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Infect Immun. Sep 2010; 78(9): 3981–3992.
Published online Jun 28, 2010. doi:  10.1128/IAI.00402-10
PMCID: PMC2937463

Immune Defenses against Batrachochytrium dendrobatidis, a Fungus Linked to Global Amphibian Declines, in the South African Clawed Frog, Xenopus laevis[down-pointing small open triangle]

Abstract

Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.

Batrachochytrium dendrobatidis is a newly described chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians (29). Growing evidence links amphibian declines in Australia, Central America, the western United States, Europe, and Africa to this emerging infectious disease (4, 9, 12, 26, 29, 34-36, 45, 65). B. dendrobatidis colonizes skin cells of adults and the keratinized mouth parts of tadpoles (3, 4, 29, 34) but does not invade other tissues. It is spread by waterborne zoospores that attach to the skin and migrate to the basal layer of the epidermis (3). The pathogen replicates within the epidermal cells and moves to the surface as the cells mature. Emerging zoospores may infect the same host or another nearby host (3, 4, 29, 34). Recent evidence supports the hypothesis that death results from impaired retention of essential ions by the skin resulting in eventual cardiac arrest (63, 64). Some species of amphibians are very resistant to lethal infections of B. dendrobatidis, whereas others are more susceptible (4, 26, 27, 38, 66-68), and the factors that determine resistance or susceptibility are not well understood. Although much is known about amphibian immunity in general (9, 14, 41), there is limited information about specific immune responses against B. dendrobatidis.

We hypothesized that resistant species have antimicrobial peptides or antibodies in the mucus that limit initial infections by B. dendrobatidis zoospores and prevent the further colonization of the same host by zoospores emerging from the skin. Previous work has shown that individual purified antimicrobial peptides (11, 44-50, 52, 68) and enriched skin peptides (48, 52, 66-68) from many species can inhibit the growth of B. dendrobatidis zoospores and mature sporangia in vitro. The skin of amphibians is also protected by the adaptive immune system. Antigens in the skin can be transported to the spleen, where an immune response involving both T cells and B cells can occur (9, 14, 41). In mammals and in fish, antibodies are present in mucosal secretions (10, 28, 31, 53), but there have been no previous studies of antibodies in amphibian mucus.

X. laevis was chosen as the species to investigate immunity to B. dendrobatidis because this species has been widely used as a model for studies of amphibian immunity since the 1960s (9, 14, 41). X. laevis is quite resistant to the lethal effects of infection with B. dendrobatidis in nature. Infections were detected in archived specimens of X. laevis as early as 1938, and the incidence of infected individuals appears to be constant (~3%) over the last 60 years (1941 to 2001) (65).

We show here that after exposure to B. dendrobatidis, immunocompetent frogs developed a mild infection that is almost completely cleared by 45 days. Antimicrobial skin peptides inhibited B. dendrobatidis growth, were present at effective concentrations in resting frogs, and increased in number when frogs were exposed to an “alarm” stress. Treatment with norepinephrine depleted skin peptide stores and increased host susceptibility to infection. X-irradiation depleted leukocytes in the spleen without altering the capacity to secrete skin peptides, and the infection intensity was significantly greater in the irradiated frogs. Immunization with heat-killed B. dendrobatidis induced significantly elevated pathogen-specific IgM and IgY in the serum detectable for at least 1 month after the last immunization. In addition to antimicrobial peptides, skin mucus samples from frogs exposed to B. dendrobatidis 5 months earlier contained antibodies of all three immunoglobulin classes that bind B. dendrobatidis. Whether the mucosal antibodies are protective will be determined in ongoing studies. Collectively, these data demonstrate that X. laevis is a good model species to study immune defenses against B. dendrobatidis, and both innate and adaptive immune mechanisms appear to be involved in the resistance to lethal infections.

MATERIALS AND METHODS

Frogs.

Outbred X. laevis frogs ranging in size from about 30 to 50 g were purchased from Xenopus I (Dexter, MI) and held in polystyrene containers at a density of about 10 frogs/16 liters of dechlorinated tap water at a temperature of about 20 to 24°C. For the first infection experiment (see results in Fig. Fig.4),4), the frogs were young postmetamorphic adults, averaging 4 to 5 g. Frogs were fed ground beef heart, and their water was changed three times weekly. All animal procedures were approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee.

FIG. 4.
Peptide depletion induced by norepinephrine increases susceptibility of X. laevis to B. dendrobatidis. (A) Infection intensity on the skin of frogs that were untreated (n = 10) or injected with 80 nmol of norepinephrine/g (peptide-depleted) ( ...

Collection and partial purification of skin peptides.

Granular gland secretion was stimulated by injection of norepinephrine-HCl (Sigma, St. Louis, MO) dissolved in amphibian phosphate buffered saline (APBS; 6.6 g of NaCl, 1.15 g of Na2HPO4, and 0.2 g of KH2PO4/liter of distilled water) as previously described (48). To collect secretions from resting frogs, they were placed directly in collection buffer without norepinephrine injection. Frogs that were given a simulated “alarm” stress were chased in collection buffer. Briefly, each frog was removed from its holding tank, rinsed with clean water, and placed in a container with 500 ml of collection buffer. The investigator reached in with a gloved hand and forced each frog to swim for 10 min and rest for 5 min more while the peptides accumulated in the buffer. Peptides were partially purified, concentrated, and quantified as previously described (48). The weight of each frog was determined at the time of peptide induction, and total peptides were divided by weight to determine peptide recovery in μg/g. To estimate the amount of peptides in mucus, we calculated the surface area of the skin according to the method of McClanahan and Baldwin [i.e., the surface area = 9.9 (weight in grams)0.56] (30). We assumed the thickness of the mucus to be 50 μm (7), and therefore the volume of mucus covering one cm2 of skin would be 5 μl. Thus, total peptides (μg) per cm2 × 200 = total μg/ml in mucus.

Collection of mucus to test for the presence of immunoglobulins.

For mucus collection, X. laevis frogs that had previously been exposed to B. dendrobatidis ~5.5 months earlier were injected with norepinephrine (80 nmol/g) and placed in 50 ml of collection buffer for 15 min, and the samples were immediately frozen on dry ice. Samples were lyophilized and resuspended in lysis buffer (8.77 g of NaCl, 1.68 g of EDTA, 1.58 g of Tris-Cl, and 10 ml of Triton X-100/liter of distilled water, supplemented with 1 ml each of 5 mM dithiothreitol, 100 μM phenylmethylsulfonyl fluoride in isopropanol, and 5 mM epsilon-aminocaproic acid). The presence of each immunoglobulin class (IgM, IgX, or IgY), the total amount of each class of antibody, and the relative amounts of antibodies binding to B. dendrobatidis in mucus samples were determined by enzyme-linked immunosorbent assay (ELISA) as described below.

Mass spectrometry.

To confirm the presence of previously described antimicrobial peptides in our samples, enriched skin peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as previously described (61, 66).

Growth inhibition assays.

Growth inhibition of B. dendrobatidis was determined as previously described with minor modifications (46, 48, 50). Briefly, zoospores of B. dendrobatidis isolate JEL 197 (29) were isolated after culture for 1 week on 1% tryptone agar at 23°C and plated (5 × 104/50 μl, five replicates) in 1% tryptone broth in 96-well flat-bottom microtiter plates with or without the addition of 50-μl serial dilutions of a natural mixture of skin peptides or individual purified skin peptides from X. laevis dissolved in sterile water. The plates were incubated for 1 week at 23°C. Positive control wells received 50 μl of sterile water without peptides. No growth (negative control) wells contained zoospores treated at 60°C for 10 min. Growth after 7 days was measured as increased optical density at 490 nm (OD490) with an MRS Microplate Reader (Dynex Technologies, Inc., Chantilly, VA). Magainin II was purchased from Sigma. Caerulein precursor fragment (CPF) and PGLa (for peptide with amino-terminal glycine and carboxyl-terminal leucinamide) were a generous gift from Michael Zasloff.

Immunosuppression regimens and exposure to B. dendrobatidis (summarized in Table Table11).

Prior to treatment, all frogs were weighed and swabbed for B. dendrobatidis to determine whether they were infected and then placed in individual sterile containers. In experiment 1, frogs were injected with 80 nmol of norepinephrine/g via the dorsal lymph sac to deplete skin peptides on day −1. Controls were not treated, and both groups were exposed to 106 B. dendrobatidis zoospores (isolate JEL 197) (29) at day 0 and again at day 21 with 106 zoospores of isolate JEL 275, known to be lethal for boreal toads (8). (After the initial exposure, it was decided that JEL 275 might be more virulent because it was isolated from a diseased toad more recently. This isolate was used in all other exposure experiments.) All frogs were swabbed at day 32 to quantify B. dendrobatidis infection intensity by real-time PCR. Experiment 2 was designed to determine the effects of norepinephrine alone (in the absence of the pathogen) on weight loss, and animals that were confirmed to be free of B. dendrobatidis by PCR were injected with norepinephrine (80 nmol/g) or with vehicle alone (APBS), and their weights were monitored approximately every 10 days for 50 days. In experiment 3, frogs were either injected with 80 nmol of norepinephrine/g or irradiated with 9 Gy X-rays, or they were injected with 80 nmol of norepinephrine/g and also irradiated with 9 Gy X-rays on day −1. Controls were not treated, and all groups were exposed to 106 JEL 275 zoospores in 500 ml of water containing 0.005 IU of penicillin and 0.005 μg of streptomycin/ml at days 0 and 21. Each frog was swabbed for B. dendrobatidis and weighed approximately every 10 days. The infection intensity was determined by real-time PCR. Frogs were observed frequently for signs of B. dendrobatidis-induced illness. Experiment 4 was designed to determine the effects of irradiation on leukocyte numbers. Two additional groups of frogs were either irradiated with 9 Gy X-rays or not irradiated, and the spleens were removed 5 days later. Total leukocytes were enriched over Ficoll, counted, and expressed as total leukocytes per gram of body weight. Skin squares from control and irradiated frogs were fixed for histology. Experiment 5 was designed to determine whether irradiation alters the capacity to secrete skin peptides. Frogs were irradiated with 9 Gy X-rays or not irradiated, injected with 2 nmol/g norepinephrine at 3 or 10 days after treatment, and placed in collection buffer for 15 min. Peptides were enriched and quantified as described previously.

Quantification of B. dendrobatidis zoospores on the skin.

X. laevis frogs were swabbed with a sterile cotton swab 10 times on the abdomen, legs, and each foot according to established protocols (6, 22). DNA was extracted according to the method of Boyle et al. (6) by adding 60 μl of PrepMan Ultra (Applied Biosystems, Foster City, CA) to each swab, along with 30 to 35 mg of zirconium/silica beads measuring 0.5 mm in diameter (Biospec Products, Bartlesville, OK). The swabs were homogenized for 45 s in a Mini Beadbeater (MP Bio, Solon, OH) and then centrifuged at 15,000 × g for 30 s. The homogenization and centrifugation were repeated before boiling the samples for 10 min and cooling them for 2 min at room temperature. Samples were centrifuged for 3 min (15,000 × g), and the supernatant containing DNA was removed for real-time PCR (6, 22). Real-time probe-based PCR assays were performed by using an Mx3000P Real-Time PCR system (Stratagene, La Jolla, CA) according to the method of Boyle et al. (6). The default Stratagene conditions (95°C for 10 min, followed by 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min) were used for 40 cycles. A standard curve based on the threshold cycle (CT) values from the control zoospore DNA was generated, and the number of zoospore equivalents in each positive sample was calculated.

Immunization and blood collection.

Prior to immunization, 20 X. laevis frogs were anesthetized, and blood was drawn by cardiac puncture to obtain preimmune plasma samples. Immunized frogs were injected intraperitoneally (10 μl/g of body weight [gbw]) with heat-killed (60°C, 20 min) B. dendrobatidis (isolate JEL 197, mixed zoospores and maturing sporangia) at a concentration of 5 × 107 cells/ml at days −42, −28, and 0 prior to the collection of serum samples. Control frogs were injected with APBS alone (10 μl/gbw). Blood was drawn at days 7, 14, 21, and 28 after the final immunization, and IgM and IgY antibodies that bind B. dendrobatidis were quantified by ELISA. Antibody titers (IgY) specific for B. dendrobatidis in sera from APBS-injected or B. dendrobatidis-immunized frogs were determined by ELISA. Serum samples were serially diluted in APBS with 0.5% bovine serum albumin (BSA) and 0.1% Tween 20 (ABT) for all preimmune (n = 20), day 7 (n = 10), day 14 (n = 10), day 21 (n = 10), and day 28 (n = 7) samples, and the titration endpoint was established as the dilution at which absorbance at 450 nm (OD450) was no longer significantly greater than the background level (ABT only).

ELISA.

B. dendrobatidis antigen-coated plates (96-well; Nunc Maxisorp, Rochester, NY) were prepared by adding 5 × 104 cells (JEL 197) at 50 μl/well as zoospores alone or as mixtures of zoospores and maturing sporangia. The plates were centrifuged (about 200 × g), and the cells were fixed by the addition of 0.25% glutaraldehyde in APBS. The fixative was removed, and the plates were washed with ABT and stored at 4°C. X. laevis sera or lyophilized mucus diluted in ABT were added to the wells, and B. dendrobatidis-specific antibodies were detected using anti-Xenopus monoclonal antibodies specific for IgM (10A9), IgY (11D5) (21), or IgX (410D9) (33), followed by horseradish peroxidase-conjugated goat anti-mouse antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL). The reactions were visualized with 3,3′,5,5′-tetramethylbenzidine substrate (Sigma). After about 60 min, the reaction was stopped by the addition of 2 M H2SO4, and the plates were read at 450 nm (OD450) in an ELISA plate reader.

To test for the presence of total IgM, IgX, or IgY in mucus, lyophilized mucus samples were diluted to a concentration of 5 mg/ml in ABT and placed in a 96-well microtiter plate (four replicates), and the plates were incubated at 37°C for 1 h. Control wells were coated with 1% normal rabbit serum in ABT. After incubation, all of the wells were blocked with 1% BSA in ABT at 37°C for 2 h. The plates were washed with ABT, and IgM, IgY, and IgX were detected using the 10A9 (anti-IgM), 11D5 (anti-IgY), and 410D9 (anti-IgX) monoclonal antibodies, followed by horseradish peroxidase-conjugated goat anti-mouse antibodies. The reactions were visualized by the addition of substrate as described above. Using purified IgM, IgY, and IgX standards, total antibodies of each class in the mucus of the frogs were quantified by ELISA.

Statistical comparisons.

All parameters compared in the present study (B. dendrobatidis growth as OD490, peptide concentrations as μg/ml of mucus, infection intensity as zoospore equivalents, leukocyte numbers, and antibody concentrations as OD450) were averaged, and the mean values ± the standard errors were compared by one-tailed or two-tailed Students t test, one-way analysis of variance (ANOVA) with planned comparisons or Tukey post hoc tests, or analysis of covariance (ANCOVA) as detailed in the figure legends. Zoospore equivalents, peptide concentrations, and OD values were log transformed as indicated in the figure legends to meet the assumptions of normal distribution and homogeneity of variances for parametric statistics. A P value of ≤0.05 was considered to be statistically significant. Error bars shown in all figures represent standard errors except in Fig. 3E and F, which show the 95% confidence intervals. If no error bar is shown, the standard error was less than the diameter of the symbol. In B. dendrobatidis growth inhibition assays, each data point represents the mean ± the standard error of five replicate wells. For other parameters, the number of animals or samples is shown in the figure legends.

FIG. 3.
Effects of “alarm” stress or norepinephrine (NE) on induction and recovery of skin peptide secretion. (A) Effect of simulated predator stress on the secretion of skin peptides by X. laevis (n = 5 in each group except 2.0 nmol of ...

RESULTS

Contribution of antimicrobial skin peptides.

We hypothesized that X. laevis is resistant to the lethal effects of infection by B. dendrobatidis because it has very effective antimicrobial peptides in the mucus that form a significant barrier to initial infection and reinfection by zoospores. To examine the role of antimicrobial peptides, we determined the pattern of expressed skin peptides by MALDI-TOF MS and assessed the growth-inhibitory activity of the natural mixtures of skin peptides or individual purified antimicrobial peptides.

Granular glands in the skin of X. laevis produce bioactive peptides, including antimicrobial peptides (1, 16, 17, 23, 25, 32, 40, 57, 60, 70). The contents of the glands are secreted onto the surface of the skin after stimulation of the local sympathetic nerves, which might occur as a result of injury or alarm (2, 13, 19, 58). This stimulation is achieved in the laboratory by injecting norepinephrine directly into the dorsal lymph sac (13). Using MALDI-TOF MS, a number of previously identified antimicrobial peptides produced in X. laevis were routinely observed in the skin secretions, including PGLa (1), magainin I and magainin II (70), CPF (16, 17, 23, 40, 60), LPF (23, 25), and XPF (16, 17, 23, 60) (Fig. (Fig.1).1). Enriched skin peptide mixtures like those shown in Fig. Fig.11 strongly inhibited growth of B. dendrobatidis zoospores (Fig. (Fig.2A).2A). Although there was some variability among the samples tested, most frogs secreted peptides that inhibited zoospore growth at concentrations above 30 to 60 μg/ml. The MICs ranged from 62.5 to 500 μg/ml, and the average percent inhibition of zoospores at 500 μg/ml was 99.4 ± 0.4 (data not shown). The skin peptides also inhibited growth of mixtures of zoospores and maturing sporangia, but the MICs were slightly increased (data not shown). Three of the purified antimicrobial peptides—CPF, PGLa, and magainin II—were also tested for their ability to inhibit zoospore growth in vitro. All three were significantly inhibitory. CPF inhibited growth at concentrations of >1.6 μM with an MIC of 12.5 μM (Fig. (Fig.2B).2B). PGLa showed inhibition at a concentration greater than 25 μM with an MIC of 50 μM (Fig. (Fig.2C).2C). Magainin II inhibited the growth of zoospores at concentrations greater than 32 μM (the MIC was approximately 162 μM) (Fig. (Fig.2D).2D). Collectively, these data show that both natural mixtures of skin peptides and individual peptides from X. laevis are potent inhibitors of the growth of B. dendrobatidis zoospores. Thus, antimicrobial peptides are likely to play a role in inhibiting colonization of the skin by B. dendrobatidis.

FIG. 1.
MALDI-TOF MS imaging profile of X. laevis skin secretions. Previously described antimicrobial peptides are labeled. PGLa, peptide with amino-terminal glycine and carboxyl-terminal leucinamide; CPF, caerulein precursor fragment; XPF, xenopsin precursor ...
FIG. 2.
Skin peptides from X. laevis inhibit B. dendrobatidis growth. B. dendrobatidis zoospores were cultured with or without dilutions of (A) natural mixtures of skin peptides from X. laevis, (B) pure synthetic CPF, (C) pure synthetic PGLa, and (D) pure synthetic ...

Effects of an “alarm” stress on secretion of skin peptides.

To test whether an “alarm” stress event leads to the secretion of physiologically relevant concentrations of skin peptides, X. laevis frogs were subjected to a simulated predator attack as described in Materials and Methods. Frogs that were stressed in this way secreted significantly higher amounts of total skin peptides (19,581 ± 7,340 μg/ml in mucus) than resting frogs (3,256 ± 345 μg/ml in mucus) (one-way ANOVA, Tukey post hoc test, P < 0.01, Fig. Fig.3A).3A). Frogs injected with a very low dose of norepinephrine (0.2 nmol/g) secreted an amount of peptides approximately equivalent to that observed with resting frogs (2,262 ± 623 μg/ml in mucus), whereas frogs that received 2 nmol of norepinephrine/g secreted more peptides (41,646 ± 11,121 μg/ml in mucus), an amount that was significantly greater than that of both resting and chased frogs (one-way ANOVA, Tukey post hoc test, P < 0.05, Fig. Fig.3A).3A). The concentrations of skin peptides recovered from both resting frogs and those subjected to the “alarm” stress were within the range of concentrations shown to inhibit B. dendrobatidis growth in vitro (Fig. (Fig.2A).2A). Therefore, it is likely that resting frogs and “alarmed” frogs can secrete a mixture of skin peptides at concentrations sufficient to inhibit colonization of the skin by B. dendrobatidis.

Depletion and recovery of skin peptides after norepinephrine induction.

To better understand the role of antimicrobial peptides in resistance to B. dendrobatidis infection, we developed a method to deplete skin peptides by norepinephrine induction. To determine the concentration of norepinephrine required to exhaust skin peptides stores, increasing concentrations of norepinephrine were injected into the dorsal lymph sac. Skin peptide secretion was induced in a dose-dependent fashion (51) (Fig. (Fig.3B).3B). Because 80 and 160 nmol/g induced approximately the same maximal amounts of recoverable peptides (Fig. (Fig.3B),3B), 80 nmol of norepinephrine/g was chosen as a dose sufficient to maximally deplete peptides.

To determine the amount of time required for the recovery of peptides in the skin after depletion, groups of frogs were injected with either the maximal dose (80 nmol/g) or a moderate dose (20 nmol/g) of norepinephrine at day 0. A subset of frogs was injected again with the same dose at later time points. After induction with 80 nmol of norepinephrine/g, peptide concentrations were severely reduced at 1 and 3 weeks after the initial stimulation. Seven to nine weeks after the initial induction, the peptide concentrations had recovered (Fig. (Fig.3C).3C). In comparison, frogs that were injected with 20 nmol of norepinephrine/g showed a significant reduction in the amount of peptides induced at 3 days but had recovered to starting concentrations by 4 to 5 weeks (Fig. (Fig.3D).3D). The initial peptide concentration and slope of recovery were plotted for 80 and 20 nmol of norepinephrine/g (Fig. 3E and F). The data suggest that after the first injection of 80 nmol of norepinephrine/g, the frogs retained the capacity to secrete approximately 19,180 μg of peptides/ml, and full recovery required approximately 50 to 104 days. In comparison, after the first injection of 20 nmol of norepinephrine/g, the frogs would be able to secrete approximately 10,394 μg of peptides/ml, and recovery required approximately 20 days. Thus, the amounts of available skin peptides were significantly reduced in X. laevis by stimulation with 80 nmol of norepinephrine/g, and peptide recovery occurred slowly allowing us to assess the importance of this innate defense in the protection of X. laevis from B. dendrobatidis infections.

Effects of skin peptide depletion on susceptibility to infection with B. dendrobatidis.

When frogs were injected with 80 nmol of norepinephrine/g to deplete skin peptides and exposed to B. dendrobatidis, the intensity of infection was significantly increased in comparison with untreated controls (one-tailed Student's t test after log transformation, P = 0.0395) (Fig. (Fig.4A).4A). Individual weight changes were monitored as a measure of morbidity due to B. dendrobatidis infection. Peptide-depleted and B. dendrobatidis-exposed frogs lost significantly more weight over the 6-week period of this experiment in comparison with the untreated, B. dendrobatidis-exposed frogs (no treatment group) (ANCOVA; P = 0.028, Fig. Fig.4B).4B). To determine the effects of norepinephrine alone on weight loss (in the absence of the pathogen), animals that were confirmed to be free of B. dendrobatidis by PCR were injected with norepinephrine (80 nmol/g) or with vehicle alone (APBS), and their weights were monitored for 50 days. Norepinephrine induced significantly greater weight loss at day 4, but the weights were not significantly different from those of the controls at later time points (Fig. (Fig.4C).4C). Thus, norepinephrine alone causes a temporary weight loss rather than the sustained weight loss observed in the B. dendrobatidis-infected and peptide-depleted frogs. These data suggest that the peptide-depleted frogs became more susceptible to B. dendrobatidis infection, carrying higher burdens of the pathogen and losing more weight compared to untreated controls.

Time course of infection and clearance of B. dendrobatidis in immunocompetent frogs.

To develop a better understanding of the time course of infection in the skin with B. dendrobatidis, we infected naive animals at days 0 and 21 and monitored the intensity of infection over a 60-day period using real-time PCR to monitor zoospores released from the skin. At 11 days after the first exposure, most (9/10) of the exposed frogs were positive for the presence of zoospores, with an average infection intensity of 7.4 ± 3.4 zoospore equivalents. By day 20, the infection intensity had increased to 272.5 ± 124.2 zoospore equivalents and decreased in subsequent weeks to a low of 5.2 ± 1.7 zoospore equivalents at 44 days after the first exposure (Fig. (Fig.5A,5A, no-treatment group). Thus, the infection was limited to very low levels by about 45 days after initial exposure.

FIG. 5.
X-irradiation increases susceptibility of X. laevis to B. dendrobatidis infection. (A) Infection intensity on the skin of frogs treated as shown and exposed to B. dendrobatidis zoospores (isolate JEL 275) at days 0 and 21 was measured by real-time PCR ...

Effects of X-irradiation on susceptibility to B. dendrobatidis infection.

To determine whether the adaptive immune system is involved in resistance to B. dendrobatidis, we treated frogs with a sublethal dose (9 Gy) of X-rays to inhibit lymphocyte functions and then exposed the irradiated frogs and controls to B. dendrobatidis 1 day later and again at day 21. X. laevis frogs that were irradiated and exposed to the pathogen had significantly greater numbers of zoospore equivalents on the skin over time than did nonirradiated and B. dendrobatidis-exposed controls (ANCOVA, P ≤ 0.001). The infection intensity declined by day 44 in the same pattern as that of the nonirradiated controls (Fig. (Fig.5A).5A). A third group of frogs that was both irradiated and peptide depleted also had significantly higher numbers of zoospores on the skin compared to the nonirradiated and B. dendrobatidis-exposed controls (ANCOVA, P ≤ 0.001) (Fig. (Fig.5A),5A), and infection intensity declined by day 44. In this experiment, peptide depletion alone did not result in increased infection intensity. The irradiated and B. dendrobatidis-infected frogs lost more weight than the nonirradiated, pathogen-exposed frogs throughout the time course of this experiment (ANCOVA; P < 0.001) (Fig. (Fig.5B).5B). The weight loss was not a side effect of the irradiation protocol, since frogs that were irradiated but not exposed to B. dendrobatidis maintained their weights throughout the experiment (Fig. (Fig.5B).5B). The doubly treated (irradiated and peptide-depleted) and pathogen-exposed frogs also lost significantly more weight throughout the course of the experiment than did the nonirradiated controls (data not shown). The animals were observed frequently, and there was no evidence of obvious skin damage. Histological sections also showed no significant differences in the appearance of the skin of irradiated animals in comparison to the controls (data not shown). Although none of the frogs in the experiment died, greater weight loss in frogs that were immunosuppressed compared to the untreated controls suggests that the increased burden of zoospores on their skin impaired their health. Frogs that were irradiated with 9 Gy X-rays had significantly reduced numbers of leukocytes in their spleens 5 days after irradiation in comparison with nonirradiated controls (two-tailed Student's t test, P = 0.0314) (Fig. (Fig.6A).6A). The numbers of B and T cells were approximately equally reduced by the irradiation (data not shown), as determined by flow cytometry using B-cell-specific (10A9) or T-cell-specific (AM22 and 2B1) (15, 24) monoclonal antibodies. To demonstrate that the X-rays did not damage the skin and compromise antimicrobial peptide production and secretion, irradiated frogs were induced to secrete peptides by injection of norepinephrine (2 nmol/g) at 3 or 10 days after irradiation. They released skin peptides at concentrations that were equivalent to those of nonirradiated frogs (Fig. (Fig.6B),6B), suggesting that this innate skin defense was not impaired by irradiation. Histological studies of the skin of irradiated frogs showed that granular glands were intact (data not shown). These data show that irradiation reduces leukocyte numbers in the spleen and increases infection intensity after exposure to B. dendrobatidis but does not affect the capacity to secrete skin peptides.

FIG. 6.
X-irradiation reduces leukocyte numbers in the spleen but does not alter capacity to secrete skin peptides. (A) Effects of X-irradiation (9 Gy) on total leukocyte numbers in the spleen 5 days after exposure (n = 3 for each treatment). *, ...

Induction of B. dendrobatidis-specific antibodies after immunization.

To determine whether a systemic immune response against B. dendrobatidis can be generated, X. laevis frogs were injected with heat-killed B. dendrobatidis three times over a 6-week period. Beginning 1 week after the final immunization, blood samples were drawn weekly for a period of 4 weeks and tested for the presence of B. dendrobatidis-specific antibodies by ELISA. Although a small number of frogs were bled prior to immunization to determine the preimmune status, most frogs were bled only once. Immunization induced robust production of IgM antibodies (data not shown) and class-switched IgY antibodies against mature sporangia and zoospores or zoospores alone (one-way ANOVA, Tukey post hoc test, P < 0.01; Fig. Fig.7A).7A). IgY binding activity was significantly greater than that of APBS-injected controls at all days tested except day 28 for the assay against mixed sporangia and zoospores (two-tailed Student's t test, P ≤ 0.005; Fig. Fig.7A).7A). Although binding was greatest at 14 days after the final immunization, significantly elevated levels of the anti-B. dendrobatidis antibodies were still detectable at days 21 and 28 (Fig. (Fig.7A).7A). IgY antibody titers were minimal (<1/800) in frogs injected with APBS at all time points but ranged from 1/800 to 1/6,400 at day 14 in frogs immunized against B. dendrobatidis (Fig. (Fig.7B).7B). Thus, a robust and specific immune response that persisted for at least 1 month was generated in frogs immunized against B. dendrobatidis.

FIG. 7.
Immunization of X. laevis against B. dendrobatidis (Bd) induces production of high-titer IgY antibody responses. After the final injection, blood was drawn by cardiac puncture weekly for 4 weeks. Blood samples drawn prior to immunization served as preimmune ...

Immunoglobulins in skin mucus.

The immunization of X. laevis against B. dendrobatidis produced a systemic adaptive immune response. However, B. dendrobatidis is a skin pathogen, and it is important to determine whether adaptive immune defenses are detectable in the skin after pathogen exposure. One potential mode of protection of the skin is the production and secretion of immunoglobulins in the mucus, as observed in fish (28, 53) and mammals (10, 31). To demonstrate whether X. laevis mucus contains immunoglobulins, frogs previously exposed to B. dendrobatidis (about 5.5 months earlier) were induced to secrete mucus by the injection of 80 nmol of norepinephrine/g. The mucosal secretions were collected and tested for the presence and quantities of total IgX, IgM, and IgY immunoglobulins by ELISA. All five individuals tested showed strong signals for all three immunoglobulin classes using monoclonal reagents specific for each class of immunoglobulin. The mean OD450 readings were significantly greater for all individuals and each immunoglobulin class except IgX in frog number 5 compared to an irrelevant serum control (1% normal rabbit serum) as determined by one-way ANOVA (Tukey post hoc test, P < 0.01) and two-tailed Student's t test, P ≤ 0.025 (Fig. (Fig.8A).8A). Using purified IgM, IgY, and IgX standards, the concentrations of each class of antibody in the mucus were determined by ELISA. The IgM concentration was approximately 98 μg/ml, the IgY concentration was approximately 800 μg/ml, and the IgX concentration was approximately 1,670 μg/ml (Fig. (Fig.8B8B).

FIG. 8.
Total and pathogen-specific antibodies in mucosal secretions. (A) Total binding activity of monoclonal antibodies (MAb) specific for Xenopus immunoglobulins in mucosal secretions (5 mg/ml) assayed by ELISA (n = 5) in comparison with an irrelevant ...

To test whether the mucosal antibodies can bind to B. dendrobatidis, the mucus samples were tested for specific binding activity against the pathogen by ELISA. Mucus samples diluted to a concentration of 1 mg/ml showed strong binding of all three classes of antibodies to B. dendrobatidis antigens fixed on ELISA plates (Fig. (Fig.8C).8C). The binding activity was significantly greater that that of an irrelevant serum control (1% normal rabbit serum) as determined by one-way ANOVA (Tukey post hoc test, P < 0.01) and two-tailed Student's t test, P ≤ 0.025. These data suggest that X. laevis is capable of secreting immunoglobulins into the skin mucus that bind to B. dendrobatidis and that these mucosal antibodies could play a role in resistance to infection. Whether B. dendrobatidis-specific immunoglobulins are increased after infection and whether these molecules are protective remains to be tested in ongoing studies.

DISCUSSION

This study is the first to systematically explore the immune defenses of a model amphibian, X. laevis, against B. dendrobatidis. B. dendrobatidis is a skin pathogen, and thus studies of immune defenses in the skin are critical for understanding disease resistance versus susceptibility. The data from our study strongly suggest that both innate and adaptive components of the immune system are involved in resistance to lethal B. dendrobatidis infections in this species.

One of the most important innate defenses of amphibian skin is the array of antimicrobial peptides secreted into the mucus. Previous studies have demonstrated that antimicrobial skin peptides from many amphibian species can inhibit B. dendrobatidis growth in vitro (11, 44-52, 66-68). We showed in the present study that natural peptide mixtures in the skin secretions of X. laevis also potently inhibited the growth of B. dendrobatidis zoospores. The MIC for the most active purified peptide (CPF) was 12.5 μM, and the MICs for enriched mixtures of peptides ranged from 62.5 to 500 μg/ml (Fig. (Fig.2).2). The zoospore is the infectious life stage of B. dendrobatidis, and it is responsible for establishing an infection on the skin surface that results in the colonization of epithelial cells for further maturation and replication (3, 4, 29, 34). By directly killing zoospores in the mucus, antimicrobial peptides may prevent zoospores from settling on the skin and colonizing epithelial cells. Continuous secretion of even small amounts of antimicrobial peptides may also inhibit reinfection of the skin by zoospores emerging from an established infection. We also showed that when X. laevis frogs were placed in a situation that mimicked an attack, they secreted significantly more skin peptides than resting, unmanipulated frogs (Fig. (Fig.3A).3A). The concentrations of peptides secreted by the resting frogs, as well as by the chased frogs, were within the range of concentrations necessary for zoospore growth inhibition (Fig. (Fig.2A).2A). This suggests that frogs exposed to B. dendrobatidis in the wild would be able to secrete skin peptides at a sufficient concentration to inhibit initial infections. Previous studies comparing the peptide effectiveness of stream-dwelling amphibian species in Australia showed that those with greatest skin peptide effectiveness against B. dendrobatidis in vitro did not decline, whereas those with poor peptide effectiveness declined (68). When four other Australian amphibian species were experimentally infected with B. dendrobatidis, those with the greatest in vitro peptide effectiveness against B. dendrobatidis were more resistant (significantly greater survival) (67). A similar comparison of eight species from a stream-dwelling assemblage in Panama showed that all except two species had relatively poor measures of peptide effectiveness against B. dendrobatidis and would be predicted to decline (66).

After secretion onto the surface of the skin, some time is required for peptide stores to be renewed. Previous data suggested that after maximal stimulation with 80 nmol of norepinephrine/g, peptides were not fully restored after 3 weeks (51). In the present study, we extended the time of analysis. By 7 to 9 weeks the peptide levels were not significantly different from those at day 0 (Fig. (Fig.3C).3C). Analysis of the slope of peptide recovery suggests that animals injected with 80 nmol of norepinephrine/g require approximately 50 to 104 days for complete recovery (Fig. (Fig.3E).3E). Skin peptide concentrations were restored to initial levels within 4 weeks after a moderate norepinephrine stimulation (20 nmol/g) (Fig. (Fig.3D).3D). These animals would be expected to restore their peptides in approximately 20.4 days (Fig. (Fig.3F).3F). When peptides were depleted by the injection of 80 nmol of norepinephrine/g, exposure to B. dendrobatidis resulted in a greater intensity of infection at 31 days after first exposure compared to intact untreated controls that were also exposed to B. dendrobatidis (Fig. (Fig.4A).4A). This is the clearest evidence that antimicrobial peptides in the mucus play a role in limiting the degree of skin infection. Although the most direct effect of injection of norepinephrine at this concentration is depletion of skin peptide reserves, the norepinephrine could also have other immunosuppressive effects, such as activation of corticosterone release (56). Because the mucus also contains immunoglobulins, it is possible that norepinephrine treatment to deplete peptides also temporarily depletes immunoglobulins from the mucus. Future studies will determine whether the maximal norepinephrine dose (80 nmol/g) depletes immunoglobulins and how long they will be reduced. This very strong norepinephrine stimulus to deplete peptides was a pharmacological treatment to impair the skin peptide defenses. However, it is unlikely that natural stresses in nature would result in such a significant long-term depletion of peptide reserves.

Although X. laevis does not die as a result of infection with B. dendrobatidis, the skin does become infected. We observed zoospores at about 11 days postinfection (approximately three cycles of replication at 20 to 24°C) (69) in untreated hosts. This number increased significantly by 20 to 30 days and then waned to very low levels at days 44 and 59 (Fig. (Fig.5A).5A). This suggests that within 30 days, immune defenses have been activated sufficiently to begin to clear the infection. The same kinetics of pathogen clearance were observed in frogs that were X-irradiated with a dose that has previously been shown to inhibit skin allograft rejection, tumor rejection, and the ability to resist infection by a ranavirus (20, 42, 43, 55), but the intensity of infection was greater in irradiated frogs (Fig. (Fig.5A).5A). Although lymphocyte defenses were impaired (Fig. (Fig.6A),6A), the capacity to secrete skin peptides was not (Fig. (Fig.6B).6B). We believe that the most direct effect of X-irradiation was destruction of leukocytes. However, it is possible that irradiation also induces a stress response and release of corticosteroids to suppress global immune functions. Although none of the frogs in these experiments died, frogs that were immunosuppressed had increased numbers of zoospores on their skin (Fig. (Fig.5A)5A) and experienced greater weight loss compared to untreated controls (Fig. (Fig.5B).5B). Significant weight losses after infection with B. dendrobatidis have been documented for other, more susceptible species, e.g., Pseudacris triseriata (37) and Rana muscosa (18). Collectively, these results suggest that both antimicrobial peptide defenses and lymphocyte-mediated defenses are involved in the clearance of B. dendrobatidis infections in X. laevis.

These findings are at variance with the prevailing view that the adaptive immune system plays no role in defense against chytridiomycosis. A recent study found that systemic immunization (via the dorsal lymph sac) with formalin-killed B. dendrobatidis was not effective in preventing disease development and mortality in Rana muscosa juveniles experimentally exposed to live B. dendrobatidis (59). However, we feel there were some important technical limitations of that study. The immunization protocol used was less rigorous than the one we used to induce serum antibodies in our studies, and the temperature of the maintenance of the frogs was quite cool (17°C). This temperature would likely delay development of an immune response. Their protocol used a starting preparation of 105 zoospores that was formalin fixed and washed three times, and the final precipitate was mixed with adjuvant. It is unknown what the final protein concentration of the preparation was. The frogs were immunized twice at day 0 and day 30 and exposed to infectious zoospores 30 days after the last immunization. In contrast, our immunization protocol used more cells and an extra immunization at warmer temperatures (105 freshly killed sporangia and zoospores per gram body weight delivered three times over 6 weeks to frogs housed at 20 to 24°C). The other investigators were unable to monitor the development of an immune response because of the lack of reagents to detect antibodies or T cells in this species. Thus, it is unclear whether an effective antibody response or T-cell-mediated response developed in the immunized R. muscosa. It is also possible that because the antigenic preparation was delivered via the dorsal lymph sac, it would generate serum antibodies but have no effect on the mucosal antibody compartment.

Our studies also contradict two recent microarray studies that found little evidence for an adaptive immune response to B. dendrobatidis in the closely related amphibian, Silurana (Xenopus) tropicalis (39, 54). We feel there were a number of technical difficulties that may explain why these other studies did not detect adaptive immune defenses. In the first study (54), the frogs were held at 18°C, which would be suboptimal for this species and would likely be mildly immunosuppressive (41). A second technical problem with that study was that there were only two time points sampled (day 3 and variable times between 10 and 21 days after infection), and three tissues sampled (skin, liver, and spleen). Because of the small spleen size, spleens were only examined at day 3 in that study. Given the slow kinetics of infection and immune responses observed in our study, we feel the microarray study chose observation points too early in the immune response. The second study (39) examined S. tropicalis exposed to B. dendrobatidis at a warmer temperature (26°C). However, the intensity of infection reported for those animals was extremely low (<3 zoospore equivalents) throughout the course of the experiment, and it is impossible to determine from the data presented whether there were significant differences between infection intensity at early and late time points. This suggests that the isolate used had low virulence for S. tropicalis. Because of the design of the experiment (too few animals), there were no significant differences in prevalence between early and late time points. Thus, it seems likely that animals experienced a very low level of infection throughout, and the adaptive immune system may not have been involved in clearance.

The adaptive immune responses against B. dendrobatidis in amphibians were largely unknown when these studies were begun. Our data showed that an irradiation-sensitive component of the adaptive immune system limits infection by B. dendrobatidis. Experimental immunization of X. laevis induced the production of high-titer antibody responses of both the IgM and the IgY classes. Although we have not yet shown the T-cell contribution to this response, it is well known that the development of high-titer IgM and class-switched IgY responses in X. laevis requires T-cell help (5, 62). Thus, the high-titer responses to B. dendrobatidis suggest that T cells are also being activated by this immunization.

In an attempt to understand how adaptive immunity may be protecting amphibians from B. dendrobatidis, we chose to look for the presence of immunoglobulins in mucosal secretions. Although mucosal antibodies have been demonstrated in fish (28, 53) and mammals (10, 31), there have been no previous reports of immunoglobulins in the mucus of amphibians. Our data show that immunoglobulins of three classes can be secreted into the skin mucus of X. laevis. When IgX-secreting B cells were discovered in X. laevis, they were shown to be highly enriched in gut epithelium and barely detectable in the liver and spleen (33). Thus, IgX is thought to be the amphibian counterpart of mammalian IgA (33). The presence of IgM and IgY antibodies in mucus, as well as IgX (Fig. (Fig.8),8), suggests that all three classes may have a role to play in protection of the skin. We showed that antibodies in the mucus can bind to B. dendrobatidis antigens in an ELISA (Fig. (Fig.8C).8C). Studies are in progress to determine whether B. dendrobatidis-exposed frogs have increased B. dendrobatidis-specific immunoglobulins in the mucus and whether these antibodies can protect. Antibodies in the mucus may play a role in neutralizing zoospores to prevent their entry into epidermal layers or to opsonize them for destruction by phagocytes or complement. If mucosal antibodies are protective, perhaps a method of skin immunization can be developed that may offer protection to more susceptible species.

Taken together, the data presented here suggest that both innate and adaptive defenses play important roles in protecting resistant amphibian species, such as X. laevis, against B. dendrobatidis. Antimicrobial peptides secreted onto the skin or mucosal antibodies may limit colonization on the skin. If B. dendrobatidis is successful at colonizing the skin, additional adaptive defenses may be mobilized to help control infection and protect the individual from death. Further studies are needed to better understand the mechanisms by which adaptive immune defenses may control B. dendrobatidis infection in the relatively resistant X. laevis and other more susceptible species.

Acknowledgments

This study was supported by the National Science Foundation grants IOS-0520847, IOS-0619536, and IOS-0843207 (to L.A.R.-S.) and DEB-0213851 (to J. Collins, subcontract to L.A.R.-S.). J.P.R. was partially supported by NHBLI Immunology of Blood and Vascular Systems training grant 5T32 HL069765-05 (to J. Hawiger).

We thank Chad O'Leary and Kimberly Henry for assistance with the growth inhibition assays and Michael Freeman for assistance with the X-irradiation experiments.

Notes

Editor: G. S. Deepe, Jr.

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 June 2010.

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