Phylogeny of Pasteurellaceae

This chapter provides an overview on the DNA based phylogeny of the family Pasteurellaceae and the genetic relatedness between taxa taking into account the various gene targets and approaches applied in the literature. The classical 16S rRNA gene based phylogeny as well as phylogenies based on house-keeping genes are described. Moreover, strength and weakness of the different trees and their topology are discussed based on the phylogenetic groups resolved. The data should help to get a clearer picture on the recent, current and future classification and also provide information to genetic characterization of members of the family. The history of phylogeny applied to the family as well as the phylogenetic history of the family is thereby presented. In this way it is the story of the search for the optimal phylogenetic marker without giving a final conclusive suggestion but it is also a resource for choosing the appropriate gene target(s) for people investigating the phylogeny of groups of Pasteurellaceae.

Identification of animal Pasteurellaceae by MALDI-TOF mass spectrometry

Species of the family Pasteurellaceae play an important role as primary or opportunistic animal pathogens. In veterinary diagnostic laboratories identification of this group of bacteria is mainly done by phenotypic assays while genetic identification based on housekeeping genes is mostly used for research and particularly important diagnostic samples. MALDI-TOF MS seems to represent a promising alternative to the currently practiced cumbersome, phenotypic diagnostics carried out in many veterinary diagnostic laboratories. We therefore assessed its application for animal associated members of the family Pasteurellaceae. The Bruker Biotyper 3.0 database was complemented with reference spectra of clinically relevant as well as commensal animal Pasteurellaceae species using generally five strains per species or subspecies and tested for its diagnostic potential with additional, well characterized field isolates. About 250 strains comprising 15 genera and more than 40 species and subspecies were included in the study, covering most representatives of the family. A high discrimination at the genus and species level was observed. Problematic discrimination was only observed with some closely related species and subspecies. MALDI-TOF MS was shown to represent a highly potent method for the diagnosis of this group of animal pathogens, combining speed, precision and low running costs.

Emended description of porcine [Pasteurella] aerogenes, [Pasteurella] mairii and [Actinobacillus] rossii

The aim of this study was to improve the definition and identification of a group of veterinarily important bacteria referred to as the [Pasteurella] aerogenes-[Pasteurella] mairii-[Actinobacillus] rossii complex. These organisms have mainly been isolated from the reproductive and intestinal tracts of pigs and in most cases have been considered as opportunistic pathogens. A collection of 87 strains were characterized by phenotypic analysis from which 41 strains were selected for 16S rRNA gene sequence comparison, out of which 23 have been sequenced in the present study. One group of 21 strains phenotyped as biovars 1, 3-5, 9-11, 19 and 25-27, including the type strain of [P.] aerogenes, showed 16S rRNA gene sequence similarities of 99.6 % or higher; another group of 18 strains including biovars 2, 6-8, 12-15, 21, 23, 24 and 26A and the type strain of [A.] rossii showed 97.8 % or higher 16S rRNA gene sequence similarity. Between the two groups, 93.8-95.7 % 16S rRNA gene sequence similarity was observed. Strains of [P.] mairii showed 99.5 % similarity, with 95.5-97.2 and 93.8-95.5 % similarity to strains of [P.] aerogenes and [A.] rossii, respectively. Four strains could not be classified with any of these groups and belonged to other members of Pasteurellaceae. Comparisons were also made to DNA-DNA hybridization data. Biovars 1, 9, 10, 11 and 19, including the type strain of [P.] aerogenes, linked at 70 % DNA reassociation, whereas strains identified as biovars 2, 6, 7, 8, 12, 15 and 21 of [P.] aerogenes linked at 81 %. The latter group most likely represents [A.] rossii based on the 16S rRNA gene sequence comparisons. DNA reassociation between the [P.] aerogenes and [A.] rossii groups was at most 37 %, whereas 47 % was the highest DNA reassociation found between [P.] aerogenes and [P.] mairii. The study showed that [P.] aerogenes, [P.] mairii and [A.] rossii can not be easily separated and may consequently be misidentified based on current knowledge of their phenotypic characteristics. In addition, these taxa are difficult to separate from other taxa of the Pasteurellaceae. A revised scheme for separation based upon phenotypic characters is suggested for the three species [P.] aerogenes emend., [P.] mairii emend. and [A.] rossii emend., with the respective type strains ATCC 27883T, NCTC 10699T and ATCC 27072T.

Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae

Phylogenies of housekeeping gene and 16S rRNA gene sequences were compared to improve the classification of the bacterial family Pasteurellaceae and knowledge of the evolutionary relationships of its members. Deduced partial protein sequences of the housekeeping genes atpD, infB and rpoB were compared in 28, 36 and 28 representative taxa of the Pasteurellaceae, respectively. The monophyly of representatives of the genus Gallibacterium was recognized by analysis of all housekeeping genes, while members of Mannheimia, Actinobacillus sensu stricto and the core group of Pasteurella sensu stricto formed monophyletic groups with two out of three housekeeping genes. Representatives of Mannheimia, Actinobacillus sensu stricto, [Haemophilus] ducreyi and [Pasteurella] trehalosi formed a monophyletic unit by analysis of all three housekeeping genes, which was in contrast to the 16S rRNA gene-derived phylogeny, where these taxa occurred at separate positions in the phylogenetic tree. Representatives of the Rodent, Avian and Aphrophilus-Haemophilus 16S rRNA gene groups were weakly supported by phylogenetic analysis of housekeeping genes. Phylogenies derived by comparison of the housekeeping genes diverged significantly from the 16S rRNA gene-derived phylogeny as evaluated by the likelihood ratio test. A low degree of congruence was also observed between the individual housekeeping gene-derived phylogenies. Estimates on speciation derived from 16S rRNA and housekeeping gene sequence comparisons resulted in quite different evolutionary scenarios for members of the Pasteurellaceae. The phylogeny based on the housekeeping genes supported observed host associations between Mannheimia, Actinobacillus sensu stricto and [Pasteurella] trehalosi and animals with paired hooves.

Phylogeny of the family Pasteurellaceae based on rpoB sequences

Sequences of the gene encoding the beta-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.

Proposal of Histophilus somni gen. nov., sp. nov. for the three species incertae sedis 'Haemophilus somnus', 'Haemophilus agni' and 'Histophilus ovis'

Earlier investigations have shown that 'Haemophilus somnus', 'Haemophilus agni' and 'Histophilus ovis' represent the same species. In the present investigation, the taxonomic position of this species is explored further by sequencing the 16S rRNA and rpoB genes of strains that were investigated previously by DNA-DNA hybridization. These results clearly support the allocation of this species to a novel genus within the family PASTEURELLACEAE: The phenotypic separation of Histophilus somni gen. nov., sp. nov. from other members of the family can, for most strains, be based on capnophilia, yellowish pigmentation and indole production. However, due to phenotypic variation, the use of a species-specific PCR test based on the 16S rRNA gene is included in the species description. This is justified by the high sequence similarity of the 16S rRNA gene within the species and the fact that the highest sequence similarity to any other taxon within the family is 93.4 %. The type strain, 8025(T)=ATCC 43625(T)=CCUG 36157(T), was isolated in the USA from a bovine brain with lesions of thromboembolic meningoencephalitis.

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.

Is characterization of a single isolate sufficient for valid publication of a new genus or species? Proposal to modify recommendation 30b of the Bacteriological Code (1990 Revision)

From 1990 to 2000, the number of published named taxa based upon new isolates at species and genus levels in International Journal of Systematic and Evolutionary Microbiology, formerly International Journal of Systematic Bacteriology, have increased by approximately four- and sevenfold, respectively. New taxa based upon characterization of only a single isolate remained at around 40% for both categories. The Bacteriological Code (1990 Revision) has no recommendations on the number of strains required for definition of new taxa. For a few groups, a minimum number of 5-10 strains has been suggested in minimal standards. Since an exponential increase in new taxa can be expected in the future, the authors discuss problems related to naming new species and genera based upon descriptions of a single isolate and suggest that this practice is re-evaluated. It is proposed that the following should be added to Recommendation 30b of the Bacteriological Code: 'Descriptions should be based on as many strains as possible (minimum five), representing different sources with respect to geography and ecology in order to be well characterized both phenotypically and genotypically, to establish the centre (from which the type strain could be chosen) and the extent of the cluster to be named. In addition, comparative studies should be performed, including reference strains that represent neighbouring species and/or genera, in order to give descriptions that are sufficiently detailed to allow differentiation from these neighbours.'

A very rare cancer in Down syndrome: medulloblastoma. Epidemiological data from 13 countries

Persons with Down syndrome (DS) uniquely have an increased frequency of leukemias but a decreased total frequency of solid tumors. The distribution and frequency of specific types of brain tumors have never been studied in DS. We evaluated the frequency of primary neural cell embryonal tumors and gliomas in a large international data set. The observed number of children with DS having a medulloblastoma, central nervous system primitive neuroectodermal tumor (CNS-PNET) or glial tumor was compared to the expected number. Data were collected from cancer registries or brain tumor registries in 13 countries of Europe, America, Asia and Oceania. The number of DS children with each category of tumor was treated as a Poisson variable with mean equal to 0.000884 times the total number of registrations in that category. Among 8,043 neural cell embryonal tumors (6,882 medulloblastomas and 1,161 CNS-PNETs), only one patient with medulloblastoma had DS, while 7.11 children in total and 6.08 with medulloblastoma were expected to have DS. (p 0.016 and 0.0066 respectively). Among 13,797 children with glioma, 10 had DS, whereas 12.2 were expected. Children with DS appear to be specifically protected against primary neural cell embryonal tumors of the CNS, whereas gliomas occur at the same frequency as in the general population. A similar protection against neuroblastoma, the principal extracranial neural cell embryonal tumor, has been observed in children with DS. Additional genetic material on the supernumerary chromosome 21 may protect against embryonal neural cell tumor development.

Public health risks of enterobacterial isolates producing extended-spectrum β-lactamases or AmpC β-lactamases in food and food-producing animals: an EU perspective of epidemiology, analytical methods, risk factors, and control options

The blaESBL and blaAmpC genes in Enterobacteriaceae are spread by plasmid-mediated integrons, insertion sequences, and transposons, some of which are homologous in bacteria from food animals, foods, and humans. These genes have been frequently identified in Escherichia coli and Salmonella from food animals, the most common being blaCTX-M-1, blaCTX-M-14, and blaCMY-2. Identification of risk factors for their occurrence in food animals is complex. In addition to generic antimicrobial use, cephalosporin usage is an important risk factor for selection and spread of these genes. Extensive international trade of animals is a further risk factor. There are no data on the effectiveness of individual control options in reducing public health risks. A highly effective option would be to stop or restrict cephalosporin usage in food animals. Decreasing total antimicrobial use is also of high priority. Implementation of measures to limit strain dissemination (increasing farm biosecurity, controls in animal trade, and other general postharvest controls) are also important.

Camel Streptococcus agalactiae populations are associated with specific disease complexes and acquired the tetracycline resistance gene tetM via a Tn916-like element

Camels are the most valuable livestock species in the Horn of Africa and play a pivotal role in the nutritional sustainability for millions of people. Their health status is therefore of utmost importance for the people living in this region. Streptococcus agalactiae, a Group B Streptococcus (GBS), is an important camel pathogen. Here we present the first epidemiological study based on genetic and phenotypic data from African camel derived GBS. Ninety-two GBS were characterized using multilocus sequence typing (MLST), capsular polysaccharide typing and in vitro antimicrobial susceptibility testing. We analysed the GBS using Bayesian linkage, phylogenetic and minimum spanning tree analyses and compared them with human GBS from East Africa in order to investigate the level of genetic exchange between GBS populations in the region. Camel GBS sequence types (STs) were distinct from other STs reported so far. We mapped specific STs and capsular types to major disease complexes caused by GBS. Widespread resistance (34%) to tetracycline was associated with acquisition of the tetM gene that is carried on a Tn916-like element, and observed primarily among GBS isolated from mastitis. The presence of tetM within different MLST clades suggests acquisition on multiple occasions. Wound infections and mastitis in camels associated with GBS are widespread and should ideally be treated with antimicrobials other than tetracycline in East Africa.

A Foraging Cost of Migration for a Partially Migratory Cyprinid Fish

Migration has evolved as a strategy to maximise individual fitness in response to seasonally changing ecological and environmental conditions. However, migration can also incur costs, and quantifying these costs can provide important clues to the ultimate ecological forces that underpin migratory behaviour. A key emerging model to explain migration in many systems posits that migration is driven by seasonal changes to a predation/growth potential (p/g) trade-off that a wide range of animals face. In this study we assess a key assumption of this model for a common cyprinid partial migrant, the roach Rutilus rutilus, which migrates from shallow lakes to streams during winter. By sampling fish from stream and lake habitats in the autumn and spring and measuring their stomach fullness and diet composition, we tested if migrating roach pay a cost of reduced foraging when migrating. Resident fish had fuller stomachs containing more high quality prey items than migrant fish. Hence, we document a feeding cost to migration in roach, which adds additional support for the validity of the p/g model of migration in freshwater systems.

When language of instruction and language of application differ: Cognitive costs of bilingual mathematics learning

Bilingual education programs implicitly assume that the acquired knowledge is represented in a language-independent way. This assumption, however, stands in strong contrast to research findings showing that information may be represented in a way closely tied to the specific language of instruction and learning. The present study aims to examine whether and to which extent cognitive costs appear during arithmetic learning when language of instruction and language of retrieving differ. Thirty-nine high school students participating in a bilingual education program underwent a four-day training on multiplication and subtraction problems in one language (German or French), followed by a test session in which they had to solve trained as well as untrained problems in both languages. We found that cognitive costs related to language switching appeared for both arithmetic operations. Implications of our findings are discussed with respect to bilingual education as well as to cognitive mechanisms underlying different arithmetic operations.

Sizing up your enemy: individual predation vulnerability predicts migratory probability

Partial migration, in which a fraction of a population migrate and the rest remain resident, occurs in an extensive range of species and can have powerful ecological consequences. The question of what drives differences in individual migratory tendency is a contentious one. It has been shown that the timing of partial migration is based upon a trade-off between seasonal fluctuations in predation risk and growth potential. Phenotypic variation in either individual predation risk or growth potential should thus mediate the strength of the trade-off and ultimately predict patterns of partial migration at the individual level (i.e. which individuals migrate and which remain resident). We provide cross-population empirical support for the importance of one component of this model—individual predation risk—in predicting partial migration in wild populations of bream Abramis brama, a freshwater fish. Smaller, high-risk individuals migrate with a higher probability than larger, low-risk individuals, and we suggest that predation risk maintains size-dependent partial migration in this system.