CONCLUSIONES FINALES
MANIFIESTO SOBRE LA OBJECIÓN DE CONCIENCIA SANITARIA
The technique used for detecting Bd presence was the swab-PCR assay described in Hyatt et al. (2007). The swabbing protocol followed was the one prepared and recommended by the Department of Conservation (Haigh, 2007). A new dry swab (MWE MW100-100) was used for each frog. Sterile gloves were used for handling each frog. If a frog had soil on the surfaces to be swabbed, the soil was gently removed using stream water. All ventral areas of the frog were swabbed, including the underside of the legs and axilla, with particular emphasis on the feet and the inguinal region. All areas were swabbed at least three times. After swabbing the frog, the swab was placed back into a sterile container and labelled with the frog ID, location (NZMG coordinates from GPS receiver), and size (snout to vent length, SVL) of the frog. In the field, swabs were kept as cool as possible. Upon returning from the field swabs were stored at 4ºC (from 1 to 12 months) to avoid growth of other non-target microorganisms prior to sending the sample to the diagnostic laboratory.
4.4.3 Testing
All swab samples were sent to a qualified laboratory (EcoGene™, Landcare Research) for testing for the presence of Bd. The method used for testing was the real-time PCR with three replicates per sample as described in Hyatt et al. (2007). Samples were either tested separately (single sample/swabs per test) or pooled (two samples per test) as shown in Appendix VIII.
4.5
Results
Sixty-one swab samples were collected from the Waitakere Ranges, and 63 from Great Barrier Island. Locations of the samples taken are shown in Figure 4.1 (detailed locations are available in Appendix VIII). All swabs tested negative for the presence of chytrid fungus. Estimated prevalence of chytridiomycosis was <5% (95% confidence level) for both populations.
Figure 4.1 Map showing all swab samples taken (dots) in; a) Waitakere Ranges and; b) Great Barrier Island populations of Hochstetter’s frog (both maps are at the same scale).
4.6
Discussion
Chytrid fungus was not detected at any location sampled in this study. Based on the number of samples tested, it is 95% certain that chytrid fungus prevalence is lower than 5%, but it is not possible to be 100% certain that the fungus is not present at lower prevalence. However, Bd prevalence, when present, is often higher than 5% (McDonald et al., 2005). Nonetheless, all Hochstetter’s frog populations sampled for Bd presence to date, including those from areas where the fungus is known to be present, have tested
negative. These results suggest that L. hochstetteri is relatively resilient to the disease, or possibly even immune. The recent discovery of the spontaneous elimination of the chytrid fungus by infected captive individuals of Leiopelma archeyi (BishopSpeare et al., 2009) raises the question of whether all native frogs might have low levels of susceptibility to the disease.
Immunity to chytridiomycosis has been found previously in some species. In Australia, where Bd is widely present some species such as Mixophyes spp. and Litoria lesueurii
appear not to have had population declines, despite being exposed to the fungus in the environment. Moreover, after an initial decline Litoria genimaculata increased to pre- decline numbers, which suggests that Bd may have become endemic after the initial epidemic wave (McDonald et al., 2005).
Further studies on the response of native frogs to Bd infection may help elucidate the level of threat that chytridiomycosis really imposes to wild populations. Knowing this is critical in effectively allocating resources available for conservation of these species. Experimental infections have proven to be useful in identifying species at higher risk of becoming infected with Bd and developing chytridiomycosis (Vazquez et al., 2009). If Hochstetter’s frogs are found effectively resistant to Bd they could also serve as a model for studying the immune mechanism, which would lead to a better understanding of the disease.
4.7
Recommendations:
1) Laboratory studies on the vulnerability of Hochstetter’s frog to Bd. While this is not clear keep surveying populations.
2) Test for presence of Bd in the introduced Litoria species where they share their distribution with native frogs.
4.8
References
Bell, B. D., Carver, S., Mitchell, N. J., & 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, A., Olsen, V., Hengstberger, S. G., Boyle, D. B., Marantelli, G., et al. (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., Marantelli, G., Skerratt, L. F., & Speare, R. (2005). Virulence of the amphibian chytrid fungus Batrachochytrium dendrobatidis varies with the strain. Diseases of Aquatic Organisms, 68, 47-50.
Berger, L., Speare, R., Daszak, P., Green, D. E., Cunningham, A. A., Goggin, C. L., et al. (1998). Chytridiomycosis Causes Amphibian Mortality Associated with Population Declines in the Rain Forests of Australia and Central America.
PNAS, 95(15), 9031-9036.
Berger, L., Speare, R., Hines, H. B., Marantelli, G., Hyatt, A. D., McDonald, K. R., et al. (2004). Effect of season and temperature on mortality in amphibians due to chytridiomycosis. Australian veterinary journal, 82(7), 434-439.
Berger, L., Speare, R., & Hyatt, A. D. (1999). Chytrid fungi and amphibian declines: overview, implications and future directions. In A. Campbell (Ed.), Declines and disappearances of Australian frogs (pp. 23-33). Canberra: Environment Australia.
Berger, L., Speare, R., & Kent, A. (1999). Diagnosis of chytridiomycosis in amphibians by histologic examination. Retrieved 1st of June, 2009, from http://www.jcu.edu.au/school/phtm/PHTM/frogs/histo/chhisto.htm
Bishop, P. J., Speare, R., Poulter, R., Butler, M., Speare, B. J., Hyatt, A., et al. (2009). Elimination of the amphibian chytrid fungus Batrachochytrium dendrobatidis by Archey's frog Leiopelma archeyi. Diseases of Aquatic Organisms, 84, 9-15. Boyle, D. G., Boyle, D. B., Olsen, V., Morgan, J. A. T., & Hyatt, A. D. (2004). Rapid
quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Diseases of Aquatic Organisms, 60, 141-148.
Daszak, P., Berger, L., Cunningham, A. A., Hyatt, A. D., Green, D. E., & Speare, R. (1999). Emerging Infectious Diseases and Amphibian Population Declines.
Daszak, P., Cunningham, A. A., & Hyatt, A. D. (2003). Infectious disease and amphibian population declines. Diversity and Distributions, 9, 141-150.
DiGiacomo, R. F., & Koepsell, T. D. (1986). Sampling for detection of infection or disease in animal populations. JAVMA, 189(1), 22-23.
Forzán, M. J., Gunn, H., & Scott, P. (2008). Chytridiomycosis in an aquarium collection of frogs: diagnosis, treatment, and control. Journal of zoo and wildlife medicine, 39(3), 406-411.
Garner, T. W. J., Garcia, G., Carroll, B., & Fisher, M. C. (2009). Using itraconazole to clear Batrachochytrium dendrobatidis infection, and subsequent depigmentation of Alytes muletensis tadpoles. Diseases of Aquatic Organisms, 83(3), 257-260. Green, D. M. (1994). Genetic and cytogenetic diversity in Hochstetter's frog, Leiopelma
hochstetteri, and its importance for conservation management New Zealand Journal of Zoology, 21, 417-424.
Haigh, A. (2007). Swabbing protocol for New Zealand frogs to detect the amphibian chytrid fungus (Version 2, 2007). Department of Conservation.
Haigh, A., Shaw, S. D., Holzapfel, S. A., & Bishop, P. J. Emergency translocation as a response to the detection of Bd into a naive threatened population of Leiopelma
archeyi in New Zealand. Retrieved September 27th, 2008, from
http://www.nzfrogs.org/site/nzfrog/files/Stephanieposter.gif
Harris, R. N., Brucker, R. M., Walke, J. B., Becker, M. H., Schwantes, C. R., Flaherty, D. C., et al. (2009). Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus. The ISME journal.
Hyatt, A. D., Boyle, D. G., Olsen, V., Boyle, D. B., Berger, L., Obendorf, D., et al. (2007). Diagnostic assays and sampling protocols for the detection of
Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms, 73, 175-192. Johnson, M. L., & Speare, R. (2003). Survival of Batrachochytrium dendrobatidis in
Water: Quarantine and Disease Control Implications. Emerging Infectious Diseases, 9(8).
Johnson, M. L., & Speare, R. (2005). Possible modes of dissemination of the amphibian chytrid Batrachochytrium dendrobatidis in the environment. Diseases of Aquatic Organisms, 65, 181-186.
Kriger, K. M., Hines, H. B., Hyatt, A. D., Boyle, D. G., & Hero, J.-M. (2006). Techniques for detecting chytridiomycosis in wild frogs: comparing histology with real-time Taqman PCR. Diseases of Aquatic Organisms, 71, 141-148. Kriger, K. M., Pereoglou, F., & Hero, J.-M. (2007). Latitudinal variation in the
prevalence and intensity of chytrid (Batrachochytrium dendrobatidis) infection in eastern Australia. Conservation Biology, 21(5), 1280-1290.
Lips, K. R., Brem, F., Brenes, R., Reeve, J., D., Alford, R. A., Voyles, J., et al. (2006). Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. PNAS, 103(9), 3165-3170.
Lips, K. R., Diffendorfer, J., Mendelson, J. R., III, & Sears, M. W. (2008). Riding the Wave: Reconciling the Roles of Disease and Climate Change in Amphibian Declines. PLoS Biol, 6(3), e72.
Longcore, J. E., Pessier, A. P., & Nichols, D. K. (1999). Batrachochytrium Dendrobatidis gen. et sp. nov., a Chytrid Pathogenic to Amphibians. Mycologia, 91(2), 219-227.
McDonald, K. R., Méndez, D., Müller, R., Freeman, A. B., & Speare, R. (2005). Decline in prevalence of chytridiomicosis in frog populations in North Queensland, Australia. Pacific Conservation Biology, 11, 114-120.
Olsen, V., Hyatt, A., Boyle, D. B., & Mendez, D. (2004). Co-localisation of
Batrachochytrium dendrobatidis and keratin for enhanced diagnosis of chytridiomycosis in frogs. Diseases of Aquatic Organisms, 61, 85-88.
Olson, D. & Ronnenberg, K. (2008) Global Bd Mapping Project: Update - 17 January 2008. Retrived 27th May, 2009, from http://www.parcplace.org/ bdmap2008update.html
Poulter, R., Busby, J., Butler, M., Bishop, P. J., & Speare, R. (2007). Chloramphenicol cures frogs with chytridiomycosis. Paper presented at the Amphibian Declines and Chytridiomycosis: Translating Science into Urgent Action; 5-7 November 2007, Tempe, Arizona. Retrieved 1st of June 2009, from www.parcplace.org/documents/Bd_Program_post-FINAL.pdf
Retallick, R. W. R., McCallum, H., & Speare, R. (2004). Endemic Infection of the Amphibian Chytrid Fungus in a Frog Community Post-Decline. PLoS Biol, 2(11), e351.
Richmond, J. Q., Savage, A. E., Zamudio, K. R., & Rosenblum, E. B. (2009). Toward Immunogenetic Studies of Amphibian Chytridiomycosis: Linking Innate and Acquired Immunity. BioScience, 59(4), 311-320.
Rollins-Smith, L. A., & Conlon, J. M. (2005). Antimicrobial peptide defenses against chytridiomycosis, an emerging infectious disease of amphibian populations.
Developmental and comparative immunology, 29(7), 589-598.
Shaw, S. D., Haigh, A., Bishop, P. J., Skerratt, L. F., Speare, R., Bell, B. D., et al. (2009, 20-22 February). Distribution and prevalence of Batrachochytrium dendrobatidis in New Zealand. Paper presented at the Second Meeting of the Australasian Societies for Herpetology, Auckland, New Zealand.
Skerratt, L. F., Berger, L., Speare, R., Cashins, S., Mc Donald, K. R., Phillott, A. D., et al. (2007). Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth, 4, 125-134.
Une, Y., Kadekaru, S., Tamukai, K., Goka, K., & Kuroki, T. (2008). First report of spontaneous chytridiomycosis in frogs in Asia. Diseases of Aquatic Organisms, 82(2), 157-160.
Vazquez, V. M., Rothermel, B. B., & Pessier, A. P. (2009). Experimental infection of North American plethodontid salamanders with the fungus Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms, 84(1), 1-7.
Waldman, B., van de Wolfshaar, K. E., Klena, J. D., Andjic, V., Bishop, P. J., & Norman, R. J. d. B. (2001). Chytridiomycosis in New Zealand frogs.
Surveillance, 28(3), 9-11.
Weldon, C., du Preez, L. H., Hyatt, A. D., Muller, R., & Speare, R. (2004). Origin of the Amphibian Chytrid Fungus. Emerging Infectious Diseases, 10(12), 2100- 2105.
Woodhams, D. C., Alford, R. A., & Marantelli, G. (2003). Emerging disease of amphibians cured by elevated body temperature Diseases of Aquatic Organisms, 55, 65-67.
Woodhams, D. C., Ardipradja, K., Alford, R. A., Marantelli, G., Reinert, L. K., & Rollins-Smith, L. A. (2007). Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Animal Conservation, 10, 409-417.