Mª José Redondo

Description of Enteromyxum scophthalmi gen. nov., sp. nov. (Myxozoa), an intestinal parasite of turbot (Scophthalmus maximus L.) using morphological and ribosomal RNA sequence data

A new Myxozoa species causing enteritis and death in cultured turbot, Scophthalmus maximus, is described at light and electron microscope levels. In addition, small subunit ribosomal RNA gene sequences (SSU rDNA) from the new species and from similar myxozoans were obtained and used for phylogenetic inference, as complementary criteria to resolve its taxonomic classification. The new parasite is closely related to Myxidium leei, another enteric histozoic species from marine fish. However, the ascription of M. leei to the genus Myxidium was based on weak morphological evidence and is not supported by our rDNA data analysis. A close relationship with Zschokkella, the other morphologically related myxozoan genus is also not supported. The combined morphological and molecular study results in the establishment of the new genus Enteromyxum to accommodate the new species E. scophthalmi, and the former M. leei, which is transferred to the new genus as Enteromyxum leei (Diamant, Lom & Dyková 1994) n. comb. This genus of marine, histozoic and enteric myxozoans includes significant parasite species for marine finfish culture.

Novel horizontal transmission route for Enteromyxum leei (Myxozoa) by anal intubation of gilthead sea bream Sparus aurata.

The aim of the present study was to determine whether Enteromyxum leei, one of the most threatening parasitic diseases in Mediterranean fish culture, could be transmitted by peranal intubation in gilthead sea bream Sparus aurata L. Fish were inoculated either orally or anally with intestinal scrapings of infected fish in 3 trials. Oral transmission failed, but the parasite was efficiently and quickly transmitted peranally. Prevalence of infection was 100% at 60 d post inoculation (p.i.) in Trial 1 under high summer temperature (22 to 25 degrees C; fish weight = 187.1 g), and 85.7% in just 15 d p.i. in Trial 3 using smaller fish (127.5 g) at autumn temperature (19 to 22 degrees C). In Trial 2, prevalence reached 60% at 60 d p.i. in the group reared at constant temperature (18 degrees C), whereas no fish was infected in the group that was kept at low winter temperature (11 to 12 degrees C), although infection appeared (46.1% at 216 d p.i.) when temperature increased in spring. The arrested development at low temperature has important epidemiological consequences, as fish giving false negative results in winter can act as reservoirs of the parasite. Histopathological examination showed a posterior-anterior intestinal gradient in the progression of the infection, in terms of both intensity and parasite maturation. Thus, per-anal intubation provides a very uniform, reliable and faster mode of transmission of E. leei than the commonly used transmission methods (cohabitation, exposure to infected effluent and oral inoculation), which require long exposure times or give variable and unpredictable results.

Light and electron microscopic studies on turbot Psetta maxima infected with Enteromyxum scophthalmi: Histopathology of turbot enteromyxosis

In the last decade, a new parasite that causes severe losses has been detected in farmed turbot Psetta maxima (L.), in north-western Spain. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. The aim of this study was to characterize the main histological changes that occur in E. scophthalmi-infected turbot. The parasite provoked catarrhal enteritis, and the intensity of the lesions was correlated with the progression of the infection and with the development of the parasite. Infected fish were classified into 3 groups, according to the lesional degree they showed (slight, moderate and severe infections). In fish with slight infections, early parasitic stages were observed populating the epithelial lining of the digestive tract, without eliciting an evident host response. As the disease progressed, catarrhal enteritis was observed, the digestive epithelium showed a typical scalloped shape and the number of both goblet and rodlet cells was increased. Fish with severe infections suffered desquamation of the epithelium, with the subsequent release of parasitic forms to the lumen. The dislodged enterocytes underwent anoikis, a mode of apoptosis triggered by the loss of anchorage, which might facilitate spreading of the parasite. Lymphohaematopoietic depletion was also observed, mainly in head kidney and spleen, which could contribute to the high virulence of this parasite.

Risk factors associated with Enteromyxum scophthalmi (Myxozoa) infection in cultured turbot, Scophthalmus maximus (L.).

An epidemiological cohort study of Enteromyxum scophthalmi in cultured turbot was performed on a farm in North Western Spain. Four different ongrowing stocks (A, B, C, D) were monitored monthly until market size. Fish from stocks C and D were divided into 2 subgroups, receiving filtered (CF and DF) or unfiltered (CUF and DUF) water. The lack of water filtration was positively associated with infection prevalence, as all fish kept in filtered water remained uninfected. Parasite abundance varied seasonally (P<0.05) in stock B and subgroup CUF. Infection was also associated (P<0.05) with host weight, and the highest prevalences and intensities were detected in 101-200 g and 201-300 g fish. Distribution pattern of E. scophthalmi in subgroups CUF and DUF had a variance higher than the mean, indicating overdispersion. The minimum period necessary for the first detection of the parasite and for the appearance of disease symptoms and mortality, varied depending on the stock and introduction date, although a long pre-patent period was always observed. Several factors, such as host density, parasite recruitment and parasite-induced fish mortality can contribute to the observed distribution pattern. Risk factors found to be associated with E. scophthalmi infection, including water quality and accumulation of infective stages in the culture tanks, should be considered when designing control strategies to prevent the introduction and spread of infective stages in the facilities.

Detection of carbohydrate terminals in the enteric parasite Enteromyxum scophthalmi (Myxozoa) and possible interactions with its fish host Psetta maxima

The existence and localisation of carbohydrate terminals in Enteromyxum scophthalmi stages was investigated at light (LM) and transmission electron microscopes (TEM) using lectin histochemistry techniques, with the aim of contributing to elucidate the participation of carbohydrate–lectin interactions in the parasite invasion and relationships with the fish host. The presence of abundant mannose and/or glucose residues was demonstrated by the intense staining by concanavalin A at both LM and TEM. The staining pattern obtained with soybean agglutinin and Bandeiraea simplicifolia I (BSI) indicated the abundance of N-acetyl-galactosamine and D-galactose at a lesser extent. The lectins wheat germ agglutinin, BSI and Ulex europaeus agglutinin produced weaker marks. Most lectins recognised structures present in both pre-sporogonic and sporogonic stages, though the glycosidic pattern and/or staining intensity varied between developmental stages. No staining was obtained with Sambucus nigra agglutinin. The TEM studies demonstrated glucose–mannose, N-acetyl-glucosamine, N-acetyl-galactosamine and α-D-galactose as dominant structures at the parasite membrane and host–parasite interface, suggesting a role in host–parasite interactions. All these terminals were also detected in the mitochondria of P cells and were scant in the S cells and nuclei. In turbot intestine, mannose–glucose terminals and N-acetyl-glucosamine were labelled on the epithelial brush border and in the mucous cells and rodlet cells. The relevance of these findings in relation to the host–parasite interaction is discussed.

An unidentified epi-epithelial myxosporean in the intestine of gilthead sea bream Sparus aurata L.

In the course of experimental infections of gilthead sea bream Sparus aurata with the myxozoan Enteromyxum leei, stages of an unidentified myxozoan were observed attached to the intestinal brush border of some fish. Infection levels of the parasite, which was named “epi-epithelial myxosporean” (EEM) were recorded, and its structure was studied by light microscopy (LM) and electron microscopy (EM). In situ hybridisation (ISH) probes specific for E. leei were developed and used to differentiate between the two parasites. The EEM parasite was observed only in epi-epithelial position on the intestine mucosa and never in any of the other tissues studied (kidney and gall bladder). Prevalence was variable, with values reaching 40.2%. With transmission EM, trophozoites displayed pseudopodia-like projections inserted in between the enterocyte microvilli, producing an intimate interface. No mucosal histopathology that could be attributed to the myxozoan was found. EEM stages did not stain with the E. leei-specific ISH probes. From the results of the LM, EM and ISH studies, we conclude that the EEM parasite found in gilthead sea bream intestine in both Mediterranean and Red Sea sites is a coelozoic myxosporean, distinct from E. leei.