Patent Grant
10849942
The present invention relates to treatment of bacterial infections in aquaculture, generally farmed production of shrimp, prawns and fish. In particular the invention relates to compositions and methods for reducing or preventing infections and/or for treating existing infections in such aquaculture.
Bacteriophages are the most numerous form of life on Earth. They can be found in all environments where bacteria grow. Bacteriophages are detected in ground and surface water, soil, food (e.g., sauerkraut, wine), sewage and sludge. They have also been isolated from humans and animals, for example from faeces, urine, saliva, spit, rumen and serum. Bacteriophages are able to penetrate different organs and tissues, including the central nervous system, and are a part of intestinal flora together with their bacterial hosts. They are responsible for 10-80% of total bacterial mortality in aquatic ecosystems and are an important factor limiting bacterial populations.
Therapeutic applications of bacteriophage are known. WO 03/093462 discloses methods for the immobilisation of viruses, in particular bacteriophages, whilst retaining their biological activity for use as antibacterial agents. Given that the natural environment of bacteriophages is aqueous it has been widely assumed that stability towards dehydration as disclosed in WO 03/093462 tends towards the natural stability in aqueous media.
Oceans and inland waters are largely fished to their limit and the supply of wild-caught fish peaked in the 1990s. With the global wild fish supply stagnant and the human population increasing, new research shows that farmed fish and shellfish production will have to increase by 133 percent between 2010 and 2050 in order to meet projected fish demand worldwide.
Nearly one-third of the world's seafood is produced by industrial aquaculture and production has increased by 6% per year from 8.7 million tons of fish in 1990 to 50 million tons in 2011 and projected to reach 80 million tons by 2030. Fish farming plants, however, often suffer from heavy financial losses due to the development of infections caused by microbial pathogens, including multidrug resistant bacteria that are easily transmitted through water and therefore able to infect a great variety of fish species.
Although pathogenic species have been described in the majority of bacterial taxonomic groups, only a relatively small number are responsible for significant economic losses. Vibriosis and photobacteriosis are primarily diseases of marine and estuarine fish, both in natural and commercial production systems throughout the world, occurring infrequently in freshwater fish. Both diseases can cause significant mortality in fish, reaching values of up to 100% in infected facilities. Vibriosis and photobacteriosis are caused by bacteria from the family Vibrionaceae. Vibriosis is caused by species of Vibrio, namely by Vibrio anguillarum.
Others species of Vibrio, such as V. alginolitycus, V. carchariae, V. salmonicida, V. damsela, V. ordalii, V. parahemolyticus and V. vulnificus, also cause important infections in several species of fish. Photobacteriosis is caused by Photobacterium damselae subsp. piscicida which is a highly pathogenic bacterium that does not seem to have host specificity, infecting a diverse range of fish species. Other bacteria including Aeromonas salmonicida, causative agent of furunculosis, Rickettsia-like bacteria, Cytophaga marina, Flavobacterium psychrophilum and Pseudomonas plecoglossicida are also important groups of fish pathogens.
Diseases like EMS (Early Mortality Syndrome) of shrimps are caused by bacterial infections in which the bacterium (Vibrio parahaemolyticus) is itself infected by a lysogenic bacteriophage which carries a toxin gene. On bacteriophage infection the bacterium incorporates the bacteriophage into its genome and expresses the toxin which leads to EMS in the shrimp.
Although vaccination is the ideal method to prevent many different kinds of infectious diseases it is not always applicable in fish species.
Chemotherapy is a rapid and effective alternative method to treat or prevent bacterial infections, but the frequent use of antibiotics has resulted in an increasing drug-resistance in pathogenic bacteria in the aquaculture, agriculture and medical areas and since few chemotherapeutic drugs are licensed for use in fisheries alternative treatments are required.
Nakai et al. Diseases of Aquatic Organisms, vol. 37, pp. 33-41, (23 Jun. 1999) describes the effect of treating Lactococcus garvieae infection in yellowtail through the use of bacteriophage to which L. garvieae is susceptible. Nakai et al. report that each of the three bacteriophage isolates they tested for stability in natural (unsterilised) sea water persisted for 3 days but had perished within 1 week. Nakai et al. also describe the use of fish food impregnated with 107.9 PFU g-1 of bacteriophage. Giving this food to fish that were subsequently challenged with 108.5 CFU of L. garvieae by anal intubation decreased the mortality rate of the challenged fish.
WO 2006/047872 discloses antibacterial compositions comprising bacteriophage that are adsorbed onto a matrix. The composition may be added to a feed for aquatic use.
Bacteriophage have been proposed for various treatments of bacterial infection. It is known, however, that bacteriophages survive only for relatively short periods in their natural environment, i.e. in water. Average decay rates of viruses in natural seawater samples can be calculated, based on well-known data, e.g. in C H Suttle (Microb. Ecol. (1994) 28: 237-243, at about 0.48 day-1. The greatly reduced survival of bacteriophages in natural aqueous environments is due to a combination of causes, significantly predation and sunlight.
Hence, there is a need for an alternative means to treat or reduce bacterial infections of farmed crustaceans, especially shrimp and prawns, and fish.
Object of the Invention
An object of the present invention is to provide compositions and uses of those compositions and methods using those compositions that offer an alternative treatment of bacterial infections in commercially reared crustaceans, e.g. prawns and shrimp, and/or fish. A further aim of particular embodiments is to provide improved such compositions, uses and methods.
A composition comprising bacteriophage covalently attached to a particle is for use in treating bacterial infection in fish or crustaceans. Edible particles are preferably used. The present invention is based upon enhancement of stability and viability of bacteriophage in aqueous environments, rendering possible treatment of bacterial infections in aquaculture.
Feed for crustaceans or fish is provided, comprising bacteriophage covalently attached to a particle for treating bacterial infection in fish or crustaceans.
A method of making fish or crustacean feed comprises mixing bacteriophages covalently attached to particles into feed components, to produce feed comprising said particles.
Bacteria infected with a lysogenic bacteriophage are provided and can be used in treating disease of fish or crustaceans.
A composition of the invention accordingly comprises bacteriophage covalently attached to a particle for use in treating bacterial infection in fish or crustaceans. After administration to the fish or crustaceans, for example via feed containing the particles, bacterial infections are treated.
The particle can be a carrier particle, made e.g. of edible material or an inert material, in which case the carrier particle is typically approximately spherical. It may have an average diameter of up to 1 mm, up to 100 microns, up to 50 microns, up to 10 microns, from 1 nm, from 10 nm, from 100 nm, from 0.5 microns or any combinations of these. In specific examples below, particles in the range 1 to 200 microns were used. The particles in general can be approximately round or spheroid; they are preferably smooth. Particles or fragments of edible material may also be of irregular shapes and sizes.
Particle size is suitably measured using methods and apparatus recognized as standard in the art. Particle sizing in dispersions can be accomplished using a variety of techniques, including laser diffraction, dynamic light scattering (DLS), disc centrifugation, and light microscopy. All of these techniques have their advantages and limitations. Laser diffraction relies on a well-controlled presentation of the sample to the measurement region and is limited to samples with narrow range of particle concentrations. Dilution is often required and this may affect the particle size, particularly in compounds with high solubility. Examples of sizing equipment are made by Malvern Instruments (UK), using laser diffraction methods. For highly irregular particles, the diameter refers to the greatest diameter in any dimension even if the particle is relatively non-spherical.
In embodiments of the invention, bacteriophages covalently attached to a plurality of particles are provided. These are preferably in relatively homogenous form, in which a large proportion, preferably substantially all, of the plurality of particles have diameters in the stated range, more preferably 80% or more, 90% or more or 95% or more of the particles with phage covalently attached have diameters in the stated range (being any range as set out above or elsewhere herein).
Particles for use in the invention to which bacteriophage are immobilised by covalent bonding are generally edible by or substantially inert to the animal to be treated. In examples, nylon particles (beads) were used. Other inert, preferably non-toxic biocompatible material may be used. In addition, the particle may be made of a biodegradable material. Suitable materials include polymethyl methacrylate, polyethylene, ethylene/acrylate copolymer, nylon-12, polyurethane, silicone resin, silica and nylon 1010. WO 2003/093462 describes further materials that the particles may be made from.
Immobilisation or attachment of bacteriophage to the particle substrate may be achieved in a number of ways. Preferably, bacteriophage are immobilised via covalent bonds formed between the bacteriophage coat protein and the carrier substrate.
Further, bacteriophage are preferably immobilised to the substrate via their head groups or nucleocapsid by activating the substrate particle before the addition and bonding of bacteriophage.
The term “activated/activating/activation” is understood to mean the activation of the substrate such as electrically, e.g. by corona discharge, or by reacting said substrate with various chemical groups (leaving a surface chemistry able to bind viruses, such as bacteriophage head or capsid groups).
Activation of said substrate may be achieved by, for example, preliminary hydrolysis with an acid, preferably HCl followed by a wash step of water and an alkali to neutralise the acid. Preferably, said alkali is sodium bicarbonate. Binding of bacteriophage via their head groups is advantageous. In the case of complex bacteriophage for example, binding via head groups leaves the tail groups, which are necessary for bacteria-specific recognition, free to infect, i.e., bind and penetrate a host bacterial cell. A plurality of various strain-specific bacteriophage may be immobilised to a substrate at any one time.
Coupling of phage to a substrate is as a result of the formation of covalent bonds between the viral coat protein and the substrate such as through an amino group on a peptide, for example a peptide bond. “Coupling Agents” that aid this process vary, and are dependent on the substrate used. For example, for coupling to nylon or other polymers with amino or carboxy surface groups the coupling agents carbodiimide or glutaraldehyde may be used.
Further details of methods and preferred methods for covalent attachment of bacteriophage to particles or pellets or feed components, retaining phage infectivity, are described in more detail in WO 2003/093462 and WO 2007/072049.
A further option is to use particles that comprise one or more targeting moiety, e.g. a protein or ligand, to direct the particles to desired targets within fish or crustaceans.
For example, particles can comprise one or more lectins to target them e.g. to fish gills for treatment e.g. of Yersinia infection.
Suitably the present invention delivers bacteriophage via feed and the particle is made of edible material. Hence it is conveniently incorporated in feed for fish/crustaceans. Bacteriophage can be attached to particles of carbohydrate (e.g. cellulose) or protein (including fish protein or animal protein) and this can be achieved using for example electric discharge methods of application to nylon beads.
Feed comprising the particles may comprise carbohydrate, protein, lipid, vitamin or a mixture of one or more of all.
The invention is of use in treatment of diseases of fish and crustaceans caused by the following bacteria:
Vibrio species:
V. harveyi
V. fluvialis
V. parahaemolyticus
V. vulnificus
V. alginolyticus
V. penaeicida
V. anguillarum
V. carchariae
V. salmonicida
V. damsela
V. ordalii
V. owensii
Aeromonas species:
A. salmonicida
A. hydrophilla
A. punctata
Yersinia ruckeri
Moritella viscosa
Rickettsia salmonis
Piscirickettsia salmonis
Lactococcus garvieae
Pseudomonas
plecoglossicida
Flavobacterium
psychrophilum
Photobacterium
damselae
In one preferred application of the invention the bacteriophage are for use in treating bacterial infection in crustaceans; more specifically, for treating infection by Vibrio bacteria species.
V. parahaemolyticus is a common inhabitant of coastal and estuarine environments all over the world. Hence they are often found naturally associated with shrimp aquaculture systems. Certain environmental conditions may be more favourable for the establishment, survival and growth of the organism such as temperature, salinity, zooplankton, tidal flushing and dissolved oxygen.
V. parahaemolyticus is closely related to shrimp pathogenic luminous bacteria such as V. harveyi, V. campbelli and V. owensii. These along with other closely related Vibrio spp form a “V. harveyi clade”. Bacteria within this clade have a very high degree of similarity at phenotypic and genotypic level. Certain strains of V. parahaemolyticus can cause gastroenteritis in humans and clinical strains are characterised by the ability to produce a thermostable direct hemolysin (TDH) or a TDH-related hemolysin (TRH). The genes encoding these hemolysins (tdh and trh genes) are generally used as markers for human pathogenic strains of V. parahaemolyticus. Human pathogenic strains possessing these markers account for 1-2 percent of environmental strains of V. parahaemolyticus. All strains (both clinical and environmental) produce a thermolabile hemolysin (TLH) encoded by tlh gene and this is generally used as a marker for V. parahaemolyticus in diagnostic tests (48). The tdh and trh genes encoding the virulence factors are present in “pathogenicity islands”, which are discrete genetic units present only in virulent strains; having a Guanine+Cytosine (G+C) content that is different from the rest of the chromosomal DNA and are generally acquired by horizontal gene transfer.
By use of the invention with bacteriophage specific to Vibrio species these infections of e.g. shrimp and prawn can now be treated.
In another preferred application of the invention, the bacteriophage are for treating bacterial infection in fish, especially for treating infection by Vibrio, Aeromonas, Yersinia, Moritella, Rickettsia, Piscirickettsia, Lactococcus, Pseudomonas, Flavobacterium or Photobacterium bacteria species. Useful bacteriophage are disclosed e.g. in US 2013/0323209.
Feed for fish and crustaceans, especially shrimp and prawns, is provided by the invention. One aspect of these embodiments of the invention hence provides feed for crustaceans or fish, comprising bacteriophages covalently attached to particles for treating bacterial infection in fish or crustaceans.
It is preferred that all of the feed is edible and so it is preferred that the particle is made of edible material, e.g. carbohydrate or protein as described elsewhere herein. Mixed in with the particles are other feed components that typically include carbohydrate, protein, lipid, vitamin or a mixture of one or more of all.
Another aspect of these embodiments of the invention hence provides feed for crustaceans or fish to which bacteriophage is covalently attached, for treating bacterial infection in fish or crustaceans. Typically, the feed contains edible feed components to which bacteriophage are covalently attached. As per previous embodiments, bacteriophage may be covalently attached to carbohydrate or protein of the feed.
In particular embodiments of the invention, illustrated in the examples below, feed pellets are provided to which the bacteriophage are covalently attached, generally to the outer surface thereof by methods in which pellets are activated then have phage attached. Suitable and preferred pellet sizes are as described elsewhere herein.
Specific pellets of the invention, with bacteriophage covalently attached are for treating infection by Vibrio bacteria species in crustaceans.
Other specific pellets of the invention, with bacteriophage covalently attached are for treating infection by Vibrio, Aeromonas, Yersinia, Moritella, Rickettsia, Piscirickettsia, Lactococcus, Pseudomonas, Flavobacterium or Photobacterium bacteria species in fish.
Bacteriophage for the invention include bacteriophage in general without limitation provided that the bacteriophage is obtainable and its host or target bacteria can be cultured and infected in culture. The bacteriophage can be ssRNA, dsRNA, ssDNA or dsDNA bacteriophage, with either circular or linear arrangement of the genetic material. The suitable bacteriophage include Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridea, Rudiviridae, Ampullaviridae, Bacilloviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fusseloviridae, Globuloviridae, Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae and Tectiviridae. Suitable phage for use in embodiments mentioned above infect and are lytic for the bacterial families and species mentioned.
Examples of how to isolate desired phage are widespread in the literature, including just by way of illustration: Gill J J and Hyman P, “ Phage choice, isolation, and preparation for phage therapy”, Curr Pharm Biotechnol., 2010, Jan.;11(1): pp2-14, and the previously mentioned “Bacteriophage Therapy” minireview by Sulakvelidze et al., Antimicrobial Agents and Chemotherapy, March 2001, pp 649-659.
The invention extends the viability of both lytic and lysogenic bacteriophages in their natural environment, sea, fresh water or other aqueous environments, by covalent immobilisation. Surprisingly increased viability and stability have been illustrated in examples below and now make possible the bacterial treatments set out herein.
Immobilisation has been found in examples set out below in more detail to make predation more difficult. Predation can occur through enzymatic digestion with extracellular or intracellular enzymes from bacteria or fungi, from ingestion by protozoa and subsequent digestion or from the digestive processes of other eukaryotic organisms.
In general, advantages of the invention stem from the unexpected extension of phage viability in saline waters, fresh waters and other predominately aqueous environments; unexpected resistance to degradation by components of natural environment; unexpected resistance to predation—achieved by the use of bacteriophage covalently attached to particles in feed as described herein.
A method of the invention comprises combining pellet components with particles to which bacteriophage are covalently attached, to form feed comprising the particles. That feed is then for use to deliver the bacteriophage to the target fish/crustaceans.
In a particular method of making fish or crustacean feed, the steps comprise mixing bacteriophages covalently attached to particles into feed components, to produce feed comprising said particles.
The method may comprise:
Heat can be used to achieve at least partial sterilization of the pellets. One method of the invention comprises:
This order of steps avoids applying heat to and thus damaging the bacteriophage component of the feed.
In certain methods the particles are added to formed pellets. This may be achieved by spraying pellets with a solution or suspension of the particles. The sprayed pellets can then be dried to adhere the particles thereto.
In an example of the method, preparation of the pellets comprises:
Typically the pellet components comprise a mixture of one or more or all of proteins, fats, carbohydrates, minerals, vitamins and water (e.g. meat or fish meal, wheat flour, rice bran, rice pollard, split peas, corn, soya meal, mill mix, fish oil, vitamin and mineral premix etc.). Similar mixes are used for both crustaceans and fish, though specific tailored mixes are also used.
The conditioning step can be used to increase the water content and/or to partially or completely cook pellet components. Steam is generally used, which effectively cooks the components and increases moisture content at the same time. Depending upon the steam heat and step duration some degree of sterilisation may also occur at this time.
Pellets are generally formed by passing the conditioned material through a pelletizing mill. Pellet size varies and the pulverising step can be of longer duration or more vigorous if the end pellets are to be of smaller sizes. Depending on the size of the fish/crustaceans, pellet diameters are typically in the range 0.1 to 30 mm, more generally 0.5 mm or greater, also more generally up to 25 mm, up to 20 mm, up to 15 mm, up to 10 mm or up to 8 mm. Pellet sizes under 2 mm normally require fairly extensive pulverisation to be carried out. Shrimp pellets are more commonly in the range approximately up to 5 mm or 8 mm, and can be smaller, say up to 2 mm or 3 mm. Fish pellets are larger and more commonly of diameter 3 mm upwards.
Pellet components usually include starch. However, in water the pellets disintegrate due to the starch swelling. Conditioning at lower temperatures has been shown to reduce the starch expansion and provide a way of maintaining pellet integrity while wet. An optional step is to add a second heating step after the pelletizing step.
Heating after milling, where conventionally the cooling process can occur, has two main effects:
The use of this post-milling conditioning step dramatically improves the stability of the pellet in water. Formulation cost is saved because less binder needs to be added and this tremendously helps digestibility for marine life.
Pellet buoyancy can be altered dependent on whether the marine life targeted are top or bottom feeders. Hollow pellets allow significantly longer flotation times.
Still further provided by the invention are methods of making fish or crustacean feed comprising covalently attaching bacteriophage to feed pellets.
As per embodiments in the examples below, which contain greater details, one such method comprises forming feed components into pellets, and treating the pellets to covalently attach bacteriophage thereto. Pellet treatment is suitably described elsewhere herein, for activation of pellets then covalent attachment of phage. Electrical based are especially suitable. In an example corona discharge has been successfully used. Activated pellets can then be combined with phage, e.g. by bringing the pellets into contact with a solution or suspension of phage.
In a separate aspect of the invention, it is possible to take advantage of cellular factors that prevent superinfection of bacteria already infected with a lysogenic bacteriophage by a second bacteriophage of the same type. Accordingly, the invention provides bacteria infected with a lysogenic bacteriophage for use in treating disease of fish or crustaceans. A method of preventing disease in fish or crustaceans comprises infecting the same with this bacteria.
In use, fish or crustaceans are hence deliberately infected with this bacteria, known to be relatively prevalent but relatively innocuous (as the bacteriophage with which it is infected is lysogenic and does not cause disease). This step, however, prevents disease caused by bacteria being subsequently infected with bacteriophage carrying a toxin gene. The presence of the first bacteriophage infection means superinfection by more pathogenic bacteriophage is reduced.
The bacteria are for example Vibrio bacteria for use in treating disease of crustaceans.
The bacteria is for example a Vibrio, Aeromonas, Yersinia, Moritella, Rickettsia, Piscirickettsia, Lactococcus, Pseudomonas, Flavobacterium or Photobacterium bacteria species for use in treating disease of fish.
Feed for crustaceans or fish, comprising these bacteria, form further embodiments of the invention.
The invention is now illustrated in the following specific embodiments with reference to the accompanying drawings in which:
We tested bacteriophage covalently attached to both plastic (nylon) and carbohydrate (cellulose) particles to prove the feasibility of using compositions of the invention in aquaculture applications.
It was first determined whether bacteriophages immobilised on to cellulose powder survive longer than free bacteriophages in both sea water and fresh water.
Procedures
Media and Methods
Table 1—All media was made and methods were performed in accordance with the appropriate standard operating procedure (SOP).
“Agar 50” SOP
Preparation of nutrient agar plates
“Broth 51” SOP
Autoclaving the media
“Immobilisation” SOP
Applicable bacteriophages
Materials required
Use of corona discharge machine (flatbed)
Washing material
Antibacterial activity assay
“Culture” SOP
Appropriate Bacteria
Equipment and Materials required
Preparation of Bacteria
Storage of Bacteria
Bacteria and Bacteriophages
Bacteria and bacteriophages were acquired from internal stores. All bacteria and bacteriophages used in this study are detailed in Table 2. All bacteria were cultured in accordance with the instructions contained in the relevant SOP
P. aeruginosa
Source of water samples
Sea water sample was sourced from Troon beach and fresh water sample was sourced from Drumpellier Lochs, Scotland, UK.
Preparation of Cellulose
Cellulose powder, average particle size 50 μm was utilised in this study. Cellulose powder to be treated with corona discharge was handled aseptically.
Immobilisation of bacteriophages onto cellulose
Cellulose powder was placed on the corona discharge table as detailed in the Immobilisation SOP. A bacteriophage solution of concentration of 1×107 PFU/ml was prepared for immobilisation. Cellulose was treated by 2× corona discharge treatments at 7.5 kV and a 10 ml bacteriophage solution was aseptically applied to the material. Cellulose powder was vacuum filtered to remove any excess bacteriophages in solution.
Preparation of 96 well plate with immobilised & non-immobilised bacteriophage in sea and fresh water environments.
Each well of the plate was filled with 200 μl final volume with equivalent volume/weight of free bacteriophages/immobilised bacteriophages 0.2 g.
Storage of samples
Each 96 well plate was incubated at 40° C. for the duration of the study to indicate an accelerated time course. 96 well plates were only removed prior to sampling.
Sampling of Bacteriophage survival
Each sample was tested in triplicate by adding the contents of a single well to 9 ml of nutrient broth and 1 ml liquid culture of the host bacterium Pseudomonas aeruginosa NCO2000. Samples were incubated at 37° C. for two hours in an orbital incubator. After incubation samples were filtered using 0.2 μm filters and serial diluted 1/10 using PBS for dilution to concentrations of 1×10-1-1×10-8. A plaque assay was performed using the soft agar overlay method, 200 μl of each concentration including ‘neat’ concentration was plated on nutrient agar plates before being inoculated. Plates were incubated at 37° C. overnight in LEEC compact incubator. Following incubation visible plaques were counted and PFU/ml was determined.
The data shown in
Table 3 shows the activity of bacteriophage covalently attached to various substrates after periods of storage—significant activity was maintained in all cases.
S. aurues
P. aeruginosa
Peptobacterium
Peptobacterium
Salmonella
Feed pellets for shrimp were made as follows:
A formulation of proteins, carbohydrates, fats, minerals and vitamins comprising 182 g/kg fish meal, 200 g/kg rice pollard, 300 g/kg mill mix, 118 g/kg wheat flour, 185 g/kg coconut meal and 15 g/kg vitamin and mineral premix was thoroughly mixed in a twin shaft mixer.
A pulverizer was used to grind the mixture into a fine powder.
A conditioner was then used to expose the fine powder to a high pressure (150 psi) steam for 30 minutes. This increased the moisture content of the powder, as well as beginning to convert the starch into a readily digestible form.
The conditioned powder then entered a pellet mill set to produce pellets of 1.5 mm diameter.
The pellets were then subjected to a second conditioning step in order to facilitate binding of the starch and/or gluten in the pellet. This step dramatically increased the stability of the pellet in water.
The pellets were then cooled and dried. Dried pellets were subsequently sprayed with an aqueous suspension of bacteriophage covalently attached to nylon particles of average diameter 100 microns at a concentration of 109 CFU ml-1 allowed to dry and then processed into containers.
Feed pellets for fish were made as follows:
A formulation of proteins, carbohydrates, fats, minerals and vitamins comprising 201 g/kg fish meal, 11 g/kg fish oil, 251 g/kg rice bran, 254 g/kg mill mix, 150 g/kg copra meal, 118 g/kg broken rice, 10 g/kg wheat flour and 5 g/kg vitamin and mineral premix was thoroughly mixed in a twin shaft mixer.
A pulverizer was used to grind the mixture into a fine powder.
A conditioner was then used to expose the fine powder to a high pressure (150 psi) steam for 30 minutes.
The conditioned powder then entered a pellet mill set to produce pellets of 5 mm diameter.
The pellets were then cooled, and dried. Dried pellets were subsequently sprayed with an aqueous suspension of bacteriophage covalently attached to cellulose particles of average diameter 50 microns at a concentration of 109 CFU ml-1 allowed to dry and then processed into containers.
Fish food pellets based on wheat germ (composition: wheat germ, derivatives of vegetable origin, fishmeal and fish derivatives, yeasts, vegetable protein extracts, molluscs and crustacean, vitamins and minerals) were subjected to two passes through a flat bed corona machine at 7.5KV. Pellets were immediately sprayed with bacteriophage solution (1×107 pfu/mL of Lin24) and air dried, and stored for two weeks at room temperature.
A lawn of Camplyobacter jejuni (ATCC12851) was prepared on Petri dishes and fragmented fish pellets retrieved from storage and placed on the surface. These were incubated at 37C for 36 hours.
Examination of the dishes showed clearing zones around the pellet fragments, illustrated in
The experiment was repeated with Maize based pellets (maize derivatives of vegetable origin, fishmeal and fish derivatives, yeasts, vitamins and minerals and Spirulina), with similar results (not shown).
We developed the following protocols for treatment of Vibrio infections of shrimp.
Isolation of Bacteriophages Displaying Lytic Activity against Vibrio parahaemolyticus
The isolation of bacteriophages displaying lytic activity against V. parahaemolyticus is undertaken using 3 methods. Environmental samples are added directly to a V. parahaemolyticus agar overlay and also incubated at 37° C. for 9 h with a culture of V. parahaemolyticus. V. parahaemolyticus samples are also subjected to 1 mg/mL of mitomycin C to induce bacteriophage replication.
The presence of lytic bacteriophages is confirmed by the formation of clear plaques in a V. parahaemolyticus agar overlay.
Characterisation of Bacteriophages
Each lytic bacteriophage isolated is characterised by determining the host range, efficiency of plating (EOP) burst size, growth curve, molecular characterisation and restriction analysis.
Host Range and Efficiency of Plating
Each bacteriophage is added to agar overlays of each isolated V. parahaemolyticus to determine the host range. The EOP is determined by adding samples of a series of dilution factors to agar overlays of each isolated V. parahaemolyticus.
Measurement of Burst Size and Growth Curves
The burst size of each bacteriophage is determined for each V. parahaemolyticus isolated by incubation of a co-culture of bacteria and bacteriophage. Samples are taken at different time points to establish the number of bacteria and the number of bacteriophages remaining in solution. The burst size is calculated using the following calculation:
Restriction Analysis
Bacteriophage DNA is subjected to digestion by restriction enzymes to ensure that genetically identical bacteriophages are used in the bacteriophage cocktail.
Testing of Antimicrobial Activity
Tank tests are used to measure the activity of immobilised bacteriophage on V. parahaemolyticus. This system consists of sterile salt water inoculated with a known bacterial concentration and a tank containing supplements to replicate pond conditions. The bacteria are added to salt water containing a known concentration of immobilised bacteriophages.
Multiplicity of infection (MOI) is varied to determine the impact at different concentrations (Table 4).
Outcomes and Success Criteria
The following are the identified outcomes:
Laboratory and Tank Testing
The aim of this stage is to further exemplify the effectiveness of the immobilised bacteriophage cocktail at treating V. parahaemolyticus by undertaking additional laboratory testing and by undertaking a tank test containing live shrimp.
Shelf life Testing
The long term shelf life of the immobilised bacteriophage formula is assessed at different storage temperatures using standard methods. This determines the recommended storage conditions and shelf life of a final product.
Infection Model with Live Shrimp
Shrimp are added to separate tanks and subjected to 3 different concentrations of V. parahaemolyticus. This determines the concentration required to elicit EMS pathology. Shrimps are assessed for V. parahaemolyticus infection of the hepatopancreas by observing differences in hepatopancreas size, overall weight vs controls and overall mortality.
Tank Test with Live Shrimp
To assess the efficacy of treatment options, shrimp are fed a specific concentration of feed containing immobilised bacteriophage and a specific concentration of cellulose containing immobilised bacteriophage. Treated shrimps are exposed to an infectious dose of V. parahaemolyticus. Shrimps are assessed for V. parahaemolyticus infection of the hepatopancreas, differences in hepatopancreas size and overall weight vs controls, and overall mortality.
Procedures
Shelf Life Testing
Immobilised material is stored at 4° C., ambient room temperature, and at 30° C. to represent a tropical climate. The shelf life of free bacteriophage solution stored at each temperature is also compared. Each material and solution are added to agar overlays of all V. parahaemolyticus isolates. Antimicrobial activity is confirmed by the presence of a zone of inhibition of bacterial growth around the material. Material is sampled at different time points until antimicrobial activity ceases.
Infection Model with Live Shrimp
A total of 20 L. vannamei shrimp are used for the infection model. A total of 5 shrimp are exposed to different concentrations of V. parahaemolyticus. Each shrimp is kept in an individual tank. The concentrations are 1×104 CFU, 1×102 CFU and 10 CFU representing sub lethal doses. V. parahaemolyticus is introduced using ingestion of shrimp food particles, reverse gavage or direct injection.
Tank Test
A total of 5 replicates containing 10 post larval stage L. vannamei shrimp are exposed to shrimp feed with immobilised bacteriophage and cellulose with immobilised bacteriophage. A total of 5 replicates containing 10 post larval stage L. vannamei shrimp are also exposed to free bacteriophage added to shrimp feed and free bacteriophage added to cellulose. All treatments are dried and incubated for 7 days at room temperature before treatment.
After a treatment dose, the shrimp are then exposed to an infectious dose of V. parahaemolyticus as determined in the infection model. V. parahaemolyticus is delivered by ingestion of shrimp food particles, reverse gavage or through direct injection. Shrimp mortality is recorded daily and upon mortality the hepatopancreas of each shrimp is measured and sampled for bacterial counts and the presence of haemocytic nodules and hyaline necrosis of the tissue. Treated shrimp are compared to control groups consisting of shrimp exposed to V. parahaemolyticus alone and shrimp exposed to each bacteriophage treatment alone. The study is conducted for 30 days or based on the results of the infection model.
Outcomes and Success Criteria
The following are the identified outcomes:
Saltwater shrimp were exposed to Vibrio parahaemolyticus, the causative agent of AHPND (Acute hepatopancreas necrosis disorder). This infection was then treated by giving the shrimp a feed comprising immobilised bacteriophages active against V. parahaemolyticus.
Acquisition and Culture of Microorganisms and Bacteriophages
V. parahaemolyticus strain designation 0004 has displayed mortality in shrimp and is available for immediate work. V. parahaemolyticus 0004 will be routinely cultured using the methods detailed above. Bacteriophage DRGS has been shown to have lytic activity against V. parahaemolyticus 0004 and will be used for the study.
Shrimp Tank Setup
Two 17 litre saltwater tanks were set up with a mature biological filter and water movement provided by a circulation pump. Salt water with a salinity of 34 ppm purified using reverse osmosis and maintained at a temperature of 26° C. was used for the study.
Saltwater Shrimp Survival and Food Uptake
A total of 20 Thor amboinensis saltwater shrimp were acquired and 10 specimens were added to 2 separate tanks. Shrimp feed measuring 1 mm in diameter manufactured by CP foods was used in this study and the uptake of the feed by shrimp was assessed for 3 days.
Immobilisation of Bacteriophage
CP feed material was disinfected by exposure to UV light for 30 min before being twice exposed to corona treatments at 7.5 kV. A total of 10 ml of a 1×108PFU stock of bacteriophage was applied to 20 grams of feed material. Each material was then washed 3 times in sterile distilled water and dried in a laminar flow cabinet. Antimicrobial activity was assessed using an agar overlay and using the culture test to determine antimicrobial activity.
Thor Amboinensis Care and Feeding Schedule
Shrimp were regularly fed twice a day on feed equivalent to 5% of their estimated body weight. One tank was fed untreated CP feed and the other tank was fed feed comprising immobilised bacteriophage. Feeding occurred for 3 days before inoculation of the tanks with V. parahaemolyticus and was maintained during inoculation.
Inoculation and Assessment of Shrimp Health
Each tank was dosed with a culture of V. parahaemolyticus to make a final volume of 1×108 CFU/mL in the tank. Shrimp health was assessed after 6 hours of exposure and each shrimp was given a rating using the criteria described in Table 5. Health was then assessed daily. A sample of tank water was taken daily to provide counts of bacteria in each tank. For the bacterial counts, a sample of tank water was subjected to 8× 1/10 serial dilutions and a sample plated onto TCBS agar. For the bacteriophage counts, a tank water sample was passed through a 0.2 μM filter and subjected to a 8× 1/10 serial dilutions and a 100 μl sample was added to a V. parahaemolyticus 0004 3% NaCl soft nutrient agar overlay.
Results
All material containing immobilised bacteriophage displayed antimicrobial activity and resulted in a 2 log reduction when directly exposed to the bacteria in solution. No shrimp casualties were observed before inoculation with V. parahaemolyticus in both tanks (Table 6;
1 × 106
1 × 104
Conclusions
Immobilised bacteriophage on shrimp feed confers a protective effect on Thor amboinensis shrimp exposed to a large infectious dose of a pathogenic strain of V. parahaemolyticus that is known to cause AHPND in aquacultured shrimp. No significant difference was found in V. parahaemolyticus numbers in the tank water which indicates the protective effect is happening locally at the site of infection. At the conclusion of the trial, 3 shrimp were sacrificed and the presence of bacteriophages in the gut confirmed.
The invention hence provides compositions and methods for treatment of bacterial infections in aquaculture, generally of shrimp, prawns and fish.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15164343 | Apr 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/058809 | 4/20/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/170013 | 10/27/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050220770 | Scott et al. | Oct 2005 | A1 |
| Number | Date | Country |
|---|---|---|
| 2006047872 | May 2006 | WO |
| WO-2006047872 | May 2006 | WO |
| 2013000093 | Jan 2013 | WO |
| WO-2013000093 | Jan 2013 | WO |
| 2014049008 | Apr 2014 | WO |
| Entry |
|---|
| Christiansen et al. Applied and Environmental Microbiology vol. 80, No. 24 Dec. 1, 2014 (Year: 2014). |
| International Search Report and Written Opinion, International Patent Application No. PCT/EP2016/058809, dated Jul. 22, 2016. |
| International Preliminary Report on Patentability, International Patent Application No. PCT/EP2016/058809, dated Jul. 12, 2017. |
| Christiansen Roi Hammershaimb et al: “Detection and quantification of Flavobacterium psychrophilum-specific bacteriophages in vivo in rainbow trout upon oral administration: implications for disease control in aquaculture” Applied and Environmental Microbiology, American Society for Microbiology, vol. 80, No. 24, pp. 7683-7693. |
| Number | Date | Country | |
|---|---|---|---|
| 20180110812 A1 | Apr 2018 | US |
