The Future of Fish Farms ????



I saw this on another forum:

http://metronews.ca/news/canada/452004/foreign-complaint-behind-audit-of-pei-lab/

".....Guy Gravelle, a spokesman for the Canadian Food Inspection Agency, said in an email Friday that an OIE member country had become concerned because the facility’s work on samples from that country were not consistent with findings from other researchers......."

You have wonder why Kibenge's lab can "find" genetic ISA virus signatures where many other labs using similar techniques can not. When you're amplifying single fragments of DNA/RNA, the potential for false positives is huge without very strict lab and analytical protocols. That seems to be basis for the independent lab audit that his lab came up short in. If it's true that sloppy lab procedures are the problem, he's done a grave disservice to the science and public debate.
 
I toured a closed containment facility in West Van a few months ago where they currently are raising several fish species via closed containment. The thing that stuck with me most was the proprietary technology related to their feed. I'm not sure of the exact components of the feed but it's an aquatic plant-based feed without any animal protein/contaminants. The feed was being produced on-site and I believe it was some sort of algae that can be easily created sustainably and turned into pellets for feed. This facility was quite advanced and they were at the financing stage needed for patents and further expansion. Anyone familiar with algae feed and if it's actually feasible? These guys (scientists/businessmen) seemed very keen on it but I haven't heard about it much elsewhere. I like the idea of having a land-based closed containment facility with an ability to grow it's own feed sustainably. Hope this takes off.

Agreed. Another externalized cost that subsidizes the fish farm industry.

Krill fishing: http://www.nature.com/news/2010/100901/full/467015a.html

China's now into it. When the krill harvest is made unsustainable due to overfishing and climate change, what's next? Charlie notes it's not likely plant protein. Even if soy worked well, do we want to burn more rain forest to grow fish feed? So what is the future of fish farming when sourcing adequate, sustainable feed stocks seem to be the limiting factor to growth?
 
You have wonder why Kibenge's lab can "find" genetic ISA virus signatures where many other labs using similar techniques can not. When you're amplifying single fragments of DNA/RNA, the potential for false positives is huge without very strict lab and analytical protocols. That seems to be basis for the independent lab audit that his lab came up short in...
The ONLY country or scientists questioning his work is Canada, which is Harper’s response - silence and discredit ALL the scientists!

Positive results for the ISA virus has been detected by the North American OIE reference lab for ISA and "TWO" other labs. DFO itself got positive ISA test results in 2004. Canada, CFIA, BC, DFO, and BC fish farmers have ALL have gotten positive results and/or symtems of the ISA "disease" and have NOT done any further follow-up testing to confirm it. Think about those implications if confirmed - no more trade with China and the U.S.! BTW... have YOU ever tested for the ISAVirus?

It is already known BC and CFIA both are using procedures and equipement not as sensitive as North American OIE reference lab for ISA. Frederick S.B. Kibenge is probably the most authoritative individuals in the world when it comes to ISA and ISAVirus. Before blindly accepting CFIA (aka Canada - aka Harper) you might want to read up on his work?
http://avc.upei.ca/oie

Might want to also listen to the interview with him:
http://www.cbc.ca/asithappens/podcasts/

You have probably had some of YOUR own Chinook die from ISAvdiseases and not even realized it. Here is portions of Center for Food Security & Public Health concerning ISA.

Quote:
... the cumulative mortality can sometimes exceed 90% if the disease remains unchecked.

Understanding of the epidemiology of ISA is still incomplete, which complicates its control. The reservoirs for the virus are not known, but experiments have shown that several species of salmonids can carry virulent ISA viruses asymptomatically. These viruses might cause outbreaks if they are transmitted to farmed Atlantic salmon. Noncultivable, apparently nonpathogenic, isolates have also been detected in wild salmonids. Small changes in these viruses, analogous to the mutations that allow low pathogenicity avian influenza viruses to become highly pathogenic, may allow them to become more virulent. Some evidence suggests that certain ISA viruses may cause illness in species other than Atlantic salmon...

The two major lineages of ISAV are the European genotype (or genotype I) and the North American genotype (or genotype II). Various clades occur within these genotypes. A small, highly polymorphic region (HPR) of the viral hemagglutinin-esterase (a surface glycoprotein encoded by genomic segment 6) can be used to classify ISAV isolates into numbered groups. HPR0 and HPR00 consist of the viruses that can be detected in fish by reverse transcription polymerase chain reaction (RT-PCR) assays, but cannot be cultured in the currently used cell lines. These viruses seem to be nonpathogenic for Atlantic salmon and other salmonids. Viruses with deletions in the HPR (e.g., those viruses classified as HPR1, HPR2, HPR3, etc.) appear to be more virulent and can be isolated in cell culture. A number of HPR genotypes have been detected.

The reservoir hosts for ISAV are unknown. In experiments, isolates that are virulent for Atlantic salmon usually infect other fish asymptomatically [meaning: asymptomatic if a patient is a carrier for a disease or infection but experiences no symptoms]. Subclinical infections [meaning: is the asymptomatic (without apparent sign) carrying of an (infection) by an individual of a virus] with these isolates have been reported in salmonids including brown trout (the freshwater resident form of Salmo trutta), sea trout (the migratory form of S. trutta), rainbow trout (the freshwater resident form of O. mykiss), steelhead trout (the migratory form of O. mykiss), chum salmon (O. keta), Chinook salmon (O. tshawytscha), coho salmon (O. kisutch) and Arctic char (Salvelinus alpinus), as well as some non-salmonids such as herring (Clupea harengus) Atlantic cod (Gadus morhua) and pollock (Pollachius virens). Non-cultivable (HPR0 or HPR00) isolates have been detected in asymptomatic wild or feral fish such as brown trout, sea trout, Atlantic salmon and rainbow trout. Salmonids including brown trout and sea trout, which can carry ISAV viruses asymptomatically for long periods, and wild Atlantic salmon, have been proposed as possible reservoir hosts.

This disease is suspected to occur in Iraq. Noncultivable, apparently nonpathogenic (HPR0 or HPR00), isolates of ISAV have been reported in wild salmonids in several countries, states or provinces including Norway, Scotland, Ireland, New Brunswick, Nova Scotia, Maine and Chile. These noncultivable isolates probably also occur in some areas where fish have not been tested.

... In Chile, ISA viruses of the European genotype have caused infectious salmon anemia in Atlantic salmon, but the virus isolated from coho salmon in 1999 was of the North American genotype. Most of the ISA viruses that have been detected in Canada and the U.S. belong to the North American lineage, but avirulent (HPR0) viruses from the European lineage have also been found in both countries.

... ISAV probably infects fish through the gills, but ingestion has not been ruled out. This virus is shed in epidermal mucus, urine, feces and gonadal fluids. In one study, virus shedding was first detected 7 days after inoculation, and rose above the minimum infective dose on day 11, two days before the first deaths occurred. Shedding peaked approximately 15 days after inoculation, when mortalities were high.

... Fish that survive the illness can shed the virus for more than a month.

Virulent isolates of ISAV can be detected in asymptomatic brown trout, sea trout, rainbow trout and herring for weeks after intraperitoneal injection. (However, replication is limited in herring.) In sea trout, viral RNA could still be found 135 days after infection. Blood from a brown trout, taken 7 months after it had been infected, caused disease when it was injected into Atlantic salmon. ISAV can also be transmitted from salmon to trout, and from trout to salmon...

... It has also been found for a short time in non-salmonids. Nucleic acids could be detected in juvenile Atlantic cod for up to 45 days after intraperitoneal injection. Pollock have been suggested as potential carriers, because they are common in and around Atlantic salmon net pens.

... It is possible that virulent ISA viruses are generated from avirulent strains that circulate among wild salmonids and other species.

In farmed Atlantic salmon, the clinical signs may include lethargy, anemia, leukopenia, ascites, exophthalmia, darkened skin and increased mortality. In some cases, the hematocrit may be nearly normal; in others, severe anemia with a hematocrit as low as 2-3% may be seen. As a result of the anemia, the gills may be pale. Hemorrhages may be found in the anterior chamber of the eye. Jaundice on the ventral portion of the body has been reported among Atlantic salmon in Chile.

... seen in the outbreak among farmed coho salmon in Chile. Jaundice, with yellowing of the base of the fins and on the abdomen, was also reported.

In Atlantic salmon, the gills may be pale and the skin can be darkened. Exophthalmia may also be seen. Yellow- or blood-tinged fluid may be found in the peritoneal and pericardial cavities. Petechiae, which may be extensive, can usually be found on various organs and tissues, including the eye, the internal organs, the visceral fat and the skeletal muscles. The spleen may be enlarged and congested. Congestion, enlargement and necrosis may also be apparent in the liver; in some cases, this organ may become dark brown or black, and it may be covered with a thin layer of fibrin. The kidney may be swollen and dark; blood and liquid may exude from the cut surface. The gastrointestinal tract may also be congested, but blood is not usually found in the intestinal lumen if the carcass is fresh. The histopathological lesions may include hemorrhagic necrosis of the liver, renal interstitial hemorrhages and tubular nephrosis, filamental sinus congestion of the gills, splenic congestion with erythrophagocytosis, and congestion of the lamina propria of the stomach and foregut.

... The salmon in Chile were recovering from piscirickettsiosis, and it is not known whether this disease may have contributed to the unusual signs. Other lesions were consistent with ISA outbreaks reported among Atlantic salmon in other countries.

In coho salmon in Chile, the gross lesions were similar to those seen in Atlantic salmon, but jaundice was apparent, and the liver and gall bladder were pale.

... The liver and spleen were congested in a minority of these fish. Unlike Atlantic salmon, relatively few rainbow trout had obvious liver necrosis or kidney necrosis on histopathology, and some trout had epicarditis, endocarditis and myocarditis.

Subclinical infections may be common among salmonids in some locations. A recent study in Norway detected ISAV in 22 of 24 Atlantic salmon smolt production sites by real-time RT PCR. In this study, more than one isolate was found in some smolt populations and at one marine site. In Norwegian rivers, the prevalence of apparently nonpathogenic strains in wild salmonids was highly variable; in some cases, the virus was detected in only a single fish, while in others, it was found in 100% of the fish tested. Similarly, the distribution of the virus was not homogeneous among wild fish in Scotland.
http://www.cfsph.iastate.edu/Factsheets/pdfs/infectious_salmon_anemia.pdf
 
Also, while I feel that Tincan has a point - e.g. that louder voices often win because scientists appear timid in what they say - I'd argue that ultimately the truth and the data wins. It sometimes takes longer than I wish but ultimately, facts and data are hard to argue with. What I'm not willing to do is to overstate a case and provide ammunition that allows others to say, that's not really true or you didn't even come close to truly proving that. If that seems timid, then so be it. It's what allows me to go to sleep happy each evening.

I wish it were true that truth and data wins out but it's hard to argue that is true when, for example, we're wiping out species on our planet at an unconscionable rate.

[vpxmTZ02FXg] http://www.youtube.com/watch?v=vpxmTZ02FXg

Since this is a fishing forum we can keep it on topic and see that despite the strong consensus in the scientific community we continue to allow behavior that wipes out countless species every year with unknown consequences. To that end I'd have to say that unfortunately truth and data does NOT win. That is why I feel we need more outspoken individuals in the scientific community and elsewhere to become leaders in our communities that will help shape policy that relies on science. Clearly not all scientists are ready and willing to step into these roles but I am hopeful that some will because they are the experts with the answers to many of the challenges we face these days.
 


I saw this on another forum:

http://metronews.ca/news/canada/452004/foreign-complaint-behind-audit-of-pei-lab/

".....Guy Gravelle, a spokesman for the Canadian Food Inspection Agency, said in an email Friday that an OIE member country had become concerned because the facility’s work on samples from that country were not consistent with findings from other researchers......."

You have wonder why Kibenge's lab can "find" genetic ISA virus signatures where many other labs using similar techniques can not. When you're amplifying single fragments of DNA/RNA, the potential for false positives is huge without very strict lab and analytical protocols. That seems to be basis for the independent lab audit that his lab came up short in. If it's true that sloppy lab procedures are the problem, he's done a grave disservice to the science and public debate.

Very convenient for Harper. They had to do two audits to come up with enough findings to attempt this smear.
To quote from the Mark Hume article in the newspaper posted elsewhere:-

“This is stunning news,” said Rick Routledge, a professor at Simon Fraser University, who sent the lab samples that showed a ISA virus was present on the Pacific coast. “This comes as a shock. . . my head is spinning. I had no idea they would take it that far,” he said."

The government is terrified of being forced by international pressure and agreements of having to shut your industry down. They will stop at absolutely nothing to prevent this. As Charlie said above DFO found ISA in 2004 and never followed up. They are deliberately supressing information, silencing critics, and removing funding and staff from scientific research.
The conservatives want to remove all wild salmon. They are inconvenient because they are hard to manage, stand in the way of total domestication of the ocean for aquaculture, will be in the way of the Northern Gateway pipeline development, and prevent damming of the Fraser river for Hydro power or selling water to the States.
This whole thing is one more attack by Harper on whom he sees as "enemies of the state". This Harper guy is incredibly dangerous.
 
I thought Cohen recommended the fish farms be removed if they were shown to pose anything more than a minimal risk to wild salmon.

I think Harper has been busy with his white out, as it now seems to read scientists that pose anything more than a minimal risk to fish farms should be removed. Cue up the attack ads seems like he might have some more targets coming up.

i'd sure like to see Trudeau, or for that matter anyone, get him in the boxing ring. We could probably eliminate our deficit with a pay per view. I'd sure love to see it.
 


I saw this on another forum:

http://metronews.ca/news/canada/452004/foreign-complaint-behind-audit-of-pei-lab/

".....Guy Gravelle, a spokesman for the Canadian Food Inspection Agency, said in an email Friday that an OIE member country had become concerned because the facility’s work on samples from that country were not consistent with findings from other researchers......."

You have wonder why Kibenge's lab can "find" genetic ISA virus signatures where many other labs using similar techniques can not. When you're amplifying single fragments of DNA/RNA, the potential for false positives is huge without very strict lab and analytical protocols. That seems to be basis for the independent lab audit that his lab came up short in. If it's true that sloppy lab procedures are the problem, he's done a grave disservice to the science and public debate.

When you post up what Canadian Food Inspection Agency (CFIA) is saying does not inspire me much.
Think xlbeef....... I was reading some "senate debates" the other day when I ran across a little information.
[FONT=Arial,helvetica]One Senator was asking what the [/FONT] CFIA was doing to fix the problems at that agency. He responded that the Harper government had hired 600 new employee to fix things. The only problem with his answer was that the Union of Public Employee's had not see any new applications for membership..... So only three things could have happened. One is that these 600 new inspectors are non-union (yea sure) or two, those 600 people do not exist. (ie its a lie)
Or three, they hired 600 new pencil pushers into management. (ie friends of the ultra far right)
Wonder if it's one, two or three?

Since when have you taken up science? your post would indicate that you have been doing some studying.
Good to see you are coming around to letting the facts speak and not ideology....
Now you need to re-look at the facts.... How are your fish at the "Indian" farm?
Still sick or are they all better....
Still having that 9% die off?
GLG
 
Since when have you taken up science? your post would indicate that you have been doing some studying.

I am always trying to learn as much as I can about it. Unfortunately it is not my position or job to fully understand it all but I do have an opinion which is subject to change. But you must understand that I have a long history of seeing right through mortons BS, even at my basic level, and at any point weather she is right or not, I will be highly suspicious and I should be. While I may not be a full fledged scientist I can recognize many assumptions posted here against salmon farming in bc. IT seems that those here apposed to it and actively apposing it aren't interested in much of a discussion which is the tradition here on this site. Even discussions here with others against salmon farming such as between seadna and others have some tones that are not so tasteful when different opinions arise.
I just posted a related article. I really did not have much to say about it but I see some more assumptions were made by that post.

As far as creative indian farm well you have to talk to them about that. I do not know what makes you think those fish are in the water they have been out for some time.
 
I am always trying to learn as much as I can about it. Unfortunately it is not my position or job to fully understand it all but I do have an opinion which is subject to change .
It is everyone’s job to fully understand any evidence and data being published on this topic, especially if you are going to offer up unsubstantiated statements which are exactly that, opinion, and not based on facts!!
But you must understand that I have a long history of seeing right through mortons BS, even at my basic level, and at any point weather she is right or not, I will be highly suspicious and I should be. .
No, on the contrary, you must understand that many opponents on here have a long history of seeing through the salmon feed lot industry BS and that offered up by yourself. We are highly suspicious and should be because your industry is totally motivated by financial considerations at the expenses of everything and everyone else.
And for you to make accusations at Morton, when she has a Ph.D. and has published scientific papers co-authored with other respected scientists in the National Academy of Science, and you have done neither is utter BS. It is worse than that, she has to suffer the jibes, attacks and snide comments from people like you all the time, and yet you have absolutely no qualifications to do so.
The stress she suffers in the role she has chosen is unbelievable, and yet you people never stop. It is sickening.
While I may not be a full fledged scientist I can recognize many assumptions posted here against salmon farming in bc. .
Please post the “assumptions” you recognise here.
IT seems that those here apposed to it and actively apposing it aren't interested in much of a discussion which is the tradition here on this site. Even discussions here with others against salmon farming such as between seadna and others have some tones that are not so tasteful when different opinions arise. .
Not true. People like Seadna and Charlie have posted links to actual scientific papers and quoted detailed sources right here. But you either do not read them, ignore them if they challenge your ill-informed “opinion” or attack the authors.

I just posted a related article. I really did not have much to say about it but I see some more assumptions were made by that post.
On the contrary, the assumptions were made by you that Dr. Kibenge, a world renowned scientist, had done “a great disservice to science and public debate”. I note that all the focus is on whether or not his lab got any “false positives” and not on why any other labs may have failed to find what he did or why those results like DFO obtained in 2004 were never followed up.
 
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As someone who's had technicians in my group do 10's of 1000's of PCR's and who's done quite a few of my own, it's not rocket science to make PCR work. While it is possible to get a positive PCR product from a negative sample (the false positive that Birdsnest mentions), false positives are generally the result in misc. crap amplifying that is not the intended product. If one only measures whether or not a PCR product is produced, then it's possible one may report false positives. HOWEVER, and this is key, one can obtain the DNA sequence of the PCR product and if it it gives a sequence of ISAv, it's not a false positive. Many of the samples that have been looked at, not only produced PCR products but the PCR products were sequenced and demonstrated to be ISAv (or ISAv-like) sequences. So, it's not appropriate to classify all positive ISAv tests as false positives. Also, just because some labs have looked for ISAv and not observed it doesn't mean it wasn't present in their samples. Given that the fairly large number of samples with ISAv positive results by PCR AND sequencing, I suspect false negative results from the non-reference gov't or industry labs (who could easily set up their PCR to be not very sensitive).
 
It is everyone’s job to fully understand any evidence and data being published on this topic, especially if you are going to offer up unsubstantiated statements which are exactly that, opinion, and not based on facts!!

Well my opinion is based on some facts. I am sorry if a scientific paper does not follow each one of my comments and I see that is a requirement for you and that is fine.

We are highly suspicious and should be because your industry is totally motivated by financial considerations at the expenses of everything and everyone else.

Well I would not go as far as you have on the statement but I too am not a huge fan of large corporations. There is no doubt that they have a not so good track record on all fronts. Unfortunately salmon farming is not really appropriate for small business so corporations it is.

And for you to make accusations at Morton, when she has a Ph.D. and has published scientific papers co-authored with other respected scientists in the National Academy of Science, and you have done neither is utter BS. It is worse than that, she has to suffer the jibes, attacks and snide comments from people like you all the time, and yet you have absolutely no qualifications to do so.
The stress she suffers in the role she has chosen is unbelievable, and yet you people never stop. It is sickening.

Your right. She chose that role. Sounds like you are close personal friends with her or know her well?

On the contrary, the assumptions were made by you that Dr. Kibenge, a world renowned scientist, had done “a great disservice to science and public debate”. I note that all the focus is on whether or not his lab got any “false positives” and not on why any other labs may have failed to find what he did or why those results like DFO obtained in 2004 were never followed up.

That whole post was cut and pasted. I did not write that but I did find it interesting. From what I understand, in science land, if other labs can not duplicate your findings then it just isn't so. But no Morton chose it to be as fact and made the big announcement without further investigation. Not very professional if you ask me but I am not a biologist so... my view is worthless to you and I am TOTALLY ok with that!

Please post the “assumptions” you recognise here.

You post your credentials first and then I will continue. What are you doing for a living as well....honestly too!
 
Come on - requiring him to post his credentials before you will explicitly discuss the assumptions you claim is a pretty weak argument.
 
As someone who's had technicians in my group do 10's of 1000's of PCR's and who's done quite a few of my own, it's not rocket science to make PCR work. While it is possible to get a positive PCR product from a negative sample (the false positive that Birdsnest mentions), false positives are generally the result in misc. crap amplifying that is not the intended product. If one only measures whether or not a PCR product is produced, then it's possible one may report false positives. HOWEVER, and this is key, one can obtain the DNA sequence of the PCR product and if it it gives a sequence of ISAv, it's not a false positive. Many of the samples that have been looked at, not only produced PCR products but the PCR products were sequenced and demonstrated to be ISAv (or ISAv-like) sequences. So, it's not appropriate to classify all positive ISAv tests as false positives. Also, just because some labs have looked for ISAv and not observed it doesn't mean it wasn't present in their samples. Given that the fairly large number of samples with ISAv positive results by PCR AND sequencing, I suspect false negative results from the non-reference gov't or industry labs (who could easily set up their PCR to be not very sensitive).

Do you think it is done on purpose?
 
Come on - requiring him to post his credentials before you will explicitly discuss the assumptions you claim is a pretty weak argument.

Its not an argument. I just want to know before I continue.
 
To assume that any foreign viruses in the north pacific are product of salmon farming is here is a pretty far stretch considering what we have done previously before salmon farms were here.


EVERYBODY should not want this to be true!!! But the truth should be told.
 
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To assume that any foreign viruses in the north pacific are product of salmon farming is here is a pretty far stretch considering what we have done previously before salmon farms were here.

EVERYBODY should not want this to be true!!! But the truth should be told.

I actually would have never made that statement. However, I will agree… I would be very surprised and not expect to find any foreign Norwegian viruses from anywhere on our Westcoast in the “North Pacific.” By definition that would be the Bering Sea and NONE of our Pacific salmon even migrate and enter the Bering Sea, unless they flat-out get lost. Therefore, you are correct it would be a “pretty (sp) far stretch” to see any “foreign viruses” by product of BC salmon lots! If they are found there, I personally would have to point my finger at Japan and/or Russia fish feed lots.
The principal arms of the Pacific Ocean are (in the north) the Bering Sea; (in the east) the Gulf of California; (in the south) Ross Sea; and (in the west) the Sea of Okhotsk, the Sea of Japan, and the Yellow, East China, South China, Philippine, Coral, and Tasman seas.
Now with that and assuming you are actually referring to Gulf of Alaska that would be a very bad and uninformed statement to make as DFO themselves have already taken samples and found ISAv in salmon in the Gulf a Alaska – see and start reading the Cohen Commission testimonies and reports! Btw…
The Gulf of Alaska, an arm of the Pacific Ocean, extends along the southeastern coastline of Alaska from the Alaska Peninsula to the Alexander Archipelago.
Then just do a search and you will find Mainstream (majority owned by the country of Norway) has already acknowledged the European strain of ISAv was transported to Chile by the Atlantic feedlot industry! It is not really hard to confirm that those feedlots transmit disease and viruses. FYI… you really believe Norway doesn’t realize they are transmitting “ALL” their viruses and diseases all over the world - That might be a little naïve.

Since this has been referring to ISA and ISAv I will gladly provide all the information your heart desires on that topic, as I have actually done quite a bit research on that myself.

I assure, and it certainly appears YOU don’t have a clue of what you are dealing with here!

SCIENTIFIC OPINION
Scientific Opinion on infectious salmon anaemia (ISA)
EFSA Panel on Animal Health and Welfare (AHAW)
European Food Safety Authority (EFSA), Parma, Italy

On request from the European Commission, Question No EFSA-Q-2012-00060, adopted on 16 November 2012.

Panel members: Edit Authie, Charlotte Berg, Anette Bøtner, Howard Browman, Ilaria Capua, Aline De Koijer, Klaus Depner, Mariano Domingo, Sandra Edwards, Christine Fourichon, Frank Koenen, Simon More, Mohan Raj, Liisa Sihvonen, Hans Spoolder, Jan Arend Stegeman, Hans-Hermann Thulke, Antonio Velarde, Ivar Vågsholm, Preben Willeberg and Stéphan Zientara. Correspondence: AHAW@efsa.europa.eu

Acknowledgement: The Panel wishes to thank the members of the Working Group on infectious salmon anaemia: Edgar Brun, Debes Christiansen, Philippe Lemey, Niels Jørgen Olesen, Rob Raynard, Espen Rimstad, Fulvio Salati, Mike Sharp (chair until July 2012), Liisa Sihvonen and Ivar Vågsholm (chair from July 2012) for the preparatory work on this scientific opinion and EFSA staff, Per Have for the support provided to this scientific opinion.

1.4. ISAV infection
The development of more sensitive methods for virus detection by PCR during the 1990s (Mjaaland et al., 1997) enabled studies providing evidence of ISA virus infection in apparently healthy wild fish (feral Atlantic salmon, brown trout and sea trout, and escaped, farmed rainbow trout) (Raynard et al., 2001; Plarre et al., 2005).Refinements to molecular methods enabled the description of genomic sequences of ISA virus in wild salmonids which were hypothesised to show a full-length sequence of the HPR of the haemagglutinin-esterase (HE) gene (Mjaaland et al., 2002b). Thus, the hypothesis that deletions of HPR0 were required for emergence of HPR variants (HPRΔ) associated with virulent forms of ISAV was derived (Mjaaland, Hungnes, et al., 2002; Nylund et al., 2003).

ISAV can be genetically differentiated on the basis of the sequence of the HPR of genomic segment 6 which encodes the HE protein. Deletions within the HPR region (HPRΔ ISAV) have been identified in all virulent isolates causing clinical ISA disease and appear to be necessary for pathogenicity.

HPR0 ISAV appears to be widely distributed, both in areas infected with and areas free from HPRΔ ISAV and clinical disease. From a disease control point of view it is important to understand the dynamics and interrelationship between HPR0 and HPRΔ ISAV and, more particularly, the likelihood of and the reasons why HPRΔ arises from a background source of HPR0 ISAV.

In view of the above, the Commission is evaluating whether it is appropriate, proportionate and necessary to apply risk management measures to HPR0 ISA, in addition to those applied to HPRΔ ISAV. EFSA has been asked for a scientific opinion on the HPR0 variant of ISAV and to assess the risks posed by HPR0 ISA for the health of aquatic animals, in particular Atlantic salmon. The terms of reference (ToRs) provided by the Commission can be formulated as three questions:

1. Can HPR0 ISA cause clinical disease?
2. What is the risk of HPR-deleted ISA emerging from HPR0 ISA and, if relevant, indicating factors for such an emergence
a. What is the risk of HPR-deleted (HPRΔ) ISAV emerging from HPR0
b. What are the factors relevant for such an emergence?

2. The capability of HPR0 ISAV to cause clinical disease (ToR1)
ISA is a systemic and lethal condition and clinical signs suggest circulatory failure. So far only HPRΔ ISAV variants have been reported to cause disease in Atlantic salmon.

No experimental infection has been carried out so far with HPR0 ISAV. However, this variant has also been detected in naturally infected salmon, most often in the gills (McBeath et al., 2009; Christiansen et al., 2011). As opposed to the systemic and severe disease caused by HPRΔ ISAV, HPR0 ISAV replicates in (D.H. Christiansen, personal communication) and causes a localized infection of salmon gills with no signs of disease and only occasional spread to other organs (Christiansen et al., 2011). Parallel testing of kidney, heart and gill tissue for the presence of HPR0 ISAV by real-time RT-PCR disclosed a significantly higher overall detection in gill tissue compared with kidney and heart. Also, the load of HPR0 ISAV virus in positive gills was generally much higher than in kidneys and hearts (Christiansen et al., 2011; Lyngstad et al., 2011). Thus, HPRΔ ISAV and HPR0 ISAV show different infection patterns and tissue tropism, a pattern similar to that found in wild aquatic birds in which low-pathogenic avian influenza virus causes a subclinical, transient, mucosal infection whereas highly pathogenic influenza causes a systemic and lethal infection in poultry.

2.4. Geographical distribution of HPR0 ISAV
The first detection of HPR0 ISAV was done on gill tissue from a wild-caught Atlantic salmon in Scotland (Cunningham et al., 2002). In addition to Scotland, HPR0 ISAV has also been detected in farmed Atlantic salmon from the Faroe Islands, Norway, Canada, Chile and Denmark (N.J. Olesen, personal communication).
HPR0 has also been detected in wild Atlantic salmon in the Faroes and Norway. Three out of 88 confirmed wild Atlantic salmon caught by a Faroese research vessel at the feeding grounds in the North Atlantic were HPR0 positive (D.H. Christiansen, personal communication). Furthermore, 4 out of 305 Atlantic salmon caught in rivers in mid-Norway were found to be positive by PCR. Viral RNA from one of them was sequenced to HPR0, clustering phylogenetically with the Faroes cluster. The amount of RNA from the other three was too scarce for sequencing, but still empirically indicated the presence of HPR0. All salmon were caught in an area with on-going ISA outbreaks with virus subtypes associating with a cluster different from that identified as HPR0. The four positive ones were all from the same river and confirmed as “wild salmon” according to fish scale examination. (R. Grøntvedt and T. Lyngstad, personal communication).

The Faroe Islands documented findings of HPR0 in Atlantic salmon in their monitoring from 2005 to 2009. HPR0 was detected on gills 1–13 months post sea transfer (mean 7.7 months). The various cohorts (49) were sampled 5–12 times each year, and the presence of HPR0 on gills showed peaked transient infection profile with peak prevalence up to 100 % lasting for 4 months. Almost all of the cohorts were positive for HPR0. No clinical disease or histopathological consequences have been reported in association with this HPR0 infection in the Faroes (Christiansen et al., 2011).

In Chile, all ISAV strains detected in 2011 were identified as HPR0 (Kibenge et al., 2012). No outbreaks were observed and HPRΔ was not detected.

In a retrospective study in Norway (Lyngstad et al., 2012), ISAV was present in 23 % of 210 cohorts of marine farmed Atlantic salmon along the coast, with no suspicion of ISA. HPR0 ISAV was confirmed in 59 % of these ISAV-positive groups. The rest of the positive groups were not sequenced due to lack of RNA, but the low titres may indicate the presence of HPR0.

The groups were sampled once and at various points in time after sea transfer. In other screening studies, HPR0 has been detected in gill samples from juvenile salmon and in brood stock in the freshwater environment (M. Devold and D. H. Christiansen, personal communication). A low level of HPR0 has also been detected in ovarian fluid of farmed Atlantic salmon (D. H. Christiansen, personal communication).
 
part 2

3. What is the risk of HPRΔ ISAV emerging from HPR0 ISAV (TOR 2a)
In order to address this question the genomic structure of ISAV is briefly described and the evidence available regarding virulence factors and phylogenetics and evolutionary dynamics is considered.

3.1. Genome of ISAV
The ISA virus belongs to the genus Isavirus of the family Orthomyxoviridae. The genome of ISAV consists of eight negative-sense single-stranded RNAs. Each segment contains one or more open reading frames (ORFs) flanked by 5 and 3 untranslated regions (Sandvik et al., 2000; Clouthier et al., 2002; McBeath et al., 2006) and together they encode 10 or 11 proteins (Mjaaland et al., 1997; Rimstad and Mjaaland, 2002; Kibenge et al., 2007a). Whereas segments 1 to 6 encode one protein each, segment 7 encodes two or three proteins and segment 8 encodes two proteins. Functional characterisation of the two viral surface proteins, the HE protein coded by segment 6 and the F protein coded by segment 5 has disclosed that ISAV possess the major functional characteristics of the influenza virus family including receptor-binding, fusion and receptor-destroying activities (Falk et al., 1997, 2004; Krossøy et al., 2001; Rimstad et al., 2001; Aspehaug et al., 2005). The sequence of segments 1, 2 and 4 have been identified to encode the RNA polymerases PB2, PB1 and PA, respectively (Krossøy et al., 1999; Clouthier et al., 2002; Snow et al., 2003a) whereas segment 3 encodes a nucleoprotein (Ritchie et al., 2001; Aspehaug et al., 2004). The unspliced mRNA of segment 7 encodes a non-structural protein and ORF2 of segment 8 encodes an RNA-binding structural protein. Both proteins have interferon antagonistic properties (Biering et al., 2002; McBeath et al., 2006; García-Rosado et al., 2008). The spliced mRNA of segment 7 encodes a putative nuclear export protein (Kibenge et al., 2007b) and ORF1 of segment 8 encodes the matrix protein (Biering et al., 2002; Falk et al., 2004).

3.2. Virulence of ISAV
ISAV isolates vary in virulence, as observed by differences in disease development and clinical signs in field outbreaks, as well as in experimental trials. In experimental infections, induced mortality for different ISAV isolates varied from 0 % to 47 % in the injected fish and from 3 % to 75 % in the cohabitant fish (Mjaaland et al., 2005).

Only a few potential virulence markers have been identified in the ISAV genome. The functional role of these markers, if any, has not yet been determined. The HE gene includes an HPR of 35 amino acids close to the transmembrane region (Devold et al., 2001; Kibenge, Kibenge, et al., 2001; Krossøy et al., 2001; Rimstad et al., 2001; Mjaaland, Hungnes, et al., 2002; Falk et al., 2004). Approximately 30 different HPR variants have been identified in Europe, North America and Chile (Nylund et al., 2006; Kibenge et al., 2009; Christiansen et al., 2011). The HPR variants can be explained as various deletions (Mjaaland, Hungnes, et al., 2002) from a putative ancestral sequence (HPR0) first identified in a wild salmon in Scotland (Cunningham et al., 2002). Whereas all ISAV isolates from ISA disease outbreaks have deletions in the HPR region with respect to the HPR0, the HPR0 subtype has not been associated with clinical or pathological signs of ISA disease (Cunningham et al., 2002; Cook-Versloot et al., 2004; Nylund et al., 2006; McBeath et al., 2009; Vike et al., 2009; Christiansen et al., 2011). This strongly suggests that the HPR region is an important virulence marker of ISAV (Mjaaland et al., 2005).

In addition, all virulent strains of ISAV examined so far have the Q266L266 amino acid substitution or short amino acid insertions immediately upstream or downstream of the putative arginine cleavage site R267 in the F protein (Devold et al., 2006; Markussen et al., 2008; Cottet et al., 2011).

However, pathogenic ISAV isolates with identical HPR deletions and mutations around the putative cleavage site R267 of the F gene vary in virulence (Mjaaland et al., 2005; Markussen et al., 2008) suggesting that other as yet unknown genetic changes have an influence on virulence. This is supported by findings in influenza viruses where specific mutations in the PB1 (Conenello et al., 2007) and PB2 (Shinya et al., 2004) genes have been linked to virulence.

3.3. Phylogenetics and evolutionary dynamics of ISAV
Based on phylogenetic analysis of segment 2 and segment 8, ISAV isolates have been divided into two major genogroups: the North American (NA) genogroup including isolates from Canada and the USA, and the European (EU) genogroup consisting of ISAV isolates from Norway and Scotland (Blake et al. 1999; Krossøy et al. 2001). Based on genetic analysis of only the extracellular region of the HE gene on segment 6, the EU isolates have been further divided into four subgroups (with HPR0 being represented in all subgroups) (Nylund et al., 2003, 2006; Devold et al., 2006; Lyngstad et al., 2008; McBeath et al., 2009; Vike et al., 2009; Christiansen et al., 2011). It should be noted that this subgrouping may not be consistent across different segments (Plarre et al., 2012).

Phylogenetic analysis of Faroese HPR0 ISAV variants identified a lineage with close genetic similarity to HPRΔ ISAV associated with the Faroese ISA disease epidemic (Christiansen et al., 2011). Lyngstad et al (2012) showed that genetic and geographic distances between pairs of HPR0-ISAV sequences are positively correlated, suggesting that the population of HPR0 ISAV is geographically structured. They also showed that virulent ISAV HE gene sequences from isolated ISA outbreaks were significantly closer in geographical distance to the genetically closest HPR0 ISAV HE gene sequences than would be expected by chance. This association between genetic and geographical distance supports the hypothesis that HPRΔ ISAV may evolve from HPR0 ISAV (Cunningham et al., 2002; Mjaaland, Hungnes, et al., 2002; Nylund et al., 2003).

Several hypotheses have been put forward to explain the origin of HPRΔ and its relationship with HPR0. The original hypothesis was that the virulent HPRΔ ISAV is derived from non-virulent HPR0 ISAV by deletions in the HPR of the HE molecule (Cunningham et al., 2002; Mjaaland, Hungnes, et al., 2002).

A postulated “insertion hypothesis” whereby virulent HPRΔ ISAV undergoes mutations involving insertions into the HPR, resulting in HPR0 ISAV and thus attenuation (Kibenge et al., 2012), is not consistent with these findings and does not explain the presence of geographical variants of HPR0.

A third hypothesis brought forward is that HPR0 is the consensus sequence of all HPRΔ sequences during virus replication (Kibenge et al., 2012). If this hypothesis is correct, fish should be HPR0 ISAV positive while being infected by a range of virulent HPRΔ ISAV (“HPRΔ cloud”), of which the combined HPR consensus sequence would be HPR0. Selection by farming would then be on a variety of HPRΔ ISAV mutants rather than on individual HPR0 ISAV sequences.

Although quasispecies are a common feature of RNA virus infections, i.e. a mixed population of mutants rather than clonally expanded virus particles, there is no experimental support of an “HPRΔ cloud” in all ISAV infected fish. Nucleotide sequences from fish found to be HPR0 positive have not reported the presence of a variety of HPRΔ sequences (Christiansen et al., 2011, Lyngstad 2012). Lyngstad et al (2012) found a geographical relationship between HPR0 and HPRΔ in the nucleotide sequences of the HE gene (excluding the HPR region) (Lyngstad et al., 2012). More recently, the use of new generation sequencing/pyrosequencing of HPR0-positive fish and of ISA-diseased fish demonstrated the presence of a very small fraction of HPRΔ in HPR0 positive fish and an even smaller fraction of HPR0 ISAV in diseased fish having one dominant HPRΔ form, thus rejecting the third hypothesis (Markussen et al., 2012). The original deletion hypothesis has, on the other hand, been supported by the findings from sequencing of viral RNA from ISA outbreaks.

To gain further insights into the process of virulence evolution, a quantitative assessment of the different evolutionary forces for ISA would be useful, in particular the relative contribution of mutation and reassortment processes. The evolutionary rate of different ISAV segments has been estimated to be about an order of magnitude lower than that of influenza A viruses (Plarre et al., 2012), but for the HE gene, the rate estimate was also lower than that obtained for a different ISAV dataset (Castro-Nallar et al., 2011). This implies that the tempo and mode of ISAV evolution may need further investigation. Although transitional viruses having only the relevant virulence mutations in either the HE or the F gene have not been identified, reassortment may also be important in generating virulent combinations of these segments. Reassortment appears to occur relatively frequently for ISA (Plarre et al., 2012), but more formal assessments (e.g., comparisons with influenza) are required to determine how pervasive the reassortment is. Finally, the fitness aspects of the virulence mutations in the HE and F gene segments remain to be determined, such as the synergistic or compensatory nature of these mutations, or the preferred evolutionary trajectory towards virulence.
 
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