Cultivation of the heart urchin Echinocardium cordatum and validation of its use in marine toxicity testing for environmental risk assessment

To study environmental risk assessment, echinoderms provide a useful model for ecotoxicological testing. However, limited knowledge of the life history of field collected heart urchins is a problem and the use of cultured urchins has been investigated here. The present study describes a culture method for the heart urchin Echinocardium cordatum under controlled laboratory conditions, providing organisms with a low biological variation. Based on our optimized growth protocol both larvae and juveniles have a growth rate comparable to E. cordatum in the wild. The toxicological response of cultured and field-collected E. cordatum was compared in standard saltwater toxicity bioassays. Using ammonium chloride as a water-soluble reference toxicant, mean 96 h LC50 values for cultured heart urchins versus field collected animals were 37.4 ± 7.6 mg NH4+/l (n = 5) versus 22.5 ± 4.9 mg NH4+/l (n = 19), respectively. Additional toxicity experiments with tributyl tin (TBT) spiked sediments revealed 14d LC50 values of 1,242 (95% confidence interval 986¿1,564) and 964 (95% confidence interval 843¿1,102) µg Sn/kg dw respectively in cultured and field collected E. cordatum. From this it was concluded that cultured heart urchins are less sensitive to TBT than field collected E. cordatum. Furthermore in whole sediment toxicity tests, survival of cultured sea urchins was higher or at least similar to that of field collected E. cordatum. The increased sensitivity of field urchins compared to cultured urchins in various toxicity tests may be due to multiple environmental stressors reducing their overall performance. Overall it was demonstrated that the use of cultured E. cordatum provides a significant advance for urchin-based bioassays for marine environmental toxicity testing, resulting in a more homogeneous, vital population with experimental data displaying reduced variability

Sphaerothecum destruens pathology in cyprinids

Sphaerothecum destruens is a significant intracellular parasite of fish which has caused disease and mortalities in cultured north American Chinook salmon Oncorhynchus tshawytscha and Atlantic salmon Salmo salar. Several hosts for S. destruens have been identified within the Salmonidae family, and the histopathology of the infection can differ between hosts. Recently, S. destruens has been associated with the most invasive cyprinid species in Europe, topmouth gudgeon Pseudorasbora parva. Accurate disease identification based on thorough descriptions of clinical signs and histopathology in this new range of hosts is thus paramount to support further epizootiological studies. In this study, the associated histopathology of S. destruens infection is described along with its pathogenesis in the endangered cyprinid sunbleak Leucaspius delineatus. Histological examination of 100 L. delineatus in a wild population in the south of England revealed the presence of S. destruens infections, with a prevalence of 5% with S. destruens, suggesting an over-dispersed distribution within the L. delineatus sample. Clinical signs of the infection were absent, but histological examination revealed the presence of both disseminated and nodular lesions in several organs

Diseases of dab (Limanda limanda): Analysis and assessment of data on externally visible diseases, macroscopic liver neoplasms and liver histopathology in the North Sea, Baltic Sea and off Iceland

In the framework of the ICON project (Integrated Assessment of Contaminant Impacts on the North Sea), common dab (Limanda limanda) from seven offshore sampling areas in the North Sea, Icelandic waters and the western Baltic Sea were examined in 2008 for the presence of externally visible diseases and parasites (EVD), macroscopic liver neoplasms (tumours) (MLN) and histopathological liver lesions (LH). Methodologies applied followed standardised ICES and BEQUALM protocols. The EDV results revealed pronounced spatial variation, with dab from the central and northern North Sea sampling areas showing the highest disease prevalence. MLN were recorded only in North Sea dab from the German Bight, Firth of Forth and Ekofisk at a low prevalence. LH results revealed a dominant prevalence of non-specific, mostly inflammatory, lesions and a low prevalence of early toxicopathic non-neoplastic lesions, tumour pre-stages (foci of cellular alteration) and liver tumours. For the analysis and assessment of spatial variation of EVD, a Fish Disease Index (FDI) was calculated for individual dab, summarising data on the presence/absence of EDV, their severity grades, effects on the host and compensating for effects of length, sex and season. FDI data confirmed that the health status of North Sea dab from the offshore areas Dogger Bank, Ekofisk and Firth of Forth was significantly worse than in dab from the German Bight, Icelandic areas and the western Baltic Sea. An assessment of the disease data following ICES/OSPAR criteria was accomplished by applying established numeric background (BAC) and ecological assessment criteria (EAC) for EDV, MLN and LH. The combined assessment of the three disease categories indicated that health effects classified as unacceptable were rare and mainly affected dab from the North Sea.Based on the findings of the present study, it is recommended to monitor wild fish diseases in the context of assessing the impact of hazardous substances and other stressors on the marine environment. The Fish Disease Index (FDI) is regarded as a strong tool for disease data analysis and assessment, suitable as ecosystem health indicator

CONTRIBUTION OF CELL MARKERS TO THE STUDY OF RMS PATHOGENESIS

Red mark syndrome (RMS) is a chronic non-lethal skin disease affecting farmed rainbow trout (O. mykiss) in U.K., Austria, Germany, Italy (Galeotti et al., 2011), Serbia and U.S.A. Histology shows a lymphocyte/macrophage infiltration in scale pockets, dermis and ipodermis. Aiming at the full comprehension of RMS aetio-pathogenesis, we focused on the mechanisms of cell recruitment/activation in the skin lesions, in order to elucidate if and how an hypothetical microbial agent might trigger the host inflammatory response. Samples of skin from infected fish were evaluated by histology, immunohistochemistry and electron microscopy. The following markers were used: rabbit to human CD3 (A-0452, Dako); rabbit to rainbow trout IgT/IgM (Prof. Sunyer); rabbit to salmonid HSP70 (AS05061A, Agrisera); rabbit to human GM-CSFR\u3b1 (sc-690, Santa Cruz Biotech.); mouse to PCNA (2586, Cell Signaling Technology); mouse to AE1/AE3 Cytokeratin (M3515, Dako); mouse to E Cadherin (M3612, Dako). Anti trout IgT and IgM marked a limited number of scattered cells in the dermis and ipodermis. Anti CD3 marked a relevant number of cells composing the skin infiltrate. HSP70 marked monocyte/macrophages, dendritic like-cells and endothelial cells, within the scale pockets involved by inflammation. GM-CSFR\u3b1 positive monocyte-macrophage were scattered in the derma, surrounding the scales. Anti-Cytokeratin and E Cadherin marked the epithelial cells. PCNA positive cells have been detected in epidermis, dermis and hypodermis, as well among infiltrating lymphocytes, stromal fibroblasts and vascular endothelial cells. HSP70 are considered not only as acute phase proteins, but also as molecules able to mediate immunity and inflammation (Pockley, 2003); they can be released by several cell populations in response to various stimuli. Briefly HSP70 could act as an \u201cantigen\u201d inducing a severe T lymphocyte response, leading to an auto-immune like reaction. Dendritic cells and APCs are stimulated by HSP70 to release TNF-\u3b1, IL1-\uf062and GM-csf. We might speculate that a microbial agent promotes HSP70 expression by macrophages/endothelial cells, within scale pockets. HSP70 might be also internalized by skin APCs. The pro-inflammatory cytokines released could then trigger the local inflammatory process. The GM-csf stimulates the development of osteoclasts. Skin APCs could express HSP70 and therefore stimulate T cell proliferation. These findings might justify the severe cell infiltration detectable in the skin of RMS affected fish