Method for vaccinating salmonidae fish to resist salmon rickettsia

Abstract

There is provided a method of protecting salmonidae fish from infection with salmon rickettsia, the method comprising: obtaining a sample of a salmon rickettsia vaccine; determining the activity of the salmon rickettsia in the sample so as to confirm that the salmon rickettsia vaccine contains the improved live salmon rickettsia; and administering the vaccine containing improved live salmon rickettsia to the salmonidae fish in need of the protection.

Description

TECHNICAL FIELD

[0001] The present invention relates generally to the field of salmon vaccines. BACKGROUND

[0002] In 2018, the total fish production reached 179 million tons, of which approximately 88% was used for direct human consumption, a proportion that has increased significantly in recent decades. World aquaculture’s contribution to global fish production has been increasing, reaching 46.0% in 2016-2018, valued at $250 billion. To date, China is the main world exporter of fish and fish products, followed by Norway, Vietnam, India, and Chile. In Chile, the production of farmed Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), and rainbow trout (Oncorhynchus mykiss) has grown dramatically, thus consolidating its position as the second largest salmon producer in the world, after Norway. Due to strong global demand for salmonids in the Americas, Europe, and Asia, and rising prices, Chile’s export earnings have continued to grow, reaching $6.6 billion.

[0003] Disease in aquatic animals is one of the most serious factors that condition the expansion and development of sustainable aquaculture under intensive aquaculture conditions.

[0004] In Chile, the National Fisheries and Aquaculture Service (SERNAPESCA, Servicio Nacional de Pesca) has identified the presence of Piscirickettsia salmonis (i.e., the causative agent of salmonid rickettsial septicaemia (SRS)) along its coast. P. salmonis is probably the most serious health problem faced by the Chilean salmon industry due to its high invasiveness, recurrent outbreaks, and wide dissemination in other farmed salmonid species. In 2018, mortality associated with P. salmonis accounted for 54.7% and 83.3% of the total mortality due to infectious causes in Atlantic salmon and rainbow trout, respectively.

[0005] SRS continues to evolve, new outbreaks occur, and these outbreaks are increasingly insidious and difficult to treat. New outbreaks tend to show increased bacterial virulence, increased clinical and pathological severity, and variable clinical presentation under similar species, age, and management conditions.

[0006] SRS has proven to be very difficult to control. The use of antibiotics, either prophylactically or in the early stages of infection, can inhibit the growth of the pathogen, but antibiotic treatment failure is common, and antibiotic treatment has been essentially unsuccessful in stopping disease outbreaks.

[0007] Improved live salmon rickettsia is commercially available. The salmon rickettsia in these vaccines is shipped to salmon farms in lyophilized form and needs to be reconstituted before use. Sometimes, due to external factors, the lyophilized salmon rickettsia loses viability. Therefore, the reconstituted vaccine from these non-viable salmon rickettsia does not adequately protect salmon from salmon rickettsia infection, causing huge losses to the farmers.

[0008] Therefore, there is a need for a method of determining the viability of attenuated salmon rickettsia for use as an antigen in an improved live salmon rickettsia vaccine. SUMMARY

[0009] In a first aspect, the disclosure provides a method of protecting salmonids from salmon rickettsia infection, the method comprising:

[0010] a. obtaining a sample of a salmon rickettsia vaccine;

[0011] b. determining the viability of salmon rickettsia in the sample, thereby confirming that the salmon rickettsia vaccine contains improved live salmon rickettsia;

[0012] c. administering the vaccine containing improved live salmon rickettsia to a salmonid in need of the protection.

[0013] In a second aspect, the disclosure provides a method of protecting salmonids from salmon rickettsia infection, the method comprising:

[0014] a. obtaining a sample of a lyophilized salmon rickettsia stock;

[0015] b. determining the viability of the lyophilized salmon rickettsia in the sample, thereby confirming that the lyophilized salmon rickettsia stock comprises live salmon rickettsia

[0016] c. formulating a vaccine by mixing the lyophilized salmon rickettsia comprising live salmon rickettsia stock or a portion thereof with a pharmaceutically acceptable carrier or diluent;

[0017] d. administering the vaccine containing an immunologically effective amount of improved live salmon rickettsia to a salmonid in need of the protection.

[0018] In certain embodiments of the first or second aspect disclosed herein, the step of determining the viability of the salmon rickettsia in the sample comprises contacting the sample with an ATP extractant, a divalent cation, a luciferase / luciferin, and optionally a divalent cation chelator; and quantifying luminescence.

[0019] In another set of embodiments of the first or second aspect disclosed herein, the step of determining the viability of the salmonid rickettsia in the sample comprises contacting the sample with a first dye that differentially stains viable and non-viable salmonid rickettsia and, optionally, a second dye that stains both viable and non-viable salmonid rickettsia. In preferred embodiments within this set, the first dye that differentially stains viable and non-viable salmonid rickettsia is propidium iodide, and wherein the step further comprises staining the salmonid rickettsia with a second dye that stains both viable and non-viable salmonid rickettsia, wherein the second dye is SYTO® 9. In particularly preferred embodiments, the viable salmonid rickettsia are quantified by fluorescence microscopy, most preferably using a live-compatible microscope.

[0020] In certain sub-set embodiments, the analysis of the fluorescence microscopy using a live-compatible microscope is performed at about 200x magnification with about 1 mm field of view. Within this sub-set, it is preferred that the analysis comprises generating a plurality of images upon exposure of the salmonid rickettsia sample to blue light under the microscope and determining the average number of live cells in the plurality of images. It is particularly preferred that the plurality is five or more, more preferably ten or more. In most preferred embodiments, the average number of live cells is five or more. DETAILED DESCRIPTION

[0021] In general, the present disclosure provides a method of determining the viability of salmonid rickettsia prior to administering a vaccine containing modified live salmonid rickettsia to salmonids. In the context of the present disclosure, the phrases "vaccine containing modified live salmonid rickettsia" and "vaccine containing an immunologically effective amount of modified live salmonid rickettsia" both refer to an average number of live cells in at least five (preferably at least ten) images generated immediately upon exposure of the salmonid rickettsia to blue light under a microscope at 200x magnification in a 1 mm field of view, wherein the average number of live cells is five or more. A vaccine containing modified live salmonid rickettsia cells in the amounts described above elicits a protective immune response against salmonid rickettsia challenge.

[0022] Thus, in one aspect, the present disclosure provides a method of protecting a salmonid from salmonid rickettsia infection, the method comprising: obtaining a sample of a salmonid rickettsia vaccine; determining the viability of the salmonid rickettsia in the sample, thereby confirming that the salmonid rickettsia vaccine contains modified live salmonid rickettsia; administering the vaccine containing modified live salmonid rickettsia to a salmonid in need of the protection.

[0023] In another aspect, the disclosure provides a method of protecting a salmonid fish from infection by a Salmonid Rickettsia, the method comprising: obtaining a sample of a lyophilized Salmonid Rickettsia stock; determining the viability of the lyophilized Salmonid Rickettsia in the sample, thereby confirming that the lyophilized Salmonid Rickettsia stock comprises viable Salmonid Rickettsia, formulating a vaccine by mixing the lyophilized Salmonid Rickettsia comprising the viable Salmonid Rickettsia stock or a portion thereof with a pharmaceutically acceptable carrier or diluent; administering the vaccine containing an immunologically effective amount of the modified viable Salmonid Rickettsia to a salmonid fish in need of the protection.

[0024] A non-limiting example of a vaccine suitable for use in the methods described herein is a vaccine containing viable modified Salmonid Rickettsia strain AL 20542. ALPH AJE CT® LiVac SRS is a vaccine containing modified viable Salmonid Rickettsia, strain AL 20542.

[0025] The determination of viability can be a binary decision (viable / non-viable), or can be quantitative (e.g., 100% of the Salmonid Rickettsia in the sample are viable, 80% are viable, 50% are viable, 20% are viable, 10% are viable, etc.). Preferably, at least 10% of the Salmonid Rickettsia in the sample used to determine viability are truly viable. The method of determining viability of the Salmonid Rickettsia should be chosen according to whether a binary or quantitative answer is desired. However, since the viability is determined prior to the fish being vaccinated, the viability test should be reliable, simple to use, and preferably does not require specialized training or expensive equipment.

[0026] In certain embodiments, the viability is determined based on the quantification of ATP present. This method is based on the need for ATP to facilitate the beetle luciferase-luciferin reaction, resulting in the production of light ("luminescence").

[0027] A sample containing or suspected of containing viable Salmonid Rickettsia is contacted with a reaction buffer, at least one ATP extractant, a divalent cation, optionally a divalent cation chelator, and a luciferase / luciferin mixture, wherein the concentration of divalent cations is sufficiently low or is sufficiently neutralized by the cation chelator to reduce the negative impact of divalent cations on the extraction of ATP.

[0028] Various ATP extractants alter cell membrane or cell wall permeability or disrupt the integrity of the membrane and / or cell wall of the microbial source (i.e., lyse or cause pore formation) to effect extraction or release of ATP. Generally, ATP extractants are known in the art and can include various agents including, but not limited to: antibiotics such as polymyxin B (e.g., polymyxin Bl and polymyxin B2), polymyxin-β-nonapeptide (PMBN), and chlorhexidine (CHEX); alkyl glucosides or alkyl thio glucosides such as octyl-β-D-l-thiopyranoglucoside (see U.S. Patent No. 6,174,704, incorporated herein by reference in its entirety); non-ionic detergents such as Triton-XlOO (TX-100); betaine detergents such as carboxypropyl betaine (CB-18); quaternary ammonium salts such as trimethyl octadecyl ammonium bromide (TMA-18); protamine; 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, phospholipase A2, phospholipase A2-activating peptide (PLAP); bacteriophages; and the like. See, e.g., Morbe et al., Microbiol. Res. (1997) vol. 152, pp. 385-394.

[0029] In one embodiment of the present application, the ATP extractant includes CTAB, a quaternary ammonium salt. In a preferred embodiment, CTAB is present in the reagent composition at a concentration of about 0.04% - 0.15% (w / v). In another embodiment, the ATP extractant can include CHEX and an ethoxylated alkyl phenol such as Triton X-100. In a preferred embodiment, CHEX is preferably between about 0.04% - 0.16% (w / v) and the ethoxylated alkyl phenol is present at between about 0.25% - 1.0% (w / v). In a particularly preferred embodiment, the reagent composition can include more than one ATP extractant. One preferred embodiment includes CHEX (between about 0.04% - 0.16% (w / v)); an ethoxylated alkyl phenol such as Triton X-100 (between about 0.25% - 1.0% (w / v)); and a quaternary ammonium salt such as CTAB (between about 0.02% - 0.08% (w / v)).

[0030] The beetle luciferase-luciferin reaction depends not only on ATP but also on divalent cations. Therefore, to promote luciferase activity, divalent cations are typically supplied (unless they are already present in the sample). Divalent cations include magnesium, calcium, and manganese. Divalent cations can be supplied in salt or halide forms such as sulfates, sulfonates, gluconates, carbonates, chlorides, and bromides. For example, magnesium cations can be supplied in the form of magnesium chloride, magnesium sulfate, magnesium gluconate, magnesium acetate, magnesium bromide, magnesium carbonate, etc. Preferably, the divalent cation is selected from magnesium chloride or sulfate. Because the permeability of certain cell membranes or walls can be negatively affected by the presence of divalent cations, the concentration of divalent cations can be empirically formulated for a given microorganism or a given extraction / detection system to provide an appropriate balance between, for example, ATP release from cells and ATP detection.

[0031] Divalent cation chelating agents include, but are not limited to, salts of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), 1,2-cyclohexanediaminetetraacetic acid (CDTA), nitrotriacetic acid (NTA), citric acid, sodium gluconate, gluconic acid, lignin sulfonate, and mixtures thereof. Preferably, the chelating agent is selected from the group consisting of EDTA, EGTA, and CDTA due to its general availability and relatively low cost. The appropriate level of the divalent cation chelating agent can be determined empirically based on providing a level sufficient to neutralize the negative impact of the divalent cation on ATP extraction, but not to the point of preventing cation-dependent, luciferase-catalyzed ATP detection. In some embodiments, the divalent cation chelating agent is EDTA.

[0032] The difference between the concentration of the divalent cation chelator and the concentration of the divalent cation in the mixture can be less than about 5 mM. Alternatively, the concentration of the divalent cation chelator can be at least half of the concentration of the divalent cation in the mixture, preferably equal to or even greater than its concentration. In cases of low divalent cation concentrations (e.g., less than about 5 mM, 2.5 mM, or 1 mM), the divalent cation chelator may be unnecessary.

[0033] Given the ability of divalent cation chelators to neutralize the negative effects of divalent cations on ATP extraction, the concentration of divalent cations can be adjusted based on the level of divalent cation chelator present in the reagent composition or reaction mixture. When the divalent cation chelator concentration is low (e.g., less than about 5 mM, 2.5 mM, or 1 mM), the divalent cation concentration will be correspondingly lower, preferably less than 2.5 mM, more preferably between 0.2 and 1 mM. However, when the divalent cation chelator concentration is high (e.g., 2 to 20 mM), the divalent cation concentration will be correspondingly higher, preferably less than or equal to the concentration of the divalent cation chelator.

[0034] At its most basic level, luciferase is defined by its ability to produce light. More specifically, luciferase catalyzes the oxidation of the substrate luciferin, thereby producing oxidized luciferin and photons. Its catalytic products include light. Luciferase provides sensitivity, detectable products, and convenient measurement of ATP. Any ATP-dependent luminescent enzyme is considered for use in the reagent compositions and methods of the present invention.

[0035] The naturally occurring substrate of beetle luciferase is firefly luciferin. Luciferin can be isolated from nature (e.g., from fireflies) or synthesized. Synthetic luciferin can have the same structure as naturally occurring luciferin, or it can be a variant or derivative, as long as it functions similarly (Bowie et al., 1973; Branchini, 2000; Craig et al., 1991; Miska and Geiger, 1987; Yang and Thomason, 1993). Exemplary luciferin derivatives used in this invention include, but are not limited to, 6-deoxyaminoluciferin, D-luciferin methyl ester, D-luciferinyl-L-phenylalanine, D-luciferinyl-LNa-arginine, D-luciferin-O-sulfate, and D-luciferin-O-phosphate (Miska and Geiger, 1987), esters of luciferases that generate luciferin through esterase hydrolysis or by action of components in a sample (Craig et al., 1991; Yang and Thomason, 1993). Other examples of available luciferin analogues include naphthylluciferin and quinolinylluciferin, which emit light in green and red spectra, respectively (Branchini et al., 1989). Luciferin is available from multiple commercial sources (e.g., Promega Corp. Madison, WI; Molecular Probes, Eugene, OR). The beetle luciferase-catalyzed reaction that produces a luminescent signal from the luciferase-luciferin reaction requires luciferase, luciferin, adenosine triphosphate (ATP), magnesium (or other divalent cations), and molecular oxygen. In the initial reaction, luciferin reacts with ATP to form luciferyl adenosine, simultaneously eliminating inorganic pyrophosphate. The luciferyl adenosine remains tightly bound to the catalytic site of luciferase. When this form of enzyme is exposed to molecular oxygen, the enzyme-bound luciferyl adenosine is oxidized to produce oxidized luciferin in an electronically excited state.

[0036] The beetle luciferase-luciferin reaction results in the production of light (“luminescence”). Because the beetle luciferase-luciferin reaction is ATP-dependent, luciferase can be used to measure ATP. The reaction is very sensitive, allowing for concentrations as low as 10... -16ATP is detected in samples containing 1 mole or less of ATP. See U.S. Patent 7,422,868. Kits for performing such methods are commercially available and include, but are not limited to, the BacTiter-Glo™ Microbial Cell Viability Assay Kit manufactured by Promega Corporation (Madison, WI, USA).

[0037] In another embodiment, the method for determining viability is based on the finding that combinations of dyes can be selected to label live and dead cells in different ways. Therefore, a method for determining the viability of salmon rickettsiae in a sample involves contacting the salmon rickettsiae with a dye that differentially stains live and dead salmon rickettsiae.

[0038] In some embodiments, the method involves contacting salmon rickettsiae with a combination of two dyes: one dye stains both live and dead salmon rickettsiae, while the other dye stains only dead salmon rickettsiae. Importantly, the two dyes differ in their spectral characteristics.

[0039] One such combination is SYTO®9 and propidium iodide. SYTO®9 is a green fluorescent nucleic acid staining agent, and propidium iodide is a red fluorescent nucleic acid staining agent. These staining agents differ in their spectral characteristics and their ability to penetrate healthy bacterial cells. When used alone, SYTO®9 staining agent typically marks all bacteria in a population, i.e., bacteria with intact membranes and bacteria with damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes, resulting in reduced fluorescence of SYTO®9 staining agent when both dyes are present.

[0040] Therefore, in a particularly preferred embodiment, the method for determining the viability of salmon rickettsiae in a sample includes contact with a green dye that stains both live and dead cells and with a red dye that stains only dead cells. In a specific embodiment, the green staining agent is SYTO®9, and the red dye is propidium iodide. Thus, using a suitable mixture of SYTO®9 and propidium iodide staining agents, bacteria with intact cell membranes stain fluorescent green, while bacteria with damaged membranes stain fluorescent red. The maximum excitation / emission values ​​of these dyes are approximately 480 / 500 nm for SYTO®9 staining agent and 490 / 635 nm for propidium iodide.

[0041] The kit using SYTO® 9 and propidium iodide is commercially available and includes LIVE / DEAD® BACLIGHT. TM Bacterial viability kit (Molecular Probes, Eugene, OR, USA).

[0042] In some embodiments, the staining results are analyzed by fluorescence microscopy. Preferably, the fluorescence microscope used for analysis is field-compatible. For the purposes of this disclosure, a "field-compatible" fluorescence microscope is portable (e.g., weighing no more than 2 kg, preferably less than 1 kg, and even more preferably less than 500 g), while providing a resolution of about 100x to about 200x, and can be connected to a data display device such as a laptop, tablet, or mobile phone. The field of view is between about 1 mm and about 2 mm.

[0043] In a particularly preferred embodiment, fluorescence microscopy analysis is performed at approximately 200x magnification with a field of view of approximately 1 mm.

[0044] If the salmon rickettsia sample does not contain live salmon rickettsia, the salmon rickettsia stock solution should be discarded. Conversely, if the salmon rickettsia sample contains live salmon rickettsia, the user can administer the vaccine containing modified salmon rickettsia to salmonids without further steps.

[0045] Alternatively, users can formulate salmon rickettsia vaccines based on the measurement results and adjust the concentration of modified live salmon rickettsia or the volume of the vaccine so that the vaccinated salmon receive an appropriate and effective dose of modified live salmon rickettsia. For example, if microscopic fluorescence measurements show only three or four live salmon rickettsia organisms in a 1 mm field of view at 200x magnification, the volume or concentration can be doubled to increase the number of live salmon rickettsia to five or more.

[0046] In some implementations, the vaccine can be administered by injection, including but not limited to intramuscular and intraperitoneal injection. Typically, the vaccine is administered in volumes between 10 and 250 microliters. The volume of a vaccine dose can be adjusted within these limits based on the amount of rickettsiae in the live salmon sample, including but not limited to 20 microliters, 50 microliters, 75 microliters, 100 microliters, 150 microliters, or 200 microliters.

[0047] There are several methods for administering vaccines to salmonids. In some implementations, commercially available fish vaccination devices may be equipped with multiple injection needles, wherein the needles are configured to be spaced apart at a desired distance. Suitable vaccination machines include the NFTT product line (Pharmaq), and specific models include NFT 20, NFT 25, and NFT 30. NFT 20 and NFT 25 deliver the vaccine intraperitoneally, but can also be reconfigured for intramuscular injection. NFT 30 features a special DNA module that enables intramuscular delivery of the DNA vaccine into the fish fillet.

[0048] These machines process fish ranging in size from 120 mm to 250 mm (20-150 grams). After completion, they sort the vaccinated fish into three different sizes. Furthermore, they have channels for misplaced, undersized, or rejected fish.

[0049] Alternatively, the vaccine can be administered manually using a syringe. See, for example, the MICRO-MATIC® syringe sold by Pharmaq. Individual syringes are available in two sizes: 0.05 ml and 0.1 ml per dose. Additionally, a replaceable 0.025 ml plunger is available for the 0.05 ml syringe if needed. These syringes are suitable for both water-based and oil-based vaccine formulations. Dosage size can be easily adjusted by + / - 10%. Records show dosage accuracy deviations of less than 3%.

[0050] In addition to modified live salmon rickettsiae, vaccines may also contain additional antigens, including but not limited to modified live or inactivated antigens, including but not limited to viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV), iridovirus, nodavirus, fish myocarditis virus (PMCV), fish ororeovirus (PRV, sometimes called HSMIV or HSMI virus) that causes inflammation of the heart and musculoskeletal system, Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio (Aliivibrio) salmonicida, Yersinia ruckeri O1, and Moritella viscosa. In other implementations, the vaccine may contain plasmids encoding antigens against pathogens such as PMCV (fish myocarditis virus), PD (pancreatic disease) virus, and fish ororeovirus (a pathogen of cardiac and musculoskeletal inflammatory syndrome).

[0051] The vaccines disclosed herein, containing modified live salmon rickettsiae and optionally one or more additional antigens, may also contain pharmaceutically acceptable carriers or diluents.

[0052] Examples of pharmaceutically acceptable carriers or diluents that can be used in this invention include water, preservatives, culture media, stabilizers such as SPGA, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing reagents such as bovine serum or skim milk, and buffers (e.g., phosphate buffer).

[0053] Vaccines may or may not contain adjuvants. Adjuvants are non-specific stimulants of the immune system. They enhance the host's immune response to the vaccine. Non-limiting examples of adjuvants known in the art are alum, vitamin E, nonionic block polymers, muramyl dipeptides, ISCOM (immunostimulatory complex), saponins, and carbomer.

[0054] The invention will be further described in the following non-limiting embodiments.

[0055] Example

[0056] Example 1

[0057] The purpose of this embodiment is to: firstly, provide a proof of principle that the activity of salmon rickettsiae in vaccine samples can be quantified, and secondly, determine a suitable assay for this purpose.

[0058] Seven measurements were used in this embodiment:

[0059] Assay 1: BACTITER-GLO™ Microbial Cell Viability Assay - Catalog No.: G8231

[0060] Measurement 2: Live / Dead BACLIGHT TM Bacterial Viability Kit - Reference L7012 - Lot No. 2480125

[0061] Assay 3: Cell Counting Kit - 8, Sigma Aldrich - Product No.: 96992, Product Code: 102522147

[0062] Assay 4: Resazurin Assay Kit (Cell Viability) - Abcam (ab129732)

[0063] Assay 5: Bacterial Count Assay Kit (Colorimetric Method) - Abcam (ab284554)

[0064] Assay 6: Bacterial Viability Assay Kit - Abcam (ab189818)

[0065] Determination 7: AQUASNAP® Total AQUASNAP® Free Probe - Hygenia

[0066] A water bath was prepared at 22.5°C, and one vial containing modified live salmon rickettsiae was collected from a -80°C freezer. ® The SRS vaccine was placed in a box containing dry ice. The vaccine was thawed in a water bath for 3 minutes and then rapidly reconstituted with NaCl by connecting the vial to the ECOFLAC® connector and then to the NaCl vial.

[0067] Squeeze the vial several times to flush the vaccine from the vial into the vial. Invert the vial three times to ensure proper mixing. Follow the same protocol for all experiments.

[0068] Dead sample preparation

[0069] Using a vaccine gun, fill a 1.5 ml Eppendorf tube with the vaccine. Place the tube on a heat block set to 70°C for 30 minutes.

[0070] Assay protocol

[0071] Unless otherwise described, all experiments were performed using protocols from the assay kit manufacturer. Different assays recommend using different numbers of bacteria in the analysis; however, in these experiments, unless otherwise described, 100 μl (one vaccine dose) was used as the input volume. All incubations were performed at room temperature.

[0072] For more accurate measurements, 10 doses of vaccine were sprayed from the vaccine gun into Eppendorf tubes, and from there 100 μl was transferred to a 96-well plate using a pipette.

[0073] For live / dead mixtures, use the following settings:

[0074] 100% active = 100 µl live vaccine; 75% active = 75 µl live vaccine + 25 µl inactivated sample

[0075] 50% active = 50 µl live vaccine + 50 µl dead sample

[0076] 25% active = 25 µl live vaccine + 75 µl dead sample

[0077] 100% mortality = 100 µl of dead sample

[0078] Live / Dead BACLIGHT TM The luminescence values ​​of the bacterial viability kit (plate reader) are provided in Tables 1 (replication) and 2 (mean).

[0079] Table 1. Luminescent, Repeatable, BACTITER-GLO™ Microbial Cell Viability Assay

[0080] 100% alive 100% alive 24 hours 75% alive 75% alive 24 hours 50% 50% 24 hours 25% alive 24 hours 100% dead 100% dead 24 hours 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 14920

[0081] Table 2. Average luminescence value and BACTITER-GLO™ microbial cell viability assay

[0082] 100% alive 100% alive 24 hours 75% alive 75% alive 24 hours 50% 50% 24 hours 25% alive 24 hours 100% dead 100% dead 24 hours 151080 66502 141200 62222 104320 45539 40112 22974 14252

[0083] For Cell Counting Kit-8, OD values ​​are provided in Table 3 (repeat) and Table 4 (mean).

[0084] Table 3. Replication of OD values ​​from the Cell Counting Kit - 8.

[0085] 100% alive 75% alive 50% alive 25% alive 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

[0086] Table 4. Cell count 8, mean OD value

[0087] 100% alive 75% alive 50% alive 25% alive 100% dead 0.017201 0.0228 0.0212 0.022 0.0216

[0088] For the Resazurin assay kit, the replicate OD values ​​are provided in Table 5, and the average OD values ​​are provided in Table 6.

[0089] Table 5. Resazurin assay kit, repeat OD values

[0090] 100% alive 75% alive 50% alive 25% alive 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

[0091] Table 6. Resazurin assay kit, average OD value

[0092] 100% alive 75% alive 50% alive 25% alive 100% dead Blank 0.279 0.274 0.280 0.701 0.492 0.479

[0093] For the bacterial count assay kit (colorimetric method) Abcam (ab284554), OD values ​​are provided in Table 7 (replication) and Table 8 (mean).

[0094] Table 7. Bacterial Count Assay Kit (Colorimetric Method) Abcam (ab284554), OD Values ​​(Repeat)

[0095] 100% alive 75% alive 50% alive 25% alive 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

[0096] Table 8. Bacterial Count Assay Kit (Colorimetric Method) Abcam (ab284554), OD Values ​​(Average)

[0097] 100% alive 75% alive 50% alive 25% alive 100% dead 0.0053 0.0027 0.0024 0.0027 -0.0003

[0098] For the bacterial viability assay kit - Abcam (ab189818), fluorescence values ​​are provided in Table 9 (repeat) and Table 10 (mean).

[0099] Table 9. Bacterial Viability Assay Kit - Abcam (ab189818), Fluorescence Value, Repeat Count

[0100] 100% alive 75% alive 50% alive 25% alive 100% dead 2132 1902 1650 1529 1605 4164 1677 1661 1434 1461 4612 2059 1881 2524 1871

[0101] Table 10. Bacterial Viability Assay Kit - Abcam (ab189818), Fluorescence Value, Average Value

[0102] 100% alive 75% alive 50% alive 25% alive 100% dead 3636 1879 1731 1829 1646

[0103] For the AQUASNAP® assay, the viability after four measurements is provided in Table 11. The AQUASNAP® assay involves the use of two assay probes—referred to as the AQUASNAP® total and AQUASNAP® free probes. ® Total probes measure both microbial ATP (from living cells and particulate matter) and free ATP (from non-microorganisms or dead cells) in solution. AQUASNAP® free probes measure non-microbial ATP (also known as dead cell ATP) in solution.

[0104] First use AQUASNAP® ® A total probe was used to record the total ATP load. Then, AQUASNAP® was used. ® The free probe measures free ATP (from dead cells) on the same sample. The difference between the total probe and the free probe indicates the number of live cells in the sample. The units in the table are expressed as a percentage based on the following formula: 100 – ((AQUASNAP® free probe / AQUASNAP® total probe) * 100).

[0105] Table 11. AQUASNAP® Viability Results, Four Measurements

[0106] 100% alive 75% alive 50% alive 25% alive 30.7 11.5 -11.5 2.1

[0107] For using Live / Dead BACLIGHT TM For the microscopic method using the bacterial viability kit, place one dose (100 μl) into an Eppendorf tube and mix with 1 μl of each staining agent (SYTO® 9 and propidium iodide (PI)). Incubate the sample in the dark for 15 minutes, then prepare a microscope slide using 20 μl of the stained sample. Assay viability using ten replicates taken simultaneously from the same vaccine bag. (Use BACLIGHT) TM The kit stains the sample and images are taken on a Leica microscope at 1000x magnification and 0.180mm field of view.

[0108] Ten images were taken for each of the ten samples. All images were then manually analyzed by first counting the total number of cells in all ten images, and then counting the number of dead cells in each image. Units in the table are percentages, as determined by the following formula: 100 – ((dead cells / total number of cells) * 100).

[0109] The results are provided in Table 12.

[0110] Table 12. Live / Dead BACLIGHTTM Bacterial viability kit, cell viability, replication

[0111] 100% dead 100% alive 75% alive 50% alive 25% alive 100% dead Measurement 1 Measurement 2 Measurement 3 Measurement 4 14.4 13.4 12.5 12.9 13.5 11.9 12.1 10.4 15.5 10.1

[0112] Conclusion: Of the seven assays tested, five were unable to effectively distinguish between live and dead salmon rickettsiae in vaccine samples. Only two assays were considered useful: the BACTITER-GLO™ microbial cell viability assay and the Live / Dead BACLIGHT assay, analyzed by fluorescence microscopy. TM Bacterial viability kit.

[0113] BACLIGHT TM The bacterial viability kit contains two dyes for staining nucleic acids. SYTO®9 can penetrate the cell membrane and stain both live and dead cells, while PI can only penetrate damaged cell membranes and therefore only stain dead cells.

[0114] SYTO®9 is excited by blue light (485 nm), and experiments on salmon rickettsia bacteria in the LIVAC® vaccine showed that exposure to blue light under a microscope kills the bacteria after 10–20 seconds. It was found that about half of the cells surviving upon exposure died within 15 seconds. This means that images (or ideally videos) need to be taken quickly before the bacteria die, and the color changes from green to red.

[0115] In LIVAC® samples, only about 10% of salmon rickettsiae exhibited the expected bright green color, while the remaining bacteria showed very weak green staining. Weakly stained green cells do not turn red upon exposure to blue light, indicating they lack metabolic activity and may still possess some membrane integrity preventing them from turning red. As a result, weakly stained bacteria can be difficult to see when examining samples under a microscope, and the same problem is observed when reviewing images taken from the samples. Image processing software is then required to enhance contrast / brightness for proper observation of the weakly stained bacteria.

[0116] Replicate 1

[0117] Further development was carried out using fluorescence microscopy. For on-site readings, the iOlight fluorescence microscope was chosen. It is field-compatible and can be connected to screens such as cordless phones, tablets, laptops, or desktop computers. Any of these devices has image analysis software. However, the microscope's magnification is only 200x, and it is unclear whether this can produce images of sufficient quality. Therefore, the purpose of the illustrative experiment was to determine whether 200x magnification was sufficient to reliably determine the presence of active salmon rickettsiae in the sample.

[0118] In these experiments, the number of live cells in images generated by field-compatible microscopes was compared with the number of live cells generated by LeicaDMRB fluorescence microscopes (i.e., more powerful but non-field-compatible microscopes).

[0119] The results are provided in Table 13.

[0120] Table 13. Comparison of viability determined by field-compatible and non-field-compatible microscopes

[0121]

[0122] As shown in the table, the number of green cells in images generated by a field-compatible microscope at 200x magnification is positively correlated with the viability measured at 1000x concentration.

[0123] In the next experiment, it was determined whether this positive correlation still existed under suboptimal thawing conditions.

[0124] Optimal conditions: All experiments were performed using the normal thawing protocol, and samples were analyzed immediately after reconstruction. The normal thawing protocol is as follows:

[0125] Remove the vial from the liquid nitrogen;

[0126] Place in a water bath at 15-25 degrees Celsius;

[0127] Thaw until a small piece of ice is still visible, up to 3 minutes;

[0128] Then immediately connect the vial to the 1 L saline (0.9% NaCl) flask using the connector, and inject the contents of the vial by repeatedly squeezing the flask until all the contents of the vial are transferred into the flask.

[0129] The vaccine is now ready and can be stored at room temperature with an expiration date set at 8 hours.

[0130] The suboptimal conditions change as follows:

[0131] Experiment 1 (-80°C to 4°C to -80°C): Remove the vial from the -80°C freezer to 4°C until thawed, then move it back to -80°C and thaw using the normal procedure.

[0132] Experiment 2 (RT thawing - 20 hours later): Thaw the vial at room temperature, reconstitute it in saline solution, and leave it at room temperature for 20 hours.

[0133] Experiment 3 (24 hours at 28°C): Thaw the vials using the normal procedure, reconstitute them in saline solution, and place them at 28°C for 24 hours.

[0134] Experiment 3 (24 hours at RT): Thaw the vials using the normal procedure, reconstitute them in saline, and leave them at room temperature for 24 hours.

[0135] Experiment 3 (24 hours at 4°C): Thaw the vials using the normal procedure, reconstitute them in saline solution, and place them at 4°C for 24 hours.

[0136] The results are summarized in Table 14.

[0137] Table 14: Effect of thawing conditions on cell viability as measured by field-compatible and off-site compatible microscopes.

[0138]

[0139] Based on these results, it is concluded that five cells per 1 mm imaging field of view appears to be an appropriate cutoff value for cell viability. To eliminate variability between images, several (preferably at least five, more preferably at least ten) images should be analyzed. If the average number of live cells is five or more, the vaccine contains live salmon rickettsiae and is suitable for administration to salmon, and if administered according to the manufacturer's instructions (e.g., if the ambient water temperature is 10°C or higher, or in other embodiments, 12°C or higher), it is expected to provide immunity against salmon rickettsiae.

[0140] Conversely, if the average number is less than five, the practitioner should discard the vaccine or repeat the staining and analysis of several images. If the average number of live cells is five or more in the second staining and analysis, the vaccine is effective. If the average number of green cells is less than five in the second staining and analysis, the thawed vaccine batch needs to be discarded.

[0141] Example 3

[0142] This confirmatory example demonstrates the applicability of the techniques described herein to field conditions. In vaccinating salmon against salmon rickettsia with the modified live salmon rickettsia vaccine (LIVAC® SRS), Pharmaq representatives traveled to multiple salmon farms in Chile and tested the viability of salmon rickettsia in the respective farm settings. Samples of the vaccine preparation were collected and processed as shown in Table 15.

[0143] Table 15

[0144] Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6 Replicate 7 Replicate 8 Replicate 9 18 Replicate 10 23 20.3 Example 2 Site 59 Vaccination service 59 21.8 Median count Sample handling notes 24 Mean count 24 15.8 Standard deviation Farm 1 59 Service 1 56 25.0 Sample 1 (no notes) Farm 1 41 Service 1 41 14.9 Sample 2 (no notes) Farm 1 22 Service 1 22 6.4 Sample 3 (no notes) Farm 2 33 Service 1 35 13.4 Sample 1, new reconstitution Farm 2 30 Service 1 33 11.8 Sample 2 (last reconstitution) Farm 3 98 Service 2 119 68.7 Sample 1, 3 hours post reconstitution Farm 3 123 Service 2 117 30.9 Sample 2, 3 hours post reconstitution Farm 3 59 Service 2 63 22.2 Sample 3, 1 hour post reconstitution Farm 4 10* Service 3 10 5.7 Sample 1 (no notes) Farm 4 10* Service 3 10 3.7 Sample 2 (no notes) Farm 4 6* Service 3 6 3.7 Sample 3 (no notes) Farm 5 4* Service 4 6 4.2 LIVAC® SRS, 1 week thawed Farm 5 18 Service 4 18 7.9 LIVAC® SRS, 1 week thawed and (bacteria inactivated at 70°C) Farm 5 20 Service 4 21 6.2 LIVAC® SRS, 1 year thawed Farm 5 45 Service 4 45 10 LIVAC® SRS, 1 year thawed (bacteria inactivated at 70°C) Farm 6 33 Service 3 36 15.9 Sample 1 (last reconstitution) Farm 6 15 Service 3 15 5.8 Sample 2 (new reconstitution) Farm 6 37 Service 3 43 23.2 Sample 3 (last reconstitution) Farm 5 0 Service 4 1 1 Live vaccine stained with Syto9*** Farm 5 21 Service 4 22 8.6 Live vaccine stained with Syto9 + PI Farm 5 43 Service 4 43 8.2 Vaccine inactivated at 70°C, stained with Syto9 Farm 5 19 Service 4 18 8.3 Vaccine inactivated at 70°C, stained with Syto9 + PI Farm 6 23 Service 3 23 7.3 Sample 1 (1 hour post reconstitution) Farm 6 12 Service 3 12 4.9 Sample 2 (1 hour post reconstitution) Farm 6 Service 3 Sample 3 (1 hour post reconstitution) Farm 5 Service 4 Sample 1 (1 hour post reconstitution) Farm 5 Service 4 Sample 2 (1 hour post reconstitution) Farm 5 Service 4 0 Sample 3, 4 hours post reconstitution 1 1.3 Farm 7 Service 5 24 Sample 1, 2 hours post reconstitution (final dose from bottle) 25 8.2 Farm 2 Service 1 5 Sample 1, 1 / 2 hour post reconstitution (new bottle) 4 2.3 Farm 8 Service 2 21 Probe sample, 18 hours post reconstitution 19 6.9 Farm 8 Service 2 11 Sample 1, 5 hours post reconstitution 13 5.7 Farm 8 Service 2 6 Sample 2, 5 hours post reconstitution 8 5.3 Farm 8 Service 2 19 Sample 3, 5 hours post reconstitution 26 15.8 Farm 3 Service 2 30 Sample 1, new reconstitution 31 10.8 Farm 3 Service 2 12 Sample 2, new reconstitution 11 3.0 Farm 3 Service 2 24 Sample 3, 3 hours post reconstitution 25 6.4 Farm 3 Service 2 48 Sample 4, 3 hours post reconstitution 51 8.9 Farm 6 Service 3 12 Sample 1, 1.5 hours post reconstitution 12 4.7 Farm 6 Service 3 16 Sample 2, 2.5 hours post reconstitution 16 7.6 Farm 6 Service 3 19 Sample 3, new reconstitution 18 9.9 Farm 6 Service 3 6 Sample 4, new reconstitution 7 2.1 ​ ​ 12 ​ 12 4.5 ​ ​ 21 ​ 27 11.9

[0145] * - The sample was diluted 5 times instead of 200 times. The count needs to be divided by 40.

[0146] ** - Reconstruction produces 10,000 batches. "New reconstruction" refers to the vaccine analyzed before any dose in the batch is administered to the fish. "Final reconstruction" refers to the remaining vaccine in the batch after substantially all doses have been administered. Generally, substantially all doses are administered to the fish within 2–5 hours (more likely 2–3 hours) after vaccine reconstruction.

[0147] *** - A control sample to check if the reagents are working properly. The two staining agents in the kit are Syto9 and PI. Syto9 is a green staining agent that will stain both live and dead cells, while PI is a red staining agent that will stain dead cells red. If Syto9 and PI are used together, PI will "remove" the green from the dead cells and stain them red.

[0148] These data confirm that inactivated or thawed samples (i.e., samples that may not contain live salmon rickettsiae) contained low bacterial counts, while samples analyzed less than 24 hours after reconstruction contained live salmon rickettsiae. Therefore, the method for testing salmon rickettsiae viability in modified live salmon rickettsiae vaccines can be performed under field conditions.

[0149] All publications (including patent and non-patent publications) referenced in this specification indicate the level of skill of a person skilled in the art to which this invention pertains. All such publications are incorporated herein by reference in their entirety, to the extent that each individual publication is specifically and individually indicated as incorporated by reference.

[0150] Although the invention described herein has been illustrated with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. Therefore, it should be understood that many modifications can be made to the illustrative embodiments, and other arrangements can be designed, without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. A method of protecting a salmonid fish from infection by Salmonid Rickettsia, the method comprising: a. obtaining a sample of Salmonid Rickettsia vaccine; b. determining the viability of Salmonid Rickettsia in the sample, thereby confirming that the Salmonid Rickettsia vaccine contains modified live Salmonid Rickettsia; c. administering the vaccine containing modified live Salmonid Rickettsia to the salmonid fish in need of the protection.

2. A method of protecting a salmonid fish from infection by Salmonid Rickettsia, the method comprising: a. obtaining a sample of a lyophilized Salmonid Rickettsia stock; b. determining the viability of the lyophilized Salmonid Rickettsia in the sample, thereby confirming that the lyophilized Salmonid Rickettsia stock comprises live Salmonid Rickettsia c. formulating a vaccine by mixing the lyophilized Salmonid Rickettsia comprising a live Salmonid Rickettsia stock or a portion thereof with a pharmaceutically acceptable carrier or diluent; d. administering the vaccine containing an immunologically effective amount of modified live Salmonid Rickettsia to the salmonid fish in need of the protection.

3. The method of claim 1 or claim 2, wherein the step of determining the viability of the Salmonid Rickettsia in the sample comprises contacting the sample with an ATP extractant, a divalent cation, a luciferase / luciferin, and optionally a divalent cation chelator; and quantifying luminescence.

4. The method of claim 1 or claim 2, wherein the step of determining the viability of the Salmonid Rickettsia in the sample comprises contacting the sample with a first dye that differentially stains live and non-live Salmonid Rickettsia, and optionally a second dye that stains both live and non-live Salmonid Rickettsia.

5. The method of claim 4, wherein the first dye that differentially stains live and non-live Salmonid Rickettsia is propidium iodide, and wherein the step further comprises staining Salmonid Rickettsia with the second dye that stains both live and non-live Salmonid Rickettsia, wherein the second dye is SYTO® 9.

6. The method of claim 5, further comprising quantifying live Salmonid Rickettsia by fluorescence microscopy.

7. The method of claim 6, wherein the fluorescence microscopy is performed by a field-compatible microscope.

8. The method of claim 6, wherein the analysis of the fluorescence microscopy is performed at about 200x magnification with about 1 mm field of view.

9. The method of claim 8, wherein the analysis comprises taking a plurality of images and determining the average number of live cells in the plurality of images when a sample of Salmonid Rickettsia is exposed to blue light under the microscope.

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 average number is five or more.

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

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