scott class presentation cohen inquiry feb 6,...

06/02/2015 1

Outline

1) DFO’s preparation for Cohen

2) Where my research fit in to the Inquiry

3) Defining the controversies

4) Disease Hearings

5) Aquaculture Hearings

6) ISAv Hearings

7) Cohen Recommendations

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Pacific Salmon Commission Bilateral Scientific Workshop to Examine the Decline in Fraser River Sockeye (June 2010)

1. Explanatory powerProductivity correlations with 17 declining stocksProductivity correlations with 2 healthy stocks

2. Direct and/or indirect evidence3. Specific Research Required4. Management Actions if validated

16 Hypotheses explored under the following themes:Predators, parasites and disease Physical oceanographic conditionsToxic algae and pollutantsFreshwater conditionsOther Factors

Report: “Synthesis of Evidence from a Workshop on the Decline of Fraser River Sockeye” (by Randall Peterman)

HypothesisTime

PeriodStrenth of evidence

Very likely Likely Possible Unlikely

Very Unlikely

2009 FairOverall Fair

2009 GoodOverall Good


2009 FairOverall Good



2009 PoorOverall Poor



2009 GoodOverall Good

2009 PoorOverall Poor


9. Competitive interactions with pink salmon are important contributors to the Fraser sockeye situation

4. Harmful algal blooms in the Strait of Georgia and/or northern Puget Sound/Strait of Juan de Fuca are an important contributor to the

5. Contaminants in the Fraser River and/or Strait of Georgia are an important contributor to the Fraser sockeye situation

6. Freshwater habitat conditions in the Fraser River watershed are an important contributor to the Fraser sockeye situation

7. Delayed density dependent mortality is an important contributor to the Fraser sockeye situation

8a. En-route mortality during upstream migration is an important contributor to the Fraser sockeye situation

8b. The effects of en-route mortality on fitness of the next generation is an important contributor to the Fraser sockeye situation

Relative likelihood that each hypothesis caused observed changes in productivity during the indicated time period

1a. Predation by marine mammals is an important contributor to the Fraser sockeye situation

1b. Unreported catch in the ocean outside of the Pacific Salmon Treaty area is an important contributor to the Fraser sockeye situation

2. Marine and freshwater pathogens (bacteria, parasites, and/or viruses), are important contributors to the Fraser sockeye situation

3a. Ocean conditions (physical and biological) inside Georgia Strait are important indicators of contributors to the Fraser sockeye situation

3b. Ocean conditions (physical and biological) outside Georgia Strait are important indicators of contributors to the Fraser sockeye situation

4

Hypothesis:

A novel viral pathogen is infecting and weakening sockeye salmon

throughout their life‐cycle and directly or indirectly enhancing mortality,

especially when salmon are faced with additional stressors

06/02/2015 5

Functional Genomics: Assess Salmon Condition and Predict Fitness

Genome‐wide scan of physiology

Identify what may be occurring within the fish, such as: Food deprived/starvingConsuming poor quality prey (low lipid content)Under stress—temperature, oxidative, hypoxia, disease, toxins, osmoticGrowing fast or growing slow Active or inactiveReady saltwater/freshwater (as smolts leave or adults return)) Mature or immature

Identify genes/pathways or conditional states associated with: Migration timing and speedPoor survival

06/02/2015 6

Salmon migrate to FW to Spawn

What can genomics tell us about factors that may be limiting the success of adult

spawning migrations?

En‐route and pre‐spawning mortality of adult sockeye salmon returning to spawn in the Fraser River has increased from 15% historically to

30‐90% over the past 15 years

06/02/2015 7

Gill biopsies sampled non‐destructively

Merging telemetry, non‐destructive biopsy sampling, and genomics to establish direct linkages between salmon condition and fate

Individual genetic stock ID tells us where they are going (adults) or where they came from (smolts)

Radio tagging allows us to track the migratory routes, timing, and

end points of individual fish

06/02/2015 806/02/2015 8

2006:3 separate

Tagging studies

SW—JS/JDFSFW—Lower River

FW—Spawning Grounds

Genomic signatures predicts migration and spawning failure of wild Canadian Salmon

Fraser River

Salmon Tagged

Non-destructive GillBiopsy

Published in ScienceJan. 14, 2011331: 214-217

Telemetry Receiver

06/02/2015 9

The same genomic signature was associated with elevated pre‐mature mortality in each tagging study

SW Tagging Lower River Tagging Spawning Tagging

13.5x lower probabilitysuccessful migration

5x lower probabilitysuccessful migration

3.7x lower probabilitysuccessful spawning

individuals

Profile associate with high mortality termed “Mortality‐related Signature” (MRS)

MRS MRS MRS

06/02/2015 10

•Strong stimulation of immunity of fish with the MRS•65% of the physiological pathways affected were known to be active during viral infections•Also noted a “leukemia‐like” signature•Dysfunctional gas exchange was also noted, but other stressors were inconsistent

Hypothesis: Many salmon dying prematurely in the river were fighting a viralinfection that was present before they entered the Fraser River

What can the genes and pathways stimulated in the MRS tell us about the potential cause?

Most Important Message of the Study

Adult migrants were physiologically compromised well before entering the river

River conditions alone may not fully explain the high, but variable mortalities experienced

Previous studies were suggestive of the roles of stress and early freshwater adaptation to poor in-river survival, but this was the first to suggest a potential mechanism

We did NOT suggest this is the only factor undermining performance of salmon in the river

06/02/2015 12Miller et al. in prep

Mortality‐related Signature is present in multiple tissues, species, and life history stages of salmon

Subsequent Studies revealed…

The potential viral origin of this signature is strengthened when we look at genes that overlapamong tissues

06/02/2015 13

What can genomics tell us about factors that may be limiting the success of smolts migrating out to sea?

Early marine survival is highly variable between years, but is generally declining for many stocks of Coho, Chinook and Sockeye Salmon

06/02/2015 14Miller et al. in prep

Mortality‐Related Signature (MRS) in smolts

50% reduction in prevalence Summer‐Fall

Smolts show signs of MRS well before they leave the river

0

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FW Parr FW Natal SW Summer SW Fall

Prevalence Unhealthy signature LiverSmolt Liver MRS

Are these reductions in prevalence of MRS due to mortality or recovery?

Genomic analysis reveals a Poor Conditional State of 2007 Smolts

Left the River highly affected by the MRS in multiple tissuesOnly weakly affected 6 weeks later in the ocean

In the ocean, signs of

Enhanced Immunosuppression

Enhanced Stress

Hypoxia (response to low oxygen availability) Tests for harmful algal bloom species heterosigma on the gills positive

Slower Growth

No indication of prolonged starvation, although reduced feeding possible

Miller et al. in prep

06/02/2015 16

After screening MRS‐positive and negative tissues with multiple known intracellular pathogens, we attempted to

concentrate viral particles in MRS‐positive tissue and applied Next Generation Sequencing to see if there might be a novel

viral agent involved

Miller et al. in prep

Looking for a pathogen associated with MRS

06/02/2015 17

Novel Salmon Parvovirus Identified

Bocavirus Genus

Dependovirus Genus

Novel Salmon Virus

What do we know about Parvoviruses?

Not previously described in fish, but present in mammals, lower vertebrates (birds and reptiles), shrimp and insects

Most can cause both acute and chronic disease

Highly virulent pandemics in dogs ongoing since the 1970’s

Replication often stimulated under conditions of stressPotentially important role of environmental conditions on viral virulence?

In mammals, feeding, growth, and coordination can be impacted

Generally most profound effects on the young

06/02/2015 19

30% of adult gills were parvovirus positive before entering the Fraser River

Num

ber o

f fish

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P arvovirus P os it ive P arvovirus Negative

Unsuccess fulS uccess ful

9.5 times lower probability of arriving to spawning grounds if positive Parvovirus

F‐Stat P=0.008

Parvovirus correlated with premature mortality of Early Shuswap stock in 2010

Is this novel Salmon Parvovirus potentially impactingSalmon Declines?

Need to establish role in early marine survivalCombining disease ecology with controlled laboratory studies

Preliminary findings:

1. Parvovirus present in multiple tissues of sockeye salmon

2. Present in smolts and adults, fresh and saltwater, with maximum prevalence in smolts leaving natal rearing areas

3. Molecular analysis shows the highest viral loads during early marine residence—consistent with onset of disease/enhanced virulence

4. Prevalence in multiple tissues declines in early ocean environment

5. Disease challenge experiment established infectivity of the virus, but more challenges required to establish whether it causes disease and its potential relationship with the MRS

How was this data received by DFO? Controversy Begins...

Immediate negative reaction from fish health scientists and veterinarians

Before we discovered the parvovirus, the “New Genomic Technology” and conclusions that can be drawn from it were hotly debated

After we discovered the virus, arguments evolved to “if we did not know about it, it is not likely to be pathogenic”

Reasons for the divide ultimately became clear:

Fish health mandate is to prove that disease is not a factor—to demonstrate “freedom from infection”, ours was to ask whether disease or other stressors may be a factor

You never start with a virus, you have to start with a disease...but when it comes to wild salmon, few actually study disease because you can’t observefish dying, especially in the ocean...

Controversy #1: Muzzling of Government Scientist

Published in ScienceJan. 14, 2011331: 214-217

When Science paper came out, DFO immediately banned my speakingto the media about this paper

Eventually, this decision became the story in the news...March 21, 2011: Cohen enquiry scientist silent but her ‘salmon leukemia’ theory still makes wavesMarch 22, 2011: Never heard of salmon leukemia? Chances are that you won’t...for a whileMarch 27, 2011: DFO silencing ground breaking researchMarch 27, 2011: DFO’s stifling of research a case of deja vuApril 27, 2011: Science writers call for an end to muzzling of scientists by Canadian governmentJune 27, 2011: Feds silence scientist over West Coast salmon studyJune 29, 2011: Scientist muzzled over missing salmon studyJune 29, 2011: Feds miss mark in scientist muzzling saga July 29, 2011June 30, 2011: Hook, line and sink her findings (CBC radio)July 28, 2011: Muzzling scientists wrongJuly 29, 2011: Gagging scientist hurts the publicJuly 29, 2011: Federal scientist unfairly silenced union saysJuly 29, 2011: Suppressing science a rights violationJuly 29, 2011: Swimming upstream against government secrecyAugust 2, 2011: Federal scientist silenced by gag orderAugust 25, 2011: Privy council office blocked scientists’ access to mediaAND ON....

06/02/2015 24

Province Newspaper July 27, 2011 Times Colonist Newspaper July 27, 2011

Muzzling of Government ScientistsMinister Ashfield’s Statement in response to “Muzzling” on July 29, 2011, after the Green party began asking questions

“We don’t want to influence the ongoing judicial proceedings”

“Our government has been very clear that judicial inquiries are not conducted through the media,”

“The research and report by Dr. Kristi Miller on Pacific salmon was not withheld from anyone... it was published in a broadly circulated science magazine “

Controversy #2: Any indication of disease in wild fish used by ENGO’s as “proof” of the negative impacts of

salmon farms

Alexandra Morton

Disease Hearings Begin: Aug 22‐25

Disease Hearings Begin: Aug 22‐25Days 1‐2Veterinarians and fish health experts spent 2 days trying hard to dispel any notion that disease could be a factor in wild salmon declines

It quickly became obvious that they had very limited knowledge about even what pathogens may be affecting wild salmon, especially in the early marine environment

One fish health scientist went so far as to negate his own research for 10 years on salmon leukemia, casting dispersions on his long held hypothesis that this disease was caused by a virus, or that it was even a disease

This same scientist wrote the commissioned review on disease, and neglected to mention aquaculture in his report– a topic of considerable discussion at Cohen

In his ranking of risk posed by various pathogens, he ranked low any pathogens or parasites that had never been studied in sockeye salmon—the vast majority of them!

Disease Hearings Begin: Aug 22‐25Controversy #3: The Body Guards“Dr. Kristi Miller, who is present at the Cohen Commission today protected by a huge security service bodyguard”“Why was Nanaimo‐based scientist Dr. Kristi Miller shadowed everywhere by two burly bodyguards with earpieces and escorted from the courtroom like a...”

“With a security guard sitting to her right, Ms. Miller watched as two of her department colleagues testified on Tuesday. Word of the researcher’s arrival quickly spread through the crowd.Approached for an interview after the hearing, Ms. Miller said she couldn’t talk with reporters prior to taking the stand. A Fisheries and Oceans spokeswoman quickly arrived to reiterate that point.”

Disease Hearings ContinueDays 2‐3I testified with a virologist from DFO

Key Controversial Questions:Was I in fact muzzled?Were farmed salmon tested for parvovirus?Was parvovirus the “smoking gun”?

First time the Parvovirus findings came to light

Cohen Commission of InquiryParticipants

Government of CanadaProvince of British Columbia (Gary Marty, Provincial Veterinarian)Pacific Salmon CommissionBC Public service alliance of CanadaRio Tinto Alcan IncBC salmon farmers associationSeafood producers associationAquaculture coalition (ENGOs) (Alexandra Morton)Area D salmon Gillnet Association, Area B harvest committeeWest coast trollers/United FishermenBC Wildlife FederationTsawwassen and Musqueam First NationsWest central coast salish first nationsFirst nations coalitionMetis Nation BCSto:lo Tribal councilLaich‐kwil‐tach treaty socieityMusgamagw Tsawataineuk Tribal CouncilHeiltsuk tribal council

Cohen Commission of InquiryParticipants

Government of CanadaJust the facts please...

Province of British Columbia (Gary Marty, Provincial Veterinarian)Exceedingly aggressive and confrontational

BC salmon farmers associationMaking it clear that our data were not suggestive of salmon farm impacts

Aquaculture coalition (ENGOs) (Alexandra Morton)Salmon leukemia, parvovirus testing in farmed salmon, “Smoking gun”

Sto:lo Tribal council“Smoking gun” backfire, why weren’t first nations informed of a new virus?

Area D salmon Gillnet Association, Area B harvest committeeMy links with aquaculture and what that suggests about my credibility

BC Wildlife FederationSupportive

06/02/2015 33

Headlines

Cohen ‘Miller‐Time’

Was Kristi Miller’s virus research obstructed by DFO colleagues?

Salmon scientist says she may have the ‘smoking gun’ that killed Fraser River sockeye

DFO ignored request to explain why test rejected

Groups rally to support controversial scientist

Scientist says fish farms may not affect wild salmon

Scientist says she was not silenced by DFO over salmon evidence

Scientist tells inquiry her work was muzzled by feds

Aquaculture Hearings Begin: Aug 25‐27What diseases affecting farmed salmon and how are farms managed and regulated?

Histopathology records from Industry Regulatory Audit program key evidenceVeterinary histopathologist Gary Marty admits that he can only diagnose the infectious agent causing mortality in 40% of cases—60% “open farm” casesEarlier, a DFO vet suggested that for all pathologies relating to sockeye salmon, the cause is known and there was “no room for novel, undescribed pathogens”

In the commissioned report on salmon farms and sockeye, they treated farms with an open diagnosis as if they were healthy farms

Alexandra Morten testified about lesions Marty reports repeatedly being highly consistent with ISA virus—a virus causing devastating mortality in Europe. These are among the “open farm” cases.

This virus, and evidence of its potential existence in BC salmon, would become the subject of an extra evidentiary hearing called in December 2011

Aquaculture HearingsControversy #4: Can DFO effectively regulate an industry thatthey are simultaneously promoting?

In the final report, Judge Cohen took a strong stand on this issue, calling for DFOto get out of the business of aquaculture promotion

There was a great divide between the “Participants”—the groups represented by lawyers—onIssues pertaining to disease and aquaculture

Lawyers for the Province, the Government of Canada, and the aquaculture industry fiercely questioned witnesses with any views on negative impacts of industry and of disease, while Lawyers for First Nations, ENGO’s, and to some degree wild fisheries took the opposite tact

ISAv HearingsControversy #5: Do BC salmon carry ISA virus?All started with Alexandra Morton and Dr. Rick Rutledge breaking the news that Dr. Fred Kibenge tested wild BC salmon (for them) and results came up positive.

As ISAv is a reportable disease, meaning that its presence can affect international trade, if truethis was a huge finding

Canadian Food Inspection Agency responsible for managing all reportable pathogens

Testing for ISAv in my lab

The hearings were among the most controversial and biggest media events of the inquiry

Scientists from four laboratories testified on their molecular testing results for ISAvThree of the four labs, including mine, had weak positives for ISAvThe only lab that did not show any positives was the CFIA Validation lab

06/02/2015 37

ISAv Testing Results from my LabOur lab recently obtained sequences from multiple segments of an Infectious Salmon Anemia (ISA)‐like Orthomyxovirus in BC salmon

ISA viruses have caused devastating mortality in the aquaculture industry in Norway and Chile, but even if there was a related strain or species in BC, one cannot assume it is pathogenic

ISA Prevalence in Livers of Sockeye smolts

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Isa7-P7 Isa-P8

HS_Summer_2007_WCVIHS_Summer_2007_FraserSOG_Summer_2008SOG_Summer_2009FW_Natal_Chilko_2010SOG Summer_2010SOG_Fall_2008SOG_Fall_2009SOG_Fall_2010SOG_July_Ricker_2010

Negative Positive

Retrospective Genomics analysis of microarray data reveals >5,000genes differentially regulated in association with weak PCR positives

Functional analysis reveals top Biological Pathway affected: INFLUENZA INFECTION

Orthomyxoviruses are “influenza viruses”!

2007

Powerful host response suggests potential for disease and pathogenicity

Prevalence of ISAv‐like detections in smolts

ISAv HearingsControversy #6: Why don’t the molecular tests match up?First 1.5 days of testimony very technical, largely focussed on how the assays work, and why different results could be shown in different labs

The labs were not using all of the same tests, although the real outlier was the CFIA

CFIA and the Provincial Health Laboratory responsible for testing industry fish were using theirown (different) unpublished “validated” assays

All three labs with weak positives were using the same published assays

The controversy heated up when the CFIA and DFO managers went on the stand It became known that a Postdoctoral fellow DFO’s Fish Health Laboratory had alsohad positive molecular tests for ISAv in 2003‐2004, but her results were questionedby the lead scientist who did not permit her to publish them.

The CFIA testing results came into question, as the scientist had reported that theywere “inconclusive” due to poor sample quality, and the managers and the Ministerreported to the media that they were all NEGATIVE.

e‐mail from a CFIA executive "It is clear that we are turning the PR tide to our favour," he writes, "and this is because of the very successful performance of our spokesmen at the Tech Briefing yesterday... Congratulations! One

battle is won, now we have to nail the surveillance piece, and we will win the war also."

ISAv Hearings

Controversy #7: The CFIA PR tide

Did not establish a single cause that explained the two‐decade decline in productivity; cumulative effects of multiple stressors most

plausible

Established “no fault” on DFO for failing to take action on any particular stressor, but felt that DFO had not focussed enough

research on this issue

Sockeye salmon face an uncertain future

Cohen Final Report: October 2012

Recommendations relevant to topics covered:3. The Government of Canada should remove from the DFO’s mandate the promotion of salmon farming as an industry and

farmed salmon as a product

12. For research purposes beyond routine monitoring, DFO should require, as a condition of licence, that … farms provide… fish

samples, including live fish or fresh silvers (recently deceased fish), in a quantity and according to a protocol specified by DFO.

Cohen makes 75 Recommendations

18. If …between now and 2020, the minister of DFO determines that net‐pen salmon farms in the Discovery Islands…

pose more than a minimal risk of serious harm to the health of migrating Fraser River sockeye salmon, he or she should promptly

order that those salmon farms cease operations.


67 The fish health research priorities of DFO should reflect its responsibility for the conservation of wild fish. To that end, DFO’s science managers should encourage innovation and new research into novel diseases and other conditions affecting wild fish, beyond the interests of specific “clients” such as the CFIA or aquaculture

management.


68 DFO should undertake or commission research into the health of Fraser River sockeye salmon…

• …what pathogens are encountered … along their entire migratory route, and the cumulative effects …;

• the hypothesis that diseases are transmitted from farmed salmon to wild sockeye;

• the hypothesis that diseases are transmitted from salmonidenhancement facility salmon to wild sockeye


To date, the Government of Canada has not officially responded to any of the

Recommendations

What I learned in this process...

With new technology and/or approaches comes a challenge to the status quo

Don’t underestimate the resistance you may face to “change”, especially when it comes to challenging the long held belief

systems of other scientists

Not all managers want to know more—especially if it could tip their balance on the tightrope they are walking across

Perseverance eventually pays off…

Beyond Cohen

Genome BC Strategic BC Salmon Health InitiativeStrategic Goal:Discover the pathogens and potential diseases that may undermine the productivity and performance of BC salmon, their evolutionary history, and the potential role of exchanges between wild and cultured salmon

Highly Multidisciplinary ApproachGenomics

EpidemiologyHistopathology

ImmunologyVirology

ParasitologySalmon EcologyBioinformatics

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P arvovirus P os it ive P arvovirus Negative

UnsuccessfulS uccessful

Assays

SamplesKristi MillerNov. 2, 2013

Salm

on T

issu

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mpl

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MicrobeHC

BioMark platform can simultaneously assess 96 quantitative RT‐PCR assays (horizontal axis) across 96 samples (vertical axis) for the same cost it takes to run 4‐6 conventional assays.

Herein, assays are run in quadruplicate, with brighter color indicative of higher microbe load.

00.050.1

0.150.2

0.250.3

0.350.4

0.45% positives in Chilko

% positives inLateShuswap

Adult tagging study reveals microbe and host immune biomarkers linkages with migration mortality to spawning grounds

Nov. 3, 2013

Chilko sockeye positive for Piscine Reoviruscarry 2.3 greater odds of dying before reaching spawning grounds

Chilko sockeye positive for Loma salmonaecarry 9.6 greater odds of dying before reaching spawning grounds

Microbes associated with fate also elicit the strongest response in the host

197 adult sockeye salmon tagged and biopsy sampled on marine approach to Fraser River

45 microbes and 58 host genes quantitatively assessed on BioMark

Smolt tagging study reveals microbe and host immune biomarker linkages with mortality of smolts en route to the ocean

197 2-year old Chilko sockeye smolts tagged and gill biopsied leaving natal Lake in 2012

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2012V5V7

Survivorship

Distance from Chilko Lake release site (km)

Chilko Chilcotin Fraser SOG JS/DP

Jeffries et al. 2014

Fish that did not survival early migration showed potential evidence of a disease state

10 stress/immune genes stimulated in fish that did not survive (strong anti-viral signals)

Fish with high loads of Infections hematopoietic necrosis virus did not make it to the ocean

60% of Chilko sockeye 2-year olds tagged in-river did not make it to the ocean

Auklet predation study identifies association between microbe carrier states and likelihood of predation

Kristi MillerNov. 3, 2013

Predated fish carried higher microbe loads

Predated fish carried higher microbe diversity

Miller et al. 2014

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% p

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TrawlPredated

79 predated smolts contrastedwith 86 smolts trawled from feeding area in Queen CharlotteStrait

Parvicapsula sp. Infection increases risk of predation

scott class presentation cohen inquiry feb 6,...

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