Chytrid run-down

As a part of our EVPP 305: Environmental Microbiology course at GMU our AWESOME professor required we investigate a microbial-relevant topic and write about it. I chose a chytrid fungus (Batrachochytrium dendrobatidis) wreaking havoc on amphibian populations globally. Enjoy!

SUMMARY

The Chytridiomycosis fungus, Batrachochytrium dendrobatidis, first emerged in the mid 1990s as a pathogen infecting large numbers of wild frog populations, particularly in Australia and Central America. Initially, scientists were unsure what this newly emerged pathogen was, as it had morphological characteristic to that of fungi of the phylum Chytridiomycosis, yet infected vertebrate organisms – something that no other chytrid had ever been observed doing. Over the last thirty years, the infections have continued to spread to global amphibian populations, but scientists have also made strides in detection methods, like real-time PCR, as well as potential treatments, like the chloramphenicol antibiotic. Nearly a third of global amphibian populations are considered impaired, so there is an urgent need for scientists to continue investigating and characterizing this pathogen.

INTRODUCTION

During the late 1990s, wild and captive amphibian populations, specifically organisms of the order Anura (i.e., frogs, often referred to as anurans), in Australia and Central America observed significant declines in population sizes due to a then unknown cause (Berger et al., 1998). These declines were also reflected in frog populations of various genera and species in American zoos, like the National Zoological Park (NZP) in Washington, D.C. (Longcore et al., 1999; Pessier et al., 1999). In the captive populations, specifically, Pessier et al. (1999) noted that nearly all of the individuals that died did not show any obvious signs that they were going to die prior to their death. This seemingly overnight fetal response to a relatively unknown fungus induced heightened concern for the well being of captive, as well as wild, amphibian populations during this time period. As a response, numerous studies were initiated to examine infected frogs that had died in a variety of these die-off events, in order to determine what exactly this pathogen was and hopefully find ways to manage its impacts. Although many of the infected amphibian populations were geographically isolated from one another, each managed to exhibit the same symptom: visible epidermal (i.e., skin) infection, prior to their demise.

INITIAL ASSESSMENT

In order to determine the specific cause of these mass die offs in frogs, as well as other amphibian populations, Longcore et al. (1999) conducted a study in which the fungus was isolated from a blue poison dart frog (Dendrobates tinctorius azureus) that had recently died at the NZP. Once isolated, the fungus was photographed during the various stages of development as it grew on PmTG nutrient agar. Additionally, captive frogs were inoculated with the disease to observe the physical response, as well as determine the amount of time needed to infect otherwise healthy individuals.

Across studies, following examination of the skin of the affected organisms under an electron microscope, the morphology of the zoospores produced by the pathogen suggested that the organism belonged to the phylum Chytridiomycota, commonly referred to as chytrids, within the kingdom Fungi (Berger et al.,1998; Longcore et al., 1999; Pessier et al., 1999). These motile fungi are typically found in water and soils, and are known to gather their nutrients by decomposing organic matter, as well as parasitizing many other fungi, plants, algae and invertebrates (Pessier et al., 1999). The unknown pathogen infecting amphibian populations was novel in that it was the first chytrid fungus to parasitize a vertebrate. During the study conducted by Longcore et al. (1999), two new characteristics previously not observed in chytrids fungi were present in the unknown pathogen, which warranted the assignment of a new genus and species name, Batrachochytrium dendrobatidis (hereafter referred to as Bd). The new name originated from the Greek word for frog, “batracho,” and the genus of the frog species the cultures were isolated from, “Dendrobates.” The two newly observed features in this chytrid were the growth of thallus and the minute (ultrastructure) details on the zoospores produced by the pathogen (Longcore et al., 1999).

CHARACTERIZATION AND DIAGNOSIS

Following the isolation of pure cultures of the unknown chytrid in the lab, transmission electron microscopy was used to examine the morphology of the fungi. Morphologically, this unknown looked like many other known chytrids with a single flagellum on the posterior of each living zoospore (Figure 1).

Berger et al. (1998) found that Bd is comprised of mostly spherical sporangia that inhabit the stratum corneum and stratum granulosum, which are essentially the two outermost layers on the skin of a frog and are responsible for releasing lipids to create a water retaining film. Not only does the fungus centralize on the outermost layers of the skin, but also it is observed most frequently within the webbing on the feet and ventral (i.e., stomach) side of the frogs. In infected organisms, the affected areas typically turn light brown in color and develop a bumpy texture, observable by the naked eye (Pessier et al., 1999).

In order to determine if an individual is infected with the Bd pathogen, cotton swabs are typically used to collect a sample of the fungus on the frog to then be analyzed using qualitative polymerase chain reaction (i.e., real-time PCR or qPCR) techniques (Young et al., 2012).

During the histological analysis of Bd, Pessier et al. (1999) characterized a variety of responses to different staining methods (Table 1). Knowing these characteristics will aid future studies in identifying the presence of this pathogen.

IMPACTS ON AMPHIBIANS

The Bd pathogen has infected at least 350 species of amphibians, on all continents except Antarctica, because this continent does not actually support any known populations of amphibians (Fisher et al., 2009).

The area of the body on the ventral side of an infected organism that the Bd concentrates on is particularly harmful to frogs, specifically, as it is a critical area known as the “drinking patch” and is the sole area responsible for absorbing water. In addition to absorbing water, this area of the body also supports exchange of necessary electrolytes for everyday life functions in the frogs (Voyles et al., 2012; Young et al., 2012). Young et al. (2012) observed extreme dehydration in frogs exposed to Bd in a controlled lab setting within 24-48 hours of inoculation with the fungus. In a wild caught Australian green tree frog (Litoria caerulea) individual infected with Bd, the outer-most layer of skin swelled to at least twelve times the normal thickness (i.e., 2-5 μm to 60 μm)(Berger et al., 1998). This swelling was a response to the Bd sporangia growing in the skin.

This pathogen threatens nearly all amphibian species, aside from a few who are naturally immune (congregated in Asia). The rapid rate of Bd development once an organism is infected contributes to the overall potential to destroy densely populated groups of amphibians before interventions can occur.

FACTORS INFLUENCING SPREAD

To add to the ominous future regarding the potential harm this pathogen may have on global amphibian populations, it is important to remember that this pathogen managed to cross entire oceans to impact populations geographically isolated from one another. The general consensus among amphibian experts is that we still do not fully understand where this pathogen emerged (Kilburn et al., 2010); however, there are two main hypotheses: the Novel Pathogen Hypothesis (NPH) and the Endemic Pathogen Hypothesis (EPH) (Rachowicz et al., 2005). The NPH essentially states that Bd is actually being transported to new geographic regions, whether that be by animal trade, human activity, etc. On the other hand, the EPH suggests that Bd has always existed in the infected locations, and is now responding to either a change in environmental conditions, an increase in available hosts, or an actual increase in the fungi’s ability to spread between hosts (i.e., pathogenicity). Compiling evidence for each hypothesis may be more challenging than it seems, for example, some studies have found evidence that Bd was present in areas like Illinois, over 100 years ago, which could support the idea that Bd has been around for a long time and just gone unnoticed until now, or perhaps areas like Illinois could actually just be a spreading center (Talley et al., 2015).

An anthropogenic source of the spread of Bd has been linked to the global food trade market for American bullfrogs, Lithobates catesbeianus. In the United States markets alone, infection rates in the frogs being traded range from 41-62% (Schloegel et al., 2012).

In Panama, a study conducted by Kilburn et al. (2012), determined that elevation (e.g., high vs. low) did not influence the frequency of occurrence of Bd in the 58 different anuran species observed. In order to determine the presence of Bd, as well as the intensity, among the anuran populations the researchers utilized qPCR. When analyzing trends in Bd infections at the level of a single species, trends can emerge geographically and temporally (Fisher et al., 2009).

CONCLUSIONS/FUTURE DIRECTIONS

Over the last two and a half decades, Bd has gained epizootic status, meaning it is a rapidly spreading disease in communities supporting amphibian populations (Kilburn et al., 2010). Since many chytrid fungi are known to spread via water, this is a good place to focus future management action. While the elevation results from the study conducted by Kilburn et al. (2012) suggested that Bd does not have elevation limitations, it also suggests that no amphibian populations is necessarily more or less safe from the pathogen, based on their elevation. This ability of Bd to persist in a wide array of environments poses significant threat to global amphibian populations. The results of this study could have important management implications in regards to prioritizing which populations to target for remediation efforts (i.e., endangered species at higher elevation do not have prioritization over equally endangered species at lower elevations).

In Europe, there is a genus of salamander, Speleomantes, which is able to persist in the presence of Bd (Pasmans et al., 2013). Future studies should focus on determining what aspects of their skin secretions aid in their resistance to this otherwise amphibian-fetal pathogen. Once the composition of these secretions are better understood, they may be used to mass create a combative agent to apply in areas heavily impacted by Bd.

Additionally, a study conducted by Young et al. (2012) demonstrated the first successful treatment of terminally ill green tree frogs (Litoria caerulea) that were inoculated with Bd in a controlled environment. Curing the frogs exposed to the pathogen in this experiment required a multistep process including: aggressive rehydration therapy to account for the fluids and electrolytes lost by the newly degraded skin, as well as applying an antibiotic treatment of chloramphenicol with heat to the infected area. The authors urged the need for further research and replications of this study in order to determine the role heat may or may not actually be contributing in the recovery of the frogs. Still, this method has the potential to aid in the recovery of captive frog populations, including in zoos and the wildlife trade; however, applying these methods to wild populations may become problematic as they rely heavily on constant monitoring and involved treatment.

References

Berger, L., Speare, R., Daszak, P., Green, D.E., Cunningham, A.A., Goggin, C.L., Slocombe, R., Ragan, M.A., Hyati, A.D., McDonald, K.R., Hines, H.B., Lips, K.R., Marantelli, G. & Parkes, H. (1998) Chytridiomycosis Causes Amphibian Mortality Associated with Population Declines in the Rain Forests of Australia and Central America. Proceedings of the National Academy of Sciences of the United States of America, 95, 9031–9036.

Fisher, M.C., Garner, T.W.J. & Walker, S.F. (2009) Global Emergence of Batrachochytrium dendrobatidis and Amphibian Chytridiomycosis in Space, Time, and Host. Annual Review of Microbiology, 63, 291–310.

Kilburn, V.L., Ibáñez, R., Sanjur, O., Bermingham, E., Suraci, J.P. & Green, D.M. (2010) Ubiquity of the Pathogenic Chytrid Fungus, Batrachochytrium dendrobatidis, in Anuran Communities in Panamá. EcoHealth, 7, 537–548.

Longcore, J.E., Pessier, A.P. & Nichols, D.K. (1999) Batrachochytrium Dendrobatidis gen. et sp. nov., a Chytrid Pathogenic to Amphibians. Mycologia, 91, 219–227.

Pasmans, F., Van Rooij, P., Blooi, M., Tessa, G., Bogaerts, S., Sotgiu, G., Garner, T.W.J., Fisher, M.C., Schmidt, B.R., Woeltjes, T., Beukema, W., Bovero, S., Adriaensen, C., Oneto, F., Ottonello, D., Martel, A. & Salvidio, S. (2013) Resistance to Chytridiomycosis in European Plethodontid Salamanders of the Genus Speleomantes (ed ID Jacobsen). PLoS ONE, 8, e63639.

Pessier, A.P., Nichols, D.K., Longcore, J.E. & Fuller, M.S. (1999) Cutaneous Chytridiomycosis in Poison Dart Frogs (Dendrobates spp.) and White’s Tree Frogs (Litoria Caerulea). Journal of Veterinary Diagnostic Investigation, 11, 194–199.

Rachowicz, L.J., Hero, J.-M., Alford, R.A., Taylor, J.W., Jess A. T. Morgan, Vredenburg, V.T., Collins, J.P. & Briggs, C.J. (2005) The Novel and Endemic Pathogen Hypotheses: Competing Explanations for the Origin of Emerging Infectious Diseases of Wildlife. Conservation Biology, 19, 1441–1448.

Schloegel, L.M., Toledo, L.F., Longcore, J.E., Greenspan, S.E., Vieira, C.A., Lee, M., Zhao, S., Wangen, C., Ferreira, C.M., Hipolito, M., Davies, A.J., Cuomo, C.A., Daszak, P. & James, T.Y. (2012) Novel, panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade: CHYTRID GENOTYPES AND THE BULLFROG TRADE. Molecular Ecology, 21, 5162–5177.

Talley, B.L., Muletz, C.R., Vredenburg, V.T., Fleischer, R.C. & Lips, K.R. (2015) A century of Batrachochytrium dendrobatidis in Illinois amphibians (1888–1989). Biological Conservation, 182, 254–261.

Voyles, J., Vredenburg, V.T., Tunstall, T.S., Parker, J.M., Briggs, C.J. & Rosenblum, E.B. (2012) Pathophysiology in Mountain Yellow-Legged Frogs (Rana muscosa) during a Chytridiomycosis Outbreak (ed B Gratwicke). PLoS ONE, 7, e35374.

Young, S., Speare, R., Berger, L. & Skerratt, L.F. (2012) Chloramphenicol with Fluid and Electrolyte Therapy Cures Terminally Ill Green Tree Frogs (Litoria caerulea) with Chytridiomycosis. Journal of Zoo and Wildlife Medicine,43, 330–337.