Using metabolic profiling to assess plant-pathogen interactions: an example using rice (<Emphasis Type="Italic">Oryza sativa)</Emphasis> and the blast pathogen <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

A metabolomics based approach has been used to study the infection of the Hwacheong rice cultivar (Oryza sativa L. cv. Hwacheong) with compatible (KJ201) and incompatible (KJ401) strains of the rice blast fungal pathogen Magnaporthe grisea. The metabolic response of the rice plants to each strain was assessed 0, 6, 12, 24, 36, and 48 h post inoculation. Nuclear Magnetic Resonance (NMR) spectroscopy and Gas and Liquid Chromatography Tandem Mass spectrometry (GC/LC-MS/MS) were used to study both aqueous and organic phase metabolites, collectively resulting in the identification of 93 compounds. Clear metabolic profiles were observed at each time point but there were no significant differences in the metabolic response elicited by each pathogen strain until 24 h post inoculation. The largest change was found to be in alanine, which was ~30% (±9%) higher in the leaves from the compatible, compared to the resistant, plants. Together with several other metabolites (malate, glutamine, proline, cinnamate and an unknown sugar) alanine exhibited a good correlation between time of fungal penetration into the leaf and the divergence of metabolite profiles in each interaction. The results indicate both that a wide range of metabolites can be identified in rice leaves and that metabolomics has potential for the study of biochemical changes in plant-pathogen interactions.     

Pudendal reflexes and effects of conditioning stimuli in cats

Evaluation of pudendal reflexes and effects of pudendal branch conditioning on those reflexes was carried out in 2 studies. In the first study of pudendal reflexes, 20 adult male and female mixed-breed cats underwent surgical isolation of the anal branch, urethral branch, and distal trunk (consisting primarily of the dorsal nerve of the penis/clitoris) of the pudendal nerve. Reflexes were tested in all possible ipsilateral and contralateral test-response combinations. Latency values and effects of increasing stimulus rate on response amplitude were recorded. Reflexes were detected in all combinations, with response latencies between 6.3 and 13.0 ms. Response amplitudes were diminished at stimulus rates of 3 to 5 Hz, and responses were apparently abolished at 4 to 16 Hz, suggesting that pudendal reflexes are polysynaptic. In the second study of conditioning effects, 9 adult male and female mixed-breed cats underwent preparation similar to that for study 1. A train of conditioning stimuli was applied to branches of the pudendal nerve prior to attempting to induce reflex responses, as performed in study 1. Conditioning completely abolished reflex responses for a period of 70 to 130 ms. Reflex responses were diminished in amplitude, compared with those observed during preconditioning trials, for 180 to 300 ms after conditioning.     

Somatostatin receptor imaging in vivo by planar scintigraphy facilitates the diagnosis of canine insulinomas

Somatostatin receptors expressed by insulinomas in 5 dogs were imaged in vivo by means of indium in 111 pentetreotide (OctreoScan) scintigraphy. The diagnosis in each dog was supported by the presence of hypoglycemia (<60 mg/dL), hyperinsulinemia (>20 microU/mL), and histopathologic review of neoplastic tissue. All insulinomas expressed high-affinity somatostatin receptors of subtype sst2, as shown by receptor autoradiography in vitro using 125I-[tyrosine3]-octreotide and 125I-[leucine8, Dtryptophan22, tyrosine25]-somatostatin-28 with an sst2 subtype-selective analogue. Scintigrams were obtained at 1, 4, 12, and 24 hours after the i.v. administration of 74-222 MBq of OctreoScan to each patient. Abnormal foci of activity were 1st observed from 1 hour after administration of the radioligand in dog 3, to 24 hours after its administration in dog 4; in dogs 1 and 2, abnormal foci of activity were visible from 12 hours. Dog 5 showed a questionable abnormal focus of activity at 12 hours, but not at 24 hours. Scintigraphy enabled accurate prediction of the anatomical location of the primary tumor in 1 of 4 dogs, but was unable to differentiate a right- from a left-pancreatic lobe tumor, or vice versa, in 3 dogs; the 5th dog had equivocal results. 111In-pentetreotide scintigraphy is a useful diagnostic adjunct to the clinical evaluation of the insulinoma patient, but is unable to localize the tumor in some cases.     

Physiologic assessment of blood glucose homeostasis via combined intravenous glucose and insulin testing in horses

OBJECTIVE: To characterize the physiologic response to i.v. bolus injection of glucose and insulin for development of a combined glucose-insulin test (CGIT) in horses. ANIMALS: 6 healthy mares and 1 mare each with pituitary adenoma and urolithiasis. PROCEDURE: Horses were given a CGIT (glucose, 150 mg/kg; insulin, 0.1 U/kg); results were compared with a singular i.v. glucose tolerance test (GTT; 150 mg/kg) and a singular i.v. insulin sensitivity test (IST; 0.1 U/kg). Healthy horses were also given a CGIT after receiving xylazine and undergoing stress. RESULTS: Physiologically, the CGIT resulted in a 2-phase curve with positive (hyperglycemic) and negative (hypoglycemic) portions; the positive phase came first (250% of baseline at 1 minute). The descending segment declined linearly to baseline by approximately 30 minutes and to a nadir at 58% of baseline by 75 minutes. After a 35-minute valley, a linear ascent to baseline began. Addition of insulin in the CGIT increased glucose utilization by approximately 4.5 times during the positive phase but not during the negative phase. The diseases' effects and experimental inhibition of insulin secretion with xylazine and stress were detectable by use of the 2 phases of the CGIT. Only a single positive phase resulted from the GTT and a single negative phase from the IST CONCLUSIONS AND CLINICAL RELEVANCE: The CGIT resulted in a consistent, well-defined glycemia profile, which can be disrupted experimentally or by a disease process. The CGIT has clinical potential because it provides integrated information and more information than either the singular GTT or IST.     

Growth inhibition of environmental mastitis pathogens during physiologic transitions of the bovine mammary gland

Ten dairy cows were infused intramammarily near drying off with concanavalin A (conA) or phytohemagglutinin (PHA). Mammary secretions were collected during physiologic transitions of the udder and were used in an in vitro microbiological assay to determine growth inhibition of mastitis pathogens. As mammary involution progressed, in vitro growth inhibition of Klebsiella pneumoniae, Escherichia coli, and Streptococcus uberis increased. Mammary secretions from conA- and PHA-treated glands had significantly increased bacterial growth inhibition. Secretions contained significantly increased concentrations of lactoferrin and a decreased citrate:lactoferrin molar ratio earlier in the dry period than did control mammary secretions. Greatest bacterial growth inhibition was observed in mammary secretions obtained 7 days before parturition. However, differences in secretion composition or bacterial growth inhibition were not found between conA- or PHA-treated and control udder halves during the prepartum period. Bacterial growth inhibition by mammary secretion decreased markedly during early lactation. A highly significant positive correlation was found between bacterial growth inhibition and concentrations of lactoferrin, serum albumin, and immunoglobulin G. A highly significant negative correlation was found in the citrate:lactoferrin molar ratio during early involution and the peripartum period.     

Granulated lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology of 10-day-old pigs

Lysozyme is a 1,4-β-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine the effect of a purified granulated lysozyme, compared with antibiotics, on growth performance, small intestinal morphology, and Campylobacter shedding in 10-d-old pigs. Forty-eight pigs (n = 16 per treatment), with an initial BW of 4.0 ± 0.1 kg (P > 0.40), were weaned at 10 d of age, blocked by litter and sex, and assigned to pens (8 pigs/pen). Each block was randomly assigned to consume 1 of 3 liquid dietary treatments for 14 d: a control diet, the control diet + lysozyme (100 mg/kg of diet), or the control diet + antibiotics (neomycin and oxytetracycline, 16 mg/kg of diet). Pigs were weighed and blood was sampled on d 0, 7, and 14. Blood was analyzed for plasma urea N and IgA. After 14 d of treatment, pigs were killed and samples of the jejunum and ileum were collected and fixed to measure villus height and crypt depth. Rectal swabs were taken on d 0, 7, and 14 of treatment, and samples of ileal and cecal contents were taken at d 14 of treatment to determine the presence of Campylobacter. Pigs consuming lysozyme and antibiotics gained BW at a faster rate than did control pigs over the course of the study (402 ± 12 and 422 ± 14 g/d, respectively, vs. 364 ± 14 g/d; P < 0.02), resulting in heavier ending BW (9.9 ± 0.3, 9.9 ± 0.3, and 9.0 ± 0.2 kg for pigs in the lysozyme, antibiotic, and control groups, respectively; P < 0.03). Immunoglobulin A decreased and plasma urea N increased over the course of the study (P < 0.1), regardless of dietary treatment (P > 0.6). Crypt depth was increased in pigs fed lysozyme- and antibiotic-treated diets, compared with pigs fed the control diet, in both the jejunum (60.0 ± 2.8 and 62.2 ± 3.0 μm, respectively, vs. 50.7 ± 3.1 μm; P < 0.03) and ileum (76.0 ± 7.5 and 72.2 ± 5.0 μm, respectively, vs. 52.4 ± 3.5 μm; P < 0.02). Villus height did not differ in the jejunum (P > 0.2) but was increased in the ileum of pigs consuming the lysozyme- and antibiotic-treated diets, compared with pigs fed the control diet (312 ± 20 and 314 ± 10 μm, respectively, vs. 263 ± 15 μm; P < 0.4). Small intestinal total mucosa and mucosal protein concentrations, as well as disaccharidase-specific activities, were not altered by lysozyme or antibiotics (P > 0.05). Campylobacter was detected in 27% of control samples but in only 5% of samples from pigs fed antibiotics and 8% of samples from pigs fed lysozyme (P < 0.01). Thus, granulated lysozyme is a suitable alternative to antibiotics for 10-d-old pigs consuming manufactured liquid diets.