ISA experts – what they actually said

The Christmas and New Years hangovers have finally subsided and we got our throbbing heads together to bring you something new.

While we were partying, we see the Cohen Commission finally got the transcripts published from the three days of ISA hearings before Christmas.

The transcript from the first day shows that what the four experts (Kristi Miller, Are Nylund, Fred Kibenge and Nellie Gagne) actually said about the ISA virus is quite different than what has been reported in the mainstream media and on anti-aquaculture blogs and websites. 

Contrary to what some say, no ISA virus has been confirmed in Pacific wild salmon, or farmed salmon in B.C.

The take-home messages from the hearings are: 

  1. No ISA virus has been confirmed in wild or farmed salmon in B.C.
  2. Something may be triggering “positive” test results; it is unclear what this is.
  3. If the “positive” results are true, they may be detecting a variant of the ISA virus which has been present in the Pacific ocean for hundreds or even thousands of years.
  4. There is no evidence of ISA in farmed Atlantic salmon in B.C. 

Here are some tidbits from the hearings. To keep things clear, we are marking statements as fact, claim, suggestion or speculation.

Fact: ISA virus and ISA disease are different.

There is a large difference between detection of the virus, or the viral genome, and the actual disease. And usually you will only find disease development in Atlantic salmon. And none of the other salmonid species are really suffering from ISA infection. You may have some disease developing in rainbow trout, or steelhead, as you call it, but most of the other species will be carriers or they will have a viremia, but they will not show any clear signs of disease.

– Dr. Are Nylund

Fact: RT-PCR tests work by amplifying a specific segment of virus RNA a scientist is looking for.

PCR is a process of specific amplification of DNA that is on specific detection of a fragment of DNA in the mixture of DNA. RT is for reverse transcription. In this case, we’re working with RNA viruses, so we need to start by extracting the RNA from, in this case, a fish tissue. And if the RNA of the virus is present in there, mixed with the RNA of the fish, where we’d try to detect it with the PCR assay. So the assay requires primers. Primers are short custom-made segments of DNA that will anneal if there’s a match with the DNA in your mixture. If the virus is in the mixture with the DNA of the fish, we would get a match, and the PCR process will amplify that segment between the two primers that you have put in your mixture. The probe is in between those primers. The probe is linked with a reporter of fluorescent molecule. So when the PCR process goes on, if there was a match with the primers first, the PCR process amplifies what’s in between those primers, so it creates a sequence, a short fragment of DNA, and the probe will be released, and what the real time RT-PCR acid detects is the fluorescence from a probe.

– Nellie Gagne

Fact: ISA virus could not be confirmed in samples which tested positive under RT-PCR tests.

I was not able to sequence any ISA virus from these samples. So I was not able to verify that this was actually ISA virus I was picking out. But you know that the assays that we are using, the real time assay we’re using are very specific, so they should only be picking out ISA virus, and maybe not all ISA virus, but most of the ISA viruses that we know.

– Dr. Are Nylund

Fact: Dr. Kristi Miller ran her own RT-PCR tests using her own method she developed by using bits and pieces of others’ methods, probes and primers, but she did not have a positive control sample to measure her results against.

We ran the assays with no positive control, which it can be problematic in that you don’t know if your assay doesn’t work.

– Dr. Kristi Miller

Speculation: based on her results, Miller believes she has discovered a previously unknown strain of ISA virus.

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. So, you know, when you develop one of these assays, you usually develop the assay and a lot of them were developed in, I guess, Nylund’s lab, and he could speak to this better than I could in terms of their development. But you have a backdrop of knowing all of the strains that you know about, all of the sequences that you know exist and you try to develop an assay that will amplify all known strains. But you can’t know things that you don’t have a sequence for, and so 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.

– Dr. Kristi Miller

Fact: Miller provided samples to virologist Dr. Kyle Garver, who was not able to replicate her results using the standard method used by DFO, but was able to replicate some results using Miller’s method.

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 preamplification.

– Dr. Kristi Miller

Fact: After researcher Molly Kibenge reported in 2004 she had discovered ISA virus in wild Pacific salmon, DFO’s lab tried to replicate her results and could not.

We had the same primers, we had the same kit that she was using, everything was matching. There was minor differences at some points, but we reran things. We had done it so many times, it was — it was not possible [to replicate Kibenge’s results].

– Nellie Gagne

Fact: In recent testing, the methods used by DFO are to international standards.

We use our validated assay, which to our knowledge, and again, we use the current information we have, the current strains of ISA that we know. We used this assay and in theory this assay is made to be universal, taking all the ISA that we know of. So to — the test is designed to be fit for a purpose. It has a good sensitivity. It picks down to plus or minus seven copies, if you look at the validation dossier, this is what it says. It is a sensitive assay. It has been used in our program and has been producing positive results. It’s not because I’m reporting only negative results right now, that’s not ever the — it’s not always the case. We’ve confirmed cases of ISA. We’ve confirmed HPR0 in the region, it’s — at the moment the assay is working for what it was designed to do. And in this situation it’s difficult. I recognize that we always — we are not trying not to detect anything. We’re doing our best to find something. And the other thing we’ve done, because it’s been reported by others, we have used the OIE primers. In some labs they don’t seem to be working the best but they were the ones, the Plarre primers and Snow are the ones that were used initially to report the first positives in Dr. Kibenge’s lab. So we used them also on the samples we received. So at this point I think we’ve done a reasonable effort to detect what was — what was claimed to be there.

– Nellie Gagne

Suggestion: Labs in Canada should submit to a double-blind test to see if their methods are actually working properly.

In my view, the best way to compare labs, if that was an issue in terms of repeatability or reproducibility of results, would be to have an experimental sample in which there is a known amount of virus, that sample to be distributed blind, so that each lab can use their methods, and that way that will be a very effective way, a very objective scientific way of comparing the labs. In which case, if they can’t have the same results, then there is a problem. But to compare labs based on field samples and particularly in this case where even the virus may be so variable that using real time on two separate segments you can’t even pick up the same fish, it becomes a bit difficult to…

– Dr. Fred Kibenge

Claim of fact: Miller says she was able to sequence an ISA virus in her samples. This has not been confirmed by other research yet.

Ultimately, gaining a genetic sequence is an ultimate validation that what you’re picking up by PCR is a real product and it’s the product that you’re expecting to be picking up. Now, it’s possible if you contaminate a PCR to sequence a positive, you know, from a contaminated product. But again, in our case, we did not have ISA virus in our lab; we had no positive control. So if the reasoning is if we’re able to pick up a PCR product and we are able to sequence it from wild fish and it sequences as ISA, it is a real ISA product from wild fish. There is no other way for us to get ISA product sequence in our lab, other than it coming from those wild fish. So that really was the ultimate validation for us, and we were able to do that with all four primer sets that amplified product in our fish.

– Dr. Kristi Miller

Claim of fact: Miller tested fish dating back to 1986 and says she found evidence of an ISA virus. She speculates the virus has been around much longer than 25 years.

We have, since then, sequenced from these 1986 samples and found that the three fixed base differences that we see, today, existed in 1986 as well, which suggests that not only has this been here for at least 25 years, but it’s been here probably quite considerably longer than that, given that there were already fixed differences that existed in 1986.
Q So are you effectively finding positive ISA PCR test results relating to Fraser sockeye from the ’80s?
DR. MILLER: Yes.

– Dr. Kristi Miller under questioning by commission lawyer Martland

Speculation: Dr. Are Nylund does not believe evidence shows ISA is in B.C. and finds Miller’s results “strange.”

But if you look at the situation in wild Pacific salmon that we’ve seen so far and the result presented by Miller here, I don’t think we have seen evidence of ISA virus in Pacific salmon, so far. No hard evidence. We have a lot of indications that the virus could be present in Pacific salmon, but there is no hard evidence. And I really would like to discuss the results presented by Miller, because I find them a bit strange, some of the results.

– Dr. Are Nylund

Speculation: Dr. Fred Kibenge believes that either ISA, or something like ISA is being detected.

Now, whether it’s ISA or ISA virus-like, you know, that depends on probably to need some more work. I know that in the virus classification, you know, ISA is put in the family Orthomyxoviridae. There’s one genus ISA virus and there’s one species, ISA — infectious salmon anaemia virus. So within that genus, I would expect that there may be ISA virus-like sequences that could be homologous – we’ve got to get picking up here – so I cannot exclude the fact that the virus that we’re detecting here may be within the genus ISA virus. It may be ISA virus sequences or it may be ISA virus-like, but I think the evidence is, to me, it’s overwhelming that there’s Orthomyxovirus here.

– Dr. Fred Kibenge

Speculation: Miller believes more research is needed, and doesn’t believe the virus being detected is causing disease.

I do think that there is sequence validation that there is an ISA-like virus here. How it gets classified I think will be determined both based on a fuller sequence and also obviously we have not established that it causes disease.

– Dr. Kristi Miller

Fact and speculation: The North American and European strains of ISA split about 100 years ago and evolved differently, and it is quite possible what is being detected in B.C. now is some form of ISA which has been here a long time.

We discovered ISA on the east coast in the late 1990s, and prior to that it was found in Norway. But we found, also, due to the divergence in sequences from the North American — what we call North American strains and European strains, we found that actually those viruses were probably coming from an original common source that separated physically, geographically, at least a hundred years and had time to evolve separately to create those two big branches if ISA; the North Americans and the Europeans. And the viruses were there in nature for more than a hundred years naturally. They were there for thousands of years and they have evolved with their host. In this case, I don’t know where we are at this point, because we don’t have enough information, but it could really be that we’re looking at another ISA that was there for a long time. And it’s an interesting theory that I would — I’m keen to see more work done on that.

– Nellie Gagne

Fact: Whatever the virus being detected is, it is not killing farmed Atlantic salmon, which are most susceptible to the disease caused by the ISA virus.

If it’s ISA, there’s several things that don’t match the picture we have right now with ISA as it is in Atlantic salmon aquaculture, because we’re talking of all below normal level that we detect in carriers, at this stage. We’re talking of an unsusceptible species. Atlantic salmon are the susceptible species of diseases. Right now, what we see, there’s none reported in Atlantic salmon, in cultured Atlantic salmon.

Nellie Gagne

Fact: Miller’s ISA research is not connected with her Fraser sockeye “genomic signature” research.

We do not see a correlation in the positives that we’re seeing with ISA with our genomic signature.

– Dr. Kristi Miller

Speculation: Dr. Are Nylund was skeptical of Miller’s testing method and suggested that she may have been detecting her own primers attaching to random bits of RNA.

I’ve never been acquainted with this method before and it’s a bit worrying the way they’re doing it, but as I said, it could lead to false positives…I think, especially that first stage where she does the pre-amplification with only the primers, they could attach to more or less random RNA or DNA, causing a segment that later could become positive in the real-time PCR.

– Dr. Are Nylund

Speculation: Nylund did not believe Miller was actually able to sequence the ISA virus because there was important data missing in her sequence.

I find it hard to believe that this could be a functional sequence. I think this could be due to unspecific annealing of the primers that are picking up something else than actually virus.

– Dr. Are Nylund

Speculation: Nylund suspected there may be a Pacific variant of ISA being detected by his and Kibenge’s labs.

And there may very well be a Pacific ISA virus that we have not yet detected and it could be very different from the North Atlantic ISA virus. But I think the method that they are using are quite good, except the one that has been used by Miller.

– Dr. Are Nylund

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5 thoughts on “ISA experts – what they actually said”

  1. You have cherry-picked a few quotes from a lot of transcript here — and the experts were clearly bending over backwards choosing their words so as not to offend any number of offendable parties. It would be great if you could get the experts to comment on the accuracy and aptness of your four conclusions. If they actually agree that that is what they meant, it would lend your argument a lot of credibility.

    1. If by “cherry-picked” you mean “selected” then yes, that is what we did. But unlike any other blogs or news reports we have seen, we have posted the words of the witnesses verbatim and provided a link for people to go read the full day’s proceedings for themselves, nor do we pretend to be “experts” on what they said (reading a lot of documents does not make one an expert).

      You think the witnesses were trying not to offend people, interesting speculation, but that is your interpretation of their words.

      If you find any statements made by the witnesses which contradict our four “take-home messages” please feel free to post them.

  2. Bravo Salmonfarmscience, thanks for the summary. Hopefully Morton and her followers will have the dignity to feel the appropriate amount of embarrassment and apologize to the public, salmon farmers and scientists they attempted to deceive and defame. I won’t hold my breath.

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