ABSTRACT:We assessed seasonal histological changes as markers of health status in mussels Mytilus spp. sampled from Southampton Water, Hampshire, UK and the River Exe, Devon, UK between November 2004 and October 2005. A total of 29 health parameters related to pathogens, inflammatory and non-specific pathologies, and reproductive and physiological condition were recorded monthly from individual mussels collected from these 2 sites. We then assessed the diffential prevalence of these health parameters according to species. M. edulis, M. galloprovincialis and their hybrids were identified using the Glu-5' gene and the ME15 and ME16 primer sets that distinguish alleles specific to M. edulis (180 bp), M. galloprovincialis (126 bp) and hybrids (180 bp/126 bp). Although no overall annual differences were observed between species with respect to median levels of adipogranular (ADG) tissue and reproductive status, specific differences in reproductive status were observed within individual months. During these months (August to October), M. edulis exhibited a relatively lower reproductive status compared to M. galloprovincialis and hybrids. With respect to all remaining health parameters (pathogens, inflammatory and non-specific pathology), principal components analysis revealed no overall differences between species throughout the year. However, greater differences were observed between species during the autumn and winter than during the spring and summer, thus indicating that species differences may be exacerbated by season. This study highlights how species can affect the accurate interpretation of histopathology data collected during biological effects monitoring programmes. Whether species can also affect the biomarker response of Mytilus mussels to contaminated environments remains to be shown. The results are discussed in the context of biological effects monitoring utilising mussels.

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) is a devastating virus of carp. Using generic primers for the DNA polymerase and the major capsid protein genes of cyprinid herpesviruses, nucleotide sequences divergent from previously described CyHV-3 were obtained. At least 3 novel groups of putative CyHV-3-like viruses were identified, sharing 95 to 98% nucleotide identity with CyHV-3 strains. Carp carrying the CyHV-3 variants did not show clinical signs consistent with CyHV-3 infection and originated from locations with no actual CyHV-3 outbreaks. These strains might represent low-or non-pathogenic variants of CyHV-3.KEY WORDS: Carp · Alloherpesviridae · Phylogeny · CyHV-3 · Variant strains Resale or republication not permitted without written consent of the publisherDis Aquat Org 107: [113][114][115][116][117][118][119][120] 2013 third member of the genus Cyprinivirus, the haema topoietic necrosis herpes virus of goldfish or cyprinid herpesvirus 2 (CyHV-2), which specifically infects goldfish Carassius auratus (Jung & Miyazaki 1995, Davison et al. 2013.The complete genome sequences of 3 CyHV-3 isolates (KHV-U, KHV-I and TUMST1) originating from different geographical locations show only limited differences among each other (Aoki et al. 2007) and share more than 99% DNA sequence identity over the complete genomes (Avarre et al. 2011). Recent papers have described some degree of genetic variability between CyHV-3 isolates based upon nucleotide polymorphisms in specific regions of the genome and variation in the variable number of tandem repeats (VNTRs; Bigarré et al. 2009, Kurita et al. 2009, Avarre et al. 2011. In general, the variability between CyHV-3 strains, particularly in the welldefined open reading frames, is limited, whereas the VNTRs could prove to be valuable markers for epidemiological studies aimed at identifying the source of disease outbreaks.In the present study, novel CyHV DNA sequences were detected in carp and koi tissues by application of generic PCR primers capable of detecting a wide range of cyprinid herpesviruses. The CyHV sequences suggest that infections have occurred with viruses closely related to CyHV-3 but yet genetically distinguishable. A preliminary molecular characterisation of the novel cyprinid herpesviruses was carried out, and the sequences were compared with other herpesviruses from fish. MATERIALS AND METHODSThe material used in this study was taken as part of a surveillance programme or routine diagnostic testing of diseased carp and koi in the UK, Austria, Italy and the Netherlands from 2006 to 2012. Gill and kidney tissues were sampled from common carp/koi carp and stored in absolute ethanol. Total DNA was extracted from approximately 20 mg of tissue using the QIAamp DNA Mini Kit (Qiagen) or 50 µl of a clarified 10% tissue homogenate prepared for virus culture using the QIAamp virus mini Kit (Qiagen) following the manufacturer's protocol. The DNA was eluted in a 50 and a 60 µl volume, respectively. Purified DNA samples were stored at −20°C until...

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.

Viral haemorrhagic septicaemia (VHS), caused by the novirhabdovirus VHSV, often leads to significant economic losses to European rainbow trout production. The virus isolates are divided into 4 distinct genotypes with additional subgroups including sublineage Ia, isolates of which are the main source of outbreaks in European rainbow trout farming. A significant portion of Danish rainbow trout farms have been considered endemically infected with VHSV since the first disease outbreak was observed in the 1950s. However, following a series of sanitary programs starting in 1965, VHSV has not been detected in Denmark since January 2009. Full-length Ggenes of all Danish VHSV isolates that were submitted for diagnostic analyses in the period 2004−2009 were sequenced and analysed. All 58 Danish isolates from rainbow trout grouped with sublineage Ia isolates. Furthermore, VHSV isolates from infected Danish freshwater catchments appear to have evolved into a distinct clade within sublineage Ia, herein designated clade Ia-1, whereas trout isolates originating from other continental European countries cluster in another distinct clade, designated clade Ia-2. In addition, phylogenetic analyses indicate that VHSV Ia-1 strains have caused a few outbreaks in Germany and the UK. It is likely that viruses have been transmitted from infected site(s) out of the Danish environment, although a direct transmission pathway has not been identified. Furthermore, VHSV Ia-2 isolates seem to have been transmitted to Denmark at least once. Interestingly, one viral isolate possibly persisted in a Danish watershed for nearly 4 yr without detection whereas other subclades of VHSV isolates appear to have been eliminated, probably because of implemented eradication procedures.

Market-sized rainbow trout Oncorhynchus mykiss were challenged by waterborne exposure to viral haemorrhagic septicaemia virus (VHSV isolate of genogroup Ia). Fish were sampled at 4 stages of infection (before onset of clinical signs, clinically affected fish, mortalities and survivors) and the viral load determined in (1) internal organs, (2) muscle tissue and (3) brain and gill tissue. Virus levels were determined by virus titration and real-time RT-PCR. VHSV was detected by either method in the majority of fish before onset of clinical signs and in the survivor group as well as in all fish in the clinically affected fish and mortality groups. Mean virus amounts per mg of tissue determined by virus titration (TCID 50 ) or real-time RT-PCR (copy number) were >10 4 in preclinical fish, >10 3.8 in clinically affected fish, >10 3.9 in mortalities and >10 1.2 in survivors. Virus levels tended to be highest in the internal organs of subclinical and clinically affected fish and in brain and gill tissue of survivors. The results demonstrate that significant levels of VHSV can be found in tissues of rainbow trout that may be marketed for human consumption, which may have relevance for the biosecurity of VHS-free areas.