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Development of diagnostic tools and vaccines for aquatic animals

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

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Development of diagnostic tools and vaccines for aquatic animals

  1. 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. 2. Outline  Introduction  Climate change and aquaculture  Development of diagnostics tests  Vaccine development  Final thoughts & conclusions
  3. 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. 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. 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. 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. 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. 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. 9. DETECTION OF PATHOGENS HOW? Morphological/Traditional Methods -culture and histology Immunological Methods Molecular Methods
  10. 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. 11. Challenges  Accuracy  Specificity  Sensitivity  Speed  Technical complexity  Cost  Availability  Robust  Affordable  Requirements differ: lab versus field
  12. 12. Examples of novel technologies with potential for use in aquaculture  Immunoassays  Molecular tests  Other technologies
  13. 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. 14. ISAV Rapid Test Kit MethodISAV Rapid Test Kit Method
  15. 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. 16. ImmunoMagnetic Reduction (IMR) -Larger, clustered magnetic beads cannot rotate very well and therefore the signal is reduced
  17. 17. Examples of Molecular Tests -Real-time qPCR in the field Mobile PCR -Genesig q16 on-site qPCR diagnostics
  18. 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. 19. Other Technologies MALDI TOF MS ID of protein profiles Need culture Rapid and low cost analysis Database for aquaculture? DNA sequencing
  20. 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. 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. 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. 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. 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. 25. Types of Vaccine • Inactivated whole cell • Adjuvanted • Sub-unit • Recombinant • Live attenuated • Synthetic (peptide) • DNA vaccines Development cost
  26. 26. Not an easy to develop a vaccine -need to identify protective antigens -like finding a needle in a haystack!
  27. 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. 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. 29. Species identification Biochemical tests Antibiotic susceptibility testing: disc diffusion, MIC Serotyping Genetic characterisation CHARACTERISATION
  30. 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. 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. 32. CUMULATIVE PERCENTAGE MORTALITY OF CARP FOLLOWING VACCINATION AND CHALLENGE -protection against challenge by different A. hydrophila isolates 98140
  33. 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. 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. 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. 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. 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. 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. 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. 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. 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. 42. TAcknowledgements All involved in case studies

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