Fecal Suspension Research Articles

Detection of turkey coronavirus in commercial turkey poults in Brazil

Poult enteritis complex has been incriminated as a major cause of loss among turkey poults in other countries. We have observed this in Brazil, associated with diarrhoea, loss of weight gain and, commonly, high mortality. In this study, we have used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect turkey coronavirus (TCoV) in sick poults 30 to 120 days of age from a particular producer region in Brazil. The RT-PCR was applied to extracts of intestine tissue suspensions, and the respective intestinal contents, bursa of Fabrícius, faecal droppings and cloacal swabs. Primers were used to amplify the conserved 3' untranslated region of the genome, and the nucleocapsid protein gene of TCoV. Histopathological and direct immunohistochemical examinations were performed to detect TCoV antigen in infected intestine and bursa slides. All the results from stained tissues revealed lesions as described previously for TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides. However, all bursa of Fabrícius tissues analysed were negative. RT-PCR findings were positive for TCoV in all faecal droppings samples, and in 27% of cloacal swabs. Finally, the best field material for TCoV diagnosis was faecal droppings and/or intestine suspensions.

Survival of Calicivirus in Foods and on Surfaces: Experiments with Feline Calicivirus as a Surrogate for Norovirus

Although there is a large body of evidence incriminating foods as vehicles in the transmission of norovirus, little is known about virus survival in foods and on surfaces. Feline calicivirus was used as a surrogate for norovirus to investigate its survival in representative foods of plant and animal origin and on metal surfaces. Known concentrations of feline calicivirus in a natural fecal suspension were deposited onto lettuce, strawberries, ham, or stainless steel and incubated for 7 days at refrigeration or room temperatures. Virus was recovered at 1-day intervals, and the titers of the virus were determined by plaque assay. Infectious virus was recoverable until day 7 from lettuce, ham, and stainless steel. Statistically higher titers of feline calicivirus (P < 0.05) were recovered from ham under all conditions than from lettuce, strawberries, or stainless steel. These data provide valuable information for epidemiological and monitoring purposes as well as for the development of food processing practices and appropriate strategies to inactivate norovirus and control its transmission via foods and surfaces.

In vitro gas formation and fermentation parameters using different substrates and pig faecal inocula affected by bile extract

Abstract The effect of bile extract on microbial gas formation and composition, production of volatile fatty acids (VFA) and methane and on reductive acetogenesis was studied in vitro . As substrates cellulose and potato starch in combination with graded levels of bile extract concentrations (0; 333; 666; 1333 mg/l) were incubated with a faecal suspension from pigs as microbial inoculum according to procedures of a modified “Hohenheim gas formation test” (HFT). Bile extract concentration of 1333 mg/l in the in vitro system reduced total gas production from cellulose significantly ( p p p p p

A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area

The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan minor groove binder probes. The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6.6 x 10(9) to 9.1 x 10(10) marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence. The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water. The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.

Tissue-type plasminogen activator prevents formation of intra-abdominal abscesses after surgical treatment of secondary peritonitis in a rat model

Optimal therapy of secondary peritonitis frequently results in the formation of residual abscesses, which bear a substantial mortality and morbidity. This study aims to prove that fibrinolytic therapy with recombinant tissue plasminogen activator (rtPA) can reduce abscess formation after surgical treatment of secondary peritonitis in a rat model, without causing unwanted side effects. Male Wistar rats received an intra-abdominal injection with a suspension of sterile feces, 10(5) cfu Escherichia coli and 10(4) cfu Bacteroides fragilis. Surgical debridement was performed 1 h after inoculation. Animals were randomized into four groups (n = 14 each). Three groups received human rtPA at 1 h (rtPA1); 1 h and 6 h (rtPA2); and 1, 6, and 24 h (rtPA3), respectively. Each dose contained 1.25 mg rtPA. Controls received saline only. Animals were killed after 5 days. rtPA treatment reduced abscess formation in surviving animals, depending on number of doses given. Animals in group rtPA3 had no abscesses in contrast to 88% of the controls (mean 3.6 +/- 2.7 abscesses per rat; p < 0.05). In the rtPA1 and rtPA2 group, frequency of abscess formation was 58 and 33%, respectively. Mortality, course of body weight, and bacteremia were not affected by rtPA and neither were peritoneal cell counts and levels of TNF-alpha, IL-1beta, IL-6 and IL-10. No bleeding complications were observed. rtPA reduces intra-abdominal abscess formation after surgical treatment of generalized peritonitis without increasing mortality or affecting the local inflammatory response.

Establishment and follow-up of bifidobacterial species in the gut of healthy bottle-fed infants of 1–4 months age

Twenty-one healthy bottle-fed infants were screened monthly (1-4 months) for bifidobacteria in their stools. Bifidobacteria were detected by culture and isolates specified by PCR. Alternatively, direct PCR in undiluted fecal suspensions was carried out for detection of bifidobacteria under the cultural detection level. All infants harbored cultivable bifidobacteria throughout the study period. Beerens medium was shown to permit a better recovery of bifidobacteria than MRS and horse blood Columbia agar. Direct PCR detection proved valuable in detecting species for which no cultural isolate could be recovered since the species were under the cultural detection level. B. bifidum, B. longum-infantis and B. breve were confirmed as dominant and stable species in infant stools while B. adolescentis and B. catenulatum group exhibited unstable colonization profiles. A trend towards B. breve decrease began at month 3 while carriage of the B. catenulatum group and B. adolescentis was rising. This observation warrants further analysis to assess a possible switch occurring at month 3 in bottle-fed infants, between so-called infant and adult bifidobacterial species.

Quantitative PCR Method for Sensitive Detection of Ruminant Fecal Pollution in Freshwater and Evaluation of This Method in Alpine Karstic Regions

A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 x 10(9) marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 10(5) marker equivalents per liter).

Biotic and Abiotic Factors Influencing In Vitro Growth of Escherichia coli O157:H7 in Ruminant Digestive Contents

The gastrointestinal tract (GIT) of ruminants is the main reservoir of enterohemorrhagic Escherichia coli, which is responsible for food-borne infections in humans that can lead to severe kidney disease. Characterization of biotic and abiotic factors that influence the carriage of these pathogens by the ruminant would help in the development of ecological strategies to reduce their survival in the GIT and to decrease the risk of contamination of animal products. We found that growth of E. coli O157:H7 in rumen fluid was inhibited by the autochthonous microflora. Growth was also reduced when rumen fluid came from sheep fed a mixed diet composed of 50% wheat and 50% hay, as opposed to a 100% hay diet. In fecal suspensions, E. coli O157:H7 growth was not suppressed by the autochthonous flora. However, a probiotic strain of Lactobacillus acidophilus inhibited E. coli O157:H7 growth in fecal suspensions. The inhibitory effect was dose dependent. These lactic acid bacteria could be a relevant tool for controlling O157:H7 development in the terminal part of the ruminant GIT, which has been shown to be the main site of colonization by these pathogenic bacteria.

Metabolic Fate of Fraxin Administered Orally to Rats

Naturally occurring fraxin (1) was administered orally to rats to investigate its metabolism. Urinary metabolites were analyzed by three-dimensional HPLC, and fraxetin-7-O-sulfate (2), fraxetin-7-O-beta-glucuronide (3), fraxetin (4), 6,7,8-trihydroxycoumarin (5), and fraxidin (6) were isolated. Fraxin (1) was extensively metabolized to 4, which was partly metabolized to 5 in a rat fecal suspension after incubation for 24 h. Urinary excretion of 4 and 5 in rats administered orally with 1 was substantially reduced when the rats were treated with antibiotics to suppress their intestinal flora. Incubation of 1 with a rat liver S-9 mixture yielded 6. These results suggest that hydrolysis and demethylation of 1 are performed by intestinal microflora, while methylation occurs in the liver.

Bifidobacteriummay affectin vitrometabolism of daidzein by faecal flora from mice and a human male equol producer

Recently, there has been great interest in the biological effects of equol, a metabolite of daidzein produced by intestinal flora. In this study, we used a newly isolated intestinal bacterium from healthy human faeces to investigate the effects of intestinal bacteria on the in vitro metabolism of daidzein by faecal flora in mice and a human male equol producer. The 16S rDNA partial sequence (1394 bp) of strain TM-20 that was isolated from infant faeces exhibited a 99% similarity to that of Bifidobacterium pseudocatenulatum . Therefore, this strain was identified as belonging to the genus Bifidobacterium . In faecal suspensions from mice, equol concentrations were significantly lower in the control faecal suspension than in the faecal suspension supplemented with the high concentration of strain TM-20. However, equol concentrations were significantly higher in the control faecal suspension than in the faecal suspension supplemented with the low concentration of strain TM- 20. In faecal suspensions from a human male equol producer, equol concentrations were lower in the control faecal suspension than in the faecal suspension supplemented with the high concentration of strain TM-20. However, equol concentrations were higher in the control faecal suspension than in the fecal suspension supplemented with the low concentration of strain TM-20. Metabolic activity of the faecal equol production in mice and humans may be changed by some types of bifidobacteria. To our knowledge, this is the first report of a Bifidbacterium sp. that is capable of changing the faecal equol production from daidzein in vitro. Key words: daidzein, equol, faecal flora, bifidobacteria

Thermal Stability of Hepatitis E Virus

The thermal stability of virulent hepatitis E virus (HEV) and hepatitis A virus (HAV) was compared. Fecal suspensions of virus were heated to temperatures between 45 degrees C and 70 degrees C, and residual infectivity was determined in a cell culture system that was permissive for both viruses. Although HEV was less stable than was HAV, some HEV would most likely survive the internal temperatures of rare-cooked meat.

Effects of aggregation and sample size on composite faecal egg counts in sheep

Composite faecal egg counts (FEC) are increasingly used to support strategic anthelmintic treatment decisions in grazing livestock. However, their accuracy as estimators of group mean FEC is affected by the number of individual samples included, how thoroughly they are mixed, and the underlying degree of parasite aggregation between individual hosts. This paper uses a Negative Binomial model for parasite aggregation, and a Poisson model for egg distribution within faecal suspensions, in order to optimise composite FEC protocol for commercial sheep flocks. Our results suggest that faecal egg density in a well-mixed composite sample from 10 sheep (3 g of faeces from each), estimated by examination of four independently filled McMaster chambers, is likely to provide an adequate estimate of group mean FEC in the majority of situations. However, extra care is needed in groups of sheep for which high levels of FEC aggregation might be expected. The implications of statistical error in FEC estimates depend on how they are used. The simulation-based approach presented here is a powerful tool for investigating the risks of error in FEC-driven treatment decisions in different situations, as well as for the statistical analysis of parasitological data in general.

Stability and biotransformation of various dietary anthocyanins in vitro

Anthocyanins, which are found in high concentrations in fruit and vegetable, may play a beneficial role in retarding or reversing the course of chronic degenerative diseases. However, little is known about the biotransformation and the metabolism of anthocyanins so far. The aim of the study was to investigate possible transformation pathways of anthocyanins by human faecal microflora and by rat liver microsomes as a source of cytochrome P450 enzymes as well as of glucuronyltransferases. Pure anthocyanins, an aqueous extract of red radish as well as the assumed degradation products were incubated with human faecal suspension. The incubation mixtures were purified by solid-phase extraction and analysed by HPLC/DAD/MS and GC/MS. Quantification was done by the external standard method. Furthermore the biotransformation of anthocyanins by incubation with rat liver microsomes in the presence of the cofactor NADPH (as a model for the phase I oxidation) and in the presence of activated glucuronic acid (as a model for the phase II glucuronidation) was investigated. Glycosylated and acylated anthocyanins were rapidly degraded by the intestinal microflora after anaerobic incubation with a human faecal suspension. The major stable products of anthocyanin degradation are the corresponding phenolic acids derived from the B-ring of the anthocyanin skeleton. Anthocyanins were not metabolised by cytochrome P450 enzymes, neither hydroxylated nor demethylated. However they were glucuronidated by rat liver microsomes to several products. The gut microflora seem to play an important role in the biotransformation of anthocyanins. A rapid degradation could be one major reason for the poor bioavailability of anthocyanins in pharmacokinetic studies described so far in the literature. The formation of phenolic acids as the major stable degradation products gives an important hint to the fate of anthocyanins in vivo.

Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate virus for norovirus

We investigated the efficacy of three ethanol-based hand rubs (Sterillium Virugard, 95% ethanol; Sterillium Rub, 80% ethanol; Desderman N, 75.1% ethanol) against feline calicivirus (FCV), the surrogate virus for norovirus, on artificially contaminated hands of healthy volunteers. The ASTM E 1838-02 standard was used. Experiments were controlled with 70% ethanol and 70% propan-1-ol which were previously found to have maximal efficacy against FCV. In the first step, three different organic loads (5% fetal bovine serum, 5% faecal suspension and the tripartite ASTM load) were compared. A significant influence of the type of organic load was found (P<0.001, ANOVA). In the second step, the hand rubs were investigated with a 5% faecal suspension as a challenging organic load. The hand rub based on 95% ethanol was more effective than those based on 70% ethanol (mean log10 reduction factor: 2.17 vs. 1.56; P=0.17) and 70% propan-1-ol (mean RF: 1.63 vs. 0.95; P=0.0003). The hand rub based on 80% ethanol was also more effective than those based on 70% ethanol (mean RF: 1.25 vs. 1.03: P=0.20) and 70% propan-1-ol (mean RF: 1.43 vs. 1.09; P=0.03). The hand rub based on 75.1% ethanol was less effective than those based on 70% ethanol (mean RF: 1.07 vs. 1.27; P=0.47) and 70% propan-1-ol (mean RF: 0.78 vs. 0.97; P=0.35). Based on our data, ethanol has superior efficacy against FCV than propan-1-ol. In addition, a higher ethanol concentration in three commercially available hand rubs was associated with better efficacy against FCV.

Effect of Fluconazole on Phagocytic Response of Polymorphonuclear Leukocytes in a Rat Model of Acute Sepsis

Recently, fluconazole (FLZ) has been shown to improve survival and reduce multiorgan failure in experimental and clinical septic shock. The mechanism by which FLZ affords protection against sepsis remains obscure. This study examines the effect of FLZ on phagocytic activity of polymorphonuclear leukocytes (PMNs) in a rat model of septic shock by inducing fecal peritonitis in male Wistar rats using intraperitoneal instillation (1 mL/kg) of fecal suspension in saline (1:1 w/v). Sham control rats received sterile fecal suspension and vehicle treatment. FLZ was administered in the doses of 0, 3, 10, and 30 mg/kg by gavage 30 minutes before fecal instillation. The samples of peritoneal fluid were collected 8 hours following fecal inoculation for the evaluation of phagocytic response of PMNs using zymosan-induced luminol-dependent chemiluminescence (CL). Fecal peritonitis caused massive infiltration of PMNs in the peritoneal cavity (ANOVA F4.45 = 6.322, P < .001). Although FLZ reduced the infiltration of PMNs, this effect was neither significant nor dose dependent. The actual CL response was significantly higher in the peritoneal fluid of rats subjected to peritonitis, which was significantly and dose-dependently attenuated by FLZ treatment (ANOVA F4.45 = 11.048, P < .001). Normalization of CL response for 1000 PMNs revealed that FLZ dose-dependently albeit insignificantly reduced the activity of PMNs. The high dose of FLZ caused 2.29-fold decrement in the area under curve (AUC) pertaining to cumulative CL response. The findings of this study suggest that FLZ protects rats against septic shock by inhibiting PMN-mediated inflammatory cascade without compromising their phagocytic activity.

Intranasal immunization with a replication-deficient adenovirus vector expressing glycoprotein H of murine cytomegalovirus induces mucosal and systemic immunity

A vaccine vector, designated AdV-gH, was constructed by inserting the complete open reading frame of MCMV gH under control of the human CMV IE-1 promoter into the E-1 region of a replication-deficient adenovirus 5. In vitro infection of QB1-293 cells and mouse embryo cells with AdV-gH resulted in expression of MCMV gH detected by IFA. Immunization of BALB/c mice with AdV-gH (1 × 10 7 PFU) given by the intranasal route induced a humoral response with antibody detected in serum of 100% of vaccines. Antibody to MCMV gH was also detected in the bronchoalveolar lavage, fecal suspensions and vaginal washings. The viral titer of lung and salivary gland of immunized mice 10 days after intranasal challenge with MCMV (1 × 10 5 PFU) were significantly reduced compared to controls, but virus infection was not prevented. Re-exposure of mice to AdV-gH 30 days after primary immunization induced a significant boost of serum antibody response. When rechallenged with MCMV intranasally, these mice had further reduction of MCMV titers in the lung and salivary glands. Such a strategy may be important in reducing horizontal transmission of CMV infections across mucosal surfaces and in altering host immunity to CMV.

Comparison of three microscopic techniques for diagnosis ofCyclospora 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 microl 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.

The relationship between concentration of a dual marker strain of Salmonella Typhimurium in bovine faeces and its probability of detection by immunomagnetic separation and culture.

To modify a strain of Salmonella serotype Typhimurium to express unique marker traits and then define how the concentration of the marker in bovine faeces affects the probability of its detection by culture preceded by immunomagnetic separation (IMS). DNA encoding for the production of green fluorescent protein (gfp) and resistance to kanamycin was inserted into the bacterial chromosome of Salm. Typhimurium. Transposon insertion was demonstrated by Southern blot hybridization. Varying amounts of one electroporant (gfpSal-1) were inoculated into suspensions of bovine faeces and attempts made to isolate gfpSal-1 using a protocol based on pre-enrichment incubation, IMS and enrichment in selective media. Isolates of gfpSal-1 were differentiated from wild strains of Salmonella using fluorescence under u.v. light and expression of kanamycin resistance. A logistic and Gompertz function each derived from the dose-response data partially explained the observations with the fit of the Gompertz function judged to be superior. The 10, 50 and 90% limits of detection from the Gompertz function were estimated to be 1.92, 2.03 and 2.27 CFU g(-1) respectively. Reliance on the traditional concept of 'limit of detection' could introduce unacceptable errors in the interpretation of test findings when the concentration of Salm. Typhimurium in bovine faeces (pooled or individual) is below ca 3 CFU g(-1) of faeces. The dose-response curve can be used to aid the design of protocols for detecting Salmonella in individual and pooled faecal specimens. The experiments demonstrate that both reporter genes in tandem are useful for studying the performance of culture-based methods for detecting pathogens in faeces.

Metabolic fate of orally administered phyllodulcin in rats.

Naturally occurring phyllodulcin (1) was orally administered to rats to investigate its metabolic fate. Urinary metabolites were analyzed by three-dimensional HPLC. Phyllodulcin-3'-O-sulfate (2), phyllodulcin-3'-O-beta-glucuronide (3), 2-[2-(3,4-dihydroxyphenyl)ethyl]-6-hydroxybenzoic acid (4), and one novel bibenzyl derivative, 2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-6-hydroxybenzoic acid (5), together with thunberginol G (6) and hydrangenol (7) were isolated from the phyllodulcin-treated urine. 1 was extensively metabolized to 4-6 by a rat fecal suspension after incubation for 24 h. Urinary excretion of 4-6 in rats administered phyllodulcin orally was substantially reduced when the rats were treated with antibiotics to suppress their intestinal flora. On the other hand, the incubation of 1 with rat liver S-9 mix showed the presence of 7 together with 4 and 5.

Effects of cleaning and disinfection in reducing the spread of Norovirus contamination via environmental surfaces

A reverse transcriptase polymerase chain reaction assay was used to study the transfer of Norovirus (NV) from contaminated faecal material via fingers and cloths to other hand-contact surfaces. The results showed that, where fingers come into contact with virus-contaminated material, NV is consistently transferred via the fingers to melamine surfaces and from there to other typical hand-contact surfaces, such as taps, door handles and telephone receivers. It was found that contaminated fingers could sequentially transfer virus to up to seven clean surfaces. The effectiveness of detergent– and disinfectant-based cleaning regimes typical of those that might be used to decontaminate faecally contaminated surfaces and reduce spread of NV was also compared. It was found that detergent-based cleaning with a cloth to produce a visibly clean surface consistently failed to eliminate NV contamination. Where there was faecal soiling, although a combined hypochlorite/detergent formulation at 5000 ppm of available chlorine produced a significant risk reduction, NV contamination could still be detected on up to 28% of surfaces. In order consistently to achieve good hygiene, it was necessary to wipe the surface clean using a cloth soaked in detergent before applying the combined hypochlorite/detergent. When detergent cleaning alone or combined hypochlorite/detergent treatment failed to eliminate NV contamination from the surface and the cleaning cloth was then used to wipe another surface, the virus was transferred to that surface and to the hands of the person handling the cloth. In contrast, were surfaces where contaminated with NV-infected faecal suspension diluted to 1 in 10 and 1 in 80, intended to simulate surfaces that have become contaminated after secondary transfer, treatment with a combined bleach/detergent formulation, without prior cleaning, was sufficient to decontaminate surfaces and prevent transfer.