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: https://www.facebook.com/alexandra.morton.1671/posts/1864901490405070.
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:
Hello
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.
alex
Begin forwarded message:
From: Alex
Morton
Subject: What ISAv tests everyone is using
Date: November 26, 2014 at 7:51:15 AM PST
Hello
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.
So.
- 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.
alex
Cohen Commission Exhibit
|
Finding
|
Lab
|
2053
|
2011 database reports 40% of the tests on farmed Chinook salmon
suffering high mortality and severe jaundice produced an ISAv response.
|
Pacific Biological Station
|
2040
|
Ct values were produced for ISAv for the same Rivers Inlet samples
included in this study
|
National Animal Health Lab, Moncton New Brunswick
|
1549
|
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
|
2052
|
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
|
2045
|
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 http://www.dfo-mpo.gc.ca/Library/282434.pd22f
|
Pacific Biological Station
|
2060
|
ISAv Ct values in replicates of samples that produced ISAv Ct values
in Moncton Lab.
|
Pacific Biological Station
|
2043
|
ISAv Ct values for Fraser River sockeye salmon
|
Pacific Biological Station
|
2051
|
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
|
2042
|
plots of ISAv data from Fraser River sockeye
|
Pacific Biological Station
|
On Nov 14, 2014, at 9:14 AM, Alex Morton
Hello
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?
alex
<ISA Saksida Exh 2055.pdf> think <Classic ISA virus
graph.jpg>
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