Thursday, 11 August 2016

NItty Gritty PCR, ISA Testing - What You Need to Know, Updated Sept 19, 2016

To give you an idea of how difficult it is to accept any comment by DFO, the CFIA, the provincial testing system, and broader scientific community, I am publishing this on-going commentary from Alex Morton from 2014 on the nitty gritty in PCR testing, but the recipients are well-versed, knowledgeable persons in the NGO, environmentalist and scientific world.

I have left out the email addressees, for their privacy. Note that Saksida is a vet that fish farms have employed. The usual practice is to find scientists who will work on their behalf and then fund science from the fish farm side of things. This is common practice in the science sector, and one reason that more journals are now requesting divestiture and sponsorship relationships.

For example, Helge Aarskogg, CEO, Marine Harvest, said in 2015 that lice were the fish farms worst problem and noted that Marine Harvest had 90 studies underway on the problem. What this does is create conflict of interest issues with a wide sector of the science world.

And, of course, look at this video of a GoPro put down in one fish farm, one net, and look at the dying, and dead and diseased fish, along with wild fish being eaten:

The point of this nitty gritty, is that the CFIA used the wrong ISA virus to test for, and thus found no ISA in BC.

Look at the table of ISA findings in BC

Now the 2014 note. Revel in the nitty gritty:


For the record.

Below is a letter I sent earlier this week.  I continue to try to figure out why some labs detect ISAv in BC and others do not.


Begin forwarded message:

From: Alex Morton
Subject: What ISAv tests everyone is using
Date: November 26, 2014 at 7:51:15 AM PST


I think I now have a better understanding of what tests everyone used/is using for ISAv in Washington and BC.  I think it is important to recognize no lab has reported a “confirmed” case of ISAv.  This debate is around the “suspect” results reported by all the labs listed in the table below.  This is a very important point. It also should be realized that surveying wild salmon for ISAv, is going to produce a different kind of result than diagnostics on farmed salmon experience mortality, where most ISAv work has been done.  Survey work will encounter low levels of the virus and most tests used today perform poorly on these kinds of samples, some like virus isolation, don’t work at all and of course virus isolation is currently required by Canada to confirm ISAv.  The only place we are going to be able to “confirm” ISAv, if it is in BC, is from moribund farmed Atlantic salmon and of course we are not allowed access to those fish.

I hope that any will correct me if I am wrong about the information below. 

Tests used?

Gary Marty 2006-Oct 2009 - Conventional PCR targeting ISAv segment 2, the largest segment in the ISA virus with low copy numbers making this a low sensitivity test. I don’t see use of this test recommended by the literature. Then Nov 2009 - mid April 2012 an undisclosed PCR protocol was apparently used to target segment 8.  According to Cohen exhibits when Marine Harvest sent 30+ samples to the Animal Health Centre beginning in the summer of 2009 specifically for ISAv testing, these were the tests that produced the negative results. 

CFIA Surveillance in wild salmonids apparently used the RT-PCR assay reported in Caraguel et al 2009 - a study for which a PCR test was developed for what appears to be Atlantic Canada farmed salmon. The ISAv variants from Atlantic Canada are quite different from the European variants, perhaps diverging perhaps  100 years ago (Krossoy et al 2001).  The paper is not clear about what salmon they were working with, but they thank the participating labs for “endless sample collection,” and all the labs are in Atlantic Canada, so perhaps they were using Canadian Atlantic salmon?  Caraguel et al (2009) reports the assay is unpublished. Was it designed to be sensitive to Atlantic Canada ISAv variants? Do we know how it performs on on EU variants?  How do we know this is the best test for ISAv surveillance in wild Pacific salmon?  On the CFIA website  they state:  

The PCR test is highly sensitive, and can sometimes produce false positive results. Therefore, positive results obtained at this stage require further confirmatory testing. Confirmatory testing involves isolating the virus using cell culture.” This statement suggests the CFIA could very well have gotten ISAv positive results similar to others, but did not report them because they failed to culture the virus.  However, no one has ever cultured ISAv from  wild fish that tested positive for ISAv via PCR (Plarre et al. 2005). In addition, both the Pacific Biological Station and Freshwater Institute printed disclaimers on their ISAv results for the CFIA stating that they would not attest to the results because the test was not validated for the species or tissue they received.   This work does not report on ISAv testing in Atlantic salmon in the Pacific, the known host for the virus.  I think it is very important to note that the CFIA describes these tests as fulfilling trade requirements and so only needed to follow the protocol established for that purpose.  Theirs was not investigative work, it was prescribed protocol for the purposes of trade.

USA Surveillance The US team published their ISAv surveillance results yesterday (attached).  They used Snow et al (2006) an OIE recommended test developed to target the most conserved regions of the ISA virus gene.  Perhaps the authors could help with the next point which states that if a suspect test result occurs significant additional testing “will be conducted.”  I am unsure if that means it was done or will be done and what type of testing that was/will be?  Are they referring to attempts to culture the virus, conventional PCR and sequencing, or simply more replicate test with the Snow et al (2006)?  They state no ISAV was detected.  However, to evaluate these results against those contained in the Cohen exhibits, we would need to know whether there were any “positive” responses by the test.  Here too no tests report on Atlantic salmon, the most likely host for this virus.   This paper compares results with the CFIA, which used a different test.  They mention the 2 positive Rivers Inlet sockeye that triggered all this, but they make no mention of all the other ISAv - positive tests results that were also tabled at the Cohen Commission.  Those tests were done by two of the same labs used by the CFIA, so they must be credible labs - see the table below.  They also made no mention of Miller’s testimony which included reference to novel deletions in the ISAv detected in her lab.  Novel deletions in the ISA virus gene are well known. If you visit the WAHID portion of the OIE website, you will see Atlantic Canada is dealing with one right now.  Researchers working with influenza viruses warn that unless PCRs are updated novel variants can escape detection (Klungthong et al. 2010). This work lists “trade” in the top two objectives “Document regional ISAV freedom status to support facilities participating in domestic or international trade. “ Of course trade is very important, but doing research to support trade is a different objective than doing research to figure out why so many labs have gotten ISAv positive responses from tests.

  • None of these three government labs used the same test, 
  • none reported if there where “suspect" positive results between replicates, even though this kind of result is the source of this debate
  • The above reporting suggests even a solid PCR positive result would not have been reported if the virus was not cultured?
  • No one ever asked to view the results I have to try to better understand what we are seeing. I have always found this odd given the enormous amount of money and effort spent responding to these tests.

When Chile, Norway, Scotland, Atlantic Canada, Maine, and the Faroe Islands report ISAv they are reporting on results from salmon farms experiencing mortality.  Here we are engaged in surveillance testing.  Diagnostic and surveillance testing are very different.  If European ISAv has entered the Pacific and a Pacific species, I think it is accurate to say we should expect mutations and our testing should respond accordingly.  The fact that conventional PCR has produced sequences that BLASTing to GenBank finds matches is a point that cannot be ignored just because no one has cultured the virus. If it is not ISAv, what is this influenza-like virus in the Pacific?

I would like to propose that if we want to learn if ISAv is here we should all use the same test on the fish most likely to be infected with ISAv, moribund Atlantic farmed salmon.  Otherwise we are not using the best science possible to determine if ISAv is in the north west Pacific.  However, clearly that is not going to happen anytime soon.

I suspect all of us have been under enormous pressure, and I don’t mean to criticize any.  I simply want to try to take this debate back into scientific terms where we are dealing with one of the most lethal salmon viruses known.  Chile ignored the early signs that ISAv was there, from that we know the industry is loath to respond until necessary.  We, fortunately, have a very different situation here as the industry is not so large, but we do have wild Pacific salmon that find themselves from time to time in highly concentrated stressful, disease promoting situations.  I hope that all of you will keep an open mind as further work is done, recognizing that some labs are doing investigative work while others are following legislated protocol to maintain open borders.  In this situation both can be right, i.e. government protocol does not confirm ISAv, but the virus could still be in BC, Washington and Alaska.

Thank you all for reading this.


Cohen Commission Exhibit


2011 database reports 40% of  the tests on farmed Chinook salmon suffering high mortality and severe jaundice produced an ISAv response. 
Pacific Biological Station
Ct values were produced for ISAv for the same Rivers Inlet samples included in this study
National Animal Health Lab, Moncton New Brunswick
numerous diagnostics by a on Atlantic farmed salmon describe; “Sinusoidal congestion is one of the classic lesions associated with ISAV infection…

BC provincial government lab
genomic profiling of BC farmed salmon with ISAv Cts values reports on “The program DAVID (a public genomics functional analysis package) identified ‘Influenza Infection’ as the most enriched pathway… This is a very strong signal indicating that fish positive for ISAv7 are responding to the virus similarly as mammals would respond to other influenza infections…suggests that the virus is causing enough damage to elicit a strong response in salmon
Pacific Biological Station
reports 100% of Cultus Lake sockeye tested positive for ISA virus by RT-PCR, a finding highly consistent with this work. No further investigation were very similar. Cultus Lake sockeye is reported at risk from extinction

Pacific Biological Station
ISAv Ct values in replicates of samples that produced ISAv Ct values in Moncton Lab.
Pacific Biological Station
ISAv Ct values for Fraser River sockeye salmon
Pacific Biological Station
internal federal report on sequencing and alignment of products from  Orthomyxovirus ISA8/7 quantitative RT-PCR (taqman) screening using Plarre primers show 95-100% match to some European ISAv isolates (small fragments) and prevalence.

Pacific Biological Station
plots of ISAv data from Fraser River sockeye
Pacific Biological Station

On Nov 14, 2014, at 9:14 AM, Alex Morton


I followed up on the CFIA announcement and requested the Complete Report offered on their website (attached).  They say only that their PCR test was “standard.”  In their survey they completely disregard all the “classic” ISAv lesions reported by the provincial pathologist in BC farmed salmon. I have those records as they were released by the Cohen Commission. Attached is a graph I did of the reporting on the two lesion identified as classic ISAv lesions in BC farmed salmon.

I feel it is not acceptable that our federal government proclaims they have done a survey, with results contrary to many government and other labs, and provide no adequate methods that would allow us to interpret those results.  There should be detailed information about what segment, primers, probes, look at Plarre et al 2005’s surveillance paper for example (attached).

There has to be a technical reason why so many labs have detected ISAv and yet the CFIA claims they can’t.  However, we will never figure this out if the CFIA is allowed to announce BC is ISAv-free in complete absence of detailed description of what tests they did. This has been preceeded by a very heavy-handed approach to suppress the work by the labs who tried to do this work.   

I hope all realize, that while the CFIA declared the Kibenge tests were non-repeatable, that in actuality they never did repeat those tests (letter to me from CFIA Dec 2103 available on request).  The only samples retested by the CFIA were the 48 Rivers Inlet samples and Nelle Gagne did get positives, along with Miller, Nylund and Kibenge.  Their results were in agreement.  In an email from Gagne, Nov 4 2011, she writes in regards to her tests; “I am not convinced it should be reported to our friends in Ottawa, guess why!” This is Canada.

The CFIA recommended stripping the Kibenge lab of OIE status and the OIE voted to do so in absence of a single contrasting result.  Dr. Brian Evans of the CFIA, who led this “investigation” into Fred’s lab is now the #2 man at the OIE.  I have written to many of them asking why they did this and they refuse to answer.

I have attached the report by Brad Davis that became a Cohen Commission exhibit regarding influenza response detected in salmon from BC that tested positive for ISAv.  This work has to either be dennounced as a complete mistake, or we have to do more to understand why ISAv sequence is being detected.  If the sequences matching ISAv in GenBank are not from ISAv then what are they attached too?  Don’t we want to know?

See the attached email exhibit stating that Sonya Saksida reported ISA positives to the CFIA, but we never hear about these. 

I also remind you that Simon Jones failed to do any follow-up when Molly Kibenge reported ISAv detection in 100% of the Cultus sockeye.  I see this as indefensible. I understand the urge to doubt these results, but no follow-up?  A reportable virus capable of killing salmon is reported in 100% of the most endangered Fraser sockeye salmon and he never goes back for a second look, never provides this report to the Cohen Commission and disallows publication.  This is science in Canada today.

If you read all the Province of BC reports to the salmon farming industry,  (and I believe I might be the only person who has done this), you will see that in 2009, Marine Harvest suddenly requested over 30 tests specifically for ISAv from the provincial vet.  Prior to this interval there had only been one such request back to 2006. Shortly thereafter, the three Norwegain companies signed an MOU to share viral information between themselves and no one officially imported eggs again. I think they scared themselves with some virus. We heard testimony from the provincial pathologist on ISAv tests on BC farmed salmon and he stated that he used an inhouse test, that we also have never seen details on.  Why is it the ISAv tests developed and used by the international community are not good enough for Canada?  There may well be a sound reason, but I think we need to hear it so that we can understand the results.  Since when do we start accepting science that does not provide methods?  I believe we allow science to be degraded in Canada when we do this.

I think BC could be pre-ISAv outbreak status, similar to the 1999-2007 interval in Chile. I think the behaviour we are witnessing may be how Chile got hit so hard, I think the virus was already out there and some condition changed and wham it went virulent.  I think we might have the opportunity to deal with this virus before it goes viral on this coast and that this opportunity is being squandered here as it was in Chile.  There is absolutely no reason it would not be here, as Canada never required certification that eggs had to be ISAv - free.  The scientific debate has been supressed, and the labs who did the recent testing for the CFIA refuse to “attest” to their results, using methods that are shrouded from view. 

Science has been handicapped creating a favourable environment for international commerce. I know commerce is important, but so is science.  Scientists are afraid to look at this virus and the public interest present and future is put at risk. It simply does not make sense that Rick Routledge found the only 2 ISAv infected salmon in BC.  If we believe this we should nominate him to the Order of Canada for stopping ISAv in BC… :)

This is Canada today, unless we push back and make a strong request to see the details on the PCR tests conducted by the 3 labs contracted by the CFIA to test for ISAv we are complicit in allowing a scientific dark ages to prevail.

These are my thoughts, I have great respect and and admiration for all of you and I think we need to show a little bravery here and support for the labs on the frontlines.  We are all involved in this, why wouldn’t we demand that the labs who did the tests for the CFIA reveal thier methods?


<ISA Saksida Exh 2055.pdf> think <Classic ISA virus graph.jpg>

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