Molecular methods for molluscan diseases and marine fish virus (VNN) Snježana Zrnčić, PhD, DVM TCDC/TCCT Consultant No 2 – Diagnostics zrncic@irb.hr

MOLECULAR METHODS FOR MOLLUSCAN DISEASES Diseases with economical impact for European shellfish culture Advantage – more sensitive in detecting low prevalence of pathogen in molluscan population Methods: PCR for detection of Marteilia refringens in digestive gland of oysters and mussels PCR for detection of Bonamia spp. in gills of oysters In situ hybridization for both parasites qRTPCR for detection of OsHV-1 in oysters

Marteilia refringens detection and characterisation by PCR-RFLP 1 Scope – confirmatory method for detection of parasite in mussels and oyster Distinguish M.refringens types (O type in oysters, M type in mussels and C type in clams) Method was developed by Le Roux et al. (2001) Steps of the method: DNA extraction, preparation of PCR mix sample dispensing amplification and gel loading

Marteilia refringens detection and characterisation by PCR-RFLP 2 Dna is extracted from piece of digestive gland using QIAamp DNA Mini Kit (QIAGEN) according to manufacturer instructions Set of primers described in Le Roux et. 2001 (M2A ansdM3AS) Positive and negative control Amplification in thermocycler Interpretation of results (positive 412 bp) PCR product are digested with HhaI which are different for O or M type In recent time we switch from RFLP to gene sequencing which enable comarison of blast and determination of type O,M or C

RESULTS OF PCR FOR Marteilia refringens IN SAMPLES OF MUSSELS

In situ hybridization for both parasites In Situ Hybridization (ISH) is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section The underlying basis of ISH is that nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached Sensitive method, advantage – it is possible to detect pathogen in paraffin blocks Steps: deparaffinisation, deproteinization (proteinase K), hybridization (with digoxigenin probe), washing and detection (anti-digoxigenin-alkaline phosphatase conjugate), counter staining and mounting, microscoping

VIRAL NERVOUS NECROSIS(VNN) Synonim Viral encephalopathy and retinopathy Betanoda virus from the family Nodaviridae SJNNV (stripped jack nervous necrosis virus) Diagnostics Clinical signs Histology, Immunohistochemistry Virus isolation on SSN 1 cell line succeeded by IFAT or Immunoperoxidase identification Conventional RT PCR Real time PCR

It is difficult to diagnose virus in carrier fish Cell culture detection is not enough sensitive even in clinical outbreaks Conventional PCR tecniques were appropriate for confirmation of disease during outbreaks Surveillance of virus in free living population requires more sensitive method Panzarin et al. (2010) developed and validated real-time TaqMan PCR for detection of low titers betanodavirus (hydrolysis probe which increases specificity of real time protocol)