A method of vaccinating salmonids against p salmonis

Abstract

Provided is a method of protecting a salmonid against P salmonis infection, the method comprising: obtaining a sample of a P salmonis vaccine; determining viability of P salmonis in said sample thereby confirming that said P salmonis vaccine contains modified live P salmonis; and administering the vaccine that contains modified live P salmonis to the salmonid in need of said protection.

Description

ZP000490 A METHOD OF VACCINATING SALMONIDS AGAINST P SALMONIS FIELD OF THE INVENTION

[0001] This invention is generally in the field of salmon vaccines. BACKGROUND

[0002] In 2018, about 88% of the 179 million tons of total fish production was utilized for direct human consumption, a share that has increased significantly in recent decades. The contribution of world aquaculture to world fish production has constantly increased, reaching 46.0% in 2016– 2018, valued at $250 billion. China is by far the main world exporter of fish and fish products, followed by Norway, Vietnam, India and Chile. In Chile, aquaculture production of Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss) has grown strongly, thus consolidating its position as second worldwide salmon producer just behind Norway. Chile has seen sustained export revenue growth on the back of strong global demand for salmonids throughout the Americas, Europe and Asia and an increase in prices, reaching export revenues of USD 6.6 billion.

[0003] Aquatic animal disease is one of the most serious constraints to the expansion and development of sustainable aquaculture under intensive aqua farming conditions.

[0004] In Chile, the National Fisheries Service (SERNAPESCA, Servicio Nacional de Pesca) identified along their coasts the presence of Piscirickettsia salmonis, the etiological agent of Salmonid Rickettsial Septicaemia (SRS). P. salmonis is probably the most serious health problem for the Chilean salmon industry, because of its highly aggressive nature, recurrent outbreaks, and widespread transmission among other cultivated salmonid species. In 2018, mortalities associated with P. salmonis represented 54.7% and 83.3% of the total mortalities attributed to infectious causes in Atlantic salmon and rainbow trout, respectively.

[0005] SRS continues to evolve and new outbreaks continually occur which are increasingly insidious and refractory to treatments. New outbreaks frequently show increased bacterial virulence, clinical and pathological severity and variable presentation under similar conditions of species, age and management measures.

[0006] SRS has proven very difficult to control. The use of antibiotics, both prophylactically and during early infection, may inhibit the growth of the pathogen, but failure of antibiotic treatment is common, and antibiotic treatments have been largely unsuccessful in stopping disease outbreaks.

[0007] Modified live P salmonis vaccines have been commercially available. P salmonis in these vaccines is shipped to salmon farms in lyophilized form and needs to be reconstituted before use. Sometimes, due to external factors, lyophilized P salmonis lose viability. Thus, the reconstituted vaccines from these non-viable P salmonis do not properly protect salmon against P salmonis infection leading to significant losses for the farmers.

[0008] Thus, there is a need for a method of determining viability of attenuated P salmonis that is used as the antigen in modified live P salmonis vaccines. SUMMARY OF INVENTION

[0009] In the first aspect, this disclosure provides a method of protecting a salmonid against P salmonis infection, the method comprising: a. obtaining a sample of a P salmonis vaccine; b. determining viability of P salmonis in said sample thereby confirming that said P salmonis vaccine contains modified live P salmonis; c. administering the vaccine that contains modified live P salmonis to the salmonid in need of said protection.

[0010] In the second aspect, the disclosure provides a method of protecting a salmonid against P salmonis infection, the method comprising: a. obtaining a sample of lyophilized P salmonis stock; b. determining viability of said lyophilized P salmonis in said sample thereby confirming that said lyophilized P salmonis stock comprises live P salmonis c. formulating a vaccine by admixing the lyophilized P salmonis comprising live P salmonis stock, or a portion thereof, with a pharmaceutically acceptable carrier or diluent;d. administering the vaccine that contains immunologically effective amount of modified live P salmonis to the salmonid in need of said protection.

[0011] In certain embodiments of the first or the second aspect disclosed herein the step of determining viability of said P salmonis in said sample comprises contacting the sample with an ATP extracting agent, a divalent cation, luciferase / luciferin and, optionally, a divalent cation chelator; and quantifying luminescence.

[0012] In another set of embodiments of the first or the second aspect disclosed herein the step of determining viability of said P salmonis in said sample comprises contacting the sample with a first dye that differentially stains viable and non-viable P salmonis, and, optionally, a second dye that stains both viable and non-viable P salmonis. In preferred embodiments in this set, the first dye that differentially stains viable and non-viable P salmonis is propidium iodide and wherein the step further comprises staining P salmonis with the second dye that stains both viable and non-viable P salmonis, wherein the second dye is SYTO® 9. In particularly preferred embodiments, quantification of viable P salmonis by fluorescent microscopy, most preferably using a microscope that is field compatible.

[0013] In certain subset embodiments, the analysis of said fluorescent microscopy using a microscope that is field compatible is conducted at about 200 x magnification with about 1 mm view. Within this subset, it is preferred that the analysis comprises creating a plurality of images upon upon exposure of P salmonis sample to the blue light under the microscope and determining the mean number of viable cells the plurality of images. Particularly preferred is the plurality of five or more, more preferably ten or more. In most preferred embodiments, the mean number of viable cells is five or more. DETAILED DESCRIPTION

[0014] In general, this disclosure provides methods of determining viability of P salmonis prior to administering vaccines containing modified live P salmonis to salmonids. In the context of this disclosure, the phrases “vaccine that contains modified live P salmonis” and “contains immunologically effective amount of modified live P salmonis” both refer to the mean number of live cells in at least five (preferably at least ten) images generated immediately upon exposureof P salmonis to the blue light under the microscope, under 200 X magnification in 1 mm field of view, wherein said mean number of live cells is five or more. Vaccines containing modified live P salmonis cells in the amount as described above elicit protective immune response against P salmonis challenge.

[0015] Thus, in one aspect, this disclosure provides a method of protecting a salmonid against P salmonis infection, the method comprising: obtaining a sample of a P salmonis vaccine; determining viability of P salmonis in said sample thereby confirming that said P salmonis vaccine contains modified live P salmonis; administering the vaccine that contains modified live P salmonis to the salmonid in need of said protection.

[0016] In another aspect, this disclosure provides a method of protecting a salmonid against P salmonis infection, the method comprising: obtaining a sample of lyophilized P salmonis stock; determining viability of said lyophilized P salmonis in said sample thereby confirming that said lyophilized P salmonis stock comprises live P salmonis formulating a vaccine by admixing the lyophilized P salmonis comprising live P salmonis stock, or a portion thereof, with a pharmaceutically acceptable carrier or diluent; administering the vaccine that contains immunologically effective amount of modified live P salmonis to the salmonid in need of said protection.

[0017] A non-limiting example of the vaccine suitable for use in the methods described herein is a vaccine containing live attenuated P salmonis strain AL 20542. ALPHAJECT® LiVac SRS is a vaccine containing modified live P salmonis, strain AL 20542.

[0018] The determination of viability may be a binary decision (Viable / Non-Viable) or it can be quantitative (e.g., 100% of P salmonis in the sample are viable, 80% are viable, 50% are viable, 20% are viable, 10% are viable and so on). It is preferred that at least 10% of the P salmonis in the sample used for determining viability are indeed viable. The method of determining viability of P salmonis should be chosen on whether the answer needs to be binary or qualitative. However, since the viability is determined before vaccinating the fish, the test for viability should be reliable, simple to use and preferably not require expert training or expensive equipment.

[0019] In certain embodiments, the viability is determined based on quantitation of the ATP present. This method is based on the requirement for ATP for facilitating beetle luciferase- luciferin reaction resulting in the generation of light ("luminescence").

[0020] The sample that contains or is suspected of containing live P salmonis is contacted with a reaction buffer, at least one ATP extracting agent, a divalent cation, optionally, a divalent cation chelator and a luciferase / luciferin mixture in which the divalent cation concentration is sufficiently low or sufficiently neutralized by cation chelator to reduce the negative effects of divalent cation on ATP extraction.

[0021] Multiple ATP extracting agents alter cell membrane or cell wall permeability or disrupt the integrity of (i.e., lyses or causes the formation of pores in) the membrane and / or cell wall of the microbial source to effect extraction or release of ATP. Generally, ATP extracting agents are known in the art and may include a variety of agents, including, but not limited to antibiotics, such as polymyxin B (e.g., polymyxin Bl and polymyxin B2), polymyxin-beta-nonapeptide (PMBN), and chlorohexidine (CHEX); alkylglucoside or alkylthioglucoside, such as Octyl- beta -D-1- thioglucopyranoside (see U.S. Patent no. 6,174,704 herein incorporated by reference in its entirety); nonionic detergents, such as Triton-XlOO (TX-100); betaine detergents, such as carboxypropylbetaine (CB-18); quarternary ammonium salts, such as trimethyloctadecyl ammonium bromide (TMA-18); protamines; amines, such as triethylamine (TEA) and triethanolamine (TeolA); and cationic, antibacterial, pore forming, membrane-active, and / or cell wall-active polymers, such as polylysine, nisin, magainin, melittin, phopholipase A2, phospholipase A2activating peptide (PLAP); bacteriophage; and the like. See e.g., Morbe et al., Microbiol. Res. (1997) vol.152, pp.385-394.

[0022] In one embodiment of the invention, the ATP extracting agent(s) include CTAB, a quaternary ammonium salt. In preferred embodiments, CTAB is present in the reagent composition at a concentration between about 0.04 percent - 0.15 percent (w / v). In another embodiment, the ATP extracting agents may include CHEX and an ethoxylated alkylphenol, such as Triton X-100. In preferred embodiments, CHEX is preferably between about 0.04 percent - 0.16 percent (w / v) and the ethoxylated alkylphenol is present between about 0.25 percent - 1.0 percent (w / v). In a particularly preferred embodiment, the reagent composition may includemore than one ATP extracting agent. One preferred embodiment includes CHEX, (between about 0.04 percent -0.16 percent (w / v)); an ethoxylated alkylphenol, such as Triton X-100 (between about 0.25 percent -1.0 percent (w / v)); and a quaternary ammonium salt, such as CTAB, (between about 0.02 percent -0.08 percent (w / v)).

[0023] The beetle luciferase-luciferin reaction is dependent not only on ATP, but also on divalent cations. Therefore, to facilitate luciferase activity, divalent cations are typically supplied (unless already present in the sample). Divalent cations include magnesium, calcium and manganese. Divalent cations may be supplied as salts or halides such as sulfate, sulfonate, gluconate, carbonate, chloride and bromide. For example, magnesium cations may be supplied as magnesium chloride, magnesium sulfate, magnesium gluconate, magnesium acetate, magnesium bromide, magnesium carbonate, etc. Preferably, the divalent cation is selected from the chloride or sulfate salts of magnesium. Because the permeability of certain cell membranes or walls may be negatively affected by the presence of divalent cations, divalent cation concentrations may be empirically formulated for a given microorganism or a given extraction / detection system to provide the proper balance between e.g., cell release of ATP and ATP detection.

[0024] Divalent cation chelator agents include, without limitation, salts of ethylenediamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and 1,2- Cyclohexanedinitrilotetraacetic acid (CDTA), nitriloacetic acid (NTA), citric acid, sodium gluconate, gluconic acid, lignosulfonates, and mixtures thereof. Preferably, the chelator agent is selected from the group consisting of EDTA, EGTA and CDTA, due to their general availability and relatively low cost. Suitable levels of divalent cation chelator may be empirically determined on the basis of providing levels sufficient to neutralize the negative effects of divalent cation on ATP extraction, but not to the extent that they prevent the cation- dependent, luciferase-catalyzed ATP detection. In certain embodiments, the divalent cation chelator is EDTA.

[0025] The difference between the divalent cation chelator concentration and the divalent cation concentration in the mixture may be less than about 5 mM. Alternatively, the divalent cation chelator concentration may be at least one-half of the divalent cation concentration in the mixture, preferably equal to or even greater in concentration. Divalent cation chelator may beunnecessary in instances where the divalent cation concentration is low (e.g. less than about about 5 mM, 2.5 mM, or 1 mM).

[0026] Given that divalent cation chelators have the ability to neutralize the negative effects of divalent cations on ATP extraction, divalent cation concentrations may be adjusted depending on the level of divalent cation chelator present in a reagent composition or reaction mixture. When divalent cation chelator is low (e.g. less than about 5 mM, 2.5 mM or 1 mM), divalent cation concentrations will be accordingly lower, preferably less than 2.5 mM, more preferably between 0.2 - 1 mM. When divalent cation chelator is higher (e.g. 2-20 mM), however, divalent cation concentration will be accordingly higher, preferably with a concentration less than or equal to the concentration of the divalent cation chelator.

[0027] At their most basic level, luciferases are defined by their ability to produce luminescence. More specifically, luciferases catalyze the oxidation of a substrate, luciferin, thereby producing oxyluciferin and photons. Luciferases, whose catalytic products include light, offer sensitivity, a detectable product, and facile measurement of ATP. Any ATP-dependent luminescence- producing enzyme is contemplated for use in the reagent compositions and methods of the present invention.

[0028] The naturally-occurring substrate for beetle luciferases is firefly luciferin. Luciferin may be isolated from nature (e.g. from fireflies) or synthesized. Synthetic luciferin can have the same structure as the naturally occurring luciferin or can be a variant or derivitization, so long as it functions analogously (Bowie et al., 1973; Branchini, 2000; Craig et al., 1991; Miska and Geiger, 1987; Yang and Thomason, 1993). Exemplary luciferin derivatives for use in the present invention include, but are not limited to, 6- deoxyaminoluciferin, D-luciferin methyl ester, D-luciferyl-L- phenylalanine, D- luciferyl-L-N a-arginine, D-luciferin-O-sulphate and D-luciferin-0-phosphate (Miska and Geiger, 1987), esters of luciferases that are hydrolyzed or acted upon by esterases to luciferin by components in a sample (Craig et al., 1991; Yang and Thomason, 1993). Other examples of useful luciferin analogs include naphthyl- and quinolylluciferin, which emit light in the green and red-light spectra respectively (Branchini et al., 1989). There are multiple commercial sources for luciferin (e.g., Promega Corp. Madison, WI; Molecular Probes, Eugene, OR).

[0055] The beetle luciferase-catalyzed reaction producing a luminescent signal from theluciferase-luciferin reaction requires luciferase enzyme, luciferin, adenosine triphosphate (ATP), magnesium (or other divalent cation), and molecular oxygen, hi the initial reaction, luciferin and ATP react to form luciferyl adenylate with the elimination of inorganic pyrophosphate. The luciferyl adenylate remains tightly bound to the catalytic site of luciferase. When this form of the enzyme is exposed to molecular oxygen, the enzyme-bound luciferyl adenylate is oxidized to yield oxyluciferin in an electronically excited state.

[0029] A beetle luciferase-luciferin reaction results in the generation of light ("luminescence"). Because the beetle luciferase-luciferin reaction is ATP- dependent, luciferase can be used to assay for ATP. The reaction is remarkably sensitive, allowing ATP to be detected in a sample containing as little as 10-16moles ATP or less. See US Patent 7422868. Kits enabling such methods have been commercially available and include, without limitations BACTITER-GLO™ Microbial Cell Viability Assay, manufactured by Promega Corporation (Madison, WI, USA).

[0030] In another embodiments, the method for determination of viability is based on a discovery that one can select dye combination that label alive and dead cells differently. Accordingly, the method of determining viability of P salmonis in the sample comprises contacting said P salmonis with a dye that stains alive and dead P salmonis differentially.

[0031] In certain embodiments, the method comprises contacting P salmonis with a combination of two dyes: one stains both alive and dead P salmonis, and the other that stains dead P salmonis only. It is important that the to dyes differ in spectral characteristics.

[0032] One such combination is SYTO®9 and propidium iodide. SYTO® 9 is a green-fluorescent nucleic acid stain and propidium iodide a red-fluorescent nucleic acid stain. These stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, the SYTO®9 stain generally labels all bacteria in a population — those with intact membranes and those with damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes, causing a reduction in the SYTO®9 stain fluorescence when both dyes are present.

[0033] Accordingly, in particularly preferred embodiments, the method of determining viability of P salmonis in the sample comprises contacting with a green dye that stains both live and dead cells and with a red dye that stains dead cells only. In a particular embodiment, the green stainis SYTO®9 and the red dye is propidium iodide. Thus, with an appropriate mixture of the SYTO®9 and propidium iodide stains, bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red. The excitation / emission maxima for these dyes are about 480 / 500 nm for SYTO®9 stain and 490 / 635 nm for propidium iodide.

[0034] Kits utilizing SYTO® 9 and propidium iodide are commercially available and include LIVE / DEAD® BACLIGHTTMBacterial Viability Kits (Molecular Probes, Eugene, OR, USA).

[0035] In certain embodiments the results of the staining are analyzed by fluorescent microscopy. It is preferred that the fluorescent microscope used for the analysis is field- compatible. For the purposes of this disclosure, “field-compatible” fluorescent microscope is portable (e.g., weighs no more than 2 kg, preferably, less than 1 kg, even more preferably, less than 500 g) and at the same time provides about 100x to about 200x resolution and can be connected to data displaying device such as a laptop, a tablet, or a cell phone. The field of view is between about 1 mm and about 2 mm.

[0036] In a particularly preferred embodiment, the analysis of the fluorescent microscopy is conducted at about 200 X magnification with about 1 mm field of view.

[0037] If the sample of P salmonis does not contain live P salmonis, the P salmonis stock should be discarded. Conversely, if the sample of P salmonis contains viable P salmonis, then the user can administer the vaccine containing modified P salmonis to salmonids without further steps.

[0038] Alternatively, the user can formulate P salmonis vaccine based on the results of the assay and adjust either the concentration of the modified live P salmonis or the volume of the vaccine so that the salmonids that are vaccinated receive the proper effective dose of the modified live P salmonis. For example, if the microscope fluorescence assay shows that there are only three or four live P salmonis organisms in 1 mm field under 200X magnification, the volume or the concentration may be doubled to raise the number of live P salmonis to five or above five.

[0039] In certain embodiments, the vaccines may be administered by injection, including without limitations, intramuscular and intraperitoneal injections. Generally, the vaccines administered in the volumes of between 10 and 250 microliters. Based on the amount of the viable P salmonis in the sample, the volume of one vaccine dose may be adjusted within these limits including,without limitations, 20 microliters, 50 microliters, 75 microliters, 100 microliters, 150 microliters or 200 microliters.

[0040] Multiple methods exist to administer the vaccines to salmonids. In certain embodiments, commercially available fish vaccination equipment may be fitted with a multi-needle injection tip, where the tips of the needles are configured to be at a desired distance from each other. Suitable vaccination machines include NFTT lines of products (Pharmaq) and the specific models include NFT 20, NFT 25 and NFT 30. NFT 20, NFT 25 deliver vaccines intraperitoneally but can be reconfigured for intramuscular injections also. NFT 30 NFT 30 has a special DNA module that is capable of delivering a DNA vaccine intramuscularly, into the filet of the fish.

[0041] The machines handle fish in the sizes from 120 mm to 250 mm (20-150 grams). Once finished, it sorts the vaccinated fish into three different sizes. In addition, it has a channel for misplaced, undersized or rejected fish.

[0042] Alternatively, the vaccine may be administered manually via a syringe. See, e.g., MICRO- MATIC® syringe sold by Pharmaq. The single syringe is available in two sizes: 0.05 ml per dose and 0.1 ml per dose. Additionally, the 0.05 ml syringe can be supplied with an interchangeable 0.025 ml piston if required. These syringes can be used for both water-based and oil-based vaccine formulations. The dose size can easily be adjusted + / - 10 %. The dose accuracy is documented to deviate less than 3 %.

[0043] In addition to the modified live P salmonis, the vaccines may further contain additional antigens, including without limitations modified live or killed antigens, including, without limitations Viral Hemorrhagic Septicemia Virus (VHSV), Infectious Hematopoietic Necrosis virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anaemia virus (ISAV), Salmon pancreatic disease virus (SPDV), Iridovirus, Nodavirus, Piscine myocarditis virus (PMCV), Piscine Orthoreovirus (PRV) which causes Heart and Skeletal Muscle Inflammation (the virus is sometimes referred to as HSMIV or HSMI virus), Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio (Aliivibrio) salmonicida, Yersinia ruckeri O1 and Moritella viscosa. In other embodiments, the vaccine may contain plasmids encoding antigens against such pathogens as PMCV (Piscine Myocarditis Virus), PD (pancreatic disease) virus, and piscine orthoreovirus (causative agent of Heart and Skeletal Muscle Inflammation syndrome).

[0044] The vaccines disclosed herein and containing modified live P salmonis and optionally one or more additional antigens may further comprise a pharmaceutically acceptable carrier or diluent.

[0045] Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include water, a preservative, culture medium, stabilizers such as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein containing agents such as bovine serum or skimmed milk, and buffers (e.g. phosphate buffer).

[0046] The vaccine may or may not comprise an adjuvant. Adjuvants are non-specific stimulators of the immune system. They enhance the immune response of the host to the vaccine. Non- limiting examples of adjuvants known in the art are Alum, vitamin E, non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulating complexes), Saponins, and Carbopol.

[0047] The invention will be further described in the following non-limiting examples. EXAMPLES Example 1

[0048] The purposes of this example were: first, to provide the proof-of principle, namely, that viability of P salmonis in vaccine samples can be quantified, and second, to identify the assays that are suitable for this purpose.

[0049] Seven assays were used in this example: Assay 1 : BACTITER-GLO™ Microbial Cell Viability Assay – Cat no: G8231 Assay 2 : Live / Dead BACLIGHTTMBacterial Viability Kit, – Ref L7012 - Lot 2480125 Assay 3 : Cell Counting Kit – 8 , Sigma Aldrich – Product no: 96992 , Pcode: 102522147 Assay 4 : Resazurin Assay Kit (Cell Viability) - Abcam (ab129732) Assay 5 : Bacterial Counting Assay Kit (Colorimetric) - Abcam (ab284554) Assay 6 : Bacterial viability assay kit - Abcam (ab189818) Assay 7 : AQUASNAP® Total and AQUASNAP® Free – Hygenia

[0050] Prepared water bath at 22,5°C, and collected 1 vial of ALPHA JECT LIVAC®SRS vaccine containing modified live P salmonis from -80°C freezer to box with dry ice. The vaccine wasthawed on water bath for 3 min, and quickly reconstituted the vaccine with NaCl by connecting the vial to the ECOFLAC® adapter and then to the NaCl bottle.

[0051] The bottle was squeezed a couple of times to flush out the vaccine from the vial into the bottle. The bottle was inverted three times to ensure proper mixing. The same protocol was followed for all experiments. Dead sample prep

[0052] Using the vaccine gun, a 1,5 ml Eppendorf tube was filled with vaccine. The tube was placed on a heat block set at 70°C for 30 min. Assay protocols

[0053] All experiments were done using the protocol from the manufacturer of the assays, unless otherwise described. The different assays recommend different number of bacteria to be used in the analysis, however in these experiments 100 µl (1 vaccine dose) was used as input, unless otherwise described. All incubations were done in room temperature.

[0054] For more precise measurements, 10 doses of vaccine were ejected from the vaccine gun to an Eppendorf tube, and from there a pipette was used to transfer 100 µl to the 96-well plate.

[0055] For Live / Dead mix the following set-up was used: 100% Viable = 100 µl Live Vaccine 75% Viable = 75 µl Live Vaccine + 25 µl Dead sample 50% Viable = 50 µl Live Vaccine + 50 µl Dead sample 25% Viable = 25 µl Live Vaccine + 75 µl Dead sample 100% Dead = 100 µl Dead sample

[0056] Luminescence values for Live / Dead BACLIGHTTMBacterial Viability Kit (plate reader) are provided in Tables 1 (replicates) and 2 (mean). Table 1. Luminescence, replicates, BACTITER-GLO™ Microbial Cell Viability Assay 75% 75% viable, 50% 25% viable, 100% 100% 100% viable 100% 24h viable 24h 50% 24h 24h dead dead, 24h 126000 60990 135200 59540 105600 56380 40050 21980 13890 156400 64760 133300 63620 106800 54510 40420 23000 14160 175100 69740 132800 62680 103500 55350 39840 22770 14380 177000 67800 176700 63050 101700 55740 39920 23510 13910 120900 69220 128000 104000 5718 40330 23610 14920Table 2. Mean luminescence values, BACTITER-GLO™ Microbial Cell Viability Assay 100% 100% 75% viable 25% viable, 100% 100% dead viable viable, 24h 75% viable 24h 50% 50% 24h 24h dead 24h 151080 66502 141200 62222 104320 45539 40112 22974 14252

[0057] For Cell Counting Kit – 8, OD values are provided in tables 3 (replicates) and 4 (mean). Table 3. Cell Counting Kit – 8 replicates of OD values. 100% viable 75% viable 50% viable 25% viable 100% Dead 0.000667 0.008667 0.003667 0.005667 0.004667 -0.00133 0.004667 0.003667 0.002667 0.004667 0.000667 0.003667 0.004667 0.005667 0.002667 0.042 0.048 0.047 0.047 0.047 0.044 0.049 0.047 0.049 0.049 Table 4. Cell Count 8, mean OD values 100% viable 75% viable 50% viable 25% viable 100% Dead 0.017201 0.0228 0.0212 0.022 0.0216

[0058] For Resazurin Assay Kit, replicate OD values are provided in table 5 and mean OD values are provided in table 6. Table 5. Resazurin Assay Kit, Replicate OD values 100% viable 75% viable 50% viable 25% viable 100% Dead Blank 0.699 0.0708 0.0704 0.708 0.708 0.678 0.068 0.068 0.0699 0.698 0.698 0.689 0.0699 0.684 0.701 0.698 0.071 0.0696 Table 6. Resazurin Assay Kit, Mean OD values 100% viable 75% viable 50% viable 25% viable 100% Dead Blank 0.279 0.274 0.280 0.701 0.492 0.479

[0059] For Bacterial Counting Assay Kit (Colorimetric), Abcam (ab284554), OD values are provided in tables 7 (replicates) and 8 (mean). Table 7. Bacterial Counting Assay Kit (Colorimetric), Abcam (ab284554), the OD values (replicates) 100% viable 75% viable 50% viable 25% viable 100% Dead 0.005167 0.002967 0.002867 0.002567 -0.00173 0.004667 0.002267 0.003267 0.003067 0.001067 0.006167 0.002767 0.000967 0.002367 Table 8. Bacterial Counting Assay Kit (Colorimetric), Abcam (ab284554), the OD values (mean) 100% viable 75% viable 50% viable 25% viable 100% Dead 0.0053 0.0027 0.0024 0.0027 -0.0003

[0060] For Bacterial viability assay kit - Abcam (ab189818), fluorescence values are provided in tables 9 (replicates) and 10 (mean). Table 9. Bacterial viability assay kit - Abcam (ab189818), fluorescence values, replicates 100% viable 75% viable 50% viable 25% viable 100% Dead 2132 1902 1650 1529 1605 4164 1677 1661 1434 1461 4612 2059 1881 2524 1871 Table 10. Bacterial viability assay kit - Abcam (ab189818), fluorescence values, mean 100% viable 75% viable 50% viable 25% viable 100% Dead 3636 1879 1731 1829 1646

[0061] For AQUASNAP® assays, viability after four measurements is provided in table 11. AQUASNAP® assay consists of using two assay probes, called AQUASNAP® Total and AQUASNAP® Free. AQUASNAP®®Total measures both microbial ATP (living cells and particulate matter) and Free ATP (non-microbial or dead cells) in solution. AQUASNAP® Free measures non-microbial ATP (aka dead cells) in a solution.

[0062] AQUASNAP®®Total was used first to record the Total ATP load. Then, AQUASNAP®®Free was used on the same sample to measure the Free ATP (from dead cells). The difference between Total and Free provides an indication of the quantity of live of cells in the sample. The units in the table are in percentages based on the following formula: 100 – ((AQUASNAP® Free / AQUASNAP® Total) * 100). Table 11. AQUASNAP® viability results, four measurements Measurement1 Measurement2 Measurement3 Measurement4 30.7 11.5 -11.5 2.1

[0063] For microscopy using the Live / Dead BACLIGHTTMBacterial Viability Kit, one dose (100 µl) was put into an Eppendorf tube and mixed with 1 µl of each stain (SYTO®9 and Propidium Iodine (PI)). Sample was incubated 15 min in the dark, and a microscopy slide was then prepared using 20 µl of stained sample. The viability was determined using ten replicates taken at the same time from the same vaccine bag. The samples were stained with the BACLIGHTTMKit, and images taken on the Leica Microscope under 1000x magnification and 0.180 mm field of view.

[0064] Ten images were taken of each of the 10 samples. All images were then analyzed manually by first counting the total number of cells in all 10 images, then the number of dead cells were counted for each image. The units in the table are in percentages, as determined by the following formula: 100 – ((dead cells / total number of cells) *100).

[0065] The results are provided in table 12. Table 12. Live / Dead BACLIGHTTMBacterial Viability Kit, cell viability, replicates rep1 rep2 rep3 rep4 rep5 rep6 rep7 rep8 rep9 rep10 14.4 13.4 12.5 12.9 13.5 11.9 12.1 10.4 15.5 10.1

[0066] Conclusion: Out of the seven assays tested, five were not able to adequately differentiate between live and dead P salmonis in the vaccine samples. The only two tests that were considered to be useful were BACTITER-GLO™ Microbial Cell Viability Assay and Live / Dead BACLIGHTTMBacterial Viability Kit, analysed by fluorescence microscopy.

[0067] The BACLIGHTTMBacterial Viability Kit contains two dyes that stain nucleic acid. SYTO®9 can enter through the cell membrane and stain both live and dead cells, while PI can only enter damaged cell membrane and thus only stain dead cells.

[0068] SYTO®9 is excited by blue light (485 nm), and for the P. salmonis bacteria in the LIVAC® vaccine the experiments show that exposure to the blue light under the microscope kills the bacteria after 10-20 seconds. It was discovered that about half of the cells that are alive at the moment of exposure become dead within 15 seconds. This means the images (or ideally video) needs to be taken quickly, before the bacteria die and the colour shifts from green to red.

[0069] In the samples of LIVAC® only around 10% of P salmonis have a bright green colour as expected, while the rest of the bacteria have a very weak green staining. The weakly stained green cells do not turn red when exposing them to the blue light, suggesting that they are not metabolically active, and they could still have some membrane integrity preventing them from turning red. The result of this is that when observing the samples in the microscope it can be challenging to see the weak stained bacteria, and the same issue observed when looking at images taken from the sample. The images then need to be processed by imaging software, to enhance the contrast / brightness to correctly observe the weak stained bacteria. Example 2

[0070] Fluorescence microscopy analysis was selected for further development. For readings in the field, iOlight Fluorescent Microscope was selected. It is field-compatible and can be connected to a screen such as a wireless phone, a tablet, a laptop or a desktop. Any of these devices have image-analysing software. However, this microscope has a magnification of only 200x, and it was not clear whether images of sufficient quality can be generated. Accordingly, the purpose of the exemplified experiments was to determine whether 200x magnification is sufficient to reliably determine whether viable P salmonis are present in the sample

[0071] In these experiments, the number of viable cells in the images generated by the field- compatible microscope was compared to the number of viable cells generated by Leica DMRB Fluorescence microscope which is a much more powerful microscope but is not field compatible.

[0072] The results are provided in Table 13.Table 13. Comparison of viability determined by field-compatible and not field-compatible microscopes Not field-compatible micriscope, 1000 X magnification Experiment Field Compatible microscope, iOlight (# of green Leica DMRB Fluorescence microscope. The viability No cells), 200X magnification 1 mm field of view was calculated as described in Example 1.1 11.02 13.03 4.54 20.05 12.76 30.5 23.9 7 23.9 12.4 8 29.3 18.0 9 27.0 16.1 10 24.0 14.0 11 21.9 9.6 mean 26.1 14.1

[0073] As the table shows, the number of green cells in the images generated at 200X magnification by the field-compatible microscope positively correlates with the viability determined at 1000X concentrations.

[0074] In the next experiment, it was determined whether this positive correlation persists if the thawing conditions were less than optimal.

[0075] Optimal conditions: All experiments were done using normal thawing protocol, and samples were analysed right after reconstitution. The normal thawing protocol is as follows: Vial is taken from liquid nitrogen; Placed in water bath at 15-25 degrees Celsius; Thawed until a small piece of ice is still visible, max 3min; Vial is then immediately connected to 1L saline (0.9% NaCl) flask using an adapter, and the contents of the vial is injected by squeezing the flask repeatedly until all the content of the vial is transferred to the flask.

[0076] The vaccine is now ready and can be kept at room temperature, expiration time is set to 8 hours.

[0077] Sub-optimal conditions varied as follows: Experiment 1 (-80°C to 4°C to -80°C): vial taken from -80 °C freezer to 4 °C, until thawed, moved back to -80 °C, then thawed using normal protocol Experiment 2 (RT thawed - 20h later): vial thawed at room temperature, reconstituted in saline, left in room temperature for 20h Experiment 3 (24h at 28°C): vial thawed at using normal protocol, reconstituted in saline, left at 28°C for 24h Experiment 3 (24h at RT): vial thawed at using normal protocol, reconstituted in saline, left at room temperature for 24h Experiment 3 (24h at 4°C): vial thawed at using normal protocol, reconstituted in saline, left at 4°C for 24h

[0078] The results are summarized in table 14. Table 14: effect of thawing conditions on cell viability measured by field-compatible and not field-compatible microscopes. iOlight (# of green cells) Leica (viability) Experiment 1 (-80°C to 4°C to -80°C) 4.9 4.4 Experiment 2 RT thawed - 20h later 5.9 5.4 laExperiment 324h at 28°C 15.4 6.4 mitpExperiment 324h at RT 17.1 9.5 o-buExperiment 324h at 4°C 21.8 11.5 S Experiment 5 23.9 12.4 Experiment 6, sample 1 21.9 9.6 Experiment 6, sample 2 24.0 14.0 Experiment 3 sample 1 27.0 16.1 laExperiment 3, sample 2 29.3 18.0 mitpExperiment 4 30.5 23.9 O

[0079] Based on these results, it was concluded that five cells per 1 mm imaging field appear to be a proper cut-off for cell viability. To eliminate image-to-image variability, several (preferably at least five, more preferably at least ten) images should be analysed. If the mean number ofviable cells is five or above, the vaccine contains live P salmonis and is suitable for administration to salmon and is expected to provide immunity against P salmonis if administered according to the manufacturing instructions (e.g., if the ambient water temperature is 10°C or above, or in other embodiments, 12 °C or above.

[0080] Conversely, if the mean number is below five, then the practitioner should either discard the vaccine or repeat the staining, followed by analysing several images. If in the second staining and second analysis the mean number of viable cells is five or above, the vaccine is efficient. If in the second staining and second analysis the mean number of green cells is below 5, then the thawed batch of the vaccine needs to be discarded. Example 3

[0081] This confirmatory example demonstrates applicability of the technology described herein to field conditions. Pharmaq representatives travelled to multiple salmon farms in Chile at the time of salmon vaccination against P salmonis with modified live P salmonis vaccine (LIVAC® SRS) and tested the viability of P salmonis at the respective farm sites. Samples of vaccine preparation were taken and handled as provided in Table 15. Table 15 Vaccination Median Mean Standard Site Service Count Sample handling notes Count Dev Farm 1 Service 1 18 Sample 1 (conditions not recorded) 23 20.3 Farm 1 Service 1 59 Sample 2 (conditions not recorded) 59 21.8 Farm 1 Service 1 24 Sample 3 (conditions not recorded) 24 15.8 Farm 2 Service 1 59 sample 1, New reconstitution 56 25.0 Farm 2 Service 1 41 sample 2 (final reconstitution**) 41 14.9 Farm 3 Service 2 22 sample 1, 3 hours after reconstitution 22 6.4 Farm 3 Service 2 33 sample 2, 3 hours after reconstitution 35 13.4 Farm 3 Service 2 30 sample 3, 1hr after reconstruction 33 11.8 Farm 4 Service 3 98 Sample 1 (conditions not recorded) 119 68.7 Farm 4 Service 3 123 Sample 2 (conditions not recorded) 117 30.9 Farm 4 Service 3 59 Sample 3 (conditions not recorded) 63 22.2 Farm 5 Service 4 10* LIVAC® SRS, 1 week defrosted 10 5.7 LIVAC® SRS, 1 week defrosted and Farm 5 Service 4 10* (bacteria inactivated 70°C) 10 3.7 Farm 5 Service 4 6* LIVAC® SRS, 1 year defrosted 6 3.7LIVAC® SRS, 1 year defrosted (bacteria Farm 5 Service 4 4* inactivated 70°C) 6 4.2 Farm 6 Service 3 18 sample 1 (final reconstitution) 18 7.9 Farm 6 Service 3 20 sample 2 (new reconstitution) 21 6.2 Farm 6 Service 3 45 sample 3 (final reconstitution) 45 10 Farm 5 Service 4 33 Live vaccine stained with Syto9*** 36 15.9 Farm 5 Service 4 15 Live vaccine stained with Syto9+PI 15 5.8 Vaccine inactivated at 70°C, stained with Farm 5 Service 4 37 Syto9 43 23.2 Vaccine inactivated at 70°C, stained with Farm 5 Service 4 0 Syto9+PI*** 1 1 Farm 6 Service 3 21 sample 1 (1hr post reconstitution) 22 8.6 Farm 6 Service 3 43 sample 2 (1hr post reconstitution) 43 8.2 Farm 6 Service 3 19 sample 3 (1hr post reconstitution) 18 8.3 Farm 5 Service 4 23 Sample 1 (1hr post reconstitution) 23 7.3 Farm 5 Service 4 12 Sample 2 (1hr post reconstitution) 12 4.9 Farm 5 Service 4 0 Sample 3 (inactivated at 70°C) 1 1.3 Farm 7 Service 5 24 Sample 1, 4 hrs. post-reconstitution 25 8.2 Farm 7 Service 5 5 Sample 2, 3 hrs. post-reconstitution 4 2.3 Sample 1, 2 hours post-reconstitution (Final Farm 2 Service 1 21 doses from the bottle) 19 6.9 Sample 2.1 / 2 hour post-reconstitution (New Farm 2 Service 1 11 bottle) 13 5.7 Farm 8 Service 2 6 probe sample, 18 hr post reconstitution 8 5.3 Farm 8 Service 2 19 sample 1, 5 hr post reconstitution 26 15.8 Farm 8 Service 2 30 sample 2, 5 hr post reconstitution 31 10.8 Farm 8 Service 2 12 sample 3, 5hr post reconstitution 11 3.0 Farm 3 Service 2 24 sample 1, New reconstitution 25 6.4 Farm 3 Service 2 48 sample 2, New reconstitution 51 8.9 Farm 3 Service 2 12 sample 3, 3 hr post reconstitution 12 4.7 Farm 3 Service 2 16 sample 4, 3hr post reconstitution 16 7.6 Farm 6 Service 3 19 sample 1, 1,5 hr post reconstitution 18 9.9 Farm 6 Service 3 6 sample 2, 2,5 hr post reconstitution 7 2.1 Farm 6 Service 3 12 sample 3, New reconstitution 12 4.5 Farm 6 Service 3 21 sample 4, New reconstitution 27 11.9 *-Sample diluted 5x instead of 200 x. the counts need to be divided by 40. ** - Reconstitution results in a 10,000 dose batch. “New reconstitution” refers to the vaccine analysed before any of the doses in the batch are administered to the fish. “Final reconstitution” refers to the vaccine remaining in the batch after substantially all doses have been administered.In general, substantially all doses are administered to fish in within 2-5 hours (more likely 2-3 hours) after the vaccine is reconstituted. *** - a control sample to check that the reagents are working correctly. The two stains in the kit are Syto9 and PI, were Syto9 is a green stain that will color both alive and dead cells, and PI is a red stain that will color dead cells red. If Syto9 and PI are used together, the PI will “push” the green out of dead cells and color them red.

[0082] These data demonstrate the samples which have been inactivated or stayed defrosted (i.e., the samples that presumably contain no live P salmonis) contained low counts of the bacteria, whereas the samples that have been analysed less than 24 hours after reconstitution contain viable P salmonis. Therefore, the methods of testing for viability of P salmonis in modified live P salmonis vaccines can be performed under field conditions.

[0083] All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0084] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

CLAIMS 1. A method of protecting a salmonid against P salmonis infection, the method comprising: a. obtaining a sample of a P salmonis vaccine; b. determining viability of P salmonis in said sample thereby confirming that said P salmonis vaccine contains modified live P salmonis; c. administering the vaccine that contains modified live P salmonis to the salmonid in need of said protection.

2. A method of protecting a salmonid against P salmonis infection, the method comprising: a. obtaining a sample of lyophilized P salmonis stock; b. determining viability of said lyophilized P salmonis in said sample thereby confirming that said lyophilized P salmonis stock comprises live P salmonis c. formulating a vaccine by admixing the lyophilized P salmonis comprising live P salmonis stock, or a portion thereof, with a pharmaceutically acceptable carrier or diluent; d. administering the vaccine that contains immunologically effective amount of modified live P salmonis to the salmonid in need of said protection.

3. The method according to claim 1 or claim 2, wherein the step of determining viability of said P salmonis in said sample comprises contacting the sample with an ATP extracting agent, a divalent cation, luciferase / luciferin and, optionally, a divalent cation chelator; and quantifying luminescence.

4. The method according to claim 1 or claim 2, wherein the step of determining viability of said P salmonis in said sample comprises contacting the sample with a first dye that differentially stains viable and non-viable P salmonis, and, optionally, a second dye that stains both viable and non-viable P salmonis.

5. The method of claim 4, wherein the first dye that differentially stains viable and non-viable P salmonis is propidium iodide and wherein the step further comprises staining P salmonis withthe second dye that stains both viable and non-viable P salmonis, wherein the second dye is SYTO® 9.

6. The method of claim 5, further comprising quantification of viable P salmonis by fluorescent microscopy.

7. The method of claim 6, wherein said fluorescent microscopy is performed by a microscope that is field compatible.

8. The method of claim 6, wherein analysis of said fluorescent microscopy is conducted at about 200 x magnification with about 1 mm view.

9. The method of claim 8, wherein the analysis comprises creating a plurality of images upon upon exposure of P salmonis sample to the blue light under the microscope and determining the mean number of viable cells the plurality of images.

10. The method of claim 9, wherein the plurality is five or more.

11. The method of claim 9, wherein the plurality is ten or more.

12. The method of claim 10 or 11, wherein the mean number is five or more.

13. The method according to any one of claims 1-12, wherein said P salmonis is P salmonis strain AL 20542.