Red mark syndrome of trout (Oncorhynchus mykiss; Walbaum, 1792): Histopathological scoring and correlation with gross lesions.

Red mark syndrome (RMS) is a skin disorder affecting rainbow trout (Oncorhynchus mykiss). The present work aimed to correlate the gross skin lesions affecting 46 fish sampled from farms surveyed for RMS with their microscopic features, identifying histological parameters that may be suggestive of disease progression. Skin lesions were grossly included in one of three categories (types I, II and III) according to the progressive degree of severity. Histological parameters and anti-proliferating cell nuclear antigen (PCNA) tissue immunoreactivity were semi-quantitatively assessed. In the dermis, PCNA-positive lymphocytes, fibroblasts and endothelial cells were indicative of active phlogosis. A significant increase in PCNA-immunoreactive lymphocytes, from gross type I to type III cases, was found only in the hypodermis. The histological parameters significantly associated with the gross lesion severity were progressive loss of the epithelium and scales, recruitment of inflammatory cells in the stratum compactum, loss of architecture of the stratum compactum, perivascular and perineural granulomatous inflammation and increase in lymphocyte infiltration of the muscular layer. In the type II and type III categories, inflammation in the hypodermis and muscle displayed a granulomatous pattern, reinforcing the hypothesis of an immunopathological mechanism. The morphological diagnosis of "deep chronic dermatitis associated to panniculitis and myositis, characterised by lympho-histiocytic and granulomatous reaction" is suggested.

Ultrastructural and biomolecular detection of Rickettsiales-like organisms in tissues of rainbow trout with Red Mark Syndrome.

Red mark syndrome (RMS) and US strawberry disease (US SD) are skin disorders affecting rainbow trout farmed in Europe and USA. The disease etiology has not yet been established. In spite of specific investigations, identifying Rickettsia-like organism (RLO)- and Midichloria-like organism (MLO)-related DNA in affected individuals, these pathogens have never been observed. We performed histological, ultrastructural and biomolecular analysis on skin and spleen samples of trout with RMS. Examination by TEM revealed the presence of intracytoplasmic microorganisms resembling Rickettsiales within macrophages, fibroblasts and erythrocytes. The microorganisms were oval or short rod shaped (400-800 nm in length and 100-200 nm in width) and often showed a cell wall similar to Gram-negative bacteria. PCR analysis for Rickettsiales supported these findings: 53% of affected trout were positive by both PCR and TEM The primers RiFCfw-RiFCrev were used to anneal both the RLO 16S DNA sequence and the MLO 16S DNA sequence. For this reason, and in agreement with previous studies confirming the presence of Rickettsiales-related DNA in trout with RMS, we assume that TEM detected microorganisms morphologically consistent with bacteria belonging to Rickettsiales order and could be considered as possible causative agents of RMS.

Rhabdomyosarcoma of Soft Tissues in an Adult Brook Trout (Salvelinus fontinalis).

This report describes a spontaneously arising rhabdomyosarcoma of soft tissues in a brook trout (Salvelinus fontinalis). The lesion was examined by means of histology, immunohistochemistry (IHC) and transmission electron microscopy (TEM). The cross-reactivity of the primary antibodies used in the IHC was investigated in silico using the Protein Blast system. Microscopically, the lesion appeared as a 'small round cell' undifferentiated sarcoma with rare myotube formation. IHC identified expression of sarcomeric actin and vimentin and these molecules showed the highest protein sequence identity. Lower protein sequence identity coincided with negative immunolabelling for desmin, MyoD1, myogenin and CD3. TEM revealed myofibrils, but without a defined sarcomeric architecture. The diagnosis of solid alveolar rhabdomyosarcoma of soft tissues was achieved on the basis of histological and ultrastructural findings.

Multidisciplinary approach for the study of an Egyptian coffin (late 22nd/early 25th dynasty): combining imaging and spectroscopic techniques.

This paper demonstrates that an educated methodology based on both non-invasive and micro invasive techniques in a two-step approach is a powerful tool to characterize the materials and stratigraphies of an Egyptian coffin, which was restored several times. This coffin, belonging to a certain Mesiset, is now located at the Museo Civico Archeologico of Bologna (inventory number MCABo EG 1963). Scholars attributed it to the late 22nd/early 25th dynasty by stylistic comparison. The first step of the diagnostic approach applied imaging techniques on the whole surface in order to select measurements spots and to unveil both original and restored areas. Images and close microscopic examination of the polychrome surface allowed selecting representative areas to be investigated in situ by portable spectroscopic techniques: X-ray Fluorescence (XRF), Fiber Optic Reflectance Spectroscopy (FORS) and Fourier Transform Infrared spectroscopy (FTIR). After the analysis of the results coming from the first step, very few selected samples were taken to clarify the stratigraphy of the polychrome layers. The first step, based on the combination of imaging and spectroscopic techniques in a totally non-invasive modality, is quite unique in the literature on Egyptian coffins and enabled us to reveal many differences in the ground layer's composition and to identify a remarkable number of pigments in the original and restored areas. This work offered also a chance to check the limitations of the non-invasive approach applied on a complex case, namely the right localization of different materials in the stratigraphy and the identification of binding media. Indeed, to dissolve any remaining doubts on superimposed layers belonging to different interventions, it was necessary to sample few micro-fragments in some selected areas and analyze them prepared as cross-sections. The original ground layer is made of calcite, while the restored areas show the presence of either a mixture of calcite and silicates or a gypsum ground, overlapped by lead white. The original pigments were identified as orpiment, cinnabar and red clay, Egyptian blue and green copper based pigments. Some other pigments, such as white lead, Naples yellow, cerulean blue and azurite were only found in the restored areas.

Differential characterization of emerging skin diseases of rainbow trout--a standardized approach to capturing disease characteristics and development of case definitions.

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example.

Effect of the disclosure of MS diagnosis on anxiety, mood and quality of life of patients: a prospective study.

BACKGROUND: In the light of the new diagnostic criteria for multiple sclerosis (MS) and currently available early treatment, this study aimed to explore whether, and to what extent, disclosure of the diagnosis of MS or clinically isolated syndrome (CIS) affects patients' anxiety, mood and quality of life (QoL). METHODS: Eligible participants were all patients referred for the first time to the Neurological Unit who had manifested symptoms suggestive of MS for no more than 6 months. All patients were evaluated for (i) QoL (SEIQoL and MS-QoL54), (ii) Anxiety (STAI) and Depression (CMDI) on study inclusion (T0), 30 days after diagnosis disclosure (T30), and after 1 (T1y) and 2 (T2y) years' follow-up. RESULTS: Two hundred and twenty-nine patients were enrolled; 93 of these were unaware of their diagnosis. Patients who already knew their diagnosis (100 with CIS and 22 with MS) were excluded from the main analyses and used to perform control analyses. At the end of the screening, an MS diagnosis was disclosed to 18 of the 93 patients, whereas a CIS diagnosis was disclosed to 62 patients (12 patients received a diagnosis other than MS or CIS). Thirty days after diagnosis disclosure, irrespective of the diagnosis disclosed, both QoL and Anxiety and Depression were significantly rated as better compared to the start of screening, (p(s) < 0.03), and this improvement remained stable over the two annual follow-ups. However, as suggested by a significant 'Time' × 'Diagnosis' interaction with regard to both QoL and Anxiety and Depression (p(s) < 0.02), the effect of the disclosure in the short term differed depending on CIS or MS diagnosis. Specifically, on MSQoL, which is a health-related QoL scale, we found a statically significant improvement, immediately after the diagnosis disclosure, in both the MS and CIS groups (p(s) < 0.01). Differently, on SEIQoL, which is a non health-related QoL measure, and on the anxiety scale, we observed a statistically significant improvement only in the group which received a MS diagnosis (p(s) < 0.03). CONCLUSIONS: This first prospective study provides objective data showing that early disclosure of MS diagnosis improves both the patient's QoL and psychological well-being. In addition, the results seem to suggest that CIS disclosure does not lead to the same favourable effects.

Attenuated total reflection micro FTIR characterisation of pigment-binder interaction in reconstructed paint films.

The interaction of pigments and binding media may result in the production of metal soaps on the surface of paintings which modifies their visible appearance and state of conservation. To characterise more fully the metal soaps found on paintings, several historically accurate oil and egg yolk tempera paint reconstructions made with different pigments and naturally aged for 10 years were submitted to attenuated total reflectance Fourier transform infrared (ATR FTIR) microspectroscopic analyses. Standard metal palmitates were synthesised and their ATR spectra recorded in order to help the identification of metal soaps. Among the different lead-based pigments, red lead and litharge seemed to produce a larger amount of carboxylates compared with lead white, Naples yellow and lead tin yellow paints. Oil and egg tempera litharge and red lead paints appeared to be degraded into lead carbonate, a phenomenon which has been observed for the first time. The formation of metal soaps was confirmed on both oil and egg tempera paints based on zinc, manganese and copper and in particular on azurite paints. ATR mapping analyses showed how the areas where copper carboxylates were present coincided with those in which azurite was converted into malachite. Furthermore, the key role played by manganese in the production of metals soaps on burnt and raw sienna and burnt and raw umber paints has been observed for the first time. The formation of copper, lead, manganese, cadmium and zinc metal soaps was also identified on egg tempera paint reconstructions even though, in this case, the overlapping of the spectral region of the amide II band with that of metal carboxylates made their identification difficult.

Evaluation of immunological parameters in farmed gilthead sea bream, Sparus aurata L., before and during outbreaks of 'winter syndrome'.

In recent years, 'winter syndrome' has caused severe economic losses in many marine farms in southern Europe. This study compared the activity of the immune system in healthy, asymptomatic and diseased gilthead sea bream. Serum protein and immunoglobulin content were evaluated as well as serum complement activity from these groups of fish. Differential leucocyte counts were also determined. Immunological assays were performed to determine intra- and extracellular 'respiratory burst' activity as non-specific immune response parameters. Diseased fish showed a significant reduction in haemolytic activity (90%), and in serum proteins and immunoglobulins in comparison with the other groups. A significant increase in the lymphocyte percentage (50%) and a decrease in the granulocyte percentage (over 70%) was found in asymptomatic fish. Respiratory burst activity was reduced in both the clinical and preclinical stages of the disease, compared with the controls. These observations confirm a severe immunodeficiency in diseased fish but also the presence of a cellular immune dysfunction in fish without clinical signs, before the onset of the disease.

Fatal cerebral haemorrhage after liver transplantation in a patient with transthyretin variant (gly53glu) amyloidosis.

Transthyretin (TTR) amyloidosis is a form of systemic amyloidosis caused by an amyloidogenetic TTR variant. The most common mutant forms of TTR are mainly produced by the liver and therefore orthotopic liver transplantation (OLT) is currently accepted as the only known curative treatment. We describe the clinical and pathological features of a patient with TTR variant (gly53glu) with TTR amyloid infiltration of the leptomeningeal vessels in whom fatal cerebral haemorrhage occurred two months after OLT, soon after severe viral pneumonia.

Epidemiology of status epilepticus in a rural area of northern Italy: a 2-year population-based study.

We performed a 2-year population-based study on status epilepticus (SE) in adults in the rural area of Lugo di Romagna, northern Italy, to verify whether an area of low-level urbanization has a lower risk of occurrence of SE (as recently suggested), different clinical features and short-term prognosis than areas of high-level urbanization. We found crude and age- and sex-adjusted annual incidence rates of SE of 16.5/100 000 and 11.6/100 000, respectively. In patients under 60 years crude incidence was 2.9/100 000 and in the elderly (>/=60 years) 38.6/100 000. Acute symptomatic SE accounted for 30% and a cerebrovascular pathology was the most frequently associated etiologic condition (60%). A history of seizures was reported in 41% of patients. The first therapeutic intervention was mainly benzodiazepines (lorazepam 46%; diazepam 33%). The 30-day case fatality was 7%. We observed that the adult population of an area with a low level of urbanization has the same risk for SE, clinical features and short-term prognosis as European urban areas. The only contrasting result is the 30-day case fatality of 7% against the 39% found in the other Italian study (Bologna), despite the similarity of the SE features in these two areas of the same region. We infer that the short-term prognosis of SE could also be considerably influenced by differences in health service organization (and hence management) possibly due to different levels of urbanization.

The effect of in vivo growth on the cellular and extracellular components of the marine bacterial pathogen Photobacterium damsela subsp. piscicida.

Photobacterium damsela subsp. piscicida, the causative agent of fish pasteurellosis, was grown in vivo. Bacterial cells and extracellular products (ECPs) were analysed via electrophoresis and immunoblot analysis, using specific sea bass antisera. Growth in vivo induced the synthesis of unique bacterial cell proteins at > 206, 206, 21.3, 18, 7.6 and < 7.6 kDa. Sea bass serum raised against live bacterial cells of the pathogen and especially a sea bass serum raised against formalin-inactivated bacterial cells grown in a specific novel medium recognized the novel antigens at > 206 (associated with iron sequestration), 21.3, 7.6 and < 7.6 kDa, suggesting that the latter medium conserves the synthesis of natural bacterial cell proteins in vitro. In vivo growth of the pathogen induced the synthesis of more toxic ECPs in comparison with in vitro growth and an inverse correlation between total protein concentration in the ECPs and toxicity per unit of protein was observed. Substrate-polyacrylamide electrophoresis revealed the presence of in vivo synthesized ECPs of the pathogen (proteases) at 175, 132, < 79 and 48.3 kDa. Histological examination of tissues isolated from fish injected with these ECPs revealed inflammatory and necrotic lesions in the spleen, liver, head kidney, intestine and heart as soon as 48 h post-introduction of the ECPs.

Investigation of media formulations promoting differential antigen expression by Photobacterium damsela ssp. piscicida and recognition by sea bass, Dicentrarchus labrax (L.), immune sera.

Photobacterium damsela ssp. piscicida (Phdp) isolates were grown in various bacteriological media, in eukaryotic cell culture media and in the presence of fish cells (resembling some aspects of in vivo growth environments). Bacterial cells, extracellular products (ECPs) and crude capsular polysaccharide were isolated and analysed by electrophoresis and Western blot using sea bass sera. Growth in bacteriological media conserved the synthesis of cell and extracellular components when these were compared with those prepared under near-in vivo growth conditions. In fact, synthesis of a larger range of cell components was induced after growth in bacteriological media. Certain media based on yeast extract and peptones from various sources and a specific salt formulation induced the synthesis of novel cell components at approximately 21.3 and 14 kDa. These antigens were recognized by sea bass sera collected after natural pasteurellosis outbreaks and other sea bass sera raised against live or inactivated Phdp cells. The ECPs of the pathogen were not good immunogens in their soluble form despite various treatments prior to immunization. The results are discussed with respect to vaccine development.

Vaccination trials of sea bass, Dicentrarchus labrax (L.), against Photobacterium damsela subsp. piscicida, using novel vaccine mixtures.

Bacterial cells of the marine fish pathogen Photobacterium damsela subsp. piscicida were grown in novel culture media. A mixture of whole cells and extracellular components was inactivated and used in bath, intraperitoneal (i.p.) and oral vaccination of sea bass, Dicentrarchus labrax, employing two sizes of fish. A commercial vaccine was used for comparative purposes. Control and immunized fish were either bath or intraperitoneally challenged 6 and 12 weeks post-vaccination. Small fish had significantly higher relative percentage survival with the novel vaccine mixture both at 6 and 12 weeks post-vaccination by bath, in comparison with the commercial vaccine. No protection was afforded at 6 or 12 weeks post-immunization by either vaccine after challenge via i.p. injection. Sea bass (1.5-2 g) intraperitoneally vaccinated with various adjuvanted vaccine mixtures were not protected against pasteurellosis. In contrast, larger sea bass (20 g) benefited from vaccination with the novel vaccine mixtures. Intraperitoneal challenge with the pathogen resulted in protection in both fish groups vaccinated with novel vaccine mixtures, whereas control fish suffered high mortalities (> 80%). Orally vaccinated fish were immersion challenged with the pathogen. At 6 and 12 weeks post-vaccination the control fish had a high mortality and the fish vaccinated with the novel vaccine mixture achieved good protection.

Accuracy of ICD-9 codes in identifying ischemic stroke in the General Hospital of Lugo di Romagna (Italy).

We assessed the sensitivity and the positive predictive value (PPV) of the ICD-9 codes in identifying ischemic strokes. The study involved the cross-sectional comparison between patients with an ischemic stroke diagnosis made by neurologists and patients with the 434 or 436 discharge codes. Sensitivity of the codes (all diagnostic levels and first level respectively) was 82% and 76%; PPV: 71% and 76%. The annual crude incidence of ischemic stroke was 2.62 per 1000 based on verified strokes and 3.03 per 1000 based on 434 or 436 coded medical records (at all diagnostic levels). Thirty-day case fatality ratio was 22.3% in verified strokes and 36.8% among patients diagnosed with codes 434 or 436 but without stroke (all levels). Our results disclosed inaccuracy in use of the ICD-9 codes in the diagnosis of ischemic stroke in the general hospital of Lugo di Romagna, Ravenna Province, Italy. The misdiagnosis of patients could be influenced by the degree of severity of clinical features. Epidemiological data and cost-analysis forecasts based only on the ICD-9 system must be considered with caution.

Pretelangiectasis and telangiectasis of the bovine liver: a morphological, immunohistochemical and ultrastructural study.

Forty-five livers from conventionally slaughtered Holstein-Friesian steers with telangiectasis were studied by histochemical methods, immunolabelling for fibronectin, laminin and type IV collagen, and transmission electron microscopy. None of the previously described changes in telangiectasis (necrosis, hepatitis, thromboembolism, dilatation of the space of Disse by glycogen extruded from hepatocytes and reduced density of the perisinusoidal reticulin framework) were evident. Pretelangiectasis (sinusoidal dilatation) and telangiectasis (blood-filled cavities) were characterized by sinusoidal barrier alterations, leading to sinusoidal capillarization; and there was progressive formation of a true basement membrane and perisinusoidal fibrosis. Comparison of bovine liver telangiectasis and human peliosis hepatis suggests that they have a similar pathogenesis. It is suggested that a primary alteration of the sinusoidal barrier is responsible for an increased deposition of basement membrane components (fibronectin, laminin, type IV collagen) in the perisinusoidal region, and fibrosis. These are likely to render the exchange of oxygen and substrates between blood and hepatocytes more difficult and to produce haemodynamic imbalances, leading to hepatocyte atrophy and eventually to sinusoidal disruption.

Response of rainbow trout gill (Na(+)+K (+))-ATPase and chloride cells to T 3 and NaCl administration.

With the aim of comparing the effects of oral T3 and NaCl administration on trout hypoosmoregulatory mechanisms, three groups of rainbow trout (Oncorhynchus mykiss Walbaum) held in freshwater (FW) were fed a basal diet (C), the same diet containing 8.83 ppm of 3,5,3'-triiodo-L-thyronine (T3) (T) or 10% (w/w) NaCl (N) respectively for 30 d. They were then transferred to brackish water (BW) for 22 d and fed on diet C. Gill (Na(+)+K(+))-ATPase activity and its dependence on ATP, Na(+) and pH, number of gill chloride cells (CC), serum T3 level as well as fish growth, condition factor (K) and mortality were evaluated. During the FW phase, as compared to C trout, T trout showed a two fold higher serum T3 level, had unchanged gill (Na(+)+K(+))-ATPase activity and increased CC number, whereas N trout showed higher gill (Na(+)+K(+))-ATPase activity and CC number. At the end of the experiment the enzyme activity was in the order T>N>C groups and all groups showed similar CC number. Both treatments changed the enzyme activation kinetics by ATP and Na(+). A transient increase in K value occurred in N group during the period of salt administration. In BW, T and N groups had higher and lower survival than C group respectively. Other parameters were unaffected by the treatments. This trial suggests that T3 administration promotes the development of hypoosmoregulatory mechanisms of trout but it leaves the (Na(+)+K(+))-ATPase activity unaltered till the transfer to a hyperosmotic environment.

Basement membrane components in mammary tumours of the dog and cat.

The distribution of the basement membrane (BM) components, laminin, type IV collagen and type VII collagen were studied immunohistochemically in benign and malignant growths of the mammary epithelium of the dog and cat. Intact BMs were found in benign growths, but in well-differentiated malignant tumours they were generally discontinuous, and missing in poorly differentiated carcinomas. An increase in the histological grade of atypia was accompanied by a more marked disruption or fading of BM. Monoclonal antibody (LH 7.2) proved useful in demonstrating type VII collagen in tumours in which massive proliferation of blood vessels made the evaluation of BM features with antibodies to laminin and type IV collagen difficult. Type VII collagen is present in BM of the mammary epithelium but not under the endothelium of blood vessels; it may therefore enhance the value of BM markers as aids in the study of neoplastic progression.

Identification of cell types present in bovine haemolymph nodes and lymph nodes by immunostaining.

Mediastinal haemolymph nodes (HN) and lymph nodes (LN) of 15 healthy calves and ten healthy steers and 53 steers with respiratory diseases (pneumonias) were studied using monoclonal antibodies (Mabs) specific to bovine B and T lymphocytes: BAQ155A (B), CH128A (BoCD2), CACT138A (BoCD4), CACT80 (BoCD8), BAQ4A (Null cells). Topographical distribution of the HN T and B cell subset appears similar to the distributions in the LN. The germinal centres contained B+, BoCD2+ and BoCD4+ cells, whereas no staining of lymphocytes could be seen in the mantle zone. BoCD2+, BoCD4+ and BoCD8+ lymphocytes were located in the parafollicular lymphatic tissue, whereas B and Null lymphocytes were scattered. The same pattern was observed in the hyperplastic haemolymphadenitis of steers with respiratory diseases with an increased positive staining for BoCD4 and BoCD8. A similar pattern was observed for HN and LN Immunoglobulin Containing Cells (ICC). Furthermore, in the calf HN, IgM positive ICC were the most frequent class (44% in HN vs. 33% in LN). It is likely that they could contribute to produce a particularly efficient primary immune response. This is the first study of the distribution of different lymphocyte phenotypes in mammalian haemolymph nodes.