Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand

AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profi les, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively. METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identi- fi cation of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confi rm phenotypic identifi cation of colonies using species-specifi c primers, capsule type using serogroup-specifi c primers and multiplex PCR, and to test for the presence of HSB and toxA genes. RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1–53% of tissue samples collected from pigs 5–6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confi rmed as P. multocida using a species-specifi c PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes. CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specifi c, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.

Concordant molecular and phenotypic data delineate new taxonomy and conservation priorities for the endangered mountain yellow-legged frog

The mountain yellow-legged frog Rana muscosa sensu lato, once abundant in the Sierra Nevada of California and Nevada, and the disjunct Transverse Ranges of southern California, has declined precipitously throughout its range, even though most of its habitat is protected. The species is now extinct in Nevada and reduced to tiny remnants in southern California, where as a distinct population segment, it is classified as Endangered. Introduced predators (trout), air pollution and an infectious disease (chytridiomycosis) threaten remaining populations. A Bayesian analysis of 1901 base pairs of mitochondrial DNA confirms the presence of two deeply divergent clades that come into near contact in the Sierra Nevada. Morphological studies of museum specimens and analysis of acoustic data show that the two major mtDNA clades are readily differentiated phenotypically. Accordingly, we recognize two species, Rana sierrae, in the northern and central Sierra Nevada, and R. muscosa, in the southern Sierra Nevada and southern California. Existing data indicate no range overlap. These results have important implications for the conservation of these two species as they illuminate a profound mismatch between the current delineation of the distinct population segments (southern California vs. Sierra Nevada) and actual species boundaries. For example, our study finds that remnant populations of R. muscosa exist in both the southern Sierra Nevada and the mountains of southern California, which may broaden options for management. In addition, despite the fact that only the southern California populations are listed as Endangered, surveys conducted since 1995 at 225 historic (1899-1994) localities from museum collections show that 93.3% (n=146) of R. sierrae populations and 95.2% (n=79) of R. muscosa populations are extinct. Evidence presented here underscores the need for revision of protected population status to include both species throughout their ranges.

Phenotypic variation in natural populations of kikuyu in Australia

The quality of tropical grasses is a major limitation to animal production in tropical and subtropical areas. This is mainly associated with the lower digestibility because C4 grasses have higher fibre levels. Any improvement in quality would require a reduction in the lignin and an increase in the digestion of the neutral detergent fibre content of these plants (Clark and Wilson 1993). Kikuyu (Pennisetum clandestinum) is an important grass for the dairy and beef industries of the subtropics of Australia, South Africa and New Zealand (Mears 1970). Increased digestibility could substantially improve animal production in these industries. These experiments investigated the variation in agronomic and quality of natural populations selected from diverse regions within Australia. Runners of 14 kikuyu selections were collected by project staff or local agronomists from areas considered to have grown kikuyu for over 30 years while Whittet and Noonan were established by seed. Entries were established as single spaced plants on a 1.5 m grid in a randomised block with 3 replicates and evaluated under irrigation at Mutdapilly (brown podsol) and Wollongbar (red ferrosol). Foliage height, forage production and runner yield were assessed along with crude protein (CP), in vitro dry matter digestibility (IVDMD), metabolisable energy (ME), acid detergent fibre (ADF) and neutral detergent fibre (NDF) content of the leaf in autumn, winter and spring.

Geographic and Phenotypic Variation in Heartwood and Essential-Oil Characters in Natural Populations of Santalum austrocaledonicum in Vanuatu.

Phenotypic variation in heartwood and essential-oil characters of Santalum austrocaledonicum was assessed across eleven populations on seven islands of Vanuatu Trees differed significantly in their percentage heartwood cross-sectional area and this varied independently of stem diameter The concentrations of the four major essential-oil constituents (alpha-santalol, beta-santalol. (Z)-beta-curcumen-12-ol, and cis-nuciferol) of alcohol-extracted heartwood exhibited at least tenfold and continuous tree-to-tree variation Commercially important components alpha- and beta-santalol found in individual trees ranged from 0 8-47% and 0-24 1%, respectively, across all populations, and significant (P < 0 05) differences for each were found between Individual populations. The Erromango population was unique in that the mean concentrations of its monocyclic ((Z)-beta-curcumen-12-ol and cis-nuciferol) sesquiterpenes exceeded those of Its bi- and tricyclic (alpha- and beta-santalol) sesquiterpenes Heartwood colour varied between trees and spanned 65 colour categories, but no identifiable relationships were found between heartwood colour and alpha- and beta-santalol, although a weak relationship was evident between colour saturation and total oil concentration These results indicate that the heartwood colour is not a reliable predictive trait for oil quality The results of this study highlight the knowledge gaps in fundamental understanding of heartwood biology in Santalum genus The intraspecific variation in heartwood cross-sectional area. oil concentration. and oil quality traits is of considerable importance to the domestication of sandalwood and present opportunities for the development of highly superior S austrocaledonicum cultivars that conform to the industry's International Standards used for S album.

Phenotypic and genotypic variation within populations of kikuyu (Pennisetum clandestinum) in Australia.

Experiments at 2 sites in subtropical eastern Australia investigated the variation in agronomic attributes, quality and genetic structure existing within: naturally-occurring populations of kikuyu ( Pennisetum clandestinum) from within Australia; selections produced from the treatment of Whittet seed with mutagenic chemicals; and available cultivars. Runners were collected from coastal areas extending from Western Australia to the Atherton Tableland in north Queensland. One experiment evaluated 10 mutagenic selections and 4 cultivars in a lattice design and the other evaluated 12 ecotypes and 3 cultivars in a randomised block design. The experimental unit was single plants, which were sown on a 1.5 m grid into a weed-free seed-bed (Mutdapilly) or a killed kikuyu stand (Wollongbar), both of which were kept clear of weeds and other kikuyu plants for the duration of the experiments. Foliage height, forage production and runner yield were assessed. Leaf material was analysed for concentrations of crude protein (CP), acid detergent fibre (ADF) and neutral detergent fibre (NDF) and for in vitro dry matter digestibility (IVDDM) in autumn, winter and spring. DNA was extracted from each plant in the ecotype comparison and subjected to a modified DAF (DNA amplification fingerprinting) analysis to determine the level of genetic relatedness. In the first experiment, none of the mutagenic lines derived from Whittet yielded significantly more or was more digestible than commercial Whittet material, although some selections were superior to the other commercial kikuyu cultivars, Noonan and Crofts, and 'common' kikuyu. However, there were significant differences in plant height and runner expansion. In the second experiment, significant differences in plant height, foliage yield, runner development, and leaf CP, ADF, NDF and IVDDM concentrations were demonstrated between the ecotypes, mutagenic selections and cultivars. There was a 4- to 6-fold difference in plant yield and a 6- to 10-fold difference in runner production between the ecotypes at the 2 sites. Quality of the leaf ranged from 200 to 270 g/kg (CP), from 700 to 770 g/kg (IVDDM), from 170 to 250 g/kg (ADF) and from 470 to 550 g/kg (NDF). Improvements in quality and agronomic attributes were not mutually exclusive. Genetic fingerprint analysis of the kikuyu lines indicated that they formed 2 broad groupings. Most of the regional ecotypes were grouped with 'common' kikuyu as represented by the material collected from Wollongbar, and the Beechmont, Atherton Tableland and Gympie ecotypes were grouped with the registered cultivars Whittet, Noonan and Crofts. Two lines produced by mutagenesis from Whittet remained closely linked to Whittet. These results suggest that there was variation between populations of kikuyu in yield, quality and genetic diversity but that mutagenesis by treating seed with sodium azide and diethylene sulphide did not achieve a significant change in the digestibility of leaf over cv. Whittet.

FTO genotype is associated with phenotypic variability of body mass index

There is evidence across several species for genetic control of phenotypic variation of complex traits1, 2, 3, 4, such that the variance among phenotypes is genotype dependent. Understanding genetic control of variability is important in evolutionary biology, agricultural selection programmes and human medicine, yet for complex traits, no individual genetic variants associated with variance, as opposed to the mean, have been identified. Here we perform a meta-analysis of genome-wide association studies of phenotypic variation using ~170,000 samples on height and body mass index (BMI) in human populations. We report evidence that the single nucleotide polymorphism (SNP) rs7202116 at the FTO gene locus, which is known to be associated with obesity (as measured by mean BMI for each rs7202116 genotype)5, 6, 7, is also associated with phenotypic variability. We show that the results are not due to scale effects or other artefacts, and find no other experiment-wise significant evidence for effects on variability, either at loci other than FTO for BMI or at any locus for height. The difference in variance for BMI among individuals with opposite homozygous genotypes at the FTO locus is approximately 7%, corresponding to a difference of ~0.5 kilograms in the standard deviation of weight. Our results indicate that genetic variants can be discovered that are associated with variability, and that between-person variability in obesity can partly be explained by the genotype at the FTO locus. The results are consistent with reported FTO by environment interactions for BMI8, possibly mediated by DNA methylation9, 10. Our BMI results for other SNPs and our height results for all SNPs suggest that most genetic variants, including those that influence mean height or mean BMI, are not associated with phenotypic variance, or that their effects on variability are too small to detect even with samples sizes greater than 100,000.

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

Chromosomal rearrangements, phenotypic variation and modularity: A case study from a contact zone between house mouse Robertsonian races in Central Italy

The Western European house mouse, Mus musculus domesticus, is well-known for the high frequency of Robertsonian fusions that have rapidly produced more than 50 karyotipic races, making it an ideal model for studying the mechanisms of chromosomal speciation. The mouse mandible is one of the traits studied most intensively to investigate the effect of Robertsonian fusions on phenotypic variation within and between populations. This complex bone structure has also been widely used to study the level of integration between different morphogenetic units. Here, with the aim of testing the effect of different karyotypic assets on the morphology of the mouse mandible and on its level of modularity, we performed morphometric analyses of mice from a contact area between two highly metacentric races in Central Italy. We found no difference in size, while the mandible shape was found to be different between the two Robertsonian races, even after accounting for the genetic relationships among individuals and geographic proximity. Our results support the existence of two modules that indicate a certain degree of evolutionary independence, but no difference in the strength of modularity between chromosomal races. Moreover, the ascending ramus showed more pronounced interpopulation/race phenotypic differences than the alveolar region, an effect that could be associated to their different polygenic architecture. This study suggests that chromosomal rearrangements play a role in the house mouse phenotypic divergence, and that the two modules of the mouse mandible are differentially affected by environmental factors and genetic makeup.

The Consequences of Phenotypic Plasticity in Cyclically Varying Environments: A Genetic Algorithm Study

By “phenotypic plasticity” we refer to the capacity of a genotype to exhibit different phenotypes, whether in the same or in different environments. We have previously demonstrated that phenotypic plasticity can improve the degree of adaptation achieved via natural selection (Behera & Nanjundiah, 1995). That result was obtained from a genetic algorithm model of haploid genotypes (idealized as one-dimensional strings of genes) evolving in a fixed environment. Here, the dynamics of evolution is examined under conditions of a cyclically varying environment. We find that the rate of evolution, as well as the extent of adaptation (as measured by mean population fitness) is lowered because of environmental cycling. The decrease is adaptation caused by a varying environment can, however, be partly or wholly compensated by an increase in the degree of plasticity that a genotype is capable of. Also, the reduction of population fitness caused by a variable environment can be partially offset by decreasing the total number of genetic loci. We conjecture that an increase in genome size may have been among the factors responsible for the evolution of phenotypic plasticity.

Phenotypic plasticity and longevity in plants and animals: cause and effect?

Immobile plants and immobile modular animals outlive unitary animals. This paper discusses competing but not necessarily mutually exclusive theories to explain this extreme longevity, especially from the perspective of phenotypic plasticity. Stem cell immortality, vascular autonomy, and epicormic branching are some important features of the phenotypic plasticity of plants that contribute to their longevity. Monocarpy versus polycarpy can also influence the kind of senescent processes experienced by plants. How density-dependent phenomena affecting the establishment of juveniles in these immobile organisms can influence the evolution of senescence, and consequently longevity, is reviewed and discussed. Whether climate change scenarios will favour long-lived or short-lived organisms, with their attendant levels of plasticity, is also presented.

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.