Embryonic development of the grass pufferfish (Takifugu niphobles): From egg to larvae.

Tetraodontidae (pufferfish) family members carry the smallest genomes among vertebrates, and these pocket-sized genomes have directly contributed to our understanding of the structure and evolution of higher animals. The grass pufferfish (Takifugu niphobles) could be considered a potential new model organism for comparative genomics and development due to the potential access to embryos, and availability of sequence data for two similar genomes: that of spotted green pufferfish (Tetraodon nigroviridis) and Fugu (Takifugu rubripes). In this study, we provide the first description of the normal embryonic development of T. niphobles, by drawing comparisons with the closely related species cited above. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored at a constant temperature consistent with natural conditions. T. niphobles development was divided into seven periods of embryogenesis: the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods; and stages subdividing these periods are defined based on morphological characteristics. The developmental stage series described in this study aims to provide the utilization of T. niphobles as an experimental model organism for comparative developmental studies.

Nonrandom cytogenetic changes in New Zealand patients with acute myeloid leukemia.

Bone marrow clones with abnormal chromosomes were observed in 56% of 66 patients with forms of acute myeloid leukemia [French-American-British (FAB) M1-M6]. Acute myeloblastic leukemia (AML, M1 and M2) was the most common form, and 65% of these patients showed chromosomal abnormalities compared with 41% of patients with acute myelomonocytic leukemia (AMMoL, M4). The recognized nonrandom chromosomal abnormalities found were trisomy 8, monosomy 5 or 7, trisomy 1q, t(6;9), t(8;21), t(15;17), and abnormalities in 17q. There was also a strong involvement of chromosome No. 11: Abnormalities were found in eight patients when their leukemia was diagnosed and in a further three patients during the course of karyotypic evolution. Six of these patients had AMMoL or AMoL. Complex or multiple clones were found in 37% of AML patients at diagnosis. Our AML patients had a reduced frequency of abnormalities in chromosome No. 5 or 7 and an increased frequency of abnormalities in chromosome No. 8 compared with studies reported in other countries (p = 0.01). This difference suggests that in New Zealand AML might be caused by factors different from those operating in more industrialized centers.

Local knowledge and rural mental health reform.

Rural mental health care reform has failed to recognize that service utilization and access is guided by the meanings and explanations that rural dwellers ascribe to symptoms and treatments for mental illness. These meanings and explanations are described here as local knowledge. It is argued that planning for reform in rural mental health service delivery must take into account the local knowledge and explanations for mental illness and its treatment in order to improve rural mental health.

Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences.

Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time.

Phylogeny of the Pasteurellaceae as determined by comparison of 16S ribosomal ribonucleic acid sequences.

Previously, virtually complete 16S ribosomal ribonucleic acid sequences were determined for 54 strains of species in the family Pasteurellaceae. The sequences for 16 additional strains have been determined, bringing the total number of strains sequenced to 70. The additional strains include: Actinobacillus hominis, A. muris, A. salpingitis, Pasteurella bettyae, P. mairii, P. testudinis, and Bisgaard taxa 2, 3, 5, 6, 7, 8, 9, 13, and 14 (2 strains). A phylogenetic tree was constructed based upon sequence similarity using the Neighbor-Joining method. The additional sequence information and phylogenetic analysis generally supported our previously described phylogenetic structure for the family Pasteurellaceae. Cluster 1, containing Haemophilus sensu stricto, was unchanged. P. mairii was closely related to P. aerogenes and Bisgaard taxon 6 was related to H. somnus in Cluster 2. A. salpingitidis and Bisgaard taxa 2, 3, 7, and 13 fell in Cluster 3 which contains Pasteurella sensu stricto. A. hominis was closely related to Actinobacillus sensu stricto species in Cluster 4A. Bisgaard taxa 5, 8, 9 and P. bettyae fell in Cluster 4B. A. muris was related to P. pneumotropica in Cluster 5. Haemophilus parainfluenzae strains branched deeply as a 6th cluster. Bisgaard taxon 14 and P. testudinis formed a 7th cluster which branched deeper than any previously described clusters in the family Pasteurellaceae. The branching was extremely complex and taxonomic division of the family into phylogenetically and phenotypically coherent genera will be difficult.

Transient leukemoid proliferation of the cytogenetically unbalanced +21 cell line of a constitutional mosaic boy.

A newborn without any signs of Down's syndrome was found to have an acute proliferation that remitted without drug therapy. Chromosomal analysis of blood, bone marrow, and skin cells revealed that the child was a constitutional mosaic with normal cells and a low number of cells in which one no. 21 chromosome was replaced by a probably isochromosome for the no. 21 long arm: 46,XY/46,XY,i(21q). The abnormal cell line of the mosaic appeared to be selectively involved in this proliferation.

Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria.

The phylogenetic structure of the bacteroides subgroup of the cytophaga-flavobacter-bacteroides (CFB) phylum was examined by 16S rRNA sequence comparative analysis. Approximately 95% of the 16S rRNA sequence was determined for 36 representative strains of species of Prevotella, Bacteroides, and Porphyromonas and related species by a modified Sanger sequencing method. A phylogenetic tree was constructed from a corrected distance matrix by the neighbor-joining method, and the reliability of tree branching was established by bootstrap analysis. The bacteroides subgroup was divided primarily into three major phylogenetic clusters which contained most of the species examined. The first cluster, termed the prevotella cluster, was composed of 16 species of Prevotella, including P. melaninogenica, P. intermedia, P. nigrescens, and the ruminal species P. ruminicola. Two oral species, P. zoogleoformans and P. heparinolytica, which had been recently placed in the genus Prevotella, did not fall within the prevotella cluster. These two species and six species of Bacteroides, including the type species B. fragilis, formed the second cluster, termed the bacteroides cluster. The third cluster, termed the porphyromonas cluster, was divided into two subclusters. The first contained Porphyromonas gingivalis, P. endodontalis, P. asaccharolytica, P. circumdentaria, P. salivosa, [Bacteroides] levii (the brackets around genus are used to indicate that the species does not belong to the genus by the sensu stricto definition), and [Bacteroides] macacae, and the second subcluster contained [Bacteroides] forsythus and [Bacteroides] distasonis. [Bacteroides] splanchnicus fell just outside the three major clusters but still belonged within the bacteroides subgroup. With few exceptions, the 16 S rRNA data were in overall agreement with previously proposed reclassifications of species of Bacteroides, Prevotella, and Porphyromonas. Suggestions are made to accommodate those species which do not fit previous reclassification schemes.

Phylogeny of Helicobacter isolates from bird and swine feces and description of Helicobacter pametensis sp. nov.

Previously, nine fecal isolates from wild birds and a domestic swine were identified as helicobacters by phenotypic characterization and reaction with a helicobacter genus-specific DNA probe. These isolates fell into three biotypes by analysis of phenotypic traits. To further characterize these isolates, full 16S rRNA sequences were determined for strains representing each biotype, and sequence comparison indicated that the strains represented three novel, phylogenetically defined Helicobacter species. Three 16S rRNA-based DNA probes were designed and used to identify the remaining strains. Probe reactivity divided the strains into the same three groups identified phenotypically. Six of the isolates represented a new species of the genus Helicobacter for which we propose the name Helicobacter pametensis sp. nov. The following phenotypic features distinguished H. pametensis from other Helicobacter and Campylobacter species: positive tests for oxidase, catalase, alkaline phosphatase, nitrate reduction, growth at 42 degrees C, and growth in the presence of 1% glycine; negative tests for urease, gamma glutamyl transpeptidase, indoxyl acetate hydrolysis, and hippurate hydrolysis; and susceptibility to nalidixic acid and cephalothin. H. pametensis cells were motile and possessed one subterminal sheathed flagellum at each end. The two additional Helicobacter species were similar to H. pametensis except that they were urease positive, hydrolyzed indoxyl acetate, and were resistant to cephalothin. Because these two additional species are phenotypically similar and are represented by only two isolates for one species and one isolate for the other, they are not formally named but are referred to as Helicobacter sp. "Bird-B" and Helicobacter sp. "Bird-C."(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular Campylobacter-like organism from ferrets and hamsters with proliferative bowel disease is a Desulfovibrio sp.

Proliferative bowel disease is an intestinal disorder of a variety of domestic animals associated with the presence of an intracellular Campylobacter-like organism (ICLO). We have identified the ICLO obtained from a ferret with proliferative colitis by 16S rRNA sequence analysis. In this ferret, proliferative bowel tissue containing the ICLO had translocated to the mesenteric lymph nodes, omentum, and liver. The 16S rRNA genes of the ICLO were amplified from an infected fragment of extraintestinal tissue by using universal prokaryotic primers. Approximately 1,480 bases of the amplified 16S rRNA gene were sequenced by cycle sequencing. Comparison of the sequence of the ICLO with those of over 400 bacteria in our data base indicated that the sequence of the ICLO was most closely related to that of Desulfovibrio desulfuricans (87.5% similarity). Phylogenetic analysis with 12 Desulfovibrio species and 20 species from related genera placed the ICLO in a subcluster within the genus Desulfovibrio with D. desulfuricans and 5 other Desulfovibrio species. We will refer to this organism as the intracellular Desulfovibrio organism (IDO). Specific primers were produced for PCR amplification of a 550-base fragment of the 16S rRNA gene of the IDO in proliferative intestinal tissue samples. This unique 550-base segment was amplified from samples of frozen intestinal tissue from nine ferrets and three hamsters with ICLO-associated disease but not in four intestinal tissue samples from animals without the ICLO-associated disease. The 550-base amplified products from the bowel tissues of one hamster and one ferret were fully sequenced. The ferret IDO partial sequence was identical to the previously determined 16S rRNA sequence over its length, and the hamster IDO sequence differed by a single base. The same intracellular organism has been identified in proliferative intestinal tissues of swine and that the organism has been successfully maintained in tissue culture. The availability of specific primers for PCR-based detection of this intracellular Desulfovibrio organism will aid in the determination of its role in the pathogenesis of proliferative bowel disease in a variety of infected hosts.

Brevinema andersonii gen. nov., sp. nov., an infectious spirochete isolated from the short-tailed shrew (Blarina brevicauda) and the white-footed mouse (Peromyscus leucopus).

A spirochete which infects short-tailed shrews (Blarina brevicauda) and white-footed mice (Peromyscus leucopus) has been shown previously to be ultrastructurally and serologically distinct from other spirochetes. Two of the original isolates from Connecticut and Minnesota and 16 new isolates obtained from shrews captured in Minnesota were characterized phenotypically and genetically in this study. A comparative analysis of the 16S rRNA sequences of two shrew isolates and one mouse isolate and the 16S rRNA sequences of 16 other spirochetes and Escherichia coli revealed that these organisms exhibited low levels of similarity (range of similarity values, 73.9 to 77.8%; average level of similarity, 74.7%). The shrew and mouse isolates which we examined formed a deeply branching subgroup that was clearly distinct from the other genera of spirochetes examined. These and other results indicated that the new spirochetes represent a unique taxon in the order Spirochaetales. Accordingly, we propose that they should be classified as members of a new genus, Brevinema. The three strains of Brevinema which we examined had 16S rRNA sequences that were nearly identical. We also compared these isolates by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fatty acid and enzyme analyses, restriction endonuclease analysis, and Southern hybridization and found that the levels of genetic and phenotypic homogeneity among the strains were very high. We concluded that the isolates which we examined were members of a single species, for which we propose the name Brevinema andersonii. The type strain of Brevinema andersonii is CT11616 (= ATCC 43811).(ABSTRACT TRUNCATED AT 250 WORDS)

Helicobacter pylori isolated from the domestic cat: public health implications.

Helicobacter pylori has been directly linked with active chronic gastritis, peptic ulceration, and gastric adenocarcinoma in humans. Although a substantial portion of the human population is colonized with H. pylori, the patterns of transmission of the organism remain in doubt, and reservoir hosts have not been identified. This study documents the isolation of H. pylori from domestic cats obtained from a commercial vendor. The isolation of H. pylori from these cats was confirmed by morphologic and biochemical evaluations, fatty acid analysis, and 16S rRNA sequence analysis. H. pylori was cultured from 6 cats and organisms compatible in appearance with H. pylori were observed in 15 additional cats by histologic examination. In most animals, H. pylori was present in close proximity to mucosal epithelial cells or in mucus layers of the glandular or surface epithelium. Microscopically, H. pylori-infected cat stomachs contained a mild to severe diffuse lymphoplasmacytic infiltrate with small numbers of neutrophils and eosinophils in the subglandular and gastric mucosae. Lymphoid follicles were also noted, particularly in the antrum, and often displaced glandular mucosal tissue. Thus, the domestic cat may be a potential model for H. pylori disease in humans. Also, the isolation of H. pylori from domestic cats raises the possibility that the organism may be a zoonotic pathogen, with transmission occurring from cats to humans.

Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents.

Helical organisms with novel ultrastructural characteristics were isolated from the intestinal mucosa of rats and mice. These bacteria were characterized by the presence of 9 to 11 periplasmic fibers which appeared as concentric helical ridges on the surface of each cell. The cells were motile with a rapid corkscrewlike motion and had bipolar tufts of 10 to 14 sheathed flagella. The bacteria were microaerophilic, nutritionally fastidious, and physiologically similar to Helicobacter species and Wolinella succinogenes but could be differentiated from these organisms by their unique cellular ultrastructure. Using 16S rRNA sequencing, we found that strain ST1T (T = type strain) was related to previously described Helicobacter species, "Flexispira rappini," and W. succinogenes. The closest relatives of strain ST1T were Helicobacter mustelae and "F. rappini" (average similarity value, 96%). On the basis of phylogenetic data, strain ST1T (= ATCC 49282T) represents a new species of the genus Helicobacter, for which we propose the name Helicobacter muridarum.

Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria.

Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.

Phylogenetic analysis of the spirochetes.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. THe borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated fromt the shew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema illini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.