RESEARCH PROJECT:

Out of Africa? On the origin of amphibian chytridiomycosis.

Claudio Soto-Azat, M.V.

Literature review and scientific paper submitted in part fulfilment of the requirements for the degree of Master of Science in Wild Animal Health, University of London, 2007. Soto-Azat: Origin of chytridiomycosis

STATEMENT OF CONTRIBUTION

The author of the present manuscript was responsible for the collection, analysis, and interpretation of all the data involved in the present project and has written up the present report. The idea and the collection permits were developed and requested by the project supervisor Dr. Andrew Cunningham.

Cover: Golden toad (Bufo periglenes) in amplexus, Monteverde, Costa Rica, 1989. © G. Dimijia and M. Dimijia.

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TABLE OF CONTENTS

1. LITERATURE REVIEW: “Chytridiomycosis: present, past and future” ……………………………………………………….... 4-15

a. The threat of emerging infectious diseases ...……………. .. 4 b. Chytridiomycosis and amphibian declines ...... ……………. 5 c. Batrachochytrium dendrobatidis a recently spread pathogen 6 d. Sampling methods and diagnostic tests …………………… 7 e. Africa: origin of chytridiomycosis? ………………………... 7 f. Future implications.………...……………………………… 10 g. Literature cited …………………………………………….. 11

2. SCIENTIFIC PAPER: “Out of Africa? On the origin of chytridiomycosis” …………………………………………… 16-29

a. Abstract …………………………………………………… 16 b. Introduction ……………………………………………….. 16 c. Material and methods ……………………………………... 18 d. Results ……………………………………………………... 20 e. Discussion …………………………………………………. 22 f. Acknowledgements ………………………………………… 25 g. Literature cited …………………………………………….. 26

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Amphibian chytridiomycosis: present, past and future

C. Soto-Azat1,2

1Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, UK. 2The Royal Veterinary College, Royal College Street, London NW1 0TU, UK.

The threat of emerging infectious diseases

Emerging infectious diseases (EIDs) have been reported increasingly as causes of death and population declines of free living wild animals (Daszak et al. 2000). These diseases, are those that have recently increased in incidence or geographic range, recently moved into new host populations, recently been discovered or are caused by newly-evolved pathogens (Daszak et al. 2000). Disease emergence most frequently results from a change in ecology of host, pathogen or both (Schrag & Wiener 1995). Some EIDs have emerged as a result of anthropogenic effects on climate, which consequently have generated an increase in frequency and severity of weather patterns (Daszak et al. 2000). Flooding and abnormally high rainfall have been followed by outbreaks of Rift Valley fever virus in humans and domestic cattle in Kenya (Linthicum et al. 1999), and the El Niño Southern Oscillation has been associated with human cholera epidemics in South America (Colwell 1996).

Other EIDs are associated with “spill-over” from domestic animals to wild animals living in proximity (Daszak et al. 2000). In West Africa, the transmission of rabies from domestic dogs to African wild dogs (Lycaon pictus) has been responsible of severe population declines (Cleaveland & Dye 1995, Kat et al. 1995). In this case, domestic animals may act as maintenance hosts, enabling the pathogen to avoid the threshold density effect and drive the smaller population of wild animals to virtual extinction, as occurred in the Serengeti in 1991, when African wild dogs became locally extinct concurrent with epizootic of canine distemper in sympatric domestic dogs (Macdonald 1992, Alexander et al. 1996).

However, in most cases the emergence of infectious pathogens has been driven by direct human intervention, via host or parasite translocations, facilitated by the globalization of agriculture, commerce and human travel (Daszak et al. 2001). This phenomenon termed “pathogen pollution” is rooted in the unprecedented globalization of the transport of domestic animals and their products, wild animals (for pets, hunting, research, food or conservation), contaminated products and materials (Daszak et al. 2001, Cunningham et al. 2003). These movements have been linked to the emergence of a series of diseases, such as West Nile virus in the Americas (Anderson et al. 1999), squirrel poxvirus in the UK (Sainsbury et al. 2000) and avian malaria in Hawaii

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(Vanriper et al. 1986), among others. These pathogen introductions may pose a significant threat to global biodiversity when disease is introduced into naïve populations (Daszak et al. 2000, Cunningham et al. 2003) and therefore has not undergone selection for resistance to them (Skerratt et al. 2007).

Chytridiomycosis and amphibian declines

Chytridiomycosis, a recently described emerging disease of amphibians caused by the non-hyphal zoosporic chytrid fungus Batrachochytrium dendrobatidis (Bd) (Berger et al. 1998, Longcore et al. 1999) has been implicated in epizootic amphibian mass mortality on a global scale (Skerratt et al. 2007). This pathogen has been associated with amphibian population declines in at least Australia, Central America, Ecuador, USA, Spain and New Zealand (Lips 1999, Bosch et al. 2001, Bradley et al. 2002, Green et al. 2002, Muths et al. 2003, Ron et al. 2003, Bell et al. 2004, Bosch & Martinez-Solano 2006). Furthermore, it is believe to be responsible of the extinction of the golden toad (Bufo periglenes) in Costa Rica, the sharp-snouted day frog (Taudactylus acutirostris) in North-East Australia, and possibly contributed to the disappearance of some species of harlequin frogs (Atelopus sp.) in Central and South America, and the Northern and Southern gastric-brooding frogs (Rheobatrachus vitellinus and R. silus, respectively) from Eastern Australia (Daszak et al. 1999, Daszak & Cunningham 1999, Wright et al. 2001, Ron et al. 2003, La Marca et al. 2005, Pounds et al. 2006, Schloegel et al. 2006). This highly pathogenic, virulent and readily transmissible emergent disease has no precedent (Skerratt et al. 2007), and it has been described as “the worst infectious disease ever recorded among vertebrates in terms of the number of species impacted, and its propensity to drive them to extinction” (ACAP 2005).

Chytrids are ubiquitous fungi found in aquatic habitats and moist soil, where they either degrade cellulose, chitin or keratin (Powell 1993). Parasitic chytridiomycetes are known to infect plants, algae, protists and invertebrates (Powell 1993). The amphibian pathogen is the first chytrid recognized as a parasite of vertebrates (Longcore et al. 1999). Batrachochytrium dendrobatidis causes an infection of the skin resulting in hyperkeratosis, sloughing, erosions of the epidermis, rarely ulcerations, and finally death due to either the release of toxins by the fungus, or disturbance in respiration, or osmoregulation through the damaged skin (Berger et al. 1999b, Daszak et al. 2001). The clinical signs are non-specific and include abnormal posture, lethargy, and loss of righting reflex (Daszak et al. 1999). The life cycle consists of aquatic motile uniflagellated zoospores which invade keratinized tissues of amphibians, especially of the hind limbs and ventral patch of adults, where they develop into a stationary, intracellular sporangium restricted to the stratum corneum and stratum granulosus of the epidermis (Berger et al. 1998). Inflammatory response to infection is negligible (Berger et al. 1998). Each sporangium produces new zoospores and subsequently releases them via discharge tubes into the environment (Berger et al. 1999b). Tadpoles appear to be unaffected by the fungus which infects their keratinised mouthparts, in fact it seems that tadpoles can act as a source of infection for later life stages, causing “post-metamorphic death syndrome” (Berger et al. 1998). Although a saprophytic stage is unknown, Bd can be found in soil and water from a wide variety of environments (Berger et al. 1999b), and may survive in sterile lake water for as long as seven weeks (Johnson & Speare 2003) and in sterile moist river sand for up to 12 weeks

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(Johnson & Speare 2005). Its occurrence in Canadian wild frogs provide evidence that the fungus may survive the cold winter temperatures of Ontario and Québec (Oullet et al. 2005). Such persistence in the environment and via aclinical infection of larval mouthparts, lowers the threshold host density for the disease and allows it to cause the observed catastrophic declines, local and global extinctions (Daszak et al. 1999).

Currently, there are three hypotheses to explain the emergence of Bd: (1) it has been introduced recently into new areas, (2) it has recently become more pathogenic to the hosts, and (3) Amphibians have a lowered resistance due to environmental changes allowing an endemic parasite to cause mass mortalities (Berger et al. 1999a). The present manuscript gives evidence to support the first theory.

Batrachochytrium dendrobatidis a recently spread pathogen

The simultaneous discovery of the pathogen in Panama and Australia, the wave- like spread of declines in Australia and Central America and the catastrophic rate of population declines, all suggest that the disease has been introduced into these areas from an area of enzootic infection elsewhere (Berger et al. 1998, Daszak et al. 1999, Lips et al. 2006). To date, chytridiomycosis is known to infect over 200 species of anurans and caudates worldwide (Hyatt et al. 2007). Infected amphibians have been found in the international amphibian trade for food, pet stores, ornamental pond stocking, zoos, laboratories, and in amphibians species introduced into Australia, Europe, North and South America (Pessier et al. 1999, Mutschmann et al. 2000, Mazzoni et al. 2003, Daszak et al. 2004, Hanselmann et al. 2004, Cunningham et al. 2005, Garner et al. 2006). Furthermore, Fisher and Garner (2007) reported the infection in 28 species of introduced amphibians. Of these, the three most widely distributed species, the African clawed frog (Xenopus laevis), the North American bullfrog (Rana catesbiana) and the cane toad (Bufo marinus), have established feral populations in the Americas, Europe, Australia, Asia and many oceanic and coastal islands. These three invasive species appear to be resistant to chytridiomycosis, as free-living chytrid- infected populations of these species have not experienced die-offs, while captive individuals of X. laevis and R. catesbiana have been shown to have low levels of infection in the absence of clinical signs. Such carriers of the infection have been demonstrated to transmit the infection to other, susceptible amphibian species resulting in their mortality (Parker et al. 2002, Daszak et al. 2004). This implies that these species may act as vectors of chytridiomycosis (Daszak et al. 2003).

Genetic analyses of Bd isolates from Australia, Canada, USA, Panama and West Africa, indicate that they are very highly conserved genetically and that the disease might have spread by clonal propagation (Morehouse et al. 2003). This also suggests, that chytridiomycosis has been rapidly and recently globally spread (Skerratt et al. 2007).

Finally, retrospective histological surveys for Bd from archived museum amphibian specimens from Canada, USA, Australia, Ecuador and Venezuela, collected before the reported population declines, showed no evidence of chytrid infection (Berger et al. 1998, Green & Sherman 2001, Bonaccorso et al. 2003, Ron et al. 2003, Oullet et al. 2005).

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It has been found that amphibians from temperate and from tropical montane areas are more susceptible to the effects of chytrid infection (Berger et al. 1998, Daszak et al. 1999, Muths et al. 2003). Furthermore, Pounds et al. (2006) and Bosh et al. (2007) suggest that widespread amphibian declines and extinctions are associated with global warming through the chytrid-thermal-optimum hypothesis, which proposes increased cloud cover resulting from warming in the highlands of Central, South America and Spain, resulting in daytime cooling and an increase in humidity, which could facilitate the effects of Bd by either promoting its spread, or increasing its virulence (Skerratt et al. 2007). Therefore, even when it seems that Bd fulfil the novel pathogen hypothesis (Daszak et al. 2003, Rachowicz et al. 2005), effects of environmental changes influencing the presentation of the disease should not be underestimated (Bosch et al. 2007).

Sampling methods and diagnostic tests

To date a range of diagnostic assays exists for the detection of Bd in live and dead tadpoles and adults (Hyatt et al. 2007). These include histopathology (Berger et al. 1998), immunohistochemistry (Berger et al. 2002, Van Ells et al. 2003, Olsen et al. 2004), electron microscopy (Berger et al. 2002), conventional polymerase chain reaction (PCR) assay (Annis et al. 2004) and quantitative real-time PCR Taqman (qPCR) assay (Boyle et al. 2004).

Additionally, a range of sampling methods has been developed for the detection of Bd. For histological examination, skin samples are taken from the areas were lesions occur predominantly, namely ventral abdomen, ventral pelvis, hind limbs and hind feet; i.e. areas that are in closest contact with water or moist substrate (Longcore et al. 1999). The sampling methods for qPCR analysis include, water baths, toe-clips, oral disc excisions and swabs (Boyle et al. 2004). When compared with histology and immunohistochemistry, qPCR has been demonstrated to be faster, cheaper and to have higher sensitivity, repeatability and reproducibility, mainly due to the ability to detect the chytrid at lower levels and at earlier stages of infection (Hyatt et al. 2007). On the other hand, when compared with standard-PCR, qPCR has been demonstrated to be faster, less prone to contamination, capable to detect such small quantities of zoospores as one present in the test sample (v/s 10 of standard-PCR), and able to give objective accurate quantitation of unknowns (Boyle et al. 2004). Although the use of toe-clips can be used to detect Bd infection earlier in the course of infection than the use of skin swabs, the latter have been demonstrated to have the highest sensitivity of all sampling protocols. Therefore the recommended sampling procedure for the detection of Bd infection is to analyse skin swabs using the qPCR assay (Hyatt et al. 2007).

Africa: origin of chytridiomycosis?

The finding of Bd in the skin of an individual of X. laevis collected in 1938, from the Western Cape Region, South Africa, and the subsequent stable prevalence of the chytrid infection until the year 2001, in specimens of three species (X. laevis, X. gilli and X. muelleri), all collected in South Africa, Lesotho and Swaziland, suggests that a stable endemic relationship between pathogen and host existed in this region (Weldon et al. 2004). Thus, Weldon et al. (2004) have proposed that the disease originated in

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Africa and was introduced to naïve amphibian populations at a global scale by anthropogenic activities.

Currently in Africa, Bd infection is known to occur in 20 anuran species from South Africa, Swaziland, Lesotho, Botswana, Tanzania, Kenya and Ghana, nevertheless, amphibian declines have not been reported (Hopkins & Channing 2002, Weldon 2002, Lane et al. 2003, Speare & Berger 2004, Weldon & Du Preez 2004, Weldon et al. 2004). It has been suggested that Xenopus spp., are among those in which the pathogen originated (Weldon et al. 2004). For this group of fully aquatic amphibians, infection has been reported from wild X. laevis, X. gilli, X. petersii, X. muelleri, captive X. tropicalis exported to USA from Ghana, and captive Hymenochirus boettgeri exported to Vancouver (Canada) from Florida (USA) (Reed et al. 2000, Raverty & Reynolds 2001, Morehouse et al. 2003, Weldon et al. 2004, Weldon 2005). Using histology, Weldon et al. (2004) have reported the earliest known historical cases of Bd infection. After the 1938 record, the next chytrid positive cases were a specimen of X. gilli collected in 1943, a species only known to exist in restricted areas of the Western Cape in South Africa, and a specimen of X. petersii collected in 1965 from Botswana (Weldon 2005). Subsequent histological examination for the detection of Bd from African museum archived amphibians have shown that until 1996, 21 chytrid-positive cases have been detected in South Africa, Swaziland and Southern Botswana (Weldon & Du Preez 2004), which suggest that Southern Africa may be the origin of the pathogen (Table 1).

Its spread from Africa seems to have been facilitated by the enormous quantities of wild X. laevis exported since the 1930s from South Africa to USA, Australasia and Europe for scientific purposes (Weldon et al. 2004, Fisher & Garner 2007). As Bd does not survive desiccation, transportation of the pathogen over long distances is most likely to occur via the movement of live amphibians (Berger et al. 1999b, Daszak et al. 2000). On the other hand, R. catesbiana is widely farmed as a food product and its trade has growth exponentially in the last decades (Weldon et al. 2004, Garner et al. 2006). In this last species, the pathogen has been identified from archived specimens since 1978 (Hanselmann et al. 2004). It is suspected that these two species plus B. marinus may become the most likely way of the widespread dissemination of the disease between regions (Daszak et al. 2003, Mazzoni et al. 2003, Weldon et al. 2004).

Outside the African continent, Bd infection first appeared in amphibian populations in North America (1961), Australia (1978), South America (1980), Central America (1986), Europe (1997) and New Zealand (1999) (Bosch et al. 2001, Waldman et al. 2001, Puschendorf 2003, Ron et al. 2003, Oullet et al. 2005, Speare & Berger 2005) (Table 2). The importance of these records relies in that all of them precede or at least are concurrent with amphibian population declines in their respective regions (Berger et al. 1998, Bosch et al. 2001, Waldman et al. 2001, Bradley et al. 2002).

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Table 1. Documented African amphibian correlative cases of Batrachochytrium dendrobatidis infection, between 1938 and 1998.

Year Species Area Country Reference

1938 Xenopus laevis Western Cape South Africa (Weldon et al. 2004) 1943 Xenopus gilli Western Cape South Africa (Weldon et al. 2004) 1965 Xenopus petersi Southern Botswana Botswana (Weldon 2005) 1969 Xenopus laevis Kanye Youth Centre Botswana (Weldon 2005) 1972 Xenopus laevis Free State South Africa (Weldon et al. 2004) 1972 Xenopus laevis Eastern Cape South Africa (Weldon et al. 2004) 1973 Xenopus laevis KwaZulu-Natal South Africa (Weldon et al. 2004) 1974 Xenopus laevis Free State South Africa (Weldon et al. 2004) 1974 Xenopus laevis Western Cape South Africa (Weldon et al. 2004) 1976 Xenopus gilli Western Cape South Africa (Weldon et al. 2004) 1981 Heleophryne regis Western Cape South Africa (Weldon 2005) 1982 Xenopus laevis Western Cape South Africa (Weldon et al. 2004) 1985 Xenopus laevis KwaZulu-Natal South Africa (Weldon et al. 2004) 1987 Xenopus laevis Free State South Africa (Weldon et al. 2004) 1991 Xenopus muelleri Swaziland Swaziland (Weldon et al. 2004) 1991 Xenopus laevis Free State South Africa (Weldon et al. 2004) 1991 Xenopus laevis Free State South Africa (Weldon et al. 2004) 1995 Xenopus laevis KwaZulu-Natal South Africa (Weldon et al. 2004) 1996 Afrana fuscigula Northern Cape South Africa (Weldon 2005) 1996 Xenopus laevis Free State South Africa (Weldon et al. 2004) 1996 Xenopus laevis Northern Cape South Africa (Weldon et al. 2004)

Table 2. Documented earliest cases of Batrachochytrium dendrobatidis infection by region.

Year Species Location Country Reference

1938 Xenopus laevis Western Cape South Africa (Weldon et al. 2004) 1961 Rana clamitans Québec Canada (Oullet et al. 2005) 1978 Litoria gracilenta South East Queensland Australia (Speare et al. 2005) 1980 Atelopus bomolochos Provincia del Cañar Ecuador (Ron et al. 2003) 1986 Atelopus varius Sarapiquí Costa Rica (Puschendorf 2003) 1997 Alytes obstetricans Peñalara National Park Spain (Bosch et al. 2001) 1999 Litoria raniformis Christchurch New Zealand (Waldman et al. 2001)

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Future implications

Currently, it seems that chytridiomycosis is widely spread globally, and while new studies are carried out, increase in the number of affected species and emergence of the pathogen in new areas, are more frequently reported (Lane et al. 2003, Mazzoni et al. 2003, Hanselmann et al. 2004, Beard & O'Neill 2005, Cunningham et al. 2005, Garner et al. 2005, Herrera et al. 2005, Lampo et al. 2006, Fisher & Garner 2007). Consequently, efforts should concentrate in the development of management measures to control the transmission of the disease into naïve susceptible amphibian populations, in order to prevent the appearance of new severe amphibian population crashes and extinctions (Berger et al. 1999a, Skerratt et al. 2007). For instance the set up of regional diagnostic centres and rapid response teams to deal with outbreaks, has been suggested (Skerratt et al. 2007).

Effective management of chytridiomycosis will depend on different countries recognising the disease as a threatening process for native amphibians and local ecosystems, and identifying the extent of spread of the disease in both wild and captive amphibians, including those species present in the food, laboratory, zoo and pet trade (Hyatt et al. 2007). Countries that are currently more affected by the effects of chytridiomycosis, are responding with the development of threat abatement plans. Australia have declared the disease as a “key threatening process”, in order to create a national strategy to manage and reduce the impacts of the disease on the environment (DEH 2005). As eradication of the disease is apparently not feasible at present, mitigation actions, focused on preventing the introduction and spreading of the pathogen into chytridiomycosis-free areas, and decreasing the impact of the disease on populations that are currently infected, should be undertaken (DEH 2005). Furthermore, Fisher and Garner (2007) stressed the importance of reduce the risk of importation, release of Bd, release of amphibians; and limit the spread of Bd once release have occurred.

Additionally, there is a necessity for chytridiomycosis to be listed as notifiable amphibian disease in the World Organization for Animal Health (OIE); furthermore, this organism should develop standards for the growing amphibian international trade, with consequent testing requirements for imports and exports of amphibians, which seems to be the most significant factor in the current pandemic of chytridiomycosis.

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LITERATURE CITED

ACAP (2005) Amphibian Conservation Summit. 17-19 September 2005, Washington D.C. Alexander KA, Kat PW, Munson LA, Kalake A, Appel MJG (1996) Canine distemper- related mortality among wild dogs (Lycaon pictus) in Chobe national park, Botswana. Journal of Zoo and Wildlife Medicine 27:426-427 Anderson JF, Andreadis TG, Vossbrinck CR, Tirrell S, Wakem EM, French RA, Garmendia AE, Van Kruiningen HJ (1999) Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut. Science 286:2331-2333 Annis SL, Dastoor FP, Ziel H, Daszak P, Longcore JE (2004) A DNA-based assay identifies Batrachochytrium dendrobatidis in amphibians. Journal of Wildlife Diseases 40:420-428 Beard KH, O'Neill EM (2005) Infection of an invasive frog Eleutherodactylus cocqui by the chytrid fungus Batrachochytrium dendrobatidis in Hawaii. Biological Conservation 126:591-595 Bell BD, Carver S, Mitchell NJ, Pledger S (2004) The recent decline of a New Zealand endemic: how and why did populations of Archey's frog Leiopelma archeyi crash over 1996-2001? Biological Conservation 120:189-199 Berger L, Hyatt AD, Olsen V, Hengstberger SG, Boyle D, Marantelli G, Humphreys K, Longcore JE (2002) Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians. Diseases of Aquatic Organisms 48:213-220 Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, Slocombe R, Ragan MA, Hyatt AH, McDonald KR, Hines HB, Lips KR, 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 Science, USA 95:9031-9036. Berger L, Speare R, Hyatt A (1999a) Chytrid fungi and amphibian declines: Overview, implications and future directions. In: Campbell A (ed) Declines and Disappearances of Australian Frogs. Environment Australia, Canberra, p 23-33 Berger L, Speare R, Hyatt A (1999b) Chytrid fungi and amphibian declines: overview, implications and future directions. In: Campbell A (ed) Declines and disappearances of Australian frogs. Environment Australia, Canberra, p 23-33 Bonaccorso E, Guayasamin JM, Méndez D, Speare R (2003) Chytridiomycosis in a Venezuelan amphibian (Bufonidae: Atelopus cruciger). Herpetological Review 34:331-334 Bosch J, Carrascal LM, Duran L, Walker S, Fisher MC (2007) Climate change and outbreaks of amphibian chytridiomycosis in a montane area of Central Spain; is there a link? Proceedings of the Royal Society B-Biological Sciences 274:253- 260 Bosch J, Martinez-Solano I (2006) Chytrid fungus infection related to unusual mortalities of Salamandra salamandra and Bufo bufo in the Penalara Natural Park, Spain. Oryx 40:84-89 Bosch J, Martinez-Solano I, Garcia-Paris M (2001) Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (Alytes obstetricans) in protected areas of central Spain. Biological Conservation 97:331-337

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Boyle DG, Boyle DB, Olsen V, Morgan JAT, Hyatt AD (2004) Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Diseases of Aquatic Organisms 60:141-148 Bradley GA, Rosen PC, Sredl MJ, Jones TR, Longcore JE (2002) Chytridiomycosis in native Arizona frogs. Journal of Wildlife Diseases 38:206-212 Cleaveland S, Dye C (1995) Maintenance of a microparasite infecting several host species: Rabies in the Serengeti. Parasitology 111:S33-S47 Colwell RR (1996) Global climate and infectious disease: The cholera paradigm. Science 274:2025-2031 Cunningham AA, Daszak P, Rodriguez JP (2003) Pathogen polution: defining a parasitological threat to biodiversity conservation. Journal of Parasitology 89(Suppl):S78-S83 Cunningham AA, Garner TWJ, Aguilar-Sanchez V, Banks B, Foster J, Sainsbury AW, Perkins M, Walker SF, Hyatt AD, Fisher MC (2005) Emergence of amphibian chytridiomycosis in Britain. Veterinary Record 157:386-387 Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE, Speare R (1999) Emerging infectious diseases and amphibian population declines. Emerging Infectious Diseases 5:735-748 Daszak P, Cunningham AA (1999) Extinction by infection. Trends in Ecology and Evolution 14:279 Daszak P, Cunningham AA, Hyatt AD (2000) Wildlife ecology - Emerging infectious diseases of wildlife - Threats to biodiversity and human health. Science 287:443-449 Daszak P, Cunningham AA, Hyatt AD (2001) Anthropogenic environmental change and the emergence of infectious diseases in wildlife. Acta Tropica 78:103-116 Daszak P, Cunningham AA, Hyatt AD (2003) Infectious disease and amphibian population declines. Diversity and Distributions 9:141-150 Daszak P, Strieby A, Cunningham AA, Longcore JE, Brown CC, Porter D (2004) Experimental evidence that the bullfrog (Rana catesbeiana) is a potential carrier of chytridiomycosis, an emerging fungal disease of amphibians. Herpetological Journal 14:201-207 DEH (2005) Threat Abatement Plan for infection of amphibians with chytrid fungus resulting in chytridiomycosis: Threat Abatement Plan, Department of Environment and Heritage, Canberra Fisher MC, Garner TWJ (2007) The relationship between the emergence of Batrachochytrium dendrobatidis, the international trade in amphibians and introduced amphibian species. Fungal biology reviews 21:2-9 Garner TWJ, Perkins MW, Govindarajulu P, Seglie D, Walker S, Cunningham AA, Fisher MC (2006) The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana. Biology Letters 2:455-459 Garner TWJ, Walker S, Bosch J, Hyatt AD, Cunningham AA, Fisher MC (2005) Chytrid fungus in Europe. Emerging Infectious Diseases 11:1639-1641 Green DE, Converse KA, Schrader AK (2002) Epizootiology of sixty-four amphibian morbidity and mortality events in the USA, 1996-2001. Annals of the New York Academy of Science 969:323-339 Green DE, Sherman CK (2001) Diagnostic histological findings in Yosemite Toads (Bufo canorus) from a die-off in the 1970s. Journal of Herpetology 35:92-103

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Morehouse EA, James TY, Ganley ARD, Vilgalys R, Berger L, Murphy PJ, Longcore JE (2003) Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently emerged clone. Molecular Ecology 12:395-403 Muths E, Corn PS, Pessier AP, Green DE (2003) Evidence for disease related amphibian decline in Colorado. Biological Conservation 110:357-365 Mutschmann F, Berger L, Zwart P, Gaedicke C (2000) Chytridiomycosis on amphibians - first report from Europe. Berliner Und Munchener Tierarztliche Wochenschrift 113:380-383 Olsen V, Hyatt AD, Boyle DG, Mendez D (2004) Co-localisation of Batrachochytrium dendrobatidis and keratin for enhanced diagnosis of chytridiomycosis in frogs. Diseases of Aquatic Organisms 61:85-88 Oullet M, Mikaelian I, Pauli BD, Rodrigue J, Green DM (2005) Historical evidence of widespread chytrid infection in North American amphibian populations. Conservation Biology 19:1431-1440 Parker JM, Mikaelian I, Hahn N, Diggs HE (2002) Clinical diagnosis and treatment of epidermal chytridiomycosis in African clawed frogs (Xenopus tropicalis). Comparative Medicine 52:265-268 Pessier AP, Nichols DK, Longcore JE, Fuller MS (1999) Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea). Journal of Veterinary Diagnostic Investigation 11:194-199 Pounds AJ, Bustamante MR, Coloma LA, Consuegra JA, Fogden MPL, Foster PN, la Marca E, Masters KL, Merino-Viteri A, Puschendorf R, Ron SR, Sanchez- Azofeifa GA, Still CJ, Young BE (2006) Widespread amphibian extinctions from epidemic disease driven by global warming. Nature 439:161-167 Powell MJ (1993) Looking at Mycology with a Janus Face - a Glimpse at Chytridiomycetes Active in the Environment. Mycologia 85:1-20 Puschendorf R (2003) Atelopus varius (harlequin frog) fungal infection. Herpetological Review 34:20 Rachowicz LJ, Hero JM, Alford RA, Taylor JW, Morgan JAT, Vredenburg VT, Collins JP, Briggs CJ (2005) The novel and endemic pathogen hypothesis: competing explanations for the origin of emerging infectious diseases of wildlife. Conservation Biology 19:1441-1448 Raverty S, Reynolds T (2001) Cutaneous chytridiomycosis in dwarf aquatic frogs (Hymenochirus boettgeri) originating from southeast Asia and in a western toad (Bufo boreas) from northeastern British Columbia. Canadian Veterinary Journal 42:385-386 Reed KD, Ruth GR, Meyer JA, Shukla SK (2000) Chlamydia pneumoniae infection in a breeding colony of African clawed frogs (Xenopus tropicalis). Emerging Infectious Diseases 6:196-199 Ron SR, Duellman WE, Coloma LA, Bustamante MR (2003) Population decline of the Jambato Toad Atelopus ignescens (Anura : Bufonidae) in the Andes of Ecuador. Journal of Herpetology 37:116-126 Sainsbury AW, Nettleton P, Gilray J, Gurnell J (2000) Grey squirrels have high seroprevalence to a parapoxvirus associated with deaths in red squirrels. Animal Conservation 3:229-233 Schloegel LM, Hero JM, Berger L, Speare R, McDonald K, Daszak P (2006) The decline of the sharp-snouted day frog (Taudactylus acutirostris): The first documented case of extinction by infection in a free-ranging wildlife species? Ecohealth 3:35-40

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Schrag SJ, Wiener P (1995) Emerging Infectious-Disease - What Are the Relative Roles of Ecology and Evolution. Trends in Ecology & Evolution 10:319-324 Skerratt L, Berger L, Speare R, Cashins S, McDonald KR, Phillott AD, Hines HB, Kenyon N (2007) Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth:1-10 Speare R, Berger L (2004) Global distribution of chytridiomycosis in amphibians. http://www.jcu.edu.au/school/phtm/PHTM/frogs/chyglob.htm. Downloaded 7 August 2007. Speare R, Berger L (2005) Chytridiomycosis in amphibians in Australia. http://www.jcu.edu.au/school/phtm/PHTM/frogs/chyspec.htm. Downloaded on 7 August 2007. Speare R, Skerratt L, L. B, Hines H, Hyatt A, Mendez D, McDonald K, Hero J, Marantelli G, Muller R, Alford R, Woods R (2005) A project that designs and trials a pilot survey to map the distribution of chytridiomycosis (caused by the amphibian chytrid) in Australian frogs, Department of Environment and Heritage, Canberra Van Ells T, Stanton J, Strieby A, Daszak P, Hyatt AD, Brown C (2003) Use of immunohistochemistry to diagnose chytridiomycosis in dyeing poison dart frogs (Dendrobates tinctorius). Journal of Wildlife Diseases 39:742-745 Vanriper C, Vanriper SG, Goff ML, Laird M (1986) The Epizootiology and Ecological Significance of Malaria in Hawaiian Land Birds. Ecological Monographs 56:327-344 Waldman B, van de Wolfshaar KE, Klena JD, Andjic V, Bishop P, Norman RJdB (2001) Chytridiomycosis in New Zealand frogs. Surveillance 28:9-11 Weldon C (2002) Chytridiomycosis survey in South Africa. Froglog 51:1-2 Weldon C (2005) Host species and localities of chytridiomycosis in Africa. http://www.puk.ac.za/fakulteite/natuur/soo/drk/aacrg/chytrid/chytrid_e.html. Downloaded 7 August 2007. Weldon C, Du Preez LH (2004) Decline of the Kihansi spray toad, Nectophrynoides asperginis, from the Udzungwa mountains, Tanzania. Froglog 62:2-3 Weldon C, du Preez LH, Hyatt AD, Muller R, Speare R (2004) Origin of the amphibian chytrid fungus. Emerging Infectious Diseases 10:2100-2105 Wright K, Berger L, Nichols DK, Speare R, Sredl MJ, Pessier A, Johnson B (2001) Roundtable: amphibian population decline. Journal of Herpetological Medicine and Surgery 11:14-27

15 Soto-Azat: Origin of chytridiomycosis

DISEASES OF AQUATIC ORGANISMS Vol. 1: 16-30, 2007 August 2007 Dis Aquat Org

Out of Africa? On the origin of amphibian chytridiomycosis

C. Soto-Azat1,2, A. A. Cunningham1, B. T. Clarke3, J. C. Poynton3

1Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, UK. 2The Royal Veterinary College, Royal College Street, London NW1 0TU, UK. 3Natural History Museum, Department of Zoology, Cromwell Rd, London SW7 5BD, UK.

ABSTRACT: Amphibian chytridiomycosis is an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). This pathogen is known to infect over 200 species of anurans and caudates, and has been associated with amphibian population declines and extinctions. Current evidence indicates that the pathogen has been recently introduced into new areas from an area of enzootic infection, and that the international trade of amphibians has been involved in the wide spread of the disease. We developed a novel, non-destructive method for the retrospective assessment of archived museum amphibians in order to test the hypothesis that Bd originated from Africa. The earliest case of Bd infection was found in a specimen of X. laevis bunyoniensis collected in 1935 in Uganda, and we suggest that by the 1930s Bd infection was widely established in sub-Saharan Africa. The finding of five cases of Bd infection from three species of Xenopus, the low levels of infection and the stable prevalence over the time detected, indicate that an historical equilibrium between Xenopus spp. and Bd infection have existed in Africa.

KEY WORDS: Batrachochytrium dendrobatidis, chytridiomycosis, Africa, Xenopus, museum specimens, emerging infectious disease.

INTRODUCTION

Emerging infectious diseases (EIDs) have been reported increasingly as causes of death and population declines of free living wild animals (Daszak et al. 2000). In most cases this emergence has been driven by direct human intervention, via host or parasite translocations (“pathogen pollution”), facilitated by the globalization of agriculture, commerce and human travel (Daszak et al. 2001, Cunningham et al. 2003). These movements have been linked to the emergence of a series of diseases, such as West Nile virus in the Americas (Anderson et al. 1999), squirrel poxvirus in the UK (Sainsbury et al. 2000) and avian malaria in Hawaii (Vanriper et al. 1986). Pathogen

16 Soto-Azat: Origin of chytridiomycosis introductions may pose a significant threat to global biodiversity when disease is introduced into naïve populations (Daszak et al. 2000, Cunningham et al. 2003) and therefore has not undergone selection for resistance to them (Skerratt et al. 2007).

Chytridiomycosis, a recently described emerging disease of amphibians caused by the non-hyphal zoosporic chytrid fungus Batrachochytrium dendrobatidis (Bd) (Berger et al. 1998, Longcore et al. 1999), has been associated with amphibian population declines in Australia, Central America, Ecuador, USA, Spain and New Zealand (Lips 1999, Bosch et al. 2001, Bradley et al. 2002, Green et al. 2002, Muths et al. 2003, Ron et al. 2003, Bell et al. 2004). Furthermore, it is believe to have been involved in the extinction of the sharp-snouted day frog (Taudactylus acutirostris) in Australia, the golden toad (Bufo periglenes) in Costa Rica and some species of harlequin frogs (Atelopus spp.) in Central and South America (Ron et al. 2003, La Marca et al. 2005, Pounds et al. 2006, Schloegel et al. 2006). The devastating effects of this highly pathogenic, virulent and readily transmissible emerging disease have no precedent in historical times (Skerratt et al. 2007), and it has been described as “the worst infectious disease ever recorded among vertebrates in terms of the number of species impacted, and its propensity to drive them to extinction” (ACAP 2005).

Batrachochytrium dendrobatidis causes an infection of the skin of post- metamorphic amphibians resulting in hyperkeratosis, sloughing, erosions of the epidermis, rarely ulcerations, and finally death due to either the release of toxins by the fungus, or disturbance in supplementary respiration or osmoregulation through the damaged skin (Berger et al. 1999, Daszak et al. 2001). The fungus can be found in soil and water from a wide variety of environments (Berger et al. 1999) and may survive in sterile lake water for as long as seven weeks (Johnson & Speare 2003) and in sterile moist river sand for up to 12 weeks (Johnson & Speare 2005). Its occurrence in Canadian wild frogs provide evidence that the chytrid may survive the cold winter temperatures of Ontario and Québec (Oullet et al. 2005). Such persistence in the environment and via aclinical infection of larval mouthparts, lowers the threshold host density for the disease and allows it to cause the observed catastrophic declines, local and global extinctions (Daszak et al. 1999).

The simultaneous discovery of the pathogen in Panama and Australia, the wave- like spread of declines in Australia and Central America and the catastrophic rate of population declines, all suggest that the fungus has been introduced into these areas from an area of enzootic infection elsewhere (Berger et al. 1998, Daszak et al. 1999, Lips et al. 2006). To date, chytridiomycosis is known to infect over 200 species of anurans and caudates worldwide (Kriger et al. 2006). Infected amphibians have been found in the international amphibian trade for food, pet stores, ornamental pond stocking, zoos, laboratories, and in amphibian species introduced into Australia, Europe, North and South America (Pessier et al. 1999, Mutschmann et al. 2000, Mazzoni et al. 2003, Daszak et al. 2004, Hanselmann et al. 2004, Cunningham et al. 2005, Garner et al. 2006). Furthermore, Fisher and Garner (2007) reported the infection in 28 species of introduced amphibians. Of these, the three most widely distributed species, the African clawed frog (Xenopus laevis), the North American bullfrog (Rana catesbiana), and the cane toad (Bufo marinus), have established feral populations in the Americas, Europe, Australia, Asia and many oceanic and coastal islands. These three invasive species appear to be resistant to chytridiomycosis as free-living chytrid infected populations of these species have not experienced die-offs, while captive individuals of X. laevis and R.

17 Soto-Azat: Origin of chytridiomycosis catesbiana have been shown to have low levels of infection in the absence of clinical signs. Such carriers of the infection have been demonstrated to transmit the pathogen to other, susceptible amphibian species, resulting in their mortality (Parker et al. 2002, Daszak et al. 2004). This implies that these species may act as vectors of chytridiomycosis (Daszak et al. 2003).

Genetic analyses of Bd isolates from Australia, Canada, USA, Panama and West Africa, indicate that they are very highly conserved genetically and that the disease might have spread by clonal propagation (Morehouse et al. 2003). This suggests that chytridiomycosis has been rapidly and recently globally spread (Skerratt et al. 2007).

Retrospective histological surveys for Bd from archived museum amphibian specimens from Canada, USA, Australia, Ecuador and Venezuela, collected before the reported population declines, showed no evidence of chytrid infection (Berger et al. 1998, Green & Sherman 2001, Bonaccorso et al. 2003, Ron et al. 2003, Oullet et al. 2005).

Currently, the earliest known case of Bd infection is in a specimen of X. laevis collected in 1938 in South Africa (Weldon et al. 2004), leading to the proposition that the disease originated in Africa and was subsequently introduced to naïve species globally by anthropogenic activities. As Bd does not survive desiccation, transportation of the pathogen over long distances is most likely to occur via the movement of live amphibians (Berger et al. 1999, Daszak et al. 2000). Between the 1930s and 1950s X. laevis was extensively exported from Africa for use in pregnancy assays for humans (Weldon et al. 2004, Rachowicz et al. 2005). More recently, Xenopus spp. have been used as experimental models for immunological, embryological and molecular biological research (Fisher & Garner 2007). On the other hand, R. catesbiana is extensively farmed as a food product and its international trade has growth exponentially in the last decades (Garner et al. 2006). It is now known that these highly traded species are widely infected with Bd, and therefore are likely to be implicated in the dissemination of the pathogen between regions (Daszak et al. 2003, Mazzoni et al. 2003, Cunningham et al. 2005, Garner et al. 2006, Fisher & Garner 2007).

In the present manuscript we describe the development of a novel, non- destructive method for the retrospective assessment of archived museum specimens for the presence of Bd infection. We use this method to test the hypothesis that Bd originated from Africa. Specifically, we test the hypotheses that: (1) Amphibians of the family Pipidae within Africa have been historically infected with bd, but amphibians of the same family outwith Africa have not; and (2) Prior to the recent global amphibian decline phenomenon, Bd infection was restricted to Xenopus spp. within Southern Africa.

MATERIAL AND METHODS

Specimens: We examined 666 frogs of the family Pipidae, archived at the Natural History Museum, London. The specimens surveyed were preserved in 70% ethanol and included tadpoles, post-metamorphic and adult frogs, of 23 species collected for purposes other than disease investigation, from South America and Africa between 1844 and 1994 (Table 1).

18 Soto-Azat: Origin of chytridiomycosis

Table 1. Summary of 666 frogs sampled at the Natural History Museum, London, for the detection of Batrachochytrium dendrobatidis.

Period of No. Species Country collection examined Hymenochirus boettgeri 1899-1963 27 Cameroon, Equatorial Guinea, Gabon, Nigeria. Hymenochirus curtipes 1920-1962 5 Congo, Dem. Rep. Congo. Hymenochirus feae 1902 2 Gabon. Pipa parva 1925 1 Venezuela. Pipa pipa 1844-1980 38 Bolivia, Brazil, French Guiana, Grenada, Guyana, Suriname, Trinidad and Tobago. Pipa carvalhoi 1947-1978 6 Brazil. Psuedohymenochirus merlini 1959-1976 4 Sierra Leone. Silurana epitropicalis 1979 4 Dem. Rep. Congo. Silurana tropicalis 1864-1952 101 Cameroon, Equatorial Guinea, Gabon, Ghana, Guinea-Bissau, Liberia, Nigeria, Sierra Leone. Xenopus clivii 1902-1934 15 Ethiopia, Sudan. Xenopus fraseri 1852-1948 38 Cameroon, Dem. Rep. Congo, Equatorial Guinea, Gabon. Xenopus gilli 1947-1985 14 South Africa. Xenopus laevis borealis 1900-1935 21 Kenya. Xenopus laevis bunyoniensis 1913-1935 68 Uganda. Xenopus laevis laevis 1855-1994 157 Dem. Rep. Congo, Eritrea, Kenya, Lesotho, Malawi, Mozambique, Namibia, South Africa, Sudan, Tanzania, Uganda. Zimbabwe. Xenopus laevis poweri 1955-1959 4 Dem. Rep. Congo, Zambia. Xenopus largeni 1972-1975 3 Ethiopia. Xenopus longipes 1984 44 Cameroon. Xenopus muelleri 1865-1940 58 Malawi, Mozambique, Nigeria, Tanzania, Zambia. Xenopus petersii 1864-1932 20 Angola, Dem. Rep. Congo, Tanzania, Zambia. Xenopus vestitus 1971-1975 12 Rwanda, Uganda. Xenopus victorianus 1904-1947 21 Dem. Rep. Congo, Uganda. Xenopus wittei 1977 3 Uganda.

Sampling: Post-metamorphic and adults specimens were firmly brushed, three to five times, against its ventral surfaces, including pelvis, hind limbs and hind feet, using a tapered interdental refill 3.2-6.0 mm brush (Oral-B Laboratories). For tadpoles the same brush was gently passed three times over the oral discs (because of its delicate nature). In order to minimize any chance of false positives, each individual specimen was sampled in a manner to prevent possible cross contamination and disposable gloves were always changed when a new jar containing frogs was opened. Once the sample was obtained, the interdental brush was deposited in a 1.5 ml eppendorf tube and stored at -80ºC prior processing.

19 Soto-Azat: Origin of chytridiomycosis

DNA extraction and real-time PCR assay: Each interdental brush was deposited in a tube containing 50 μl of PrepMan Ultra (Applied Biosystems) and between 30 and 40 mg of Zirconium/silica beads of 0.5 mm diameter (Biospec Products). The extraction was completed as described by Boyle et al. (2004) and 1/10 dilutions were stored at -80ºC. Diluted nucleic acid extractions were subsequently subjected to a quantitative real-time polymerase chain reaction Taqman assay (qPCR) of the ITS-1/5.8S ribosomal DNA region of Bd, using a Prism 7700 Sequence Detection System (Applied Biosystems) (Boyle et al. 2004, Hyatt et al. 2007). For each sample, the diagnostic assay was performed in duplicate, and standards of known zoospores concentrations were included with each PCR plate as were negative controls. As we were using dilutions as test samples, we corrected the results obtained by a factor of 10. A result was considered to be positive when: (1) amplification (i.e. a clearly sigmoid curve) occurred in both replicated PCR reactions, and (2) values higher than 0.1 genomic equivalents (GE) were obtained from both replicated reactions.

Validation of the brush sampling method: In order to validate the use of the interdental brush as a valid sampling method for the detection of Bd by qPCR, and because there was some concern that previous use of metallic swabs might have caused inhibition of the PCR reaction (Garner pers. comm.) (the interdental brushes incorporated a metal shaft), we conducted two experiments with known Bd positive frogs.

Expt 1––Ethanol fixed frogs: Six midwife toads (Alytes obstetricans), naturally infected with the chytrid fungus, and that were stored in 70% ethanol for a period of two years, were sampled on the same areas described above using three different methodologies: (1) swabbing, with a standard sterile swab; (2) brushing, with an interdental brush; and (3) scraping, with a Nº 20 sterile scalpel. All samples were subjected to the same DNA extraction and qPCR assay protocols, and its interpretation was performed as already explained. These frogs had been previously diagnosed as being infected with Bd by toeclip-qPCR.

Expt 2 ––Formalin fixed frogs: Two Cape clawed frogs (X. gilli) and four Mallorcan midwife toads (A. muletensis), naturally infected with the chytrid fungus, and that were stored in neutral buffered formalin for 16 ± 2 years, were sampled, analyzed and their results were interpreted in the same manner as described in experiment 1. These frogs had been previously diagnosed as being infected with Bd by histopathology.

RESULTS

Detection of Bd from archived Pipids: The overall prevalence of chytrid infection was 0.8%. None of the South American specimens were found to be positive. Five African frogs tested positive for Bd. These corresponded to three species and were from considerably distant areas within Africa. The earliest historical case was an adult X. laevis bunyoniensis collected in Uganda in 1935. The next cases were an adult X. fraseri and a tadpole X. laevis laevis collected in Cameroon and Malawi, in 1948 and 1969, respectively. Two adult frogs X. gilli simultaneously collected in 1985 from the Cape Point Nature Reserve, South Africa, in 1985, were also positive for Bd infection. Overall, infections were considered slight, and the average GE obtained was 3.7 ± 4.4 (Table 2).

20 Soto-Azat: Origin of chytridiomycosis

Table 2. Genomic equivalents (GE) of five museum archived amphibians, found positive to Batrachochytrium dendrobatidis infection. Note that specimen codes include the year of collection.

Specimen Species Age Origin GE SD 1935.10.10.295 X. laevis bunyoniensis adult Lake Bunyoni, Uganda. 10.3 7.1 1948.1.8.74 X. fraseri adult Batouri District, Cameroon. 1.2 0.0 1969.436 X. laevis laevis tadpole Near Lilongwe, Malawi. 0.5 0.2 1985.1373 X. gilli adult Cape Point, South Africa. 6.4 1.5 1985.1374 X. gilli adult Cape Point, South Africa. 0.2 0.0

Table 3. Results and genomic equivalents (GE) obtained from six midwife toads (A. obstetricans) using Batrachochytrium dendrobatidis-specific quantitative real-time Taqman PCR (qPCR) assay recovered from swabs, interdental brushes and scrapes. These frogs were previously diagnosed as chytrid positives by toeclip-qPCR.

Swab Interdental brush Scrape Specimen Result GE Result GE Result GE AU05122 negative 4.8x10-4 positive 143,599 positive 8.7 CN05015 positive 348 positive 696 positive 5.1 AU05121 positive 113,138 positive 99,669 positive 91,273 CN05023 negative - negative - positive 144 CN05021 positive 7.3 positive 58 positive 20 AU05117 negative - negative - negative -

In addition, two other specimens gave inconclusive results. An adult X. laevis laevis collected in 1934 from the Lake Musyhe, South West Uganda, had a titre of 39.0 ± 18.8 GE, but its amplification curves were not clearly sigmoid. And an adult X. laevis bunyoniensis collected in 1935 from the Lake Bunyoni, South West Uganda (collected simultaneously with the first positive case detected), had a titre of 0.3 ± 0.4 GE, but the GE values were higher than 0.1 from just one of the replicate analyses. Both specimens failed to give positive results on repetition of the qPCR assay.

Validation of the brushing sampling method. Expt 1––Ethanol fixed frogs: Results and GE obtained from the three different sampling methods are summarized in table 3. The qPCR was able to detect 3, 4 and 5 positives, when swabbing, brushing and scraping were performed, respectively. Only specimen number 6 was negative to the three different sampling methodologies (Table 3). Genomic equivalents obtained from the brush-qPCR technique (average of 40,670), were considerably higher than those obtained from swab-qPCR (18,916) and scrape-qPCR (15,242) methods.

Expt 2––Formalin fixed frogs: Batrachochytrium dendrobatidis could not be detected in any of the chytrid infected frogs fixed in neutral buffered formalin, regardless of the sampling method employed.

21 Soto-Azat: Origin of chytridiomycosis

DISCUSSION

By screening the pipid frog collection of the Natural History Museum, London, we were able to survey an extensive area of South America and Africa for the historical presence of Bd (Table 1). The absence of the chytrid infection from South American pipids, along with the presence in its African relatives, supports the hypothesis that Africa was the origin of the disease. The earliest case of chytridiomycosis obtained in our study, a specimen of X. laevis bunyoniensis collected in 1935 in Uganda, has extended in 3 years the date of the first reported case worldwide. Prior to this study, the earliest case was a specimen of X. laevis collected in the Western Cape coastal lowland, South Africa, in 1938 (Weldon et al. 2004). Our next historical record obtained, a X. fraseri collected from East Cameroon, demonstrates that Bd probably was widely distributed across sub-Saharan Africa by 1948. Our third record of Bd, obtained from a X. laevis laevis tadpole, collected in 1969 from Malawi, at the best of our knowledge is the first report of chytridiomycosis detected from a museum archived tadpole. The data obtained in the present study, plus that reported by Weldon et al. (2004), make possible to draw a new map about the distribution of Bd infection Africa, prior to the mergence of the global amphibian decline phenomenon (Fig. 1).

Recent studies have shown that amphibian Bd infection is present in 20 African anuran species from South Africa, Swaziland, Lesotho, Botswana, Tanzania, Kenya and Ghana, nevertheless, amphibian declines have not been reported (Hopkins & Channing 2002, Weldon 2002, Lane et al. 2003, Speare & Berger 2004, Weldon & Du Preez 2004, Weldon et al. 2004, Weldon 2005). Most of these cases of chytrid infection in Africa have been described in pipid frogs (Weldon 2005). For this group of fully aquatic anurans, infection has been reported from wild X. laevis, X. gilli, X. petersii, X. muelleri, captive X. tropicalis exported to USA from Ghana, and from a group of captive Hymenochirus boettgeri exported to Vancouver (Canada) from Florida (USA), but in this case the definitive origin of the frogs was not determined (Reed et al. 2000, Raverty & Reynolds 2001, Morehouse et al. 2003, Weldon et al. 2004). Our finding of the chytrid in X. fraseri is the first report of Bd infection for this species. As some of these Xenopus species (X. fraseri, X. laevis, X. muelleri, X. petersii and X. tropicalis) are also considered very abundant in their natural habitats and population declines have not been reported, it is possible that these species may act as natural hosts of the pathogen. Furthermore, the prevalence obtained for Bd infection in our study (0.8%) as well as than that reported by Weldon et al (2004) (2.7%), remained steady over time, which suggests that a stable endemic relationship between pathogen and host have existed at least for the last 70 years in this region. In order to make the prevalence of both studies comparable, we obtained a 3.1% when just X. gilli, X. muelleri and X. laevis from South Africa, Lesotho and Swaziland were included, which suggest that our methodology is as sensitive as that employed by Weldon et al. (Weldon et al. 2004), if no better.

It has been found that amphibians from temperate and from tropical montane areas are more susceptible to the effects of chytrid infection (Berger et al. 1998, Daszak et al. 1999, Muths et al. 2003). It is interesting to note, therefore, that the chytrid- positive animals from tropical Africa found in our study were collected at altitudes between 650 to 2,000 m. Additionally, since our findings are the first reports of chytrid infection for Malawi, Uganda and Cameroon, we think that efforts to assess the current presence of Bd in tropical and sub-tropical Africa are warranted.

22 Soto-Azat: Origin of chytridiomycosis

1948

1935

1969

1935. X. laevis bunyoniensis. 1938. X. laevis. 1943. X. gilli. 1948. X. fraseri. 1938 1969. X. laevis laevis. 1943 1985. X. gilli (two cases). 1985

Figure 1. Map of Africa showing the historical distribution of Batrachochytrium dendrobatidis infection. Cases reported in the present study (grey circles) and the other two earliest cases reported by Weldon et al. (2004) (white circles).

We found evidence of Bd infection in two X. gilli, collected in 1985 from the Cape Peninsula. Weldon et al. (2004), also described two cases of Bd infection in X. gilli from this region. Xenopus gilli is an endangered species which is known only to inhabit a restricted area of less than 500 km2 in the coastal lowlands of the Cape Peninsula and Cape Agulhas in South Africa (Channing 2001). Whether or not, Bd infection in addition to the already present habitat destruction and hybridization with X. laevis, signifies a further threat for remaining X. gilli populations is unclear. Further studies on species susceptibility to chytridiomycosis should be undertaken in order to define for which species this disease is a conservation threat, and which species might act as reservoir hosts and disease vectors.

Evidence shows that outside the African continent, chytridiomycosis first appeared in amphibian populations in North America (1961), Australia (1978), South America (1980), Central America (1986), Europe (1997) and New Zealand (1999) (Bosch et al. 2001, Waldman et al. 2001, Puschendorf 2003, Ron et al. 2003, Oullet et al. 2005, Speare & Berger 2005) (Table 4). The importance of these records relies in that

23 Soto-Azat: Origin of chytridiomycosis

Table 4. Documented global appearance of chytridiomycosis by region.

Year Species Location Country Reference 1938 Xenopus laevis Western Cape South Africa (Weldon et al. 2004) 1961 Rana clamitans Québec Canada (Oullet et al. 2005) 1978 Litoria gracilenta South East Queensland Australia (Speare et al. 2005) 1980 Atelopus bomolochos Provincia del Cañar Ecuador (Ron et al. 2003) 1986 Atelopus varius Sarapiquí Costa Rica (Puschendorf 2003) 1997 Alytes obstetricans Peñalara National Park Spain (Bosch et al. 2001) 1999 Litoria raniformis Christchurch New Zealand (Waldman et al. 2001)

all of them precede or at least are concurrent with amphibian population declines in their respective regions (Berger et al. 1998, Bosch et al. 2001, Waldman et al. 2001, Bradley et al. 2002).

The results of the first validation experiment performed showed that the skin scrape, followed by the brush, were more sensitive than the swab method. This possibly occurred, due to the fact that the scalpel followed by the brush, were able to get a deeper skin tissue, and consequently were capable to detect earlier stages of the disease. However, unexpectedly, the diagnostic assay using the brushing method was able to detect more GE than when the skin scraping method was employed. A possible explanation could be that swabbing, brushing and scraping were performed following the same order, and maybe by the stage when the scalpel was used to sample the frogs, less zoospores were left in the frog, and consequently less GE were obtained.

Regardless of the method used to sample the chytrid infected frogs stored in formalin, the real-time PCR assay failed to detect infection. It is well known that formalin causes irreversible DNA damages which are greater with prolonged fixation time (Pavelic et al. 1996). The apparent incapacity of qPCR to detect the chytrid from formalin fixed specimens is the main problem for the widespread use of this diagnostic method for museum amphibian specimens and might have precluded the detection of infected animals in this study. It is likely that many of the specimens collected post- 1920 were originally fixed in formalin before being transferred to alcohol for long-term preservation. Since we do not know what proportion of our samples were initially fixed in this way, or for how long they were kept in formalin before being transferred to ethanol, we cannot evaluate the likelihood of this affecting our results in the specimens collected post-1920 (Prior to 1920 formalin was not used as a fixative and all specimens were fixed directly in ethanol).

To date just histology and immunohistochemistry has been carried out to diagnose chytridiomycosis from museum archived amphibians (Weldon et al. 2004, Oullet et al. 2005). Samples for this purpose include toe-clips and excised skin from the pelvic patch, hind limbs and the interdigital webbing (Green & Kagarise Sherman 2001, Lips et al. 2003, Puschendorf 2003, Weldon et al. 2004, Oullet et al. 2005, Speare & Berger 2005). Although histopathology is a good and reliable method for chytrid detection when performed by a trained pathologist, its destructive character makes it unsuitable for use with valuable museum specimens.

24 Soto-Azat: Origin of chytridiomycosis

The qPCR assay is an excellent tool for the diagnosis of Bd (Kriger et al. 2006). This analysis is able to detect chytrid zoospores recovered from oral disc excisions, toe- clips, water baths and swabs (Hyatt et al. 2007). When compared with histology and immunohistochemistry, qPCR has been demonstrated to be faster, cheaper and to have higher sensitivity, repeatability and reproducibility, mainly due to the ability to detect the chytrid at lower levels and at earlier stages of infection (Hyatt et al. 2007). To date, the non-invasive swab-qPCR technique is considered the preferred method to diagnose the chytrid fungus from wild amphibians (Kriger et al. 2006). The interdental brush technique is a modification of the swabbing technique which appears to have higher sensitivity than conventional swabbing but which leaves no visible sign of a sample having been taken. We, therefore, recommend the use of this technique for the non- destructive sampling of post-mortem specimens, including reference specimens.

Based on the GE obtained, all Bd infections were considered mild. This could be explained by: (1) As this species probably are not clinically affected by the disease, they may act as carriers for the chytrid, therefore low levels of infection are expected; and/or (2) As the specimens have been stored for a long period of time, fungal DNA material may have been degraded with time, and just small amount of detectable DNA remained in each positive specimen.

Batrachochytrium dendrobatidis isolates from distinct parts of the world have shown low levels of diversity, which makes the qPCR assay a reliable method for the diagnosis of chytridiomycosis. However, if Africa is effectively the origin of chytridiomycosis, as has been proposed, African isolates should show an increased allelic variation, and decreased association among loci when compared with outside Africa genotypes (Rachowicz et al. 2005). If this is the case, there could be an underestimation of the real prevalence levels of Bd infection in amphibians from Africa tested using qPCR (Boyle et al. 2004).

In conclusion, we established that the brush-qPCR diagnostic method is a valid, non-invasive alternative for retrospective chytridiomycosis studies using archived tadpoles, post-metamorphic and adult amphibians. We were able to report the first historical case of Bd infection from a X. laevis specimen collected in Uganda in 1935, and we suggest that Bd infection appears to have been widely established in sub- Saharan Africa by the 1930s. Further studies are needed in order to determine how other African amphibian families within and outwith Africa have been historically infected with Bd, and if so, to determine whether or not there has been an historical host-pathogen equilibrium for Bd and African frogs.

Acknowledgements: This study was carried out in fulfilment of the Wild Animal Health MSc degree (C.S.) at the Royal Veterinary College and the Zoological Society of London. The authors thank S. F. Walker and Dr. M. C. Fisher for providing us the Batrachochytrium dendrobatidis infected frogs fixed in ethanol and formalin. We would like also to extend our gratitude to M. W. Perkins for assistance with the laboratory analyses, and Dr. T. W. J. Garner for his valuable comments.

25 Soto-Azat: Origin of chytridiomycosis

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