LABORATORY ISSUES – AQUATIC ANIMAL DISEASE, DIAGNOSIS AND GLOBAL TRENDS *Fred S.B. Kibenge 1 , Marcos G. Godoy 2 , &

1. 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)

2. 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.

3. Outline • 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

4. Aquatic Animal Diseases (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

5. 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

6. Aquatic 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.

7. Flow of Biological Aquatic Material to Chile Modified from M. Godoy & F. Kibenge, November 2008

8. Aquatic 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.

9. Aquatic Animal Diseases (global trends & spread & emerging threats)

10. Aquatic Animal Diseases (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)

11. Global distribution of viral haemorrhagic septicemia virus ?<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)

12. Salmon alphaviruses From Intervet Schering-Plough Animal Health PD Technical Manual

13. PD outbreaks in Norway in Year 2011 Nord-Trøndelag 1 Møre og Romsdal 1 Sogn og Fjordane 5 12 Hordaland 5 Rogaland

14. Prevalence of Cardiomyopathy syndrome (CMS) & Heart and skeletal muscle inflammation (HSMI) CMS recorded CMS suspected HSMI recorded HSMI suspected

15. Current status of ISA

16. First-time Outbreaks of ISA 2000 1984 * * 1996 2009 * * * * 1998 2009 2001 * 2007

17. ISAV Strain identification 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

18. Prevalence of ISA outbreaks in Norway (1984 to August 2010) Fuente

19. Update on ISA situation in Scotland: 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

20. Update on ISA situation in Faroe Islands: ISAV European genotype

21. Update on ISA situation in Chile: 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

22. Recent introduction of ISAV to Chile: was it single or multiple introductions?

23. Phylogeny of concatenated F and HE genes fromGenotype 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

24. Prevalence of ISAV (virulent and HPR0) positive cases in Chile (July 2007 to April 2011) 260 Cases

25. Update on ISA situation in Canada and USA: 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

26. First-time ISAV HPR0 Reports 2006 2002 * * 2004 * * * * 2002 2004 * 2008

27. ISAV HPR0 Characteristics 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

28. ISAV HPR0 viruses: Challenges • 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?

29. 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

30. 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.

31. Laboratory Diagnostic Tests 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

32. General challenges faced by diagnostic labs 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.

33. Specific diagnostic challenges • Turnaround time of diagnostic laboratory • Distance from farm site to diagnostic laboratory • Quality of sample Diagnostic labs Farm sites

34. Specific diagnostic challenges: - mixed infections Norway: HSMI, PD, CMS, Sea lice Chile: SRS, BKD, Caligus, ISAV-HPR0

35. Specific diagnostic challenges: - 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.

36. Conclusions • 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.

37. Thank you