Molecular identification of Eimeria species in Spanish bats

This is the first study reporting the detection and molecular characterization of Eimeria in bats in Spain, specifically in 12 of 32 chiropteran species described in the Iberian Peninsula. A total of 76 faecal samples were collected from different bat roosting sites across Spanish territory. The DNA was extracted from the samples and sequenced by targeting the Eimeria SSU-rRNA gene. Two Eimeria species were detected in 29 of the 76 faecal samples (38%), and the bat-specific Eimeria rioarribaensis and rodent-specific Eimeria jerfinica were detected in 4 (5%) and 25 (33%) of the samples, respectively. This is the first report of E. rioarribaensis in the bats Rhinolophus euryale, Myotis myotis and Nyctalus lasiopterus, extending the host and geographical ranges for this bat coccidian parasite. The identification of the rodent-specific parasite species E. jerfinica in bats indicates the occurrence of this species in Spain, although its presence has not previously been reported in wild rodents in this country. Considering that most of the Eimeria spp. reported in bats were described only on the basis of morphometric data, molecular studies are required to determined which Eimeria species occur in bats, to complete the identification of these species and to clarify the phylogeny.     

Enzyme electrophoresis,genetic identity and description of a new genus and species of heteronemertean (Nemertea,Anopla) from northwestern Spain and North Wales

The anoplan order Heteronemertea, particularly the genera Cerebratulus, Lineus and Micrura, contains a very large number of nominate species, many of which are inadequately described. As a consequence, systematic difficulties are encountered with the identification of many taxa in this group, especially those originally established primarily on the basis of their external features. The present paper concerns heteronemerteans collected from two locations, the Foz Estuary (north-western Spain) and Llandudno (North Wales). The Spanish collection included specimens identified as Lineus longissimus (Gunnerus), whilst samples from Llandudno contained large numbers of Lineus viridis (Müller); samples of a third similar but apparently undescribed species were found at both locations. Starch gel electrophoresis showed that samples of the apparent third species were genetically almost identical from each of the two locations, but were clearly different from the two described Lineus species. Histological studies of the unknown specimens revealed anatomical characters, including the unique feature of a proboscis epithelium ciliated throughout its length, which exclude it from any known heteronemertean taxon; it is accordingly placed in a new genus and species, for which the name Riseriellus occultus is proposed.     

Isolation of Legionella pneumophila from Cooling Tower Water by Filtration

Methods are described for detection of Legionella pneumophila in cooling tower water or other water sources by direct fluorescent-antibody staining. A procedure for isolation of Legionella bacteria from water samples by guinea pig inoculation is described. Two different serogroups of L. pneumophila were isolated repeatedly from one of the cooling towers.     

Wolbachia strains in cryptic species of the Anastrepha fraterculus complex (Diptera,Tephritidae) along the Neotropical Region

Infection by Wolbachia was described previously in eleven species of Anastrepha fruit flies some of which are important pests of fruticulture. One such species is the nominal Anastrepha fraterculus, the South American fruit fly, which actually comprises a complex of cryptic species. The suggestions of using Wolbachia for the control of these pest species, make imperative a more precise characterization of the existing strains of the bacteria. In this study, population samples of the A. fraterculus complex from Brazil, Argentina, Peru, Ecuador, Colombia, Guatemala and Mexico were analyzed for Wolbachia infection. The bacteria were genotyped by the MLST and WSP Typing methodologies. All samples were infected with Wolbachia of supergroup “A”. For each of the five MLST genes, unique as well as already known alleles were detected. Nineteen sequence types for the concatenated sequences of the five MLST genes, and twenty wsp alleles were found in the samples. Host-specific haplotypes, shared strains among distinct hosts, and more than one strain of Wolbachia were found in some population samples. Recombination among the MLST genes and intragenic recombination between wsp haplotypes was rare. Phylogenetic analysis showed a great similarity among the Wolbachia strains in the A. fraterculus complex. However, some strains of Wolbachia are found throughout the Neotropical Region and there are specific strains in determined geographical areas.     

Larvae of phoronids (Phoronida) from Terpeniya Bay (Southeastern Sakhalin)

Phoronid larvae were found in planktonic samples from the northern coast of Terpeniya Bay. In some samples, their density was up to 220 specimens/m³. Larval stages having 10, 12, 16, 20, and 22 tentacles are described. Larvae were identified as Actinotrocha branchiata and belong to the species Phoronis muelleri Selys-Longchamps, 1903. However, unlike the Ph. muelleri larvae described in the literature, the larvae we found are smaller (not more than 900 μm) before metamorphosis and have fewer tentacles (24). They lack paired vacuolated diverticula of the stomach, which are characteristic of Ph. muelleri larvae. However, judging by all other characters, namely transparency, the absence of coelomic cylinder in the preoral lobe, and the presence of adult tentacle primordia, one pair of blood cell aggregations, and a pyriform organ, these larvae are similar to the previously described larvae of Ph. muelleri. Adult forms of Ph. muelleri were previously found in Terpeniya Bay and described by Mamkaev (1962) and Emig (1984).     

Secondary pollen types characteristic of Louisiana honeys

Nine secondary pollen types (those comprising 16–45% of the pollen sample) were recovered from 54 honey samples (10 g) collected from the major beekeeping areas of Louisiana. The pollen types in order of decreasing importance are:Berchemia scandens, rattan vine;Rubus spp., blackberry and dewberry;Salix spp., willow;Trifolium repens, white clover;Glycine max, soybean;Parthenocissus quinquefolia, Virginia creeper; Rosaceae, rose family;Cephalanthus occidentalis, buttonbush; andCornus spp., dogwood. The first four secondary types also appeared as dominant pollen (over 45%) in some of the Louisiana honey samples investigated and were previously reported. The remaining five pollen types and the “bee plants” associated with each pollen type are identified and described.     

Distribution and Morphology of Longidorus breviannulatus Norton &Hoffman, 1975 and Longidorus fragilis Thorne, 1974 (Nematoda: Longidoridae) from North America

In a survey of ecotypes for longidorids, primarily from the rhizosphere hardwood trees growing in sandy soil along stream banks, 828 soil samples were collected from 37 Arkansas counties in 1999-2001. Eight populations of Longidorus breviannulatus were identified from the Arkansas survey samples. A total of 19 populations from California, Illinois, Iowa, Kansas, Michigan, Nebraska, New Jersey, New York, and Wisconsin were identified from the collection of the second author. A few males were found in New York and Nebraska populations and are described herein. Seven populations of L. fragilis were identified in the Arkansas survey samples, and one population was found from Indiana. Four juvenile stages of L. fragilis are present, and data are given for them herein.     

Gut Microbiota Patterns Associated with Colonization of Different Clostridium difficile Ribotypes

C. difficile infection is associated with disturbed gut microbiota and changes in relative frequencies and abundance of individual bacterial taxons have been described. In this study we have analysed bacterial, fungal and archaeal microbiota by denaturing high pressure liquid chromatography (DHPLC) and with machine learning methods in 208 faecal samples from healthy volunteers and in routine samples with requested C. difficile testing. The latter were further divided according to stool consistency, C. difficile presence or absence and C. difficile ribotype (027 or non-027). Lower microbiota diversity was a common trait of all routine samples and not necessarily connected only to C. difficile colonisation. Differences between the healthy donors and C. difficile positive routine samples were detected in bacterial, fungal and archaeal components. Bifidobacterium longum was the single most important species associated with C. difficile negative samples. However, by machine learning approaches we have identified patterns of microbiota composition predictive for C. difficile colonization. Those patterns also differed between samples with C. difficile ribotype 027 and other C. difficile ribotypes. The results indicate that not only the presence of a single species/group is important but that certain combinations of gut microbes are associated with C. difficile carriage and that some ribotypes (027) might be associated with more disturbed microbiota than the others.     

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.     

Health evaluation of wild gentoo penguins (Pygoscelis papua) in the Antarctic Peninsula

Historically wildlife conservation was based on habitat protection and exploitation control. Only recently have diseases been considered an important issue. However, pathogens are usually described during or after disease outbreaks, but to determine which pathogens may be emerging, surveys of wildlife health are critical in a given time. This study deals with the health status of gentoo penguins Pygoscelis papua in two localities at the Antarctica Peninsula and one at Ardley Island off the South Shetland Islands. Cloacal swaps, fresh fecal samples, ectoparasites, and blood smears were collected. We examined and dissected 14 penguin corpses found dead. Fecal samples were positive for Campylobacter, Escherichia coli and in the carcasses four endoparasitic species were found: Diphyllobothrium sp. and Parorchites zederi, Corynosoma shackletoni and Stegophorus adeliae. The tick Ixodes uriae occurred in five of the examined penguins, and the louse Austrogoniodes gressitti on six birds. From the colony grounds, we collected 1,184 I. uriae. We recorded antibiotic-resistant bacteria, such as E. coli, in ecosystems where gentoo penguins breed. Cloacal samples (300) were negative for Chlamydia, as well as for Salmonella, Campylobacter, E. coli, Newcastle and Influenza viruses.     

The determination of bicarbonate in nanoliter samples

A methodological system is described which permits the determination of bicarbonate ions in samples of a few nanoliters. The samples were collected anaerobically and were then equilibrated in oil of different CO₂ tensions. The pH was measured with antimony microelectrodes after each equilibration. The $\text{pH}\text{log}$, P_CO2 curve thus obtained represented the buffer curve. Using the intratubular pH measured in situ prior to the sampling, the corresponding P_CO2 was obtained from the buffer curve. Using the Henderson-Hasselbalch equation, the bicarbonate concentration was calculated for that particular P_CO2. Some theoretical and practical aspects on these determinations are discussed.     

Diatoms of the genus Attheya west, 1860 from the Sea of Japan

This paper documents the species diversity of the diatom genus Attheya from the Sea of Japan. Two species (A. arenicola and A. longicornis) were studied in field samples and in culture. Their morphology is described using light and electron microscopy; the ecology and the distribution data are provided. A. arenicola is a new record for the seas of Russia.     

Comparative Genomic Analysis and Phenotypic Characterization of Bronchoscope-Associated Klebsiella aerogenes

Bronchoscopes have been linked to outbreaks of nosocomial infections. The phenotypic and genomic profiles of bronchoscope-associated Klebsiella aerogenes isolates are largely unknown. In this work, a total of 358 isolates and 13 isolates were recovered from samples after clinical procedures and samples after decontamination procedures, respectively, over the five months. Antimicrobial susceptibility testing found seven K. aerogenes isolates exhibiting a low-level resistance to antimicrobial agents. Among seven K. aerogenes isolates, we found five sequence types (STs) clustered into three main clades. Collectively, this study described for the first time the phenotypic and genomic characteristics of bronchoscope-associated K. aerogenes.     

High Prevalence of Co-Infections by Invasive and Non-Invasive Chlamydia trachomatis Genotypes during the Lymphogranuloma Venereum Outbreak in Spain

The evolution of Chlamydia trachomatis is mainly driven by recombination events. This fact can be fuelled by the coincidence in several European regions of the high prevalence of non-invasive urogenital genotypes and lymphogranuloma venereum (LGV) outbreaks. This scenario could modify the local epidemiology and favor the selection of new C. trachomatis variants. Quantifying the prevalence of co-infection could help to predict the potential risk in the selection of new variants with unpredictable results in pathogenesis or transmissibility. In the 2009-2013 period, 287 clinical samples with demonstrated presence of C. trachomatis were selected. They were divided in two groups. The first group was constituted by 137 samples with C. trachomatis of the LGV genotypes, and the second by the remaining 150 samples in which the presence of LGV genotypes was previously excluded. They were analyzed to detect the simultaneous presence of non-LGV genotypes based on pmpH and ompA genes. In the first group, co-infections were detected in 10.9% of the cases whereas in the second group the prevalence was 14.6%, which is the highest percentage ever described among European countries. Moreover, bioinformatic analyses suggested the presence among men who have sex with men of a pmpH-recombinant variant, similar to strains described in Seattle in 2002. This variant was the result of genetic exchange between genotypes belonging to LGV and members of G-genotype. Sequencing of other genes, phylogenetically related to pathotype, confirmed that the putative recombinant found in Madrid could have a common origin with the strains described in Seattle. Countries with a high prevalence of co-infections and high migration flows should enhance surveillance programs in at least their vulnerable population.     

Barnacles (Cirripedia: Thoracica) from the equatorial East Pacific

Previously unidentified barnacles (Cirripedia: Thoracica) that were collected during the ninth cruise of the R/V Akademik Mstislav Keldysh in the equatorial zone of the East Pacific Ocean from depths of 1422–1490 m are examined. The following species were found in two samples we studied: Amigdoscalpellum costellatum, A. elegans, Arcoscalpellum michelottianum, Annandaleum gruvelii gruvelii, Cristallinaverruca allisoni, and Metaverruca recta. These species are described, illustrated, and compared to the published data. This study extends the previously known areas of distribution of some species and adds further data on their morphology.     

A method for preventing sorbitol interference with the determination of inorganic phosphate

A technique is described for preventing interference of sorbitol with the assay of P₁ by modifying the procedure of B. N. Ames (1966, in Methods in Enzymology, E. F. Neufeld and V. Ginsburg, eds., Vol. 8, pp. 115–118, Academic Press, New York). The new method relies on the ability of precipitated protein to bind phosphomolybdate and so allow separation of the P₁ from the soluble sorbitol. The conditions for the formation and precipitation of phosphomolybdate-protein complex and for the subsequent assay of P₁ are described. No unique set of conditions could be found which prevented interference at all sorbitol concentrations tested. Instead, conditions for the elimination of interference by particular sorbitol concentration ranges were established. The application of the procedure to samples containing 0–150 nmol of P₁ and 10–100 μmol of sorbitol is described. Complete recovery of P₁ was achieved after precipitation. Standard plots were linear. Coefficients of variation ranged from 9% with low amounts of P₁ (≤25 nmol) to 2.5% at higher levels (150 nmol). One hundred nanomoles of P₁ gave an absorbance at 700 nm of 0.87. Modifications are described to extend the technique to different sorbitol concentration ranges and other applications of the method are mentioned.     

A quantitative study of adipokinetic hormone of the firebug,Pyrrhocoris apterus

The development of an enzyme-linked immunoassay (ELISA) for the adipokinetic neuropeptide hormone, Pya-AKH, from the firebug Pyrrhocoris apterus L. is described. The ELISA measures as little as 20 fmol of Pya-AKH. Tested against a range of synthetic peptides, the assay has a high sensitivity for peptides containing the C-terminal motif FTPNWamide. The amounts of Pya-AKH in the brain, corpora cardiaca, suboesophageal ganglia, and fused thoracic and abdominal ganglionic mass are very small, with only the corpora cardiaca containing appreciable levels of the hormone (ca. 4 pmol per bug). Preliminary estimates of the persistence of the hormone in the haemolymph are consistent with values determined for AKHs in other insects, and suggest that Pya-AKH has a rapid turnover with a half-life of ca. 18 min. Measurements of circulating titres of AKH in Pyrrhocoris are only possible in the ELISA described here by using pooled samples of haemolymph, and after preliminary clean-up of the haemolymph samples. The titre of Pya-AKH in resting reproductive female Pyrrhocoris is ca. 1 fmol/μl.     

ESR and TL investigations on gamma irradiated linden (Tilia vulgaris)

Electron spin resonance (ESR) and thermoluminescence (TL) signals induced by gamma irradiation in linden (Tilia vulgaris) were studied for detection and dosimetric purposes. Before irradiation, linden leaf samples exhibit one singlet ESR signal centred at g = 2.0088. Besides this central signal, in spectra of irradiated linden samples, two weak satellite signals situated about 3 mT left (g = 2.0267) and right (g = 1.9883) were observed. Dose–response curves for the left satellite signal and the central single signal were constructed, and it was found that both of these curves can be described best by the combination of two exponential saturation functions. Variable temperature and fading studies at room temperature showed that the radiation-induced radicals in linden leaf samples are very sensitive to temperature. The stabilities of the left satellite (g = 2.0267) and the central single (g = 2.0088) signal at room temperature over a storage period of 126 days turned out to be best described by a sum of two first-order decay functions. The kinetic features of the left satellite signal were studied over the temperature range of 313–373 K. The results indicate that the isothermal decay curves of the left satellite ESR signal also proved to be best fitted by the combination of two first-order decay functions. Fading and annealing studies suggested the existence of two different radiation-induced free radical species. At the same time, Arrhenius plots evidenced two different kinetic regimes with two different activation energies. TL investigation of polyminerals from the linden samples allowed to discriminate clearly between irradiated and unirradiated samples even 75 days after irradiation.     

Distribution and diversity of members of the bacterial phylum Fibrobacteres in environments where cellulose degradation occurs

The Fibrobacteres phylum contains two described species, Fibrobacter succinogenes and Fibrobacter intestinalis, both of which are prolific degraders of cellulosic plant biomass in the herbivore gut. However, recent 16S rRNA gene sequencing studies have identified novel Fibrobacteres in landfill sites, freshwater lakes and the termite hindgut, suggesting that members of the Fibrobacteres occupy a broader ecological range than previously appreciated. In this study, the ecology and diversity of Fibrobacteres was evaluated in 64 samples from contrasting environments where cellulose degradation occurred. Fibrobacters were detected in 23 of the 64 samples using Fibrobacter genus-specific 16S rRNA gene PCR, which provided their first targeted detection in marine and estuarine sediments, cryoconite from Arctic glaciers, as well as a broader range of environmental samples. To determine the phylogenetic diversity of the Fibrobacteres phylum, Fibrobacter-specific 16S rRNA gene clone libraries derived from 17 samples were sequenced (384 clones) and compared with all available Fibrobacteres sequences in the Ribosomal Database Project repository. Phylogenetic analysis revealed 63 lineages of Fibrobacteres (95% OTUs), with many representing as yet unclassified species. Of these, 24 OTUs were exclusively comprised of fibrobacters derived from environmental (non-gut) samples, 17 were exclusive to the mammalian gut, 15 to the termite hindgut, and 7 comprised both environmental and mammalian strains, thus establishing Fibrobacter spp. as indigenous members of microbial communities beyond the gut ecosystem. The data highlighted significant taxonomic and ecological diversity within the Fibrobacteres, a phylum circumscribed by potent cellulolytic activity, suggesting considerable functional importance in the conversion of lignocellulosic biomass in the biosphere.     

Entosthodon mouretii (Corb.) Jelenc new to Europe

Entosthodon mouretii (Corb.) Jelenc, a species related to E. fascicularis (Hedw.) C. Müll., is referred for the first time in Europe, from samples collected in Spain. The Spanish specimens are described and illustrated.