1. Advanced surveillance for oyster diseases
Marty Deveney, Jessica Buss, Kathryn Wiltshire
AAHL Fish Diseases Laboratory
Kevin Ellard DPIPWE
16 August 2019

2. Marine molecular surveillance
Filtration
Extraction control
Sample control
Preserve buffer
Collection-Plankton Tows
Marine molecular surveillance
Inhibition control
Interpretation
DNA Extraction
Plankton samples are spiked with the positive control, filtered on board the sampling vessel or in the laboratory, DNA is extracted from the sample, tested by qPCR and this provides results on presence or absence of the target organism in the sample.
These results need to be interpreted in a biologically meaningful context, bearing in mind the pest status of the test site, seasonality, sampling sensitivity, water currents and other environmental factors.
qPCR
-target(s)
-3 controls
Results

3. OsHV-1 environmental surveillance
Hobart Port / Derwent River samples January 2016
Tested after February 2016 OsHV-1 outbreak
3 confirmed positives from 30 samples
Assess utility of environmental surveillance

5. Adelaide ‘control’ from 2016 to the end of 2017
But then . . .
One indeterminate (1/2 tests positive) re-tested negative (0/4)
600+ oyster samples tests negative

6. 2018 – POMS in Port Adelaide
February 2018 OsHV-1 microvariant outbreak
Growing areas free
Port River oyster tissue surveillance Feb 18-Jan 19
Sample Date
Detections
Samples
Apparent prevalence
 21-Feb-18 16 25
64.0%
 02-Mar-18
143
208
68.8%
 24-Apr-18 32
50.0%
 28-May-18 17
53.1%
 26-Jun-18 12 31
38.7%
 14-Aug-18
37.5%
12-Sep-18 11 37
29.7%
 23-Oct-18 6 46
13.0%
 26-Nov-18 13 33
39.4%
 20-Dec-18 23
71.9%
 22-Jan-19 7 36
19.4%
Bayesian LCM both tests ‘or’ rule
DSe 0.9 (95%CI 0.81 – 0.94)
DSp 0.94 (95%CI 0.86 – 1.00)

7. Plankton surveillance Nov 17-Jan 19
Population
Season
Negative
Martenot only
Jenkins only
Both positive
Apparent occurrence
SA - wild
Summer 65 2 1
5.8%
Autumn 38 3
15.6%
Winter 15
0.0%
Spring 52
7.1%
Tas - farm 39 16 5
35.0% 14 9
44.0% 20
4.8% 33 4
15.4%
Tas - wild 36 7
23.4% 25 8
32.4%
3.8% 27
Detections when transmission is occurring
Bayesian LCM both tests ‘or’ rule
DSe 0.83 (95%CI 0.57 – 0.95)
DSp 0.91 (95%CI 0.86 – 0.96)

8. Samples to detect 7% prevalence
Target SSE
Test applied
Number of samples
Mean (95% CI)
0.8
Jenkins
98 (187 – 60)
Martenot
28 (42 – 24)
Both tests
27 (38 – 23)
0.95
182 (348 – 111)
52 (78 – 44)
49 (71 – 43)
Basis of an area surveillance method using eDNA
Need to understand analytical sensitivity better

9. Flow cytometry test for OsHV-1
James Paterson, Sarah Giles, Jim Mitchell – Flinders University
Designed around Merck Muse

10. Flow cytometry test for OsHV-1
Adapted for OsHV-1
Optimised for detection
Validating

11. Field PCR for OsHV-1 Biomeme Franklin No technical background needed
Sarah Catalano, Kathryn Wiltshire, Jessica Buss, Marty Deveney
Biomeme Franklin
No technical background needed
Results in <2h

12. Field PCR for OsHV-1 Duplexed OIE/EMAI tests Chemistry works
Validating against samples tested with lab qPCR

13. Conclusions eDNA is a viable method for surveillance for OsHV-1
area surveillance
assessing viral load
assessing risk posed by shipping at ports
Flow cytometry method: counts in water (experienced operators)
Field PCR provides analytical capacity for anyone

14. Thanks Future Oysters CRC-P, DIIS, FRDC, DAWR
Sarah Culloty, Sharon Lynch (University College Cork, Ireland)
DAWR / The National System / NIMPIS, Ingo Ernst, Peter Stoutjestijk, James Forwood, Susie Kropman et al.
NBC, SA, Qld, NT, NSW, TAS Governments, NBC
MISA, Staff at SARDI Aquatic Sciences (Marine Ecosystems) and SARDI Crop Science (Molecular Diagnostics)
Biosecurity SA, Flinders Ports, AMLR and KI NRM Boards
MolTools, ICMB X organisers
US DoA CoastalSEES, EU ICON programs
MPI NZ, Brian Jones, Jeannine Fischer, Jen Brunton, Richard Fraser, Anjali Pande
Cawthron Institute, Susie Wood
CSIRO Mark Crane
Joshua Mackie
AMSA, AMSA
ABC Four Corners

15. Risk OsHV-1 prevalence in biofouling Pacific Oysters 8-25%
Pacific Oysters common in biofouling communities

16. When biosecurity works nothing happens