In this paper, we combine field observations of highly statistically significant co-occurrence with histopathological, ultrastructural and molecular phylogenetic analyses, to provide evidence for extreme morphological plasticity in a microsporidium parasite infecting the musculature of marine crabs. The parasite appears to alternate between lineages that culminate in production of either bizarre needle-like spores in the peripheral sarcoplasm of heart and skeletal muscle fibres (reminiscent of Nadelspora canceri infecting Cancer magister) or alternatively, Ameson-like spores with pronounced surface projections, in the skeletal muscles (as for Ameson pulvis, previously described infecting Carcinus maenas). Both lineages occur in direct contact with the cytoplasm of host muscle cells and can exist simultaneously within the same cell. Pathological data appears to reveal a remarkable shift in morphology during pathogenic remodelling of host tissues. Sequence analysis of multiple clones derived from amplification of the ssrRNA gene from infected regions of the heart and skeletal muscles appear to confirm the genetic identity of the two lineages. Furthermore, derived ssrRNA gene sequences are more similar (>99%) to N. canceri than to the coparasite Ameson michaelis infecting Callinectes sapidus (93%). Although molecular phylogenetic data support transfer of A. pulvis into the genus Nadelspora, the expansion in the generic description required to include such widely divergent characteristics is so significant as to be unfeasible within the current taxonomic framework of the phylum Microsporidia. At present, it is preferable to propose that the parasite infecting C. maenas forms a clade with other morphologically diverse but phylogenetically and ecologically similar muscle-infecting microsporidians from marine crustacean hosts. Given the strong evidence for significant plasticity in morphology amongst members of the phylum Microsporidia, novel approaches to phylogeny, based predominantly upon the informed use of molecular sequence data, are now deemed a necessity.

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.

A PCR protocol (McIntosh et al., 1996. Appl. Environ. Microbiol. 62, 3929-2932), designed to detect the P57 gene of Renibacterium salmoninarum, causative agent of Bacterial Kidney Disease, was modified slightly by redesign of the forward primer and recommended conditions to prevent possible false positives observed when tested against pure cultures of Yersinia ruckeri. The modified PCR, in combination with an improved DNAzol (TM)-based DNA extraction technique, was very specific and sensitive (detecting between 5 and 72 R. salmoninarum cfu per mg head kidney tissue). It could detect infected fish using only 50 mg head kidney material, making the technique Suitable for sampling small fish. It was also shown that samples Could be pooled from up to five rainbow trout prior to PCR without noticeably affecting the sensitivity of the assay, providing at least 50 mg head kidney tissue was included from each of the fish that were pooled. Crown Copyright (c) 2008 Published by Elsevier B.V. All rights reserved.

A cross-sectional survey of Renibacterium salmoninarum infection in farmed rainbow trout (RBT) and wild fish populations was carried out in 10 farms and six river catchments, respectively, in England and Wales. The majority of the wild fish were sampled in 1998 and the farmed fish in 2000. Grayling, Thymallus thymallus, and brown trout, Salmo trutta, were the main wild species sampled. Two fish, one grayling and one salmon, Salmo salar, were R. salmoninarum culture-positive, compared with 40 confirmed polymerase chain reaction-positive wild fish. The highest prevalence of R. salmoninarum infection was found in grayling in rivers with RBT farms with a history of R. salmoninarum infection. One hundred and fifty fish were sampled from each RBT farm, but none of the fish was found to be R. salmoninarum-positive. Evidence was found, for the first time, for the presence of R. salmoninarum in an eel, Anguilla anguilla.

Tissue samples from 150 rainbow trout (Oncorhynchus mykiss) from a commercial fish farm in southern England were tested for Renibacterium salmoninarum in each of two consecutive years. Head-kidney samples from each fish were swabbed onto each of five culture media (KDM-2, KDM-C, MHC, S-KDM, and Ten-M) to compare their ability to culture salmoninarum from naturally infected fish. Renibacterium salmoninarum was isolated from 80 of the 300 fish. The percentage of positives identified using each medium varied: 80% on Ten-M; 42.5% on S-KDM; 17.5% on KDM-C; 10% on KDM; and 5% on MHC. The study was extended in the second year to compare sampling locations in the kidney. Swabs from the head-kidney and anterior-kidney of the 150 fish were plated onto S-KDM. Renibacterium salmoninarum was isolated from the anterior-kidney of only 3 fish compared to 20 from the head-kidney.

Between 1995 and 1998, marine fish from around the coast of the UK were collected and samples analysed for viral haemorrhagic septicaemia virus (VHSV) using cell culture isolation methods. In 1997 and 1998 the samples were also analysed for VHSV by reverse transcription PCR (RT-PCR). A total of 1867 fish of 11 species were tested, but VHSV was isolated on only 1 occasion, from herring Clupea harengus, in 1996. However, despite VHSV not being isolated in 1997 and 1998, in both years samples of herring from the west and south coasts of England produced positive signals in the RT-PCR, and in 1997 cod from the east coast of England also produced positive signals in the RT-PCR. These results are believed to be true indications of the presence of VHSV nucleic acid in the fish. In 1997, birnaviruses from Serogroup B1 were isolated from herring (a previously unrecorded host for the virus) and cod Gadus morhua, and a birnavirus from Serogroup A2 was also isolated from cod. In 1998, an aquareovirus was isolated from haddock Melanogrammus aeglefinus, a previously unrecorded host for the virus.

ABSTRACT A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS 994 . Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS 994 profiles.