1. 980 prawns selected from 98 ponds on 13 Farms.
2 Ponds which were previously positive were sampled in the North Qld farm.(pos on 31/10/15)
980 prawns selected from 98 ponds on 13 Farms.
There were no detections of the pirA toxin gene by qPCR.
As already discussed by Matt there were no positive detections of the pirA toxin from any of the samples we tested. In the 98 ponds and 13 farms we did sample 2 ponds which were previously positive in October 2015 event that was outlined in the OIE notification
Indicates positive locations from OIE notification

2. So if you know the results what am I going to discuss
So if you know the results what am I going to discuss ? I asked members of your executive committee for suggestions of what prawn farmers may find interesting? Answers were fishing stories
, beer
and chickens.
Needless to say I was dissapointed.

3. What is that Cost?
Direct $ to fix an incorrect result
(false negative or false positive)
Or even worse
you may make long term investment decisions based on incorrect results and then additionally need to direct $ on what should have been done in the first place
(over or underestimating the importance of an agent in genetic selection programs by using the wrong assays)
The farmers guide to accepting real time polyermase chain
Reaction (qPCR) results:
“If you think the cost of understanding qPCR is excessive you may pay the price of its absence”
I was certain the farmers guide to understanding real time polymersase change reaction qPCR results would rate a mention.
But seriously with qPCR being the most commonly used assay in Australia for the study and detection of pathogens of prawns the prawn farming industry in particular is becoming increasingly reliant on the outputs from qPCR analysis.
If you are going to be basing business and industry decisions on the results of qPCR assays it makes sense that you guys as an industry have someone in your group or on your farm that understands aspects of qPCR.
“If you consider the cost of understanding qPCR excessive you may pay the price of its absence”.
What will that cost be? Well short term either direct costs of being on the end of an incorrect result.
Or what could be even worse is making long term investment decisions based on incorrect results and then additionally needing to direct $ onto what you should have done in the first place. An example of this could be over or underestimating the importance of an agent in genetic selection programs.

4. Results Target Assay Sample Testing process What did you look for ?
How well does that test work ?
Assay
What is in place to monitor the test is working?
Sample
Testing process
AS with any process the quality of a qPCR result depends on the quality of its component parts. It is important that the following components are understood:
What was your target? Is that considered best practise for the detection of that disease agent?
What test or assay did you use to find that target?
How well does that assay work?
What is the smallest and largest answer the assay will accurately provide?
And Is that answer reliable? What systems are to ensure that assay is working?
What sample did you analyse to look for that target?
Is that the sample that provides the most accurate answer ?
Does that testing system produce consistently accurate, sensitive, specific results?
What samples were used?
How well did that work ?

5. Results Target What was not detected?
Im going outline the answers to those questions in our case:
What was our target.
Considering there were no reported disease outbreaks we tried to detect the cause of the disease

6. AHPND caused by a toxin gene which is called :
“PirAB –like”
P = Photorhabdus luminescens
ir = insect related: AB, TC, McF
pirB
AB = the binary toxin (2 parts namely A + B
McF=‘Makes caterpillars floppy”
AHPND is caused by a toxin. The gene that encodes that toxin is called pirAB like. The P refers to the bacteria Photorhabdus luminescens which has the toxin producing gene. This bacteria lives in the gut of a soil inhabiting nematode worm parasite.
The bacteria recognizes when it is inside the insect and then activates its Insect related toxin producing genes…hence the ir in the name.
The bacteria have 4 types of insect related toxin genes…with complex names and acronyms such as Mcf pirAB TC. The mcf toxins full name is makes caterpillars floppy. The toxin that causes APHND is most like the AB toxin which is a binary toxin made up of 2 parts A and B.
Its called “like” because its not exactly the same.
It has ~30% protein identity to the pirA/b proteins of a number of bacteria some of which are used as biological insecticides. In the case of AHPND the toxin gene is associated with a few species of Vibrio bacteria.
like = not exactly the same
~30% protein homology to PirAB. Other bacteria have similar and are used to make insecticides

7. Doesn’t matter if it is was in the plasmid or in the bacterial genome.
pirB
Doesn’t matter if it is was in the plasmid or in the bacterial genome.
We decided to part of the genome that coded for the toxin component A. By targeting this it became irrelevant what species of bacteria was hosting the gene and whether or not the gene was in a bacterial genome or a plasmid.
If a positive is found: where it was can be worked out later

8. Assay
A real time PCR (qPCR) had been developed overseas in the lab of Donald Lightner.
It has been reported to detect down to 10 copies of the toxin gene
by detecting a small component of the gene.

9. Pir copies/uL Ct AP 10 34.26 1000000 16.83 Neg Undetected 100 30.37
33.86
16.81
30.42
33.81
16.94
33.51
33.61
16.8
15 samples
The AFDL at AAHL, essentially the Australian team of doing this type of work had previously adapted this assay into their suite of tests.
With a transfer of legal contracts they provided us with method instructions and 15 samples to test.
AFDL knew the results of those samples.
Samples were sent from Geelong to Townsville. JCU project staff did the testing on those samples and submitted the results back to AAHL.

10. Plasmid JCU AFDL copies/uL AP KC AP SB AP
101
31.98
32.38
33.53
1.35
106
15.62
15.87
16.11
0.37
Neg
Undet
102
29.59
29.38
29.7
0.22
29.26
29.72
0.29
32.82
33.14
33.09
0.11
16.41
16.31
16.04
-0.32
29.2
29.9
29.84
32.16
33.11
33.16
0.52
15.86
16.14
16.21
0.21
32.77
32.86
32.89
0.08
32.49
32.55
32.95
0.43
16.06
16.1
-0.08
Plasmid
JCU
AFDL
copies/uL
Condon
Bochow
101
33.34
32.97
34.26
1.11
106
17.16
16.97
16.83
-0.23
Neg
Undet
102
30.47
30.36
30.37
-0.04
30.75
30.57
-0.29
34.06
34.38
33.86
-0.36
17.68
17.79
16.81
-0.93
30.95
30.45
30.42
-0.28
33.8
33.81
-0.12
17.29
16.94
-0.54
34.07
33.89
33.51
-0.47
33.41
33.68
33.61
0.06
17.7
16.98
16.8
We obtained the same negative results as AFDL
and our positive results were very similar to the results obtained by the AFDL. The JCU results were considered acceptable and we were able to plan analysis of samples.

11. Sample Needed to increase the bacterial numbers present in samples
Confirm the presence of bacteria in the samples
Have samples in formats that would allow bacterial cultures to be grown if there was a positive detection.
Have samples in formats that could be sent to BQ and AFDL for repeat testing
The sampling protocol for AHPND is different to that which is usually required for PCR. In our case we needed to increase the bacterial numbers present in the samples,
confirm the presence of bacteria in the samples,
have samples in formats that would both allow culture if there was a positive
and also be stored in formats that were as close to as possible similar to those tested at JCU for repeat testing at BQ and AFDL AAHL.
A sample of stomach and hepatopancreas was collected , in bacterial broth that was stored at ~ 28-32C for hours before being cooled and shipped to JCU. At JCU an aliquot of the sample was removed for testing, a duplicate aliquot stored in bacterial media with glycerol and the original sample frozen.

12. Sample
We then analysed the samples for the presence of the pirA gene by qPCR
and then before accepting those results as valid conducted another PCR which confirmed that the samples were processed in a way that preserved bacterial DNA. Without this it is difficult to prove that the negative qPCR results weren’t due to poorly collected or handled samples.
The bacterial genome PCR results weren’t standardised so they can’t be interpretted but it was interesting to observe the different results from different prawns in different ponds on different farms. This is a snapshot of some of the bacterial 16s pcr results.

13. Results Target Assay Sample Testing process What did you look for ?
How well does that test work ?
Assay
What is in place to monitor the test is working?
Sample
Testing process
So with all of the monitoring and quality control in place in the various components of the qPCR process how well does the assay perform?
What samples were used?
How well did that work ?

14. Results
Does the assay to detect the pirA toxin gene perform consistently, accurately and specifically to detect the pirA gene ?
Test
performance
SO if you have all the components of the testing applied with Quality Controls: How well does this testing perform?

15. No contamination was detected. No PirA was detected
Ct values for the detection of the pirA toxin gene in NQCs
Analysis Title
Corbett Rotor Gene: n=11
Qiagen Rotor Gene: n=13
NQC 1
NQC 2
Farm A
23.55
30.54
21.3
27.89
23.57
30.19
21.47
28.2
Farm B
23.66
30.35
21.82
27.91
23.72
30.32
28.19
24.11
30.72
23.78
30.48
Farm C
30.89
21.81
27.97
23.73
30.57
21.57
29.07
Farm D
21.54
28.47
21.49
28.07
Farm E
22.28
28.45
22.25
28.99
Farm F
23.93
30.53
28.04
23.63
30.83
21.38
28.24
Farm G
23.91
30.1
23.83
30.2
Ct values for the detection of the pirA toxin gene in NQCs
Analysis Title
Corbett Rotor Gene: n=11
Qiagen Rotor Gene: n=13
NQC 1
NQC 2
Farm H
23.69
30.66
21.68
28.73
23.6
30.59
21.63
28.42
Farm I
23.61
30.6
21.82
27.91
23.62
30.37
21.47
28.19
Farm J
22.64
29.76
22.66
29.9
Farm K
23.34
29.79
21.7
28.07
23.28
30.08
21.48
28.44
Farm L
23.95
30.55
21.74
28.27
23.89
30.93
21.55
28.28
Farm M
23.9
21.65
23.79
30.53
21.76
Every qPCR analysis that was conducted included 2 known positives for the pirA gene. These were the Network Quality Controls NQC 1 & 2. NQC 1 contained copies of the genome and NQC 2 contained 100 copies.
As you can see by glancing down the columns when repeated over repeat analysis runs during the project the result on each of those same samples were very consistent.
There were no false positive results.
There were no contamination issues.
There was no pirA detected in any samples from prawns
Bacteiral DNA was detected in all cases.
According to current best practise the pirA toxin gene was not detectable in the prawn samples.
Yes the assay performed consistently to detect pirA over the duration of the project.
No contamination was detected.
No PirA was detected
Bacterial 16s was detected

16. Sampling Teams: ProAqua: Alistair Dick
Thank you:
Biosecurity QLD: without their consent we could not have been involved in this project.
FRDC: funding to APFA
APFA: funding to JCU staff, travel, sample collection and reagents for testing. Helen Jenkins for logistical arrangements and purchasing.
AFDL AAHL (CSIRO): Method instructions, positive controls, equivalence testing panel. (Nick Moody, David Cummins, Lynette Williams, John Hoad).
JCU Townsville: In kind access to equipment and laboratories (College of Public Health, Medical and Veterinary Sciences)
Sampling Teams: ProAqua: Alistair Dick
Ridley Australia: Matt Briggs and Caitlin Riguet
BIARC: Brian Paterson
JCU: Shaun Bochow, Kelly Condon
APFs that hosted our sampling teams: Australian Prawn Farms, Seafarm, Tru Blu, Gold Coast Marine Aquaculture, Pacific Reef, Rocky Point Prawn Farm, Truloff Fresh Prawns, David Lin, Campwin Beach Prawn Farm, GI Rural Pty Ltd., Paradise Prawn Farm.
This was very much a group effort to get the testing completed quickly so there is a large number of people to thank.
BQ: without consent of BQ we could not have completed the testing. This was particularly sensitive because another university in QLD had conducted the Panama type 4 testing that was later shown to be incorrect.

17. Questions ?

20. Cost of a False Negative? Biosecurity Failure
Equine Influenza: Horse racing $7 Billion industry: EI: in the billions. Costs bore by the Australian government Equated to every Australian tax payer paying $35 each. (disease detected in an area covering 3.5% of Australias landmass affecting 8% of the susceptible horse population) (AVJ 2011)
Cost of a False Positive:
Oyster mortality: Oyster annually $90 million. TAS $26 M direct cost of POMS $12M (May 2016) TAS population 0.5 million. Long term costs to industry of missing an infected co-hort of animals and shipping them to naïve populations.
Australian Prawn Farming Industry: current value $87.7 million (APFA 2016).
The cost of a false positive result: A banana farmer that bore the money costs, social stigma and significant person stress of being falsely diagnosed with Panama race 4 disease…cost the QLD government $ in compensation payment.
Conversely the cost of a false negative result: The 2007 Equine influenza response is considered an example of a successful biosecurity response to an exotic disease incursion. This good response cost economy several billion dollars. The cost in government payments alone equated to every Australian tax payer paying $35 on EI. This was from a disease that only affected 8% of the susceptible horses in Australia and occurred in an area covering 3.5% of our land mass…imagine the cost of EI if the qPCR results that came out of that lab were incorrect and the disease spread further.
A present aquaculture example is the POMS outbreak in Tasmanian oysters. The total oyster industry is worth $90million of which the Tas industry valued at $26M has up to May 2016 suffered $12M in losses. One can only guess the long term costs to industry of shipping a co-hort of animals that are carrying POMS to a naïve population elsewhere in Australia because of a false negative qPCR result.
When we set out to do this testing we took great comfort in knowing the Australian Prawn Farming industry was only worth $87.7 million.

22. Photorhabdus luminescens
As a quick side note:
In some biochemical tests used to identify Photorhabdus lumunescens mistakenly key the bacteria to be another organism such as Shewanella putrefaciens, Pseudomonas and Providencia. Important to note if you get a gross skin lesion.
Photorhabdus generally isn’t a risk to humans. In fact it is proposed to be the bacteria that caused Angels Glow which was noted during the Am

24. Determine the risk: What do we know about viral disease in P
Determine the risk: What do we know about viral disease in P.monodon from WA/NT?
Total tested:
: GAV 60
2010: WSSV and YHV: 90 wild and 60 farmed.
Broome
15: 2000
23: 2001
JBG
19:
Onslow:
Carnarvon:
Sources: data: (Jones) FRDC 98/212 Determination of the disease status of Western Australian commercial prawn stocks
2010 data: East National survey to demonstrate freedom from WSSV and YHV.

25. Determine the risk: What do we know about viral disease in P
Determine the risk: What do we know about viral disease in P.monodon from WA/NT?
Virus + %
IHHNV
10/151 7
MBV
2/151 1
HPV
24/151 16
SMV 2
MoV
65/153 43
GAV
111/155 72
YHV 7
3/155
YHV 6
7/60 12
Virus + %
IHHNV
5/50 10
MBV
1/50 2
HPV
7/50 14
SMV
0/50
MoV
2/67 3
GAV
10/52 20
YHV 7
1/52
Virus + %
IHHNV
4/136 3
MBV
0/136
HPV
7/136 5
SMV
2/136 2
Mo V
4/150
GAV
73/271 26
YHV 7
25/271 9
GAV 2/20
YHV 7 1/5
YHV 7 0/8
GAV 1/8
GAV: 2009: 33 pools of 166 prawns all neg.
GAV 1/6
YHV 7 0/6
GAV 7/29
YHV 7 0/29
GAV 14/44
YHV 7 1/44
GAV 41/51
YHV 7 0/51
GAV 0/4
YHV 7 0/4
GAV 56/60
YHV 7 2/60
YHV 7 from QLD re-tested from 2008: 13/161 + Ct value ranging from Previously Gav loads associated with heatlhy P.mondon are (10 x 5) 20, Moribund shrimp = Ct of 7 or 10 x 9 copies. Source of cut-off values: Pathogenicity of gill-associated virus and Mourilyan virus during mixed infections of black tiger shrimp (Penaeus monodon) Dang T. H. Oanh,1 3 Marielle C. W. van Hulten,1 4 Jeff A. Cowley1 and Peter J. Walker1,2 Journal of General Virology 2011.
YHV 6 7/60
Cowley et al. (2015) Viral presence, prevalence and disease management in wild populations of the Australian black tiger prawn (P.monodon). FRDC 2013/036.