Fecal Suspension Research Articles

Comparison of three microscopic techniques for diagnosis of Cyclospora cayetanensis

Cyclospora cayetanensis oocysts in the feces of humans from Kathmandu, Nepal were identified on the basis of their size and other morphological characteristics. We compared the detection of C. cayetanensis oocysts in the feces using three microscopic techniques such as formalin–ether sedimentation, sucrose centrifugal floatation, and direct smear. Standard procedures were used for the formalin–ether sedimentation and the sucrose centrifugal floatation techniques using 0.5 g of feces, however, the direct smear technique was performed using 10 μl of fecal suspension (0.005 g of feces) and observed under the fluorescent microscope. Of the 403 samples examined, 21 samples were positive for oocysts by all three techniques. Therefore, in these 21 samples, the number of oocysts recovered by the three techniques were compared. The highest number of oocyst was obtained by the sucrose centrifugal floatation technique. In contrast, the formalin–ether sedimentation technique was found to be the least reliable concentration technique for the detection of Cyclospora in human feces. Surprisingly, the direct smear technique was found to be an effective and rapid technique for diagnosis of C. cayetanensis making it a technique of choice for routine epidemiological investigation of the prevalence of this infection in human populations.

Pharmacokinetic Difference between Licorice and Honey-processed Licoricein Rabbits

Objectives. Licorice and honey-processed licorice are widely used in clinical Chinese medicine for a variety of conditions and ailments. In this study, we investigated the difference in pharmacokinetics between unprocessed licorice and honey-processed licorice. Methods. Six rabbits were given decoctions of unprocessed licorice and honey-processed licorice in a crossover design. The concentrations of glycyrrhizin and glycyrrhetic acid in serum as well as glycyrrhetic acid in feces suspension were determined by HPLC methods. The pharmacokinetic parameters were calculated by a noncompartment model and statistical comparison was made by paired Student's t test. Results. No significant difference in pharmacokinetics of glycyrrhizin was found between the two treatments; however, licorice processed with honey increased the AUC of glycyrrhetic acid by 48%. The hydrolysis of glycyrrhizin to glycyrrhetic acid in feces suspension was greatly enhanced by the addition of honey. Conclusions. Honey processing of licorice affected the presystemic metabolism of glycyrrhizin and resulted in the elevation of blood levels of glycyrrhetic acid, which indicates that honey might increase the efficacy and toxicity of licorice. ( Mid Taiwan J Med 2004;9:73-9 )

The suitability of a faecal suspension of sheep as inocula for the estimation of utilizable crude protein of feeds by in vitro incubation

The objective of the experiments was to study the suitability of using a faecal suspension of sheep for the estimation of the utilizable crude protein (uCP) of feeds for sheep by an in vitro incubation. Twenty-four single feeds and eight feed mixtures were used as incubation substrates. In Experiment 1, the gas production after the in vitro incubation with rumen fluid or with a faecal suspension of a sheep were compared using the Hohenheim gas test. It was found that there were significant linear regression between the 24, 48 and 72 h gas production with rumen fluid and those with faecal suspensions of 35, 50, 100 and 150 g wet faeces of sheep (which were 18.6, 23.5, 52.0 and 70.5 g faeces DM, respectively) per litre McDougall's buffer (P < 0.0001). The highest regression coefficient (r2) was calculated between the gas production after inoculation with a suspension of 100 g wet faeces per litre McDougall's buffer (x, ml·200 mg − 1 feed DM) for 48 h and the gas production after inoculation with rumen fluid (y, ml·200 mg − 1 feed DM) for 24 h: Based on these results, in Experiment 2 the estimation of utilizable crude protein (uCP) of feeds was compared by using the in vitro incubation technique of Zhao and Lebzien (2000), where feeds were inoculated either with rumen fluid or with a faecal suspension (100 g wet faeces of sheep, i.e. 52 g faeces DM per litre McDougall's buffer). The results indicated that there were no significant differences of the estimated uCP after inoculation with rumen fluid or the faecal suspension (P > 0.05). A significant regression was found between the uCP after incubation for 48 h with 100 g wet faeces (x, g·kg − 1 DM) and the uCP after incubation for 24 h with rumen fluid (y, g·kg − 1 DM): Although this regression was significant, the coefficient r2 was not high. Therefore, further research is needed before sheep faeces could replace rumen fluid as an inocula for the estimation of uCP by the in vitro incubation technique.

Improvement of human faecal flora-associated mouse model for evaluation of the functional foods

Animal models are required for evaluation of the functional foods such as pro/prebiotics exerting effects through the metabolism of the intestinal microflora. The object of this study was to establish new human flora-associated mice reflecting the environment of the human intestinal tract. We inoculated a human faecal suspension into segmented filamentous bacteria (SFB) monoassociated mice as a model system. In both human flora (HF) and SFB-associated mouse (HF-SFB mouse), intestinal characteristics such as the composition of intraepithelial lymphocytes, the expression of major histocompatibility complex (MHC) class II molecules and the number of immunoglobulin A-producing cells in the mucosa was closer to those of conventionally reared mice than was case with human flora-associated mice (HF mice) lacking SFB. Several predominant bacterial groups except lactobacilli in human flora were found in faeces of HF-SFB mice. Lactobacilli established small populations in the gut of HF-SFB mice when administered before inoculation with the human flora. Faecal enzymatic activities and organic acid concentration of HF-SFB mice proportionally reflected those of the donor subject. We established a new human flora-associated mouse (HF-SFB mouse), in which intestinal characteristics are normally developed and their major microbial composition reflect the human. HF-SFB mice are a valuable model for studying pro/prebiotic effects on the human intestine.

Zymosan-induced luminol-dependent chemiluminescence response of circulating and extravasated leukocytes in experimental sepsis.

This study examines a concurrent profiling of circulating and extravasated polymorphonuclear leukocytes (PMNs) in a rat model of experimental sepsis. Fecal peritonitis was induced in Wistar male rats by intraperitoneal instillation of a fecal suspension in saline (1:1 w/v). Blood and peritoneal fluid were collected 8 h following fecal inoculation for the evaluation of inflammatory response of PMNs using zymosan-induced luminol-dependent chemiluminescence. Fifty microliters of pre-diluted blood or peritoneal fluid samples were mixed with 150 microl of reaction mixture (4 x 10(-4) M luminol+50 microg opsonized zymosan+0.1% gelatin in Hank's balanced salt solution) and the chemiluminescence signal was measured in a luminometer at 37 degrees C. Fecal peritonitis caused a significant leukocytopenia (3540+/-297 mm(-3) versus control value of 7525+/-711 mm(-3), p < 0.001) accompanied by massive infiltration of PMNs in the peritoneal cavity (34700+/-4006 versus 7325+/-425 mm(-3), p < 0.001). The phagocytic activity of circulating blood PMNs was down-regulated whereas a significant up-regulation was observed in the activity of PMNs from peritoneal fluid. In conclusion, this study clearly demonstrates sepsis-induced alterations in both blood and peritoneal fluid PMNs and their quantitative assessment may be helpful in disease evaluation and designing effective therapies.

A deglucosylated metabolite of paeoniflorin of the root of Paeonia lactiflora and its pharmacokinetics in rats.

Paeoniflorin is a bioactive monoterpene glucoside in Paeoniae Radix (PR), the roots of Paeonia lactiflora (Ranunculaceae). By oral administration to rats with the decoction of PR, the metabolism and pharmacokinetics of paeoniflorin were investigated in this study. A deglucosylated metabolite of paeoniflorin, paeoniflorgenin (PG), in serum was identified based on HPLC/MS and NMR spectral data. HPLC/UV methods were developed for determining PG in serum and feces suspension. A non-compartment model was used for the calculation of pharmacokinetic parameters. Moreover, the metabolism of paeoniflorin by various types of feces was investigated as well. The paeoniflorin levels in serum were below the detection limit throughout the study. The C (max), t (max), and AUC (0-t) of PG were 8.0 microg/mL, 10 min and 487.0 microg min/mL, respectively. Paeoniflorin was found to be hydrolyzed into PG through incubation with feces of rabbit, rat, pig or human. Similar profiles of PG were shown for various types of feces, except for rabbit. In conclusion, paeoniflorin was not absorbed per se, whereas its aglycone paeoniflorgenin was absorbable and circulating in the bloodstream. Rat and pig are appropriate models for investigating the metabolism and pharmacokinetics of paeoniflorin.

Fluconazole attenuates lung injury and mortality in a rat peritonitis model

Acute lung injury following peritonitis constitutes an enigmatic clinical problem with no specific therapy. Recently, immunomodulators such as azole compounds have been shown to attenuate shock-related tissue injury. The present investigation was undertaken to study the effect of fluconazole on acute lung injury and survival following faecal peritonitis in rats. Male Wistar rats weighing 225-235 g. Faecal peritonitis (Fp) was produced in four groups of adult male Wistar rats by intraperitoneal administration of non-sterile faecal suspension (1:1 w/v saline). A fifth group of rats was given sterile faecal material (SFM), which served as control. Rats in Fp groups were given fluconazole in doses of 0 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg by gavage 30 min before induction of peritonitis. The control animals received an equal volume of distilled water. Survival over a period of 72 h, oxidative stress, neutrophil activity, and lung injury were measured. This study showed a 90% survival in the fluconazole-treated group compared to only 20% survival in untreated rats (P<0.008 log-rank test). The lungs of animals with Fp showed massive pathological changes including intraalveolar oedema, fibrosis, and mixed inflammatory cell infiltrate. These changes were dose-dependently attenuated by fluconazole. Enhanced oxidative stress (P<0.001) and neutrophil activity in the peritoneal fluid and lung (P<0.001) in Fp animals was dose-dependently reduced by fluconazole. This study clearly suggests the role of neutrophils in Fp-induced tissue injury/mortality, which may be dose-dependently, attenuated by fluconazole.

Serotype-specific mucosal immune response and subsequent poliovirus replication in vaccinated children.

A total of 32 unimmunized children (median age 5 months) living in an orphanage in Minsk, Belarus, were vaccinated with three doses of oral poliovirus vaccine (OPV) with a 60-day interval between the doses. Blood samples were drawn before the immunizations and 45-50 days after each vaccine dose. Excretion of the vaccine viruses was followed by examining fecal specimens collected weekly after each vaccine dose. All children seroconverted by the second dose of OPV at the latest to all three serotypes of poliovirus but differences were seen regarding the intestinal responses. The strongest responses in both neutralizing antibodies and virus-binding IgA in fecal suspensions were seen toward poliovirus Type 2. Consistent with this, relatively little poliovirus Type 2 excretion was seen after the second and third doses of OPV as compared to that of poliovirus Type 1 or Type 3. The delayed development of functional intestinal immunity against the latter serotypes was associated with relatively weaker intestinal antibody responses compared to poliovirus Type 2. In the case of poliovirus Type 3, about 10% of children were still excreting the vaccine virus 9 weeks after administering the third dose. These results are consistent with epidemiological observations of the serotype-specific efficacy of OPV immunizations. The proportion of specimens showing nonpolio enteric viruses gradually increased through the 6-month study period. This may reflect the possibility that after the first dose of OPV, intensive replication of the vaccine viruses may out compete the nonpolio viruses in the intestines of the vaccinees or, alternatively, mask nonpolio viruses during the cell culture isolation procedure.

Mucosal immunization with a replication-deficient adenovirus vector expressing murine cytomegalovirus glycoprotein B induces mucosal and systemic immunity

The murine cytomegalovirus (MCMV) glycoprotein B (gB) gene was expressed in an adenovirus replication-deficient vector. This virus, designated Ad-gB, was used to immunize BALB/c and B6 mice by the intranasal (i.n.) route to induce an immune response. Following primary immunization, antibody was detected in serum of 100% of vaccinees, as well as the bronchoalveolar lavage, fecal suspensions and vaginal washings. The viral titer of lung and salivary gland of vaccinees 10 days after intranasal challenge with MCMV at 10 5 or 10 3 plaque forming units (PFU) were significantly reduced compared to controls. Re-exposure of vaccinees to Ad-gB 30 days after primary immunization induced a remarkable boost of serum and mucosal antibody responses and further reduction of MCMV titers in the lung and salivary glands. The ability to induce both a systemic and mucosal immune response to a specific gene product may be important in reducing horizontal transmission of CMV infections across mucosal surfaces and in altering host immunity to CMV.

Biotransformation of pinoresinol diglucoside to mammalian lignans by human intestinal microflora, and isolation of Enterococcus faecalis strain PDG-1 responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol.

By anaerobic incubation of pinoresinol diglucoside (1) from the bark of Eucommia ulmoides with a fecal suspension of humans, eleven metabolites were formed, and their structures were identified as (+)-pinoresinol (2), (+)-lariciresinol (3), 3'-demethyl-(+)-lariciresinol (4), (-)-secoisolariciresinol (5), (-)-3-(3", 4"-dihydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butane-1, 4-diol (6), 2-(3', 4'-dihydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butane-1, 4-diol (7), 3-(3"-hydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butane-1, 4-diol (8), 2-(3', 4'-dihydroxybenzyl)-3-(3"-hydroxybenzyl)butane-1, 4-diol (9), (-)-enterodiol (10), (-)-(2R, 3R)-3-(3", 4"-dihydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butyrolactone (11), (-)-(2R, 3R)-2-(3', 4'-dihydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butyrolactone (12), (-)-(2R, 3R)-3-(3"-hydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butyrolactone (13), 2-(3', 4'-dihydroxybenzyl)-3-(3"-hydroxybenzyl)butyrolactone (14), 2-(3'-hydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butyrolactone (15) and (-)-(2R, 3R)-enterolactone (16) by various spectroscopic means, including two dimensional (2D)-NMR, mass spectrometry and circular dichroism. A possible metabolic pathway was proposed on the basis of their structures and time course experiments monitored by thin-layer chromatography. Furthermore, a bacterial strain responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol was isolated from a human fecal suspension and identified as Enterococcus faecalis strain PDG-1.

Simultaneous determination of glycyrrhizin metabolites formed by the incubation of glycyrrhizin with rat feces by semi-micro high-performance liquid chromatography.

A method for semi-micro high-performance liquid chromatography (HPLC) has been established for the simultaneous determination of 3alpha-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid together with glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glucuronide formed by incubation of glycyrrhizin with rat feces. The analysis was accomplished within 25 min with a TSKgel ODS-80TsQA (150 x 2.0 mm i.d.) column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile at a flow rate of 0.2 ml.min(-1), a thermostatic oven at 25 degrees C, and detection at 254 nm. The detection limits of these compounds were 0.2 pmol per injection (5 microl). The metabolites of glycyrrhizin, by anaerobic or aerobic incubation with rat fecal suspension over 48 h, were determined. Glycyrrhizin was almost completely converted to metabolite glycyrrhetic acid, and metabolites 3alpha-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid in negligible amounts in anaerobic conditions. However, the metabolic time courses of 3-dehydroglycyrrhetic acid when incubated in aerobic conditions revealed that it apparently continued increasing during the whole incubation period.

An adapted ImmunoMagnetic cell Separation method for use in quantification of Escherichia coli O157:H7 from bovine faeces

Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture- and PCR detection, pre-enrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only. If the actual numbers of E. coli O157:H7 are to be quantified, growth enrichment must be excluded and the organisms isolated directly from the sample of interest. Such quantification is necessary, for example, to determinate contamination levels on beef carcasses and for determination of bacterial numbers in in vivo gene expression studies. In the present study, it was not possible to recover organisms from bovine faecal suspensions using the customary IMS system and so a range of alternative buffers and other paramagnetic beads was tested. Combination of a 6.2-μm diameter bead with a detergent-based buffer gave optimal recovery of E. coli O157:H7 organisms from faecal suspensions. This system was validated for recovery of E. coli O157:H7 by comparing it with that obtained with the standard Dynabeads® IMS protocol, using both the traditional broth enrichment method and a quantitative detection approach. We conclude that a 6.2-μm diameter Aureon bead can be used for quantitative isolation of E. coli O157:H7 directly from bovine faeces and, for this purpose, is preferred to the 2.8-μm diameter Dynal bead.

Influence of a propionic acid feed additive on performance of turkey poults with experimentally induced poult enteritis and mortality syndrome

Poult enteritis and mortality syndrome (PEMS) has multiple etiological agents associated with its occurrence, including two viruses and at least three Escherichia coli isolates. Myco Curb (MC) contains organic acids and is used as a feed additive to inhibit growth of many bacteria and toxin-producing molds but not viruses. Studies evaluating the influence of MC on BW, feed conversion, and mortality indicate that turkey poults tolerate MC at 1.25% but not 2.50%, but higher MC content in feed provides greater suppression of growth of bacterial isolates commonly associated with PEMS. In two PEMS experiments, 1.25% MC was blended into poult starter feed and was maintained in the feed for the duration of the 3-wk experiments. In these experiments, 1-d-old commercial poults were placed into battery brooders and were given turkey starter feed and water ad libitum. At 6 d posthatch, PEMS-designated poults were given a 1-mL oral gavage of a 10% suspension of feces from PEMS-infected poults. BW depression due to PEMS was not alleviated by MC, although there was less variation in mean BW of the MC-fed poults, and there was a highly significant reduction in mortality (68% in PEMS-exposed with MC vs. 32.5% in PEMS-exposed without MC). The reduction in mortality in the MC-fed poults was attributed to decreased bacterial content of the gut and to maintenance of packed cell volume and hemoglobin content. It was concluded that MC might be a potential nutritional intervention during PEMS.

A survey of Blastocystis sp. in livestock, pets, and zoo animals in Japan

The prevalence of Blastocystis sp. was examined in fecal samples collected from cattle, pigs, dogs, and a variety of zoo animals (primates, carnivores, herbivores, pheasants, and ducks) by direct observation of fresh fecal suspensions or cultured materials, using light microscopy. The cattle and pigs were randomly sampled from 11 and 12 commercial farms, respectively, located in the western region of Japan. The dog material used in this study was obtained from pets housed in an animal shelter in the city of Osaka. Zoo animals were chosen based on housing conditions that minimized the possibility of intra-zoo transmission of the organism. The prevalence rate among the groups varied greatly. A high prevalence of infection was observed in the farm animal group, ranging from 95% (58/61) in pigs to 71% (39/55) in cattle, whereas the dog fecal samples were completely free of the organism. Prevalence of the organism in the zoo animal were 85% (29/34) in primates, 80% (8/10) in pheasants, 56% (9/16) in ducks, and 0% (0/58) in various carnivores and herbivores. Among the zoo animals infected with Blastocystis, eight species of primates, eight species of pheasants, and four species of ducks were confirmed as new hosts. Since Blastocystis organisms isolated from various animals were morphologically indistinguishable from Blastocystis hominis by light microscopy, further genomic studies are required for analysis of the zoonotic potential or etiological significance of these isolates.

Metabolism of constituents in Huangqin-Tang, a prescription in traditional Chinese medicine, by human intestinal flora.

In the course of studies on the metabolism of active components of Huangqin-Tang by human intestinal flora (HIF), Huangqin-Tang and all individual herbs in the decoctions were incubated with a human fecal suspension separately. By using a high-performance liquid chromatographic (HPLC) method which was previously established in our laboratory, the metabolites in both the compound prescription and all the single herb decoctions were identified and determined both qualitatively and quantitatively. We found that the constituents of Huangqin-Tang, incluing baicalin (baicalein 7-glucuronide; BG), wogonoside (wogoninoglucuronide; WG), oroxylin-A-glucuronide (OG) from Scutellariae Radix, paeoniflorin (PF) from Paeoniae Radix, liquiritin (liquiritigenin 4'-O-glucoside; LG), isoliquirtin (isoliquiritigenin 4-glucoside; ILG) and glycyrrhizic acid (GL) from Glycyhhizea Radix, were converted to their metabolites baicalein (B), wogonin (W), oroxylin-A (O), paeonimetabolin-I (PM-I), liquiritigenin (L), isoliquiritigenin (IL) and glycyrrhetinic acid (GA) by HIF. The contents of the metabolites in Huangqin-Tang and in each single herb decoction increased significantly after incubation with intestinal flora. Comparing with single herb decoctions, the transformation of BG, WG, OG, LG and ILG in the compound prescription was promoted, however, that of PF and GL was inhibited. All the results suggested that the glycosides of many medicinal herbs could be converted to aglycones by HIF, and the metabolism of most glycosides was improved in the compound prescription.

Composition of human intestinal flora analysed by fluorescent in situ hybridisation using group-specific 16S rRNA-targeted oligonucleotide probes

Cultivation methods classically used to describe the faecal flora composition are often too selective for certain groups of bacteria. This study was conducted to determine the human faecal flora composition by fluorescent in situ hybridisation, a direct and culture-independent method. Four group-specific probes and a domain probe Eub 338 targeting the 16S rRNA were used to analyse fixed faecal bacterial suspensions from nine healthy adult volunteers. Epifluorescence microscopy and image analysis were performed to evaluate the relative proportion of cells from each group. After optimisation of hybridisation conditions, the reproducibility of the protocol was evaluated to validate the FISH procedure. The domain probe Eub 338 labelled an average of 80 ± 11% total faecal bacteria. The panel of four probes revealed more than 75% of the flora. The Clostridium coccoides-Eubacterium rectale group was the most represented, accounting for 36 ± 7% of the bacteria. The three other probes used, Bacto 1080, Bif 164 and Fprau 645 labelled 17 ± 5%, 15 ± 9% and 23 ± 5% of the total flora, respectively. The two dominant groups belonging to Clostridium and relatives constituted nearly 60% of the total flora. FISH coupled with image analysis is a direct and powerful molecular tool to assess the composition of the human faecal flora.

Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs

The objective of this study was to assess the risk of transmission of swine hepatitis E virus (swine HEV) to naı̈ve pigs by inoculation with tissues or feces collected from pigs infected experimentally with swine HEV. Seventy-five, 3-week-old pigs were assigned randomly to 24 groups of 3–4 pigs and inoculated with homogenates of tissues (liver, heart, pancreas, or skeletal muscle) or a suspension of feces from swine HEV-infected pigs collected at 3, 7, 14, 20, 27, or 55 days post inoculation (DPI). Each inoculum was prepared as a 10% suspension (w/v) in PBS buffer and tested by a semi-quantitative RT-PCR for swine HEV RNA and by the swine bioassay. The inoculation route was intravenous for liver, heart and pancreas, and via stomach tube for skeletal muscle and fecal suspension. The liver homogenate inocula and feces collected at 3–7 and 14–20 DPI were positive for swine HEV RNA by RT-PCR. The pigs inoculated with liver homogenates collected at 3–7 and 14–20 DPI developed anti-HEV antibodies and swine HEV RNA was detected in their sera. Pigs inoculated with heart, pancreas, skeletal muscle homogenates or fecal suspensions failed to develop anti-HEV antibodies. These findings suggest that there is a potential risk of transmission of swine HEV via liver tissue from infected pigs in the early stages (3–20 DPI) of infection and the in vitro RT-PCR assay correlates well with the swine bioassay.

Foodborne viruses and fresh produce.

Raw and minimally processed fruits and vegetables are typically sold to the consumer in a ready-to-use or ready-to-eat form. These products do not generally contain preservatives or antimicrobial agents and rarely undergo any heat processing prior to consumption. For many years raw fruits and vegetables have been implicated as vehicles for transmission of infectious micro-organisms. Although fresh produce can support the growth and/or survival of many pathogenic bacteria there is little published information on the stability of human pathogenic viruses on these food products. Viruses cannot grow in or on foods but may sometimes be present on fresh produce as a result of faecal contamination. This contamination can arise at source in the growth and harvesting area from contact with polluted water and inadequately or untreated sewage sludge used for irrigation and fertilization. Alternatively, fruits or vegetables handled by an infected person might become contaminated with virus and transmit infection. The most frequently reported foodborne viral infections are viral gastroenteritis and hepatitis A: both have been associated with the consumption of fresh fruit or vegetables.

Use of Steam Condensing at Subatmospheric Pressures To Reduce Escherichia coli O157:H7 Numbers on Bovine Hide

This study used a laboratory-scale apparatus to apply subatmospheric steam to bovine hide pieces inoculated with Escherichia coli O157:H7 in maximum recovery diluent (MRD) and in high–liquid content and low–liquid content fecal suspensions (HLC fecal and LLC fecal, respectively). The survival of the organism in fecal clods, which were stored for 24 days in a desiccated state, was assessed. Inoculated fecal clods were also treated with subatmospheric steam. Steam treatment at 80 ± 2°C for 20 s reduced E. coli O157:H7 concentrations on hide inoculated to initial concentrations of approximately 7 log10 CFU/g by 5.46 (MRD inoculum), 4.17 (HLC fecal inoculum), and 5.99 (LLC fecal inoculum) log10 CFU/g. The reductions achieved in samples inoculated with LLC feces were larger than in samples inoculated with HLC feces (P < 0.05). Treatment at 80 ± 2°C for 10 s resulted in significantly smaller reductions (P < 0.05) on hide pieces of 2.54 (MRD), 1.94 (HLC fecal), and 2.15 (LLC fecal) log10 CFU/g. There were no significant differences among the reductions observed in all inoculum types in samples treated for 10 s. E. coli O157:H7 inoculated in fecal clods to 7.78 log10 CFU/g and stored at 4 or 15°C survived for at least 24 days. Steam treatment (20 s) of 3-day-old clods reduced surviving E. coli O157:H7 numbers from 4.20 log10 CFU/g to below the limit of detection of the assay used (1.20 log10 CFU/g). This study shows that steam condensing at or below 80 ± 2°C can reduce E. coli O157:H7 when present on bovine hide, reducing the risk of cross contamination to the carcass during slaughter and dressing.

RT-PCR based analysis of cell culture negative stools samples from poliomyelitis suspected cases.

Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several serotypes of enterovirus which possess the potential to cause neurological disorders are not easily isolated in the cell culture systems used for the AFP diagnosis. Our goal was to look into the presence of enterovirus genomes in fecal suspensions previously considered negative by cell culture procedures, using RT-PCR. One hundred and seventy-three fecal samples collected from AFP cases and contacts occurring in Brazil, Peru and Bolivia and tested negative regarding viral isolation, after inoculation in the cell lines RD and Hep2C, were analyzed by RT-PCR using a pair of primers which specifically detects enterovirus genome RNA's. Twenty-six samples (15%) showed amplicons compatible with those observed for enterovirus RNA amplification. The identity of these amplicons were confirmed by nucleotide sequencing. By using RT-PCR directly in the fecal suspensions we were able to detect enterovirus RNA's in twenty-six additional samples. These samples would be considered as negative if only the standard cell-culture-based methodology had been utilized. No polioviruses were detected among the positive samples.