Due the ISA virus positive test results myself and others have gotten from international laboratories, the Cohen Inquiry into the Fraser sockeye decline is re-opening December 15, 16 and 19. We don't yet know if ISA virus has been accidentally imported to BC, what strains exactly we have, or if the positive results mean ISA virus has always been here. What we do know is this virus has caused a lot of salmon deaths worldwide, that it cannot be cured, but the damage it does can be minimized. This report contains some of what we know about the virus, what government has said about the virus under oath previously at the inquiry and the lab reports. I am not allowed to comment at the moment about the DFO paper reported widely in the media. I have written this posting because the government of Canada has not included crucial information regarding fresh, high-quality samples from the Fraser River that have tested positive in recent weeks.
I am grateful to Justice Cohen and his staff for re-opening the inquiry, even as all of us would rather be preparing for family gatherings and Christmas. They have called excellent witnesses that include the scientists who have done the ISAv positive tests and a DFO co-author on the controversial paper. If you plan to attend please note the new address:
Asia Pacific Hall at the Morris J Wosk Centre for Dialogue
580 West Hastings Street
(enter via Seymour Street courtyard entrance)
They have also re-opened the public comments on their website: www.cohencommission.ca
Infectious Salmon Anemia virus (ISAv) is an internationally reportable pathogen (OIE). ISAv is an infectious, influenza C virus (NMFS, 2002) known to spread horizontally fish-to-fish, but also through movement of contaminated equipment and vertically via eggs (Vike et al. 2009). Outbreaks can initially cause low mortalities, but can rise to exceed 90% if left unchecked. ISAv devastated the Faroe Islands salmon farming industry in 2000, cost Scotland $32 million in an unsuccessful attempt to eradicate it, caused $5 million in losses in New Brunswick (OIE, 2010). The U.S. spent $8 million in 2002/3 trying to eradicate it from Maine when it spread southward from New Brunswick (NMFS, 2002). In Chile, ISAv caused $2 billion in losses from 2007-2009 (Asche et al. 2010). Every country that gets ISAv does not want it, but cannot get rid of it despite expensive efforts.
ISAv was unknown before 1984, when it became a virulent pathogen in Norwegian salmon farms. There are two major genotypes; European (genotype I) and North American (genotype II), with many different strains exhibiting varying levels of virulence (Mjaaland et al. 2005). ISAv is highly prevalent in Norwegian salmon farms. A 2007 study, found 22 / 24 farmed Atlantic salmon smolt populations screened positive for ISAv (Nylund et al. 2007). BC received 8 million Atlantic salmon eggs from Iceland and Iceland received eggs from Norway (Powerpoint by Dr. Kibenge). slide 7. BC also received over 15,000,000 eggs from Washington State where the source remains unclear, DFO website.
The European genotype appears to have originated in the rivers of Norway as a benign virus. European strain ISAv has been found in Nova Scotia (Nylund et al. 2007). In 1999, North American ISAv isolates were found in Chilean coho salmon farms and the strong match suggested the virus had been introduced from the North Atlantic shortly before detection (Nylund et al. 2007). In 2007, the European strain ISAv appeared in Atlantic salmon farms in Chile and became highly virulent sweeping through the industry (Vike et al. 2009).
The ISAv HPR0 avirulent type is asymptomatic and cannot be cultured (McBeath et al. 2009) and so will never be “confirmed” according to Canadian regulation. As well, cell culture is less sensitive than PCR and unable to detect low levels of the virus (Devold et al. 2000). While the HPR0 strain appears avirulent to Atlantics, its impact on sockeye salmon and Pacific non-salmonids is unknown. HPR0 is considered the original wild form (McBeath et al 2009, Plarre et al. 2005). If the virulent forms of ISAv are arising from ISAv HPR0 it is a dangerous pathogen to maintain in a culture environment. Dangerously, it is allowed it to exist unreported because it does not meet detection requirements implemented by Canada. Plarre et al. (2005) found HPR0 in 60% of farm salmon in Norway.
There is no cure or effective vaccine or ISAv. It is managed through strict containment of bloodwater , which is not done in BC (Figure 1), single age-class sites and maintaining distance between farms. One strain is linked to a jaundice illness among coho salmon farms in Chile (Godoy et al. 2008) and a highly virulent strain has caused disease in rainbow trout (OIE, 2010).
ISA is an RNA virus that exhibits “error-prone” replication. This creates potential for rapid mutation. While it has not been possible to calculate the speed of its “molecular clock,” we know the culture environment increases virulence. ISAv’s high-mutation rate makes it possible calculate how long it has been in a new location and because each original strain is distinctive ISAv can be traced to source.
When first detected by PCR in Chile, it was not obvious that it was ISAv because the symptoms were atypical. It was called Icterus Syndrome and “virus isolation was not always possible” (Godoy et al. 2008). In 2007, persistent, low-level mortality prompted a request to Dr. Fred Kibenge at the Atlantic Veterinary College in PEI to rule out ISAv. Dr. Kibenge’s lab is one of two World of Animal Health Organisation (OIE) reference labs for ISA virus OIE LinkThe second lab is in Norway. He diagnosed European strain ISA virus in the Chilean farm salmon. There was resistance to his diagnosis, a year-long delay in responding (Asche et al. 2010) and the ISAv spread with unforeseen speed, killing 70% of Chilean farm salmon. Some of the deaths were due culling. The “closest relative” to the Chilean ISAv was an isolate from a marine salmon farm in central Norway (Vike et al. 2009).
A report by the aquaculture industry publication INTRAFISH (Carvajal, 2002) on the Chilean epidemic describes the failure to respond to the earliest indications of ISAv as an aggravating factor in the scale of the epizootic. There are comments that the virus was behaving differently in Chile than Norway, that it was under control and would not become a problem. How it arrived in Chile is debated. Some blame Marine Harvest, others felt the 1999 ISAv strain had mutated, but eventually it was accepted that European genotype ISAv had indeed been recently introduced via eggs (Godoy et al. 2008, Vike et al. 2009).
Marine Harvest’s farms were hit first and their Chilean CEO suggests sufficient measures were not used to control the virus and that perhaps any farm with even a single positive test should have been destroyed at the outset. An industry representative is quoted saying there was a delay in adopting effective measures (Carvajal, 2002).
ISAv unreported in British Columbia
Canada does not include ISA virus reporting on the egg import forms used to import Atlantic salmon eggs into BC (Cohen Inquiry, exhibit#1683) and ISAv was not made a reportable disease in BC salmon farms until January 2011.
There had been no reports of ISAv made to the CFIA as of April 2011 (CFIA per com). During the Cohen Commission hearings several witnesses testified ISAv has not been detected in BC.
DR. MacWILLIAMS: There has been no indication of ISA or ISAV on this coast in B.C (Transcript 2011-08-22 pg 9, line 23)(DFO Fish Health)
DR. KENT: Yeah, I see that. Thank you. Thanks for clarifying that ISA has not been seen in B.C. (Transcript 2011-08-23, pg 46, line 24) (ex-director of DFo’s Pacific Biological Station)
DR. MARTY: And so that gives me a great deal of confidence that we don't have ISAV in British Columbia. (Transcript 2011-08-43, Pg 56, line 32)
CLARE BACKMAN: The level of surveys done in the country of origin and then again, the quarantine and follow-up sampling here in British Columbia has been successful in preventing any exotic disease, including this particular one, ISAV. (Transcript 2011-09-07, Pg 44, line 20)
PETER MCKENZIE: we have over 5,000 tests for ISAV, all are negative, and that gives us an extremely high level of confidence that our industry is free from ISAV. (Transcript 2011-08-31)
There were documents released by the Cohen Commission reporting ISAv has not been found in BC.
2007 August confidential Briefing to Minister Agriculture and Lands by BC vet Mark Sheppard…”ISA virus is considered a Reportable Disease by the OIE…The presence of ISA would profoundly impact a company’s business due to initial slaughter orders of entire populations and subsequent bio-containment activity…The Ministry remains confident that farmed fish in BC are free of ISA disease….BCMAL is well aware of ISA.” Mark Sheppard, Exhibit # 1680
Press Release by Mainstream Canada: “Thousands of tests have shown that there is no ISA virus in B.C. salmon, farmed or wild… “As the chief veterinarian of B.C. said today, it defies logic and common sense to assume that a few samples taken at random from the coast would indicate the presence of ISA when thousands of controlled tests of good-quality samples indicate no such thing,” said Dr. Peter McKenzie, Mainstream Canada’s fish health manager and professional veterinarian.
Since 2003, farmed salmon in B.C. have been tested by regulators for the ISA virus. Nearly 5,000 farmed salmon have been tested, and all were negative for the virus. Wild fish are also tested for the virus. In 2011 alone, nearly 1,200 samples of farmed and wild fish have been tested for the virus and have all come back negative. As well, in reaction to recent speculation that ISA might be in B.C. these 1,200 samples were retested by the B.C. Animal Health Centre and again showed no sign of the virus.
However, for weeks, while the CFIA conducted its investigation, the world was whipped into a frenzy through the malicious activities of a small group of anti-salmon farming activists, the same group who submitted the samples for testing. These activists have made it clear their mission is to shut down the B.C. salmon farming industry, at any cost. The activists’ approach was emotional, fear-driven and ignored any good science which disagreed with their pre-conceived conclusions.”. Mainstream Canada press release 2011-11-08
Definition of ISA virus confirmation
The OIE considers a case “suspect” for ISA virus if it meets any of the following criteria (OIE, 2010):
1. Clinical signs or lesions consistent with the disease
2. Isolation in cell culture
3. Two independent tests suggest (RT-PCR and IFAT)
4. Antibodies to the virus found
ISA virus is confirmed if:
1. The illness is consistent with this disease
2. Viral antigens have been detected in tissues
3. The virus has been either cultured, or nucleic acids have been detected by RT-PCR from at least one fish
Four diagnostic tests are used to confirm ISAV: histology, reverse transcriptase-polymerase chain reaction (RT-PCR), cell culture and indirect fluorescent antibody testing (IFAT). But these tests are imperfect as HPR0 cannot cultured, ( McBeath et al 2009) and ISAV 7 does not produce high levels of antibodies (Mjaaland et al. 2005).
BC has passed two of these diagnostic milestones, but BC has not been identified as a “suspect” region by the OIE (Go to top of web page click "Aquatic," select "infectious salmon anemia"). I do not understand why the OIE has thus far not responded with a listing as a result of its own reference lab's positive RT-PCR results with a "suspect" listing for British Columbia. There have been 8 positive RT-PCRs in 5 wild salmon. The classic ISAv lesions diagnosed in 2,040 times in Atlantic farm salmon, 84 times in farm Pacific salmon and 4 times in Sablefish (100% examined) source - BC Ministry of Agriculture and Lands (BCMAL) 2006 through April 2010, at which time the salmon farmers ended the BCMAL audits (Cohen Commission Exhibit #1549).
“Weak positive” PCR results for ISA virus do occur and confound diagnosis (Ritchie and Gagne 2006). This is caused by the strains that also cannot be cultured. The avirulent HPR0 strain may be the original wild strain (Nylund et al. 2004) and appears to travel in the eggs (Nylund et al. 2004, Vike et al. 2009), and cannot be cultured, but its presence is considered a concern in Scotland because of its capacity for mutation towards virulence (McBeath et al 2009).
Clinical signs of ISA virus are: pale gills, slight swelling of the liver, which may turn very dark, there may be swelling of the spleen, generally extensive petechial hemorrhages on the pyloric caecae, mesenteric fat and swimbladder (Cipriano 2002), hemorrhages in the eyes (Winton, J.R) and “proliferative gill inflammation (McBeath et al 2009).”
Sampling of BC wild salmon
In late May 2011, approximately 300 sockeye smolts were collected in Rivers Inlet by Dr. Rick Routledge’s lab (SFU). Rivers Inlet sockeye returns fell to below 1% of historic abundance in the late 1990’s and have failed to rebuild consistently since then. The spring 2011 out-migration appeared to be exceptionally poor. The smolts were collected and frozen for stomach content analysis, which was not optimal for virus preservation. In October, the hearts of forty-eight of the smolts of the smaller smolts were sent to Dr. Kibenge’s lab on ice to rule out ISAV. 2/48 tested positive for ISAV, European strain. Gills from the same 48 smolts samples were forwarded to Dr. Are Nylund, U. of Bergen. Nylund tested only gills and Kibenge tested only hearts.
On October 12, 2011, nine salmon carcasses were sampled in the Harrison Mills area (tributary to the Fraser River). There were 2 chinook, 1 chum and 6 coho. Fresh heart and gill tissue were shipped on ice to Dr. Kibenge at the Atlantic Vet College and placed in the preservative RNALater and shipped cool to Dr. Are Nylund at the U. of Bergen.
October 20, 2011, five juvenile herring were caught from the dock in Sointula Harbour where the fish have been seen whirling and sinking on their sides. Their hearts were preserved in RNALater and some went to Nylund and some went to Kibenge.
In October, hearts and gills were extracted from five sockeye smolts captured in May in the Discovery Islands preserved in RNALater and sent to Nylund and Kibenge.
November 4, 2011, ten sockeye carcasses were sampled from the Harrison Mills area. Fresh heart and gills were sent to Dr. Kibenge and frozen at -80C, for about 10 days before testing and RNALater preserved heart and gill tissue were sent to Dr. Nylund.
Further sampling has been done in many rivers of Vancouver Island, no results yet.
The ISA virus tests results
There have been eight positive tests for ISAv in coho, sockeye, chinook and chum. (Table 1)
Table 1: PSM = prespawn mortality. While the fish were found at Harrison Mills they were drifting downriver so it remains uncertain what stock they were.
Table 2: RI = Rivers Inlet, HM = Harrison Mills, DI = Discovery Island, herring were from Sointula area. PSM = Prespawn mortality. While Dr. Kibenge tests segment 8 and 6, Dr. Nylund tests segment 8 and 7
The PCR cycle threshold (ct) is the number of cycles required for detection. Cts ≤ 29 indicate abundant target nucleic acid - a strong positive. Cts of 30 – 37 indicate moderate amounts of target nucleic acid - a positive. Cts of 38-40 indicate minimal amounts of target nucleic acid and represent an “infection state or contamination.”
Diagnostic Aids Select RealTime PCRs
Description of the ISAv positive fish
The sockeye smolts sent for testing were selected on the basis of poor condition (Nicole Gerbrandt pers. com.).
Coho (#3) was 61.5 cm, female and appeared in good condition, was a prespawn mort.
Chinook (#5) was 92 cm, female, prespawn mort, yellow tint to exterior, redness at base of dorsal and pelvic fins, cartilage of the head was yellow, yellow liver (Figure 2).
Chum (#6) was 56.5 cm, female, prespawn silver-bright female, growth on gills, liver pale, spleen very dark with crisp edges.
DI sockeye smolts (#7) were 9 – 13 cm long many with bleeding in eyes and base of fins
Herring were generally bleeding at the base of their fins
Sockeye (#9) 61 cm, 2.4 kg female, prespawn mort, gills – pale with fungus-like appearance, kidney dark, mottled with grey, liver very yellow with red specks, spleen dark with sharp edges, heart very pale, thin belly wall
Sockeye (#14) 65 cm, 2.55kg, prespawn mort, gill pink, kidney dark red (normal), liver brown, spleen very dark, heart pink (normal)
The CFIA took possession of the samples in Dr. Kibenge’s lab and also the remainder of the sockeye smolts from Dr. Routledge. On November 8, the CFIA, DFO and BCMAL held a phone-in press conference. They reported the DFO Moncton lab was unable to reproduce any positive of the ISAV results. The representative for the CFIA said the Nylund result was “inconclusive” and a “negative” because it was “not repeatable” so “technically from the CFIA point of view the Nylund results are negative”.
The CFIA went on to say there is “no evidence that ISAV occurs in fish of British Columbia.” In an interview Dr. Nylund states "Our results are not conclusive, but do suggest ... that an ISA virus is present in wild populations of O. nerka (Pacific sockeye) Seattle Times)
The CFIA has made no specific mention of any other samples that tested positive, in particular the coho heart - positive for European strain ISAV. The spokespeople pointed out that a positive PCR is not definitive, that the virus must be sequenced and cultured. BCMAL Minister, Don McRae said:
“Reckless allegations based on incomplete science can be devastating to these communities and unfair to the families that make a living from the sea. Since Premier Clark is currently on a trade mission to China, I have personally asked her to reassure our valued trading partners that now as always BC can be relied upon as a supplier of safe, sustainable seafood.”
When asked for information, the provincial fish farm vet, Dr. Gary Marty from the Animal Health Centre in Abbotsford BC, did not reveal what segment of the ISAV RNA he targeted in his PCR tests on farm salmon with ISAV histology. PCR tests are highly specific different PCRs will give different results.
There has been no comment from the DFO Pacific Region regarding the ISAV positives, which is unprecedented in regards to aquaculture issues. There has been no mention of a testing wild, farmed or hatchery salmon for ISAV.
The SFU press conference on the ISA virus positive results has been criticized as premature, but was held out of a sense of obligation to advise the scientific community. The samples we had at that time were degraded, but freezer-burn is not known to trigger an ISAV positive result. We felt it was important that the scientific community be made aware so that the proper response could begin in hopes of isolating the source and beating the ISAV tendency towards increased virulence in the captive environment. As a participant of the Cohen Inquiry I read hundreds of DFO emails there was no mention of ISAv positive results.
My response has been to test immediately as widely as possible and split the samples between the Kibenge and Nylund labs as each use slightly different results. I inform both the labs and the CFIA of the results. I post the lab reports on my blog: https://alexandramorton.typepad.com and I have attached them as appendices to this report. I do not think fish disease should be a federal secret and people who better understand this virus need all the information possible.
We have 8 positive PCR test results by two different labs, in two generations and five species of wild salmon caught 600km apart. Infection in the gill only might suggest very recent infection, or detection of the HPR0 strain. Detection in the heart suggests a more systemic infection. The adult sockeye, coho and chum looked healthy externally. The Chinook was approximately 25 pounds, white-fleshed, with jaundice-yellow skin and liver. Prespawn mortality in the Harrison Mills region is high and the second ISAv positive location (River’s Inlet) sockeye are reduced to approximately 1% of historic abundance.
PCRs target small, highly specific portions of the ISAv RNA and so until the BC strain/s are sequenced there remains a possibility these PCR positives are an ancient North Pacific strain similar enough to the European strain to trigger a PCR positive. It is possible that the positive results are reading some other virus, or that BC has a new mutation of ISAv. However, there is no reason to remain in doubt, all that is required is a high quality sample of the suspect pathogen delivered to a lab with ISAv culturing and sequencing experience.
The situation in BC is highly reminiscent of Chile from 1999-2007. ISAv has been detected, but it is being ignored because it is not yet killing millions of farm salmon. This is an unconscionable situation because we know ISAv will become virulent in salmon farms, this has been the experience globally. An epidemic of a virulent ISAv strain among wild Pacific salmon is an unprecedented risk at a catastrophic level. ISAv cannot be allowed to replicate in the environment where it is known to mutate into higher virulence.
We have over 2,000 histological reports of “classic” ISAv-type lesions in farm salmon. While the salmon farming industry and the government agencies tasked to promote aquaculture report ISAv is not causing these lesions, it biologically preposterous that European strain ISAv would only exist in Pacific salmon and not in the millions of European farm salmon, with symptoms of the disease, being held in net pens on the migration routes of the infected Pacific salmon.
Hatcheries and salmon farms are dangerous habitat to allow ISAv to reside and replicate in.
Rolland and Winton (2003) challenged steelhead, chum, Chinook and coho salmon with ISAv and report these Pacific species were resistant to the strains used, but they state “the potential for ISAv to adapt to Oncorhynchus spp. should not be ignored.” When it became apparent a coordinated effort was required to control the Chilean epidemic it was “discovered that the government had no tools to coordinate the effort” (Asche et al. 2010). This appears to be the case in BC as well. When the BC salmon farming industry refused to allow government any further fish health audits in April 2010, there was no legal recourse for government to demand access to farm salmon (Cohen Commission Exhibit #1637). Therefore, even as evidence mounts that the European strain of the most lethal salmon disease known is in BC, there may not be any mechanism to test Atlantic salmon. The legal decision Morton vs the Province of BC raised the question, who truly owns farm salmon in marine net pens. The decision found it was the same ocean inside and outside the pens. Under the constitution of Canada no one may privatize ocean spaces, nor own a fish in the ocean.
Enough evidence exists to begin screening of wild and hatchery for ISAv and to isolate Atlantic salmon from Pacific salmon. Oweekeno Lake should be checked because the sockeye smolts that tested positive for ISAv had departed the lake only about 10 days prior. Enhancement hatcheries are an excellent source of high-quality samples and it will be important that they do not amplify the virus.
Heart tissue retains ISA virus the longest, kidney contains the most virus during active infection and the gills capture the HPR0 strain. RNALater is considered the best preservative, but 96% ethanol and storage at -80C are also used. Fresh samples on ice are the best possible samples for culture.
There is history of political and industry resistance to positive ISAv diagnosis in new regions. The BCMAL minister’s comments that he reported BC’s ISAv-free status to China seems premature and an indication that this is a serious trade barrier issue. But we have to ask – why wouldn’t we want to know if ISAv is here?
This is a highly successful virus that benefits from artificial salmon populations. If we use everything we know about ISAv we won’t eradicate it, but we might to stop it from killing fish.
Dr. Fred Kibenge's Lab report on River's Inlet Sockeye: Download Ref Lab form for Laboaratory results submitting data to OIE - BC Sockeye salmon smolts - complete.pdf (319.5K)
Dr. Fred Kibenge's Lab report on Oct. 12, 2011 Coho and chum salmon: Download Alexandra Morton Samples (SOCKEYE CHINOOK and COHO)_VT10142001_OCTOBER20 2011.pdf (45.9K)
Dr. Are Nylund's report on Rivers Inlet sockeye: Download Report 021111.pdf (22.0K)
Dr. Are Nylund's report on Nov. 4, 2011 sockeye: Download Report231111.pdf (15.9K)
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Carvajal, 2002, ISA and the reshaping of Chile’s salmon industry INTRAFISH
Cipriano 2002 Infectious Salmon Anemia Virus. US Geological Survey, Fish Disease Leaflet #85
Cohen Commission Exhibit # 1637, 1688 https://www.cohencommission.ca
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OIE 2010 Infectious Salmon Anemia www.cfsph.iastate.edu/Factsheets/pdfs/infectious_salmon_anemia.pdf
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Winton, J.R. Fish Health Management, Chapter
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