La-10-101 25.30

Using amphibians in laboratory studies: precautions against the emerging infectious disease chytridiomycosis Dirk S Schmeller1, Adeline Loyau1, Tony Dejean2 and Claude Miaud2 1Station d’Ecologie Expe´rimentale du CNRS a` Moulis, USR 2936, 09200 Saint Girons, France; 2Laboratoire d’Ecologie Alpine,UMR CNRS 5553, Universite´ de Savoie 73376, Le Bourget-du-Lac cedex, FranceCorresponding author: D S Schmeller. Email: ds@die-schmellers.de AbstractThe African clawed frog Xenopus laevis is by far the most widely used amphibian species in laboratories. In the wild, X. laevis is an asymptomatic carrier of an emerging infectious disease called chytridiomycosis. The vector is the chytrid fungus Batrachochytrium dendrobatidis (Bd), which has devastating effects on wild amphibian populations around the world. The impact of Bd on the metabolism of X. laevis has not been comprehended yet. However, even if asymptomatic, an infection is likely to affect the individual’s physiology, immunology, development, reproduction and overall response to stress from a purely medical point of view, which will introduce noise and therefore increase variance within experimental groups of X. laevis. This could have implications on the scientific results from studies using this species. Here, we review the current knowledge on treatments of infected amphibians and propose a hygiene protocol adapted to laboratory populations and amphibian husbandry. Following the presented sanitation guidelines could further prevent the spread of Bd and probably of other amphibian pathogens. The sanitation guidelines will help to reduce the impact of amphibian husbandry on natural populations and must be considered a crucial contribution to amphibian conservation, as today 32% of all amphibians are Keywords: Hygiene, chytridiomycosis, disinfection, Xenopus Laboratory Animals 2011; 45: 25 – 30. DOI: 10.1258/la.2010.010101 It is now widely recognized that research should be con- Hyla, Ambystoma, and particularly Xenopus spp. (X. laevis ducted on healthy, pathogen-free, animals and products to and X. tropicalis).6 However, the control of pathogens and ensure reliable data and allow comparison and interpret- infectious diseases in amphibian facilities is rather new.
ation of research results. For example, infection can have Only a few breeding facilities use sentinels for the detection either a positive or a negative impact on a trial.1 Even sub- of diseases. Even large institutions such as the Federation of clinical infections have been pointed out to potentially con- European Laboratory Animal Science Associations in found research results.2 As a consequence, during the 1980s, Europe (FELASA) or the National Research Council Guide bioexclusion protocols have been developed and specific for the Care and Use of Laboratory Animals7 –9 in the USA pathogen-free (SPF) animals have been bred (or requested have not yet established health monitoring recommen- to suppliers) to eradicate infectious diseases in animal facili- dations for amphibians as they exist for rodents10 and ties. Despite stringent procedures, several emerging and only recently a description of amphibian diseases was pub- re-emerging pathogens have been recently identified in lished.11 As such, extensive routine health monitoring is rodent laboratory colonies, including the murine noro- usually not performed in amphibians, and real SPF amphi- virus.3,4 The threat represented by emerging pathogens led bians do not exist. It has been estimated that still one-third to the organization of a workshop dedicated to the of X. laevis used are taken from the wild,12 where they are ‘Detection, Impact and Control of Specific Pathogens in likely to encounter various pathogens. This might be Animal Resource Facilities’ in 2009. One major conclusion especially true for several newly emerging infectious dis- of this workshop was the recommendation of ‘the improve- ment of communications of disease outbreaks and potential Batrachochytrium dendrobatidis (Bd).11,13 Bd is the vector for risks for animal models for the scientific community’.5 the disease chytridiomycosis, which is known to be a proxi- Amphibian species are widely used in developmental, mate driver of rapid amphibian species declines and extinc- cell and molecular biology, as well as in genetic and tions on five continents14 (despite the still unclear origin of genomic research. This includes the genera Rana, Bufo, the disease14–16), and it has been placed on the OIE Wildlife Disease List in 2001. The fungus infects keratinized more delicate due to the potential negative effects of disin- epidermal cells of amphibian species and causes a hyperker- fectants on amphibian species. Further, many amphibian atotic and hyperplastic response of the stratum corneum and facilities are conventional animal facilities in which the stratum granulosum.17 The causes of death of amphibians risk of cross contamination is elevated as tanks often are related to Bd are still unknown, but may include disruption interlinked, allowing Bd zoospores to be transported from of osmoregulation and toxin release.11,18,19 In tadpoles usually the keratinized mouthparts are infected, while the Here, we review the current knowledge on disinfection infection spreads further during and after metamorphosis techniques for amphibian facilities, treatments of amphibian specimen, the effect of treatments on some amphibian In the wild, X. laevis is subclinically infected by Bd, carry- species, and propose a sanitation protocol adapted to lab- ing infection but does not develop lethal chytridiomycosis oratory populations and amphibian husbandry.
likely due to magainin, an antibiotic, antifungal, antiparasi-tic and antiviral, which has been found on its skin.21However, even if subclinical, an infection is likely to affect the physiology, immunology, development, reproduction and overall response to stress from a purely medical pointof view. Hence, noise will be introduced increasing variance Disinfectants have been tested on Bd cultures and live amphi- within experimental groups of X. laevis, which could have bian specimens. So far, Bd cultures have shown few cases of implications on the scientific results from studies using resistance to known disinfectants active against other patho- X. laevis.2 This might be especially important, given the dif- genic fungi.25 In a series of in vitro tests, Johnson et al.25 deter- ficulty to interpret signs of illness expressed by X. laevis.12 mined the efficacy of the following chemical disinfectants: Hence, diseases might become established unrecognized sodium chloride, sodium hypochlorite (household bleach), po- and pathogenesis may develop to a high degree before tassium permanganate, formaldehyde solution, didecyl ill-effects become obvious.22 It is therefore imperative to reg- dimethyl ammonium chloride (DDAC; Path-XTM agricultural ularly test captive populations and disinfect them if needed.
More recently, the closely related species X. tropicalis is Queensland, Australia), quaternary ammonium compound replacing X. laevis in laboratories. In contrast to X. laevis, it 128 (James Varley & Sons, St Louis, MO, USA), Mancozeb has been reported to be severely affected by the Bd fungus, (Dithane, Dow AgroSciences, Indianapolis, IA, USA), with more than 80% of mortality during one epizootic Virkonw (DuPont, Wilmington, DE, USA), ethanol and ben- event.23 These individuals were likely infected by X. laevis zalkonium chloride. The time of exposure was critical for the individuals.23 Indeed, X. tropicalis normally lives at higher efficacy of most chemicals (Table 1). Webb et al.26 tested three temperatures than X. laevis, a requirement that is not always fulfilled in laboratories and may explain Bd outbreaks due to (Betadine Antiseptic Liquid, Faulding Pharmaceuticals, a lowered ability of immune responses of X. tropicalis in low- Adelaide, Australia), and multicomponent products containing temperature environments.24 In addition, stressful captive con- polymeric biguanide hydrochloride, DDAC and dimethyl ditions and the wide use of immunosuppressed Xenopus indi- benzyl ammonium chloride (TriGene Virucidal Disinfectant viduals largely increases the probability of developing an Cleaner, Medichem International, Sevenoaks, Kent, UK) or ben- zalkonium chloride and biguanide (F10 Super Concentrate The absence of clinical signs of Bd infection and the wide Disinfectant, Lomb Scientific, Taren Point, New South Wales, use of X. laevis leading to naturalization of many popu- lations worldwide was hypothesized as fatal for global Johnson et al.25 tested the efficacy of UV light, heat and amphibian diversity.15 The transmission to natural popu- desiccation on Bd cultures. Complete desiccation within lations was possible, because individual frogs did either a 3 h time window at room temperature led to the death of escape laboratories and breeding facilities or were liberated all Bd cultures, which also showed a high sensitivity to intentionally. Due to the unawareness of the deadly disease, heating. The 100% death of zoospores and sporangiae precautions were not put in place. Therefore, to protect wild occurred after 4 h at 378C, 30 min at 478C and 5 min at populations of any kind of amphibians, strict hygiene rules 608C. UV light (at 1000 mW m2 with a wavelength of need to be employed to mitigate the further spread of the 254 nm) was ineffective at killing Bd in culture.
disease. Here in particular it is noteworthy that UV treat- Few tests were conducted on infected amphibian individ- ment of waste water before release, as is usually employed uals. Banks and McCracken28 report the inefficacy of in Xenopus facilities, does not kill Bd and therefore is a Plistopurw, a combination of copper phosphate, acriflavin potential source of Bd transmission.25 In amphibian facilities HCl and P-chlorophenoxetol (Sera, Heinsberg, North with the aim to provide SPF individuals, the usually Rhine-Westphalia, Germany) followed by griseofulvin employed safety measures might be sufficient (use of (Grisovin, Sigma Pharmaceuticals, Clayton South, Victoria, safety locks, autoclaving of equipment) as these usually are effective against Bd. However, contamination with dayfrog tadpoles. Schmidt et al.29 assessed the effect of food has currently not been ruled out. This is particularly household bleach and Virkonw S on tadpole performance difficult in amphibian breeding facilities as live food needs and zooplankton abundance in a factorial experiment.
to be provided, making autoclaving of food impossible.
They found that bleach at a concentration of 2% killed all Further, once Bd has found a host the treatment becomes tadpoles of Rana temporaria and Bufo bufo. Virkonw S (10% Schmeller et al. Treatment of chytridiomycosis in amphibian captive breeding Effectiveness of disinfectants on Batrachochytrium dendrobatidis (Bd) culture, their minimal effective concentration for 100% kill, time of exposure for 100% kill and the corresponding reference organic acids and an inorganicbuffer system DDAC, and dimethyl benzylammonium chloride solution) did not show any detectable negative effect on tad- poles and zooplankton, but was effective against Bd. Two When setting up a new facility, all equipment should be studies analysed the efficacy of itraconazole (Sporanoxw, properly sterilized before use. If uncertainty remains, sterili- Janssen Pharmaceutica, Beerse, Belgium). Nichols and zation should be performed, so that the facility can be con- Lamirande30 used a 0.01% itraconazole solution in 0.6 saline sidered pathogen free. If facility users move among several over 11 days of 5 min treatments each day and successfully breeding rooms or outdoor sites, the sterilization or disinfec- eliminated infection in juvenile blue-and-yellow poison dart tion protocols should be applied. When handling amphi- frogs Taudactylus acutirostris. In subadult and adult individ- bians, disposable, powder-free gloves should be used and uals of the same species, an eight-day treatment of dumped in biological hazard garbage. However, there are itraconazole baths resolved the infection.31 Itraconazole was some reports on negative effects of certain gloves, leading also successfully used in Alytes muletensis tadpoles, but led to mortalities in larval and adult amphibians.33,34 In addition, we advise that equipment should be regularly(every week) disinfected or sterilized within the housingplace as a general precautious principle.
The entry to any amphibian husbandry should be pro- tected by a safety lock. Despite the fact that chytridiomyco- Virkonw S is a broad-spectrum disinfectant, based on per- sis does not pose a threat to humans (no zoonoses) and oxygen compounds ( potassium peroxymonosulphate), sur- therefore regulations do not (yet) require an elevated secur- factant (sodium dodecylbenzenesulphonate), organic acids ity of facilities, a safety lock is highly recommendable to (sulphamic acid) and an inorganic buffer system, with avoid any exit or entry of Bd and infected amphibians to known capability at killing bacteria, fungi and viruses, and from any breeding facility. Laboratory coats and including Bd. It achieves deactivation and/or destruction shoes should be provided for each person working in the of the target organism through general oxidative disruption breeding facility, for visitors disposable paper shoe protec- of key structures and compounds vital to normal activity tion is recommended. Shoes should be disinfected at entry (e.g. proteins and lipids). We recommend the use of this dis- and exit using a footbath or cushions soaked with either infectant for equipment, because of its low environmental 1% Virkonw S solution (10 g Virkonw S in 1 L of water; impact and apparently low toxicity on amphibians,29 but renewed every day) or any other available surface disinfec- lack of toxicity is not guaranteed by the manufacturer.
tant (renewed at least once per week). Hands and laboratory Release to water bodies before inactivity ( pale pink colour coats need to be disinfected when exiting the facility.
of solution) should however be avoided and disposalthrough sewer systems is recommended by the manufac-turer. As Virkonw S is irritant to the skin and may cause serious damage to eyes, direct contact with the skin andeyes should be avoided by wearing suitable protective cloth- Whenever possible, amphibians captured in the wild should ing, gloves, eye and face protection (in accordance with BS be housed individually. Preventing co-housing of amphi- EN 166). Product label instructions and information includ- bians during collection limits contacts and disease trans- ing the precautionary statements should be read and fol- mission among animals. As amphibians bred in captivity are not systematically tested for Bd, no one can be sure that new individuals purchased or exchanged with other (no rinsing required). Small material in contact with amphi- laboratories are Bd negative. Any new individual entering bians (e.g. scalpels, scissors, etc.) should be immersed in a a laboratory facility should therefore be placed in quarantine 1% Virkonw S solution and stored in disposable plastic and tested negative before being included with the rest of bags or in a special storage room to assure that they the group. Individuals should be regularly tested for Bd cannot be re-contaminated. After sterilization of equipment, using, i.e. the realtime polymerase chain reaction Taqman assay published earlier.17 With this assay the accurate detec- Recommendations of local Occupational Health and Safety tion and quantification of one zoospore in a diagnostic specialists should be sought to adapt the choice to local situ- sample is possible, using an MGB probe and two specific ations. Finally, laboratory clothes should be disinfected by primers, ITS-1 and 5.8S. Generally, there are two possibili- washing them at 608C and any disposable items (gloves, ties for achieving reliable test results: (i) testing all individ- bags, etc.) should be collected in biological waste containers.
uals at regular intervals (i.e. every 6 months) and (ii)keeping a sentinel population, which receives water fromall the tanks in the breeding facility and test the individuals once per month as suggested for fishes.35 A sentinel popu- Itraconazole is a triazole antifungal drug usually used for lation will reduce the costs and increase the probability of the treatment of systemic fungal infections of dogs, cats detecting even low concentrations of infections and should and humans and comes as capsules or a liquid solution. It be preferred, but which tank contains infected individuals is effective against all filamentous fungi, dimorphic fungi will remain unclear. Once infection is detected, it is not suf- and yeasts, such as blastomycosis, histoplasmosis, aspergil- ficient to treat only the individual and disinfect its tank, but losis and cryptococcosis. It may also be used against some all equipment and tanks it could have come in contact with.
yeast and dermatophyte (ringworm) infections. Triazoles Hence, a complete disinfection or sterilization of tanks, are a subgroup of the azole group of drugs. These drugs equipment and water in the location where Bd has been are fungistatic at the concentrations used systemically and fungicidal at the concentrations that may be achievedtopically. The mechanism of action is through the disruptionof the oxidative enzymes of the fungal organism.36 Itraconazole is a potent cytochrome P450 3A4 isoenzyme A standard sterilization protocol for equipment, shoes and system (CYP3A4) inhibitor and may increase plasma con- coats comprise the following steps. A fresh 1% Virkonw S centrations of drugs metabolized by this pathway.
solution (10 g/L) should be prepared every day and the sol- Detailed handling instructions and precautions are pro- ution should not be used when the colour is not at least a vided by the manufacturer and should be consulted before medium pink. A spray bottle can be used to squirt Virkonw S solution on all used equipment, which might Treatment of individual amphibians and tadpoles need to have been in contact with water and wait for 5 min before take into consideration potential negative effects of the re-use, and preferably until the equipment has dried different drugs (Table 2). So far, itraconazole promises the Effectiveness of disinfectants in treatments of live animals (in vivo) and their side-effects, their minimal effective concentration for 100% kill, time of exposure for 100% kill, the days of treatment (N days), the species and life stage, and the corresponding reference mexicanum (adult)Potymotyphluskaupii (adult) Schmeller et al. Treatment of chytridiomycosis in amphibian captive breeding highest efficacy.30– 32 It is therefore recommended that 6 Gurdon J. Introductory comments: Xenopus as a laboratory animal. In: Bd-infected individuals be treated using itraconazole by Tinsley RC, Kobel HR, eds. The Biology of Xenopus. Oxford: OxfordUniversity Press, 1996:3–6 being bathed in an itraconazole solution. Forza´n et al.37 7 Clark J, Gebhart G, Gonder J, Keeling M, Kohn D. Special report: The 1996 guide for the care and use of laboratory animals. ILAR J Lamirande30,31 using a 0.1% solution in 0.6% saline for 5 min treatments each day over a period of 11 days and 8 Bayne K. Revised guide for the care and use of laboratory animals reported no side-effects. Garner et al.32 reduced the number available. American Physiological Society. Physiologist 1996;39:199 9 Bayne K. Potential for unintended consequences of environmental of treatments to seven days (Table 2), but reported depigmen- enrichment for laboratory animals and research results. ILAR J tation of tadpole skin in their species.32 The latter treatment is preferable; as it reduces the number of times individuals are 10 Alworth L, Harvey S. IACUC issues associated with amphibian research – handled and stressed, but side-effects need to be taken into use of amphibians in the research, laboratory, or classroom setting. ILAR J account and the individuals need to be closely observed 11 Densmore CL, Green DE. Diseases of amphibians. ILAR J 2007;48:235 – 54 during the treatment. A second test for Bd should be per- 12 Reed B. Guidance on the Housing and Care of the African Clawed Frog – formed two weeks after the end of the topical treatment to Xenopus laevis. London: Royal Society of the Prevention of Cruelty to exclude the possibility that re-infection has occurred.
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