Routine phenotypic identification of bacterial species of the family Pasteurellaceae isolated from animals

Pasteurellaceae are bacteria with an important role as primary or opportunistic, mainly respiratory, pathogens in domestic and wild animals. Some species of Pasteurellaceae cause severe diseases with high economic losses in commercial animal husbandry and are of great diagnostic concern. Because of new data on the phylogeny of Pasteurellaceae, their taxonomy has recently been revised profoundly, thus requiring an improved phenotypic differentiation procedure to identify the individual species of this family. A new and simplified procedure to identify species of Actinobacillus, Avibacterium, Gallibacterium, Haemophilus, Mannheimia, Nicoletella, and Pasteurella, which are most commonly isolated from clinical samples of diseased animals in veterinary diagnostic laboratories, is presented in the current study. The identification procedure was evaluated with 40 type and reference strains and with 267 strains from routine diagnostic analysis of various animal species, including 28 different bacterial species. Type, reference, and field strains were analyzed by 16S ribosomal RNA (rrs) and rpoB gene sequencing for unambiguous species determination as a basis to evaluate the phenotypic differentiation schema. Primary phenotypic differentiation is based on beta-nicotinamide adenine dinucleotide (beta-NAD) dependence and hemolysis, which are readily determined on the isolation medium. The procedure divides the 28 species into 4 groups for which particular biochemical reactions were chosen to identify the bacterial species. The phenotypic identification procedure allowed researchers to determine the species of 240 out of 267 field strains. The procedure is an easy and cost-effective system for the rapid identification of species of the Pasteurellaceae family isolated from clinical specimens of animals.

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.

Habitat selection of three cryptic Plecotus bat species in the European Alps reveals contrasting implications for conservation

Assessing the ecological requirements of species coexisting within a community is an essential requisite for developing sound conservation action. A particularly interesting question is what mechanisms govern the stable coexistence of cryptic species within a community, i.e. species that are almost impossible to distinguish. Resource partitioning theory predicts that cryptic species, like other sympatric taxa, will occupy distinct ecological niches. This prediction is widely inferred from eco-morphological studies. A new cryptic long-eared bat species, Plecotus macrobullaris, has been recently discovered in the complex of two other species present in the European Alps, with even evidence for a few mixed colonies. This discovery poses challenges to bat ecologists concerned with planning conservation measures beyond roost protection. We therefore tested whether foraging habitat segregation occurred among the three cryptic Plecotus bat species in Switzerland by radiotracking 24 breeding female bats (8 of each species). We compared habitat features at locations visited by a bat versus random locations within individual home ranges, applying mixed effects logistic regression. Distinct, species-specific habitat preferences were revealed. P. auritus foraged mostly within traditional orchards in roost vicinity, with a marked preference for habitat heterogeneity. P. austriacus foraged up to 4.7 km from the roost, selecting mostly fruit tree plantations, hedges and tree lines. P. macrobullaris preferred patchy deciduous and mixed forests with high vertical heterogeneity in a grassland dominated-matrix. These species-specific habitat preferences should inform future conservation programmes. They highlight the possible need of distinct conservation measures for species that look very much alike.

Divergent evolutionary processes associated with colonization of offshore islands

Oceanic islands have been a test ground for evolutionary theory, but here, we focus on the possibilities for evolutionary study created by offshore islands. These can be colonized through various means and by a wide range of species, including those with low dispersal capabilities. We use morphology, modern and ancient sequences of cytochrome b (cytb) and microsatellite genotypes to examine colonization history and evolutionary change associated with occupation of the Orkney archipelago by the common vole (Microtus arvalis), a species found in continental Europe but not in Britain. Among possible colonization scenarios, our results are most consistent with human introduction at least 5100 bp (confirmed by radiocarbon dating). We used approximate Bayesian computation of population history to infer the coast of Belgium as the possible source and estimated the evolutionary timescale using a Bayesian coalescent approach. We showed substantial morphological divergence of the island populations, including a size increase presumably driven by selection and reduced microsatellite variation likely reflecting founder events and genetic drift. More surprisingly, our results suggest that a recent and widespread cytb replacement event in the continental source area purged cytb variation there, whereas the ancestral diversity is largely retained in the colonized islands as a genetic ‘ark’. The replacement event in the continental M. arvalis was probably triggered by anthropogenic causes (land-use change). Our studies illustrate that small offshore islands can act as field laboratories for studying various evolutionary processes over relatively short timescales, informing about the mainland source area as well as the island.

How does the taxonomic status of allopatric populations influence species richness within African cichlid fish assemblages?

Aim  Current estimates of species richness within rapidly evolving species flocks are often highly dependent on the species status of allopatric populations that differ in phenotypic traits. These traits may be unreliable indicators of biological species status and systematists may have inconsistently assigned species among lineages or locations on the basis of these traits, thus hampering comparative studies of regional species richness and speciation rates. Our aim was to develop a method of generating standardized estimates of regional species richness suitable for comparative analysis, and to use these estimates to examine the extent and consistency of species assignment of allopatric populations within rapidly evolving cichlid fish flocks present in three east African lakes. Location  Lakes Malawi, Victoria and Tanganyika. Methods  Using published taxon co-occurrence data, a novel approach was employed to calculate standardized ‘minimum’ estimates of regional species richness for hard substrate associated complexes of cichlids within each of the lakes. Minimum estimates were based on an explicit assumption that if taxa present on equivalent habitats have disjunct distributions, then they are allopatric forms of the same species. These estimates were compared with current observed ‘high-end’ regional species richness estimates for those complexes to determine the consistency of species assignment of allopatric populations between lineages within a lake. A ‘sympatry’ index was developed to enable comparisons of levels of species assignment of allopatric populations between-lakes to be made. Results  Within each lake, the minimum and high-end estimates for species richness were significantly correlated across complexes, indicating that the complexes that contain more recognized species contain the most genuine biological species. However, comparisons of complexes among lakes revealed considerable differences. For equivalent geographical areas, substantially higher proportions of recognized species were totally allopatric within the studied Lake Malawi and Lake Victoria complexes, than those of Lake Tanganyika. Main Conclusions  Among African lakes, levels of assignment to species status of allopatric populations were found to be distinctly different. It is unclear whether the discrepancies are a consequence of differences between the lake faunas in degrees of phenotypic divergence among allopatric populations, or are simply the result of inconsistent taxonomic practices. In either case, these results have considerable wider relevance for they emphasize that quantitative measures of regional and beta diversity are critically dependent on the species status of allopatric populations, an issue usually neglected in comparative studies of species richness. The technique introduced here can be used to standardize measures of regional diversity of lineages for comparative analyses, potentially enabling more accurate identification of processes influencing rates of speciation.

Ecosystem impacts of feral rabbits on World Heritage sub-Antarctic Macquarie Island: A palaeoecological perspective

The introduction and establishment of non-indigenous species through human activities often poses a major threat to natural biodiversity. In many parts of the world management efforts are therefore focused on their eradication. The environment of World Heritage sub-Antarctic Macquarie Island has been severely damaged by non-indigenous species including rabbits, rats and mice, introduced from the late AD 1800s. An extensive eradication programme is now underway which aims to remove all rabbits and rodents. To provide a long-term context for assessing the Island's pre-invasion state, invasion impacts, and to provide a baseline for monitoring its recovery, we undertook a palaeoecological study using proxies in a lake sediment core. Sedimentological and diatom analyses revealed an unproductive catchment and lake environment persisted for ca. 7100 years prior to the introduction of the invasive species. After ca. AD 1898, unprecedented and statistically significant environmental changes occurred. Lake sediment accumulation rates increased >100 times due to enhanced catchment inputs and within-lake production. Total carbon and total nitrogen contents of the sediments increased by a factor of four. The diatom flora became dominated by two previously rare species. The results strongly suggest a causal link between the anthropogenic introduction of rabbits and the changes identified in the lake sediments. This study provides an example of how palaeoecology may be used to determine baseline conditions prior to the introduction of non-indigenous species, quantify the timing and extent of changes, and help monitor the recovery of the ecosystem and natural biodiversity following successful non-indigenous species eradication programmes.

Real-Time RT-PCR for the Detection of Lyssavirus Species

The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

Worldwide evidence of a unimodal relationship between productivity and plant species richness

The search for predictions of species diversity across environmental gradients has challenged ecologists for decades. The humped-back model (HBM) suggests that plant diversity peaks at intermediate productivity; at low productivity few species can tolerate the environmental stresses, and at high productivity a few highly competitive species dominate. Over time the HBM has become increasingly controversial, and recent studies claim to have refuted it. Here, by using data from coordinated surveys conducted throughout grasslands worldwide and comprising a wide range of site productivities, we provide evidence in support of the HBM pattern at both global and regional extents. The relationships described here provide a foundation for further research into the local, landscape, and historical factors that maintain biodiversity.

How many species of cichlid fishes are there in African lakes?

The endemic cichlid fishes of Lakes Malawi, Tanganyika and Victoria are textbook examples of explosive speciation and adaptive radiation, and their study promises to yield important insights into these processes. Accurate estimates of species richness of lineages in these lakes, and elsewhere, will be a necessary prerequisite for a thorough comparative analysis of the intrinsic and extrinsic factors influencing rates of diversification. This review presents recent findings on the discoveries of new species and species flocks and critically appraises the relevant evidence on species richness from recent studies of polymorphism and assortative mating, generally using behavioural and molecular methods. Within the haplochromines, the most species-rich lineage, there are few reported cases of postzygotic isolation, and these are generally among allopatric taxa that are likely to have diverged a relatively long time in the past. However, many taxa, including many which occur sympatrically and do not interbreed in nature, produce viable, fertile hybrids. Prezygotic barriers are more important, and persist in laboratory conditions in which environmental factors have been controlled, indicating the primary importance of direct mate preferences. Studies to date indicate that estimates of alpha (within-site) diversity appear to be robust. Although within-species colour polymorphisms are common, these have been taken into account in previous estimates of species richness. However, overall estimates of species richness in Lakes Malawi and Victoria are heavily dependent on the assignation of species status to allopatric populations differing in male colour. Appropriate methods for testing the specific status of allopatric cichlid taxa are reviewed and preliminary results presented.

Explosive speciation rates and unusual species richness in haplochromine cichlid fishes: Effects of sexual selection

Ancient lakes are often unusually species rich, mostly as a result of radiation and species-flock formation having taken place in only one or a few of many taxa present. Understanding why some taxa radiate and others do not is at the heart of understanding biodiversity. In this chapter I discuss possible explanations for disproportionally large species numbers in some cichlid fish lineages in East African Great Lakes: the halochromine cichlid fishes in Lakes Victoria and Malawi. I show that speciation rates in this group are higher than in any other lacustrine fish radiation. Against this background, I review hypotheses put forward to explain diversity in cichlid species flocks. The evolution of species diversity requires three processes: speciation, ecological radiation and anatomical diversification, and it is wrong to consider hypotheses that are relevant to different processes as alternatives to each other. The African cichlid species flocks show unusually high ecological species packing in several phylogenetic groups and unusually high speciation rates in haplochromines. Therefore, it maybe concluded that at least two evolutionary models are required to explain the difference between cichlid diversity and other fish diversity in East African Lakes: one for speciation in haplochromines and one for coexistence. Subsequently I review work on speciation in haplochromines, and in particular studies aimed at testing the hypothesis of speciation by sexual selection. Haplochromines have a polygynous mating system, conducive to sexual selection, but other polygynous cichlids are not particularly species rich. This suggests that more than just strong sexual selection is required to explain haplochromine species richness. Recent palaeoecological evidence undermines the previously popular hypotheses that explained the species richness of Lake Victoria in terms of speciation under varying natural or sexual selection regimes in satellite lakes or in isolated lake basins. I summarize experimental and comparative studies, which provide evidence for two mechanisms of sympatric speciation by disruptive sexual selection on polymorphic coloration. Such modes of speciation may explain (i) the high speciation rates in colour polymorphic lineages of haplochromine cichlids under conditions where colour variation is visible in clear water, and (ii) in combination with factors that affect population survival, the unusual species richness in haplochromine species flocks. I argue that sexual selection, if disruptive, can accelerate the pace of adaptive radiation because the resultant genetic population fragmentation allows a much increased rate of differential response to disruptive natural selection. Hence, the ecological pattern of diversity resembles that produced by disruptive natural selection, with the difference that disruptive sexual selection continues to cause (gross) speciation even after niche space is saturated. This may explain the unusually high numbers of very closely related and ecologically similar species in haplochromine species flocks. The role of disruptive sexual selection is twofold: it not only causes speciation, but also maintains reproductive isolation in sympatry between species that have evolved in sympatry or allopatry. Therefore, the maintenance of diversity in species flocks that originated through sexual selection depends on the persistence of the selection regime within the environmental signal space under which that diversity evolved.

Pinus nigra (European black pine) as the dominant species of the last glacial pinewoods in south-western to central Iberia: a morphological study of modern and fossil pollen

Aim Our aim was to discriminate different species of Pinus via pollen analysis in order to assess the responses of particular pine species to orbital and millennial-scale climate changes, particularly during the last glacial period. Location Modern pollen grains were collected from current pine populations along transects from the Pyrenees to southern Iberia and the Balearic Islands. Fossil pine pollen was recovered from the south-western Iberian margin core MD95-2042. Methods We measured a set of morphological traits of modern pollen from the Iberian pine species Pinus nigra, P. sylvestris, P. halepensis, P. pinea and P. pinaster and of fossil pine pollen from selected samples of the last glacial period and the early to mid-Holocene. Classification and regression tree (CART) analysis was used to establish a model from the modern dataset that discriminates pollen from the different pine species and allows identification of fossil pine pollen at the species level. Results The CART model was effective in separating pollen of P. nigra and P. sylvestris from that of the Mediterranean pine group (P. halepensis, P. pinea and P. pinaster). The pollen of Pinus nigra diverged from that of P. sylvestris by having a more flattened corpus. Predictions using this model suggested that fossil pine pollen is mainly from P. nigra in all the samples analysed. Pinus sylvestris was more abundant in samples from Greenland stadials than Heinrich stadials, whereas Mediterranean pines increased in samples from Greenland interstadials and during the early to mid-Holocene. Main conclusions Morphological parameters can be successfully used to increase the taxonomic resolution of fossil pine pollen at the species level for the highland pines (P. nigra and P. sylvestris) and at the group of species level for the Mediterranean pines. Our study indicates that P. nigra was the dominant component of the last glacial south-western/central Iberian pinewoods, although the species composition of these woodlands varied in response to abrupt climate changes.

Phenotypic plasticity is a negative, though weak, predictor of the commonness of 105 grassland species

Aim The usual hypothesis about the relationship between niche breadth and range size posits that species with the capacity to use a wider range of resources or to tolerate a greater range of environmental conditions should be more widespread. In plants, broader niches are often hypothesized to be due to pronounced phenotypic plasticity, and more plastic species are therefore predicted to be more common. We examined the relationship between the magnitude of phenotypic plasticity in five functional traits, mainly related to leaves, and several measures of abundance in 105 Central European grassland species. We further tested whether mean values of traits, rather than their plasticity, better explain the commonness of species, possibly because they are pre-adapted to exploiting the most common resources. Location Central Europe. Methods In a multispecies experiment with 105 species we measured leaf thickness, leaf greenness, specific leaf area, leaf dry matter content and plant height, and the plasticity of these traits in response to fertilization, waterlogging and shading. For the same species we also obtained five measures of commonness, ranging from plot-level abundance to range size in Europe. We then examined whether these measures of commonness were associated with the magnitude of phenotypic plasticity, expressed as composite plasticity of all traits across the experimental treatments. We further estimated the relative importance of trait plasticity and trait means for abundance and geographical range size. Results More abundant species were less plastic. This negative relationship was fairly consistent across several spatial scales of commonness, but it was weak. Indeed, compared with trait means, plasticity was relatively unimportant for explaining differences in species commonness. Main conclusions Our results do not indicate that larger phenotypic plasticity of leaf morphological traits enhances species abundance. Furthermore, possession of a particular trait value, rather than of trait plasticity, is a more important determinant of species commonness.

A STUDY OF ILLNESS AND RESERVOIRS ASSOCIATED WITH GIARDIA LAMBLIA IN RURAL EGYPT

A longitudinal investigation of the health effects and reservoirs of Giardia lamblia was undertaken in forty households located in a rural Nile Delta region of Egypt. Stool specimens obtained once weekly for six months from two to four year old children were cyst or trophozoite-positive in 42 percent of the 724 examined. The mean duration of excretion in all but one Giardia-negative child was seven and one-half weeks with a range of one to 17 weeks. Clinical symptoms of illness were frequently observed within a month before or after Giardia excretion in stool of children, but a statistical inference of association was not demonstrated.^ Seventeen percent of 697 specimens obtained from their mothers was Giardia-positive for a mean duration of four weeks and a range of one to 18 weeks. Mothers were observed to excrete Giardia in stool less frequently during pregnancy than during lactation.^ Nine hundred sixty-two specimens were collected from 13 species of household livestock. Giardia was detected in a total of 22 specimens from cows, goats, sheep and one duck. Giardia cysts were detected in three of 899 samples of household drinking water.^ An ELISA technique of Giardia detection in human and animal stool was field tested under variable environmental conditions. The overall sensitivity of the assay of human specimens was 74 percent and specificity was 97 percent. These values for assay of animal specimens were 82 percent and 98 percent, respectively.^ Surface antigen studies reported from the NIH Laboratory of Parasitic Diseases show that antigens of three Egyptian human isolates are different from each other and from most other isolates against which they were tested.^ The ubiquity of human and animal fecal contamination combined with estimates of ill days per child per year in this setting are substantial arguments for the introduction of a suggested mass parasite control program to intervene in the cyclical transmission of agents of enteric disease. ^

A study of the association between timing of solid food introduction in infants and overweight and obesity in pre-school aged children in NHANES 2003-2008

Background. Previous studies suggest an association between timing of introduction of solid food and increased risk of obesity in pre-school aged children, but no study included a representative sample of US children. We sought to examine whether there was any association between the timing of solid food introduction and overweight/obesity in pre-school aged children. Design/methods. Cross-sectional study of a nationally representative sample (N=2050) of US children aged 2 to 5 years with information on infant feeding practices and measured weight and height from the National Health and Nutrition Examination Survey 2003–2008. The main outcome measure was BMI for age and sex ≥ 85th percentile. The main exposure was timing of solid food introduction at < 4, 4–5, or ≥ 6 months of age. Binomial logistic regression was used in the analysis controlling for child's sex, birth weight and breastfeeding status as well as maternal age at birth, smoking status and socio-demographic variables. Results. Two thousand and fifty children were included in the sample; 51% male and 49% female; 57.1% Non-Hispanic White, 21.9% Hispanic, 14.0% Non-Hispanic Black, and 7% other race/ethnicity. Twenty-two percent of the children were overweight or obese. Sixty-nine percent were breastfed or fed breast milk at birth and 36% continued breastfeeding for ≥ six months. Solid foods were introduced before 4 months of age for 11.2% of the children; 30.3% received solid foods between 4 to 5 months; with 58.6% receiving solid foods at 6 months or later. Timing of solid food introduction was not associated with weight status (OR= 1.36, 95% CI [0.83–2.24]). Formula-fed infants and infants breastfed for < 4 months had increased odds of overweight and obesity (OR=1.54, 95% CI [1.05–2.27] and OR= 1.60, 95% CI [1.05–2.44], respectively) when compared to infants breastfed for ≥ 6 months. Conclusion. Timing of solid food introduction was not associated with weight status in a national sample of US children ages 2 to 5 years. More focus should be placed on promoting breastfeeding and healthy infant feeding practices as strategies to prevent obesity in children. ^

(Appendix) Benthic foraminifera distribution of upper Creatceous and Paleocene at DSDP Hole 72-516F

Benthic foraminifers of the Coniacian-Santonian through the Paleocene were recovered from a continuous pelagic carbonate section from Hole 516F on the Rio Grande Rise. Sixty-five genera and 153 species have been identified, most of which have been reported from other localities. Bathyal depths are reflected in the benthic assemblages dominated by gavelinellids (Gavelinella beccariiformis, G. velascoensis), Nuttallides truempyi, and various gyroidinids and buliminids. Rapid subsidence during the Coniacian-Santonian from nearshore to upper to middle bathyal depths was followed by much reduced subsidence, with the Campanian-Paleocene interval accumulating at middle bathyal to lower bathyal depths. A census study based on detailed sampling reveals major changes in benthic faunal composition at the Cretaceous/Tertiary boundary transition. It was a time of rapid turnover, with the extinctions of numerous species and the introduction of many new species. Overall, species diversity decreases about 20%, and approximately one-third of latest Maestrichtian species do not survive to the end of the Cretaceous. This shift indicates a significant environmental change in the deep sea, the precise nature of which is not apparent from the foraminifers or their enclosing sediments.