The first report of viral haemorrhagic septicaemia in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in the United Kingdom.

Viral haemorrhagic septicaemia (VHS) was diagnosed in rainbow trout in the UK in May 2006. VHS virus (VHSV) was isolated from fingerlings showing typical histopathological lesions at a single rainbow trout farm site experiencing high mortality. The virus was confirmed as VHSV by serological and molecular biological tests. Phylogenetic analysis based on the complete glycoprotein gene sequence revealed that the isolate was closely related (99% nucleotide identity) to several Danish isolates from 1991 to 2000 and was assigned to VHSV genogroup Ia. The pathogenicity of the isolate was determined in infection experiments using rainbow trout fry. Following waterborne challenge, cumulative mortalities reached 96.67-100% by 12 days post-infection. This represents the first isolation of a pathogenic freshwater VHSV in the UK.

Identification of the 5-iodoacetamidofluorescein reporter site on the Na,K-ATPase.

5-Iodoacetamidofluorescein (5-IAF) labels the catalytic (alpha) subunit of dog kidney Na,K-ATPase without inhibiting enzymatic activity and is thus a useful fluorescent reporter of enzyme conformation under conditions of enzyme turnover. In this study conditions for labeling a unique sulfhydryl group are described, and this residue is identified in the cDNA-derived sequence. Reaction with iodoacetate (IAA) prior to fluorescent labeling lowers the stoichiometry of 5-IAF incorporation from 2.1 to 1.2 mol/mol alpha beta protomer, and increases the conformationally dependent fluorescence changes by 40-50%, consistent with the elimination of nonspecific labeling. IAA/IAF-enzyme has the same catalytic activity as the IAF-enzyme. In contrast, treatment with iodoacetamide prior to labeling with 5-IAF abolishes all fluorescence responses, although activity is retained. IAA/IAF-enzyme was digested by extensive trypsinolysis, and the fluorescent peptides released from the membrane were purified by high performance liquid chromatography and sequenced. Several fluorescent peptides were found, containing all or part of the sequence Cys-Ile-Glu-Leu-Cys-Cys-Gly-Ser-Val-Lys, corresponding to residues 452-461 in the sheep alpha subunit. The major site of modification is the second of the vicinal cysteine residues, Cys-457. Phenylarsine oxide, a reagent specific for vicinal sulfhydryl groups, prevents fluorophore incorporation, thereby confirming the identification of the IAF site from the sequence data.

Accessibility of tryptophan residues in Na,K-ATPase.

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.

Cervical cytology and Chlamydia trachomatis infection.

We found, as have others, a strong correlation between indirect immunofluorescence techniques and cell culture for the diagnosis of Chlamydia trachomatis infection in material obtained from the cervix. Five epithelial cell types indicative of Chlamydia trachomatis infection (indicator cells) were found in smears in patients with positive immunofluorescence. An attempt to diagnose Chlamydia trachomatis infection in 50 routine smears based on the presence of these indicator cells showed false positives and false negatives so that the diagnosis of Chlamydia trachomatis still requires confirmation by immunofluorescence or culture techniques. The indicator cells frequently had the same morphometric features as cells seen with cervical intraepithelial neoplasia, which may explain why some smears revert to normal after patients are given antibiotics.

Distribution of immunoglobulin-containing cells in the bronchial mucosa of patients with chronic respiratory disease.

Bronchi biopsy specimens of 42 patients with chronic respiratory diseases were studied for immunoglobulin-containing cells, using an indirect immunoperoxidase technique. The number of immunocytes was expressed per unit tissue section surface. The cells were located directly under the epithelium. In most biopsy specimens IgA-, IgG- and IgM-containing cells were demonstrable, with IgA- and IgG-containing cells in the majority. IgE-containing cells were found only in small numbers in biopsy specimens of a few patients. The presence of IgE-containing cells was nearly always accompanied by a high amount of mast cell-bound IgE in the bronchial mucosa. No correlation could be found between the number and class of immunocytes and clinical data.

Demonstration of calcitonin and carcinoembryonic antigen (CEA) in medullary carcinoma of the thyroid (MCT) by immunoperoxidase technique.

The presence of calcitonin and of carcinoembryonic antigen (CEA) was studied in six cases of medullary carcinoma of the thyroid using an immunoperoxidase technique. In five cases the material was obtained surgically and in one at autopsy. Tissue from primary tumours as well as from metastases was studied. Calcitonin and CEA were identified within all the tumours studied, although their pattern of distribution and staining intensity varied both within the cells and within the tumour. Some parts of the tumour contained both CEA and calcitonin, while others stained positively only for one of these substances. In some parts of the tumour there was no positive staining for either substance. Within the cells, CEA showed a typical linear distribution along cell surfaces, while calcitonin showed a more even cytoplasmic distribution and the deposits were more granular. Normal tissue surrounding tumour deposits did not show positive staining. It is considered that cells of medullary carcinoma of the thyroid contain both calcitonin and CEA. Identification of CEA and calcitonin in tumour tissue can be used as a diagnostic aid to identify medullary carcinoma of the thyroid. Iit is considered that these substances are being produced by this tumour and can be used as tumour markers.