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
Nucleic acid-based diagnostic assays
Case study of an emerging virus in Australia
Antibody-based assays for the detection of
pathogen antigens in animal tissues.
Recombinant proteins
Killed or inactivated pathogen
ELISA using monoclonal/polyclonal
polyclonal antibodies directed against
White spot virus/yellowhead virus.
Slater J. et al., personal communication.
Western blot using monoclonal antibody
directed against White spot virus.
Immuno-histochemistry for the detection of
Pisciricketsia salmonis in Atlantic salmon liver.
Corbeil et al., DAO 64:37-44, 2005.
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.
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).
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.
PCR
Detection of a Tasmanian rickettsia-like organism in
Atlantic salmon tissues using a single step PCR assay.
A
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
B
C
D
E
F
G
264 bp
Advantages: Fast, specific, DNA sequencing allows identification of variants,
relatively low cost.
Disadvantage: Potential cross contamination between samples.
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.
Cloning of pathogen target gene
Target gene
T7
SP6
Restriction
sites
DNA
plasmid
Plasmid ori
Kanamycin
Plac
lacZα ccdβ
Molecular titer can be established using the real-time
assay (e.g. number of viral gene copies/g).
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
B2c
B1c
F1
5’ F1c
F2
3)
B2
B1c
F1c
F1
B1
F2c
5)
4)
5’
B1c
B2
B2c
B1
B1c
F1c
F1
F2
3’
F1
C
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.
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.
In situ hybridisation of abalone herpesvirus in nerve cords
Advantages: Specific, sensitive, allows localization of virus in tissues.
Disadvantages: Labour intensive and time consuming.
Luminex technology
2 Dyes are used to create 100
unique color combinations
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.
Luminex xTAG beads
A uniquely designed 24 oligonucleotide tag/anti-tag sequence for
each bead type provides isothermal conditions across all bead sets
and sequences.
Pilot study on non-aquatic viruses conducted at CSIRO/AAHL
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)
Currently developing at CSIRO a multiplex assay for
aqua-reoviruses and aqua-birnaviruses.
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.
EMERGENCE OF AN ABALONE HERPES VIRUS IN
AUSTRALIA
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
Australia
Histopathological examination of
moribund animals indicated a
ganglioneuritis.
Examination by electron microscopy
revealed the presence of a herpes-like
virus in the pleuropedal ganglion.
Moribund abalone
Pleuropedal ganglion and
pedal ganglionated cords
Development of specific conventional PCR
assays for detection and identification of AbHV
1
2
486 bp
Virus sequence obtained from Savin et al.,
Virol Journal 7:308, 2010.
3
4
5
Real-time TaqMan PCR assay ORF-49
Corbeil et al., DAO 92(1) 1-10, 2010.
Cloning of target gene
AbHV ORF-49
126 bp target gene
AbHV can be titered using the real-time TaqMan
assay (number of virus gene copies).
In situ hybridisation assay
Diagnostic applications
Detection and confirmation of AbHV as aetiological agent.
Surveillance.
Broodstock screening.
Research applications
Variants pathogenicity.
Early detection in host.
AbHV stability in sea water at different temperatures.
AbHV susceptibility to various disinfectants.
Research applications
Evaluation of carrier state in abalone survivor from previous
outbreaks.
Evaluation of disease resistance of abalone
family lines.
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