ZEBRAFISH Volume 13, Supplement 1, 2016 ª Mary Ann Liebert, Inc. DOI: 10.1089/zeb.2015.1206
Biosecurity and Health Monitoring at the Zebrafish International Resource Center Katrina N. Murray,1 Zolta´n M. Varga,1 and Michael L. Kent1,2
Abstract
The Zebrafish International Resource Center (ZIRC) is a repository and distribution center for mutant, transgenic, and wild-type zebrafish. In recent years annual imports of new zebrafish lines to ZIRC have increased tremendously. In addition, after 15 years of research, we have identified some of the most virulent pathogens affecting zebrafish that should be avoided in large production facilities, such as ZIRC. Therefore, while importing a high volume of new lines we prioritize safeguarding the health of our in-house fish colony. Here, we describe the biosecurity and health-monitoring program implemented at ZIRC. This strategy was designed to prevent introduction of new zebrafish pathogens, minimize pathogens already present in the facility, and ensure a healthy zebrafish colony for in-house uses and shipment to customers. Introduction
T
he Zebrafish International Resource Center (ZIRC) has been operating in a stand-alone aquatic facility on the University of Oregon campus since 2000. Our mission is to acquire, maintain, and redistribute zebrafish resources to the international research community. ZIRC also operates a diagnostic pathology and health consultation service. Fulfilling ZIRC’s mission necessitates a high volume of line imports and exports. Originally, this entailed importing individual lines as adult fish into the Quarantine Room. These fish were spawned in the Quarantine Room and their surface-sanitized embryos were moved to the Main Fish Room. The lines were then maintained and propagated in the Main Fish Room to satisfy pending customer requests and to cryopreserve them for longterm storage. As researchers began producing new mutant and transgenic lines in large-scale projects,1–3 ZIRC had to adopt a new importation strategy to accommodate a higher volume. We have shifted priorities from importing and maintaining new lines as live fish to importing and banking their cryopreserved sperm.4 From some facilities, lines are imported directly as cryopreserved sperm and others are still imported as live fish and cryopreserved in the ZIRC Quarantine Room. Using this approach, we have exponentially increased the number of zebrafish lines imported and available from ZIRC (Fig. 1). There are several demonstrated and potential impacts of pathogens on zebrafish in research.5 Pathogens that are most common or are of greatest concern are summarized in Table 1.6–23 In addition to virulent pathogens causing signifi1 2
cant mortality, infections in apparently healthy fish can cause reduced fecundity6 and confuse research endpoints such as behavior,7 development, and disease studies.24,25 At least one zebrafish pathogen, Mycobacterium marinum, is recognized as a cause of disease in humans working with fish.16,26 Increasing the volume of imported lines has also had important implications for the ZIRC biosecurity program, particularly because several zebrafish pathogens are highly virulent and many survive freezing (e.g., cryopreservation) in fish tissue and in media for long-term storage.13,27–29 Along with banking and redistributing resources for the research community, we place a high priority on animal health, monitoring, and disease prevention. We are constantly evaluating the risk of importing and moving pathogens along with fish resources. The microsporidian Pseudoloma neurophilia, a common pathogen of zebrafish, is capable of vertical transmission within ova,8 and the ability of other virulent pathogens to be maternally transmitted or survive in cryopreserved sperm samples is not fully understood. Given the existing knowledge, our biosecurity program is designed to identify and minimize these risks while ensuring a high level of animal welfare and animal and human health. Here, we provide an overview of major operations at ZIRC and identify key risk points for biosecurity. We describe our program for importation of new lines as live fish and cryopreserved sperm while minimizing risks for simultaneously importing pathogens. Finally, we describe the extensive health monitoring undertaken at ZIRC, which allows us to constantly assess individual, tank, and colony health.
Zebrafish International Resource Center, University of Oregon, Eugene, Oregon. Department of Microbiology, Oregon State University, Corvallis, Oregon.
S-30
BIOSECURITY AND HEALTH MONITORING AT ZIRC
S-31
FIG. 1. Annual number of new lines imported to ZIRC from 2001 to 2014. ZIRC, Zebrafish International Resource Center.
Facility Overview
Zebrafish lines are maintained in three separate areas at ZIRC; the Main Fish Room, the Freezer Room, and the Quarantine Room. The Main Fish Room operates on recirculating water systems with mechanical and biological filtration (Fig. 2). Municipal water is purified by reverse osmosis units, which have mechanical and carbon prefilters. Instant Ocean salt and Aragonite are added, conditioning the water to a conductivity of 500 lS/cm and pH between 7.2 and 7.6. Approximately 10% of the water is exchanged per day. The Main Fish Room water and air temperature are maintained at 28.5C. System and water parameters, including conductivity and pH, are monitored electronically.30 Separate recirculating water systems supply each half of the Main Fish Room (designated sides A and B). UV sterilizers are present on each system, delivering a minimum dose of 132,000 lWsec/cm2 (at max water flow rate of 200 gal/min, 80% lamp life, and 98% water transmissibility). Pressurized sand filters and optional bag filters on a bypass loop were installed to reduce fine particulates, which might shield pathogens from UV irradiation. Bag filters are used following acute increases in fine particulates resulting from electrical and mechanical disruptions that alter system water flow through the fluidized sand beds. The Freezer Room holds 6 MVE TEC 300 vapor-phase liquid nitrogen freezers for storing cryopreserved sperm. Six empty freezers are available for future storage. The Quarantine Room is in the same building, but physically separate from the Main Fish Room. A flow-through water system feeds the Quarantine Room. Municipal water runs through reverse osmosis filters and is treated with Instant
Ocean salt and Aragonite for conditioning. Effluent drains to the municipal sewer system. The room temperature is set to 28.5C. Husbandry Overview
All embryos are surface-sanitized with 30 ppm sodium hypochlorite for 10 min. New sodium hypochlorite solution is prepared daily using refrigerated laboratory grade sodium hypochlorite (Fisher Scientific) and reverse osmosis water. To minimize potential for transmission of pathogens between stocks at spawning, wild-type fish that have been used for out-cross are not returned to their original tank. They are euthanized or maintained in a separate 1-gallon (3.8 L) tank to be reused for breeding with the same mutant or transgenic stock. Separate anesthesia and recovery solutions are used for each tank of fish. Stocks are generally maintained until 1 year of age. Fish are euthanized by hypothermal shock or overdose of tricaine Methanesulfonate31 (S-Tricaine, Western Chemical). All protocols have been approved by the University of Oregon Institutional Animal Care and Use Committee. Fish are housed in 1-gallon plastic or 20-gallon (76 L) glass aquaria in the Main Fish Room. Only 1-gallon plastic tanks are used in the Quarantine Room. Twenty-gallon tanks hold 150–250 fish. One-gallon tanks hold up to 25 fish. Snails have never been used in ZIRC tanks. The light cycle is set for 14 h of light and 10 h of dark. Fish are fed Great Salt Lake brine shrimp, Artemia salina, nauplii (Artemia International) in the morning and a powdered feed mix (powder feed recipes are available on-line http://zebrafish.org/documents/protocols.php) in the afternoon. Juvenile fish and males being conditioned for sperm
S-32
Chronic inflammation. Low mortality.
Severe, systemic disease. Zoonotic risk for employees. Severe, chronic inflammation. High mortality. Severe, chronic enteritis and severe emaciation. Moderate to high mortality.
Mycobacterium chelonae
Mycobacterium marinum
Observed in renal ducts. Lesions are rare.
Myxidium streisingeri
ZIRC, Zebrafish International Resource Center.
Severe systemic disease. High mortality.
Edwardsiella ictaluri
Pseudocapillaria tomentosa
Mycobacterium haemophilum
Emaciation, reduced fecundity, and behavior changes. Low mortality.
Pathology and impact
Pseudoloma neurophilia
Name
No. Likely requires alternate worm host to complete life cycle.
Unknown. Not observed within eggs.
Unknown. Numerous bacilli observed in ovaries and associated with testes. No. Possible contamination of zebrafish embryos with feces containing P. tomentosa eggs during spawning.
Unknown. Bacilli observed in ovaries and occasionally testes. Unknown. Bacilli rarely observed within eggs.
Yes. Documented intraovum transmission.
Maternal transmission
ZIRC risk category Low risk. Widespread in zebrafish facilities. Presently at ZIRC. Control measures are in place. Spores survive freezing and chlorine surface disinfection of embryos. Low risk. Widespread in zebrafish facilities. Presently at ZIRC where it is generally limited to a low prevalence of asymptomatic fish. Survives freezing. High risk. Not at ZIRC. Common in aquarium fishes and occasionally seen in zebrafish facilities. Survives freezing. High risk. Not at ZIRC. Occasionally seen in zebrafish facilities. Results in significant morbidity and mortality. Survives freezing. High risk. Not at ZIRC. Occasionally seen in zebrafish facilities. Can result in significant morbidity and mortality. Disease is more severe in zebrafish lines with AB background. Ability to survive freezing is unknown, but unlikely. Eggs tolerate chlorine surface disinfection of embryos. High risk. Not at ZIRC. Not widespread in zebrafish facilities, but has been diagnosed in several aquarium fishes, particularly Danio species. Results in significant disease and mortality. Ability to survive freezing is unknown. Low risk. Not at ZIRC. Occasionally seen in zebrafish facilities.
Table 1. Pathogens and Risks for ZIRC
23
J. Shelley (pers. comm.) and Refs. 20–22
18,19
14,17
14–16
11–13
6–10
References
BIOSECURITY AND HEALTH MONITORING AT ZIRC
FIG. 2.
S-33
Diagram of the ZIRC Main Fish Room water filtration system.
cryopreservation receive an additional midday powder feeding. Larval fish are fed paramecia, cultured in-house, until day 10 when they are moved from static to flowing water and are fed powdered feed and brine shrimp, both twice daily. When large stocks are in the nursery, the larval diet may be supplemented with Branchionus plicatilis l-type rotifers (Reed Mariculture, Inc.). When 1-gallon tanks require cleaning fish are transferred to a new clean tank. Glass 20-gallon aquaria are cleaned with the fish in the tank. Tanks are scrubbed with a scouring pad and in-flowing water is increased to flush liberated tank debris and biofilm out the tank drain, minimizing fish exposure.11 Clean, autoclaved scrubbers and nets are used on every tank. Between fish populations, 1-gallon and 20-gallon tanks are scrubbed to remove visible debris, soaked in 0.15% bleach solution for at least 15 min, neutralized in 0.002% (w/v) sodium thiosulfate solution, rinsed in a tunnel washer with 82C water, and air-dried.
After spawning, their surface-sanitized embryos may be moved to the Main Fish Room or reared in the Quarantine Room. When lines are imported as cryopreserved sperm, the samples are stored in a vapor-phase liquid nitrogen freezer designated for samples frozen outside of ZIRC. Following customer orders, sperm from the freezer is thawed for in vitro fertilization (IVF) with AB eggs from the Main Fish Room. Resultant embryos are surface-sanitized and reared in the Main Fish Room, shipped to customers, or reared in the Quarantine Room. In the Main Fish Room, fish are spawned and surfacesanitized embryos may be shipped to customers or reared in the Main Fish Room. Adult zebrafish are also shipped to customers. Sperm from mutant and transgenic lines is cryopreserved and stored in designated vapor-phase liquid nitrogen freezers for future regeneration of lines. Biosecurity Risk Points and Response
Operational Overview
New lines come to ZIRC as live fish or cryopreserved sperm (Fig. 3). Live fish are imported into the Quarantine Room. For mutant and transgenic lines, we request 10 adult males be sent. They are acclimated for at least 2 weeks then anesthetized with tricaine methanesulfonate (168 mg/L) and sperm is obtained for cryopreservation. For wild-type lines, males and females are imported into the Quarantine Room.
Preventing introduction of new pathogens into the ZIRC Main Fish Room colony is a major priority of our health and biosecurity program. This includes pathogens already present at ZIRC in certain populations (e.g., M. chelonae and P. neurophilia) and those that are not (e.g., M. marinum, M. haemophilium, Pseudocapillaria tomentosa, and Edwardsiella ictaluri). Kent et al. (2009)32 provided recommendations for control of pathogens in fish laboratories. We
FIG. 3. Operational overview of movement of zebrafish adults, embryos, and sperm into and out of ZIRC rooms.
S-34
apply most of these approaches with certain modifications given the unique situation at ZIRC in which numerous fish are being both imported and exported on a monthly basis. We have identified points in our flow of operations that represent the greatest risks for pathogen introduction and have devised the plan described below to minimize these risks. Quarantine Room
The most likely route of new pathogen introduction into the facility is through live fish imported into the Quarantine Room. For most lines, only males are imported for cryopreservation of sperm. Following a customer request, the line is regenerated by IVF with AB eggs from the Main Fish Room. By prioritizing cryopreservation of lines over spawning and raising surface-sanitized embryos, we have limited the number of lines reared in the Main Fish Room. Moving lines through the liquid nitrogen freezers also provides an additional point at which pathogens associated with the imported stock can be reduced. While in the Quarantine Room, all stocks are monitored daily for morbidity and mortality. Fish displaying clinical signs of disease (physical or behavioral) are euthanized and a portion of liver and the spleen are dissected and frozen
MURRAY, VARGA, AND KENT
at -80C. The remaining carcass is fixed in Dietrich’s fixative and processed for histological sectioning and staining with hematoxylin and eosin (H&E). If mycobacteriosis is diagnosed in the tissue sections, the frozen organs are sent to the Oregon State University Veterinary Diagnostic Lab (OSU VDL) for a quantitative polymerase chain reaction (PCR) assay to identify the species of Mycobacterium in the sections. After acclimation, clinically normal fish in the Quarantine Room are anesthetized to obtain sperm samples for cryopreservation. When sufficient sperm samples have been obtained, the fish are euthanized and fixed for histopathology. If mycobacteriosis is diagnosed in tissue sections, the paraffin block with embedded fish tissue is sent to OSU VDL for the Mycobacterium PCR panel. We use the results of diagnostic testing of all fish in the Quarantine Room to assess the risk that pathogens may contaminate the frozen sperm samples (described below and Fig. 4). When wild-type lines are imported, our preference is to have them be the only line in the Quarantine Room at that time, which allows us the opportunity to disinfect the water system and equipment before they arrive. Fish are spawned when they arrive to avoid the possibility that adjustment to new facility water parameters could repress breeding. If
FIG. 4. Flow chart for responding to biosecurity risks posed by cryopreserved sperm samples when used to regenerate a line at ZIRC.
BIOSECURITY AND HEALTH MONITORING AT ZIRC
insufficient embryos are produced to propagate the line, the adults are acclimated in the Quarantine Room for 2 weeks then spawned again. When sufficient fertilized embryos are recovered, the adults are euthanized, fixed, and submitted for histological processing, sectioning, and H&E staining. The embryos are surface-sanitized and held in isolation pending histopathology results on the adults. If the adults are free of virulent pathogens (Table 1), the progeny are moved into the Main Fish Room. If virulent pathogens are detected in the adults, the progeny are reared in the Quarantine Room until they can be spawned and submitted for diagnostic testing while their embryos are held in isolation. Alternatively, the line may be reimported, spawned, and parents tested as described above. Cryopreserved sperm
Some facilities submitting large numbers of lines are shipping cryopreserved sperm, in lieu of adult males, directly to ZIRC. These sperm samples are stored in vapor-phase liquid nitrogen freezers designated for sperm originating outside of ZIRC. Varying numbers of sperm samples per fish line are submitted. Initial customer orders for these lines may be filled by thawing sperm samples for IVF with AB eggs from the Main Fish Room. These embryos are surfacesanitized and shipped to customers. The ZIRC Animal Health Report, which is available on-line, notifies customers that for embryos generated from frozen sperm, the paternal stock may not have been reared at ZIRC and its health status is unknown. When banked sperm samples reach a minimum number, the line must be regenerated and reared at ZIRC so that additional sperm samples can be obtained and cryopreserved. Contamination of cryopreserved bovine semen samples with bacterial and fungal pathogens, including a Mycobacterium, have been documented.33–35 We have detected P. neurophilia in zebrafish sperm samples10 and have observed mycobacterial infections in the testis and within the mesonephric duct in zebrafish tissue sections. We therefore consider cryopreserved sperm samples a biosecurity risk that should be evaluated based on health information from the submitting facility. We request health information from all facilities submitting cryopreserved sperm. We enquire what type of health monitoring is performed, frequency of sampling, and results of diagnostic tests. We also request that a sample of 20–30 fish from the same water system as the submitted lines be euthanized, fixed, and sent to us ahead of the sperm samples. Our preference is that the fixed fish be the actual males that contributed to the sperm samples. If that is not possible, then we request moribund fish or fish exposed to pooled effluent either from a prefiltration sentinel tank or a sump. These fish are submitted for histopathology and diagnostic results determine whether the sperm will be used in IVF and embryos reared in the Main Fish Room or in the Quarantine Room (Fig. 4). Sperm from an outside facility with no detected pathogens and sperm from fish reared in the ZIRC Main Fish Room are cleared for IVF and progeny can be reared in the Main Fish Room. Having fixed males from which the sperm samples were obtained gives us the most accurate information about the biosecurity risk of the sperm samples. If low virulence pathogens are detected in these fish, like Myxidium strei-
S-35
singeri, P. neurophilia, and M. chelonae, the sperm samples are marked with an attention flag in the database, the sperm is cleared for IVF, and the progeny can be reared on side B of the Main Fish room. P. neurophilia and M. chelonae have been previously identified in the ZIRC Main Fish Room colony.10,11 Water system UV sterilizers, equipment disinfection protocols, and husbandry and breeding procedures are in place to minimize these pathogens in the ZIRC colony. However, given the widespread presence of these pathogens within the zebrafish research community, excluding fish lines from facilities with these pathogens would greatly limit the number of lines that could enter the ZIRC Main Fish Room. Our hope is that going through cryopreserved sperm, instead of live spawns, when these lines are introduced will reduce the potential transfer of these pathogens to the progeny. However, we do recognize the risk that new introductions pose and therefore require that they be reared on side B of the Main Fish Room. We reserve side A for lines that have been maintained for several generations at ZIRC. Sperm samples coming from facilities with virulent pathogens are treated differently. Based on the high degree of morbidity and mortality associated with M. marinum, M. haemophilum, E. ictaluri, and P. tomentosa, we classify these pathogens as highly virulent.15,17,18,20 Moreover, mycobacteria are well recognized to survive freezing.36,37 If these pathogens are identified in submitted fixed fish or if a submitting facility has a history of these pathogens, then sperm samples can be used in IVF, but progeny are reared in the Quarantine Room. If health information and/or fixed fish are not available from a submitting facility, we treat the sperm samples as if they came from a facility with virulent pathogens and rear the progeny in the Quarantine Room. When these fish are sexually mature, sperm samples are obtained and cryopreserved and all contributing males are submitted for histopathology. Staff movement and training
The ZIRC has several protocols in place to prevent people from inadvertently carrying fish pathogens into the Main Fish Room. The key element in our prevention strategy is education. All staff members and student employees are trained to identify moribund fish and in operational and facility protocols, including the biosecurity reasoning behind their implementation. We want all staff members to feel invested in protecting fish health and feel that education and involvement are central to the success of our biosecurity program. We routinely edit and update facility standard operating procedure (SOPs) and changes are discussed in facility meetings. New SOPs are distributed to all staff members and training sessions scheduled when appropriate. Upon entering the ZIRC building, everyone changes into a pair of dedicated facility shoes, which are disinfected monthly, or dons shoe covers. Separate shoes or a second set of shoe covers are worn in the Quarantine Room. At every doorway adhesive floor mats are present to minimize tracking of debris between rooms. Floors are also bleached twice weekly with 600 ppm bleach solution. Employees with access to the Quarantine Room are limited to a few full-time staff members. These employees organize their work schedules such that they do not work with fish in
S-36
the Main Fish Room after completing tasks in the Quarantine Room. All equipment used is dedicated for Quarantine Room use only and gloves are required for all activities. Main Fish Room Health Monitoring
The biosecurity program at ZIRC includes extensive monitoring and diagnostic sampling in the Main Fish Room, allowing us to assess individual, tank, and colony health status. This program should also facilitate early detection of and response to any new pathogens in the colony. Fish are monitored for physical and behavioral signs of morbidity and mortality daily. ZIRC health-monitoring protocols are available on-line (http://zebrafish.org/documents/ protocols.php#ZIRC%20Health%20Monitoring%20SOPs). The majority of moribund fish are euthanized, fixed, and submitted for histopathology. Employees isolate and label moribund fish in 1-gallon tanks in a dedicated area of the fish room and notify the veterinarian. If they are not able to immediately remove moribund fish from a tank, they can label the tank with a waterproof flag (Post-its, 3M) and it will be removed during the next room health check. The ZIRC health-monitoring program also includes quarterly sampling of pre- and postfiltration sentinel fish. On each water system we have 3 1-gallon tanks set up in pre- and postfiltration locations. Tanks represent 3 months, 6 months, and 1 year of exposure. Sentinel tanks are stocked with 3–4 month-old AB wild-type fish from a source tank that is prescreened for P. neurophilia infection. Evaluating sentinel data has the advantage of comparing pre- and postfiltration and UV exposure in fish of known age, genetic background, and environmental experiences. However, we have found that evaluation of moribund fish is more valuable in terms of early detection of pathogens. After 3 years of P. neurophilia being below our level of detection by both histopathology and PCR,10 we detected it in a moribund fish 7 months before it appeared in prefiltration sentinel fish. The time required for prefiltration sentinel fish to receive an infective dose is dependent upon the amount of pathogen being shed in the water system and exposure of the sentinel fish, which is affected by water flow and drainage dynamics in the sentinel tank. For systems running large volumes of water, reliance on a sentinel program alone for assessing colony health could result in an underestimate of pathogen presence. For example, while screening wild-type lines we found that 15 of 34 20-gallon tanks tested on 1 water system were positive for P. neurophilia. Testing occurred over a 3-month period and positive populations were removed upon diagnosis. Sentinel fish receiving effluent from the system during that time period were clear of the infection after 3 months. Sentinel fish were tested again after an additional 3 months, giving time for potentially early infections to become more fulminant, but remained negative. Furthermore, absence of pathogens like P. neurophilia and M. chelonae in sentinel fish at one sampling point does not indicate eradication of the pathogen from a main system, but only whether or not fish were exposed to an infective dose during the exposure period. Continuous sampling of moribund fish, therefore, gives better evidence of what pathogens are present. Following the reemergence of P. neurophilia in moribund and sentinel fish at ZIRC, we increased our monitoring and
MURRAY, VARGA, AND KENT
sampling of wild-type lines in an effort to identify and minimize infected populations. A sample of fish from all tanks is tested by histopathology or PCR at 8-months-of-age. Wildtype fish are maintained in 20-gallon tanks at a stocking density of 150–250 fish. Sampling five fish per tank allows us to detect P. neurophilia infections at a prevalence of at least 50% with 95% confidence.38,39 Because AB fish are used more heavily (spawned more frequently, used for outcrossing, and supply eggs for IVF), we sample 11 fish from all AB tanks, which allows us to detect a minimum prevalence of 25% with 95% confidence. The 8-month sampling age was set after an initial screen of all wild-type tanks. During follow-up testing we noted false negative results for tanks originally tested before 8 months. Results of health monitoring in the Main Fish Room are available online (http://zebrafish.org/documents/health_report .php). We have recently changed our online Animal Health Report to include results of all testing, not just sentinel results. The information we provide about the ZIRC water system, type and frequency of health monitoring, infectious pathogens diagnosed in the past 3 months, 1 year, and 3 years mirror the information that we request from facilities sending new lines to ZIRC. We promote open sharing of facility health information with the purpose of allowing importing facilities to adopt appropriate biosecurity protocols and monitoring strategies to prevent the inadvertent co-importation of pathogens. Conclusion
The ZIRC imports a high volume of new zebrafish lines to cryopreserve, maintain, and redistribute to the research community. We place a high priority on fish health and avoidance of specific pathogens, and hence we have implemented a biosecurity program to mitigate the risks of introduction and transmission of pathogens. New lines are imported as live fish or cryopreserved sperm. Mutant and transgenic lines are cryopreserved for future regeneration based on customer orders. When sperm samples are depleted to the point that the line needs to be reared at ZIRC and sperm refrozen, we assess the biosecurity risk posed by the sperm samples and decide whether the resultant embryos should be reared in the Main Fish Room or the Quarantine Room. Risk is evaluated by looking at fish health history from the submitting facility, diagnostic results from fixed fish from the submitting facility, and ideally from diagnostic results from the males that contributed to the frozen sperm samples. Protocols and equipment are also in place to minimize potential for people to serve as fomites and inadvertently move pathogens into the Main Fish Room and also between stocks and tanks. In the Main Fish Room we perform daily monitoring for morbidity and mortality and submit the majority of moribund fish for diagnostic histopathology. Sentinel fish representing 3 months, 6 months, and 1 year of exposure are sampled and tested quarterly. In addition, all 20-gallon tanks of wild-type fish are screened for P. neurophilia at 8 months. Several aspects of the ZIRC biosecurity program can be adapted for application in facilities of different sizes and program goals. Requesting fish health history information from a sending facility before importing new fish is an important first step in formulating a strategy to contain and control pathogens that could be imported with the fish.
BIOSECURITY AND HEALTH MONITORING AT ZIRC
Responses may range from quarantining new imports in a separate room, on a separate rack, or just implementing increased monitoring of the tank. Results of diagnostic testing of imported fish can determine frequency and means of monitoring the health of the progeny. ZIRC performs histopathology on all imported fish before clearing progeny to be raised in our Main Fish Room. Other facilities may opt to test a subset of imported fish, fix or freeze back fish for possible future testing and monitor the progeny, or do selective testing for pathogens on an exclusion list. In addition, the ZIRC health-monitoring strategy can be scaled to fit different facilities. Education and active participation of facility staff in recognizing and removing moribund fish are central elements to any effective monitoring strategy. Removing moribund fish can limit the spread of infectious pathogens within a tank population. The amount and type of diagnostic testing of moribund fish will vary between facilities. Similarly, sentinel programs can vary in sample size and frequency of diagnostic testing. The ZIRC biosecurity program will have to change and evolve within our own facility as well. In particular, as we identify new zebrafish pathogens, gain additional understanding about modes of pathogen transmission, and develop new diagnostic assays, our protocols for pathogen monitoring and testing along with assessing and responding to risks will change. As a resource center, we also need to adjust to meet the needs and challenges arising from new scientific developments in the zebrafish field. For example, the improvement and increasing popularity of CRISPR/Cas940 technology for generating mutant lines will likely increase the number of labs submitting to ZIRC. Some of these labs will provide adult fish while others will want to submit lines as frozen sperm. Receiving samples for diagnostic testing for lines submitted as cryopreserved sperm may be challenging if sperm is frozen off-site and if considerable time elapses between when the line is cryopreserved and when it is shipped to ZIRC. We will continue to adapt our biosecurity program to factor in these and future unforeseen issues. Prioritizing fish health, welfare, and biosecurity will allow importing increasingly higher volumes of new lines while maintaining a healthy in-house fish colony for propagation at ZIRC and distribution to the international research community. Acknowledgments
We would like to thank Andrzej Nasiadka and David Lains for reading portions of the article and providing helpful comments and suggestions. The Zebrafish International Resource Center is supported by the NIH Office of Research Infrastructure Programs (award P40OD011021). Disclosure Statement
No competing financial interests exist. References
1. Kettleborough RN, Busch-Nentwich EM, Harvey SA, Dooley CM, de Bruijn E, van Eeden F, et al. A systematic genome-wide analysis of zebrafish protein-coding gene function. Nature 2013;496:494–497.
S-37
2. Clark KJ, Balciunas D, Pogoda HM, Ding Y, Westcot SE, Bedell VM, et al. In vivo protein trapping produces a functional expression codex of the vertebrate proteome. Nat Methods 2011;8:506–515. 3. Varshney GK, Lu J, Gildea DE, Huang H, Pei W, Yang Z, et al. A large-scale zebrafish gene knockout resource for the genome-wide study of gene function. Genome Res 2013; 23:727–735. 4. Carmichael C, Westerfield M, Varga ZM: Cryopreservation and in vitro fertilization at the Zebrafish International Resource Center. In: Zebrafish, Methods in Molecular Biology. Lieschke GJ, Oates AC, and Kawakami K, (eds), pp. 45–65. Humana Press, a part of Springer Science + Business Media, LLC, Clifton, NJ 2009. 5. Kent ML, Harper C, Wolf JC. Documented and potential research impacts of subclinical diseases in zebrafish. ILAR J 2012;53:126–134. 6. Ramsay JM, Watral V, Schreck CB, Kent ML. Pseudoloma neurophilia infections in zebrafish Danio rerio: effects of stress on survival, growth, and reproduction. Dis Aquat Organ 2009;88:69–84. 7. Spagnoli S, Xue L, Kent ML. The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): implications for the zebrafish as a model organism. Behav Brain Res 2015;291:351–360. 8. Sanders JL, Watral V, Clarkson K, Kent ML. Verification of intraovum transmission of a microsporidium of vertebrates: pseudoloma neurophilia infecting the Zebrafish, Danio rerio. PLoS One 2013;8:e76064. 9. Ferguson JA, Watral V, Schwindt AR, Kent ML. Spores of two fish microsporidia (Pseudoloma neurophilia and Glugea anomala) are highly resistant to chlorine. Dis Aquat Organ 2007;76:205–214. 10. Murray KN, Dreska M, Nasiadka A, Rinne M, Matthews JL, Carmichael C, et al. Transmission, diagnosis, and recommendations for control of Pseudoloma neurophilia infections in laboratory zebrafish (Danio rerio) facilities. Comp Med 2011;61:322–329. 11. Murray KN, Bauer J, Tallen A, Matthews JL, Westerfield M, Varga ZM. Characterization and management of asymptomatic Mycobacterium infections at the Zebrafish International Resource Center. J Am Assoc Lab Anim Sci 2011;50:675–679. 12. Whipps CM, Matthews JL, Kent ML. Distribution and genetic characterization of Mycobacterium chelonae in laboratory zebrafish Danio rerio. Dis Aquat Organ 2008; 82:45–54. 13. Mediel MJ, Rodriguez V, Codina G, Martin-Casabona N. Isolation of mycobacteria from frozen fish destined for human consumption. Appl Environ Microbiol 2000;66:3637–3638. 14. Whipps CM, Lieggi C, Wagner R. Mycobacteriosis in zebrafish colonies. ILAR J 2012;53:95–105. 15. Ostland VE, Watral V, Whipps CM, Austin FW, St-Hilaire S, Westerman ME, et al. Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio. Dis Aquat Organ 2008;79:107–118. 16. Johnson MG, Stout JE. Twenty-eight cases of Mycobacterium marinum infection: retrospective case series and literature review. Infection 2015; 43:655–662. 17. Whipps CM, Dougan ST, Kent ML. Mycobacterium haemophilum infections of zebrafish (Danio rerio) in research facilities. FEMS Microbiol Lett 2007;270:21–26.
S-38
18. Murray KN, Peterson TS. Pathology in practice. P tomentosa infection in zebrafish. J Am Vet Med Assoc 2015; 246:201–203. 19. Maley D, Laird AS, Rinkwitz S, Becker TS. A simple and efficient protocol for the treatment of zebrafish colonies infected with parasitic nematodes. Zebrafish 2013;10:447–450. 20. Hawke JP, Kent M, Rogge M, Baumgartner W, Wiles J, Shelley J, et al. Edwardsiellosis caused by Edwardsiella ictaluri in laboratory populations of Zebrafish Danio rerio. J Aquat Anim Health 2013;25:171–183. 21. Kent ML, Lyons JM. Edwardsiella ictaluri in the green knife fish, Eigemannia virescens. Fish Health News 1982; 11:ii. 22. Waltman WD, Shotts EB, Blazer VS. Recovery of Edwardsiella-Ictaluri from Danio (Danio-Devario). Aquaculture 1985;46:63–66. 23. Whipps CM, Murray KN, Kent ML. Occurrence of a myxozoan parasite Myxidium streisingeri n. sp. in laboratory zebrafish Danio rerio. J Parasitol 2015;101:86–90. 24. West K, Miles R, Kent ML, Frazer JK. Unusual fluorescent granulomas and myonecrosis in Danio rerio infected by the microsporidian pathogen Pseudoloma neurophilia. Zebrafish 2014;11:283–290. 25. Whipps CM, Moss LG, Sisk DM, Murray KN, Tobin DM, Moss JB. Detection of autofluorescent Mycobacterium chelonae in living zebrafish. Zebrafish 2014;11:76–82. 26. Cheung JP, Fung B, Wong SS, Ip WY. Review article: mycobacterium marinum infection of the hand and wrist. J Orthop Surg (Hong Kong) 2010;18:98–103. 27. Lobb CJ, Ghaffari SH, Hayman JR, Thompson DT. Plasmid and serological differences between Edwardsiella ictaluri strains. Appl Environ Microbiol 1993;59:2830–2836. 28. Gao LY, Manoranjan J. Laboratory maintenance of Mycobacterium marinum. Curr Protoc Microbiol 2005;Chapter 10:Unit 10B 1. 29. Brady YJ, Vinitnantharat S. Communications: viability of bacterial pathogens in Frozen Fish. Aquat Anim Health 1990;2:2. 30. Varga ZM. Aquaculture and husbandry at the zebrafish international resource center. Methods Cell Biol 2011;104: 453–478.
MURRAY, VARGA, AND KENT
31. Matthews M, Varga ZM. Anesthesia and euthanasia in zebrafish. ILAR J 2012;53:192–204. 32. Kent ML, Feist SW, Harper C, Hoogstraten-Miller S, Law JM, Sanchez-Morgado JM, et al. Recommendations for control of pathogens and infectious diseases in fish research facilities. Comp Biochem Physiol C Toxicol Pharmacol 2009;149:240–248. 33. Thibier M, Guerin B. Hygienic aspects of storage and use of semen for artificial insemination. Anim Reprod Sci 2000; 62:233–251. 34. Shu Z, Weigel KM, Soelberg SD, Lakey A, Cangelosi GA, Lee KH, et al. Cryopreservation of Mycobacterium tuberculosis complex cells. J Clin Microbiol 2012;50:3575–3580. 35. Bielanski A, Bergeron H, Lau PC, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003;46:146–152. 36. Heckly RJ. The preservation of stock cultures of Mycobacterium tuberculosis by freezing. Am Rev Tuberc 1950;62:99–100. 37. Iivanainen E, Martikainen PJ, Katila ML. Effect of freezing of water samples on viable counts of environmental mycobacteria. Lett Appl Microbiol 1995;21:257–260. 38. Simon RC, Schill WB. Tables of sample size requirements for detection of fish infected by pathogens: three confidence levels for different infection prevalence and various population sizes. J Fish Dis 1984;7:6. 39. AFS-FHS: FHS Blue Book: Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens. AFS-FHS, Bethesda, Maryland, 2007. 40. Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res 2014;24:142–153.
Address correspondence to: Katrina N. Murray, DVM, PhD Zebrafish International Resource Center University of Oregon 1307 Franklin Boulevard Eugene, OR 97403-5274 E-mail: [email protected]
Biosecurity and Health Monitoring at the Zebrafish International Resource Center Katrina N. Murray,1 Zolta´n M. Varga,1 and Michael L. Kent1,2
Abstract
The Zebrafish International Resource Center (ZIRC) is a repository and distribution center for mutant, transgenic, and wild-type zebrafish. In recent years annual imports of new zebrafish lines to ZIRC have increased tremendously. In addition, after 15 years of research, we have identified some of the most virulent pathogens affecting zebrafish that should be avoided in large production facilities, such as ZIRC. Therefore, while importing a high volume of new lines we prioritize safeguarding the health of our in-house fish colony. Here, we describe the biosecurity and health-monitoring program implemented at ZIRC. This strategy was designed to prevent introduction of new zebrafish pathogens, minimize pathogens already present in the facility, and ensure a healthy zebrafish colony for in-house uses and shipment to customers. Introduction
T
he Zebrafish International Resource Center (ZIRC) has been operating in a stand-alone aquatic facility on the University of Oregon campus since 2000. Our mission is to acquire, maintain, and redistribute zebrafish resources to the international research community. ZIRC also operates a diagnostic pathology and health consultation service. Fulfilling ZIRC’s mission necessitates a high volume of line imports and exports. Originally, this entailed importing individual lines as adult fish into the Quarantine Room. These fish were spawned in the Quarantine Room and their surface-sanitized embryos were moved to the Main Fish Room. The lines were then maintained and propagated in the Main Fish Room to satisfy pending customer requests and to cryopreserve them for longterm storage. As researchers began producing new mutant and transgenic lines in large-scale projects,1–3 ZIRC had to adopt a new importation strategy to accommodate a higher volume. We have shifted priorities from importing and maintaining new lines as live fish to importing and banking their cryopreserved sperm.4 From some facilities, lines are imported directly as cryopreserved sperm and others are still imported as live fish and cryopreserved in the ZIRC Quarantine Room. Using this approach, we have exponentially increased the number of zebrafish lines imported and available from ZIRC (Fig. 1). There are several demonstrated and potential impacts of pathogens on zebrafish in research.5 Pathogens that are most common or are of greatest concern are summarized in Table 1.6–23 In addition to virulent pathogens causing signifi1 2
cant mortality, infections in apparently healthy fish can cause reduced fecundity6 and confuse research endpoints such as behavior,7 development, and disease studies.24,25 At least one zebrafish pathogen, Mycobacterium marinum, is recognized as a cause of disease in humans working with fish.16,26 Increasing the volume of imported lines has also had important implications for the ZIRC biosecurity program, particularly because several zebrafish pathogens are highly virulent and many survive freezing (e.g., cryopreservation) in fish tissue and in media for long-term storage.13,27–29 Along with banking and redistributing resources for the research community, we place a high priority on animal health, monitoring, and disease prevention. We are constantly evaluating the risk of importing and moving pathogens along with fish resources. The microsporidian Pseudoloma neurophilia, a common pathogen of zebrafish, is capable of vertical transmission within ova,8 and the ability of other virulent pathogens to be maternally transmitted or survive in cryopreserved sperm samples is not fully understood. Given the existing knowledge, our biosecurity program is designed to identify and minimize these risks while ensuring a high level of animal welfare and animal and human health. Here, we provide an overview of major operations at ZIRC and identify key risk points for biosecurity. We describe our program for importation of new lines as live fish and cryopreserved sperm while minimizing risks for simultaneously importing pathogens. Finally, we describe the extensive health monitoring undertaken at ZIRC, which allows us to constantly assess individual, tank, and colony health.
Zebrafish International Resource Center, University of Oregon, Eugene, Oregon. Department of Microbiology, Oregon State University, Corvallis, Oregon.
S-30
BIOSECURITY AND HEALTH MONITORING AT ZIRC
S-31
FIG. 1. Annual number of new lines imported to ZIRC from 2001 to 2014. ZIRC, Zebrafish International Resource Center.
Facility Overview
Zebrafish lines are maintained in three separate areas at ZIRC; the Main Fish Room, the Freezer Room, and the Quarantine Room. The Main Fish Room operates on recirculating water systems with mechanical and biological filtration (Fig. 2). Municipal water is purified by reverse osmosis units, which have mechanical and carbon prefilters. Instant Ocean salt and Aragonite are added, conditioning the water to a conductivity of 500 lS/cm and pH between 7.2 and 7.6. Approximately 10% of the water is exchanged per day. The Main Fish Room water and air temperature are maintained at 28.5C. System and water parameters, including conductivity and pH, are monitored electronically.30 Separate recirculating water systems supply each half of the Main Fish Room (designated sides A and B). UV sterilizers are present on each system, delivering a minimum dose of 132,000 lWsec/cm2 (at max water flow rate of 200 gal/min, 80% lamp life, and 98% water transmissibility). Pressurized sand filters and optional bag filters on a bypass loop were installed to reduce fine particulates, which might shield pathogens from UV irradiation. Bag filters are used following acute increases in fine particulates resulting from electrical and mechanical disruptions that alter system water flow through the fluidized sand beds. The Freezer Room holds 6 MVE TEC 300 vapor-phase liquid nitrogen freezers for storing cryopreserved sperm. Six empty freezers are available for future storage. The Quarantine Room is in the same building, but physically separate from the Main Fish Room. A flow-through water system feeds the Quarantine Room. Municipal water runs through reverse osmosis filters and is treated with Instant
Ocean salt and Aragonite for conditioning. Effluent drains to the municipal sewer system. The room temperature is set to 28.5C. Husbandry Overview
All embryos are surface-sanitized with 30 ppm sodium hypochlorite for 10 min. New sodium hypochlorite solution is prepared daily using refrigerated laboratory grade sodium hypochlorite (Fisher Scientific) and reverse osmosis water. To minimize potential for transmission of pathogens between stocks at spawning, wild-type fish that have been used for out-cross are not returned to their original tank. They are euthanized or maintained in a separate 1-gallon (3.8 L) tank to be reused for breeding with the same mutant or transgenic stock. Separate anesthesia and recovery solutions are used for each tank of fish. Stocks are generally maintained until 1 year of age. Fish are euthanized by hypothermal shock or overdose of tricaine Methanesulfonate31 (S-Tricaine, Western Chemical). All protocols have been approved by the University of Oregon Institutional Animal Care and Use Committee. Fish are housed in 1-gallon plastic or 20-gallon (76 L) glass aquaria in the Main Fish Room. Only 1-gallon plastic tanks are used in the Quarantine Room. Twenty-gallon tanks hold 150–250 fish. One-gallon tanks hold up to 25 fish. Snails have never been used in ZIRC tanks. The light cycle is set for 14 h of light and 10 h of dark. Fish are fed Great Salt Lake brine shrimp, Artemia salina, nauplii (Artemia International) in the morning and a powdered feed mix (powder feed recipes are available on-line http://zebrafish.org/documents/protocols.php) in the afternoon. Juvenile fish and males being conditioned for sperm
S-32
Chronic inflammation. Low mortality.
Severe, systemic disease. Zoonotic risk for employees. Severe, chronic inflammation. High mortality. Severe, chronic enteritis and severe emaciation. Moderate to high mortality.
Mycobacterium chelonae
Mycobacterium marinum
Observed in renal ducts. Lesions are rare.
Myxidium streisingeri
ZIRC, Zebrafish International Resource Center.
Severe systemic disease. High mortality.
Edwardsiella ictaluri
Pseudocapillaria tomentosa
Mycobacterium haemophilum
Emaciation, reduced fecundity, and behavior changes. Low mortality.
Pathology and impact
Pseudoloma neurophilia
Name
No. Likely requires alternate worm host to complete life cycle.
Unknown. Not observed within eggs.
Unknown. Numerous bacilli observed in ovaries and associated with testes. No. Possible contamination of zebrafish embryos with feces containing P. tomentosa eggs during spawning.
Unknown. Bacilli observed in ovaries and occasionally testes. Unknown. Bacilli rarely observed within eggs.
Yes. Documented intraovum transmission.
Maternal transmission
ZIRC risk category Low risk. Widespread in zebrafish facilities. Presently at ZIRC. Control measures are in place. Spores survive freezing and chlorine surface disinfection of embryos. Low risk. Widespread in zebrafish facilities. Presently at ZIRC where it is generally limited to a low prevalence of asymptomatic fish. Survives freezing. High risk. Not at ZIRC. Common in aquarium fishes and occasionally seen in zebrafish facilities. Survives freezing. High risk. Not at ZIRC. Occasionally seen in zebrafish facilities. Results in significant morbidity and mortality. Survives freezing. High risk. Not at ZIRC. Occasionally seen in zebrafish facilities. Can result in significant morbidity and mortality. Disease is more severe in zebrafish lines with AB background. Ability to survive freezing is unknown, but unlikely. Eggs tolerate chlorine surface disinfection of embryos. High risk. Not at ZIRC. Not widespread in zebrafish facilities, but has been diagnosed in several aquarium fishes, particularly Danio species. Results in significant disease and mortality. Ability to survive freezing is unknown. Low risk. Not at ZIRC. Occasionally seen in zebrafish facilities.
Table 1. Pathogens and Risks for ZIRC
23
J. Shelley (pers. comm.) and Refs. 20–22
18,19
14,17
14–16
11–13
6–10
References
BIOSECURITY AND HEALTH MONITORING AT ZIRC
FIG. 2.
S-33
Diagram of the ZIRC Main Fish Room water filtration system.
cryopreservation receive an additional midday powder feeding. Larval fish are fed paramecia, cultured in-house, until day 10 when they are moved from static to flowing water and are fed powdered feed and brine shrimp, both twice daily. When large stocks are in the nursery, the larval diet may be supplemented with Branchionus plicatilis l-type rotifers (Reed Mariculture, Inc.). When 1-gallon tanks require cleaning fish are transferred to a new clean tank. Glass 20-gallon aquaria are cleaned with the fish in the tank. Tanks are scrubbed with a scouring pad and in-flowing water is increased to flush liberated tank debris and biofilm out the tank drain, minimizing fish exposure.11 Clean, autoclaved scrubbers and nets are used on every tank. Between fish populations, 1-gallon and 20-gallon tanks are scrubbed to remove visible debris, soaked in 0.15% bleach solution for at least 15 min, neutralized in 0.002% (w/v) sodium thiosulfate solution, rinsed in a tunnel washer with 82C water, and air-dried.
After spawning, their surface-sanitized embryos may be moved to the Main Fish Room or reared in the Quarantine Room. When lines are imported as cryopreserved sperm, the samples are stored in a vapor-phase liquid nitrogen freezer designated for samples frozen outside of ZIRC. Following customer orders, sperm from the freezer is thawed for in vitro fertilization (IVF) with AB eggs from the Main Fish Room. Resultant embryos are surface-sanitized and reared in the Main Fish Room, shipped to customers, or reared in the Quarantine Room. In the Main Fish Room, fish are spawned and surfacesanitized embryos may be shipped to customers or reared in the Main Fish Room. Adult zebrafish are also shipped to customers. Sperm from mutant and transgenic lines is cryopreserved and stored in designated vapor-phase liquid nitrogen freezers for future regeneration of lines. Biosecurity Risk Points and Response
Operational Overview
New lines come to ZIRC as live fish or cryopreserved sperm (Fig. 3). Live fish are imported into the Quarantine Room. For mutant and transgenic lines, we request 10 adult males be sent. They are acclimated for at least 2 weeks then anesthetized with tricaine methanesulfonate (168 mg/L) and sperm is obtained for cryopreservation. For wild-type lines, males and females are imported into the Quarantine Room.
Preventing introduction of new pathogens into the ZIRC Main Fish Room colony is a major priority of our health and biosecurity program. This includes pathogens already present at ZIRC in certain populations (e.g., M. chelonae and P. neurophilia) and those that are not (e.g., M. marinum, M. haemophilium, Pseudocapillaria tomentosa, and Edwardsiella ictaluri). Kent et al. (2009)32 provided recommendations for control of pathogens in fish laboratories. We
FIG. 3. Operational overview of movement of zebrafish adults, embryos, and sperm into and out of ZIRC rooms.
S-34
apply most of these approaches with certain modifications given the unique situation at ZIRC in which numerous fish are being both imported and exported on a monthly basis. We have identified points in our flow of operations that represent the greatest risks for pathogen introduction and have devised the plan described below to minimize these risks. Quarantine Room
The most likely route of new pathogen introduction into the facility is through live fish imported into the Quarantine Room. For most lines, only males are imported for cryopreservation of sperm. Following a customer request, the line is regenerated by IVF with AB eggs from the Main Fish Room. By prioritizing cryopreservation of lines over spawning and raising surface-sanitized embryos, we have limited the number of lines reared in the Main Fish Room. Moving lines through the liquid nitrogen freezers also provides an additional point at which pathogens associated with the imported stock can be reduced. While in the Quarantine Room, all stocks are monitored daily for morbidity and mortality. Fish displaying clinical signs of disease (physical or behavioral) are euthanized and a portion of liver and the spleen are dissected and frozen
MURRAY, VARGA, AND KENT
at -80C. The remaining carcass is fixed in Dietrich’s fixative and processed for histological sectioning and staining with hematoxylin and eosin (H&E). If mycobacteriosis is diagnosed in the tissue sections, the frozen organs are sent to the Oregon State University Veterinary Diagnostic Lab (OSU VDL) for a quantitative polymerase chain reaction (PCR) assay to identify the species of Mycobacterium in the sections. After acclimation, clinically normal fish in the Quarantine Room are anesthetized to obtain sperm samples for cryopreservation. When sufficient sperm samples have been obtained, the fish are euthanized and fixed for histopathology. If mycobacteriosis is diagnosed in tissue sections, the paraffin block with embedded fish tissue is sent to OSU VDL for the Mycobacterium PCR panel. We use the results of diagnostic testing of all fish in the Quarantine Room to assess the risk that pathogens may contaminate the frozen sperm samples (described below and Fig. 4). When wild-type lines are imported, our preference is to have them be the only line in the Quarantine Room at that time, which allows us the opportunity to disinfect the water system and equipment before they arrive. Fish are spawned when they arrive to avoid the possibility that adjustment to new facility water parameters could repress breeding. If
FIG. 4. Flow chart for responding to biosecurity risks posed by cryopreserved sperm samples when used to regenerate a line at ZIRC.
BIOSECURITY AND HEALTH MONITORING AT ZIRC
insufficient embryos are produced to propagate the line, the adults are acclimated in the Quarantine Room for 2 weeks then spawned again. When sufficient fertilized embryos are recovered, the adults are euthanized, fixed, and submitted for histological processing, sectioning, and H&E staining. The embryos are surface-sanitized and held in isolation pending histopathology results on the adults. If the adults are free of virulent pathogens (Table 1), the progeny are moved into the Main Fish Room. If virulent pathogens are detected in the adults, the progeny are reared in the Quarantine Room until they can be spawned and submitted for diagnostic testing while their embryos are held in isolation. Alternatively, the line may be reimported, spawned, and parents tested as described above. Cryopreserved sperm
Some facilities submitting large numbers of lines are shipping cryopreserved sperm, in lieu of adult males, directly to ZIRC. These sperm samples are stored in vapor-phase liquid nitrogen freezers designated for sperm originating outside of ZIRC. Varying numbers of sperm samples per fish line are submitted. Initial customer orders for these lines may be filled by thawing sperm samples for IVF with AB eggs from the Main Fish Room. These embryos are surfacesanitized and shipped to customers. The ZIRC Animal Health Report, which is available on-line, notifies customers that for embryos generated from frozen sperm, the paternal stock may not have been reared at ZIRC and its health status is unknown. When banked sperm samples reach a minimum number, the line must be regenerated and reared at ZIRC so that additional sperm samples can be obtained and cryopreserved. Contamination of cryopreserved bovine semen samples with bacterial and fungal pathogens, including a Mycobacterium, have been documented.33–35 We have detected P. neurophilia in zebrafish sperm samples10 and have observed mycobacterial infections in the testis and within the mesonephric duct in zebrafish tissue sections. We therefore consider cryopreserved sperm samples a biosecurity risk that should be evaluated based on health information from the submitting facility. We request health information from all facilities submitting cryopreserved sperm. We enquire what type of health monitoring is performed, frequency of sampling, and results of diagnostic tests. We also request that a sample of 20–30 fish from the same water system as the submitted lines be euthanized, fixed, and sent to us ahead of the sperm samples. Our preference is that the fixed fish be the actual males that contributed to the sperm samples. If that is not possible, then we request moribund fish or fish exposed to pooled effluent either from a prefiltration sentinel tank or a sump. These fish are submitted for histopathology and diagnostic results determine whether the sperm will be used in IVF and embryos reared in the Main Fish Room or in the Quarantine Room (Fig. 4). Sperm from an outside facility with no detected pathogens and sperm from fish reared in the ZIRC Main Fish Room are cleared for IVF and progeny can be reared in the Main Fish Room. Having fixed males from which the sperm samples were obtained gives us the most accurate information about the biosecurity risk of the sperm samples. If low virulence pathogens are detected in these fish, like Myxidium strei-
S-35
singeri, P. neurophilia, and M. chelonae, the sperm samples are marked with an attention flag in the database, the sperm is cleared for IVF, and the progeny can be reared on side B of the Main Fish room. P. neurophilia and M. chelonae have been previously identified in the ZIRC Main Fish Room colony.10,11 Water system UV sterilizers, equipment disinfection protocols, and husbandry and breeding procedures are in place to minimize these pathogens in the ZIRC colony. However, given the widespread presence of these pathogens within the zebrafish research community, excluding fish lines from facilities with these pathogens would greatly limit the number of lines that could enter the ZIRC Main Fish Room. Our hope is that going through cryopreserved sperm, instead of live spawns, when these lines are introduced will reduce the potential transfer of these pathogens to the progeny. However, we do recognize the risk that new introductions pose and therefore require that they be reared on side B of the Main Fish Room. We reserve side A for lines that have been maintained for several generations at ZIRC. Sperm samples coming from facilities with virulent pathogens are treated differently. Based on the high degree of morbidity and mortality associated with M. marinum, M. haemophilum, E. ictaluri, and P. tomentosa, we classify these pathogens as highly virulent.15,17,18,20 Moreover, mycobacteria are well recognized to survive freezing.36,37 If these pathogens are identified in submitted fixed fish or if a submitting facility has a history of these pathogens, then sperm samples can be used in IVF, but progeny are reared in the Quarantine Room. If health information and/or fixed fish are not available from a submitting facility, we treat the sperm samples as if they came from a facility with virulent pathogens and rear the progeny in the Quarantine Room. When these fish are sexually mature, sperm samples are obtained and cryopreserved and all contributing males are submitted for histopathology. Staff movement and training
The ZIRC has several protocols in place to prevent people from inadvertently carrying fish pathogens into the Main Fish Room. The key element in our prevention strategy is education. All staff members and student employees are trained to identify moribund fish and in operational and facility protocols, including the biosecurity reasoning behind their implementation. We want all staff members to feel invested in protecting fish health and feel that education and involvement are central to the success of our biosecurity program. We routinely edit and update facility standard operating procedure (SOPs) and changes are discussed in facility meetings. New SOPs are distributed to all staff members and training sessions scheduled when appropriate. Upon entering the ZIRC building, everyone changes into a pair of dedicated facility shoes, which are disinfected monthly, or dons shoe covers. Separate shoes or a second set of shoe covers are worn in the Quarantine Room. At every doorway adhesive floor mats are present to minimize tracking of debris between rooms. Floors are also bleached twice weekly with 600 ppm bleach solution. Employees with access to the Quarantine Room are limited to a few full-time staff members. These employees organize their work schedules such that they do not work with fish in
S-36
the Main Fish Room after completing tasks in the Quarantine Room. All equipment used is dedicated for Quarantine Room use only and gloves are required for all activities. Main Fish Room Health Monitoring
The biosecurity program at ZIRC includes extensive monitoring and diagnostic sampling in the Main Fish Room, allowing us to assess individual, tank, and colony health status. This program should also facilitate early detection of and response to any new pathogens in the colony. Fish are monitored for physical and behavioral signs of morbidity and mortality daily. ZIRC health-monitoring protocols are available on-line (http://zebrafish.org/documents/ protocols.php#ZIRC%20Health%20Monitoring%20SOPs). The majority of moribund fish are euthanized, fixed, and submitted for histopathology. Employees isolate and label moribund fish in 1-gallon tanks in a dedicated area of the fish room and notify the veterinarian. If they are not able to immediately remove moribund fish from a tank, they can label the tank with a waterproof flag (Post-its, 3M) and it will be removed during the next room health check. The ZIRC health-monitoring program also includes quarterly sampling of pre- and postfiltration sentinel fish. On each water system we have 3 1-gallon tanks set up in pre- and postfiltration locations. Tanks represent 3 months, 6 months, and 1 year of exposure. Sentinel tanks are stocked with 3–4 month-old AB wild-type fish from a source tank that is prescreened for P. neurophilia infection. Evaluating sentinel data has the advantage of comparing pre- and postfiltration and UV exposure in fish of known age, genetic background, and environmental experiences. However, we have found that evaluation of moribund fish is more valuable in terms of early detection of pathogens. After 3 years of P. neurophilia being below our level of detection by both histopathology and PCR,10 we detected it in a moribund fish 7 months before it appeared in prefiltration sentinel fish. The time required for prefiltration sentinel fish to receive an infective dose is dependent upon the amount of pathogen being shed in the water system and exposure of the sentinel fish, which is affected by water flow and drainage dynamics in the sentinel tank. For systems running large volumes of water, reliance on a sentinel program alone for assessing colony health could result in an underestimate of pathogen presence. For example, while screening wild-type lines we found that 15 of 34 20-gallon tanks tested on 1 water system were positive for P. neurophilia. Testing occurred over a 3-month period and positive populations were removed upon diagnosis. Sentinel fish receiving effluent from the system during that time period were clear of the infection after 3 months. Sentinel fish were tested again after an additional 3 months, giving time for potentially early infections to become more fulminant, but remained negative. Furthermore, absence of pathogens like P. neurophilia and M. chelonae in sentinel fish at one sampling point does not indicate eradication of the pathogen from a main system, but only whether or not fish were exposed to an infective dose during the exposure period. Continuous sampling of moribund fish, therefore, gives better evidence of what pathogens are present. Following the reemergence of P. neurophilia in moribund and sentinel fish at ZIRC, we increased our monitoring and
MURRAY, VARGA, AND KENT
sampling of wild-type lines in an effort to identify and minimize infected populations. A sample of fish from all tanks is tested by histopathology or PCR at 8-months-of-age. Wildtype fish are maintained in 20-gallon tanks at a stocking density of 150–250 fish. Sampling five fish per tank allows us to detect P. neurophilia infections at a prevalence of at least 50% with 95% confidence.38,39 Because AB fish are used more heavily (spawned more frequently, used for outcrossing, and supply eggs for IVF), we sample 11 fish from all AB tanks, which allows us to detect a minimum prevalence of 25% with 95% confidence. The 8-month sampling age was set after an initial screen of all wild-type tanks. During follow-up testing we noted false negative results for tanks originally tested before 8 months. Results of health monitoring in the Main Fish Room are available online (http://zebrafish.org/documents/health_report .php). We have recently changed our online Animal Health Report to include results of all testing, not just sentinel results. The information we provide about the ZIRC water system, type and frequency of health monitoring, infectious pathogens diagnosed in the past 3 months, 1 year, and 3 years mirror the information that we request from facilities sending new lines to ZIRC. We promote open sharing of facility health information with the purpose of allowing importing facilities to adopt appropriate biosecurity protocols and monitoring strategies to prevent the inadvertent co-importation of pathogens. Conclusion
The ZIRC imports a high volume of new zebrafish lines to cryopreserve, maintain, and redistribute to the research community. We place a high priority on fish health and avoidance of specific pathogens, and hence we have implemented a biosecurity program to mitigate the risks of introduction and transmission of pathogens. New lines are imported as live fish or cryopreserved sperm. Mutant and transgenic lines are cryopreserved for future regeneration based on customer orders. When sperm samples are depleted to the point that the line needs to be reared at ZIRC and sperm refrozen, we assess the biosecurity risk posed by the sperm samples and decide whether the resultant embryos should be reared in the Main Fish Room or the Quarantine Room. Risk is evaluated by looking at fish health history from the submitting facility, diagnostic results from fixed fish from the submitting facility, and ideally from diagnostic results from the males that contributed to the frozen sperm samples. Protocols and equipment are also in place to minimize potential for people to serve as fomites and inadvertently move pathogens into the Main Fish Room and also between stocks and tanks. In the Main Fish Room we perform daily monitoring for morbidity and mortality and submit the majority of moribund fish for diagnostic histopathology. Sentinel fish representing 3 months, 6 months, and 1 year of exposure are sampled and tested quarterly. In addition, all 20-gallon tanks of wild-type fish are screened for P. neurophilia at 8 months. Several aspects of the ZIRC biosecurity program can be adapted for application in facilities of different sizes and program goals. Requesting fish health history information from a sending facility before importing new fish is an important first step in formulating a strategy to contain and control pathogens that could be imported with the fish.
BIOSECURITY AND HEALTH MONITORING AT ZIRC
Responses may range from quarantining new imports in a separate room, on a separate rack, or just implementing increased monitoring of the tank. Results of diagnostic testing of imported fish can determine frequency and means of monitoring the health of the progeny. ZIRC performs histopathology on all imported fish before clearing progeny to be raised in our Main Fish Room. Other facilities may opt to test a subset of imported fish, fix or freeze back fish for possible future testing and monitor the progeny, or do selective testing for pathogens on an exclusion list. In addition, the ZIRC health-monitoring strategy can be scaled to fit different facilities. Education and active participation of facility staff in recognizing and removing moribund fish are central elements to any effective monitoring strategy. Removing moribund fish can limit the spread of infectious pathogens within a tank population. The amount and type of diagnostic testing of moribund fish will vary between facilities. Similarly, sentinel programs can vary in sample size and frequency of diagnostic testing. The ZIRC biosecurity program will have to change and evolve within our own facility as well. In particular, as we identify new zebrafish pathogens, gain additional understanding about modes of pathogen transmission, and develop new diagnostic assays, our protocols for pathogen monitoring and testing along with assessing and responding to risks will change. As a resource center, we also need to adjust to meet the needs and challenges arising from new scientific developments in the zebrafish field. For example, the improvement and increasing popularity of CRISPR/Cas940 technology for generating mutant lines will likely increase the number of labs submitting to ZIRC. Some of these labs will provide adult fish while others will want to submit lines as frozen sperm. Receiving samples for diagnostic testing for lines submitted as cryopreserved sperm may be challenging if sperm is frozen off-site and if considerable time elapses between when the line is cryopreserved and when it is shipped to ZIRC. We will continue to adapt our biosecurity program to factor in these and future unforeseen issues. Prioritizing fish health, welfare, and biosecurity will allow importing increasingly higher volumes of new lines while maintaining a healthy in-house fish colony for propagation at ZIRC and distribution to the international research community. Acknowledgments
We would like to thank Andrzej Nasiadka and David Lains for reading portions of the article and providing helpful comments and suggestions. The Zebrafish International Resource Center is supported by the NIH Office of Research Infrastructure Programs (award P40OD011021). Disclosure Statement
No competing financial interests exist. References
1. Kettleborough RN, Busch-Nentwich EM, Harvey SA, Dooley CM, de Bruijn E, van Eeden F, et al. A systematic genome-wide analysis of zebrafish protein-coding gene function. Nature 2013;496:494–497.
S-37
2. Clark KJ, Balciunas D, Pogoda HM, Ding Y, Westcot SE, Bedell VM, et al. In vivo protein trapping produces a functional expression codex of the vertebrate proteome. Nat Methods 2011;8:506–515. 3. Varshney GK, Lu J, Gildea DE, Huang H, Pei W, Yang Z, et al. A large-scale zebrafish gene knockout resource for the genome-wide study of gene function. Genome Res 2013; 23:727–735. 4. Carmichael C, Westerfield M, Varga ZM: Cryopreservation and in vitro fertilization at the Zebrafish International Resource Center. In: Zebrafish, Methods in Molecular Biology. Lieschke GJ, Oates AC, and Kawakami K, (eds), pp. 45–65. Humana Press, a part of Springer Science + Business Media, LLC, Clifton, NJ 2009. 5. Kent ML, Harper C, Wolf JC. Documented and potential research impacts of subclinical diseases in zebrafish. ILAR J 2012;53:126–134. 6. Ramsay JM, Watral V, Schreck CB, Kent ML. Pseudoloma neurophilia infections in zebrafish Danio rerio: effects of stress on survival, growth, and reproduction. Dis Aquat Organ 2009;88:69–84. 7. Spagnoli S, Xue L, Kent ML. The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): implications for the zebrafish as a model organism. Behav Brain Res 2015;291:351–360. 8. Sanders JL, Watral V, Clarkson K, Kent ML. Verification of intraovum transmission of a microsporidium of vertebrates: pseudoloma neurophilia infecting the Zebrafish, Danio rerio. PLoS One 2013;8:e76064. 9. Ferguson JA, Watral V, Schwindt AR, Kent ML. Spores of two fish microsporidia (Pseudoloma neurophilia and Glugea anomala) are highly resistant to chlorine. Dis Aquat Organ 2007;76:205–214. 10. Murray KN, Dreska M, Nasiadka A, Rinne M, Matthews JL, Carmichael C, et al. Transmission, diagnosis, and recommendations for control of Pseudoloma neurophilia infections in laboratory zebrafish (Danio rerio) facilities. Comp Med 2011;61:322–329. 11. Murray KN, Bauer J, Tallen A, Matthews JL, Westerfield M, Varga ZM. Characterization and management of asymptomatic Mycobacterium infections at the Zebrafish International Resource Center. J Am Assoc Lab Anim Sci 2011;50:675–679. 12. Whipps CM, Matthews JL, Kent ML. Distribution and genetic characterization of Mycobacterium chelonae in laboratory zebrafish Danio rerio. Dis Aquat Organ 2008; 82:45–54. 13. Mediel MJ, Rodriguez V, Codina G, Martin-Casabona N. Isolation of mycobacteria from frozen fish destined for human consumption. Appl Environ Microbiol 2000;66:3637–3638. 14. Whipps CM, Lieggi C, Wagner R. Mycobacteriosis in zebrafish colonies. ILAR J 2012;53:95–105. 15. Ostland VE, Watral V, Whipps CM, Austin FW, St-Hilaire S, Westerman ME, et al. Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio. Dis Aquat Organ 2008;79:107–118. 16. Johnson MG, Stout JE. Twenty-eight cases of Mycobacterium marinum infection: retrospective case series and literature review. Infection 2015; 43:655–662. 17. Whipps CM, Dougan ST, Kent ML. Mycobacterium haemophilum infections of zebrafish (Danio rerio) in research facilities. FEMS Microbiol Lett 2007;270:21–26.
S-38
18. Murray KN, Peterson TS. Pathology in practice. P tomentosa infection in zebrafish. J Am Vet Med Assoc 2015; 246:201–203. 19. Maley D, Laird AS, Rinkwitz S, Becker TS. A simple and efficient protocol for the treatment of zebrafish colonies infected with parasitic nematodes. Zebrafish 2013;10:447–450. 20. Hawke JP, Kent M, Rogge M, Baumgartner W, Wiles J, Shelley J, et al. Edwardsiellosis caused by Edwardsiella ictaluri in laboratory populations of Zebrafish Danio rerio. J Aquat Anim Health 2013;25:171–183. 21. Kent ML, Lyons JM. Edwardsiella ictaluri in the green knife fish, Eigemannia virescens. Fish Health News 1982; 11:ii. 22. Waltman WD, Shotts EB, Blazer VS. Recovery of Edwardsiella-Ictaluri from Danio (Danio-Devario). Aquaculture 1985;46:63–66. 23. Whipps CM, Murray KN, Kent ML. Occurrence of a myxozoan parasite Myxidium streisingeri n. sp. in laboratory zebrafish Danio rerio. J Parasitol 2015;101:86–90. 24. West K, Miles R, Kent ML, Frazer JK. Unusual fluorescent granulomas and myonecrosis in Danio rerio infected by the microsporidian pathogen Pseudoloma neurophilia. Zebrafish 2014;11:283–290. 25. Whipps CM, Moss LG, Sisk DM, Murray KN, Tobin DM, Moss JB. Detection of autofluorescent Mycobacterium chelonae in living zebrafish. Zebrafish 2014;11:76–82. 26. Cheung JP, Fung B, Wong SS, Ip WY. Review article: mycobacterium marinum infection of the hand and wrist. J Orthop Surg (Hong Kong) 2010;18:98–103. 27. Lobb CJ, Ghaffari SH, Hayman JR, Thompson DT. Plasmid and serological differences between Edwardsiella ictaluri strains. Appl Environ Microbiol 1993;59:2830–2836. 28. Gao LY, Manoranjan J. Laboratory maintenance of Mycobacterium marinum. Curr Protoc Microbiol 2005;Chapter 10:Unit 10B 1. 29. Brady YJ, Vinitnantharat S. Communications: viability of bacterial pathogens in Frozen Fish. Aquat Anim Health 1990;2:2. 30. Varga ZM. Aquaculture and husbandry at the zebrafish international resource center. Methods Cell Biol 2011;104: 453–478.
MURRAY, VARGA, AND KENT
31. Matthews M, Varga ZM. Anesthesia and euthanasia in zebrafish. ILAR J 2012;53:192–204. 32. Kent ML, Feist SW, Harper C, Hoogstraten-Miller S, Law JM, Sanchez-Morgado JM, et al. Recommendations for control of pathogens and infectious diseases in fish research facilities. Comp Biochem Physiol C Toxicol Pharmacol 2009;149:240–248. 33. Thibier M, Guerin B. Hygienic aspects of storage and use of semen for artificial insemination. Anim Reprod Sci 2000; 62:233–251. 34. Shu Z, Weigel KM, Soelberg SD, Lakey A, Cangelosi GA, Lee KH, et al. Cryopreservation of Mycobacterium tuberculosis complex cells. J Clin Microbiol 2012;50:3575–3580. 35. Bielanski A, Bergeron H, Lau PC, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003;46:146–152. 36. Heckly RJ. The preservation of stock cultures of Mycobacterium tuberculosis by freezing. Am Rev Tuberc 1950;62:99–100. 37. Iivanainen E, Martikainen PJ, Katila ML. Effect of freezing of water samples on viable counts of environmental mycobacteria. Lett Appl Microbiol 1995;21:257–260. 38. Simon RC, Schill WB. Tables of sample size requirements for detection of fish infected by pathogens: three confidence levels for different infection prevalence and various population sizes. J Fish Dis 1984;7:6. 39. AFS-FHS: FHS Blue Book: Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens. AFS-FHS, Bethesda, Maryland, 2007. 40. Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res 2014;24:142–153.
Address correspondence to: Katrina N. Murray, DVM, PhD Zebrafish International Resource Center University of Oregon 1307 Franklin Boulevard Eugene, OR 97403-5274 E-mail: [email protected]
