General course objectives
The course aims to provide the participants with knowledge on the most used methods for diagnosis of important fish viruses. The course will focus on; 1) basic cell cultivation techniques, production of cells for different purposes (IFAT, diagnostic trays, titration, etc.), cell susceptibility testing, inoculation of samples and sub-cultivation procedures, reading of cell cultures (including CPE) and virus titration, 2) providing the participants with knowledge on the most used methods for diagnosis of important fish pathogens, 3) Real-Time PCR protocols validated for surveillance of listed fish diseases, 4) genotyping the most important viral pathogens by sequencing and blasting and 5) information on the underlying principles of the tests and how to critically review them in order to assess pitfalls.
The major focus will be on the viral fish diseases using VHS as model. The course will provide a forum where pre-knowledge, experience and examples can be discussed between participants and teachers, and hereby raise the awareness of pitfalls when using the various techniques.
This 5 day course is primarily based on practical work (hands on) in combination with theoretical presentations. This year the course will focus on the comparison between diagnostic techniques for listed fish diseases by evaluating pros and cons of cell culture and Real-Time PCR methods.
At the first day of the course all participants will take part in a fish farm visit. The trip will be supervised by the Danish Veterinary and Food Administration, Section for Aquaculture, that will provide guidelines and protocols for inspection, necropsy and sample collection. Organ material will be collected on site for processing in the lab the following days. During the farm visit sampling procedures will be demonstrated and afterwards conducted by each participant. Starting from day 2, theoretical class room teaching will be given on methodologies, pitfalls and troubleshooting. After the introduction, the participants will be divided into groups. Each group will start processing the samples collected at the farm. This process will include cell culture preparation, inoculation on monolayers, observation of cell culture and assessment of cytopathogenic effect. All cell cultures used for isolation of the listed viruses will be demonstrated and procedures will be conducted by the participants.
Participants will initially be introduced to basic cell culture work: 24-well plate preparation for diagnostic purpose as well as 96-well plate for titration and flask maintenance will be demonstrated and subsequently prepared by the participants. Inoculation of diagnostic samples on cell cultures will be practised. The CPE of different viruses will be shown and the participant will practise reading of diagnostic trays. Titration procedures will be demonstrated and afterwards participants will have the opportunity to practise by themselves. Finally, course participants will calculate virus titres.
The application of novel validated and accredited Real-Time PCR protocols suitable for surveillance will presented and discussed with the participants. This year there will be more focus on genetic characterization of listed fish viruses with theoretical lectures and practical exercises on sequencing, Blast tool and phylogenetic analysis. For this reason each participant is required to bring his/her own laptop.
The course is dialogue-based and sufficient time will be given for discussions throughout the course and for the evaluation of test results. Quality assurance, cleaning, disinfection, etc. will be an integral part of the practical demonstrations. The taught methods will primarily focus on the protocols given within the EU legislation and OIE guidelines from the Manual of Aquatic Animal Diseases, and include how to select proper controls, the typical pitfalls and troubleshooting, etc. A social dinner will be organized on the second evening. Further details are provided in the invitation letter.
A student who has met the objectives of the course will be able to:
- Sample and process material for diagnostic purpose
- Maintain, cultivate, inoculate and read most used cell lines (BF-2, EPC, CCB and ASK) for fish disease diagnostic purposes
- Prepare cell cultures for different purposes, e.g. diagnosis, IFAT and virus titration
- Inoculate and sub-cultivate diagnostic samples
- Read diagnostic trays
- Titrate virus
- Apply Real-Time PCR for surveillance purposes
- Genotype important viral isolates by sequencing and blasting
- Assess pitfalls and errors in test performances and designs.