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Department of Biological Sciences, University of Maine, Orono, Maine 04469
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ABSTRACT |
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Batrachochytrium dendrobatidis is a pathogen of amphibians that has been implicated in severe population declines on several continents. We investigated the zoospore activity, physiology and protease production of B. dendrobatidis to help understand the epidemiology of this pathogen. More than 95% of zoospores stopped moving within 24 h and swam less than 2 cm before encysting. Isolates of B. dendrobatidis grew and reproduced at temperatures of 4–25 C and at pH 4–8. Growth was maximal at 17–25 C and at pH 6–7. Exposure of cultures to 30 C for 8 d killed 50% of the replicates. B. dendrobatidis cultures grew on autoclaved snakeskin and 1% keratin agar, but they grew best in tryptone or peptonized milk and did not require additional sugars when grown in tryptone. B. dendrobatidis produced extracellular proteases that degraded casein and gelatin but had no measurable activity against keratin azure. The proteases were active against azocasein at temperatures of 6–37 C and in a pH range of 6–8, with the highest activity at temperatures of 23–30 C and at pH 8. The implications of these observations on disease transmission and development are discussed.
Key words: chytridiomycosis, Chytridiomycota, disease, fungal proteases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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has been detected in dead or dying anurans in North America, Australia, Central America and Europe and causes chytridiomycosis, which is implicated as the cause of amphibian deaths and some population declines (Berger et al 1998, Lips 1999, Bosch et al 2000, Bradley et al 2002). The spherical thalli of B. dendrobatidis live within keratinized epidermal cells of amphibians (Pessier et al 1999). Inoculation with B. dendrobatidis led to the death of poison dart frogs in a test of Koch's postulates (Nichols et al 2001); however, the direct cause of death from chytridiomycosis is uncertain (Berger et al 1998). Not all amphibians develop chytridiomycosis or die when experimentally or naturally infected with B. dendrobatidis (P. Daszak, pers comm). Environmental factors also may affect the rate of mortality because many deaths from chytridiomycosis occur during the cooler time of the year at a given location, or in populations in cool, high-altitude regions (Berger et al 1998, Lips 1998, 1999, Bosch et al 2000, Bradley et al 2002). The pH of the aquatic environment also has been suggested as a possible cofactor in the development of chytridiomycosis (Berger et al 1998, Bosch et al 2000).
Chytridiomycosis is considered an emerging infectious disease (Daszak et al 2000). The effects of B. dendrobatidis on some populations of amphibians have been devastating, and herpetologists, ecologists and epidemiologists are investigating its role in amphibian declines. Longcore et al (1999) reported on the morphology and development in pure culture and the zoospore ultrastructure of B. dendrobatidis, but additional physiological information about the fungus is needed. Herein we present data on the effects of temperature, pH, nutrient preferences, zoospore longevity, swimming ability and enzyme production for B. dendrobatidis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Four (10 cm diam) 0.5% tryptone-agar plates were flooded with 3 mL of sterilized pond water. One drop of a zoospore suspension (
65 000 zoospores) was added to one side of each plate. The control plate was dried immediately and incubated. The other plates were covered and left in the hood 24 h. After 24 h the plates were dried and incubated at 23 C for 1 wk. After 1 wk, plates were photographed, and the distances of the growing colonies from the site of the initial drop were measured.
Inoculation of cultures and measurement of growth – All cultures were grown in 30 mL of the appropriate liquid medium in 50 mL screw-top, Corning polypropylene centrifuge tubes (Corning, New York). Inoculum was 1 mL of a 2-wk-old liquid culture standardized with distilled water to an optical density of 0.100 or 0.050 at 495 nm. Growth was measured by absorbance at 495 nm of 1 mL of a gently shaken culture. Growth was measured at the end of the incubation periods unless otherwise noted. Cultures for all experiments were screened microscopically to check for live zoospores and contamination. In all growth experiments, each treatment consisted of four replicates and each experiment was repeated at least once.
Temperature experiments – Batrachochytrium dendrobatidis cultures in TG medium were incubated at 10, 17, 23, 25 and 28 C. Beginning on day 0 and every 3 d thereafter for 3 wk, four cultures per isolate were removed and growth was measured as above. Cultures also were grown at 4 C for 6 mo. Four cultures per isolate were removed and measured monthly for their growth.
Experiments were designed to examine the effect of exposure of B. dendrobatidis to 30 C. After inoculation, all cultures were incubated at 23 C for 4 d to establish actively growing colonies. Growth then was measured, and half of the culture tubes were transferred to 30 C; the rest were kept at 23 C as controls. Four replicates were measured for each temperature treatment at 2, 4, 6 and 8 d after transfer. The condition of the cultures was evaluated microscopically and by inoculating TGhL plates with 1 mL of culture from each replicate and incubating the plates at 23 C for 6–10 d to observe colony growth.
Effect of pH – Preliminary experiments indicated that several buffers affected the growth of B. dendrobatidis. Growth of the chytrid in unbuffered TG liquid medium changed the pH of the medium less than 0.5 pH units after 2 wk of incubation. We used unbuffered media in pH experiments adjusted with 1 N HCl or 1 M NaOH to the required pH and unadjusted TG medium (pH 6.8–7.0) served as the control. We tested growth at pH 4.0, 5.0, 6.0, 7.0 and 8.0. Inoculum was 1 mL of culture standardized to an optical density of 0.050 absorbance units at 495 nm. Cultures were incubated 2 wk at 23 C and shaken once.
Nitrogen and carbon sources – Isolate 274 of B. dendrobatidis (from Colorado) was used in experiments to test the effect of nitrogen source on growth. This isolate had not been growing on artificial media as long as isolates 197 or 215 and therefore was expected to be the least adapted to tryptone of the three isolates. Each treatment contained 0.3% glucose and 1% of one of these nitrogen sources: asparagine, gelatin hydrolysate (Sigma), yeast extract (Difco), peptonized milk (BBl or Oxoid), malt extract (Difco), peptone (Difco) or tryptone. The control was 0.3% glucose with no added nitrogen source. Isolate 274 also was used to test the effect of the addition of carbohydrates to tryptone medium on growth. Each treatment consisted of 1% tryptone plus 0.3% of one of the following: sucrose, maltose, sorbitol, mannose, glucose, glycerol or lactose. The control was 1% tryptone with no added sugars. To determine the effect of different concentrations of glucose on growth, we incubated isolate 274 with 1% tryptone and 0, 0.15, 0.3, 0.9, 1.8 or 3.6% glucose. The effect of tryptone concentration on growth also was tested on 0.3% glucose medium supplemented with 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0 or 3.0% tryptone. Growth of the cultures was measured after incubation for 2 wk at 23 C.
Isolate 197 of Batrachochytrium was inoculated onto 1% keratin agar (from scleroproteins, ICN, Costa Mesa, California) and into snakeskin medium (1 g macerated snakeskin in 75 mL ddH2O) to test its ability to grow on complex protein sources. To examine the salt tolerances of B. dendrobatidis, isolate 197 was grown in 1% tryptone liquid medium with 0.5 or 1% NaCl for 2 wk at 23 C.
Protease production –
Evidence for protease activity was determined initially by inoculating protein-substrate agar plates. We inoculated TGhL plates with 1 mL of liquid culture of isolate 197, dried them in the laminar flow hood 1 h and then incubated them at 23 C for 4–6 d, until dense growth and live zoospores were visible. Ten mm diam plugs containing colonies from these plates were placed culture-side down on protease assay plates, which contained 1% agarose and either 1% skim milk (Carnation) or 1% gelatin (Sigma). Each assay plate received four plugs: three with B. dendrobatidis colonies and one uninoculated control. Assay plates were incubated at 23 C for 2–4 d. Clear zones in the medium surrounding the plugs from the breakdown of proteins indicated protease activity (Karaup et al 1994).
Further tests for protease activity used culture supernatants of isolate 197 grown in 75 mL of 1% skim milk powder in water inoculated with 1 mL of a 2-wk-old liquid culture. Cultures were incubated 2–4 d at 23 C, until the skim milk became clear. Cultures were centrifuged at 6000 rpm for 20 min to remove cells, and the supernatant was frozen at -80 C. Samples were freeze-dried, resuspended to one-tenth of their original volume in 50 mM Tris-HCl (pH 7.0) and dialyzed against membranes (5 KDa pores) at 6 C in three changes of buffer (buffer volume greater than 20 times the volume of the samples). After dialysis, the 10x concentrated supernatants were stored in 1.5 mL aliquots at -20 C until used in the following tests to measure the temperature and pH response of the extracellular proteases.
Temperature and pH response of extracellular proteases – The pH and temperature ranges of extracellular proteases were measured with an azo dye bonded to casein (Sigma). The reaction mixture for measuring the temperature range consisted of 500 µL of 0.2 M CaCl2 and 500 µL of 5% azocasein in 50 mM Tris HCl (pH 7.5) in 1.7 mL microfuge tubes. To the reactions, 200 µL of water (as control) or 10x concentrated supernatant were added, and reactions were incubated 36 h at 6, 15, 23, 30 or 37 C. Reactions were stopped with 5% trichloro acetic acid (TCA) and centrifuged at 11 000 rpm for 2 min to remove precipitated proteins. Absorbance of the supernatant was measured at 440 nm. Reactions with distilled water served as the blank, and boiled culture supernatant served as the control. Each temperature treatment consisted of three replicates with unaltered supernatant, three with boiled supernatant and three with water. The pH range of extracellular proteases was measured in reaction mixtures of 1 mL 5% azocasein adjusted to pH 6, 6.5, 7, 7.5 or 8 with Tris-HCl buffer and 200 µL of 10x concentrated supernatant. Reactions were incubated 36 h at 23 C then stopped with 200 µL of 5% TCA and measured as above.
Statistical analysis of data – The Kruskal-Wallis rank test was used to detect significant differences. Differences between isolates or between treatments for an isolate in temperature experiments were tested at two points—during logarithmic growth and during stationary growth phase. Differences were considered significant if P < 0.050.
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RESULTS |
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Temperature effects on growth – Preliminary observations indicated that colonies of B. dendrobatidis on TGhL agar could grow for up to 5 mo at approximately 5 C. In experiments to test the growth of B. dendrobatidis at low temperature, isolates 197, 215 and 274 were alive and producing zoospores after 6 mo of incubation at 4 C (Fig. 1). In both repetitions of the experiment, isolate 215 grew significantly less than the other isolates after 6 mo at 4 C.
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Effect of pH on growth – In all experiments, isolates 197, 215 and 274 grew most at pH 6–7, with less growth at pH 8 and minimal growth at pH 4 and 5 (Fig. 3). Isolate 215 grew less at pH 6–7 than the other two isolates. At the end of the experiment, swimming zoospores were present in all cultures grown at all pH.
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), dilute salts solution (Fuller and Jaworski 1987) and yeast nitrogen base plus 1% glucose and thiamine (Sigma), did not support growth of B. dendrobatidis. Therefore, more complex media were used to determine the ability of isolate 274 to grow on different nitrogen and carbon sources. Nitrogen sources strongly influenced the growth of B. dendrobatidis (Fig. 4). The chytrid grew most in 1% tryptone in distilled water and second best in 1% peptonized milk; however, the growth was significantly less than that on tryptone. All other media supported less growth than the control, which contained 0.3% glucose, plus any nutrients that were transferred with the inoculum. Malt extract, yeast extract and asparagine supported trace amounts of growth. After 2 wk of incubation, live thalli were found in all tested media except asparagine and all except asparagine and gelatin hydrolysate media contained motile zoospores. Different carbon sources added to liquid medium with 1% tryptone did not increase the growth of B. dendrobatidis as compared to the control, which contained 1% tryptone. B. dendrobatidis grew less on glycerol than on other added carbon sources. All cultures contained live zoospores and thalli at the end of the experiments.
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Isolates of B. dendrobatidis grew and produced zoospores on 1% keratin agar and in snakeskin liquid medium after 1 wk. Growth in snakeskin medium was sparse compared to growth in TG medium. Colonies were flatter and larger in diameter when grown on keratin agar than on TGhL medium. The fungus grew and formed motile zoospores in media supplemented with 0.5% NaCl and grew slowly in media containing 1% NaCl. Growth in media containing 0.5% NaCl was less robust than in 1% tryptone alone.
Production of extracellular enzymes – After 2–4 d of incubation, distinct clear zones were visible around each plug containing B. dendrobatidis colonies on skim milk and gelatin assay plates, indicating the production of extracellular proteases (Fig. 5). Control plugs were devoid of activity. Skim milk assay plates supported the growth of numerous thalli and the production of zoospores.
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DISCUSSION |
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); however, zoospore longevity in pure culture may differ from that in the presence of bacteria, ingestive protists and other microbiota found in the environment.
In still water, zoospores of B. dendrobatidis swam less than 2 cm before they encysted, suggesting that zoospores are unable to swim long distances to find a host. On the skin of moderately infected amphibians, thalli are in clusters of infected skin cells (Longcore, unpubl obs) rather than being spread more evenly over the surface of the skin. This distribution probably develops because many zoospores encyst and infect cells in the immediate area from which they were released. Batrachochytrium dendrobatidis may spread from amphibian to amphibian by close or direct contact during mating, schooling of larvae or other aggregative behaviors. Zoospores could be spread longer distances if carried in water currents, but this also would decrease the chances of a zoospore contacting a host, because the spores would be diluted to low concentrations. The chance of zoospores finding an amphibian may be increased if the zoospores are attracted to their host, as with some other parasitic chytrids (Sparrow 1960, Held 1974, Muehlstein et al 1988, Deacon and Saxena 1997). We tested tryptone, gelatin hydrolysate, glucose and lactose as potential attractants because they were in media used to culture chytrid; keratin and gelatin were tested because they are similar to components of amphibian skin. Our experiments did not reveal evidence of chemotaxis to the tested compounds (Piotrowski 2002). However, we did not test amphibian skin. If B. dendrobatidis is attracted to amphibian skin or compounds released from it, zoospores may swim farther than our results suggest.
Even though we used inoculum of a standard age and optical density, significant variation was observed in the growth of isolates between repeats of an experiment. We believe this variation was due to an "inoculum effect" caused by the ratio of zoospores to thalli in liquid cultures changing from day to day. Because we measured inoculum by optical density, batches of inoculum were not identical. When liquid medium is inoculated with a portion of a stock culture, it could contain mostly zoospores, mostly thalli or varying proportions of each. This may affect the length of time before cultures achieve logarithmic and stationary phases of growth. Several of the experiments differed among repetitions, which we believe resulted from an inoculum effect, but overall growth trends were similar.
Batrachochytrium dendrobatidis grows within a wide range of temperatures (4–25 C) and grows optimally at 17–25 C. This wide range of permissive and optimum temperatures should let this pathogen persist in many environments. The ability to persist and even grow slowly at 4 C would let B. dendrobatidis overwinter in its hosts, even in mid-latitude, temperate climates where temperatures of the aquatic environments are low. As temperatures rise in the environment, the chytrid then may reproduce rapidly, as it did when cultures were transferred to 23 C after incubation at 4 C.
Batrachochytrium dendrobatidis does not grow well above 25 C, and higher temperatures do not favor epidemics (Berger et al 1998, Bosch et al 2000). Unless some isolates have different temperature constraints, outbreaks of chytridiomycosis in the tropics probably will be limited to cooler areas, as has been observed in Australia and Panama (Berger et al 1998, Lips 1998). In temperate zones, outbreaks could occur in montane areas in warmer months (Bosch et al 2000) or lowlands during the winter (Bradley et al 2002).
At temperatures of 28 C or above, or below 10 C, B. dendrobatidis does not grow or grows slowly; infections at these temperatures may not be fatal because growth of the fungus is not favored. Pure cultures that did not grow at 28 C revived when returned to optimal temperatures (Longcore et al 1999), and the same may happen when B. dendrobatidis is within skin cells. Although exposure to 30 C killed cultures of B. dendrobatidis, half the replicates still were alive even after 8 d at 30 C. If a species of amphibians can survive elevated temperatures, exposure to temperatures above 30 C for more than 8 d may be an effective treatment for chytridiomycosis if the fungus is not protected from this temperature extreme by being within skin cells.
Outbreaks of chytridiomycosis may be affected by pH, but the pH optimum (pH 6–7) for B. dendrobatidis is not outside common pHs of freshwater systems. Although the fungus grows poorly below pH 6, its zoospores can live at that pH, and once inside the host, the fungus may be buffered from external conditions. It is not surprising that all the isolates have similar physiological requirements; different genera of chytrids, even in different orders, have similar temperature and pH tolerances as B. dendrobatidis (Barr 1969, 1970a, b).
Nitrogen source has a strong effect on the growth of B. dendrobatidis. This may be a result of the micronutrient, carbon and nitrogen levels of the nitrogen sources tested. B. dendrobatidis grew more on tryptone than on peptone, both of which are digests of casein protein with similar amino nitrogen content, total nitrogen content and carbohydrate content (product information from BD, Franklin Lakes, New Jersey). However, they differ in that peptone has only 0.1 µg/g thiamine compared to 0.4 µg/g for tryptone. It is not unusual for chytrids to require exogenous thiamine (Barr 1969, 1970a, b), and this difference between the two media may account for differences in growth. The chytrid grew almost as much on peptonized milk as on tryptone. Even though both are digests of casein, peptonized milk has less than half the total nitrogen of tryptone (product information from Oxoid Ltd., Hampshire, England).
The pH and amount of carbohydrates in the different nitrogen sources may have had an effect on growth. Gelatin hydrolysate (pH 5.7) and malt-extract (pH 5.6) media have pH slightly below the growth optimum for B. dendrobatidis. The high sugar content of malt extract (60 to 63% reducing sugars) and yeast extract (17.5% carbohydrate) (product information from BD, Franklin Lakes, New Jersey) compared to tryptone (7.7%) may explain the lower growth in these liquid media. The high level of nitrogen and low pH (4.5) or the limited nutrient complexity of asparagine medium could explain the poor growth in this medium. Although growth was sparse in media other than tryptone and peptonized milk, live zoospores were present in all media except asparagine and gelatin hydrolysate, suggesting that the chytrid can grow, but not thrive, on many different nitrogen sources. We suggest 0.5 or 1% tryptone liquid or solid (1% agar) medium for culturing B. dendrobatidis.
The results from the carbon/nitrogen ratio experiments suggest that B. dendrobatidis does not require sugars other than those in tryptone and that high percentages of sugar or tryptone (greater than 2%) hinder growth. Although B. dendrobatidis grew on snakeskin and keratin media, we cannot conclude that it was using the keratin or producing a keratinase, because some of the keratin might have been degraded by autoclaving.
Batrachochytrium dendrobatidis produces extracellular proteases that degraded casein and gelatin but did not degrade keratin azure. However, many types of keratin exist and the form of keratin in the keratin azure might be more resistant to the chytrid's protease attack than the keratin in amphibian skin. Although the chytrid is found only in the keratinized cells of amphibians, it is uncertain if it actually degrades the keratin. It is possible that B. dendrobatidis is found in keratinized epidermal cells because these cells are dead and easier to invade.
Isolate 197 was the only isolate studied for enzyme production. Preliminary experiments, however, showed that isolates 215 and 274 also produced casein-degrading proteases. Different isolates may produce different levels or types of proteases, and the differences may make some isolates more virulent. The temperature and pH ranges of the enzymes are similar to the temperature and pH optima for growth of B. dendrobatidis on defined media. The proteases produced by B. dendrobatidis may be nonspecific because they can degrade skim milk proteins, gelatin and snakeskin. This might let the chytrid survive saprobically on protein substrates in the environment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication March 31, 2003.
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LITERATURE CITED |
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———. 1970a Phlyctochytrium reinboldtae (Chytridiales): morphology and physiology. Can J Bot 48:479-484
———. 1970b Phlyctochytrium arcticum n. sp. (Chytridiales): morphology and physiology. Can J Bot 48:2279-2283
Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, Slocombe R, Ragan MA, Hyatt AD, McDonald KR, Hines HB, Lips KR, Marantelli G, Parkes H., 1998 Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc Natl Acad Sci USA 95:9031-9036
Bosch J, Martinez-Solano I, Garcia-Paris M., 2000 Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (Alytes obstetricans) in protected areas of central Spain. Biol Conserv 97:331-337
Bradley GA, Rosen PC, Sredl MJ, Jones TR, Longcore JE., 2002 Chytridiomycosis in native Arizona frogs. J Wildl Dis 38:206-212[Abstract]
Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE, Speare R., 2000 Emerging infectious diseases and amphibian population declines. Emerging Infectious Diseases 5:1-25
Deacon JW, Saxena G., 1997 Orientated zoospore attachment and cyst germination in Catenaria anguillilae, a facultative endoparasite of nematodes. Mycol Res 101:513-522
Fuller MS., 1986 The flagellated fungal spore. In: Sutton BC, ed. A century of mycology. Cambridge, UK: Cambridge University Press. p 161–192
———, Jaworski A., 1987 Zoosporic fungi in teaching and research. Athens, Georgia: Southeastern Publishing Co. p 294
Held AA., 1974 Attraction and attachment of zoospores of the parasitic chytrid Rozella allomycis in response to host-dependent factors. Arch Microbiol 95:97-114
Karaup T, Olson LW, Heldt-Hansen HP., 1994 Some characteristics of extracellular proteases produced by members of the Chytridiales and the Spizellomycetales (Chytridiomycetes). Can J Microbiol 40:106-112
Lips K., 1998 Decline of a tropical montane amphibian fauna. Conserv Biol 12:106-117
———. 1999 Mass mortality and population declines of anurans at an upland site in western Panama. Conserv Biol 13:117-125
Longcore JE, Pessier AP, Nichols DK., 1999 Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 91:219-227
Muehlstein LK, Amon JP, Leffler D., 1988 Chemotaxis in the marine fungus Rhizophydium littoreum. Appl Environ Microbiol 54:1668-1672
Nichols DK, Lamirande EW, Pessier AP, Longcore JE., 2001 Experimental transmission of chytridiomycosis in dendrobatid frogs. J Wildl Dis 37:1-11[Abstract]
Pessier AP, Nichols DK, Longcore JE, Fuller MS., 1999 Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frog (Litoria caerulea). J Vet Diag Invest 11:194-199
Piotrowski JS., 2002 Physiology, enzyme production, and zoospore behavior of Batrachochytrium dendrobatidis, a chytrid pathogenic to amphibians [master's thesis]. Orono, Maine: University of Maine
Sparrow FK., 1960 Aquatic Phycomycetes. 2nd ed. Ann Arbor: University of Michigan Press. p 65–71
Stevens RB., 1974 Mycology guidebook. Seattle, WA: University of Washington Press
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