The design of fish holding facilities for toxicological, biomedical and behavioral studies is often based upon the welfare requirements for maintenance of the fish in terms of water source and volume, flow rates, temperature and environmental enrichment requirements. However, consideration also needs to be given to the prevention and control of infectious disease.
Fish housing systems are typically configured in one of three main designs; static, flow-through, or recirculating. Systems can also be configured using a combination of these designs. Recirculating and static configurations are most commonly used for small tropical fish like zebrafish and medaka, where as flow-through is most commonly used for larger more traditional aquaculture species like salmon and trout. (Ostrander, 2000
; Wedemeyer, 2001
; Astrofsky et al., 2002a
; Courtland, 2002
; Stoskopf, 2002
) The design of fish holding facilities, including the rooms, corridors and tanks is crucial for effective bio-containment, prevention and control of infectious disease.
Water sources for these systems can come from municipal sources (tap water), unprotected sources (surface water from lakes or rivers), protected sources (wells, aquifers, or springs), or from artificial sources (reverse osmosis or distillation). The latter source is used most commonly in zebrafish and medaka facilities, whereas municipal or protected sources are used most commonly with larger aquatic research facilities and aquaculture facilities. Regardless of the source selected, the water must be adequately pre-treated (particulate filtration, degassing, activated carbon filtration, sand filtration, and UV sterilized) to remove unwanted agents (e.g., chlorine, chloramine, heavy metals, excess dissolved gasses, etc.) before it should come in contact with the fish species to be housed (Harms, 2003
; Wedemeyer, 2001
; Courtland, 2002
). If a process like reverse osmosis or distillation to pre-treat water is used, then salts, minerals, and electrolytes will have to be added after these filtration processes. There should be enough on-site holding capacity of pre-treated water or the ability to provide a constant supply, depending upon your systems configurations, especially in the event of emergencies.
Tank rooms should be located away from laboratory areas that may be used for disease diagnostic purposes and should have controlled access for permitted laboratory staff only. Standard procedures for entering the facility (logging in and donning protective clothing) need to be established before entering specific rooms. Careful logging of materials entering (and leaving) the facility should also be maintained. Individual tank rooms may be used for different purposes, based on the size and shape of tanks within the rooms or the nature of the work to be undertaken. For efficient cleaning and disinfection, the floors, walls and even ceiling finishes should be non-porous, impervious, non-corrosive, and resistant to disinfectants. Light fixtures should be sealed and be of waterproof construction. If natural light is not provided from windows, then lights should be on programmable timers in order to provide adequate photoperiods for the fish species housed. Light timers must not be routinely overridden and if access to the animal housing area is required during the dark portion of the light cycle red lights can be used to help minimize interruptions in established photoperiods. Electrical outlets should be individually ground fault interrupted (GFI), be connected to specific GFI circuits and located in areas in which their exposure to water is minimized. In areas where this is not possible, outlets should have appropriate protective covers (plastic bubble type or self closing type). For each room, there should be a hand-washing sink or hand sanitizers for staff entering and leaving the room. If necessary there should be facilities for disinfecting footwear before entering and leaving. This additional measure depends upon the type of work done within the facility and the biosecurity measures required. Within each tank room, the positioning of each tank is usually determined by the space available and the original designed layout of the room, particularly for insulated tanks containing larger volumes of water. Care must be taken to avoid cross contamination by splashing. Moreover, fish pathogens can also be spread by aerosols created by aeration (Wooster and Bowser, 1996
; Roberts-Thomson et al., 2006
). Adjacent tank lids should be kept down and if practical, physical barriers be installed between tanks. Each tank room needs an area of benching for manipulation of aquaria, etc. Because of the liberal use of disinfectants and possible need to conduct experiments with marine fishes, fittings should be stainless steel or plastic wherever possible. To facilitate cleaning and disinfection of the room, storage of consumables and equipment within tank rooms should be minimized. A dedicated feed storage area outside the tank rooms but within the bio-secure area should be provided. For large species, a post mortem room or area should be similarly provided with easily disinfected surfaces and stainless steel benches.
The size and shape of tanks can be extremely variable and custom designed systems are often built. In general, tank systems for larger fish often utilize round opaque and insulated tanks between 50 and 1000 l capacity with continuous flow-through, whereas toxicological or behavioral studies frequently use small fish species in glass or plastic aquaria maintained on rack systems in temperature-controlled facilities with limited water exchange depending on the experiment. Glass should be minimized due to its extra weight and the issues and dangers associated with breakage. Zebrafish and other small tropical species racks, with self contained recirculating systems have become popular. Large facilities have begun incorporating large recirculating systems with sometimes redundant capacity in which the same water system may be used for hundreds of tanks. These are equipped with industrial grade biofiltration systems (like fiuidized sand filters) and multi-lamp UV sterilizers. Regardless of the size or scope of the systems, it is critical to perform and record routine scheduled maintenance on each systems associated components at least as per manufactures recommendations. It is very important to adequately pre-filter the water to remove particulate waste, maintain the clarity of the quartz sleeves and to change the UV bulbs at the recommended schedules to optimize the effectiveness of UV sterilizers. UV dose is defined as intensity × time and is expressed in units of microwatts per square centimeter per second (mW/cm2
/s). The required dose is related to the size and transparency of the organism to UV radiation (Wedemeyer, 1996
). Recommended UV dosages needed to inactivate common fish pathogens are as follows. For bacteria like Aeromonas
spp., and Pseudomonas
spp. between 4000 and 5000 is required. As low as 2000 is required for viruses like Infectious Hematopoietic Necrosis Virus (IHNV) and Channel Catfish Virus (CCV), but agents like Infectious Pancreatic Necrosis Virus(IPNV) 150,000 is required. For myxozoans (e.g., Myxobolus cerebralis
) 27,600 is required. To inhibit the growth of fungal hyphae from Saprolegnia
spp. requires at least 230,000 (Wedemeyer, 1996
). Microsporidia (now also considered relatives of fungi) are more fragile. A three log reduction in viability of Encephalitozoon
spp. spores requires between 6 and 19,000 (Huffman et al., 2002
; Marshall et al., 2003
). Indeed, our observations from one large zebrafish facility suggest that 30–50,000 reliably kills spores of Pseudoloma neurophilia.
A principle design feature should be that cleaning and maintenance can be easily carried out. Invariably, this means that adequate space is provided between tanks or runs of tanks. There are compromises to be made regarding maintenance of pipe work, valves, etc. and the need for disinfection and cleaning. Each tank or water system must have its own net(s) and other equipment as required with facilities within the room for initial disinfection of these.
All facilities should have a dedicated quarantine room, or at least a separated quarantine area. As the name implies, strict separation of fish, water, and equipment must be maintained between the quarantine area and the main facility. This is accomplished by having dedicated equipment and only staff trained in sterile or aseptic techniques and other policies for avoiding pathogen transmission that are allowed in the area. Rigid policies should be in place to ensure staff working inside the quarantine area does not transfer potential pathogens outside of the area or into the main facility. Policies should include having designated protective clothing, dedicated husbandry and care equipment for use in the quarantine facility, prohibiting staff from moving directly from quarantine to the main facility, hand washing after leaving the facility, and thorough disinfection of equipment and wastes required to leave quarantine. For the former chemical disinfection may be adequate but for the latter, autoclave disinfection may be advised (Wedemeyer, 2001
; Harms, 2003
Appropriate training of husbandry and investigative staff in understanding the importance of control of infectious diseases and procedures to avoid pathogen exposures is critical. Prior work experience of fish husbandry staff in many research facilities comes from caring for pet or ornamental fish, which can be entirely different than caring for large numbers of valuable research animals. At least one staff member should have some training in fish health, and this person could be the key point to interact with the institution’s laboratory animal veterinarian and outside diagnostic laboratories. Several short courses on fish health are available and many are geared towards aquarists and aquaculturists, fish health professionals and veterinarians. The staff should follow written standard operating procedures (SOPs). These documents are considered “living documents” in that they should be modified or updated as frequently as necessary by the manager or supervisor such that these SOPs reflect what is actually being done by personnel within the laboratory and animal facilities.