In vitro modeling of Batrachochytrium dendrobatidis infection of the amphibian skin

Autoři: Elin Verbrugghe aff001;  Pascale Van Rooij aff001;  Herman Favoreel aff002;  An Martel aff001;  Frank Pasmans aff001
Působiště autorů: Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium aff001;  Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium aff002
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225224


The largest current disease-induced loss of vertebrate biodiversity is due to chytridiomycosis and despite the increasing understanding of the pathogenesis, knowledge unravelling the early host-pathogen interactions remains limited. Batrachochytrium dendrobatidis (Bd) zoospores attach to and invade the amphibian epidermis, with subsequent invasive growth in the host skin. Availability of an in vitro assay would facilitate in depth study of this interaction while reducing the number of experimental animals needed. We describe a fluorescent cell-based in vitro infection model that reproduces host-Bd interactions. Using primary keratinocytes from Litoria caerulea and the epithelial cell line A6 from Xenopus laevis, we reproduced different stages of host cell infection and intracellular growth of Bd, resulting in host cell death, a key event in chytridiomycosis. The presented in vitro models may facilitate future mechanistic studies of host susceptibility and pathogen virulence.

Klíčová slova:

Amphibians – Apoptosis – Cell cultures – Cell staining – Fluorescence microscopy – Fungal pathogens – Host cells – Host-pathogen interactions


1. Scheele B, Pasmans F, Skerratt LF, Berger L, Martel A, Beukema W, et al. Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity. Science 2012; 363: 1459–1463.

2. Skerratt LF, Berger L, Speare R, Cashins S, McDonald KR, Phillott AD, et al. Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth 2007; 4: 125.

3. Lips KR. Overview of chytrid emergence and impacts on amphibians. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2016; 371: 20150465. doi: 10.1098/rstb.2015.0465 28080989

4. Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin L, et al. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. P. Natl. Acad. Sci. USA 1998; 95: 9031–9036.

5. Martel A, Spitzen-van der Sluijs A, Blooi M, Bert W, Ducatelle R, Fisher M C, et al. Batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians. P. Natl. Acad. Sci. USA 2013; 110: 15325–15329.

6. Voyles J, Berger L, Young S, Speare R, Webb R, Warner J, et al. Electrolyte depletion and osmotic imbalance in amphibians with chytridiomycosis. Dis. Aquat. Organ. 2007; 77: 113–118. doi: 10.3354/dao01838 17972752

7. Voyles J, Young S, Berger L, Campbell C, Voyles WF, Dinudom A, et al. Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines. Science 2009; 326: 582–585. doi: 10.1126/science.1176765 19900897

8. Carver S, Bell BD. Does chytridiomycosis disrupt amphibian skin function? Copeia 2010; 3: 487–495.

9. Marcum RD, St-Hilaire S, Murphy PJ, Rodcnick KJ. Effects of Batrachochytrium dendrobatidis infection on ion concentrations in the boreal toad Anaxyrus (Bufo) boreas boreas. Dis. Aquat. Organ. 2010; 91: 17–21. doi: 10.3354/dao02235 20853738

10. Brutyn M, D’Herde K, Dhaenens M, Van Rooij M, Verbrugghe E, Hyatt AD, et al. Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin. Fungal Genet. Biol. 2012; 49: 830–837. doi: 10.1016/j.fgb.2012.07.002 22903040

11. Berger L, Speare R, Skerratt LF. Distribution of Batrachochytrium dendrobatidis and pathology in the skin of green tree frogs Litoria caerulea with severe chytridiomycosis. Dis. Aquat. Organ. 2005; 68: 65–70 doi: 10.3354/dao068065 16465835

12. Young S, Speare R, Berger L, Skerratt LF. Chloramphenicol with fluid and electrolyte therapy cures terminally ill green tree frogs (Litoria caerulea) with chytridiomycosis. J. Zoo Wildl. Med. 2012; 43: 330–337. doi: 10.1638/2011-0231.1 22779237

13. Cramp RL, McPhee RK, Meyer EA, Ohmer ME, Franklin CE. First line of defence: the role of sloughing in the regulation of cutaneous microbes in frogs. Conserv. Physiol. 2014; 2: cou012. doi: 10.1093/conphys/cou012 27293633

14. Ohmer ME, Cramp RL, White CR, Franklin CE. Skin sloughing rate increases with chytrid fungus infection load in a susceptible amphibian. Funct. Ecol. 2015; 29: 674–682.

15. Bovo RP, Andrade DV, Toledo LF, Longo AV, Rodriguez D, Haddad CF, et al. Physiological responses of Brazilian amphibians to an enzootic infection of the chytrid fungus Batrachochytrium dendrobatidis. Dis. Aquat. Organ. 2016; 117: 245–252. doi: 10.3354/dao02940 26758658

16. Grogan LF, Skerratt LF, Berger L, Cashins SD, Trengove RD, Gummer JP. Chytridiomycosis causes catastrophic organism-wide metabolic dysregulation including profound failure of cellular energy pathways. Sci. Rep. 2018; 8: 8188. doi: 10.1038/s41598-018-26427-z 29844538

17. Russo CJ, Ohmer ME, Cramp RL, Franklin CE. A pathogenic skin fungus and sloughing exacerbate cutaneous water loss in amphibians. J. Exp. Biol. 2018; 221: jeb167445. doi: 10.1242/jeb.167445 29752415

18. Wu NC, Cramp RL, Ohmer ME, Franklin CE. Epidermal epidemic: unravelling the pathogenesis of chytridiomycosis. J. Exp. Biol. 2019; 222: jeb191817 doi: 10.1242/jeb.191817 30559300

19. Berger L, Longcore J, Hyatt AD. Life cycle stages of Batrachochytrium dendrobatidis (Longcore), the amphibian chytrid. Dis. Aquat. Organ. 2005; 62: 51–63.

20. Van Rooij P, Martel A, D’Herde K, Brutyn M, Croubels S, Ducatelle R, et al. Germ tube mediated invasion of Batrachochytrium dendrobatidis in amphibian skin is host dependent. Plos One 2012; 7: e41481. doi: 10.1371/journal.pone.0041481 22911798

21. Greenspan SE, Longcore JE, Calhoun AJ. Host invasion by Batrachochytrium dendrobatidis: fungal and epidermal ultrastructure in model anurans. Dis. Aquat. Organ. 2012; 100: 201–210. doi: 10.3354/dao02483 22968788

22. Fites JS, Ramsey JP, Holden WM, Collier SP, Sutherland DM, Reinert LK, et al. The invasive chytrid fungus of amphibians paralyzes lymphocyte responses. Science 2013; 342: 366–369. doi: 10.1126/science.1243316 24136969

23. Van Rooij P, Martel A, Brutyn M, Maes S, Chiers K, Van Waeyenberghe L, et al. Development of in vitro models for a better understanding of the early pathogenesis of Batrachochytrium dendrobatidis infections in amphibians. ATLA-Altern. Lab. Anim. 2010; 38: 519–528.

24. Boyle DG, Boyle DB, Olsen V, Morgan JAT, Hyatt AD. Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis. Aquat. Organ. 2004; 60: 141–148. doi: 10.3354/dao060141 15460858

25. Farrer RA, Weinert LA, Bielby J, Garner TWJ, Balloux F, Clare F et al. Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. P. Natl. Acad. Sci. USA 2011; 108: 18732–18736.

26. Rafferty K.A. Mass culture of amphibian cells: methods and observations concerning stability of cell type. In: edited by Mizell M, Biology of amphibian tumors,. New York: Springer-Verlag, 1969, pp 52–81.

27. Blooi M, Laking AE, Martel A, Haesebrouck F, Jocque M, Brown T, et al. Host niche may determine disease-driven extinction risk. PLoS ONE 2017; 12: e0181051. doi: 10.1371/journal.pone.0181051 28704480

28. Thomas V, Blooi M, Van Rooij P, Van Praet S, Verbrugghe E, Grasselli E, et al. Recommendations on diagnostic tools for Batrachochytrium salamandrivorans. Transbound. Emerg. Dis. 2018; 65: e478–e488. doi: 10.1111/tbed.12787 29341499

29. Garner TW, Schmidt BR, Martel A, Pasmans F, Muths E, Cunningham AA, et al. Mitigating amphibian chytridiomycoses in nature. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2016; 371: 20160207. doi: 10.1098/rstb.2016.0207 28080996

30. Fisher MC, Garner TWJ, Walker SF. Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. Annu. Rev. Microbiol. 2009; 63: 291–310. doi: 10.1146/annurev.micro.091208.073435 19575560

31. Kilpatrick AM, Briggs CJ, Daszak P. The ecology and impact of chytridiomycosis: an emerging disease of amphibians. Trends Ecol. Evol 2010; 25: 109–118. doi: 10.1016/j.tree.2009.07.011 19836101

32. Van Rooij P, Martel A, Haesebrouck F, Pasmans F. Amphibian chytridiomycosis: A review with focus on fungus-host interactions. Vet. Res. 2015; 46:137. doi: 10.1186/s13567-015-0266-0 26607488

33. Russel WMS, Burch RL. The Principles of Humane Experimental Technique. London, UK: Methuen; 1959.

34. Vredenburg VT, Knapp RA, Tunstall TS, Briggs CJ. Dynamics of an emerging disease drive large-scale amphibian population extinctions. Proc. Natl. Acad. Sci. U S A. 2010; 107: 9689–94. doi: 10.1073/pnas.0914111107 20457913

35. Garner T, Walker S, Bosch J, Leech S, Rowcliffe JM, Cunningham AA, et al. Life history Tradeoffs influence mortality associated with the amphibian pathogen Batrachochytrium dendrobatidis. Oikos 2009; 118: 783–91.

36. Kriger KM, Hero J-M. Large-scale seasonal variation in the prevalence and severity of chytridiomycosis. J. Zool. 2007; 271: 352–59.

37. Pasmans F, Van Rooij P, Blooi M, Tessa G, Bogaerts S, Sotgiu G, et al. Resistance to Chytridiomycosis in European Plethodontid Salamanders of the Genus Speleomantes. Plos One 2013; 8: e63639. doi: 10.1371/journal.pone.0063639 23703511

38. Ramsey JP, Reinert LK, Harper LK, Woodhams DC, Rollins-Smith LA. Immune Defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, in the South African clawed frog, Xenopus laevis. Infect. Immun. 2010; 78: 3981–3992. doi: 10.1128/IAI.00402-10 20584973

39. Rollins-Smith LA. The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines. Biochim. Biophys. Acta. 2009; 1788: 1593–1599. doi: 10.1016/j.bbamem.2009.03.008 19327341

40. Rollins-Smith LA, Ramsey JP, Pask JD, Reinert LK, Woodhams DC. Amphibian immune defenses against chytridiomycosis: impacts of changing environments. Integr. Comp. Biol. 2011; 51: 552–562. doi: 10.1093/icb/icr095 21816807

41. Smith HK, Pasmans F, Dhaenens M, Deforce D, Bonte D, Verheyen K, et al. Skin mucosome activity as an indicator of Batrachochytrium salamandrivorans susceptibility in salamanders. Plos One 2018; 13: e0199295 doi: 10.1371/journal.pone.0199295 30020936

42. Woodhams DC, Ardipradja K, Alford RA, Marantelli G, Reinert LK, Rollins-Smith LA. Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Anim. Conserv. 2007; 10: 409–417.

43. Bates KA, Clare FC, O’Hanlon S, Bosch J, Brookes L, Hopkins K, et al. Amphibian chytridiomycosis outbreak dynamics are linked with host skin bacterial community structure. Nat. Commun. 2018; 9: 693. doi: 10.1038/s41467-018-02967-w 29449565

44. Bletz MC, Kelly M, Sabino-Pinto J, Bates E, Van Praet S, Bert W, et al. Disruption of skin microbiota contributes to salamander disease. Proc. R. Soc. B 2018; 285: 20180758. doi: 10.1098/rspb.2018.0758 30135150

45. Brannelly LA, Roberts AA, Skerratt LF, Berger L. Epidermal Cell death in frogs with chytridiomycosis. PeerJ 2017; 5: e2925. doi: 10.7717/peerj.2925 28168107

46. Schloegel LM, Toledo LF, Longcore JE, Greenspan SE, Vieira CA, Lee M et al. Novel, panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade. Mol. Ecol. 2012; 21: 5162–5177. doi: 10.1111/j.1365-294X.2012.05710.x 22857789

47. O'Hanlon SJ, Rieux A, Farrer RA, Rosa GM, Waldman B, Bataille A, et al. Recent Asian origin of chytrid fungi causing global amphibian declines. Science 2018; 360: 621–627. doi: 10.1126/science.aar1965 29748278

Článek vyšel v časopise


2019 Číslo 11