Atypical Aeromonas salmonicida vapA type V and Vibrio spp

16 Healthy and / or moribund farmed and wild ballan wrasse, Labrus bergylta (>0.5 to 900 g) 17 were sampled from hatcheries (n= 2) and Atlantic salmon cage sites (n= 8) in Scotland 18 between February 2016 and October 2018. Less than half of the sampled individuals (n= 43, 19 32.3 %) had been vaccinated (autogenous polyvalent vaccine; dip and / or injection) against 20 atypical furunculosis (type V and VI) while 20 (15.0 %) fish were not vaccinated and the 21 rest (70 individuals, 52.7 %) were of unknown vaccination status. Swab samples from skin 22 lesions, gill, liver, spleen and kidney were inoculated onto a variety of bacteriological agar 1 plates and bacteriology identification and sequencing analysis was performed on significant 2 bacterial colonies. Atypical Aeromonas salmonicida (aAs) vapA type V was the predominant 3 bacterial species (70/215 bacteria isolates; 32.5 % of bacteria samples – 43/117 positive 4 individual fish; 36.8 %) isolated in this survey followed by Vibrio species which were the 5 most geographically prevalent bacteria. Photobacterium indicum/profundum was also 6 isolated from L. bergylta for the first time during this study. The collection of these bacterial 7 isolates provides useful information for disease management. Identifying the aAs isolates 8 involved in disease in ballan wrasse could provide vital information for improving / updating 9 existing autogenous vaccines. 10


INTRODUCTION
for the species L. bergylta in order to improve their welfare in aquaculture and to enable 1 their efficient performance as cleaner fish in salmon pens. Health screening and 2 characterisation of these bacterial pathogens is essential for successful vaccine formulation. 3 Thus, in the current study, a real-time health survey was conducted to determine the bacterial 4 pathogens present in both farmed ballan wrasse hatcheries and Atlantic salmon cage sites 5 (wild and farmed fish) in Scotland between February 2016 and October 2018, in order to 6 identify the most prevalent bacterial pathogens of ballan wrasse. originating form site A and had been vaccinated by two immersions (prime; ca. 0.5 g and 16 booster vaccination; ca. 2 g) and / or injection at ca. 15 g and all the fish in the batches from 17 which these individuals originated from had been vaccinated with the same practice. There 18 was one more individual that had been vaccinated however, no information has been 19 provided and whether the rest of the cleaner fish on site had been vaccinated. Furthermore, 20 16 (12.0%) fish were unvaccinated and the rest of the individuals (n=74, 55.6%) were of 21 unknown vaccination status (farmed or wild origin). Swab samples from skin lesions, gills, 22 liver, and kidney were inoculated onto Marine Agar, Tryptone Soya Agar (TSA), and TSA 23 + 5% Defibrinated Horse Blood + 1.5% NaCl, and incubated at 22°C for 24 -72h for primary 24 bacterial isolation. Pure colonies were then picked on the basis of morphology, predominance and prevalence, streaked onto fresh plates and incubated, as described before, 1 for purification. Passaged isolates were then tested by Gram's staining (bioMerieux) and 2 Catalase (catalse reagents. VWR UK)/ Oxidase (oxidase strips, Oxoid UK) tests for purity 3 confirmation and primary identification.  (Table 1). PCR reactions consisted of each primer at 10 μM, 1 unit 10 of GoTaqG2 master mix (Promega), 5 μL of DNA sample and milliQ water to reach a final 11 reaction volume of 25 μL. The following thermal cycling conditions were used in G-storm 12 thermocycler: 1 cycle at 95°C for 5 min, 35 cycles at 95°C for 30 sec, 55°C (gyrB) and 44°C 13 (16 rRNA) for 30 sec and 73°C for 1 min, followed by 1 cycle at 73°C for 7 min. The PCR 14 product was then purified using QIAquick PCR Purification Kit (Qiagen, Germamy) as 15 described by the manufacturer and 3.5 μL of the clean-up were mixed with 2.5 μL of each 16 of the forward and reverse primers in a separate nuclease free Eppendorf tube and 1.5 μL of 17 nuclease free water to reach a total volume of 7.5 μL. Products sent for sequencing to GATC 18 (Eurofins) and obtained sequences were compared to known sequences using an in silico 19 nucleotide alignment tool 'BLAST' (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Isolates that 20 were recognised as presumed aAs by the naked eye; small, friable colonies, non-motile 21 coccobacilli (prior to 16S confirmation) or by PCR testing (16S) were then confirmed to be 22 aAs using the A-layer membrane -vapA primer sets (Gulla et al. 2016) to determine the vapA 23 strain type (Table 1) as described by Gulla et al. (2016). The PCR product was then purified 24 using QIAquick PCR Purification Kit (Qiagen, Germamy) and samples mixed with forward 25 and reversed primers as described above and sent for sequencing to GATC (Eurofins).
Sequences were analysed with Clustal Omega at EMBL-EBI (https://www.ebi.ac.uk/) 1 against the published type strain sequences.   2). The majority of aAs vapA type V had been isolated from liver (25) and kidney (32), while 19 the least aAs recovery was noted from fin (5), skin (4) and gill (2) samples. Also the aAs 20 vapA type VI isolates (2) were from skin, liver and kidney of deployed ballan wrasse. The Pseudoalteromonas sp. and Moritella viscosa which were isolated only in at least one of the 10 following skin lesions, gills and / or fins.

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No external disease signs were noted on the fish with a few exceptions. The majority of fish 12 sampled from site A had fin rot and fish were lethargic. Internally, in some cases, the 13 following clinical signs were observed: granulomas in the liver and/or kidney, ascites and 14 empty gut which in some individuals was red. A suspected atypical As outbreak was active    here to be the most predominant strain in Scotland whereas strain type VI appears to be 16 mainly in Norway.

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Atypical strains of As were isolated from 6 out of 10 sites that took part in this health 18 screening survey and the bacterium was the most prevalent in 4 out of 10 sites. The results 19 from this survey suggest that aAs was the most prevalent bacterial species at these sites 20 between February 2016 and October 2018. It is worth noting that the aAs vapA type VI 21 isolates in this survey originated from two deployed individuals in sea cages and were 22 speculated to be related to a secondary infection following immune suppression and /or be 23 indicative of virulence adaptation of type VI against the host. Although currently, antibiotic 24 treatments are successfully applied for controlling disease outbreaks in hatcheries and cage sites, As is known carry plasmids linked with antibiotic resistance. For instance As resistance 1 to oxytetracycline, tetracycline and chlorafenicol has been previously reported (Adams et al. can be helpful on identifying differences within the aAs strains that belong to the same type.

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This information can then be used to improve/update existing autogenous vaccines.  (Reid et al. 2003, Paillard, 2004. Although, juvenile ballan wrasse (approx. 30 g) were not susceptible to these bacteria species during cohabitation challenge and only i.p.

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Photobacterium indicum/profundum, also reported in this screening, has not previously been 17 associated with fish disease outbreaks but has been isolated from moribund lobster and Overall, aAs was the most prevalent bacterial species isolated form ballan wrasse on the farm 1 sites considering the number of individuals sampled in total, followed by Vibrio species 2 which were the most geographically prevalent bacteria. Understanding the prevalence of 3 these pathogens is vital for mitigating disease outbreaks by optimising fish husbandry and 4 biosecurity practices. Furthermore, the collection of these bacterial isolates provides useful 5 information for disease management. Also, characterisation of the aAs vapA types could 6 provide important information for improving/updating existing autogenous vaccines.    Defibrinated Horse Blood + 1.5% NaCl) for phenotypic and sequencings identification. Pie chart for Site E is not shown as only atypical Aeromonas 1 salmonicida was isolated in a single sampling (2/2). Site F, single sampling point with no bacteria recovery from individuals sampled. 2). Swabs from skin lesions, gills, liver and kidney plated on variety of agar plates (Marine Agar, Tryptone Soya Agar (TSA), and TSA + 5% Defibrinated