Aquaculture 437 (2015) 10–20

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Protection of Pacific oyster (Crassostrea gigas) spat frommortality due toostreid herpesvirus 1 (OsHV-1 μVar) using simple treatments ofincoming seawater in land-based upwellers

Richard J. Whittington a,⁎, Paul M. Hick a, Olivia Evans a, Ana Rubio a, Bruce Alford b,Navneet Dhand a, Ika Paul-Pont a

a Faculty of Veterinary Science, The University of Sydney, Camden, NSW 2570, Australiab Broken Bay Oysters, Mooney Mooney, NSW 2488, Australia

⁎ Corresponding author at: Faculty of Veterinary SciencWerombi Road, CamdenNSW2570, Australia. Tel.:+612 9

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

http://dx.doi.org/10.1016/j.aquaculture.2014.11.0160044-8486/© 2014 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 7 October 2014Received in revised form 11 November 2014Accepted 15 November 2014Available online 21 November 2014

Keywords:Crassostrea gigasOstreid herpesvirus 1Disease preventionWater temperatureIncubation period

The microvariant genotype of Ostreid herpesvirus 1 (OsHV-1 μVar) has severely disrupted the production ofPacific oysters Crassostrea gigas in Europe, New Zealand, and Australia since its first detection in France in2008. The disease occurs in the warmer months, recurs annually, and requires new management strategies.Larvae and spat are the most susceptible life history stages, which poses a threat to hatchery production. Thisis the first study to examine strategies to enable survival of spat in treated seawater in an OsHV-1 μVar endemicarea. In 2013 and 2014, seven controlled experiments were conducted in which potentially infected estuarinewater was pumped through an upweller nursery system housing spat. Controls died of OsHV-1 infection in 6of 7 experiments. Filtration of seawater to 5 μm was protective, whereas filtration to 30 μm or 55 μm was not.UV irradiation was not required for protection after 5 μm filtration. Chilling water by 2–3 °C delayed the onsetand reduced the mortality rate. Aging seawater for 48 h prior to use prevented mortality, but the mechanism,either sedimentation of particles or inactivation of OsHV-1, was not determined. Some oysters in treatments inwhich mortality was prevented contained low quantities of OsHV-1 DNA, suggesting that infections may haveoccurred. The results support a hypothesis that OsHV-1 is carried on particles rather than being uniformlydistributed in water; the removal of the putative particulate vector of OsHV-1 from seawater using aging/sedimentation of water or filtration to 5 μm enabled C. gigas spat to survive despite the presence of OsHV-1μVar in the water supply.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The pathogen Ostreid herpesvirus 1 microvariant (OsHV-1 μVar) hasseverely disrupted the production of Crassostrea gigas in Europeancountries since its first detection in France in 2008 (EFSA, 2010). Inthe southern hemisphere disease associated with OsHV-1 μVar first oc-curred in New Zealand in April 2010 (Bingham et al., 2013; Keelinget al., 2014) leading to severe production losses. By November 2010,the disease was recognized in farmed oysters in the Georges River estu-ary at Botany Bay, Australia (Jenkins et al., 2013), and from January2013, it has caused cessation of C. gigas farming in the HawkesburyRiver system, 50 km north of that location (Paul-Pont et al., 2014). InAustralia, the disease was referred to as Pacific oyster mortality syn-drome (POMS) to avoid negative consumer sentiment associated withherpesvirus terminology. There is a strong seasonal pattern of disease

e, The University of Sydney, 4253511619; fax:+6129351 1618..J. Whittington).

occurrence in all these countries. A threshold temperature of 16 °C to17 °C for onset of mortality due to OsHV-1 has been well establishedthrough observational and experimental research in France (Pernetet al., 2012; Petton et al., 2013), and the disease is often referred to assummer mortality for that reason. The response to the threat of OsHV-1 μVar in Europe, NewZealand, andAustralia includes research on selec-tion of lines of Pacific oysters with genetic resistance to disease causedby OsHV-1. Complementary strategies to develop an improved under-standing of the biology of OsHV-1 and the epidemiology of the diseasewill enable the design of husbandry systems suitable for farming Pacificoysters in OsHV-1 endemic areas.

Experience in France and other European countries is that OsHV-1affects spat more severely than adults (Peeler et al., 2012) and lossescan be near total (Segarra et al., 2010). Observations in Australia andNew Zealand confirm age-related differences in susceptibility but sug-gest that oysters from spat through to adult stages can be highly suscep-tible (Bingham et al., 2013; Paul-Pont et al., 2013a, b; Paul-Pont et al.,2014). For this reason, research has been directed to devise ways toavoid losses throughout the production cycle. It is nowknown thatmor-tality in adults can be kept below 50% by placing stock in intertidal

11R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

cultivation structures at a specified height to reduce immersion time inseawater (Paul-Pont et al., 2013b).

The commercial production cycle of Pacific oysters in Australia de-pends on hatchery production of triploid and diploid spat, predomi-nantly in Tasmania. Spat are shipped for grow out to farms locatedmainly in Tasmania, New South Wales, and South Australia. The modeof spread of OsHV-1 over long geographic distances is unknown, al-thoughmovements of infected spat fromhatcheries are almost certainlyresponsible for some of the spread observed in Europe (EFSA, 2010). Asit is unclear howOsHV-1 arrived in Australia to affect aquaculture in theGeorges River estuary (Jenkins et al., 2013) or how it spread to theHawkesbury River system (Paul-Pont et al., 2014), hatcheries inOsHV-1 free zones like Tasmania are considered to be at risk of infection.If outbreaks of POMS occur on hatcheries, there will be severe impactson spat production, whichwill have an immediate and devastating eco-nomic impact on production of Pacific oysters throughout Australia. Asno husbandry or genetic advances yet allow for reasonable survival ofspat exposed to OsHV-1 μVar, the need for identification of safe rearingtechniques that may enable hatcheries to produce and farmers to holdspat in land-based facilities for prolonged periods becomes increasinglyimportant. The trials described in this report are the first to examinestrategies to enable survival of spat in an OsHV-1 μVar endemic area.

It has been established through laboratory challenge experiments inEurope and Australia that OsHV-1 is shed from infected oysters into thesurroundingwater and that it can be transmitted to healthy oysters by in-oculation of the water in which they are kept (Paul-Pont et al., in press;Schikorski et al., 2011b). Logically, some kind of water treatmentmethodwould be required to prevent infection of oysters. However, there are nopublished data on the duration of viability of OsHV-1 in seawater or onmeans of disinfection. Anecdotal reports from France suggest spat raisedin ponds in the systemknown as “pousse en claire” do not suffer from thedisease but themechanisms are unknown (Richez, 2012). Factors such ashigh water temperature and natural UV irradiation in shallow water arepossible explanations. In France, OsHV-1 has been reported to be a prob-lem only when water temperatures are in the range 16–24 °C, and inthese ponds, temperature can exceed 24 °C (Richez, 2012).

During seasonal OsHV-1 μVar outbreaks in Australia in summer2011–2012, detailed epidemiological observations were made of thepatterns of mortality leading to a hypothesis that OsHV-1 was carriedon particles rather than being free and uniformly distributed in water(Paul-Pont et al., 2013a). In fact, it was distributed in a manner

Fig. 1. Layout of upwellers in treatment tanks. Top left, detail of upweller. Bottom left, treatmentand at the rear of the main holding tank; the UV unit is wall mounted.

consistent with an association with plankton (Suthers and Rissik,2009). It was shown also that the rate of detection of OsHV-1 in naturalseawater samples can be increased two-fold by low speed centrifuga-tion and testing of the pellet in PCR, consistent with the virus being as-sociated with particles N~10 μm (Evans et al., 2014). The aim of thisstudy was to test the hypothesis that the inactivation of OsHV-1 or re-moval of the putative particulate vector from seawater using aging orsedimentation/filtration respectively would enable C. gigas spat to sur-vive despite the presence of OsHV-1 μVar in the water supply.

2. Materials and methods

2.1. Oyster culture system

Transmission of OsHV-1 μVar and occurrence of disease in spat heldin land-based upwellers (Fig. 1) were compared for naturally infectedestuarine water and water that was treated in different ways: control(untreated, potentially naturally infected water), filtered, aged (48 h),or chilled.

Upweller tankswere installed in a large enclosed farm shed adjacentto the estuarine lower Hawkesbury River at Mooney Mooney, NewSouth Wales, Australia, in March 2013. The entire system was disman-tled and rebuilt in a different farm shed in September 2013 (Fig. 1).For both systems, four main treatment tanks each containing 4upwellers were installed within a large fibreglass holding tank whichwas connected to waste for discharge of all water from the upwellersto the river. All of the treatment tankswere in close proximity to one an-other, and no attempt was made to control aerosols from the tanks ormovement of personnel and other equipment into and within thefarm sheds during the experiments.

The water supply for the aged water treatment was independent ofthe others (Fig. 2); itwas drawnbygravity fromholding tanks thatwerefilled intermittently using a pump (Silencer, Davey, Australia). The inletfoot valve for this pump was located about 4 m from a submersiblecontinuous-flow pump (Dynapond, Davey, Australia) that suppliedwater to the other treatments. The foot valve and the submersiblepump were 0.5 m below the water surface at all times and couldmove up and down with the 2 m tidal movement as each was attachedto a float mechanism on a wooden post; the two posts were about 4 mapart.

tanks for trial 1. Right, treatment tanks for trials 2 to 7; filters are shown in the foreground

Fig. 2. Schematic physical design of the facility constructed to investigate treatment of incoming seawater. The treatments shown differed between experiments (see Table 1 for details).Each mixing tank had an overflow to waste that is not shown. Not to scale.

12 R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

The reticulation for the other treatments comprised the submers-ible pump in the river, pipework, a mixing tank and a manifold fromwhich three individual treatment tanks were supplied. The controltank was supplied from the middle off-take on the manifold for theduration of the experimental period; immediately prior to trial 4,the filters on the first and third off-takes were swapped to avoid po-sitional bias.

Each mixing tank (118 × 91 × 43 cm, L ×W × D, 460 L) had an over-flow to waste and a separate pump to distribute water to the upwellers(Fig. 2). Flow rate to the upwellers from the pump was controlled bybalancing the water flow to two destinations using a ball valve on eachoutlet: (1) recirculation of water back into the mixing tank and (2) flowto the treatment tank containing the upwellers. By this means, excessflow and pressure through filters was prevented.

Upwellers were 140 mm high × 100 mm diameter with 1 mm meshscreens. Four were included per treatment tank (81 × 60 × 35 cm,L × W × D, 170 L) (Fig. 1). Typical flow rates used in all trials excepttrial 4 were 0.5 to 1 L per min per upweller. For the control and filteredwater treatments in trial 4, flow rates of 1 to 4 L per min per upwellerwere used. Water flow rates in filtration treatments were nominal; theywere adjusted after cleaning filters, but as the filters became obstructed,the flow rate reduced. Flow rates did not exceed the maximum ratingfor the filters of 38 L/min. Filters were cleaned each day. Water supplyto the upwellers was stopped during cleaning of the filters, which wasachieved using fresh water under moderate pressure from a domesticwater supply tap. Paper/polyester filter cartridges were replaced weeklyin trials 1, 2, 4, 5 and 6, daily in trial 7, but were not replaced in trial 3.

Therewas a 5month interval between trials 1 and 2, duringwhich thetanks and apparatus were dry. The upwellers were disinfected withVirkon®(Antec International Limited) prior to trials 2, 3 and4; treatmenttanks were rinsed out, mixing tanks were cleaned to remove sediments,and the pipework was physically vibrated to remove adherent debris inthe lumens. There was a 3-week interval between trials 3 and 4 duringwhich the treatment and mixing tanks were dry. There was an 11-daygap between trials 4 and 5 and tanks were cleaned but not disinfectedprior to trial 5. No disinfection or cleaning was undertaken prior to trials6 or 7.

2.2. Experimental treatments

Seven consecutive experimentswere conducted between April 2013andMay 2014, that is during autumn2013 and summer–autumn2013–2014 when the virus was known to be active in the estuary system. Thespecific water treatments that were tested varied from trial to trial(Table 1). Treatments were chosen to identify the simplest and least ex-pensive method of reducing OsHV-1 transmission risk. Thus, the chillertreatment was used only once and UV irradiation was included in trials1 and 2 only. The duration of each trial was determined either by theonset of mortality and loss of spat due to mortality, or by the reductionin the numbers of spat due to sampling.

2.2.1. River controlControl oysters were placed in 2 mm mesh socks within a

6 mm mesh floating basket (BST, Australia) in the river. The basketwas tethered to a rope and could move on a 1.5 m radius; this allowedit to be as close as 0.5 m to the water intake points for the othertreatments.

2.2.2. Upweller controlWater was pumped continuously from the river to the mixing tank

and supplied to the upweller tank via the manifold, without anytreatment.

2.2.3. ChillerWater was pumped continuously from the river to the mixing tank

then via the manifold to a stainless steel heat exchange coil immersedin a cold water bath then fed to the upweller tank; the temperature re-duction was 2–3 °C in comparison with river temperature.

2.2.4. Filtration with or without UVWater was pumped continuously from the river to the mixing tank

then via the manifold to the filters, then in series (if required, trials1 and 2 only) to a UV light source (VGX48VH, Atlantic-Ultraviolet)and then supplied to the upweller tank. Pre-filtration using either a

Table1

Outlin

eof

triald

esignan

dtheresu

ltsof

qPCR

teststo

detect

OsH

V-1

DNA.D

ataarethegreatest

viralloa

d(cop

iespe

rmg)

detected

andthehigh

estn

umbe

rof

poolswithde

tectab

leDNA(inpa

renthe

ses)

onan

yda

y.Viral

load

saretheav

erag

eof

4po

ols(trial1)

or5po

ols(trials2–

7)of

spat

(poo

lswithno

DNAde

tected

wereex

clud

edfrom

thecalculationof

theav

erag

e).Treatmen

tsin

which

mortalitieswereob

served

aresh

ownin

bold.The

freq

uenc

yof

detectionof

OsH

V-1

DNA(n

umbe

rof

test

days)in

treatm

ents

witho

utmortalityisgive

nin

theresults.

Trial

Startda

teDurationof

trial(

days)

Mortalityfirst

observed

(day

)Interval

betw

een

PCRtests

OsH

V-1

DNA

firstde

tected

(day

)

Max

imum

viralloa

d(n

umbe

rof

positive

pools)

Rive

rco

ntrol

Upw

eller

control

Age

dwater

Filtration

(μm)

Chilled

water

100/5+

UV

100/5

55/5

100/30

55

16Apr

1339

203–

4da

ys17

5.4×

105(4)

6.3×

105(4)

BLOQ(2

1)

BLOQ(1

1)

––

––

4.2×

105(3)

230

Oct

1328

151da

y12

1.9×

106(5)

1.7×

106(5)

BLOQ(4

1)

BLOQ(3

2)

BLOQ(1

1)

––

––

327

Nov

1328

201da

y15

2.8×

105(5)

2.1×

105(5)

BLOQ(2

1)

––

–BL

OQ(1

1)

8.3×

104(5)

–

415

Jan14

1514

1–4da

ys11

2.0×

105(5)

4.5×

105(5)

BLOQ(1

)–

–BL

OQ(1

1)

6.0×

104(5)

––

526

Feb14

28Nil

3–4da

ys28

Not

detected

Not

detected

Not

detected

–BL

OQ(1

1)

Not

detected

––

–

626

Mar

1449

244–

5da

ys22

2.0×

105(5)

5.5×

103(1

)Not

detected

–Not

detected

BLOQ(1

1)

––

–

714

May

1415

154–

5da

ys15

6.5×

104(5)

Not

detected

––

–Not

detected

––

–

–,n

otinclud

edin

trial.

BLOQ,D

NAde

tected

butlev

elbe

low

limitof

quan

tification

.1Allpo

olswithde

tectab

leDNAwereinconc

lusive

—i.e.,on

lyon

eof

tworeplicateDNAex

tracts

yielde

daCt.

2Tw

oof

threepo

olswereinco

nclusive

.

13R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

100 μmor a 55 μmstacked polypropylene disc filter (1 inch short; Arkal,Israel) was used in all filtration treatments. The post-filtration used insome treatments was a 5 μm pleated paper cartridge filter in a plasticflow through canister housing (Omnifilter BF7, USA) (trials 1, 2, 3, 4,5, and 6) or a 5 μm pleated polyester fabric cartridge filter (Puretec,Australia) in the same housing (from 25th April 2014, trials 6 and 7).In trials 3 and 4, a 30 μmpleated paper cartridge post-filter (Omnifilter)was used after pre-filtration instead of a 5 μm filter. In trial 3, a 55 μmfilter treatment without post-filtration was tested. Filter pore sizes arenominal, as specified by themanufacturers. Notation used in the resultsdescribes the filter combination used in each treatment, i.e., 55, 55/5,100/5, and 100/30 μm.

2.2.5. Aged waterWater was pumped from the river when required and stored in two

opaque green polyethylene holding tanks (20,000 L trial 1; 10,000 L, tri-als 2 to 6). The water was not circulated or mixed except during fillingso that sedimentation would occur. Water was supplied from an outlet10 cm from the base of each holding tank by continuous gravity feed toa dedicatedmixing tank and then via a pump to the upwellers. The fill-ing of the holding tanks with river water and the supply of water fromthem to the upwellers was alternated between the two holding tanks,ensuring that a 48 h aging timewas always observed between tank fill-ing and drawing water.

2.3. Oysters

Triploid Pacific oyster spat (Shellfish Culture, Tasmania) that werecertified by the competent authority to be free of OsHV-1 based onPCR testing (trial 1 batch SPL12FT 4 month old; trials 2, 3, and 4 batchSPL13B, 2.5, 3.5, and 5 months old, respectively; trials 5, 6, and 7batch SPL13C 3.5, 4.5, and 6.5 months old, respectively) were placedin each of the upwellers in the treatment tanks and the floating basketin the river at the start of each trial. A new cohort was recruited directlyfrom Tasmania for each trial unless otherwise stated. Spat were heldout of water for b24 h during transport and placement in the trial.There were 500 spat per upweller (2000 spat per treatment) unlessotherwise stated. There were 2000 spat in the river control. No addi-tional food was supplied to spat in upwellers.

Due to high mortality by day 23, the river control in trial 6 wasreplenished with 2000 new spat on day 28.

The spat used for trial 7were received at the commencement of trial6 and were held in untreated river water in the mixing tank that sup-plied water to the upweller control and filter treatment, from 26thMarch, i.e., throughout trial 6.

Spat were monitored for OsHV-1 infection, survival, and growth.

2.4. Random sampling and detection of mortality

Oysters in each upweller and the river basket were examined dailyexcept in trial 4 where they were checked three times weekly. Spatwere tipped onto a tray or table for close inspection. Spat were consid-ered to be dead if the valveswere empty orwere open or opened duringhandling. Not every dead oyster was expected to be able to be detectedin this way. Themortality rate was assessed bymanually counting deadspat in each treatment. At least 40 apparently live spat per treatment(10 per upweller) were sampled at these times (Fig. 3). As active sam-pling led to a reduction in the number of spat per treatment over time,this was taken into consideration when calculating the cumulativemortality as previously described (Paul-Pont et al., 2013b). Conditions

Fig. 3.Mortality patterns of spat in each treatment group and average daily water temper-ature. Panel A, trial 1; panel B, trial 2; panel C, trial 3; panel D, trial 4; panel E, trial 5; panelF, trial 6; panel G, trial 7. The arrow indicates the daywhenOsHV-1DNAwasfirst detectedin spat from the river control and/or the upweller control. Temperatures are for probes inthe control upweller tank.

14 R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

15R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

that would eliminate the risk of cross contamination between samplesfrom different treatment groups were not practical in the farm shed en-vironment, but the operator washed hands using soap and water andrinsed the table or tray using freshwater between the inspections ofeach treatment group.

2.5. Detection of OsHV-1 using qPCR

Oyster spat were pooled for homogenisation and testing. For trial 1,four pools of 10 oysters were tested per treatment per time point. Fortrials 2 to 7, five pools of oysters were tested per treatment per timepoint; each pool contained 300–500 mg of whole spat if they wereb6 mm in length, or 500 mg of tissues removed from the shell if theywere N6mm long. The number of spat per pool ranged from 5 to 10 de-pending on the size of the spat. Oysters were pooled into 2 mL safe lockmicro test tubes (VWR, Australia) containing 1 mL of Ultrapure water(Life Technologies, Australia) and two 3 mm stainless steel beads. Oys-ters were homogenised using mechanical disruption via high-speedshaking in a TissueLyser II machine (Qiagen®, Australia). The resultingsuspension was 500mg/mL. The presence of shell appeared to enhancethe homogenisation process, but the effects of shell on assay sensitivityhave not been investigated.

Nucleic acids were purified using a 5XMagMAX™-96 Viral RNA Iso-lation Kit (Ambion®, Life Technologies™ Australia) andMagMAX™ Ex-press 96 magnetic particle processor (Applied Biosystems™, LifeTechnologies™ Australia) according to manufacturer's instructions fora 50 μL sample volume protocol using the AM1836 deep-well standardprogram (Ambion®, Life Technologies™ Mulgrave, Australia). Purifiednucleic acids were tested for OsHV-1 using a quantitative real-time po-lymerase chain reaction (qPCR) prepared using the Ag-Path IDOne-stepRT-PCR kit (Life Technologies) based on the method of Martenot et al.(2010) as previously described (Paul-Pont et al., 2013b). The detectionlimit was evaluated at 3 copies per mg of tissue and the quantificationlimit at 12 copies per mg tissue based on guidelines previously pub-lished (Bustin et al., 2009; Martenot et al., 2010). Two replicates ofeach DNA extract were tested. A plasmid DNA standard curve serieswas included on each plate. A valid run was defined as a run exhibitingno amplification of the negative control, amplification of the positivecontrol (Ct within range of the standard curve) and a standard curvewith r2 N 0.95 and efficiency between 90% and 110%. The threshold set-ting for each run was calculated using the amplification-based algo-rithm (Mx300 Software, Stratagene) for the standard curve series andmanually locked for analysis of the experiment samples. A sample wasdefined as positive when both replicates exhibited an exponential in-crease in the ROX normalised baseline corrected FAM fluorescenceand a threshold cycle (Ct) valuewas assignedwhen the fluorescent sig-nal exceeded the threshold. The quantity of OsHV-1 was inferred forpositive samples as the number of genome copies from the standardcurve. Samples with a valid Ct below the limit of quantification wereregarded as containing trace quantities of OsHV-1 DNA and were re-ported as positive, below the limit of quantification. An inconclusive re-sult was assigned for samples when only one replicate had a valid Ct.Experimental samples were reported as negative when amplificationdid not occur and a Ct value could not be assigned for either replicate,indicating that OsHV-1 was not present within the limits of detectionof the assay.

2.6. Prevalence of infection

The prevalence of OsHV-1 infection in each treatment group at eachtimewas estimated from the results of pooled testing of live oysters ran-domly selected from the upwellers or river basket after the removal ofdead individuals, based on the proportion of positive pools, using calcu-lators available at http:/epitools/ausvet.com.au. These are based onCowling et al. (1999). Method 7, the Bayesian method, requires specifi-cation of priors for prevalence, sensitivity, and specificity.We specified a

uniformbeta prior for prevalence (alpha=1 andbeta=1), an informa-tive prior for sensitivity assuming amedian of 0.90with lower 5% inter-val of 0.70 (alpha = 15.03 and beta = 2.56), and an informative priorfor specificity assuming a median of 0.995 with a lower 5% interval of0.95 (alpha = 1137.5 and beta = 6.7). In trial 1, there were 4 poolswith 10 spat per pool. In trials 2 and 3, there were five pools with fivespat per pool. In trials 4 to 7, there were six to 10 spat per pool. For sim-plicity, the term “low” is used to describe prevalences≤13%whichwerevalues obtained for mean prevalence for 1 positive pool out of 4 or 5pools tested for the range of pool sizes used. Corresponding figures forall 4 or 5 pools positive were ≥60% and are referred to as “high”.

2.7. Water temperature monitoring

A temperature probe (Thermocron® temperature logger,Thermodata Pty Ltd) was placed into each experimental tank, as wellas the floating basket in the river and each water aging/holding tank,to record temperature every hour.

2.8. Growth

The shell lengths of spat in each treatment were assessed at the timeof analysis of samples of the oysters in the laboratory. The pools of spatwere placed on 2mmsquare ruled graph paper, photographed and thenindividual spat were measured after enlarging the images.

3. Results

3.1. Mortality

The patterns of cumulativemortality in each trial are shown in Fig. 3.In general, when disease occurred, it was consistent with the patternsexpected in OsHV-1 infection of spat, that is, a very rapid increase inmortality over a few days.

Trial 1.Mortality was detected in the upweller control tank suppliedwith untreatedwater on day 21 and reached 100% on day 28. On thesame day, the first mortalities were observed in the river controlspat, and the cumulative mortality increased rapidly to 88.3% (day31) then 97.2% by the end of the experiment on day 39. Mortalityprogressed more slowly in the chiller treatment from day 29 untilthe end of the experiment when cumulative mortality reached42.3%. No mortality was observed in the spat in the aged water,100/5 μm filter, or filter + UV treated water groups.Trial 2. Mortality was detected on day 15 in both the river controland the upweller control, reaching 60% and over 80%, respectively,within 1 day, and then progressed very gradually. No mortalitywas observed in the spat in the aged water, 100/5 μm filter, or100/5 μm filter + UV treatments.Trial 3.Mortality commenced on day 20 in spat in the upweller con-trol and on day 21 in spat in the river control, andmost of these spatwere dead within 3 days. Mortality was first detected 4 days later inspat in the upwellers suppliedwith 55 μm filtered water. The cumu-lative mortality plateaued at 60% for several days before completionof observations. No mortality was observed in the spat in the agedwater or 100/5 μm filter treatments.Trial 4.Mortality beganon day14 and reached 100% in the spat in theriver control, the upweller control and in the upwellers suppliedwith 100/30 μm filtered water within 2 days. No mortalities wereobserved in spat in the aged water or 55/5 μm filter treatments dur-ing this time.Trial 5.Nomortalities were observed in spat in any of the treatmentsor controls during the 28 day trial.

A

B

Fig. 4.Water temperature in thenorthern-most 10,000 Lwater holding tank (panel A) andthe upweller control tank (panel B) in trial 4. Bars show the temperature range(minimum-maximum) while the black symbols show the daily mean temperaturebased on recordings every 60 minutes.

16 R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

Trial 6.Mortality began on day 23 in the river control, and 95% diedwithin 1 day. The floating basket in the river was replenished with2000 new spat on day 28. Thirteen dead oysters were observed inthe river control between days 47 and 49, with the additional finalcumulative mortality being 0.1% at the end of the trial. No mortalitywas observed in the upweller control; therefore, comparison withfiltration treatments cannot be made. No mortality was observedin the aged water treatment.Trial 7. Minor mortality was noticed in the spat in the river controltowards the end of the experiment, with final mortality of 1.4%,but no mortalities were observed in spat in any of the other treat-ments or controls during the 15-day trial.

3.2. Detection of OsHV-1 DNA

Quantitative PCR (qPCR) tests to detect OsHV-1 DNAwere conduct-ed on samples collected from each treatment each day in trials 2 and 3,but for trials 1, 4, 5, 6, and 7, the tests for OsHV-1 DNA were done onsamples from every 2nd to 4th day (Table 1).

In treatment groups where there was high mortality (≥60%), theviral load exceeded 105 copies per mg of spat tissue prior to or at thetime of mortality, and all of the pools of spat that were testedcontained detectable OsHV-1 DNA, suggesting a high prevalence ofinfection. This applied to the river control in trials 1, 2, 3, 4, and 6;the upweller control in trials 1, 2, 3, and 4; the 55 μm filter treat-ment in trial 3; and the 100/30 μm filter treatment in trial 4. Thespat in the river control had just begun to die on the last day oftrial 7, and they contained 6.5 × 104 DNA copies per mg averagedover the 5 pools.

Among trials, OsHV-1 DNAwas detected up to 5 days before the onsetof mortality in the river control and upweller control; this time could bedetermined accurately in trials 2 and 3where daily samples were collect-ed and tested and represent the incubationperiod for the disease. Howev-er, when the infection was first detected in these treatments theprevalence was low (1 of the pools positive) and viral load was alsolow, below limits of detection (BLOD) or below limits of quantification(BLOQ) to 102 copies per mg. As testing was not conducted daily inother trials, the incubation period for them is uncertain.

In the chiller treatment in trial 1, therewas a delay in the onset ofmor-tality and a more progressive increase in mortality than in the controlgroups; cumulative mortality reached only 42% (Fig. 3). OsHV-1 DNAwas first detected on day 25, and mortality began on day 29 when viralload exceeded 105 copies per mg.

In treatments in which there was no mortality, OsHV-1 DNA was ei-ther not detected, or was detected intermittently, typically at low preva-lence and low viral load (below the level of quantification of the qPCRassay) (Table 1). The only exception to this was in trial 6 where spat inthe upweller control remained apparently healthy throughout the49 day experiment but on day 14 one of 5 pools contained 5.5 × 103 cop-ies per mg. Further evidence of low viral load was the inconsistent detec-tion of OsHV-1 template DNA in the two replicate assays conducted oneach DNA extract (Table 1) (Bustin et al., 2009). Intermittent detectionof OsHV-1 DNA was common: in trial 1, in the aged water and 100/5 μm filter + UV treatments, OsHV-1 DNA was detected in spat on day25 but not before that (i.e., on days 2, 5, 9, 13, 17, or 21) or after that(i.e., on days 29, 33, or 37). In trial 2, OsHV-1 DNA was detected in spatin the 100/5 μm filter + UV treatment group on only 4 days out of the28 for which samples were collected and tests conducted. Similarly forthe 100/5 μm filter treatment, it was for 3 test days out of 28, and foraged water 4 test days out of 28. The corresponding results for trial 3were aged water 3 of 28 test days; 100/30 μm filter 1 of 28 test days. Intrial 5, the only detection of OsHV-1 DNA over 8 test days was fromspat in the 100/5 μm filter treatment on day 15. In trial 6, viral DNA wasdetected on 1 of 8 test days in the 55/5 μm filter treatment group, andin trial 7, it was not detected in spat on any of 4 test days.

3.3. Water temperature

As the water was pumped from the river, the temperature in the ex-periment followed the natural daily and seasonal fluctuations of watertemperature observed in the river. Mean daily water temperatureswere above 17 °C throughout the trials. The temperature profiles ofthe river control, upweller control and filter treatments were similar;those for the upweller control are shown in Fig. 4. The average dailywater temperature was between 20 °C and 25 °C in trials 1 (April–May), 2 (October–November), and 3 (November–December); around25 °C in trials 4 (January–February) and 5 (February–March), decreasedprogressively in trial 6 (April) from about 25 °C to about 17 °C, and wasbetween 17 °C and 20 °C in trial 7 (May).Minimumdailywater temper-ature was 2–3 °C below daily average; maximum daily temperatureswere up to about 5 °C warmer than average daily temperatures (datanot shown). Temperatures were reduced by 2–3 °C in the chiller treat-ment in trial 1. Temperature profiles in the holding tankswere differentfrom those in the other treatments andwere influenced by degree offill,ambient air temperature, and solar exposure as the tanks were notshaded (Fig. 4). Daily maximum temperatures in the water holdingtanks exceeded 30 °C on three occasions in trial 1, three occasions intrial 4 and on one occasion in trial 6. In trial 2, no data were collectedfrom 30/10/13 to 5/11/13 due to probe and computer faults.

3.4. Growth of oysters

In the first trial, there was noticeable retardation of growth of thespat in the aged water and filter water treatments relative to the rivercontrol, upweller control, and chiller treatments. Measurements of thelength of spat confirmed these observations in trials 1 to 7 (Fig. 5). Ingeneral, spat kept in treatments in which water was filtered to 5 μm,and those in the aged water treatment tended not to grow while spatkept in untreated water or water filtered to 55 μm or 30 μm grew inlength.

A B

DC

E

G

F

Fig. 5. Length of oyster spat in each treatment group. Panel A, trial 1, panel B, trial 2; panel C, trial 3; panel D, trial 4; panel E, trial 5; panel F, trial 6; panel G, trial 7.Measurements are for thefirst time point and for the last time point at which animals in every treatment could be measured. Data are the mean and standard deviation for samples of 25 to 40 oysters in eachtreatment.

17R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

4. Discussion

This studywas conducted during the summer–autumn in an estuaryinwhich natural exposure of oysters to OsHV-1 μVarwas likely to occur.Water temperatures recorded throughout the trials exceeded thethreshold for onset of mortality associated with OsHV-1 of 16 °C thathas been observed in France (Pernet et al., 2012) and were within therange observed in summer2012–2013when thefirst outbreakoccurredin theHawkesbury river system (Paul-Pont et al., 2014). Observations ofdisease in spat held in untreated control water or in the river itself indi-cated that transmission of OsHV-1 μVar and disease occurred on 6 occa-sions. This enabledmeaningful study of the effectiveness of agingwaterfor 48 h or filtration to reduce the incidence of transmission of OsHV-1μVar. Indeed, for a total population time at risk of 202 days, i.e., the com-bined duration of the 7 trials, the total cumulative mortality observedfor spat held in aged water or water filtered to 5 μm was zero.

In trials 1, 2, 3, and 4, highmortality rates (60–100%) were observedin spat in both the river control and the upweller control, enabling validcomparison with all other treatments. No mortality was observed inspat in the upweller control in trials 5, 6, and 7, so it was not possibleto make robust conclusions about the filtration treatments in these tri-als, although general observations remain relevant. High mortality oc-curred in spat in the river control in trial 6, enabling comparison withspat in the aged water treatment.

In trials 1, 2, 3, and 4, therewere strikingdifferences inmortality pat-terns of spat in different treatment groups, and consistent results wereobtained among trials (Fig. 3). There was a high mortality rate in spatin both the river control and in the upweller control associated withOsHV-1 infection; it developed suddenly and peaked within a fewdays. No mortalities were observed in spat that were kept in waterthat had been aged for 48 h or in spat that were kept in water filteredto 5 μm. In the third trial, spat kept in water filtered to 30 μm survivedbut in the fourth trial 30 μm filtration failed to prevent mortality. Filtra-tion to 55 μm did not prevent mortality in the third trial. UV irradiationof water did not appear to be necessary to preventmortality after filtra-tion of water to 5 μm, based on a comparison of filtration treatmentswith and without UV irradiation in the second trial.

In trials 2 and 3 in which daily sampling and PCR tests were under-taken, the incubation period (the interval between the first detection ofOsHV-1 and the onset of mortality) in controls was 3 to 5 days. This iscomparable to the estimate of 4 daysmade from epidemiological obser-vations during the first natural epizootic in the Hawkesbury river estu-ary in January 2013 (Paul-Pont et al., 2014).

Thefindings of the trials described here have important practical im-plications for the farming of Pacific oysters. First, it appears to be possi-ble to treat water using simple physical methods such as aging water ina holding tank for 48 h, or filteringwater to 5 μm, and in this way renderestuarinewater safe for maintaining spat. Based on research on OsHV-1

18 R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

from Europe, the spat used in these trials are believed to be the mostsusceptible life history stage (Peeler et al., 2012; Renault et al., 1995).Therefore, the findings should be relevant to older and larger classesof oysters. Second, the findings should be applicable to design of waterintakes for commercial hatcheries because similar filtration and waterholding systems can be used in hatcheries. Third, it may be possible todevise shore-based or pontoon-based upweller systems to hold spatwithin an OsHV-1 infected estuary during the seasonal risk period forOsHV-1. Fourth, the water treatments described here should also be ap-plicable for holding broodstock and other valuable resources. The caveatis that artificial nutrition will be required for optimal maintenance andgrowth of oysters kept in treated water.

PCR data confirmed the presence of high levels of OsHV-1 within thetissues of spat during periods of highmortality, and in the case of the sev-enth trial, as mortality was beginning. The average viral loads amongpools of oysters at these times in these treatments were above thethreshold of 1 × 104 copies per mg of oyster tissue that has been associ-ated withmortality in France and Australia (Oden et al., 2011; Paul-Pontet al., 2013b; Paul-Pont et al., 2014). Therefore, it is reasonable to assumethat the observed mass mortalities were caused by the virus.

Across all trials, there were 15 treatment groups containing spat inwater filtered to 5 μm or in aged water. While mortality was not ob-served, OsHV-1 was detected by qPCR at some time points in samplesfrom 10 of these treatment groups (Table 1). The interpretation ofqPCR results from spat in treatment groups that did not experiencemortality is unclear because (i) cross contamination from other treat-ments inwhich highmortality occurred could easily have occurred dur-ing some of the trials, and (ii) the PCR provides no indication aboutviability of virus, i.e., DNA from inactivated virus could be detected.When OsHV-1 DNA was detected in treatment groups without mortal-ity, it was typically associated with only a small proportion of spat andin a low quantity that was below the limit of quantification of theqPCR assay. Cross contamination of samples between treatment groupswas possible and could possibly explain the detection of OsHV-1DNA inapparently healthy spat. Alternatively, these spat may have been ex-posed to sub-lethal doses of live virus, inactivated virus, or viral nucleicacid. If exposure to live virus had in fact occurred, the infection did notprogress under the conditions of the trial. Trial 6 provided an exampleof detection of OsHV-1 DNA in a low proportion of spat kept in untreat-ed water in the upweller control at a time when there was nomortalityin this treatment group (day 14). This demonstrated an incident of virusexposure from naturally infectious seawater without progression tohigh mortality disease.

It is important to note that even if the spat in treatments in whichmortality did not occur had consistently had negative qPCR results, itwould not have confirmed that infectious OsHV-1 had been eliminatedfrom treated water, due to low sample sizes, short duration of trials andthe nature of the PCR data. There remains some uncertainty about theinterpretation of negative results because sample sizes of 40 spat pertreatment per day were not sufficient to prove beyond reasonabledoubt that virus had been excluded; the daily confidence would be95% that prevalence of infection was less than about 10% (Cannon andRoe, 1982), although because testing was repeated over a number ofdays the effective prevalence limit would be lower than this. To confirmfreedom of infection in spat raised in treated water would require a dif-ferent trial design so as to prevent cross contamination from controls,larger sample sizes, and long-term monitoring.

The mechanisms of action of aged water and 5 μm filtration treat-ments to prevent mortality were not investigated but may be relatedto the elimination of OsHV-1 attached to particles that were removedfrom the water column by sedimentation or filtration, respectively.This is consistent with the hypothesis about particulate attachment ofOsHV-1 (Evans et al., 2014; Paul-Pont et al., 2013a). However, in thecase of aged water, the protective effect might also be related to lossof infectivity of OsHV-1 over 48 h. OsHV-1 is a member of the orderHerpesvirales (Davison et al., 2009); it is a double stranded DNA virus

with a lipid envelope (Hwang et al., 2013; Renault et al., 1994). In gen-eral enveloped viruses are less resistant than non-enveloped viruses todamage due to environmental exposure (Quinn andMarkey, 2001). Theonly data available on the stability of a pathogenic marine herpesvirusconcerns the abalone herpesvirus AbHV, which was stored in seawaterunder ideal conditions in a laboratory; it retained infectivity and viru-lence for abalone for only a few days at 15 °C (Corbeil et al., 2012).The large enveloped double stranded DNA virus white spot syndromevirus, a pathogen of shrimp, was infective for up to 12 days in seawater(Satheesh Kumar et al., 2013). In freshwater, koi herpesvirus lost infec-tivity within approximately 3 days at temperatures above 15 °C(Shimizu et al., 2006). Studies of other aquatic pathogenic virusessuch as viral hemorrhagic septicemia virus suggest that factors such astemperature and salinity are likely to have substantial effects on viralsurvival and infectivity in seawater (Hawley and Garver, 2008). In thecase of AbHV, survival was significantly less at 25 °C compared to 15°C (Corbeil et al., 2012). Further research is required to understand themechanism through which the aging of seawater protects C. gigas spatfrom OsHV-1-related mortality.

The physical design of the experiment involved water distributedfrom a common manifold that supplied both the control oysters andthe filtration or chiller treatments. Therefore, the potential for expo-sures to OsHV-1 for all these treatments was directly comparable.There was delayed onset of mortality in spat in the chilled water treat-ment in trial 1, which may relate to physiological influences on thepathogenesis of the disease. It is possible that a small reduction inwater temperature (in this experiment it was about 2–3 °C)may favourthe oyster and not viral replication. This will require confirmation in anexperimental infection model under controlled environmental condi-tions. In trial 3, the delayed onset of mortality compared to controls inspat placed in 55 μmfilteredwatermay relate to a reduction in exposuredose due to partial removal.

The aged water treatment was different to the other treatments be-cause the water was intermittently sourced from the river. It was ob-tained during about 3 h every 2 days to fill the holding tank, while theother treatments were based on a continuous supply of water fromthe river. It is known that OsHV-1 is not uniformly distributed in seawa-ter, either spatially (small scale—m, and large scale—km) or temporally(Paul-Pont et al., 2013a). For this reason, the aged water treatment isnot directly comparable to the other treatments. It is possible thatwater obtained from the river at the times the pump was activated tofill a holding tank may not have contained OsHV-1. Depending on thedegree of fill, solar radiation and ambient air temperature, the probesin the holding tanks sometimes recorded daily ranges greater thanthose in other treatments. For these reasons, the experiment withaged water was conducted six times in order to providemany opportu-nities for the treatment to fail. In five of the 6 trials in which aged waterwas included as a treatment, the controls in the river died. However theriver controls may not be a complete proxy for the water used to fill theholding tanks as the spatial and temporal clustering of OsHV-1 in theriver could be at a very fine level (meters, hours). This was illustratedin trial 1 as the river controls died about a week after the upweller con-trols and in trials 6 and 7 when only the river controls died, suggestingexposure of the two treatments at different times, despite the closeproximity of the submersible pump and the floating basket in the river.

Although the experiments were of relatively short duration, growthof spatwas retarded in the agedwater and filter treatment groups com-pared to the river controls and upweller controls. Hatcheries may al-ready use filtration and supply cultured algae and rotifers to spat toenable their growth. However, design and maintenance specificationsfor filtration need to be checked and more consideration of spat nutri-tion will be required for long-term husbandry if rearing spat in treatedwater is attempted commercially.

The different nutritional and/or physiological state of oysters in theaged water and 5 μm filtered treatments, which was evident in theirlow growth rate, might also be important in explaining the absence of

19R.J. Whittington et al. / Aquaculture 437 (2015) 10–20

mortality in these treatments. As well-fed oysters died due to OsHV-1and poorly fed oysters did not, it is reasonable to question whetherthe removal of food from water per se was responsible for protectionof spat. That this was unlikely to be the case is suggested by several ob-servations: (i) in laboratory transmission studies, unfed oysters exposedto OsHV-1 by co-habitation or intramuscular injection have been re-ported to suffer high mortality rates (Paul-Pont et al., in press;Schikorski et al., 2011a; Schikorski et al., 2011b); (ii) well-fed oystershad higher energy reserves and decreased risk of mortality due toOsHV-1 (Pernet et al., 2014); and (iii) in this study, OsHV-1 was not de-tected in most of the oysters in treatments in which mortality did notoccur (those poorly fed), suggesting that infection was usually avoidedin these treatments. There was no evidence for the alternative, namely,that exposure and infection occurred but disease failed to express. Thedesign of these experiments did not permit investigation of growthand mortality if artificial feed was provided or of compensatory growthandmortality if natural feedingwas allowed to resume, but the absenceof infection in themajority of samples of spat from agedwater and 5 μmfilter treatments suggests that disease would be avoided in the absenceof renewed exposure to OsHV-1. In the case of summer mortality in Pa-cific oysters, a condition which has been discussed by some authors in acontext other than that of OsHV-1 infection, correlations have been re-ported between plane of nutrition and mortality. In particular, therewas lower cumulative mortality where nutrition was restricted(Cotter et al., 2010; Delaporte et al., 2007). In contrast, observations inthe field indicated that mobilisation of energy reserves during OsHV-1μVar infection was correlated with improved survival (Pernet et al.,2014). As the published literature is ambiguous on the role of nutritionin summer mortality, the impact of restricted nutrition and subsequentchanges in physiology under the conditions in this study warrants fur-ther investigation.

During these trials, there was considerable opportunity for cross con-tamination of treatments. The treatment tanks were in close proximity,there would have been aerosols from the tanks and pumps, and therewas movement of personnel and other equipment into and within theshed. However, based on whether or not disinfection and cleaning ofthe systemwas conducted between trials (see Section 2.1 in thematerialsandmethods), some observations relevant to the stability of the virus arepossible. Based on the interval of 11 days between trial 4 where OsHV-1infection and mortality occurred in controls, and trial 5 where there wasnomortality in any treatment, it is reasonable to conclude that the persis-tence of infectivity (sufficient to induce mortality) in a cleaned culturesystem was less than 11 days. Trial 3 commenced immediately aftertrial 2, after disinfection using Virkon. Based on the absence of mortalityin control upwellers in trial 3 within a recognised incubation period forOsHV-1 mortality (4–10 days), it is reasonable to conclude that Virkonis an effective disinfectant for OsHV-1when applied to aquaculture infra-structure. An alternative to these conclusions is that infection mecha-nisms other than virus derived from contaminated surfaces is required(for example, ingestion of particle-attached virus).

A further consideration is that hatcheries at risk of OsHV-1 infectionfor example due to importation of broodstock from other locationsmaybe required to implement effluent controls to prevent infection of localwild stocks of Pacific oysters. Effective methods to minimize the risk ofOsHV-1 spread from hatcheries through effluent control are currentlyunknown and at present would have to be based on general principlesof virology anddisinfection rather than actual data. The design of the ex-periments described here does not allow conclusions about effluenttreatment, because particles to which OsHV-1 may attach may not bepresent in effluent water and therefore OsHV-1 may not be able to beremoved by filtration or sedimentation. However, the findings suggesta useful experimental design for conducting research on effluent con-trols for hatcheries.

In conclusion, the findings of this study support the hypothesis thatthe removal of the putative particulate vector of OsHV-1 from seawaterusing sedimentation or filtration would enable C. gigas spat to survive

despite the presence of OsHV-1 μVar in an estuarine water supply.These results mandate investment by oyster hatcheries in simple andpractical methods to treat incoming estuarine water to enable safe pro-duction of spat in infected estuaries during the risk period for OsHV-1μVar disease expression. These water treatments would also allowhatcheries or farmers to hold spat in land-based facilities during the sea-sonalwindow of infection, provided that nutritionwas addressed to en-able maintenance of body condition or growth of spat.

Acknowledgments

The experiments were funded by the Fisheries Research and Devel-opment Corporation and the University of Sydney, with co-fundingfrom the Tasmanian Oyster Research Committee (trial 1) and OystersAustralia via the Seafood CRC (trials 2 to 7). Mrs. Alison Tweedie, Mrs.Vickie Patten, Mrs. Ann-Michele Whittington, and Ms. Chloe Englishprovided skilled technical assistance. Thanks are due to Mr. RegRichards (deceased) and Mr. Steve Jones who gave access to sheds atMooney Mooney in which the infrastructure for the trials was installed.Special thanks are due to Shellfish Culture Tasmania for supplying thespat.

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