Batrachochytrium dendrobatidis

Introduction

Figure 1. Chytrid fungus, Batrachochytrium dendrobatidis.⁴

Fungal disease has emerged as an increasing threat to a number of different organisms, especially amphibian species. Batrachochytrium dendrobatidis (Figure 1), a Chytridiomycota fungus that causes the infectious disease chytridiomycosis has been found to be the major cause of amphibian death caused by fungal infection. B. dendrobatidis infects the superficial, keratin-containing layers of amphibian skin⁹. The infection then spreads across the skin, causing it to thicken and slough off. This thickening interferes with osmoregulation and eventually leads to death¹. It has been found to affect at least 93 amphibian species in frogs, toads (Anura) and salamanders (Caudata)². Evidence of B. dendrobatidis has been found worldwide and is believed to have originated in Africa and spread through the trade of Xenopus laevis³.

B. dendrobatidis has been responsible for the epidemic illness and death of amphibian populations in Australia, New Zealand, Europe, Central America, and the United States⁴. It has been found to affect at least 93 amphibian species in frogs, toads (Anura) and salamanders (Caudata)². Evidence of B. dendrobatidis has been found worldwide and is believed to have originated in Africa and spread through the trade of Xenopus laevis³. B. dendrobatidis has been responsible for the epidemic illness and death of amphibian populations in Australia, New Zealand, Europe, Central America, and the United States⁴. The spread of B. dendrobatidis has larger environmental implications as well. In a study of the common toad (Bufo bufo) and the midwife toad (Alytes obstetricans) in Peñalara Natural Park it was found that after chytridiomycosis nearly eliminated the midwife toad, the common toad significantly increased its number of breeding sites²⁵. Other studies have found that tree frogs (Hyla chrysoscelis developed slower when reared with B. dendrobatidis, but only when predatory newts (Notophthalmus viridescens) were present ².

Batrachochytrium dendrobatidis

Chytridiomycota

The Chytridiomycota make up a monophyletic group of zoosporic fungi phylogenetically related to the true fungi, in contrast to pseudofungi ¹⁶. There is a single class, Chytridiomycetes, and five orders: Chytridiales, Spizellomycetales, Blastocladiales, Monoblepharidales, and Neocallimastigales ¹⁷. Collectively, the Chytridiomycota are often referred to as chytrids. Chytrid fossils are known from the Devonian Rhynie Chert, which is a 480 milllion year old sedimentary deposit¹⁸. Chytrid fungi are found primarily in soil and water. They are saprobes, which use substrates like detritus and keratin. Some members of the chytrid family are facultative or obligate anaerobes. Many are parasites of other fungi, algae, vascular plants, nematodes, or insects. Many also live in freshwater as aquatic saprobes. Chytrids have mitochondria with flattened cristae, and cell walls composed of glucan and chitin ²⁴.

Physiology

The survival, growth and reproduction of B. dendrobatidis is highly temperature dependent. Temperature has an effect on mortality rates associated with B. dendrobatidis. B. dendrobatidis can grow in 4-25^oC, but grows optimally in 17-25^oC. Nitrogen has a strong effect on the growth of B. dendrobatidis. It is possible that B. dendrobatidis grow in keratinized epidermal cells because they are dead and easier to invade ⁵.
More than 95% of zoospores stopped moving within 24 h and swam less than 2 cm before encysting⁵. B. dendrobatidis cultures grew on autoclaved snakeskin, as well as 1% keratin agar. Cultures grew optimally in tryptone or peptonized milk. No additional sugars were required when grown in tryptone. B. dendrobatidis produces extracellular proteases that degraded casein and gelatin. The proteases were active against azocasein at temperatures between 6–37 ^oC and in a pH range of 6–8. The highest level of activity occurred at temperatures of 23–30 ^oC and at pH 8⁵.

Morphology

B. dendrobatidis is identified by its intracellular flask-shaped sporangia (spore containing bodies) and septate thalli. The fungus produces smooth-walled zoosporangia (zoospore containing bodies), which are generally spherical in shape. Each zoosporangium (5.2 ± 0.72 μm⁶) produces a single discharge tube, which penetrates the skin and releases mature zoospores. The zoospores (3-5 μm in diameter) are oval shaped and have a single posterior flagellum (19–20 μm in length), which allows it to move in water. There is little morphological difference in B. dendrobatidis strains^4,7

Figure 2. Life Cycle of Chytrid Fungus, Batrachochytrium dendrobatidis ¹⁴

Life Cycle

B. dendrobatidis has two stages of it’s life cycle. The first is a motile waterborne zoospore in which it disperses. The second is a stationary monocentric thallus which develops into a single zoosporangia (container for zoospores)⁸. In Figure 2, the sporangium (contents of the zoosporangium) divides into new zoospores that exit the sporangium through one or more papillae. B. dendrobatidis has not been shown to reproduce sexually. It is well adapted to living in the epidermis. Sporangia live inside the superficial epidermis (stratum granulosum and stratum corneum) in amphibians. Their rate of development matches that of the epidermal cells they inhabit as they move outwards and also keratinize⁸. The cells they initially infect are living, but soon grow into dead keratinized cells. The papillae merge with and dissolve the epidermal cell membrane and open onto the surface of the cell facing outwards from the body. The duration of the life cycle in vitro is 4 to 5 at 22°C and due to the similarity in life cycles in vivo and in vitro, duration is assumed to be the same in both⁸. Temperatures higher than 25 °C increase the rate of epidermal turnover and reduce growth of B. dendrobatidis⁵. In warm conditions some frogs may recover from infection¹⁹ because the fungus does not have time to complete its life cycle before being shed with the epidermal layer⁸.

Pathogenicity

Figure 3. Dead Bd-infected Atelopus limosus at Sierra Llorona¹³

Chytridiomycosis

Responses to infection by B. dendrobatidis vary widely in nature and in the lab. Some individuals become infected and recover, others die²⁰ and the time to death varies as well⁴. Variation in host resistance, virulence of the pathogen and the environment all contribute to the range of responses to B. dendrobatidis infection ²¹. B. dendrobatidis also has different effects on the same species at different stages in its life cycle. In the larval stage Anaxyrus boreas is more susceptible than it is in the metamorphic stage²³. In Rana cascadae, the post metamorphic life stages were more susceptible to infection. Symptoms of chytridiomycosis include abnormal posture, loss of righting reflex, lethargy, and rapid progression to death²². Increased epidermal sloughing, epidermal ulceration and reddening of the skin (Figure 3) around the toes and front pelvic area have been reported ⁴.
B. dendrobatidis infects superficial layers of the skin in amphibians which contain keratin. During metamorphosis skin becomes increasingly keratinized, which allows the infection to spread over the organism. In juveniles and adults the digits and pelvic patch of skin involved in osmoregulation are the most effected areas. As the infection continues the skin thickens and sloughs off. The skin in amphibians is intimately tied to regulation of hydration state, which can have compounding negative effects on the organism. The cause of death due to B. dendrobatidis, is cardiac arrest due to improper electrolyte blood levels⁹. Mortality rate and time till death by B. dendrobatidis varies with fungal dose, temperature, age of amphibian, and host species^1,10

Spread

B. dendrobatidis originated in Africa and was transported to other countries through global trade of Xenopus laevis³. Infection has been documented in amphibians from exposure to infected soil or water. B. dendrobatidis can survive in tap water for 3 weeks and deionized water for 4 weeks. In lake water infectivity was observed for 7 weeks after introduction⁴. The bullfrog is the most commonly farmed amphibian and accidental introduction of captive bullfrogs into the wild populations is very common. B. dendrobatidis is asymptomatic in bullfrogs, making it hard to detect without performing tests. B. dendrobatidis has been detected in all introduced species of bullfrogs excluding Japan, and native bullfrogs from Eastern Canada¹¹.

Methods of Control

Figure 4. Table 2 showing the effect of different UV light and Dessication treatments on Batrachochytrium dendrobatidis survival¹² (Reproduced by permission of Inter-Research)

A number of physical and chemical disinfectants can cause 100% mortality in B. dendrobatidis. The fungus is highly sensitive to heat and died within 4 hours at 37^oC. Figure 4, desiccation also proved successful for some of its treatments. UV light exposure did not kill 100% of the B. dendrobatidis for any of the tested exposure times. Many chemical methods proved useful. Ethanol and Virkon both killed 100% of B. dendrobatidis within 30 seconds. Sodium hyperchlorite was useful, as was Didecyldimethylammonium Chloride (DDAC). DDAC is promoted as being environmentally safe and is used in agriculture and in forestry. Dithane and Formaldehyde were not as effective. Sodium chloride and potassium permanganate were also not as effective¹².

Conclusion

The Amphibian Conservation Summit declared amphibian chytridiomycosis, “the worst infectious disease ever recorded among vertebrates in terms of the number of species impacted, and its propensity to drive them to extinction.”¹⁵ Although there are antifungal treatments available for organisms infected with B. dendrobatidis in captivity, there remains no solution to the problem presented by the spread of B. dendrobatidis in the wild. The incredible speed with which chytridiomycosis caused by B. dendrobatidis can damage a population and the widespread prevalence of this fungus adds to the difficulty in attempts to control further infection and aid already infected organisms. Many unique and invaluable amphibians species are at risk from not only this fungus, but from habitat loss, and urgent conservation efforts would benefit worldwide amphibian populations.

References

1. Berger, L., Speare, R., Daszak, P., Green, D. E., Cunningham, A. A., Goggin, C. L., Slocombe, R., Ragan, M. A., Hyatt, A. D., McDonald, K. R., Hines, H. B., Lips, G. M., and Parkes, H. "Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America." Proceedings of the National Academy of Sciences 95.15 (1998): 9031-9036

2. Parris, M. J., and Beaudoin, J. G. "Chytridiomycosis impacts predator-prey interactions in larval amphibian communities." Oecologia 140.4 (2004): 626-632.

3. Webb, R., Mendez, D., Berger, L., and Speare, R "Virulence of the amphibian chytrid fungus Batrachochytrium dendrobatidis varies with the strain." Diseases of Aquatic Organisms 68.1 (2005): 47.

4. Johnson, M. L., and Speare. "Survival of Batrachochytrium dendrobatidis in water: quarantine and disease control implications." Emerging Infectious Diseases 9.8 (2003): 922.

5. Piotrowski, J. S., Annis S. L., and Longcore J. E. "Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians." Mycologia 96.1 (2004): 9-15.

6. Weldon, C., du Preez, L. H., Hyatt, A. D., Muller, R., and Speare, R. "Origin of the amphibian chytrid fungus." Emerging infectious diseases 10.12 (2004): 2100.

7. Longcore, J. E., Pessier A. P., and Nichols, D. K. "Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians." Mycologia (1999): 219-227.

8. Berger, L., Hyatt, A. D., Speare, R., and Longcore, J. E. "Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis." Diseases of aquatic organisms 68 (2005): 51-63

9. Voyles, J., Young, S., Berger, L., Campbell, C., Voyles, W. F., Dinudom, A., Cook, D., Webb, R., Alford, R. A., Skerratt, L. F., and Speare, R. "Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines." Science 326.5952 (2009): 582-585.

10. Rachowicz, L. J., and Vredenburg, V. T. "Transmission of Batrachochytrium dendrobatidis within and between amphibian life stages." Diseases of aquatic organisms 61 (2004): 75-83.

11. Garner, T. W., Perkins, M. W., Govindarajulu, P., Seglie, D., Walker, S., Cunningham, A. A., and Fisher, M. C. "The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana." Biology Letters 2.3 (2006): 455-459.

12. Johnson, M. L., Berger, L., Phillips, L., and Speare, R. "Fungicidal effects of chemical disinfectants, UV light, desiccation and heat on the amphibian chytrid, Batrachochytrium dendrobatidis." Diseases of aquatic organisms 57 (2003): 255-260.

13. Photo by Brian Gratwicke

14. Illustration by Alison E. Burke

15. ACAP, 2005. Amphibian Conservation Summit, 17–19 September 2005, Washington DC.

16. Cavalier‐Smith, Thomas. "A revised six‐kingdom system of life." Biological Reviews 73.3 (1998): 203-266.

17. Barr, D. J. S. "Chytridiomycota." Systematics and Evolution. Springer Berlin Heidelberg, (2001): 93-112.

18. Speer, B. R. "Introduction to the Chytridiomycota." Introduction to the Chytridiomycota. University of California, Berkeley- University of California Museam of Paleontology, 26 Jan. 1998. Web. 17 Apr. 2013.

19. Berger, L. R., Speare, H. B., Hines, G., Marantelli, A. D., Hyatt, K. R., McDonald, L. F., Skerratt, V., Olsen, J. M., Clarke, G., Gillespie, M., Mahony, N., Sheppard, C., Williams, M. J., Tyler. Effect of season and temperature on mortality in amphibians due to chytridiomycosis Australian Veterinary Journal 82 (7)(2004): 434 - 439

20. Retallick R. W. R., H. McCallum H, R. Speare. Endemic infection of the amphibian chytrid fungus in a frog community post-decline. PLoS Biology 2(11) (2004): 1965–1971

21. Retallick, R. W. R. & V. Miera. Strain differences in the amphibian chytrid Batrachochytrium dendrobatidis and non-permanent, sub-lethal effects of infection. Diseases of Aquatic Organisms 75 (2007): 201 – 207.

22. Simoncelli, F., A. Fagotti, R. Dall’’Olio, D. Vagnetti, R. Pascolini and I. Di Rosa. Evidence of Batrachochytrium dendrobatidis Infection in Water Frogs of the Rana esculenta Complex in Central Italy EcoHealth 2 (2005): 307–312.

23. Garcia, T. S. J. M. Romansic, A. R. Blaustein. Survival of three species of anuran metamorphs exposed to UV-B radiation and the pathogenic fungus Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 72 (2006): 163–169.

24. Norvell, Lorelei L. "Biology Reference." Fungi. Biology Reference, n.d. Web. 18 Apr. 2013.

25. Bosch, Jaime, and Pedro A. Rincón. "Chytridiomycosis‐mediated expansion of Bufo bufo in a montane area of Central Spain: an indirect effect of the disease." Diversity and Distributions 14.4 (2008): 637-643

Edited by Claire Forster