Emerging Infectious Disease and the Trade in Amphibians

Amphibians are the most threatened class of vertebrate, and rates of species decline and extinction far exceed those seen historically. Habitat loss, climate change, over-exploitation and emerging infectious disease have all been identified as threatening processes. The trade in amphibians has been implicated in over-exploitation through the harvesting of wild animals, and as an important pathway for the global spread of the fungal pathogen, Batrachochytrium dendrobatidis (Bd). However, there are no analyses of how Bd may spread through the trade chain. This thesis addresses this issue by (1) determining the prevalence of Bd at different stages of the trade chain; (2) examining knowledge, husbandry protocols and biosecurity among retailers and (3) assessing the risk of Bd dissemination into the wild in the UK. Approximately 20,000 amphibians from at least 11 countries enter the UK annually via Heathrow Animal Reception Centre. Overall Bd prevalence was 3.6%, but was confined to six of the 43 genera encountered, and only detected in shipments from the USA and Tanzania. Amphibians were sold by 30% of the estimated 3500 livestock retailers in the UK, but made a low contribution to overall income. Disease awareness and knowledge in retailers was found to be lacking, although husbandry standards were deemed to be appropriate. Mortality appeared to be influenced by restocking methods and number of species held, but mass die-offs as a result of disease were generally uncommon. Screening of over 2000 amphibians from 148 retailers for Bd revealed a prevalence of 5.8%, but the geographic distribution of infection in the UK was patchy, and was more prominent in aquatic species. A risk assessment conducted according to the framework set out by the World Organisation of Animal Health, identified regions and sections of the trade that pose the greatest threats in terms of introducing Bd, and assessed various mitigation measures. The consequences of novel strains of Bd and a second, recently discovered Batrachochytrium species were found to pose a risk to both native UK and captive amphibians. As trade bans are unlikely to be feasible or effective, alternative measures to mitigate the impact of disease are evaluated

Detection of Batrachochytrium dendrobatidis in amphibians imported into the UK for the pet trade

There is increasing evidence that the global spread of the fungal pathogen Batrachochytrium dendrobatidis (Bd) has been facilitated by the international trade in amphibians. Bd was first detected in the UK in 2004, and has since been detected in multiple wild amphibian populations. Most amphibians imported into the UK for the pet trade from outside the European Union enter the country via Heathrow Animal Reception Centre (HARC), where Bd positive animals have been previously detected. Data on the volume, diversity and origin of imported amphibians were collected for 59 consignments arriving at HARC between November 2009 and June 2012, along with a surveillance study to investigate the prevalence of Bd in these animals. Forty three amphibian genera were recorded, originating from 12 countries. It was estimated that 5000 – 7000 amphibians are imported through HARC into the UK annually for the pet trade. Bd was detected in consignments from the USA and Tanzania, in six genera, resulting in an overall prevalence of 3.6%. This suggests that imported amphibians are a source of Bd within the international pet trade

Recent Asian origin of chytrid fungi causing global amphibian declines

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

T.W.J.G., M.C.F., D.S.S., A.L., E.C., F.C.C., J.B., A.A.C., C.M., F.S., B.R.S., S.O., were supported through the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity (NERC standard grant NE/K014455/1 and NE/E006701/1; ANR-08-BDVA-002-03). M.C.F., J.S., C.W., P.G. were supported by the Leverhulme Trust (RPG-2014-273), M.C.F., A.C., C.W. were supported by the Morris Animal Foundation. J.V. was supported by the Bolyai János Research Grant of the Hunagrian Academy of Sciences (BO/00597/14). F.G. and D.G. were supported by the Conservation Leadership Programme Future Conservationist Award. C.S.A. was supported by Fondecyt (No. 1181758). M.C.F. and A.C. were supported by. Mohamed bin Zayed Species Conservation Fund Project (152510704). GMR held a doctoral scholarship (SFRH/BD/69194/2010) from Fundação para a Ciência e a Tecnologia. L.F.T., C.L., L.P.R. K.R.Z., T.Y.J., T.S.J. were supported by São Paulo Research Foundation (FAPESP #2016/25358-3), the National Counsel of Technological and Scientific Development (CNPq #300896/2016–6) and a Catalyzing New International Collaborations grant from the United States NSF (OISE-1159513). C.S.A. was supported by Fondecyt (No. 1181758). T.M.D. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282).Peer reviewedPublisher PD

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

© The Author(s) 2018.Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.T.W.J.G., M.C.F., D.S.S., A.L., E.C., F.C.C., J.B., A.A.C., C.M., F.S., B.R.S., S.O., were supported through the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity (NERC standard grant NE/K014455/1 and NE/E006701/1; ANR-08-BDVA-002-03). M.C.F., J.S., C.W., P.G. were supported by the Leverhulme Trust (RPG-2014-273), M.C.F., A.C., C.W. were supported by the Morris Animal Foundation. J.V. was supported by the Bolyai János Research Grant of the Hunagrian Academy of Sciences (BO/00597/14). F.G. and D.G. were supported by the Conservation Leadership Programme Future Conservationist Award. C.S.A. was supported by Fondecyt (No. 1181758). M.C.F. and A.C. were supported by. Mohamed bin Zayed Species Conservation Fund Project (152510704). GMR held a doctoral scholarship (SFRH/ BD/69194/2010) from Fundação para a Ciência e a Tecnologia. L.F.T., C.L., L.P.R. K.R.Z., T.Y.J., T.S.J. were supported by São Paulo Research Foundation (FAPESP #2016/25358-3), the National Counsel of Technological and Scientifc Development (CNPq #300896/2016–6) and a Catalyzing New International Collaborations grant from the United States NSF (OISE-1159513). C.S.A. was supported by Fondecyt (No. 1181758). T.M.D. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282).Peer Reviewe

Clinically healthy amphibians in captive collections and at pet fairs : a reservoir of Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis is the cause of the fungal disease chytridiomycosis, a potentially lethal skin disease of amphibians. Asymptomatically infected amphibians may pose a risk for environmental pathogen pollution. This study therefore assessed the role of healthy, captive amphibians as a reservoir of Batrachochytrium dendrobatidis. Samples were collected from captive amphibians in Belgium, the Netherlands, Germany and France (559 from anurans, 330 from urodelans and 4 from gymnophians) from private owners, zoos, and laboratories. In addition to which, 78 anurans from 19 living collections were sampled during a pet fair in the Netherlands. Nearly 3% of the captive amphibians were infected by B. dendrobatidis, and 13.6% of the collections yielded at least one positive result. At the fair, 7 out of 78 anurans, representing 2 collections were positive. None of the animals that tested positive showed any obvious health problems at the time of sampling. Our results demonstrate the potential of the amphibian pet trade as a vehicle for the spread of B. dendrobatidis

West Africa - A Safe Haven for Frogs? A Sub-Continental Assessment of the Chytrid Fungus (Batrachochytrium dendrobatidis)

A putative driver of global amphibian decline is the panzootic chytrid fungus Batrachochytrium dendrobatidis (Bd). While Bd has been documented across continental Africa, its distribution in West Africa remains ambiguous. We tested 793 West African amphibians (one caecilian and 61 anuran species) for the presence of Bd. The samples originated from seven West African countries - Bénin, Burkina Faso, Côte d'Ivoire, Ghana, Guinea, Liberia, Sierra Leone - and were collected from a variety of habitats, ranging from lowland rainforests to montane forests, montane grasslands to humid and dry lowland savannahs. The species investigated comprised various life-history strategies, but we focused particularly on aquatic and riparian species. We used diagnostic PCR to screen 656 specimen swabs and histology to analyse 137 specimen toe tips. All samples tested negative for Bd, including a widespread habitat generalist Hoplobatrachus occipitalis which is intensively traded on the West African food market and thus could be a potential dispersal agent for Bd. Continental fine-grained (30 arc seconds) environmental niche models suggest that Bd should have a broad distribution across West Africa that includes most of the regions and habitats that we surveyed. The surprising apparent absence of Bd in West Africa indicates that the Dahomey Gap may have acted as a natural barrier. Herein we highlight the importance of this Bd-free region of the African continent - especially for the long-term conservation of several threatened species depending on fast flowing forest streams (Conraua alleni (“Vulnerable”) and Petropedetes natator (“Near Threatened”)) as well as the “Critically Endangered” viviparous toad endemic to the montane grasslands of Mount Nimba (Nimbaphrynoides occidentalis)

Detailed maps of West Africa.

<p>From top to bottom, depicting the most western positive records of <i>Bd</i> (black) and the negative records (transparent grey) (2a). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056236#pone-0056236-g002" target="_blank">Figure 2b</a> indicates in white transparent lines the transport system (roads) of the region. If <i>Bd</i> is transported via humans, the area around Accra (Ghana) is most likely to be the point of introduction (well connected via transportation routes and highly suitable environment). Further shown (2c) are the extents of the potentially forest regions (green) with the Upper Guinea Forests west of the Dahomey Gap <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056236#pone.0056236-Burgess1" target="_blank">[after 131]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056236#pone.0056236-Daszak1" target="_blank">[2a]</a>. In 2d the known point localities of <i>Conraua alleni</i> (transparent yellow), <i>Petropedetes natator</i> (transparent blue) (light green = overlapping localities), and <i>Nimbaphrynoides occidentalis</i> (dark green) are depicted.</p

Map of confirmed records of <i>Bd</i> on the African continent (black dots).

<p>Grey transparent dots represent the West African localities with negative <i>Bd</i> records. The hollow black circles indicate <i>Bd</i> positive localities <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056236#pone.0056236-SotoAzat1" target="_blank">[87]</a> which were not used for modelling. The three red colours represent the geographical extent of three different models, predicting the potential distribution of <i>Bd</i>. Modelling is based on the conditions of sites with confirmed presence of the pathogen (light red = maximum; red = mean; dark red = minimum; for niche parameters see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056236#pone-0056236-t002" target="_blank">Table 2</a>).</p