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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: [email protected]
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:
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