In vitro susceptibility to pentavalent antimony in Leishmania infantum strains is not modified during in vitro or in vivo passages but is modified after host treatment with meglumine antimoniate

Background: Leishmaniasis is a common parasitic disease in Southern Europe, caused by Leishmania infantum. The failures of current treatment with pentavalent antimonials are partially attributable to the emergence of antimony-resistant Leishmania strains. This study analyses the in vitro susceptibility to pentavalent antimony of intracellular amastigotes from a range of L. infantum strains, derived from the same infected animal, during in vitro and in vivo passages and after host treatment with meglumine antimoniate. Results: SbV-IC50 values for strains from two distinct isolates from the same host and one stock after two years of culture in NNN medium and posterior passage to hamster were similar (5.0 ± 0.2; 4.9 ± 0.2 and 4.4 ± 0.1 mgSbV/L, respectively). In contrast, a significant difference (P < 0.01, t test) was observed between the mean SbV-IC50 values in the stocks obtained before and after treatment of hosts with meglumine antimoniate (4.7 ± 0.4 mgSbV/L vs. 7.7 ± 1.5 mgSbV/L). Drug-resistance after drug pressure in experimentally infected dogs increased over repeated drug administration (6.4 ± 0.5 mgSbV/L after first treatment vs. 8.6 ± 1.4 mgSbV/L after the second) (P < 0.01, t test). Conclusions: These results confirm previous observations on strains from Leishmania/HIV co-infected patients and indicate the effect of the increasing use of antimony derivatives for treatment of canine leishmaniasis in endemic areas on the emergence of Leishmania antimony-resistant strains.

Antimicrobial peptide genes in Bacillus strains from plant environments

The presence of the antimicrobial peptide (AMP) biosynthetic genes srfAA (surfactin), bacA (bacylisin), fenD (fengycin), bmyB (bacyllomicin), spaS (subtilin), and ituC (iturin) was examined in 184 isolates of Bacillus spp. obtained from plant environments (aerial, rhizosphere, soil) in the Mediterranean land area of Spain. Most strains had between two and four AMP genes whereas strains with five genes were seldom detected and none of the strains had six genes. The most frequent AMP gene markers were srfAA, bacA, bmyB, and fenD, and the most frequent genotypes srfAA-bacA-bmyB and srfAAbacA-bmyB-fenD. The dominance of these particular genes in Bacillus strains associated with plants reinforces the competitive role of surfactin, bacyllomicin, fengycin, and bacilysin in the fitness of strains in natural environments. The use of these AMP gene markers may assist in the selection of putative biological control agents of plant pathogens

A new disruption vector (pDHO) to obtain heterothallic strains from both Saccharomyces cerevisiae and Saccharomyces pastorianus

Yeasts are responsible for several traits in fermented beverages, including wine and beer, and their genetic manipulation is often necessary to improve the quality of the fermentation product. Improvement of wild-type strains of Saccharomyces cerevisiae and Saccharomyces pastorianus is difficult due to their homothallic character and variable ploidy level. Homothallism is determined by the HO gene in S. cerevisiae and the Sc-HO gene in S. pastorianus. In this work, we describe the construction of an HO disruption vector (pDHO) containing an HO disruption cassette and discuss its use in generating heterothallic yeast strains from homothallic Saccharomyces species.

Molecular dating of caprines using ancient DNA sequences of Myotragus balearicus, an extinct endemic Balearic mammal

Background: Myotragus balearicus was an endemic bovid from the Balearic Islands (Western Mediterranean) that became extinct around 6,000-4,000 years ago. The Myotragus evolutionary lineage became isolated in the islands most probably at the end of the Messinian crisis, when the desiccation of the Mediterranean ended, in a geological date established at 5.35 Mya. Thus, the sequences of Myotragus could be very valuable for calibrating the mammalian mitochondrial DNA clock and, in particular, the tree of the Caprinae subfamily, to which Myotragus belongs. Results: We have retrieved the complete mitochondrial cytochrome b gene (1,143 base pairs), plus fragments of the mitochondrial 12S gene and the nuclear 28S rDNA multi-copy gene from a well preserved Myotragus subfossil bone. The best resolved phylogenetic trees, obtained with the cytochrome b gene, placed Myotragus in a position basal to the Ovis group. Using the calibration provided by the isolation of Balearic Islands, we calculated that the initial radiation of caprines can be dated at 6.2 ± 0.4 Mya. In addition, alpine and southern chamois, considered until recently the same species, split around 1.6 ± 0.3 Mya, indicating that the two chamois species have been separated much longer than previously thought. Conclusion: Since there are almost no extant endemic mammals in Mediterranean islands, the sequence of the extinct Balearic endemic Myotragus has been crucial for allowing us to use the Messinian crisis calibration point for dating the caprines phylogenetic tree.

Unexpected genomic variability in clinical and environmental strains of the pathogenic yeast Candida parapsilosis

Invasive candidiasis is the most commonly reported invasive fungal infection worldwide. Although Candida albicans remains the main cause, the incidence of emerging Candida species, such as C. parapsilosis is increasing. It has been postulated that C. parapsilosis clinical isolates result from a recent global expansion of a virulent clone. However, the availability of a single genome for this species has so far prevented testing this hypothesis at genomic scales. We present here the sequence of three additional strains from clinical and environmental samples. Our analyses reveal unexpected patterns of genomic variation, shared among distant strains, that argue against the clonal expansion hypothesis. All strains carry independent expansions involving an arsenite transporter homolog, pointing to the existence of directional selection in the environment, and independent origins of the two clinical isolates. Furthermore, we report the first evidence for the existence of recombination in this species. Altogether, our results shed new light onto the dynamics of genome evolution in C. parapsilosis.

A new disruption vector (pDHO) to obtain heterothallic strains from both Saccharomyces cerevisiae and Saccharomyces pastorianus

Yeasts are responsible for several traits in fermented beverages, including wine and beer, and their genetic manipulation is often necessary to improve the quality of the fermentation product. Improvement of wild-type strains of Saccharomyces cerevisiae and Saccharomyces pastorianus is difficult due to their homothallic character and variable ploidy level. Homothallism is determined by the HO gene in S. cerevisiae and the Sc-HO gene in S. pastorianus. In this work, we describe the construction of an HO disruption vector (pDHO) containing an HO disruption cassette and discuss its use in generating heterothallic yeast strains from homothallic Saccharomyces species.

Laboratory diagnosis of brucellosis in a rural endemic area in northeastern Spain

Sera obtained from 62 patients from four mountain counties in Catalonia (Northeastern Spain), in whom brucellosis had been diagnosed on the basis of clinical evidence and/or personal history, were analyzed using the rose Bengal test, standard serum agglutination test (SAT), Coombs" test, ELISA, and complement fixation. The diagnosis was further confirmed through blood cultures. Clinical evidence, epidemiology, and the results from serologic tests were used to assign patients to one of two groups: group 1 (n = 38) patients had primary infections, whereas group 2 (n = 24) patients had been previously exposed to the microorganism, i.e. re-infection of group 2 individuals occurred after long periods of time during which no active infection by Brucella had been detected. Receivingoperating charts (ROC) were used to determine the diagnostic value of the different tests and to establish discriminant values. Blood culture was a valuable diagnostic tool in group 1 (0.92 sensitivity) but was inappropriate in group 2 (0.08). The combination of positive rose Bengal test and agglutination ≥1/160 was valid for diagnosis in group 1. In group 2, agglutination < 1/160 (including negative agglutination) did not rule out brucellosis. The combination of positive rose Bengal test and Coombs" test ≥1/320 was the best diagnostic criterion (0.8 specificity; 1 sensitivity). ELISA (for IgG, IgM, or both) did not improve diagnostic accuracy

Two different epidemiological scenarios of border disease in the populations of Pyrenean chamois (Rupicapra p. pyrenaica) after the first disease outbreaks

Since 2001 several outbreaks of a new disease associated with Border disease virus (BDV) infection have caused important declines in Pyrenean chamois (Rupicapra pyrenaica) populations in the Pyrenees. The goal of this study was to analyze the post-outbreak BDV epidemiology in the first two areas affected by disease with the aim to establish if the infection has become endemic. We also investigated if BDV infected wild and domestic ruminants sharing habitat with chamois. Unexpectedly, we found different epidemiological scenarios in each population. Since the disease outbreaks, some chamois populations recuperated quickly, while others did not recover as expected. In chamois from the first areas, prevalence was high (73.47%) and constant throughout the whole study period and did not differ between chamois born before and after the BDV outbreak; in all, BDV was detected by RT-PCR in six chamois. In the other areas, prevalence was lower (52.79%) and decreased during the study period; as well, prevalence was significantly lower in chamois born after the disease outbreak. No BDV were detected in this population. A comparative virus neutralisation test performed with four BDV strains and one Bovine viral diarrhoea virus (BVDV) strain showed that all the chamois had BDV-specific antibodies. Pestivirus antibodies were detected in all the rest of analyzed species, with low prevalence values in wild ruminants and moderate values in domestic ruminants. No viruses were detected in these species. These results confirm the hypothesis that outbreaks of BDV infection only affect the Pyrenean chamois, although other wild ruminants can occasionally be infected. In conclusion, two different scenarios have appeared since the first border disease outbreaks in Pyrenean chamois: on the one hand frequent BDV circulation with possible negative impact on population dynamics in some areas and on the other, lack of virus circulation and quick recovery of the chamois population.

Molecular dating of caprines using ancient DNA sequences of Myotragus balearicus, an extinct endemic Balearic mammal

Background: Myotragus balearicus was an endemic bovid from the Balearic Islands (Western Mediterranean) that became extinct around 6,000-4,000 years ago. The Myotragus evolutionary lineage became isolated in the islands most probably at the end of the Messinian crisis, when the desiccation of the Mediterranean ended, in a geological date established at 5.35 Mya. Thus, the sequences of Myotragus could be very valuable for calibrating the mammalian mitochondrial DNA clock and, in particular, the tree of the Caprinae subfamily, to which Myotragus belongs. Results: We have retrieved the complete mitochondrial cytochrome b gene (1,143 base pairs), plus fragments of the mitochondrial 12S gene and the nuclear 28S rDNA multi-copy gene from a well preserved Myotragus subfossil bone. The best resolved phylogenetic trees, obtained with the cytochrome b gene, placed Myotragus in a position basal to the Ovis group. Using the calibration provided by the isolation of Balearic Islands, we calculated that the initial radiation of caprines can be dated at 6.2 ± 0.4 Mya. In addition, alpine and southern chamois, considered until recently the same species, split around 1.6 ± 0.3 Mya, indicating that the two chamois species have been separated much longer than previously thought. Conclusion: Since there are almost no extant endemic mammals in Mediterranean islands, the sequence of the extinct Balearic endemic Myotragus has been crucial for allowing us to use the Messinian crisis calibration point for dating the caprines phylogenetic tree.

Phenotipic comparison of clinical and plant beneficial strains of Pantoea agglomerans

Certain strains of Pantoea are used as biocontrol agents for the suppression of plant diseases. However, their commercial registration is hampered in some countries because of biosafety concerns. This study compares clinical and plant-beneficial strains of P. agglomerans and related species using a phenotypic analysis approach in which plant-beneficial effects, adverse effects in nematode models, and toxicity were evaluated. Plant-beneficial effects were determined as the inhibition of apple fruit infection by Penicillium expansum and apple flower infection by Erwinia amylovora. Clinical strains had no general inhibitory activity against infection by the fungal or bacterial plant pathogens, as only one clinical strain inhibited P. expansum and three inhibited E. amylovora. By contrast, all biocontrol strains showed activity against at least one of the phytopathogens, and three strains were active against both. The adverse effects in animals were evaluated in the plant-parasitic nematode Meloidogyne javanica and the bacterial-feeding nematode Caenorhabditis elegans. Both models indicated adverse effects of the two clinical strains but not of any of the plant-beneficial strains. Toxicity was evaluated by means of hemolytic activity in blood, and genotoxicity with the Ames test. None of the strains, whether clinical or plant-beneficial, showed any evidence of toxicity

Experimental Identification of Actinobacillus pleuropneumoniae Strains L20 and JL03 Heptosyltransferases, Evidence for a New Heptosyltransferase Signature Sequence.

We experimentally identified the activities of six predicted heptosyltransferases in Actinobacillus pleuropneumoniae genome serotype 5b strain L20 and serotype 3 strain JL03. The initial identification was based on a bioinformatic analysis of the amino acid similarity between these putative heptosyltrasferases with others of known function from enteric bacteria and Aeromonas. The putative functions of all the Actinobacillus pleuropneumoniae heptosyltrasferases were determined by using surrogate LPS acceptor molecules from well-defined A. hydrophyla AH-3 and A. salmonicida A450 mutants. Our results show that heptosyltransferases APL_0981 and APJL_1001 are responsible for the transfer of the terminal outer core D-glycero-D-manno-heptose (D,D-Hep) residue although they are not currently included in the CAZY glycosyltransferase 9 family. The WahF heptosyltransferase group signature sequence [S(T/S)(GA)XXH] differs from the heptosyltransferases consensus signature sequence [D(TS)(GA)XXH], because of the substitution of D(261) for S(261), being unique.

Spiders on a Hot Volcanic Roof: Colonisation Pathways and Phylogeography of the Canary Islands Endemic Trap-Door Spider Titanidiops canariensis (Araneae, Idiopidae)

Studies conducted on volcanic islands have greatly contributed to our current understanding of how organisms diversify. The Canary Islands archipelago, located northwest of the coast of northern Africa, harbours a large number of endemic taxa. Because of their low vagility, mygalomorph spiders are usually absent from oceanic islands. The spider Titanidiops canariensis, which inhabits the easternmost islands of the archipelago, constitutes an exception to this rule. Here, we use a multi-locus approach that combines three mitochondrial and four nuclear genes to investigate the origins and phylogeography of this remarkable trap-door spider. We provide a timeframe for the colonisation of the Canary Islands using two alternative approaches: concatenation and species tree inference in a Bayesian relaxed clock framework. Additionally, we investigate the existence of cryptic species on the islands by means of a Bayesian multi-locus species delimitation method. Our results indicate that T. canariensis colonised the Canary Islands once, most likely during the Miocene, although discrepancies between the timeframes from different approaches make the exact timing uncertain. A complex evolutionary history for the species in the archipelago is revealed, which involves two independent colonisations of Fuerteventura from the ancestral range of T. canariensis in northern Lanzarote and a possible back colonisation of southern Lanzarote. The data further corroborate a previously proposed volcanic refugium, highlighting the impact of the dynamic volcanic history of the island on the phylogeographic patterns of the endemic taxa. T. canariensis includes at least two different species, one inhabiting the Jandia peninsula and central Fuerteventura and one spanning from central Fuerteventura to Lanzarote. Our data suggest that the extant northern African Titanidiops lineages may have expanded to the region after the islands were colonised and, hence, are not the source of colonisation. In addition, T. maroccanus may harbour several cryptic species.

Genotypic comparison of Pantoea agglomerans plant and clinical strains

Pantoea agglomerans strains are among the most promising biocontrol agents for avariety of bacterial and fungal plant diseases, particularly fire blight of apple and pear. However, commercial registration of P. agglomerans biocontrol products is hampered because this species is currently listed as a biosafety level 2 (BL2) organism due to clinical reports as an opportunistichuman pathogen. This study compares plant-origin and clinical strains in a search for discriminating genotypic/phenotypic markers using multi-locus phylogenetic analysis and fluorescent amplified fragment length polymorphisms (fAFLP) fingerprinting.Results: Majority of the clinical isolates from culture collections were found to be improperly designated as P. agglomerans after sequence analysis. The frequent taxonomic rearrangements underwent by the Enterobacter agglomerans/Erwinia herbicola complex may be a major problem in assessing clinical associations within P. agglomerans. In the P. agglomerans sensu stricto (in the stricter sense) group, there was no discrete clustering of clinical/biocontrol strains and no marker was identified that was uniquely associated to clinical strains. A putative biocontrol-specific fAFLP marker was identified only in biocontrol strains. The partial ORF located in this band corresponded to an ABC transporter that was found in all P. agglomerans strains. Conclusion: Taxonomic mischaracterization was identified as a major problem with P.agglomerans, and current techniques removed a majority of clinical strains from this species. Although clear discrimination between P. agglomerans plant and clinical strains was not obtained with phylogenetic analysis, a single marker characteristic of biocontrol strains was identified whichmay be of use in strain biosafety determinations. In addition, the lack of Koch's postulate fulfilment, rare retention of clinical strains for subsequent confirmation, and the polymicrobial nature of P. agglomerans clinical reports should be considered in biosafety assessment of beneficial strains in this species