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

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

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

Climate Change and Aquaculture Alters risk of disease-pathogen distribution-pathogen prevalence-pathogen virulence

Impact will vary (+ve and –ve)

-Climatic regionstropicalsub-tropicaltemperate-Different environmentsfreshwatermarinebrackishwater

Some examples of climate sensitive diseases

Disease TypeEpizootic 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 diseaseFish viral disease

Fish parasitic diseases

Fish bacterial disease

Shrimp viral diseaseShrimp viral diseaseShrimp viral disease

Shrimp bacterial disease

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

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

Diagnostic Tests-Detection of Pathogens Screening:

presumably healthy individuals

Diagnostics test: diseased animals

WHERE? In infected fish clinically & sub-

clinically

In the environment

DETECTION OF PATHOGENS HOW?Morphological/Traditional Methods -culture and histology

Immunological Methods

Molecular Methods

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!

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

Examples of novel technologies with potential for use in aquaculture

Immunoassays Molecular tests Other technologies

Lateral Flow Device (LFD)- Immunochromatography

User-friendly formatVery specificVery sensitive Very rapidLong-term stability over a wide range of temperaturesRelatively inexpensive to make

ISAV Rapid KitSensitivity = PCR

-ve

+ve

ISAV Rapid Test Kit MethodISAV Rapid Test Kit Method

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

ImmunoMagnetic Reduction (IMR)-Larger, clustered magnetic beads cannotrotate very well and therefore the signal is reduced

Examples of Molecular Tests-Real-time qPCR in the field

Mobile PCR-Genesig q16 on-site qPCR diagnostics

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

Other TechnologiesMALDI TOF MSID of protein profiles Need cultureRapid and low cost analysisDatabase for aquaculture?

DNA sequencing

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

7 NorwegianNorwegianSalmonSalmonProduction, Production, Use of Pure Antibiotics and theUse theEffectEffectofofVaccinesVaccines010203040506019811982198319841985198619871988198919901991199219931994199519961997199819992002001200220032004USE of antibiotics (MT)050100150200250300350400450500550600650Salmon 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 has Antibiotic usage has reduced by 99.5%reduced by 99.5%

Atlantic salmon production in Scotland

AnnuallyIn Scotland ~20 million trout and ~40 million

salmon vaccinated Globally ~90 million trout and 418 million

salmon vaccinated

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

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

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

Development cost

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

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

Case Study 1: Whole cell vaccineRainbow 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

Species identification

Biochemical tests

Antibiotic susceptibility testing: disc diffusion, MIC

Serotyping

Genetic characterisation

CHARACTERISATION

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

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

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

98140

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

MVRKGEKKLAKPPTTKAANPQPRRRANNRRRSNRTDAPVSKASTVTGFGRGTNDVHLSGMSRISQAVLPAGTGTDGYVVVDATIVPDLLPRLGHAARIFQRYAVETLEFEQPMCPANTGGGYVAGFLPDPTDNDHTFDALQATRGAVVAKWWESRTVRPQYTRTLLWTSSGKEQRLTSPGRLILLCVGNNTDVVNVSVLCRSVRLSVPSLETPEETTAPIMTQGSLYNDSLSTNDFKSILLGSTPLDIAPDGAVFQLDRPLSIDYSLGTGDVDRAVYWHLKKFAGNAGTPAGWFRWGIWDNFNKTFTDGVAYYSDEQPRQILLPVGTVCTRVDSEN

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

PepScanPepScan

Antibodies were incubated with the Antibodies were incubated with the bead-peptidebead-peptide

Bio-Plex system Bio-Plex system

Samples were read with the dual laser set of the Bio-Plex Samples were read with the dual laser set of the Bio-Plex systemsystem

Data exported to Data exported to Excell and Excell and analysedanalysed

Synthetic peptides were coupled to the Synthetic peptides were coupled to the fluorescent beadsfluorescent beads

Bead-peptide-Ab was incubated with Bead-peptide-Ab was incubated with reporter molecule (ab conjugated with reporter molecule (ab conjugated with

PE)PE)

0

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M F

IPolyclonal Antibodies

0500

1000150020002500300035004000

M F

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5G10 3B10 4A12 4C3

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SB 1 SB2 SB3 SB4 SB7 SB9 SB10 SB15 SB17

Mouse, rabbit and Mouse, rabbit and fish ab recognised fish ab recognised

peptide 20peptide 20

Peptide 3 is Peptide 3 is recognised just recognised just by mouse and by mouse and

rabbit 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

Epitope Mapping Results Amino acid sequence

of the ‘epitope’ (part of the molecule) that binds to Nodavirus-specific fish antibody

Region 191-202 is the Region 191-202 is the major immunogenic major immunogenic domain for Nodavirusdomain for Nodavirus

-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 antigensCarbohydrate antigens?

CASE STUDY 4: Reverse Vaccinology

UOS: Sean Monaghan, Carol McNair, Randolph Richards, James

Bron & Sandra Adams

CommercialisationRoute 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.

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

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.

TAcknowledgementsAll involved in case studies