Comparison of ELISA with shell vial cell culture method for the detection of human rotavirus in fecal specimens

The aim of the study was to compare an enzyme immunoassay method with shell vial cell culture method for detection of rotavirus in fecal specimens. In addition, the correlation between laboratory results and clinical scores of patients with gastroenteritis was evaluated. A total of 219 fecal specimens from children (ages 3 weeks to 5 years) with acute gastroenteritis submitted to pediatric emergency room were evaluated by both ELISA and shell vial cell culture. A Vesikari score was used for assessing the severity of the illness. Among 219 stool samples tested, 107 (48.9%) were determined to be positive. Two specimens were positive by shell vial cell culture method while they were ELISA negative. According to these results the calculated sensitivity, specificity, PPV, and NPV of ELISA were 98.1%, 100%, 100%, and 98.2%, respectively. The mean severity score for the 107 episodes of rotavirus diarrhoea was 11.0 +/- 3.6 compared to 4.5 +/- 1.9 for the 112 episodes of non-rotavirus diarrhea in the same population. Our study indicates that ELISA, which is easier to perform, faster and cheaper than cell culture methods may be suitable for routine diagnosis of rotavirus infections. The severity of rotavirus positive gastroenteritis was significantly higher than that of rotavirus negative patients.     

Comparison of methods for rotavirus detection in water and results of a survey of Jerusalem wastewater.

Methods for the detection of viable rotaviruses and rotavirus antigen in water were developed and compared. The methods included laboratory-developed enzyme-linked immunosorbent assays (ELISAs) with chromogenic and luminescent substrates, commercial Rotazyme and Enzygnost ELISAs, and an indirect immunofluorescent assay. Of the methods tested, the immunofluorescent assay and the Enzygnost ELISA were the most sensitive for the simian rotavirus SA-11. All of the methods were positive for human rotavirus from clinical specimens. Seeded SA-11 rotavirus was concentrated from water by absorption to and elution from Zeta Plus filters followed by organic flocculation. Interference with the assays by components of the wastewater concentrates was minimal for the ELISAs, although the undiluted organic flocs were cytotoxic for the immunofluorescent assay. A survey of Jerusalem wastewater was carried out over the course of 1 year, and samples were assayed for rotaviruses and enteroviruses. Although enteroviruses were found in almost all of the samples, all samples were negative for rotaviruses. The concentration of rotaviruses in the wastewater was thus below the detection limit of the method used.     

Antagonistic effect exerted by three strictly anaerobic strains against various strains of Clostridium perfringens in gnotobiotic rodent intestines

Our purpose was to study bacterial antagonism between a limited number of strictly anaerobic strains and Clostridium perfringens in the intestinal tract of gnotobiotic rodents. Gnotobiotic mice harboring a Bacteroides thetaiotaomicron, a Fusobacterium necrogenes, and a Clostridium sp. strain were protected against pathogenic B, C, and D C. perfringens serotypes. A drastic antagonistic effect of this three-strain association was also observed against a nonpathogenic C. perfringens serotype A (CpA). It was less efficient in gnotobiotic rats than in mice and less efficient in gnotobiotic mice fed an autoclaved diet than in mice fed the same diet sterilized by irradiation. No diffusible inhibitory substances against CpA were detected in feces of gnotobiotic mice harboring the three antagonistic strains, and no nutrient depletion was demonstrated in filtrates prepared from 10-fold diluted feces of these mice. In vitro mixed cultures of the three antagonistic strains failed to inhibit growth of CpA, whereas CpA did not multiply in a 10-fold diluted feces from gnotobiotic mice. A reverse correlation between the initial number of antagonistic strains and the division number of CpA was determined using serially diluted fecal suspensions. Thus, large numbers of viable cells of both antagonistic strains were required to inhibit the target strain in fecal suspensions as was also found in gnotobiotic mice intestines. However, no diffusible inhibitory substance was detectable nor could depletion of growth factors be identified as causing antagonism. Whatever factors that may be responsible for antagonism were found to be influenced by the host and its diet.     

Comparison of methods for rotavirus detection in water and results of a survey of Jerusalem wastewater

Methods for the detection of viable rotaviruses and rotavirus antigen in water were developed and compared. The methods included laboratory-developed enzyme-linked immunosorbent assays (ELISAs) with chromogenic and luminescent substrates, commercial Rotazyme and Enzygnost ELISAs, and an indirect immunofluorescent assay. Of the methods tested, the immunofluorescent assay and the Enzygnost ELISA were the most sensitive for the simian rotavirus SA-11. All of the methods were positive for human rotavirus from clinical specimens. Seeded SA-11 rotavirus was concentrated from water by absorption to and elution from Zeta Plus filters followed by organic flocculation. Interference with the assays by components of the wastewater concentrates was minimal for the ELISAs, although the undiluted organic flocs were cytotoxic for the immunofluorescent assay. A survey of Jerusalem wastewater was carried out over the course of 1 year, and samples were assayed for rotaviruses and enteroviruses. Although enteroviruses were found in almost all of the samples, all samples were negative for rotaviruses. The concentration of rotaviruses in the wastewater was thus below the detection limit of the method used.     

B组轮状病毒RT—PCR来源的cDNA探针核酸诊断方法的建立

近十年来,国内外学者相继报道在人以及牛、羊、猪、大鼠的腹泻粪样中检出Ｂ组轮状病毒〔１－３〕。洪涛等于１９８３年首次报道成人腹泻轮状病毒（ＡＤＲＶ）属于Ｂ组轮状病毒〔４〕。目前,轮状病毒Ｂ组已被公认为引起人及不同动物腹泻的流行病学的重要因素。由于Ｂ组轮...     

Effect of dietary protein, alfalfa, and zeolite on excretory patterns of 5',5',7',7'-[3H]zearalenone in rats

A series of experiments was conducted to determine how dietary protein, alfalfa, or zeolite influence the excretory patterns of zearalenone (Z), a uterotropic mycotoxin synthesized by Fusarium fungi. Rats were fed diets containing 16.3% casein, 40% casein, 11.2% casein + 25% alfalfa, or 25% casein + 25% alfalfa. Also fed were diets containing 0, 1, 2, or 5% anion exchange zeolite. Tracer doses of [3H]Z were administered either as a constituent of the diet or as a topical application on the skin at the base of the skull. When Z was administered orally, no differences were seen in the fraction of the dose excreted in urine or feces as a result of varying dietary levels of alfalfa and protein. Topical doses resulted in rats fed 25% casein + 25% alfalfa or 40% casein excreting more Z in urine than those fed 25% alfalfa or 16.3% casein. Fecal excretion of Z was greatest for rats fed 25% casein + 25% alfalfa whereas rats fed 40% casein excreted more fecal Z than those fed 16.3% casein. Feeding Z to rats receiving dietary zeolite resulted in a positive correlation between dietary zeolite and fecal excretion of Z but a negative correlation with urinary excretion of Z. Topical administration of Z produced a positive correlation between dietary zeolite and fecal Z excretion but no effect on urinary excretion. It may be concluded that protein and alfalfa treatments alleviate Z toxicosis through increased metabolism whereas zeolite binds Z in the digestive tract to prevent absorption.     

The mouse bioassay for the detection of estrogenic activity in rodent diets: I. A standardized method for conducting the mouse bioassay

A standardized procedure was developed for conducting the mouse bioassay for detecting estrogenic activity in rodent diets. Studies were conducted with CD-1 mice to determine the appropriate weaning age and length of bioassay period. Uterine growth curves were generated from mice weaned at 15 days of age and fed a negative control diet until 28 days of age. These mice showed slow regular increases in uterine weights from 15 22 days of age followed by rapid uterine growth in some mice from 24 to 28 days of age. Estrogenic bioassays using female mice weaned at 15 days of age and fed the positive control diets containing 4 or 6 ppb diethylstilbestrol (DES) demonstrated significant (P less than 0.05) increases in uterine weight and in uterus to body weight (U:BW) ratios over those of mice fed the negative control diet without DES for 5, 7 or 9 days after weaning. In contrast, mice weaned at 17 days of age showed significant (P less than 0.05) increases in uterine weight and in U:BW ratios only at 5 days after weaning. Six ppb DES was required in the positive control diet to produce a 1.5 fold increase in the U:BW ratio over those of mice fed the negative control diet. It was concluded that mice should be weaned at 15 days of age and that the bioassay period should be terminated at 7 days, when the mice are 22 days old, for best reproducible results. The criteria for a valid bioassay should include the demonstration of a significant statistical increase in the U:BW ratios of mice fed the DES positive diet over those of mice fed the negative control diet.     

用反转录—聚合酶链反应检测B组轮状病毒

轮状病毒（Ｒｏｔａｖｉｒｕｓ）是属于呼肠病毒科（Ｒｅｏｖｉｒｉｄａｅ）的双链ＲＮＡ（ｄｓＲＮＡ）病毒。至今已将轮状病毒分为七个组（Ａ～Ｇ）。已经发现的Ｂ组轮状病毒分别来自人、大鼠、牛、猪、羊。近十年来,通过轮状病毒的研究,轮状病毒Ｂ组已被公认为引起人...     

Effects of chitosan intake on fecal excretion of bisphenol A and di(2-ethyl)phthalate in rats

We evaluated the effects of chitosan intake on fecal excretion of bisphenol A (BPA) and di(2-ethyl)phthalate (DEHP) in rats. The rats were fed a chitosan diet (CHI group) or a control diet (control group) for 10 d and orally administrated BPA or DEHP (100, 500 mg/kg body weight, respectively) on day 4. Feces were collected and the rates of fecal excretion of BPA and DEHP were calculated. Fecal excretion rates of BPA and DEHP were significantly higher in the CHI group than in the control group. A significant negative correlation was observed between the fecal excretion rates of BPA and DEHP and apparent fat digestibility. Furthermore, the CHI group showed not only increased but also accelerated BPA excretion into the feces. In conclusion, we found that that chitosan intake significantly increased the fecal excretion of BPA, DEHP, and fat, suggesting that it might be useful for reducing adverse effects caused by lipophilic xenobiotics.     

Effect of dietary copper sulfate, Aureo SP250, or clinoptilolite on ureolytic bacteria found in the pig large intestine.

The predominant ureolytic bacteria in the pig large intestine were determined while growing pigs were fed a basal diet or basal diet plus copper sulfate, Aureo SP250, or clinoptilolite. Fecal samples were collected from four pigs fed each diet at 3, 9, and 14 weeks and analyzed for total colony counts and percent ureolytic bacteria. Fecal urease activity, ammonia nitrogen, and identity of the ureolytic bacteria were determined at 14 weeks. Copper sulfate and Aureo SP250 reduced the number of ureolytic organisms, with a marked decrease occurring in the Streptococcus spp., which made up 74% of the ureolytic isolates from the pigs on the basal diet. Other ureolytic species detected at lower concentrations were Staphylococcus spp., Selenomonas ruminantium, Bacteroides multiacidus, and Eubacterium limosum. Copper sulfate also reduced fecal urease activity (P less than 0.10). Fecal ammonia concentrations were not different between pigs fed the various diets. These data suggest that the streptococci are the most numerous ureolytic species in the pig intestinal tract and are significantly reduced by copper sulfate and Aureo SP250; however, only copper sulfate reduced intestinal urease activity.     

Effect of dietary copper sulfate, Aureo SP250, or clinoptilolite on ureolytic bacteria found in the pig large intestine

The predominant ureolytic bacteria in the pig large intestine were determined while growing pigs were fed a basal diet or basal diet plus copper sulfate, Aureo SP250, or clinoptilolite. Fecal samples were collected from four pigs fed each diet at 3, 9, and 14 weeks and analyzed for total colony counts and percent ureolytic bacteria. Fecal urease activity, ammonia nitrogen, and identity of the ureolytic bacteria were determined at 14 weeks. Copper sulfate and Aureo SP250 reduced the number of ureolytic organisms, with a marked decrease occurring in the Streptococcus spp., which made up 74% of the ureolytic isolates from the pigs on the basal diet. Other ureolytic species detected at lower concentrations were Staphylococcus spp., Selenomonas ruminantium, Bacteroides multiacidus, and Eubacterium limosum. Copper sulfate also reduced fecal urease activity (P less than 0.10). Fecal ammonia concentrations were not different between pigs fed the various diets. These data suggest that the streptococci are the most numerous ureolytic species in the pig intestinal tract and are significantly reduced by copper sulfate and Aureo SP250; however, only copper sulfate reduced intestinal urease activity.     

beta-Aspartyl-epsilon-lysine, a peptide of the fecal contents of axenic mice

Small quantities of low-molecular weight peptides have been characterized in the feces of axenic mice. In fecal material of axenic mice fed an autoclaved synthetic (SN) diet, we isolated a dipeptide and characterized its structure as beta-aspartyl-epsilon-lysine. This product was also present in the feces of gnotobiotic mice harbouring Clostridium perenne. We could not detect the product in the fecal contents of holoxenic mice, Clostridium difficile-contaminated mice or axenic mice fed the irradiated SN diet. The peptide, beta-aspartyl-epsilon-lysine, was produced by heating proteins during sterilization, causing the formation of a pseudopeptide bond between the epsilon-amino group of lysine and the amide group of asparagine. The intestinal strains seemed to differ in their ability to split this pseudopeptide bond in vivo.     

Adult diarrhea rotavirus in Jilin, China

In May 1986, an outbreak of epidemic acute diarrhea occurred in one city and three counties in Yanbian area. The diarrheal cases were seen in all age groups, the majority of the cases were seen in adult age group. No bacterial pathogens were isolated in 22 fecal samples examined, however rotavirus like particles of 52-68 nm in diameter were found in 50% (11/22) of fecal samples by the immunoelectron microscopy using convalescent sera. Examination by the adult diarrhea rotavirus (ADRV) ELISA kit showed positive reaction in 86% (6/7) of fecal extracts, however in all 22 fecal extracts examination using ELISA kit for detection of group A rotavirus showed negative reaction. The PAGE patterns of viral RNAs similar to those of ADRV were seen in 13 out of 22 fecal extracts. The detection rate by PAGE analysis was 59%. Based on the above data, the etiological agent of the epidemic acute diarrhea, which occurred in Yanbian area, was identified to be ADRV.     

Species variation in the fecal metabolome gives insight into differential gastrointestinal function

The metabolic composition of fecal extracts provides a window for elucidating the complex metabolic interplay between mammals and their intestinal ecosystems, and these metabolite profiles can yield information on a range of gut diseases. Here, the metabolites present in aqueous fecal extracts of humans, mice and rats were characterized using high-resolution (1)H NMR spectroscopy coupled with multivariate pattern recognition techniques. Additionally, the effects of sample storage and preparation methods were evaluated in order to assess the stability of fecal metabolite profiles, and to optimize information recovery from fecal samples. Finally, variations in metabolite profiles were investigated in healthy mice as a function of time. Interspecies variation was found to be greater than the variation due to either time or sample preparation. Although many fecal metabolites were common to the three species, such as short chain fatty acids and branched chain amino acids, each species generated a unique profile. Relatively higher levels of uracil, hypoxanthine, phenylacetic acid, glucose, glycine, and tyrosine amino acids were present in the rat, with beta-alanine being unique to the rat, and glycerol and malonate being unique to the human. Human fecal extracts showed a greater interindividual variation than the two rodent species, reflecting the natural genetic and environmental diversity in human populations. Fecal composition in healthy mice was found to change over time, which might be explained by altered gut microbial presence or activity. The systematic characterization of fecal composition across humans, mice, and rats, together with the evaluation of inherent variation, provides a benchmark for future studies seeking to determine fecal biomarkers of disease and/or response to dietary or therapeutic interventions.     

Stimulation of fecal fat excretion and the disposal of protoporphyrin in a murine model for erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is characterized by toxic accumulation of the hydrophobic compound protoporphyrin (PP). Ferrochelatase-deficient (fch/fch) mice are an animal model for human EPP. Recently, we have demonstrated that the accumulation of another hydrophobic compound, unconjugated bilirubin, could effectively be treated by stimulation of fecal fat excretion. We investigated whether stimulation of fecal fat excretion enhanced the disposal of PP in fch/fch mice. Fch/fch mice were fed for 8 wk with a high-fat diet (16 wt% fat; control) or with the high-fat diet mixed with either a nonabsorbable fat (sucrose polyester) or the intestinal lipase inhibitor orlistat. The effects of the treatments on fecal excretion of fat and PP and on hepatic PP concentrations were compared with control diets. Fecal fat excretion in fch/fch mice on a high-fat diet was higher than in mice on a low-fat diet (+149%, P < 0.05). Sucrose polyesters and orlistat increased fecal fat excretion even more, up to sixfold of control values. However, none of the different treatments affected fecal PP excretion or hepatic PP concentration. Treatment of fch/fch mice with a high-fat diet, a nonabsorbable fat diet, or with orlistat increased the fecal excretion of fat but did not increase fecal PP excretion or decrease hepatic PP concentration. The present data indicate that accumulation of PP is not amenable to stimulation of fecal fat excretion.     

The influence of exogenous antigenic stimulation on the specificity repertoire of background immunoglobulin-secreting cells of different isotypes

The total number of spontaneously occurring ("background") IgM-, IgG-, and IgA-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of IgM-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Ac146 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG- and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG- and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG- and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG- and IgA-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation.     

猪和牛轮状病毒的分离与鉴定

应用MA-104细胞、胰蛋白酶处理培养物和旋转培养技术,成功地从患腹泻仔猪和犊牛的粪便样品中,分离到6株猪轮状病毒和4株牛轮状病毒。经形态学、血清学和病毒RNA电泳分析等鉴定,确为典型轮状病毒。经MA-104细胞连续传代,这10株轮状病毒均成为细胞适应毒株,并在该细胞上产生明显的细胞病变。     

THE DIET OF GALÁPAGOS SPERM WHALES PHYSETER MACROCEPHALUS AS INDICATED BY FECAL SAMPLE ANALYSIS

Fecal samples were collected while following sperm whales ( Physeter macrocephalus ) off the Galápagos Islands, Ecuador. They contained 133 upper beaks and 164 lower beaks of cephalopods. Analysis of the lower beaks suggests that the sperm whales fed primarily on three genera of cephalopods; Histioteutbis (62%), Ancistrocbeirus (16%), and Octopoteutbis (7%). The beak dimensions indicate that the cephalopods ranged in mantle length from 5 to 54 cm and in mass from 12 to 650 g. Fecal samples varied significantly between five study years and over different parts of the study area, but the number of beaks collected per sample did not correlate significantly with defecation rate (a measure of feeding success). Using beak material from fecal samples gives a biased estimate of sperm whale diet, reducing the frequencies of very small and very large cephalopods. However, all other available methods of assessing sperm whale diet also possess biases.     

轮状病毒灭活疫苗和减毒活疫苗序贯免疫的体液免疫应答效果

目的:评价采用轮状病毒灭活疫苗进行初始免疫,减毒活疫苗进行加强免疫的序贯免疫方案的体液免疫应答效果。方法:将实验小鼠随机分为4组(口服疫苗组、序贯疫苗组、口服对照组及序贯对照组),按相应方案免疫后,ELISA检测血清轮状病毒特异性IgG和IgA、肠道轮状病毒特异性IgA;微量中和实验检测血清病毒特异性中和抗体;同时采用ELISA分析口服活疫苗后病毒排出情况。结果:与对照组相比,序贯疫苗组小鼠产生的轮状病毒特异性血清IgG、IgA、中和抗体及肠道IgA水平显著升高。与口服疫苗组相比,序贯疫苗组的免疫方案诱发的轮状病毒特异性血清IgG、IgA、中和抗体水平显著升高,肠道IgA水平两组间没有显著差异。同时,与口服疫苗组相比,序贯疫苗组中轮状病毒灭活疫苗进行的初始免疫未影响第一次口服活疫苗后病毒的排出量和排出时间,但序贯疫苗组第二次口服活疫苗后病毒的排出量迅速减少,排毒时间快速缩短,与口服疫苗组第三次服苗后病毒的排出量和排出时间相似。结论: 轮状病毒灭活疫苗和减毒活疫苗序贯免疫可有效诱发小鼠全身和黏膜局部的体液免疫应答,该方案将有可能成为轮状病毒疫苗临床应用的候选方案。