Extraction and recovery technique for myxozoan parasites from the Piaractus mesopotamicus kidney embedded in paraffin

Tissues fixed and embedded in paraffin for histopathological studies keep their cell characteristics. There are several protocols for extracting genetic material from tissue embedded in paraffin, but there is no protocol for material aimed at the direct identification of parasites. The lack of techniques which describe the recovery of parasites from tissue embedded in paraffin has led us to test a technique for recovering myxosporean parasites found in Piaractus mesopotamicus kidney fragments embedded in paraffin, for a rapid, direct, and economic identification. Once the excess paraffin was removed from the kidney fragment, this was deparaffinized in xylene, hydrated in 70% alcohol, placed in an Eppendorf tube containing 70% alcohol, and left under vigorous and constant agitation in a vortex until the tissue was disintegrated. The precipitated material was mixed with the 70% alcohol and 20 µL were collected for preparing the smears, which were stained with Giemsa. Myxobolus sp. spores at many developmental stages were observed by light microscopy. The technique has proved to be useful for recovering myxosporean parasites from tissue embedded in paraffin and it constitutes an effective tool for prevalence studies when the myxosporean parasites are not detected in fresh mounts.

parasites from tissue embedded in paraffin has led us to test a technique for recovering myxosporean parasites found in Piaractus mesopotamicus kidney fragments embedded in paraffin, for a rapid, direct, and economic identification. Once the excess paraffin was removed from the kidney fragment, this was deparaffinized in xylene, hydrated in 70% alcohol, placed in an Eppendorf tube containing 70% alcohol, and left under vigorous and constant agitation in a vortex until the tissue was disintegrated. The precipitated material was mixed with the 70% alcohol and 20 µL were collected for preparing the smears, which were stained with Giemsa. Myxobolus sp. spores at many developmental stages were observed by light microscopy. The technique has proved to be useful for recovering myxosporean parasites from tissue embedded in paraffin and it constitutes an effective tool for prevalence studies when the myxosporean parasites are not detected in fresh mounts.
Key words: Extraction technique; Myxozoa; Pacu; Parasitology Protocols for genetic extraction of parasitized tissue fixed in a formalin solution or Bouin and embedded in paraffin were registered by Ribeiro-Silva and Garcia (2008). Despite there is a long list of extraction protocols, few studies consider and describe the reuse of this material for parasite identification. The technique of fixing organs in Bouin and embedding them in paraffin has been replaced by fixing organs in a formalin solution and embedding them in glycol methacrylate, due to improved outcomes in terms of using less artifacts and obtaining a better resolution by light microscopy (COLE, SYKES, 1974;JUNQUEIRA, 1995). However, several laboratories use this technique with paraffin due to economic reasons and easy handling to preserve the morphological cell characteristics, as well as the compatibility to use antibodies in immune histochemical reactions (LEHMANN; KREIPE, 2001).
Myxozoan are among the most important pathogens observed in marine and freshwater fish species which can parasitize many organs, causing economic losses (SARDELLA et al., 1987;CASAL et al.,2002;MARTINS, 2004;ESZTERBAUER et al., 2006). They are found free into the cavities (celozoic), within the cells and tissues (histozoic), and into the blood vessels. The main hosts are aquatic vertebrates and, in some cases, invertebrates. Their pathogenicity is related to lesions caused by plasmodia in host tissues (LOM; DYKOVÁ, 1992) or to the production of enzymes which degenerate the tissue (KAWAI et al.,2012). According to Eszterbauer et al. (2006), 52 genera and 1,350 parasitic myxozoan species have been observed. In South America, only 31 species were registered, and 28 species belong to the Myxobolus genus (EIRAS et al., 2008).
Using these techniques allows a posterior observation and identification of spores for morphological and morphometric determination (CAMPOS et al., 2008;2011). The technique enables identification at the genus level (LOM; NOBLE, 1984). Considering the lack of similar studies, aimed at recovering parasites from organs embedded in paraffin, this study describes a technique for recovering myxozoan parasites from fixed tissue.
A paraffin block with the Piaractus mesopotamicus kidney fixed in a Bouin solution for 6 h was selected for 5 µm thick histological sections and stained with hematoxylin-eosin and Ziehl-Neelsen for histopathological analysis (as approved by the Research Ethics Committee of CEUA/UNESP, under the Protocol 020092/2009). When parasites were observed, the excess paraffin was removed from the block with a scalpel, to get a 3 x 3 x 3 mm cube, to which hot paraffin (60ºC) was added to remove the tissue. After this procedure, the tissue was transferred to Eppendorf (2.0 mL), then, xylene was added and heated in a bath at 60ºC for 15 min. The tube was agitated in a vortex for 15 min, centrifuged at 14,000 G for 10 min, and the supernatant was discharged. This procedure was repeated twice. We added 2 mL of 70% alcohol to the sample and it was agitated in a vortex for 30 min, in order to disintegrate the tissue. Then, the tube was centrifuged at 14,000 G for 15 min and 20 µL of precipitated was collected and mounted between a glass and a cover slip. Other samples were obtained; the smears were stained with Giemsa, they underwent direct observation by light microscopy (Olympus BX51), and the images were registered using a camera DP72 (Software Cell Sens v 1.5).
In the smears, Myxobolus sp. Spores were observed at many developmental stages, as well as polar capsules and nuclei (Figures 1A and 1B). tube was agitated in a vortex for 15 min, centrifuged at 14,000 G for 10 min, and the supernatant was discharged. This procedure was repeated twice. We added 2 mL of 70% alcohol to the sample and it was agitated in a vortex for 30 min, in order to disintegrate the tissue. Then, the tube was centrifuged at 14,000 G for 15 min and 20 µL of precipitated was collected and mounted between a glass and a cover slip. Other samples were obtained; the smears were stained with Giemsa, they underwent direct observation by light microscopy (Olympus BX51), and the images were registered using a camera DP72 (Software Cell Sens v 1.5).
In the smears, Myxobolus sp. Spores were observed at many developmental stages, as well as polar capsules and nuclei ( Figures 1A and 1B).  We may conclude that this technique was able to recover the parasite from the tissue embedded in paraffin, allowing the detection of parasites which were not found in fresh mounts.