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Evaluation of Common Disinfectants Effective against Ophidiomyces ophiodiicola , the Causative Agent of Snake Fungal Disease

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Efficacy of disinfectants used in veterinary, wildlife, and environmental settings were tested on Ophidiomyces ophiodiicola, the cause of snake fungal disease. Bleach and several common household cleaners are effective disinfectants; chlorhexidine, Simple Green, and spectricide were not. This information can be used to prevent transmission of this fungus between snakes.
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DOI: 10.7589/2016-01-012 Journal of Wildlife Diseases, 52(3), 2016, pp. 759–762
ÓWildlife Disease Association 2016
Evaluation of Common Disinfectants Effective against Ophidiomyces
ophiodiicola, the Causative Agent of Snake Fungal Disease
Marta Rzadkowska,
1
Matthew C. Allender,
1,4
Miranda O’Dell,
2
and Carol Maddox
3
1
Wildlife Epidemiology
Laboratory, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, 2001 South
Lincoln Avenue, Urbana, Illinois 61802, USA;
2
Veterinary Diagnostic Laboratory, College of Veterinary Medicine,
University of Illinois, 2001 South Lincoln Avenue, Urbana, Illinois 61802, USA;
3
Department of Pathobiology, College of
Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, Illinois 61802, USA;
4
Corresponding
author (email: mcallend@illinois.edu)
ABSTRACT: Efficacy of disinfectants used in vet-
erinary, wildlife, and environmental settings were
tested on Ophidiomyces ophiodiicola, the cause of
snake fungal disease. Bleach and several common
household cleaners are effective disinfectants;
chlorhexidine, Simple Green, and spectricide
were not. This information can be used to prevent
transmission of this fungus between snakes.
Snake fungal disease caused by Ophidio-
myces ophiodiicola, a keratinophilic fungus,
infects snakes as a primary pathogen (Allender
et al. 2015b). It was first observed in 2006 in
timber rattlesnakes (Crotalus horridus) (Clark
et al. 2011) and in 2008 in eastern massasau-
gas (Sistrurus catenatus; Allender et al. 2011).
This fungus causes dermal infections in
captive and wild snakes (Allender et al.
2015b). Clinical signs include skin swelling,
crusts, and nodules of the skin (Allender et al.
2011, 2015b). The mode of transmission is
unknown, but is speculated to occur with
direct contact between individuals or with the
contaminated environment (Allender et al.
2015b).
There are no current protocols for disin-
fecting equipment used in the field, in the
laboratory, and in animal husbandry facilities
for this fungus. Protocols established for other
pathogens threatening reptiles and amphibi-
ans, such as ranaviruses (Bryan et al. 2009)
and Batrachochytrium (Johnson et al. 2003),
have not been evaluated for Ophidiomyces.A
simple disinfection protocol for the inhibition
of O. ophiodiicola is necessary in field and
laboratory settings to prevent transmission of
fungal spores that may lead to disease. We
tested the efficacy of several disinfectants in
free-ranging reptile and amphibian protocols.
An isolate of O. ophiodiicola was cultured
in 2013 from the face of an eastern massa-
sauga, confirmed morphologically and by 18S
rRNA sequencing and quantitative PCR (Al-
lender et al. 2015a). Cultures were propagated
at room temperature (20 C) on potato
dextrose agar slants (Remel, Thermo Scientif-
ic, Lenexa, Kansas, USA). Spores were
scraped from the slants and suspended in 10
mL sterile saline, filtered with sterile glass
wool in a funnel under vacuum, and diluted to
a turbidity of 1 McFarland. Plate counts with
saline averaged 541,600 colony-forming units/
mL in the 1 McFarland suspension. We added
100 lL of the filtrate to 100 lLof11
disinfectants (Table 1). After 0.5-, 2-, 5-,
and/or 10-min exposures, the mixture was
centrifuged at 5 3G for 10 min and all but 20
lL of the disinfectant was removed. Each
sample was resuspended in 380 lL of saline.
Serial dilutions were made to obtain five final
dilutions of 1:20 to 1:200 that would result in
30–300 colonies for the untreated saline
control following 10–14 d of incubation (data
not shown).
One hundred microliters of each dilution
were spiral plated on Sabouraud dextrose agar
Emmons plates (Remel) using a T-spreader
and incubated at room temperature. Disin-
fectants were evaluated along with a sterile
saline positive control to determine the
number of colony-forming units present in
each disinfectant treatment compared to no
disinfectant. All procedures for treatment and
control were performed in triplicate and
results averaged (Table 2).
Several products resulted in 100% inactiva-
tion of Ophidiomyces in vitro, but there were
759
time-dependent relationships for many of the
trials. Disinfectants were chosen based on
availability and common use in similar studies.
Based on preliminary data we used 3% bleach
as a starting point; lower concentrations might
still be effective in field settings, but were not
tested. The fungus was sensitive to disinfec-
tants that may be good alternatives for
laboratory and veterinary applications. Lysol
products, CLR, and 409 (vendors provided in
Table 1) are ready-to-use disinfectants and are
easily accessible as common household clean-
ers. Process NPD, a germicidal detergent
used for broad antimicrobial disinfection, is
practical in laboratory settings.
Bleach was effective at inactivating Ophi-
diomyces and is commonly used in the field.
Equipment can be cleaned and disinfected to
prevent spread of the fungus between individ-
uals using either a 3% or 10% solution at 2-, 5-,
and 10-min contact times. Because ranaviruses
and chytrid fungi are effectively inactivated
with concentrations of bleach lower than those
tested in this study, the use of bleach at these
concentrations will be effective at disinfecting
all three pathogens (Table 2). Including bleach
in shoe-washing stations for visitors has merit
in high-risk areas and may prevent spread of
the fungus. Care must be taken as even 3%
bleach is corrosive and irritating to skin.
Quaternary ammonia products (NPD) were
as effective as bleach when used at 10 min and
are less corrosive.
Three products were ineffective against
Ophidiomyces even with a 10-min contact
time. Chlorhexidine, a bactericide and viru-
cide, is frequently used in veterinary settings
because of its spectrum of activity and low risk
of adverse effects (Zoetis 2015). Chlorhexi-
dine decreased fungal growth but did not
prevent it. Simple Green, promoted to be an
environmentally safe disinfectant, was also not
efficacious. Colony counts were lower than
with both saline and chlorhexidine, but
TABLE 1. Disinfectants used to test inactivation of Ophidiomyces ophiodiicola in vitro.
Product Active ingredient Manufacturer
Lysol Power Bathroom Cleaner Citric acid, 2.5% Recikk Benckiser, Parsippany, New
Jersey, USA
Lysol All-Purpose Cleaner Alkyl (C12, 67%; C14, 25%; C16,
1%) dimethyl benzyl ammonium
saccharinate, 0.1%
Recikk Benckiser, Parsippany, New
Jersey, USA
CLR Bath &Kitchen Cleaner Lactic acid, 5–10% Jelmar, LLC, Skokie, Illinois, USA
Simple Green All-Purpose Cleaner Ethoxylated alcohol mixture, ,5% Sunshine Makers, Inc., Huntington
Beach, California, USA
409 Alkyl (C12, 40%; C14, 50%; C16,
10%) dimethyl benzyl
ammonium chloride, 0.3%
The Clorox Company, Oakland,
California, USA
Nolvasan Chlorhexidine, 2% Fort Dodge Laboratories, Fort
Dodge, Iowa, USA
Bleach Sodium hypochlorite, 3% and 10% The Clorox Company, Oakland,
California, USA
Ethanol Ethanol, 70% Sigma-Aldrich, St. Louis, Missouri,
USA
Process NPD Quaternary ammonium, 0.4% Steris, St. Louis, Missouri, USA
Benzalkonium chloride Benzalkonium chloride, 0.16% Sigma-Aldrich, St. Louis, Missouri,
USA
High Spectracide Immunox
Fungus Plus Insect Control for
Lawns
Propiconazole, 1.45% Chemsico, St. Louis, Missouri,
USA
Low Spectracide Immunox Fungus
Plus Insect Control for Lawns
Propiconazole, 1.45% Chemsico, St. Louis, Missouri,
USA
760 JOURNAL OF WILDLIFE DISEASES, VOL. 52, NO. 3, JULY 2016
growth was not inhibited at even the lowest
dilution of spores. Spectracide, a product
intended for residential purposes, did not
inhibit growth even at the higher concentra-
tion suggested for tree and shrub care. The
active ingredient, propiconazole, was regis-
tered in 1981 and is used as an antifungal in
landscape and agricultural settings (EPA
2006). In 2006, the US Environmental Pro-
tection Agency approved it for disease control
in crops such as vegetables, berries, tree nuts,
cereals, and grains (EPA 2006). While its use
and relationship to the emergence of snake
fungal disease cannot be determined, future
studies should investigate the role that envi-
ronmental use of this and other fungicides has
on O. ophiodiicola.
TABLE 2. Summary of average Ophidiomyces ophiodiicola colony forming units between days 10 and 14 for
each disinfectant at five dilutions of spores after exposure to respective disinfectant. Disinfectants without a
specified time were tested at 10 min.
Disinfectant
Dilutions of spore suspension
a
1:20 1:65 1:110 1:155 1:200
3% Bleach
10 min 0 0 0 0 0
5min 00000
2min 00000
10% Bleach
10 min 0 0 0 0 0
5min 00000
2min 00000
70% Ethanol
10 min 0 0 0 0 0
2min 00000
30s 43210
NPD
10 min 0 0 0 0 0
2 min 47 29 16 11 6
30s 83211
Benzalkonium chloride 0 0 0 0 0
Lysol Power Bathroom Cleaner 0 0 0 0 0
Lysol All Purpose Cleaner
10 min 0 0 0 0 0
2 min 148 109 122 110 67
30 s TNTC 238 171 146 94
CLR Bath &Kitchen Cleaner 0 0 0 0 0
409
10 min 0 0 0 0 0
2min 21110
30s 11110
Simple Green All-Purpose Cleaner 34 25 15 8 5
2% chlorhexidine
10 min 90 49 21 16 6
2 min TNTC TNTC TNTC TNTC TNTC
Spectracide Immunox High TNTC 444 232 173 120
Spectracide Immunox Low 625 537 319 225 112
Saline TNTC 183 134 95 64
a
TNTC ¼too numerous to count.
LETTERS 761
We only tested spores, and while unlikely,
the hyphae may be resistant to these methods.
Studies of the effect on hyphae and of the
presence of organic debris in field settings are
needed.
In conclusion, a 2-min exposure to at least
3% bleach or 70% ethanol or a 10-min
exposure to 0.16% Roccal, Lysol products,
CLR, NPD, or 409 are recommended for
disinfection of O. ophiodiicola. Furthermore,
in areas that may have risk of chytrid or
ranavirus exposure, at least 3% bleach is
recommended. We recommend mud or leaf
litter be removed from shoes and field
equipment before application of disinfectant
to ensure adequate exposure to bleach. These
methods should reduce the spread of this
often-fatal disease in free-ranging and captive
snakes, but much work is still needed.
Funding for this project was provided
through a Competitive State Wildlife Grant
from the Fish and Wildlife Service.
LITERATURE CITED
Allender MC, Baker S, Wylie D, Loper D, Dreslik MJ,
Phillips CA, Maddox C, Driskell EA. 2015a. Devel-
opment of snake fungal disease after experimental
challenge with Ophidiomyces ophiodiicola in cotton-
mouths (Agkistrodon piscivorous). PLoS One 10:
e0140193.
Allender MC, Dreslik M, Wylie S, Phillips C, Wylie DB,
Maddox C, Delaney MA, Kinsel MJ. 2011. Chrys-
osporium sp. infection in eastern massasauga rattle-
snakes [letter]. Emerg Infect Dis 17:2383–2384.
Allender MC, Raudabaugh D, Gleason F, Miller A.
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Johnson ML, Berger L, Philips L, Speare R. 2003.
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Submitted for publication 14 January 2016.
Accepted 16 February 2016.
762 JOURNAL OF WILDLIFE DISEASES, VOL. 52, NO. 3, JULY 2016
... A 70% ethanol solution with a contact time of 2 min also was effective, as were the commercial disinfectants Roccal, Lysol products, CLR, Process NPD and 409 for 10 min. Chlorhexidine, Simple Green, and Spectracide were not found to be effective against O. ophidiicola (Rzadkowska et al. 2016). When working with this pathogen in the field or in the laboratory, it is important to use appropriate biosecurity and disinfection protocols to prevent contamination of samples and transmission between animals. ...
... When working with this pathogen in the field or in the laboratory, it is important to use appropriate biosecurity and disinfection protocols to prevent contamination of samples and transmission between animals. Organic matter should be removed from all equipment before disinfection to maximize product efficiency (Rzadkowska et al. 2016). One study attempting to identify an effective treatment for ophidiomycosis found that the fungus Rhodococcus rhodochrous and synthetic volatile compounds reduced the growth of O. ophidiicola in vitro (Cornelison et al. 2016), but neither approach has been tested in live animals with ophidiomycosis, nor on a larger scale. ...
... Given the range of O. ophiodiicola found in this study, it is possible that ophidiomycosis is more widespread throughout Pennsylvania. Biosecurity measures are warranted when handling free-ranging snakes to reduce risk of human-mediated disease transmission, such as incorporating appropriate decontamination protocols for equipment between handling individuals (Rzadkowska et al. 2016). Further studies to determine the long-term effects of ophidiomycosis on these populations are needed due to the sensitive state status of C. horridus. ...
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The efficacy of a number of disinfection treatments was tested on in vitro cultures of the fungus Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis in amphibians. The aim was to evaluate the fungicidal effects of chemical disinfectants, sterilising ultraviolet (UV) light, heat and desiccation, using methods that were feasible for either disinfection in the field, in amphibian husbandry or in the laboratory. The chemical disinfectants tested were: sodium chloride, household bleach (active ingredient: sodium hypochlorite), potassium permanganate, formaldehyde solution, Path-X agricultural disinfectant (active ingredient: didecyl dimethyl ammonium chloride, DDAC), quaternary ammonium compound 128 (DDAC), Dithane, Virkon, ethanol and benzalkonium chloride. In 2 series of experiments using separate isolates of B. dendrobatidis, the fungicidal effect was evaluated for various time periods and at a range of chemical concentrations. The end point measured was death of 100% of zoospores and zoosporangia. Nearly all chemical disinfectants resulted in 100%, mortality for at least one of the concentrations tested. However, concentration and time of exposure was critical for most chemicals. Exposure to 70% ethanol, 1 mg Virkon ml(-1) or 1 mg benzalkonium chloride ml(-1) resulted in death of all zoosporangia after 20 s. The most effective products for field use were Path-X and the quaternary ammonium compound 128, which can be used at dilutions containing low levels (e.g. 0.012 or 0.008%, respectively) of the active compound didecyl dimethyl ammonium chloride. Bleach, containing the active ingredient sodium hypochlorite, was effective at concentrations of 1% sodium hypochlorite and above. Cultures did not survive complete drying, which occurred after <3 h at room temperature. B. dendrobatidis was sensitive to heating, and within 4 h at 37 degrees C, 30 min at 47 degrees C and 5 min at 60 degrees C, 100% mortality occurred. UV light (at 1000 mW m(-2) with a wavelength of 254 nm) was ineffective at killing B. dendrobatidis in culture.
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Ophidiomyces ophiodiicola, the causative agent of snake fungal disease, is a serious emerging fungal pathogen of North American-endemic and captive snakes. We provide a detailed literature review, introduce new ecological and biological information and consider aspects of O. ophiodiicola that need further investigation. The current biological evidence suggests that this fungus can persist as an environmental saprobe in soil, as well as colonizing living hosts. Not unlike other emerging fungal pathogens, many fundamental questions such as the origin of O. ophiodiicola, mode of transmission, environmental influences, and effective treatment options still need to be investigated.
Material Safety Data Sheet (MSDS)
  • Zoetis
Zoetis. 2015. Material Safety Data Sheet (MSDS). Nolvasan. https://www.zoetisus.com/contact/pages/ product_information/msds_pi/msds/nolvasan_skin_ and_wound_cleanser.pdf. Accessed April 2016. Submitted for publication 14 January 2016. Accepted 16 February 2016.