-hour time intervals.

2. The bacteriophage for use according to claim 1, wherein an infection in fish farming is the infection with pathogenic strains of Aeromonas sp. and Pseudomonas sp., especially the strains of Aeromonas hydrophila, Aeromonas salmonicida or Pseudomonas fluorescens, wherein used bacteriophage is the bacteriophage strain selected from the group deposited in the Polish Collection of Microorganisms under the following deposition numbers: F/00096 (strain 25AhydR2PP), F/00094 (strain 50AhydR13PP), F/00098 (strain 22PfluR64PP), F/00099 (strain 67PfluR64PP), F/00100 (strain 71PfluR64PP), F/00095 (strain 98PfluR60PP) and F/00101 (strain 60AhydR15PP).

3. A bacteriophage for use in stimulating fish immunity against infections by stimulating both innate and humoral immune systems.

4. The bacteriophage for use according to claim 3, wherein the used bacteriophage strain is selected from the group deposited in the Polish Collection of Microorganisms under the following deposition numbers: F/00096 (strain 25AhydR2PP), F/00094 (strain 50AhydR13PP), F/00098 (strain 22PfluR64PP), F/00099 (strain 67PfluR64PP), F/00100 (strain 71PfluR64PP), F/00095 (strain 98PfluR60PP) and F/00101 (strain 60AhydR15PP).

5. A bacteriophage strain selected from the group deposited in the Polish Collection of Microorganisms under the following deposition numbers: F/00096 (strain 25AhydR2PP), F/00094 (strain 50AhydR13PP), F/00098 (strain 22PfluR64PP), F/00099 (strain 67PfluR64PP), F/00100 (strain 71PfluR64PP), F/00095 (strain 98PfluR60PP) and F/00101 (strain 60AhydR15PP).

Description

[0032] In order the invention becomes more evident, it is illustrated on the attached figures.

[0033] FIG. 1 presents the results of analysis of susceptibility of A. hydrophila 7966 strain for bacteriophages and bacteriophage preparations. 1A. hydrophila 7966 with 25AhydR2PP; 2A. hydrophila 7966 with BAFADOR II; 3A. hydrophila 7966 with BAFADOR III; 4A. hydrophila 7966 with BAFADOR IV; 5the growth control of A. hydrophila 7966.

[0034] FIG. 2 presents the results of analysis of susceptibility of A. hydrophila 7965 strain for bacteriophages and bacteriophage preparations. 1A. hydrophila 7965 with 13AhydR10PP; 2A. hydrophila 7965 with 14AhydR10PP; 3A. hydrophila 7965 with 85AhydR10PP; 4A. hydrophila 7965 with BAFADOR II; 5the growth control of A. hydrophila 7965.

[0035] FIG. 3 presents the results of analysis of susceptibility of A. hydrophila 49140 strain for bacteriophages and bacteriophage preparations. 1A. hydrophila 49140 with 50AhydR13PP; 2 A. hydrophila 49140 with BAFADOR II; 3A. hydrophila 49140 with BAFADOR III; 4A. hydrophila 49140 with BAFADOR IV; 5the growth control of A. hydrophila 49140.

[0036] FIG. 4 presents the results of analysis of susceptibility of A. hydrophila 33658 strain for bacteriophages and bacteriophage preparations. 1A. hydrophila 33658 with 60AhydR15PP; 2A. hydrophila 33658 with BAFADOR II; 3A. hydrophila 33658 with BAFADOR III; 4A. hydrophila 33658 with BAFADOR IV; 5the growth control of A. hydrophila 33658.

[0037] FIG. 5 presents the results of analysis of susceptibility of P. fluorescens 8B/UWM strain for bacteriophages and bacteriophage preparations. 1 P. fluorescens 8B/UWM with 22PfluR64PP; 2 P. fluorescens 8B/UWM with 67PfluR64PP; 3 P. fluorescens 8B/UWM with 71PfluR64PP; 4 P. fluorescens 8B/UWM with BAFADOR II; 5 P. fluorescens 8B/UWM with BAFADOR III; 6P. fluorescens 8B/UWM with BAFADOR IV; 7the growth control of P. fluorescens 8B/UWM.

[0038] FIGS. 6-9 show restriction profiles of selected bacteriophages.

[0039] FIG. 6 presents the restriction profile of bacteriophage 60AhydR15PP obtained after digestion with the following restriction enzymes: DraI (lane 2), SspI (lane 4), AseI (lane 6). Lanes 1 and 8DNA ladder (1 kb).

[0040] FIG. 7 presents restriction profiles of bacteriophages 22PfluR64PP (lane 2), 67PfluR64PP (lane 3) and 71PfluR64PP (lane 4) obtained after digestion with EcoRI restriction enzyme. Lane 1DNA ladder (1 kb).

[0041] FIG. 8 presents the restriction profile of bacteriophage 50AhydR13PP obtained after the digestion with SspI restriction enzyme (lane 2) and the restriction profile of bacteriophage 98PfluR60PP obtained after the digestion with EcoRI restriction enzyme (lane 3). Lane 1DNA ladder (1 kb).

[0042] FIG. 9 presents the restriction profile of bacteriophage 25AhydR2PP (lane 2) obtained after the digestion with EcoRI restriction enzyme. Lane 1DNA ladder (1 kb).

EXAMPLE 1. ISOLATION AND CHARACTERISTIC OF BACTERIOPHAGES

[0043] Preparation of Bacterial Strains Collection of the Aeromonas Spp. and Pseudomonas sp. Genus Isolated from People and Farm Animals.

[0044] Initially, the collection of 82 bacterial strains of the Aeromonas spp. and Pseudomonas sp. was prepared (Table 1). These strains were used to test the specificity of isolated bacteriophages. The collection includes both reference strains available in public repositories and isolates obtained from the Adam Mickiewicz University in Poznan and from the Department of Fish Pathology and Immunology of Inland Fisheries Institute in Olsztyn, and University of Warmia and Mazury in Olsztyn (Table 2).

TABLE-US-00001 TABLE 1 Bacterial strain collection of Aeromonas sp., Pseudomonas sp., Yersinia sp., Renibacterium sp. and Enterococcus sp. Code Strain R1 Yersinia ruckeri 29473 R2 Aeromonas hydrophila 7966 R3 Aeromonas hydrophila 1206101 R4 Yersinia ruckeri 5304100 R5 Aeromonas sobria R6 Aeromonas hydrophila 49140 R7 Yersinia ruckeri 29473 R9 Aeromonas hydrophila 35654 R10 Aeromonas hydrophila 7965 R11 Aeromonas hydrophila 5247167 R12 Aeromonas hydrophila 7965 (290158) R13 Aeromonas hydrophila 49140 R14 Aeromonas hydrophila 33658 (788242) R15 Aeromonas hydrophila 33658 R16 Aeromonas hydrophila 35654 R21 Aeromonas hydrophila RK 70363 R22 Aeromonas hydrophila SK 3 R23 Aeromonas hydrophila ATCC 49140 R24 Aeromonas hydrophila LMG 13656 R25 Aeromonas hydrophila AK 44 R26 Aeromonas hydrophila ATCC 7966.sup.T R27 Aeromonas sobriaL MG 13469 R28 Aeromonas sobria CIP 7433.sup.T R29 Aeromonas salmonicida LMG 14900.sup.T R30 Aeromonas salmonicida LMG 3782.sup.T R31 Aeromonas salmonicida CDC 0434-84 R32 Aeromonas salmonicida AK 46 R33 Aeromonas salmonicida LMG 3780.sup.T R34 Aeromonas salmonicidaLMG 13450 R40 1B/IRS/03/13_Aeromonas hydrophila R41 2B/IRS/03/13_Aeromonas hydrophila R42 3B/IRS/03/13_Aeromonas hydrophila R43 4B/IRS/03/13_Aeromonas hydrophila R44 5B/IRS/04/13_Aeromonas hydrophila R45 6B/IRS/05/13_Aeromonas hydrophila R46 7B/IRS/05/13_Aeromonas hydrophila R47 8B/IRS/05/13_Aeromonas hydrophila R48 9B/IRS/05/13_Aeromonas hydrophila R49 10B/IRS/05/13_Aeromonas hydrophila R50 11B/IRS/05/13_Aeromonas hydrophila R51 12B/IRS/06/13_Aeromonas hydrophila R52 13B/IRS/06/13_Aeromonas hydrophila R53 1B/IRS/04/14K_Aeromonas hydrophila R54 2B/IRS/04/14K_Aeromonas hydrophila R55 3B/IRS/04/14K_Aeromonas hydrophila R56 4B/IRS/04/14P_Aeromonas hydrophila R57 1B/UWM/03/13_Yersinia ruckeri R58 2B/UWM/03/13_Pseudomonas fluorescens R59 3B/UWM/03/13_Aeromonas hydrophila R60 4B/UWM/03/13_Pseudomonas fluorescens R61 5B/UWM/03/13_Pseudomonas fluorescens R62 6B/UWM/03/13_Pseudomonas fluorescens R63 7B/UWM/03/13_Pseudomonas fluorescens R64 8B/UWM/03/13_Pseudomonas fluorescens R65 9B/UWM/03/13_Aeromonas hydrophila R66 10B/UWM/03/13_Yersinia ruckeri R67 11B/UWM/03/13_Aeromonas hydrophila R68 13B/UWM/03/13_Pseudomonas fluorescens R69 14B/UWM/03/13_Yersinia ruckeri R70 15B/UWM/03/13_Yersinia ruckeri R71 16B/UWM/04/13_Aeromonas hydrophila/caviae R72 17B/UWM/06/13_Yersinia ruckeri R73 18B/UWM/06/13_Aeromonas salmonicida subsp. salmonicida R74 19B/UWM/06/13_Aeromonas salmonicida subsp. salmonicida R75 20B/UWM/06/13_Aeromonas hydrophila R76 21B/UWM/06/13_Yersinia ruckeri R77 22B/UWM/06/13_Aeromonas sobria R78 23B/UWM/06/13_Aeromonas hydrophila R79 24B/UWM/06/13_Renibacterium salmonicidum R80 25B/UWM/07/13_Aeromonas sobria R81 26B/UWM/07/13_Aeromonas hydrophila R82 27B/UWM/07/13_Aeromonas hydrophila R83 28B/UWM/07/13_Aeromonas sobria R84 29B/UWM/07/13_Pseudomonas fluorescens R85 30B/UWM/06/14_Enterococcus R86 1/14P/UWM_Yersinia ruckeri R87 2/14P/UWM_Yersinia ruckeri R88 3/14P/UWM_Yersinia ruckeri R89 31B/UWM/08/14_Aeromonas hydrophila R90 32B/UWM/08/14_Aeromonas hydrophila R91 33B/UWM/08/14_Pseudomonas fluorescens R92 34B/UWM/08/14_Yersinia ruckeri

TABLE-US-00002 TABLE 2 Bacterial strains of Aeromonas sp., Pseudomonas sp., Yersinia sp., Renibacterium sp. and Enterococcus sp. Number of No Bacteria strains Source 1 Aeromonas hydrophila 6 UAM 38 UWM 2 Aeromonas salmonicida 6 UAM 2 UWM 3 Aeromonas sobria 2 UAM 4 UWM 4 Pseudomonas fluorescens 9 UWM 5 Renibacterium salmonicidum 1 UWM 6 Enterococcus 1 UWM 7 Yersinia ruckeri 13 UWM
Isolation of Bacteriophages Active Against Selected Strains of Aeromonas Spp. and Pseudomonas sp. from Environmental Samples.

[0045] Bacteriophages were isolated from samples taken from the intake manifolds, representing an initial stage of the wastewater treatment process, received from the Main Sewage Treatment Plant (GO) in Lodz or from samples of water obtained from the Inland Fisheries Institute (IRS) in abieniec (Table 3).

TABLE-US-00003 TABLE 3 Isolated bacteriophages and their hosts. No Bacteriophage Source Host 1 11AhydR10PP GO Aeromonas hydrophila 7965 3 13AhydR10PP GO Aeromonas hydrophila 7965 4 14AhydR10PP GO Aeromonas hydrophila 7965 5 25AhydR2PP GO Aeromonas hydrophila 7966 6 50AhydR13PP GO Aeromonas hydrophila 49140 7 53AhydR13PP GO Aeromonas hydrophila 49140 8 60AhydR15PP GO Aeromonas hydrophila 33658 9 62AhydR11PP GO Aeromonas hydrophila 5247167 10 80AhydR10PP IRS Aeromonas hydrophila 7965 11 82AhydR10PP IRS Aeromonas hydrophila 7965 12 85AhydR10PP IRS Aeromonas hydrophila 7965 13 86AhydR10PP IRS Aeromonas hydrophila 7965 14 72AsobR5PP IRS Aeromonas sobria 15 75AsobR5PP IRS Aeromonas sobria 16 76AsobR5PP IRS Aeromonas sobria 17 19AhydR15PP GO Aeromonas hydrophila 33658 18 22PfluR64PP GO Pseudomonas fluorescens 8B/UWM/03/13 19 23PfluR64PP GO Pseudomonas fluorescens 8B/UWM/03/13 20 67PfluR64PP GO Pseudomonas fluorescens 8B/UWM/03/13 21 69PfluR64PP GO Pseudomonas fluorescens 8B/UWM/03/13 22 70PfluR64PP GO Pseudomonas fluorescens 8B/UWM/03/13 23 71PfluR64PP GO Pseudomonas fluorescens 8B/UWM/03/13 24 88PfluR61PP IRS Pseudomonas fluorescens 5B/UWM/03/13 25 98PfluR60PP GO Pseudomonas fluorescens 4B/UWM/03/13

[0046] All bacteriophages used in further experiments were purified by a serial passage to a single plaque on plates with Luria-Bertani (LB) medium. This procedure required at least 5-fold passage.

[0047] The specificity of bacteriophages isolated with the plate method was initially determined on the basis of the lytic capacity of phages against selected strains of Aeromonas spp., and Pseudomonas sp., isolated from diseased fish, obtained from the Department of Fish Pathology and Immunology of Inland Fisheries Institute in Olsztyn (IRS) and the University of Warmia and Mazury in Olsztyn and against selected strains of Aeromonas spp., and Pseudomonas sp. which constitute the extension of the collection of exemplary strains isolated from patients, obtained from the University of Adam Mickiewicz University in Pozna.

[0048] In order to confirm the results, the study of specificity of the isolated phages was repeated 3 times (Tables 4 and 5).

TABLE-US-00004 TABLE 4 The specificity of selected bacteriophages against selected model and environmental strains of Aeromonas spp. (Proteon Pharmaceuticals bacterial strain collection). Bacteriophages Bacterial strains 11AhydR10PP 13AhydR10PP 14AhydR10PP 19AhydR15PP 25AhydR2PP 50AhydR13PP 53AhydR13PP A. hydrophila R2 cl R6 cl R9 cl + R10 cl cl R11 R12 cl cl R13 cl R14 cl cl R15 cl + R21 cl R22 cl R23 cl R24 cl R25 cl R26 cl R40 cl R41 cl R48 cl cl R52 cl cl R53 R55 + R59 R65 cl R71 A. salmonicida R30 cl R31 cl R32 cl R33 A. sobria R5 R28 cl R80 Bacteriophages Bacterial strains 60AhydR15PP 62AhydR11PP 80AhydR10PP 82AhydR10PP 85AhydR10PP A. hydrophila R2 R6 + R9 + R10 R11 cl R12 cl R13 cl R14 cl cl R15 R21 R22 R23 R24 cl cl R25 cl cl R26 R40 R41 R48 cl cl R52 cl cl R53 + R55 + R59 cl R65 cl R71 + A. salmonicida R30 cl cl R31 cl cl R32 R33 cl cl A. sobria R5 cl R28 R80 + Bacteriophages Bacterial strains 86AhydR10PP 72AsobR5PP 75AsobR5PP 76AsobR5PP A. hydrophila R2 R6 R9 R10 R11 R12 R13 R14 R15 R21 R22 R23 R24 R25 R26 R40 R41 R48 cl R52 cl + R53 + R55 + R59 R65 R71 + A. salmonicida R30 R31 R32 R33 A. sobria R5 R28 R80 + + + cltotal lysis; +growth inhibition; no effect

TABLE-US-00005 TABLE 5 Specificity of selected bacteriophages against chosen environmental strains of Pseudomonas sp. (Proteon Pharmaceuticals bacterial strain collection). Bacteriophages Bacterial strain 22PfluR64PP 23PfluR64PP 67PfluR64PP 68PfluR64PP 69PfluR64PP P. fluorescens R60 R61 cl cl cl cl R64 cl cl cl R68 cl cl R91 cl cl Bacteriophages Bacterial strain 70PfluR64PP 71PfluR64PP 88PfluR61PP 98PfluR60PP P. fluorescens R60 cl R61 cl cl cl cl R64 cl R68 R91 cl cltotal lysis; +growth inhibition; no effect

[0049] Isolated bacteriophages were propagated using a host strain as a production strain. These samples were subjected to genomic DNA isolation of bacteriophages based on the modified method of Su et al. [MT Su, 1998].

Genetic Characteristics of Bacteriophages

[0050] Isolated DNA of bacteriophages was used to perform restrictive analysis with enzymes: AseI, DraI, SspI and EcoRI. Obtained restriction profiles allowed to define initial genetic characteristic of bacteriophages (FIGS. 6, 7, 8 and 9). Subsequently, after genomes sequencing, more detailed genetic characteristics of bacteriophages was done. Received sequences were analyzed by comparison to genomes of bacteriophages available in BLAST database, then by designation of potential open reading frames in Artemis program and by searching homology to described bacteriophages' proteins using blastp algorithm.

[0051] On the basis of performed analysis it was showed that: [0052] Bacteriophage 60AhydR15PP, classified to Myoviridae family (Caudovirales order), contains linear double-stranded DNA (circular form of genome) in size of approximately 165 kbp and shows high similarity to the group of lytic bacteriophages T4, specific against many bacteria from Aeromonas sp. [0053] Bacteriophage 25AhydR2PP shows high homology to phage AS7, belonging to T7-like family. It is characterized by linear double-stranded DNA in size of approximately 42 kbp. It belongs to lytic phages. [0054] Bacteriophage 50AhydR13PP shows high homology to phage AS7, belonging to T4-like family. Its genome has size of approximately 165 kbp. [0055] Bacteriophages 22PfluR64PP, 67PfluR64PP, 71PfluR64PP were classified to Podoviridae family (Caudovirales order) with short, unshrinkable tails and icosaedral capsid containing linear double-stranded DNA in size of approximately 40 kbp. They show high similarity to lytic bacteriophages of T7 group specific to many bacteria of the Pseudomonas sp. [0056] Sequence of phage 98PfluR60PP did not show similarity to previously known phages families. However, a detailed comparative analysis of particular proteins allowed to find homology with the typical phage proteins necessary to perform a lytic cycle. The genome of 98PfluR60PP is 74 kb in size.

EXAMPLE 2. PREPARATION PRODUCTION

Determination and Optimization of Conditions for the Propagation of Bacteriophages in a Laboratory Scale.

[0057] Optimization was carried out for each bacteriophage strain using the host bacterial strain.

[0058] The following cultivation conditions were optimized: volume of inoculum of both bacterial and bacteriophage culture, time of cultivation of pure culture and incubation of the infected culture, the cultivation temperature, aeration rate and the type of a growth medium. YES medium at pH 7.0 was selected as the growth medium. The optimum volume of the bacterial inoculum was estimated to be 210.sup.9 CFU per 0.5 liter of the culture medium. Depending on a bacteriophage strain, cultures were adjusted to an optical density OD.sub.620=0.2-0.8. The optimal growth temperature of the bacterial culture was set to 25 C. Optimized aeration rate for cultivation was reached at 140 rpm in a shaker Ecotron from Infors company. In the process of optimization, it was observed that the addition of 1% by volume of a phage in titer of 10.sup.9 PFU/ml (5 ml per 0.5 l of culture) was the optimum inoculum of the bacteriophage.

Development of Technology for the Production and Purification of Bacteriophage Suspension.

Stages of Production

[0059] 1. Amplification in Bioreactor

[0060] The first step in the production line is a amplification of the particles of bacteriophages that specifically destroy bacterial cells of selected strains of Aeromonas spp., or Pseudomonas sp. This is achieved by inoculation of growth medium with the bacterial production strain and cultivation until the appropriate optical density is obtained, then the bacteriophage inoculum is added and the process of proliferation of bacteriophage particles is carried out (conditions discussed above). Once the amplification process is finished, the culture is transferred in a sterile manner using of a peristaltic pump to the next stage of the production process. Each strain of bacteriophages is amplificated as a separate culture. In our research, we used 5-liter (4 liter working volume) airlift bioreactor whose main advantage is the use of modern, disposable amplification bags.

[0061] 2. Biomass Removal

[0062] A completion of the process of amplification of bacteriophages requires the removal of remains of bacteria form a culture broth. For this purpose, the tangential microfiltration is performed using a membrane of a pore size of 0.45 m, and then microfiltration using a membrane of a pore size of 0.22 m. This procedure ensures to obtain a sterile suspension with very little decline in titer of phage particles.

[0063] 3. Assay of the Activity of Manufactured Component

[0064] After completion of the filtration process, the phage suspension is subjected to an activity assay expressed as PFU/ml units (plaque forming unit/ml). Determination of the activity is carried out in accordance with the procedure Enumeration of Bacteriophages in Suspension by Double Agar Overlay Plaque Assay validated in Proteon Pharmaceuticals SA (Certificate of Good Laboratory Practice No. 10/2015/DPL).

[0065] 4. Production of the Final Bacteriophage Preparation

[0066] In this step, the manufactured components are mixed. Before mixing, the volumes of respective components are calculated, assuring the equal amount of each component in the preparation. Calculations are based on previously determined activity (PFU/ml). The final formulation is then aliquoted and stored at temp. 2-8 C.

EXAMPLE 3. STUDIES OF EFFICIENCY AND SAFETY OF BACTERIOPHAGE PREPARATION

[0067] In the conducted studies 3 bacteriophage preparations of the following compositions were used: [0068] BAFADOR II: 60AhydR15PP, 62AhydR11PP, 13AhydR10PP, 14AhydR10PP, 85AhydR10PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP, [0069] BAFADOR III: 60AhydR15PP, 25AhydR2PP, 50AhydR13PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP, 98PfluR60PP [0070] BAFADOR IV: 60AhydR15PP, 25AhydR2PP, 50AhydR13PP, 22PfluR64PP, 98PfluR60PP

[0071] All above preparations were characterized by equivalent amounts of components and activity of 10.sup.8 PFU/ml.

[0072] Bacteriophage preparations were prepared in such a way that each bacteriophage was subjected to the optimized procedure of amplification, removal of bacterial biomass by microfiltration and determination of its activity in PFU/ml. The suspensions of manufactured bacteriophages were mixed in equal amounts obtaining the final bacteriophage preparation. These preparations tested for microbiological purity did not indicate a presence of bacteria.

In Vitro Studies

[0073] Based on measurements of optical density (OD.sub.620) of bacterial strains, the ability of developed bacteriophage preparations and bacteriophage components to reduce the number of bacterial cells was tested.

[0074] 3 bacteriophage preparations (BAFADOR II, BAFADOR III and BAFADOR IV) and 11 different bacteriophages (13AhydR10PP, 14AhydR10PP, 25AhydR2PP, 50AhydR13PP, 60AhydR15PP, 62AhydR11PP, 85AhydR10PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP and 98PfluR60PP) were used in the studies.

[0075] 5 bacterial strains were used as a test system: A. hydrophila 7966, A. hydrophila 7965, A. hydrophila 49140, A. hydrophila 33658 and P. fluorescens 8B/UWM.

[0076] All experiments were performed in triplicates on 96-well plates. Bacterial cultures of optical density around 0.2 were mixed with suspensions of bacteriophages in 1:1 volume ratio (100 l:100 l). Mixtures were incubated at 25 C. for 21 hours. OD.sub.620 values were recorded every 20 min.

[0077] Obtained results are presented on FIGS. 1-5.

[0078] Based on obtained results, it was found that mixtures of bacteriophages were much more advantageous in eradication of bacterial strains than individual bacteriophage component. Moreover, these studies confirmed better efficiency of BAFADOR III and BAFADOR IV preparations over BAFADOR II preparation.

In Vivo Studies

The Assessment of Safety of a Prototypical Bacteriophage Preparation in Protection of Farmed Fish Against Bacterial Pathogens.

[0079] The studies were carried out in collaboration with the University of Warmia and Mazury.

The Experimental Procedure 1

[0080] The experimental material were 20 carps, 20 rainbow trouts and 20 European catfish kept in separate tanks and treated with bacteriophage preparation BAFADOR II at the concentration of 10.sup.5 PFU/ml for 1 hour via immersion. The assessment of selected hematological and biochemical parameters of fish blood was conducted before administration of bacteriophage preparation BAFADOR II and 1, 2 and 3 days after application.

TABLE-US-00006 TABLE 6 The influence of bacteriophage preparation administered via immersion on selected hematological and biochemical parameters in carp (n = 20, mean values standard deviation; *statistical significance p < 0.05) Days of blood sampling (days after immersion) Before Measured parameters immersion 1 2 3 Erythrocytes count 1.5 0.4 1.6 0.5 1.7 0.3 1.6 0.3 (RBC) (mln/mm) Hematocrit (Ht) (%) 32.5 3.2 34.5 3.4 34.9 3.2 33.4 2.9 Hemoglobin (Hb) (g %) 10.6 1.4 11.4 1.4 11.6 1.6 10.8 1.5 Mean corpuscular 58.4 7.5 56.5 8.4 55.9 7.5 57.9 8.5 hemoglobin (g/L) Mean corpuscular 25.6 5.5 26.4 4.8 27.6 5.2 26.8 4.9 hemoglobin concentration (g/L) Cortisol (ng/L) 179 27 185 32 191 45 187 35 Glucose (mg/L) 110 15 115 14 114 12 118 16 Aspartate transaminase 84.2 12.5 86.5 13.8 87.2 14.5 88.9 13.3 activity (AST) (U/L) Alanine transaminase 2.5 0.8 2.7 0.7 2.8 0.6 2.9 0.8 activity (ALT) (U/L)

TABLE-US-00007 TABLE 7 The influence of bacteriophage preparation administered via immersion on selected hematological and biochemical parameters in rainbow trout (n = 20, mean values standard deviation; *statistical significance p < 0.05). Days of blood sampling (days after immersion) Before Measured parameters immersion 1 2 3 Erythrocytes count 2.4 0.5 2.8 0.6 2.7 0.5 2.6 0.4 (RBC) (mln/mm) Hematocrit (Ht) (%) 39.8 4.5 40.5 4.1 41.6 3.8 42.5 3.9 Hemoglobin (Hb) (g %) 26.5 3.8 28.2 3.2 27.8 2.9 28.9 3.6 Mean corpuscular 58.4 7.5 56.5 8.4 55.9 7.5 57.9 8.5 hemoglobin (g/L) Mean corpuscular 31.5 5.2 32.8 4.5 34.2 4.8 33.6 4.2 hemoglobin concentration (g/L) Cortisol (ng/L) 192 34 198 32 197 35 191 38 Glucose (mg/L) 185 23 192 26 193 27 189 25 Aspartate transaminase 96.5 22.4 98.5 2.5 97.8 24.2 98.5 24.4 activity (AST) (U/L) Alanine transaminase 4.6 1.2 4.9 1.5 4.8 1.4 4.7 1.7 activity (ALT) (U/L)

TABLE-US-00008 TABLE 8 The influence of bacteriophage preparation administered via immersion on selected hematological and biochemical parameters in catfish (n = 20, mean values standard deviation; *statistical significance p < 0.05). Days of blood sampling (days after immersion) Before Measured parameters immersion 1 2 3 Erythrocytes count 1.5 0.5 1.7 0.5 1.8 0.5 1.6 0.5 (RBC) (mln/mm) Hematocrit (Ht) (%) 19.7 1.5 20.8 1.1 21.4 1.8 20.3 1.9 Hemoglobin (Hb) (g %) 21.5 2.8 22.4 2.2 23.8 2.8 22.7 2.6 Cortisol (ng/L) 142 31 148 34 147 29 141 27 Glucose (mg/L) 165 20 162 19 163 21 168 22

[0081] Based on the obtained results, it was demonstrated that bacteriophage preparation BAFADOR II had no negative effect on selected hematological parameters (erythrocyte count, hematocrit, hemoglobin), liver enzymes activity: AST, ALT and glucose level up to 3 days after administration in carp (Table 6), rainbow trout (Table 7) and catfish (Table 8). Also, no significant changes in a cortisol level, a hormone secreted during stress, were observed.

The Experimental Procedure 2

[0082] The experimental material were 20 carps, 20 rainbow trouts and 20 European catfish kept in separate tanks and treated with bacteriophage preparation BAFADOR II at the concentration of 10.sup.5 PFU/ml for 1 hour via immersion. The assessment of selected parameters of humoral and cellular immunity in fish blood was conducted before administration of bacteriophage formulation BAFADOR II and 3, 5 and 7 days after application.

TABLE-US-00009 TABLE 9 The influence of bacteriophage preparation administered via immersion on selected immune parameters in carp (n = 20, mean values standard deviation; *statistical significance p < 0.05) Days of blood sampling (days after immersion) Measured parameters 0 3 5 7 Respiratory burst 0.46 0.03 0.58 0.5* 0.75 0.05* 0.85 0.04* activity of phagocytes (RBA, OD 620 nm) Potential killing 0.38 0.04 0.49 0.5* 0.60 0.04* 0.75 0.05* activity of phagocytes (PKA, OD 620 nm) Proliferative activity 0.49 0.05 0.62 0.5* 0.86 0.04* 0.91 0.05* of lymphocytes stimulated by ConA (OD 620 nm) Proliferative activity 0.32 0.04 0.56 0.7* 0.69 0.07* 0.79 0.05* of lymphocytes stimulated by LPS (OD 620 nm) Lysosyme activity in 1.8 0.4 2.9 0.6* 3.6 0.4* 4.1 0.4* serum (mg/L) Ceruloplasmin activity 64.5 5.9 72.5 4.6* 73.5 4.8* 74.0 5.2* in serum (IU) Total serum protein 43.5 4.0 50.3 3.5* 51.0 4.5* 50.8 4.2* (g/L) Ig in serum (g/L) 7.5 0.6 8.9 0.7* 9.6 0.8* 10.5 0.7*

TABLE-US-00010 TABLE 10 The influence of bacteriophage preparation administered via immersion on selected immune parameters in rainbow trout (n = 20, mean values standard deviation; *statistical significance p < 0.05) Days of blood sampling (days after immersion) Measured parameters 0 3 5 7 Respiratory burst 0.46 0.03 0.58 0.5* 0.75 0.05* 0.85 0.04* activity of phagocytes (RBA, OD 620 nm) Potential killing 0.38 0.04 0.49 0.5* 0.60 0.04* 0.75 0.05* activity of phagocytes (PKA, OD 620 nm) Proliferative activity 0.49 0.05 0.62 0.5* 0.86 0.04* 0.91 0.05* of lymphocytes stimulated by ConA (OD 620 nm) Proliferative activity 0.32 0.04 0.56 0.7* 0.69 0.07* 0.79 0.05* of lymphocytes stimulated by LPS (OD 620 nm) Lysosyme activity in 1.8 0.4 2.9 0.6* 3.6 0.4* 4.1 0.4* serum (mg/L) Ceruloplasmin activity 64.5 5.9 72.5 4.6* 73.5 4.8* 74.0 5.2* in serum (IU) Total serum protein 43.5 4.0 50.3 3.5* 51.0 4.5* 50.8 4.2* (g/L) Ig in serum (g/L) 7.5 0.6 8.9 0.7* 9.6 0.8* 10.5 0.7*

TABLE-US-00011 TABLE 11 The influence of bacteriophage preparation administered via immersion on selected immune parameters in catfish (n = 20, mean values standard deviation; *statistical significance p < 0.05) Days of blood sampling (days after immersion) Measured parameters 0 3 5 7 Respiratory burst 0.39 0.05 0.58 0.4* 0.72 0.05* 0.79 0.04* activity of phagocytes (RBA, OD 620 nm) Potential killing 0.30 0.04 0.47 0.4* 0.58 0.05* 0.67 0.05* activity of phagocytes (PKA, OD 620 nm) Proliferative activity 0.41 0.04 0.56 0.5* 0.69 0.06* 0.75 0.04* of lymphocytes stimulated by ConA (OD 620 nm) Proliferative activity 0.32 0.04 0.47 0.4* 0.61 0.05* 0.70 0.05* of lymphocytes stimulated by LPS (OD 620 nm) Lysosyme activity in 2.6 0.4 3.4 0.5 4.2 0.6* 4.9 0.5* serum (mg/L) Ceruloplasmin activity 61.0 6.5 72.5 4.5* 74.0 5.5* 73.0 4.5* in serum (IU) Total serum protein 41.5 3.0 50.0 3.5 51.5 4.0* 52.0 3.5* (g/L) Ig in serum (g/L) 6.8 0.5 7.9 0.7 8.8 0.5* 9.5 0.5*

[0083] Based on the obtained results, it was demonstrated that the preparation BAFADOR II caused statistically significant increase in measured parameters of innate cellular immunity (respiratory burst activity and potential killing activity of phagocytes, proliferative activity of lymphocytes) and humoral immunity (lysozyme and ceruloplasmin activity, total serum protein and Ig in serum) in treated fish species. These changes were observed just after 3 days of administration of bacteriophage preparation.

The Assessment of Effectiveness of a Prototypical Bacteriophage Preparation in Protection of Farmed Fish Against Bacterial Pathogens.

[0084] The studies were carried out in collaboration with the University of Warmia and Mazury.

Aim of the Study:

[0085] The assessment of possibilities of applying bacteriophages to prevent bacterial infections in fish caused by Pseudomonas sp.

[0086] The experimental material was carp experimentally infected by intraperitoneal injection of environmental strain Pseudomonas fluorescens isolated from infected fish and identified on biochemical level by API test. Fish were infected with bacterial suspension at a concentration of 610.sup.8 CFU/ml (dose 0.2 ml per fish). Bacteriophage preparations (BAFADOR II, III and IV) were administered via immersion for one hour.

The Experimental Procedure 3

[0087] The experimental material were 100 carps randomly divided into 5 equal groups kept in separate tanks. Fish from 2, 3, 4 and 5 groups were experimentally infected by intraperitoneal injection of environmental strain Pseudomonas fluorescens isolated from infected fish and identified using the API test. Fish were infected with bacterial suspension at a concentration of 610.sup.8 CFU/ml (dose 0.2 ml per fish). Bacteriophage preparation (BAFADOR II) was administered via immersion at a concentration of 10.sup.5 PFU/ml for one hour.

TABLE-US-00012 TABLE 12 Scheme of application of bacteria and bacteriophages. Number No of fish Description of experiment 1 20 Negative control not infected and not treated with bacteriophage preparation 2 20 Positive control infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) 3 20 Group infected with P. fluorescens: at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR II) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation in concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 24 h after infection 4 20 Group infected with P. fluorescens: at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR II) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation in concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 48 h after infection 5 20 Group infected with P. fluorescens: at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR II) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation in concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 24 h and 48 h after infection

[0088] Mortality rate of fish was estimated during the experiment (Table 13). Based on obtained results, it was demonstrated that bacteriophage preparation caused decrease in a death rate of fish in groups treated with bacteriophages both after 24 (group 3), and 48 hours (group 4) after experimental infection with Pseudomonas fluorescens (20 and 30% of deaths, respectively). The strongest therapeutic effect was observed after double administration of preparation by immersion 24 and 48 hours after infections (group 5; 15% of deaths).

TABLE-US-00013 TABLE 13 The mortality of farmed carp after experimental infection with P. fluorescens and administration of bacteriophage preparation (BAFADOR II). No of group Date 1 2 3 4 5 2 Oct. 2015 0 0 0 0 0 3 Oct. 2015 0 1 0 0 0 4 Oct. 2015 0 3 1 2 0 5 Oct. 2015 0 3 1 2 1 6 Oct. 2015 0 3 1 1 1 7 Oct. 2015 0 1 1 1 1 8 Oct. 2015 0 0 0 0 0 Mortality 0 11 4 6 3 (in pieces) Total mortality 0% 55% 20% 30% 15%

The Experimental Procedure 4

[0089] The experimental material were 100 carps randomly divided into 5 equal groups kept in separate tanks. Fish from 2, 3, 4 and 5 groups were experimentally infected by intraperitoneal injection of environmental strain Pseudomonas fluorescens isolated from infected fish and identified using the API test. Fish were infected with bacterial suspension at a concentration of 610.sup.8 CFU/ml (dose 0.2 ml per fish). Bacteriophage preparation (BAFADOR III) was administered by immersion at a concentration of 10.sup.5 PFU/ml for one hour.

TABLE-US-00014 TABLE 14 Scheme of application of bacteria and bacteriophages Number No of fish Description of experiment 1 20 Negative control not infected and not treated with bacteriophage preparation 2 20 Positive control infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) 3 20 Group infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR III) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation at a concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 24 h after infection 4 20 Group infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR III) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation in concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 48 h after infection 5 20 Group infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR III) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation at a concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 24 h and 48 h after infection

[0090] Mortality rate of fish was estimated during the experiment (Table 15). Obtained results show that bacteriophage preparation of the present invention reduced mortality of fish in groups treated with bacteriophages, both after 24 (group 3), and 48 hours (group 4) after experimental infection with Pseudomonas fluorescens (15 and 25% of deaths, respectively). The strongest therapeutic effect was observed after double administration of preparation by immersion 24 and 48 hours after infections (group 5; 10% of deaths).

TABLE-US-00015 TABLE 15 Mortality rate of carp culture after experimental infection with P. fluorescens and treatment with bacteriophage preparation (BAFADOR III). No of group Date 1 2 3 4 5 12 Oct. 2015 0 0 0 0 0 13 Oct. 2015 0 1 0 0 0 14 Oct. 2015 0 3 1 1 0 15 Oct. 2015 0 3 1 2 1 16 Oct. 2015 0 3 1 1 1 17 Oct. 2015 0 0 0 1 0 18 Oct. 2015 0 0 0 0 0 Mortality 0 10 3 5 2 (in pieces) Total mortality 0% 50% 15% 25% 10%

The Experimental Procedure 5

[0091] The experimental material were 100 carps randomly divided into 5 equal groups kept in separate tanks. Fish from 2, 3, 4 and 5 groups were experimentally infected by intraperitoneal injection of environmental strain Pseudomonas fluorescens isolated from infected fish and identified using biochemical test API. Fish were infected with bacterial suspension at a concentration of 610.sup.8 CFU/ml (dose 0.2 ml per fish). Bacteriophage preparation (BAFADOR IV) was administered via immersion at a concentration of 10.sup.5 PFU/ml for one hour.

TABLE-US-00016 TABLE 16 Scheme of application of bacteria and bacteriophages Number No of fish Description of experiment 1 20 Negative control not infected and not treated with bacteriophage preparation 2 20 Positive control infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) 3 20 Group infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR IV) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation in concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 24 h after infection 4 20 Group infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR IV) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation in concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 48 h after infection 5 20 Group infected with P. fluorescens at a concentration of 6 10.sup.8 CFU/ml (dose 0.2 ml/fish) and treated with bacteriophage preparation (BAFADOR IV) at a concentration of 10.sup.5 PFU/ml (25 ml of preparation in concentration of 10.sup.8 PFU/ml per 2.5 L of water, 1 h bath) 24 h and 48 h after infection

[0092] Mortality rate of fish was estimated during the experiment (Table 17). Obtained results show that bacteriophage preparation of the present invention reduced mortality of fish in groups treated with bacteriophages, both after 24 (group 3), and 48 hours (group 4) after experimental infection with Pseudomonas fluorescens (15 and 25% of deaths, respectively). The strongest therapeutic effect was observed after double administration of preparation by immersion 24 and 48 hours after infection (group 5; 10% of deaths).

TABLE-US-00017 TABLE 17 The mortality of farmed carp after experimental infection with P. fluorescens and treatment with bacteriophage preparation (BAFADOR IV). No of group Date 1 2 3 4 5 22 Oct. 2015 0 0 0 0 0 23 Oct. 2015 0 1 0 0 0 24 Oct. 2015 0 3 1 1 0 25 Oct. 2015 0 3 1 2 1 26 Oct. 2015 0 2 1 1 0 27 Oct. 2015 0 1 0 1 1 28 Oct. 2015 0 0 0 0 0 Mortality 0 11 3 5 2 (in pieces) Total mortality 0% 55% 15% 25% 10%

[0093] Based on conducted experiments, it was demonstrated that a death rate of fish was significantly reduced in groups treated with bacteriophages, both in 24 and 48 hours after experimental infection with Pseudomonas fluorescens. The strongest therapeutic effect was observed after double administration of preparation by immersion 24 and 48 hours after infection. Moreover, it was observed that fish mortality was the smallest in the experiments in which bacteriophage preparations BAFADOR III and BAFADOR IV were applied. In these studies, a death rate after double administration of preparations was at the level of 10% while in case of BAFADOR II at the level of 15%.

[0094] Summary of results concerning safety and efficiency of bacteriophage preparations in farmed fish. [0095] 1. Bacteriophage preparation does not affect biochemical and hematological blood parameters in farmed fish. [0096] 2. Bacteriophage preparation stimulates both innate cellular and humoral immune systems in farmed fish. [0097] 3. Bacteriophage preparation reduces mortality of farmed fish infected with a pathogenic bacterial strain.

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