ISA positive test results in BC

The Rivers Inlet Sockeye samples – ground zero – in the BC debate


The fish that ignited the debate - is ISA virus in BC - were 2 out of 48 Rivers Inlet sockeye smolts submitted to Dr. Kibenge by Dr. Rick

Routledge of Simon Fraser University. Routledge wanted to rule out ISA virus in an inexplicably declining population of wild salmon

that he is studying. Simon Fraser University held a press conference to get these results into the public domain, seeing this as a serious,

time-sensitive situation and hoping to elicit scientific investigation. At that time, the public was unaware of any ISA positive test results.

Government and industry had testified under oath only weeks earlier in the Cohen Commission that ISA virus had never been detected in

BC. It is ironic the debate was ignited with these fish because they were highly-degraded, having been stored for months in a householdtype

freezer for stomach content analysis. The viral work was an after-thought.

While it is commonly presented that DFO could not reproduce Dr. Kibenge’s positive result in these salmon, below is a November 4, 2011

email from Ms. Nellie Gagne, DFO Moncton lab, sometimes called the National Reference Lab, to Dr. Peter Wright (DFO) and others

titled: – Good News? Bad News

“Hi,

The homogenates (19, heart and gills) were done and one very weak (almost 38) showed up, in one duplicate only.

It is not one of the positives of AVC...

I am not convinced it should be reported to our friends in Ottawa, guess why !”

We do not like to see a Ct like this, but this is the type of Ct that is equivalent to the finding by Nylund, i.e. I can’t conclude anything from

it.

Nellie” (Cohen Exhibit 2040)

A list of ISA positives found in British Columbia salmon in 7 different labs

1.) Nellie Gagne (DFO/CFIA) weak positive among the 48 Rivers Inlet sockeye. There has been no other reporting on results from

samples she has been sent that also tested positive in Dr. Kibenge’s lab. The media continues to report on these 48 samples without

mention of the growing list of positives in BC farm salmon.

2.) Cohen exhibit 2053, Farmed chinook salmon, Clayoquot Sound. Creative Salmon provided samples to Dr. Kristi Miller (DFO),

Pacific Biological Station, to figure out why their fish were experiencing unexplained persistent mortality and jaundice condition. In

Cohen Exhibit 2053, test results are given for 47 farm salmon, from two different salmon farms, 12 tested positive for ISAv.

3.) Cohen exhibit 2060, lists many ISAv positives from the Miller lab (DFO) in wild sockeye salmon from a large number of

commercially and socially important BC salmon rivers. Samples were collected in 2007-2010. Miller reports some primers work better

than others: “So I believe that what we have in B.C. is a somewhat divergent strain of ISA that is not universally picked up with all --

with the assays that are presently in use... there is always the possibility that you will develop an assay that doesn't pick other variants

that you didn't know about. And I believe that that's what's happening here.” (Cohen Commission Testimony, Dec 15, 2011, Page 22)

4.) Sonja Saksida, BC Centre for Aquatic Health Services, reported ISA PCR positives to the CFIA in the farm chinook salmon

from Creative Salmon, sequence confirmed for ISA-P7. The only mention of this reporting is in an email from Miller to Stephen Stephens

(DFO, OTTAWA) Cohen Exhibit 2055.

5.) Cohen Exhibit 2043, 2056 – positive results by Dr. Kyle Garver, Pacific Biological Station DFO described by Dr. Miller: “So he

ran basically the validated assay that Nellie uses, and the ISA-7 Plarre assay and he was able -- he was not able to pick up any positives

using the DFO validated assay, but he did pick up a positive of ISA-7 using our assay with our pre-amplification.”

6.) Cohen Exhibit 2045, a 2004 draft paper coauthored by Drs. Molly and Fred Kibenge and Drs. Simon Jones and Garth

Traxler (DFO) reporting 115 ISA virus positive results. These results demonstrate up to 99.7% identity to an ISAv isolate from Norway.

Sequence was produced. These samples included Atlantic salmon, wild salmon from Alaskan waters, throughout BC and 100% of the

Cultus Lake sockeye tested, a Fraser sockeye population that has been declining despite efforts to restore it. It is unclear whether the

Americans or the CFIA were informed. This work was never provided to the Cohen Commission by DFO. Dr. Fred Kibenge provided this

document to the Commission when he was asked by the Commission to provide all information on ISAv in BC.

7.) Kibenge, AVC, results on 2/48 River’s Inlet sockeye smolts positive for European ISAv genotype

8.) Are Nylund, U. Bergen weak ISA virus positives among the 48 sockeye smolts and ongoing positives in BC samples I am providing

to his lab.

9.) Kibenge, AVC, results on 3/11 salmon that died in the Fraser River without spawning positive for European strain ISAv; chinook,

coho, sockeye. I am the owner of these samples and they were provided to the CFIA

10.) Kibenge, AVC, results on HPR5 sequenced from a female chum salmon in the Vedder River, a tributary to Cultus Lake. I am owner

of these samples and they were provided to the CFIA

11.) Kibenge, AVC, positive results on 19 farm salmon bought in supermarkets in the city of Vancouver. Many are HRP5, one HPR7b,

this work is ongoing and will be more fully reported when complete. I am the owner of these samples and they were provided to the

CFIA.

Despite all these labs, reporting ISA virus positive results, including 3 DFO labs, the DFO position remains - no salmon in BC have tested

positive for ISA virus.

Dec. 4, 2012 “In recent years, the Government of Canada and B.C. have tested more than 5,000 wild and farmed salmon in B.C. for

infectious salmon anemia. None has ever tested positive.”

http://www.ottawacitizen.com/news/confirmed+disease+salmon/7646808/story.html

There is currently only one lab known to have tested BC salmon for ISA virus reporting 100% negative results. The provincial lab run by

Dr. Gary Marty, BC’s Provincial Animal Health Centre reports ISA virus “negative” results for 7,002 BC farm salmon. (Cohen Exhibit

2146). It is unclear what protocol he is using.

Inconsistent results

A common finding throughout ISA virus positive results for BC is that they are “inconsistent.” Real time PCR positive results come and go

between replicates. Dr. Miller’s work demonstrates that different probes behave differently (Cohen Exhibit 2060). This suggests that the

trouble identifying this virus may not be a failure by the labs, but rather the unique nature of this variant of virus.

As Greg McDade, a participant lawyer for the Cohen Commission stated in his final argument:

“The independent findings by Dr. Nylund and Dr. Kibenge in the initial Rivers Inlet group of 48 fish provided them with the confirmation

of each other, and on a statistical basis eliminate any reasonable possibility of contamination or other laboratory error.

The subsequent findings of Dr. Miller are again confirmatory of both Dr. Kibenge and Dr. Nylund. The fact that Dr. Miller used different

primers, a different machine, and a different methodology, but also produced positive findings, itself provides another level of

confirmation that reduces any possibility of a consistent error. Dr. Miller’s genetic approach is novel and advanced, but this is not

grounds to reject it. Dr. Kibenge, the OIE designated expert on ISA, considered her results to be credible. Dr. Miller is a senior DFO

scientist, in charge of the genomics lab, and it would not serve DFO or Canada well to contest the credibility of her findings.

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