Encephalomyelitis associated with microsporidian infection in farmed greater amberjack, Seriola dumerili (Risso).

An outbreak of a disease characterized by a peculiar spiral movement in farmed greater amberjack, Seriola dumerili (Risso), occurred in Kagoshima Prefecture, Japan, in May 2008, immediately after importing the fish from China. Although neither bacteria nor viruses were detected in routine diagnostic tests, histopathological observations of the affected fish revealed severe inflammation in the tegmentum of the brain including the medulla oblongata and the anterior part of the spinal cord. In addition, a microsporidian parasite was observed in the nerve cell bodies or axons in the inflamed tissues. We identified a microsporidian small subunit rRNA gene (SSU rDNA) from the lesion, and the sequence showed 96.1% identity with that of Spraguea lophii. Subsequent in situ hybridization using probes presumably specific to the SSU rRNA confirmed that the parasite observed in histopathology harboured the identified SSU rRNA. Apparently degenerated microsporidian cells or spores were also frequently observed in tissue sections. Thus, the disease was most probably caused by the infection of a hitherto unknown microsporidian parasite that has a genetic affinity to the genus Spraguea, in the central nervous system of the amberjack.

Francisella halioticida sp. nov., a pathogen of farmed giant abalone (Haliotis gigantea) in Japan.

AIMS: In 2005, a Francisella sp. was isolated from diseased cultured giant abalone (Haliotis gigantea) in Japan. The aim of this study was to clarify the taxonomic status of this Francisella sp. Shimane-1 isolate in relation to the four described Francisella species. METHODS AND RESULTS: The 16S rRNA gene and several housekeeping genes of the Shimane-1 were compared to isolates of the four recognized species within the Francisella genus. DNA-DNA hybridization (DDH) and biochemical profile comparison were performed with the two phylogenetically closely related species, Francisella philomiragia and Francisella noatunensis. Results show that the Shimane-1 is genetically different from all described Francisella species and differs phenotypically from F. philomiragia and F. noatunensis. The average DDH similarity of Francisella sp. Shimane-1 to F. noatunensis ssp. noatunensis (NCIMB14265(T)) and to F. philomiragia (DSM7535(T)) was 49·2 and 61%, respectably, clearly supporting the establishment of Shimane-1 as a new species within the Francisella genus. CONCLUSIONS: The phenotypic and genetic results presented in this study suggest the establishment of Shimane-1 as a novel species, for which the name Francisella halioticida sp. nov. (=LMG26062(T), =DSM23729(T)) is proposed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study clarifies the taxonomic position and characteristics of a novel mollusc pathogenic Francisella species.

Elevation of Francisella philomiragia subsp. noatunensis Mikalsen et al. (2007) to Francisella noatunensis comb. nov. [syn. Francisella piscicida Ottem et al. (2008) syn. nov.] and characterization of Francisella noatunensis subsp. orientalis subsp. nov., two important fish pathogens.

AIMS: This study was conducted to clarify the taxonomic status of Francisella sp. strain Ehime-1, a fish pathogen, in relation to the fish pathogens F. piscicida and F. philomiragia subsp. noatunensis and to F. philomiragia subsp. philomiragia. METHODS AND RESULTS: Francisella sp. Ehime-1 was compared to F. piscicida, F. philomiragia subsp. noatunensis and several F. philomiragia subsp. philomiragia isolates through sequencing of the 16S rRNA-gene and several house-keeping genes and determination of biochemical and phenotypic properties. Results show that F. piscicida is indistinguishable from F. philomiragia subsp. noatunensis by sequence and phenotypic traits. Francisella sp. Ehime-1 and F. philomiragia subsp. noatunensis are clearly separated from F. philomiragia. Francisella sp. Ehime-1 is biochemically, phenotypically and genetically different from F. philomiragia subsp. noatunensis (=F. piscicida), but DNA-DNA hybridization does not clearly support establishment as a separate species (level of relatedness 64% and 73.4%, mean 68.7%). CONCLUSIONS: We propose to elevate F. philomiragia subsp. noatunensis to species rank as F. noatunensis comb. nov., while F. piscicida is considered a heterotypic synonym of F. noatunensis comb. nov. Evidence suggests that Francisella sp. Ehime-1 represents a novel subspecies of F. noatunensis, for which the name F. noatunensis subsp. orientalis subsp. nov. is proposed (=DSM21254(T), = LMG24544(T)). SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the taxonomy and characteristics of fish-pathogenic Francisella spp.

Histopathology of Antarctic krill, Euphausia superba, bearing black spots.

Small black spots have been noticed on the cephalothorax of Antarctic krill, Euphausia superba, since January, 2001. To study the nature of the black spots, the krill were sampled in the winter of 2003, 2006, and 2007 in the South Georgia region, the Antarctic Ocean. Histological observations revealed that the black spots were melanized nodules that were composed of hemocytes surrounding either bacteria or amorphous material. In the 2007 samples, 42% of the krill had melanized nodules. Most of the nodules had an opening on the body surface of the krill. A single melanized nodule often contained more than one type of morphologically distinct bacterial cell. Three bacteria were isolated from these black spots, and classified into either Psychrobacter or Pseudoalteromonas based on the sequences of 16S rRNA genes. More than three bacterial species or strains were also confirmed by in situ hybridization for 16S rRNA. The melanized nodules were almost always accompanied by a mass of atypical, large heteromorphic cells, which were not observed in apparently healthy krill. Unidentified parasites were observed in some of the krill that had melanized nodules. These parasites were directly surrounded by the large heteromorphic cells. Histological observations suggested that these heteromorphic cells were attacking the parasites. These results suggest the possibility that the krill had been initially affected by parasite infections, and the parasitized spots were secondary infected by environmental bacteria after the parasites had escaped from the host body.

Expression of YAV proteins and vaccination against viral ascites among cultured juvenile yellowtail.

Yellowtail ascites virus (YAV) is a member of the family Birnaviridae and causes viral ascites among juvenile yellowtail (Seriola quinqueradiata). We have reported the cloning and expression of two viral cDNAs, the first being segment A encoding a polyprotein of viral capsid proteins (VP2 and VP3) and a protease (NS), and the second being VP2-epitope encoding serotype-specific epitope region on VP2, using a baculovirus expression system. Another viral cDNA encoding a polyprotein of NS and VP3 was cloned and expressed in this study. For the expression of NS/VP3 (YAV nt 1626 to 3066) in insect cells a 31-kDa protein, corresponding to VP3 was detected, indicating an appropriate posttranslational processing of NS/VP3 polypeptide by NS protease itself. The analysis of the N-terminal amino acid sequence of this protein showed that NS protease may cleave an Ala-Ser bond. A study of the potential for vaccination of yellowtail fry by injection of insect cell lysates infected with baculovirus, containing either cDNA of segment A, VP2-epitope, or NS/VP3 was undertaken. Only a vaccination with cell lysates infected with a recombinant virus carrying the full length of YAV segment A gene demonstrated approximately the same effect as that of inactivated YAV. This result suggested that all proteins VP2, VP3, and NS are required for an effective vaccination.

Complete nucleotide sequences of the genes encoding translation elongation factors 1 alpha and 2 from a microsporidian parasite, Glugea plecoglossi: implications for the deepest branching of eukaryotes.

Complete nucleotide sequences of the genes putatively encoding translation elongation factors 1 alpha (EF-1 alpha) and 2 (EF-2) from a mitochondrion-lacking protozoan, Glugea plecoglossi, that belongs to microsporidians were determined. The deduced amino acid sequences of the putative EF-1 alpha and EF-2 of Gl. plecoglossi showed very unusual features compared with typical eukaryotic sequences. The degree of divergence was especially great in the EF-1 alpha sequence, although it clearly showed a eukaryotic feature when aligned with homologs from the three primary kingdoms. Phylogenetic analyses of EF-1 alpha and EF-2 on the basis of the maximum likelihood method of protein phylogeny clearly and consistently suggested that among eukaryotic species being analyzed, Gl. plecoglossi and another mitochondrion-lacking protozoan, Giardia lamblia, respectively represent the earliest and the second earliest offshoots of eukaryotes. When the EF-1 alpha and EF-2 phylogenies were totally evaluated, the earliest divergence of Gl. plecoglossi in eukaryotes became more clearly confirmed. If the phylogenetic relationship inferred from the present analysis are correct, microsporidians might be extremely ancient eukaryotes that diverged before the occurrence of mitochondrial symbiosis.

Protein phylogeny of translation elongation factor EF-1 alpha suggests microsporidians are extremely ancient eukaryotes.

Partial regions of the mRNA encoding a major part of translation elongation factor 1 alpha (EF-1 alpha) from a mitochondrion-lacking protozoan, Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1 alpha's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed, G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis.

Phylogenetic position of kinetoplastid protozoa inferred from the protein phylogenies of elongation factors 1alpha and 2.

Partial regions of the mRNA encoding a major part of translation elongation factor 2 (EF-2) from a kinetoplastid protozoan, Trypanosoma cruzi, were amplified by means of polymerase chain reaction and their primary structures were analyzed. The deduced amino acid sequence was aligned with those of other eukaryotic and archaebacterial EF-2s, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) method. ML analyses of EF-2 phylogeny using six different stochastic models of amino acid substitutions consistently suggested that the phylogenetic position of T. cruzi is likely to be closer to higher eukaryotes than that inferred from the phylogeny of small subunit ribosomal RNA (SrRNA). These results are consistent with those for the elongation factor 1alpha (EF-1alpha) phylogeny. When the EF-1alpha and EF-2 phylogenies were totally evaluated, it became much clearer that the divergence of T. cruzi occurred later than that of a mitochondrion-lacking protozoan, Entamoeba histolytica, although this is not conclusive.

Phylogenetic place of mitochondrion-lacking protozoan, Giardia lamblia, inferred from amino acid sequences of elongation factor 2.

Partial regions of the mRNA encoding a major part of translation elongation factor 2 (EF-2) from a mitochondrion-lacking protozoan, Giardia lamblia, were amplified by polymerase chain reaction, and their primary structures were analyzed. The deduced amino acid sequence was aligned with other eukaryotic and archaebacterial EF-2's, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) and the maximum parsimony (MP) methods. The ML analyses using six different models of amino acid substitutions and the MP analysis consistently suggest that among eukaryotic species being analyzed, G. lamblia is likely to have diverged from other higher eukaryotes on the early phase of eukaryotic evolution.

Phylogenetic place of a mitochondria-lacking protozoan, Entamoeba histolytica, inferred from amino acid sequences of elongation factor 2'.

Partial DNA regions encoding a major part of translation elongation factor 2 (EF-2) from a mitochondria-lacking protozoan, Entamoeba histolytica, were amplified by polymerase chain reaction and their primary structures were analyzed. The deduced amino acid sequence was aligned with other eukaryotic and archaebacterial EF-2's, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) method. The ML analyses using four different stochastic models of amino acid substitutions consistently suggest that among eukaryotic species being analyzed, E. histolytica is likely to have diverged from other higher eukaryotes on the early phase of eukaryotic evolution.