Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae.

Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3-14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada's agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

Beneficial effect of lidocaine on ventricular electrical stability and spontaneous ventricular fibrillation during experimental myocardial infarction.

Several studies have questioned the efficacy of lidocaine in reducing the incidence of ventricular fibrillation shortly after acute myocardial infarction when arrhythmogenic mechanisms may be different from those operative several hours later. To determine whether lidocaine inhibits the occurrence of early ventricular fibrillation, the left anterior descending and septal coronary arteries were occluded at their origins in open chest anesthetized dogs. Fourteen of 16 control dogs died with ventricular fibrillation. Fifteen dogs received two different dose regimens of lidocaine before coronary occlusion. Of the 11 treated dogs maintaining lidocaine bl), 6 survived (P less than 0.05). Five dogs received the larger dose; all died, four having blood levels of 6.3 mug/ml or greater at the time of death. Ventricular fibrillation threshold also increased in six of eight dogs when lidocaine was administered after coronary occlusion. It is concluded that lidocaine at blood levels of 1.2 to 5.5 mug/ml significantly reduces the incidence of ventricular fibrillation early after coronary occlusion. Administration of this agent therefore may be of particular value in the early phase of acute myocardial infarction.

Ultrasound contrast-detail analysis: a comparison of low-contrast detectability among scanhead designs.

Contrast-detail (CD) analysis was used to compare the low-contrast detection capabilities of expert observers using different array-type scanhead technologies. Five expert observers viewed five different contrast targets to obtain CD curves for each scanhead. Differences in CD curves are interpreted in terms of the image contrast, resolution, and noise. It was found that differences in low-contrast detectability were due to differences in beam properties. Clinical images obtained during patient examinations are used to show how some clinically relevant tasks are distributed in their contrast and size.

Ultrasound contrast-detail analysis: a preliminary study in human observer performance.

Results of a human observer performance study using a new ultrasound contrast-detail (CD) phantom are reported. The new phantom allows estimates of the fundamental statistical uncertainties involved in CD analysis. Results of these experiments show that observers generally considered to be "trained" require experience at the specific task of low-contrast target detection under signal known exactly (SKE) conditions, and that observers require an orientation period at the beginning of each observation session to obtain stable responses. The results obtained in this study can be used to estimate the number of independent images, observers, and repeat observation sessions required for a desired uncertainty in CD curves, and show that 10% fractional standard deviations are obtainable with modest observer effort.

The effects of lossy compression on the detection of subtle pulmonary nodules.

We examined the ability of radiologists to detect pulmonary nodules in computed radiographic (CR) chest images subjected to lossy image compression. Low-contrast 1-cm diameter targets simulating noncalcified pulmonary nodules were introduced into clinical images and presented to ten radiologists in a series of two-alternative forced-choice (2AFC) observer experiments. The percentages of correct observer responses obtained while viewing noncompressed images (1:1) were compared with those obtained for the same images compressed 7:1, 16:1, 44:1, and 127:1. The images were compressed using a standard full-frame discrete cosine transform (DCT) technique. The degree of compression was determined by quantizing Fourier components in various frequency channels and then Huffman encoding the result. The data show a measurable decline in performance for each compression ratio. Through signal-to-noise ratio (SNR) analysis, we found that the reduction in performance was due primarily to the compression algorithm that increased image noise in the frequency channels of the signals to be detected.

Ultrasonic measurement of glomerular diameters in normal adult humans.

The average size of acoustic scatterers and the integrated backscatter coefficient of kidney cortex were measured in vivo from 2-4 MHz for a group of 50 normal adult volunteers. Our goal was to determine the sensitivity of the ultrasonic measurements under clinical conditions by identifying biologic sources of estimation uncertainty. Based on 10 measurements on each kidney of each volunteer, the average glomerular diameter for the group was found to be 216 +/- 27 microns (SD). Glomerular size was found to correlate with body surface area (r = 0.4), and the ratio of glomerular surface area to body surface area (GSA/BSA) was found to be constant throughout normal adult life with GSA/BSA = (8.24 +/- 1.35) x 10(-8) (SD). These results are consistent with histologic analyses found in the literature. Within an individual, 7% standard errors in GSA/BSA are typical. Biologic variability dominates the variance in scatterer size estimates in a group not matched for BSA, where it accounts for 47% of the variance. In a group of individuals matched for BSA, biologic variability accounts for only 21% of the variance; day-to-day variability accounts for 35% of the variance; and experimental parameters account for the remainder. If a deviation greater than 2 x SD is considered abnormal, then this technique can potentially detect changes in glomerular diameter as small as 30 microns within an individual. To detect abnormal GSA/BSA values for an individual, GSA/BSA would have to differ from the mean for that group by about 3.6 x 10(-8) or about 40%. Therefore, at this time scatterer size estimates appear most reliable for tracking the progression of disease and treatment for an individual over time.

Effects of endothelin-1 on renal microvasculature measured using quantitative ultrasound.

Renal vascular resistance is an important feature of kidney function and disease. To maintain adequate blood flow, renal vascular resistance varies in response to changes in systemic pressure. Vascular resistance is largely determined by arteriolar diameter, which is regulated by local and systemic factors. We used quantitative ultrasound techniques to follow renal vascular changes in anesthetized dogs during local intraarterial infusion of a potent vasoconstrictor, endothelin-1 (ET-1). Average arteriolar diameters were estimated by analyzing echo-signal spectra (5-15 MHz) obtained from renal cortex in vivo before, during, and after ET-1 infusion. At calculated arterial concentrations of 0.01 nM, 0.1 nM, and 1.0 nM, ET-1 reduced the average arteriolar diameter of 38 +/- 2 microns by 2%, 63%, and 91%, respectively, without producing a significant change in systemic blood pressure. Changes in scatterer size were consistent with the observed changes in renal hemodynamics detected using Doppler techniques. In addition, acoustic attenuation was found to increase with ET-1 concentration. These data suggest that quantitative ultrasound methods are sensitive to changes in renal arteriolar diameter, and may be a new noninvasive method for continuously monitoring changes in vascular resistance.

Use of gonadotropin-releasing hormone for hastening ovulation in transitional mares.

Natural GnRH and its analog have potential for hastening ovulation in mares. A study was conducted to evaluate the efficacy of a GnRH agonist given either as an injectable or s.c. implant for induction of ovulation in mares. Forty-five seasonally anestrous mares (March) were assigned to one of three groups (n = 15/group): 1) untreated controls; 2) i.m. injection of the GnRH agonist buserelin at 12-h intervals (40 micrograms/injection for 28 d or until ovulation) and 3) GnRH agonist administered as a s.c. implant (approximately 100 micrograms/24 h for 28 d). Six mares per group were bled on d 0, 7, 14 and 21 after injection or insertion of implant. Samples were taken at -1, -.5 and 0 h and at .5, 1, 1.5, 2, 4, 6 and 8 h after GnRH. Additional daily samples were drawn for 28 d after injection or until ovulation. Samples were assayed for concentration of LH and FSH. Progesterone concentrations were determined in samples collected on d 4, 6 and 10 after ovulation. Number and size of follicles and detection of ovulation were determined by ultrasonography. Number of mares induced to ovulate within 30 d was 0 of 15, 7 of 15 and 9 of 15 for groups 1, 2 and 3, respectively. During treatment, follicle sizes were smaller for mares in group 3 (implant). The LH response to GnRH agonist (area under curve) was similar among groups at d 0 but was greater (P less than .05) for mares in group 3 on d 7 and 14 and groups 2 and 3 on d 21 than for controls. A similar pattern was detected for peak concentrations of LH after GnRH on d 0, 7, 14 and 21. Daily concentrations of LH remained low in untreated control mares compared with GnRH-treated mares throughout the sampling period. Concentrations of LH for mares in group 3 that ovulated were elevated greatly above those for group 2 mares, whereas concentrations of FSH were similar in both treatment groups prior to ovulation.

Gonadotropin response by postpartum mares to gonadotropin-releasing hormone.

We hypothesized that the LH response to GnRH would be greater as the interval from foaling increases, whereas the FSH response would decrease, and that corpus luteum function after the first ovulation would be similar to that after the second ovulation. At parturition, mares were assigned to receive GnRH (2 micrograms/kg) intravenously on 1) d 3 postpartum (n = 6); 2) d 6 postpartum (n = 6); 3) d 1 of first postpartum estrus (foal estrus) and again on d 1 of second postpartum estrus (n = 8). Blood was collected through an indwelling cannula at -2, -1 and 0 h relative to GnRH stimulation (basal concentrations) and at .25, .5, .75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h post-GnRH. Samples were assayed for concentrations of LH and FSH. Basal concentrations of LH were lower (P less than .05) for mares given GnRH on d 3 postpartum than for mares on d 1 of foal estrus. A rise in concentrations of LH was noted within 30 min in all groups, but the response to GnRH on d 1 of the first estrus was less (P less than .05) than on d 1 of second postpartum estrus. As the interval from parturition increased, the amount of LH secreted in response to GnRH increased. The maximum response to GnRH was greater (P less than .05) during d 1 of the first estrus than on d 3 or 6 postpartum and was greater on d 1 of cycle 2 than on d 1 of cycle 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrasonographic studies on the reproductive tract of mares after parturition: effect of involution and uterine fluid on pregnancy rates in mares with normal and delayed first postpartum ovulatory cycles.

During breeding of mares, ultrasonographic detection of uterine fluid accumulations in the first postpartum ovulatory period was associated with significantly decreased pregnancy rates, when compared with rates in control mares (P less than 0.005). The previously gravid uterine horn was recognized as the larger horn, when assessed for size by ultrasonography, for a mean of 21 days (range, 15 to 25 days) after parturition. On the basis of similar measurements obtained during 3 ultrasonographic scans (5-day period), uterine involution was determined to be completed in a mean of 23 days (range, 13 to 29 days). Progestin treatment did not affect uterine size, fluid accumulation, or rate of involution after parturition. However, delaying the first postpartum ovulation with 8 days of progestin treatment significantly improved pregnancy rates (P less than 0.05). More (P less than 0.05) mares became pregnant (23 of 28, 82%) when ovulation occurred after day 15 in the first postpartum ovulatory period, compared with those mares that ovulated before day 15 (6 of 12, 50%). We concluded that ultrasonographic detection of uterine fluid and postpartum progestin treatment can be used to manipulate breeding strategies and to improve pregnancy rates in mares bred during the first postpartum ovulatory period.

Microbial degradation of cellulose polymers used in cosmetics and toiletries.

Synopsis Cellulose polymers which are commonly found in cosmetics can act as potential targets for microbial attack and sometimes support extensive growth under suitable conditions. As a result, cellulosic substances can be converted from a stiff gel into a running liquid, thereby rendering the cosmetic unfit for use. To provide an understanding of the biodeterioration of cellulose in cosmetics, this paper reviews the structure of cellulose, mechanism of enzyme degradation and effects of structural properties of cellulose on the rate of hydrolysis. At least three different types of enzymes (exo-beta-1, 4-glucanase, endo-beta-1,4-glucanase and beta-glucosidase) are involved in the degradation of crystalline cellulose. Enzyme production by three fungi belonging to the genera Aspergillus, Fusarium and Penicillium is described.

Abnormal spinal curvature in the fetus.

We reviewed 20 cases of sonographically detected abnormal fetal spinal curvature to help determine the significance of this finding. Both marked (13 cases) and mild (7 cases) curves were diagnosed sonographically. Sonography showed associated anomalies in 19 fetuses, including neural tube and ventral wall defects. Outcomes were poor, with only three infants surviving. Isolated scoliosis was identified prenatally in one neurologically normal infant with hemivertebrae. We conclude that abnormal spinal curvature in the fetus is a significant finding, whether mild or severe. Even as an isolated finding, it is significant, as prenatal detection assists postnatal management.

Transposon mutagenesis and tagging of fluorescent Pseudomonas: Antimycotic production is necessary for control of Dutch elm disease.

Antimycotic-producing strains of Pseudomonas syringae are being tested as Dutch elm disease control agents. We examined the role of antimycotic production in disease control. Transposon Tn903 was used to mutagenize the antimycotic-producing strain MSU174. Eighty-one mutants that did not inhibit fungal growth were identified among 15,000 Tn903-containing derivatives. Linkages between Tn903 insertions and defects in antimycotic metabolism were established. Three Tn903-containing strains (two antimycotic producers and one nonproducer) were individually introduced into American elm seedlings. The seedlings were subsequently challenged with Ceratocystis ulmi, the causal agent of Dutch elm disease. Protection of the elms was observed with the two antimycotic-producing strains but not with the nonproducing strain. The introduced strains could be readily recovered from the seedlings after two growing seasons. They were unequivocally identified by the Tn903 insertions they contain.

Value of radiopaque markers in identifying partial small bowel obstruction.

Confirming partial small bowel obstruction is often a diagnostic challenge. In this case report, 4-mm solid radiopaque markers were used in 4 patients to show partial small bowel obstruction. Results of enteroclysis were normal in 2 of the 4 patients, and the markers were used to challenge suspected partial obstruction. The markers coalesced in the region of the partial obstruction, which was confirmed at surgery. Enteroclysis is the examination of choice in the diagnosis of partial small bowel obstruction. However, examinations with false-negative results can occur, particularly with adhesive and/or intermittent obstructions. The use of radiopaque markers in these cases proved an effective and useful method of establishing the diagnosis of partial small bowel obstruction, particularly in the 2 cases in which enteroclysis results were normal. Prospective studies are needed to establish the feasibility of this novel technique.

Urinary IgA in IgA nephropathy and Henoch-Schoenlein purpura.

To determine the concentrations and molecular forms of urinary IgA in IgA nephropathy and Henoch-Schoenlein purpura, we studied 29 patients with these IgA-associated renal diseases (IgAN). Control groups comprised 10 patients with other diverse renal disease and 11 healthy volunteers. Urinary IgA and IgG concentrations were higher in IgAN than in either control group and correlated positively with the serum creatinine concentration as well as the urinary protein excretion (P less than 0.01). However, IgA/IgG ratios did not differ among the three groups. Polymeric IgA (p-IgA) in the urine predominated only in normals; in IgAN and patients with other renal diseases, monomeric IgA (m-IgA) occurred almost exclusively. Serum IgA concentrations were generally normal in IgAN; four patients had concentrations greater than 500 mg/dl. Although the fraction of p-IgA in serum (median, 18%) was increased above normal (5-10%) in 13 of 16 (81%) subjects, neither the concentration of IgA or IgG nor the amount of p-IgA correlated with the serum creatinine concentration. These data suggest that the molecular form and concentration of urinary IgA are not discriminating for IgAN and are independent of these characteristics of serum IgA.

Image calibration of laser digitizers, printers, and gray-scale displays.

Laser film digitizers, interactive gray-scale monitors, and laser film printers are necessary to transmit digital image information. These devices must be standardized so that hard- and soft-copy images are as similar as possible. Standardization of appropriate calibration procedures is necessary to attain this goal. Radiographs are converted into digital data representations by a laser film digitizer. These representations (and those obtained with other modalities) are transferred to a laser film printer or to an interactive monitor with gray-scale display. To obtain the best gray-level fidelity, printer output optical densities should be identical to those of the input film. Laser printers should be calibrated regularly to ensure uniform results. A gray-scale controller functions as an adjunct to the host computer and can automate the calibration process. Gray-scale controller functions may someday be incorporated into an accelerator or array-processor board.

Abdominal wall hernias: review of herniography and correlation with cross-sectional imaging.

Herniography is an accurate means of identifying groin hernias when the clinical diagnosis is uncertain. Its role in evaluation of other types of ventral hernias is less clear; however, with minor modifications in technique, herniography is also useful in these cases. This article reviews the technique of herniography, normal variations in anatomy, and interpretation of herniograms of the groin and anterior abdominal wall on the basis of 72 patients studied over 3 1/2 years. Herniography is also compared with cross-sectional imaging techniques. Inguinal and femoral hernias are best shown and classified with herniography. Ventral hernias are well demonstrated by both herniography and cross-sectional imaging; however, herniation of only fat and supine imaging may lead to misdiagnosis with computed tomography or ultrasound. Spigelian hernias are probably best depicted with cross-sectional imaging. Diagnostic pitfalls of herniography are reviewed, including the need for obtaining postexercise radiographs, oblique radiographs, and tangential radiographs.