1. BIOTECHNOLOGY AND THE DIAGNOSIS AND SURVEILLANCE OF AQUATIC ANIMAL PATHOGENS SERGE CORBEIL, L WILLIAMS and M ST.J. CRANE. Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong, Victoria, Australia.

2. Antiboby-based diagnostic assays Nucleic acid-based diagnostic assays Case study of an emerging virus in Australia Overview:

3. Antibody-based assays for the detection of pathogen antigens in animal tissues. Recombinant proteins Killed or inactivated pathogen

4. Slater J. et al., personal communication. ELISA using monoclonal/polyclonal polyclonal antibodies directed against White spot virus/yellowhead virus.

5. Western blot using monoclonal antibody directed against White spot virus.

6. Corbeil et al., DAO 64:37-44, 2005. Immuno-histochemistry for the detection of Pisciricketsia salmonis in Atlantic salmon liver.

7. Lateral flow immunoassay Advantages: No need for sophisticated equipment and highly trained staff. Hand held device can be used in the field. Disadvantage: Low sensitivity.

8. Molecular assays for the detection of nucleic acids in animal tissues. -We require target DNA sequence information for the design of oligonucleotides (primers and/or probes). -Based on amplification/detection or hybridization of nucleic acid sequence specific to the target pathogens.

9. Polymerase chain reaction (PCR) Thermal cycle conditions Initial denaturation  94°C for 15 min.  35 cycles  94°C30 sec.  52°C30 sec.  72°C30 sec. Final extension  72°C for 5 min.

10. PCR

11. 264 bp A B C D E F G A)Hafnia alvei (negative control) B)Aeromonas salmonicida C)Yersinia ruckeri D)Vibrio anguillarum E)Rickettsia-like organism Tasmania F)Piscirickettsia salmonis ATL-4-91 (positive control) G)100 bp DNA ladder Detection of a Tasmanian rickettsia-like organism in Atlantic salmon tissues using a single step PCR assay. Advantages: Fast, specific, DNA sequencing allows identification of variants, relatively low cost. Disadvantage: Potential cross contamination between samples.

12. Real-time PCR (e.g.TaqMan/SYBR Green) Advantages: Faster, specific, very sensitive, high throughput, quantitative, can multiplex, no cross contamination. Disadvantage: More costly than conventional PCR.

13. Cloning of pathogen target gene Target gene lacZα ccdβ Plasmid ori Kanamycin SP6 Plac Restriction sites T7 DNA plasmid

14. Molecular titer can be established using the real-time assay (e.g. number of viral gene copies/g).

15. Loop-mediated isothermal amplification (LAMP) 5’3’ B2B1F2cF1c F2 F1C Primer BIP 2) 5’ B2B1 F2cF1c F1 F2 5’ B1cB2c B2 B1C Primer BIP 1)

16. F1c F2c B2 B1c B1 3) F1 4) F1c B1cB2c B1B2B1c 5’ F1 C F1 F2 3’ 5)

17. Advantages: -Fast (early detection of infection) -Specific -More sensitive than PCR -Does not require thermo-cycler. Disadvantages: -Primers design can be difficult. -Can not multiplex.

18. In situ hybridisation (ISH) ISH uses a labeled complementary DNA or RNA (i.e. probe) to localize a pathogen specific nucleic acid in a section of animal tissue.

19. In situ hybridisation of abalone herpesvirus in nerve cords Disadvantages: Labour intensive and time consuming. Advantages: Specific, sensitive, allows localization of virus in tissues.

20. Luminex technology 2 Dyes are used to create 100 unique color combinations

21. Bead based technology measures multiple analytes (e.g. DNA sequences from viruses) simultaneously in a single reaction vessel. Beads are coated with linker DNA sequences specific to various viruses, or variants of a given virus, and then mixed to make an array. Luminex technology

22. A uniquely designed 24 oligonucleotide tag/anti-tag sequence for each bead type provides isothermal conditions across all bead sets and sequences. Luminex xTAG beads

23. Detection of different pathogens in a single assay 1.Influenza virus 2.Newcastle disease virus (NDV) 3.Infectious bursal disease virus (IBDV) 4.West Nile virus (WNV) Pilot study on non-aquatic viruses conducted at CSIRO/AAHL

24. Currently developing at CSIRO a multiplex assay for aqua-reoviruses and aqua-birnaviruses.

25. Advantages: Specific, sensitive, high throughput, fast, cost effective, high multiplexing capability. Disadvantages: Sophisticated equipment and requires trained technical staff, optimisation of assays is difficult. Luminex technology

26. EMERGENCE OF AN ABALONE HERPES VIRUS IN AUSTRALIA

27. Conduct diagnosis and surveillance to meet the needs of those trading in animal both nationally and internationally AAHL – a national facility Undertake research to manage the risks from endemic and exotic animal diseases Australian Animal Health Laboratory (AAHL)

28. Australia

30. Histopathological examination of moribund animals indicated a ganglioneuritis. Examination by electron microscopy revealed the presence of a herpes-like virus in the pleuropedal ganglion.

31. Moribund abalone

32. Pleuropedal ganglion and pedal ganglionated cords

33. Development of specific conventional PCR assays for detection and identification of AbHV 12345 486 bp Virus sequence obtained from Savin et al., Virol Journal 7:308, 2010.

34. Real-time TaqMan PCR assay ORF-49 Corbeil et al., DAO 92(1) 1-10, 2010.

35. Cloning of target gene AbHV ORF-49 126 bp target gene

36. AbHV can be titered using the real-time TaqMan assay (number of virus gene copies).

37. In situ hybridisation assay

38. Diagnostic applications Broodstock screening. Detection and confirmation of AbHV as aetiological agent. Surveillance.

39. Research applications Variants pathogenicity. Early detection in host. AbHV stability in sea water at different temperatures. AbHV susceptibility to various disinfectants.

40. Evaluation of disease resistance of abalone family lines. Evaluation of carrier state in abalone survivor from previous outbreaks. Research applications

41. Acknowledgements Fisheries Research and Development Corporation Projects No. 07/006 and 09/032: Aquatic Animal Health Subprogram CSIRO Joanne Slater, Nick Gudkovs, Nick Moody, Alex Hyatt, Sandra Crameri, Jeff Cowley, Hans Heine, Vicky Boyd, John White. Depeartment of Primary Industries Victoria S Warner, K Savin, F Wong, M Fegan, N Kvalheim, I Mohamad, T Sawbridge, N Cogan, D Savage, M Vardy, BG Cocks. Victorian Abalone Divers Association for providing infected abalone Great Southern Waters Inc. for providing healthy, uninfected abalone