Experimental Exposures of Boreal Toads (Bufo boreas) to a Pathogenic Chytrid Fungus...

Experimental Exposures of Boreal Toads (Bufo boreas) to aPathogenic Chytrid Fungus (Batrachochytrium dendrobatidis)

Cynthia Carey,1 Judsen E. Bruzgul,1 Lauren J. Livo,1 Margie L. Walling,2 Kristin A. Kuehl,1

Brenner F. Dixon,1 Allan P. Pessier,3 Ross A. Alford,4 and Kevin B. Rogers5

1Department of Integrative Physiology, University of Colorado, Boulder, CO 80309-03542Department of Environmental and Radiological Health Sciences/Epidemiology, Colorado State University, Ft. Collins, CO 805213Department of Pathology, Zoological Society of San Diego, PO Box 120551, San Diego, CA 92112-05514School of Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia5Colorado Division of Wildlife, PO Box 775777, Steamboat Springs, CO 80477

Abstract: One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic

chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen

and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until

death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The

significant relation between dosage and the number of days survived (dose-response curve) supports the

hypothesis that the degree of infection must reach a particular threshold of about 107–108 zoosporangia before

death results. Variation in air temperature between 12�C and 23�C had no significant effect on survival time.

The infection can be transmitted from infected to healthy animals by contact with water containing zoospores;

no physical contact between animals is required. These results are correlated with observations on the pop-

ulation biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified.

Key words: Batrachochytrium dendrobatidis, amphibian pathogen, Bufo boreas, chytrid fungus, chytridiomy-

cosis

INTRODUCTION

Populations of many species of amphibians have experi-

enced serious declines over the last several decades (Alford

and Richards, 1999; Stuart et al., 2004). Although factors

such as habitat destruction and introduction of invasive

species have contributed to these declines, infectious dis-

ease has been identified as another significant cause (Berger

et al., 1998; Carey, 2000; Carey et al., 1999, 2003a, 2003b;

Daszak et al., 1999, 2003). A recently discovered chytrid

fungus (Batrachochytrium dendrobatidis), initially isolated

from captive amphibians suffering from a mycotic skin

disease (Longcore et al., 1999; Nichols et al., 1998; Pessier et

al., 1999), has now been linked to mass mortalities of wild

amphibian populations in many areas, including Europe,

South America, Central America, Australia, New Zealand,

and North America (Berger et al., 1998; Bishop, 2000;

Bosch et al., 2001; Bradley et al., 2002; Green et al., 2002;

Green and Kagarise Sherman, 2001; Lips et al., 2003; Muths

et al., 2003; Ron et al., 2003; Ron and Merino, 2000). For a

recent list of geographic localities and amphibian species onPublished online: January 18, 2006

Correspondence to: Cynthia Carey, e-mail: [email protected]

EcoHealth 3, 5–21, 2006DOI: 10.1007/s10393-005-0006-4

Original Contributions

� 2006 EcoHealth Journal Consortium

which this chytrid fungus has been found, see Carey et al.,

(2003a) or Speare and Berger (2000). This pathogen,

thought to have recently emerged (Daszak et al., 2003),

demonstrates little genetic diversity among isolates col-

lected in various locations around the world (Morehouse et

al., 2003). The closest chytrid relative from which this

pathogenic form evolved is currently not known (James et

al., 2000). Although the geographic origin of B. dendro-

batidis has not been proven, evidence from museum

specimens suggests it may have originated in Africa (Wel-

don et al., 2004).

A wide variety of bacterial, viral, and fungal agents are

normally found on the skin, in the digestive tract, and in

other tissues of amphibians (Granoff, 1969; Taylor, 2001;

Taylor et al., 2001). However, this chytrid fungus and a

group of ranaviruses are the only pathogens for which there

is a demonstrated correlation between the degree of

infection observed in laboratory animals and similar de-

grees of infection from animals captured in nature,

including those from populations experiencing mass mor-

tality events and population declines (Carey et al., 2003a,

2003b; Green et al., 2002; Jancovich et al., 1997; Nichols et

al., 2001). Although most members of the phylum Chy-

tridiomycota typically meet their nutritional requirements

by breaking down organic matter in aquatic systems, some

species are parasitic upon selected invertebrates, such as

insects (Longcore et al., 1999). B. dendrobatidis is the first

chytrid fungus known to be parasitic upon a vertebrate

host. Zoospores of this fungus preferentially attack kerati-

nocytes in the skin of metamorphosed amphibians

(Longcore et al., 1999; Pessier et al., 1999). Although

amphibian larvae appear to lack keratin in their epidermis,

this pathogen has been identified in the keratinized

mouthparts of tadpoles and toes of premetamorphic tad-

poles of a few species (Berger et al., 1998; Carey et al.,

2003a, 2003b; Fellers et al., 2001; Rachowicz and Vreden-

burg, 2004; Marantelli et al., 2004).

In the few years since its discovery, some progress has

been made in understanding the interactions of this fungal

pathogen with amphibians (see reviews by Berger et al.,

2004; Carey et al., 2003a, 2003b; Daszak et al., 2003;

Davidson et al., 2003; Longcore et al., 1999; McDonald et

al., 2005; Nichols et al., 2001; Pessier et al., 1999; Retallick

et al., 2004). Nichols et al., (2001) experimentally exposed

captive Dendrobates tinctorius and D. auratus to the type

isolate of B. dendrobatidis by dripping a solution containing

zoospores onto the back and legs of these amphibians once

a day for 30 days or 4–5 days per week for 4 weeks or by

immersing them in water containing chytrid zoospores in

their cages. All exposed animals died with skin infections,

whereas control animals did not develop infections. Some

amphibian species, such as bullfrogs (Rana catesbeiana)

and tiger salamanders (Ambystoma tigrinum), apparently

carry the pathogen on their epidermis without developing

lethal infections; therefore, they can serve as reservoirs for

the transmission of chytridiomycosis to susceptible species

(Daszak et al., 2003; Davidson et al., 2003; Hanselmann et

al., 2004; Weldon et al., 2004). Further, B. dendrobatidis can

persist in an endemic state in healthy frogs once an epi-

demic wave has passed through amphibian populations

(McDonald et al., 2005; Retallick et al., 2004). To date,

however, many questions remain about the interaction of

this pathogen with amphibians, including what constitutes

the minimal infective dose of zoospores necessary to cause

a lethal infection, how various environmental factors affect

the ability of zoospores to cause an infection, and how

various amphibian species differ in susceptibility to infec-

tion by this pathogen. Much more information about these

interactions is needed to develop effective methods for

preventing further amphibian mortalities and population

declines.

We here report on a series of experiments designed to

examine the interaction of B. dendrobatidis with the boreal

toad (Bufo boreas), a species that is known to be susceptible

to infection by this pathogen. Boreal toads, widely dis-

tributed throughout many parts of the western United

States, suffered severe population declines and extinctions

in the southeastern part of their range (Carey, 1993; Corn

et al., 1989) in the late 1970s to early 1980s. As a result, this

species is classified as ‘‘endangered’’ in Colorado (Goettl,

1997) and New Mexico, a ‘‘species of special concern’’ in

Wyoming (Keinath and Bennett, 2000), and a ‘‘sensitive

species’’ in Utah (Utah Division of Wildlife Resources,

1998); it is classified as ‘‘warranted but precluded’’ for

federal listing as a threatened or endangered species by the

U.S. Fish and Wildlife Service. Since 1996, B. dendrobatidis

has been associated with mortalities in some of the few

remaining populations of toads in Colorado (Muths et al.,

2003). Its presence in a few museum specimens collected in

Colorado during the 1970s and the similarities between the

patterns of historical declines and current mass mortalities

suggest, but do not conclusively prove, that the historical

die-offs of this species in Colorado in the 1970s were due to

this pathogen (Carey, 1993; Carey et al., 1999). The boreal

toad is an ideal model system for examining the interac-

tions between Batrachochytrium and amphibians because

6 Cynthia Carey et al.

chytrid-free boreal toadlets can be obtained from a Colo-

rado Division of Wildlife hatchery and because an isolate of

this fungal pathogen from a boreal toad in a Colorado

population is available for experimentation.

These experiments were designed to investigate (1)

whether lethal infections due to chytridiomycosis can be

induced experimentally in boreal toadlets, (2) how vari-

ations in temperature and body mass affect survival of

boreal toadlets exposed to B. dendrobatidis zoospores, (3)

whether uninfected boreal toadlets can become infected by

exposure to water in which B. dendrobatidis-infected

toadlets have been housed, and (4) how the duration of

exposure to, and the dosage of B. dendrobatidis zoospores

affect the postexposure survival of boreal toadlets. An-

swers to these questions are important for identifying

factors that might contribute to the ability of B. dendro-

batidis to infect and kill boreal toads in nature. Although

the interactions between zoospores and amphibians may

differ in nature and in the laboratory, experimental

measurements of the number of zoospores necessary to

initiate a fatal infection and the amount of exposure time

necessary for infection to occur, as well as experimental

determination of whether or not amphibians can become

infected by contact with water containing zoospores in the

laboratory, are valuable steps in understanding these

phenomena in nature. In addition, it is important to

understand how environmental factors, such as tempera-

ture, affect the dynamics of infection of amphibians by

B. dendrobatidis.

MATERIALS AND METHODS

General Husbandry

Because boreal toads are designated as endangered or

threatened throughout the southeastern part of their geo-

graphic distribution, our experiments were necessarily re-

stricted to toadlets that were provided from the Colorado

Division of Wildlife’s John W. Mumma Native Aquatic

Species Restoration Facility (NASRF). The toadlets used in

these experiments were raised at NASRF from eggs or

tadpoles collected at five different localities in the moun-

tains of Colorado in late May through June. Because of the

relative synchronicity of egg laying at various boreal toad

breeding locations, the age of toadlets used in this study

probably differed by less than 6 weeks, regardless of the

locality of origin. Because the development of a polymerase

chain reaction (PCR) assay for B. dendrobatidis occurred

after the experiments described here were conducted, we

were unable to verify that all the toadlets were free of

chytrid infection at the start of each experiment. However,

the toadlets were raised by the hatchery in a manner that

minimized exposure to B. dendrobatidis and no outbreaks

of this pathogen have been reported for hatchery toads.

Subsequent PCR analysis of samples from the hatchery

indicates that the hatchery currently is free from contam-

ination with this pathogen. Therefore, we feel confident

that the toadlets had not been exposed to B. dendrobatidis

prior to their use in these experiments.

After transport from NASRF to the University of

Colorado in Boulder, young-of-the-year toadlets were

maintained at 23�C, the temperature shown to be optimal

for B. dendrobatidis growth (Piotrowski et al., 2004), on a

12-hour/12-hour light/dark cycle in troughs containing tap

water. Toadlets were fed Drosophila or pin-head crickets

(Fluker Farms, Port Allen, LA) three times a week. The

crickets were fed Fluker’s Calcium Fortified Cricket

Quencher in order to provide vitamins and minerals to the

toadlets. During experiments, toadlets were housed in

walk-in environmental chambers in which temperature,

light, and feeding conditions were controlled as described

above, unless specified otherwise in individual experiments.

In each experiment, however, toadlets were held in 20%

Holtfreter’s solution (pH 6.5, the optimal pH for B. dend-

robatidis growth [Piotrowski et al., 2004]), made with

double-distilled water to minimize exposure of chytrid

zoospores to contaminants in tap water.

B. dendrobatidis Cultures and Exposures

Isolate JEL#275 of B. dendrobatidis was used to expose

toadlets to this pathogen. This strain was originally isolated

from an infected boreal toad from Clear Creek County,

Colorado, by Joyce Longcore. Cultures were grown in H-

broth (10 g tryptone and 3.2 g glucose/liter distilled water)

at 23�C for 4–7 days prior to the beginning of an experi-

ment. On the day that toadlets were to be exposed to B.

dendrobatidis, zoospores were filtered through a sterile 20

lm nylon mesh filter (Spectra/Mesh�; Spectrum Labora-

tories, Rancho Dominguez, CA) to remove sporangia and

then an aliquot of the filtrate was counted with a hemacy-

tometer to determine the concentration of zoospores per

milliliter in the filtrate. A solution containing the desired

concentration of zoospores was made by diluting the filtered

H-broth culture with 20% Holtfreter’s solution (pH 6.5)

and sufficient penicillin/streptomycin (Sigma, St. Louis,

Exposure of Boreal Toads to B. dendrobatidis 7

MO) to comprise 1% of the final volume. Toads in the

exposure groups were placed in aliquots of this solution.

Because the toadlets were exposed to the chytrid zoospores

in a nonsterile environment, penicillin/streptomycin was

necessary to minimize bacterial growth on the nutrients in

the H-broth. Toads in control groups were exposed to

identical proportions of B. dendrobatidis-free H-broth and

penicillin/streptomycin in 20% Holtfreter’s solution.

Detection of B. dendrobatidis DNA in Toad Skin

These experiments were conducted before a PCR test for B.

dendrobatidis DNA was developed. In anticipation of the

development of this test, samples were collected during or

at the end of these experiments by scraping the ventral skin

with a sharpened wooden dowel, which was then placed in

1 ml of 0.25 M EDTA (pH 8) saturated with NaCl. These

samples were subsequently analyzed by Pisces Molecular

(Boulder, CO) for DNA specific to B. dendrobatidis using

PCR primers developed by Annis et al., (2004). Toad car-

casses were stored in 10% formalin. Sections of skin from

the ventral pelvic region (pelvic or drink patch) were

processed for histological examination and embedded in

paraffin. Sections (5 lm) were stained with hematoxylin

and eosin.

Experiment 1: Can Infections with

Batrachochytrium Be Experimentally Induced in

Boreal Toadlets, and How Do Temperature and

Body Mass Affect the Survival of These Toadlets

after Exposure?

Our first experiment was designed to test whether boreal

toadlets could be infected experimentally with the B.

dendrobatidis isolate JEL#275. Each of 160 toadlets was

weighed, placed in an individual 236 ml plastic Ziploc

container with a perforated lid, and randomly assigned to

one of four groups: control or exposed, each maintained at

either 23�C or 12�C. Mass ranged from 1.6–38.0 g (aver-

age ± standard error [SE] mass = 12.41 ± 0.56 g). Analysis

of variance (ANOVA) revealed no significant differences in

mean initial mass for toadlets among groups (F = 0.170,

P = 0.6806) or between the two temperature treatments

(F = 2.324, P = 0.1294). The group · temperature inter-

action was also nonsignificant (F = 44.701, degree of free-

dom [DF] = 1, P = 0.3430).

For toadlets in exposure groups, we used what we

believed to be a large dose (106 zoospores/toadlet daily) and

a relatively long exposure time (72 hours) to maximize the

chance of producing skin infections and mortality under

laboratory conditions. Toads in the exposure groups were

exposed individually to this dose of zoospores in 20 ml of

Holtfreter’s solution constituted as described above. This

volume was adequate to cover the floor of the Ziploc

container and to immerse the ventral side of the toadlet

throughout the exposure period. Control toadlets were

individually exposed to an identical volume of the sham

exposure solution. Fresh solutions containing zoospores

were made from B. dendrobatidis cultures each day. After

24 and 48 hours, the initial exposure solutions were dis-

carded from each container and replaced with the same

volume of identically constituted solutions. After 72 hours,

all toadlets were transferred from the small containers and

placed individually in 15 · 30 · 11 cm plastic containers

with 200 ml of B. dendrobatidis-free 20% Holtfreter’s

solution (pH 6.5). Holtfreter’s solutions in the cages were

replaced three times per week when the toadlets were fed.

The containers were held flat so that the ventral surfaces of

the toadlets in a normal sitting posture were in continuous

contact with the solution.

In order to examine the time course between exposure

and the appearance of detectable infection, five toadlets

from each group were sacrificed by cervical dislocation,

followed by spinal and cerebral pithing, on a random

sampling schedule on postexposure days 1, 3, 8, 14, and 21.

Skin scrapes were collected for PCR analysis, and carcasses

were preserved for histological examination.

Thirty-two of the 80 exposed toadlets died on or prior

to their assigned sampling date. On the assumption that

these toadlets died from B. dendrobatidis infections (as-

sessed after death with histology and, later, PCR), experi-

ment 1 afforded additional opportunities to analyze the

effects of two variables, temperature variation and toadlet

body mass, that could plausibly affect the number of days

to death. The relation of the number of days to death as a

function of body mass was evaluated with least-squares

regression. The average time of survival of toadlets that

died prior to their scheduled sampling date in two tem-

perature treatments, 23�C and 12�C, was evaluated using

Student’s t-test. Tissue samples from all toadlets that died

prior to their scheduled sampling date were analyzed by

PCR.

As the experiment progressed, we noted several

behavioral and physiological differences between control

and exposed toadlets at each temperature. Anecdotal

observations of behavioral differences were not statistically


analyzed. However, on the last sampling date, the number

of respirations per minute of all surviving exposed toadlets

(n12�C = 6, n23�C = 5) and randomly selected control

toadlets (n12�C = 6, n23�C = 4) were counted; the mean

number of respirations per minute of control and exposed

animals was compared with two-way ANOVA, with treat-

ment (control or exposed) and temperature (12�C or 23�C)

as the independent variables.

When the experiment was terminated 3 weeks after the

initial exposure, some control and all of the remaining ex-

posed animals were sacrificed. Holtfreter’s solution and the

containers in which exposed and control animals had been

sitting for 24 hours were immediately used in experiment 2.

Experiment 2: Can Uninfected Boreal Toadlets

Become Infected by Exposure to Liquid (i.e.,

Holtfreter’s Solution) in Which Infected Toadlets

Have Been Housed?

This experiment was designed to determine whether chy-

tridiomycosis could be transmitted to uninfected animals

through exposure to Holtfreter’s solutions used in the

previous experiment to house infected toadlets. Twenty-

one control animals remained at the end of experiment 1:

11 had been maintained at 12�C and 10 at 23�C. These

toadlets were assigned randomly to containers from

experiment 1 which had housed either control or exposed

toadlets in 200 ml Holtfreter’s solution that had been in the

container the preceding 24 hours. Toadlets for experiment

2 remained in the experiment 1 Holtfreter’s solution for 48

hours, after which the liquid was replaced with clean, B.

dendrobatidis-free, 20% Holtfreter’s solution (pH 6.5). This

solution was then changed three times per week during

feedings. Survival of toadlets was monitored daily for 34

days. When a toadlet died, the date of death was recorded

and skin scrapes were collected for PCR analysis. A log-

rank test on censored survival data (StatXact; Cytel Statis-

tical Software, Cambridge, MA) was used to assess whether

the patterns of survival of control and exposed groups

differed significantly.

Body masses of toadlets used this experiment ranged

between 6 and 30 g. Average body masses of control and

exposed toadlets, compared using two-way ANOVA, did

not differ significantly (P = 0.6782, DF = 1) and there was

no significant interaction term (P = 0.6636, DF = 1).

However, the average mass of the combined control and

exposed toadlets at 23�C was significantly heavier than the

control and exposed body masses at 12�C.

Experiments 3 and 4: How Do the Time of Exposure

and the Dosage of B. dendrobatidis Zoospores Affect

the Survival of Boreal Toadlets?

As noted below (see Results), PCR analysis and histology of

tissues from exposed toadlets in experiment 1 indicated

that exposure to 106 zoospores per day for 3 days was

sufficient to cause chytridiomycosis and death. Experiment

3 examined the effects of a variety of dosages and durations

of exposure on the number of days survived. The experi-

ment was designed as a 2 · 5 factorial, with one factor

being exposure duration (1 or 3 days) and the other being

dosage of zoospores/20 ml (0 [control], an estimated 1

zoospore, 100, 104, or 106 zoospores], resulting in a total of

10 experimental groups with 15 toadlets each. Toadlets

were weighed and randomly assigned to one of the 10

groups. Mean initial toadlet mass did not differ signifi-

cantly among groups (F = 0.576, DF = 9, P = 0.8154).

Individual toadlets from control and varying dosage

groups designated for 1-day exposure were placed in 236

ml Ziploc containers containing 20% Holtfreter’s solution

(pH 6.5) with either B. dendrobatidis-free broth (controls)

or broth containing various concentrations of zoospores

for 24 hours, after which the solutions were replaced daily

with 20 ml Holtfreter’s solution (pH 6.5). After 3 days,

toadlets were placed in individual 15 · 30 · 11 cm plastic

boxes for the duration of the experiment. Even though

these toadlets were exposed to B. dendrobatidis zoospores

for only 1 day, this procedure on days 2 and 3 ensured that

handling of the toadlets in the 1-day and 3-day exposures

was identical. Toadlets that were exposed for 3 days and

their controls were kept in the small Ziploc containers for 3

days and given fresh B. dendrobatidis-containing or B.

dendrobatidis-free doses of Holtfeter’s solution daily. At the

end of the 3-day exposure, these groups were also placed

individually in large plastic boxes at 23�C for the 42-day

duration of the experiment. Toads were fed and water was

changed three times per week; mortality was monitored

daily. Skin scrapes for PCR analysis were collected at death

or, for toadlets that survived the entire 42-day period, at

the end of the experiment.

In experiment 4, we examined the effect of 1-day

exposure to a different set of zoospore concentrations (0

[control], 20, 40, 60, 100, and 103 zoospores/20 ml) on

survivability with groups of 10 Bufo boreas toadlets each.

This experiment was also conducted at 23�C and moni-

tored for 42 days under the same housing and care con-

ditions as in experiment 3. No significant differences in


initial toadlet mass existed among groups (F5,54 = 1.725,

P = 0.1445). At the end of the experiment, skin scrapes

were collected for PCR analysis.

Survival curves of control and exposure groups in

experiments 3 and 4 were evaluated by the StatXact log-

rank test for censured survival data. This test indicated

whether the rates of death and percentage of animals in

each group that died differed significantly among dosages

and (in experiment 3) the duration of exposure. Addi-

tionally, the known fate modeling procedure in program

MARK (White and Burnham, 1999) was used to isolate the

most parsimonious models from suites of candidate models

containing all possible first-order combinations of dose,

exposure duration (in experiment 3), and body mass to

determine their relative importance in determining the

length of survival. Program MARK uses second-order

Akaiki information criteria (AICc; Burnham and Anderson,

2002) to identify the model that best describes the data

without sample overparameterization. Using this approach

for model selection is superior to traditional hypothesis

testing for this data set because it allows simultaneous

comparison of multiple candidate models, balances preci-

sion and bias when selecting the appropriate model

(Burnham and Anderson, 2002), and is not restricted to

parametric data.

In addition, we examined whether the results of these

experiments were consistent with a simple model of the

disease process, based on the assumption that populations

of B. dendrobatidis on the host grow exponentially until a

threshold density is reached that produces host mortality.

Exponential population growth can be represented as a

linear increase of log(number of parasites) with time. A

linear function has two parameters, the slope and the

intercept. The intercept for these population growth lines is

the number of zoospores that settled on the first day of

exposure, day 1 of this experiment. For treatments in which

toadlets were exposed to an estimated single zoospore, this

must equal log10(1) for successful infections. For other

treatments, this number should be equal to log10(np), where

n is the number of zoospores to which the toadlets in each

treatment were exposed and p is the proportion of zoosp-

ores that colonize a toadlet in 1 day of exposure. For

experiment 3, we assumed that p was constant across all

treatments, estimated p using two methods, and used the

average of these estimates for our model. One estimate of p

was calculated as the proportion of toadlets that were ex-

posed to an estimated single zoospore for 1 day that became

infected. The second was calculated for the treatment in

which individuals were exposed to 100 zoospores as the

probability of colonization per zoospore that made the zero

term of the binomial distribution equal to the observed

proportion of failures to infect. We also assumed that all

population growth lines had the same slopes. If all the

growth lines had the same slopes, differing only in inter-

cepts, and mortality occurs when these growth lines cross a

threshold number, then the common slope of the growth

lines can be estimated by dividing 2 (the difference in ele-

vation in log units between the lines for initial exposures to

102, 104, and 106 zoospores) by the differences in time to

mortality averaged between all possible pairs of individuals

in the treatments exposed for 1 day to 102 or 104 and to 104

or 106 zoospores. We compared the average time to mor-

tality predicted by these four lines with the observed time to

mortality for toadlets exposed to zoospores for 1 day. We

cross-checked these results with those for individuals ex-

posed to zoospores for 3 days. We assumed that the slopes

and intercepts of the B. dendrobatidis population growth

lines remained the same as those for individuals exposed for

1 day. We calculated the total estimated B. dendrobatidis

population on individuals in each treatment as the sum of

the three population growth lines for the populations star-

ted on each day of exposure and determined estimated times

to mortality by inspecting the values of these sums.

We also examined how well our growth models fit the

data from experiment 4. We assumed that the slopes of

population growth lines remained the same as those esti-

mated for the third experiment since the conditions under

which toadlets were maintained following infection were

identical. We estimated the intercepts of the growth lines

for each exposure level in the same manner but used a value

of p derived from the observed rates of infection in this

experiment, by calculating the p values necessary to make

the zero term of the binomial distribution equal to the

observed proportion of failures to infect for the 20, 40, and

60 zoospore treatments and averaging these values.

RESULTS

Experiment 1: Can Infections with B. dendrobatidis

Be Experimentally Induced in Boreal Toadlets, and

How Do Temperature and Body Mass Affect the

Survival of These Toadlets after Exposure?

Both histological examination of ventral skin and PCR

analysis for B. dendrobatidis DNA in ventral skin confirmed

that exposure of boreal toadlets to 106 zoospores for 3 days


caused chytridiomycosis. Histological lesions observed in

exposed toadlets were moderate epidermal hyperplasia

characterized by increased epidermal thickness and disor-

ganization of keratinocytes and mild to moderate ortho-

keratotic hyperkeratosis (Fig. 1). Within keratinocytes in

the stratum corneum, there were moderate numbers of

characteristic chytrid thalli, including zoosporangia with

developed zoospores and septate (colonial) thalli charac-

teristic of B. dendrobatidis. Chytrid thalli were evident in

histological sections as early as 9 days following infection.

Tissues of only 3 of the 80 exposed animals tested negative

for B. dendrobatidis DNA; these were sampled either on day

1 or day 3 following exposure. Additionally, 32 exposed

animals died on or prior to their scheduled sampling date.

Because both histological analysis and PCR indicated the

presence of B. dendrobatidis in the skin of these animals, we

concluded that 3-day exposure to 106 zoospores was suf-

ficient to cause death in boreal toadlets.

One control toadlet died on the first day of the 21-

day experiment and was found to be free of B. dendro-

batidis infection by PCR analysis. Tissues of 6 of the 20

control toadlets submitted for PCR analysis were weakly

positive for B. dendrobatidis DNA, probably due to con-

tamination during the sampling procedure at the end of

the study. Although instruments were cleaned with etha-

nol between samples, this cleaning procedure may not

have been sufficient to remove all B. dendrobatidis DNA,

resulting in contamination. No other control animals,

including those toadlets nominally positive for B. dend-

robatidis DNA, died prematurely before their assigned

sampling date.

The mean number of days survived by toadlets that

died before their scheduled sampling date was 14.0 ± 0.8

days (n = 14) and 13.8 ± 0.8 days (n = 18) at 12�C and

23�C, respectively. These values did not differ significantly

(t = 0.188, P = 0.8525, DF = 30), indicating that tem-

perature variation over this range had no significant effect

on the survival time of these boreal toadlets.

Body mass of all toadlets in experiment 1 ranged be-

tween 1–38 g, but the masses of those that died on or before

their scheduled sampling date ranged only between 4–19 g.

Our regression analysis indicated that toadlet body mass

within this range had a significant effect on the number of

days survived. Because temperature had no significant ef-

fect on survival time, the days of survival of toadlets held at

12�C and 23�C were pooled and analyzed; a highly signif-

icant correlation existed between the number of days sur-

vived following exposure to B. dendrobatidis zoospores and

toadlet body mass (r = 0.795, n = 30, P < 0.0001; Fig. 2).

The least-squares regression equation best describing this

relationship is as follows: Days survived = 8.79 + 0.545 ·Mass (g) (F = 49.798, P < 0.0001). An SAS t-test (Satt-

erthwaite method) showed a highly significant relationship

between mass and survival time, with shorter survival times

associated with smaller masses (t = 3.62, P = 0.0018).

Control and exposed toadlets exhibited several

behavioral differences as the experiment progressed. Spe-

cifically, exposed toadlets held their bodies out of water as

Figure 1. Histopathology of Bufo boreas experi-

mentally infected with B. dendrobatidis. A: Skin

from an uninfected control, magnified ·40. B:

Skin from a toad exposed to B. dendrobatidis 12

days previously. The epidermis is thickened with

disorganized keratinocytes (hyperplasia), and

there is a cluster of chytrid thalli within the

superficial keratinized layers (stratum corneum,

center), magnified ·40. C: Detail of chytrid thalli

within the stratum corneum. Numerous devel-

opmental stages are present including a flask-

shaped zoosporangium containing numerous

discrete zoospores.


much as possible by climbing on the walls of their con-

tainer or by adopting a four-legged posture that raised their

ventral surface above the water on the bottom of the cage.

Sometimes exposed toadlets elevated their toes out of the

water. In comparison, control toadlets usually sat in the

water and were rarely observed with ventral body surfaces

out of the water.

ANOVA indicated that control toadlets had a signifi-

cantly higher respiration rate than exposed toadlets by the

end of the 3-week experiment (F = 10.6, DF = 1, P =

0.0047; Table 1). Temperature had no significant effect on

respiration rates of exposed and control toadlets held at

12�C and 23�C (F = 3.592, DF = 1, P = 0.0752), nor did the

interaction between treatment and temperature (F = 2.770,

DF = 1, P = 0.1144).

Experiment 2: Can Uninfected Boreal Toadlets

Become Infected by Exposure to Water in Which

Infected Toadlets Have Been Housed?

Exposure to water in which toadlets infected with B.

dendrobatidis had been living for 24 hours caused signifi-

cant chytridiomycosis and death of boreal toadlets (Fig. 3).

A log-rank test for censored survival data indicated a sig-

nificant difference in the pattern of survival of control and

Figure 2. Number of days survived by boreal toadlets (Bufo boreas) as a function of body mass following exposure to 106 zoospores of

B. dendrobatidis for 3 days. Each point represents data for one toad.

Table 1. Mean Breaths per Minute of Control (Nonexposed)

Boreal Toadlets (Bufo boreas) or Toads Exposed to Chytrid Fungal

Zoospores (Batrachochytrium dendrobatidis) at Two Constant

Temperatures

Treatment and temperature Mean ± SE breaths/min n

Control (12�C) 147 ± 5.4 6

Exposed (12�C) 125 ± 9.1 6

Control (23�C) 195 ± 9.0 4

Exposed (23�C) 128 ± 24.5 5

Figure 3. Changes in the percentage survival of groups of boreal

toadlets (Bufo boreas) as a function of time (days) during a 34-day

experiment following placement in water in which uninfected toads

(control group) or toads infected with B. dendrobatidis (exposed

group) had been held. n = 10 for controls (5 at 23�C, 5 at 12�C), n =

11 for exposed (5 at 23�C, 6 at 12�C).


exposed toadlets (two-sided exact inference P = 0.0001).

Mortalities of exposed toadlets began on day 16. Exposed

toadlets held at 12�C and 23�C survived an average of

23.8 ± 2.42 (n = 6) and 25.45 ± 2.56 (n = 4) days,

respectively, during the 34-day experiment. Only one of the

exposed toadlets (at 23�C) survived the full 34 days of the

experiment. All of the exposed toadlets tested positive for

B. dendrobatidis DNA by PCR analysis. In contrast, 9 of the

10 toadlets (5 at 12�C and 5 at 23�C) transferred to water

previously used by control (nonexposed) toadlets survived

the entire 34 days of the experiment. One control toadlet

(at 23�C) died on day 34. None of the control toadlets,

including the one that died on day 34, tested positive for B.

dendrobatidis DNA.

During the course of this experiment, toadlets placed

in water previously occupied by B. dendrobatidis-exposed

toadlets exhibited avoidance of water and decreased rates of

respiration as noted in exposed toadlets from experiment 1.

Experiments 3 and 4: How Do the Time of Exposure

and the Dosage of B. dendrobatidis Zoospores Affect

the Survival of Boreal Toadlets?

Both the number of zoospores to which a toadlet was ex-

posed (dosage) and the number of days of exposure

strongly affected the duration of survival of exposed boreal

toadlets (log-rank test two-sided exact inference P =

0.0001). The average number of days survived by toadlets

exposed to 106 zoospores (15.5 ± 1.9 and 16.4 ± 3.9 days

for the 1- and 3-day exposure groups, respectively) did not

differ significantly (Fig. 4). At lower dosages (an estimated

1, 100, or 10,000 zoospores), both dosage and duration of

exposure to B. dendrobatidis zoospores significantly affected

the mean length of survival, percent mortality, and per-

centage of toadlets actually infected by the exposure treat-

ment (Table 2).

The duration of exposure had no effect on the per-

centage of toadlets surviving at high dosages: all toadlets in

groups exposed to 10,000 and 106 zoospores died by the

end of the test (Table 2). At lower dosages, only 13% of

toadlets exposed for 3 days to an estimated single zoospore

survived, whereas 93% of those exposed to that dosage for

only 1 day survived the 42-day experiment (Table 2).

Finally, both the time of exposure and the dosage af-

fected the percentage of toadlets in each group infected by

B. dendrobatidis. For example, only 38% of the toadlets

became infected when exposed to an estimated single

zoospore for 24 hours, yet 100% were infected by a 3-day

exposure (Table 2).

Duration of exposure, dose, and toadlet mass were

factors available for analysis for their effects on survival

time in experiment 3. These factors were evaluated by

ranking a suite of candidate models in program MARK

using the logit link function (Table 3). The model that

included exposure duration and dose (model A) had

nearly three times the support (AICc weight = 0.572) than

the one that also included mass (model B, AICc weight =

0.225). Of the top four models (models A-D), those that

included exposure duration as a variable (A and B) were

almost four times more strongly supported than those

without (C and D). Dose was selected in all four of the

top models, demonstrating its importance in predicting

survival of toadlets. The most parsimonious model was as

follows:

Figure 4. Experiment 3: Changes in the per-

centage survival of groups of boreal toadlets (Bufo

boreas) exposed to different dosages of B.

dendrobatidis zoospores for 1 or 3 days in a

42-day experiment. n = 15 for each group.


S ¼ expð4:987 þ 0:459DUR � 0:684DOSEÞ1 � expð4:987 þ 0:459DUR � 0:684DOSEÞ

where S is daily survival, DUR is the exposure duration, and

DOSE is the dose used.

Toadlets were exposed to 100 zoospores for 1 day in

both experiments 3 and 4. The percentage of toadlets sur-

viving the 42-day experiments was similar (27% in exper-

iment 3 and 30% in experiment 4). A t-test showed no

significant difference in mean survival time for toadlets at

this common dosage level (mean survival(experiment 3) =

34.5 ± 1.6 days, mean survival(experiment 4) = 33.0 ± 2.3

days; t = 0.562, P = 0.5796, DF = 23). These results give us

confidence that the conditions under which the experi-

ments were run, although offset in time, were sufficiently

similar that the results can be compared between the two

experiments.

Except for the 100-zoospore exposure replicate, toadlets

were exposed to a different set of dosages in experiment 4.

All toadlets exposed to 1,000 zoospores died within 42 days.

At dosages lower than 1,000 zoospores, the percentage of

toadlets surviving the 42-day experiment and the percentage

infected with B. dendrobatidis varied among groups (Ta-

ble 2, Fig. 5); some toadlets exposed to lower dosages lived

the full 42 days (Fig. 5, Table 2). One control animal also

tested weakly positive for B. dendrobatidis DNA (Table 2).

A similar suite of candidate models were ranked by

program Mark for experiment 4 (Table 4). Because

experiment 4 had only a single exposure duration and the

doses were within an order of magnitude, the contribution

Table 3. Models Considered by Program MARK to Predict Survival (S) in Toadlets from Experiment 3 and Their Ranking by AICc:

Parameters Considered Include Exposure Duration (Dur), Dose, and Mass

Model Parameters AICc

Delta

AICc

AICc

Weight

Model

Likelihood Par Deviance

A S(DurDose) 835.965 0.00 0.572 1.0000 3 829.960

B S(DurDoseMass) 837.832 1.87 0.225 0.3933 4 829.823

C S(Dose) 838.727 2.76 0.144 0.2514 2 834.724

D S(DoseMass) 840.476 4.51 0.060 0.1048 3 834.470

E S(Dur) 913.565 77.60 0.000 0.0000 2 909.562

F S(DurMass) 915.209 79.24 0.000 0.0000 3 909.203

G S(.) 917.353 81.39 0.000 0.0000 1 915.352

Par, number of parameters; S(.), daily survival, was the only parameter used in the model.

Table 2. Proportion (%) of Toadlets Surviving 42 Days following Exposure to Varying Dosages of Chytrid Fungi Zoospores and

Proportion That Tested Positive for B. dendrobatidis by PCR: Control Groups Were Not Exposed to the Fungus

Experiment 3 dosages (n = 15) Control 1 100 10,000 1,000,000

3-Day exposure

Surviving 42 days 100% 13% 0% 0% 0%

Positive 0% 100% n/a n/a n/a

1-Day exposure

Surviving 42 days 100% 93% 27% 0% 0%

Positive 0% 38% n/a n/a n/a

Experiment 4 dosages (n = 10) Control 20 40 60 100 1000

1-Day exposure

Surviving 42 days 100% 50% 60% 40% 30% 0%

Positive 10% 100% 60% 90% 90% 100%

n/a, not applicable.


of mass was more than three times that for dose in terms of

toadlet survival (model I AICc weight = 0.375 vs. model J

AICc weight = 0.118). Even though a small range of masses

was used in this experiment, a size-related effect on survival

was clearly demonstrated, with increasing mass resulting in

increased survival time. This mass effect can be summa-

rized as follows:

S ¼ expð2:568 þ 0:155MASSÞ1 þ expð2:568 þ 0:155MASSÞ

For model I, S is daily survival and MASS is given in grams.

We used the data from experiments 3 and 4 to address

the question of whether death occurs when the number of B.

dendrobatidis sporangia infecting a toadlet exceeds a

threshold. Our models of the growth of B. dendrobatidis

populations on individuals fit the data closely (Table 5) and

predict that the threshold for death is about 107–108 zoo-

sporangia per toadlet. Using the models fitted for 1-day

exposures in experiment 3 produced predicted times to

mortality that were close to the actual times experienced by

individuals exposed to zoospores over 3 days. The intercepts

estimated for experiment 4 using the proportion of animals

successfully infected are low, suggesting that rates of suc-

cessful establishment of zoospores on hosts were lower in

this experiment than in experiment 3. However, the models

also fit the data for this experiment reasonably well. The

relationship between observed and predicted median dates

of mortality for experiments 3 and 4 is shown in Figure 6.

The fact that the regression accounts for most of the vari-

ation in the data and has a slope almost identical to 1

suggests that our models are a good reflection of the actual

disease process in boreal toads.

DISCUSSION

We believe that this study has a number of significant

findings: (1) chytridiomycosis can be experimentally in-

Figure 5. Experiment 4: changes in the per-

centage survival of groups of boreal toadlets

(Bufo boreas) exposed to different dosages of

B. dendrobatidis zoospores for 1 day in a 42-day

experiment. n = 10 for each group.

Table 4. Models Considered by Program MARK to Predict Survival (S) in Toadlets from Experiment 4 and Their Ranking by AICc:

Parameters Considered Include Dose and Mass

Model Parameters AICc

Delta

AICc

AICc

Weight

Model

Likelihood Par Deviance

H S(DoseMass) 304.472 0.00 0.441 1.0000 3 298.455

I S(Mass) 304.797 0.33 0.375 0.8497 2 300.789

J S(Dose) 307.117 2.65 0.118 0.2664 2 303.108

K S(.) 308.280 3.81 0.066 0.1489 1 306.278

Par, number of parameters; S(.), daily survival, was the only parameter used in the model.


duced in boreal toadlets; (2) the duration of exposure

and the dosage of B. dendrobatidis play important roles

in determining the length of survival; (3) our model

predicts that the level of infection must reach a threshold

to cause death; (4) larger toadlets live longer given a

particular dosage than smaller toadlets, at least within the

size range of toadlets used in this study; (5) housing

exposed animals at air temperatures of 12�C and 23�C

has no significant effect on the length of survival fol-

lowing exposure; and (6) lethal chytridiomycosis can be

transmitted through the water in which infected toadlets

have been sitting. Following a discussion of each of these

Table 5. Data and Parameters of Models of Exponential Amphibian Chytrid Population Growth on Hosts Fitted to Data from

Experiments 3 and 4a

Number of zoospores Time to death (days) Population growth lineLog10(estimated

Days exposed n Log10(n) Median Mean difference Predicted Slope Intercept fatal threshold)

Experiment 3

1 1 0.000 31 2.909 27 0.295 0.000 8.851

1 100 2.000 27 6.860 25 0.295 0.665 8.336

1 10,000 4.000 22 6.697 18 0.295 2.665 8.861

1 1 · 106 6.000 14 12 0.295 4.665 8.501

3 1 0.000 26 2.445 27 0.295 0.000 7.622

3 100 2.000 23 4.919 24 0.295 0.665 7.403

3 10,000 4.000 17 3.510 18 0.295 2.665 7.633

3 1 · 106 6.000 15 11 0.295 4.665 9.042

Experiment 4

1 20 1.301 28 5.050 27 0.295 0.072 8.333

1 40 1.602 25.5 0.583 27 0.295 0.044 7.715

1 60 1.778 24.5 -3.976 27 0.295 0.117 7.493

aFatal threshold is the number of B. dendrobatidis estimated to be present on hosts at the median day of mortality for each treatment. Mean difference

represents the average of all pairwise differences in time to mortality between individuals in a treatment and individuals in the treatment in the same

experiment with the next higher number of zoospores per 20 ml. Fatal threshold B. dendrobatidis numbers represent the number calculated to be in the

average population on hosts in the given treatment at the median day of mortality; for hosts exposed over 3 days, these are the sum of the three growth

curves for those initial populations.

Figure 6. Modeled survival by mass of boreal

toadlets at low dosages ranging from 20 to 100

zoospores of B. dendrobatidis. Comparison of

observed median days survival postinfection of

infected animals in each treatment in experi-

ments 3 and 4, with median days survival

predicted using models assuming that amphib-

ian chytrid populations grow at equal rates on all

hosts and produce mortality when a threshold

number per host is reached. Dashed line

indicates line of equality, solid line is regression

line, y = 0.994x – 1.02.


findings, the findings of this study will be correlated with

observations of the population biology of boreal toad

populations experiencing mass mortalities associated with

B. dendrobatidis.

Chytridiomycosis Can Be Experimentally Induced

in Boreal Toadlets

This study showed that lethal chytridiomycosis can be

experimentally induced in the laboratory in boreal toadlets

by exposure to B. dendrobatidis zoospores and, in most

instances, death followed exposure within 5–7 weeks. In

our first experiment, exposure to 106 zoospores for 72

hours caused infections in 96% of boreal toadlets in that

group. These results coupled with those of Nichols et al.,

(2001) indicate that chytridiomycosis can be induced

readily in susceptible amphibian species.

Behavioral and physiological changes in the toadlets

were noted as the experiment progressed. First, infected

toadlets tried to avoid contact with water in the bottom of

their cages. The avoidance of water, regardless of the

motivational cause underlying the behavior, should pro-

long survival following infection because the rate of rein-

fection from zoospores released from the skin surface

would be low whenever the ventral surface was dry. Second,

the rate of respirations decreased as the severity of the

infections increased. These results may reflect a gradual

inhibition of metabolism by pathological changes in the

skin caused by B. dendrobatidis. These observations do not

support the hypothesis that B. dendrobatidis kills amphib-

ians by blocking oxygen uptake through the skin. If this

hypothesis were true, lung ventilation should increase if

cutaneous respiration is curtailed by B. dendrobatidis

infection. However, our data show the opposite trend.

Duration of Exposure and Dosage of B. dendrobatidis

Play Important Roles in the Length of Survival

The results of experiments 3 and 4 indicate that the initial

dosage of zoospores to which toadlets were exposed and the

length of exposure have significant effects on the number of

toadlets infected and the number of days survived (Figs. 4

and 5). Low dosages and a 1-day exposure fostered longer

average survival times than high dosages and a 3-day

exposure duration. However, average mortality of groups

exposed to 1 or 3 days of the highest dosage level of 106

zoospores did not differ significantly. The significance of

these data will be discussed in the next section.

Although toadlets were exposed for 24 hours to their

individual dose of zoospores in a small (20 ml) amount of

solution, not all individuals became infected at low expo-

sures. Our estimates of initial sizes of B. dendrobatidis

populations on toadlets (Table 5) suggest that approxi-

mately 4% of the zoospores present in the solution used for

exposures became established on the host during 24 hours

in experiment 3, while only 1% became established during

24 hours of exposure in experiment 4. Therefore, it appears

that a relatively high proportion (96–99%) of the zoospores

present in the immediate vicinity of toadlets fail to colonize

them successfully during a 24-hour period.

Reinfection by Zoospores Released by an Individual

Is Necessary for the Level of Infection to Reach a

Threshold Necessary to Cause Death

In experiment 1, both PCR and histological analysis of

toadlets killed at intervals throughout the experiment

showed increases in the amount of B. dendrobatidis

present and indicated that death occurred with moderate

to heavy infections. In this study, even a nominal dose of

1 zoospore in the water may be sufficient to result

eventually in a lethal infection. Our data show an inverse

correlation between dosage and length of survival: the

higher the number of zoospores in the initial exposure,

the shorter the time to death. The finding that the

number of days survived by boreal toadlets following

exposure to B. dendrobatidis zoospores is directly related

to dosage supports the contention that heavy infections

are needed to cause mortality. Exposure to a few zoosp-

ores causes infection, but time is needed for multiple

reinfections to reach the threshold required to kill the

animal. Exposure to a large number of zoospores causes

death more rapidly than a small number because a large

number of zoospores can relatively quickly produce the

critical degree of infection necessary to cause death.

Similarly, the length of exposure is also important for

reaching the critical level of infection to cause death. With

the exception of toadlets infected with 106 zoospores, the

average days of survival was lower in toadlets exposed for

3 days than for 1 day for a given dosage.

Our growth model (Fig. 6) indicates that the approxi-

mate threshold for infection to cause death ranges between

107 and 108 zoosporangia (thalli). When a zoospore invades

amphibian skin, it invades a keratinocyte and transforms into

a sporangium, which produces zoospores. Mature zoospores

exit from the sporangium through a discharge tube into the


water adjacent to the epidermis. The zoospores then can

reinfect the same host or find other hosts. The mechanism by

which B. dendrobatidis kills amphibians is as yet unknown,

but two hypotheses have been advanced (Berger et al., 1998;

Carey et al., 2003a) that are consistent with the idea that a

certain level of infection must be reached to cause death.

First, the fungus might produce a toxin that diffuses from the

epidermis into the body and causes lethal tissue damage. A

heavy concentration of sporangia would produce more toxin

than a light one. Second, the presence of this chytrid in the

epidermis causes hyperplasia and hyperkeratosis, or excess

production of skin layers. Thickening of the skin layers in the

ventral skin of the pelvic area is hypothesized to cause lethal

disruptions in water and ion balance. A heavy infection

would result in a greater degree of ionic and/or water

imbalance than a light one.

Larger Toadlets Will Live Longer Given a Particular

Dosage than Smaller Toadlets, at Least within the

Size Range of Toadlets Used in This Study

Body size was a significant factor in determining the length

of survival of an infected toadlet in experiments 1, 3, and 4.

The size range of toadlets used in these experiments does

not represent the total variation in body mass observed in

field populations because these toadlets were young of the

year. Some boreal toads, particularly females, can grow to

over 80 g (Carey et al., 2005), but the endangered status of

this toad precludes use of large boreal toads in laboratory

experiments. Therefore, the significant association between

the duration of survival of boreal toadlets and body mass

may not necessarily hold for larger toads.

It is unknown why smaller animals died more rapidly

than larger ones. We have demonstrated that exposing

toadlets of similar sizes to different doses of zoospores leads

to differences in time to death consistent with the existence

of a threshold number of thalli necessary to cause death. It

seems likely that the size of this threshold is directly related

to the surface area of the animal. The threshold of small

animals, therefore, is both smaller and reached more

quickly than the threshold for larger animals. The effect

may relate to the fact that small toadlets have a relatively

small surface area of ventral skin. Alternatively, the skin of

smaller toadlets might be easier to penetrate than that of

larger ones. Also, there might be size-, and therefore age-,

related differences in maturation of skin immune defenses,

such as the ability to synthesize and secrete antimicrobial

peptides.

Housing Exposed Animals at Air Temperatures of

12�C and 23�C Has No Significant Effect on Length

of Survival Following Exposure

Numerous anecdotal accounts suggest that temperature can

be an important cofactor contributing to the success of B.

dendrobatidis in causing mortality of amphibians. Specifi-

cally, a number of amphibian deaths linked to B. dendro-

batidis coincide with the onset of seasonally cold

temperatures (Berger et al., 2004; Carey, 2000; Carey et al.,

1999; McDonald et al., 2005; Retallick et al., 2004). Further,

studies of a tropical frog indicate that B. dendrobatidis

infection may be cured by exposure to a temperature of

37�C (Woodhams et al., 2003).

These previous studies document that very high and

very low temperatures affect the interaction between B.

dendrobatidis and its hosts. This study, conducted at mod-

erate temperatures, found that housing exposed animals at

12�C or 23�C had no significant effect on survival in both

experiments 1 and 2. Most physiological and cellular rate

processes increase two- to threefold for every 10�C rise in

temperature (the Q10 rule) (Rome et al., 1992). Several

important factors could be temperature-dependent,

including the growth rate of sporangia, the rate of zoospore

release, and the swimming speed, penetration rate, and

keratinocyte invasion rate by zoospores. However, this study

shows that the overall length of time survived by B. dend-

robatidis-infected boreal toadlets does not obey the Q10 rule.

Additional research on the effect of temperature on the

interactions between B. dendrobatidis and its hosts is needed.

Lethal Chytridiomycosis Can Be Transmitted

through the Water in Which Infected Boreal

Toadlets Have Been Sitting

Experiment 2 confirms the findings of Marantelli et al.,

(2004) that direct body contact is not necessary for chy-

tridiomycosis to be transmitted. Within 21 days after

infection with 106 zoospores per day for 3 days, toadlets

shed sufficient zoospores into Holtfreter’s solution to kill

previously uninfected toadlets. A recent study indicates that

water can remain infective for up to 7 weeks after the

introduction of B. dendrobatidis (Johnson and Speare,

2003). Therefore, this pathogen, shed by an infected indi-

vidual in a breeding pond in June, could be transmitted to

an uninfected individual coming into contact with the

water at this location for much of the summer activity

period, even if no other infected amphibians were present.


Correlations of the Findings of This Study with

Observations on Populations of Boreal Toads in

Colorado Experiencing Mass Mortalities Associated

with B. dendrobatidis

These studies were conducted in environmental conditions

that differed considerably from those in the habitat of

boreal toads, and as a result, they cannot be used to predict

the survival times of boreal toads infected with B. dendro-

batidis in nature. The conditions in these experiments were

optimal for B. dendrobatidis: 23�C and pH 6.5 are within

the optimal ranges for growth of this fungus in laboratory

culture (Piotrowski et al., 2004). Furthermore, in this

study, toads were held in continuous contact with water.

Since toads can reinfect themselves with zoospores pre-

sumably only when they are in water or on a moist sub-

strate, constant contact with water undoubtedly promoted

reinfection and shortened the survival time relative to the

probable survival time if the toads had been periodically

allowed to dry their skin. In contrast, boreal toad body

temperatures, the pH of the water with which they come

into contact, and the amount of daily contact with water

vary considerably in their native habitat in ways that should

promote longer survival times of infected toads in the field

than in these experimental conditions. Body temperatures

of boreal toads fluctuate between near freezing and almost

30�C daily during their 3- to 4-month summer activity

period (Carey, 1978). Toads spend the other 7–8 months of

each year in hibernation under snow and ice, where their

body temperatures remain near freezing. The pH of the

water in which these toads come into contact varies widely

from location to location and throughout the year (Jones et

al., 2001). Finally, nonbreeding individuals of this species

spend at least part of each day during their summer active

period away from contact with water.

Despite these differences, the results of this study can

contribute to our understanding of some of the obser-

vations that we have made on boreal toad populations

experiencing mass mortalities associated B. dendrobatidis.

During these events, population sizes decrease either to

extinction or to very low levels (Carey, 1993; Muths et

al., 2003). We have noticed that larger toads (adults)

tended to persist in the population longer than smaller

ones (postmetamorphic juveniles) and that adult females

tended to survive more years than adult males following

the initial documentation of the presence of B. dendro-

batidis and subsequent mortality of most toads in the

population.

The range of body sizes used in this study was small

compared to the full range (up to more than 80 g) of body

masses found in the field (Carey et al., 2005). Because

approximately 4–6 years are required to grow to adult body

size, typical boreal toad populations have a broad spectrum

of body sizes. Although further study is necessary to verify

that body size has an effect on survival in large boreal toads,

our data are consistent with observations that juveniles and

smaller males died out before larger females in infected

populations (Carey, personal observation).

We presume that most boreal toads become infected

with B. dendrobatidis in aquatic environments, such as

breeding ponds, hibernacula, or moist substrate on which

adults sit and bask in the sun during the summer. This

pathogen, shed by an infected individual breeding in June,

could be transmitted to an uninfected individual coming

into contact with the water in this location for much of the

summer active period, even if no other infected amphibians

were present. The number of zoospores released in the field

from an infected boreal toad is not known, but we antici-

pate that a lightly infected individual would shed fewer

zoospores than a heavily infected one. Therefore, the dos-

age to which an uninfected animal would be exposed in the

field would vary with the number of infected individuals

present, the severity of their infections, the proximity of the

healthy individual to the infected one, the life span of

zoospores, and probably other factors.

Adult females exhibit several behaviors that could

minimize their risk of infection by B. dendrobatidis

zoospores and that support our observations that adult

females in infected populations may live longer generally

than adult males during an outbreak of B. dendrobatidis.

Adult males are likely to spend several weeks during

breeding each spring in continuous contact with water and

to have frequent skin-to-skin contact with other adult

males. The risk of infection in breeding ponds is minimized

for females because they spend less than 1 day at a breeding

site during egg laying and because they do not breed every

year (Carey et al., 2005). Females may also minimize the

risk of infection by hibernating more frequently as solitary

individuals than males, some of whom have been observed

to hibernate communally (Carey, personal observation).

Most boreal toad populations in Colorado became ex-

tinct in the late 1970s through early 1980s (Carey, 1993;

Corn et al., 1989). One hypothesis to explain why the few

relict populations survived these mass extinction events was

that they might have possessed some sort of immune resis-

tance against whatever pathogen might have caused the mass


mortalities, thought at the time to be Aeromonas hydrophila

(Carey, 1993). Although the exact causes of the 1970s

mortalities will never be proven conclusively, the presence of

chytridiomycosis in museum specimens collected in Colo-

rado during the mid-1970s and the similarities between the

pattern of mass mortalities in the 1970s and 1990s (the latter

known to be linked to B. dendrobatidis; Muths et al., 2003)

suggest that this pathogen was the infectious agent (Carey et

al., 1999). Toadlets in this study originated from five pop-

ulations located in different mountain ranges in the Colo-

rado Rockies. Because individuals from each of these

populations proved to be susceptible to this pathogen under

the conditions of these experiments, we conclude that the

persistence of these relict populations was not likely related

to immunity to this pathogen.

Acknowledgments

This study was supported by a National Science Founda-

tion grant (Integrated Research Challenges in Environ-

mental Biology, DEB-0213851). We thank the Colorado

Division of Wildlife, especially Mark Jones, Chuck Loeffler,

and Craig Fetkavich, for facilitating use of animals from the

John W. Mumma Native Aquatic Species Restoration

Facility for this research. Joyce E. Longcore generously

provided B. dendrobatidis cultures and advice throughout

the course of these experiments. John Wood of Pisces

Molecular, Inc. made several recommendations concerning

PCR sampling, for which we are grateful.

REFERENCES

Alford RA, Richards SJ (1999) Global amphibian declines: aproblem in applied ecology. Annual Review of Ecology andSystematics 30:133–165

Annis SL, Dastoor F, Ziel H, Daszak P, Longcore JE (2004) ADNA-based assay identifies Batrachochytrium dendrobatidis inamphibians. Journal of Wildlife Diseases 40:420–428

Berger L, Speare R, Daszak P, Green DE, Cunningham AA, GogginCL, et al. (1998) Chytridiomycosis causes amphibian mortalityassociated with population declines in the rain forests of Aus-tralia and Central America. Proceedings of the National Academyof Sciences USA 95:9031–9036

Berger L, Speare R, Hines HB, Marantelli G, Hyatt AD, McDonaldKR, et al. (2004) Effect of season and temperature on mortalityin amphibians due to chytridiomycosis). Australian VeterinaryJournal 82:434–439

Bishop P (2000) Chytrid fungi identified from dying frogs in NewZealand. FrogLog 39:1–2

Bosch J, Martınez-Solano I, Garcıa-Parıs M (2001) Evidence of achytrid fungus infection involved in the decline of the commonmidwife toad (Alytes obstetricans) in protected areas of centralSpain. Biological Conservation 97:331–337

Bradley GA, Rosen PC, Sredl MJ, Jones TR, Longcore JE (2002)Chytridiomycosis in native Arizona frogs. Journal of WildlifeDiseases 38:206–212

Burnham KP, Anderson DR (2002) Model Selection and Multi-model Inference: A Practical Information-Theoretic Approach,New York: Springer

Carey C (1978) Factors affecting body temperatures of toads.Oecologia 35:197–219

Carey C (1993) Hypothesis concerning the causes of the disap-pearance of boreal toads from the mountains of Colorado.Conservation Biology 7:355–362

Carey C (2000) Infectious disease and worldwide declines ofamphibian populations, with comments on emerging diseases incoral reef organisms and in humans. Environmental HealthPerspectives 108(Suppl 1):1–8

Carey C, Bradford DF, Brunner JL, Collins JP, Davidson EW,Longcore JE, et al. (2003a) Biotic factors in amphibian popu-lation declines. In: Amphibian Decline: An Integrated Analysis ofMultiple Stressor Effects, Linder G, Krest SK, Sparling DW(editors), Pensacola, FL: Society of Environmental Toxicologyand Chemistry, pp 153–208

Carey C, Cohen N, Rollins-Smith L (1999) Amphibian declines:an immunological perspective. Developmental and ComparativeImmunology 23:459–472

Carey C, Corn PS, Jones MS, Livo LJ, Muths E, Loeffler CW (2005)Environmental and life history factors that limit recovery insouthern Rocky Mountain populations of boreal toads (Bufo boreaboreas). In: Status and Conservation of US Amphibians, Lannoo MJ(editor), Berkeley: University of California Press, pp 222–236

Carey C, Pessier AP, Peace AD (2003b) Pathogens, infectiousdisease, and immune defenses. In: Amphibian Conservation,Semlitch RD (editor), Washington DC: Smithsonian Institu-tion, pp 125–136

Corn PS, Stolzenburg W, Bury RB (1989) Acid precipitationstudies in Colorado and Wyoming: interim report of surveys ofmontane amphibians and water chemistry. US Fish and WildlifeService Biological Report 80:1–56

Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE,Speare R (1999) Emerging infectious diseases and amphibianpopulation declines. Emerging Infectious Diseases 5:735–748

Daszak P, Cunningham AA, Hyatt AD (2003) Infectious diseaseand amphibian population declines. Diversity and Distributions9:141–150

Davidson EW, Parris M, Collins JP, Longcore JE, Pessier AP,Brunner J (2003) Pathogenicity and transmission of chytridi-omycosis in tiger salamanders (Ambystoma tigrinum). Copeia2003:601–607

Fellers GM, Green DE, Longcore JE (2001) Oral chytridiomycosisin the mountain yellow-legged frog (Rana muscosa). Copeia2001:945–953

Goettl JP Jr (1997) Boreal toad (Bufo boreas) recovery plan(southern Rocky Mountain population), Denver: ColoradoDivision of Wildlife

Granoff A (1969) Viruses of amphibia. Current Topics in Micro-biology and Immunology 50:107–137

Green DE, Converse KA, Schrader AK (2002) Epizootiology ofsixty-four amphibian morbidity and mortality events in the


USA, 1996–2001. Annals of the New York Academy of Sciences969:323–339

Green DE, Kagarise Sherman C (2001) Diagnostic histologicalfindings in Yosemite toads (Bufo canorus) from a die-off in the1970s. Journal of Herpetology 35:92–103

Hanselmann R, Rodrıguez A, Lampo M, Fajardo-Ramos L,Aguirre AA, Kilpatrick AM, et al. (2004) Presence of anemerging pathogen of amphibians in introduced bullfrogsRana catesbeiana in Venezuela. Biological Conservation 120:115–119

James TY, Porter D, Leander CA, Vilgalys R, Longcore JE (2000)Molecular phylogenetics of the Chytridiomycota supports theutility of ultrastructural data in chytrid systematics. CanadianJournal of Botany 78:336–350

Jancovich JK, Davidson EW, Morado JF, Jacobs BL, Collins JP(1997) Isolation of a lethal virus from the endangered tigersalamander Ambystoma tigrinum stebbinsi. Diseases of AquaticOrganisms 31:161–167

Johnson ML, Speare R (2003) Survival of Batrachochytriumdendrobatidis in water: quarantine and disease control impli-cations. Emerging Infectious Diseases 9:922–925

Jones MS, Livo LJ, Holland AA (2001) Boreal Toad ResearchProgress Report 2000, Denver: Colorado Division of Wildlife

Keinath D, Bennett J (2000) Distribution and Status of the BorealToad (Bufo boreas boreas) in Wyoming, Cheyenne: U.S. Fish andWildlife Service

Lips KR, Green DE, Papendick R (2003) Chytridiomycosis in wildfrogs from southern Costa Rica. Journal of Herpetology 37:215–218

Longcore JE, Pessier AP, Nichols DK (1999) Batrachochytriumdendrobatidis gen. et sp. nov., a chytrid pathogenic toamphibians. Mycologia 91:219–227

Marantelli G, Berger L, Speare R, Keegan L (2004) Distribution ofthe amphibian chytrid Batrachochytrium dendrobatidis andkeratin during tadpole development. Pacific Conservation Biol-ogy 10:173–179

McDonald KR, Mendez D, Muller R, Freeman AB, Speare R(2005) Decline in the prevalence of chytridiomycosis in frogpopulations in North Queensland, Australia. Pacific Conserva-tion Biology 11:114–120

Morehouse EA, James TY, Ganley ARD, Vilgalys R, Berger L,Murphy P, et al. (2003) Multilocus sequence typing suggests thechytrid pathogen of amphibians is a recently emerged clone.Molecular Ecology 12:395–403

Muths E, Corn PS, Pessier AP, Green DE (2003) Evidence fordisease-related amphibian decline in Colorado. Biological Con-servation 110:357–365

Nichols DK, Lamirande EW, Pessier AP, Longcore JE (2001)Experimental transmission of cutaneous chytridiomycosis indendrobatid frogs. Journal of Wildlife Diseases 37:1–11

Nichols DK, Pessier AP, Longcore JE (1998) Cutaneous chy-tridiomycosis in amphibians: an emerging disease? In: Pro-ceedings of the American Association of Zoo Veterinarians/

American Association of Wildlife Veterinarians, Joint Conference,Media, Pennsylvania: American Association of Zoo Veteri-narians, pp 269–271

Pessier AP, Nichols DK, Longcore JE, Fuller MS (1999) Cutaneouschytridiomycosis in poison dart frogs (Dendrobates spp.) andWhite’s tree frogs (Litoria caerulea). Journal of VeterinaryDiagnostic Investigation 11:194–199

Piotrowski JS, Annis SL, Longcore JE (2004) Physiology of Ba-trachochytrium dendrobatidis, a chytrid pathogen of amphibians.Mycologia 96:9–15

Rachowicz LJ, Vredenburg VT (2004) Transmission of Batracho-chytrium dendrobatidis within and between amphibian lifestages. Diseases of Aquatic Organisms 61:75–82

Retallick RWR, McCallum H, Speare R (2004) Endemic infectionof the amphibian chytrid fungus in a frog community post-decline. Public Library of Science Biology 2:e351

Rome LC, Stevens ED, John-Alder HB (1992) The influence oftemperature and thermal acclimation on physiological function.In: Environmental Physiology of the Amphibians, Feder ME,Burggren WW (editors), Chicago: University of Chicago Press,pp 183–205

Ron SR, Duellman WE, Coloma LA, Bustamante MR (2003)Population decline of the Jambato toad Atelopus ignescens(Anura: Bufonidae) in the Andes of Ecuador. Journal of Her-petology 37:116–126

Ron SR, Merino A (2000) Amphibian declines in Ecuador: over-view and first report of chytridiomycosis from South America.FrogLog 42:2–3

Speare R, Berger L (2000) Global distribution of chytridiomycosisin amphibians. Available: http://www.jcu.edu.au/school/phtm/PHTM/frogs/chyglob.htm [accessed June 31, 2005]

Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fis-chman DL, et al. (2004) Status and trends of amphibian declinesand extinctions worldwide. Science 306:1783–1786

Taylor SK (2001) Mycoses. In: Amphibian Medicine and CaptiveHusbandry, Wright KM, Whitaker BR (editors), Malabar, FL:Krieger, pp 181–191

Taylor SK, Green DE, Wright KM, Whitaker BR (2001) Bacterialdiseases. In: Amphibian Medicine and Captive Husbandry, WrightKM, Whitaker BR (editors), Malabar, FL: Krieger, pp 159–179

Utah Division of Wildlife Resources (1998) Western toad. In:Inventory of Sensitive Vertebrate and Invertebrate Species: A Pro-gress Report, pp 159–161. Available: http://dwredc.nr.utah.gov/ucdc/ViewReports/vertrpt.pdf [accessed December 15, 2005]

Weldon C, du Preez LH, Hyatt AD, Muller R, Speare R (2004)Origin of the amphibian chytrid fungus. Emerging InfectiousDiseases 10:2100–2105

White GC, Burnham KP (1999) Program MARK: survival esti-mation from populations of marked animals. Bird Study46(Suppl):120–138

Woodhams D, Alford RA, Marantelli G (2003) Emerging diseaseof amphibians cured by elevated body temperature. Diseases ofAquatic Organisms 55:65–67


Experimental Exposures of Boreal Toads (Bufo boreas) to a Pathogenic Chytrid Fungus...

Documents

Transcript of Experimental Exposures of Boreal Toads (Bufo boreas) to a Pathogenic Chytrid Fungus...