Hepatic steatosis associated with microsporidiosis in teleost fishes from Marajó Island, Brazil

A total of 40 specimens of the teleost fish Gobioides grahamae Palmer & Wheeler, 1955 were obtained from the municipality of Salvaterra on Marajó Island in the Brazilian state of Pará. Their livers were removed and processed for light microscopy. Overall, 90% of the specimens presented some degree of steatosis of the liver, which was invariably associated with the presence of Microsporidium sp. The present study confirms the occurrence of steatosis in G. grahamae associated with parasitic infections by Microsporidium. The findings indicate that the condition of otherwise healthy fishes in their natural environment may be affected negatively by parasites.


INTRODUCTION
The vertebrate liver has a number of different functions, in particular the storage of reserves of glycogen and lipids (Bucke et al. 1984). The metabolism of lipids, including the synthesis and degradation of fatty acids, is regulated primarily by the liver, and metabolic imbalances may modify both the morphology and function of the organ, a pathological process known as fatty degeneration or infiltration, steatosis or lipidosis (Coelho 2002, Caballero et al. 2004. Morphological modifications, such as the vacuolization of the hepatocytes, glycogen depletion, inflammations, changes in the shape of the sinusoid vessels, and neoplasms may be interpreted as a response to environmental stress or a pathological process caused by infections or parasites (Thomas 1990, Köhler et al. 1992, Teh et al. 1997. Microsporidiosis is caused by microorganisms of the phylum Microsporidia Balbiani, 1882. These organisms exhibit characteristics of both eukaryotes (presence of a nucleus) and prokaryotes (typical ribosomal units), but are included in the Fungi due to their molecular traits (Matos et al. 2003 They parasitize organisms of a number of different taxonomic groups, in particular animals, including humans (Polonais et al. 2010, Ghosh et al. 2006, Brasil et al. 1997) and insects (Matos et al. 2006).
Microsporidians may be found in a number of different types of organs, including the liver, kidneys, gills, gonads, intestine, and skin, in which they cause major alterations to the tissue. The present study describes the presence of hepatic steatosis in the teleost fish Gobioides grahamae Palmer & Wheeler, 1955, associated with parasitic infection by Microsporidium sp.
A total of 40 specimens of G. grahamae were examined. The specimens (15 females and 25 males) had a mean standard length of 16 cm (range: 12-20 cm) and mean weight of 70 g (range: 50-90 g), and were collected in the municipality of Salvaterra (00°45'21" S, 48°30'54" W) on the island of Marajó, in the northern Brazilian state of Pará, between June and December, 2011. The fishes were kept in aerated plastic bags for transportation to the Carlos Azevedo Research Laboratory at UFRA in the capital of the state of Pará, Belém. where they were anesthetized with MS 222 (Sandoz Laboratories), prior to necropsy.
The necropsies on the hosts began with opening the abdominal cavity using a pair of anatomical scissors, in order to gain access to the viscera. The organs were examined under a stereomicroscope.
For light microscopy, small fragments (0.5 cm) of the liver tissue parasitized by microsporidia were fixed in Davidson's solution (formaldehyde, ascetic acid, 95% ethanol, and distilled water) for 24 hours and then embedded in paraffin. Sections of 3 µm were stained with Masson's trichrome and hematoxylin-eosin (Luna 1968). The stained sections were analyzed and photographed using a Nikon E-200 microscope, with Micrometrics ® -SE Premium imaging software.
Most (90% or 36/40) of the specimens examined presented hepatic steatosis, a pathological process associated with infection by Microsporidium sp. Macroscopically, the livers of these specimens were characterized by an increase in volume and yellow blemishes, as described by Coelho (2002). Microscopically, the dislocation of the layers of hepatocytes and a large quantity of adipocytes could be observed in the hepatic stroma. The histopathological analysis revealed modifications of the liver cells, which were disorganized, with signs of accentuated and diffused steatosis, moderate colestasis, and multifocal necrosis.
This study highlights the prevalence of hepatic steatosis higher than that described by Rosenthal et al. (2013), who studied the occurrence of hepatic steatosis in Oncorhynchus mykiss from cultivation, finding that 70.6% of the fish had lesions consistent with hepatic steatosis.
The hepatic steatosis was characterized by the presence of clear, rounded cytoplasmatic vacuoles, with hepatocytes of increased volume and dislocated nuclei (Figure 1). Cysts of Microsporidium sp. surrounded by leucocytes were also present. Staining with Masson's trichrome revealed the presence of a fine capsule of collagen surrounding the cysts which, in some specimens, had been ruptured, releasing spores into the threads of hepatocytes (Figure 2). The spores are ellipsoid or ovoid in shape, and were the only form of the parasite observed in the specimens analyzed in the present study. No microsporidian spores were found in the specimens without steatosis, indicating that the presence of the parasite is the factor determining the occurrence of this disorder in G. grahamae.
Diseases of the liver include infectious conditions, such as those caused by fungi (as in the present study, associated with microsporidian cysts), bacteria, viruses, and protozoans, and noninfectious disorders, all of which are generally characterized by lesions and an increase in the volume of the liver, which is easily observed macroscopically. Francisco (2006) and Campos et al. (2008) observed steatosis in fishes maintained in contaminated environments, although Thomas (1990), Silva (2004), Santos et al. (2004), and Camargo and Martinez (2007 found no lesions whatsoever, even in polluted environments. It is important to note that the release of toxins by microorganisms -in the present case, related to the presence of Microsporidium sp. -is one of the main causes of steatosis, due to the fact that it reduces the production of proteins by the rugose endoplasmatic reticulum, which has a direct effect on the metabolism of lipids by the hepatocytes (McGavin and Zachary 2007). The present study found that the condition of otherwise healthy fishes in their natural environment may suffer adverse effects from parasitism.