Optimization of efficacy of a live attenuated Flavobacterium psychrophilum immersion vaccine

Accepted Manuscript Optimization of efficacy of a live attenuated Flavobacterium psychrophilum immersion vaccine Ponnerassery S. Sudheesh, Kenneth D. Cain PII:

S1050-4648(16)30424-7

DOI:

10.1016/j.fsi.2016.07.004

Reference:

YFSIM 4063

To appear in:

Fish and Shellfish Immunology

Received Date: 26 April 2016 Revised Date:

16 June 2016

Accepted Date: 8 July 2016

Please cite this article as: Sudheesh PS, Cain KD, Optimization of efficacy of a live attenuated Flavobacterium psychrophilum immersion vaccine, Fish and Shellfish Immunology (2016), doi: 10.1016/ j.fsi.2016.07.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Optimization of efficacy of a live attenuated Flavobacterium psychrophilum immersion

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vaccine

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Ponnerassery S. Sudheesh a, Kenneth D. Cain a*

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Department of Fish and Wildlife Sciences, College of Natural Resources, University of Idaho, Moscow, ID 83844-1136, USA

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*Corresponding author: +1-208-885-7608; Fax: +1-208-885-9080

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E-mail: [email protected] (Kenneth D. Cain)

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Abstract This study was aimed to optimize the efficacy of a recently developed live attenuated

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immersion vaccine (B.17-ILM) as a promising vaccine against bacterial coldwater disease

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(BCWD) caused by Flavobacterium psychrophilum in salmonids. Rainbow trout (RBT) fry

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were vaccinated by immersion, and different parameters affecting vaccination such as fish

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size, vaccine delivery time, dose, duration of protection, booster regimes and vaccine growth

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incubation time were optimized. Specific anti-F.psychrophilum immune response was

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determined by ELISA. Protective efficacy was determined by challenging with a virulent

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strain of F. psychrophilum (CSF-259-93) and calculating cumulative percent mortality

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(CPM) and relative percent survival (RPS). All vaccinated fish developed significantly

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higher levels of serum antibody titers by week 8 when compared to their respective controls.

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Immersion vaccination for 3, 6 and 30 min. produced significant protection with comparable

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RPS values of 47 %, 53 % and 52 %, respectively. This vaccine provided significant

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protection for fish as small as 0.5 g size with an RPS of 55 %; larger fish of 1 g and 2 g size

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yielded slightly higher RPS values of 59 % and 60 %, respectively. Fish vaccinated with

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higher vaccine doses of ~1010 and 108 colony forming units mL-1 (cfu ml-1) were strongly

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protected out to at least 24 weeks with RPS values up to 70 %. Fish vaccinated with lower

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doses (106 and 105 cfu mL-1) had good protection out to 12 weeks, but RPS values dropped to

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36 % and 34 %, respectively by 24 weeks. Vaccine efficacy was optimum when the primary

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vaccination was followed by a single booster (week 12 challenge RPS = 61 %) rather than

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two boosters (week 12 challenge RPS = 48 %). Vaccination without a booster dose resulted

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in a lower RPS (13 %) indicating the necessity of a single booster dose to maximize efficacy.

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This study presents key findings that demonstrate the efficacy and commercial potential for

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this live attenuated BCWD vaccine.

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ACCEPTED MANUSCRIPT Key words: Coldwater disease, Flavobacterium psychrophilum, live attenuated vaccine,

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immersion vaccination, rainbow trout, iron limited medium.

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ACCEPTED MANUSCRIPT 1. Introduction

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Bacterial coldwater disease (BCWD) is a significant bacterial disease of salmonids and other

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species grown in both public and commercial aquaculture sectors [1-4]. The disease is caused

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by a Gram-negative fastidious bacterial pathogen, Flavobacterium psychrophilum, which

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inflicts heavy losses especially in sensitive species such as rainbow trout (Oncorhynchus

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mykiss), coho salmon (O. kisutch) and ayu (Plecoglossus altivelis) [1,5-12]. Although the

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disease can affect all life stages of fish, it is most severe in fry and juveniles when water

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temperatures range between 10 °C and 14 °C [2,3,13].

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The mode of transmission, host entry and dissemination mechanisms are not well understood,

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but horizontal transmission [14] and suspected vertical transmission of the pathogen [15-21],

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make the disease extremely difficult to treat and control. Hatcheries adopt different methods

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involving strict biosecurity and management practices as well as treating the disease with

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approved antibiotics such as Aquaflor® and Terramycin® (www.fda.gov/cvm, accessed on

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June 15, 2016). Among other problems, development of antibiotic resistant strains [22-24] is

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a major concern when considering continuous use of antibiotics to control BCWD. Clearly,

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alternative control and prevention methods need to be developed and available for

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aquaculturists. Vaccination has the potential to prevent BCWD, but despite concerted

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research efforts an approved vaccine is still not commercially available. A rifampicin

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resistant strain of F. psychrophilum (259-93-B.17) has been developed as a potential live

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attenuated vaccine to control BCWD [25]. When this vaccine strain is grown in TYES

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medium containing 50 µM of a known iron chelator, 2,2, dipyridyl, protection in fish is

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enhanced following vaccination. This modified formulation of 259-93-B.17 grown in iron

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limited medium was designated 259-93-B.17-ILM (B.17-ILM) and it was shown that in

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immersion vaccination trials in Coho salmon relative percent survival (RPS) was 73 % when

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fish were challenged with the virulent parent 259-93 strain [26]. In that study, the high levels

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ACCEPTED MANUSCRIPT of protection via immersion vaccination were achieved in fish that were adipose fin clipped

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immediately prior to vaccination and the delivery duration time was 1 h. From a practical

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standpoint, such procedures would be labor intensive, costly and difficult to implement for

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mass vaccination of fish in the field.

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When developing an efficient fish vaccine for commercialization, in addition to potency and

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safety testing, there are several factors related to the host, pathogen, environment and the

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method of antigen delivery that need to be considered [27,28]. It is essential to develop a

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robust vaccination method that can produce the desired immune response, protect fish for a

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prolonged period with a defined minimum antigen dose, and that can be delivered efficiently

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in a short period of time. As BCWD is frequently observed in fry and fingerlings [2,3,13], it

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is also important to know the minimum size of fish that can be vaccinated to produce a

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sufficient immune response and protection. Therefore, this study was carried out to determine

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the optimum vaccine delivery time, minimum size of fish that can be effectively vaccinated,

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effective dosage, duration of protection, and booster vaccination requirements for this new

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B.17-ILM formulation.

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2. Materials and methods

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2.1. Bacteria and culture conditions

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The virulent strain, F. psychrophilum CSF-259-93 was grown for 72 h at 15 ˚C in tryptone

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yeast extract salts broth (TYES; 0.4 % tryptone, 0.04 % yeast extract, 0.05 % calcium

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chloride, 0.05 % magnesium sulfate, pH 7.2) following published protocols [29]. To harvest

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bacteria for challenge experiments, 72 h grown broth culture was centrifuged (4300 x g) for

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15 min, the supernatant was poured off, and pellet was resuspended in sterile phosphate

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buffered saline (PBS, pH 7.0) to the appropriate optical density value at 525 nm (OD525). To

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ACCEPTED MANUSCRIPT estimate colony forming units-1 (CFU mL-1), a 6 x 6 drop plate method was used [30]. Plates

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were incubated at 15 ˚C for 96 h and colonies were counted.

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2.2. Preparation of iron limited B.17 vaccine (ILM)

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The original parent vaccine strain, F. psychrophilum- 259-93-B.17 was stored at -80 ˚C as a

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master seed stock in TYES broth medium with 15 % glycerol. A cryo-tube from the master

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seed stock was withdrawn, thawed and 200 µL of this stock culture was used to inoculate 10

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mL of TYES medium and incubated at 15 ˚C for 72 h with shaking at 80 rpm. To grow the

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strain under iron limiting conditions (ILM), the whole 10 mL culture was inoculated to 250

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mL TYES medium containing the iron chelator, 2, 2-dipyridyl (DPD) (Sigma Aldrich, St.

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Louis, MO, USA) at a final concentration of 50 µM. The culture was allowed to grow at 15

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˚C for 96 h with shaking at 80 rpm. The culture was scaled up to 1 L and then to 5 L volumes

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in TYES containing 50 µM DPD and incubated at 15 ˚C for 96 h with shaking at 80 rpm.

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This final vaccine culture broth was mixed with sterile glycerol to a final concentration of 15

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% glycerol, mixed well and aseptically poured into sterile 1 L serum bottles (Genesis

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Industries Inc, WI, USA), closed with sterile rubber stopper and sealed with aluminum seals

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using a crimper. All bottles were labeled and stored at -80 ˚C until vaccinated.

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2.3. Experimental animals

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Unless otherwise stated, healthy rainbow trout (O. mykiss) (RBT) with a mean weight of 2.5

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g were obtained from the Aquaculture Research Institute of University of Idaho, Moscow,

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USA. Fish were acclimated for one week in 500 L tanks with continuous aeration in a

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dechlorinated municipal water flow-through system at 12 ˚C with 5 L min−1 water flow and

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12:12 h day-night cycle at the wet lab facilities of the Department of Fish and Wildlife

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Sciences, University of Idaho, Moscow, USA. The fish were fed a commercial pellet feed

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(Skretting, USA) at 3 % of body weight per day in two divided doses until used in the

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vaccination trials. In order to check for any previous exposure to F. psychrophilum infection,

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ACCEPTED MANUSCRIPT a sample of 10 fish were withdrawn from culture tank, anesthetized in 100 mg mL-1 tricaine

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methanesulfonate (MS-222; Argent Chemicals, Redmond, WA, USA) and blood was

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collected by caudal venipuncture to separate sera and test for F. psychrophilum specific

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antibodies by using an enzyme linked immunosorbant assay (ELISA). In addition, kidney

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and spleen tissue samples from all 10 fish were dissected out and aseptically plated on TYES

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agar plates to check for any growth of F. psychrophilum.

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2.4. Ethics

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All experimental procedures involving live fish were carried out with prior approval from the

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Institutional Animal Care and Use Committee, University of Idaho (IACUC # 2012-30).

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2.5. Vaccination

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Unless otherwise stated in specific experiment, initial and booster vaccination for all

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experiments were carried out in an identical manner as follows. The vaccine bottles stored at

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-80 ˚C were thawed at room temperature overnight and immediately before vaccinating fish,

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the vaccine was diluted 1:10 in a total volume of 20 L using clean rearing water and the fish

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were immersion vaccinated for 3 min or otherwise indicated in specific experiment.

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Vaccinated fish were immediately moved back to their rearing tanks. Two weeks after initial

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vaccination, all fish were booster vaccinated in an identical manner as primary vaccination.

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Control groups were immersed in a 1:10 dilution of TYES media in an identical manner as

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the vaccinated fish. Unless otherwise stated each treatment group consisted of a minimum

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number of 150 fish and all fish in a treatment group were vaccinated together.

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2.6. Fish challenge

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Eight weeks following initial vaccination (or later depending on specific experiment) 75 fish

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(triplicate groups of 25 each) from each treatment group were challenged with a virulent

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ACCEPTED MANUSCRIPT strain of F. psychrophilum (CSF-259-93) following a standard challenge method [26]. A

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subset of fish (n = 25) was injected with sterile PBS to serve as the mock infected control.

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Mortalities were monitored on a daily basis for 28 days and re-isolation of F. psychrophilum

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was attempted by sampling 20 % of the daily mortality and streaking kidney, liver, and

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spleen samples on TYES agar and incubating plates at 15 ˚C for 96 h. Presumptive

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identification of isolates as F. psychrophilum was based on colony color (yellow) and

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morphology (convex with smooth morphology or convex with a thin spreading margin).

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2.7. Serum collection and enzyme linked immunosorbent assay (ELISA)

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Fish used for collecting serum were randomly netted out from tanks and anesthetized by

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using tricaine methanesulfonate (MS-222; Argent Chemicals, Redmond, WA, USA). Blood

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was collected by venipuncture of the caudal vein by using a sterile ½ cc insulin syringe with

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27G needle (Terumo, Elkton, MD). Blood from very small fry was collected using 0.1 mL

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hematocrit tubes (Fisher Scientific, Pittsburgh, PA, USA). Blood was placed in sterile 0.5

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mL tubes and allowed to clot overnight at 4 ˚C. The next day, separated sera was collected by

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centrifugation at 15,000 x g. Sera samples were stored at -20 ˚C until used in the ELISA.

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Sera collected from 5 fish in each treatment group at the beginning of the experiment before

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vaccination (one pool of 5 fish per treatment/control group for each experiment) served as

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negative control for respective treatment/ control group.

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Specific anti-F. psychrophilum immune response was determined by an ELISA procedure

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described earlier [26]. Serum samples were diluted from 1:50 to 1:102,400 in a series of

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doubling dilutions in PBS containing 0.02 % sodium azide. Titer was set as the reciprocal of

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the highest dilution that had an optical density at least two times greater than the negative

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control.

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2.8. Experimental design

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ACCEPTED MANUSCRIPT 2.8.1. Optimization of vaccine delivery time

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This experiment was carried out to determine the effect of different delivery times on the

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efficacy of this vaccine. Briefly, groups of fish (mean size, 2.0 g; 200 fish/group) were

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vaccinated as described in section 2.5, except that each treatment group of fish was subjected

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to different immersion delivery times of 1.5 min (1.5 min-B.17-ILM), 3 min (3 min-B.17-

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ILM), 6 min (6 min-B.17-ILM) and 30 min (30 min-B.17-ILM). Respective control fish for

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each vaccine delivery time period were immersed in TYES media. All treatment groups were

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booster vaccinated with the corresponding delivery times two weeks post initial

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immunization as described in section 2.5. Respective control fish were boosted using the

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respective delivery times by immersing in TYES media. The mean size of fish in different

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treatment groups at the time of booster vaccination was 2.95 g. Initial vaccination dose was ~

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5.0 X 1010 cfu ml-1 and booster vaccine dose was ~ 4.0 X 1010 cfu ml-1. Fish from each

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treatment/control group were challenged with ~ 1.46 x 107 cfu fish-1 of CSF-259-93 eight

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weeks post initial immunization as described in section 2.6. The ELISA was performed on 5

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individual sera samples prepared from 5 fish collected from each treatment/control group at

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0, 2, 4, 6 and 8 weeks of the experiment.

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2.8.2. Optimization of size at first vaccination

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This experiment was carried out to determine the optimum fish size at first vaccination. Fish

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from the same spawning group were segregated into approximate size groups (150

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fish/group) of mean size 2 g (2 g-B.17-ILM), 1g (1 g-B.17-ILM) and 0.5 g (0.5 g-B.17-ILM)

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and vaccinated as described in section 2.5. Respective control fish for each size group were

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immersed in TYES media. Fish were booster vaccinated two weeks after initial vaccination

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as described in section 2.5, when the mean size of fish in 2.0 g, 1.0 g and 0.5 g size groups

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reached 3.30 g, 1.67 g and 0.78 g, respectively. Respective control groups were immersed in

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TYES media. Initial vaccination dose was ~ 9.0 X 109 cfu ml-1 and booster vaccination dose

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ACCEPTED MANUSCRIPT was ~ 7.0 X 109 cfu ml-1. Fish size groups of 2 g, 1 g and 0.5 g were challenged by CSF-259-

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93 eight weeks post initial vaccination at doses of ~ 5.18 x 107 cfu fish-1, 3.45 x 107 cfu fish-1

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and 1.73 x 107 cfu fish-1, respectively as described in section 2.6. The ELISA was performed

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on 5 individual sera samples prepared from 5 fish collected from each treatment/control

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group at 0, 2, 4, 6 and 8 weeks of the experiment.

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2.8.3. Optimization of dose and duration of protection

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This experiment was carried out to determine the effect of different vaccine doses on the

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efficacy and also to determine the duration of protection offered by the vaccine beyond 8

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weeks post initial vaccination. Four different doses of the vaccine were prepared by thawing

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vaccine stocks with a viable count of ~ 1.58 x 1010 cfu mL-1 (1010-B.17-ILM) and diluting

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and adjusting to lower doses of ~ 3.0 x 108 cfu mL-1 (108-B.17-ILM), 1.0 x 106 cfu mL-1 (106-

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B.17-ILM) and 2.5 x 105 cfu mL-1 (105-B.17-ILM). Cell free supernatants obtained by

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centrifugation (8000 x g) of B.17-ILM vaccine culture broth was used to dilute and re-

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suspend the cells of lower doses of the vaccine in order to keep the uniformity of the

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composition of the extracellular fraction of the vaccine. Treatment groups of fish (mean size,

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2.65 g; 400 fish/group) were initially vaccinated with each of the above four doses of the

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vaccine as described in section 2.5. Control fish were immersed in TYES media. Fish were

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booster vaccinated two weeks after initial vaccination as described in section 2.5 when they

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reached a mean size of 3.01 g. The dose for booster vaccinations in each vaccinated group

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were ~ 9.17 x 109 cfu mL-1 (1010 -B.17-ILM), 2.90 x 108 cfu mL-1 (108 -B.17-ILM), 1.10 x 106

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cfu mL-1 (106 -B.17-ILM) and 2.20 X 105 cfu mL-1 (105 -B.17-ILM). Control fish were

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immersed in TYES medium. To determine the efficacy and duration of protection beyond the

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typical 8 week challenge, fish in each treatment and control group were challenged at 8, 12

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and 24 weeks post initial vaccination using the virulent CSF-259-93 stain of F.

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psychrophilum at doses of ~ 3.0 x 107 cfu fish-1, 2.29 x 107 cfu fish-1 and 3.40 x 107 cfu fish-1,

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ACCEPTED MANUSCRIPT respectively as described in section 2.6. The ELISA was performed on 5 individual sera

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samples prepared from 5 fish collected from each treatment/control group at 0, 2, 4, 8, 12 and

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24 weeks of the experiment.

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2.8.4. Optimization of booster frequency

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This experiment was carried out to determine the effect of different booster regimes on the

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protective efficacy of the B.17-ILM vaccine. Three groups of fish (mean size, 2.6 g; 400

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fish/group) were initially vaccinated using the B.17-ILM vaccine as described in section 2.5.

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Control fish were immersed in TYES media. The viable count of cells in the initial

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vaccination was ~ 1.58 x 1010 cfu mL-1. Two weeks after initial immunization, one group of

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fish was given a single booster vaccination (one booster-B.17-ILM), and the second group

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was boostered two times on 2nd and 4th week of initial immunization (two booster-B.17-

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ILM) as described in section 2.5. A third group of fish was maintained without any booster

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vaccination (no booster B.17-ILM). The unvaccinated control group was immersed in TYES

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medium. The viable count of bacteria in the first and second booster vaccinations was ~ 9.17

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X 109 cfu mL-1 and 1.28 X 1010 cfu mL-1, respectively. The mean size of fish at first and

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second booster vaccinations was 3.08 g and 4.27 g, respectively. Fish in each treatment and

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control group were challenged at 8 and 12 weeks post initial vaccination with CSF-259-93 at

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doses of ~ 3.0 x 107 cfu fish-1 and 2.29 x 107 cfu fish-1, respectively as described in section

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2.6. The ELISA was performed on 5 individual sera samples prepared from 5 fish collected

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from each treatment/control group at 0, 2, 4, 8, and 12 weeks of the experiment.

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2.8.5. Optimization of incubation time of vaccine production

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This experiment was carried out to determine the effect of two different incubation times (72

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h and 96 h) of the bacteria during the production of B.17-ILM vaccine on its protective

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efficacy. Two sets of vaccine broth cultures of B.17-ILM were set up and the vaccine was

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produced as described in section 2.2, except that one set of cultures were incubated for 72 h

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and the other was incubated for 96 h.

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fish/group) were vaccinated using either the 72 h cultures of vaccine (72 h-B.17-ILM) or the

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96 h vaccine cultures (96 h-B.17-ILM) as described in section 2.5. Control fish were

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immersed in TYES media. Fish were booster vaccinated two weeks following initial

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vaccination as described in section 2.5 when they reached a mean size of 3.0 g. Viable counts

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of bacteria present in the 72 h vaccine cultures used in the primary and booster vaccinations

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were ~ 3.38 x 108 cfu mL-1 and 2.70 x 108 cfu mL-1, respectively. Viable counts in the 96 h

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vaccine cultures used in the primary and booster vaccinations were ~ 1.58 X 1010 cfu mL-1

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and 9.17 X 1010 cfu mL-1, respectively. Fish in each treatment and control group were

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challenged at 8 and 12 weeks post initial vaccination by CSF-259-93 at doses of ~ 3.0 x 107

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cfu fish-1 and 2.29 x 107 cfu fish-1, respectively as described in section 2.6. The ELISA was

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performed on 5 individual sera samples prepared from 5 fish collected from each

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treatment/control group at 0, 2, 4, 8, and 12 weeks of the experiment.

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2.8.6. Statistical analyses

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Cumulative percent mortality (CPM) of each treatment or control group during 28 days after

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challenge was calculated for all experiments. The relative percent survival (RPS) was

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calculated for the vaccinated groups using the following formula:

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(1 – [CPM in vaccinated group/CPM in unvaccinated group]) x 100 [31]. Differences in CPM

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and antibody titer (log10 transformed) were determined using a one-way ANOVA (α = 0.05)

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after confirming residuals were normally distributed and variances were equal for both data

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sets. If the differences were significant (P < 0.05), a Tukey’s post-hoc test was carried out to

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determine which groups were different. Statistical analysis was done using GraphPad®Prism

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v5.03 (GraphPad, San Diego, CA, USA).

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Each treatment group (mean size, 2.6 g; 400

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ACCEPTED MANUSCRIPT 3. Results

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Initial screening of experimental fish for previous exposure to F. psychrophilum both by an

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ELISA assay and by culturing of kidney and spleen tissue samples from 10 fish did not show

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any detectable anti-F. psychrophilum antibodies in serum or viable cells in sampled tissues

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(data not shown).

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3.1.1. Vaccine delivery time

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Specific immune response in all vaccinated fish at all sampling points (2, 4, 6 and 8 weeks)

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were significantly higher (P < 0.05) than the control fish immersed in TYES medium (Fig. 1

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A-D). Specific immune response was not detected at week 0 in all treatment groups.

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Cumulative percent mortality (CPM) in all vaccinated groups except the 1.5 min group was

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significantly lower (P < 0.05) than the control groups (Fig. 2). As shown in Table 1, the

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relative percent survival (RPS) was just above 50 % in the fish groups vaccinated for 6 min

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and 30 min, followed by the 3 min vaccinated group (47 %) and the lowest was (37 %) in the

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1.5 min vaccinated group.

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3.1.2. Size at first vaccination

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The specific immune response as measured by ELISA titers in different treatment groups at

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specific sampling periods are summarized in Fig. 3A-D. Except for the 1 g size group at week

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2, all three vaccinated size groups at all sampling periods showed significantly higher ELISA

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titers (P < 0.05) compared to respective control groups. Specific immune response was not

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detected at week 0 in all treatment groups. Cumulative percent mortality (CPM) in all

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vaccinated groups were significantly lower (P < 0.05) than the unvaccinated control group

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(Fig. 4). The RPS values of different size groups of vaccinated fish are given in table 2. The

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ACCEPTED MANUSCRIPT RPS in 2 g and 1 g size vaccinated fish were about 60 % compared to a slightly lower RPS

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value of 55 % in the 0.5 g vaccinated fish.

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3.1.3. Dose and duration of protection

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All fish groups vaccinated with different doses of the vaccine at all sampling periods showed

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significantly higher antibody titers (P < 0.05) compared to TYES control groups (Fig. 5A-E).

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At week 4, the mean ELISA titers of fish in the 1010-B.17-ILM dose group was significantly

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higher (P < 0.05) than that of 106-B.17-ILM and 105-B.17-ILM vaccinated fish. Similarly, in

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the same week mean ELISA titer of 108-B.17-ILM was significantly higher (P < 0.05) than

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that of 105-B.17-ILM. The ELISA titers among all vaccinated groups did not differ

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significantly in all other weeks of sampling. Specific immune response was not detected at

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week 0 in all treatment groups.

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Cumulative percent mortality (CPM) in the TYES control group after challenge at week 8

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was significantly higher (P < 0.05) than all vaccinated groups (Fig. 6A). The CPM in the low

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dose vaccinated groups (106-B.17-ILM and 105-B.17-ILM) were significantly higher (P <

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0.05) than that of 1010-B.17-ILM vaccinated fish. At the same time, CPM in 108-B.17-ILM,

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106-B.17-ILM and 105-B.17-ILM vaccinated groups did not differ significantly among each

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other (Fig. 6A).

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When subgroups from the same treatment groups of fish were challenged at week 12, the

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CPM in all vaccinated groups were not significantly different from each other (Fig. 6B).

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However, the CPM in the TYES control group was significantly higher than all vaccinated

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groups (P < 0.05). When subgroups of fish were challenged again at 24 week after initial

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vaccination, the CPM in the high dose vaccinated groups, 1010-B.17-ILM and 108-B.17-ILM

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were significantly lower (P < 0.05) than that of control fish (Fig. 6C). However, the CPM in

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ACCEPTED MANUSCRIPT the low dose vaccinated groups (106-B.17-ILM and 105-B.17-ILM) were not significantly

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different from that of control fish.

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The RPS values of vaccinated fish are summarized in Table 3. Fish initially vaccinated with

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the highest dose of vaccine at ~ 1.58 x 1010 (1010-B.17-ILM) provided maximum level of

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protection with RPS values of 67 %, 61 % and 70 %, respectively, when challenged at 8, 12

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and 24 week after initial immunization. The protection was found slightly reduced at week 12

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and then reached highest levels at week 24 after initial immunization. Vaccination with the

307

next lower dose of ~ 3.0 x 108 cfu mL-1 (108-B.17-ILM) yielded RPS values of 40 %, 48 %

308

and 55 % at week 8, 12 and 24, respectively. Fish vaccinated with further lower doses (~ 1.0

309

x 106 cfu mL-1 and 2.5 x 105 cfu mL-1) yielded somewhat similar RPS values; however, their

310

RPS values were lower than that of fish vaccinated with higher doses of the vaccine at all

311

three challenges. The RPS values of fish groups vaccinated with lower doses (~ 1.0 x 106 cfu

312

mL-1 and 2.5 x 105 cfu mL-1) reached highest levels at 12 week challenge and then the

313

protection was found slightly reduced at 24 week challenge.

314

3.1.4. Booster frequency

315

Analysis of specific immune response by ELISA showed that all three fish groups vaccinated

316

with different booster frequencies produced significantly higher titers (P < 0.05) than the

317

unvaccinated control group at week 2, 4 and 8 (Fig. 7). ELISA titers among the three

318

vaccinated groups were not significantly different at all sampling points. However, the

319

ELISA titers of fish vaccinated without any booster and those boostered twice at week 12

320

were not significantly different from that of the unvaccinated control group. The specific

321

antibody response in the fish boostered twice did not increase appreciably over time despite a

322

second booster vaccination at week 4. Also there was some increase in the ELISA titers of

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ACCEPTED MANUSCRIPT the control fish group by week 12 (Fig. 7). Specific immune response was not detected at

324

week 0 in all treatment groups.

325

Figure 8A & B show CPM in fish challenged at week 8 and 12, respectively after initial

326

immunization. At week 8, CPM in all vaccinated groups were significantly lower (P < 0.05)

327

than that of control. Fish group receiving a single booster vaccination showed the lowest

328

CPM, which was significantly lower (P < 0.05) than that of fish boostered twice or not

329

boostered at al. Fish group that did not get any booster vaccination suffered heavy mortality

330

with a CPM of 80 %. Fish that were boostered twice had a significantly higher CPM (P <

331

0.05) than those boostered only once.

332

When challenged at week 12, the CPM in the TYES control group was significantly higher (P

333

< 0.05) than that of one booster-B.17-ILM and two booster-B.17-ILM. However, fish that

334

was not boostered again suffered heavy mortality with a CPM of 80 % and was not

335

significantly different from that of TYES control group. More interestingly, the CPM in fish

336

groups that were boostered once and twice did not differ significantly each other with lower

337

CPM values of 36 % and 48 %, respectively. The RPS values after challenge at week 8 and

338

12 was highest in the one booster-B.17-ILM group (67 % and 61 %), followed by the two

339

booster-B.17-ILM (56 % and 48 %), and was lowest in the fish group that did not receive any

340

booster vaccination (17 % and 13 %) (Table 4). Administration of second booster vaccination

341

produced higher CPM and a lower RPS than a single booster in both the challenges at week 8

342

and 12.

343

3.1.5. Incubation time of vaccine production

344

Specific immune response measured by ELISA showed that fish vaccinated by B.17-ILM

345

vaccine either grown for 72 h or 96 h produced significantly higher ELISA titers than

346

unvaccinated control fish at 2, 4, 8 and 12 weeks after initial immunization (Fig. 9A-D).

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ACCEPTED MANUSCRIPT However, ELISA titers between fish groups vaccinated by 72 h and 96 h grown vaccine did

348

not differ significantly during the entire period of vaccination up to 12 weeks. Specific

349

immune response was not detected at week 0 in all treatment groups.

350

The CPM after challenge at week 8 and 12 in the unvaccinated control fish was significantly

351

higher (P < 0.05) than that of both 72 h-B.17-ILM and 96 h-B.17-ILM vaccinated fish (Fig.

352

10A & B). The CPM at week 8 in the 72 h-B.17-ILM was significantly higher (P < 0.05) than

353

that of 96 h-B.17-ILM (Fig. 10A). However, the CPM at week 12 challenge did not differ

354

significantly between the two vaccinated groups (Fig. 10B). As shown in Table 5, the RPS

355

values of 96 h-B.17-ILM group challenged at week 8 and 12 were higher (67 % and 61 %,

356

respectively) than that of 72 h-B.17-ILM group (40 % and 48 %, respectively).

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4. Discussion

359

Vaccination has become a critical component of the modern fish farming industry. When

360

developing an aquaculture vaccine, optimizing multiple parameters associated with safety

361

and efficacy is essential and required to produce a cost-effective product that can be licensed

362

and commercially marketed. Recently, a live attenuated vaccine capable of protecting fish

363

from BCWD has been developed [25,26,32]. This study provides results from optimization

364

trials testing different parameters affecting the efficacy of vaccination in RBT such as size of

365

fish at first vaccination, vaccine delivery time, dose, duration of protection, booster

366

frequency, and vaccine culture incubation time. Fish were immersion vaccinated with the

367

B.17-ILM vaccine and efficacy was determined by measuring specific antibody responses as

368

well as CPM and RPS following pathogen challenge with a known virulent strain of F.

369

psychrophilum.

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ACCEPTED MANUSCRIPT Experiments involving a range of immersion delivery times showed that fish vaccinated with

371

B.17-ILM for different immersion time periods developed protective immunity against

372

BCWD challenge with RPS values ranging from 36 % to 52 %. An earlier vaccination trial

373

with the B.17-ILM vaccine elicited significant protection in Coho salmon following

374

immersion [26]. However, the delivery time in that trial was 1 h, and fish had their adipose

375

fin clipped just prior to vaccination in an attempt to enhance antigen uptake through the skin

376

opening. This would likely be impractical under a field vaccination situation, but the present

377

results demonstrate that shorter delivery duration is effective and there is no need to adipose

378

fin clip fish prior to vaccination to obtain significant protection. Results indicate that

379

immersion vaccination for 1.5 min is probably too short to provide sufficient protection

380

against BCWD, and immersion for 3 min or longer provides the greatest protection.

381

Commercial immersion vaccines may be administered by dipping fish in the vaccine solution

382

for shorter periods of 30 to 60 s [33,34]; however, several studies have demonstrated that

383

prolonged immersion can enhance the immune response in trout [35-39]. This is in agreement

384

with our results, but it should be realized that immersion times longer than 3 min did not

385

appear to provide a practical advantage at the dosage tested. It is possible that longer

386

immersion times may be beneficial if vaccine dose was lower, but from the data presented

387

here we would recommend at least a 3 min dip for vaccinating fish with this live attenuated

388

vaccine.

389

Another consideration for successful vaccination of fish is to determine the minimum size of

390

fish that can be vaccinated effectively. This has the advantage of increasing the numbers of

391

smaller size fish that may be vaccinated per unit of vaccine, and providing earlier protection

392

for fish that may be susceptible to BCWD. It was found that 0.5 g, 1.0 g and 2.0 g RBT were

393

able to mount a specific antibody response against F. psychrophilum, and they were

394

significantly protected from pathogen challenge. The vaccine provided a similar level of

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ACCEPTED MANUSCRIPT protection for 1.0 g and 2.0 g fish, but RPS values were slightly lower for 0.5 g fish. It is well

396

understood that development of immune related organs and production of cells and molecules

397

associated with nonspecific and specific immunity in fish begins at very early stages of life

398

[40-43]. It has been shown that the expression of humoral molecules such as IgM and a true

399

functionally competent specific immune response involving immunological memory appears

400

to develop in fish larvae of 20-30 mm in size or later in most freshwater species [42]. From a

401

practical point of view, the minimum size at which maximum protective immunity can be

402

achieved in salmonid fish is generally considered to be between 1.0 to 2.5 g [44].

403

Interestingly, the B.17-ILM vaccine was able to confer protection even to 0.5 g fish, which is

404

important considering the dynamics of BCWD and the potential to vaccinate fish at a very

405

early size.

406

Another important practical consideration for optimizing fish vaccines is to optimize dose

407

and determine the duration of protection. Fish vaccinated in this study with four different

408

doses of the B.17-ILM vaccine developed significantly higher levels of specific anti-

409

F.psychrophilum antibodies than unvaccinated control fish by two weeks post initial

410

immunization. Antibody levels were sustained through 24 weeks, and fish vaccinated with

411

the lowest dose of vaccine (~ 2.5 x 105 cfu mL-1) had antibody responses comparable to fish

412

vaccinated with higher doses. Different degrees of protection were obtained in a somewhat

413

dose dependent manner when subgroups of fish from each treatment group were challenged

414

at week 8, 12 and 24. The highest RPS values were obtained in fish vaccinated with highest

415

dose (~ 1.58 x 1010) and the lowest RPS values in fish vaccinated with lower doses (~ 1.0 x

416

106 cfu mL-1 and 2.5 x 105 cfu mL-1) of the vaccine at all three challenges. As antigen uptake

417

during immersion vaccination is mainly through the skin [45,46], gill [46,47] and gut [48,49]

418

of fish, the number of viable cells of the vaccine present in the vaccine solution or the

419

administered dose may have a direct bearing on efficiency of uptake [50]. Earlier reports of

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ACCEPTED MANUSCRIPT oral immunization studies used high doses of vaccine. In one study, rainbow trout fed

421

formalin treated freeze-dried vibrio vaccine at a dose of 1012 cfu fish-1 resulted in 78 % RPS,

422

whereas, feeding 1011 cfu fish-1 and lower doses resulted in much lower levels of protection

423

[51]. In another study an RPS of up to 70 % was obtained when rainbow trout were fed an

424

oral vaccine at a dose of 1012 cfu 100 g fish-1 [52]. In adult and immature ayu, immunization

425

against vibriosis was found most effective when a dose of 3 x 109 cfu fish-1 was given daily

426

for 8-14 days [53]. It was even suggested that a Vibrio anguillarum vaccine dose of less than

427

108 cfu fish-1 may be too low to produce immunity by the oral route [54]. In the case of

428

immersion vaccination, the amount of antigen taken up by each fish is considered small and

429

is in the range of 0.01-0.2 % of the initial vaccine bath concentration [55]. One study has

430

demonstrated that uptake of BSA-conjugated fluorescent latex microspheres from bath

431

suspensions by 1.5–3.0 g rainbow trout is logarithmically proportional to particle

432

concentration in suspensions [56]. Thus, increasing vaccine dose in the immersion medium

433

can provide more antigens available for uptake by the fish. It has been observed that reducing

434

the antigen dose in immersion bath vaccinations will require proportionally longer delivery

435

times for antigen uptake to occur [57].

436

The protection offered by all doses of the vaccine for a prolonged period of time (up to 24

437

weeks post initial vaccination) is important and demonstrates that protection can be achieved

438

through the most susceptible periods and through critical growout stages. Vaccinating fish

439

with B.17-ILM vaccine grown for 96 h resulted in better protection than with 72 h vaccine

440

cultures despite comparable specific immune response produced by both vaccines. This

441

enhanced protection may be due to the different antigenic profiles of the live vaccine when

442

grown for 96 h; however, this was not confirmed. It is possible that changes in gene

443

expression led to altered antigenicity of the vaccine, or changes in receptor expression on the

444

surface of the bacteria may have enhanced binding of the live attenuated F. psychrophilum

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ACCEPTED MANUSCRIPT cells and increased antigen uptake. Correlations between in vitro growth phase and changes

446

in gene expression, including virulence factors have been well recognized in other bacterial

447

species [58-61].

448

Our results clearly demonstrate that a booster vaccination is required for maximum protection

449

from BCWD; however, an additional booster does not provide an advantage. Protection was

450

not adequate without a booster vaccination, despite the development of anti-F. psychrophilum

451

antibodies. This is not completely surprising, as specific serum antibody levels do not always

452

parallel the level of protection offered by this vaccine [26,32]. Durbin et al. [62] have

453

observed low antibody titers and high protection in fish vaccinated with killed bacteria grown

454

in ILM versus killed bacteria grown in regular media. These and other findings [63,64]

455

support our findings, but in general if specific antibody titers are highly elevated we would

456

expect to see a protective response. One thing to consider is that vaccination with live

457

attenuated vaccines may elicit other aspects of the immune response. Attenuated vaccines

458

have been shown to produce significantly greater T-cell proliferation as compared to B-cells,

459

resulting in enhanced cell mediated [64-66] and innate immunity [67]. More recently, it has

460

been shown that expression levels of secretory IgD and IgT in gills are significantly

461

upregulated in RBT immersion vaccinated with B.17-ILM vaccine [68], which suggests that

462

mucosal immunity may be important. We only monitored serum antibody levels in this study.

463

In conclusion, this study demonstrates that this B.17-ILM vaccine can provide strong

464

protection from BCWD in RBT when administered for 3 min by immersion. This would be

465

practical in a hatchery setting using standard vaccination protocols. Furthermore, we have

466

shown that fish as small as 0.5 g can be effectively protected against BCWD, which usually

467

affects smaller sized fish. Immersion vaccination with the B.17-ILM vaccine provides

468

protection out to at least 24 weeks post initial immunization. A vaccine dose as low as ~105

469

cfu mL-1 can provide significant protection for fish, but higher doses appear to provide longer

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ACCEPTED MANUSCRIPT protection. Taken together, this study provides important optimization criteria that supports

471

the need to continue development of this live attenuated B.17-ILM vaccine as a critical tool

472

for combating BCWD in aquaculture facilities.

473

Acknowledgement

474

We gratefully acknowledge the financial support provided by the Idaho Department of

475

Commerce through the Idaho Global Entrepreneurial Mission (IGEM grant # 13587). We

476

thank Scott Williams at the Aquaculture Research Institute, University of Idaho for providing

477

fish used in this study.

478

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479

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[51] E. Anders, Experimental oral immunization of rainbow trout and eel against Vibrio anguillarum, Forschung Wissenshaftliche Schriereine 16 (1978) 59-60.

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against vibriosis: Comparison of an extract antigen with whole cell bacteria by oral and

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intra-peritoneal routes, J. Fish Dis. 6 (1983)129-134.

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[53] K. Kawai, R. Kusuda, Field testing of oral Vibrio anguillarum bacterin in pond-cultured ayu, Fish Pathol. 20 (1985) 413-419.

[54] R. Campbell, A. Adams, M. F, Tatner, M. Chair, P. Sorgeloos, Uptake of Vibrio anguillarum vaccine by Artemia salina as a potential oral delivery system to fish fry,

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Fish Shellfish Immunol. 3 (1993) 451-459.

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[55] M.F. Tatner, M.T. Horne, Factors influencing the uptake of 14C-labelled Vibrio anguillarum vaccine in direct immersion experiments with rainbow trout, Salmo

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gairdneri Richardson, J. Fish Biol. 22 (1983) 585–591.

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[56] J.D. Moore, M. Ototake, T. Nakanishi, Particulate antigen uptake during immersion

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immunisation of fish: The effectiveness of prolonged exposure and the roles of skin and

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gill, Fish Shellfish Immunol. 8(6) (1998) 393-408.

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[57] M.F. Tatner, The quantitative relationship between vaccine dilution, length of immersion

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time and antigen uptake, using a radiolabeled Aeromonas salmonicida bath in direct

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immersion experiments with rainbow trout, Salmo gairdneri, Aquaculture 62 (1987)

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173–185.

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[58] M.M. Nakamura, S-Y. Liew, C.A. Cummings, M.M. Brinig, C. Dieterich, D.A. Relman,

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Growth Phase- and Nutrient Limitation-Associated Transcript Abundance Regulation in

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Bordetella pertussis, Infect.Immun. 74(10) (2006) 5537-5548. [59] T.L. Nicholson, A.M. Buboltz, E.T. Harvill, S.L. Brockmeier, Microarray and functional

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analysis of growth phase-dependent gene regulation in Bordetella bronchiseptica, Infect.

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Immun. 77(10) (2009) 4221-31.

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[60] I. Sitkiewicz, J.M. Musser, Analysis of growth-phase regulated genes in Streptococcus agalactiae by global transcript profiling, BMC Microbiol. 10 (2009) 9-32.

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[61] S. Klumpp, T. Hwa, Bacterial growth: global effects on gene expression, growth feedback and proteome partition, Current Opin. Biotechnol. 28 (2014) 96–102.

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[62] M. Durbin, D. McIntosh, P.D. Smith, R. Wardle, B. Austin, Immunization against

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furunculosis in rainbow trout with iron-regulated outer membrane protein vaccines:

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relative efficacy of immersion, oral, and injection delivery, J. Aquat. Anim. Health 11

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(1999) 68-75.

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[63] S. Vervarcke, F. Ollevier, R. Kinget, A. Michoel, Mucosal response in African catfish after administration of Vibrio anguillarum O2 antigens via different routes, Fish

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Shellfish Immunol. 18 (2005) 125-133.

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[64] P.I. Angelidis, D. Karagiannis, E.M. Crump, Efficacy of a Listonella anguillarum (syn.

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Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax, Dis. Aquat.

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Organ. 71(1) (2006) 19-24.

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[65] M.J. Marsden, L.M. Vaughan, T.J Foster, C.J. Secombes, A live (delta aroA) Aeromonas

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salmonicida vaccine for furunculosis preferentially stimulates T-cell responses relative to

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B-cell responses in rainbow trout (Oncorhynchus mykiss), Infect. Immun. 64 (1996)

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3863-9.

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[66] C. H. Moral, E. F. Del Castillo, P. L. Fierro, A.V. Cortés, J.A. Castillo, A.C. Soriano,

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S.M. Salazar, R.B. Peralta, N.G. Carrasco, Molecular characterization of the Aeromonas

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hydrophila aroA gene and potential use of an auxotrophic aroA mutant as a live

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attenuated vaccine, Infect Immun. 66 (1998) 1813-21. [67] R. Russo, C.A. Shoemaker, V.S. Panangala, P.H. Klesius, In vitro and in vivo interaction

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of macrophages from vaccinated and non-vaccinated channel catfish (Ictalurus

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punctatus) to Edwardsiella ictaluri, Fish Shellfish Immunol. 26 (2009) 543-52.

[68] M. Makesh, P.S. Sudheesh, K.D. Cain, Systemic and mucosal immune response of trout

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to immunization by different routes, Fish Shellfish Immunol. 44(1) (2015) 156-163.

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Figure 4.

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ACCEPTED MANUSCRIPT Figure captions

702

Fig. 1. Biweekly mean ELISA titer ± SEM (n = 5) of RBT vaccinated with B.17-ILM vaccine

703

for different vaccine delivery periods. A: Week 2, B: Week 4, C: Week 6 and D: Week 8 post

704

initial vaccination. Asterisk indicate significant difference (P < 0.05) in ELISA titers of

705

vaccinated fish from respective controls. Initial (Week-0) ELISA titers were 0 for all

706

treatments.

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Fig. 2. Cumulative percent mortality (CPM) ± SEM (n = 75) of RBT vaccinated with B.17-

709

ILM vaccine for different immersion delivery periods and then challenged with CSF-259-93

710

at week 8. Asterisk indicate significant difference (P < 0.05) between vaccinated and

711

respective control groups.

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708

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Fig. 3. Biweekly mean ELISA titer ± SEM (n = 5) in different size groups of RBT vaccinated

714

with B.17-ILM vaccine. A: Week 2, B: Week 4, C: Week 6 and D: Week 8 post initial

715

vaccination. Asterisk indicate significant difference (P < 0.05) in ELISA titers of vaccinated

716

fish from respective controls. Initial (Week-0) ELISA titers were 0 for all treatments.

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Fig. 4. Cumulative percent mortality (CPM) ± SEM (n = 75) in different size groups of RBT

719

vaccinated with B.17-ILM vaccine and then challenged with CSF-259-93 at week 8. Asterisk

720

indicate significant difference (P < 0.05) between vaccinated and respective control groups.

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721 722

Fig. 5. Biweekly mean ELISA titer ± SEM (n = 5) of RBT vaccinated with different doses of

723

B.17-ILM vaccine. A: Week 2, B: Week 4, C: Week 8, D: Week 12 and D: Week 24 post

724

initial vaccination. Different letters indicate significant difference (P < 0.05) and shared or

41

ACCEPTED MANUSCRIPT 725

same letters indicate no significant difference between treatment groups. Initial (Week-0)

726

ELISA titers were 0 for all treatments.

727

Fig. 6. Cumulative percent mortality (CPM) ± SEM (n = 75) of RBT vaccinated with

729

different doses of B.17-ILM vaccine and then challenged with CSF-259-93 at A: Week 8, B:

730

Week 12 and C: Week 24 post initial vaccination. Different letters indicate significant

731

difference (P < 0.05) and shared or same letters indicate no significant difference between

732

treatment groups.

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733

Fig. 7. Biweekly mean ELISA titer ± SEM (n = 5) of RBT vaccinated and boostered with

735

B.17-ILM vaccine at different frequencies. A: Week 2, B: Week 4, C: Week 8 and D: Week

736

12 post initial vaccination. Different letters indicate significant difference (P < 0.05) and

737

shared or same letters indicate no significant difference between treatment groups. Initial

738

(Week-0) ELISA titers were 0 for all treatments.

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Fig. 8. Cumulative percent mortality (CPM) ± SEM (n = 75) of RBT vaccinated and

741

boostered with B.17-ILM vaccine at different frequencies and then challenged with CSF-259-

742

93 at A: Week 8 and B: Week 12 post initial vaccination. Different letters indicate significant

743

difference (P < 0.05) between treatment groups.

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Fig. 9. Biweekly mean ELISA titer ± SEM (n = 5) of RBT vaccinated with B.17-ILM vaccine

746

previously grown for 72 h and 96 h. A: Week 2, B: Week 4, C: Week 8 and D: Week 12 post

747

initial vaccination. Different letters indicate significant difference (P < 0.05) between

748

treatment groups. Initial (Week-0) ELISA titers were 0 for all treatments.

749

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ACCEPTED MANUSCRIPT Fig. 10. Cumulative percent mortality (CPM) ± SEM (n = 75) of RBT vaccinated with B.17-

751

ILM vaccine previously grown for 72 h and 96 h and then challenged with CSF-259-93 at A:

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Week 8 and B: Week 12 post initial vaccination. Different letters indicate significant

753

difference (P < 0.05) between treatment groups

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Table 1. Relative percent survival (RPS) of RBT vaccinated with B.17-ILM vaccine for

756

different immersion delivery periods and then challenged with CSF-259-93 at week 8.

RPS (%)

30 min vaccinated

52

6 min vaccinated

53

3 min vaccinated

47

1.5 min vaccinated

36

757

758

759

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Treatments

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763

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Table 2. Relative percent survival (RPS) in different size groups of RBT vaccinated with

765

B.17-ILM vaccine and then challenged with CSF-259-93 at week 8. 766

Treatments

RPS (%)

2 g-ILM

60 768

1 g-ILM

59

0.5 g-ILM

55

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772

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Table 3. Relative percent survival (RPS) of RBT vaccinated with different doses of B.17-

774

ILM vaccine and then challenged with CSF-259-93 at 8, 12 and 24 week post initial

775

vaccination.

Week 12

Week 24

1010-B.17-ILM

67

61

70

108-B.17-ILM

40

48

55

106-B.17-ILM

35

43

36

105-B.17-ILM

29

45

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Treatment groups

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RPS (%)

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Table 4. Relative percent survival (RPS) of RBT vaccinated and boostered with B.17-ILM

778

vaccine at different frequencies and then challenged with CSF-259-93 at 8 and 12 week post

779

initial vaccination. 780

Treatments Week 12

One booster-0.3-B.17-ILM

67

61

Two booster-0.3-B.17-ILM

56

48

No booster-0.3-B.17-ILM

17

13

781

782

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Week 8

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RPS (%)

783

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Table 5. Relative percent survival (RPS) of RBT vaccinated with B.17-ILM vaccine

789

previously grown for 72 h and 96 h and then challenged with CSF-259-93 at 8 and 12 week

790

post initial vaccination. -1

RPS (%)

Treatments

Week 8

Week 12

67

61

3.0 x 10

72 h-B.17-ILM

3.0 x 10

7

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7

96 h-B.17-ILM

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Challenge dose (cfu fish )

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48

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ACCEPTED MANUSCRIPT
B.17-ILM vaccine can provide strong protection from bacterial coldwater disease in rainbow trout when administered for 3 min by immersion.


Rainbow trout as small as 0.5 g can be effectively protected against bacterial

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coldwater disease, which usually affects smaller sized fish.

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24 weeks post initial immunization.

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Immersion vaccination with the B.17-ILM vaccine provides protection out to at least


A vaccine dose as low as ~105 cfu mL-1 can provide significant protection for fish, but higher doses appear to provide longer protection.

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A booster dose is required to provide optimum protection and the vaccination approach would be practical in a hatchery setting using standard vaccination

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protocols.