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Over the last half-century, plastic production has exploded resulting in environmental pollution. Accumulated plastics can be degraded into smaller pieces known as microplastics (MPs) found everywhere, and especially in the food chain. MPs have been detected in human feces and colon biopsies attesting of their transit through the gastro-intestinal tract (GIT). However, to date, little is known about the fate and potential effects of MPs in the human digestive sphere, particularly in the infant at-risk population. During their GIT travel, MPs can interact with physico-chemical digestive parameters, as well as gut microbiota, intestinal epithelium and its covering mucus layer. This joint PhD between MEDIS (Clermont-Ferrand) and Toxalim (Toulouse) aimed to investigate the fate of virgin MPs of polyethylene (PE), the most manufactured plastic polymer worldwide, in the adult and infant gut. This was studied using an original in vitro approach combining a human colon model and intestinal cells in culture. This doctoral research started with evaluating the impact of various stool conservative methods (48-h freezing -80°C, 48-h freezing -80°C with glycerol or lyophilization with maltodextrin/trehalose) on gut microbiota composition and activity in the Mucosal Artificial Colon (M-ARCOL), set-up under adult conditions. We showed that inoculating with raw frozen stool (48-h at -80°C) was the best option among those tested compared to fresh stools. Next, the M-ARCOL model was adapted to the specific toddler conditions in a new configuration termed Tm-ARCOL. This adaptation allowed to reproduce in vitro the main particularities associated to infant microbiota, such as lower bacterial diversity and higher abundance of Bifidobacteriaceae, together with higher concentrations of acetate and lower amounts of propionate and butyrate, as compared to adults. Then, we assessed the effects of a chronic ingestion of PE MPs on adult and infant microbiota using M-ARCOL and Tm-ARCOL, respectively, as well as the indirect impact of gut microbe metabolites after PE MP exposure on the intestinal barrier simulated by a co-culture of Caco-2 and mucus-secreting HT29-MTX cells. Interestingly, the effect of PE MP exposure on the gut microbiota was donor-dependent and resulted in an increase in potential pathobionts, like Enterobacteriaceae, regardless of age conditions. Regarding the activity of the gut microbiota, we showed for the first time that PE MP exposure was associated with a significant decrease in butyrate production in infant conditions, while skatole production increased significantly in adults. In contrast, no significant impact of PE MPs on the intestinal barrier, as mediated by changes in gut microbial metabolites, was evidenced.This PhD work provided pioneering and significant insights into the interactions of PE MPs with the adult and infant human gut microbiota and the intestinal barrier, filling some of the gaps in knowledge on MP behavior in the human GIT. This work contributes to a better understanding of MP health risk assessment for public policies. In the near future, our in vitro approach could be used for further investigations on more representative MP types (forms, sizes, aged and/or contaminated particles), in healthy but also disturbed situations including an altered gut barrier and dysbiotic microbiota (e.g. obesity, inflammatory bowel syndrome or inflammatory bowel diseases).; Au cours des 50 dernières années, la production plastique a fortement augmenté, conduisant à une pollution environnementale massive. Les plastiques accumulés peuvent être dégradés en particules de plus petites tailles appelées microplastiques (MPs) et retrouvées tout au long de la chaîne alimentaire. Chez l’Homme, les MPs ont été détectés dans les selles et le côlon, attestant de leur transit le long du tractus gastro-intestinal (TGI). Cependant, à ce jour, peu de travaux ont évalué le devenir et les effets potentiels des MPs dans la sphère digestive humaine, en particulier chez les enfants. Au cours de leur transit gastrointestinal, les MPs peuvent interagir avec les processus physico-chimiques liés à la digestion, le microbiote intestinal, l'épithélium intestinal, ou encore le mucus. Cette thèse, menée conjointement entre MEDIS (Clermont-Ferrand) et Toxalim (Toulouse), visait à étudier le devenir de MPs vierges de polyéthylène (PE), polymère plastique le plus fabriqué au monde, dans l’environnement digestif de l'adulte et de l'enfant. Cette étude a été réalisée grâce à une approche in vitro originale, combinant un modèle de côlon humain et des cellules intestinales en culture. Ces travaux de thèse ont tout d’abord évalué l'impact de différentes méthodes de conservation des selles (congélation à -80°C/48h, congélation à -80°C/48h avec du glycérol ou lyophilisation maltodextrine/tréhalose) sur la composition et l'activité du microbiote intestinal dans le modèle Mucosal Artificial Colon (M-ARCOL), en conditions adultes. Nous avons montré que l'inoculation du bioréacteur avec des selles congelées brutes (-80°C/48-h) était la meilleure option parmi celles testées comparativement aux selles fraîches. Ensuite, le dispositif M-ARCOL a été adapté aux conditions spécifiques du côlon chez l’enfant dans une nouvelle configuration appelée Tm-ARCOL. Cette adaptation a permis de reproduire in vitro les principales particularités associées au microbiote des enfants, comme une diversité bactérienne plus faible et une abondance plus importante des Bifidobacteriaceae, ainsi que des concentrations plus élevées en acétate et plus faibles en propionate et butyrate, comparativement aux adultes. Nous avons ensuite évalué les effets d'une ingestion chronique de MPs de PE sur le microbiote de l’adulte et de l’enfant en utilisant respectivement les modèles M-ARCOL et Tm-ARCOL, ainsi que l’effet indirect des surnageants de fermentation (contenant les métabolites issus des microbiotes intestinaux) sur la barrière intestinale mimée par la mise en œuvre d’une co-culture de cellules Caco-2 et HT29-MTX productrices de mucus. Les effets de l’exposition aux MPs de PE sur le microbiote intestinal se sont révélés donneur-dépendants. Les MPs induisent une augmentation de souches potentiellement pathogènes, comme les Enterobacteriaceae, et ce indépendamment des conditions d’âge. Concernant l'activité du microbiote intestinal, nous avons montré pour la première fois que l'exposition aux MPs de PE était associée à une diminution significative de la production de butyrate chez les enfants, tandis que la production de scatole augmentait chez les adultes. En revanche, aucun impact significatif des MPs de PE sur la barrière intestinale, régi par les modifications des métabolites microbiens, n'a été mis en évidence.

Background Gastrointestinal symptoms are common in COVID-19 patients and SARS-CoV-2 RNA has been detected in the patients’ feces, which could lead to fecal–oral transmission. Therefore, fecal sample testing with real-time RT-PCR is highly recommended as a routine test for SARS-CoV-2 infection. However, varying rates of detection in fecal sample have been reported. The aim of this study was to provide insights into the detection rates of SARS-CoV-2 in COVID-19 patients’ fecal sample by using four real-time RT-PCR kits and two pretreatment methods (inactive and non-inactive). Results The detection rate of Trizol pretreatment group was slightly higher than that of Phosphate Buffered Saline (PBS) groups, showing that pretreatment and inactivation by Trizol had no influence to SARS-CoV-2 nucleic acid test (NAT) results. 39.29% detection rate in fecal sample by DAAN was obtained, while Bio-germ was 40.48%, Sansure 34.52%, and GeneoDx 33.33%. The former three kits had no significant difference. The DAAN kit detection rates of ORF1ab and N gene were nearly equal and Ct value distribution was more scattered, while the Bio-germ kit distribution was more clustered. The positive rate of SARS-COV-2 in fecal samples correlated with the severity of the disease, specifically, severe cases were less likely to be identified than asymptomatic infection in the DAAN group (adjusted OR 0.05, 95%CI = 0.00 ~ 0.91). Conclusions Trizol should be of choice as a valid and safe method for pretreatment of fecal samples of SARS-CoV-2. All real-time RT-PCR kits assessed in this study can be used for routine detection of SARS-CoV-2 in fecal samples. While DAAN, with high NAT positive rate, could be the best out of the 4 kits used in this study. SARS-CoV-2 positive rate in fecal sample was related to the severity of illness.

Objective: To determine the diet of the bighorn sheep and identify differences in its composition between sexes and periods (reproductive and segregation). Design/methodology/approach: The study was conducted at the UMA Rancho Noche Buena, Hermosillo, Sonora, Mexico. To determine the plant species in the bighorn sheep feces the micro histological technique and a cell catalog of plants from the study area were used. From the diet information, the relative frequency, the Shannon-Weaver diversity index and the Kulczynski similarity index by sex and period (reproductive and segregation) were determined. Results: The diet of the bighorn sheep included 40 plant species, being herbaceous (36.14.4%) and grasses (26.88.9%) the most common. The male diet during the segregation period was mainly composed of grasses (36.2%) and female diet by herbaceous (30%) and grasses (29.8%). There were no differences in the diversity of the diets in males and females during the segregation period (H ́1.0), overall, their diets were very similar (80%). Limitations/implications: Collect a greater number of fecal samples by sex and period (reproductive and segregation) and to analyze the nutritional content of the plants consumed by bighorn sheep. Findings/conclusions: In this study, the sexual segregation exhibited by the bighorn sheep was not due to food preferences

The gut microflora play a very important role in the life of animals. Although an increasing number of studies have investigated the gut microbiota of birds in recent years, there is a lack of research work on the gut microbiota of wild birds, especially carnivorous raptors, which are thought to be pathogen vectors. There are also a lack of studies focused on the dynamics of the gut microbiota during development in raptors. In this study, 16S rRNA gene amplicon high-throughput sequencing was used to analyze the gut microbiota community composition of a medium-sized raptor, the Eurasian Kestrel (Falco tinnunculus), and to reveal stage-specific signatures in the gut microbiota of nestlings during the pre-fledging period. Moreover, differences in the gut microbiota between adults and nestlings in the same habitat were explored. The results indicated that the Eurasian Kestrel hosts a diverse assemblage of gut microbiota. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the primary phyla shared within the guts of adults and chicks. However, adults harbored higher abundances of Proteobacteria while nestlings exhibited higher abundances of Firmicutes and Actinobacteria, and consequently the majority of dominant genera observed in chicks differed from those in adults. Although no significant differences in diversity were observed across the age groups during nestling ontogeny, chicks from all growth stages harbored richer and more diverse bacterial communities than adults. In contrast, the differences in gut microbial communities between adults and younger nestlings were more pronounced. The gut microbes of the nestlings in the last growth stage were converged with those of the adults. This study provides basic reference data for investigations of the gut microbiota community structure of wild birds and deepens our understanding of the dynamics of the gut microflora during raptor development.

The gut microflora play a very important role in the life of animals. Although an increasing number of studies have investigated the gut microbiota of birds in recent years, there is a lack of research work on the gut microbiota of wild birds, especially carnivorous raptors, which are thought to be pathogen vectors. There are also a lack of studies focused on the dynamics of the gut microbiota during development in raptors. In this study, 16S rRNA gene amplicon high-throughput sequencing was used to analyze the gut microbiota community composition of a medium-sized raptor, the Eurasian Kestrel (Falco tinnunculus), and to reveal stage-specific signatures in the gut microbiota of nestlings during the pre-fledging period. Moreover, differences in the gut microbiota between adults and nestlings in the same habitat were explored. The results indicated that the Eurasian Kestrel hosts a diverse assemblage of gut microbiota. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the primary phyla shared within the guts of adults and chicks. However, adults harbored higher abundances of Proteobacteria while nestlings exhibited higher abundances of Firmicutes and Actinobacteria, and consequently the majority of dominant genera observed in chicks differed from those in adults. Although no significant differences in diversity were observed across the age groups during nestling ontogeny, chicks from all growth stages harbored richer and more diverse bacterial communities than adults. In contrast, the differences in gut microbial communities between adults and younger nestlings were more pronounced. The gut microbes of the nestlings in the last growth stage were converged with those of the adults. This study provides basic reference data for investigations of the gut microbiota community structure of wild birds and deepens our understanding of the dynamics of the gut microflora during raptor development.

Background DNA extraction is an important factor influencing the microbiome profile in fecal samples. Considering that the QIAamp DNA Stool Mini Kit, one of the most commonly used DNA extraction kits, is no longer manufactured, this study aimed to investigate whether a new commercially available kit, the QIAamp PowerFecal Pro DNA Kit, yields comparable microbiome profiles with those previously obtained using the QIAamp DNA Stool Mini Kit. Results We extracted DNA from fecal samples of 10 individuals using three protocols (protocol P of the QIAamp PowerFecal Pro DNA Kit, and protocols SB and S of the QIAamp DNA Stool Mini Kit with and without an additional bead-beating step, respectively) in triplicate. Ninety extracted DNA samples were subjected to 16S rRNA gene sequencing. DNA quality measured by 260/280 absorbance ratios was found to be optimal in protocol P. Additionally, the DNA quantity and microbiome diversity obtained using protocol P were significantly higher than those of protocol S, however, did not differ significantly from those of protocol SB. Based on the overall microbiome profiles, variations between protocol P and protocol SB or S were significantly less than between-individual variations. Furthermore, most genera were not differentially abundant in protocol P compared to the other protocols, and the number of differentially abundant genera, as well as the degree of fold-changes were smaller between protocols P and SB than between protocols P and S. Conclusions The QIAamp PowerFecal Pro DNA Kit exhibited microbiome analysis results that were comparable with those of the QIAamp DNA Stool Mini Kit with a bead-beating step. These results will prove useful for researchers investigating the gut microbiome in selecting an alternative protocol to the widely used but discontinued kit.

International audience; In vitro gut models, such as the mucosal artificial colon (M-ARCOL), provide timely and cost-efficient alternatives to in vivo assays allowing mechanistic studies to better understand the role of human microbiome in health and disease. Using such models inoculated with human fecal samples may require a critical step of stool storage. The effects of preservation methods on microbial structure and function in in vitro gut models have been poorly investigated. This study aimed to assess the impact of three commonly used preserving methods, compared with fresh fecal samples used as a control, on the kinetics of lumen and mucus-associated microbiota colonization in the M-ARCOL model. Feces from two healthy donors were frozen 48 h at − 80°C with or without cryoprotectant (10% glycerol) or lyophilized with maltodextrin and trehalose prior to inoculation of four parallel bioreactors (e.g., fresh stool, raw stool stored at − 80°C, stool stored at − 80°C with glycerol and lyophilized stool). Microbiota composition and diversity (qPCR and 16S metabarcoding) as well as metabolic activity (gases and short chain fatty acids) were monitored throughout the fermentation process (9 days). All the preservative treatments allowed the maintaining inside the M-ARCOL of a complex and functional microbiota, but considering stabilization time of microbial profiles and activities (and not technical constraints associated with the supply of frozen material), our results highlighted 48 h freezing at − 80°C without cryoprotectant as the most efficient method. These results will help scientists to determine the most accurate method for fecal storage prior to inoculation of in vitro gut microbiome models. Key points • In vitro ARCOL model reproduces luminal and mucosal human microbiome. • Short-term storage of fecal sample influences microbial stabilization and activity. • 48 h freezing at − 80°C: most efficient method to preserve microbial ecosystem. • Scientific and technical requirements: influencers of preservation method.

Introduction Consensus in sample preparation for untargeted human fecal metabolomics is lacking. Objectives To obtain sample preparation with broad metabolite coverage for high-throughput LC–MS. Methods Extraction solvent, solvent ratio and fresh frozen-vs-lyophilized samples were evaluated by metabolite feature quality. Results Methanol at 5 mL per g wet feces provided a wide metabolite coverage with optimal balance between signal intensity and saturation for both fresh frozen and lyophilized samples. Lyophilization did not affect SCFA and is recommended because of convenience in normalizing to dry matter. Conclusion The suggested sample preparation is simple, efficient and suitable for large-scale human fecal metabolomics.

Fecal immunochemical tests (FITs) for hemoglobin (Hb) are increasingly used for colorectal cancer (CRC) screening. Most FIT manufacturers instruct that fecal samples from multiple parts of one bowel movement should be obtained. Our aim was to compare the FIT diagnostic performance based on fecal samples from just one versus two different sites of one bowel movement. A total of 1141 participants of screening colonoscopy provided two fecal samples from two different sites of a single bowel movement for FIT analyses. There was no statistically significant difference in the diagnostic performance of the FIT when either one or both fecal samples were used for analysis, with area under the curve (AUC) for detecting CRC ranging from 0.94 (95% confidence interval (CI) 0.84&ndash, 0.99) for one FIT to 0.95 (95%CI 0.86&ndash, 0.99) for a geometric mean of two FITs. The manufacturers&rsquo, recommendation of sampling multiple sites of the stool aims to reduce intra-individual Hb variability and improve diagnostic performance. If no such improvement can be achieved, the recommendation for multiple-site sampling might have potential adverse effects on population adherence to FIT-based CRC screening. Our results point to a potential of increasing adherence to FIT screening by simplifying instructions for fecal sampling at no loss of the diagnostic performance.

The present project is focused on the study of a prevalence of the gut protist, Giardia intestinalis, in human and animal samples using two diagnostic approaches: (i) coproscopical diagnostics, specifically Sheather flotation technique, and (ii) PCR. First, three PCR protocols for different genes (triosephosphate isomerase, beta-giardin and small ribosomal subunit) were optimized by gradient PCR. Based on these result, the gene TPI was chosen as the best option for molecular diagnostics of G. intestinalis in the collected stool and fecal samples. Giardia was detected only in the animal samples (2/41, dog and rabbit samples), all the analysed human samples remained negative. Further, the TPI protocol showed low sensitivity for diagnostics of G. intestinalis.

We report a preliminary evaluation of the NG-Test CARBA 5 immunochromatographic assay for detecting carbapenemases directly from rectal swabs on the same day of sampling. Thirty fecal swabs were examined for carbapenemase-producing organisms (CPOs) by conventional culture, PCR, and NG-Test CARBA 5. Each sample was tested by the immunochromatographic assay five times, including direct testing and incubation in trypticase soy broth for 1, 2, 3, and 4 h. Twenty patients yielded CPOs by culture. Immunochromatographic and PCR results were concordant and detected the same 25 carbapenemases (11 KPC, 8 VIM, and 6 NDM). In five cases, we detected co-carriage of KPC and VIM. Compared with PCR, the sensitivity of NG-Test CARBA 5 for the detection of KPC, VIM, and NDM was 80% without incubation, 88% with one hour, 92% with two, and 100% with three hours incubation, while specificity was 100% for all time points. All samples containing adequate fecal content were detected by NG-Test CARBA 5 concordantly with PCR, without incubation. NG-Test CARBA 5 is a reliable test that rapidly detects the presence of carbapenemases at the same day of sampling, directly from rectal swabs. It thus provides early information to guide antimicrobial treatment and infection control interventions.

This study presents the pathological, immunohistochemical, and molecular findings associated with the extra-intestinal detection of canine kobuvirus (CaKV) in a 5-month-old Chihuahua puppy, that had a clinical history of bloody-tinged feces. Principal pathological findings were interstitial pneumonia, necrotizing bronchitis, and parvovirus-induced enteritis. Molecular diagnostic methods identified CaKV within the cerebellum, cerebrum, lung, tonsil, and liver. CaKV and rotavirus were not identified within the feces and intestine. Immunohistochemistry (IHC) assays detected antigens of CDV and CAdV-1 in the lungs. These results confirmed the extra-intestinal detection of CaKV in this puppy and represent the first extra-intestinal detection of CaKV in a dog.

Background The number of large-scale studies on the gut microbiota in human cohorts is rapidly increasing. However, the few and expensive options for storage of fecal samples at room temperature have been an obstacle for large-scale metagenomic studies and the development of clinical/commercial personal metagenomic sequencing. Results In this study, we systematically tested a novel N-octylpyridinium bromide-based fecal sample preservation method and compared it with other currently used storage methods. We found that the N-octylpyridinium bromide-based method enabled preservation of the bacterial composition in fecal samples transported and stored at room temperature for up to at least 14 days. Conclusions We describe a novel chemical stabilizer that allows cost-effective transportation and storage at room temperature for several days with preservation of bacterial composition. This method will facilitate sample collection even in remote area and also enable transport via normal commercial transportation routes. Electronic supplementary material The online version of this article (10.1186/s40168-018-0429-0) contains supplementary material, which is available to authorized users.

International audience; AbstractCurrent Johne’s disease control programs primarily focus on decreasing transmission of Mycobacterium avium subsp. paratuberculosis (MAP) from infectious adult cows to susceptible calves. However, potential transmission between calves is largely overlooked. The objective was to determine the extent of MAP infection in calves contact-exposed to infectious penmates. Thirty-two newborn Holstein–Friesian calves were grouped into 7 experimental groups of 4, consisting of 2 inoculated (IN) calves, and 2 contact-exposed (CE) calves, and 1 control pen with 4 non-exposed calves. Calves were group housed for 3 months, with fecal samples were collected 3 times per week, blood and environmental samples weekly, and tissue samples at the end of the trial. The IN calves exited the trial after 3 months of group housing, whereas CE calves were individually housed for an additional 3 months before euthanasia. Control calves were group-housed for the entire trial. All CE and IN calves had MAP-positive fecal samples during the period of group housing; however, fecal shedding had ceased at time of individual housing. All IN calves had MAP-positive tissue samples at necropsy, and 7 (50%) of the CE had positive tissue samples. None of the calves had a humoral immune response, whereas INF-γ responses were detected in all IN calves and 5 (36%) CE calves. In conclusion, new MAP infections occurred due to exposure of infectious penmates to contact calves. Therefore, calf-to-calf transmission is a potential route of uncontrolled transmission on cattle farms.

Aim: The aim of this study was to screen the suspected samples by polymerase chain reaction (PCR) and epidemiological analysis of positive cases of canine parvovirus type2. Materials and Methods: Fecal samples were collected from dogs suspected for canine parvovirus type 2 (CPV-2) and viral DNA was extracted. Primers were designed, and PCR was done with all extracted DNA samples. Age, sex and breed wise distribution of positive cases were analyzed. Results: Out of a total 44 collected fecal samples, 23 were found to be positive for CPV-2 by developed PCR. The disease was found to be more common in Labrador male pups of 3-6 months of age. The percentage of positive cases in vaccinated dogs was found to be around 17.4%. Conclusion: Almost half (52.3%) of total collected samples were found to be positive by PCR. However, number of field samples are needed to further validate this test and additionally sequence analysis needs to be done to ensure the prevalent field strain of CPV-2.

Recently, outbreaks associated with equine coronavirus (ECoV) have occurred in Japan and the United States. While ECoV is likely to be pathogenic to horses, it has not been shown that experimental inoculation of horses with ECoV produces clinical signs of disease. In this study, we inoculated three Japanese draft horses with an ECoV-positive diarrheic fecal sample to confirm infection after inoculation and to investigate the clinical course and virus shedding patterns of ECoV. Virus neutralization tests showed that all three horses became infected with ECoV. Two of the three horses developed clinical signs similar to those observed during ECoV outbreaks, including fever, anorexia, and gastrointestinal dysfunction. All horses excreted a large amount of virus into their feces for more than 9 days after inoculation regardless of the presence or absence of clinical signs, which suggests that feces are an important source of ECoV infection. ECoV was also detected in nasal swabs from all horses, suggesting that respiratory transmission of ECoV may occur. Both symptomatic horses developed viremia, while the asymptomatic horse did not. White blood cell counts and serum amyloid A concentrations changed relative to the clinical condition of the inoculated horses; these may be useful markers for monitoring the clinical status of horses infected with ECoV. This is the first report of induction of clinical signs of ECoV infection in horses by experimental inoculation. These clinical and virological findings should aid further investigation of the pathogenesis of ECoV.

The risk to public health from the large number of dog stools present on streets of urban areas is cause for concern. Dog faeces may be a serious hazard because they may contain microorganisms that are both pathogenic to humans and resistant to several classes of antibiotics. The aim of this study was to evaluate the potential for zoonotic infections and for the presence of antibiotic resistant bacteria in canine faeces which contaminates the urban environment. A total of 418 canine faecal samples were collected from streets in seven areas of Bari, Southern Italy. We have isolated multi-drug resistant Enterococci and meticillin-resistant Staphylococcus aureus from these dog faecal samples. The presence of the resistant bacteria in an urban environment may represent a public health hazard which requires control measures by competent authorities. No Salmonella, Yersinia or Campylobacter species were isolated. Giardia cysts were detected in 1.9% of the samples. The predominant Enterococcus species were E. faecium (61.6%), E. gallinarum (23.3%) and E. casseliflavus (5.5%). Other species, including E. faecalis were also isolated. These strains were resistant to clindamycin (86.3%), tetracycline (65.7%), erythromycin (60.27%) and ampicillin (47.9%). High-level aminoglycoside resistance (HLAR) was found in 65.7% of enterococci. Resistance to three or more antibiotics and six or more antibiotics were observed in 67.12% and 38.4% of Enterococcus spp., respectively. Resistance to vancomycin and teicoplanin was not detected in any of the Enterococcus spp. isolated. Methicillin-resistant Staphylococcus aureus was isolated in 0.7% of the faecal samples. Canine faeces left on the streets may represent a risk factor for transmission of microorganisms and a reservoir of multidrug- resistant bacteria thus contributing to the spread of resistance genes into an urban area.

Bovine torovirus (BToV) is recognized as an enteric pathogen of calves, but its etiological role in diarrhea and epidemiological characterization in adult cows remain unclear. In 2007-2008, three outbreaks of epidemic diarrhea occurred in adult cows at three dairy farms in Niigata Prefecture, Japan. BToV was the only enteric pathogen detected in these outbreaks, as determined by electron microscopy, reverse transcription-PCR, bacteria and parasite tests of fecal samples, and antibody tests with paired sera. The epidemiological features of the three outbreaks were similar to those of bovine coronavirus infection, except for the absence of bloody diarrhea, with diarrhea spreading among most adult cows, but not in calves, within several days and diarrhea lasting for 3-5 days with anorexia. Decreased milk production and mild respiratory symptoms were also observed in two of the outbreaks. Nucleotide sequence analysis of the BToV nucleocapsid, spike, and hemagglutinin-esterase (HE) genes revealed a close relatedness among the detected BToV strains from each outbreak and those of Japanese BToV strain Aichi/2004. Furthermore, we isolated a BToV strain, designated Niigata (TC), from a fecal sample using a human rectal tumor cell line. Sequence analysis of this isolate and Aichi/2004 indicated that both strains have truncated HE genes with deletions in the 3' region that occurred through cell culture-adaptation. The short projections that are believed to be formed by the HE protein on virus particles were not observed in these cultured strains by electron microscopy. Taken together, these results suggest that BToV causes epidemic diarrhea in adult cows and should be included in the differential diagnosis of diarrhea in adult cows. In addition, our findings indicate that the HE protein of BToV may not be necessary for viral replication.

When reconstructing the diets of primates, researchers often rely on several well established methods, such as direct observation, studies of discarded plant parts, and analysis of macrobotanical remains in fecal matter. Most of these studies can be performed only on living primate groups, however, and the diets of extinct, subfossil, and fossil groups are known only from proxy methods. Plant microremains, tiny plant structures with distinctive morphologies, can record the exact plant foods that an individual consumed. They can be recovered from recently deceased and fossil primate samples, and can also be used to supplement traditional dietary analyses in living groups. Here I briefly introduce plant microremains, provide examples of how they have been successfully used to reconstruct the diets of humans and other species, and describe methods for their application in studies of primate dietary ecology.

Diagnostic metagenomics is a rapidly evolving laboratory tool for culture-independent tracing of foodborne pathogens. The method has the potential to become a generic platform for detection of most pathogens and many sample types. Today, however, it is still at an early and experimental stage. Studies show that metagenomic methods, from sample storage and DNA extraction to library preparation and shotgun sequencing, have a great influence on data output. To construct protocols that extract the complete metagenome but with minimal bias is an ongoing challenge. Many different software strategies for data analysis are being developed, and several studies applying diagnostic metagenomics to human clinical samples have been published, detecting, and sometimes, typing bacterial infections. It is possible to obtain a draft genome of the pathogen and to develop methods that can theoretically be applied in real-time. Finally, diagnostic metagenomics can theoretically be better geared than conventional methods to detect co-infections. The present review focuses on the current state of test development, as well as practical implementation of diagnostic metagenomics to trace foodborne bacterial infections in fecal samples from animals and humans.

Summary. Porcine respiratory coronavirus (PRCV), a spike (S) gene deletion mutant of Transmissible gastroenteritis virus (TGEV), causes mild or subclinical respiratory infections in pigs. The shedding of PRCV/TGEV was studied at different days post-arrival in fecal and nasal swabs from PRCV/TGEV seronegative sentinel pigs introduced into a PRCV seropositive herd with questionable TGEV serology and diarrhea. Nasal shedding of PRCV was detected in 57% and 63% of samples by nested-RT-PCR and cell culture immunofluorescence (CCIF), respectively. However fecal shedding of PRCV was detected in 37% of the samples by nested-RT-PCR and 19% by CCIF. Four respiratory and 5 fecal PRCV strains were isolated in swine testicle cells including nasal/fecal PRCV pairs (isolated at the same time) from 3 pigs. Comparison of nasal/fecal PRCV pairs from individual pigs revealed different deletions in the spike (S) gene (648 or 681 nt) in 2 pairs and a consistent change in nt 790/791 (aa T to V) for all pairs. In preliminary studies, inoculation of gnotobiotic pigs with each plaque-purified pair of the nasal and fecal PRCV isolates, revealed no clinical disease but different tropisms. The nasal isolate was shed both nasally and in feces, but the fecal isolate was shed only marginally in feces, and not nasally. Our results show that nested-RT-PCR was as sensitive as CCIF for PRCV detection in nasal swabs, but was more sensitive than CCIF for PRCV detection in fecal samples; alternatively PRCV shed in feces was more labile with loss of infectivity. The S-gene sequence differences found between the fecal and respiratory PRCV isolates may influence their tissue tropism. These new PRCV isolates should be useful to understand the molecular basis of coronavirus tropism and evolution in infected swine.

The risk to public health from the large number of dog stools present on streets of urban areas is cause for concern. Dog faeces may be a serious hazard because they may contain microorganisms that are both pathogenic to humans and resistant to several classes of antibiotics. The aim of this study was to evaluate the potential for zoonotic infections and for the presence of antibiotic resistant bacteria in canine faeces which contaminates the urban environment. A total of 418 canine faecal samples were collected from streets in seven areas of Bari, Southern Italy. We have isolated multi-drug resistant Enterococci and meticillin-resistant Staphylococcus aureus from these dog faecal samples. The presence of the resistant bacteria in an urban environment may represent a public health hazard which requires control measures by competent authorities. No Salmonella, Yersinia or Campylobacter species were isolated. Giardia cysts were detected in 1.9% of the samples. The predominant Enterococcus species were E. faecium (61.6%), E. gallinarum (23.3%) and E. casseliflavus (5.5%). Other species, including E. faecalis were also isolated. These strains were resistant to clindamycin (86.3%), tetracycline (65.7%), erythromycin (60.27%) and ampicillin (47.9%). High-level aminoglycoside resistance (HLAR) was found in 65.7% of enterococci. Resistance to three or more antibiotics and six or more antibiotics were observed in 67.12% and 38.4% of Enterococcus spp., respectively. Resistance to vancomycin and teicoplanin was not detected in any of the Enterococcus spp. isolated. Methicillin-resistant Staphylococcus aureus was isolated in 0.7% of the faecal samples. Canine faeces left on the streets may represent a risk factor for transmission of microorganisms and a reservoir of multidrug- resistant bacteria thus contributing to the spread of resistance genes into an urban area.

Commercial swab transport systems are used for collection and transporting of fecal and other microbiological samples. This system must maintain viability and contribute to survival of microorganisms during transport to the laboratory. Four swab transport systems have been compared, eSwab, Σ- Transwab and fecalSwab, all three with flocked swabs, and Copan Venturi Transystem with rayon swabs. The study followed the recommendations from the Clinical Laboratory Standard Institute; document M40-A for recovery of Samonella, Shigella, Yersinia and Neisseria gonorrhoeae after storage for 0, 6, 24, 48 and 72 h at room- (22-25°C) and refrigerated (2-8ºC) temperature. A fecal sample has also been inoculated with Salmonella or Shigella to simulate a fecal sample positive for Salmonella or Shigella. Recovery of all strains was higher with eSwab, Σ- Transwab and fecalswab than with Copan Venturi Transystem stored at both temperatures. A heavy growth was observed with all transport systems after storage for 24, 48 and 72 h at room temperature, except for Neisseria gonorrhoeae, and Shigella. The number of CFU for all strains was constant up to 72 h at refrigerated temperature with Copan Venturi Transystem. In the experiment with fecal sample recovery of Salmonella and Shigella was best with fecalSwab at both storage temperatures. ESwab, Σ- Transwab and fecalSwab are equivalent and can be used as an alternative to Copan Venturi Transystem with better survival of enteric pathogens and Neisseria gonorrhoeae. Fecal samples should be refrigerated in order to avoid heavy overgrowth of fecal flora.

International audience; The expression of most virulence factors during pathology is the result of the bacterial response to environmental changes. The understanding of the conditions affecting gene expression and bacterial physiology in the digestive tract is a clue toward control of pathology. We developed tools with the luciferase gene from Vibrio harveyii as reporter, which allows the measure of promoter activity in digestive tract. These tools can be used in most G+ bacteria such as Streptococci, Clostridii and aerobe Bacilli. In this work, Lactococcus lactis was chosen as model because it is genetically the best known bacteria of the streptococcal group. Transcriptional fusions were constructed between luciferase and different promoters active in various metabolic conditions. The luciferase activities were compared in culture broth and in mouse digestive tract. Three promoters were chosen for this study: P his , P ald , and P mleA the features of which are presented in Table 1. P his , the promoter of histidine biosynthesis operon, can be active during stringent response, is closed in media containing histidine and open under histidine starvation (Delorme under publication). Two forms of the P his promoter were used, the first one is an intact and fully regulated promoter, the second one drives a constitutive expression. P ald , the promoter of acetolactate decarboxylase is open in rich media and closed in stringent conditions (Goupil under publication). P mleA , the promoter of malate fermentation genes is closed in absence of malate and open when malate is added to the medium, it is not sensitive to stringent control (Renault under publication)

Post mortem or even normal changes during life occurring in major gut bacterial populations are not known. We investigated Bacteroides sp., Bifidobacterium sp., Clostridium leptum, Clostridium coccoides, Streptococcus sp., Lactobacillus sp. and Enterobacteriacaea ratios in 7 fecal samples from healthy volunteers and in 61 autopsies rectum and cecum samples and studied the effect of post mortem time using quantitative real-time PCR. Bacterial ratios in stool samples from volunteers and rectum samples from autopsy cases were similar and did not change significantly up to 5 days post mortem. In cecum, significant post mortem time-dependent differences were observed in ratios of Bacteroides sp. (p = 0.014) and Lactobacillus sp. (p = 0.024). Our results showed that ratios of Bacteroides sp., Bifidobacterium sp., Clostridium leptum, Clostridium coccoides, Streptococcus sp., Lactobacillus sp. and Enterobacteriacaea can be investigated in autopsy rectum samples up to 5 days after death. BioMed Central open access

Summary The immunoelectron microscopy (IEM) technique has been used for the detection of a rabbit enteric coronavirus (RECV). Immune serum was prepared in guinea pigs; the viral antigen used for the immunization procedure was obtained from the caecum of a sick rabbit, concentrated by centrifugation and purified on Percoll gradient. In order to identify the viral particles used in the immunization procedure, the protein pattern of the particles was determined by electrophoresis and compared with the pattern of other known coronaviruses. Analysis of structural polypeptides of the purified viral particles revealed a pattern similar to that reported for other coronaviruses. These polypeptides cross reacted with two other coronavirus specific immune sera (IBV and TGE). IEM assay of fecal samples collected from healthy and sick rabbits showed the presence of immune aggregates in specimens from both sick and healthy rabbits. Those aggregates contained viral particules sharing morphological characteristics with other coronaviruses. Furthermore, IEM assay was shown to be more sensitive than a direct EM procedure to detect coronavirus particles in rabbit feces. This assay also allowed the detection of a larger number of chronic carriers.