1. SAMPLE SUBMISSION AND OTHER REQUIREMENTS FOR DIAGNOSTICS Snježana Zrnčić, PhD, DVM TCDC/TCCT Consultant No 2 – Diagnostics zrncic@irb.hr

2. REQUIREMENTS FOR ACCURATE DIAGNOSIS AT THE SAMPLING SPOT Proper choice of fish/molluscs specimen at the cultivation facility Timing of sampling (for VHS/IHN water temperature should not exceed 14 o C, for KHV it should not be under 22 o C) Molluscs for bonamiosis in spring, for marteiliosis in the autumn before decrease of sea temp. to 17 o C If lab personnel is not present during sampling all those data together with location of sampling, farm registration, number of specimen in sample, date of sampling, and responsible person should be supplied on the document accompanied purcell

3. PACKING AND SHIPPING  Veterinarians authorised for sampling on aquaculture establishment should be well educated and trained for sampling, packing and mode of shipping of samples  All samples (whole fish, molluscs or organs in transport media) should be protected of contamination  Each pool of fish/molluscs should be packed in separate package (plastic bag) properly marked  liquids should not pour our of package  Package (cooling box or styrofoam box) should maintain the temperature (must not exceed 10 o C)  Purcell must be supplied with enough freezing blocks to secure this temperature during the whole transportation  Regular post must be avoid

4.  If there is no possibility for vets to transport samples to diagnostic lab, reliable courier service with experience of transportation of cold purcells should be chosen  Some bad experience with some courier services during summer months  Purcell must be properly tagged (name, address and phone number of sender’s organisation, address, phone etc of laboratory, compulsory conditions, time of delivery)  Document describing the purcell must be properly protected of slubberness and wetting

6. UPON ARRIVAL  Accompanied documentation must be checked to register the sample  Temperature inside the purcell must be measured be check  If those conditions are satisfactory, it is possible to deliver samples to the diagnostic laboratory  In the lab – registration of samples, opening of the required protocols according to the sort of samples  Determination of the priorities

9. PROCESSING OF SAMPLES According to the sample  Organs in transport media (EMEM with 10% FBS and pen/strep buffered with HEPES or TRIS) for virological examination shoul be subjected to the virological testing on the cell lines no later that 48 hours after sampling  Organs are homogenized in stomacher or mortar and pestle with sterile sand, diluted with transport media in ratio 1:10, centrifuged on 5000 rpm at 40C for 20 minutes, filtered through 450 μm and incubate for 4 hours at 150C or overnight at 40C  In the exceptional situation period between sampling and inoculation of cell lines should be prolonged to 72 hours  During some unpredictible situation exceptionally, a sample can be freeze at -800C and the inoçulation of cell line can be prolonged for 15 days

10. TARGET ORGANS FOR VIROLOGICAL EXAMINATION OF SALMONIDS Anterior kidney Heart Spleen

11. TARGET ORGANS FOR VIROLOGICAL EXAMINATION OF SALMONIDS HEART

12. PROCESSING OF SAMPLES  According to the sample  Organs in transport media (EMEM with 10% FBS and pen/strep buffered with HEPES or TRIS) for virological examination shoul be subjected to the virological testing on the cell lines no later that 48 hours after sampling  Organs are homogenized in stomacher or mortar and pestle with sterile sand, diluted with transport media in ratio 1:10, centrifuged on 5000 rpm at 4 0 C for 20 minutes, filtered through 450 μm and incubate for 4 hours at 15 0 C or overnight at 4 0 C  In the exceptional situation period between sampling and inoculation of cell lines should be prolonged to 72 hours  During some unpredictible situation exceptionally, a sample can be freeze at -80 0 C and the inoçulation of cell line can be prolonged for 15 days

13. PROCESSING OF ORGAN SAMPLES FOR CELL CULTURE

14. PROCESSING OF MUSSELS FOR CYTOLOGICAL AND MOLECULAR DIAGNOSTICS

15. STORAGE OF SAMPLES  Samples submitted for laboratory diagnostics must be stored until the written report has been issued  According to lab protocols about service to customers all negative samples are stored two weeks after completing the examination and positive samples are stored a month  Samples for virological testing on cell cultures are stored at - 80 o C  Samples submitted for cytologycal (stained tissue imprints )and histological testing for molluscan diseases are stored one year after completing testing

16. LABORATORY ORGANISATION AND EQUIPMENT  Laboratory should be organised in the manner that there is no possibility for cross contamination – it means that dissection should be 1st step, cell culture propagation should be performed in separate LAF with no possible contact with infectious material and work with fish viruses in another facility – IMPORTANT TO ENSURE ACCURATE RESULTS OF TESTING  Equipment must be calibrated (pipettes), termometers, incubators and they should operate in allowed range, centrifuges should be calibrated for speed and temperature, spectrophotometers should be calibrated in proper periods

17. MARKING AND TRACKING  Traceability is the very important element during the whole process  The same mark should be traceable from submission of the sample, during all step of laboratory procedure until reporting  Used cell lines (number of passage, date of subcultivation) should be marked with aim to give us possibility to go back through the whole process in the case of any suspicious of the correctness of the results  All used consumables (media, sera, chemicals etc. must be registered and marked

18. SECURITY MEASURES Cleaning and disinfection protocols must be followed to avoid any possibility of contamination or spreading the pathogen from the lab All equipment should be properly cleaned and disinfected regularly, all disinfection procedures should be registered and efficacy should be controlled regularly (working surfaces, LAF, incubators, refrigerators etc.) Laboratory stuff should wear protective clothes, gloves, and shoes which are used only for lab work It is not allowed to wear same coats during dissection and cell propagation, Material for testing should be disposed according in the manner which will not harm environment or allow spreading of infective material or possilitity of chemical contamination