Detectability vs. time and costs in pooled DNA extraction of cutaneous swabs: a study on the amphibian chytrid fungi
In: Amphibia-ReptiliaAbstract
Epidemiology relies on understanding the distribution of pathogens which often can be detected through DNA-based techniques, such as quantitative Polymerase Chain Reaction (qPCR). Typically, the DNA of each individual sample is separately extracted and undergoes qPCR analysis. However, when performing field surveys and long-term monitoring, a large fraction of the samples is generally expected to be negative, especially in geographical areas still considered free of the pathogen. If pathogen detection within a population – rather than determining its individual prevalence – is the focus, work load and monetary costs can be reduced by pooling samples for DNA extraction. We test and refine a user-friendly technique where skin swabs can be pooled during DNA extraction to detect the amphibian chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans (Bsal). We extracted pools with different numbers of samples (from one to four swabs), without increasing reaction volumes, and each pool had one sample inoculated with a predetermined zoospore amount. Pool size did not reduce the ability to detect the two fungi, except if inoculated with extremely low zoospore amounts (one zoospore). We confirm that pooled DNA extraction of cutaneous swabs can substantially reduce processing time and costs without minimizing detection sensitivity. This is of relevance especially for the new emerging pathogen Bsal, for which pooled DNA extraction had so far not been tested and massive monitoring efforts in putatively unaffected regions are underway.
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Allender, M., Bunick, D., Dzhaman, E., Burrus, L., Maddox, C. (2015): Development and use of a real-time polymerase chain reaction assay for the detection of Ophidiomyces ophiodiicola in snakes. J. Vet. Diagnostic Investig. 27: 217-220.
-
Aw, T.G., Rose, J.B. (2012): Detection of pathogens in water: from phylochips to qPCR to pyrosequencing. Curr. Opin. Biotechnol. 23: 422-430.
-
Bai, C., Garner, T.W.J., Li, Y. (2010): First evidence of Batrachochytrium dendrobatidis in China: discovery of chytridiomycosis in introduced American bullfrogs and native amphibians in the Yunnan Province, China. Ecohealth 7: 127-134.
-
Belden, L.K., Hughey, M.C., Rebollar, E.A., Umile, T.P., Loftus, S.C., Burzynski, E.A., Minbiole, K.P.C., House, L.L., Jensen, R.V., Becker, M.H., Walke, J.B., Medina, D., Ibáñez, R., Harris, R.N. (2015): Panamanian frog species host unique skin bacterial communities. Front. Microbiol. 6: 1171.
-
Bletz, M., Rosa, G., Andreone, F., Courtois, E., Schmeller, D., Rabibisoa, N., Rabemananjara, F., Raharivololoniaina, L., Vences, M., Weldon, C., Edmonds, D., Raxworthy, C., Harris, R., Fisher, M., Crottini, A. (2015): Widespread presence of the pathogenic fungus Batrachochytrium dendrobatidis in wild amphibian communities in Madagascar. Sci. Rep. 5: 8633.
-
Bletz, M.C., Rebollar, E.A., Harris, R.N. (2015): Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis. Dis. Aquat. Organ. 113: 1-8.
-
Blooi, M., Martel, A., Haesebrouck, F., Vercammen, F., Bonte, D., Pasmans, F. (2015): Treatment of urodelans based on temperature dependent infection dynamics of Batrachochytrium salamandrivorans. Sci. Rep. 5: 8037.
-
Blooi, M., Pasmans, F., Spitzen-van der Sluijs, A., Vercammen, F., Martel, A. (2013): Duplex real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in amphibian samples. J. Clin. Microbiol. 51: 4173-4177.
-
Boyle, D., Boyle, D., Olsen, V., Morgan, J., Hyatt, A. (2004): Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis. Aquat. Organ. 60: 141-148.
-
Brewster, J., Paoli, G. (2013): DNA extraction protocol for rapid PCR detection of pathogenic bacteria. Anal. Biochem. 442: 107-109.
-
Churchill, G.A., Giovannoni, J.J., Tanksley, S.D. (1993): Pooled-sampling makes high-resolution mapping practical with DNA markers. Genetics 90: 16-20.
-
Crawford, A., Lips, K., Bermingham, E. (2010): Epidemic disease decimates amphibian abundance, species diversity, and evolutionary history in the highlands of central Panama. Proc. Natl. Acad. Sci. U.S.A. 107: 13777-13782.
-
DiRenzo, G.V., Campbell Grant, E.H., Longo, A.V., Che-Castaldo, C., Zamudio, K.R., Lips, K.R. (2018): Imperfect pathogen detection from non-invasive skin swabs biases disease inference. Methods Ecol. Evol. 9: 380-389.
-
Esteves, F., Gaspar, J., de Sousa, B., Antunes, F., Mansinho, K., Matos, O. (2012): Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies. Clin. Microbiol. Infect. 18: e177-e184.
-
Fisher, M.C., Henk, D.A., Briggs, C.J., Brownstein, J.S., Madoff, L.C., McCraw, S.L., Gurr, S.J. (2012): Emerging fungal threats to animal, plant and ecosystem health. Nature 484: 186-194.
-
Fox, J., Weisberg, S. (2011): An R Companion to Applied Regression. SAGE Publications, Thousand Oaks, CA.
-
Garner, T.W.J., Schmidt, B.R., Martel, A., Pasmans, F., Muths, E., Cunningham, A.A., Weldon, C., Fisher, M.C., Bosch, J. (2016): Mitigating amphibian chytridiomycoses in nature. Philos. Trans. R. Soc. London B Biol. Sci. 371: 20160207.
-
He, Q., Wang, J.-P., Osato, M., Lachman, L.B. (2002): Real-time quantitative PCR for detection of Helicobacter pylori. J. Clin. Microbiol. 40: 3720-3728.
-
Hensel, M., Shea, J., Gleeson, C., Jones, M. (1995): Simultaneous identification of bacterial virulence genes by negative selection. Science 269: 400.
-
Hyatt, A., Boyle, D., Olsen, V., Boyle, D., Berger, L., Obendorf, D., Dalton, A., Kriger, K., Hero, M., Hines, H., Phillott, R., Campbell, R., Marantelli, G., Gleason, F., Colling, A. (2007): Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. Dis. Aquat. Organ. 73: 175-192.
-
Iwanowicz, D., Schill, W., Olson, D., Adams, M., Densmore, C., Cornman, R., Adams, C., Chester Figiel, J., Anderson, C., Blaustein, A., Chestnut, T. (2017): Potential concerns with analytical methods used for the detection of Batrachochytrium salamandrivorans from archived DNA of amphibian swab samples, Oregon, USA. Herpetol. Rev. 48: 352-355.
-
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. Dis. Aquat. Organ. 71: 141-148.
-
Langwig, K., Frick, W., Reynolds, R., Parise, K., Drees, K., Hoyt, J., Cheng, T., Kunz, T., Foster, J., Kilpatrick, A. (2015): Host and pathogen ecology drive the seasonal dynamics of a fungal disease, white-nose syndrome. Proc. R. Soc. London B Biol. Sci. 282: 20142335.
-
Livak, K., Flood, S., Marmaro, J., Giusti, W., Deetz, K. (1995): Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. Genome Res. 4: 357-362.
-
Longcore, J., Pessier, A., Nichols, D. (1999): Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 91: 219-227.
-
Longo, A., Rodriguez, D., Leite, D., Toledo, L., Almeralla, C., Burrowes, P., Zamudio, K. (2013): ITS1 copy number varies among Batrachochytrium dendrobatidis strains: implications for qPCR estimates of infection intensity from field-collected amphibian. PLoS ONE 8: e59499.
-
Martel, A., Blooi, M., Adriaensen, C., Van Rooij, P., Beukema, W., Fisher, M.C., Farrer, R.A., Schmidt, B.R., Tobler, U., Goka, K., Lips, K.R., Muletz, C., Zamudio, K.R., Bosch, J., Lotters, S., Wombwell, E., Garner, T.W.J., Cunningham, A.A., Spitzen-van der Sluijs, A., Salvidio, S., Ducatelle, R., Nishikawa, K., Nguyen, T.T., Kolby, J.E., Van Bocxlaer, I., Bossuyt, F., Pasmans, F. (2014): Recent introduction of a chytrid fungus endangers Western Palearctic salamanders. Science 346: 630-631.
-
Martel, A., Van Rooij, P., Vercauteren, G., Baert, K., Van Waeyenberghe, L., Debacker, P., Garner, T.W.J., Woeltjes, T., Ducatelle, R., Haesebrouck, F., Pasmans, F. (2011): Developing a safe antifungal treatment protocol to eliminate Batrachochytrium dendrobatidis from amphibians. Med. Mycol. 49: 143-149.
-
Martel, A., Spitzen-van der Sluijs, A., Blooi, M., Bert, W., Ducatelle, R., Fisher, M.C., Woeltjes, A., Bosman, W., Chiers, K., Bossuyt, F., Pasmans, F. (2013): Batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians. Proc. Natl. Acad. Sci. U.S.A. 110: 15325-15329.
-
Pallister, J., Gould, A., Harrison, D., Hyatt, A., Jancovich, J., Heine, H. (2007): Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses. J. Fish 30: 427-438.
-
R Core Team (2017): R: a Language and Environment for Statistical Computing.
-
Rachowicz, L.J., Knapp, R.A., Morgan, J.A.T., Stice, M.J., Vredenburg, V.T., Parker, J.M., Briggs, C.J. (2006): Emerging infectious disease as a proximate cause of amphibian mass mortality. Ecology 87: 1671-1683.
-
Rao, R.U., Huang, Y., Bockarie, M.J., Susapu, M., Laney, S.J., Weil, G.J. (2009): A qPCR-based multiplex assay for the detection of Wuchereria bancrofti, Plasmodium falciparum and Plasmodium vivax DNA. Trans. R. Soc. Trop. Med. Hyg. 103: 365-370.
-
Sabino-Pinto, J., Bletz, M., Iturriaga, M., Vences, M., Rodríguez, A. (2017): Low infection prevalence of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Chytridiomycetes: Rhizophydiales) in Cuba. Amphibia-Reptilia 38: 1-7.
-
Skerratt, L., Berger, L., Speare, R., Cashins, S., McDonald, K., Phillott, A., Hines, H., Kenyon, N. (2007): Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. Ecohealth 4: 125-134.
-
Spitzen-van der Sluijs, A., Martel, A., Asselberghs, J., Bales, E.K., Beukema, W., Bletz, M.C., Dalbeck, L., Goverse, E., Kerres, A., Kinet, T., Kirst, K., Laudelout, A., Marin, L.F., Nöllert, A., Ohlhoff, D., Sabino-Pinto, J., Schmidt, B.R., Speybroeck, J., Spikmans, F., Steinfartz, S., Veith, M., Vences, M., Wagner, N., Pasmans, F., Lötters, S. (2016): Expanding distribution of lethal amphibian fungus Batrachochytrium salamandrivorans in Europe. Emerg. Infect. Dis. 22: 1286-1288.
-
Spitzen-van der Sluijs, A., Spikmans, F., Bosman, W., de Zeeuw, M., van der Meij, T., Goverse, E., Kik, M., Pasmans, F., Martel, A. (2013): Rapid enigmatic decline drives the fire salamander (Salamandra salamandra) to the edge of extinction in the Netherlands. Amphibia-Reptilia 34: 233-239.
-
Stegen, G., Pasmans, F., Schmidt, B., Rouffaer, L., van Praet, S., Schaub, M., Canessa, S., Laudelout, A., Kinet, T., Adriaensen, C., Haesebrouck, F., Bert, W., Bossuyt, F., Martel, A. (2017): Drivers of salamander extirpation mediated by Batrachochytrium salamandrivorans. Nature 544: 353-356.
-
Thomas, V., Blooi, M., Van Rooij, P., Van Praet, S., Verbrugghe, E., Grasselli, E., Lukac, M., Smith, S., Pasmans, F., Martel, A. (2018): Recommendations on diagnostic tools for Batrachochytrium salamandrivorans. Transbound. Emerg. Dis. 65: e478-e488.
-
Woodhams, D., Kilburn, V., Reinert, L., Voyles, J. (2008): Chytridiomycosis and amphibian population declines continue to spread eastward in Panama. Ecohealth 5: 268-274.
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Detectability vs. time and costs in pooled DNA extraction of cutaneous swabs: a study on the amphibian chytrid fungi
In: Amphibia-ReptiliaAbstract
Epidemiology relies on understanding the distribution of pathogens which often can be detected through DNA-based techniques, such as quantitative Polymerase Chain Reaction (qPCR). Typically, the DNA of each individual sample is separately extracted and undergoes qPCR analysis. However, when performing field surveys and long-term monitoring, a large fraction of the samples is generally expected to be negative, especially in geographical areas still considered free of the pathogen. If pathogen detection within a population – rather than determining its individual prevalence – is the focus, work load and monetary costs can be reduced by pooling samples for DNA extraction. We test and refine a user-friendly technique where skin swabs can be pooled during DNA extraction to detect the amphibian chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans (Bsal). We extracted pools with different numbers of samples (from one to four swabs), without increasing reaction volumes, and each pool had one sample inoculated with a predetermined zoospore amount. Pool size did not reduce the ability to detect the two fungi, except if inoculated with extremely low zoospore amounts (one zoospore). We confirm that pooled DNA extraction of cutaneous swabs can substantially reduce processing time and costs without minimizing detection sensitivity. This is of relevance especially for the new emerging pathogen Bsal, for which pooled DNA extraction had so far not been tested and massive monitoring efforts in putatively unaffected regions are underway.
| All Time | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1017 | 241 | 14 |
| Full Text Views | 202 | 12 | 2 |
| PDF Views & Downloads | 69 | 22 | 4 |