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BIOTECHNOLOGY AND THE DIAGNOSIS AND SURVEILLANCE OF AQUATIC ANIMAL PATHOGENS

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. Overview:. Antiboby-based diagnostic assays.

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BIOTECHNOLOGY AND THE DIAGNOSIS AND SURVEILLANCE OF AQUATIC ANIMAL PATHOGENS

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

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

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

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

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

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

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

  9. Polymerase chain reaction (PCR) • Thermal cycle conditions • Initial denaturation • 94°C for 15 min. • 35 cycles • 94°C 30 sec. • 52°C 30 sec. • 72°C 30 sec.Final extension • 72°C for 5 min.

  10. PCR

  11. Detection of a Tasmanian rickettsia-like organism in Atlantic salmon tissues using a single step PCR assay. A B C D E F G • Hafnia alvei (negative control) • Aeromonas salmonicida • Yersinia ruckeri • Vibrio anguillarum • Rickettsia-like organism Tasmania • Piscirickettsia salmonis ATL-4-91 (positive control) • 100 bp DNA ladder 264 bp 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 SP6 T7 Restriction sites lacZαccdβ DNA plasmid Plac Kanamycin Plasmid ori

  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) 1) 5’ B2 B1 F1c F2c 3’ F2 Primer BIP F1C 2) 5’ B2 B1 F1c F2c Primer BIP B1C B2 F1 B2c B1c 5’ F1c F2

  16. 3) B2 B1c F1c B1 F1 F2c 5) 4) 5’ B1c B2 B1 F1c B2c B1c F1 F2 3’ F1C

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

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

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

  21. Luminex technology 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.

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

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

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

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

  26. EMERGENCE OF AN ABALONE HERPES VIRUS IN AUSTRALIA

  27. Australian Animal Health Laboratory (AAHL) 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

  28. Australia

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

  30. Moribund abalone

  31. Pleuropedal ganglion and pedal ganglionated cords

  32. Development of specific conventional PCR assays for detection and identification of AbHV 1 2 3 4 5 486 bp Virus sequence obtained from Savin et al., Virol Journal 7:308, 2010.

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

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

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

  36. In situ hybridisation assay

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

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

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

  40. 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

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