Detailed protocol for the detection of chytrid fungus using qPCR
What SAVE THE FROGS! is doing to stop the chytrid fungus
Articles by SAVE THE FROGS! Founder Dr. Kerry Kriger
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The chytrid fungus (Batrachochytrium dendrobatidis) is the most significant threat to the world's montane amphibian populations.
In terms of its effect on biodiversity, chytridiomycosis is quite possibly the worst disease in recorded history. First identified in 1998, this potentially lethal skin disease is caused by the chytrid fungus Batrachochytrium dendrobatidis, which has been detected on at least 287 species of amphibians from 36 countries. Chytridiomycosis has caused amphibian population declines in Australia, South America, North America, Central America, New Zealand, Europe, and Africa, and is likely responsible for over 100 species extinctions since the 1970's.
In this video SAVE THE FROGS! Founder Dr. Kerry Kriger discusses his Ph.D. research on the ecology of chytridiomycosis in Eastern Australia and discusses ways we can stop the spread of this deadly disease. With special guest Susan Jewell of the U.S. Fish and Wildlife Service's Fish and Aquatic Conservation division, and amphibian biologist Jonathan Kolby, a former wildlife inspector for the US Fish and Wildlife Service. Recorded 11-Sep-2013 as a free online SAVE THE FROGS! Academy class attended by 98 online participants.
"Your video on chytrid fungus is really good. Thanks for condensing the information and presenting it in a helpful manner"
-- Buck O'Brien, West Virginia
The US Fish & Wildlife Service recently solicited comments on its proposal to list amphibians as injurious wildlife UNLESS certified free of chytrid fungus. The proposal, if successful, will significantly reduce the spread of chytrid fungus via a reduction in the intercontinental trade of amphibians for use as pets and food. You can read our letter to the USFWS here.
Humans ship millions of amphibians around the world each year. When an infected frog arrives in a new location, its disease can spread to native populations if (1) it escapes captivity, (2) it is intentionally set free, or (3) water from its holding tank is released into the environment. Native amphibian populations generally have no evolved defenses against the new pathogen, and an epidemic that results in population decline or extinction can occur. The largely unregulated pet and food trades are the two most common sources of disease introduction into naïve amphibian populations. For instance, the skin disease chytridiomycosis has been detected in Mexican axolotls Ambystoma mexicanum in Australian pet shops, and in American bullfrogs Lithobates catesbeiana being farmed for international trade in Uruguay. Infected frogs are also unintentionally exported internationally via the zoo trade and laboratory animal trade. Find out what SAVE THE FROGS! is doing to stop the spread of the chytrid fungus.
Recommended reading: "Minimising exposure of amphibians to pathogens during field studies" by Philott et al (2011).
Dead and dying frogs generally have disorders of the epidermis, and often exhibit behavioral changes such as lethargy and loss of righting reflex. Chytrid zoosporangia live in the heavily keratinized stratum corneum and stratum granulosum of the frogs’ pelvic patch, digits, and ventral body, and in the keratinized mouthparts of tadpoles. Associated epidermal changes included irregular cell loss, hyperkeratosis, and excessive sloughing of the skin. Infected frogs begin to die roughly 21 days post-infection, and though larvae are susceptible to infection, deaths are generally restricted to metamorphosed individuals (this is because larvae do not have much keratin). Though chytridiomycosis is unlikely to cause larval mortality, it can significantly decrease body mass at metamorphosis and increase the duration of the larval period, both of which are likely to negatively affect the amphibian's survivorship in the long run. Two leading hypotheses have been put forward to explain how Batrachochytrium dendrobatidis directly kills its host: 1) toxic, proteolytic enzymes are released by the fungus, and 2) loss of electrolytes negatively affects osmoregulation and/or oxygen uptake, two primary functions of amphibian skin.
Chytridiomycosis-related declines are typified by “post-metamorphic death syndrome”, in which large numbers of metamorphosed individuals are found dead or dying (or not found at all), while tadpoles and embryos still survive at the site in normal numbers. This is to be expected: B. dendrobatidis is keratinophilic, and thus is absent in embryos (which lack keratin) and is generally restricted to the mouthparts of larvae, which until the latter stages of their development are the only keratinized areas of their body. Upon metamorphosis, the keratinized layer begins to cover the entire body, and infection can become lethal, potentially resulting in population decline and local extinction.
While methods exist for curing laboratory animals infected with Batrachochytrium dendrobatidis, it is not currently possible to eradicate the fungus from wild amphibian populations. Nor is it possible to protect a natural wilderness area prior to the arrival of the chytrid fungus. It is thus of vast importance that the spread of the fungus due to human activities be halted. This will require international cooperation, as countries will need to implement stringent quarantine procedures and diagnostic testing, and severely restrict the transportation of amphibians. We recommend that you do not purchase amphibians unless you are certain they were (1) captive-bred in disease-free conditions and (2) raised locally. If you cannot confirm both of these things, you may be inadvertently contributing to the spread of diseases to native, susceptible populations. Currently, there are very few pet dealers that provide thorough disease testing of their amphibians, as the price is generally prohibitive.
A correct diagnosis generally requires laboratory testing by an experienced pathologist because many infected frogs show no clinical symptoms of infection, and conversely many sick frogs have illnesses other than chytridiomycosis. There are no symptoms that necessarily implicate chytridiomycosis is the culprit. The most reliable methods for diagnosis are quantitative polymerase chain reaction (qPCR, or “real-time” PCR) and histology.
qPCR is a technique used by molecular biologists to detect the presence and quantity of B. dendrobatidis DNA on a sample. The sample can either be a cotton swab that was run across the amphibian or a piece of amphibian skin, the former method being preferred for live amphibians as (a) it is harmless, and (b) it samples a larger portion of the amphibian’s body and thus yields a more reliable result. qPCR analysis can cost anywhere from US$6 to US$40 per sample depending on various factors. qPCR is generally the preferred method of testing live amphibians for chytridiomycosis, as it has a low chance of yielding false negatives or false positives, it is rapid, cost-effective, and harmless to amphibians.
Here's a one-minute video demonstrating how to swab a frog for chytrid fungus:
The frog in the video above is quite large. If you need to swab a smaller frog, you can hold it as in the photo below (which was taken by fatty acid expert Dave Hall).
qPCR does have its drawbacks: expensive equipment is required, and many precautions must be taken to ensure that contamination (resulting in false-positives) does not occur and that inhibition (resulting in false-negatives) is detected if it occurs. Furthermore, the mere presence of B. dendrobatidis on amphibian skin does not necessarily imply that the fungus was the cause of death. To determine this, a more complete autopsy would be necessary, and this is generally done with histology.
Histology relies on the microscopic examination of amphibian skin to detect the chytrid fungus. Histology allows the diagnostician to determine the cause of death in an individual, as the degree of damage to the skin can be assessed, and other potential causes of death ruled out. Though histology is an excellent method for detecting Batrachochytrium dendrobatidis on dead or dying frogs, it is not recommended for live, apparently healthy individuals, or biologically important individuals such as those in endangered species in captive breeding programs. This is due to the necessity of skin samples, which can harm the amphibian and raise ethical concerns. Furthermore, histology has a high chance of yielding a false negative result, due to the difficulty in testing a large portion of the amphibian’s skin (the fungus may be present on the amphibian, but not on the skin sample).
The examination of oral disc deformities in larval anurans has been suggested as a quick, reliable, non-invasive method of chytrid diagnosis as B. dendrobatidis can cause the deterioration of the keratinized mouthparts of tadpoles. However, this method but must be used with caution as DDT intoxication and temperature changes can similar mouthpart changes. Furthermore, the reliability of visual examinations of oral disc deformities has only been tested on a small number of species, and thus the generality of its usefulness is unknown.
The thermal and hydric requirements of the chytrid fungus are the most important determinants of its geographical distribution, and also which amphibians are most likely to be affected by it. The chytrid fungus prefers cooler temperatures: it grows best in the laboratory between 17oC to 23oC, and tends to die above about 28oC. Additionally, the fungus has waterborne zoospores and cannot survive desiccation. Chytridiomycosis is thus most problematic in amphibians living in cool, wet areas. In the lowlands of eastern Australia, chytrid infections in stony creek frogs (Litoria lesueuri complex) are most severe at more southerly, cooler latitudes, and at sites that receive high rainfall. Further, infection prevalence and severity exhibits strong seasonality, increasing in the cooler months of early spring and winter. Temperatures change drastically across altitudes, and as expected based on its preference for cooler temperatures, Batrachochytrium dendrobatidis has its most serious effect on amphibians living in montane regions: virtually all of the amphibian declines caused by B. dendrobatidis have taken place at high altitudes. On a smaller geographic scale, B. dendrobatidis is most likely to affect amphibians that breed in permanent flowing water (as opposed to amphibians that breed in ephemeral water bodies, ponds, or leaf-litter). This is because the chytrid fungus (1) cannot survive desiccation, and thus prefers permanent water bodies; and (2) prefers streams, as they tend to be cooler than ponds and are able to transport the waterborne zoospores of the fungus long distances.
This slideshow, prepared by SAVE THE FROGS! Founder Dr. Kerry Kriger, details the results of his research on The Ecology of Chytridiomycosis in Eastern Australia. Please feel free to embed this slideshow on your own website (and add a link to http://savethefrogs.com while you're at it!). You can find the embed code here.
Not all chytridiomycosis-infected amphibian species at a site decline; nor do all species at an infected site become infected. Species vary in their inherent susceptibility to the fungus. This may be due to (1) differences in their ability to mount a sufficient immune response via anti-microbial peptides, or (2) differing microbiota on their skin: bacteria that live on amphibian skin can compete with or kill B. dendrobatidis, but the bacterial community on amphibians varies between populations and species. Furthermore, certain species may have life history traits that predispose them to infection, such as a tendency to breed in permanent flowing water, or an avoidance of microhabitats that could render their skin less hospitable to the chytrid fungus (which grows best in cool temperatures). Species also vary in fecundity (some females lay 20 eggs and others lay 20,000) and thus their resilience (the ability to recover after a disturbance, such as the introduction of a new pathogen.) Finally, species vary in the rate at which they can evolve defenses against newly introduced pathogens. Little is known about the rate at which species evolve resistance. Several endangered species in northern Australia (Litoria rheocola, L. nannotis, Nyctimistes dayi and Taudactylus eungellensis) were extirpated from upland areas by the chytrid fungus decades ago. Though their lowland populations have persisted relatively unaffected, the species have never been able to re-colonize their former upland habitats, where chytridiomycosis is presumably still a significant threat to which they have not adapted. Though it has become the poster child of evolved resistance, the Eungella Day Frog Taudactylus eungellensisis remains critically endangered to this day, and exists in large numbers on only one very small stretch of stream in Eungella National Park. This species has by no means evolved sufficient resistance to chytridiomycosis. Likewise, Central American amphibian species that were decimated by chytridiomycosis have yet to show significant signs of recovery. (Photo of T. eungellensis by K. Kriger)
Though our understanding of amphibian diseases has increased dramatically in the last decade, we still have an incomplete understanding of the manner in which diseases interact with other stressors (i.e. climate change, pollutants, invasive species) to exacerbate population declines. A synergistic effect is when the damage done by multiple stressors is greater than the sum of their individual parts. Climate change can alter the dynamics of parasite communities by making conditions more hospitable to growth of the pathogen, and it can also decrease the immune defenses of stressed amphibians that are not coping with sub-optimal climatic conditions. Pesticides, coal combustion residues and other pollution can also decrease amphibian immune response, rendering them more susceptible to infectious disease.
Please read the suggestions of the OIE the international body that issues recommendations regarding wildlife disease issues.
Infectious diseases are a primary cause of amphibian declines and loss of biodiversity, and the successful eradication of disease from the wild is currently impossible. The continued inter-continental trade and transport of amphibians will inevitably lead to the spread of novel pathogens, followed by numerous extinctions. Legislation to prevent the emergence of new diseases is urgently required to protect global amphibian biodiversity.
SAVE THE FROGS! Founder Dr. Kerry Kriger is quoted in this new Scientific American article, which details the mass die-off of salamanders in The Netherlands, and the description of another species of chytrid fungus, Batrachochytrium salamandrivorans.
Please don't purchase frogs as pets, and if you do, be certain they were certified disease-free by a reputable laboratory. Never eat frog legs. Never release frogs or tadpoles into any location other than that from which they came. And please donate to SAVE THE FROGS! so that we can tackle the global issues that facilitate the emergence of chytridiomycosis. Nobody else does what we do, and we can't do it without you! Thanks!
If you would like to contribute a section on ranavirus, trematodes, or any topic not yet covered here in detail, please contact us. Your contribution will be gratefully acknowledged.
Emerging infectious diseases are diseases that have recently increased in any of the following:
Incidence: The disease affects more individuals now than it did in the past.
Impact: The disease now causes more damage to the infected individual, either due to decreased immune defenses in the individual, or increased virulence of the pathogen
Pathogenicity: The pathogen has evolved to be more virulent, i.e. causes more damage to the infected individual
Geographical Range: The disease has spread to locations outside of its native range.
Host Range: The disease now infects more species than it did in the past.
This video of the chytrid fungus under a microscope was made by the Amphibian Avenger, Lucy Cooke, on a recent trip to Colombia.
"Dear Dr. Kriger, My name is Emily, I am a senior at Saratoga Springs High School ( NYS). I studied The Presence of Batrachochytrium dendrobatidis in the Adirondacks. I know you're busy traveling and with the up coming Save The Frogs Day but I wanted to share my results with you. Your website has helped me so much with my research. Thanks, Emily Boivin"