Scottish Marine and Freshwater Science Volume 4 Number 3: Epidemiology and Control of an Outbreak of Viral Haemorrhagic Septicaemia in Wrasse Around Shetland Commencing 2012

Appendix 2

Wild Fish Sampling Protocol

I.S. Wallace

Sampling Strategy

This uses the approximate proportions of the main species present in the catch to determine numbers to sample. This basis for choosing this strategy is that VHSV appears to be ubiquitous in species of wild marine fish therefore targeting specific species may result in under detection in the local populations.

Sampling Location

Sampling will be undertaken in a laboratory at the NAFC Marine Centre with the fish being delivered to the back door each day.

Target Tissues

Kidney, spleen, heart and brain will be taken from each fish into VTM.

Pool Size

Fish will be pooled in tens.

Tissue Sampling Protocol

• Sort fish into respective species outside and count the number of each (roughly)
• Based on proportions of each species present decide on the number of each to sample
• Bring the fish inside
• Wearing sterile disposable gloves, lay 10 (depending on fish size) fish on a stainless steel dissecting tray overlaid with paper towel. Place round fish with head to the left and flat fish eyes up (will depend on if left or right eyed)
• Complete the sample record sheet
• Using 70% ethanol solution and paper towel surface sterilize each fish
• Using a sterile disposable scalpel cut open the body and cranial cavities of each fish in the pool (scalpel 1)
• Change gloves and scalpel (scalpel 2)
• Open the body cavity of the first fish using a gloved hand, remove samples of spleen, kidney and heart tissue and place in a labelled VTM tube (scalpel 2)
• Repeat for each fish in the pool
• Using the same scalpel (scalpel 2) remove a sample of brain tissue and place in the same labelled VTM tube
• Repeat for each fish in the pool
• Note: Final tissue volumes in each pool must be between the minimum and maximum marks on the virus reference tubes
• Used scalpels and forceps go into a sharps container
• All sampled fish are to be double bagged in heavy duty black bin liners
• Used towel roll and gloves go into a separate black bin liner
• Clean the tray with a solution of 70% ethanol
• The labelled VTM tube goes into a stainless steel rack contained within a polystyrene cool box and freezer blocks
• When the bags are full, or at the end of the sampling day, they must be put into the freezer adjacent to the laboratory and labelled as to the contents.

Additional

• As the site adjacent to the sampling laboratory has a movement order for VHS in place careful bio-security must be implemented at all times.
• On arrival at the laboratory, prior to any sampling taking place, all sampling surfaces within the sampling laboratory are to be disinfected using a solution of virkon.
• Warning signs will be displayed on the outside of the sampling laboratory doors to inform non-sampling personnel of the nature of the work being undertaken and to stay out.
• Bring into the laboratory only what is necessary to undertake the sampling. Store un-used consumables within the vehicles until required.
• On completion of sampling all bench surfaces and the floor are to be cleaned and disinfected using a solution of virkon.
• Any equipment being returned to the MSS laboratory must be cleaned and disinfected prior to leaving the site and again on return to the MSS laboratory comprising: stainless steel dissecting trays; ethanol spray bottles; polythene boxes used to transport samples and any personal protective equipment such as wellington boots and oil skins. Used sharps containers must be locked shut and tied in clear polythene bags for incineration.
• Waste bags containing sampled fish and used consumables are to be incinerated at the local Shetland Islands Council energy recovery plant.