Development of diagnostic tools and vaccines for aquatic animals presentation by Sandra Adams, University of Stirling, Stirling, United Kingdom

1. Development of Diagnostic Tools
and Vaccines for
Aquatic Animals
Symposium on Agricultural Biotechnologies, 15-17 February 2016
Alexandra (Sandra) Adams
Institute of Aquaculture
University of Stirling, Scotland, UK
Institute of AquacultureInstitute of Aquaculture

2. Outline
 Introduction
 Climate change and aquaculture
 Development of diagnostics tests
 Vaccine development
 Final thoughts & conclusions

3. Fish Disease -disease is considered a major
constraint to aquaculture production globally
 Bacterial
 Fungal
 Viral
 Parasitic
Photograph courtesy of Peter Dixon, CEFAS
Control of disease is complex:
Pathogen detection, disease diagnosis, treatment,
prevention and general health management

4. Climate Change and Aquaculture
 Alters risk of disease
-pathogen distribution
-pathogen prevalence
-pathogen virulence
 Impact will vary (+ve
and –ve)
-Climatic regions
tropical
sub-tropical
temperate
-Different environments
freshwater
marine
brackishwater

5. Some examples of climate
sensitive diseases
Disease Type
Epizootic Ulcerative Syndrome-EUS)
Koi herpes virus disease (KHVD)
Viral Encephalopathy & Retinopathy
Fish Ectoparasites
Streptococcus infections
White Spot Disease (WSD)
Infectious Myonecrosis
White tail disease
Shrimp AHPND
Fish fungal disease
Fish viral disease
Fish viral disease
Fish parasitic diseases
Fish bacterial disease
Shrimp viral disease
Shrimp viral disease
Shrimp viral disease
Shrimp bacterial disease

6. Climate Change and Aquaculture
 Climate change will affect movement and spread
of diseases
 Need to have in place
-Relevant rapid diagnostic tests
-Appropriate vaccines to prevent diseases
Amoebic Gill Disease
-no vaccine
Furunculosis
-effective vaccine

7. Rapid Diagnosis
 Speed of pathogen detection important
- to prevent spread of disease
 Significant progress in development of
rapid methods to detect pathogens
- adaption and optimisation of clinical and
veterinary methods for use in
aquaculture

8. Diagnostic Tests
-Detection of Pathogens
 Screening:
presumably healthy
individuals
 Diagnostics test:
diseased animals
WHERE?
 In infected fish
clinically & sub-
clinically
 In the environment

9. DETECTION OF PATHOGENS
HOW?
Morphological/Traditional Methods -culture and histology
Immunological Methods
Molecular Methods

10. Combination of Methods
 A combination of methods is often
required for a definitive diagnosis
of disease
 Good sample collection is important
Selection of the methods depends on a variety of factors
-- each method has its merits and disadvantages
Which methods should be applied in aquaculture?
Methods need to be robust yet sensitive!

11. Challenges
 Accuracy
 Specificity
 Sensitivity
 Speed
 Technical complexity
 Cost
 Availability
 Robust
 Affordable
 Requirements differ: lab versus field

12. Examples of novel technologies with
potential for use in aquaculture
 Immunoassays
 Molecular tests
 Other technologies

13. Lateral Flow Device (LFD)
- Immunochromatography
User-friendly format
Very specific
Very sensitive
Very rapid
Long-term stability over a
wide range of temperatures
Relatively inexpensive to make
ISAV Rapid Kit
Sensitivity = PCR
-ve
+ve

14. ISAV Rapid Test Kit MethodISAV Rapid Test Kit Method

15. Nanotechnology
- magnetic beads (coated with antibody) rotate under a magnetic
- this gives a signal
- when the pathogen binds to the beads they form clusters

16. ImmunoMagnetic Reduction (IMR)
-Larger, clustered magnetic beads cannot
rotate very well and therefore the signal is reduced

17. Examples of Molecular
Tests
-Real-time qPCR in the field
Mobile PCR
-Genesig q16 on-site qPCR diagnostics

18. Isothermal amplification
 Nucleic acid amplification at a single temperature
 o More suitable for use in the field
 LAMP – Loop-mediated isothermal amplification
 Other formats (NASBA, RPA etc..)
 Assays developed for many livestock pathogens

19. Other Technologies
MALDI TOF MS
ID of protein profiles
Need culture
Rapid and low cost analysis
Database for aquaculture?
DNA sequencing

20.  Control significant diseases
 Save costs
 Reduce concerns over residue levels and
environmental impacts
 Reduce the need for antibiotics and
chemicals
 Reduce problems with antibiotic resistance
Vaccines

21. 7
NorwegianNorwegianSalmonSalmonProducti
on, Production, Use of Pure Antibiotics and
theUse
theEffectEffectofofVaccinesVaccines0102030
405060198119821983198419851986198719
881989199019911992199319941995199619
97199819992002001200220032004USE of
antibiotics
(MT)05010015020025030035040045050055
0600650Salmon production (1,000
MT)Vibriosis vaccineFurunculosis
vaccineOil-basedFurunc.
vaccineCombination vaccines
From: Fish Vaccination – A brief overview. Dr Marian McLoughlinFrom: Fish Vaccination – A brief overview. Dr Marian McLoughlin
Antibiotic usage hasAntibiotic usage has
reduced by 99.5%reduced by 99.5%

22. Atlantic salmon production
in Scotland
Annually
In Scotland ~20 million trout and ~40 million
salmon vaccinated
Globally ~90 million trout and 418 million
salmon vaccinated

23.  Increase in commercially available
vaccines
 BUT there are still diseases where no are
vaccines available
 AND some existing vaccine do not
perform well
Fish Vaccines

24.  Safety
 Cost-effectiveness
 Long term protection
 Serotypic/genetic variation of the pathogen
 Time-/age when fish most susceptible to disease
 Species
 Route of administration
 Method of vaccine preparation
Primary considerations

25. Types of Vaccine
• Inactivated whole cell
• Adjuvanted
• Sub-unit
• Recombinant
• Live attenuated
• Synthetic (peptide)
• DNA vaccines
Development
cost

26. Not an easy to develop a vaccine
-need to identify protective antigens
-like finding a needle in a haystack!

27.  Identification and inclusion of all
important serotypes/genotypes
 In vivo expression & immuno-
proteomics
 Epitope mapping
 Reverse vaccinology
 DIVA vaccines?
Approaches for the Identification of
Protective Antigens in Fish Vaccine
Development

28. Case Study 1: Whole cell vaccine
Rainbow Trout Fry Syndrome (RTFS)
-caused by Flavobacterium psychrophium
Need a vaccine that protects against the many different field
isolates BUT…
-difficult because F. psychrophilum very heterogeneous
-fry are susceptible to infection so necessary to develop
immersion or oral vaccine to provide protection at small size
Rowena Hoare, Thao Ngo, Kerry Bartie, Sung-Ju Jung, Alexandra Adams

29. Species identification
Biochemical
tests
Antibiotic susceptibility testing:
disc diffusion, MIC
Serotyping
Genetic characterisation
CHARACTERISATION

30. Case Study 2: Recombinant vaccine
Aeromonas hydrophila
• Gram negative, opportunistic bacterium
• Affects variety of fish species world wide
• Difficult to develop a vaccine because of
antigenic diversity
• Immunoproteomics approach taken after
growing bacteria under different
conditions
Outer membrane
profiles of different
A. hydrophila
isolates

31. VACCINE DEVELOPMENT
Virulence studiesVirulence studies
(Artificial infection)
AEROMONAS HYDROPHILA (14 STRAINS)
Immunological
Analysis
(Antibody
response)
Protein profile analysis
bacteria grown in vitro/in vivo)
Identification of common
potential antigens by 2D
electrohoresis and WB
Recombinant vaccine production
Analysing the protection of antigen in fish against A. hydrophila

32. CUMULATIVE PERCENTAGE MORTALITY OF CARP
FOLLOWING VACCINATION AND CHALLENGE
-protection against challenge by different A. hydrophila
isolates
98140

33. Fish Nodavirus
 Infects the central nervous system
of fish (eg sea bass, sea bream)
 Small (25-34 nm) icosahedral,
single stranded viruses with
positive sense RNA genome
CASE STUDY 3 - Epitope
Mapping
Costa, J.Z.; A. Adams; J.E. Bron; K.D. Thompson; W.G. Starkey
and R.H. Richards
Identification of B-cell epitopes on the betanodavirus capsid
protein
Journal of Fish Diseases 30 (7), pp 419-426, 2007

34. MVRKGEKKLAKPPTTKAANPQPRRRANNRRRSNRTDAPVSKASTVTGFGRGTNDVHLSGMSRISQAVLPAGTGTDGYVVVDATIVPDLLPRLGHAARIFQRYAVETLEFEQPMCPANTGGGYVAGFLPDPTDNDHTFDALQA
TRGAVVAKWWESRTVRPQYTRTLLWTSSGKEQRLTSPGRLILLCVGNNTDVVNVSVLCRSVRLSVPSLETPEETTAPIMTQGSLYNDSLSTNDFKSILLGSTPLDIAPDGAVFQLDRPLSIDYSLGTGDVDRAVYWHLKKFA
GNAGTPAGWFRWGIWDNFNKTFTDGVAYYSDEQPRQILLPVGTVCTRVDSEN
Betanodavirus coat protein sequenceBetanodavirus coat protein sequence
MVRKGEKKLAKPPTTKAANPQPRRRANNRRRSNRTDAPVSKASTVTGFGRGTNDVHLSGMSRISQAVLPAGTGTDGYVVVDA
-- Pep 1 ---
-- Pep 2 --
-- Pep 3 ---
-- Pep 4 ---
-- Pep 5 ---
-- Pep 6 ---
-- Pep 7 ---Overlapping peptidesOverlapping peptides
 The peptides are the linked to
fluorescent beads
 Each bead is slightly different
and can be identified
Epitope Mapping

35. PepScanPepScan
Antibodies were incubated with the bead-Antibodies were incubated with the bead-
peptidepeptide
Bio-Plex systemBio-Plex system
Samples were read with the dual laser set of the Bio-Plex systemSamples were read with the dual laser set of the Bio-Plex system
Data exported toData exported to
Excell and analysedExcell and analysed
Synthetic peptides were coupled to the fluorescentSynthetic peptides were coupled to the fluorescent
beadsbeads
Bead-peptide-Ab was incubated with reporterBead-peptide-Ab was incubated with reporter
molecule (ab conjugated with PE)molecule (ab conjugated with PE)

36. 0
250
500
750
1000
1250
1500
MFI
Polyclonal Antibodies
0
500
1000
1500
2000
2500
3000
3500
4000
MFI
5G10 3B10 4A12 4C3
0
500
1000
1500
2000
2500
MFI
SB 1 SB2 SB3 SB4 SB7 SB9 SB10 SB15 SB17
Mouse, rabbit and fishMouse, rabbit and fish
ab recognised peptideab recognised peptide
2020
Peptide 3 isPeptide 3 is
recognised just byrecognised just by
mouse and rabbitmouse and rabbit
All species reveal high
recognition of the region
between peptide 19-21
Sea bass has high binding
to peptides 15-16, 10
and 1

37. Epitope Mapping Results
 Amino acid sequence
of the ‘epitope’ (part
of the molecule) that
binds to Nodavirus-
specific fish antibody
 Region 191-202 is theRegion 191-202 is the
major immunogenicmajor immunogenic
domain for Nodavirusdomain for Nodavirus

38. -relies on the combined use of immunological and genomic
information to identify relevant protein antigens
Cellular immunity: identification of the epitopes recognized by
CD4+ T cell or CD8+ T cells can be utilized in ‘‘reverse’’ as a tool
to identify new antigens
Carbohydrate antigens?
CASE STUDY 4: Reverse Vaccinology
UOS: Sean Monaghan, Carol
McNair, Randolph Richards, James
Bron & Sandra Adams

39. Commercialisation
Route from research
to commercialisation
can be long and
expensive
Many vaccines
developed through
research but not taken
forward or ‘stuck in the
pipeline’-this needs to
be addressed.

40. Conclusions and challenges
 Potential for development of novel rapid diagnostic
tests for lab and field use
 Many methods for vaccine development, but very
difficult for parasite diseases
 Still challenges e.g. understanding mucosal
immunity
 Route from research to commercialisation can be
long and expensive

41. Final thoughts
 Climate change will affect the movement and spread of
diseases in the aquatic environment – need relevant rapid
tests and vaccines in place.
 Not possible to develop vaccines against all diseases.
 In some cases vaccines too expensive to use
 Thus, alternatives to vaccines also need to be considered so
that antibiotic and chemical usage does not increase.
 Continued education and training is also important -some
regions of the world do not currently have wide acceptance
of the use of vaccines as a fish health control method.

42. TAcknowledgements
All involved in case studies