A method to measure an indicator of viraemia in Atlantic salmon using a reporter cell line.

RTG-P1 is a transgenic fish cell line producing luciferase under the control of the IFN-induced Mx rainbow trout gene promoter. This cell line was used to measure viraemia of Salmonid alphavirus (SAV), the cause of Salmon Pancreas Disease (SPD), a serious disease in farmed Atlantic salmon. Two SAV genotype 1 (SAV1) isolates were used in this study, F93-125 (tissue-culture adapted, from Ireland) and 4640 (from a field case in Scotland). The kinetics and magnitude of luciferase activity were monitored versus the time of infection. During a direct infection experiment, the induction of luciferase significantly increased 16- and 4-fold after incubation for 6 days with F93-125 at 15 and 20°C, respectively. Filtration and heat treatment experiments demonstrated that the luciferase induction in RTG-P1 was dependent on viral replication. Unlike many cell lines used in fish viral diagnostic, RTG-P1 is not sensitive to salmonid serum, therefore, viraemia could be successfully monitored on serum collected from fish during a cohabitation challenge with 4640 isolate. A peak of viraemia could be detected 16 days post IP inoculation of the shedders. This novel cost-effective method to measure viraemia does not rely on development of cytopathic effect (CPE) in culture, is compatible with non-lethal blood collections in fish and can be used to assign emerging diseases to a viral aetiology.

Atlantic salmon (Salmo salar L.) serum vitellogenin neutralises infectivity of infectious pancreatic necrosis virus (IPNV).

Vitellogenin is a phosphoglycoprotein which represents the main precursor of the egg yolk in teleost fish. This reproductive protein was also demonstrated to play an important role in innate immunity by acting as a pattern recognition molecule capable of binding to bacteria, fungi and enhancing macrophage phagocytosis. The presented results demonstrate that, egg homogenate, ovarian fluid and serum of mature female Atlantic salmon have high neutralising ability for infectious pancreatic necrosis virus (IPNV). Vitellogenin from mature female Atlantic salmon serum, purified by immuno-affinity on a column matrix coated with monoclonal anti-Atlantic salmon vitellogenin antibody, was able to neutralise between 9.1 x 10(4) and 3.09 x 10(5) TCID(50) IPNV mg(-1) of protein. To the author's knowledge, this is the first time that the neutralising activity of vitellogenin on a teleost virus has been demonstrated. The results may explain why IPNV is difficult to detect by culture methods in ovarian fluid and egg homogenates from carrier mature females and suggest a possible means of vertical transmission via the egg.

Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification.

We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.

Radial artery as conduit for distal renal artery reconstruction.

UNLABELLED: Reconstruction of the renal artery with both saphenous vein and prosthetic material as bypass graft is durable in atherosclerotic disease. Extensive experience with saphenous vein grafts in pediatric patients and patients without atherosclerosis reveals a disturbing incidence of vein graft aneurysm degeneration. Distal renal artery reconstruction involving small branch vessels is generally not amenable to prosthetic reconstruction. We report a new approach to distal renal artery bypass grafting to avert these limitations. CASE: A 43-year-old man with previously normal blood pressure had malignant hypertension, which proved difficult to control despite use of a beta-blocker and an angiotensin II inhibitor. At renal angiography a fusiform aneurysm was revealed in a posterior branch of the right renal artery. The renal artery aneurysm was resected, and the left radial artery was harvested and used as a sequential aortorenal bypass graft to the two branch renal arteries. The postoperative course was uneventful, and the patient now has normal blood pressure with a calcium channel blocker for maintenance of the radial artery graft. Pathologic analysis revealed a pseudoaneurysm with dissection between the media and external lamella, consistent with fibromuscular dysplasia. CONCLUSION: Autologous artery is the preferred conduit for renal reconstruction in the pediatric population. On the basis of cardiac surgery experience, we used the radial artery and found it to be a technically satisfactory conduit for distal renal reconstruction in a patient without atherosclerosis.

Lethal neonatal and severe late infantile forms of carnitine palmitoyltransferase II deficiency associated with compound heterozygosity for different protein truncation mutations.

We describe a lethal neonatal form of carnitine palmitoyltransferase II (CPT II) deficiency with compound heterozygosity for 2 truncation mutations (Q413fs and 109AGC --> GCAGC). A new phenotype for a severe late infantile form of CPT II deficiency with hypoglycemia is associated with compound heterozygosity for the severe Q413fs mutation and a mild point mutation (P50H).

Phenotypic variability among first-degree relatives with carnitine palmitoyltransferase II deficiency.

Carnitine palmitoyltransferase (CPT) II deficiency disorders are clinically very variable. To examine the cause(s) of variable symptoms in first-degree relatives with CPT II deficiency, four sisters with various combinations of mutations and polymorphisms in the CPT2 gene were studied, together with 20 sedentary and 24 trained healthy female subjects. One sister, whose symptoms began at age 7 years, was more severely affected than her older sister, whose symptoms began at age 16 years; both were compound heterozygotes for the common S113L mutation and Q413fs, and for the common CPT2 polymorphisms, V3681 and M647V. A third sister became hypoglycemic with fasting, was heterozygous for the S113L mutation, and homozygous for the polymorphism variants. The fourth sister was asymptomatic, heterozygous for the Q413fs mutation, and homozygous for the normal polymorphisms. Residual CPT II activity in skeletal muscle and cultured skin fibroblasts from the two myopathic sisters, and palmitate oxidation in fibrobasts, were abnormally low; cellular and total body fat oxidation were also diminished. Muscle function and fat oxidation were nomal at rest, but a switch to carbohydrate utilization occurred at lower exercise intensities than in sedentary and trained individuals, respectively. Reliance on carbohydrates during stress and hormonal alterations may explain, in part, the variance in ages of onset and serverity of symptoms in myopathic patients.