Development and validation of a range of endogenous controls to support the implementation of practical Taqman real-time PCR-based surveillance for fish diseases within aquaculture.

The use of Taqman real-time PCR-based technology has recently become more frequent in the detection of pathogens in the aquaculture industry. This interest has necessitated the development of robust and reliable pathogen-detection assays. The development of a range of endogenous control assays to be run alongside these diagnostic assays works to further increase confidence in the latter. This study describes the design of a range of endogenous control assays based on the elongation factor 1-α (EF1-α) gene specific to a range of fish species including Atlantic salmon, Salmo salar; rainbow trout, Oncorhynchus mykiss; brown trout, Salmo trutta; cod, Gadus morhua; haddock, Melanogrammus aeglefinus; saithe, Pollachius virens; whiting, Merlangius merlangus; Norway pout, Trisopterus esmarkii; carp (family Cyprinidae), roach, Rutilus rutilus; European eel, Anguilla anguilla; and herring, Clupea harengus, as well as a number of fish cell lines. Evidence is provided of the validation of these assays for specific species, a range of tissue types and cell lines as well as an example of the potential uses of these assays.

Application of a sensitive, specific and controlled real-time PCR assay to surveillance indicates a low prevalence of viral haemorrhagic septicaemia virus (VHSV) in wild herring, Clupea harengus L., in Scottish waters.

Surveillance data on the distribution of viral haemorrhagic septicaemia virus (VHSV) in the North Sea (UK), targeting Atlantic herring in areas with previous virus detection, were obtained from research cruises conducted during 2005. The sensitive molecular approach of real-time RT-PCR (qRT-PCR) was applied alongside a newly developed endogenous positive control assay specific for herring (elongation factor 1α) to ensure integrity of template. Three hundred and five pools from 1937 individual herring were tested, and no evidence of VHSV in association with wild Atlantic herring was detected. Samples were obtained from Scottish waters where marine aquaculture is conducted. The results confirm that previous tissue culture studies have most likely not significantly underestimated the prevalence of carrier herring in this area. The significance of migratory species such as herring as a reservoir species for VHSV, with the potential to translocate virus genotypes between geographical areas, is discussed.

Intramembrane proteolysis of signal peptides: an essential step in the generation of HLA-E epitopes.

Signal sequences of human MHC class I molecules are a unique source of epitopes for newly synthesized nonclassical HLA-E molecules. Binding of such conserved peptides to HLA-E induces its cell surface expression and protects cells from NK cell attack. After cleavage from the pre-protein, we show that the liberated MHC class I signal peptide is further processed by signal peptide peptidase in the hydrophobic, membrane-spanning region. This cut is essential for the release of the HLA-E epitope-containing fragment from the lipid bilayer and its subsequent transport into the lumen of the endoplasmic reticulum via the TAP.

Therapeutic effects of cysteine protease inhibition in allergic lung inflammation: inhibition of allergen-specific T lymphocyte migration.

OBJECTIVE AND DESIGN: We have evaluated the effects of the broad-spectrum cysteine protease inhibitor E64 on allergic lung inflammation in the mouse ovalbumin model of human asthma. We have also characterised membrane-associated cathepsin enzyme activity on a range of cell types. MATERIALS: Balb/C mice, E64 and CA074, various cell lines. TREATMENT: E64 was administered by subcutaneous minipump into ovalbumin-sensitised mice prior to intranasal ovalbumin challenge. The effect of E64 on ovalbumin-induced inflammation in vivo and ovalbumin-specific T cell proliferation in vitro and ex vivo was examined. Membrane-associated cathepsin activity on various cell types was measured. RESULTS: E64 treatment (0.36-0.48 mg/day) led to a significant reduction in eosinophil numbers and lung weights in the mouse model. Histological examination of lungs confirmed the anti-inflammatory effect. E64 greatly reduced ovalbumin-specific T cell numbers in the lymph nodes draining the lung following intranasal challenge whilst an accumulation of these T cells was found in the 'priming' lymph nodes. An analysis of various cells involved in lymphocyte priming and migration revealed that monocytes, dendritic cells and endothelial cells express high levels of membrane-associated cathepsin B activity. CONCLUSIONS: Since E64 is not cell permeable and does not inhibit antigen-induced T cell proliferation in vitro or in vivo, the data indicate that membrane-associated cysteine proteases, possibly cathepsin B, may regulate T lymphocyte migration in vivo.

The subjective experience of acute pain. An assessment of the utility of 10 indices.

Sixty-nine postoperative patients indicated the severity of their pain using eight measures designed to assess pain intensity and two designed to measure pain affect. The utility and validity of the 10 measures were evaluated according to two criteria: (a) the magnitude of the relationship between each scale and a linear combination of the pain measures, and (b) relative rates of incorrect responding. The results indicate that each of the measures of pain intensity is adequately valid. In addition, this sample of patients failed to differentiate pain intensity and pain affect using the present measures, suggesting the need for additional research to explore the validity of the affective measures employed in the study. The 11-point Box Scale (BS-11) of pain intensity demonstrated the strongest relationship to a linear combination of all of the measures employed and was responded to correctly by each subject in the sample. All else being equal, these results suggest that the BS-11 scale may be the most useful clinical index of pain intensity among postoperative patients.

Electron microscope study of experimental enteric infection in neonatal dogs with a canine coronavirus.

Neonatal dogs, inoculated orally with coronavirus 1-71, grown in canine kidney cell cultures, developed diarrhea and a severe enteritis characterized by atrophy of the villi, changes in the enterocytes, and accelerated epithelial cell loss. Electron microscopy of the mucosal epithelium, 4 days after challenge, showed that the virus penetrated into the enterocytes between microvilli, possibly by pinocytotic mechanism. In the enterocytes, virions were most often enclosed, singly or in groups, in cytoplasmic vesicles. They were less frequently found in the cisternae of the Golgi apparatus, the endoplasmic reticulum, or in the dilated perinuclear space and only rarely, free in the cytoplasm. Virions replicated by budding only on the smooth.membranes of the cytoplasmic vesicles. The infected cells showed a variety of cytopathic effects, some nonspecific, such as disruption of the microvilli, loss of density of the cytoplasm, presence of lipid inclusions, alteration of mitochondria, and dilation of the endoplasmic reticulum and Golgi cisternae and of the perinuclear space. Other cytopathic effects, characteristic of the coronavirus infection, consisted of formation of dense filamentous structures and of membrane-bound bodies. Progeny virions appeared to discharge into the gut lumen through the disrupted cell membranes of infected enterocytes still in situ or following their premature shedding.