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SHORT COMMUNICATION |
1 Department of Pathobiology, University of Illinois, 2001 South Lincoln Avenue, Urbana, Illinois 61802, USA
2 Program in Ecology and Evolutionary Biology, University of Illinois, 235 NRSA, 607 East Peabody Drive, Champaign, Illinois 61820, USA
3 Corresponding author (email: tlgoldbe{at}uiuc.edu)
ABSTRACT:
Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis, was found in 24 of 109 (22%) frogs from Kibale National Park, western Uganda, in January and June 2006, representing the first account of the fungus in six species and in Uganda. The presence of B. dendrobatidis in an equatorial African montane forest raises conservation concerns, considering the high amphibian diversity and endemism characteristic of such areas and their ecological similarity to other regions of the world experiencing anuran declines linked to chytridiomycosis.
Key words: Africa, amphibians, Anura, Batrachochytrium dendrobatidis, Chytridiomycota, Uganda.
Chytridiomycosis, an emerging infectious disease caused by the fungus Batrachochytrium dendrobatidis, is a major cause of amphibian population declines (Daszak et al., 1999). Its global distribution, responsiveness to microenvironmental conditions affected by climate change, and association with anuran extinctions in Central America and Australia make it a disease of special concern for conservation (Daszak et al., 1999; Pounds et al., 2006). Despite growing information about B. dendrobatidis elsewhere in the world, comparatively little is known about the fungus in Africa, where it is thought to have originated (Weldon et al., 2004).
This report presents the results of a survey for B. dendrobatidis in Kibale National Park, Uganda. Kibale is a 795-km2 park located in western Uganda near the foothills of the Rwenzori Mountains (0°13'–0°41'N, 30°19'–30°32'E), consisting primarily of moist semideciduous and evergreen forest, transitional between lowland rainforest and montane forest (elevation ranging from approximately 1,100 to 1,600 m), and interspersed with grassland, woodland, lakes and wetlands, colonizing forest, and plantations of exotic trees (Chapman et al., 1997; Chapman and Lambert, 2000). Mean daily minimum and maximum temperatures in Kibale were recorded as 14.9 C and 20.2 C, respectively, from 1990 to 2001, with mean annual rainfall during the same period of 1749 mm, distributed across distinct, bimodal wet and dry seasons (Chapman et al., 1999, 2005). Kibale has experienced marked climate change over the last approximately 30 yr, with increasing annual rainfall, increasing maximum mean monthly temperatures, and decreasing minimum mean monthly temperatures (Chapman et al., 2005).
Medium- and high-altitude equatorial African forests such as Kibale hold a great diversity of herpetofauna, including many endemic anuran species (Channing and Howell, 2006). Surveys by Vonesh between 1995 and 1997 identified 28 anuran, 15 lizard, and 32 snake species in Kibale, an assemblage that shows an affinity with the herpetofauna of the Guinea–Congo-lean rain forests to the west (Vonesh, 2001). The herpetofauna of Kibale is markedly different from that of the coastal mountains of Tanzania, which contain many local endemic anuran species, and where B. dendrobatidis notably contributed to the recent decline of the Kihansi spray toad, Nectophrynoides asperginis (Krajick, 2006; Lee et al., 2006). To our knowledge, ours is the first multispecies survey for B. dendrobatidis in Kibale or in any similar equatorial African montane forest site.
Frogs were collected from locations near Makerere University Biological Field Station (elevation approximately 1500 m) in Kibale National Park in January and June 2006. Sites were chosen that were known to have high amphibian densities, both from anecdotal local reports and from previous amphibian surveys (Vonesh, 2001). Frogs were captured by hand or with hand nets, placed individually into single-use plastic bags until a toe clip was obtained from the right rear foot, and released at the site of capture. Toe clips were placed in approximately 5 volumes of RNAlater®stabilization solution (Ambion, Inc., Austin, Texas, USA) and stored at 4 C in the field and at –20 C after transport to the laboratory. Equipment and hands were disinfected with 10% bleach and alcohol wipes, respectively, between individuals.
Total DNA was extracted from toe clips with the use of the ZR Genomic DNA II KitTM (Zymo Research Corp., Orange, California, USA). For the tissue homogenization step, a rotor-stator homogenizer with single-use individually sterilized probes was used to prevent cross-contamination. Negative-control DNA extractions were also performed with every 10 toe clip extractions. To test for B. dendrobatidis, a previously described real-time quantitative PCR was used that can detect a single zoospore in a diagnostic sample (Boyle et al., 2004). Samples were run in triplicate, with the use of 1 µl of extracted DNA template per reaction.
To confirm positive results, a nested PCR was used that targets the fungal cysteinyl tRNA synthase (ctsyn1) gene (Morehouse et al., 2004; Garner et al., 2006), although with newly designed internal PCR primers (ctsyn1aF: 5'-TCAGCTGCCGTCGTTTGTGAATTG-3' and ctsyn1aR: 5'-CCAGAGCAGTTTG-CCAGCATCAAA-3'). Amplicons were electrophoresed in 1.5% agarose gels, which were stained with ethidium bromide and visualized under ultraviolet light. Amplicons were purified from gels with the use of the ZymoClean Gel DNA Recovery Kit (Zymo Research) and were sequenced directly and in both directions with primers ctsyn1aF and ctsyn1aR. Sequencing was performed at the University of Illinois Roy J. Carver Biotechnology Center.
A total of 109 frogs from seven species were collected (Table 1
). Frogs of all species were adults, with the exception of Leptopelis christyi and L. kivuensis, for which approximately 50% of frogs were post-metamorphic juveniles. All frogs appeared clinically normal at the time of sampling. Batrachochytrium dendrobatidis was detected in 24/109 (22.0%) toe clip samples from six species (Table 1). Prevalence estimates varied between 0% and 60% among species, although sample sizes within species were too small for meaningful statistical comparisons. Among infected frogs, there was no significant association between intensity of infection and species (Kruskal-Wallis rank sum test statistic=10.92; P=0.09). Batrachochytrium dendrobatidis was not detected in any negative control samples, indicating that laboratory contamination did not affect these results.
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This survey represents the first account of B. dendrobatidis in the six positive species listed in Table 1
. It is also the first account of B. dendrobatidis in Uganda, and it thereby significantly expands the known geographic range of the fungus in Africa. Kibale is approximately 800 km from the nearest other location where frogs infected with B. dendrobatidis have been documented (P. anchietae in Nairobi, Kenya; Speare and Berger, 2000). Kibale is approximately 1,200 km from Kihansi Gorge, Tanzania, where B. dendrobatidis contributed to the decline of N. asperginis (Krajick, 2006; Lee et al., 2006), and over 2,000 km from sites in southern Africa where infected Xenopus spp. have existed since at least as early as 1938 (Weldon et al., 2004).
It has been suggested that B. dendrobatidis may have emerged through the export of X. laevis from southern Africa for human pregnancy testing beginning in the 1930s (Weldon et al., 2004). We note that the prevalence of B. dendrobatidis in X. wittei in Kibale (25%) is higher than that reported in archived specimens of X. laevis, X. meulleri, and X. gilli collected from southern Africa between 1879 and 1999 (2.7%; Weldon et al., 2004), but is within the range of location-specific prevalence values for Xenopus spp. collected from South Africa more recently (Weldon, 2005). It is unclear whether this pattern represents a recent increase in B. dendrobatidis prevalence across Africa, or whether it merely reflects typical differences in prevalence among spatially and temporally separated populations of ecologically similar species.
No accounts exist of mass anuran mortality events in Kibale or surrounding areas. Because long-term historical records are lacking for anuran populations in Kibale and in equatorial Africa in general, it is currently unclear how B. dendrobatidis might be affecting amphibian health and conservation in the region. Considering the remarkable biodiversity and endemism of equatorial Africa’s medium- and high-altitude forests, and considering their general ecological similarity to forests in Central America and Australia that have been profoundly affected by chytridiomycosis, an improved understanding of the disease in equatorial Africa is exigent.
We gratefully acknowledge the assistance in the field of I. Rwego, T. Gillespie, C. Chapman, L. Chapman, the personnel of the Kibale EcoHealth Project, and the staff of Makerere University Biological Field Station. We also thank J. Vonesh for helping with amphibian identifications, V. Beasley, J. Levengood, and J. Longcore for providing fungal isolates, M. Fisher and T. Garner for providing information about PCR primers and protocols, and T. Garner and one anonymous reviewer for helpful comments on the manuscript. The Uganda Wildlife Authority and the Uganda National Council for Science and Technology kindly granted us permission to conduct this research. This work was supported by the University of Illinois, the Geraldine R. Dodge Foundation Frontiers for Veterinary Medicine Program, and by the Morris Animal Foundation under Award D04ZO–67.
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