Restrictions on the Importation of Zebrafish into Canada Associated with Spring Viremia of Carp Virus

SVCV in cyprinids

The common carp (Cyprinus carpio) is the primary SVCV host. ⁸ SVCV is present in Europe and can cause epidemic mortalities primarily in young carp (<1 year of age) during the spring. ⁸ SVCV was first detected in North America in 2002 in carp present in Cedar Lake, Wisconsin, ¹² and since then has also been identified in Brazil, Canada, and China, among other countries.^7–9,13 The reported outbreaks of SVCV in the United States have been limited to wild or commercial fish, ¹⁴ with no occurrence detected in research facilities. Outbreak severity correlates with water temperature, fish age, health status, population density, and numerous stress factors. ⁸ The first reported detection of SVCV in Canada was in 2006 when wild carp were sampled from Hamilton Harbor in Lake Ontario. ¹⁵ Clinical disease in infected fish is most predominant when fish are immunosuppressed and outbreaks are related to low water temperature, a known immunosuppressant in fish. ⁸ SVCV outbreaks generally occur in water temperatures ranging between 11 and 17°C, with a robust decline of disease-related mortality in adult fish at temperatures above 22°C. ⁷ Rearing fish in a controlled environment with elevated temperatures (typically 19–20°C or greater) can prevent or stop clinical outbreaks.^7,16 Fish may be asymptomatic carriers of the virus, and aquatic arthropods may also be vectors of disease.^7,8 The virus is stable and can survive for long periods of time (up to 5 weeks) in the environment in temperatures less than 10°C. ⁷

Viral transmission is horizontal through excretion in the feces and urine. Fish become infected when the virus enters the gill epithelium. The virus then enters the blood and spreads to the liver, kidney, spleen, and digestive tract with high viral titers occurring in the liver and kidney. ⁷ Vertical (via germ plasm) transmission has not been demonstrated as a source of infection in any species. However, the OIE does not rule out this route because the virus has been isolated from carp ovarian fluid. ⁷ Infected fish may become moribund and exhibit external hemorrhaging on the skin, pale gills, and ascites.^8,9 Upon histopathological analysis, enteritis, peritonitis, edema, and petechial bleeding in various organs may be observed.

As in almost every realm of biomedicine, zebrafish serve as a powerful model system to understand infectious disease etiology. In the laboratory, zebrafish have been used to study and model vertebrate immunity and genetic response to infectious disease, including SVCV infection.^17–21 The goal of zebrafish SVCV infection models is to improve disease management or develop treatments for SVCV in commercial fish stocks. Embryos, larvae, and adult zebrafish have been used to study fish responses to the SVCV virus with embryos demonstrating resistance to infection. ¹⁸ Larvae [>3 days postfertilization (dpf)] can be manipulated to SVCV susceptibility using artificially high viral doses (≥10⁵ PFU/mL) through intracoelomic microinjection or prolonged immersion.^18–20 Adult zebrafish have similarly been manipulated to SVCV susceptibility through intracoelomic injection, intravenous injection, and prolonged immersion with artificially high viral doses (typically ≥10⁵ PFU/mL).^10,17,21 Clinically affected zebrafish were anorexic, listless, and exhibited gross abnormalities, including epidermal petechial to ecchymotic hemorrhages and death. On histopathology, bronchitis, branchial necrosis, and hepatic and splenic necrosis were observed. ¹⁷ Similar to what has been found with carp, clinical manifestation of the disease in zebrafish requires a lower water temperature. For example, Sanders et al. (2003) found the general trend that mortality rates increased from 30% at 24°C to 55% and 60% at 20 and 15°C, respectively. ¹⁷ The authors postulated that the unnaturally low temperatures also make zebrafish more susceptible to infection, morbidity, and mortality because of immunosuppression. ¹⁷ This is plausible since it is generally accepted that low temperatures affect immune responses.^22,23

OIE criteria for listing zebrafish as susceptible to SVCV

Based on their own risk assessment, the OIE placed zebrafish on the SVCV-susceptible species list in the Manual, but not the Code. The OIE's Aquatic Code chapter on SVCV states that any regulation of OIE-listed susceptible species by member countries should only be implemented following a risk assessment performed by each competent authority: “When considering the importation or transit of aquatic animals … of a species not covered in (the Code), but which could reasonably be expected to pose a risk of spread of SVC, the Competent Authority should conduct a risk analysis in accordance with the recommendations in the “Import Risk Analysis” chapter of the Code. The Competent Authority of the exporting country should be informed of the outcome of this assessment” (Article 10.9.3.3). ⁶ Assessing risk must take into account the entire pathway from identifying the potential pathogen to assessing how the pathogen would enter the environment, to estimating the probability of exposure to humans and animals, and finally to determining the socioeconomic consequences of exposure on health and the environment. ⁶ Before assessing risk, a hazard (i.e., pathogenic agent of concern) must first be identified that is appropriate to the species being imported (Article 2.1.2). ⁶ To link a pathogen with an imported aquatic species, it must be demonstrated that the species is susceptible to the pathogen. The OIE process for determining susceptibility involves three stages ⁶ :

Risk of natural infection of zebrafish with SVCV

No natural SVCV infections have been reported in zebrafish to date, including in the wild, in the hobbyist community wherein zebrafish are prolific, and in the laboratory setting. Regarding the latter, there are at least two possible explanations for why this is the case: First, zebrafish are tropical freshwater fish that are bred and reared in research facilities where the holding water temperature is maintained at 26–28°C for their entire life cycle; this high temperature suppresses SVCV propagation and spread. Second, as mentioned above, transmission of SVCV is horizontal. ⁷ In fact, zebrafish embryos up to 3 dpf, immersed in high-titer SVCV baths, showed no signs of infection even with their chorion removed. ¹⁹ In case eggs may act as fomites (recall SVCV has been found in carp ovarian fluid ⁷ ), egg disinfection has been discussed as an option to further reduce the risk of virus introduction.

Bleaching zebrafish embryos before shipment is a best practice for biosafety and biosecurity and is already a standard practice in the international zebrafish community to reduce or prevent the spread of pathogenic agents that may impact colony health. The standard bleach treatment is 30–100 ppm chlorine for 5–10 min,^24,25 well below the dose recommended by the OIE to inactivate SVCV (540 ppm chlorine for 20 min). ⁷ Discussions with the CFIA are still ongoing regarding the bleaching of embryos as a disinfection method to prevent SVCV introduction. However, the CFIA will not accept chlorine as an embryo disinfection method until the effective chlorine concentration and contact time required to render viable SVCV-free embryos are determined.

While the Manual does not discuss bleaching embryos as an acceptable SVCV disinfection method, it does accept iodophor-treated embryos for prevention of infection in other species. Iodophor treatment of zebrafish embryos is not standard practice; however, it has been used to create a germ-free zebrafish model. Furthermore, recent publications have explored its use for preventing spread of Mycobacterium spp. infection in zebrafish.^26,27 While the published protocol for generation of gnotobiotic zebrafish treats embryos less than 12 h old with 0.01% (100 ppm) free iodine for 2 min, the authors acknowledge that this treatment can result in increased death and the parameters need to be tested in each individual laboratory before use. ²⁶ A recent publication demonstrated that povidone–iodine at 25 ppm (0.0025%) for 5 min was more effective than bleaching and other embryo disinfection procedures at eliminating Mycobacterium spp. ²⁷ However, the doses utilized in both studies are below the 0.01% (100 ppm) for 10 min recommended for inactivation of SVCV. ⁸ Given that the iodophor treatment needs to occur soon after fertilization, exporting laboratories would need to complete the protocol before shipping zebrafish to Canada. Both the sensitivity of the assay and the inexperience of the community with the protocol make this an onerous request that many exporters will not accept.

The OIE risk analysis process (Article 2.1.3.6 of the Code) states that risk increases with increasing volume of commodity imported. It is unclear what the socioeconomic risk from extremely low volumes of zebrafish importation would be to human/animal health in Canada, especially considering that the vast majority of imported zebrafish for research are housed in laboratories within the academic setting with extremely restricted access. Currently, exemptions are in place for pet owners, another low-volume importer, to import zebrafish as long as they are declared because the CFIA recognizes that some species of susceptible aquatic animals held in aquaria represent a low risk for spreading diseases. ²⁸ Furthermore, goldfish imported as pets are also exempt from CFIA importation restrictions, despite the fact that they are SVCV-susceptible species under natural conditions.^7,28 It is unclear why pet fish with essentially unrestricted access are not subject to the same importation standards as fish in research, which pose less risk of spreading SVCV.

Another potential concern falling under the CFIA mandate to safeguard Canada's natural aquatic animal resources is the escape of infected zebrafish into Canadian waters where lower temperatures are more permissive of viral propagation and shedding. One field study reported that zebrafish could survive in temperatures of ∼10°C (and in some cases as low as 5°C). ²⁹ However, the evidence in this study indicates that as many as 70%–80% of the fish died within a single day of exposure to these temperatures and that none of the fish survived by the end of the experiment ∼1 month later. ²⁹ Thus, temperatures in Canadian waters that are optimal for SVCV would potentially kill embryos, larvae, and most adults, particularly in the winter. While elevated summer water temperatures may be permissive for infected zebrafish survival, the higher temperatures would also reduce SVCV outbreaks.

Conclusions of the CFIA scientific assessment

The OIE Aquatic Code (Article 10.9.3.3) recommends that competent authorities complete a risk assessment before regulating potentially susceptible species that are not listed in the Code, even if it is listed in the Manual. ⁶ The CFIA completed a scientific assessment rather than an OIE-based risk assessment of zebrafish susceptibility to SVCV. The rationale for imposing new importation restrictions for research animals without conducting a formal risk assessment as recommended by the OIE has not been articulated to the scientific community. As no natural infections have been reported, the CFIA reasoned that evidence of infection by noninvasive experimental procedures (i.e., immersion) supports listing zebrafish as susceptible to SVCV, citing several studies as providing evidence.^17–21 However, the CFIA categorization of the tests conducted on zebrafish as noninvasive procedures is incorrect since the zebrafish were exposed to low temperatures not encountered in their natural or culture environment (15–24°C), which leads to immunosuppression affecting host susceptibility (defined by the OIE as invasive). As mentioned above, a trend of increased mortality was observed with lowered temperature in Sanders et al. (2003). Furthermore, there is no evidence demonstrating that the high SVCV titers used for immersion experiments are actually present in cases of natural transmission of the virus. The concentration of virus used for bath immersion was typically ≥10⁵ PFU/mL, providing an unnaturally high load of pathogen, ³⁰ which the OIE also defines as an invasive procedure.

Despite these discrepancies and unknowns, the CFIA categorized the bath immersion conditions used as noninvasive and mimicking natural pathways for disease transmission. Since Stage 1 criterion was considered fulfilled, the CFIA proceeded with their scientific assessment of pathogen identification by diagnostic tests and isolation of the virus from infected animals. The CFIA concluded from the study by Sanders et al. (2003) that the last two stages of OIE-defined susceptibility assessment were also met as the virus could be detected by various testing methodologies and was correlated with clinical signs of diseases. From this scientific assessment, without a formal risk assessment, the CFIA decided that all three OIE-defined susceptibility criteria were met, and zebrafish has become regulated as a susceptible species in Canada. This scientific assessment was communicated to the stakeholder community in 2016, roughly 3 years after the restriction was enforced.

Other data to consider in SVCV scientific assessment

It is important to note that the OIE does not list zebrafish as a susceptible species in the Aquatic Code (Article 10.9.2), but states that “where there is insufficient evidence to demonstrate susceptibility … information will be included in the relevant disease-specific chapter in the Aquatic Manual.” ⁶ As mentioned above, zebrafish were added as a susceptible species to the Manual as a result of a study in which experimental infection was induced by intracoelomic injection, which we emphasize is not a natural route of infection. ⁷ The Code also states “if there is insufficient evidence to demonstrate susceptibility of species, the Competent Authority should assess the risk of spread of the pathogen under consideration … before the implementation of import health measures.” ⁶ The CFIA scientific assessment is meant to demonstrate this susceptibility, but, as discussed above, deviates from OIE recommendation in the definition of invasive procedures and neglects to address the OIE Aquatic Code (Article 1.5.4) where it states that “consideration should be given to environmental factors as these may affect host resistance or transmission of the pathogen.” ⁶ As SVCV is already present in Canadian waters, ¹⁵ consideration needs to be given to what additional risk zebrafish imports pose since OIE restrictions prevent countries from placing barriers on trade that are above health standards of the importing country.

Based on the OIE guidelines, other countries have completed more extensive formal risk assessments for SVCV and, in contrast to the CFIA, concluded that zebrafish are not susceptible to SVCV. For example, another OIE member country, New Zealand, has strict import regulations for SVCV-susceptible species. However, after performing a risk assessment (which references the Manual as listing zebrafish as SVCV susceptible), it does not list zebrafish as susceptible to SVCV. The New Zealand risk assessment conclusions cite the lack of reports of natural SVCV infections in zebrafish and the fact that other SVCV-susceptible species inhabit significantly colder waters than zebrafish do. ³¹ Similarly, the United States has restrictions on the importation of live fish, fertilized eggs, and gametes for species considered susceptible to SVCV in accordance with the OIE standards, but they do not include zebrafish in the list of restricted fish species. ³² We acknowledge that each country bases its conclusions on potentially different assessment methodologies and possibly different reference data; however, we think it is important to highlight the variance in conclusions and resulting regulations.

The stark contrast in conclusions between Canada and other OIE member countries suggests that the CFIA scientific assessment was lacking essential information that led them to different conclusions from other countries. For example, none of the studies using zebrafish as an SVCV infection model have demonstrated that infected fish can transmit the disease to other fish. Other key knowledge gaps include determining the prevalence of SVCV in Canada; testing susceptibility of zebrafish under natural conditions; determining health status of tested zebrafish to see if there is any effect of coinfections on susceptibility; and identifying the socioeconomic risk of importing zebrafish. We argue that investigation into these knowledge gaps should preclude restriction of zebrafish importation until it is justified. Regardless, conventional research facilities have highly regulated environmental conditions and an increasing number of facilities are implementing formal health surveillance by their staff to effectively ensure that natural SVCV infection in zebrafish, if it occurs at all, would be prevented from spreading. Nonetheless, zebrafish in research facilities are constantly monitored for clinical signs of disease since multiple research personnel use these animals frequently for breeding and other experiments. Moreover, both influent and, due to use of recirculating systems, effluent are often subject to UV sterilization. In addition, influent is often reverse osmosis filtered, and effluent is subjected to 50 μm filtration, which removes feces and other debris. Furthermore, fish health is monitored by veterinary staff, and temperatures at which zebrafish are housed are in line with those required by the OIE to prevent and limit infection and spread of SVCV. ⁷ Finally, there is no assessment of any possible risk from importing SVCV-infected zebrafish to human/animal health or socioeconomic impact greater than what is currently present in Canadian waters. ¹⁵

To summarize, the authors were encouraged to see that the CFIA sought to align their scientific assessment approach with OIE standards for determining susceptibility. However, considering the review of the same aforementioned evidence (both existing and missing) regarding the likelihood of zebrafish becoming infected, imported, and subsequently contaminating Canadian waters with significant amounts of SVCV, we think the evidence firmly supports the decision made by most other OIE member countries to exclude zebrafish from the SVCV susceptibility list.

Development of quarantine for importation of zebrafish into Canada

We next describe the efforts to work with the CFIA requirements. It is important to note that such efforts are viewed as significant and the partial success of such efforts in no way subverts the conclusion above that zebrafish should not be included on the SVCV susceptibility list.

There are three options available for importing zebrafish into Canada with respect to containment standards: (1) CFIA negotiates an export certificate from the country of origin, which certifies that the zebrafish are SVCV free. In this case, no containment is necessary. This option is not feasible, however, since virtually none of the exporting countries list zebrafish as a susceptible species and thus do not test for SVCV during routine health inspections. The CFIA has attempted to negotiate export certificates with other countries (e.g., Germany) with the aim of having exporters test for SVCV, but to our knowledge, none of the contacted countries have responded positively to the CFIA request to establish export certification practices. One exception was an arrangement made between the CFIA and the ZIRC. ZIRC agreed to test for SVCV at their expense, but after about a year, it decided that it was not economical and did not renew the annual testing (Zoltan Varga, pers. comm.). Determining the SVCV status of animals before import will likely require individual researchers to pay thousands of dollars for each shipment of animals. (2) Imported animals can be permanently housed in a level-2 aquatic containment facility (AQC2). As AQC2 facilities are intended for pathogens that are known to be present (e.g., as part of research), there is tightly restricted movement of imported animals and tissue. This is a prohibitive option for most institutions as special barriers and arduous changes to daily practices of zebrafish husbandry and use would need to be implemented. Indeed, costs for treating effluent and implementing other containment measures would constitute a large portion of researcher grant money. (3) Quarantine is an option for housing imported animals that meet most researcher needs. Unlike zebrafish housed in AQC2, animals in quarantine can be moved to noncontainment areas once CFIA issues a release based on evidence of SVCV-negative status. Alternatively, if all animals are to remain in quarantine until euthanasia, research must be conducted within the quarantine area. Unlike a traditional zebrafish quarantine facility, the CFIA requires that effluent be decontaminated before release using a CFIA-approved method (which is often based on OIE standards). As with AQC2, there is a requirement for more secure access control, dedicated equipment/clothing and holding units, biosecure waste disposal, and the development of associated standard operating procedures (SOPs). These features are not currently present at Canadian institutions and in most cases would be very difficult and expensive to retrofit.

Containment facility-related concerns with Canadian zebrafish importation regulations

With the establishment of the three importation options for zebrafish, many Canadian zebrafish facilities, in consultation with the CFIA, attempted to mitigate the negative impact of the importation regulations by adhering to the requirements for quarantine and, if releasing from quarantine, disease-free testing of zebrafish. Unfortunately, this process was slow and frequently unsuccessful. During the transition to a new importation regime, it was common for researchers to wait up to a year for approval after submitting their application. The primary causes of delay were the lack of clarity in the permit process, inconsistencies in the compliance requirements (which were made on a case-by-case basis), and the substantial costs and planning associated with modifying existing infrastructure to accommodate the new quarantine requirements. This had a significant negative impact on research productivity for all researchers who intended to use previously developed zebrafish models, mutant lines, or even standard genetically defined strains obtainable from non-Canadian breeding facilities. This also led to an unpredictable consequence on grant applications whereby reviewers would either cite concerns as to whether the Canadian zebrafish researcher could obtain the zebrafish lines listed in the proposal and/or negatively rank a grant that was proposing to remake a zebrafish line known to be available outside Canada.

Canadian zebrafish researchers have common needs regarding the importation sources and use of imported zebrafish. However, each zebrafish facility faces challenges unique to its physical infrastructure and user base when trying to meet CFIA importation regulations. First, most facilities need the ability to propagate imported zebrafish lines long term. For this to happen, imported zebrafish need to breed and the offspring maintained before they are sacrificed for SVCV testing. In addition, researchers must be able to move zebrafish embryos from a designated quarantine area to the main housing area for long-term maintenance of lines. Second, facilities must be able to import zebrafish from all sources, including both stock centers and independent zebrafish researchers, regardless of country of origin.

The first issue most facilities face is whether to apply for the AQC2 or quarantine biocontainment status classification as defined by the CFIA and described previously. There are two types of AQC2 systems that allow breeding and manipulation of live animals: small-scale in vivo and large-scale in vivo AQC2. The major difference between the two is that the limited water volume in a small-scale in vivo AQC2 biocontainment facility allows for manual decontamination of effluent; however, this significantly restricts the number of tanks a facility can have and therefore the number of fish that can be imported. ³³ The limiting factor with both types of AQC2 facilities is that all zebrafish tissue in an AQC2 facility can only be moved to another AQC2 facility. This makes long-term propagation of zebrafish lines impossible because of the inability to move zebrafish embryos to non-AQC2 main housing areas for long-term maintenance. This includes the movement of bleached embryos, the standard method of moving zebrafish from a quarantine facility to a main facility. While a small-scale in vivo AQC2 option may work for very small research laboratories that do not need to import many zebrafish and do not intend to keep them past their current generation, AQC2 is not a viable option for the majority of Canadian zebrafish facilities.

Applying for quarantine status is generally more feasible and has two main advantages over AQC2: (1) large numbers of zebrafish can be imported and (2) imported fish are free to move into other facilities. Whether applying for AQC2 or quarantine biocontainment status, a CFIA inspection of the facility is required, including review of SOPs. However, quarantine certification also requires a strategy for SVCV testing before any release of imported fish from quarantine. These requirements present three common challenges that need to be dealt with in a facility-specific manner. First, facilities and the CFIA must determine how to best test imported animals while sparing enough fish to breed for long-term propagation. Second, quarantine space should be set up to maximize the number of zebrafish that can be imported. Finally, SOPs need to be updated to reflect the testing methods, processes, and physical changes made to the facility. Once these changes are in place, facilities have the potential to import zebrafish within the current CFIA guidelines, but still require inspection before importation.

Testing for SVCV must take place before imported zebrafish tissue of any type is released from quarantine. However, the small size of adult zebrafish, along with the common practice of shipping embryos rather than adults, makes testing imported zebrafish while leaving enough individuals for long-term use difficult. Tissue samples are initially tested for SVCV by a government laboratory using quantitative RT-PCR. If results are positive, attempts are made to isolate the virus as a secondary test. The required number of fish to be tested each time is based on the sensitivity and specificity (98% and 100%, respectively) to detect the pathogen with 95% confidence if prevalence is 2%. For example, if 100 adult fish were imported from a single source, 79 would need to be tested; if only 25 adults were imported, all animals would be submitted for testing. As it is most common to import up to 10 adults, or 50–100 embryos, all imported fish would frequently be sacrificed leaving no zebrafish for long-term propagation. To address this, the CFIA and zebrafish researchers have discussed several testing options. For example, it was suggested that facilities hold sentinel zebrafish in the quarantine facility that would be sacrificed for testing once they were in contact with the imported zebrafish for a designated period of time. Another idea was that wild-type zebrafish would be imported from the same facility as the desired zebrafish lines and they would be sent for testing instead. Testing the offspring of imported zebrafish was discussed, as was environmental testing. Most options were rejected by the CFIA; however, one testing method approved by the CFIA, which we will call the brood stock method, allows for long-term propagation of zebrafish lines, but requires coordination between the CFIA and the importing zebrafish facility. In this case, imported adult or larval zebrafish are bred when they reach sexual maturity. Both the founders and the offspring are maintained in the quarantine facility until there are enough to propagate the line at which point all the original imported zebrafish are sent for SVCV testing. Negative test results release offspring from quarantine, meaning the adults and the embryos are free to move to other facilities. Positive test results require all offspring, any remaining imported zebrafish, and any zebrafish sharing the same water supply to be culled. The brood stock method of SVCV testing means that imported zebrafish remain in quarantine at least a full generation longer than normal, making space and timing of importation an issue. However, this method allows importation from all sources and the ability to propagate zebrafish for future use indefinitely, two essential components of a quality importation plan.

If a feasible SVCV disease-free testing method has been identified between a facility and the CFIA, the focus can shift to arranging quarantine space operations to maximize the numbers of zebrafish that can imported. Proper quarantine requires a clean zone at the room entry, clear separation of animals, equipment, food, water treatment, and control of vectors to prevent cross-contamination. Within the quarantine area, the brood stock method requires space to maintain imported zebrafish and offspring for breeding (if desired). If the quarantine is a single space, further importation must be suspended while offspring are being generated (2–4 months) and testing (1 month) is taking place. Another option is to divide the quarantine space into multiple working zones with a single clean zone. For example, a quarantine room with two working zones (A and B), each with independent access to the clean zone, might contain two racks in Zone A and two racks in Zone B. Separation of racks into multiple quarantine zones is possible in zebrafish facilities with recirculating water systems if each rack has its own filtration system because, in this case, each rack is considered an independent population. Zones can be clearly divided by tape or physically separated by walls, but each zone must contain a sink, its own set of nets and tanks, and a separate feeding setup.

Other significant changes to quarantine procedures are required to meet CFIA quarantine biocontainment standards. In the multiple zone approach, gowning is required in the clean zone and movement between quarantine zones requires regowning and cleaning feet and hands in the clean zone. In the quarantine zone(s), tanks must be autoclaved between uses and feeding tools bleached. Meticulous records must be kept of importation details, testing results, and other line maintenance information. Other required operational changes such as updates to training protocols, sterilization procedures, and most importantly, effluent treatments must be approved by the CFIA before implementation. Of all the noted changes, effluent treatment is the most problematic because it is not standard in many zebrafish facilities and there is ambiguity in the CFIA requirements. According to the Manual, to kill SVCV, heat, chlorine (or other chemicals), high or low pH, or UV can be used, but in many cases, the CFIA places the onus on researchers to determine the method and amount to use in their facility. ⁷

In most facilities, effluent results from reservoir overflow that is released into drains. Depending on the volume of water, the amount and timing of water release vary. Modifying a facility with an effluent heating system that maintains water at 56°C for 30 min ⁷ is often prohibitively expensive. A standard, automated treatment system using chlorine or pH treatment is not currently available for systems releasing effluent many times over the course of the day and these materials can be toxic to the technicians handling the material regularly. However, for facilities that release effluent much less frequently, or have the space to store and treat large volumes of water for treatment, high pH or chlorine treatment may be inexpensive, viable effluent treatment methods if the municipality allows it. UV treatment is an attractive treatment method because it is relatively inexpensive and nontoxic. The CFIA currently requires a UV dose of 186 mj/cm² to be used to achieve a log 4 reduction in pathogen, ³⁴ which may make this a reasonable effluent treatment system for many facilities. The type of prefiltration used will decrease the dosage of UV required (e.g., up to 2.0 log credit given for certain filtration types).

Impact of the restrictions on the Canadian research community

The process of meeting CFIA zebrafish importation requirements has negatively impacted Canadian zebrafish research by creating significant hurdles for importation of zebrafish. However, many detailed discussions with the CFIA and zebrafish researchers have led to possible solutions that are tenable, although still restrictive considering the paucity of data implicating SVCV as a naturally occurring infective agent in zebrafish. For smaller facilities, the prohibitive cost of setting up a quarantine area means that there is no workable option of being able to import zebrafish for their research. The brood stock method of testing imported fish is time-consuming, space restrictive, and requires significant cost for facility modifications and SVCV testing. However, it does allow the importation of zebrafish again, at least by some facilities, which is a step in the right direction.

Even once all CFIA requirements are met, experience from the only institution with an approved quarantine biocontainment facility shows that challenges may still persist. The zebrafish import permit it was issued is only valid for 6 months, making it difficult to coordinate with the supplier's breeding schedules, their 4-month quarantine cycle, and the limited seasonal windows to safely ship the fish (i.e., trying to avoid the coldest and warmest months of the year). Once the fish arrive, they still have to arrange to have a local CFIA inspector on site to observe receipt and unpacking of the fish. In addition, a local inspector needs to be present for the collection of sentinel fish for disease testing even though the local inspectors have no training in handling fish or access to euthanizing materials.

There is reason to be optimistic that Canadian researchers may be able to begin importing zebrafish again in the near future. However, it remains to be seen whether these changes are possible in smaller Canadian institutions that may not be able to address an abundance of costly modifications required for their facilities. As it stands now, the regulations are too onerous, and despite years of effort, no research laboratory in Canada has managed to fully establish such a facility and those that are in the process are doing so at a significant expense. Future discussions are needed to find a more inclusive solution to the Canadian zebrafish importation problem, whether this is a change in the regulations or the establishment of importation partners among the Canadian zebrafish facilities.

Impact of the restrictions on the Canadian public

As with all model organisms in biomedical research, zebrafish in research facilities have great potential to positively impact the health and socioeconomic well-being of Canadians. Unfortunately, the list of research areas negatively impacted by zebrafish importation restrictions also includes priority concerns for the CFIA and thus of the Canadian Public. ³⁵ These include, but are not limited to, the safety of Canadian food, the integrity and well-being of livestock and other animal resources, the monitoring of aquatic animal health as it pertains to natural resources, and the environmental impact of toxins. For example, the occurrence of Bovine Spongiform Encephalopathy (Mad Cow Disease, a prion disease) has had intense socioeconomic impact on Canadian farmers. The international community of prion researchers has sought out zebrafish researchers, acknowledging the unique power of the zebrafish model system to bring insights in disease etiology and provide a platform for drug discovery.^36–38

Ongoing efforts to develop therapies using zebrafish drug screen platforms have been dramatically hampered by the ban on zebrafish importation. Similarly, toxicology researchers use zebrafish to monitor and predict environmental impact.^39,40 Various molecular and behavioral endpoints in zebrafish are contributing to international standards. Prominent efforts impacting Canada include deploying zebrafish to measure toxicology of nanoparticles ⁴¹ and to discern between mitigating strategies in oil sand and oil spill industrial contamination. ⁴² In fact, as discussed previously, the Canadian importation restrictions resulted from use of zebrafish to study the etiology and treatment of finfish diseases such as SVCV that concern the aquaculture industry. These examples show how restricting Canadian zebrafish importation poses risks not only to Canadian basic research but also innovation areas that impact Canadians themselves.

Communication between the Canadian zebrafish research community and the CFIA

Subsequent to the late notification and implementation of the new regulations, the Canadian scientific community corresponded with the CFIA on several occasions to better understand why the restrictions are warranted and how the import requirements are to be met. This was achieved through letters, teleconferences, and webinars. As the CFIA had not worked out all the details of the import process before implementing the regulations, they provided what limited information they could on the containment and disease-free testing requirements for imported animals.

In the early stages, each affected institution was largely communicating independently with the CFIA. Since the CFIA response to inquiries and concerns from the academic community did not result in sufficient clarification of compliance requirements or removal of restrictions, the U15 (representative organization for Canada's 15 largest universities) was asked by several of its members to assist. As a result, the U15 was able to arrange a meeting with CFIA in November 2015. The aim was to further discuss the aforementioned concerns and determine how to best move forward to address them. It became apparent through the meeting that the CFIA was not going to remove the import restrictions unless the OIE Aquatic Code and/or Manual was changed to indicate that zebrafish should not be considered a risk. It was also agreed that the two parties had to work more closely together to determine what import process-related requirement institutions would need to be compliant and thus be able to apply for import permits. Other organizations such as the Canadian Association of Laboratory Animal Medicine are also corresponding with the CFIA about similar concerns regarding the import regulations. An offer to contribute to this article was made to the CFIA, but they respectfully declined. The CFIA did express, however, that it is committed to engage with the U15. At the Fall 2015 meeting, it was agreed that the best path forward for open and transparent communication with all members of the U15 would be an annual face-to-face forum to discuss the wide range of issues and concerns (pers. comm.; February 2016).

Moving forward

The Canadian scientific community and the CFIA differ in their view regarding the risk of imported zebrafish acting as a vector for SVCV introduction and spread within Canada. The scientists support, however, the government's commitment to safeguarding Canada from experiencing disease outbreaks that could have a devastating effect on Canadian fisheries. What is needed is more evidence-based policies and consultation (and possibly collaboration) between the CFIA and regulated parties so that this objective can be achieved while minimizing the negative impact that any new regulations have on Canadian research.

Regarding the SVCV situation, it is hoped that the import process can be further streamlined by establishing CFIA-approved embryo treatment protocol(s) that can be performed before shipping or on arrival. This embryo treatment protocol could also be applied to all embryos exiting quarantine facilities within Canada. This would minimize both costly (CFIA-specific) quarantine measures and the time required to start working with the imported lines. Indeed, this is the number one priority of zebrafish researchers, and approving embryo treatment protocols alone would obviate most problems with current CFIA import restrictions. However, the biggest issue with CFIA import regulations would still remain: the paucity of evidence supporting the inclusion of zebrafish as an SVCV-susceptible species. In working toward minimizing the burden of the new regulations, the Canadian zebrafish community is working together to share their experiences and information regarding the CFIA approval process such as CFIA-approved SOPs and quarantine facility specifications.

The CFIA and the scientific community have recently sought to improve communication lines and have both committed to more open ongoing dialog in the future. These changes leave us optimistic that the level of disconnect experienced with the SVCV regulations is unlikely to happen again going forward.