TRS-based PCR as a potential tool for inter-serovar discrimination of Salmonella Enteritidis,S. Typhimurium,S. Infantis,S. Virchow,S. Hadar,S. Newport and S. Anatum

Salmonella enterica subsp. enterica comprises a number of serovars, many of which pose an epidemiological threat to humans and are a worldwide cause of morbidity and mortality. Most reported food infection outbreaks involve the serovars Salmonella Enteritidis and Salmonella Typhimurium. Rapid identification to determine the primary sources of the bacterial contamination is important to the improvement of public health. In recent years, many DNA-based techniques have been applied to genotype Salmonella. Herein, we report the use of a manual TRS-PCR approach for the differentiation of the Salmonella enterica subspecies enterica serovars in a single-tube assay. One hundred seventy Salmonella strains were examined in this work. These consisted of serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum. Five of the TRS-primers, N₆(GTG)₄, N₆(CAC)₄, N₆(CGG)₄, N₆(CCG)₄ and N₆(CTG)₄, perfectly distinguished the S. Enteritidis and S. Typhimurium serovars, and the N₆(GTG)₄ primer additionally grouped the other five frequently isolated serovars. In our opinion, the TRS-PCR methodology could be recommended for a quick and simple DNA-based test for inter-serovar discrimination of Salmonella strains.     

Comparative studies on the reproductive systems ofCardamine flexuosa,C. impatients,C. scutata,andC. lyrata,cruciferae

The reproductive systems of fourCardamine species were compared.C. flexuosa ssp.flexuosa, a self-pollinated winter annual in fallow paddy fields, produces numerous small seeds.C. flexuosa ssp.fallax, a self-pollinated year-long annual in gardens and roadsides, also produces many small seeds.C. impatiens, a self-pollinated annual in forest margins, similarly produces numerous small seeds.C. scutata is a self-pollinated perennial in ill-drained paddy fields and creeks; its vegetative reproduction is not predominant, and it produces relatively many seeds.C. lyrata, a cross-pollinated perennial with self-incompatibility in ill-drained paddy fields and creeks, produces fewer and heavier seeds, but it does produce many ramets. Moreover, in seed-dispersal mechanisms and germination behavior of fourCardamine species several adaptive characteristics to their habitats are found. These observations on the reproductive systems of fourCardamine species apparently present different characteristics of life-history strategy for each of them.     

Redescription of Atopospira galeata (Kahl, 1927) nov. comb. and A. violacea (Kahl, 1926) nov. comb. with redefinition of Atopospira Jankowski, 1964 nov. stat. and Brachonella Jankowski, 1964 (Ciliophora,Armophorida)

The taxonomy of the Metopidae (Ciliophora, Armophorida) remains poorly understood since most of its members have not been studied by modern morphologic and molecular methods. Recent molecular investigations have indicated that the two most species-rich genera, Metopus and Brachonella, are likely nonmonophyletic with at least one well-supported 18S rDNA clade comprised of a species from each of these genera (Brachonella galeata and Metopus violaceus). We investigated these two species with silver impregnation and scanning electron microscopy. Both taxa share important morphologic characteristics not described in other species of Metopus or Brachonella. These synapomorphies include: (1) a diplostichomonad paroral membrane, (2) a bipartite adoral zone with a short buccal part composed of ordinary membranelles and a longer distal part composed of much smaller membranelles bearing a single cilium or none and extending the same length as the perzonal ciliary stripe. We transfer Brachonella galeata (Kahl, 1927) Jankowski, 1964 and Metopus violaceus Kahl, 1926 to genus Atopospira Jankowski, 1964 nov. stat. Pending detailed morphologic and molecular characterization, Brachonella campanula, B. cydonia and B. pyriforma, B. intercedens, and B. lemani remain in Brachonella Jankowski 1964.     

A comparative cytogenetic analysis of five pine species from North America, Pinus banksiana, P. contorta, P. monticola, P. resinosa, and P. strobus

The genus Pinus comprises more than 100 species, which are widely distributed in the Northern hemisphere. Cytogenetic information on North American pines is very limited despite their economic importance. In the present study, a detailed comparative cytogenetic analysis is presented for five pine species from North America, P. resinosa, P. monticola, P. contorta, P. banksiana, and P. strobus. Morphometric analysis and physical mapping of rDNA probes were performed. The karyotype of P. monticola was considered ancestral with small difference in relative chromosome lengths. P. banksiana, P. contorta, and P. strobus karyotypes were considered semi-asymmetrical and less ancestral type. P. banksiana showed five secondary constrictions, P. strobus six, and P. contorta eight. P. resinosa karyotype was semi-asymmetrical and derived with 14 secondary constrictions identified on eight different chromosomes. Karyological data were consistent with molecular cytogenetic information. A significant association was observed between the number and locations of secondary constrictions and the 18S-5.8S-26S rDNA sites detected using fluorescence in situ hybridization. The two methods were used to establish a reliable comparative karyotype of the selected pines. In general, karyotype and chromosome evolution were not related to genetic relationships among pine species studied.     

Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. “Species-identifying” biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within ±5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) “strains” composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.     

Burkholderia species in human infections in Mexico: Identification of B. cepacia,B. contaminans,B. multivorans,B. vietnamiensis,B. pseudomallei and a new Burkholderia species

BackgroundBurkholderia sensu stricto is comprised mainly of opportunistic pathogens. This group is widely distributed in the environment but is especially important in clinical settings. In Mexico, few species have been correctly identified among patients, most often B. cepacia is described.Methodology/Principal findingsIn this study, approximately 90 strains identified as B. cepacia with the VITEK2 system were isolated from two medical centers in Mexico City and analyzed by MLSA, BOX-PCR and genome analysis. The initial identification of B. cepacia was confirmed for many strains, but B. contaminans, B. multivorans and B. vietnamiensis were also identified among clinical strains for the first time in hospitals in Mexico. Additionally, the presence of B. pseudomallei was confirmed, and a novel species within the B. cepacia complex was documented. Several strains misidentified as B. cepacia actually belong to the genera Pseudomonas, Stenotrophomonas and Providencia.Conclusions/SignificanceThe presence of different Burkholderia species in Mexico was confirmed. Correct identification of Burkholderia species is important to provide accurate treatment for immunosuppressed patients.     

Produits neutres et alcaloides de Myrtopsis macrocarpa,M. myrtoidea,M. novae-caledoniae et M. sellingii

Stems and leaves of Myrtopsis macrocarpa, M. myrtoidea, M. novae-caledoniae and M. sellingii yielded terpenes, sterols, coumarins, alkaloids (furoquinolines and quinolones) and amides. A new quinolone (8-methoxy flindersine) occurs in Myrtopsis macrocarpa, a new amide (N-benzoyltryptamine) in M. myrtoidea, two new coumarins (myrsellin and myrsellinol) and a new dihydrofuroquinoline (myrtopsine) in M. sellingii. Structures of the new compounds are proposed from chemical and spectroscopic evidence.     

Longitudinal qPCR Study of the Dynamics of L. crispatus,L. iners,A. vaginae, (Sialidase Positive) G. vaginalis,and P. bivia in the Vagina

Background

To obtain more detailed understanding of the causes of disturbance of the vaginal microflora (VMF), a longitudinal study was carried out for 17 women during two menstrual cycles.

Methods

Vaginal swabs were obtained daily from 17 non-pregnant, menarchal volunteers. For each woman, Gram stains were scored, the quantitative changes of 5 key vaginal species, i.e. Atopobium vaginae, Lactobacillus crispatus, L. iners, (sialidase positive) Gardnerella vaginalis and Prevotella bivia were quantified with qPCR and hydrogen-peroxide production was assessed on TMB+ agar.

Results

Women could be divided in 9 subjects with predominantly normal VMF (grades Ia, Ib and Iab, group N) and 8 with predominantly disturbed VMF (grades I-like, II, III and IV, group D).VMF was variable between women, but overall stable for most of the women. Menses were the strongest disturbing factor of the VMF. L. crispatus was present at log7–9 cells/ml in grade Ia, Iab and II VMF, but concentrations declined 100-fold during menses. L. crispatus below log7 cells/ml corresponded with poor H₂O₂-production. L. iners was present at log 10 cells/ml in grade Ib, II and III VMF. Sialidase negative G. vaginalis strains (average log5 cells/ml) were detected in grade I, I-like and IV VMF. In grade II VMF, predominantly a mixture of both sialidase negative and positive G. vaginalis strains (average log9 cells/ml) were present, and predominantly sialidase positive strains in grade III VMF. The presence of A. vaginae (average log9 cells/ml) coincided with grade II and III VMF. P. bivia (log4–8 cells/ml) was mostly present in grade III vaginal microflora. L. iners, G. vaginalis, A. vaginae and P. bivia all increased around menses for group N women, and as such L. iners was considered a member of disturbed VMF.

Conclusions

This qPCR-based study confirms largely the results of previous culture-based, microscopy-based and pyrosequencing-based studies.     

The adenylate kinase genes of M. voltae,M. thermolithotrophicus,M. jannaschii,and M. igneus define a new family of adenylate kinases

The adenylate kinase genes (adkA) were cloned from four closely related methanogenic members of the Archaea: the mesophile Methanococcus voltae (Mv), the thermophile M. thermolithotrophicus (Mt) and the hyperthermophiles M. jannaschii (Mj) and M. igneus (Mi). All four genes encode a protein of 192 amino acids (aa), and the four enzymes were closely related, with 68–81% aa identity in pairwise comparisons. It is anticipated that the enzyme set will provide the basis for studies that can establish the structural basis for ADK thermal stability. Mj and Mi contained a gene homologous to M. vannielii secY upstream of adkA, while Mv and Mt contained an unidentified, yet conserved, upstream open reading frame (ORF). Mt, Mj and Mi, but not Mv, contained an unidentified, yet highly conserved, ORF directly downstream of adkA. Based on their size, predicted secondary structure and phylogenetic relation to bacterial and eukaryotic adenylate kinases (ADK), it was concluded that the archaeal adkA genes encoded a unique class of ADK, and suggested that Euryarchaeotal and Crenarchaeotal branches of the Archaea contain separate subclasses of the enzyme.     

Loricate choanoflagellates (Acanthoecida) from warm water seas. II. Bicosta,Apheloecion, Campyloacantha,Crucispina, Calliacantha and Saroeca

The main outcome of this and subsequent papers is to provide a baseline survey of heterotrophic protist diversity from warm water marine ecosystems, exemplified by loricate choanoflagellates (Acanthoecida). Genera in focus here (i.e. Bicosta, Apheloecion, Campyloacantha, Crucispina, Calliacantha and Saroeca) possess anterior spines or projections and a posterior pedicel, and have 0, 1 or 2 transverse costae. Longitudinal costae are, with the exception of Campyloacantha, external to transverse costal elements across all genera examined here. We describe here Apheloecion eqpacia sp. nov. and Calliacantha magna sp. nov., both of which are so far distributionally confined to warm water habitats. A ‘form A’ of Bicosta minor is introduced to facilitate the distinction between B. minor sensu stricto and a presumed warm water adapted variant with a posterior lorica chamber twist of the longitudinal costae.     

Analysis of morphological characters of Chara baltica, C. hispida, C. horrida, and C. rudis from Europe

Morphological characters of Chara baltica, C. hispida, C. horrida, and C. rudis from different localities in Central Europe and Scandinavia were analyzed. The plants were preliminarily classified using specific qualitative features into four species: C. baltica, C. hispida, C. horrida, and C. rudis. Of the 14 characters, all were used in principal-component and cluster analyses, and 12 were applied in a discriminant analysis. In the discriminant analysis, the most useful characters for distinguishing particular species were selected. The methods (canonical discriminant and canonical analyses) used herein offered good differentiation between brackish water species C. horrida and C. baltica and fresh water species C. hispida and C. rudis. The analysis showed that the two morphologically very similar species C. hispida and C. rudis are only partially distinguished.     

Morphology,bioacoustics, and ecology of Tibicen neomexicensis sp. n., a new species of cicada from the Sacramento Mountains in New Mexico,U.S.A. (Hemiptera,Cicadidae, Tibicen)

Tibicen neomexicensis sp. n., a new species of cicada found in the Sacramento Mountains of southcentral New Mexico, is described. Tibicen neomexicensis closely resembles Tibicen chiricahua Davis morphologically, but males of the two species have highly distinct calling songs that differ in phrasal structure, amplitude burst rates, and pulse structure. Unlike Tibicen chiricahua, male Tibicen neomexicensis use conspicuous dorso-ventral abdominal movements to modulate the amplitude and frequency of their calls. Tibicen neomexicensis is also smaller on average than Tibicen chiricahua, and differences in the color patterns of the wing venation identify these two species morphologically. Both species are dependent on pinyon-juniper woodlands and have similar emergence phenologies. These species appear to be allopatric, with Tibicen chiricahua found west of the Rio Grande in New Mexico, Arizona, and Mexico, and Tibicen neomexicensis so far known only from New Mexico, east of the Rio Grande. Tibicen chiricahua and Tibicen neomexicensis males share a common genitalic structure that separates them from all other species of Tibicen, and the possible evolutionary and biogeographic history of these likely sister species is also discussed.     

New Species of Boletellus Section Boletellus (Boletaceae,Boletales) from Japan,B. aurocontextus sp. nov. and B. areolatus sp. nov.

We describe and illustrate two new species of Boletellus section Boletellus, B. aurocontextus sp. nov. and B. areolatus sp. nov., which are generally assumed to be B. emodensis. In this study, we reconstructed separate molecular phylogenetic trees of section Boletellus using the nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, the largest subunit (RPB1) and the second-largest subunit (RPB2) of nuclear RNA polymerase II gene and mitochondrial cytochrome oxidase subunit 3 (cox3) gene. We also examined the morphologies of B. emodensis sensu lato (s.l.) and other related species for comparison. The molecular phylogenetic tree inferred from the sequences of nuclear DNA (ITS, and combined dataset of RPB1 and RPB2) indicated that three genetically and phylogenetically well-separated lineages were present within B. emodensis s.l. These three lineages were also distinguished on the basis of the molecular phylogenetic tree constructed using the sequences of mitochondrial DNA (cox3), suggesting distinct cytonuclear disequilibria (i.e., evidence of reproductive isolation) among these lineages. Therefore, these three lineages can be treated as independent species: B. aurocontextus, B. areolatus, and B. emodensis. Boletellus aurocontextus and B. areolatus are also distinct from B. emodensis by the macro- and microscopic morphologies. Boletellus aurocontextus is characterized by a pileus with bright yellow to lemon yellow context, which can be observed through a gap in the scales, and basidiospores with relatively large length (mean spore length, 21.4 μm; quotient of spore length and width, 2.51). In contrast, B. areolatus is characterized by a pileus with floccose to appressed thin scaly patches, a stipe with pallid or pale cream color at the upper half, and basidiospores with relatively small length (mean spore length, 16.5 μm; quotient of spore length and width, 1.80).     

Structure of ovaries in scale insects. i. pseudococcidae,kermesidae, eriococcidae,and cryptococcidae (INSECTA,hemiptera, COCCINEA)

Developing ovaries of scale insects (Hemiptera : Coccinea) Nipaecoccus nipae (Pseudococcidae) and Cryptococcus fagisuga (Cryptococcidae) contain clusters of interconnected cells (cystocytes) that are arranged into rosettes; polyfusomes occur in the centres of the rosettes. Ovaries of the investigated adult scale insects are composed of numerous short telotrophic ovarioles. Tropharia (trophic chambers) of Dysmicoccus newsteadi (Pseudococcidae), Eriococcus buxi (Eriococcidae), Cryptococcus fagisuga and Pseudochermes fraxini (Cryptococcidae) comprise only trophocytes (nurse cells), whereas those of Kermes quercus (Kermesidae) and Gossyparia spuria (Eriococcidae) also contain arrested oocytes. The latter probably degenerate. It is suggested that during evolution of scale insects a gradual reduction of germ cells to 4 per cluster (3 trophocytes and 1 oocyte) took place. In light of the obtained results, anagenesis of scale insects ovarioles is discussed.     

Pseudangularia gonzalezi n. sp. and Gibsonilepis swifti (Singh, 1952) n. g., n. comb. (Cestoda, Dilepididae) from the House Swift, Apus affinis (J.E. Gray) (Aves, Apodiformes) from Franceville, Republic of Gabon

Nine specimens of Apus affinis (J.E. Gray) were studied at Franceville, Haut-Ogooué Province, Republic of Gabon, for the presence of helminth parasites. Two cestode species of the family Dilepididae were recorded. Pseudangularia gonzalezi n. sp. is distinguished from the most similar species P. europaea Georgiev & Murai, 1993 by having elliptical cirrus-sac, longer vagina, longer rostellar sheath and greater diameter of suckers. An identification key to the species of the genus Pseudangularia Burt, 1938 is proposed. The present study is the first record of the genus Pseudangularia in the Afrotropical Region. Our study confirms that, in dilepidids with vaginal sclerites from swifts, breaking off the cirrus after copulation is a frequent phenomenon. The genus Gibsonilepis n. g. is erected as monotypic for Vitta swifti Singh, 1952 (originally described from the same host species in India) and Gibsonilepis swifti n. comb. is proposed. Gibsonilepis n. g. is distinguished from Vitta Burt, 1938 by its highly elongate rostellum, rostellar sheath much bigger than rostellum, relatively small rostellar hooks possessing strongly developed guard, disc-shaped suckers with weak peripheral musculature and flat or convex central part, long and well-expressed neck, highly lobed two-winged ovary (lobes rounded) and presence of a band consisting of rows of spine-like microtriches along posterior margin of each proglottis. This is the first record of G. swifti in the Afrotropical Region. The separation of G. swifti from the genus Vitta (parasites of swallows) suggests that, contrary to previous opinions, no dilepidid genera are shared by Apodidae (swifts) and Hirundinidae (swallows).     

Syntrophomonas wolfei gen. nov. sp. nov., an Anaerobic, Syntrophic, Fatty Acid-Oxidizing Bacterium

An anaerobic, nonphototrophic bacterium that β-oxidizes saturated fatty acids (butyrate through octanoate) to acetate or acetate and propionate using protons as the electron acceptor (H₂ as electron sink product) was isolated in coculture with either a non-fatty acid-degrading, H₂-utilizing Desulfovibrio sp. or methanogens. Three strains of the bacterium were characterized and are described as a new genus and species, Syntrophomonas wolfei. S. wolfei is a gram-negative, slightly helical rod with round ends that possesses between two to eight flagella laterally inserted along the concave side of the cell. It has a multilayered cell wall of the gram-negative type. The presence of muramic acid, inhibition of growth by penicillin, and increased sensitivity of the cells to lysis after treatment with lysozyme indicate that peptidoglycan is present in the cell wall. Cells of S. wolfei contain poly-β-hydroxybutyrate. Isoheptanoate was degraded to acetate, isovalerate, and H₂. Carbohydrates, proteinaceous materials, alcohols, or other tested organic compounds do not support growth. Common electron acceptors are not utilized with butyrate as the electron donor. Growth and degradation of fatty acids occur only in syntrophic association with H₂-using bacteria. The most rapid generation time obtained by cocultures of S. wolfei with Desulfovibrio and Methanospirillum hungatei is 54 and 84 h, respectively. The addition of Casamino Acids but neither Trypticase nor yeast extract stimulated growth and resulted in a slight decrease in the generation time of S. wolfei cocultured with M. hungatei. The addition of H₂ to the medium stopped growth and butyrate degradation by S. wolfei.     

The burrows of the miocene beaver palaeocastor,Western Nebraska,U.S.A

Daimonelix is a name given to terrestrial lebensspuren of the late Oligocene—early Miocene beaver genus Palaeocastor, and is not a plant or fresh-water sponge as was originally believed by Barbour. Palaeocastor belongs to a lineage of castorids always found in upland habitat, never near evidence of ponded water. Daimonelices are found in high concentrations in the Harrison Formation of western Nebraska and eastern Wyoming, which represents a semiarid, upland paleoenvironment of sandy substrate. Assignment of a large sample of contemporaneous Daimonelix to the species P. fossor is based on a series of incisor width measurements and on mean shaft diameters. The remarkable preservation of daimonelices, which permits detailed analysis of events in beaver burrow construction, is shown to be due to rapid silicification of plant roots invading the burrows. Some aspects of Palaeocastor ecology and ethology are also clarified. The spatial distribution of Daimonelix consists of scattered towns of high burrow density. One such colony is shown in a detailed paleogeographic map. Criteria for time equivalency of such burrow aggregations are established. The entoptychine gopher Gregorymys, the carnivore Zodiolestes, and the larger beaver P. magnus are also shown to be occasional inhabitants of Palaeocastor fossor colonies.     

Variation and taxonomy of Betula uber,B. Lenta,and B. alleghaniensis

Morphological variation was analyzed in the endangeredBetula uber and sympatric dark-barked tree birches,B. lenta andB. alleghaniensis, to determine the extent of differences among the three taxa. Univariate and multivariate analyses of leaf and fruit characters indicated that the three taxa are morphologically distinct, thatB. uber is more similar toB. lenta than toB. alleghaniensis, that intermediates betweenB. uber andB. lenta do not occur, and that leaves are necessary for absolute separation ofB. uber fromB. lenta because of overlap of fruit characters. Field observations of the bark indicate no differences betweenB. uber andB. lenta while those of growth form suggest thatB. uber may have a more compact crown thanB. lenta. The evidence presented here supports an alliance ofB. uber with the dark-barked tree birches (seriesCostatae).     

Fomitiporia pentaphylacis and F. tenuitubus spp. nov. (Hymenochaetales, Basidiomycota) from Guangxi, southern China

Two specimens of Hymenochaetaceae were collected from Guangxi, southern China, during a recent field trip in August 2011. They are described and illustrated here as two new species, Fomitiporia pentaphylacis and F. tenuitubus, based on a combination of morphological and phylogenetic (ITS and nLSU sequences) data. The two species share subglobose to globose, hyaline and thick-walled basidiospores with strongly cyanophilous in cotton blue and dextrinoid reaction in Melzer??s reagent. These characters are typical for Fomitiporia, differing from other genera in Hymenochaetaceae. F. pentaphylacis resembles F. pusilla in field by its minute basidiocarps, but F. pusilla has distinctly multiple tube layers and smaller basidiospores. F. tenuitubus is similar to F. erecta in both macro- and micro-morphological features, except its larger basidiocarps, smaller pores, and slightly shorter basidiospores. In the phylogenetic perspective, the two species nested within the Fomitiporia clade, but were separated from other sampled species as well as from each other.