Myxobolus spp. (Cnidaria: Myxobolidae) in the circulating blood of fishes from Goiás and Mato Grosso States, Brazil: case report Myxobolus

Myxosporidiosis is an infectious disease caused by myxozoans of the Phylum Cnidaria, Class Myxosporea, and Order Bivalvulida, considered a common parasite in fresh and saltwater fishes that parasitize many organs, especially gills. In the present study, 49 specimens of fishes belonging to eight genera: Tetragonopterus , Leporinus , Myleus , Pirinampus , Rhapiodon , Pygocentrus , Ageneiosus , and Serrasalmus were collected and blood smears were made, fixed with absolute methanol, and stained with Giemsa 10% to survey hemoparasites. However, myxospores were found in the circulating blood of five (10.20%) fishes belonging to genus Tetragonopterus , Myleus, and Pygocentrus. Two morphological types of Myxobolus spp. were identified in all the five fish specimens analyzed. Usually, investigations on myxozoans in fish are carried out with the search for plasmodia or cysts in the fish organs and observation of the cavity of organs. Nevertheless, this study highlights the importance of also examining the blood of these animals, since these parasites can cause severe pathogenic diseases in fish. Thus, the blood analyses can proportionate preventive sanitary control for commercial fish avoiding economic loss.


Introduction
Brazil has a large and dense hydrographic network distributed in eight major basins, with a considerable diversity of fishes and lucrative economy around farmed fish (Graça and Pavanelli, 2007). However, pathogenic parasites can cause fish diseases resulting in substantial economic losses to aquaculture and fisheries (Okamura et al., 2015).
Among the diseases reported in fishes, infections with myxozoans are frequently observed (Feist and Longshaw, 2006). These parasites belong to the Phylum Cnidaria Hatschek, 1888 and Order Bivalvulida Shulman 1959; and until now, more than 2,400 species have been described (Eiras et al., 2014;Vidal et al., 2017). Regarding the genus Myxobolus Bütschili 1882, about 900 species were described so far, which approximately 60 were described from Brazilian fishes (Lom and Dyková, 2006;Okamura et al., 2015).
The pathogenesis reported so far, includes reduced respiratory capacity (Adriano et al., 2005), intestinal

Discussion
In the literature, studies on myxozoans infecting fishes have been developed using morphological, morphometric, and molecular diagnostic tools, with a great number of new species being reported over the years. Regarding the molecular tool, normally, the DNA extraction is made direct from parasite cyst or plasmodia, which contains myxospores (Lom and Arthur, 1989;Úngari et al., 2019). In this study, few myxospores were found in the circulating blood of fishes, thus the parasite DNA extraction from the blood, and consequently its amplification did not succeed. Therefore, there is a need for a standardized DNA extraction method from the blood for myxozoans.
Regarding the myxobolid group, Myxobolus and Henneguya Thélohan, 1892, are commonly reported in fishes. The main morphological difference between these genera is that Henneguya spp. have spore projections (Lom and Dyková, 2006;Úngari et al., 2019). Thus, through morphological analysis, it was possible to state the infection with Myxobolus spp. in the circulating blood of the five positive fishes from this study.
Myxozoans parasitize various organs of fresh and saltwater fishes; can be found in intracellular spaces, inside cells, gills, spleen, liver, kidney, and musculature (Maciel et al., 2011). According to Eiras et al. (2006), myxozoans can also be found in circulating blood and blood vessels. Maciel et al. (2011) reported myxozoans in blood smears of tambaqui, Colossoma macropomum Cuvier, 1818 (Osteichthyes, Characidae). Although less frequent, the spores present in circulating blood of fish can be explained as a part of the life cycle of some Myxobolus species, reported by Molnár (2002). Another possible explanation is that blood tissue is also considered a form of a dispersion of these pathogens to other organs of the fishes (Maciel et al., 2011). Lom et al. (1983) reported the presence of Sphaerospora molnari Lom, Dyková and Pavlásková, 1983 in the circulating blood of Cyprinus carpio Linnaeus, 1758 from Europa. The blood contained numerous developmental stages, beginning with early pseudoplasmodia containing one sporogonic cell (Lom et al., 1983). In this study, only mature myxospores of Myxobolus spp. were observed.
Notwithstanding, it has been suggested that the screening of spores in the fish blood is questionable since the low-intensity parasitosis and few reports in the literature (Eiras et al., 2006;Maciel et al., 2011). However, the circulating blood and tissue blood should also be investigated for the presence of myxozoans since necrosis (Alvarez-Pellitero et al., 2008), longitudinal compressions of the body (Longshaw et al., 2003), degenerative cardiomyopathy (Yokoyama et al., 2005), and significant mortalities in wild and farmed fishes (Naldoni et al., 2009).
The fishes were captured by fishing rod and were submitted to blood collection with disposable and sterile 1 mL syringes and needles (13 x 4.5 mm), by puncturing the caudal vein. Three blood smears per specimen were made, fixed with methanol, stained with Giemsa Eosin Methylene Blue (Eisen and Schall, 2000), and examined using an optical microscope (Leica DMLB 5000, Leica Microsystems, Wetzlar, Germany) at 40x and 100x magnification.
Morphometric measurements of spore length, spore width, polar capsule length, polar capsule width, sporoplasm length, and sporoplasm width from myxospores were taken from digital images at 1000x magnification, under light microscopy with Leica software application suite LAS V3.8 (Leica Microsystems).
blood can be part of its life cycle. Also, the blood smear analysis is low cost and can be used as a preventive sanitary control for commercial fish avoiding economic loss.
In conclusion, this study reported 10.20% prevalence of myxobolid mature spores infecting the circulation blood of Brazilian fishes from Goiás and Mato Grosso States, being the first record of myxozoans parasitizing a species of the genus Tetragonopterus and the first record of Myxobolus sp. parasitizing P. nattereri.