Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters

Equine Actinobacillus species were analysed phylogenetically by 16S rRNA gene (rrs) sequencing focusing on the species Actinobacillus equuli, which has recently been subdivided into the non-haemolytic A. equuli subsp. equuli and the haemolytic A. equuli subsp. haemolyticus. In parallel we determined the profile for RTX toxin genes of the sample of strains by PCR testing for the presence of the A. equuli haemolysin gene aqx, and the toxin genes apxI, apxII, apxIII and apxIV, which are known in porcine pathogens such as Actinobacillus pleuropneumoniae and Actinobacillus suis. The rrs-based phylogenetic analysis revealed two distinct subclusters containing both A. equuli subsp. equuli and A. equuli subsp. haemolyticus distributed through both subclusters with no correlation to taxonomic classification. Within one of the rrs-based subclusters containing the A. equuli subsp. equuli type strain, clustered as well the porcine Actinobacillus suis strains. This latter is known to be also phenotypically closely related to A. equuli. The toxin gene analysis revealed that all A. equuli subsp. haemolyticus strains from both rrs subclusters specifically contained the aqx gene while the A. suis strains harboured the genes apxI and apxII. The aqx gene was found to be specific for A. equuli subsp. haemolyticus, since A. equuli subsp. equuli contained no aqx nor any of the other RTX genes tested. The specificity of aqx for the haemolytic equine A. equuli and ApxI and ApxII for the porcine A. suis indicates a role of these RTX toxins in host species predilection of the two closely related species of bacterial pathogens and allows PCR based diagnostic differentiation of the two.

Phylogenetic analysis of Prevotella nigrescens, Prevotella intermedia and Porphyromonas gingivalis clinical strains reveals a clear species clustering

Prevotella nigrescens, Prevotella intermedia and Porphyromonas gingivalis are oral pathogens from the family Bacteroidaceae, regularly isolated from cases of gingivitis and periodontitis. In this study, the phylogenetic variability of these three bacterial species was investigated by means of 16S rRNA (rrs) gene sequence comparisons of a set of epidemiologically and geographically diverse isolates. For each of the three species, the rrs gene sequences of 11 clinical isolates as well as the corresponding type strains was determined. Comparison of all rrs sequences obtained with those of closely related species revealed a clear clustering of species, with only a little intraspecies variability but a clear difference in the rrs gene with respect to the next related taxon. The results indicate that the three species form stable, homogeneous genetic groups, which favours an rrs-based species identification of these oral pathogens. This is especially useful given the 7% sequence divergence between Prevotella intermedia and Prevotella nigrescens, since phenotypic distinction between the two Prevotella species is inconsistent or involves techniques not applicable in routine identification.

Species richness, but not phylogenetic diversity, influences community biomass production and temporal stability in a re-examination of 16 grassland biodiversity studies

* Hundreds of experiments have now manipulated species richness (SR) of various groups of organisms and examined how this aspect of biological diversity influences ecosystem functioning. Ecologists have recently expanded this field to look at whether phylogenetic diversity (PD) among species, often quantified as the sum of branch lengths on a molecular phylogeny leading to all species in a community, also predicts ecological function. Some have hypothesized that phylogenetic divergence should be a superior predictor of ecological function than SR because evolutionary relatedness represents the degree of ecological and functional differentiation among species. But studies to date have provided mixed support for this hypothesis. * Here, we reanalyse data from 16 experiments that have manipulated plant SR in grassland ecosystems and examined the impact on above-ground biomass production over multiple time points. Using a new molecular phylogeny of the plant species used in these experiments, we quantified how the PD of plants impacts average community biomass production as well as the stability of community biomass production through time. * Using four complementary analyses, we show that, after statistically controlling for variation in SR, PD (the sum of branches in a molecular phylogenetic tree connecting all species in a community) is neither related to mean community biomass nor to the temporal stability of biomass. These results run counter to past claims. However, after controlling for SR, PD was positively related to variation in community biomass over time due to an increase in the variances of individual species, but this relationship was not strong enough to influence community stability. * In contrast to the non-significant relationships between PD, biomass and stability, our analyses show that SR per se tends to increase the mean biomass production of plant communities, after controlling for PD. The relationship between SR and temporal variation in community biomass was either positive, non-significant or negative depending on which analysis was used. However, the increases in community biomass with SR, independently of PD, always led to increased stability. These results suggest that PD is no better as a predictor of ecosystem functioning than SR. * Synthesis. Our study on grasslands offers a cautionary tale when trying to relate PD to ecosystem functioning suggesting that there may be ecologically important trait and functional variation among species that is not explained by phylogenetic relatedness. Our results fail to support the hypothesis that the conservation of evolutionarily distinct species would be more effective than the conservation of SR as a way to maintain productive and stable communities under changing environmental conditions.

Molecular characterization and phylogenetic analysis of small ruminant lentiviruses isolated from Canadian sheep and goats

Background: Small Ruminant Lentiviruses (SRLV) are widespread in Canadian sheep and goats and represent an important health issue in these animals. There is however no data about the genetic diversity of Caprine Arthritis Encephalitis Virus (CAEV) or Maedi Visna Virus (MVV) in this country. Findings: We performed a molecular and phylogenetic analysis of sheep and goat lentiviruses from a small geographic area in Canada using long sequences from the gag region of 30 infected sheep and 36 infected goats originating from 14 different flocks. Pairwise DNA distance and phylogenetic analyses revealed that all SRLV sequences obtained from sheep clustered tightly with prototypical Maedi visna sequences from America. Similarly, all SRLV strains obtained from goats clustered tightly with prototypical US CAEV-Cork strain. Conclusions: The data reported in this study suggests that Canadian and US SRLV strains share common origins. In addition, the molecular data failed to bring to light any evidence of past cross species transmission between sheep and goats, which is consistent with the type of farming practiced in this part of the country where single species flocks predominate and where opportunities of cross species transmissions are proportionately low.

Phylogenetic analysis of "Candidatus Mycoplasma turicensis" isolates from pet cats in the United Kingdom, Australia, and South Africa, with analysis of risk factors for infection

Two hemotropic mycoplasmas have been recognized in cats, Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum." We recently described a third feline hemoplasma species, designated "Candidatus Mycoplasma turicensis," in a Swiss cat with hemolytic anemia. This isolate induced anemia after experimental transmission to two specific-pathogen-free cats and analysis of the 16S rRNA gene revealed its close relationship to rodent hemotropic mycoplasmas. The agent was recently shown to be prevalent in Swiss pet cats. We sought to investigate the presence and clinical importance of "Candidatus Mycoplasma turicensis" infection in pet cats outside of Switzerland and to perform the molecular characterization of isolates from different countries. A "Candidatus Mycoplasma turicensis"-specific real-time PCR assay was applied to blood samples from 426 United Kingdom (UK), 147 Australian, and 69 South African pet cats. The 16S rRNA genes of isolates from different countries were sequenced and signalment and laboratory data for the cats were evaluated for associations with "Candidatus Mycoplasma turicensis" infection. Infections were detected in samples from UK, Australian, and South African pet cats. Infection was associated with the male gender, and "Candidatus Mycoplasma haemominutum" and M. haemofelis coinfection. Coinfected cats exhibited significantly lower packed cell volume (PCV) values than uninfected cats. Phylogenetic analyses revealed that some Australian and South African "Candidatus Mycoplasma turicensis" isolates branched away from the remaining isolates. In summary, "Candidatus Mycoplasma turicensis" infection in pet cats exists over a wide geographical area and significantly decreased PCV values are observed in cats coinfected with other feline hemoplasmas.

Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

Proposed minimal standards for the description of genera, species and subspecies of the Pasteurellaceae

Principles and guidelines are presented to ensure a solid scientific standard of papers dealing with the taxonomy of taxa of Pasteurellaceae Pohl 1981. The classification of the Pasteurellaceae is in principle based on a polyphasic approach. DNA sequencing of certain genes is very important for defining the borders of a taxon. However, the characteristics that are common to all members of the taxon and which might be helpful for separating it from related taxa must also be identified. Descriptions have to be based on as many strains as possible (inclusion of at least five strains is highly desirable), representing different sources with respect to geography and ecology, to allow proper characterization both phenotypically and genotypically, to establish the extent of diversity of the cluster to be named. A genus must be monophyletic based on 16S rRNA gene sequence-based phylogenetic analysis. Only in very rare cases is it acceptable that monophyly can not be achieved by 16S rRNA gene sequence comparison. Recently, the monophyly of genera has been confirmed by sequence comparison of housekeeping genes. In principle, a new genus should be recognized by a distinct phenotype, and characters that separate the new genus from its neighbours should be given clearly. Due to the overall importance of accurate classification of species, at least two genotypic methods are needed to show coherence and for separation at the species level. The main criterion for the classification of a novel species is that it forms a monophyletic group based on 16S rRNA gene sequence-based phylogenetic analysis. However, some groups might also include closely related species. In these cases, more sensitive tools for genetic recognition of species should be applied, such as DNA-DNA hybridizations. The comparison of housekeeping gene sequences has recently been used for genotypic definition of species. In order to separate species, phenotypic characters must also be identified to recognize them, and at least two phenotypic differences from existing species should be identified if possible. We recommend the use of the subspecies category only for subgroups associated with disease or similar biological characteristics. At the subspecies level, the genotypic groups must always be nested within the boundaries of an existing species. Phenotypic cohesion must be documented at the subspecies level and separation between subspecies and related species must be fully documented, as well as association with particular disease and host. An overview of methods previously used to characterize isolates of the Pasteurellaceae has been given. Genotypic and phenotypic methods are separated in relation to tests for investigating diversity and cohesion and to separate taxa at the level of genus as well as species and subspecies.

Worldwide occurrence of feline hemoplasma infections in wild felid species

While hemoplasma infections in domestic cats are well studied, almost no information is available on their occurrence in wild felids. The aims of the present study were to investigate wild felid species as possible reservoirs of feline hemoplasmas and the molecular characterization of the hemoplasma isolates. Blood samples from the following 257 wild felids were analyzed: 35 Iberian lynxes from Spain, 36 Eurasian lynxes from Switzerland, 31 European wildcats from France, 45 lions from Tanzania, and 110 Brazilian wild felids, including 12 wild felid species kept in zoos and one free-ranging ocelot. Using real-time PCR, feline hemoplasmas were detected in samples of the following species: Iberian lynx, Eurasian lynx, European wildcat, lion, puma, oncilla, Geoffroy's cat, margay, and ocelot. "Candidatus Mycoplasma haemominutum" was the most common feline hemoplasma in Iberian lynxes, Eurasian lynxes, Serengeti lions, and Brazilian wild felids, whereas "Candidatus Mycoplasma turicensis" was the most prevalent in European wildcats; hemoplasma coinfections were frequently observed. Hemoplasma infection was associated with species and free-ranging status of the felids in all animals and with feline leukemia virus provirus-positive status in European wildcats. Phylogenetic analyses of the 16S rRNA and the partial RNase P gene revealed that most hemoplasma isolates exhibit high sequence identities to domestic cat-derived isolates, although some isolates form different subclusters within the phylogenetic tree. In conclusion, 9 out of 15 wild felid species from three different continents were found to be infected with feline hemoplasmas. The effect of feline hemoplasma infections on wild felid populations needs to be further investigated.

Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri

The Mycoplasma mycoides cluster consists of six pathogenic mycoplasmas causing disease in ruminants, which share many genotypic and phenotypic traits. The M. mycoides cluster comprises five recognized taxa: Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), M. mycoides subsp. mycoides Large Colony (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp). The group of strains known as Mycoplasma sp. bovine group 7 of Leach (MBG7) has remained unassigned, due to conflicting data obtained by different classification methods. In the present paper, all available data, including recent phylogenetic analyses, have been reviewed, resulting in a proposal for an emended taxonomy of this cluster: (i) the MBG7 strains, although related phylogenetically to M. capricolum, hold sufficient characteristic traits to be assigned as a separate species, i.e. Mycoplasma leachii sp. nov. (type strain, PG50(T) = N29(T) = NCTC 10133(T) = DSM 21131(T)); (ii) MmmLC and Mmc, which can only be distinguished by serological methods and are related more distantly to MmmSC, should be combined into a single subspecies, i.e. Mycoplasma mycoides subsp. capri, leaving M. mycoides subsp. mycoides (MmmSC) as the exclusive designation for the agent of contagious bovine pleuropneumonia. A taxonomic description of M. leachii sp. nov. and emended descriptions of M. mycoides subsp. mycoides and M. mycoides subsp. capri are presented. As a result of these emendments, the M. mycoides cluster will hereafter be composed of five taxa comprising three subclusters, which correspond to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii.

Enemy damage of exotic plant species is similar to that of natives and increases with productivity

1 .In their colonized ranges, exotic plants may be released from some of the herbivores or pathogens of their home ranges but these can be replaced by novel enemies. It is of basic and practical interest to understand which characteristics of invaded communities control accumulation of the new pests. Key questions are whether enemy load on exotic species is smaller than on native competitors as suggested by the enemy release hypothesis (ERH) and whether this difference is most pronounced in resource-rich habitats as predicted by the resource–enemy release hypothesis (R-ERH). 2. In 72 populations of 12 exotic invasive species, we scored all visible above-ground damage morphotypes caused by herbivores and fungal pathogens. In addition, we quantified levels of leaf herbivory and fruit damage. We then assessed whether variation in damage diversity and levels was explained by habitat fertility, by relatedness between exotic species and the native community or rather by native species diversity. 3. In a second part of the study, we also tested the ERH and the R-ERH by comparing damage of plants in 28 pairs of co-occurring native and exotic populations, representing nine congeneric pairs of native and exotic species. 4. In the first part of the study, diversity of damage morphotypes and damage levels of exotic populations were greater in resource-rich habitats. Co-occurrence of closely related, native species in the community significantly increased the probability of fruit damage. Herbivory on exotics was less likely in communities with high phylogenetic diversity. 5. In the second part of the study, exotic and native congeneric populations incurred similar damage diversity and levels, irrespective of whether they co-occurred in nutrient-poor or nutrient-rich habitats. 9. Synthesis. We identified habitat productivity as a major community factor affecting accumulation of enemy damage by exotic populations. Similar damage levels in exotic and native congeneric populations, even in species pairs from fertile habitats, suggest that the enemy release hypothesis or the R-ERH cannot always explain the invasiveness of introduced species.

Central European plant species from more productive habitats are more invasive at a global scale

Aim: Accumulating evidence indicates that species may be pre-adapted for invasion success in new ranges. In the light of increasing global nutrient accumulation, an important candidate pre-adaptation for invasiveness is the ability to grow in nutrient-rich habitats. Therefore we tested whether globally invasive species originating from Central Europe have come from more productive rather than less productive habitats. A further important candidate pre-adaptation for invasiveness is large niche width. Therefore, we also tested whether species able to grow across habitats with a wider range of productivity are more invasive. Location: Global with respect to invasiveness, and Central European with respect to origin of study species. Methods  We examined whether average habitat productivity and its width across habitats are significant predictors of the success of Central European species as aliens and as weeds elsewhere in the world based on data in the Global Compendium of Weeds. The two habitat productivity measures were derived from nutrient indicator values (after Ellenberg) of accompanying species present in vegetation records of the comprehensive Czech National Phytosociological Database. In the analyses, we accounted for phylogenetic relatedness among species and for size of the native distribution ranges. Results: Species from more productive habitats and with a wider native habitat-productivity niche in Central Europe have higher alien success elsewhere in the world. Weediness of species increased with mean habitat productivity. Niche width was also an important determinant of weediness for species with their main occurrence in nutrient-poor habitats, but not for those from nutrient-rich habitats. Main conclusions: Our results indicate that Central European plant species from productive habitats and those species from nutrient-poor habitat with wide productivity-niche are pre-adapted to become invasive. These results suggest that the world-wide invasion success of many Central European species is likely to have been promoted by the global increase of resource-rich habitats.

Non-pathogenic Actinobacillus isolates antigenically and biochemically similar to Actinobacillus pleuropneumoniae: a novel species?

Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae.

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

Molecular phylogenetic analyses identify Alpine differentiation and dysploid chromosome number changes as major forces for the evolution of the European endemic Phyteuma (Campanulaceae)

Phyteuma is a chromosomally and ecologically diverse vascular plant genus and constitutes an excellent system for studying both the role of chromosomal change for species diversification and the evolution of high-mountain biota. This kind of research is, however, hampered by the lack of a sound phylogenetic framework exacerbated by the notoriously low predictive power of traditional taxonomy with respect to phylogenetic relationships in Campanulaceae. Based on a comprehensive taxon sampling and analyses of nuclear and plastid sequence and AFLP fingerprint data, Phyteuma is confirmed as a monophyletic group sister to the monotypic Physoplexis, which is in line with their peculiar flower morphologies. Within Phyteuma two clades, largely corresponding to previously recognized sections, are consistently found. The traditional circumscription of taxonomic series is largely rejected. Whereas distinctness of the currently recognized species is mostly corroborated, some interspecific relationships remain ambiguous due to incongruences between nuclear and plastid data. Major forces for diversification and evolution of Phyteuma are descending dysploidy (i.e., a decrease in chromosome base number) as well as allopatric and ecological differentiation within the Alps, the genus' center of species diversity. (C) 2013 Elsevier Inc. All rights reserved.

Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection

Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools.