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Simple Summary: Inflammatory bowel disease (IBD) has become more common among humans and awareness of its prevalence in animals, including horses, has also increased during the past few decades. Equine IBD is problematic as it decreases the performance of horses and causes diarrhea, weight loss, and pain-related behaviors. Apart from high levels of uncertainty in diagnosing IBD, it is stressful for the horse and expensive for the owner as it typically requires transportation to the horse hospital, fasting, endoscopy, and biopsy assessment. Therefore, there is a need for a non-invasive and affordable method to screen for IBD. Fecal microbiota composition has been shown to change in humans and other animals with IBD; therefore, we used fecal samples to study whether equine IBD is also reflected by changes in the composition of gut microbiota. We show that gut microbiota composition was different and that the abundances of certain bacterial groups were significantly increased or decreased in horses with IBD compared to healthy horses. We further used a machine learning model to predict IBD based on the microbiota composition of the fecal samples. The model produced correct predictions with 100% accuracy in our dataset. We conclude that, in the future, studying the microbiota composition of fecal samples could become an accurate, cheap, and non-invasive method in screening for equine IBD. Gastrointestinal disorders are common and debilitating in horses, but their diagnosis is often difficult and invasive. Fecal samples offer a non-invasive alternative to assessing the gastrointestinal health of horses by providing information about the gut microbiota and inflammation. In this study, we used 16S sequencing to compare the fecal bacterial diversity and composition of 27 healthy horses and 49 horses diagnosed with inflammatory bowel disease (IBD). We also measured fecal calprotectin concentration, a marker of intestinal inflammation, in healthy horses and horses with IBD. We found that microbiota composition differed between healthy horses and horses with IBD, although less than five percent of the variation in microbiota composition was explained by individual health status and age. Several differentially abundant bacterial taxa associated with IBD, age, or body condition were depleted from the most dominant Firmicutes phylum and enriched with the Bacteroidota phylum. An artificial neural network model predicted the probability of IBD among the test samples with 100% accuracy. Our study is the first to demonstrate the association between gut microbiota composition and chronic forms of IBD in horses and highlights the potential of using fecal samples as a non-invasive source of biomarkers for equine IBD. [ABSTRACT FROM AUTHOR]

Caprine paratuberculosis (PTB) is a progressive, debilitating and production-limiting disease that causes significant economic losses and raises public health concerns. The goal was to study active surveillance and associated epidemiological risk factors of caprine PTB in selected district of Odisha, India. The 818 goats of various ages, sexes and breeds were randomly screened in ten different districts for a year based on history, clinical signs and fecal smear examination using the Ziehl-Neelsen stain, yielding an overall prevalence of 38.75%, with clinical and sub-clinical PTB at 8.06 and 30.68%, respectively. A molecular tool, IS900 polymerase chain reaction, was also used to confirm the disease. With Mycobacterium avium subsp. paratuberculosis (MAP) bacilli and endoparasite infections, the majority of affected goats (69.08%) were low shedders. Puri coastal district had the highest prevalence (52.29%) followed by Sambalpur (48.61%), while Khordha had the lowest prevalence (26.41%). Caprine PTB was more common in goats over 2 years old (51.23%), in the Ganjam breed (42.30%), in females (39.17%) and in goats housed on earthen floors (55.83%) according to chi-square analysis. The current study concluded that higher (30.68%) observations of subclinical PTB were cause of real concern due to its insidious spread as well as its zoonotic significance with potential human consequences, which requires immediate attention at all levels. Because of the public health importance of this hidden killer disease, the current findings would be useful in developing a roadmap for implementing prevention and control policies, prompting provision for adequate funding with elaborative research. [ABSTRACT FROM AUTHOR]

Background Stool consistency is an important outcome measure to evaluate in the investigation of several gastrointestinal diseases. The Bristol Stool Scale (BSS) is one of the most commonly used tools for evaluation of stool consistency. BSS ranges from 1-7 and each score is assigned to a given consistency of the feces. Self-reported characterizations can differ from an expert evaluation, and the reliability of BSS is unclear. We aimed to evaluate the reliability of BSS by comparing patient scores with expert scores. Methods Patients with inflammatory bowel disease collected stool samples throughout a 3-year follow-up. The stool´s consistency was evaluated with BSS by the patients and matched with an expert score. Agreement between patient and expert scores was assessed using Cohen’s kappa. Results BSS scores from 2280 fecal samples collected from 992 patients at up to five time points were included. When all samples were compared, there was good to substantial agreement between patient and expert scores (Cohen’s weighted kappa: 0.66-0.72). When the BSS scores were simplified and categorized as 1 (scores 1-2), 2 (scores 3-5) or 3 (scores 6-7), the agreement improved slightly (Cohen’s weighted kappa: 0.73-0.77). When the scores from the first sample per patient were compared, the experts were more likely to assign higher scores compared to the patient. The proportion of the lowest assigned scores (1-2) was 12.1% for patients and 8.1% for experts. Conclusions The agreement between patient and expert BSS scores is good to substantial, especially when the BSS scores are simplified into three categories.

ABSTRACT Microbiome studies are becoming larger in size to detect the potentially small effect that environmental factors have on our gut microbiomes, or that the microbiome has on our health. Therefore, fast and reproducible DNA isolation methods are needed to handle thousands of fecal samples. We used the Chemagic 360 chemistry and Magnetic Separation Module I (MSMI) instrument to compare two sample preservatives and four different pre-treatment protocols to find an optimal method for DNA isolation from thousands of fecal samples. The pre-treatments included bead beating, sample handling in tube and plate format, and proteinase K incubation. The optimal method offers a sufficient yield of high-quality DNA without contamination. Three human fecal samples (adult, senior, and infant) with technical replicates were extracted. The extraction included negative controls (OMNIgeneGUT, DNA/RNA shield fluid, and Chemagic Lysis Buffer 1) to detect cross-contamination and ZymoBIOMICS Gut Microbiome Standard as a positive control to mimic the human gut microbiome and assess sensitivity of the extraction method. All samples were extracted using Chemagic DNA Stool 200 H96 kit (PerkinElmer, Finland). The samples were collected in two preservatives, OMNIgeneGUT and DNA/RNA shield fluid. DNA quantity was measured using Qubit-fluorometer, DNA purity and quality using gel electrophoresis, and taxonomic signatures with 16S rRNA gene-based sequencing with V3V4 and V4 regions. Bead beating increased bacterial diversity. The largest increase was detected in gram-positive genera Blautia, Bifidobacterium, and Ruminococcus. Preservatives showed minor differences in bacterial abundances. The profiles between the V3V4 and V4 regions differed considerably with lower diversity samples. Negative controls showed signs from genera abundant in fecal samples. Technical replicates of the Gut Standard and stool samples showed low variation. The selected isolation protocol included recommended steps from manufacturer as well as bead beating. Bead beating was found to be necessary to detect hard-to-lyse bacteria. The protocol was reproducible in terms of DNA yield among different stool replicates and the ZymoBIOMICS Gut Microbiome Standard. The MSM1 instrument and pre-treatment in a 96-format offered the possibility of automation and handling of large sample collections. Both preservatives were feasible in terms of sample handling and had low variation in taxonomic signatures. The 16S rRNA target region had a high impact on the composition of the bacterial profile.IMPORTANCENext-generation sequencing (NGS) is a widely used method for determining the composition of the gut microbiota. Due to the differences in the gut microbiota composition between individuals, microbiome studies have expanded into large population studies to maximize detection of small effects on microbe–host interactions. Thus, the demand for a rapid and reliable microbial profiling is continuously increasing, making the optimization of high-throughput 96-format DNA extraction integral for NGS-based downstream applications. However, experimental protocols are prone to bias and errors from sample collection and storage, to DNA extraction, primer selection and sequencing, and bioinformatics analyses. Methodological bias can contribute to differences in microbiome profiles, causing variability across studies and laboratories using different protocols. To improve consistency and confidence of the measurements, the standardization of microbiome analysis methods has been recognized in many fields.

Abstract Background Opisthorchis viverrini infection is traditionally diagnosed using the Kato–Katz method and formalin ethyl–acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O. viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)–clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA–CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA–CRISPR/Cas assay to detect O. viverrini infection in field-collected human feces. Methods To detect O. viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA–CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA–CRISPR/Cas12a assay for detecting O. viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA–CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. Results The Ov-RPA–CRISPR/Cas12a assay exhibited high specificity for detecting O. viverrini DNA. On the basis of the detection limit, the assay could detect O. viverrini DNA at concentrations as low as 10−1 ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1 ng. To validate the Ov-RPA–CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O. viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA–CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. Conclusions The Ov-RPA–CRISPR/Cas12a assay developed in this study can successfully detect O. viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O. viverrini infection, marking an initial step in the development of point-of-care diagnosis. Graphical abstract

Abstract The lack of standardization in the methods of DNA extraction from fecal samples represents the major source of experimental variation in the microbiome research field. In this study, we aimed to compare the metagenomic profiles and microbiome–phenotype associations obtained by applying two commercially available DNA extraction kits: the AllPrep DNA/RNA Mini Kit (APK) and the QIAamp Fast DNA Stool Mini Kit (FSK). Using metagenomic sequencing data available from 745 paired fecal samples from two independent population cohorts, Lifelines-DEEP (LLD, n = 292) and the 500 Functional Genomics project (500FG, n = 453), we confirmed significant differences in DNA yield and the recovered microbial communities between protocols, with the APK method resulting in a higher DNA concentration and microbial diversity. Further, we observed a massive difference in bacterial relative abundances at species-level between the APK and the FSK protocols, with > 75% of species differentially abundant between protocols in both cohorts. Specifically, comparison with a standard mock community revealed that the APK method provided higher accuracy in the recovery of microbial relative abundances, with the absence of a bead-beating step in the FSK protocol causing an underrepresentation of gram-positive bacteria. This heterogeneity in the recovered microbial composition led to remarkable differences in the association with anthropometric and lifestyle phenotypes. The results of this study further reinforce that the choice of DNA extraction method impacts the metagenomic profile of human gut microbiota and highlight the importance of harmonizing protocols in microbiome studies.

Background: Opisthorchis viverrini infection is traditionally diagnosed using the Kato–Katz method and formalin ethyl–acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O. viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)–clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA–CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA–CRISPR/Cas assay to detect O. viverrini infection in field-collected human feces. Methods: To detect O. viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA–CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA–CRISPR/Cas12a assay for detecting O. viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA–CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. Results: The Ov-RPA–CRISPR/Cas12a assay exhibited high specificity for detecting O. viverrini DNA. On the basis of the detection limit, the assay could detect O. viverrini DNA at concentrations as low as 10−1 ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1 ng. To validate the Ov-RPA–CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O. viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA–CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. Conclusions: The Ov-RPA–CRISPR/Cas12a assay developed in this study can successfully detect O. viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O. viverrini infection, marking an initial step in the development of point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

The lack of standardization in the methods of DNA extraction from fecal samples represents the major source of experimental variation in the microbiome research field. In this study, we aimed to compare the metagenomic profiles and microbiome–phenotype associations obtained by applying two commercially available DNA extraction kits: the AllPrep DNA/RNA Mini Kit (APK) and the QIAamp Fast DNA Stool Mini Kit (FSK). Using metagenomic sequencing data available from 745 paired fecal samples from two independent population cohorts, Lifelines-DEEP (LLD, n = 292) and the 500 Functional Genomics project (500FG, n = 453), we confirmed significant differences in DNA yield and the recovered microbial communities between protocols, with the APK method resulting in a higher DNA concentration and microbial diversity. Further, we observed a massive difference in bacterial relative abundances at species-level between the APK and the FSK protocols, with > 75% of species differentially abundant between protocols in both cohorts. Specifically, comparison with a standard mock community revealed that the APK method provided higher accuracy in the recovery of microbial relative abundances, with the absence of a bead-beating step in the FSK protocol causing an underrepresentation of gram-positive bacteria. This heterogeneity in the recovered microbial composition led to remarkable differences in the association with anthropometric and lifestyle phenotypes. The results of this study further reinforce that the choice of DNA extraction method impacts the metagenomic profile of human gut microbiota and highlight the importance of harmonizing protocols in microbiome studies. [ABSTRACT FROM AUTHOR]

Gastrointestinal disorders are common and debilitating in horses, but their diagnosis is often difficult and invasive. Fecal samples offer a non-invasive alternative to assessing the gastrointestinal health of horses by providing information about the gut microbiota and inflammation. In this study, we used 16S sequencing to compare the fecal bacterial diversity and composition of 27 healthy horses and 49 horses diagnosed with inflammatory bowel disease (IBD). We also measured fecal calprotectin concentration, a marker of intestinal inflammation, in healthy horses and horses with IBD. We found that microbiota composition differed between healthy horses and horses with IBD, although less than five percent of the variation in microbiota composition was explained by individual health status and age. Several differentially abundant bacterial taxa associated with IBD, age, or body condition were depleted from the most dominant Firmicutes phylum and enriched with the Bacteroidota phylum. An artificial neural network model predicted the probability of IBD among the test samples with 100% accuracy. Our study is the first to demonstrate the association between gut microbiota composition and chronic forms of IBD in horses and highlights the potential of using fecal samples as a non-invasive source of biomarkers for equine IBD.

ABSTRACTThe impact of therapeutic interventions on the human gut microbiota (GM) is a clinical issue of paramount interest given the strong interconnection between microbial dynamics and human health. Orally administered antibiotics are known to reduce GM biomass and modify GM taxonomic profile. However, the impact of antimicrobial therapies on GM functions and biochemical pathways has scarcely been studied. Here, we characterized the fecal metaproteome of 10 Helicobacter pylori-infected patients before (T0) and after 10 days (T1) of a successful quadruple therapy (bismuth, tetracycline, metronidazole, and rabeprazole) and 30 days after therapy cessation (T2), to investigate how GM and host functions change during the eradication and healing processes. At T1, the abundance ratio between microbial and host proteins was reversed compared with that at T0 and T2. Several pathobionts (including Klebsiella, Proteus, Enterococcus, Muribaculum, and Enterocloster) were increased at T1. Therapy reshaped the relative contributions of the functions required to produce acetate, propionate, and butyrate. Proteins related to the uptake and processing of complex glycans were increased. Microbial cross-feeding with sialic acid, fucose, and rhamnose was enhanced, whereas hydrogen sulfide production was reduced. Finally, microbial proteins involved in antibiotic resistance and inflammation were more abundant after therapy. Moreover, a reduction in host proteins with known roles in inflammation and H. pylori-mediated carcinogenesis was observed. In conclusion, our results support the use of metaproteomics to monitor drug-induced remodeling of GM and host functions, opening the way for investigating new antimicrobial therapies aimed at preserving gut environmental homeostasis.

Food availability plays a key role affecting almost every aspect of bird ecology, including bird population, distribution, reproduction, and behavior. Understanding the functional relationship between prey availability and insectivorous birds’ diet is important in the current scenario of habitat loss and declining of insect and bird populations. We used a fecal metabarcoding approach coupled with prey availability sampling (arthropod abundance and biomass) to test for evidence of selective foraging in a shrub-steppe passerine assemblage over the 2017–2019 breeding seasons in semi-natural steppes of central Spain. The results showed that the six bird species selected specific arthropod taxa. Heteroptera, Orthoptera, Araneae, Lepidoptera and some Coleoptera families were the main selected prey taxa, while Formicidae appeared to be avoided, suggesting that shrub-steppe passerines tend to select prey items providing essential nutrients and energy during the breeding season, while poor quality ones are neglected. Our findings highlight the utility of metabarcoding dietary data to assess prey selection patterns in insectivorous passerines and provide valuable information for the development of conservation and management programs to ensure the long-term availability of crucial food resources for shrub-steppe birds and other insectivores.

A coprological survey was conducted to determine the types and prevalence of internal parasites in captive African lions (Panthera leo) in Egypt. Individual fecal samples were collected from 30 lions of different ages and gender from Giza Zoo and different circuses over a 1-year period. Young, aged lions were suffered from weight loss, lethargy, and mild decrease in appetite with mild gastrointestinal symptoms and vomiting with presence of adult Toxascaris leonina in vomitus of 3 of them while adult ones show normal body condition without any symptoms. Fecal samples were subjected for macroscopic examination then examined using flotation and sedimentation techniques to assess the presence of parasite eggs and oocysts. The overall prevalence of helminth infections was 33.33% (10/30). Eggs of Toxascaris leonina were found in the feces of 6 (6/10, 60%) lions and Taenia species in 4 (4/10, 40%) lions. In conclusion, due to the increased number of lions in Egypt either for the purpose of acquisition or for work in show as in circus and in the zoo, therefore, diseases that affect lions must be taken into consideration. Parasitic disease is of veterinary importance for lions, but future work including other parasitic, bacterial and viral infections, epidemiology, disease distribution, line of prevention and control for each problem are required. [ABSTRACT FROM AUTHOR]

The Djungarian hamster (Phodopus sungorus) is a prominent model organism for seasonal acclimatization, showing drastic whole-body physiological adjustments to an energetically challenging environment, which are considered to also involve the gut microbiome. Fecal samples of hamsters in long photoperiod and again after twelve weeks in short photoperiod were analyzed by 16S-rRNA sequencing to evaluate seasonal changes in the respective gut microbiomes. In both photoperiods, the overall composition was stable in the major superordinate phyla of the microbiota, with distinct and delicate changes of abundance in phyla representing each <1% of all. Elusimicrobia, Tenericutes, and Verrucomicrobia were exclusively present in short photoperiod hamsters. In contrast to Elusimicrobium and Aneroplasma as representatives of Elusimicrobia and Tenericutes, Akkermansia muciniphila is a prominent gut microbiome inhabitant well described as important in the health context of animals and humans, including neurodegenerative diseases and obesity. Since diet was not changed, Akkermansia enrichment appears to be a direct consequence of short photoperiod acclimation. Future research will investigate whether the Djungarian hamster intestinal microbiome is responsible for or responsive to seasonal acclimation, focusing on probiotic supplementation. [ABSTRACT FROM AUTHOR]

The feces of captive red gorals(Naemorhedus baileyi) in Shanghai Zoo were collected and hormones were detected to estimate the reproductive cycles. Four methods were used to preserve the fecal samples to evaluate the influence of storage methods on hormone detection over time. The results showed that drying and freezing groups showed better effects on estradiol detection, and ethanol immersion and freezing group showed better effects on progesterone, and the freezing group was better on testosterone. The antibiotic storage was only effective within 15 days. Taking all methods into account, the freezing preservation effect is the best, ethanol immersion is suitable for short-term sample transportation and storage, and drying method should be an alternative method when samples cannot be frozen. The detection of fecal reproductive hormone showed that estradiol peaked at early January and whole December 2018, reached a high level from February to April 2019, and peaked from October to December 2019. The progesterone had multiple peaks in January, November and December 2018, January 2019, and remained high level in February and fluctuated from October to the middle of December. Testosterone peaked from November 2018 to February 2019, as well as in October and November 2019. According to data collected, we predicted that the reproductive period of female N. baileyi starts from October to the end of March of the next year, in which December to January is the peak period of estrous, and the oestrous period of male N. baileyi is from October to the end of February of the next year. It is suggested that the cage merging could be extended (from October to March of the next year), and the observation and recording of animal behavior during cage merging period should be strengthened, so as to improve the reproductive success. [ABSTRACT FROM AUTHOR]

Reintroduction is an effective strategy in the conservation of endangered species under scientific monitoring. Intestinal flora plays an important role in the envir onmental adaptation of endangered Père David’s deer (Elaphurus davidianus). In this study, 34 fecal samples from E. davidianus were collected from different habitats in Tianjin city of China to investigate differences in the intestinal flora under captive and semi-free-ranging conditions. Based on 16S rRNA high-throughput sequencing technology, a total of 23 phyla and 518 genera were obtained. Firmicutes was dominant in all individuals. At the genus level, UCG-005 (13.05%) and Rikenellaceae_RC9_gut_group (8.94%) were dominant in captive individuals, while Psychrobacillus (26.53%) and Pseudomonas (11.33%) were dominant in semi-free-ranging individuals. Alpha diversity results showed that the intestinal flora richness and diversity were significantly (P < 0.001) higher in captive individuals than in semi-free-ranging individuals. Beta diversity analysis also showed a significant difference (P = 0.001) between the two groups. In addition, some age- and sex-related genera such as Monoglobus were identified. In summary, the structure and diversity of intestinal flora showed significant habitat variation. This is the first time an analysis has been undertaken of the structural differences of the intestinal flora in Père David’s deer, under different habitats in the warm temperate zone, providing a reference basis for the conservation of endangered species. [ABSTRACT FROM AUTHOR]

Fecal diagnostics are a mainstay of feline medicine, and fecal identification markers help to distinguish individuals in a multi-cat environment. However, the impact of identification markers on the fecal microbiota are unknown. Given the increased interest in using microbiota endpoints to inform diagnosis and treatment, the objective of this study was to examine the effects of orally supplemented glitter and crayon shavings on the feline fecal microbiota (amplicon sequencing of 16S rRNA gene V4 region). Fecal samples were collected daily from six adult cats that were randomized to receive oral supplementation with either glitter or crayon for two weeks, with a two-week washout before receiving the second marker. No adverse effects in response to marker supplementation were seen for any cat, and both markers were readily identifiable in the feces. Microbiota analysis revealed idiosyncratic responses to fecal markers, where changes in community structure in response to glitter or crayon could not be readily discerned. Given these findings, it is not recommended to administered glitter or crayon shavings as a fecal marker when microbiome endpoints are used, however their clinical use with other diagnostics should still be considered.

The aim of the study was to check the prevalence of Escherichia coli in some captive avian species, seasonal effect on the E.coli prevalence and analysis of nucleotide sequences of E.coli. A total of 132 samples, 33 from Turkey (Meleagris gallopavo), 33 form Pheasant (Phasianus colchicus), 33 from Budgerigar (Melopsittacus undulates) and 33 from Chukar partridge (Alectoris chukar) were collected from Conservation and Research Center, UVAS, Ravi Campus, Pattoki. Colony forming units was quantified for each sample. E. coli confirmation was done by biochemical and molecular characterization. 16S rRNA was amplified and sequenced. 16S rRNA sequence was submitted to NCBI under the accession number MN841017, MN841018 and MN841019.Descriptive statistics showed the mean ± SEM value for E. coli CFU/ml of fecal sample from Turkey 1.91 × 108 ± 4.4 × 107, for Pheasants, the mean ± SEM was 1.55 × 108 ± 5.2 × 107 CFU/ml of fecal sample. The mean ± SEM of the fecal sample for Budgerigars and Chukar were 2.12 × 108 ± 3.3 × 107 CFU/ml and 1.6 × 108 ± 4.5 × 107 CFU/ml respectively. Inferential statistics showed that regardless of the bird species, there was almost a similar frequency of E. coli CFU/ml of fecal sample (p = 0.74). However, the incidence of E. coli fluctuates significantly depending on the season in the case of turkey and pheasants, and the impact was statistically significant (p

The dietary patterns are closely associated with gut microbiota, which has been proved associated with kidney stones. To assess the association among the dietary patterns, gut microbiota, and kidney stones, patients with calcium oxalate stones and participants without kidney stones were recruited in West China Hospital and were divided into the low nephrolithiasis risk (LNR) and high nephrolithiasis risk (HNR) dietary pattern group based on the results of food frequency questionnaires. The genomic DNA of the fecal samples were extracted for 16S ribosomal RNA gene sequencing. The non-kidney stone (NS) group comprised 39 LNR and 45 HNR individuals, while the kidney stone (KS) group consisted of 19 LNR and 50 HNR individuals. The distribution of oxalate in urine (p < 0.01) but not calcium (p = 0.741) was significantly varied among the four groups. Significant difference was found in the dietary patterns of people with KS and NS controls (X2 = 5.744, p = 0.017). Forty-six discriminative bacteria were found among different dietary patterns groups in KS patients and NS controls. Not only gut bacteria such as Pseudomonas, Sphingomonas, Hydrogenoanaerobacterium, Faecalitalea, etc., but also metabolic pathways associated with inflammation, lipid, and mineral metabolism were found more abundant in KS patients with HNR dietary pattern. It is noteworthy that g__Prevotellaceae_UCG_001, g__hgcI_clade, and g__Bradyrhizobium were negatively related to water intake but instead had a positive correlation with salt and meat intake. Our study revealed that gut microbiota with significantly different abundance existed in the HNR dietary patterns compared to the LNR counterparts in both calcium oxalate KS and NS individuals. The dietary patterns may affect the prevention and management of calcium oxalate stones by regulating the homeostasis of gut microbiota. [ABSTRACT FROM AUTHOR]

The Djungarian hamster (Phodopus sungorus) is a prominent model organism for seasonal acclimatization, showing drastic whole-body physiological adjustments to an energetically challenging environment, which are considered to also involve the gut microbiome. Fecal samples of hamsters in long photoperiod and again after twelve weeks in short photoperiod were analyzed by 16S-rRNA sequencing to evaluate seasonal changes in the respective gut microbiomes. In both photoperiods, the overall composition was stable in the major superordinate phyla of the microbiota, with distinct and delicate changes of abundance in phyla representing each Akkermansia muciniphila is a prominent gut microbiome inhabitant well described as important in the health context of animals and humans, including neurodegenerative diseases and obesity. Since diet was not changed, Akkermansia enrichment appears to be a direct consequence of short photoperiod acclimation. Future research will investigate whether the Djungarian hamster intestinal microbiome is responsible for or responsive to seasonal acclimation, focusing on probiotic supplementation.

The brown bear (Ursus arctos) is a second-class protected animal in China. Species identification of brown bear is an important research field of biological resource investigation and diversity conservation, and is also one of the important technical means to solve human-wildlife conflict and to crack down on illegal poaching of rare animals. We designed a pair of specific PCR primers for species identification of brown bears and established a specific PCR method for identification of brown bears based on fecal materials. The results showed that the designed specific primers could identify different subspecies of Tibetan brown bear U. a pruinosus, Northeastern brown bear U. a lasiotus and Himalayan brown bear U. a. isabellinus with high specificity, and could efficiently and specifically identify Tibetan brown bear from fecal materials, providing a technical method for wild animal investigation and identification of brown bear. And this method provides technical support for the conservation and identification of brown bears. [ABSTRACT FROM AUTHOR]

Bottom-up proteomics is a powerful method for the functional characterization of mouse gut microbiota. To date, most of the bottom-up proteomics studies of the mouse gut rely on limited amounts of fecal samples. With mass-limited samples, the performance of such analyses is highly dependent on the protein extraction protocols and contaminant removal strategies. Here, protein extraction protocols (using different lysis buffers) and contaminant removal strategies (using different types of filters and beads) were systematically evaluated to maximize quantitative reproducibility and the number of identified proteins. Overall, our results recommend a protein extraction method using a combination of sodium dodecyl sulfate (SDS) and urea in Tris–HCl to yield the greatest number of protein identifications. These conditions led to an increase in the number of proteins identified from gram-positive bacteria, such as Firmicutes and Actinobacteria, which is a challenging task. Our analysis further confirmed these conditions led to the extraction of non-abundant bacterial phyla such as Proteobacteria. In addition, we found that, when coupled to our optimized extraction method, suspension trap (S-Trap) outperforms other contaminant removal methods by providing the most reproducible method while producing the greatest number of protein identifications. Overall, our optimized sample preparation workflow is straightforward and fast, and requires minimal sample handling. Furthermore, our approach does not require high amounts of fecal samples, a vital consideration in proteomics studies where mice produce smaller amounts of feces due to a particular physiological condition. Our final method provides efficient digestion of mouse fecal material, is reproducible, and leads to high proteomic coverage for both host and microbiome proteins. [ABSTRACT FROM AUTHOR]

Abstract 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.

Most emerging and re-emerging viruses causing infectious diseases in humans and domestic animals have originated from wildlife. However, current knowledge of the spectrum of RNA viruses in the Qinghai-Tibet Plateau in China is still limited. Here, we performed metatranscriptomic sequencing on fecal samples from 56 birds and 91 small mammals in Tibet and Qinghai Provinces, China, to delineate their viromes and focused on vertebrate RNA viruses. A total of 184 nearly complete genome RNA viruses belonging to 28 families were identified. Among these, 173 new viruses shared <90% amino acid identity with previously known viral sequences. Several of these viruses, such as those belonging to genera Orthonairovirus and Hepatovirus , could be zoonotic viruses. In addition, host taxonomy and geographical location of these viruses showed new hosts and distribution of several previously discovered viruses. Moreover, 12 invertebrate RNA viruses were identified with <40% amino acid identity to known viruses, indicating that they belong to potentially new taxa. The detection and characterization of RNA viruses from wildlife will broaden our knowledge of virus biodiversity and possible viral diseases in the Qinghai–Tibet Plateau. [ABSTRACT FROM AUTHOR]

Abstract 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.

Microbiome studies are becoming larger in size to detect the potentially small effect that environmental factors have on our gut microbiomes, or that the microbiome has on our health. Therefore, fast and reproducible DNA isolation methods are needed to handle thousands of fecal samples. We used the Chemagic 360 chemistry and Magnetic Separation Module I (MSMI) instrument to compare two sample preservatives and four different pre-treatment protocols to find an optimal method for DNA isolation from thousands of fecal samples. The pre-treatments included bead beating, sample handling in tube and plate format, and proteinase K incubation. The optimal method offers a sufficient yield of high-quality DNA without contamination. Three human fecal samples (adult, senior, and infant) with technical replicates were extracted. The extraction included negative controls (OMNIgeneGUT, DNA/RNA shield fluid, and Chemagic Lysis Buffer 1) to detect cross-contamination and ZymoBIOMICS Gut Microbiome Standard as a positive control to mimic the human gut microbiome and assess sensitivity of the extraction method. All samples were extracted using Chemagic DNA Stool 200 H96 kit (PerkinElmer, Finland). The samples were collected in two preservatives, OMNIgeneGUT and DNA/RNA shield fluid. DNA quantity was measured using Qubit-fluorometer, DNA purity and quality using gel electrophoresis, and taxonomic signatures with 16S rRNA gene-based sequencing with V3V4 and V4 regions. Bead beating increased bacterial diversity. The largest increase was detected in gram-positive genera Blautia, Bifidobacterium, and Ruminococcus . Preservatives showed minor differences in bacterial abundances. The profiles between the V3V4 and V4 regions differed considerably with lower diversity samples. Negative controls showed signs from genera abundant in fecal samples. Technical replicates of the Gut Standard and stool samples showed low variation. The selected isolation protocol included recommended steps from manufacturer as well as bead beating. Bead beating was found to be necessary to detect hard-to-lyse bacteria. The protocol was reproducible in terms of DNA yield among different stool replicates and the ZymoBIOMICS Gut Microbiome Standard. The MSM1 instrument and pre-treatment in a 96-format offered the possibility of automation and handling of large sample collections. Both preservatives were feasible in terms of sample handling and had low variation in taxonomic signatures. The 16S rRNA target region had a high impact on the composition of the bacterial profile., Importance: Next-generation sequencing (NGS) is a widely used method for determining the composition of the gut microbiota. Due to the differences in the gut microbiota composition between individuals, microbiome studies have expanded into large population studies to maximize detection of small effects on microbe-host interactions. Thus, the demand for a rapid and reliable microbial profiling is continuously increasing, making the optimization of high-throughput 96-format DNA extraction integral for NGS-based downstream applications. However, experimental protocols are prone to bias and errors from sample collection and storage, to DNA extraction, primer selection and sequencing, and bioinformatics analyses. Methodological bias can contribute to differences in microbiome profiles, causing variability across studies and laboratories using different protocols. To improve consistency and confidence of the measurements, the standardization of microbiome analysis methods has been recognized in many fields., Competing Interests: E.M. is currently working as Medical Advisor for BioCodex Nordics. A.J.H. reports receiving personal fees for lectures from BioCodex, Merck, and Pfizer. All other authors declare no competing interests.

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. [ABSTRACT FROM AUTHOR]

Organic acids (OAs) play important roles in a variety of intracellular metabolic pathways, such as the tricarboxylic acid cycle, fatty acid oxidation, glycolysis. The accurate detection of OAs in fecal samples was crucial for comprehending the metabolic changes associated with various metabolic disease. However, the analytical protocol detecting OAs profiling in feces have received scant attention. In this work, an optimized protocol based on chromatography-mass spectrometry for simultaneous quantification of 23 OAs in rat feces was developed. The optimal conditions involved using a 40-mg fecal sample mixed with isopropyl alcohol, acetonitrile, and deionized water (3:2:2 vol ratio) with a total volume of 1500 μL, followed by ultrasonic extraction and a derivatization reaction with an 80 μL derivative agent. The protocol showed an acceptable linearity (R2 ≥ 0.9906), the satisfactory precision (RSD% ≤ 14.87%), the low limits of detection (0.001 to 1 μg/mL) and the limit of quantification (0.005 to 1.5 μg/mL). Moreover, the dried residues of the extracted solution showed the better stability of OAs at −20 °C, which was more suitable for a large-scale sample analysis. Finally, the developed protocol was successfully applied to compare the difference of OAs profiling in fecal samples harvested from normal and nonalcoholic fatty liver disease rats, which was beneficial to find out the metabolic change of OAs profiling and explain the related mechanism of the disease. • A protocol for accurate measurement of 23 organic acids in feces was developed. • The response surface methodology was used to optimize the protocols. • The OAs was more stable when stored in nitrogen-blown dried residues. • The protocol distinguished nonalcoholic fatty liver rats from normal rats. [ABSTRACT FROM AUTHOR]

Norovirus is a leading cause of acute gastroenteritis in humans. This paper presents the development of a novel dual-mode aptasensor for detecting norovirus using colorimetry and electrochemical methods. The initial colorimetric method utilizes gold nanoparticles (AuNPs) and sodium chloride to establish a positive correlation between the concentration of norovirus in a solution and the absorbance ratio A 650 /A 520. The naked eye can detect concentrations as low as 0.1 μg/mL, corresponding to a Ct value of 33 (2.2 copies/μL, CT = 34.102–3.2185·lgX), allowing for qualitative and semi-quantitative analysis. For more accurate trace analysis, a gold electrode is modified with a thiol-modified aptamer and closed with 6-Mercapto-1-hexanol. After incubation with norovirus, the virus specifically binds to the aptamer, causing changes in its spatial structure and distance from the electrode surface. These changes can then be detected using electrochemical square wave voltammetry (SWV). Under optimal reaction conditions, the peak current from SWV exhibits a strong linear relationship with the logarithm of norovirus concentrations between 10−9 μg/mL and 10−2 μg/mL. The regression equation Y = 14.76789 + 1.03983·lgX, with an R2 value of 0.987, accurately represents this relationship. The limit of detection was determined to be 1.365 × 10−10 μg/mL. Furthermore, the aptasensor demonstrated high specificity for norovirus in fecal samples, making it a promising tool for detecting norovirus in various sample types. [Display omitted] • We propose a dual-mode aptasensor for the detection of norovirus in feces. • A colorimetric method based on nanogold and an electrochemical method based on aptamer-modified gold electrodes. • Qualitative and semi-quantitative detection by colorimetric method, quantitative detection by electrochemistry. • The assay exhibits a low detection limit and a wide linear range. • The dual-mode aptasensor provides excellent virus detection performance in both test and real samples. [ABSTRACT FROM AUTHOR]

In nature, wild animals live in an enormous space and usually have very low genetic resistance against parasitic infection mainly due to low exposure towards the parasites themselves. However, when herds of these wild animals are kept in captivity, or in zoological gardens, parasitic infections might be worse and pose a serious threat to endangered species. The present study was conducted to observe the occurrence of gastrointestinal parasites in large felines in a Malaysian zoo. Ten faecal samples were collected from pumas (Puma concolor, n = 5), African lions (Panthera leo, n = 3), a spotted leopard (Panthera pardus, n = 1), and a black panther (Panthera onca, n = 1). All faecal samples were examined for parasite eggs, larvae, and oocysts by simple faecal floatation and formalin - ether sedimentation technique. All large felines in the zoo were infected with gastrointestinal parasites. A total of six species of gastrointestinal parasites were recovered including four nematodes (Toxocara cati, Ancylostoma spp., Toxascaris leonina, and Oxyuris sp.), a cestode (Spirometra sp.), and a protozoan (Isospora sp.). Half (n=5/10) of the large felines had mixed infections with Toxocara cati and Ancylostoma spp. [ABSTRACT FROM AUTHOR]

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: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens of humans and animals and are transmitted among the environment, wildlife, livestock, and humans. The aim of this study was to investigate the rate of isolation and antimicrobial susceptibility of NTM in wildlife. In total, 178 samples of feces (n=131) and tissues (n=47) were collected from 11 wildlife species in Gifu Prefecture and Mie Prefecture, Japan, between June 2016 and October 2018. We isolated NTM from 15.3% (20/ 131) of fecal samples using Ogawa medium, and isolates were identified by sequencing the rpoB and hsp65 genes. The rpoB sequences were compared with those from other strains of human and environmental origin. The NTM isolates were obtained from sika deer (Cervus nippon), wild boar (Sus scrofa), Japanese monkey (Macaca fuscata), raccoon dog (Nyctereutes procyonoides), masked palm civet (Paguma larvata), and Japanese weasel (Mustela itatsi) and were classified as rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM). The 12 RGM identified were Mycolicibacterium peregrinum (n=5), Mycolicibacterium fortuitum (n=3), Mycolicibacterium septicum (n=3), and Mycolicibacterium thermoresistibile (n=1), and the eight SGM were Mycobacterium paraense (n=4), Mycolicibacter arupensis (n=2), Mycolicibacter virginiensis (n=1), and Mycobacterium nebraskense (n=1). The NTM from wildlife showed ≥99% similarity with strains from different sources including humans. The RGM were susceptible to the antimicrobial agents tested except for M. fortuitum, which was resistant to azithromycin and clarithromycin. The SGM showed multiple drug resistance qualities but were susceptible to amikacin, clarithromycin, and rifabutin. These results indicate that wildlife may be reservoir hosts of NTM in Japan. The presence of antimicrobial-resistant NTM in wildlife suggests that the trends of NTM antimicrobial susceptibility in wildlife should be monitored. [ABSTRACT FROM AUTHOR]

Objectives: Emerging evidence suggests that gut microbiota dysbiosis plays a critical role in chronic kidney disease (CKD). However, the relationship between altered gut microbiome profiles and disease severity remains unclear. In this study, we sought to characterize the gut microbiota in CKD patients compared to healthy controls, and to explore potential relationships between gut microbiota composition and disease severity. Methods: Fecal samples were collected from 95 patients at different stages of CKD (non-dialysis patients from stage 1 to 5) and 20 healthy controls. Bacterial DNA was extracted for 16S ribosomal DNA sequencing targeting the V3-V4 region. The diversity and relative abundance of gut microbiota were analyzed as outcome indicators. Results: Differences were observed in the microbial composition and diversity of fecal samples from CKD patients and healthy controls. Specifically, disease severity was found to alter gut microbiota composition. Compared to that in healthy controls, CKD patients showed an increased abundance of Proteobacteria and decreased Synergistetes, most notably in disease stage 5. Lower levels of butyrate-producing bacteria and higher levels of potential pathogens were also detected in CKD patients. Further, Pyramidobacter and Prevotellaceae_UCG-001 were significantly decreased in the CKD1 group compared with healthy controls. Notably, nine microbial genera, including Escherichia-Shigella, Parabacteroides, Roseburia, rectale_group, Ruminococcaceae_NK4A214_group, Prevotellaceae_UCG.001, Hungatella, Intestinimonas, and Pyramidobacter, identified using a random forest model, distinguished between patients with CKD and healthy controls with high accuracy. Functional analysis also revealed that fatty acid and inositol phosphate metabolism were enriched in the CKD group, while aminoacyl-tRNA biosynthesis, oxidative phosphorylation, phenylalanine, tyrosine, and tryptophan biosynthesis, thiamine metabolism, pantothenate, and CoA biosynthesis, as well as valine, leucine, and isoleucine biosynthesis were enriched in healthy controls. Conclusion: Gut microbiota composition and function are associated with CKD severity. And, specific gut microbes are potentially helpful for CKD early diagnosis and prognosis monitoring. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We conducted a diet analysis of Japanese boars (Sus scrofa leucomystax) in Hachioji and Kanayama hilly areas located in Ota city of Japan from August to November 2018. Fifteen fecal samples were collected directly from the colon of culled boars and were tested for the DNA metabarcoding-based protocol to describe the plant and animal diet composition of wild boars using rbcL and CO1 markers, respectively. In conclusion, the barcoding method is a very effective tool for gaining a more detailed understanding of the impacts of boars on agricultural activity and environment conservation. [ABSTRACT FROM AUTHOR]

Abstract Current 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.

The impact of therapeutic interventions on the human gut microbiota (GM) is a clinical issue of paramount interest given the strong interconnection between microbial dynamics and human health. Orally administered antibiotics are known to reduce GM biomass and modify GM taxonomic profile. However, the impact of antimicrobial therapies on GM functions and biochemical pathways has scarcely been studied. Here, we characterized the fecal metaproteome of 10 Helicobacter pylori -infected patients before (T0) and after 10 days (T1) of a successful quadruple therapy (bismuth, tetracycline, metronidazole, and rabeprazole) and 30 days after therapy cessation (T2), to investigate how GM and host functions change during the eradication and healing processes. At T1, the abundance ratio between microbial and host proteins was reversed compared with that at T0 and T2. Several pathobionts (including Klebsiella , Proteus , Enterococcus , Muribaculum , and Enterocloster ) were increased at T1. Therapy reshaped the relative contributions of the functions required to produce acetate, propionate, and butyrate. Proteins related to the uptake and processing of complex glycans were increased. Microbial cross-feeding with sialic acid, fucose, and rhamnose was enhanced, whereas hydrogen sulfide production was reduced. Finally, microbial proteins involved in antibiotic resistance and inflammation were more abundant after therapy. Moreover, a reduction in host proteins with known roles in inflammation and H. pylori -mediated carcinogenesis was observed. In conclusion, our results support the use of metaproteomics to monitor drug-induced remodeling of GM and host functions, opening the way for investigating new antimicrobial therapies aimed at preserving gut environmental homeostasis.

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.

Wild bird avian influenza type A virus (AIV) surveillance is important for the early detection of highly pathogenic AIVs and for providing early warnings to the poultry industry and veterinary services to implement more effective control measures against these viruses. Some field samples are often found to contain more than two kinds of AIV. Correct determination of the HA/NA subtype and complete nucleotide sequences of the component viruses in those samples are often critical for timely and accurate understanding of the field situation, but it is not easy to define the genomic structure of the constituent viruses unambiguously because AIV has eight segmented genomes. In this study, with immunomagnetic beads incorporating polyclonal antibodies of chicken for subtype-specific viral enrichment, we could selectively decrease the density of one of the two constituent viruses in a sample of different subtypes, H5 and H9, artificially generated; this was represented in the changes of Ct values with subtype specific real-time RT-PCR. Following this, with NGS, we could recover nearly complete genomic sequences and arrange the consensus sequences of gene segments of the constituent viruses confidently with the quantitative variable like genome coverage, linked along the gene segments and associated with the number of viral copies in a sample. • Wild bird feces can be cross-contaminated with multiple avian influenza viruses due to the gathering and defecation of bird flocks. • The genomic characterization of avian influenza viruses mixed in a wild bird fecal sample is challenging, but it would be crutial in cases of highly pathgenicity avian influenza virusses. • The two different avian influenza viruses could be selectively purified using the immunomagnetic separation method in an HA subtype-specific manner. • The effectiveness of this assay was confirmed by the successful determination of the subtypes and genomic sequences of the component viruses through next-generation sequencing. [ABSTRACT FROM AUTHOR]