Host-parasite relationship during Epistylis sp. (Ciliophora: Epistylididae) infestation in farmed cichlid and pimelodid fish

The objective of this work was to describe the host‑ Epistylis  sp. relationship during infestation on farmed fish. Five Nile tilapia ( Oreochromis niloticus ) and ten hybrid surubim catfish ( Pseudoplatystoma reticulatum x P. corruscans ), all diseased, were used for in vivo morphological analysis of sessile peritrichs by contrast microscopy. Fragments of infected tissues were subjected to histological processing and scanning electron microscopy. Epistylis  sp. caused hemorrhagic ulcer disease, and cichlids were more prone to develop infestations throughout the body surface due to the attachment of the colonies to the scales, which did not occur with pimelodids. Multifocal granulomatous dermatitis was observed, associated with the hydropic degeneration of the epithelium and to ulcerative areas of necrosis. Microscopic examination showed the presence of bacterial microflora associated to Epistylis  sp. peduncles. Therefore, this species can be considered a portal of entry for opportunistic bacteria.

In Brazil, the exotic Nile tilapia (Oreochromis niloticus) is the most cultivated species, presenting economic relevance worldwide, and, in South America, catfishes from the Pseudoplatystoma genus are one of the main native fish reared in captivity (Boletim estatístico da pesca e aquicultura, 2011). The intensification of production of both species increases the infections by parasites and bacteria. Parasites, such as Protozoa, Metazoa, and Myxozoa, were identified in Nile tilapia (Pantoja et al., 2012) and Pseudoplatystoma spp. (Campos et al., 2008;Jerônimo et al., 2013); however, data on the host-pathogen relationship are still scarce. The present study shows new information on the pathogenesis of Epistylis sp. infestation, featuring the pathological process that occurs in two freshwater fish of economic importance.
The objective of this work was to describe the host-Epistylis sp. relationship during infestation on farmed fish.

Materials and Methods
The following fish were examined: five diseased Nile tilapia juveniles from the GIFT lineage, weighing 30 to 50 g, collected in 2012 from cages located in the Tiete River, in the municipality of Arealva, in the state of São Paulo, Brazil (22º05'16"S, 48º51'45"W); and ten diseased hybrid surubim catfish (Pseudoplatystoma reticulatum x P. corruscans juveniles), weighing 10 to 20 g, collected in 2010 from a raceway located in the municipality of Terenos, in the state of Mato Grosso do Sul, Brazil (20º25'58"S, 55º17'09"W).
Moribund fish collected by net were observed for pathological and parasitological diagnosis in loco. All body surface, fins, and gills of each fish were scraped separately (using glass slides) for light microscope observation, according to Eiras et al. (2006). Diseased fish were transported to the laboratory of invertebrate morphology of Universidade Estadual de São Paulo Júlio de Mesquita Filho for scraping observations of the living organisms with differential interference contrast microscopy, using an Axio Imager Z2 microscope (Carl Zeiss, Oberkochen, Germany). Parasite measurements were performed on living parasites, following methods normally used for this parasite (Ma & Overstreet, 2006;Kühner et al., 2016) and using the software Image Pro Plus (Media Cybernetics Inc., Rockville, MD, USA). Measurements are presented as mean±standard deviation (minimum-maximum, and number of structures measured). This was only possible for parasites of Nile tilapia, since catfish analysis was carried out far from the laboratory and the facility did not have an available photomicroscope.
Skin and fin fragments with multifocal Epistylis sp. colonies visible to the naked eye, considered as score 4 according to Pádua et al. (2013a), were fixed in 10% buffered formalin solution, processed according to usual techniques for histopathology, embedded in paraplast block, sectioned at 5.0 µm, and stained with hematoxylin and eosin. The slides were analyzed, and photomicrographs were obtained using a photomicroscope (model Eclipse E200, Nikon Imaging Sales Co., Ltd., Shanghai, China) equipped with the Moticam 2300 image capture system (Quimis, Diadema, SP, Brazil).

Results and Discussion
All sampled Nile tilapia showed mixed parasitic infestation by trichodinids and occasionally by Gyrodactylus cichlidarum on the tegument and gills. Epistylis sp. was the most predominant parasite of all fish, affecting mainly the body surface and rarely gill tissues, with a wide distribution on scales and fins. Hybrid surubim presented mixed infestation by Trichodina sp. and Epistylis sp., in which the colonies affected only the rigid structures of the fish surface, such as fin rays, stingers, head, operculum, and lips; however, no infestation was observed in the gill tissue. In Nile tilapia, the scales facilitated the development of parasitism, affecting large extensions of the tegument, which did not occur with pimelodids. Similar findings were described in the Astacus leptodactylus crayfish (Hüseyin & Selcuk, 2005), which have extensive carapace, allowing the colonization by these ciliates. Although pimelodids are less susceptible to skin infestations, recent studies have shown a high incidence of the parasite Epistylis sp. on South American catfish, especially on intergeneric and interspecific hybrids, which are more susceptible when compared to pure species (Pádua et al., 2013a). Epistylis sp. presented 8 to 58 zooids showing conical-elongated format (Figure 1 A, B, and C). The morphology was characteristic of the Epistylis genus: the peduncle was dichotomously branched and noncontractile; there was one contractile vacuole located in the zooid apical portion; the macronucleus was transverse C-shaped; and the cytoplasm was full of food vacuole (Ma & Overstreet, 2006). The peduncle of this protozoan was colonized by bacilli bacteria and occasionally by filamentous bacteria, arranged in cords (Figure 1 D). Free telotroch were observed, showing ciliature formed in the equatorial region of the cell wall and C-shaped macronucleus (Figure 1 E). All morphometric data for Epistylis sp. collected from Nile tilapia are described in Table 1. However, species identification remains difficult. Currently, molecular techniques have been developed to support the systematics of these Protozoa (Utz & Eizirik, 2007;Jiang et al., 2016).
Nile tilapia with high infestation levels, corresponding to score 4 (Pádua et al., 2013a), presented petechial hemorrhage in the area where the colony was attached to, which resulted in corrosion and loss of scales, and in the progression of the most severe lesions to multifocal ulcers. Similarly, hybrid surubim catfish showed ulcerative and bleeding lesions associated with colonies attached to the tegument in severely infested fish.
Histological sections showed focal hyperplasia in the epithelium injured by the parasite, as well as hydropic degeneration with multifocal necrosis adjacent to the colony, proliferation of claviform and mucous cells, and moderate mononuclear and granulocytic inflammatory infiltrate with the presence of giant cells, featuring multifocal granulomatous dermatitis (Figure 2 A and B). Moreover, parasites were observed adjacent to the ulceration area without penetrating the host skin, and several bacteria were associated to the colony (Figure 2 C and D). However, the development of colonies may result in local irritation, since multifocal granulomatous dermatitis was found in infested fish, characteristic of a chronic pathological process (Ulrich et al., 2007). Mononuclear infiltration and giant cell formation have also been reported in the evolution of chronic inflammatory response in teleost fish (Belo et al., 2005(Belo et al., , 2012Sakabe et al., 2013), including peritrich infestation (Valladão et al., 2015).
The scanning electron microscopy analysis showed fixed colonies on scales and fins, with partially contracted zooids, which rarely showed slightly protuberant epistomial disc (Figure 3 A, B, and C). Abundant bacterial microflora -with circular shape, in streaks, and without penetrating the host skin -was observed on the peduncle and on the attachment surface ( Figure 3 D and E). The bacterial association with Epistylis sp. colonies in fish was first described by Hazen et al. (1978), who registered Aeromonas hydrophila as the primary causative agent of red-sore disease in North American cichlid. In the present study, abundant bacterial microflora was associated to the peduncle surface of Epistylis sp., and most of the pathological changes may result from bacterial proteolytic enzymes (Martins et al., 2015). Recently, Valladão et al. (2015) have also proposed that tissue damage may have occurred due to the action of immune cells at the site of parasitism, because these cells are provided with hydrolytic molecules capable of causing cell damage.
In order to avoid parasite dissemination and host susceptibility, the best management practices allied to regular fish health knowledge should be the most important measures adopted in rearing facilities.

Conclusions
1. Epistylis sp. infestation in cichlid and pimelodid fish may cause mixed diseases by opportunistic bacteria with pathological lesions on the hosts and, therefore, can be considered a portal of entry for these bacteria.
2. Severe histological changes were observed on injured tissue, such as focal hyperplasia and hydropic degeneration with multifocal necrosis adjacent to the colony, characterizing multifocal granulomatous dermatitis.