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Tracking down the killer chytrid of amphibians

Posted on: Thursday, 7 August 2003, 06:00 CDT

Most species of fungi in the Phylum Chytridiomycota (chytrids) are saprophytic decomposers of organic matter. Some are known to be parasites of other fungi, plants, or invertebrate animals. Until recently, no chytrids had ever been recognized as pathogens in vertebrate animals. However, one chytrid species, Batrachochytrium dendrobatidis, has now been shown to cause fatal skin disease in frogs and toads. This disease has had devastating impacts on captive collections of amphibians and is believed to be a significant factor in the declines of many wild anuran populations. The following is an account of the role I played in the discovery of this novel amphibian pathogen.

After completing a residency in veterinary pathology at the National Zoological Park (NZP) in 1987, I accepted a position as a staff pathologist at the National Institutes of Health (NIH) in Bethesda, Maryland. My work at NIH mostly involved rodents and other standard laboratory animals. However, my primary professional interests continued to be the diseases of exotic animals and wildlife, especially reptiles and amphibians. Therefore, while employed at NIH, I also had a part-time consulting pathology service for several zoos and other institutions that housed non-domestic animals.

It was as a consulting pathologist that I received three dead formalin-fixed California Arroyo Toads (Bufo microscaphus californicus) in early November 1991. These animals had been part of a captive colony consisting of approximately 120 animals kept at the University of California-Santa Barbara. A disease outbreak of unknown cause had already led to the deaths of approximately 60% of the toads in this colony during the two months before the carcasses were sent to me. On histologic examination of all major organs from these toads, I discovered that all three had a skin disease characterized by moderate to marked thickening of the epidermis, especially the outer keratinized layers (Figs. 1A-B). Within these lesions (primarily in the keratinized layers) were numerous microscopic single-cell eukaryotic organisms unlike anything I had ever seen before (Figs. 1B, 2). Other organs from the toads did not contain any significant lesions. Therefore, I concluded that the skin disease was responsible for the deaths of the toads by interfering with normal skin function, which in amphibians includes maintenance of hydration and electrolyte bal-ance, thermoregulation, and even respiration to varying extents. I also concluded that the microscopic organisms were most likely the cause of the skin lesions.

FIG. 1A (top). Photomicrograph of normal skin from a California Arroyo Toad (Bufo microscaphus californicus). The epidermis (between arrows) is three to four cell layers thick and only the outermost layer is keratinized. A circular mucous gland (m) is located in the dermis just beneath the epidermis. Magnification is 200x. 1B (bottom). Photomicrograph of skin from an affected California Arroyo Toad. The epidermis is diffusely thickened, especially the keratinized layers (between arrowheads). Within the keratinized layers are numerous variably-sized round nucleated organisms. Two mucous glands (m) and one serous gland (S) are located beneath the epidermis. Magnification is 120x.

I then began a quest to determine exactly what those microorganisms were; it would eventually take more than five years to satisfy my curiosity. As the first step in this process, I performed a thorough search of the published literature and I did not find any reports of similar organisms affecting the skin of amphibians or any other animal.

My initial impression from examining standard histology slides of the toad skins was that the organisms most closely resembled sporozoan-type protozoa. Therefore, I next showed the slides to an expert on protozoal parasites, Dr. Chris Gardiner, who was stationed at the Armed Forces Institute of Pathology (AFIP) located in the Walter Reed Army Medical Complex in Washington, DC. Following his suggestions, I had additional histology slides prepared and stained with several different "special stains" (e.g., Gram, acid-fast, and periodic acid-Schiff). We also examined the organisms using transmission electron microscopy (TEM). After examining all this material, Dr. Gardiner concluded that the organisms definitely were not protozoa; he suggested that they were probably algae or fungi.

I then showed the histology slides and photographs from the TEM examinations to Dr. Maria Faust. She is an expert on algae at the Smithsonian's National Museum of Natural History. Her conclusion was that the organisms definitely were not algae, but were probably fungi or protozoa.

At this point, I concluded that the organisms must be some type of fungi. However, their morphology and staining characteristics in the special stains were unlike any fungal pathogens commonly encountered in human or veterinary medicine. To try to determine what kind of fungi they were, I next traveled to the National Library of Medicine located on the NIH campus to consult textbooks on the morphology of fungi by TEM. Unfortunately, the samples that I had from the arroyo toads had undergone some post-mortem decomposition and had been fixed in formalin, which is not an ideal fixative solution for TEM. Thus, these samples had many artifacts in them that interfered with interpretation. Also unfortunate was the fact that entire arroyo toad colony had died by this time, making it impossible to collect fresh samples and fix them in a proper TEM fixative.

The TEM examinations did clearly demonstrate that at one stage of development, the organisms consisted of cystic structures containing flagellated spore-like forms (Fig. 3). However, no types of fungi had flagellated spores in the TEM textbooks that I consulted; much later I realized that the fact that all of these books were written in the mid-to-late 1980's was very important (see footnote below).

At this point, I still had no idea what the organisms were; I could only say what they were not: fungi, algae, protozoa, animals, plants, or bacteria. Because I could not obtain further samples for study, I decided to give up trying to identify them for a while.

In the fall of 1991, I accepted a staff position as Associate Pathologist and returned to NZP. Shortly thereafter, I performed a retrospective search of the pathology files at NZP to see if any similar organisms had been seen in amphibians from the zoo's collection. My search revealed three cases of skin disease in frogs associated with organisms that closely resembled those in the ar- royo toads: a White's Tree Frog (Litoria caerulea) that died in 1988, an Ornate Horned Frog (Ceratophrys ornata) that died in January 1990, and another L. caerulea that died in September 1990. I then retrieved archived tissue samples from these cases and had the same special stains done on them as were done before; results were the same as with the arroyo toad samples. The results of TEM were also similar to before, including the artifacts that prevented definitive identification of the organisms.

FIG. 2. Higher magnification of Fig. 1B demonstrating three forms of the organisms in the epidermis at the junction between the non- keratinized and keratinized layers. One form has multiple nuclei separated by lacey cytoplasm (solid arrow). Multiple spore-like forms are present in a cystic structure; this cyst has a flat papilla (arrowhead) through which the spores are released. A clump of discharged spores (open arrow) is also present. Magnification is 480x.

Because this disease had occurred in NZP collection animals on at least three separate occasions, I reasoned that it might occur again. Over the next several years, whenever an amphibian was presented for necropsy, I instructed the pathology residents to collect numerous samples of skin for histologic examination. If I was asked why, my reply was "Because I am looking for something and I don't really know what it is."

By the spring of 1996, I had just about given up on finding the organisms again. Therefore, I decided that I would present the information that I had on this new amphibian disease at the annual conference of the American Association of Zoo Veterinarians (AAZV) that year. While preparing my abstract for the meeting, I stumbled across a textbook titled Handbook of Protoctista (Margulis et al. 1990). This 900+ page book contains detailed descriptions (including photographs of morphology by TEM) for each group of organisms in the Kingdom Protoctista (aka Kingdom Protista).

In this book were several phyla of aquatic organisms that produce flagellated spores and were originally classified as fungi; these so- called "zoosporic fungi" consisted of the Phyla Chytridiomycota, Hyphochytridiomycota, Oomycota, Labyrinthulomycota, and Plasmodiophoromycota. The morphology of the amphibian pathogens resembled those of these "zoosporic fungi" closely enough that I felt confident in calling them "aquatic fungal-like protists" in the paper that I presented at the 1996 AAZV meeting (Nichols et al. 1996). However, the arti-facts in the TEM material from the organisms still prevented determination of which "zoosporic fungi" they were.

The event that finally allowed me to determine exactly what these organisms are came in the Fall of 1996. In September that year, an outbreak of the skin disease began in the NZP collection animals. Over the next seve\ral months, this outbreak resulted in the deaths of 24 juvenile Blue Poison Dart Frogs (Dendrobates azureus), 4 Green- and-Black Poison Dart Frogs (D. auratus), 3 White's Tree Frogs, and 1 Ornate Horned Frog. Once we realized that there was an ongoing outbreak, we were able to collect skin samples from freshly dead frogs and place them in a proper fixative solution for TEM. Morphology of these organisms by TEM closely resembled those in the Phylum Chytridiomycota.

Dr. Allan Pessier, our pathology resident at the time, then ran an Internet search and found the webpage of one of the few chytrid experts in the world, Dr. Joyce Longcore at the University of Maine. We sent her photographs of our TEM findings and she confirmed that the morphology of the organisms was consistent with a chytrid species (Pessier et al. 1999). We then sent her fresh skin samples from an affected Dendrobates azureus and she was able to isolate the organisms into culture. She also isolated the identical organism from an infected D. auratus, and later from a Litoria caerulea. Subsequent studies of the organisms by Longcore confirmed that not only was this a new chytrid species but it represented a novel genus as well. We eventually named this organism Batrachochytrium dendrobatidis, which has its roots in Greek and translates roughly as "frog chytrid of dendrobatids" (Longcore et al. 1999).

While Allan, Joyce, and I were characterizing this organism and the disease it causes, I received word in 1997 that other groups had seen something very similar associated with marked declines in wild populations of anurans in Australia and Central America. Drs. Rick Speare, Alex Hyatt, and Lee Berger headed the group in Australia and the group working on the disease in Central America consisted of Drs. Karen Lips, Peter Daszak, Andrew Cunningham, and David E. Green. My correspondence with these groups revealed that they were indeed seeing an identical disease and this was associated with organisms that appeared to be very similar to the chytrids we were studying. Although these groups had not been able to culture the organisms, they had concluded that the organisms were chytrids through the use of TEM and molecular genetics (Berger et al. 1998). Suddenly the interesting disease that I had been seeing in captive amphibians had taken on much more importance!

FIG. 3. Transmission electron photomicrograph of the epidermis from an affected California Arroyo Toad. Within one epidermal cell, there is a thick-walled cyst-like structure with an undulating interior border. Three round spores are present within the cyst (the cell membranes of two of these spores overlap). Flagella (arrows) are present, indicating that the spore stages of the organisms are aquatic. Magnification is 27,308x.

Cutaneous chytridiomycosis appears to be an emergent fatal disease of both captive and wild amphibians. Many now consider it to be one of the leading causes for the global declines of wild amphibian populations (Berger et al. 1998; Bosch et al. 2001; Daszak et al. 1999; Lips 1999; Ron and Merino 2000). Within the last six years, this disease has been recognized as the cause of death of captive and/or wild amphibians in Australia, New Zealand, North America, Central America, South America, Africa, and Europe (Speare and Berger 2002). The reasons for the apparent sudden appearance of this disease in so many areas of the world remain subjects for conjecture and ongoing research.

Joyce Longcore has more than 70 isolates of chytrids from various outbreaks of amphibian chytridiomycosis and from multiple amphibian species. So far, all of these have been Batrachochytrium dendrobatidls, indicating that this single chytrid species is responsible for causing the disease worldwide. Molecular studies are underway to determine how much genetic drift has occurred among the different isolates in an effort to establish when the organisms might have been dispersed and their geographical origin. Other researchers are looking at preserved amphibian specimens in museum archives to determine when chytrids first appeared in various amphibian populations. Efforts to develop sensitive tests for detecting chytrid infections in amphibians are also underway.

Early reports that cutaneous chytrid infections were fatal to amphibians and were a cause for declines in wild populations were met with some skepticism. In response to this, we conducted several experiments in our lab at NZP to prove the pathogenicity of the chytrids. We have consistently been able to infect recently metamorphosed dendrobatid frogs of three different species and these infections have resulted in 100% mortality (Nichols et al. 2001 ; Nichols and Lamirande, unpubl. data).

In other studies, we have shown that dendrobatid tadpoles can be infected and remain asymptomatic for as long as 87 days, but then 100% of them die at the last stages of metamorphosis or shortly thereafter (Lamirande and Nichols 2002). We have also developed treatment protocols that can be used to cure chytrid infections in captive frogs (Nichols and Lamirande 2000) and tadpoles (Nichols and Lamirande, unpubl. data).

Much remains to be discovered about Batrachochytrium dendrobatidis and the factors that influence the development of chytridiomycosis in amphibians. Other chytrid-related research that I hope to be able to conduct someday includes looking at the effects of temperature on the disease, determining how long the chytrids can survive in environments without the presence of amphibian hosts, and determining exactly what causes chytrid-infected amphibians to die.

Footnote.-Organisms in the Phylum Chytridiomycota were originally classified as unique types of fungi. In the early 1980s, taxonomists agreed that these organisms, along with the other phyla of "zoosporic fungi" should be removed from the Kingdom Fungi and placed in the Kingdom Protista. Molecular DNA studies conducted in the late 1980s and early 1990s resulted in the Phylum Chytridiomycota being placed once again in with the fungi. The other phyla of "zoosporic fungi" remain classified as organisms other than fungi.

In 1991-92, when I was looking for information on fungal morphology by TEM, I consulted textbooks published in the mid-to- late 1980s. Chytrids were not included because they were not considered to be fungi at the time the books were written. Ironically, if I had used older "out-of-date" texts on fungi, chytrids would have been included, and thus I might have saved myself five years of frustration in trying to identify the organisms.

Herpetological Review, 2003, 34(2), 101-104.

(C) 2003 by Society for the Study of Amphibians and Reptiles

SELECTED REFERENCES

BERGER, L., R. SPEARE, P. DASZAK, D. E. GREEN, A. A.CUNNINGHAM, C. L. GOGGIN, R. SLOCOMBE, M. A. RAGAN, D. HYATT, K. R. MCDONALD, H. B. HINES, K. R. LIPS, G. MARANTELLI, AND H. PARKES. 1998. Chytridiomyeosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Sciences 95:9031- 9036.

BOSCH, J., I. MARTINEZ-SOLANO, AND M. GARCIA-PARIS. 2001. Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (Alytes obstetricans) in protected areas of central Spain. Biological Conservation 97:331-337.

DASZAK, P., L. BERGER, A. A. CUNNINGHAM, A. D. HYATT, D. E. GREEN, AND R. SPEARE. 1999. Emerging infectious diseases and amphibian population declines. Emerging Infectious Diseases 5(6):735- 748.

LAMIRANDE, E. W. AND D. K. NICHOLS. 2002. Effects of host age on susceptibility to cutaneous chytridiomycosis in blue-and-yellow poison dart frogs (Dendrobates tinctorius). Pp. 3-16 In Proceedings: Sixth International Symposium on the Pathology of Reptiles and Amphibians, University of Minnesota Printing Services, St. Paul, Minnesota.

LIPS, K. R. 1999. Mass mortality and population declines of anurans at an upland site in western Panama. Conservation Biology 13(1):117-125.

LONGCORE, J. E., A. P. PESSIER, AND D. K. NICHOLS. 1999. Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 91:219-227.

MARGULIS, L., J. O. CORLIS, M. MELKONIAN, AND D. J. CHAPMAN (EDS.). 1990. Handbook of Protoctista: The Structure, Cultivation, Habitats and Life Histories of Eukaryotic Microorganisms and Descendants Exclusive of Animals, Plants and Fungi. Jones & Bartlett, Boston, Massachusetts.

NICHOLS, D. K., AND E. W. LAMIRANDE. 2000. Treatment of cutaneous chytridiomycosis in blue-and-yellow poison dart frogs (Dendrobates tinctorius). Pp. 51 In Proceedings: Getting the Jump! on Amphibian Disease, Cairns, Australia, 26-30 August 2000.

_____,_____, A. P. PESSIER, AND J. E. LONGCORE. 2001. Experimental transmission of cutaneous chytridiomycosis in dendrobatid frogs. Journal of Wildlife Diseases 37(1): 1-11.

_____, A. J. SMITH, AND C. H. GARDINER. 1996. Dermatitis of anurans caused by fungal-like protists. Pp. 220-222 In Proceedings: American Association of Zoo Veterinarians, Puerto Vallarta, Mexico, 3-8 November 1996.

PESSIER, A. P., D. K. NICHOLS, J. E. LONGCORE, AND M. S. FULLER. 1999. Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea). Journal of Veterinary Diagnostic Investigation 11:194-199.

RON, S. R., ANDA. MERINO. 2000. Amphibian declines in Ecuador: overview and first report of chytridiomycosis from South America. Froglog 42:2-3.

SPEARE, R., AND L. BERGER. 2002. Global distribution of chytridiomycosis in amphibians. World Wide Web - http:// www.jcu.edu.au/school/phtm/ PHTM/frogs/chyglob.htm.

DONALD K. NICHOLS, DVM DACVP

Department of Pathology, National Zoological Park 3001 Connecticut Avenue, NW, Washington, DC 20008, USA

e-mail: nicholsd@nzp.si.edu

Copyright Society for the Study of Amphibians and Reptiles Jun 2003

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