Aquaculture 458 (2016) 21–28

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Distribution of Ostreid herpesvirus-1 (OsHV-1) microvariant in seawaterin a recirculating aquaculture system

Olivia Evans, Paul Hick, Richard J. Whittington ⁎Faculty of Veterinary Science, The University of Sydney, Camden, NSW 2570, Australia

⁎ Corresponding author at: Faculty of Veterinary SciencWerombi Road, Camden, NSW 2570, Australia.

E-mail address: richard.whittington@sydney.edu.au (R

http://dx.doi.org/10.1016/j.aquaculture.2016.02.0270044-8486/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 December 2015Received in revised form 19 February 2016Accepted 19 February 2016Available online 23 February 2016

Ostreid herpesvirus-1 (OsHV-1) microvariant presents a significant threat to the commercial production ofCrassostrea gigasworldwide. Investigating the transmission of the virus in the estuarine environment where oys-ters are grown has proven to be difficult due to the complexity of the ecosystem. The aims of this trial were to(1) investigate the distribution of OsHV-1 in seawater over time in a recirculation systemduring an experimentaldisease outbreak; (2) assess the effects of the biofiltration and ultraviolet light components of the recirculationsystem on the detection of OsHV-1; and (3) assess the potential for OsHV-1 to associate with particles in the ar-tificial seawatermatrix. The source of viruswas experimentally infected donor and cohabitating recipient oysters.Both the presence of infected oysters and the biofilter andUV light source significantly affected the detection rateof OsHV-1 in seawater. It was detected more frequently after high dose exposure (n= 6 donors per tank) com-pared with low dose exposure (n= 2 donors per tank), and when the biofilter and UV were disconnected fromthe treatment tanks. The odds of detecting OsHV-1 in the treatment tanks, whichwere periodically disconnectedfrom the biofilter and UV, were 2.56 that of the system sumps which were always connected to the biofilter andUV. It may not be possible to remove OsHV-1 completely from the seawater of a recirculation system during adisease event using biofiltration and UV irradiation alone. The presence of food did not significantly affect the de-tection rate of OsHV-1 in seawater. The pellet produced by low speed centrifugation of seawater had ~2 to 5 foldgreater odds of being positive for OsHV-1 by qPCR, in comparison to unprocessed seawater and the supernatantfraction. These results are consistent with the hypothesis that OsHV-1may be attached to particles, which in thismodelmay include algal feed, oyster faeces, fragments of oyster tissues ormicroorganisms introduced to the sys-tem with the oysters, but confirmation of this is required.Statement of relevance: It is important to understand the nature and persistence of OsHV-1 microvariant withinseawater in order to undertake aquaculture in hatchery environments, where water reticulation, recirculation,biofiltration, UV and otherwater treatmentsmay be used. Understanding the distribution andmode of transmis-sion of OsHV-1within seawater has provendifficult. Experimental studies in controlled environments are impor-tant to furthering this understanding.

© 2016 Elsevier B.V. All rights reserved.

Keywords:OsHV-1SeawaterRecirculationBiofiltrationUVParticle

1. Introduction

Ostreid herpesvirus-1 (OsHV-1)microvariants have been responsiblefor mass mortalities in commercially produced Pacific oysters(Crassostrea gigas) in Europe since 2008 (Segarra et al., 2010;Martenot et al., 2011; Martenot, 2013) and in New Zealand andAustralia since 2010 (Jenkins et al., 2013; Keeling et al., 2014;Paul-Pont et al., 2014). Microvariant genotypes of OsHV-1 have beendetected in this species in Japan (Shimahara et al., 2012) and Korea(Hwang et al., 2013) in the absence of recurrent, seasonal disease out-breaks. In China mass mortalities due to the virus have occurred in

e, The University of Sydney, 425

.J. Whittington).

other mollusc species, the Zhikong scallop (Chlamys farreri) and theblood clam (Scapharca broughtonii) (Bai et al., 2015; Xia et al., 2015).In 2012 the Pacific oyster industry generated the highest contribution(608,000 t — US$1.3 billion) to the worldwide production of oysters inaquaculture (4.7 million tonnes — US$3.8 billion) (FAO, 2012), andlosses due to OsHV-1 have been severe in affected countries (EFSA,2010; Paul-Pont et al., 2014). All age and size classes of Pacific oysterare known to be affected by the disease (Nicholas et al., 1992; Renaultet al., 1995; Arzul et al., 2002; Garcia et al., 2011; Paul-Pont et al.,2013b; Whittington et al., 2015a) but oysters less than 1 year of ageare the most susceptible (Renault et al., 1995; Schikorski et al., 2011b;Garcia et al., 2011; Whittington et al., 2015a). Despite the wide geo-graphic range of the virus and the dramatic impact that it has had onthese highly valuable stocks, the mode of transmission of the virus inthe estuarine environment is uncertain (Garcia et al., 2011; Paul-Pontet al., 2013a; Barbosa-Solomieu et al., 2015).

Fig. 1. Reticulation systems from which seawater samples were collected. Schematicphysical design of each reticulation system (one of four is shown). Each systemconsisted of 3 treatment tanks, a sump, a canister filter used for biofiltration, a waterchiller, a UV unit and an external air pump which supplied air to all of the tanks. To feedthe oysters the positions of taps 1 and 2 were reversed to cause water to bypass thetreatment tanks and recirculate through the sump, biofilter, chiller and UV; tanks werestatic during feeding. Each system had a matching standalone negative control tank.Seawater was sampled from each treatment tank, sump and negative control tank. Notto scale.

22 O. Evans et al. / Aquaculture 458 (2016) 21–28

The results of aquarium studies have suggested that seawater mayact as a medium in the horizontal transmission of OsHV-1 (Vigneronet al., 2004; Sauvage et al., 2010; Schikorski et al., 2011a; Evans et al.,2015). Cohabitation of naive oysters, i.e. those not previously exposedto the virus, with infected oysters in 1 μm filtered seawater wasshown to be an effective method for transmitting OsHV-1 and inducingclinical disease, with viral DNA detected in tankwater at concentrationsof between 101–103 DNA copies per μL over a sampling period of 168 h(Schikorski et al., 2011a). Based on studies of natural transmissionevents in the Georges River estuary, Australia it was suggested that anaquatic mechanical vector or particle is involved in the transmission ofthe virus (Paul-Pont et al., 2013a; Whittington et al., 2015b). The non-random, spatial clustering of oyster mortalities described by these au-thors was similar to that typically seen in planktonic aggregations andcommunities (Paul et al., 1993; Kingsford and Suthers, 1994; Suthersand Rissik, 2009; Benoit-Bird and Mcmanus, 2012) and it washypothesised that the virus may be attached to such particles (Paul-Pont et al., 2013a). Studies by Evans et al. (2014) suggest that OsHV-1may be present in seawater in a range of forms including free virus, floc-culated or aggregated virus, virus adsorbed to particles the same size asplant or animal cells (or larger), or various viral components at differingstages of the replication process, including free viral DNA. While it isplausible that OsHV-1 is a waterborne communicable virus capable ofassociating with particles, the nature and function of these particles re-main unclear.

The seawater samples examined by Evans et al. (2014) were collect-ed from thenatural environment of the Georges River estuary, Australia.Open estuarine environments contain highly complex ecological com-munities which are constantly cycling, growing and changing overboth the spatial and temporal scales (Malone and Mcqueen, 1983;Rissik et al., 2009; Suthers and Rissik, 2009; Guizien et al., 2014). Seawa-ter samples collected from these systemswould be as complex and var-iable as the environment from which they were taken (Suthers andRissik, 2009). Thus, the sample matrix within which OsHV-1 is to be de-tected is inherently complicated. Simplification of this matrix in con-trolled laboratory studies may allow the forms of OsHV-1 withinseawater, and particle association, to be assessed.

From a practical perspective, it is important to understand thenature and persistence of OsHV-1 in seawater in order to undertakeaquaculture in hatchery environments, where water reticulation, recir-culation, biofiltration, UV and other water treatments are routinely ap-plied. Recently, Hick et al. (2015) showed that OsHV-1 remainsinfectious in seawater for 48 h at 20 °C, which was consistent with theresults of other studies in which infected oyster tissue homogenateswere stored at 16 °C and 25 °C (Martenot et al., 2015), and in which in-fected oyster tissues were stored in water based media at several tem-peratures (Vigneron et al., 2004). Furthermore, it was shown thatOsHV-1 could be inactivated by heating to 50 °C for 5min, and by expo-sure to a high dose of ultraviolet (UV) light (N1000 mW/cm2) (Hicket al., 2015). In a separate study on the prevention of mortality due toOsHV-1 in spat, UV light exposure was unnecessary after filtration ofseawater to 5 μm (Whittington et al., 2015b). However, in the contextof a recirculation system, the effects of biofiltration and UV light treat-ment on the presence of OsHV-1 are unknown.

In a recent study Evans et al. (2015) examined the effects of feedingan algae concentrate on OsHV-1 transmission and assessed the dose–response effect represented by the number of OsHV-1-infected donoroysters cohabitated with healthy naive oysters. Both the addition offood and the number of donor oysters were important risk factors forOsHV-1 transmission and clinical disease. Seawater samples were col-lected during the second experiment described by Evans et al. (2015)and these were used in the current study. The aims of the currentstudy were to: i) study the presence and distribution of OsHV-1 inseawater over time in a recirculation system during an experimentaldisease event; ii) assess the effects of the biofiltration and ultravioletlight components of the recirculation system on the detection of

OsHV-1 in seawater; iii) assess the potential for OsHV-1 particulate as-sociation within an artificial seawater matrix.

2. Materials and methods

Five hundred and sixty individual artificial seawater samples werecollected over a 14-day period from the 11th–24th February 2015 dur-ing the second cohabitation experiment described by Evans et al. (2015)at the University of Sydney laboratories in Camden, NSW, Australia.

2.1. Oysters and experimental design

The experiment was undertaken in a physical containment level 2(PC2) aquatic animal facility. Four separate recirculation systems wereutilised (Fig. 1) each having a sump (250 L) that was connected to abiofilter (Fluval 406 canisterfilter), a chiller unit (22 °C, HC-300A,HaileaAquarium chiller) and an ultra-violet light (Smart UV Steriliser; 40W—65LMP/3900LPH at 30mJ/cm2 (90% UVT) 40mm connections, EmperorAquatics). The total system volume when open and recirculating, in-cluding the tanks and plumbing apparatus, was approximately 300 L.Negative controls (oysters that were not intentionally exposed toOsHV-1) were maintained in separate aerated 15 L tanks, but with norecirculation, biofilter or UV systems (Fig. 1). All treatment and controltanks were supplied with artificial seawater (Red Sea; 30–31 ppt salin-ity; prepared with unfiltered water from the municipal supply) andaeration, andweremaintained at 22 °C andpH range of 8.2–8.8, and am-monia, nitrite and nitrate levels b0.25 ppm. All water quality parame-ters were monitored daily and adjustments were performed asrequired. A photoperiod of 12 h of lightness (07:00–19:00 h) and 12 hof darkness (19:00–07:00 h) was used for the duration of theexperiment.

Briefly, 608 healthy triploid Pacific oysters were acquired fromOsHV-1 free populations in the Shoalhaven River (Goodnight Oysters,Greenwell Point, NSW, Australia; juveniles 9 month old, 5.5 ± 2.1 gand adults 18 month old, 32.3 ± 7.3 g) in January 2015. These

23O. Evans et al. / Aquaculture 458 (2016) 21–28

populations were confirmed as negative for OsHV-1 DNA by real-timeqPCR prior to use (n = 30), and prior to transfer from the hatchery inTasmania the batch had been certified by the Government laboratoryto be negative for OsHV-1. Oysters were fed under different regimes.The Always Fed (AF) group was fed from days 1 to 14, the Fed thenStarved (FS) group was fed from days 1 to 4 only, the Starved then Fed(SF) group was fed from days 5 to 14 only and the Always Starved (AS)group was not fed; negative control oysters received the same feedingregimes. Thirty juvenile oysters were randomly allocated to each of the3 tanks for each of the feeding treatments, and to 4 separate tanks forthe controls (Fig. 1). The adult oysters were allocated for intramuscularinjection of OsHV-1 positive inoculum (n = 96), to be used as infecteddonor oysters. The OsHV-1 positive inoculum contained approximately1 × 104 viral copies/μL (Evans et al., 2015). Donors injected with OsHV-1 negative control tissue homogenate (n=32)were used for the controlgroups. The first exposure was from days 1–4 (n = 2 donor oysters pertank) (period 1, P1), and the second was from days 9–12 (n = 6 donorsper tank) (period 3, P3); all donors were removed on days 4 and 12, re-spectively. The time between P1 and P3was called period 2 (P2), and thetime after period 3 was called period 4 (P4). Thus donor oysters werepresent during P1 and P3 and absent during P2 and P4 (Fig. 2). Deaddonor oysters were not removed until the days specified above. Deadcohabitating oysters were removed on the day they were first observed.

2.2. Feeding

Oysters in nominated tanks were fed twice daily with InstantAlgae® Shellfish Diet 1800 (Reed Mariculture, USA), a concentrate of

Fig. 2. Pattern of OsHV-1 DNA detection in artificial seawater samples each day for 14 days. Leftclosed system with biofiltration and UV off-line. Shaded cells indicate that OsHV-1 DNA was dsample processed: unprocessed seawater (solid black), pelleted fraction (striped), supernatanwere 6 donor oysters per tank. No donors were present during P2 or P4. The presence of dtreatment tanks, system sumps and negative control tanks. Over time oysters exposed to OsH(AS); and the same feeding regimes were applied to the non-exposed controls (see materials a

marine microalgae (5–20 μm) containing a mix of Isochrysis, Pavlova,Thalassiosira weissflogii and Tetraselmis according to the regime de-scribed by Evans et al. (2015). During the feeding period all 12 treatmenttanks were isolated from the recirculation, biofilter and UV for 2 h sothat the tanks were undisturbed. However, water in the sumps wasrecirculated via the biofilter, chiller and UV units (Fig. 1). Negative con-trol tanks were not connected to recirculation so each tank received atotal water exchange after each feeding session. All tanks were suppliedwith constant aeration from an air stone, including during feeding.

2.3. Reticulation patterns

Seawater samples were collected from each treatment tank (15 L)(n = 12), system sump (250 L) (n = 4) and negative control tank(15 L) (n = 4) twice daily corresponding to the times when the tankshad very different reticulation patterns: 1) at the start of each dayprior to feeding when the water had been exposed to the biofilter andUV by recirculation overnight (open system) and 2) at the end of the af-ternoon feeding session when there had been no recirculation, UV orbiofiltration connected to the treatment tanks for 2 h (closed system).

2.4. Collection and method of processing artificial seawater samples

A 50 mL polypropylene tube (Falcon) was submerged beneath thesurface of the water in the centre of each tank or sump and circledaround the tank or sump once to collect the seawater sample. Sampleswere placed in an insulated box on wet ice and transferred to the labo-ratory for immediate processing. Samples were processed according to

panel, open systemwith biofiltration and UV in-line with the treatment tanks; right panel,etected by qPCR in that sample on that day. The pattern of shading indicates the type oft fraction (dots). During period P1 there were 2 donor oysters per tank; during P3 thereonor oysters is indicated by solid grey shading. Seawater samples were collected fromV-1 were always fed (AF), fed then starved (FS), starved then fed (SF) or always starvednd methods).

24 O. Evans et al. / Aquaculture 458 (2016) 21–28

themethods described by Evans et al. (2014). Briefly, 50mL of seawaterwas collected from each tank and system sump at each sampling.A 15 mL aliquot was placed into a sterile 15 mL polypropylene tube(Falcon). A 1 mL aliquot was taken and placed into a 1.5 mL tube con-taining 0.4 g of silica–zirconia beads (Daintree Scientific) (unprocessedsample), while the remaining 14mL sample was centrifuged for 20minat 1000 ×g. A 1 mL sample of the supernatant was placed into a 1.5 mLtube containing 0.4 g of silica–zirconia beads (supernatant sample). Thesupernatant was decanted to leave approximately 1 mL and the pellet.Samples were shaken vigorously for 30 s to re-homogenise the pelletand a 1 mL sample was placed into a 1.5mL tube containing 0.4 g of sil-ica–zirconia beads (pelleted sample). All sampleswere stored at−80 °Cuntil homogenisation and DNA extraction.

2.5. Nucleic acid extraction and real-time quantitative PCR for the detectionof OsHV-1 DNA

Nucleic acids were purified from the seawater samples (unpro-cessed, pelleted and supernatant) after homogenisation according tothe procedures described by Evans et al. (2015).

Samples were analysed in duplicate in a 25 μL total reaction volumeusing the SYBR® green qPCR method described by Evans et al. (2015).This assay was modified from the fluorescent probe assay describedbyMartenot et al. (2010). A valid PCR runwas defined as one exhibitingno amplification of negative controls, amplification of both replicates ofthe positive control with a Ct within the range of the standard curve(100–107), a standard curve with r2 N 0.95 and efficiency between 90and 110%. The fluorescence threshold for each run was calculatedusing the amplification-based threshold algorithm (Stratagene) for thestandard curves, and applied to all samples. Samples were defined aspositive when one or both replicates exhibited an exponential increasein SYBR fluorescence signal and a cycle threshold of b40, with a dissoci-ation curve (Tm) that conformed to that of the positive control. Thequantification limit of the assaywas 12 viral copies per μL and the detec-tion limitwas 3 viral copies per μL (Bustin et al., 2009). Samples that sat-isfied the criteria for detection but had a Ct value below the limit ofquantification of the assay were described as positive below the limitof quantification (BLOQ). Detection of OsHV-1DNA in this assaywas de-fined as a proxy for detection of OsHV-1, given that the origin of viruswas known-infected oysters and the water samples were collected inclose spatial and temporal proximity to these oysters, and processedto avoid degradation.

2.6. Statistical analysis

Analysis of seawater sample data was conducted using theGeneralised Linear Mixed Model (GLMM) method of Genstat 15th

Table 1Detailed description of the fixed term variables included in the Generalised Linear Mixed Mod

Fixed term variable Abbreviation Categories

Water processing method Method UnprocessedPelletedSupernatant

Infection trial period Period P1P2P3P4

Reticulation system System OpenClosed

Location of sampling Location Treatment tanksSystem sumps

Feeding status Feeding AFFSSFAS

Edition (©2000–2015VSN International Ltd., UK) utilising a binary out-come for OsHV-1 detection by qPCR (0 = negative, 1 = positive). Theseawater processing technique (method), time of sample collection rel-ative to the presence and number of donor oysters (period), status ofthe reticulation system as open or closed (system), site from whichthe seawater was sampled (location) and feeding status of the oysters(feeding) were all accounted for in the fixed model while the effect oftank was accounted for in the random model. Terms are defined inTable 1.

3. Results

3.1. Source and detection of virus in seawater

Mortalities occurred in the infected donor oysters (removedfrom the systemon days 4 and 12 only) and the oysters that cohabitatedwith these (removed each day) as described by Evans et al. (2015).Most mortalities occurred during and after the second exposureperiod when more donor oysters were used. Mean viral loads indead donors ranged between 4.62 × 103–4.52 × 105 DNA copies/mg tis-sue and in dead cohabitated oysters ranged from 3.50 × 102 to3.56 × 107 DNA copies/mg tissue (Evans et al., 2015). Logically, theseoysters were the source of viral DNA that was detected in the seawatersamples.

After the introduction of infected donor oysters to tanks, OsHV-1was detected in artificial seawater throughout the trial but at very lowlevels, below the quantification limit of the qPCR assay (Fig. 2). In fact,it was detected at quantifiable concentrations in only 3 samples:unprocessed seawater collected from the closed system on day 11 at2.69 × 101 ± 3.02 DNA copies/μL and 2.48 × 101 ± 4.41 DNA copies/μL in the Always Fed (AF) and Starved then Fed (SF) oyster treatmentsrespectively, and on day 12 at 2.18 × 101 ± 2.42 DNA copies/μL in theSF treatment.

3.2. Effects of donor oysters and food on the detection of OsHV-1 inseawater

The number of donor oysters significantly affected the detection ofOsHV-1 in seawater (P b 0.001) (Table 2), with 16.2% of seawater sam-ples collected during P1 (2 donors per tank), 10.2% collected during P2,26.6% collected during P3 (6 donors per tank) and 28.1% collected dur-ing P4 testing positive (values include the results of all 3 processingmethods for both the treatment tanks and sumps) (Fig. 3). These higherdetection rates in seawater in the latter two periods corresponded togreater levels of mortality associated with the higher number of donors(see Evans et al., 2015).

el (GLMM) analysis. The effect of tank was accounted for in the randommodel.

Category details (where applicable)

………Days 1–4, n = 2 donors per tankDays 5–8, no donors presentDays 9–12, n = 6 donors per tankDays 13–14, no donors presentRecirculation, biofiltration and UV connected to treatment tanks overnightRecirculation, biofiltration and UV disconnected from the tanks for 2 h……Always fedFed then starvedStarved then fedAlways starved

Table 2Results of Generalised Linear Mixed Model (GLMM) analysis of artificial seawater with the effects of the water processing method (method), period of the infection trial (period),reticulation system status (system), seawater sampling location (seawater) and feeding status of the oysters (feeding) accounted for in the fixed model. Tank was accounted for in therandom model.

Fixed term variable P value Standard error Category Parameter estimate Odds ratio Lower 95% CI Upper 95% CI

Method b0.001 0.31 Unprocessed −0.66 … … …Pelleted 0 … … …Supernatant −0.36 … … …

Period b0.001 0.39 P1 0 … … …P2 −1.09 … … …P3 0.89 … … …P4 0.13 … … …

System 0.007 0.27 Open −0.24 … … …Closed 0 … … …

Location 0.015 0.31 Treatment tank 0.94 2.56 1.95 3.17System sumpa 0 … … …

Feeding regime 0.085 0.37 AF 0.00 … … …FS −0.64 … … …SF −0.75 … … …AS −1.13 … … …

Method × Period b0.001 0.44 Unprocessed × P1 0 1.94 1.16 2.27Pelleted × P1b 0 … … …Supernatant × P1 0 1.43 0.66 2.21Unprocessed × P2 0.57 1.10 0.33 1.87Pelleted × P2b 0 … … …Supernatant × P2 0.77 0.66 −0.11 1.44Unprocessed × P3 0.26 1.49 0.72 2.27Pelleted × P3b 0 … … …Supernatant × P3 −0.93 3.63 2.86 4.41Unprocessed × P4 1.86 0.14 −0.63 0.91Pelleted × P4b 0 … … …Supernatant × P4 0.31 1.05 0.28 1.82

Method × System 0.002 0.30 Unprocessed × Open −1.08 5.73 5.20 6.26Pelleted × Openb 0 … … …Supernatant × Open −0.43 2.20 1.67 2.73Unprocessed × Closed 0 1.94 1.41 2.46Pelleted × Closedb 0 … … …Supernatant × Closed 0 1.43 0.91 1.96

Note: “a” and “b” represent the reference category.95% confidence intervals containing the value 1.00 within their range indicate that there is no significant difference between that given category and the reference category.AF: Always Fed, FS: Fed then Starved, SF: Starved then Fed, AS: Always Starved.P1: days 1–4 (exposure period 1, n = 2 donors per tank), P2: days 5–8 (no donors present).P3: days 9–12 (exposure period 2, n = 6 donors per tank), P4: days 13–14 (no donors present).Open system: recirculation, biofiltration, and UV light connected to treatment tanks overnight.Closed system: recirculation, biofiltration and UV light disconnected from treatment tanks for 2 h.

a Odds ratios shown where reference category is represented by “a” are the odds of given category testing positive for OsHV-1 DNA by qPCR compared with the reference category.b Odds ratios where reference category is represented by “b” are the odds of the reference category testing positive for OsHV-1 DNA by qPCR compared with the other given category.

Fig. 3. The effect of the presence and number of infected donor oysters on the detectionof OsHV-1 in seawater. Data are the percentage of seawater samples from which OsHV-1 was detected by qPCR (includes both the treatment tanks and system sumps).Standard errors are shown. P1: days 1–4 (exposure period 1, n = 2 donors per tank);P2: days 5–8 (no donors present); P3: days 9–12 (exposure period 2, n = 6 donors pertank); P4: days 13–14 (no donors present).

25O. Evans et al. / Aquaculture 458 (2016) 21–28

The presence of food did not affect the detection of OsHV-1 in sea-water (P = 0.085) (Table 2).

3.3. Effects of the recirculation system components on the detection ofOsHV-1 in seawater

OsHV-1 was detected in seawater in the treatment tanks and in thesystem sumps from days 1–14 (Fig. 2). The odds of detecting OsHV-1 inartificial seawater sampled from the treatment tanks were 2.56 that ofthe system sumps (P= 0.015) (Table 2). Overall, 21.9% of the seawatersamples from the treatment tanks and 10.7% from the sumpswere pos-itive (includes results of all 3 processing methods) (Fig. 4). The higherdetection rates corresponded to the tanks, which were not always con-nected to the biofilter and UV treatments. Similarly, OsHV-1 DNA wasdetected more frequently when the system was closed (biofilter andUV off-line) (23.7% positive samples, values include the results of all 3processingmethods for both the treatment tanks and sumps) comparedto when the systemwas open (biofilter and UV in-line) (14.6% positivesamples, values include the results of all 3 processing methods for boththe treatment tanks and sumps) (P = 0.002) (Fig. 5). Even when therewere few donor oysters and low mortalities in the cohabitating oysters(P1 and P2) OsHV-1 DNA was detected in seawater in the sumps, de-spite constant biofiltration and UV irradiation (Fig. 2).

Fig. 4. Percentage of seawater samples from treatment tanks and reticulation systemsumps from which OsHV-1 was detected by qPCR (includes the results of all 3 seawaterprocessing methods: unprocessed, pelleted and supernatant). Standard errors are shown.

26 O. Evans et al. / Aquaculture 458 (2016) 21–28

3.4. Partitioning of OsHV-1 in seawater

The partitioning of OsHV-1 in artificial seawater was studied by cen-trifugation of the samples. For samples collected when donor oysterswere present (periods P1 and P3), the odds of detecting OsHV-1 DNAweremore than 1.9 fold greater (range from1.94 to 3.63) in the pelletedfraction of seawater than in the supernatant fraction or in unprocessedseawater samples (P b 0.001) (Table 2). However, there was a signifi-cant interaction of seawater processing method with the period(P b 0.001) (Table 2) so that when donor oysters were not present(P2 and P4) there was either no effect of low-speed centrifugation orit was detrimental (Table 2 and Fig. 3), implying that the form of thevirus was different depending on whether or not donor oysters werepresent. The odds of detecting OsHV-1 in the pelleted fraction of seawa-ter in an open system (biofilter and UV in-line to treatment tanks) were

Fig. 5. The effect of recirculation on the detection of OsHV-1 in seawater. Data are thepercentage of seawater samples from which OsHV-1 was detected by qPCR (includesboth the treatment tanks and system sumps). Open system: recirculation, biofilter, andUV light connected to treatment tanks overnight; Closed system: recirculation, biofilterand UV light disconnected from treatment tanks for 2 h. Standard errors are shown.

2 to 5 fold that of unprocessed seawater and the supernatant fraction,while in a closed system (biofilter and UV off-line) the odds of detectingOsHV-1 in the pelleted fractionwere 1.94 that of unprocessed seawater(Table 2).

4. Discussion

4.1. Detection of OsHV-1 in seawater in a recirculation system during anexperimental disease event

This is the first study to examine the detection of OsHV-1 in seawa-ter over time in a recirculation aquaculture system. A study of artificialseawater in a controlled laboratory trial provides insights into the hori-zontal transmission of OsHV-1. The biomass of infected oysters (includ-ing donors intramuscularly injected with OsHV-1 and cohabitatingoysters exposed to the donors) provided the source of virus, as clinicallyinfected, moribund and dead oysters release OsHV-1 into the seawaterin proportion to their biomass (Schikorski et al., 2011a; Evans et al.,2015; Paul-Pont et al., 2015; Petton et al., 2015). Previous studies havesuggested that viral quantities in the tissues of oysters infected via thecohabitation model increase to levels greater than 104–105 DNA copiesper ng of total DNA extracted from tissues within 96 h of exposure, withmortality increasing steadily from day 1 to day 10 post-exposure(Schikorski et al., 2011a). In contrast, oysters injected intramuscularlywith OsHV-1 tend to have a sudden onset of mortality 48 to 96 h postinjection, with similar high viral loads (Schikorski et al., 2011a;Paul-Pont et al., 2015). Viral loads in dead cohabitating oysters in thistrial ranged between 3.50 × 102 and 3.56 × 107 DNA copies/mg tissue,while in donors ranged between 4.62 × 103 and –4.52 × 105 DNA cop-ies/mg tissue (Evans et al., 2015), and OsHV-1 DNAwas detected in sea-watermore often during or after the second exposure period,whichhada larger number of donor oysters per tank (n= 6 donors per tank) andhigher mortalities compared with the first exposure period (n = 2 do-nors per tank) (Evans et al., 2015). It is logical to assume that moreOsHV-1 was being shed into the seawater by infected oysters after thesecond exposure.

No mortality was recorded in any of the donor or cohabitating oys-ters in the negative control treatment, but as reported previously(Evans et al., 2015) OsHV-1 DNA was detected in the tissues of severalcohabitating control oysters (101–103 DNA copies/mg tissue) that hadnot been purposely infected with OsHV-1. Therefore it was not surpris-ing that low levels of OsHV-1 DNA (below the quantification limits ofthe qPCR assay) were intermittently detected in the seawater of thecontrol tanks (Fig. 2). The likely source of the virus was cross-contami-nation from the nearby treatment tanks due to aerosols from the finebubbles created by the airstones that were present in each tank, as hasbeen demonstrated directly and indirectly in aquatic microbiology(Sharoni et al., 2015). Organisms such as Emiliania huxleyi virus,Mycobacterium intracellulare, faecal coliforms and algae have been pre-viously shown to spread in aerosols generated by gas bubbles burstingin surface water (Hatcher and Parker, 1975; Gruft et al., 1975; Wendtet al., 1980; Parker et al., 1983; Sharoni et al., 2015).

4.2. Effects of the recirculation system components on the detection rate ofOsHV-1 DNA

Previous studies involving stagnant 25 L aquaria in which experi-mentally infected oysters were maintained, or of water based-mediaused for the storage of infected oyster tissues in tubes, have shownthat OsHV-1 DNA can be detected in seawater by qPCR at concentra-tions of 101–103 DNA copies/μL (Vigneron et al., 2004; Schikorskiet al., 2011a; Paul-Pont et al., 2015). Although the viral quantities de-tected in the current trial were much lower, they were consistent withthose previously reported for natural seawater samples collected fromthe Georges River estuary during disease outbreaks in cultured Pacificoysters (Evans et al., 2014). The large water volume used in the

27O. Evans et al. / Aquaculture 458 (2016) 21–28

recirculation systems (300 L total system volume when recirculating)and the biofiltration and UV light treatment may explain the low viralquantities detected in the present work compared with prior aquariumstudies. The biofiltration units used in the current trialwere seededwithnitrifying bacteria and aged for 8 weeks in marine aquaria, in which sil-ver sweep (Scorpis lineolata) was housed, prior to use. Each biofiltercontained 3 types of filtration media: foam screen framing for filtrationof large particles, porous ceramic rings for biological filtration and bac-terial growth, and polyester polishing pads for removal of fine particles(Fluval). The odds of detectingOsHV-1DNA in the treatment tanksweremore than twice that of the system sumps and it is reasonable to assumethat the biofiltration and UV light, which were constantly connected tothe system sumps, caused viral degradation and removed some OsHV-1from the system. This outcome is reflected also in the significantlyhigher percentage of OsHV-1 positive samples in the closed system(biofilter and UV off-line) compared with the open system (biofilterand UV in-line) (Fig. 5). The authors of several prior studies haveshown that UV light inactivates OsHV-1 (Schikorski et al., 2011b; Hicket al., 2015), however the results of the present work suggest that stan-dard biofiltration and UV irradiation methods commonly used in recir-culation aquaculture systems are not sufficient to completely removeOsHV-1 from seawater during a disease outbreak.

4.3. Partitioning of OsHV-1 in seawater

This is the first study to assess the detection rate of OsHV-1 DNAbased on viral partitioning in artificial seawater sampled from aquariaand recirculation systems in a controlled laboratory trial. The centrifu-gationmethods first described by Evans et al. (2014) were used. The re-sults showed that low speed centrifugation of artificial seawatersamples and testing the resultant pellet significantly improved theodds of detecting OsHV-1 DNA by qPCR (by ~2 to 5 fold) in comparisonto unprocessed seawater and the supernatant fraction. This suggeststhat some OsHV-1 and/or OsHV-1 DNA must have been aggregated orflocculated, or associated with particles in the order of 10 μm or largerwhich can be pelleted at 1000 ×g in 20 min (Lawrence and Steward,2010; Evans et al., 2014). These results reinforce the suitability of thecentrifugation method for the application to epidemiological studieson OsHV-1 (Evans et al., 2014).

The results provide further support for the hypothesis, based on fieldobservations, that OsHV-1 is associated with particles in seawater(Paul-Pont et al., 2013a). In this aquariummodel the particles may con-sist of feed particles, faeces, fragments of oyster tissues or microorgan-isms introduced to the system with the oysters. In the present studyOsHV-1 DNA was also detected in unprocessed artificial seawater, aswell as in the supernatant fractions. These findings are consistent withthe results of Evans et al. (2014) for natural seawater samples, and sug-gest thatOsHV-1may bepresent in a range of forms including free virus,flocculated or aggregated virus, virus adsorbed to particles, and/or freeviral DNA. As real-time qPCR was the only method applied to detect ev-idence of the virus, and as the low viral quantities detected (below thequantification limit of the qPCR) did not allow for further analysiswith other methods, the specific form/s of OsHV-1 being detectedcould not be ascertained. However, the spatial and temporal proximityof the water samples to actively infected oysters, and careful handlingof water samples to avoid degradation,makes it likely that intact virionswere being detected indirectly using the qPCR. Recently a propidiummonoazide real-time PCR assay, which utilises a photo-reactive dyethat preferentially binds to double-stranded DNA, was used to differen-tiate free OsHV-1 DNA from viral DNA contained within a capsid(Moreau et al., 2015), but validation data for large DNA viruses are notavailable. Amethod such as this would be beneficial to future investiga-tions on OsHV-1 in seawater.

Whether seawater was collected during periodswhen donor oysterswere present or absent had a significant effect on the partitioning ofthe viral signal. When infected donor oysters were present in each

tank (P1: days 1–4 and P3: days 9–12), viral signal was more likely tobe detected in the pellet fraction, however when no donors were pres-ent (P2: days 5–8 and P4: days 13–14) the viral signal was nomore like-ly found in the pellet than in the other fractions. While the form/s ofOsHV-1 could not be determined, it is probable that the proportions ofOsHV-1 in its possible forms differed between periods when infecteddonors were present or absent and this may have contributed tothe method × period interaction observed in the statistical model(Table 2). Further investigation of the effects of the exposure and incu-bation period on the detection rate of OsHV-1 in seawater is required tobetter understand the importance of the pattern noted in the currentstudy.

4.4. Effect of feed on detection of OsHV-1 in seawater

The addition of algal food to the tanks did not affect the detectionrate of OsHV-1 in seawater even though Evans et al. (2015) foundfood addition to be an important risk factor for OsHV-1 transmissionand clinical disease in this recirculation aquariummodel.

5. Conclusion

This is the first study to investigate the presence and distribution ofOsHV-1 in a recirculation system during an experimental disease eventand to assess the effects of the recirculation system components on thedetection rate of OsHV-1. Donor oysters intramuscularly injected withOsHV-1 and cohabitating oysters exposed to the donors provided thesource of virus detected in the tank water. The presence and numberof donors oysters, the reticulation system (biofilter, UV), and the contacttime of water with the biofilter and UV all significantly affected the de-tection of OsHV-1 in the tank water. As was the case for natural seawa-ter, low speed centrifugation of artificial seawater at 1000 ×g for 20minand testing the resultant pellet improved the odds of detecting OsHV-1DNA by qPCR by ~2 to 5 fold, in comparison to unprocessed seawaterand the supernatant fraction. These results are consistent with the hy-pothesis that OsHV-1 is attached to particles that are important fortransmission of the virus. However the type and function of the particlesare yet to be ascertained. In the present study they may have been feed,faeces, fragments of oyster tissues or microorganisms introduced to thesystems with the oysters. Unfortunately the standard biofiltration andUV irradiation components of a recirculation aquaculture system didnot remove all detectable OsHV-1 DNA from seawater. This findingmay have important implications for the prevention and control ofOsHV-1 disease events on-farm.

Acknowledgements

This work was funded by the Australian Government through theFisheries Research and Development Corporation (Aquatic AnimalHealth Subprogram: 2012/032). Slavicka Patten, Craig Kristo and StuartGlover are thanked for their technical assistance. Navneet Dhand isthanked for his statistical guidance. Oysters were kindly provided byShellfish Culture Tasmania. Leon and Angela Riepsamen provided ex-pert care for oysters in the Shoalhaven River.

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