Chapter 28 Diagnosis and Control of Amphibian Chytridiomycosis
Chytridiomycosis is an emerging infectious disease associated with global amphibian population declines.8,16,20,38,42 The causative agent, Batrachochytrium dendrobatidis (Bd), is a chytrid fungus with a broad host range documented to infect the skin of over 300 different frog and salamander species to date (www.spatialepidemiology.net/bd-maps). Infection with Bd may be subclinical, with minimal skin lesions in some species such as the American bullfrog (Lithobates catesbeianus),13 or cause severe skin disease in highly susceptible neotropical species such as the Panamanian golden frog (Atelopus zeteki).21 Subclinically infected animals are potential sources of infection for more highly susceptible species and may serve as vectors for the introduction of Bd to new geographic regions. There continues to be debate about whether mortality caused by chytridiomycosis results from the introduction of Bd to naïve amphibian populations or whether Bd is a commensal parasite of amphibians that has become virulent as the result of environmental cofactors such as climate change (novel versus endemic pathogen hypothesis).7,22,23 Supporting evidence for the novel pathogen hypothesis includes demonstration of minimal genetic variation among geographically distinct Bd isolates and ecologic studies that show amphibian mortality and population decline only after the arrival of Bd at new locations.
The impact of chytridiomycosis and Bd infection is not limited to wild amphibian populations. Infection and disease outbreaks are observed in zoo and aquarium collections,9,25,30,31 laboratory animals,27 and amphibians in the pet and food trades.40 Veterinarians who include amphibians in their practice must be able to provide accurate diagnostic testing and treatment for Bd-infected animals and to make recommendations for control and prevention of this disease in amphibian conservation and reintroduction programs.32 This chapter expands on an overview of amphibian chytridiomycosis presented by Pessier,29 with an emphasis on new information needed by zoo and wildlife veterinarians for animal management.
Chytridiomycosis and Amphibian Survival Assurance Populations
The forward to the 2007 World Conservation Union (IUCN) Amphibian Conservation Action Plan (ACAP) stated that in the last decades of the 20th century, the amphibian extinction rate exceeded the mean extinction rate of the last 350 million years36 by at least 200 times.12 These declines are attributable to a multitude of factors, such as habitat loss, climate change, and species exploitation, but many of the most rapidly occurring losses of amphibian biodiversity are associated with chytridiomycosis.23 Unfortunately, it is possible that many amphibians will become extinct long before the political measures and scientific progress needed to mitigate the declines are implemented. This has resulted in the development of survival assurance populations that bring representatives of imperiled amphibian species into captivity with the goal of establishing healthy and sustainable populations that may be returned to the wild at a later time. The Amphibian Ark (www.amphibianark.org), formed by a cooperative effort of the World Association of Zoos and Aquariums (WAZA), the IUCN Conservation Breeding Specialist Group (CBSG), and the IUCN Amphibian Specialist Group has estimated that over 500 amphibian species could immediately require the type of ex situ intervention represented by survival assurance populations.
Although there are no recognized methods for eliminating or controlling chytridiomycosis in wild amphibian populations or their environments, it has been demonstrated that some amphibian populations that have experienced chytridiomycosis-associated declines subsequently persist at reduced densities with endemic Bd infection.34 Another encouraging line of research has demonstrated that in some species, colonization of the skin by the bacterium Janthinobacterium lividum reduces morbidity and mortality caused by chytridiomycosis.2,14 These observations require additional investigation, but highlight the possibility that some affected amphibian populations could eventually recover or that methods will be developed to mitigate the effects of infection on wild populations. Furthermore, they provide hope that the creation of survival assurance populations could be a viable conservation strategy for chytridiomycosis-associated population declines.
To maintain healthy and viable assurance populations, the methods used to control chytridiomycosis and other infectious diseases in captive settings have become critically important. A major emphasis has been on improving the biosecurity practices used in zoos and conservation programs to reduce the introduction and transmission of disease to captive animals and to prevent movement of infectious diseases from captive animals to wild amphibian populations as part of reintroduction programs.30,32 Some of the most important biosecurity practices identified include keeping amphibian survival assurance populations as close as possible to the native range of the species (e.g., the maintenance of Panamanian golden frog assurance populations in Panama), keeping survival assurance populations in long-term isolation from cosmopolitan zoo collections that have species from different parts of the world in close proximity, and use of husbandry protocols that reduce the possibility of transmission of infectious diseases in captive breeding facilities.
Chytridiomycosis and the World Organization for Animal Health
In 2008, the World Association for Animal Health (OIE) listed two amphibian infectious diseases, chytridiomycosis and ranaviral disease, as notifiable under the Aquatic Animal Health Code.39 These reporting requirements developed in response to evidence that trade and shipment of amphibians for a variety of purposes have the potential to introduce these significant amphibian pathogens to new locations. Examples of the anthropogenic movement of Bd include exports of African clawed frogs (Xenopus laevis) for use in human pregnancy diagnosis and biomedical research,47 imports of farmed American bullfrogs (Lithobates catesbeianus),40 and introduction of Bd to a wild population of Mallorcan midwife toads (Alytes muletensis) as the result of a captive breeding program.45
The implication of the OIE listing of chytridiomycosis for captive amphibian breeding or conservation programs is that the importation of live amphibians will eventually require an official International Aquatic Animal Health Certificate that certifies that animals are free of Bd infection or have been treated in a manner that eliminates infection. Alternatively, infected animals could be imported and quarantined in biosecure facilities for the purpose of creating Bd-specific pathogen-free populations. The regulatory requirements related to Bd, such as OIE reporting and oversight of amphibian import and export, fall to the chief veterinary officer of each country that is a signatory to the OIE (e.g., the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service [APHIS]). Details on the OIE requirements related to chytridiomycosis may be found in the Aquatic Animal Health code online (www.oie.int/eng/normes/fcode/en_chapitre_1.8.1.htm).
Pathogenesis of Chytridiomycosis
Lesions associated with lethal chytridiomycosis in postmetamorphic amphibians are limited to the keratinizing epithelium of the skin. Because the skin is a physiologically important organ for osmoregulation in amphibians, disruption of normal cutaneous function has been frequently cited as a probable cause of death in animals with chytridiomycosis. Recent studies using the White’s tree frog (Litoria caerulea) as a model species have demonstrated that Bd-infected animals have reduced electrolyte transport across the epidermis, with subsequent terminal hyponatremia and hypokalemia.44 This suggests that supportive treatment with electrolytes may be helpful for affected animals in clinical settings. Emerging research on the virulence of Bd will examine the roles of the serine protease and fungalysin metallopeptidase gene families, which play a role in the pathogenesis of other fungi.38 Investigation of the mechanisms of host defense against Bd has focused on antimicrobial peptides produced by the granular glands in amphibian skin, and evidence has suggested that differences in peptide excretion may influence species resistance to chytridiomycosis.7,37 As noted, other recent research has explored the role of specific cutaneous bacteria such as J. lividum that secrete antifungal compounds and function as components of innate cutaneous immunity.2,14
Diagnostic Methods
The commonly used diagnostic methods for Bd infection either examine samples of the skin microscopically for characteristic fungal organisms (e.g., cytology, histopathology), or use the polymerase chain reaction (PCR) assay to amplify sequences of deoxyribonucleic acid (DNA) specific for Bd.29 Cytology and histopathology are most useful for the diagnosis of sick animals because large numbers of organisms are usually present and easily detected. In addition, histopathology allows evaluation of the severity of associated skin lesions (e.g., epidermal hyperplasia and hyperkeratosis) and helps determine whether infection was likely to have been clinically significant. In contrast, the PCR-based methods detect much smaller numbers of organisms and are essential for detecting subclinical Bd infections. PCR assays also have the advantage of a requiring a minimally invasive sample (i.e., skin swabs) for testing. The application of PCR-based diagnostic methods and proper interpretation of PCR results are critical for the control of chytridiomycosis in captive amphibian populations and will be discussed in greater detail.
Polymerase Chain Reaction–Based Methods of Diagnosis
Both conventional and real-time Taqman PCR techniques have been described for the diagnosis of Bd infection and are used in research and commercial diagnostic laboratories.1,6,15,41 PCR testing is the diagnostic procedure of choice for the following: (1) quarantine screening of new animals that enter collections; (2) surveys of captive or wild amphibian populations for the presence of Bd; (3) evaluating of the success of antifungal treatment of Bd infection; and (4) screening of captive animals prior to reintroduction to the wild or translocation of wild animals.
When properly validated in the laboratory, the Taqman PCR technique has advantages over conventional PCR,32 including the following:
Sample Collection
A wide variety of different samples has been used to perform Bd PCR assays, including collection of skin swabs, immersion in a water bath, and collection of tissue (e.g., toe clips).15 The skin swab procedure is most commonly used and is minimally invasive while allowing for sampling of multiple areas of potentially infected skin. Samples are collected with a sterile commercially available applicator stick (swab) applied with a gentle sweeping motion to ventral skin surfaces of the feet, legs, and body. Most sampling protocols call for three to five passes with the swab over each general region of the skin. Because PCR techniques may detect very small amounts of DNA, it is important to avoid cross-contamination of samples from different animals that could cause false-positive test results. Techniques for reducing cross-contamination include the use of a new pair of disposable gloves for every animal and avoiding contact of swabs with surfaces other than the skin of the animal being tested.24,32,33
Factors related to sample collection and sample storage may influence the performance and outcome of Bd PCR testing. For instance, a specific fine-tipped rayon swab with a plastic handle (Dryswab Fine Tip MW113, Medical Wire & Equipment, Corsham, Wiltshire, England) is optimal for the recovery of Bd DNA using the Taqman technique.6,15,32 Other factors that improve recovery of Bd DNA include storage of swab samples dry rather than preservation in an ethanol solution and avoiding exposure of swabs to very high environmental temperatures (>38° C) prior to analysis.43 Although dry swab samples may be stable at room temperature (23° C) for as long as 18 months after collection, storage under refrigeration (4° C) or freezing (−20° C or below) is suggested if there will be a delay in sample processing.42 Pooling of samples in the laboratory from multiple animals into a single PCR test reaction has been used as a method for reducing the costs associated with testing large numbers of animals, but may result in reduced test sensitivity when samples from more than five animals are combined. Because of these considerations, the laboratory analyzing samples for Bd PCR should always be consulted for their preferences regarding sample collection and preservation.
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