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Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Shaw, 1802), bullfrog
AQUA-631041; No of Pages 4 Aquaculture xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Shaw, 1802), bullfrog M. Hipolito ⁎, A.M.C.R.P.F. Martins, L.L. Cassiano, R.B. Sousa, R.C. Tedesco, A. Reis, C.M. Ferreira Instituto Biológico/SP, APTA/SAA, Av. Conselheiro Rodrigues Alves, 1252, São Paulo, SP CEP 04014-002, Brazil
a r t i c l e
i n f o
Article history: Received 30 January 2014 Received in revised form 6 February 2014 Accepted 10 February 2014 Available online xxxx Keywords: Chytridiomycosis Histology Amphibian Pathology Disease Fungus
a b s t r a c t In 1998 an emerging disease, chytridiomycosis, was diagnosed, caused by the fungus, Batrachochytrium dendrobatidis, capable of infecting amphibians (Urodela and Anura) and it was considered to be the main cause of the global decline of these animals. Continuing the research work on the presence of this fungus in commercial flocks, tadpoles of bullfrogs were studied, where its presence was possibly found in the mouth of these animals using special histological techniques like historesin. Starting from the visual observation of the mouth of the tadpoles, it was observed that there was a discoloration of the partial structures of the denticles, which were typically dark in color and appeared whitish instead. The mouth of tadpoles with denticles was collected, and fixed in 10% buffered formalin. The inclusion of the biological material in glycol methacrylate (GMA) has been widely used due to a number of advantages over the usual histological methods, such as faster processing, water soluble resins eliminate the use of solvents, easy handling, obtaining semifine sections from 0.5 to 5 μm, highest resolution light microscope, and material is processed at room temperature thereby reducing distortions and artifacts. We used the commercial kit historesin FONTOVIT 7100 (Kulzer)®. After fixation in cacodylate buffer, dehydration increased in ethanol alcohol, pre-infiltrated with historesin diluted in alcohol, infiltrated with historesin pure and polymerized. Following mounting in acrylic and cut with automatic microtome. The staining can be performed with techniques used for inclusion in paraffin and is not necessary to hydrate or diaphanized cuts and color dyes suitable for fungi such as cotton blue and Congo red and hematoxylin–eosin. Slides were covered with Entellan® and coverslip. Characteristics of fungal structures, such as zoosporangia, single or grouped globular structures with discharge pipes for the removal of spores, were observed. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In the past, the fungus chitridium was considered as being predominately saprophytic of free live, with few species capable of infecting only invertebrates and plants. A new species, Batrachochytrium dendrobatidis — Bd — (fungi, Chytridiomycota, Rhizophydiales) was discovered in 1998, which showed capability to infect amphibians and cause a disease many times fatal, known as chytridiomycosis. Later studies showed still that the Bd was associated with the decline of amphibian populations in all continents habited by them, which connected to the loss of hundreds of species in the whole world (Berger and Speare, 1998, 2000; Berger et al., 1998, 1999a, b; Daszak et al., 1999; Langcore et al., 1999; Paré, 2003; Vredenburg et al., 2008; Young et al., 2007). The chytridiomycosis, is a dermal mycosis, superficial, attacking the amphibians, developing only on the outside layer of the keratinized ⁎ Corresponding author. Tel.: +55 1150871760. E-mail address: [email protected] (M. Hipolito).
epidermis, and still can attack the parts of keratinized epithelium of the mouth of tadpoles (Padgett-Flohr and Goble, 2007; Rachowicz and Vredenburg, 2004). Currently, it is recognized as a disease of big importance on the world scenery, and included in the official list of epizooties of WOA/OIE (WOAH/OIE, 2012). It has been observed in bullfrog of free life (Rana catesbeiana, now known as Lithobates catesbeianus, Frost et al., 2006) in the United States and in Venezuela (Hanselmann et al., 2004) and in bullfrog created commercially in Uruguay (Mazzoni et al., 2003) and recently too in Brazil (Hipolito et al., 2011, 2012; Schloegel et al., 2009, 2012). In Brazil, it has already been observed in native amphibians of free life and in zoo museum's collections as presented by Carnaval et al. (2006), Haddad (2005) and Toledo et al. (2006a, b), and these same authors report the need for new and extended studies in the presence of this fungus. Among these new and expanded studies, given the need for pressing diagnosis, is the use of histological techniques for the visualization of the fungus in the affected tissues, and in the oral structure of tadpoles, one of these locations. This work is intended to complement the histological
http://dx.doi.org/10.1016/j.aquaculture.2014.02.016 0044-8486/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Hipolito, M., et al., Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Sh..., Aquaculture (2014), http://dx.doi.org/10.1016/j.aquaculture.2014.02.016
2
M. Hipolito et al. / Aquaculture xxx (2014) xxx–xxx
Fig. 1. Presence of the Batrachochytrium dendrobatidis in oral tissue from bullfrog tadpoles (arrow).
diagnosis with the use of modern procedures, faster and less polluting material, as historesin, suppressing the use of organic solvents such as xylene, widely used when embedding in paraffin (Lambertini et al., 2013; Pretlow and Pretlow, 1998; Santos et al., 2004). The inclusions of tissues in glycol methacrylate (GMA) have been widely used due to a number of advantages over the usual histological methods, such as faster processing, water soluble resins are eliminating the use of solvents, easy to handle and getting semifine cuts from 0.5 to 5 μm. Moreover, it has better resolution in light microscope and the material is processed at room temperature thus reducing distortions and artifacts. In the case of search for structures of the fungus in the oral epidermis of tadpoles, after making historesin, the own colorations to these agents may also be used, such as cotton blue (Mazzoni et al., 2003), and Congo red, according to Briggs and Burgin (2003). 2. Materials and methods From the collection of oral fragments of tadpoles, collected in a commercial frog farm in the Vale do Rio Paraiba, State of São Paulo, Brazil, indicated by discoloration and the presence of fungus (Fig. 1), fragments were plated on tryptone agar for culture (Lambertini et al., 2013; Vieira and Toledo, 2012) and part of the tissue was fixed in neutral 10% formalin. The technical procedure using historesin was started, in isolated fungal colonies from pure growth on agar plates (Fig. 2) and the fragments in formalin using the commercial kit FONTOVIT 7100 (Kulzer®), with the preparation of solutions of infiltration and inclusion, with the infiltration solution (A) composed of historesin 50 mL
Fig. 2. Colony of Batrachochytrium dendrobatidis fungus growing on tryptone agar, used for mounting in historesin.
Fig. 3. Colony of Batrachochytrium dendrobatidis, in historesin, showing the typical structures of the fungus, globular zoosporangius and discharge papilla. The resin did not affect the morphology of the fungus. Staining by Congo red. 630×.
and activator powder 0.5 g. The solution of inclusion (B) composed of infiltration solution (A) 15 mL and the accelerator with 1 mL followed. The tissue fixation was by immersion in 4% in paraformaldehyde and 2.5% GMA in 0.1 M sodium cacodylate buffer pH 7.4, over a period of 2 h at 4 °C. Dehydration was carried out by immersion in a series in ethyl alcohol (ethanol) 50%, 70%, 90% and 100% with two exchanges in each pass, at room temperature and under slow and constant stirring. Pre-infiltration after the previous step, was by immersion in a solution of ethanol and GMA (infiltration solution (A)) equally at room temperature for a period of 12 h, overnight under slow and constant stirring. The infiltration was with solution (A), pure, at room temperature for 4 h. The addition was done by placing the tissue fragments into suitable plastic molds containing the solution of inclusion (B), which was prepared only at the time of inclusion, since the polymerization takes place rapidly (in a time of approximately 2 h). After complete polymerization, blocks were bonded with the admixture of assembly (C) composed of acrylic polymethylmethacrylate powder with 2 parts and methylmethacrylate liquid with 1 part by the appropriate support, this procedure being carried out quickly. The cut of 5 μm was made in automatic microtome using disposable steel blades, thus obtaining a better thickness of the slice. These were stretched in a container with distilled water at room temperature and “caught” with the aid of glass slides. Drying took place in oven at 60 °C. Then, in a few slides stained by routine hematoxylin–eosin, other blades were taken, Congo red, 0.05% in phosphate buffer pH 7.4 for 30 min (Briggs and Burgin, 2003). After drying, the coverslip was placed using Entellan®.
Fig. 4. Histological section mounted in resin, showing the presence of the fungus (arrow). Staining by hematoxylin–eosin. 400×.
Please cite this article as: Hipolito, M., et al., Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Sh..., Aquaculture (2014), http://dx.doi.org/10.1016/j.aquaculture.2014.02.016
M. Hipolito et al. / Aquaculture xxx (2014) xxx–xxx
3
References
Fig. 5. Other histologic cut mounted, also in resin, showing the presence of the fungus (arrow). Staining by hematoxylin–eosin. 200×.
Slides were observed and documented with light microscopy equipment Axio Scope A1 Zeiss® microscope and Axio Vision® program 4.8.2.
3. Results and discussion The cuts made with the fungal colony, stained with Congo red showed structures characteristics of the chytrid fungus group, with globose sporangia in various sizes and typical discharge papilla for the elimination of mobile spores, the zoospores (Fig. 3). The recognition that the structure of the fungus has not been damaged or altered by the action of historesin formations indicates that sporangia are next to oral tissue of tadpoles, and numerous were located. The originality of historesin technique is its simplicity combined with high efficiency to produce well oriented cuts and much thinner than the conventional technique in paraffin and preserve more efficiently by not requiring the cells, and the cuts are dehydrated and diaphanous. Another application is a three-dimensional visualization with physical serial sections, allowing the reconstruction of the structure. The efficiency of the latter application is heavily dependent on the quality of the embedded specimen which often leads to artifacts that complicate the analysis (De Smet et al., 2004). Results were consistent with the expected absence of structural changes of the fungus that may compromise their visualization in the oral epidermis of tadpoles being similar to those observed in common histology (Hipolito et al., 2011, 2012; Ramalho et al., 2013), as shown in Figs. 4 and 5. No references to the use of historesins in the histopathological diagnosis of Bd fungus were found, but the results showed that this technique is feasible and can be routinely used, avoiding the use of products that are potentially manipulative and aggressive to the environment, such as xylene, without interfering with the results. Like every technique that begins its use it may still need necessary adjustments and continuity of this work will bring refinement.
4. Conclusion The use of historesin to histopathological diagnosis of Batrachochytrium dendrobatidis in the mouth of bullfrog tadpole is perfectly feasible, collaborating with a firm diagnosis. Acknowledgments This work was supported by FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo, Projects FAPESP 2011 50009-9 and 2013 15659-8.
Berger, L., Speare, R., 1998. Chytridiomycosis—a new disease of amphibians. ANZCCART News 11 (4), 1–3. Berger, L., Speare, R., 2000. Global distribution of chytridiomycosis in amphibians. (Available in http://www.jcu.edu.au/school/PHTM/frogs/chyglob.htm Captured in October 2008). Berger, L., Speare, R., Daszak, P., Green, D.E., Cunningham, A.A., Goggins, C.L., Slocombe, R., Ragan, M.A., Hyatt, A.D., MacDonald, K.R., Hines, H.B., Lips, K.R., Morantelli, G., Parkes, H., 1998. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc. Natl. Acad. Sci. U. S. A. 95, 9031–9036. Berger, L., Speare, R., Hyatt, A.D., 1999a. Chytrid fungi and amphibian declines: overview, implications and future directions. (Available in http://www.jcu.edu.au/school/ phtm.frogs Captured in October 2006). Berger, L., Speare, R., Kent, A., 1999b. Diagnosis of chytridiomycosis in amphibians by histologic examination. 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Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Hass, A., Haddad, C.F.B., De Sa, R., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Gree, D.N., Wheeler, W.C., 2006. The amphibian tree of life. Bull. Am. Mus. Nat. Hist. 297, 1–370. Haddad, C., 2005. Encontrado fungo letal no Brasil. Pesq. FAPESP 114, 36. Hanselmann, R., Rodriguez, A., Lampo, M., Fajardo-Ramos, L., Aguirre, A., Kilpatrick, M., Rodríguez, J.P., Daszak, P., 2004. Presence of an emerging pathogen of amphibians in introduced bullfrogs Rana catesbeiana in Venezuela. Biol. Conserv. 120, 115–119. Hipolito, M., Martins, A.M.C.R.P.F., Ferreira, C.M., Schoegel, L., Daszak, P., Teixeira, P.C., Dias, D.C., 2011. Presence of Batrachochytrium dendrobatidis, emerging pathogen in Lithobates catesbeianus (Rana catesbeiana Shaw, 1802), bullfrog in the State of São Paulo, Brazil. Proceedings of the Aquaculture Europe 2011. EAS European Aquaculture Society, Rhodes, Greece. Hipolito, M., Martins, A.M.C.R.P.F., Ferreira, C.M., Schoegel, L., Daszak, P., Teixeira, P.C., Dias, D.C., 2012. Batrachochytrium dendrobatidis, presente in mouth of tadpoles of Lithobates catesbeianus (Rana catesbeiana Shaw, 1802), bullfrog, in São Paulo, Brazil. Proceedings of the Aquaculture Europe 2012. EAS - European Aquaculture Society and WAS - World Aquaculture Society, Prague, Czech Republic. Lambertini, C., Rodrigues, D., Brito, F.B., Leite, D.S., Toledo, L.F., 2013. Diagnóstico do Fungo Quitrídio: Batrachochytrium dendrobatidis. Herpetol. Bras. 2, 12–17. Langcore, 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. Mazzoni, R., Cunningham, A., Daszak, P., Apolo, A., Perdomo, E., Speranza, G., 2003. Emerging pathogen of wild amphibians in frogs (Rana catesbeiana) farmed for international trade. Emerg. Infect. Dis. 9 (8), 2003 (Available in http.//www.cdc.gov/ncidod/EID/vol9no8/ 03-0030.htm. Captured in August 2003). Padgett-Flohr, G.E., Goble, M.E., 2007. Evaluation of tadpole mouthpart depigmentation as a diagnostic test for infection by Batrachochytrium dendrobatidis for four California anurans. J. Wildl. Dis. 43 (4), 690–699. Paré, J.A., 2003. Fungal diseases of amphibians: an overview. Vet. Clin. Exot. Anim. 6, 315–326. Pretlow, T.P., Pretlow, T.G., 1998. Putative preneoplastic changes identified by enzyme histochemical and immunohistochemical techniques. J. Histochem. Cytochem. 46 (5), 577–583. Rachowicz, L.J., Vredenburg, V.T., 2004. Transmission of Batrachochytrium dendrobatidis within and between amphibian life stages. Dis. Aquat. Org. 61, 75–83. Ramalho, A.C.O., De Paula, C.D., Catão-Dias, J.L., Vilarin ho, B., Brandão, R.B., 2013. First record of Batrachochytrium dendrobatidis in two endemic Cerrado hylids, Bokermannohyla pseudopseudis and Bokermannohyla sapiranga, with comments on chytridiomycosis spreading in Brazil. N.West. J. Zool. 9 (art.131504. http://biozoojournals.3x.ro/nwjz/ index.html). Santos, F.C.A., Corradi, L.S., Leite, R.P., Góes, R.M., Taboga, S.R., 2004. A modified method for the selective staining of elastic system fibers in methacrylate tissue sections. Braz. J. Morphol. Sci. 21 (2), 73–79. Schloegel, L.M., Ferreira, C.M., James, T.Y., Hipolito, M., Langcore, J.E., Hyatt, A.D., Yabsley, M., Martins, A.M.C.R.P.F., Mazzoni, R., Davies, A.J., Daszak, P., 2009. The North American bullfrog as a reservoir for the spread of Batrachochytrium dendrobatidis in Brazil. Anim. Conserv. 13 (Suppl.1), 1–9. http://dx.doi.org/10.1111/j.1469-1795.2009.00307.x (2010). Schloegel, L.M., Toledo, L.F., Langcore, J.E., Greenspan, S.H., Vieira, C.A., Lee, M., Zhao, S., Wangen, C., Ferreira, C.M., Hipolito, H., Davies, A.J., Cuomo, C.A., Daszak, P., James, T.Y., 2012. Novel, panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade. Mol. Ecol. http://dx.doi.org/10.1111/j.1365294X.2012.05710.x. Toledo, L.F., Brito, F.B., Araújo, O.G.S., Giasson, L.M.O., Haddad, C.F.B., 2006a. The occurrence of Batrachochytrium dendrobatidis in Brazil and the inclusion of 17 new cases of infection. South Am. J. Herpetol. 1 (3), 185–191.
Please cite this article as: Hipolito, M., et al., Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Sh..., Aquaculture (2014), http://dx.doi.org/10.1016/j.aquaculture.2014.02.016
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Toledo, L.F., Haddad, C.F.B., Carnaval, A.O.C.Q., Brito, F.B., 2006b. A Brazilian anuran (Hylodes magalhaesi: Leptodactylidae) infected by Batrachochytrium dendrobatidis: a conservation concern. Amphibian Reptile Conserv. 4 (1), 17–21. Vieira, C.A., Toledo, L.F., 2012. Isolamento, cultivo e armazenamento do fungo quitrídio: Batrachochytrium dendrobatidis. Herpetol. Bras. 1, 18–19. Vredenburg, V., Drewes, R., Hayes, T., Swain, K., 2008. Worldwide amphibian declines: how big is the problem, what are the causes and what can be done? (Available in: http://amphibiaweb.org. Captured in June 2008).
WOAH/OIE, 2012. Aquatic Animal Health Code 2012. Infection with Batrachochytrium dendrobatidis (http://www.oie.int/fileadmin/home/eng/health_standards/aahm/ current/2.1.01_inf_BATRACHOCHYRIUM.pdf). Young, S., Berger, L., Speare, R., 2007. Amphibian chytridiomycosis: strategies for captive management and conservation. Int. Zoo Yearb. 41, 1–11.
Please cite this article as: Hipolito, M., et al., Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Sh..., Aquaculture (2014), http://dx.doi.org/10.1016/j.aquaculture.2014.02.016
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Shaw, 1802), bullfrog M. Hipolito ⁎, A.M.C.R.P.F. Martins, L.L. Cassiano, R.B. Sousa, R.C. Tedesco, A. Reis, C.M. Ferreira Instituto Biológico/SP, APTA/SAA, Av. Conselheiro Rodrigues Alves, 1252, São Paulo, SP CEP 04014-002, Brazil
a r t i c l e
i n f o
Article history: Received 30 January 2014 Received in revised form 6 February 2014 Accepted 10 February 2014 Available online xxxx Keywords: Chytridiomycosis Histology Amphibian Pathology Disease Fungus
a b s t r a c t In 1998 an emerging disease, chytridiomycosis, was diagnosed, caused by the fungus, Batrachochytrium dendrobatidis, capable of infecting amphibians (Urodela and Anura) and it was considered to be the main cause of the global decline of these animals. Continuing the research work on the presence of this fungus in commercial flocks, tadpoles of bullfrogs were studied, where its presence was possibly found in the mouth of these animals using special histological techniques like historesin. Starting from the visual observation of the mouth of the tadpoles, it was observed that there was a discoloration of the partial structures of the denticles, which were typically dark in color and appeared whitish instead. The mouth of tadpoles with denticles was collected, and fixed in 10% buffered formalin. The inclusion of the biological material in glycol methacrylate (GMA) has been widely used due to a number of advantages over the usual histological methods, such as faster processing, water soluble resins eliminate the use of solvents, easy handling, obtaining semifine sections from 0.5 to 5 μm, highest resolution light microscope, and material is processed at room temperature thereby reducing distortions and artifacts. We used the commercial kit historesin FONTOVIT 7100 (Kulzer)®. After fixation in cacodylate buffer, dehydration increased in ethanol alcohol, pre-infiltrated with historesin diluted in alcohol, infiltrated with historesin pure and polymerized. Following mounting in acrylic and cut with automatic microtome. The staining can be performed with techniques used for inclusion in paraffin and is not necessary to hydrate or diaphanized cuts and color dyes suitable for fungi such as cotton blue and Congo red and hematoxylin–eosin. Slides were covered with Entellan® and coverslip. Characteristics of fungal structures, such as zoosporangia, single or grouped globular structures with discharge pipes for the removal of spores, were observed. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In the past, the fungus chitridium was considered as being predominately saprophytic of free live, with few species capable of infecting only invertebrates and plants. A new species, Batrachochytrium dendrobatidis — Bd — (fungi, Chytridiomycota, Rhizophydiales) was discovered in 1998, which showed capability to infect amphibians and cause a disease many times fatal, known as chytridiomycosis. Later studies showed still that the Bd was associated with the decline of amphibian populations in all continents habited by them, which connected to the loss of hundreds of species in the whole world (Berger and Speare, 1998, 2000; Berger et al., 1998, 1999a, b; Daszak et al., 1999; Langcore et al., 1999; Paré, 2003; Vredenburg et al., 2008; Young et al., 2007). The chytridiomycosis, is a dermal mycosis, superficial, attacking the amphibians, developing only on the outside layer of the keratinized ⁎ Corresponding author. Tel.: +55 1150871760. E-mail address: [email protected] (M. Hipolito).
epidermis, and still can attack the parts of keratinized epithelium of the mouth of tadpoles (Padgett-Flohr and Goble, 2007; Rachowicz and Vredenburg, 2004). Currently, it is recognized as a disease of big importance on the world scenery, and included in the official list of epizooties of WOA/OIE (WOAH/OIE, 2012). It has been observed in bullfrog of free life (Rana catesbeiana, now known as Lithobates catesbeianus, Frost et al., 2006) in the United States and in Venezuela (Hanselmann et al., 2004) and in bullfrog created commercially in Uruguay (Mazzoni et al., 2003) and recently too in Brazil (Hipolito et al., 2011, 2012; Schloegel et al., 2009, 2012). In Brazil, it has already been observed in native amphibians of free life and in zoo museum's collections as presented by Carnaval et al. (2006), Haddad (2005) and Toledo et al. (2006a, b), and these same authors report the need for new and extended studies in the presence of this fungus. Among these new and expanded studies, given the need for pressing diagnosis, is the use of histological techniques for the visualization of the fungus in the affected tissues, and in the oral structure of tadpoles, one of these locations. This work is intended to complement the histological
http://dx.doi.org/10.1016/j.aquaculture.2014.02.016 0044-8486/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Hipolito, M., et al., Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Sh..., Aquaculture (2014), http://dx.doi.org/10.1016/j.aquaculture.2014.02.016
2
M. Hipolito et al. / Aquaculture xxx (2014) xxx–xxx
Fig. 1. Presence of the Batrachochytrium dendrobatidis in oral tissue from bullfrog tadpoles (arrow).
diagnosis with the use of modern procedures, faster and less polluting material, as historesin, suppressing the use of organic solvents such as xylene, widely used when embedding in paraffin (Lambertini et al., 2013; Pretlow and Pretlow, 1998; Santos et al., 2004). The inclusions of tissues in glycol methacrylate (GMA) have been widely used due to a number of advantages over the usual histological methods, such as faster processing, water soluble resins are eliminating the use of solvents, easy to handle and getting semifine cuts from 0.5 to 5 μm. Moreover, it has better resolution in light microscope and the material is processed at room temperature thus reducing distortions and artifacts. In the case of search for structures of the fungus in the oral epidermis of tadpoles, after making historesin, the own colorations to these agents may also be used, such as cotton blue (Mazzoni et al., 2003), and Congo red, according to Briggs and Burgin (2003). 2. Materials and methods From the collection of oral fragments of tadpoles, collected in a commercial frog farm in the Vale do Rio Paraiba, State of São Paulo, Brazil, indicated by discoloration and the presence of fungus (Fig. 1), fragments were plated on tryptone agar for culture (Lambertini et al., 2013; Vieira and Toledo, 2012) and part of the tissue was fixed in neutral 10% formalin. The technical procedure using historesin was started, in isolated fungal colonies from pure growth on agar plates (Fig. 2) and the fragments in formalin using the commercial kit FONTOVIT 7100 (Kulzer®), with the preparation of solutions of infiltration and inclusion, with the infiltration solution (A) composed of historesin 50 mL
Fig. 2. Colony of Batrachochytrium dendrobatidis fungus growing on tryptone agar, used for mounting in historesin.
Fig. 3. Colony of Batrachochytrium dendrobatidis, in historesin, showing the typical structures of the fungus, globular zoosporangius and discharge papilla. The resin did not affect the morphology of the fungus. Staining by Congo red. 630×.
and activator powder 0.5 g. The solution of inclusion (B) composed of infiltration solution (A) 15 mL and the accelerator with 1 mL followed. The tissue fixation was by immersion in 4% in paraformaldehyde and 2.5% GMA in 0.1 M sodium cacodylate buffer pH 7.4, over a period of 2 h at 4 °C. Dehydration was carried out by immersion in a series in ethyl alcohol (ethanol) 50%, 70%, 90% and 100% with two exchanges in each pass, at room temperature and under slow and constant stirring. Pre-infiltration after the previous step, was by immersion in a solution of ethanol and GMA (infiltration solution (A)) equally at room temperature for a period of 12 h, overnight under slow and constant stirring. The infiltration was with solution (A), pure, at room temperature for 4 h. The addition was done by placing the tissue fragments into suitable plastic molds containing the solution of inclusion (B), which was prepared only at the time of inclusion, since the polymerization takes place rapidly (in a time of approximately 2 h). After complete polymerization, blocks were bonded with the admixture of assembly (C) composed of acrylic polymethylmethacrylate powder with 2 parts and methylmethacrylate liquid with 1 part by the appropriate support, this procedure being carried out quickly. The cut of 5 μm was made in automatic microtome using disposable steel blades, thus obtaining a better thickness of the slice. These were stretched in a container with distilled water at room temperature and “caught” with the aid of glass slides. Drying took place in oven at 60 °C. Then, in a few slides stained by routine hematoxylin–eosin, other blades were taken, Congo red, 0.05% in phosphate buffer pH 7.4 for 30 min (Briggs and Burgin, 2003). After drying, the coverslip was placed using Entellan®.
Fig. 4. Histological section mounted in resin, showing the presence of the fungus (arrow). Staining by hematoxylin–eosin. 400×.
Please cite this article as: Hipolito, M., et al., Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Sh..., Aquaculture (2014), http://dx.doi.org/10.1016/j.aquaculture.2014.02.016
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References
Fig. 5. Other histologic cut mounted, also in resin, showing the presence of the fungus (arrow). Staining by hematoxylin–eosin. 200×.
Slides were observed and documented with light microscopy equipment Axio Scope A1 Zeiss® microscope and Axio Vision® program 4.8.2.
3. Results and discussion The cuts made with the fungal colony, stained with Congo red showed structures characteristics of the chytrid fungus group, with globose sporangia in various sizes and typical discharge papilla for the elimination of mobile spores, the zoospores (Fig. 3). The recognition that the structure of the fungus has not been damaged or altered by the action of historesin formations indicates that sporangia are next to oral tissue of tadpoles, and numerous were located. The originality of historesin technique is its simplicity combined with high efficiency to produce well oriented cuts and much thinner than the conventional technique in paraffin and preserve more efficiently by not requiring the cells, and the cuts are dehydrated and diaphanous. Another application is a three-dimensional visualization with physical serial sections, allowing the reconstruction of the structure. The efficiency of the latter application is heavily dependent on the quality of the embedded specimen which often leads to artifacts that complicate the analysis (De Smet et al., 2004). Results were consistent with the expected absence of structural changes of the fungus that may compromise their visualization in the oral epidermis of tadpoles being similar to those observed in common histology (Hipolito et al., 2011, 2012; Ramalho et al., 2013), as shown in Figs. 4 and 5. No references to the use of historesins in the histopathological diagnosis of Bd fungus were found, but the results showed that this technique is feasible and can be routinely used, avoiding the use of products that are potentially manipulative and aggressive to the environment, such as xylene, without interfering with the results. Like every technique that begins its use it may still need necessary adjustments and continuity of this work will bring refinement.
4. Conclusion The use of historesin to histopathological diagnosis of Batrachochytrium dendrobatidis in the mouth of bullfrog tadpole is perfectly feasible, collaborating with a firm diagnosis. Acknowledgments This work was supported by FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo, Projects FAPESP 2011 50009-9 and 2013 15659-8.
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Please cite this article as: Hipolito, M., et al., Use of historesin for viewing Batrachochytrium dendrobatidis in the mouth of Lithobates catesbeianus tadpoles (Rana catesbeiana Sh..., Aquaculture (2014), http://dx.doi.org/10.1016/j.aquaculture.2014.02.016