SOP: VI.483 Edition 6 (26.11.2018)
1. Introduction
1.1 Purpose and scope of this procedure
1.1.1 To describe methods for collecting samples for the following testing regimes – virus isolation in eggs/cell culture, extraction for virus detection and pathological examination of avian carcasses.
1.2 Background information
1.2.1 Avian carcasses are submitted for diagnosis of Avian Avulavirus type 1 (AOAV-1) and Influenza virus infection. This requires tissues being taken from carcasses and used for virus detection methods.
1.2.2 The avian carcasses are placed in a microbiological Class I/III safety cabinet on arrival and opened up using the necessary instruments.
Pieces of tissue from a variety of organs are taken, finely minced and placed in antibiotic-PBS or formalin based fixative.
2. Safety
2.1 It is the responsibility of the individual laboratory to ensure all work described in this protocol is conducted to a high safety standard. This includes an awareness of risks relating to e.g. dangerous or toxic chemicals, potentially hazardous procedures etc. Here local safety rules in your laboratory should be understood by all relevant members of staff.
2.2 Areas within this procedure which refer to Safety Critical activities are denoted in the paragraph number column with the sign to highlight these areas to users.
2.3 All sample material should be treated as potentially hazardous, wearing gloves and working in Microbiological Safety Cabinet class I/III when preparing material for further testing. Particular care should be taken when handling infected tissue from avian exotic species- from known or suspected disease premises. Before commencing work for each submission the safety requirements, including working in a Microbiological Safety Cabinet (MSC) in Class I or Class III mode, should be assessed. Several key factors should be taken into account including the type of submission/samples and the different procedures or methods being used at each stage, and their respective safety requirements.
2.4 Refer to BAAC 2006/06 and other local specific building and equipment risk assessments (RAs) and procedures before commencing work.
2. 5 Prepare or read risk assessment for the Preparation and use of Formalin Based Fixatives
2.6 Prepare or read risk assessment for the filtering of clinical material
3. Materials
3.1 Documentation and software
3.1.1 Procedure for: Virus isolation in eggs
3.1.2 Procedure for: Virus isolation in cell culture
3.1.3 Follow equipment-specific manuals for the use of equipment
3.1.4 Procedure for: Operation of TriMAT Microbiological Safety Cabinets in Class III mode.
3.1.5 Procedure for: Extraction of nucleic acids from swabs using the QIAGEN BioRobot Universal.
3.1.6 Risk assessment for: Preparation and use of Formalin Based Fixatives.
3.1.7 Guidelines for the post mortem investigation and sampling of poultry in the event of a notifiable disease investigation.
3.2 Chemicals and reagents
3.2.1 Antibiotic phosphate buffered saline (PBS)
3.2.2 Formalin for fixation of tissues.
3.2.3 70% Ethanol (replenish once a month). NB: for use as a cleaning agent, not to be used for surface decontamination.
3.2.4 Virkon S – use at 1/100 (1% w/v) made up with water. Replenish every 5 days, or if colour is not a strong pink colour. Contact time 10 minutes.
3.3 Equipment
3.3.1 Microbiological Safety Cabinet class I/III
3.3.2 Sterile bone cutters, scissors, scalpels and forceps – one set of instruments per tissue type per epidemiological unit (if in class III one set of instruments per tissue)
3.3.3 Sterile universals, falcon tubes and bijou approved for freezer use
3.3.4 Sterile disposable petri dishes
3.3.5 Disposable gloves
3.3.6 Cut resistant gloves
3.3.7 Centrifuge including buckets, lids and ‘O’ rings – capable of being maintained at the speeds specified in this SOP.
3.3.8 PPE - Disposable apron and over sleeves (if in class I).
3.3.9 Autoclave bags, poly bags and ties
3.3.10 2ml, 5ml Luer-lok syringes
3.3.11 0.8µm, 0.45µm and 0.2µm sterile single use filters.
3.3.12 Sharps box
3.3.13 Fumigation strip
3.3.14 Absorbent tissue
4. Procedure: method
4.1 Sample preparation
4.1.1 Four standard tissue pools are routinely taken from each carcass during PM; 1) brain, 2) lung and trachea, 3) pooled viscera (heart, liver, spleen and kidney) and 4) pooled intestines. The pooled intestines should include caecal tonsil, jejunum and colon contents. Tissue pools from a maximum of four carcasses from the same epi group may be pooled together. Senior management may request that a duplicate set of standard tissue pools are prepared for differential diagnosis of avian disease.
4.1.2 If original tissues are required (i.e. for further downstream testing or for
archive) the following individual tissues should be taken; brain, lung, trachea, heart, kidney, spleen, liver and intestines. Intestines should include caecal tonsil, jejunum and colon contents. Senior management may request additional tissues in some circumstances. These original tissues and any additional tissues should be collected into containers suitable for freezing and stored at -80°C.
4.1.3 If the carcass is too large to fit into an MSC, senior management will decide on the best course of action i.e. whether swabbing of the cloaca and/or oropharyngeal areas is to be carried out instead of tissue collection, or whether the carcass is to be PM’d. If the carcass has been opened prior to post mortem seek clarification from senior management to confirm the sampling method – tissues or swabs.
4.1.4 Take one universal of antibiotic PBS for each tissue pool required and label each with a LIMS generated label if available. If not available, ensure the universal is uniquely identified. Label up petri dishes for the different tissue types.
4.1. 5 Set up an MSC cabinet (preferably a large cabinet). Take in the following equipment – white roll, autoclave bags, ties, labelled universals + rack, sterile instruments – forceps, scissors and scalpel (one set per tissue pool) and carcasses.
4.1.6 Tissue samples for virus detection must be taken out using separate sterile instruments. Each tissue type must be kept in separate petri dishes to reduce the risk of cross contamination. The order of the tissue sampling for report cases is: Pool 1 – Brain, Pool 2 - Viscera- heart, liver, spleen and kidney, Pool 3 - lung and trachea and pool 4 – intestines (caecal tonsil, jejunum and colon contents).
4.1.7 Wearing cut-proof gloves on both hands with disposable gloves on top, place the bird on its back and make an incision is made across the skin of the head to reveal the skull. Open up the skull and extract the brain and place in a sterile petri dish. The rest of the carcass is opened by cutting up the front of the breastbone around both sides and opening up the rib cage. Take out the relevant tissues in the order as described in 4.1.6.
4.1.8 Gross examination of the bird is performed and tissue samples are also taken for pathological analysis.
4.1.9 If original tissues are required split each tissue type in two; half for the universal containing antibiotics-PBS and half for a sterile bijoux. Before placing the tissues into antibiotic PBS finely mince the tissues in the labelled sterile petri dishes using scissors and/or a scalpel.
4.1.10 An approx.10-20% (w/v) suspension of tissue in antibiotic PBS is required, so the volume of antibiotics in the universal may need to be adjusted prior to adding the tissue depending on the amount of tissue to be added. The tissue in bijoux must be clearly labelled and stored at - 80°C, ensuring the relevant freezer inventory is updated.
4.1.11 Allow the processed tissue suspensions to stand for 1-2 hours at room temperature. Suspensions should be processed as soon as possible after this incubation period. If immediate processing is impracticable, samples should be stored at 4oC for up to 4 days.
4.1.12 To process, centrifuge the tissue suspensions in centrifuge buckets with lids on for 5 minutes at 2000 rpm. Ensure that universals are suitable for use in the centrifuge being utilised and use adaptors where necessary. Return the centrifuge buckets to the cabinet, open them in the cabinet and take out the tubes, ensuring they are kept upright
4.1.13 Tissue pool supernatants are ready for the extraction of nucleic acids for PCR testing – Extraction of nucleic acids
4.1.14 If virus isolation is to be carried out, and there is a suspicion of bacterial contamination, the tissues pool supernatants are filtrated to reduce the risk of contamination in eggs. Use a 2 or 5ml luer-lok syringe to take up some supernatant and filter into a labelled bijoux/universal through a 0.45μm filter. This must be carried out in a class III cabinet.
4.1.15 The filtrate is ready for inoculation into eggs or tissue culture – refer to relevant procedure for Virus isolation in eggs and Virus isolation in cell culture.
4.2 Sample storage
4.2.1 Tissue pools in antibiotics-PBS are stored in the fridge until the tests is complete. All original tissues must be frozen down in a designated freezer.
4.2.2 Any positive egg passage material or tissue culture fluid must be frozen down in a secondary container.
4.3 Disposal
4.3.1 Carcasses must be placed into doubled bags and tied and a specified method of disposal followed.