Development of a diffusion-weighted MRI protocol for multicentre abdominal imaging and evaluation of the effects of fasting on measurement of apparent diffusion coefficients (ADCs) in healthy liver.

OBJECTIVE: To assess the effect of fasting and eating on estimates of apparent diffusion coefficient (ADC) in the livers of healthy volunteers using a diffusion-weighted MRI protocol with b-values of 100, 500 and 900 s mm(-2) in a multicentre study at 1.5 T. METHODS: 20 volunteers were scanned using 4 clinical 1.5-T MR scanners. Volunteers were scanned after fasting for at least 4 h and after eating a meal; the scans were repeated on a subsequent day. Median ADC estimates were calculated from all pixels in three slices near the centre of the liver. Analysis of variance (ANOVA) was used to assess the difference between ADC estimates in fasted and non-fasted states and between ADC estimates on different days. RESULTS: ANOVA showed no difference between ADC estimates in fasted and non-fasted states (p = 0.8) nor between ADC estimates on different days (p = 0.8). The repeatability of the measurements was good, with coefficients of variation of 5.1% and 4.6% in fasted and non-fasted states, respectively. CONCLUSION: There was no significant difference in ADC estimates between fasted and non-fasted measurements, indicating that the perfusion sensitivity of ADC estimates obtained from b-values of 100, 500 and 900 s mm(-2) is sufficiently low that changes in blood flow in the liver after eating are undetectable beyond the variability in the measurements. ADVANCES IN KNOWLEDGE: Assessment of the effect of prandial state on ADC estimates is critical, in order to determine the appropriate patient preparation for biological validation in clinical trials.

Effects of increasing concentrations of corn distillers dried grains with solubles on the egg production and internal quality of eggs.

A study was conducted to determine the effect of feeding high concentrations of corn distillers dried grains with solubles (DDGS) on egg production and the internal quality of eggs from laying hens. Four diets were formulated to contain 0, 17, 35, or 50% corn DDGS. A total of two hundred forty 54-wk-old Single-Comb White Leghorn laying hens were randomly allotted to 2 birds per cage with 3 consecutive cages representing an experimental unit (EU). Each EU was assigned to 1 of 4 dietary treatments according to a completely randomized design. Hens were fed for a 24-wk experimental period after transition feeding to gradually increase corn DDGS inclusion over a 4-wk period. Two sets of experimental diets were formulated, and each diet was fed for 12 wk. Egg production, feed consumption, egg component, yolk color, Haugh unit during storage times, and shell breaking strength were measured. Egg production, egg weight, egg mass, feed intake, and feed efficiency were adversely affected by the highest level of DDGS in the diet (50%) during the first 12-wk period. Once diets were reformulated to include an increased concentration of both lysine and methionine, differences among the dietary treatments were reduced, as the performance of the 50% DDGS diets was greatly improved. Over the last 6 wk of study, no differences in egg production, egg weight, and feed intake among DDGS treatments were found. The DDGS diets positively affected the internal quality of eggs during storage. Improved yolk color and Haugh unit were observed as the dietary DDGS levels increased, but the increase for Haugh unit was significant only when the DDGS level was 50%. Shell weight percentage was increased in the 50% DDGS diet, but no differences in yolk and albumen percentage were observed. It was concluded that up to 50% of DDGS could be included in the layer's diet without affecting egg weight, feed intake, egg mass, feed efficiency, and egg production as long as digestible amino acids were sufficient in DDGS-added diets.

Prevalence of Clostridium difficile-associated diarrhoea among hospitalized Jordanian patients.

We investigated stool specimens of 400 patients at Jordan University Hospital (300 patients with clinical diarrhoea and 100 controls without diarrhoea) for the presence of Clostridium difficile or its toxin. We found a 9.7% prevalence rate of C. difficile or its toxin in stools of patients with diarrhoea. The prevalence of other potential enteric pathogens, such as Salmonella spp. (2.3%), Shigella spp. (1.0%) and Entamoeba histolytica (2.7%), was significantly less. Prevalence of C. difficile or its toxin in controls was 3.0%. Toxin A was detected in 93.1% of C. difficile-associated diarrhoea cases using an enzyme immunoassay. Our study indicates that C. difficile-associated diarrhoea is mostly observed among hospitalized patients aged > or = 50 years, in association with antimicrobial treatment.

Prevalence of Clostridium difficile-associated diarrhoea among hospitalized Jordanian patients

We investigated stool specimens of 400 patients at Jordan University Hospital [300 patients with clinical diarrhoea and 100 controls without diarrhoea] for the presence of Clostridium difficile or its toxin. We found a 9.7% prevalence rate of C. difficile or its toxin in stools of patients with diarrhoea. The prevalence of other potential enteric pathogens, such as Salmonella spp. [2.3%], Shigella spp. [1.0%] and Entamoeba histolytica [2.7%], was significantly less. Prevalence of C. difficile or its toxin in controls was 3.0%. Toxin A was detected in 93.1% of C. difficile-associated diarrhoea cases using an enzyme immunoassay. Our study indicates that C. difficile-associated diarrhoea is mostly observed among hospitalized patients aged > or = 50 years, in association with antimicrobial treatment

Plasma levels of the anaphylatoxins C3a and C4a in patients with IgA nephropathy/Henoch-Schönlein nephritis.

Measurements of plasma anaphylatoxins C3a and C4a were carried out as an indicator of in vivo complement activation in 46 patients suffering from IgA nephropathy/Henoch-Schönlein nephritis. There was a significant correlation between plasma levels of C4a and plasma creatinine and urea. We also found a significant correlation of plasma levels of C3a with plasma creatinine. We propose that the measurement of anaphylatoxin levels provides a sensitive indicator of in vivo complement activation and may serve as an additional method for monitoring the progress of disease in these patients.

Plasma levels and mode of excretion of the anaphylatoxins C3a and C4a in renal disease.

Plasma levels of the anaphylatoxins C3a and C4a were examined in patients suffering with various renal diseases. Raised levels were observed in a considerable number of patients; although such elevation could be ascribed to in vivo complement activation in cases of immune complex glomerulonephritis, we found that raised plasma C4a levels appeared to be related to impaired renal function, suggesting that C4a anaphylatoxin is cleared by the kidneys. No such relationship was found in the case of the anaphylatoxin C3a suggesting the possible existence of another mechanism of elimination of C3a.

Determination of virginiamycin in combination with chlortetracycline in feeds by anion-exchange chromatography.

Standard additions experiments for chlortetracycline hydrochloride and virginiamycin at a ratio of 16:1 showed a positive bias for both the plate and turbidimetric methods. The bias was eliminated by anion-exchange chromatography in both the presence and absence of feed components. When chlortetracycline and virginiamycin pre-mixes were used for fortification of laboratory-prepared feeds at chlortetracycline to virginiamycin ratios of 8 or 16:1, the recovery of virginiamycin, without treatment, was 93-100% using the plate assay and 181-236% with the turbidimetric method. The corresponding values obtained using anion-exchange chromatography were 89-99 and 85-91%, respectively. The anion-exchange technique is necessary if the turbidimetric method is used for the assay.

Effect of volume of solution per cylinder on estimation of antibiotic potency in diffusion assay.

The volume of antibiotic solutions in cylinders used for diffusion assays is assumed to have no significant effect on estimation of potency. The size of zones of inhibition from cylinders containing 0.10, 0.20, and 0.30 mL of sample solution was compared with zones of inhibition from cylinders containing 0.20 mL of standard solutions. For zinc bacitracin, chlortetracycline.HCl, oxytetracycline, lincomycin.HCl, monensin Na, neomycin sulfate, K penicillin, streptomycin sulfate, and tylosin, the percent recovery (95-102) was optimum when both standard and sample cylinders contained the same volume (0.20 mL/cylinder). At 0.30 mL/cylinder for sample and 0.20 mL for standard solutions, there was a positive bias in potency of about 50%. At 0.10 mL/cylinder, there was a negative bias of approximately 25% except for neomycin, monensin, and bacitracin. For these antibiotics, the bias was about -50%. For hygromycin B, variation in volume of solution per cylinder has little effect on assay results. Experiments on commercial feeds and premixes gave essentially the same results as for the standard solutions experiments.

Evaluation of AOAC microbial diffusion procedure for analysis of chlortetracycline in high mineral feeds.

The performance of the AOAC microbial diffusion assay procedure for the analysis of chlortetracycline was evaluated in mixed feeds and premixes as well as laboratory-prepared feeds, all with various mineral contents. In 100 mixed feeds with a calcium content ranging from less than 1 to greater than 22%, no relationship was shown between calcium content and incidence of chlortetracycline deficiency. In 106 premixes, a relationship was shown between chlortetracycline content and adherence to guarantee: below 10 g/lb, the incidence of deficiency was high; in premixes containing 20 g chlortetracycline and above, the incidences of deficiency were less than 5%. In laboratory-prepared feeds containing 12-15% calcium, grinding and storage at both 4 degrees C and at room temperature (20 degrees C) caused no decrease in the chlortetracycline concentration. Only storage of the ground feed at 37 degrees C caused a distinct loss of activity. The temperature-related loss was attributed to the epimerization of chlortetracycline.

Bacitracin determination in feeds: evaluation of methods.

Assay of bacitracin activity in feeds is subject to many sources of variation. Bacitracin standards are complex mixtures and may contain various amounts of less active forms of bacitracin. Test organisms may respond differently to these mixtures. Collaborative studies should involve the use of one standard, perhaps USP zinc bacitracin. The use of different types of bacitracin standards (regular or zinc) in a laboratory may contribute to variation in sample potency. The pyridine extraction method is subject to serious operational difficulties. Among the contributing factors are incomplete evaporation of pyridine (causing positive bias), allowing the temperature to increase too much during evaporation (causing negative bias), and the numerous manipulative steps in the technique (causing large variability). Because of these factors and the toxic properties of pyridine, the method finds little use. Methanol extraction offers a better substitute for determining zinc or methylene disalicylate bacitracin in premixes and complete feeds by the plate method.

Evaluation of methanol extraction for determination of bacitracin in feeds.

Five laboratories evaluated methanol extraction for the determination of zinc and methylene disalicylate (MD) bacitracn in swine and poultry feeds at 100 g/ton and 2 premixes labeled 50 g/lb (MD bacitracin) and 40 g/lb (zinc bacitracin). Bacitracin activity was determined on each of 2 days with 2 dilutions on each day. No significant difference was found between dilutions within a day or between days for each sample. The kind of bacitracin or type of feed did not significantly affect results.

Some factors affecting assay variability in the determination of chlortetracycline in premix feeds.

There are many sources of error in the microbiological determination of high levels of chlortetracycline in premix. Among the critical factors that can be controlled by the analyst are careful weighing of the reference standard and careful dosing of cylinders. A spectrophotometric approach and automatic filling of cylinders are suggested to ensure proper monitoring of these operations. Among factors that cannot be controlled by the analyst is the assay design. A 3-point design which is a modified version of the (3+3) design is considered, and its advantages are illustrated.

Microbiological assays for antibiotics and vitamins: considerations for assuring accuracy.

Factors that may influence the accuracy and precision of microbiological manual and semiautomated turbidimetric methods as well as diffusion assays are discussed. Influence of kind of equipment, media, test bacteria, sample preparation, form of dose response lines, operations, and personnel on quality of assays is examined with the objective of reducing to insignificance those factors under control of the analyst that are responsible for low quality assays.

Determination of virginiamycin in feeds.

Virginiamycin was extracted from the feed by ethanol-pH 2.5 phosphate buffer (1 + 1). The pH during extraction was adjusted (when necessary) to between 4 and 5. Sample dilutions and the standard dose response line were prepared to contain ethanol pH 6 phosphate buffer (2 + 8), and the test organism was Sarcina lutea. Three feeds (a poultry ration, a swine finishing ration, and a swine starter ration) showed virginiamycin recovery of 88.8--108.9% when standard solutions were added at concentrations of 4.54--90.8 g/ton. The coefficient of variation (4--20%) was larger for low potency feeds (10 g/ton) compared to the higher feeds (100 g/ton). Similarly, excellent recovery was obtained when the swine starter feed was fortified by a commercial premix. Amprolium, roxarsone, and monensin can be present at 20 times the concentration of virginiamycin with little or no interference in the antibiotic determination. Lasalocid at 10 times the concentration of virginiamycin caused a slightly positive bias (recovery, 107.4%).

Pyridine extraction method for determination of bacitracin in feeds: collaborative evaluation.

Seventeen laboratories evaluated the pyridine extraction method and neomycin-sensitized agar for the determination of zinc and MD bacitracin in swine and broiler rations at 10 and 100 g/ton. The method was also applied to the analysis of 2 premixes labeled 50 g/lb (MD bacitracin) and 40 g/lb (zinc bacitracin). Bacitracin activity was determined on each of 2 days with 2 dilutions on each day. No significant difference was found between dilutions within a day or between days for each sample. The type of bacitracin or type of feed did not significantly affect results. The difference in results between MD and zinc bacitracin in premixes approached significance. The large coefficients of variation for premixes (ca 13%) and complete feeds (ca 15--30%) indicate operational problems. The main difficulty was evaporation of pyridine. Some laboratories were not able to evaporate it completely, whereas others lost bacitracin activity, probably due to high temperature of drying. The pyridine extraction method as in 42.200 and 42.204 should be discontinued.

Microbiological plate and turbidimetric assays of chlortetracycline in feeds: collaborative study.

The manual and automated turbidimetric assays and a modified official plate assay for chlortetracycline (CTC-HCl) in feed were collaboratively studied. Three feed samples (swine feed, 100 g CTC-HCl/ton; premix I, 20 g each of CTC-HCl and sulfamethazine/lb, and 10 g penicillin/lb; and premix II, 50 g CTC-HCl/lb) were analyzed at 2 dilutions. Twelve laboratories conducted the plate assay; 8 laboratories the manual turbidimetric method; and 7 laboratories, the Autoturb analysis. Within a method, there was no significant difference between dilutions. Between methods, there was a significant difference between the manual turbidimetric plate assays only for swine feed. However, the same sample dilutions or the average values of the 2 dilutions for both methods showed no statistical difference. Among the collaborators, the slope of CTC-HCl standard curve varied between about 2.0 and 3.0 for the plate method. The turbidimetric assay has been adopted as official first action for feeds containing larger than or equal to 20 g CTC-HCl/lb.

Turbidimetric and diffusion assay of bacitracin in feeds.

A manual turbidimetric method for determining zinc or methylene disalicylate bacitracin in feeds was developed. When zinc ions (10-4M) were added to the medium and the phosphate ion concentration in standard solutions was decreased to 1%, the median response of Streptococcus faecalis was about 0.038 unit. Feeds analyzed by the turbidimetric assay should be prewashed with petroleum ether and extracted with pyridine as in 42.204. Zinc bacitracin standards added to swine and broiler rations were recovered at 93.1-102.6% by the turbidimetric method. Excellent agreement between the manual turbidimetric method and the plate assay was also obtained for finished feeds containing the zinc or methylene disalicylate salt of the antibiotic. The turbidimetric method appears to have high accuracy and precision. It is more rapid and less costly than the plate assay.

Modified assay medium for the turbidimetric assay of chlortetracycline in feeds.

In previous experiments, the turbidimetric method for determining chlortetracycline-HCI (CTC-HCI) in feeds showed lower recovery than the AOAC plate method. Although the addition of vitamins to the turbidimetric medium improved results, values by the turbidimetric method remained about 10% lower than by the plate method. A modified (1.7X the weight recommended by the manufacturer) turbidimetric assay medium decreased assay sensitivity but did not significantly change the slope of S. aureus response to CTC-HCI. There was no evidence that vitamin fortification of the modified medium had any significant effect on the growth rate of test organism. Examination of about 100 samples of commercial feeds containing CTC-HCI showed excellent agreement in results between the turbidimetric and plate methods.