A survey of symbiotic nitrogen fixation by white clover grown on metal contaminated soils

There is conflicting evidence about toxic effects of heavy metals in soil on symbiotic nitrogen fixation. This study was set-up to assess the general occurrence of such effects. Soils with metal concentration gradients were sampled from six established field trials, where sewage sludge or metal salts have been applied, or from a transect in a sludge treated soil. Additional contaminated soils were sampled near metal smelters, in floodplains, in sludge amended arable land and in a metalliferous area. Symbiotic nitrogen fixation was measured with ¹⁵N isotope dilution in white clover (Trifolium repens L.) grown in potted soil that was not re-inoculated, and using ryegrass (Lolium perenne L.) as reference crop. The fraction nitrogen in clover derived from fixation (N_dff) varied from 0 to 88% depending on soil. Pronounced metal toxicity on N_dff was only confirmed in a sludge treated soil where nitrogen fixation was halved from the control value at soil total metal concentration of 737 mg Zn kg⁻¹, 428 mg Cu kg⁻¹ and 10 mg Cd kg⁻¹. The N_dff was significantly reduced by increasing metal concentration in soils from two other sites where N_dff was low throughout and where these effects might be attributed to confounding factors. No significant effects of metals on N_dff were identified in all other gradients even up to elevated total metal concentration (e.g. 55 mg Cd kg⁻¹). The variation of N_dff among all soils (n=48), is mainly explained by the number of rhizobia in the soil (log MPN, log (cells g⁻¹ soil)), whereas correlations with total or soil solution metal concentrations were weak (R²<0.25). The is significantly affected by the presence or absence of the host plant at the sampling site. No effects of metals were identified at even at total Zn concentrations of about 2000 mg Zn kg⁻¹, whereas metal toxicity could be identified at lower most probable number (MPN) values. This survey shows that the metal toxicity on symbiotic nitrogen fixation cannot be generalized and that survival of a healthy population of the microsymbiont is probably the critical factor.     

Challenges in measuring insoluble dietary fiber

Objectives of this review are to define criteria for evaluating insoluble dietary fiber (IDF) methods, discuss their relevance in meeting the nutritional needs of ruminants and herbivores, describe problems with empirical IDF methods, and assess their relative merits. The challenge for the researcher, nutritionist, and analyst is to select fiber methods that are relevant and reproducible. Without relevance, there is no reason to measure IDF, and without reproducibility, there is no value in doing so. Insoluble dietary fiber is a complex matrix of chemical components, and there are no primary standards that can be used to establish the validity of methods. Thus, the definition of fiber is crucial in determining method relevance. For ruminants and nonruminant herbivores, the appropriate physiological definition for selecting IDF methods may be as follows: the organic fraction of the diet that is indigestible or slowly digesting and occupies space in the gastrointestinal tract. Crude fiber does not match this definition, and its use should be abandoned. Acid detergent fiber does not measure all IDF but is useful when included with other dietary fiber methods to describe some feeds. Several current methods, including amylase-treated neutral detergent fiber (aNDF) and enzymatic-gravimetric methods, are relevant for measuring IDF. In a collaborative study, aNDF obtained a standard deviation of reproducibility (SD(R)) of 1.3%. Enzymatic-gravimetric methods of measuring IDF have been evaluated using too few feed materials to make statistically valid conclusions, but the SD(R)f IDF, for the few feeds evaluated, were similar to aNDF (0.9 to 2.4%). The enzymatic-chemical method of measuring IDF as the sum of insoluble nonstarch polysaccharides and lignin agrees with NDF, but the SDR of neutral sugar analysis using acid hydrolysis and chromatography is greater (3.2%) than other dietary fiber methods. Empirical methods--such as those used to measure IDF, although based on nutritional concepts--actually define the fraction being measured and must be followed exactly, without modification. The selection of a suitable method for IDF depends on the purpose of analysis. Analysis of sugars in insoluble polysaccharides provides more information but is less reproducible and more expensive to obtain. For routine nutritive evaluation of feeds and formulation of rations, aNDF seems to be a reasonable choice for measuring IDF based on relevance and reproducibility.     

Ganglioradiculitis (sensory neuronopathy) in a dog: clinical,morphologic, and immunohistochemical findings

A 9-year-old Labrador Retriever was diagnosed with ganglioradiculitis (sensory neuronopathy). This idiopathic disease of mature dogs is characterized by a profound loss of sensory nerve function due to mononuclear inflammatory infiltration of peripheral ganglia and spinal nerve roots, with destruction of sensory neurons. Immunohistochemistry demonstrates that the infiltrating cells are primarily T lymphocytes and that immunoglobulins are not present on the cell membranes of affected neurons. The pathogenesis of ganglioradiculitis remains unclear, but the evidence points to a cell-mediated immune mechanism.     

Effects of delmadinone acetate on pituitary-adrenal function, glucose tolerance and growth hormone in male dogs

Objective To characterise the effects of delmadinone acetate on the pituitary-adrenal axis, glucose tolerance and growth hormone concentration in normal male dogs and dogs with benign prostatic hyperplasia.
Design A prospective study involving nine normal male dogs and seven with prostatic hyperplasia.
Procedure Delmadinone acetate was administered to six normal male dogs and seven dogs with benign prostatic hyperplasia at recommended dose rates (1.5 mg/kg subcuta-neously at 0, 1 and 4 weeks). Three normal controls received saline at the same intervals. Blood concentrations of ACTH, cortisol, glucose, insulin and growth hormone were measured over 50 days. Intravenous glucose tolerance and ACTH response tests were performed before and after treatment in the nine normal animals.
Results A substantial suppression of basal and 2 h post-ACTH plasma cortisol secretion was demonstrated after one dose in all dogs given delmadinone acetate. Individual responses after the second and third administration varied between recovery in adrenal responsiveness to continued suppression. Plasma ACTH concentration was also diminished after one treatment. No effects were evident on glucose tolerance or serum growth hormone concentrations.
Conclusion Delmadinone acetate causes adrenal suppression from inhibition of release of ACTH from the pituitary gland. Treated dogs may be at risk of developing signs of glucocorticoid insufficiency if subjected to stressful events during or after therapy. Neither glucose intolerance nor hyper-somatotropism seems likely in male dogs given delmadinone acetate at the recommended dose rate, but the potential for excessive growth hormone secretion in treated bitches remains undetermined.     

Plasma cortisol responses to three cortico-trophic preparations in normal dogs

Objective To compare cortisol responses to three corticotrophic preparations in normal dogs.
Animals Eight clinically normal dogs (four intact males, four intact females) of medium size.
Procedures Each dog received four treatments on four separate occasions in a duplicated Latin square pattern. Treatments were two adrenocorticotrophin (ACTH) preparations given intramuscularly at 2.2 U/kg, one of the ACTH preparations given intramuscularly at 1 U/kg and a synthetic polypeptide with ACTH-like activity (tetracosactrin, cosyntropin) given intravenously at 5 μg/kg. Plasma samples were taken for cortisol assay before and at 0.5, 1, 2 and 4 h after treatment.
Results Plasma cortisol concentrations were similar with the two ACTH preparations and at both dose rates. Tetracosactrin produced smaller mean peak cortisol concentrations, which tended to occur earlier than with ACTH, and smaller values for the area under the curve of plasma cortisol concentration from zero time to 4 h.
Conclusion The findings suggest that canine adrenal function can be tested adequately by giving ACTH intramuscularly at 1 U/kg and measuring plasma cortisol in samples taken at 0 and 2 h, or by giving tetracosactrin intravenously at 5 μg/kg and determining cortisol concentration at 0 and 1 h.     

SMALL INTESTINAL EMPTYING TIME IN NORMAL BEAGLE DOGS

Gastric emptying time and small intestinal transit time in dogs are frequently discussed. However, it is often of interest to the radiologist to know what normal small intestinal emptying times should be. A total of 15 upper gastrointestinal studies was performed on five internal parasite-free, normal, standard Beagle dogs with three studies on each dog, 6 days apart. The ages and weights of the dogs ranged from 2–8 years and from 12.4–13.7 kg, respectively. Following 24-hour fasting, a dose of 10 ml/kg bw of 60% wt/vol barium sulfate suspension was administered through a stomach tube. Then, sequential radiographs were made at 30-minute intervals until the entire contrast medium column was in the colon and cecum. The mean, standard deviation, and range of gastric emptying time, small intestinal transit time, and small intestinal emptying time were 76 ± 16.7 (30–120), 73 ± 16.4 (30–120), and 214 ± 25.1 (180–300) minutes, respectively. This study offers the possibility that small intestinal emptying time may be used to further evaluate patients with suspected small intestinal partial obstruction, pseudo-obstruction, ischemia, or lymphangiectasia.     

Stimulated activity of the soil nitrifying community accelerates community adaptation to Zn stress

Field data have shown that soil nitrifying communities gradually adapt to zinc (Zn) after a single contamination event with reported adaptation times exceeding 1 year. It was hypothesized that this relatively slow adaptation relates to the restricted microbial diversity and low growth rate of the soil nitrifying community. This hypothesis was tested experimentally by recording adaptation rates under varying nitrification activities (assumed to affect growth rates) and by monitoring shifts in community composition. Soils were spiked at various Zn concentrations (0-4000 mg Zn kg⁻¹) and two NH₄⁺-N doses (N1, N2) were applied to stimulate growth. A control series receiving no extra NH₄⁺-N was also included. Soils were incubated in pots under field conditions with free drainage. The pore water Zn concentration at which nitrification was halved (EC50, mg Zn l⁻¹) did not change significantly during 12 months in the control series (without NH₄⁺-N applications), although nitrification recovered after 12 months at the highest Zn dose only. The EC50 after 12 months incubation increased by more than a factor 10 with increasing NH₄⁺-N dose (p < 0.05) illustrating that increased activity accelerates adaptation to Zn. Zinc tolerance tests confirmed the role of Zn exposure, time and NH₄⁺-N dose on adaptation. Zinc tolerance development was ascribed to the AOB community since the AOB/AOA ratio (AOB = ammonia oxidizing bacteria; AOA = ammonia oxidizing archaea) increased from 0.4 in the control to 1.4 in the most tolerant community. Moreover, the AOB amoA DGGE profile changed during Zn adaptation whereas the AOA amoA DGGE profile remained unaffected. These data confirm the slow but pronounced adaptation of nitrifiers to Zn contamination. We showed that adaptation to Zn was accelerated at higher activity and was associated with a shift in soil AOB community that gradually dominated the nitrifying community.     

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10⁴ (ANU) and 4.2 × 10³ (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.