Fish maintenance, immunisation and sampling

4.3.3.1.Fish source, maintenance conditions, and experimental setup

Apparently healthy rainbow trout fry, with a mean weight of 0.1 g and no known previous exposure to pathogens, were obtained from the University of Idaho’s Aquaculture Research Institute (ARI), Moscow, ID, USA. They were held in 500 L tanks supplied with de-chlorinated, single-pass, filtered municipal water at 14°C for three weeks, and fed a commercially available trout pellet feed (Rangen, Idaho, USA). Fish were grown to a mean weight of 3.1 g and then separated into treatment groups. Treatments consisted of intraperitoneal (IP) injection or oral administration of alginate-microencapsulated live B17, or an appropriate control treatment (Table 4.1). Each treatment group was comprised of 136 randomly selected fish held in a 500 L tank, with one tank allocated per treatment. On Day 56 of the trial, fish from each treatment were randomly allocated to one of 48 identical 9 L tanks, with 25 fish per tank. Each treatment group comprised of four replicate tanks, of which one was excluded from bacterial challenge (mock-infected) to provide challenge controls, as summarised in Table 4.1.

4.3.3.2.Oral immunisation

Oral immunisation was initiated on day 1 of the trial, coinciding with administration of intraperitoneal injection immunisation. Commercially available trout pellet feed was used to prepare all oral treatments. Unbroken microcapsule emulsion was mixed with feed at a ratio of 1:5 (w/w) and stirred thoroughly to coat feed with emulsion (B17-µ-Oral). Coated feed was prepared daily, and stored at 4°C until use. Using the same additive to feed ratio (1:5 w/w) control treatments were prepared by coating feed with non-microencapsulated B17 (B17-non-µ-Oral) or blank

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microcapsules suspended in canola oil – PBS emulsion (blank-Oral), as summarised in Table 4.1. A placebo/treatment-control consisted of feed similarly coated with only canola oil-PBS emulsion (con-Oral). Feed was withheld from fish for 24 h prior to initiation of oral immunisation. Each oral treatment was administered at an actual feed weight of 3% of total biomass per tank per day over 54 days. To minimise potential oral tolerance, oral treatments were administered as per a staggered 18- day feeding regimen comprised of 7 days of treated feed followed by 11 days of untreated feed, repeated thrice before bacterial challenge.

Samples of microencapsulated bacterial emulsion were enumerated on TYES agar plates in triplicate to determine CFU mL-1, but subjected to citrate digestion of microcapsules prior to plating. This was achieved by adding each sample to an equal volume of 0.1 M aqueous sodium citrate solution, which was vortexed at high speed for 1 min and allowed to stand for 10 min at 4°C before serial-dilution and plating. In preliminary tests, 10 min of exposure to citrate buffer at 4°C resulted in approximately 10-fold reduction in viability of B17 (data not shown), and this reduction was accounted for in calculating viability of bacteria post-microencapsulation. Samples of unencapsulated bacterial suspension used to prepare immunisation treatments were similarly enumerated on TYES plates, without inclusion of the citrate-digestion step.

4.3.3.3.Injection immunisation

Fish were injected intraperitoneally (IP) with microencapsulated B17 or one of three controls. Initial administration of treatments occurred on day 1 of the trial, and corresponded with the commencement of oral treatments. An identical booster immunisation was administered to coincide with commencement of the final 18-day oral immunisation regimen (day 37). To prepare microencapsulated IP treatment (B17-µ-IP), microcapsule emulsion was broken and microcapsules recovered as in section 2.2.2. Washed B17-loaded microcapsules were suspended in PBS (OD525 ≈ 0.39) by vortexing gently for 1 min. Control treatments were prepared by suspending non- microencapsulated bacteria (OD525 ≈ 0.42; B17-non-µ-IP) or blank microcapsules (OD525 ≈ 0.31;

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blank-IP) in PBS, and are summarised in Table 4.1. A placebo/treatment-control was also prepared, using only PBS for injection (con-IP). Samples of each treatment containing bacteria were enumerated on TYES plates in triplicate to determine CFU mL-1. Prior to plating, the microencapsulated treatment samples were subjected to citrate digestion as described in section 2.3.2.

Table 4.1: Summary of oral and intraperitoneally (IP) injected immunisation treatments (and controls) administered to rainbow trout before challenge with F. psychrophilum, and abbreviated labels used in text

Treatment Delivery

method Group Label Fish/Tank No. of Tanks

Microencapsulated B17 Oral B17-µ-Oral 25 3 (+1 mock infected)

Non-microencapsulated B17 Oral B17-non-µ-Oral 25 3 (+1 mock infected)

Blank microcapsules Oral blank-Oral 25 3 (+1 mock infected)

Control/Placebo (feed coated

with canola oil/PBS ) Oral con-Oral 25 3 (+1 mock infected) Micro-encapsulated B17 IP injection B17-µ-IP 25 3 (+1 mock infected)

Non-encapsulated B17 IP injection B17-non-µ-IP 25 3 (+1 mock infected)

Blank microcapsules IP injection blank-IP 25 3 (+1 mock infected)

Control/Placebo (PBS only) IP injection con-IP 25 3 (+1 mock infected)

Feed was withheld from fish for 24 h before immunisation. Prior to injection, fish were anaesthetised with 50 ppm tricaine methanesulfonate (MS-222, Argent, Redmond, WA, USA) until loss of swimming equilibrium was evident. Each treatment (25 µL; B17-µ-IP: 1.8 x 107 CFU fish-1 and B17-non-µ-IP: 1.6 x 108 CFU fish-1) was administered intra-peritoneally and fish were placed in a recovery bath before being returned to the appropriate tank. Commencing 24 h post immunisation, fish were fed untreated commercial pellet feed at 3% of total biomass per tank per day until challenged.

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4.3.4.Sampling

Samples were collected before initial immunisation on day 1 and at day 19, 37 and 55. At each time point 12 fish were sampled from each treatment. Each fish was euthanised by lethally anaesthetising with an overdose of MS-222, and then exsanguinated by severing the caudal peduncle. Blood was collected in a 1.5 mL centrifuge tube and allowed to clot overnight at 4°C. The next day, samples were centrifuged (15000 xg) for 5 min and sera collected. Serum samples were stored at -80°C until used to evaluate immune response through an enzyme-linked immunosorbent assay (ELISA). Serum collected from fish prior to immunisation was pooled and used as the negative control for ELISA. Kidney, spleen and liver samples were also obtained from each fish, inoculated onto TYES agar plates and incubated for 96 h at 15˚C to test for any pre-existing F. psychrophilum infection. Reisolation of F. psychrophilum was attempted from at least 20% of daily mortalities per tank throughout the challenge period by inoculating TYES plates with kidney, spleen and liver samples, and incubating for 96 h at 15˚C.

4.3.5.F. psychrophilum challenge

Bacterial challenge was initiated on day 57, at a mean fish weight of 9.7 g, following previously published methods [201]. Fish from the challenge control tanks were mildly anaesthetised as described, and injected with 50 μL of PBS subcutaneously posterior to the adipose fin. All other fish were similarly anaesthetised and challenged with 50 μL of previously prepared F. psychrophilum culture (at 8 ×108 CFU per fish) injected subcutaneously posterior to the adipose fin. Feeding with untreated commercial pellet feed was resumed 48 h post-challenge at 1% total biomass per tank per. Tanks were monitored for mortalities, which were sampled as described, for 21 days post-challenge. Cumulative percent mortality (CPM) from each treatment was used to calculate the relative percent mortality (RPS) as:

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