Journal of the American Association for Laboratory Animal Science Copyright 2017 by the American Association for Laboratory Animal Science
Vol 56, No 4 July 2017 Pages 412–424
Comparison of Antemortem and Environmental Samples for Zebrafish Health Monitoring and Quarantine Marcus J Crim,1,2,* Christian Lawrence,3 Robert S Livingston,1 Andrei Rakitin,4 Shane J Hurley,3 and Lela K Riley1 Molecular diagnostic assays offer both exquisite sensitivity and the ability to test a wide variety of sample types. Various types of environmental sample, such as detritus and concentrated water, might provide a useful adjunct to sentinels in routine zebrafish health monitoring. Similarly, antemortem sampling would be advantageous for expediting zebrafish quarantine, without euthanasia of valuable fish. We evaluated the detection of Mycobacterium chelonae, M. fortuitum, M. peregrinum, Pseudocapillaria tomentosa, and Pseudoloma neurophilia in zebrafish, detritus, pooled feces, and filter membranes after filtration of 1000-, 500-, and 150-mL water samples by real-time PCR analysis. Sensitivity varied according to sample type and pathogen, and environmental sampling was significantly more sensitive than zebrafish sampling for detecting Mycobacterium spp. but not for Pseudocapillaria neurophilia or Pseudoloma tomentosa. The results of these experiments provide strong evidence of the utility of multiple sample types for detecting pathogens according to each pathogen’s life cycle and ecological niche within zebrafish systems. In a separate experiment, zebrafish subclinically infected with M. chelonae, M. marinum, Pleistophora hyphessobryconis, Pseudocapillaria tomentosa, or Pseudoloma neurophilia were pair-spawned and individually tested with subsets of embryos from each clutch that received no rinse, a fluidizing rinse, or were surface-disinfected with sodium hypochlorite. Frequently, one or both parents were subclinically infected with pathogen(s) that were not detected in any embryo subset. Therefore, negative results from embryo samples may not reflect the health status of the parent zebrafish. Abbreviations: MU, University of Missouri
Both clinical and subclinical naturally occurring infections are well established to introduce potentially confounding variability that can lead to invalid or misinterpreted experiments in mammalian animal models.1 There is growing awareness within the research community that this same dynamic exists for zebrafish.6,8,18,21,37,45 Moreover, evidence is mounting that both genetic background39 and stress37 influence infection phenotypes in zebrafish, as has been established for rodent models.30 Therefore, as is the case for rodents and other mammalian model species, the exclusion of infectious agents from zebrafish colonies is a critical component of reducing confounding variability in biomedical research and is facilitated by the use of purpose-bred pathogen-free animals, approved vendor lists, pathogen exclusion lists, quarantine practices, disinfection, routine sentinel health monitoring, and environmental monitoring. Sentinel zebrafish, as conventionally described,22,48 are analogous to rodent soiled-bedding sentinels, whose routine evaluation has historically been the cornerstone of health monitoring in rodent colonies—even though it has long been recognized that not all pathogens transmit easily to soiledbedding sentinels.2,31,43,49 Consequently, recent experiments have demonstrated that a comprehensive approach incorporating both sentinel data and environmental sample types, such as cage swabs (cage level) and exhaust-air debris (cage, row, or rack level), provides a more complete representation of rodent population health.2 Pathogen transmission in zebrafish colonies Received: 05 Aug 2016. Revision requested: 14 Oct 2016. Accepted: 12 Jan 2017. 1IDEXX BioResearch, Columbia, Missouri; 2Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri; 3Boston Children’s Hospital, Boston, Massachusetts; and 4IDEXX Laboratories, Westbrook, Maine *Corresponding author. Email:
[email protected]
more closely resembles the situation in conventionally housed rodent colonies than that of rodent colonies housed in ventilated racks or static microisolation cages, although ventilated rodent racks where sentinels receive exhaust air from the colony in addition to soiled bedding4 may be a better approximation of zebrafish sentinel exposure. In contrast to rodents, where cagelevel biocontainment is relatively common,4,7,51 most zebrafish are housed in recirculating aquaculture systems, where water provides an effective medium for pathogen transmission.6,20,30 When zebrafish colonies are housed in recirculating systems, detritus accumulates in small amounts on the floor of individual tanks and in larger amounts in the sump, where the effluent water from the colony is collected for filtration prior to UV treatment. Tank or system detritus—sometimes referred to as sediment or sludge—is a mixture of feces, uneaten food, and other debris. A wide variety of bacteria, including mycobacteria and cyanobacteria, as well as fungi, algae, oomycetes, protozoa, and micro- and macroinvertebrates can inhabit detritus in zebrafish systems. Detritus, which has been used as a real-time PCR sample type for other aquatic species,10,47 is similarly a very attractive environmental sample for zebrafish health monitoring because it is easy to collect; can be obtained at the tank, rank, or system level; and includes fecal material along with a variety of microscopic particulates that are coated with biofilms. In most recirculating systems designed to house zebrafish, water passes through tanks that are plumbed in parallel (to reduce between-tank horizontal pathogen transmission), and the effluent water from individual colony tanks is collected and supplies one or more tanks containing rack- or system-sentinel zebrafish. Recirculating systems typically include zebrafish of different genetic backgrounds and may include the colonies of
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Samples for health monitoring of zebrafish
multiple investigators. The potential consequences of unrecognized pathogens are increased for large recirculating systems that house large numbers of fish, colonies belonging to multiple principal investigators, and more genetically diverse fish. Immunologically robust fish populations, which may include wild-type zebrafish lines, or in some cases, other fish species such as Japanese medaka (Oryzias latipes)23 may act as pathogen reservoirs, subclinically harboring and shedding pathogens that can cause disease in less robust or immunocompromised lines of fish housed on the same system. Prior to the availability of PCR-based assays, zebrafish health monitoring primarily involved histopathology of whole adult zebrafish,5 such as intentionally placed sentinels, escapees (that is, sump fish), older fish, retired breeders, and moribund colony zebrafish. However, diagnostic real-time PCR analysis offers key advantages over histopathology, including exquisite sensitivity, identification of mycobacteria to the species level, and the ability to test ‘found dead’ zebrafish that exhibit postmortem autolysis. In addition, PCR analysis can be used to evaluate a wide variety of sample types, including biofilms, detritus (mixture of feces, uneaten food, and debris), embryos, feces, filter materials, live feeds, manufactured feeds, microbial cultures, sperm, surface swabs, and water. The ability to test environmental samples with increased sensitivity provides a useful adjunct to sentinel testing,6 as has been demonstrated for rodent colonies,2,7,17 and can assist in the identification of potential sources of contamination, the stage in a process where contamination occurs, and evaluation of the extent of contamination that has occurred. As does zebrafish health monitoring, quarantine for zebrafish colonies presents unique challenges. Many institutions have adopted a fertilized ‘eggs-only’ policy,20 only allowing the entry of surface-disinfected embryos into main systems to reduce the introduction of new pathogens. Purpose-bred zebrafish are shipped typically as several breeding pairs or as a clutch of embryos. Genetically modified zebrafish that infrequently survive to adulthood or do not spawn well may be supplied as a few very valuable adults, for which antemortem tests would be advantageous. Moreover, chlorine toxicity to zebrafish embryos is greater at 24 h after fertilization than at 6 h afterward,19 such that embryo survival might be unacceptably low as a result of surface disinfection by the receiving institution at the time of arrival. Accepting embryos that are surface-disinfected at another institution is risky for several reasons. Protocols vary, and the efficacy of surface disinfection with sodium hypochlorite (by far the most commonly used surface disinfectant for zebrafish embryos), is concentration-, contact time-, and pH-dependent.11 The spores of the most commonly detected pathogen of zebrafish, Pseudoloma neurophilia, can be transmitted vertically through the inclusion of infectious spores within unfertilized and fertilized zebrafish eggs41 and are resistant to surface disinfection with sodium hypochlorite.11 In addition, pathogens inside eggs are shielded from surface disinfection by the surrounding organic material, regardless of whether that particular embryo survives. In addition, mycobacteria are chlorine-resistant, especially when incorporated into biofilms, and clump together due to high cell-surface hydrophobicity.26,46 Many institutions therefore raise incoming embryos to adulthood during quarantine, to surface-disinfect the progeny inhouse. The parents can then be submitted for diagnostic evaluation while the second generation remains in quarantine. If pathogens are identified in either generation, the process often is repeated for another generation, with some lines remaining in quarantine for as long as 6 mo. Holding zebrafish in quarantine
for months is problematic because of space constraints and because it can delay research or, in institutions that allow it, require research to be conducted in quarantine. Research in quarantine is a biosecurity risk and increases zebrafish manipulations and human traffic in the quarantine area, where multiple zebrafish lines, sources, and various pathogens may be present. Therefore more efficient ways to evaluate zebrafish held in quarantine are needed urgently. Few antemortem diagnostic tests have historically been available for fish.20 PCR-based diagnostics have recently become available to the zebrafish community and allow the analysis of a wide variety of possible sample types. In addition, realtime PCR analysis can be used to detect Pseudoloma neurophilia in zebrafish eggs, sperm, and embryos in addition to filtered spawn-water samples.38 However, to our knowledge, the use of antemortem zebrafish sample types such as eggs and sperm for molecular analysis to detect other pathogens has not been evaluated. In the present study, we compared the utility of several antemortem and environmental sample types for pathogen detection by real-time PCR analysis. For valuable zebrafish lines received as breeding pairs, easy-to-collect antemortem samples include embryos and feces. For zebrafish received as embryos, a subset of received embryos might be tested as a diagnostic sample representing either the remaining embryos in the shipment or the parents (and by extension, the facility of origin). Because zebrafish supplied as embryos are often surface-disinfected with sodium hypochlorite, we further sought to compare surface-disinfected embryos with embryos that were not surface-disinfected, which either were untreated or underwent fluidized rinsing in disinfected fish water.
Materials and Methods
Environmental and fecal experiments. Animals and husbandry. Animals in the environmental and fecal experiments were housed in the AAALAC-accredited facility at IDEXX BioResearch (Columbia, MO) in accordance with the guidelines set forth by the Guide for the Care and Use of Laboratory Animals,16 and all procedures were approved by the University of Missouri–Columbia Animal Care and Use Committee (protocol no. 7554). All zebrafish were housed in the same room in static or flow-through aquaria supplied with reverse-osmosis–purified water that was remineralized by using a commercially available product (Replenish, Seachem Laboratories, Madison, GA). These tanks were plumbed so that they could be operated as flow-through or static tanks. During the months leading up to the study, both tanks were set to flow-through, but they were both operated as static tanks during both trials. Two colonies of zebrafish (populations A and B), each enzootically infected with multiple zebrafish pathogens, were maintained on a 14:10-h light:dark cycle at approximately 0.7 fish per liter in separate but adjacent 75.7-L glass aquaria (Aqueon Products, Franklin, WI) on the same rack, with each aquarium containing approximately 50 adult, mixed-sex zebrafish. Fish were fed at least once daily with a combination of manufactured feeds (Golden Pearls, 5 to 50 μm, High-Protein Fry Green Granules, Kens Fish, Taunton MA) and dehydrated decapsulated brine shrimp cysts (Kens Fish, Taunton, MA) that were chemically decapsulated by using a concentrated chlorine solution and dehydrated as part of the manufacturing process. To aid in stable maintenance of waterquality parameters (Table 1), the aquaria were equipped with individual air-stone aeration and biologic filtration, including power filters (AquaClear 20 Power Filter, Rolf C Hagen, Mansfield, MA) containing plastic-foam filter material (AquaClear 20 Foam Filter Inserts, Rolf C. Hagen, Mansfield, MA), which with 413
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Table 1. Water-quality parameters for zebrafish husbandry in statica and recirculatingb,c systems Environmental and fecal experimenta Parameter pH
Range
Testing frequency
Embryo and surface-disinfection experimentb,c Range
Testing frequency
Stable at 7.2–7.8
Weekly
Stable at 6.8–8.5
Continuous
Stable at 22–23 °C
Daily
Stable at 26–28 °C
Continuous
Total ammonia nitrogen
0 ppm (mg/L)
Weekly
0 ppm (mg/L)
Weekly
Nitrite
0 ppm (mg/L)
Weekly
0 ppm (mg/L)
Weekly
Nitrate
