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Congratulations to the OIE for the adoption of Resolution 18/2011, officially recognizing that all 198 countries of the world with rinderpest‑susceptible animal populations are free of the disease (79th General Session of the OIE, 22‑27 May 2011).

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Congratulations to the OIE for the adoption of Resolution 18/2011, officially recognizing that all 198 countries of the world with rinderpest‑susceptible animal populations are free of

the disease (79th General Session of the OIE, 22‑27 May 2011)


LABORATORY ISSUES – AQUATIC ANIMAL DISEASE, DIAGNOSIS AND GLOBAL TRENDS*Fred S.B. Kibenge1, Marcos G. Godoy2, & Molly J.T. Kibenge1

1OIE Reference Laboratory for ISA, Department of Pathology & Microbiology, Atlantic Veterinary College, UPEI, Charlottetown, P.E.I., Canada.

2ETECMA, Puerto Montt, X Region, Chile.


Outline GLOBAL TRENDS

  • Introduction to Aquatic Animal Diseases

  • Current Status of ISA

  • Current Trends in Lab Diagnosis and Pathogen Surveillance

  • Challenges Faced by Diagnostic Labs for Aquatic Animal Diseases

  • Conclusions


Aquatic Animal Diseases GLOBAL TRENDS

(global trends & spread & emerging threats)

  • Aquaculture is the world’s fastest-growing animal-food producing sector

    • Annual growth rate of 8.4% since 1970; reached 65.8 million tonnes in 2008

    • Aquaculture now accounts for almost half of the total fish supply for human consumption, and is likely to continue increasing.

      • FAO predicts that by 2030 there will be an additional 2 billion people to the world population; & an additional 37 million tonnes of fish/year will be needed to maintain current levels of fish consumption.

  • China supplies 61.5% of global aquaculture production (29.5% from rest of Asia) – mostly Carp

    • 3.6% from Europe & 2.2% from South America – salmonids

    • 1.5% from North America – even production across the species groups

    • 1.4% from Africa – tilapias

    • 0.3% from Oceania – shrimps & prawns

Hall et al., 2011. Blue Frontiers: Managing the Environmental Costs of Aquaculture. The WorldFish Center, Penang, Malaysia


Factors leading to the discovery of new & emerging aquatic animal diseases

  • Increased aquaculture production

    • through translocation of cultured live animals or shipment of eggs to new destinations

  • Expanding range of “new” farmed aquatic animal species

    • such as Atlantic halibut, Arctic char, sablefish, Atlantic cod, crustaceans, molluscs

  • New production approaches

    • such as integrated multi-trophic aquaculture

  • Improved diagnostic & surveillance efforts

    • the more you look (with better technology) the more you find


Aquatic Animal Diseases animal diseases

(global trends & spread & emerging threats)

  • The spread of diseases is the most feared threat to aquaculture. It is a matter of global concern especially with increased trade & movement of live aquatic animals & their products across national borders. Examples include:

    • White spot syndrome disease in shrimp spread to 22 countries via trade in post-larvae

    • Taura syndrome spread from Americas to Asia via live shrimp movements

    • Gyrodactylus salaris spread from Sweden to Norway via live juvenile salmon for stock enhancement

    • First case of Sleeping disease in UK was linked to imported trout fillets

    • EHN virus spread from Germany to Finland via live farmed sheatfish imports

    • First cases of SVC in Switzerland, USA, Denmark were linked to koi carp imports

    • Koi herpesvirus has been linked to international koi carp trade

    • ISA outbreaks in Atlantic salmon in Chile in 2007: ISA virus was most similar to isolates from Norway.


Flow of Biological Aquatic Material to Chile animal diseases

Modified from M. Godoy & F. Kibenge, November 2008


Aquatic Animal Diseases animal diseases

(global trends & spread & emerging threats)

  • One of the most important challenges facing aquaculture is ability to control disease

    • Disease constitutes the largest single cause of economic losses in aquaculture

    • Value of world aquaculture production in 2008: USD 98.4 billion

    • Global estimate of disease losses to aquaculture by World Bank (1997) was ~ USD 3 billion

    • Current estimates suggest between 1/3rd to 1/2 of farmed fish & shrimps are lost to poor health management before they reach marketable size (Tan et al., 2006).

    • Some endemic diseases remain a challenge for aquaculture. For example:

      • SRS (Piscirickettsia salmonis) in Chile remains one of the most important causes of mortality in trout and Coho salmon in seawater, & was in Atlantic salmon before June 2007; It is the main cause of antibiotic use.

      • Pancreas diseases & sea lice in Norway, and Caligus in Chile are huge sanitary problems.



Aquatic Animal Diseases threats)

(global trends & spread & emerging threats)

  • Viral haemorrhagic septicaemia (VHS)

  • Pancreas disease (PD)

  • Cardiomyopathy syndrome (CMS)

  • Heart and skeletal muscle inflammation (HSMI)

  • Infectious salmon anaemia (ISA)


Global distribution of viral haemorrhagic septicemia virus threats)

?<2003?

Dark: VHSV isolated from marine species

Light: classical freshwater rainbow trout pathogenic VHSV isolates

Modified from Skall et al., J. Fish Dis. 2005, 28:509-529

Presence of VHSV, an RNA virus with high potential for mutation & adaptation, in wild marine fish represents a continuous potential threat to marine-farming of VHSV-susceptible species (Einer-Jensen et al., 2004)


Salmon alphaviruses threats)

From Intervet Schering-Plough Animal Health PD Technical Manual


PD outbreaks in Norway in Year 2011 threats)

Nord-Trøndelag

1

Møre og Romsdal

1

Sogn og Fjordane

5

12

Hordaland

5

Rogaland


Prevalence of Cardiomyopathy syndrome (CMS) & threats)

Heart and skeletal muscle inflammation (HSMI)

CMS recorded

CMS suspected

HSMI recorded

HSMI suspected



First time outbreaks of isa
First-time Outbreaks of ISA threats)

2000

1984

*

*

1996

2009

*

*

*

*

1998

2009

2001

*

2007


ISAV Strain identification threats)

2 basic genotypes/

serotypes

2-to-3 genogroups

HPR20

North American

HPR21

EU-G1

ISAV

real-European

EU-G2

EU-G3

European

European-in-North America

EU-G2

Nylund et al., 2007

Kibenge et al., 2001

Kibenge et al., 2007



Update on ISA situation in Scotland: threats)

ISAV European genotype

  • First ISA outbreak in 1998. Disease was erradicated in 1999

  • ISAV from different sites was 100% identical on segment 8, suggesting a single point source

    • ISAV HPR7b

  • Suspected case in November 2004

    • ISAV HPR0

  • Second ISA outbreak in southwest Shetland in January 2009

    • Infection started after June 2008

    • ISAV HPR10; from unknown source


Update on ISA situation in Faroe Islands: threats)

ISAV European genotype


Update on ISA situation in Chile: threats)

ISAV European genotype

  • First ISA outbreak occurred in June 2007 on Atlantic salmon seawater farm site in central Chiloé in Region X following recovery from an outbreak of Pisciricketsiosis.

  • ISAV was most similar to isolates from Norway.

    • it acquired mutations in surface envelope proteins

    • predominant pathogenic type was ISAV HPR7b until March 2010



Phylogeny of concatenated F and HE genes from multiple introductions?Genotype I:Genogroup 2 Clade 2.2 (Norway II) ISAV isolates

new Chile isolates,

Clade 2.2.2.1.2 (Chile)

Clade 2.2.2.1

Cottet et al., 2010

Norway 1997 isolates,

Clade 2.2.2.1.1 (Norway)

Clade 2.2.2

EU-G1 isolates

Norway HPR0 isolates

Clade 2.2

EU-G2 isolates: Clades 2.2.1.1. & 2.2.1.2

Clade 2.2.1


Prevalence of ISAV ( multiple introductions?virulent and HPR0) positive cases in Chile

(July 2007 to April 2011)

260 Cases


Update on ISA situation in Canada and USA: multiple introductions?

ISAV North American & European genotypes

  • First ISA outbreak outside of Norway was in New Brunswick, Canada, in 1996; virus might have been present by 1995

  • A single ISA outbreak occurred in Nova Scotia, Canada, in 2000.

  • ISA first confirmed in Maine, USA, in 2001

  • A single ISA outbreak occurred in Prince Edward Island, Canada, in 2009.

  • ISAV HPR0 has now completely replaced the virulent ISAV in both New Brunswick and Maine.

Farm site


First time isav hpr0 reports
First-time ISAV HPR0 Reports multiple introductions?

2006

2002

*

*

2004

*

*

*

*

2002

2004

*

2008


Isav hpr0 characteristics
ISAV HPR0 Characteristics multiple introductions?

ISAV without any deletion/insertion in HPR is designated HPR0 to indicate “full-length HPR”

All ISAV isolated to date from clinical disease have deletions in HPR relative to HPR0. HPR0 is considered the putative ancestral virus.

Cytoplasmic

tail

HPR

HE

Protein

ORF

-COOH

NH2-

Transmembrane

domain

N-terminal

region


Isav hpr0 viruses challenges
ISAV HPR0 viruses: Challenges multiple introductions?

  • Do not grow in cell culture; no CPE

    • How can they be used in experimental infections?

    • Is there a “reverse genetics system for ISAV”?

  • Do not cause disease; are non-pathogenic

    • Can they cause sub-clinical infection (e.g., immunosuppression)?

    • Are they a risk factor for developing ISA?

    • Are ISAV HPR0 viruses immunogenic?

      • Can they interfere with or boost ISAV vaccines?

  • Can only be detected by RT-PCR followed by sequencing; known only through genomic sequence fragments

    • Can cause diagnostic confusion. A challenge for prevention & control programs.

    • Can complicate surveillance efforts.

    • Can increase costs of depopulation control programs.

    • Are there other reservoirs of HPR0 viruses?


Current trends in laboratory diagnosis and pathogen surveillance

  • focusing primarily on the nucleic acid-based assays and their utility for pathogen discovery, surveillance, and confirmatory diagnosis


Historical overview of laboratory diagnosis from culture-based assays to nucleic acid-based diagnosis

From http://www.genengnews.com/gen-articles/improving-diagnostics-related-informatics/3521 downloaded January 03, 2011.

Historically, lab diagnosis has relied on culture of pathogen and/or measurement of antibodies in sera.

Early detection of infection has relied on development of rapid & sensitive diagnostic methods.

There is a need for assays that can allow unbiased analysis of pathogens in a sample, since differential diagnosis is difficult in early infection before appearance of clinical signs.


Laboratory Diagnostic Tests culture-based assays to nucleic acid-based diagnosis

NUCLEIC ACID-

BASED ASSAYS

BIASED

Singleplex PCR/RT-PCR

Multiplex PCR/RT-PCR;

Luminex;

High density qPCR/RT-qPCR;

Microarrays

Deep sequencing

CONFIRMATORY

PATHOGEN DETECTION

SCREENING/

SURVEILLANCE

DISCOVERY

UNBIASED


General challenges faced by diagnostic labs culture-based assays to nucleic acid-based diagnosis

for aquatic animal diseases

  • Statistically relevant disease surveillance & monitoring requires large numbers of aquatic animals

    • reliable detection of pathogen is difficult if clinically sick aquatic animals are not available or only low percentage of aquatic animals is infected

    • constant need to increase throughput (automation, miniaturization, etc)

    • effective monitoring requires quantitative methods that inform on pathogen load in aquatic animals or the environment

  • Need cost-effective, fast, highly sensitive & specific methods that allow unbiased pathogen detection

  • No perfect method. Assay development is never-ending

  • Prevalence of aquatic animal diseases change depending on:

    • time of year & water temperature (Plumb 1999)

    • success in disease management

  • Need for QA (Ring tests)

    • effective way in establishing national/international cut-offs of (highly sensitive) nucleic acid-based assays.


Specific diagnostic challenges culture-based assays to nucleic acid-based diagnosis

  • Turnaround time of diagnostic laboratory

  • Distance from farm site to diagnostic laboratory

  • Quality of sample

Diagnostic labs

Farm sites


Specific diagnostic challenges: culture-based assays to nucleic acid-based diagnosis

- mixed infections

Norway: HSMI, PD, CMS, Sea lice

Chile: SRS, BKD, Caligus, ISAV-HPR0


Specific diagnostic challenges: culture-based assays to nucleic acid-based diagnosis

- Standardization of diagnostic tests

  • Positive controls are expensive & not easy to get

  • Cut-off determination is complicated (criteria not defined; could be related to culture of pathogen or to clinical situation of the aquatic animal or farm, etc)

  • Case definition may be different for different countries.


Conclusions culture-based assays to nucleic acid-based diagnosis

  • Aquaculture is important now and in the future as a principal source of animal protein for human consumption

  • Aquatic animal disease is part and parcel of aquaculture

    • Intensification of aquaculture is accompanied by increased stress resulting in a significant proportion of stock becoming infected.

    • Unbiased pathogen detection from carrier aquatic animals is essential for effective disease control in the global aquaculture industry.

    • Improved diagnostic & surveillance efforts will result in the discovery of new & emerging aquatic animal diseases.

    • Nucleic acid-based assays, particularly multiplex assays such as microarrays are well suited for pathogen detection, typing, & discovery in aquatic animal populations

    • Diagnostic labs for aquatic animal diseases have challenges inherent in the nature of the aquaculture industry and require the involvement of the OIE.


Thank you culture-based assays to nucleic acid-based diagnosis


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