Employment of 16S rDNA gene sequencing techniques for improved identification of difficult-to-identify bacterial veterinary pathogens

To employ 16S rDNA PCR and automated sequencing techniques to identify a collection of bacterial veterinary pathogens from avian, equine, canine and ovine sources, that have proven difficult to identify, employing conventional cultural techniques. Universal or “broad-range” eubacterial PCR was performed on a collection of 46 difficult-to-identify bacterial isolates originating from clinical veterinary specimens. 16S rDNA PCR was performed using two sets of universal primers to successfully generate a composite amplicon of 1,068 bp, which was sequenced to obtain each isolate’s identity. Sequence analysis was able to identify all isolates examined with relative ease. Where the use of molecular identification methods is justified, such as in outbreak control or bioterrorism in animal health, employment of partial 16S rDNA PCR and sequencing employing universal or “broad-range” 16S rDNA, provides a valuable and reliable method of identification of such pathogens.     

Conversion of Lesquerolic Acid to 14-Oxo-11(<Emphasis Type="Italic">Z</Emphasis>)-Eicosenoic Acid by Genetically Variable <Emphasis Type="Italic">Sphingobacterium multivorum</Emphasis> Strains

This study focuses on the isolation and characterization of a high cadmium (Cd)-resistant bacterial strain, and possible exploitation of its Cd-accumulation and Cd-induced siderophore production property to improve plant growth in cadmium-contaminated soil through root colonization. The bacterial strain could tolerate up to 8 mM of Cd and could accumulate Cd intracellularly. The strain showed Cd-induced siderophore production maximally at 1.75 mM of Cd concentration under culture condition. It stimulated the growth of mustard and pumpkin plants in Cd-added soil through its establishment in rhizosphere. Through biochemical characterization and 16S rDNA sequence analysis, the strain KUCd1, as the name given to it, was identified as a strain of Pseudomonas aeruginosa.     

Dissociation between cardiomyocyte function and remodeling with beta-adrenergic receptor blockade in isolated canine mitral regurgitation

The low-pressure volume overload of isolated mitral regurgitation (MR) is associated with increased adrenergic drive, left ventricular (LV) dilatation, and loss of interstitial collagen. We tested the hypothesis that beta1-adrenergic receptor blockade (beta1-RB) would attenuate LV remodeling after 4 mo of MR in the dog. beta1-RB did not attenuate collagen loss or the increase in LV mass in MR dogs. Using MRI and three-dimensional (3-D) analysis, there was a 70% increase in the LV end-diastolic (LVED) volume-to-LV mass ratio, a 23% decrease in LVED midwall circumferential curvature, and a >50% increase in LVED 3-D radius/wall thickness in MR dogs that was not attenuated by beta1-RB. However, beta1-RB caused a significant increase in LVED length from the base to apex compared with untreated MR dogs. This was associated with an increase in isolated cardiomyocyte length (171+/-5 microm, P<0.05) compared with normal (156+/-3 microm) and MR (165+/-4 microm) dogs. Isolated cardiomyocyte fractional shortening was significantly depressed in MR dogs compared with normal dogs (3.73+/-0.31 vs. 5.02+/-0.26%, P<0.05) and normalized with beta1-RB (4.73+/-0.48%). In addition, stimulation with the beta-adrenergic receptor agonist isoproterenol (25 nM) increased cardiomyocyte fractional shortening by 215% (P<0.05) in beta1-RB dogs compared with normal (56%) and MR (50%) dogs. In summary, beta1-RB improved LV cardiomyocyte function and beta-adrenergic receptor responsiveness despite further cell elongation. The failure to attenuate LV remodeling associated with MR could be due to a failure to improve ultrastructural changes in extracellular matrix organization.     

A Tachyglossid-Like Humerus from the Early Cretaceous of South-Eastern Australia

A partial right humerus has been recovered from the Early Cretaceous (Albian) Eumeralla Formation at Dinosaur Cove in south-eastern Australia. General morphology, size and the presence of a single epicondylar foramen (the entepicondylar) suggest that the bone is from a mammal or an advanced therapsid reptile. The humerus is similar in size, shape and torsion to the equivalent bone of extant and late Neogene echidnas (Tachyglossidae) but, contrary to the situation in extant monotremes, in which the ulna and radius articulate with a single, largely bulbous condyle, it bears a shallow, pulley-shaped (i.e. trochlear-form) ulnar articulation that is confluent ventro-laterally with the bulbous radial condyle. This form of ulnar articulation distinguishes this bone from the humeri of most advanced therapsids and members of several major groups of Mesozoic mammals, which have a condylar ulnar articulation, but parallels the situation found in therian mammals and in some other lineages of Mesozoic mammals. As in extant monotremes the distal humerus is greatly expanded transversely and humeral torsion is strong. Transverse expansion of the distal humerus is evident in the humeri of the fossorial docodont Haldanodon, highly-fossorial talpids and some clearly fossorial dicynodont therapsids, but the fossil shows greatest overall similarity to extant monotremes and it is possible that the peculiar elbow joint of extant monotremes evolved from a condition approximating that of the fossil. On the basis of comparisons with Mesozoic and Cainozoic mammalian taxa in which humeral morphology is known, the Dinosaur Cove humerus is tentatively attributed to a monotreme. However, several apparently primitive features of the bone exclude the animal concerned from the extant families Tachyglossidae and Ornithorhynchidae and suggest that, if it is a monotreme, it is a stem-group monotreme. Whatever, the animal's true affinity, the gross morphology of its humerus indicates considerable capacity for rotation-thrust digging.     

Identification and reciprocal introgression of a QTL affecting body mass in mice

The aim of this study was to examine the effects of a QTL in different genetic backgrounds. A QTL affecting body mass on chromosome 6 was identified in an F₂ cross between two lines of mice that have been divergently selected for this trait. The effect of the QTL on mass increased between 6 and 10 weeks of age and was not sex-specific. Body composition analysis showed effects on fat-free dry body mass and fat mass. To examine the effect of this QTL in different genetic backgrounds, the high body mass sixth chromosome was introgressed into the low body mass genetic background and vice versa by repeated marker-assisted backcrossing. After three generations of backcrossing, new F₂ populations were established within each of the introgression lines by crossing individuals that were heterozygous across the sixth chromosome. The estimated additive effect of the QTL on 10-week body mass was similar in both genetic backgrounds and in the original F₂ population (i.e., ~0.4 phenotypic standard deviations); no evidence of epistatic interaction with the genetic background was found. The 95% confidence interval for the location of the QTL was refined to a region of approximately 7 cM between D6Mit268 and D6Mit123.     

Relative Ability of Orally Administered Lactobacillus murinus To Predominate and Persist in the Porcine Gastrointestinal Tract

Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n = 4), either singly or as a combination at ~10¹⁰ CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at ~10⁷ to 10⁸ CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at ~10⁵ CFU/g and was excreted at a significantly lower level than both L. murinus strains. Fecal L. murinus DPC6003 counts were also significantly higher than both Lactobacillus salivarius DPC6005 and Lactobacillus pentosus DPC6004 (~10⁶ CFU/g). The L. murinus strains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted ~10⁷ CFU of the administered strains/g, with L. murinus predominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general, L. murinus DPC6002 and DPC6003 and L. pentosus DPC6004 predominated in the feces and the cecum while P. pentosaceus DPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecal Enterobacteriaceae counts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration, Enterobacteriaceae counts were reduced by ~87 to 98% in pigs fed L. salivarius DPC6005, P. pentosaceus DPC6006, L. pentosus DPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.     

Identification of genomic regions that affect grain-mould incidence and other traits of agronomic importance in sorghum

Grain-mould is a major problem in grain sorghum utilization as mouldy grain has a reduced quality due to the deterioration of the endosperm and reduced embryo viability. Here, our objective was to use genome mapping to improve knowledge of genetic variation and co-variation for grain-mould incidence and other inter-related agronomic traits. Grain-mould incidence, kernel-milling hardness, grain density, plant height, panicle peduncle length, foliar-disease incidence, and plant color were measured on 125 F₅ genotypes derived from a cross of Sureño and RTx430. Quantitative trait loci were detected by means of 130 mapped markers (44 microsatellites, 85 AFLPs, one morphological-trait locus) distributed among ten linkage groups covering 970 cM. One to five QTLs affected each trait, with the exception of grain density for which no QTLs were detected. Grain-mould incidence was affected by five QTLs each accounting for between 10 and 23% of the phenotypic variance. The effects and relative positions of QTLs for grain-mould incidence were in accordance with the QTL distribution of several inter-related agronomic traits (e.g., plant height, peduncle length) and with the correlation between these phenotypic traits and grain-mould incidence. The detection of QTLs for grain-mould incidence was dependent on the environment, which is consistent with heritibility estimates that show strong environmental and genotype × environment effects. Several genomic regions affected multiple traits including one region that affected grain-mould incidence, plant height, panicle peduncle length, and grain-milling hardness, and a second region that influenced grain-mould (in four environments) and plant height. One genomic region, which harbors loci for plant color, influenced the severity of foliar disease symptoms and the incidence of grain-mould in one environment. Collectively QTLs detected in the present population explained between 10% and 55% of the phenotypic variance observed for a given trait.     

Plasma and salivary 6beta-hydroxycortisol measurements for assessing adrenocortical activity in patients with adrenocortical adenomas.

The aim of this study was to examine and compare the potential usefulness of plasma and salivary 6beta-hydroxycortisol measurements for assessing adrenocortical activity in patients with adrenocortical adenomas. Plasma and salivary cortisol as well as 6beta-hydroxycortisol determinations were performed by radioimmunoassay after extraction with ethyl acetate followed by chromatographic separation using a modified paper chromatographic system. Samples were obtained from 36 control subjects and 37 patients with non-hyperfunctioning adrenocortical adenomas in the morning at 8 a.m. after a low-dose of dexamethasone and after stimulation with synthetic depot ACTH. Basal and post-dexamethasone hormone levels were also measured in plasma and salivary samples of 4 patients with Cushing's syndrome from adrenal adenomas. In the baseline state, patients with non-hyperfunctioning adrenocortical adenomas had significantly higher plasma and salivary 6beta-hydroxycortisol levels (mean+/-SE, 79.0+/-7 and 17.1+/-2.2 ng/dl, respectively) compared to those measured in controls (62.0+/-4 and 7.7+/-0.6 ng/dl, respectively), whereas baseline plasma and salivary cortisol levels (9.6+/-0.5 microg/dl and 342+/-39 ng/dl, respectively) were similar to those measured in the control group (9.9+/-0.4 microg/dl and 366+/-24 ng/dl, respectively). In all groups, the changes in plasma and salivary 6beta-hydroxycortisol concentrations after dexamethasone suppression and ACTH stimulation were similar to the changes in plasma and salivary cortisol levels, although the differing ratios of 6betaOHF to cortisol indicated potentially important variations in the induction of 6beta-hydroxylase activity between the three groups. In patients with Cushing's syndrome, baseline plasma and salivary 6beta-hydroxycortisol concentrations (754+/-444 and 104+/-88 ng/dl, respectively) were more markedly increased than plasma and salivary cortisol levels (24.8+/-6.7 microg/dl and 1100+/-184 ng/dl, respectively), and all remained non-suppressible after dexamethasone administration. These results suggests that plasma and salivary 6beta-hydroxycortisol determinations may precisely detect not only overt increases of cortisol secretion in patients with Cushing's syndrome but also mild glucocorticoid overproduction presumably present in patients with non-hyperfunctioning adrenocortical tumors.     

Regeneration and micrografting of lentil shoots

Summary A protocol for regeneration and micrografting of shoots of lentil (Lens culinaris Medik) was developed. Multiple shoots (4–5) were regenerated from cotyledonary node explants on Murashige and Skoog (MS) medium containing 8.8 μM 6-benzylaminopurine. In vitro regenerated shoots were micrografted on rootstocks with 96% efficiency. The successful grafts were transplanted to pots in Redi-earth^TM, hardened off and were grown to maturity with 100% success. The success of the micrografting was independent of the nature and concentration of growth regulator used in shoot initiation medium and the time period for induction of shoots. The protocol was successful with several cultivars of lentil. The advantages of micrografting over in vitro rooting are discussed.