Pathways of amphibian chytrid fungus dispersal: global biosecurity and conservation implications

Kolby, Jonathan E. (2016) Pathways of amphibian chytrid fungus dispersal: global biosecurity and conservation implications. PhD thesis, James Cook University.

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Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) poses the greatest emerging threat to global amphibian biodiversity. Bd's low host species specificity allows the disease it causes — chytridiomycosis — to affect many of the 7,000 species of amphibians and drive population declines and extinctions worldwide. Although discovered nearly 20 years ago, the origin of Bd and catalyst of the seemingly recent global disease event remain obscure. Today, this international epizootic continues to advance virtually uncontrolled.

Modes of global Bd dispersal are not well understood, hampering the development and implementation of targeted biosecurity efforts to reduce spread. Bd is an aquatic pathogen most often associated with amphibian species that live in or near permanent bodies of water. It can neither survive desiccation nor extended exposure to elevated temperatures, but few environmental barriers appear to impede the spread of Bd. It has crossed oceans, infected terrestrial direct-developing amphibians that do not live in water, and been introduced to every continent (except Antarctica). Although low densities of Bd have been found in the environment outside of a host, amphibians consistently carry the highest pathogen loads and appear to be the primary host organisms that vector Bd.

The international trade in live amphibians transports millions of animals annually. Most previous research has focused on this anthropogenic activity as the primary pathway of global Bd dispersal. This is a sensible assumption—the highly visible movement of Bd hosts together with the lack of disease control suggests that Bd-positive animals are commonly transported in these shipments. Unfortunately, all previous surveillance efforts that aimed to demonstrate this phenomenon were performed in animal markets in Bd-positive countries where contamination from domestic Bd could not be excluded as a potential source of infection.

Upon close examination of global Bd distribution patterns, I found that regions of Bd presence do not exclusively overlap those of notable amphibian trade, raising questions as to the sources and pathways involved in pathogen dispersal. For instance, despite the absence of commercial amphibian importation to the islands of Dominica and Montserrat, chytridiomycosis drove the near-extinction of the Mountain Chicken frog (Leptodactylus fallax). Likewise, chytridiomycosis emerged in remote wilderness areas in Central America and Australia, again with no clear link to amphibian trade activity. Thus, despite the similar absence of commercial amphibian importation in Madagascar, it seems unlikely that this alone prevents the introduction of Bd. Meanwhile despite intensive field and market surveillance in Hong Kong — a global amphibian trade hub — Bd has neither been detected nor have amphibian declines been observed. Therefore, I hypothesized that additional pathways of Bd dispersal exist in the absence of commercial amphibian trade that can also transport this pathogen global distances.

The aims of this study were to characterize the likelihood and consequence of multiple pathways of amphibian pathogen dispersal, help identify Bd mitigation targets likely to reduce the greatest amount of international disease spread, and suggest potential mitigation activities. I investigated the presence of five potential Bd dispersal pathways that might concurrently contribute towards contemporary global pathogen spread. Because Bd is present both in areas heavily affected by human activity and remote wilderness areas, I explored avenues of dispersal that involved anthropogenic-assisted spread, spread by wildlife, and spread by environmental phenomena. The five pathways I evaluated include: 1. Commercial amphibian trade, 2. Amphibian hitchhikers (unintentional amphibian trade), 3. Atmospheric transport events, 4. Amphibian locomotion, and 5. Fomites. Through my series of studies, I examined high-risk environments where each pathway was most likely to be detected if an active avenue of Bd spread existed.

My results demonstrate that global Bd dispersal does involve multiple simultaneous pathways, with all five pathways having been successfully observed, with each varying in frequency and quantity of pathogen spread. At a field site in Cusuco National Park in Honduras, I discovered evidence for both atmospheric and terrestrial dispersal of Bd, previously undocumented for this aquatic pathogen. Bd was detected in rainwater in 5% of the storm events sampled and was present at a low density (1 Bd zoospore/L). Because Bd viability cannot be confirmed from the qPCR test results alone, it remains uncertain whether Bd-positive rainwater is infectious to amphibians. Even in the absence of infection, aerial dispersal can produce Bdpositive field samples and may occasionally be responsible for enigmatic isolated records of Bd detection.

Another way that Bd spreads in the absence of human assistance, and in higher densities, is through amphibian locomotion. Although infected animals spread Bd to one another in captive laboratory setting, the likelihood of this pathway was not previously measured in the wild in the natural environment. Because some frogs develop high infection loads as they undergo metamorphosis from tadpole to froglet, I studied whether seasonal mass emergences from aquatic to terrestrial habitats commonly transports Bd across habitat boundaries. In a sample of 52 recently emerged frogs, I detected the presence of Bd on 76.1% (35/46) of terrestrial vegetation surfaces where a Bd-positive frog had been resting. As previously mentioned, the viability of Bd cannot be discerned from qPCR results alone, but the cool air temperature, closed canopy, and high humidity of this cloud forest provides favourable conditions for protection from desiccation and extended Bd persistence. Furthermore, the average Bd zoospore equivalent loads that I detected on affected leaf surfaces (average = 40.48 and range was 0.12–1,040.45) compared to those measured in adjacent Bd-positive river water samples (average was 0.23 and range was 0.03–0.57) show that exposure to affected foliage may pose a greater threat of Bd transmission to terrestrial amphibians than would a comparable period of exposure to nearby river water.

I performed several studies to measure and evaluate the presence of Bd in amphibians commercially imported into the United States. Overall, I detected a moderately high prevalence of Bd in amphibians sampled immediately upon importation in the USA, validating prior studies on trade being a major route of spread. Bd was detected in 11.7% of 265 exotic pet trade amphibians from Hong Kong, 58.8% of 102 food trade bullfrogs from the Dominican Republic, 0/35 food trade bullfrogs sampled from Taiwan, and 0.5% of 565 exotic pet trade amphibians from Madagascar. In addition, this trade activity also caused the spread of Bd-contaminated shipping materials; e.g. 59.0% (62/105) of cardboard boxes carrying bullfrogs from the Dominican Republic and 5/8 bags of water carrying amphibians from Hong Kong tested positive for Bd.

Despite previous lack of detection, my surveillance data confirmed the presence of Bd in material exported from Madagascar and Hong Kong, and for the first time suggested the pathogen might already have been introduced to those countries' wild amphibian populations. In response, I designed and performed targeted rapid response investigations to determine whether Bd was present and identify potential introduction pathways. In 2013, I detected Bd in Hong Kong in Asian bullfrogs (Hoplobatrachus chinensis) sampled upon importation (8/26), in African clawed frogs (Xenopus laevis) at domestic pet stores (3/60) and in native free-ranging amphibians (2/268). In Hong Kong, I observed trade behaviors that are accompanied by a high risk of pathogen spillover - most notably the release of non-native animals into local amphibian habitats. In contrast, I did not detect the presence of Bd in 508 amphibians and 68 water filter samples tested in Madagascar, although this does not preclude its presence in very limited distributions and/or very low prevalence. I inspected the wildlife trade facility from where my previous Bd-positive frogs originated and did not observe any obvious opportunities for non- Malagasy Bd contamination –animals from other countries do not enter the facility. Despite the absence of commercial amphibian importation, I identified an incursion of Asian toads likely introduced as hitchhikers inside ocean shipping containers. It remains unknown whether these toads have recently introduced foreign pathogens to Madagascar, but this invasion clearly demonstrates how the absence of intentional amphibian trade does not entirely reduce risk of exposure to Bd. Data produced by both rapid response field studies suggests that a virulent strain of Bd (e.g. Bd-GPL) is not yet established in either Hong Kong or Madagascar, despite evidence of Bd introduction pathways, presence of suitable climatic refugia, and an abundance of susceptible host species. Therefore, I believe it remains plausible to prevent Bd-associated declines within these countries if appropriately targeted biosecurity measures are implemented.

Applying all information developed and collated to characterize the five potential Bd dispersal pathways, I then performed a risk matrix analysis to compare the amount of risk associated with each in order to better assist management decision-making. Each pathway was assigned a numerical score reflecting the combined likelihood of Bd spread and the severity of the outcome. These values represent the estimated relative contributions of each pathway to the global emergence of Bd. The international trade in live amphibians creates the most consistent and predictable opportunities for long-distance spread of viable and potentially infectious Bd and was ranked the highest risk pathway. My data shows that in the absence of this activity, Bd appears unlikely to frequently cross geographic and biotic boundaries to its survival, and that the emergence of this pandemic is likely due to human-assisted trade.

I conclude that biosecurity regulations to reduce the amount of Bd dispersed by the international wildlife trade is the most imperative action to slow the current global spread of Bd and to reduce the likelihood of additional disease emergences. Although methods to control the trade-driven spread of Bd were recommended by the World Organisation of Animal Health nearly five years ago, few countries have formally adopted these practices or require any actions specifically to reduce Bd introduction. My study confirms that the trade in live amphibians is an engine of global pathogen pollution unrivalled by other identified pathways of Bd dispersal and reinforces the need for disease management actions at least to the level commonly offered to livestock. Moving forward, proactive disease surveillance and biosecurity measures must target the international wildlife trade if we are to protect global biodiversity from novel emerging infectious diseases of wildlife.

Item ID: 52649
Item Type: Thesis (PhD)
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This is a thesis by publication. Published articles included in chapters 3, 5 and 9 have been redacted from the thesis due to copyright restrictions. The full thesis may either be requested via document delivery at your local library or viewed in the Eddie Koiki Mabo Library at JCU, Townsville.

Eight publications arising from this thesis are stored in ResearchOnline@JCU, at the time of processing. Please see the Related URLs. The publications are:

Chapter 2 (paper 1): Kolby, Jonathan E. (2014) Presence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in native amphibians exported from Madagascar. PLoS ONE, 9 (3).

Chapter 2 (paper 2): Kolby, Jonathan E., Smith, Kristine M., Berger, Lee, Karesh, William B., Preston, Asa, Pessier, Allan P., and Skerratt, Lee F. (2014) First evidence of amphibian chytrid fungus (Batrachochytrium dendrobatidis) and Ranavirus in Hong Kong amphibian trade. PLoS ONE, 9 (3). pp. 1-6.

Chapter 3 (paper 1): Kolby, Jonathan E., Smith, Kristine M., Ramirez, Sara D., Rabemananjara, Falitiana, Pessier, Allan P., Brunner, Jesse L., Goldberg, Caren S., Berger, Lee, and Skerratt, Lee F. (2015) Rapid response to evaluate the presence of amphibian chytrid fungus (Batrachochytrium dendrobatidis) and ranavirus in wild amphibian populations in Madagascar. PLoS ONE, 10 (4). pp. 1-21.

Chapter 3 (paper 2): Kolby, Jonathan E., and Skerratt, Lee F. (2015) Amphibian chytrid fungus in Madagascar neither shows widespread presence nor signs of certain establishment. PLoS ONE, 10 (10). pp. 1-6.

Chapter 3 (paper 3): Kolby, Jonathan E. (2014) Ecology: Stop Madagascar's toad invasion now. Nature, 509 (7502). p. 563.

Chapter 5 (paper 1): Kolby, Jonathan E., Ramirez, Sara D., Berger, Lee, Griffin, Dale W., Jocque, Merlijn, and Skerratt, Lee F. (2015) Presence of amphibian chytrid fungus (Batrachochytrium dendrobatidis) in rainwater suggests aerial dispersal is possible. Aerobiologia, 31 (3). pp. 411-419.

Chapter 7 (paper 1): Kolby, Jonathan E., Ramirez, Sara D., Berger, Lee, Richards-Hrdlicka, Kathryn, Jocque, Merljin, and Skerratt, Lee F. (2015) Terrestrial dispersal and potential environmental transmission of the amphibian chytrid fungus (Batrachochytrium dendrobatidis). PLoS ONE, 10 (4). pp. 1-13.

Chapter 9 (paper 1): Martel, A., Blooi, M., Adriaensen, C., Van Rooij, P., Beukema, W., Fisher, M.C., Farrer, R.A., Schmidt, B.R., Tobler, U., Goka, K., Lips, K.R., Muletz, C., Zamudio, K.R., Bosch, J., Lötters, S., Wombwell, E., Garner, T.W.J., Cunningham, A.A., Spitzen-van der Sluijs, A., Salvidio, S., Ducatelle, R., Nishikawa, K., Nguyen, T.T., Kolby, J.E., Van Bocxlaer, I., Bossuyt, F., and Pasmans, F. (2014) Recent introduction of a chytrid fungus endangers Western Palearctic salamanders. Science, 346 (6209). pp. 630-631.

Date Deposited: 22 Feb 2018 06:44
FoR Codes: 05 ENVIRONMENTAL SCIENCES > 0502 Environmental Science and Management > 050202 Conservation and Biodiversity @ 50%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070704 Veterinary Epidemiology @ 50%
SEO Codes: 96 ENVIRONMENT > 9604 Control of Pests, Diseases and Exotic Species > 960405 Control of Pests, Diseases and Exotic Species at Regional or Larger Scales @ 50%
96 ENVIRONMENT > 9604 Control of Pests, Diseases and Exotic Species > 960401 Border Biosecurity (incl. Quarantine and Inspection) @ 50%
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