The phylogeography of salmonid proliferative kidney disease in Europe and North America

Salmonid proliferative kidney disease (PKD) is caused by the myxozoan Tetracapsuloides bryosalmonae. Given the serious and apparently growing impact of PKD on farmed and wild salmonids, we undertook a phylogeographic study to gain insights into the history of genealogical lineages of T. bryosalmonae in Europe and North America, and to determine if the global expansion of rainbow trout farming has spread the disease. Phylogenetic analyses of internal transcribed spacer 1 sequences revealed a clade composed of all North American sequences plus a subset of Italian and French sequences. High genetic diversity in North America and the absence of genotypes diagnostic of the North American clade in the rest of Europe imply that southern Europe was colonized by immigration from North America; however, sequence divergence suggests that this colonization substantially pre-dated fisheries activities. Furthermore, the lack of southern European lineages in the rest of Europe, despite widespread rainbow trout farming, indicates that T. bryosalmonae is not transported through fisheries activities. This result strikingly contrasts with the commonness of fisheries-related introductions of other pathogens and parasites and indicates that fishes may be dead-end hosts. Our results also demonstrate that European strains of T. bryosalmonae infect and induce PKD in rainbow trout introduced to Europe.

Investigation of the mechanism of agonist and inverse agonist action at D-2 dopamine receptors

This study investigated, for the D-2 dopamine receptor, the relation between the ability of agonists and inverse agonists to stabilise different states of the receptor and their relative efficacies. K-i values for agonists were determined in competition, versus the binding of the antagonist [H-3]spiperone. Competition data were fitted best by a two-binding site model (with the exception of bromocriptine, for which a one-binding site model provided the best fit) and agonist affinities for the higher (K-h) (G protein-coupled) and lower affinity (K-l) (G protein-uncoupled) sites determined. Ki values for agonists were also determined in competition versus the binding of the agonist [H-3]N-propylnorapomorphine (NPA) to provide a second estimate of K-h,. Maximal agonist effects (E-max) and their potencies (EC50) were determined from concentration-response curves for agonist stimulation of guanosine-5'-O-(3-[S-32] thiotriphosphate) ([S-35]GTPgammaS) binding. The ability of agonists to stabilise the G protein-coupled state of the receptor (K-l/K-h, determined from ligand-binding assays) did not correlate with either of two measures of relative efficacy (relative E-max, Kl/EC50) of agonists determined in [S-35]GTPgammaS-binding assays, when the data for all of the compounds tested were analysed For a subset of compounds, however, there was a relation between K-l/K-h and E-max.. Competition-binding data versus [H-3]spiperone and [H-3]NPA for a range of inverse agonists were fitted best by a one-binding site model. K-i values for the inverse agonists tested were slightly lower in competition versus [H-3]NPA compared to [H-3]spiperone. These data do not provide support for the idea that inverse agonists act by binding preferentially to the ground state of the receptor. (C) 2004 Elsevier Inc. All rights reserved.

Quantitative and qualitative differences in morphological traits revealed between diploid Fragaria species

Background and Aims: The aims of this investigation were to highlight the qualitative and quantitative diversity apparent between nine diploid Fragaria species and produce interspecific populations segregating for a large number of morphological characters suitable for quantitative trait loci analysis. Methods: A qualitative comparison of eight described diploid Fragaria species was performed and measurements were taken of 23 morphological traits from 19 accessions including eight described species and one previously undescribed species. A principal components analysis was performed on 14 mathematically unrelated traits from these accessions, which partitioned the species accessions into distinct morphological groups. Interspecific crosses were performed with accessions of species that displayed significant quantitative divergence and, from these, populations that should segregate for a range of quantitative traits were raised. Key Results: Significant differences between species were observed for all 23 morphological traits quantified and three distinct groups of species accessions were observed after the principal components analysis. Interspecific crosses were performed between these groups, and F2 and backcross populations were raised that should segregate for a range of morphological characters. In addition, the study highlighted a number of distinctive morphological characters in many of the species studied. Conclusions: Diploid Fragaria species are morphologically diverse, yet remain highly interfertile, making the group an ideal model for the study of the genetic basis of phenotypic differences between species through map-based investigation using quantitative trait loci. The segregating interspecific populations raised will be ideal for such investigations and could also provide insights into the nature and extent of genome evolution within this group.

Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers

To study the potential involvement of inhibin A (inhA), inhibin B (inhB), activin A (actA) and follistatin (FS) in the recruitment of follicles into the preovulatory hierarchy, growing follicles (ranging from 1 mm to the largest designated F1) and the three most recent postovulatory follicles (POFs) were recovered from laying hens (n=11). With the exception of <4 mm follicles and POFs, follicle walls were dissected into separate granulosa (G) and theca (T) layers before extraction. Contents of inhA, inhB, actA and FS in tissue extracts were assayed using specific two-site ELISAs and results are expressed per mg DNA. InhB content of both G and T followed a similar developmental pattern, although the content was >4-fold higher in G than in T at all stages. InhB content was very low in follicles <4 nun but increased - 50-fold (P<0.0001) to peak in 7-9 mm follicles, before falling steadily as follicles entered and moved up the follicular hierarchy (40-fold; 8 mm vs F2). In stark contrast, inhA remained very low in prehierarchical follicles (&LE; 9 mm) but then increased progressively as follicles moved up the preovulatory hierarchy to peak in F1 (&SIM; 100-fold increase; P<0.0001); In F1 >97% of inhA was confined to the G layer whereas in 5-9 mm follicles inhA was only. detected in the T layer. Both inhA and inhB contents of POFs were significantly reduced compared with F1. Follicular actA was mainly confined to the T layer although detectable levels were present in G from 9 nun; actA was low between 1 and 9 mm but increased sharply as follicles entered the preovulatory hierarchy (&SIM;6-fold higher in F4; P<0.0001); levels then fell &SIM;2-fold as the follicle progressed to F1. Like actA, FS predominated in the T although significant amounts were also present in the G of prehierarchical follicles (4-9 mm), in contrast to actA, which was absent from the G. The FS content of T rose &SIM;3-fold from 6 mm to a plateau which was sustained until F1. In contrast, the FS content of G was greatest in prehierarchical follicles and fell &SIM;4-fold in F4-F1 follicles. ActA and FS contents of POFs were reduced compared with F1. In vitro studies on follicle wall explants confirmed the striking divergence in the secretion of inhA and inhB during follicle development. These findings of marked stage-dependent differences in the expression of inhA, inhB, actA and FS proteins imply a significant functional role for these peptides in the recruitment and ordered progression of follicles within the avian ovary.

Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria

Identification of Fusarium species has always been difficult due to confusing phenotypic classification systems. We have developed a fluorescent-based polymerase chain reaction assay that allows for rapid and reliable identification of five toxigenic and pathogenic Fusarium species. The species includes Fusarium avenaceum, F. culmorum, F. equiseti, F. oxysporum and F. sambucinum. The method is based on the PCR amplification of species-specific DNA fragments using fluorescent oligonucleotide primers, which were designed based on sequence divergence within the internal transcribed spacer region of nuclear ribosomal DNA. Besides providing an accurate, reliable, and quick diagnosis of these Fusaria, another advantage with this method is that it reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of fluorescent dyes in place of ethidium, bromide. Apart from its multidisciplinary importance and usefulness, it also obviates the need for gel electrophoresis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Bacteroides coprosuis sp nov., isolated from swine-manure storage pits

Two Gram-negative, anaerobic, non-spore-forming, rod-shaped organisms were isolated from a swine-manure storage pit. Based on morphological and biochemical criteria, the strains were tentatively identified as belonging to the genus Bacteroides but they did not appear to correspond to any recognized species of the genus. Comparative 16S rRNA gene sequencing studies showed that the strains were related closely to each other and confirmed their placement in the genus Bacteroides, but sequence divergence values of > 10% from reference Bacteroides species demonstrated that the organisms from manure represent a novel species. Based on biochemical criteria and molecular genetic evidence, it is proposed that the unknown isolates from manure be assigned to a novel species of the genus Bacteroides, as Bacteroides coprosuis sp. nov. The type strain is PC139(T) (=CCUG 50528(T)=NRRL B-41113(T)).

Clostridium bolteae sp. nov., isolated from human sources

Seven obligately anaerobic, gram-positive, rod-shaped, spore-forming organisms isolated from human sources were characterized using phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed that the strains were genetically highly related to each other (displaying >99% sequence similarity) and represent a previously unknown sub-line within the Clostridium coccoides rRNA group of organisms. Strains of the unidentified bacterium used carbohydrate as fermentable substrates, producing acetic acid and lactic acid as the major products of glucose metabolism. The closest described species to the novel bacterium corresponded to Clostridium clostridioforme, although a 16S rRNA sequence divergence of 3% demonstrated they represent different species. Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and Clostridium clostridioforme. Based on phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium, be classified as Clostridium bolteae sp. nov. The type strain of Clostridium bolteae is WAL 16351(T) (= ATCC(T) = BAA-613(T), CCUG(T) = 46953(T)).

Reclassification of Bacteroides putredinis (Weinberg et al., 1937) in a New Genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp nov., from human sources

During studies on the bacteriology of appendicitis in children, we often isolated from inflamed and non-inflamed tissue samples, an unusual bile-resistant pigment-producing strictly anaerobic gram-negative rod. Phenotypically this organism resembles members of Bacteroides fragilis group of species, as it is resistant to bile and exhibits a special-potency-disk pattern (resistance to vancomycin, kanamycin and colistin) typical for the B. fragilis group. However, the production of brown pigment on media containing haemolysed blood and a cellular fatty acid composition dominated by iso-C15:0, suggests that the organism most closely resembles species of the genus Porphyromonas. However, the unidentified organism differs from porphyromonads by being bile-resistant and by not producing butyrate as a metabolic end-product. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism represents a distinct sub-line, associated with but distinct from, the miss-classified species Bacteroides putredinis. The clustering of the unidentified bacterium with Bacteroides putredinis was statistically significant, but they displayed >4% sequence divergence with each other. Chromosomal DNA-DNA pairing studies further confirmed the separateness of the unidentified bacterium and Bacteroides putredinis. Based on phenotypic and phylogenetic considerations, it is proposed that Bacteroides putredinis and the unidentified bacterium from human sources be classified in a new genus Alistipes, as Alistipes putredinis comb. nov. and Alistipes finegoldii sp. nov., respectively. The type strain of Alistipes finegoldii is CCUG 46020(T) (= AHN2437(T)).

Streptococcus castoreus sp nov., isolated from a beaver (Castor fiber)

A previously undescribed, Gram-positive, catalase-negative, Streptococcus-like organism originating from a European beaver (Castor fiber) was subjected to a taxonomic study. The organism displayed beta-haemolytic activity and gave a positive reaction with Lancefield group A antisera. Based on the results of biochemical testing, the organism was tentatively identified as a member of the genus Streptococcus, but it did not correspond phenotypically to any recognized species of this genus. Comparative 16S rRNA gene sequencing studies confirmed this assignment, with the bacterium forming a hitherto unknown subline within the genus. Sequence divergence values of greater than 3% from other reference streptococcal species, however, demonstrated that the unidentified coccus-shaped organism represents a hitherto unknown species. Based on phenotypic and molecular phylogenetic evidence, it is therefore proposed that the unknown organism from a beaver be classified as a novel species, Streptococcus castoreus sp. nov. The type strain is M605815/03/2(T) (=CCUG 48115(T) = CIP 108205(T)).

Streptococcus halichoeri sp nov., isolated from grey seals (Halichoerus grypus)

Phenotypic and phylogenetic studies were performed on six unidentified, Gram-positive, catalase-negative, chain-forming Streptococcus-like organisms recovered from grey seals. Biochemically the six strains were highly related to each other, but they did not appear to correspond to any recognized species of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies confirmed that phylogenetically the strains were members of the genus Streptococcus, but sequence divergence values of greater than 3 % compared with reference streptococcal species demonstrated that the organisms from seals represent a novel species. SDS-PAGE analysis of whole-cell proteins confirmed the phenotypic distinctiveness of the seal organisms. Based on biochemical criteria and molecular chemical and genetic evidence, it is proposed that the unknown organism from seals be classified as a novel species, Streptococcus halichoeri sp. nov., the type strain of which is CCUG 48324(T) (=CIP 108195(T)).

Streptococcus marimammalium sp nov., isolated from seals

Two strains of an unidentified, Gram-positive, catalase-negative, chain-forming, coccus-shaped organism recovered from seals were characterized using phenotypic and molecular taxonomic methods. Based on morphological and biochemical criteria the strains were tentatively identified as streptococci but they did not appear to correspond to any recognized species of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies showed that the strains were closely related to each other and confirmed their placement in the genus Streptococcus. Sequence divergence values of > 5 % with reference streptococcal species demonstrated the organisms from seals represent a novel species. SDS-PAGE analysis of whole-cell proteins confirmed that the two organisms were closely related to each other but were different from all currently defined streptococcal species. Based on biochemical criteria, molecular chemical and molecular genetic evidence, it is proposed that the unknown isolates from seals be assigned to a novel species of the genus Streptococcus, Streptococcus marimammalium sp. nov. The type strain is M54/01/(T) (=CCUG 48494(T)=CIP 108309(T)).

Jeotgalicoccus pinnipedialis sp nov., from a southern elephant seal (Mirounga leonina)

A previously unknown Gram-positive, catalase-positive, facultatively anaerobic, non-spore-forming, coccus-shaped bacterium (A/G14/99/10(T)), originating from the mouth of a female southern elephant seal, was subjected to a taxonomic analysis. Comparative 16S rRNA gene-sequencing showed that the organism formed a hitherto unknown subline within the catalase-positive, low-G+C, Gram-positive cocci, exhibiting a specific association with species of the genus Jeotgalicoccus. Sequence divergence values of approximately 7%, together with phenotypic differences, showed the unknown bacterium to be distinct from the two described species of this genus, Jeotgalicoccus halotolerans and Jeotgalicoccus psychrophilus. Based on phenotypic and phylogenetic considerations, it is proposed that strain A/G14/99/10(T)=CCUG 42722(T)=CIP 107946(T) from the mouth of a seal be classified as the type strain of a novel species of the genus Jeotgalicoccus, Jeotgalicoccus pinnipedialis sp. nov.

Subdoligranulum variabile gen. nov., sp nov from human feces

During studies on the microflora of human feces we have isolated a strictly anaerobic, non-spore-forming, Gram-negative staining organism which exhibits a somewhat variable coccus-shaped morphology. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism is phylogenetically a member of the Clostridium leptum supra-generic rRNA cluster and displays a close affinity to some rDNA clones derived from human and pig feces. The nearest named relatives of the unidentified isolate corresponded to Faecalibacterium prausnitzii (formerly Fusobacterium prausnitzii) displaying a 16S rRNA sequence divergence of approximately 9%, with Anaerofilum agile and A. pentosovorans the next closest relatives of the unidentified bacterium (sequence divergence approximately 10%). Based on phenotypic and phylogenetic considerations, it is proposed that the unusual coccoid-shaped organism be classified as a new genus and species, Subdoligranulum variabile. The type strain of S. variabile is BI 114(T) (= CCUG 47106(T) = DSM 15176(T)). (C) 2004 Elsevier Ltd. All rights reserved.

Actinomyces dentalis sp nov., from a human dental abscess

A previously undescribed filamentous, beaded, Gram-positive, rod-shaped bacterium was isolated from pus of a human dental abscess. Based on its cellular morphology end the results of biochemical testing the organism was tentatively identified as a member of the genus Actinomyces, but it did not correspond to any currently recognized species of this genus. Comparative 16S rRNA gene sequencing studies showed the bacterium represents a distinct subline within the genus Actinomyces, clustering within a group of species that includes Actinomyces bovis, the type species of the genus. Sequence divergence values of >8% with other recognized species within this phylogenetic group clearly demonstrated that the organism represents a hitherto unknown species. Based on biochemical and molecular phylogenetic evidence, it is proposed that the unidentified organism recovered from a dental abscess be classified as a novel species, Actinomyces dentalis sp. nov. The type strain is R18165(T) (= CCUG 48064(T) = CIP 108337(T)).

Actinomyces nasicola sp. nov., isolated from a human nose

A previously undescribed facultatively anaerobic, catalase-negative, Actinomyces-like bacterium was isolated from the nose of a human. On the basis of its cellular morphology and the results of biochemical testing, the micro-organism was tentatively identified as a member of the genus Actinomyces, but it did not correspond to any currently recognized species. Comparative 16S rRNA gene sequencing studies showed the bacterium to be a hitherto unknown subline within the genus Actinomyces, displaying sequence divergence values of more than 6% with respect to recognized species of the genus. On the basis of biochemical, molecular chemical and molecular phylogenetic evidence, it is proposed that the unknown organism, strain R2014(T) (=CCUG 46092(T)=CIP 107668(T)), be classified as the type strain of a novel species, Actinomyces nasicola sp. nov.