WO2015188230A1 - Bacteriophage production method - Google Patents

Abstract

A method of producing a bacterial replication host for a bacteriophage is provided, including selecting a bacterial species, strain, serotype or isolate that is different to a pathogenic bacterium as a host for replication of the bacteriophage. The bacterial replication host provides a safe, efficient non-toxic vehicle for propagating bacteriophage that are capable of infecting a pathogenic bacterium that is of a different bacterial species, strain, serotype or isolate to the replication host. The pathogenic bacterium may be of the genus Vibrio, Aeromonas or Pseudomonas. Particular Vibrio bacteria are pathogens of shrimps or prawns, such as Vibrio parahaemolyticus. The invention may therefore have particular applicability to aquaculture.

Description

TITLE

BACTERIOPHAGE PRODUCTION METHOD TECHNICAL FIELD THIS INVENTION relates to bacteriophage. More particularly, this invention relates to a method for large scale production of bacteriophage that may be used as antiseptic or antibacterial agents.

BACKGROUND

Bacteriophage are viruses which are capable of infecting and replicating in bacteria, ultimately leading to bacterial cell death. Bacteriophage were discovered in 1915 by Frederick Twort. It was fairly quickly realized that bacteriophage could be used as a powerful anti-bacterial therapy, as first pioneered by Felix d'Herelle in France in the 1920's and 1930's. Typically, bacteriophage therapy involves the targeted application of bacteriophages that, upon encounter with specific pathogenic bacteria, can infect and kill them. The phage then lyse the bacteria, releasing virion progeny that can continue the infection and replication cycle, including migrating to other sites of infection anywhere in the body. The actual bacteriophage-mediated bacterial killing occurs well prior to the lysis step, such as in the first minutes of infection for a phage such as phage T4, as the phage subverts the bacterial cell into making new phages. Bacteriophage are unique among antibacterial agents in their ability to increase their numbers when in the presence of bacterial targets while only minimally impact non- target bacteria or body tissues.

With the development and growth of antibiotics after World War II, the development of phage therapy in the Western world waned, while its application continued to increase within the Soviet Union and eastern Europe. However, since the 1980's the rise of antibiotic-resistant pathogenic bacteria and the inability to produce new and more powerful antibiotics has generated new interest in bacteriophage as potential antibacterial agents in the Western world.

If bacteriophage are to become a more standard and widely-used antibacterial agent, industrial scale processes for their production need to be improved. Of particular concern is that bacteriophage produced on an industrial scale must be safe for administration to humans, while being produced at an economic cost that makes large scale bacteriophage production commercially viable. SUMMARY

The present invention is broadly directed to the selection and use of bacteria as optimal hosts for producing bacteriophage. Typically, the bacterial hosts are non- pathogenic bacteria and allow production of bacteriophage that target pathogenic bacteria. Together with the careful selection of suitable minimal growth media, the bacterial hosts disclosed herein may may facilitate efficient, large scale production of bacteriophage for a variety of subsequent antibacterial uses while minimizing the risk that the bacteriophage are contaminated with any pathogenic bacteria or components thereof.

In one aspect, the invention provides a method of producing a bacterial replication host for a bacteriophage, said method including the step of selecting a bacterial species, strain, serotype or isolate that is different to a pathogenic bacterium as host for replication of the bacteriophage.

In another aspect, the invention provides a method of producing bacteriophage including the step of propagating a bacteriophage that is capable of infecting a pathogenic bacterium in a host bacterial species, strain, serotype or isolate that is different to the pathogenic bacterium under conditions that promote propagation of the bacteriophage. .

In yet another aspect, the invention provides bacteriophage produced by the method of the second aspect.

In still yet another aspect, the invention provides a method of producing a bacteriophage composition including the steps of: preparing a bacteriophage that is capable of infecting a pathogenic bacterium by propagation in a different host bacterial species, strain, serotype or isolate under conditions that promote propagation of the bacteriophage; and forming a composition comprising the isolated bacteriophage.

In a further aspect, the invention provides a bacteriophage composition produced according to the method of the aforementioned aspect.

A still further aspect provides use of the bacteriophage composition as an antiseptic or antibacterial agent.

In some embodiments, the pathogenic bacterium is of the genus Vibrio, Aeromonas or Pseudomonas.

In particular embodiments, the pathogenic bacterium of the genus Vibrio is a pathogen of shrimp or prawns, such as Vibrio parahaemolyticus although without limitation thereto.

Throughout this specification, unless otherwise indicated, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non- stated integers or groups of integers.

It will also be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" may refer to one element, one or more elements or a plurality of elements.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an example of desirable bacteriophage growth characteristics that include the ability to produce clear and distinct plaques (clearance zones) and an absence of "halo", where there are areas of partial clearing around the plaques.

FIG. 2 shows that there may be agglutination where the susceptible bacteria "group"' together following exposure to bacteriophage

FIG. 3 shows turbidity or cloudiness that can indicate the presence of undesired temperate phage.

FIG. 4 shows a Mitomycin C growth curve for Vibrio parahaemolyticus isolate # 1129 screened against 25, 50 and 100 ng/mL Mitomycin C

FIG. 5 shows a Mitomycin C growth curve for Vibrio parahaemolyticus isolate # 1128 screened against 25, 50 and 100 ng/mL Mitomycin C.

FIG. 6 shows a Mitomycin C growth curve for Vibrio parahaemolyticus isolate # 1333 screened against 25, 50 and lOOng/mL Mitomycin C.

FIG. 7 shows a Mitomycin C growth curve for Vibrio parahaemolyticus isolate # 1440 screened against 25, 50 and lOOng/mL Mitomycin C.

FIG. 8 shows PCR results for confirmation of EMS pathogen

(+): Positive control; 27-20, 80-88, 98-65, 104-112: V.parahaemolyticus isolates; (-): Negative control. M: DNA molecular weight maker (IQ2000™ AHPNS/EMS specific sequence amplification kit).

FIG. 9 shows time-mortality relationship of V.parahaemolyticus challenging shrimp.

FIG.10 shows abnormal in colours of hepatopancrea of experimental shrimp. FIG. 11 shows changes of HP tubule structure of EMS shrimp: hemocytic infiltration, lack of B,R, F cells in HP tubules and bacterial infection.

DETAILED DESCRIPTION

Biological systems including agricultural, aqua-culture, veterinary and human are prone to bacterial infection, infestation or contamination, as are synthetic systems such as formulated personal care, medications, water delivery, fluid circulation and the like, disease, dysfunction and spoilage. In the past, antibiotics and antiseptics have been used to successfully address these infestations and contamination events however many bacterial strains are now resistant to such treatment regimes. The invention disclosed herein provides bacteriophage isolated from contaminated or infected systems, where a pathogenic bacterium is present. The pathogen bacterium while susceptible to the phage is not always the most appropriate replication host, for reasons of the need for complex growth media, difficulties in culture and the precautions required during handling of pathogenic bacteria. This invention disclosed herein provides a method where by bacteriophage are produced using a "replication host" bacterium that has been selected because of its "user friendly" nature. The method disclosed herein includes selection of both replication host bacteria and bacteriophage that overcome a number of rate limiting and labour intensive steps typically associated with production and purification of bacteriophage. The method is essentially a two phase, complementary process whereby replication host bacteria and bacteriophage can be pre-selected and thus optimised for subsequent use in the method. This enables rapid scale-up, resulting in high purity, high titer bacteriophage of >10⁷ to >10^u pfu/mL that may be used to create safe and effective antibacterial and/or antiseptic compositions and formulations.

In one aspect, the invention provides a method of producing a bacterial replication host for a bacteriophage, said method including the step of selecting a bacterial species, strain, serotype or isolate that is different to a pathogenic bacterium as a host for replication of the bacteriophage.

In another aspect, the invention provides a method of producing bacteriophage including the step of propagating a bacteriophage that is capable of infecting a pathogenic bacterium in a host bacterial species, strain, serotype or isolate that is different to the pathogenic bacterium under conditions that promote propagation of the bacteriophage. In yet another aspect, the invention provides bacteriophage produced by the method of this aspect.

As broadly used herein the term "bacteriophage" includes and encompasses any virus that is capable of infecting and replicating in a bacterium. Bacteriophage may have a DNA or RNA genome comprising single-stranded or double-stranded DNA or RNA. The genome is typically packaged or encapsulated by proteins encoded by the bacteriophage genome. Bacteriophage may be enveloped (e.g encapsulated by bacterial host-derived lipids, glycolipids and/or lipoproteins) or may be non- enveloped. Bacteriophage may exhibit a lytic cycle or a lysogenic cycle (e.g "temperate" phage) associated with replication in a bacterial host. Particular, non- limiting examples of bacteriophage include Caudovirales such as Myoviridae, Siphoviridae and Podiviridae, Ligamenvirales such as Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae and Fuselloviridae and other families such as Globuloviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae and Techtiviridae, although without limitation thereto.

By way of example only: Cystoviridae are RNA phages as are Leviviridae which have a linear, positive-sense single-stranded RNA genome and infect Enterobacter and Pseudomonas bacteria; Myoviridae are characterized by complex contractile tails and include, as examples, bacteriophage mu, PL P2, and T4 and the "T4-like" bacteriphage; Podoviridae are characterized by short, non-contractile tails and include, as examples, bacteriophages N4, P22, T3, and T7; Siphoviridae are characterized by long, non-contractile tails and include, as examples, hk022, λ, T5, and BF23 bacteriophage, Corticoviridae comprise icosahedral, lipid-containing, non- enveloped bacteriophages including bacteriophage PM2; Inoviridae comprise rod- shaped or filamentous bacteriophage of single-stranded DNA and infect Enterobacter, Pseudomonas, Vibrio and Xanthomonas bacteria; Microviridae comprise lytic bacteriophages that infect Enterobacter, Vibrio and Chlamydia bacteria and include isometric single-stranded DNA bacteriophage such as G4 and φΧ174; and Techtiviridae comprise lipid-containing bacteriophages with double capsids, such as bacteriophage PRD 1.

Suitably, the bacteriophage is capable of infecting and killing a pathogenic bacterium. Suitably, the bacteriophage is not a filamentous or temperate bacteriophage. Preferably, the bacteriophage is a lytic bacteriophage or at least displays a lytic cycle in a replication host. Other preferred characteristics of the bacteriophage include that: the bacteriophage should generate clear and distinct plaques during in vitro propagation in bacteria that are substantially free from halo, aggregation or turbidity on solid media; and/or rapid growth characteristics.

It will be appreciated that according to the invention, the bacteriophage is typically isolated from an environmental or natural source wherever a susceptible bacterial host may be present. Non-limiting examples of environmental or natural sources include contaminated or infected water systems, aquaculture systems, industrial waste, sewage, mining waste, soil, medical waste, water and human and other animal materials such as sputum, wound fluid, urine, blood, faeces and throat swabs, although without limitation thereto.

Suitably, the bacteriphage are capable of infecting a pathogenic bacterium and being propagated and/or replicated in a bacterium (referred to herein as a "replication host") that is different to the pathogenic bacterium. It will be appreciated that bacteriophage produced according to the methods disclosed herein may be capable of infecting bacteria ⁽ .e pathogenic bacteria and replication hosts) inclusive of gram positive and gram negative bacteria, rods, cocci, non-motile and motile bacteria, anaerobes, facultative anaerobes, aerobes and photosynthetic bacteria, although without limitation thereto. By way of example, the bacteria may be of a genus or other taxonomic group such as Aeromonas, Altermonas, Cytophaga, Flavobacterium, Lactococcus, Mycoplasma, Photobacterium, Proteobacteria, Vagococcus, Achromobacter, Actinomyces, Staphylococcus, Bacillus, Yersinia, Hemophilus, Helicobacter, Alphaproteobacteria, Mycobacterium, Streptococcus, Neisseria, Klebsiella, Brucella, Rickettsia, Bordatella, Clostridium, Listeria, Legionella, Vibrio, Enlerobacter, Proteus, Pasteurella, Bacteroides, Campylobacter, Morganella, Edwardsiella, Lactococcus, Diplococcus, Pseudomonas, Borrelia, Citrobacter, Corynebacterium, Moraxella, Neisseria, Escherichia, Gammaproteobacteria Salmonella, Norcardia, Proteus, Propionihacterium, Treponema, Shigella, Xanthomonas, Chlamydia, Enterococcus or Leptospirex, although without limitation thereto.

Suitably, pathogenic bacteria or bacteria that otherwise cause or are associated with one or more diseases of humans, animals or plants include Staphylococcus aureus, Staphylococcus epidermidis, Helicobacter pylori, Achromobacter anitratum Actinobacillus lignieresi, Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes faecalis, Bordatella bronchiseptica, Brucella ovis, Bacillus anthracis, Bordatella pertussis, Borrelia burgdorferi, Campylobacter jejuni, Campylobacter novyi, Campylobacter fetus, Chlamydia psittaci ovis, Citrobacter freundii, Clostridium chauvoei, Clostridium colinum, Clostridium hemolyticum, Clostridium perfringens, Clostridium septicum, Corynebacterium pseudotuberculosis, Corynebacterium pyogenes, Corynebacterium renale Corynebacterium diptheriae, Corynebacterium pseudotuberculosis, Clostridium tetani, Clostridium botulinum, Dichelobacter nodosus, Diplococcus pneumoniae, Edwardsiella ictaluri, Edwardsiella tarda, Enterobacter aerogenes, Erysipelothrix insidiosa, Flavobacterium branchiophilum, Flavobacterium columnarae, Flavobacterium psychrophila, Listeria monocytogenes. Hemophilus influenzae, Hemophilus suis, Hemophilus bovis, Hemophilus gallinarum, Histophilus (Hemophilus) somnus, Pasteurella multicida, Shigella dysenteriae, Leptospira autumnalis, Leptospira canicola, Leptospira grippothyphosa, Leptospira hyos, Leptospirea icteohaemorrhagiae, Leptospira icterohaemorrhagica, Leptospira pomona, Leptospira sejroc phosa, Mannheimia haemolytica, Pasteurella haemolytica, Melissococcus plutonius, Moraxella bovis, Morganella morganii, Mycobacterium avium, Mycobacterium avium subsp. Paratuberculosis, Mycobacterium chelonei, Mycobacterium tuberculosis, Mycobacterium, leprae, Mycobacterium, asiaticwn, Mycobacterium intracellulare, Mycoplasma pneumoniae, Mycoplasma hominis, Neisseria meningitidis, Neisseria gonorrhoeae, Nocardia asteroides, Rickettsia rickettsii, Brucella, abortis, Brucella can is, Brucella suis, Legionella pneuophila, Klebsiella pneumoniae, , Propionibacterium acnes, Paenibacillus larvae, Pasteurella multocida, Photobacterium damselae subsp. Damselae, Piscirickettsia salmonis, Proteus mirabilis, Pseudoalteromonas ruthenica, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas pseudomallei, Renibacterium salmoninarum, Riemerella anatipestifer, Treponema pallidum, Treponema pertanue, Chlamydia trachomatis, Treponema carateum, Escherichia coli, Salmonella typhimuriurn, Salmonella typhi, Salmonella anatum, Salmonella give, Salmonella infantis, Salmonella lille, Salmonella liverpool, Salmonella livingstone, Salmonella agona, Salmonella arizona, Salmonella cholerae-suis, Salmonella dublin, Salmonella gallinarum, Salmonella havana, Salmonella Johannesburg, Salmonella kottbus, Salmonella mbandaka, Salmonella muenchen, Salmonella newport, Salmonella ohio, Salmonella orion, Salmonella ouakam, Salmonella potsdam, Salmonella pullorum, Salmonella scwarzengrund, Salmonella Singapore, Salmonella tennessee, Salmonella zanaibar, Streptococcus pneumoniae, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguis, Streptococcus pyogenes, Streptococcus viridans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus equisimilis, Streptococcus uberis, Streptococcus uberus, Streptococcus zooepidemicus; Sphaerophorus necrophorus, Tenacibaculum maritinum, Vibrio cholerae, Vibrio harveyi, Vibrio alginolyticus, Vibrio penaeicida, Vibrio alginolyticus, Vibrio anguillarium, Vibrio anguillarum, Vibrio coli, Vibrio costicola, Vibrio parahaemolyticus, Vibrio pectinicida, Vibrio salmonicida, Vibrio splendidus, Vibrio tubiashi, Vibrio vulnificus, Yersinia ruckeri and Yersinia pestis, although without limitation thereto.

The replication host bacteria are suitably selected according to one or more criteria that include: they do not harbour a pro-phage; are capable of supporting bacteriophage replication; have prolific and reliable growth characteristics in vitro; do not produce toxins, or produce minimal toxins; are non-pathogenic; and/or have a requirement for inexpensive and/or minimal growth media. Non-pathogenicity and an ability to grow on inexpensive and/or minimal media are particularly desirable.

As will be described in more detail in the Examples, a variety of different replication host bacteria have been identified according to the invention, including Vibrio spp, Aeromonas spp and Pseudomonas spp, although without limitation thereto.

One feature of the invention is that bacteriophage are selected and grown under advantageous conditions that improve or enhance plaque selection and in vitro growth. Furthermore, the bacteriophage are harvested in "low complexity" media that reduce the need for complex, downstream purification. In his regard, the media preferably comprise one or more salts that include at least one monovalent cation. These may include sodium, potassium, rubidium and/or caesium. A preferred concentration is between 0.001% and 15% (w/v) or any ranges therebetween such as 0.005% and 10% (w/v); 0.02% and 5% (w/v); 0.05% and 2% (w/v); and 0.01% and 1%) (w/v), although without limitation thereto. The media preferably further comprise one or more salts that include at least one divalent cation. The divalent cation is preferably a metal cation that is water-soluble. Non-limiting examples of metals are selected from magnesium, calcium, strontium, iron, manganese, cobalt, nickel, copper, zinc and silver. A preferred concentration is between 0.003 to 3% (w/v) or ranges therebetween such as 0.005% and 1% (w/v); 0.01% and 0.5% (w/v); 0.02% and 0.2% (w/v); and 0.05% and 0.1 % (w/v), although without limitation thereto. Preferred harvest media typically comprise water, sodium, magnesium and/or calcium salts. A particularly preferred solution will comprise purified water and 5.8g/L sodium chloride plus 1.5g/L magnesium sulfate with the pH adjusted to the range 7.2 to 7.5.

Accordingly, by selection of a suitable replication host and growth in minimal media lacking exogenous materials that can cause safety concerns, the method disclosed herein provides an efficient means of producing bacteriophage in relatively large quantities for use in any of a variety of different applications. More particularly, the bacteriophage produced by the method may be substantially free of pathogenic bacteria-derived molecules and by-products that could prevent subsequent use of the bacteriophage.

Therefore, the invention also provides a bacteriophage composition or formulation that may be useful for any of a variety of different applications.

In another aspect, the invention provides a method of producing a bacteriophage composition including the steps of: preparing a bacteriophage that is capable of infecting a pathogenic bacterium by propagation in a different bacterium under conditions that promote propagation of the bacteriophage; and forming a composition comprising the isolated bacteriophage.

In a further aspect, the invention provides a bacteriophage composition produced or formulated according to the method of the aforementioned aspect.

In particular embodiments, the bacteriophage composition is suitable for use as an antibacterial agent or an antiseptic agent. Such agents may be used prophylactically, remedially or therapeutically to inhibit, prevent, remove, remediate or treat bacterial infection and/or contamination.

In some embodiments, the composition may comprise a plurality of different bacteriophage that are respectively capable of infecting and killing different bacterial pathogens.

Applications of such bacteriophage compositions include agriculture, aqua- culture, medications for veterinary and human health, personal care products, water supply, decontamination of foods, food utensils, food preparation surfaces and food storage containers, decontamination of medical and veterinary equipment and workspaces and the like. Particular applications include aquaculture uses such as in fish farming to target Aeromonas spp and prawn farming to target pathogenic bacteria such as Vibrio spp, medical and veterinary treatment infections caused by antibiotic- resistant bacteria such as MRSA and Pseudomonas spp, although without limitation thereto. Particular examples of veterinary applications include Early Mortality Syndrome (EMS) or Acute Hepatopancreas Necrosis Disease Syndrome (AHPNS), bovine respiratory disease (BRD) in feedlot cattle, mastitis in dairy cattle, Streptococcus, Staphylococcus and Salmonella in feed mills, laying hens and other intensively reared animals.

The compositions and formulations disclosed herein may comprise one or more other components such as suitable carriers, diluents or excipients. These may include one or more of water, saline, buffers, binders, fillers, lubricants, alcohols and/or polyols, stabilizers, sugars, sugar alcohols and other carbohydrates, lipids, preservatives, peptides and proteins and/or any other substances that facilitate formulation, storage, preservation and/or delivery of the bacteriophage composition. Such compositions and formulations may be suitable for delivery by spraying or aerosolization (e.g as a dilutable concentrate), topical application as a cream, lotion or paint, as a bandage, dressing or swab impregnated with the bacteriphage, by enteral or parenteral application such as by a capsule or tablet or by delivery as a solid powder or granule, although without limitation thereto.

In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES EXAMPLE 1

Biological and industrial systems including agricultural, aqua-culture, veterinary and human health are prone to microbial and particularly bacterial infestation. Overgrowth of microbial organisms can cause disease, dysfunction and spoilage. In past systems, antibiotics and antiseptics have been used to successfully address these problems, however many bacteria are now resistant to such treatment regimes and as a result, new treatment and management regimes are sought.

Phage therapies based on viral infection of a bacterial pathogen are one means of addressing this problem. One of the advantage of this approach is that bacteriophage are highly bacterial host- specific, although as a consequence, systems for rapidly growing and supplying high purity phage are lacking because the bacterial host might not be the optimal host for large scale production of the bacteriophage.

The method disclosed herein describes how to find the optimal phage for use in remedial regimes by strategically eliminating undesirable traits from both the phage and replication host. As a consequence, scale up methods that produce high titre results and high purity are provided.

This method is used to isolate and identify both bacteria and phage that overcome a number of rate limiting and labour intensive steps typically associated with production and purification of biological agents such as bacteriophage.

The method is essentially a two phase, complementary process whereby bacteria and phage can be pre-selected and thus optimised for subsequent synergistic use. This enables rapid scale-up, resulting in high purity, high titer phage of >10⁷ to >10ⁿ pfu/mL.

Phase 1: Bacteriophage selection

Bacteriophage can be isolated from contaminated or infected systems, wherever a susceptible bacterial host is present. Phage to be isolated via this method are intended to be specific for particular bacteria, and thus are only suitable for particular antibacterial applications.

Filamentous phage typically produce toxins and as a consequence, are typically unsuitable for therapeutic or remedial uses without significant downstream processing. Similarly, temperate phage cycle between lytic and lysogenic cycles, and as a consequence cycle between deleterious and recovery relationships with their host, which is an undesirable trait in therapeutic and remedial phage.

Step 1

The first step in this method is to ensure that neither filamentous nor temperate phage are selected. Critical to this method is the selection of phage that are virulent (i.e obligate lytic phage), as these phage do not integrate their DNA into the host cell DNA and kill their bacterial host by lysis. This is a preferred phage property well suited for antibacterial remediation.

A reliable way of reducing the likelihood of isolation of non-virulent phage is to source potential candidates only from 'environmental' sources and not directly from their bacterial hosts. Collection of 'environmental samples' from sources such as water, sputum, wounds, sewerage and animals, preferentially selects phage that adopt virulent reproduction methods.

Where swabs are collected, swabs dilution or dispersion into a sterile fluid may be required followed by filtration (0.45 um, 0.2 um) such that viable bacteria are retained and smaller, viral particles are passed through. Such filtrates are collected and screened.

Where larger volume, more dilute samples are collected, for example water samples; such samples may be added in equal volume of double strength microbial nutrient media, such that the final volume is at lx concentration. The resultant solution is then allowed to incubate nominally for 24 to 48 hours such that the resident bacteria can replicate (as well as any phage present in the sample). The solution is then filtered, with bacteria retained and phage passed through for further screening.

Thereafter phage can be further selected for "scale-up" performance by ensuring that their in vitro performance on solid growth media is optimal. This optimisation has a dual purpose: it enables identification and rejection of phage that may have temperate characteristics; and also identifies phage that may be more rapid or powerfully infective of a host bacterium. Step 2

Screening takes place on solid media that provides nutrients supportive of the phage host. A lawn of suitable optical density of the host bacterium is established on such media. Then aliquots of the filtrate, in a range of dilutions, are introduced with the aim of identifying "clear and distinct plaques" (or clearing of the optimal density). Phage that do not exhibit optimal in vitro characteristics on solid media are not progressed as this indicates that they are unsuitable for simplified production of high quality phage.

Desirable characteristics include the ability to produce clear and distinct plaques (clearance zones) and an absence of "halo", where there are areas of partial clearing around the plaques, as shown in FIG. 1. As shown in FIG. 2, there also may be agglutination where the susceptible bacteria "group"' together following exposure to phage. As shown in FIG. 3, turbidity or cloudiness can indicate the presence of temperate phage or in any other circumstance where other than clear and distinct clearance is observed. Rapid plaque development is a desirable characteristic that is preferentially selected for. Acceptable plates examined will typically have 95% bacterial cell clearance.

Optimal in vitro characteristics screening can also be used help to differentiate between various phage contained within an environmental sample. These include the following:

1. large sized phage will typically have a smaller clearance zone, because of their migration through solid media (the use of softer lower density media can remedy this)

2. conversely, smaller sized phage will tend to migrate a greater distance through solid media, and result in larger plaques

3. moderately sized phage result in moderately sized plaques. Once the initial screening of phage on solid media has taken place and poorly suitable candidates determined and excluded, a number of potentially suitable phage candidates can be selected for promotion to purification regimes.

Step 3

The third step relates to purification regimes that should include careful selection and serial dilution of individually selected high performing plaques. The plaques continue to be examined for optimal in vitro performance on solid media as the purification takes place.

To do this, a single plaque is selected using a sterile instrument such as a sterile pipette tip; the 'plug' is then dispensed into a 1.5 mL vial and suspended in 500uL low nutrient aqueous solution, typically comprising water, sodium, magnesium and/or calcium salts.

A preferred solution will comprise purified water and 5.8g/L sodium chloride plus 1.5g/L magnesium sulfate, with the pH adjusted to the range 7.2 to 7.5. (referred to as PD(Neb))

Some other buffers may also make a suitable solution, with certain exceptions; namely some phosphate buffers, which result in the precipitation of insoluble magnesium phosphate or calcium phosphate, depending on the cations selected. Tris (hydroxymethyl)aminomethane) is generally avoided as it contains a primary amine, which in the presence of other amine groups can result in the formation of nitrosamines, which are carcinogenic. Good's Buffers are typically appropriate. Solutions and media used will be essentially free from phosphates and amines and will contain salts added such as: at least one monovalent cation is present in the solution including Sodium, Potassium, Rubidium and/or Caesium at a concentration of between 0.001 and 15% w/v); and/or at least one divalent cation selected from the metals group is available in solution; especially favourable will include one or more selected from magnesium, calcium, strontium, iron, manganese, cobalt, nickel, copper, zinc, silver at a concentration of between 0.003 to 3% (w/v).

The plaque and solution is then mixed and allowed to stand (or incubate) typically overnight, during which time the phage migrate from the plaque into the solution. The vial may then be centrifuged and/or the supernatant passed through a sub-micron filter (0.45, 0.2 um), and the filtrate collected.

The filtrate is then subject to serial dilution and the process repeated twice over, so that on each occasion a single plaque is selected and its in vitro performance and purity verified. A particularly useful method of doing this is a modified Miles and Misra plate regime such as hereinbefore described.

Phage that have been selected by this method can then be assessed for their potential against a number of bacterial isolates (host range analysis), and subjected to more specific and discrete verification regimes such as TEM before validation and assessment of suitability for remedial or therapeutic use via the production of high titre phage.

Step 4

To enter and thus infect a host cell, phage must attach to a specific receptor or region of the bacterial cell. Phage are obligate cellular parasites and rely on random encounters as well as affinity with their host in order to achieve infection. Host growth conditions as well as the presence or absence of phage receptors will influence the phage's ability to be infective, and thus be useful in this technology. This part of the process selects for phage that have a wide range of susceptible hosts, and as a consequence are more attractive as remedial or therapeutic agents.

Bacterial hosts are actively source and maintained as a library as described in

Phase 2.

A selection of host bacteria are used to determine the range of hosts that may be susceptible to the isolated phage. Phage are identified as being especially active against a wide array of hosts and/or especially active against specifically desirable hosts (for example antibiotic- resistant bacteria or specific strains or isolates of known bacterial pathogens)

The opportunity for host infection can be preferentially influenced, especially in aquaculture systems through the use of attractants. Crude attractants attractants such as squid meal or oil, egg, worm meal and molasses can interfer with the shelf life and vitality of the phage where as highly purified, discrete attractants such as those described in Chemical Communication in Crustaceans ISBN 978-0-387-77100- 7 Thomas Breithaupt 1 Martin Thiel Editors have attractant capability without diluting or adversely effecting phage vitality and/or shelf life.

These might include metabolites such as isophorone and 6-methyl-5-hepten-2- one, Chlorodesmin, Pachydictyol A, Dictyol E, Pteroenone, 'hair crab ceramide', Isatin, Tyrosol, attractin pheromone, also free amino acids especially ASP, GLU, ASN, SER, THR, GLN, HIS, GLY, ARG, 0-ALA, TAUR, TYR, A ABA, VAL. TRP, PHE, ILE, LEU, ORN and LYS) in low concentrations (< ImM) and small peptides, including Crustacean peptide pheromones, kairomones, and substituted amino sugar kairomones nucleotides, small Similarly volatile info chemicals such as diemthyl sulfide can provide chemically mediated trophic effect.

The addition or combination of additions will be tailored for the species to be dosed; a preferred addition for the management of EMS in shrimp is Uridine diphosphate (to a maximum of 100 μg/L).

Phase 2: Selection of bacterial replication hosts

When a pathogenic bacterium is susceptible to a particular phage, it might not necessarily be the most appropriate replication host. This may be for reasons of the requirement for complex growth media, that it is difficult to culture the bacterial pathogen and/or the precautions required during handling the bacterial pathogen. This method disclosed herein whereby virulent phage are produced using a "replication host" bacterium provides a much more "user friendly" system for producing bacteriophage.

Suitable 'replication host' characteristics will ideally include

(a) does not include a pro-phage;

(b) capable of supporting phage replication;

(c) prolific and reliable growth characteristics in vitro; (d) does not excreting toxins;

(e) non-pathogenic; and/or

(f) requirement for inexpensive / minimal growth media. Non-pathogenicity and ability to grow on inexpensive and/or minimal media are particularly desirable.

Step 1

As an initial step, bacterial pathogens that cause known diseases are identified. Non-limiting examples will be described in more detail.

In salt water systems, Vibrio parahaemolyticus, is a known causative agent for EMS when characterised as being 'PCR positive'. There are however a number of strains that are not considered pathogens (or environmental risks) that are more ideally suited a role as a replication host. Vibrio species including parahemalytica isolates have been obtained from commercial and wild sources (for example crustaceans and their commercial pond environments) as well as commercially available specimens such as those available via ATCC (such as ATCC 17802)

Pathogenic Aeromonas species are responsible for gastroenteritis and wound infections. Antibiotic resistance poses a potential problem in antimicrobial therapy of these infections. While most strains are susceptible to chloramphenicol, ciprofloxacin, co-trimoxazole and the aminoglycosides, the activity of amoxycillin/clavulanate and the acylureidopenicillins is inconsistent. These organisms are ubiquitous in fresh water environments and over growth in same can also be a cause of disease in aquiculture environments. Aeromonas isolates have been collected from a number of "wild" environments including hatcheries, dams, septic systems, waterways, as well as wounds of animals and humans. In addition to this, characterised cultures have been screened.

Pseudomonas are also opportunistic pathogens and potentially suitable isolates have been identified in samples obtained from crayfish, dogs lungs, horse uterus, human wounds and aquatic environments.

Similarly cultures of other pathogens such as Streptococcus and particularly

Staphylococcus (the causative agent in mastitis and other dermal infections) have been identified from environmental and characterised samples.

The approach to this process is typically disease driven, and environmentally isolated sources are most keenly sought after as they will most likely be susceptible to similarly located phage. Experience has shown that most typically the most predominant bacterial species in a sample is the causative agent of disease or infestation.

On one occasion, sputum samples were obtained from which were obtained a number of cultures identified as:

(1) Gram +ve cocci

(2) Gram +ve rod

(3) Gram -ve rod

(4) Gram -ve rod

(5) Gram -ve rod

(6) Gram -ve coccobacillus

(7) Gram -ve rod

(8) Gram -ve rod

The purpose of the isolation was to obtain Pseudomonas isolates, and as a consequence on samples 3, 4, 5, 7 and 8 were progressed to identification phase. Biolog GEN III system analysis indicated that the isolates were most likely (respectively) Pseudomonas aeruginosa, Pseudomonas fluorescense Biotype G, Pseudomonas citronellolis and Pseudomonas Nitroreducens/azelaica. The Pseudomonas citronellolis (#7 isolate) was excluded from immediate consideration as it is an unlikely human pathogen. The remaining isolates were progressed for additional analysis.

Step 2

The presence of a prophage is an unacceptable trait in isolates selected for production of high purity, high titre phage, and thus hosts of such phage must be eliminated. At certain concentrations, the antibiotic substance Mitomycin C stresses bacterial hosts to the extent that prophage production can be visualised by measuring the optical density of growth media and observing particular trends in the data over ~ 8 hours.

Table 1 : Typical Mitomycin C screening range in ng/mL

organism Mitomycin C of Mitomycin C

Staphylococcus sp 100 400

Streptococcus 100 400

Vibrio sp 25 100

Aeromonas sp 100 400

Pseudomonas sp 25 400

Trending can be observed at certain concentrations (dependant on the susceptibility of the bacterial host) and thus the presence of a prophase forecast. Typically a prophage can be identified as a 'dip' and recovery in the optical density, indicating that the population begins to reduce (as a consequence of lysis brought on by the prophage) and then recovers as the prophage exits this cycle and reincorporates into the host DNA.

Vibrio parahaemolyticus isolate # 1129 screened against 25, 50 and 100 ng/mL Mitomycin C results in essentially clear and progressive growth curves and hence is unlikely to contain a pro-phage. This is shown in FIG 4

Vibrio parahaemolyticus isolate # 1128 screened against 25, 50 and 100 ng/mL Mitomycin C results in smooth growth curves, but has a dip just before 6 hours (at 25 ng/mL), and similar possibility at 50ng/mL between 7 and 8 hours) indicates the possibility of a pro-phage so is eliminated from further progression. This is shown in FIG. 5

Vibrio parahaemolyticus isolate # 1333 screened against 25, 50 and lOOng/mL Mitomycin C results in fluctuating growth curves, which may contain a pro-phage, dips after 1 hour . This is shown in FIG. 6

Vibrio parahaemolyticus isolate # 1440 screened against 25, 50 and lOOng/mL Mitomycin C results in clear and progressive growth curves and hence is likely to contain a pro-phage (dip between 7 and 8 hours). This is shown in FIG. 7

Screening of a group of Aeromonas bacteria using the above methods resulted in the identification and elimination of 4 of the 13 isolates assessed. Conversely, on one occasion, 7 out of seven Pseudomonas isolates (obtained from water) indicated the presence of prophage like behaviour. Pseudomonas are particularly difficult to evaluate via this method, due to the presence of pigments and significant care is required in the handling of these samples. Step 3

Antibiotic-resistant strains are driving the need for alternative therapies to be developed and as a consequence phage that are especially effective against antibiotic stains are especially valuable. For this reason it is important to have access to antibiotic resistant strains that can be used to select especially valuable phage.

Where isolates are selected from a clinical source (e.g a human or animal that has previously been treated with antibiotics) or else their immediate environment (for example animal bedding and excreta), antimicrobial resistance screening is undertaken. Most typically, Staphylococcus isolates will be screened for resistance using challenges with Ampicillin (lOug), Cefoxitin 30ug, Oxacillin lug, Penicillin lOug, Ciproxin 5ug, Amibacin 30ug and Erythromycin 15ug as well as, potentially Cephalosporins at suitable dilutions.

In the examination of 10 different Staphylococcus isolates derived from clinical environments, six out of 10 exhibited resistance to at least three of the above antibiotics. Each of these six isolates were progressed as being suitable phage challenge hosts in future phage validation. The remainder were considered for their future potential to act as replication hosts. Step 4

Bacterial isolates are typically subject to strategic biochemical screening using both commercially available (such as MicroSys V36, Biogen III Microplate) and specifically designed biochemical-screening regimes {i.e. media variations), with the objective of maximising growth and minimising media complexity. Usually biochemical screening is undertaken for species identification purposes, however on this occasion the results are also used to identify isolates that have discrete and clearly identifiable nutritional needs.

This resulted in the identification of a number of isolates that were able to provide suitable growth profiles in defined growth media.

In one example a number of V.parahemalytic strains that had previously been identified as having optimally low nutrition requirements (stored on beads at -80°C) were resuscitated in Nutrient Broth + 2% NaCl, and then incubated at 30°C for 48 hours; there after aliquots were transferred to 'PVSS' broth (comprising 5g/L bacteriological peptone, 1 g/L yeast extract and 33g/L synthetic sea salts) and allowed to grow to early exponential phase. The quality and quantity of growth was recorded for each bacterial isolate.

Additional trials were undertaken for a range of isolates using increasing less complex media, each time -selecting the isolates based on their ability to withstand and/or thrive in low nutrient environments. A best outcome was identified where the balance between growth and media simplicity was achieved. In most cases this was obtained using fully synthetic media +/- various carbohydrate sources, +/- critical amino acids. Where these strategies were unsuccessful, processes were adopted using increasingly low concentrations of non-specific nutrient sources such as Yeast agar, bacteriological peptone, nutrient broth etc. Isolates were identified that required up to -90% less concentrated or complex solutions that is traditionally reported in relevant publications.

Where a Vibrio isolate is identified as having optimal growth capacity coupled with minimal or reduced nutrition requirements it is sequestered for further investigation for its ability to act as a growth substrate (replication/ surrogate host) for virulent phage.

Step 5

In the next step, purified phage were then introduced to the potential replication hosts. These co-cultures then each contained a bacterial isolates with known minimal nutritional needs and a phage known to be effective against a bacterial pathogen. The cultures were allowed to progress for a defined period and thereafter the degree of clearance for these cultures was observed and measured, with a score of 3+ being maximum clearance (that is high level phage production) down to 0 (phage appeared to be produced or effective against that host).

Validation of the process was confirmed by introducing the resultant filtrate containing the virulent phage to the pathogenic bacteria and observing significant (>70%) reduction in optical density over 8 hours.

Using this system over a number of sequential events; a library of phage and matching optimised replication hosts and standardised minimal was developed, where the media used is a low as practically possible in complex biomolecules. The reduction and/or virtual absence of these complex bio-molecules allows a simplified removal of bacterial biomass (via 0.45 um filtration) to be undertaken in many cases, and thus eliminates much of the time, complexity and expense of many downstream processing regimes.

By way of example, Aeromonas replication hosts can be identified in a similar way, for future remedial use in aquatic, terrestrial and industrial systems. In this case, the potential presence of lytic prophage in forecast replication hosts need to be determined. This can be achieved by growing the isolates in Tryptone soy broth and then screening of isolates against Mitomycin C, which acts to induce prophage production (an undesirable trait). Cultures that do not survive the Mitomycin C challenge are then progressed to further screening.

Phage-resistant colonies can then be determined via screening on solid media, such as Tryptone soy agar, using growth of the cultures to a clear lawn density and then application of various dilution of phage. Phage resistant colonies are of particular interest as they represent a specific challenge to be satisfied in phage library development and optimisation.

The samples where allowed to incubate and then susceptible and resistant strains identified. Resistant strains were then discarded and susceptible stained subject to strategic screening for absolute of discretionary requirement of various complex nutrition sources such as are described in Bergey. Potentially suitable kits can be obtained from HIMEDIA or Pliva-Lachema Diagnostika (for example ENTEROtest 24 - kit for identification of Gram- negative fermentative rods). Again on this occasion the purpose of using the biochemical screening kit was not to identify the host, but was rather to determine the absolute requirements for media composition.

Using this regime a library of matched phage and minimal media replication hosts was created. These phage can now be grown "on demand" and supplied as needed in responses to specific infection and/or contamination events. These might include for example fmgerliiig hatcheries, food manufacturing and other water rich environments.

To date optimised synergic systems have been developed for a number of key pathogenic bacteria.

Table 2: Mat ched phag e and bacteri al replicatio n hosts

Pathogen Average Ph age litres # lead Use of replication candidates h st(s) MRS A (Staph) 1 x 10 pfu/ml through 2 Yes to

1 x 10¹⁰pfu/mL

Pseudomonas (multiple 1 x 10 pfu/ml through 15 Yes

clinical isolates) to

1 x 10¹⁰pfu/ml

Vibrio 1 x 10 pfu/ml though to >2 Vibrio

parahaemolyticus 1 x 10⁹pfu/ml parahaemolyticus (PCR Positive for (PCR Negative) EMS)

Aeromonas (aqua- 1 x io⁹pfu7mi through Multiple Yes

culture infection) to

1 x 10ⁿpfu/ml

DETAILED METHODOLOGY

Amplification of Bacteriophage from Water

General

Bacteriophage are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. However, one of the densest natural sources for bacteriophage and other viruses is sea water, where up to 9>< 10⁸ virions per/ml have been found in microbial mats at the surface. Recent investigations have revealed that bacteriophage are much more abundant in the water column of both freshwater and marine habitats than previously thought. Due to the density of phage found in these locations, water sources are seen as prime habitats for phage localisation, discovery and amplification.

Materials and Methods

Obtaining and filtering water samples • Water samples used for phage amplification should be obtained from the environment in which the bacteria are most likely to be found, e.g - freshwater, marine water, sewerage, etc.

• Water samples should be separated into four 750ml centrifuge bottles so that each vessel contains the same amount and weight of water.

• Samples are to undergo centrifugation at 5000g, 10°C for one hour.

• Samples are then filtered through three 90mm class one filter papers laid on top each other in a capture vessel and the dry vacuum pump. The mixture is filtered in to a one litre shot bottle. This will be the water filtrate used for phage amplification.

Bacterial cultivation of amplification isolates

• The host strains to be used for phage amplification should be cultivated on a suitable agar medium under optimal incubation conditions to obtain a fresh overnight grown culture. Streak out the pure culture on an agar plate in a way that distinct colonies will be obtained. Isolates retrieved from cryopreservation must be cultivated twice after retrieval to ensure optimal results.

• Pick a single colony of the isolate off a plate using a sterile swab or sterile loop and suspend in 10ml of the appropriate selectable liquid medium in a 50mL falcon tube or similar. Label all tubes with strain and date. Incubate isolates until they are in the exponential growth phase.

Preparation of amplification mixture

• For each of the amplification strains, a 250ml flask must be prepared and labeled with amplification strain and date.

• Into each of the flasks, add 50ml of 2X appropriate selectable liquid growth medium (+) (e.g - 2X Nutrient Broth, 2X Nutrient Broth and Marine Salts, 2X Tryptic Soy Broth, etc) containing 20μΜ MgS0₄ and 20μΜ CaCl₂.

• Following this add 50ml of the filtered water to each of the amplification flasks.

• Finally add 5ml of the amplification strains to each of the appropriately labeled flasks.

• Incubate cultures with shaking at optimal growth conditions for 18-24 hours.

Isolation of bacteriophage

· Separate all labeled cultures into 50ml falcon tubes containing equal amounts.

• Centrifuge tubes at 5000g, 10°C for 30 minutes.

• Remove the majority of the supernatant from the tubes and filter each into separate sterile vessels using a 0.45 μΜ filter and a 10ml syringe. This is the phage filtrate. Test for bacterial contamination

• Each of the phage filtrates are spotted (20μ1) onto a suitable agar medium and left to incubate overnight under optimal growth conditions to test for bacterial contamination. Should bacterial contamination occur, the phage is to be filtered again through a 0.45 μΜ filter. B - In some cases, phage filtrates may need to be treated with 0.5% chloroform and then centrifuged at 5000g, 10°C for 15mins before filtering.

Modified Miles and Misra plates to determine phage filtrate titer

General

The Miles and Misra Method (or surface viable count) is a technique used in to determine the number of colony forming units in a bacterial suspension or homogenate. The technique was first described in 1938 by Miles, Misra and Irwin at the London School of Hygiene and Tropical Medicine. The Miles and Misra method has been shown to be precise. A modified procedure has been developed by the inventors based on this method. Its purpose is to measure plaque forming units/ml of a phage filtrate/cocktail against a bacterial isolate. This procedure has been termed a modified Miles and Misra technique. Materials and Methods

Modified Miles and Misra plates to determine phage filtrate titre

Bacterial cultivation of trial isolates for bacterial lawns

• The organisms to be used in the trial are cultivated on a suitable agar medium under optimal incubation conditions to obtain a fresh overnight grown culture. Streak out the pure culture on an agar plate in a way that distinct colonies will be obtained. Isolates retrieved from cryopreservation are cultivated twice after retrieval to ensure optimal results.

• Pick a single colony of the isolate off a plate using a sterile swab or sterile loop and suspend in 10ml of the appropriate selectable liquid medium in a 50mL falcon tube or similar. Label all tubes with strain and date. Incubate isolates until they are in the exponential growth phase. Plate format

• Each phage filtrate is designated one standard petri dish. The plate is first divided into eight separate sections of approximately the same size. A prepared template is available to assist with this but it is also possible to do so using a ruler and a permanent marker.

• The 10^"1 dilution should be marked in the appropriate section to help with interpretation of the results.

Preparation of bacterial lawn

• Using the appropriate bacterial isolate, dispense 700μΕ of the mixture onto the

selected trial isolate petri dish. Distribute the mixture evenly across the agar surface. Remove the excess medium and leave to dry in a sterile environment. This should take 20-30 minutes.

Preparation of phage filtrate dilutions

• Using 1.2ml library tubes, add 900μΙ. of appropriate media (+) (e.g - Nutrient Broth, Nutrient Broth and Marine Salts, Tryptic Soy Broth, etc) containing 20μΜ MgS0₄ and 20μΜ CaCl₂ to each of the tubes up to a 10^"8 dilution. Eight tubes are used for each phage filtrate.

• Using a pipette and the neat mixture of the phage filtrate of interest, remove ΙΟΟμΙ. and place it in the first library tube.

· Once all phage filtrates are placed in the primary tubes, mix the tubes gently by

pipetting up and down.

• Once again, remove ΙΟΟμΙ. from the primary tubes and place it into the secondary tubes.

• Once all phage filtrates are placed in the secondary tubes, mix the tubes gently by pipetting up and down.

• Remove ΙΟΟμΙ. from the secondary tubes and place it into the tertiary tubes.

• Continue the process until all dilutions are complete.

Spotting of phage filtrates onto bacterial lawn

· Select the appropriately labeled petri dish containing the dried trial bacterial lawn.

• 20μ1 of the phage filtrate dilutions will be spotted onto each of the divided sections of the plate beginning with the highest dilution (10^"8).

• Continue to spot dilutions from 10^"8 through to the neat mixture which is placed in the center of the plate.

• Leave plates to dry in a sterile environment. Incubate overnight at optimal growth conditions. Plate interpretation

• Plate results are interpreted the following day under an inverted microscope.

• The highest dilution showing clear plaques is evaluated and the total number of plaques counted is recorded. This is done for each of the plates. Information recorded should include phage filtrate, bacterial lawn, dilution observed and number of plaques counted.

• From this information, phage titre can be calculated as plaque forming units per milliliter (pfu/ml)

• (Plaques counted x 50) x inverse of dilution observed.

Bacteriophage Filtrate Host Range Analysis General

To enter a host cell, bacteriophages must attach to specific receptors on the surface of bacteria, including lipopolysaccharides, teichoic acids, proteins, or even flagella. This specificity means a bacteriophage can infect only certain bacteria bearing receptors to which they can bind, which in turn determines the phage's host range. Host growth conditions also influence the ability of the phage to attach and invade them. As phage virions do not move independently, they must rely on random encounters with the right receptors when in solution (blood, lymphatic circulation, irrigation, soil water, etc). Adsorption is a key stage in virus recognition of a sensitive host cell, i.e. specificity of phage infection is defined at this moment. Since bacteriophage, like any other virus, are obligate intracellular parasites, successful penetration into the bacterial cell is an essential condition for continuation of their life cycle.

Materials and Methods

Bacteriophage filtrate host range analysis

Bacterial cultivation of trial isolates • The organisms to be used in the trial should be cultivated on a suitable agar medium under optimal incubation conditions to obtain a fresh overnight grown culture. Streak out the pure culture on an agar plate in a way that distinct colonies will be obtained. NB - Isolates retrieved from cryopreservation must be cultivated twice after retrieval to ensure optimal results.

• Pick a single colony of the isolate off of a plate using a sterile swab or sterile loop and suspend in 10ml of the appropriate selectable liquid medium in a 50ml falcon tube or similar. Label all tubes with strain and date. Incubate isolates until they are in the exponential growth phase.

Plate format

• Each of the trial isolates is designated one standard petri dish. The plate is allocated areas for each of the phage filtrates to be tested. These areas can be marked using a permanent marker on the underside of the petri dish.

Preparation of bacterial lawn

· Using the appropriate bacterial isolate, dispense 700μ1 of the mixture onto the selected trial isolate petri dish. Distribute the mixture evenly across the agar surface.

Remove the excess medium and leave to dry in a sterile environment. NB - This should take 20-30 minutes.

Spotting of phage filtrates onto bacterial lawn

· Select the appropriately labeled petri dish containing the dried trial bacterial lawn.

• 20μ1 of each of the phage filtrates to be tested against the isolate will be spotted onto each of the marked areas

• Leave plates to dry in a sterile environment. Incubate overnight at optimal growth conditions.

Plate interpretation

• Plate results are interpreted following 24 hours by observing for zones of clearance located near each individual phage filtrate spot. Each zone of clearance is given a numerical value with 3+ correlating with complete clearance to 0 which indicates no clearance at all. These results should be recorded on the appropriate host range data sheet.

Biolog GEN III system

Bacterial isolates were identified by an assay using the Biolog Gen III Microplate identification system. Test data are separated into a colour coded spreadsheet for the purpose of pattern observation in which we can group similar isolates and discern different ones.

The Biolog Gen III microplate analyses a microorganism in 94 phenotypic tests: 71 carbon source utilisation assays and 23 chemical sensitivity assays. Results for the 71 carbon source assays from within the Biolog identification system have been recorded and colour coded accordingly. Briefly, Tetrazolium redox dyes are used to colourimetrically indicate utilisation of the carbon sources. All of the wells start out colourless when inoculated. During incubation there is increased respiration in the wells where cells can utilize a carbon source and/or grow. Increased respiration causes reduction of the tetrazolium redox dye, forming a purple colour. Negative wells remain colourless, as does the negative control well with no carbon source. There is also a positive control well used as a reference. After incubation, the phenotypic fingerprint of purple wells is compared to an extensive species library. If a match is found, a species identification of the isolate is made.

The colour densities in wells of the carbon source utilization assays are referenced against the negative control well. All wells visually resembling the control well should be scored as "negative" (-) and all wells with a noticeable purple colour should be scored as "positive" (+). Weils with extremely faint colour, or with small purple flecks or clumps are best scored as "borderline" (\). Most species give dark, clearly discernible "positive" reactions. However, it is normal for the "positive" reactions of certain genera to be light or faint purple.

Performing Mitomycin C Trials to Detect for Presence of Prophage in Bacterial Isolates

General

A prophage is a phage (viral) genome inserted and integrated into the circular bacterial DNA chromosome or existing as an extrachromosomal plasmid. This is a latent form of a bacteriophage, in which the viral genes are present in the bacterium without causing disruption of the bacterial cell. Upon detection of host cell damage, such as UV light or certain chemicals, the prophage is excised from the bacterial chromosome in a process called prophage induction. After induction, viral replication begins via the lytic cycle. To determine if isolated bacteria were hosts for temperate bacteriophage, bacteria are stressed using mitomycin C with the hope of inducing the phage to enter the lytic cycle.

Materials & Methods

Preparation of mitomycin trial to detect presence of prophage in bacterial isolates

Preparation of mitomycin C dilutions

• Mitomycin C dilutions must be made as follows - lOOng/ml, 200ng/ml and 400 ng/ml. mitomycin C is currently purchased in vials containing 2mg of dried mitomycin C. Begin by adding 5ml of filtered dH₂0 to the vial to make a stock 400ug/ml solution. This must be kept in a lightproof vial at 4°C to reduce deterioration. Lower dilutions should not be kept for storage purposes. B - The presence of purple crystal formation in the solution indicates that the product is compromised.

• Add 500ul of (400ug/ml) mitomycin C stock to 4.5ml of sterile dH₂0 to make a 40ug/ml standard solution. This will be the solution used in the experiment. The standard mixture should prepared just prior to its addition to the bacterial broth. Bacterial cultivation of trial isolates

• The organisms to be used in the trial should be cultivated on a suitable agar medium under optimal incubation conditions to obtain a fresh overnight grown culture. Streak out the pure culture onto an agar plate in a way that distinct colonies will be obtained.

• Isolates retrieved from cryopreservation must be cultivated twice after retrieval to ensure optimal results.

• Pick a single colony of the isolate off of a plate using a sterile swab or sterile loop and suspend in 10ml of the appropriate selectable liquid medium in a 50ml falcon tube or similar. Label all tubes with strain and date. Incubate overnight with shaking under optimal incubation conditions to obtain a fresh overnight broth culture.

Obtaining optimal growth curve conditions for trial isolates

• Label 7x 250ml conical flasks with strain or isolate designation and date of preparation using a permanent marker. Prepare one flask per isolate. • To each of the 250ml conical flasks add 100ml of the appropriate selectable liquid medium.

• From one of the 10ml overnight cultures remove lOOul of bacterial suspension and place it in a 1.2ml cuvette along with 900ul of appropriate selectable liquid medium. Measure the absorbance of the cuvette at 600nm and calculate its optical density from the dilution performed.

• Determine amount of bacterial broth which needs to be added to the 100ml in the flasks to reach an optical density of 0.2 (CI x VI = C2 x V2).

• Allow the isolates to incubate for a further 30mins with shaking under optimal incubation conditions. Separate each of the individual bacterial isolate mixtures into four 50ml falcon tubes (20ml each) labeled control, lOOng/ml, 200ng/ml and 400ng/ml.

Addition of mitomycin C

· Control tubes for each isolate shall be left unaltered.

• To make a lOOng/ml sample, add 50ul of the (40ug/ml) standard solution to 20ml of each appropriately labeled bacterial broth.

• To make a 200ng/ml sample, add lOOul of the (40ug/ml) standard solution to 20ml of each appropriately labeled bacterial broth.

· To make a 400ng/ml sample, add 200ul of the (40ug/ml) standard solution to 20ml of each appropriately labeled bacterial broth.

These concentrations may need to be modified dependent on the organism

Preparation of 96 well microtiter plate

· Remove 1ml of each of the similarly treated isolates and place in separate lanes of an 8 lane reagent reservoir.

• Using a multichannel pipette, remove 200ul of each bacterial mixture and add in triplicate to each of the wells of a 96 well flat bottom plate. Use appropriate selectable liquid medium for blanks.

· Place the lid back on the plate once it is prepared and seal with parafilm. Ensure the bottom of the plate is free from streaks and place it in the Spectrostar Nano plate reader for analysis using the mitomycin C program. EXAMPLE 2

The objective of this study is to establish the capability of selected isolates of Vibrio parahaemolyticus to infect Pacific white shrimp Litopenaeus vannamei with EMS/AHPND in a challenge trial.

Materials and Methods

/. Indicator animals

Litopenaeus vannamei shrimp of 2.5 ± 0.55 gram which were tested for free of White Spot Syndrome virus, Yellow Head virus, Monodon Baculovirus, Taura virus and free of Vibrio parahaemolyticus have been used in the study. Shrimp were kept in 160 individual tanks holding 2 litres of 20%o chlorine-treated sea water which was free of Vibrio sp. The shrimp were maintained in the allocated aquarium tanks for 5 days to adapt experimental conditions. During the first and second of day of this period, 8 shrimp had died and they were replaced by 8 new shrimp from the same batch. From day 3 to day 5 of adaptation, 7 shrimp died in tank and 10 others jumped out and died. Those 17 shrimp were not replaced and not involved in challenge experiments.

2. Preparation of V. parahaemolyticus and bacterial coating feed pellets

As a preliminary test, PCR (Sirikharin et al, 2014) for EMS were carried out on the following isolates from natural EMS-infected L. vannamei shrimp : 27-20, 80- 88, 98-65, and 104 -112 (Figure 8). All shrimp were positive for EMS. As a consequence, V. parahaemolyticus strain 27-20 isolated from L. vannamei shrimp at EMS outbreak event in 2012 at Dam Doi district, Ca Mau province, Viet Nam had been chosen to use for a challenge experiment. This V. parahaemolyticus was inoculated in 2 flasks of Tryptic Soy Broth adding 2% sodium chloride. After 18 hours of incubation, two batches of culture provided 4.042 x 10⁹ cfu ml^"1 and 3.502 x 10⁹ cfu ml^"1 respectively. They were diluted 4 to 6 times in PBS and mixed well with shrimp feed pellets (Tom Boy No2) with a size range of 518 particle per gram. Then, the mixed food was incubated on ice in 15 minutes for absorbance of bacteria into food and coated with squid oil to minimize the loss of bacteria into the water during feeding. By this preparation, experimental food were contained about 7.8 x 10⁶; 7.8 x 10⁵ and 7.8 x 10⁴ cfu/particle for the first meal and 6. 8 x 10⁶, 6.8 x 10⁵ and 6.8 x 10⁴ cfu for the second meal respectively.

3. Experimental design

During the initial 3 final days of adaptation period, the average numbers of feed particles that the shrimp ate in the morning and afternoon were 24.7 and 19.2 and the number of particles likely to be eaten by 91.6% and 90.2% of shrimp were 12 in the morning and 10 in the afternoon. Those numbers was used as doses to treat experimental shrimp on the first day of challenge. After the first day, the shrimp were fed with normal feed (not coated with bacteria). The experiment was carried out with 4 treatments:

(1) Negative Control. Tl . These shrimp were not exposed to any of the V.

parahaemolyticus isolates during challenge trial. They were treated in the same way as the challenge groups.

(2) Challenge Test Treatment T2. Shrimp were orally challenged with V.

parahaemolyticus isolate 27-20 in the concentration of approximately 10⁴ cfu/feed pellet.

(3) Challenge Test Treatment T3. Shrimp were challenged with V. parahaemolyticus Isolate 27-20 in the concentration of about 10⁵ cfu/feed pellet.

(4) Challenge Test Treatment T4. Shrimp were challenged with V. parahaemolyticus isolate 27-20 in a concentration of about 10⁶ cfu/feed pellet.

Four treatments were set up in four blocks of tanks and those blocks were separated from each other by plastic walls to minimize cross contamination and the chance of human error. The tanks were aerated with a single air-stone to maintain dissolved oxygen levels above 4 ppm and evaporative loss of water from tanks will be made up using chlorine- free drinking water. The temperature in the tanks was about 27-28°C which was maintained by the ambient air temperature using the temperature control of the air conditioner in the laboratory.

On the first day of the challenge experiment, number of animals participated in treatment Tl, T2, T3 and T4 were 36, 34, 37 and 36 respectively. The experiment occurred over 10 days.

4. Aquarium facilities The tank water salinity, pH and ammonia levels will be monitored from a representative sample of randomly selected tanks within the laboratory every day.

Only authorised persons are permitted to enter the aquarium room (wet lab) and they are to wear footwear that can be properly rinsed in the foot-bath solution. Ethanol- based hand rinse dispensers will also be located in each room, and persons entering and leaving the room will be required to sterilise their hands. Temperature/salinity/pH/DO electrodes are to be rinsed in formalin and rinsed with sterile/bottled water before being taken into the aquarium room, and again before being taken out of the aquarium room.

5. Maintenance of shrimp

Shrimp are fed twice daily with a high quality, pelleted shrimp feed (Tomboy, No. 2, 38% minimum Crude Protein). Feeding rate is to be regulated to minimise the quantity uneaten feed but as consistently as possible across the experiment (see 2. Challenge treatment).

Uneaten feed and faeces are removed from the tanks using a fine scoop net 2 hours after feeding to help maintain water quality. Nets are labeled for each tank (or treatment) and after use, they will be dipped into BKC solution and then clean water before being left to dry inside the wet lab. The nets are not to be removed from the room.

A routine of maintaining tanks in order of increased bio-security risk is adopted. The sequence of maintenance is Treatments Tl, then T2, then T3 and then T4.

During the bioassay period, shrimp are checked every 2 hours during the day and as frequently as practical during the night. Moribund, freshly dead and clearly dead shrimp are removed from the tanks and placed in labelled specimen jars and fixed according to standard techniques, for histological examination. Details including treatment, tank number, date and time of removal and condition of the shrimp (moribund, freshly dead, dead) are included on the label. These shrimp are examined, using histology when necessary, to determine, as precisely as possible, the cause of death. As a planning number, a minimum of 10 dead shrimp, if there are that many, are to be examined using histology and PCR to determine cause of death or presence of EMS/AHPNS. At the end of the experiment, samples of surviving shrimp from each treatment are taken for PCR and histological examination for signs of EMS/AHPNS.

In addition V.parahaemolyticus from some of the hepatopancrea of tested shrimp will be isolated and cultured on ChromoAgar to obtain a sample of the virulent/pathogenic strain of the bacteria.

Results

1. Average initial weights of shrimp

30 individual shrimp were individual weighed prior to stocking tanks and their average weight was about 2.5±0.55gram/shrimp.

2. 5 days of adaptation period

Shrimp were transferred to the experiment tank and during the first two days of the adaptation period, the shrimp ate about 8.1 and 12.0 feed particles for morning and afternoon meal. 8 shrimp died at this stage and were replaced by new 8 shrimp from the same batch. In next three days, the amount of consumed food increased to an average of 24.7 and 19.3 particles for morning and afternoon meal. This indicated that the shrimp adapted well to experimental condition. During this period, the death of 7 shrimp was observed, most of them were dead after moulting. 10 more shrimp jumped out of the tanks and died when a lab worker tried to remove shrimp faeces and uneaten food after 2 hours of feeding. Experience in caring for experimental shrimp was gained and this phenomenon did not happen during the challenge period. Details of shrimp health status and number of consumed feed particles for each meal during adaptation period are summarized in Table 3.

Table 3: Shrimp mortality during the 5-day adaptation period

Day Shrimp death Note

Number of shrimp Tank number

1 (20/9/2014) 5 52, 120, 112, Dead in tank and replace by new

116, 49 shrimp 2 (21/9/2014) 3 96, 146, 156

3 (22/9/2014) 4 41, 2, 1,84 Dead in tank and not replace

3 58, 39, 44 Jumped out and dead, not replace

4 (23/9/2014) 1 95 Dead in tank and not replace

3 20, 49, 50 Jumped out and dead, not replace

5 (24/9/2014) 2 97, 145 Dead in tank and not replace

4 6, 86, 87, 110

3. Challenge experiment

• The first day of challenging shrimp with V.parahaemolyticus

On the first day of challenging shrimp with bacterially-coated food pellets, the food consumption was different among treatments. Most of the shrimp in the control group were able to finish 22 particles which was equivalent to about 2% body weight of shrimp per day. However, the number of intake feed pellets were less in shrimp from other groups. Based on the consumption of food on the first day, some shrimp were discarded and 30 shrimp of each treatment continued to be used in the experiment. The consumption data are summarized in Table 4.

Table 4: The consumption of experimental feed on the first day of challenge

Treatment Number of Consumption of experimental feed particles

animal and ratio

22 particles: 30/30 (100.0%)

20 particles: 2/30 (10.0%)

21 particles: 2/30 (10.0%)

22 particles: 26/30 (80.0%) 18 particles: 5/30 (16.7%) 20 particles 1/30 (0.3%)

21 particles 1/30 (0.3%)

22 particles (23/30) (76.7%)

17 particles 5/30 (16.7%)

18 particles 1/30 (0.3%)

19 particles 1/30 (0.3%)

21 particles 1/30 (0.3%)

22 particles 22/30 (73.3%)

• Mortality

Mortality of experimental shrimp began at day 2 and gradually increased to day 10 of inoculation. The highest percentage of shrimp mortality corresponding for 46.7%) belonged to treatment 4, in which shrimp were treated with the highest dose of bacteria concentration. Lower accumulative mortality was observed in treatment 3, 2 and treatment 1 counting for 33.7, 17.3 and 20.0% respectively (Figure 9).

The results from trail suggested that high concentration of bacteria in food caused more problems to shrimp than at a lower concentration. While no mortality occurred in shrimp that ingested less than 22 bacterial coated feed particles in treatment 2, this was 10.0% and 21.4% in treatment 3 and 4 respectively.

Most of the dead shrimp in treatment 1 (83.3%) and 2 (60.0%) had soft shell and this was the only clinical sign for death shrimp in control group - treatment 1. However, soft shell was observed in small percentage of dead shrimp in treatment 3 (40.0%)) and 4 (35.7%). Besides, the change in colour of shrimp hepatopancrea was observed in 20.0%, 60% and 100% shrimp of treatment 2, 3 and 4 (Figure 11). This might indicate the effect of intake V.parahaemolyticus on shrimp hepatopancrea. The results from paired-sample T-test analysis showed that there were no differences in average mortality between treatment 1 and 2. Other treatments showed significant differences in mortality (T test, p<0.05).

• Bacterial isolation and histology analysis at the end of experiment The results for V.parahaemolyticus isolation and histological analysis of shrimp hepatopancrea showed that the bacteria were absent from all shrimp in treatment 1 and the infection rate increased from 26.7% in treatment 2 to 76.7% in treatment 4. This means that the more bacteria consumed the more chance shrimp can get infection. Moreover, the bacteria were isolated more form dead shrimp than from live shrimp (Table 5).

Table 5: The results of bacterial isolation and histological analysis

The presence of V.parahaemolyticcus and changes in shrimp heapatopancrea (HP) structure were taken into account to define EMS/AHP D cases (Figure 11). Changes in HP were based on definition advised by Lightner et al. (2012). The analysis indicated that no EMS case had been identified in treatment 1 and 2; only a small percentage of shrimp got EMS in treatment 3 and 4 counting for 20. 0% and 36.7%). 100%) of EMS cases were from dead animals; however, not all dead shrimp in those treatments had EMS.

4. Water exchange and water quality during experimental periods

On the first 3 days of adaptation period, some tanks which had water loss due to evaporative and cleaning process were added with small amount of chlorinated free drinking water to keep the same water lever with other experimental tanks. On day 4 post adaptation, 5 tanks had high turbidity were siphoned, clean and exchanged 50% of water volume. This aimed to improve water quality and to test whether shrimp were able to adapt with conditions after changing water. The day after, shrimp in those tanks were still alive, ate well and did not show any clinical signs. Therefore, all experiment tanks were siphoned, cleaned and exchanged 50% of water volume on the last day of commencement period.

Table 6: Details of water exchange in experimental tanks

Water quality in all experiment tanks was kept in suitable levels for shrimp development; In which, temperature, pH, ¾, N0₂ levels remained in the range of 27-28°C, 8.0 - 8.1, 0-1 mg/1 and 0-0.2 mg/1, respectively.

Conclusion In shrimp, the different natural routes of infection by virulent bacterial isolates are theorically oral, trans-cuticular or caused by wounds, by an imbalance in the natural bacterial flora, or by vertical transmission of the pathogen. To AHP D, immersion and injection methods have been sucessfully used for challenge test to shrimp. However, up to date, there was no report on testing pathogenicity of V. parahaemolyticus in term of AHPND by oral route.

In a first challenge experiment, oral challenge of P.vannamei shrimp of about 2.5 gram with V. parahaemolyticus coated in Tomboy feed pellets (No2) could lead to mortality of 46.7% and 33.7% at a bacterial-consuming concentration of about 10⁶ and 10⁵ cfu and more than 90% of those dead shrimp exhibited the signs of EMS/AHPND. At lower concentration of bacteria (10⁴ cfu), low mortality was shown but not significantly with the control group and those dead shrimp did not show signs of EMS/AHPND.

In a second challenge experiment, P. vannamei weighing about 2 gram inoculated with V. parahaemolyticus at 3 different bacterial concentrations of about 10⁵, 10⁶, 10⁷ cfu/feed pellet, showed mortality at day two of challenge and at the end of the experiment mortalities were 36.0%, 60.0% and 80.0% respectively. Most of the dead shrimp in those treatments (44.0%; 66.7%; 73.9%) showed signs of AHPND in their hepatopancrea and were positive for V. parahaemolyticus by bacterial isolation and PCR detection.

EXAMPLE 3

The objective of this study will be to determine the effectiveness of a phage preparation to reduce the incidence of EMS/AHPND in Pacific white shrimp Litopenaeus vannamei that have been challenged with one or more strains or isolates of Vibrio parahaemolyticus, such as that in Example 2, which cause cause significant mortality in shrimp through EMS/AHPND.

Initial in vitro studies using seven (7) different AHPNS positive V. parahaemolyticus isolates (Pl/1; 5 HP; TWOl ; RN; RY02; KM; Malay08) which had been previously been identified to cause AHPNS in P. vannamei post-larvae were tested for susceptibility against a phage cocktail. Growth curve patterns suggest that these bacteria which cause AHPNS contain some strain variation between isolates of the same species. These seven bacterial isolates were obtained from different locations around Asia, by analyzing the data obtained when challenging the suspect isolates with the bacteriophage cocktail, it was observed that there was a difference in efficiency of the phage cocktail between the isolates. This contrast in responses is a good indicator of strain variability between isolates. Isolate Pl/1 was identified as an AHPNS isolate suitable for subsequent challenge experiments due to its susceptibility to bacteriophage. Isolate 27-20 identified in Example 2 was also shown to be an AHPNS isolate susceptible to bacteriophage that may be useful for challenge experiments.

Propagation of bacteriophage will be performed using non-pathogenic Vibrio replication hosts. Isolation of phage from the replication host will also be performed essentially as described in Example 1.

It is expected that treatments will be essentially as follows:

(1) Control (C). These shrimp will not be exposed to any phage or V. parahaemolyticus during challenge trial. They will be treated in the same way as the treatment and challenge groups. Other controls may include exposure to non- AHPND bacteria as a negative control.

(2) Phage Treatment (P). Shrimp will be challenged with phage that will be added to the tank water to give an initial concentration in the water of 10⁸ pfu/mL. This will be done one hour after the completion of the morning feeding on the first day and then again after each morning feeding for a total of five days. (3) Vibrio Treatment (V). Shrimp will be challenged with V. parahaemolyticus V. parahaemolyticus bacteria will be given to the shrimp through feed pellets that have been briefly dipped in PBS with a bacterial concentration 10⁶ cfu/mL. These feed pellets will only be given at both feedings on Day 1 of the Challenge Trial (4) Phage/Vibrio Challenge Treatment 1. (PV). Shrimp will be challenged with V. parahaemolyticus bacteria through feed pellets as in the Vibrio Treatment. However, after feeding has finished phage will be added to the tanks to give an initial concentration in the tank water of approximately 10⁸ pfu/mL. The phage will be added at the same dosage once each day at about the same time for a total of five days.

The tanks will be filled with filtered seawater at a salinity of about 20 ppt that has been prepared from previously concentrated and sterilised seawater. Evaporative loss of water from tanks will be made up using de-ionised water or if de-ionised water is not available, with chlorine- free drinking water. This strategy may reduce the necessity for more detailed monitoring of salinity.

The tanks will not have a continuous flow through of water but will be considered "static". The tanks will be aerated with a single air-stone to maintain dissolved oxygen levels above 5 ppm.

The temperature in the tanks will be at about 28°C which will be maintained by the ambient air temperature using the temperature control of the air conditioner in the laboratory.

The tank water salinity, pH and ammonia levels of treated water source will be monitored at the beginning. If these parameters are not acceptable, they should be improved accordingly. During a course of experiment, salinity, pH and ammonia levels will be monitored at a representative sample of randomly selected tanks within the laboratory every two days. If there are any indications that the pH and ammonia levels are moving towards unacceptable levels, more detailed sampling will be carried out and water exchanges will be made to restore water quality.

At the end of the experiment, samples of surviving shrimp from each treatment will be taken for PCR and histological examination for signs of EMS/AHPNS.

In addition consideration will be given to taking V.parahaemolyticus from some of the hepatopancrea of tested shrimp to be isolated and cultured on ChromoAgar to obtain a sample of the virulent/pathogenic strain of the bacteria.

In initial experiments, it was found that following initial administration of the phage, the toxin-producing Vibrio parahaemolyticus bacterial isolate Pl/1 has limited growth capabilities. In theory, during this time bacteria upon which the phage has no effect, should replicate unhindered and outcompete the toxin-producing bacterial isolate Pl/1. By using multiple bacteria to take advantage of the available nutrient, it was anticipated that the toxin-producing bacteria Pl/1 would have a reduced capability to replicate to sufficient numbers to cause death in the prawns. As it would be optimal to use fast-growing bacteria which do not contribute to the effect of AHPNS, the bacteria used to compete for nutrient against the AHPNS isolate Pl/1 were Vibrio parahaemolyticus which had been previously identified not to cause death/AHPNS in prawns. Six (6) non-AHPNS causing V. parahaemolyticus isolates were tested against the phage cocktail to identify those which were not susceptible and those which were. Two isolates which were not susceptible to the phage (MC and VplO) were used in a subsequent challenge trial along with one bacterial isolate to which the phage was effective (Vp5). Including the non-AHPNS causing isolate Vp5, which was susceptible to the phage cocktail, was an attempt at giving the phage an additional, non-pathogenic replication host in which to replicate to maintain high concentrations throughout the experiment.

Further experiments will be required to test the ability of a phage cocktail to protect Pacific white shrimp Litopenaeus vannamei from challenge with one or more AHPNS strains or isolates of Vibrio parahaemolyticus. This will also include better understanding bacterial defence mechanisms (such as superinfection immunity, changing phage receptor sites) that may lead to resistance to, or recovery from, phage infection.

Throughout this specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated herein without departing from the broad spirit and scope of the invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference in their entirety.

Claims

1. A method of producing a bacterial replication host for a bacteriophage, said method including the step of selecting a bacterial species, strain, serotype or isolate that is different to a pathogenic bacterium as a host for replication of the bacteriophage.

2. A method of producing bacteriophage including the step of propagating a bacteriophage that is capable of infecting a pathogenic bacterium in a host bacterial species, strain, serotype or isolate that is different to the pathogenic bacterium under conditions that promote propagation of the bacteriophage.

3. A method of producing a bacteriophage composition including the steps of: preparing a bacteriophage that is capable of infecting a pathogenic bacterium by propagation in a different host bacterial species, strain, serotype or isolate under conditions that promote propagation of the bacteriophage; and forming a composition comprising the isolated bacteriophage.

4. The method of any preceding claim, wherein the bacterial species, strain serotype or isolate that is different to the pathogenic bacterium is characterized by one or more of: does not harbour a pro-phage; is capable of supporting bacteriophage replication; has prolific and reliable growth characteristics in vitro; does not produce toxins, or produces minimal toxins; is non-pathogenic; and/or has a requirement for inexpensive and/or minimal growth media.

5. The method of any preceding claim, wherein the bacteriophage exhibits lytic growth in the bacterial species, strain, serotype or isolate that is different to the pathogenic bacterium.

6. The method of any preceding claim, wherein the pathogenic bacterium is of the genus Vibrio, Aeromonas or Pseudomonas.

7. The method of Claim 7, wherein the pathogenic bacterium of the genus Vibrio is a pathogen of shrimp or prawns.

8. The method of Claim 7 or Claim 8, wherein the pathogenic bacterium is Vibrio parahaemolyticus .

9. A bacterial replication host produced according to the method of any one of Claims 1 or Claims 4-8.

10. Bacteriophage produced according to the method of any one of Claims 2 or 4- 8.

11. A bacteriophage composition comprising the bacteriophage of Claim 11.

12. Use of the bacteriophage composition of Claim 12 as an antiseptic or antibacterial agent.

13. Use according to Claim 13, as an antibacterial agent in aquaculture.