Definition of a list of fish diseases to aid health management in Spain

Trabajo presentado en la 14th EAFP International Conference (European Association of Fish Pathologists), celebrada en Praga (República Checa), del 14 al 19 de septiembre de 2009An expert working group used a risk ranking technique to compile a list of fish disease hazards (pathogens) of relevance to Spanish aquaculture. It was possible to divide the list into three groups: I-high national risk; II-regional risk; III-low risk. The three groups were as follows: Group I (high risk) Aphanomyces invadans (EUS)**, spring viraemia of carp virus (SVCV), koi herpes virus (KHV)¿ and infectious haematopoietic necrosis virus (IHNV)¿ Group II (regional risk) Enteromyxum spp. (leei and scophthalmi), Aquabirnaviridae (incl. IPNV), viral encephalopathy and retinopathy virus (VERV), Streptococcus iniae, Philasterides dicentrarchi and Aeromonas salmonicida (in the marine environment). Group III (low risk) Sparicotyle chrysophrii/Microcotylidae, Flavobacterium maritimus, Photobacterium piscicida, Togaviridae, Sphaerospora testicularis, Edwardsiella tarda, Birnavirus (no-EVE), Lactococcus garviae, viral haemorrhagic septicaemia virus (VHSV)¿, Tenacibaculum maritimum, epizootic haematopoietic necrosis virus (EHNV)**, Renibacterium salmoninarum (BKD) and Gyrodactylus salaris. The exercise formed part of a project concerning aquaculture health management (Jacumar-GESAC¿) in Spain and is being used to help define sampling plans for disease monitoring using epidemiological and risk-based criteria. **The diseases caused by these pathogens are notifiable and exotic according to Directive 2006/88/EC ¿The diseases caused by these pathogens are notifiable and non-exotic according to Directive 2006/88/EC ¿Plan Nacional de Cultivos Marinos; Gestión sanitaria de la acuicultura: Adaptación a la nueva normativa (GESAC) ¿ financed by the Junta Nacional Asesora de Cultivos Marinos (Jacumar

Isolation of Enteromyxum parasites using fluorescence activated cell sorter analysis (FACS)

Poster presentado en el 15th International Conference on Diseases of Fish and Shellfish celebrado en Croacia del 12 al 16 de septiembre de 2011Enteromyxum scophthalmi and E. leei are myxozoan parasites producing severe diseases in marine aquaculture. These intestinal parasites cause catarrhal enteritis and emaciative syndromes and they have direct, fish-to-fish transmission, and wide host spectrum. E. scophthalmi infection typically results in 100% loses in turbot stocks, and E. leei produces chronic losses in sea bream culture by a rather slow-progressing enteritis. Due to the lack of efficacious treatments only management measures such as hygiene and culling of infected stocks are currently available. Identification of relevant Enteromyxum genes, expressed in the course of their development in the fish, would provide essential information related to the mechanisms of invasion, proliferation, maturation and virulence. These data would offer new opportunities to interfere in the parasites development and to design strategies for enteromyxoses control. Next-generation sequencing techniques are able to generate large amounts of genetic data and constitute an ideal platform for gene discovery in myxozoan parasites. The objective of the current work was to develop methods for parasite purification for downstream analysis and identification of expressed genes. Enteromyxum spp. are histozoic parasites developing in close contact with host cells within the intestinal mucosa and cannot be grown in vitro. Previous attempts to isolate developmental stages by mechanical and chemical techniques have been unsuccessful. A method was developed to generate cell suspensions containing free parasites from Enteromyxum-infected intestines by adapting protocols for purification of GALT lymphocytes [1, 2]. Briefly, pieces of intestine were cut and the mucosal layer was detached and cells disaggregated in buffer containing DTT and EDTA. The resulting cell suspension was washed and filtered through cell strainers (40 µm) and cell viability was checked by trypan blue dye exclusion. Enteromyxum-specific probes were tested for live-cell fluorescence staining of parasites using microscopy and flow cytometry. Selective live staining of E. scophthalmi was achieved using AlexaFluor-Conjugated SBA lectin, and for E.leei stages the best results were obtained by indirect immunofluorescence with a commercial anti-caspase3 pAb. Poor results were obtained using lab-made rabbit anti-E. scophthalmi and anti-E.leei antisera, although these result in good staining of parasites by immunohistochemistry [3]. Cell suspensions from infected and uninfected tissues were analyzed by flow cytometry, and parasite cells subpopulations identified by morphology/complexity and labeling patterns. Live staining of Enteromyxum spp. with these probes allows efficient automated purification of stages using a fluorescence-activated cell sorter (FACS). Sorting masks are being optimized for selective purification of parasites, prior to sequencing and characterization of genes expressed by Enteromyxum spp.This work was funded by MICINN project AGL2009-13282-C02-01. Additional funding was obtained from Generalitat Valenciana (PROMETEO 2010/006). M.E. A-N received a PhD fellowship from MICINN (FP1 program).Peer Reviewe

A new intranuclear microsporidium, Enterospora nucleophila n. sp., causing an emaciative syndrome in a piscine host (Sparus aurata), prompts the redescription of the family Enterocytozoonidae

The presence of a new microsporidium is believed to be responsible for an emaciative syndrome observed in farmed gilthead sea bream (Sparus aurata) from different facilities along the Spanish coast. Infected fish were approximately half the average weight and significant mortality was attributed to the condition in some facilities. Clinical signs included anorexia, cachexia and pale internal organs. The microsporidium was found mainly in the intestinal mucosa and occasionally in the submucosa. Morphological, histopathological, ultrastructural and molecular phylogenetic studies were conducted to characterise this organism. This microsporidium undergoes intranuclear development in rodlet cells and enterocytes, and cytoplasmic development mainly in enterocytes and macrophages. The nucleus-infecting plasmodium contains several diplokarya and displays polysporous development which occurs without an interfacial envelope. In the host cell cytoplasm, the parasite develops within a membrane-bound matrix. In both infection locations, the polar tube precursors appear as disks, first with lucent centres, then as fully dense disks as they fuse to form the polar filament, all before division of the plasmodium into sporoblasts. Up to 16 intranuclear spores result from the sporogonic development of a single plasmodium, whereas more than 40 spores result from several asynchronous reproductive cycles in the cytoplasmic infection. Fixed spores are ellipsoidal and diplokaryotic, with five to six coils of an isofilar polar filament in a single row. ssrDNA-based molecular phylogenetic inference places this parasite as a sister clade to crustacean-infecting species of the Enterocytozoonidae and closer to Enterocytozoon bieneusi than to other fish-infecting microsporidians presenting intranuclear development, i.e. Nucleospora, Paranucleospora and Desmozoon. Our studies result in the erection of a new species, Enterospora nucleophila, within the family Enterocytozoonidae, and the description of this family is amended accordingly to accommodate the features of known species assigned to it. Severe histopathological damage occurs in intense infections and this microsporidian is considered a serious emerging threat in sea bream production. © 2013 Australian Society for Parasitology Inc.Peer Reviewe

Defenses of susceptible and resistant Chinook salmon (Onchorhynchus tshawytscha) against the myxozoan parasite Ceratomyxa shasta

We investigated intra-specific variation in the response of salmon to infection with the myxozoan Ceratomyxa shasta by comparing the progress of parasite infection and measures of host immune response in susceptible and resistant Chinook salmon Oncorhynchus tshawytscha at days 12, 25 and 90 post exposure. There were no differences in invasion of the gills indicating that resistance does not occur at the site of entry. In the intestine on day 12, infection intensity and Ig+ cell numbers were higher in susceptible than resistant fish, but histological examination at that timepoint showed more severe inflammation in resistant fish. This suggests a role for the immune response in resistant fish that eliminates some parasites prior to or soon after reaching the intestine. Susceptible fish had a higher IFNγ, IL-6 and IL-10 response at day 12, but all died of fatal enteronecrosis by day 25. The greatest fold change in IFNγ expression was detected at day 25 in resistant Chinook. In addition, the number of Ig+ cells in resistant Chinook also increased by day 25. By day 90, resistant Chinook had resolved the inflammation, cytokine expression had decreased and Ig+ cell numbers were similar to uninfected controls. Thus, it appears that the susceptible strain was incapable of containing or eliminating C.shasta but resistant fish: 1) reduced infection intensity during early intestinal infection, 2) elicited an effective inflammatory response in the intestine that eliminated C.shasta, 3) resolved the inflammation and recovered from infection. © 2014 Elsevier Ltd.Peer Reviewe