Morphogenesis of salmonid gill poxvirus associated with proliferative gill disease in farmed Atlantic salmon (Salmo salar) in Norway.

Proliferative gill disease (PGD) is an emerging problem in Norwegian culture of Atlantic salmon (Salmo salar). Parasites (Ichthyobodo spp.) and bacteria (Flexibacter/Flavobacterium) may cause PGD, but for most cases of PGD in farmed salmon in Norway, no specific pathogen has been identified as the causative agent. However, Neoparamoeba sp. and several bacteria and viruses have been associated with this disease. In the spring of 2006, a new poxvirus, salmon gill poxvirus (SGPV), was discovered on the gills of salmon suffering from PGD in fresh water in northern Norway. Later the same year, this virus was also found on gills of salmon at two marine sites in western Norway. All farms suffered high losses associated with the presence of this virus. In this study, we describe the entry and morphogenesis of the SGP virus in epithelial gill cells from Atlantic salmon. Intracellular mature virions (IMVs) are the only infective particles that seem to be produced. These are spread by cell lysis and by "budding" of virus packages, containing more that 100 IMVs, from the apical surface of infected cells. Entry of the IMVs appears to occur by attachment to microridges on the cell surface and fusion of the viral and cell membranes, delivering the cores into the cytoplasm. The morphogenesis starts with the emergence of crescents in viroplasm foci in perinuclear areas of infected cells. These crescents consist of two tightly apposed unit membranes (each 5 nm thick) that seem to be derived from membranes of the endoplasmic reticulum. The crescents develop into spheres, immature virions (IVs), that are 350 nm in diameter and surrounded by two unit membranes. The maturation of the IVs occurs by condensation of the core material and a change from spherical to boat-shaped particles, intracellular mature virions (IMVs), that are about 300 nm long. Hence, the IMVs from the SGP virus have a different morphology compared to other vertebrate poxviruses that are members of the subfamily Chordopoxvirinae, and they are more similar to members of subfamily Entomopoxvirinae, genus Alphaentomopoxvirus. However, it is premature to make a taxonomic assignment until the genome of the SGP virus has been sequenced, but morphogenesis clearly shows that this virus is a member of family Poxviridae.

Transmission of infectious salmon anaemia virus (ISAV) in farmed populations of Atlantic salmon (Salmo salar).

In the present study, 24 smolt production sites were screened for the presence of infectious salmon anaemia virus (ISAV) with the help of a specific real-time RT PCR assay, and 22 of these sites had smolts that were positive. If these smolt production sites are representative for the prevalence of ISAV in Norwegian smolts, then most marine production sites must be considered to be positive for ISAV. In addition, 92 European ISAV isolates have been genotyped based on the hemagglutinin-esterase gene (HE), and their distribution pattern was analysed. This pattern has been coupled to information about the origin of smolt, eggs, and broodfish in those cases where it has been possible to obtain such information, and with information about ISAV in neighbouring farms. The pattern suggests that an important transmission route for the ISAV could be that the salmon farming industry in Norway is circulating some of the isolates in the production cycle, i.e. some sort of vertical or transgenerational transmission may occur. It has also been shown that avirluent ISAV isolates are fairly common in Norwegian farmed salmon. Based on this, it is hypothesized that the change from avirulent to virulent ISAV isolates is a stochastic event that is dependent on the replication frequency of the virus and the time available for changes in a highly polymorphic region (HPR) of the HE gene to occur. This, and the possibility that only avirluent ISAV isolates are vertically transmitted, may explain why ISA most often occurs at marine sites and why no more than about 15 farms get ISA every year in Norway.

Parvicapsula pseudobranchicola (Myxosporea) in farmed Atlantic salmon Salmo salar. Tissue distribution, diagnosis and phylogeny.

Parvicapsula pseudobranchicola infections in farmed Atlantic salmon in Norway are associated with low-grade to significant mortalities. The parasite is found as mature spores in pseudobranchs, but has also been detected in the gills, liver and kidney. Diagnosis has relied on the detection of Parvicapsula spores, with the pseudobranch being the preferred organ. A better understanding of the epizootiology of this myxosporean is a prerequisite for appropriate management and control. Hence, early detection of infections and life cycle studies are needed. We sequenced the small subunit (ssu) rDNA (18S) from P. pseudobranchicola and developed a sensitive diagnostic PCR protocol. This allowed us to (1) identify appropriate tissues for diagnostic assays, (2) examine the intraspecific variation in ssu rDNA in the parasite's Norwegian range, (3) examine annelid potential primary hosts and (4) obtain additional ssu rDNA sequences of marine Parvicapsula species to perform a phylogenetic study. Primers were constructed targeting the ssu rDNA from P. minibicornis. With these we obtained a partial ssu sequence of the P. pseudobranchicola type isolate. A new set of primers (PCF3/PCR3) was constructed for diagnostic purposes. These were tested against DNA from the host and several myxozoan species infecting Norwegian salmon. The primers give a positive product of 203 bp and pick out P. pseudobranchicola in salmnonids. They also amplify the congeners P. unicornis and P. asymmetrica infecting unrelated fish. The PCR protocol developed showed a greater sensitivity than light microscopy. The pseudobranchs were always positive and are the recommended organ for PCR diagnostics. There was no sequence variation between geographic isolates from farmed salmon. Preliminary examinations of marine polychaetes and oligochaetes collected from farm sites with parvicapsulose problems were negative. A comparison of the sequence of the ssu rDNA from P. pseudobranchicola with that of other myxozoans shows that it groups closely together with P. unicornis and P. asymmetrica. The closest relative to this group is P. minibicornis.

Effect of cervical and vaginal insemination with liquid semen stored at room temperature on fertility of goats.

The effect of vaginal and cervical deposition of liquid semen stored at room temperature on the fertility of goats was tested in a field trial in which 217 Norwegian Dairy goats aged between 6 months and 7.5 years from 14 farms were inseminated after natural oestrous. Cervical insemination with 200 x 10(6) spermatozoa resulted in 25-day non-return and kidding rates of 87.0 and 78.0%, and vaginal insemination gave 85.5 and 74.3%, respectively. There was no significant difference between the cervical and vaginal inseminations (P = 0.59 for the 25-day non-return and P = 0.40 for the kidding rates). Farm had a significant effect on the 25-day non-return rate (P = 0.03) but not on the kidding rate (P = 0.07). There were no significant differences between the fertility rates for different bucks (P = 0.36 for the 25-day non-return and P = 0.15 for the kidding rates). Fertility results after vaginal insemination were encouragingly high. Vaginal insemination is a simple, less costly and time consuming technique compared to others, also bringing into focus the animal welfare aspects of the artificial insemination procedure. As the final goal is to establish a technique that could be applied similarly on a large scale by all farmers, vaginal insemination must be considered as a method that would simplify the use of liquid buck semen in Norway.