Fecal Extracts Research Articles

Engineered Nanoparticles inside a Microparticle Oral System for Enhanced Mucosal and Systemic Immunity.

Antigen delivery through an oral route requires overcoming multiple challenges, including gastrointestinal enzymes, mucus, and epithelial tight junctions. Although each barrier has a crucial role in determining the final efficiency of the oral vaccination, transcytosis of antigens through follicle-associated epithelium (FAE) represents a major challenge. Most of the research is focused on delivering an antigen to the M-cell for FAE transcytosis because M-cells can easily transport the antigen from the luminal site. However, the fact is that the M-cell population is less than 1% of the total gastrointestinal cells, and most of the oral vaccines have failed to show any effect in clinical trials. To challenge the current dogma of M-cell targeting, in this study, we designed a novel tandem peptide with a FAE-targeting peptide at the front position and a cell-penetrating peptide at the back position. The tandem peptide was attached to a smart delivery system, which overcomes the enzymatic barrier and the mucosal barrier. The result showed that the engineered system could target the FAE (enterocytes and M-cells) and successfully penetrate the enterocytes to reach the dendritic cells located at the subepithelium dome. There was successful maturation and activation of dendritic cells in vitro confirmed by a significant increase in maturation markers such as CD40, CD86, presentation marker MHC I, and proinflammatory cytokines (TNF-α, IL-6, and IL-10). The in vivo results showed a high production of CD4+ T-lymphocytes (helper T-cell) and a significantly higher production of CD8+ T-lymphocytes (killer T-cell). Finally, the production of mucosal immunity (IgA) in the trachea, intestine, and fecal extracts and systemic immunity (IgG, IgG1, and IgG2a) was successfully confirmed. To the best of our knowledge, this is the first study that designed a novel tandem peptide to target the FAE, which includes M-cells and enterocytes rather than M-cell targeting and showed that a significant induction of both the mucosal and systemic immune response was achieved compared to M-cell targeting.

Ban-Lan-Gen Granule Alleviates Dextran Sulfate Sodium-Induced Chronic Relapsing Colitis in Mice via Regulating Gut Microbiota and Restoring Gut SCFA Derived-GLP-1 Production.

PurposeGLP-1 based therapy represents a new treatment option for inflammatory bowel disease. Ban-Lan-Gen (BLG) granule, a known anti-viral TCM formulation, exhibits potential anti-inflammatory activities in treating various kinds of inflammation. However, its anti-inflammatory effect on colitis and the underlying mechanisms remain unknown.MethodsDextran sulfate sodium (DSS)-induced chronic relapsing colitis in mice was established. The disease activity index, histological sign of damage, and levels of proinflammatory cytokines were performed to assess the protective effects of BLG. Serum GLP-1 level and colonic Gcg, GPR41 and GRP43 expression, the community compositions of gut microbiota, the levels of SCFAs in the feces and GLP-1 release from primary murine colon epithelial cells were performed to characterize the effects of BLG on gut microbiota and gut SCFA derived-GLP-1 production.ResultsBLG treatment significantly alleviated body weight loss, DAI, colon shortening, colon tissue damage, and pro-inflammatory cytokine levels of TNF-α, IL-1β and IL-6 in the colon tissues. Moreover, BLG treatment could observably restore colonic Gcg, GPR41 and GRP43 expression and serum GLP-1 level of colitic mice, as well as correct the alteration of gut microbiota in colitic mice by increasing the abundances of SCFA-producing bacteria, eg, Akkermansia and Prevotellaceae_UCG-001, and decreasing the abundances of bacteria, eg, Eubacterium_xylanophilum_group, Ruminococcaceae_UCG-014, Intestinimonas, and Oscillibacter. Furthermore, BLG treatment could markedly increase the levels of SCFAs in feces of colitic mice. In parallel, ex vivo assay also showed that and the extract of feces from BLG-treatment mice could greatly stimulate the secretion of GLP-1 from primary murine colon epithelial cells.ConclusionThese findings suggest that the anti-colitis effects of BLG are achieved at least partly by regulating gut microbiota and restoring gut SCFA derived-GLP-1 production, and BLG has the potential to be developed as a promising agent for the treatment of chronic relapsing colitis.

Reevaluation of Piezo1 as a gut RNA sensor

Piezo1 is a stretch-gated ion channel required for mechanosensation in many organ systems. It is considered a “professional” mechanosensor, impervious to endogenous chemical matter. Recently, a provocative article described a new role for Piezo1 in the gut, suggesting that it is a sensor of microbial single-stranded RNA (ssRNA). If true, this would redefine the scope of Piezo biology. Here, we sought to replicate the central finding that fecal ssRNAs are natural agonists of Piezo1. While we observed that fecal extracts stimulate calcium influx in certain cell lines, this response was independent of both Piezo1 and ssRNA.

A bioanalytical screening method for Enterococcus faecalis RNPP-type quorum sensing peptides in murine feces.

Background: Bacteria coordinate their behavior as a group via communication with their peers, known as 'quorum sensing'. Enterococcus faecalis employs quorum sensing via RNPP-peptides which were not yet reported to be present in mammalian biofluids. Results: Solid phase extraction of murine feces was performed, followed by ultra high performance liquid chromatography (UHPLC-MS/MS) in multiple reaction monitoring (MRM) mode (in total <90min/sample) for the nine known RNPP peptides. Limits of detection ranged between 0.045 and 52nM. Adequate identification criteria allowed detection of RNPP quorum sensing peptides in 2/20 wild-type murine feces samples (i.e., cAM373 and cOB1). Conclusion: A fit-for-purpose UHPLC-MS/MS method detected these RNPP peptides in wild-type murine feces samples.

Faecal proteomics in the identification of biomarkers to differentiate canine chronic enteropathies

Canine chronic enteropathy (CCE) is a collective term used to describe a group of idiopathic enteropathies of dogs that result in a variety of clinical manifestations of intestinal dysfunction. Clinical stratification into food-responsive enteropathy (FRE) or non-food responsive chronic inflammatory enteropathy (CIE), is made retrospectively based on response to treatments. Faecal extracts from those with a FRE (n = 5) and those with non-food responsive chronic inflammatory enteropathies (CIE) (n = 6) were compared to a healthy control group (n = 14) by applying TMT-based quantitative proteomic approach. Many of the proteins with significant differential abundance between groups were pancreatic or intestinal enzymes with pancreatitis-associated protein (identified as REG3α) and pancreatic M14 metallocarboxypeptidase proteins carboxypeptidase A1 and B identified as being of significantly increased abundance in the CCE group. The reactome analysis revealed the recycling of bile acids and salts and their metabolism to be present in the FRE group, suggesting a possible dysbiotic aetiology. Several acute phase proteins were significantly more abundant in the CCE group with the significant increase in haptoglobin in the CIE group especially notable. Further research of these proteins is needed to fully assess their clinical utility as faecal biomarkers for differentiating CCE cases. SignificanceThe identification and characterisation of biomarkers that differentiate FRE from other forms of CIE would prove invaluable in streamlining clinical decision-making and would avoid costly and invasive investigations and delays in implementing effective treatment. Many of the proteins described here, as canine faecal proteins for the first time, have been highlighted in previous human and murine inflammatory bowl disease (IBD) studies initiating a new chapter in canine faecal biomarker research, where early and non-invasive biomarkers for early clinical stratification of CCE cases are needed. Pancreatitis-associated protein, pancreatic M14 metallocarboxypeptidase along with carboxypeptidase A1 and B are identified as being of significantly increased abundance in the CCE groups. Several acute phase proteins, were significantly more abundant in the CCE group notably haptoglobin in dogs with inflammatory enteropathy. The recognition of altered bile acid metabolism in the reactome analysis in the FRE group is significant in CCE which is a complex condition incorporating of immunological, dysbiotic and faecal bile acid dysmetabolism. Both proteomics and immunoassays will enable the characterisation of faecal APPs as well as other inflammatory and immune mediators, and the utilisation of assays, validated for use in analysis of faeces of veterinary species will enable clinical utilisation of faecal matrix to be fully realised.

Fecal microRNAs, Fecal microRNA Panels, or Combinations of Fecal microRNAs with Fecal Hemoglobin for Early Detection of Colorectal Cancer and Its Precursors: A Systematic Review.

Simple SummaryScreening for colorectal cancer is effective for the reduction of both CRC incidence and mortality in the population at average risk. The use of innovative and robust biomarkers to enhance the potential of noninvasive CRC screening remains desirable. We aimed to conduct a systematic literature review on the diagnostic performance of fecal miRNA markers for CRC and its precursors. Several studies have reported quite promising results, in particular by combining fecal miRNA measurements with fecal hemoglobin. However, current evidence is limited by substantial heterogeneity in the methodology from study design to biosample analysis. Our review is intended to provide a valuable reference for future biomarker studies in early colorectal cancer detection. Looking at fecal miRNAs, we draw attention to the various biases to be avoided or at least minimized, by applying a harmonized methodology including true screening settings and comparable sample pre-analytics, as well as the validation of biomarkers.Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer mortality globally. Fecal miRNAs have been suggested to be promising biomarkers for CRC early detection. We aimed to conduct a systematic literature review on the diagnostic performance of fecal miRNA markers for CRC and its precursors. PubMed and Web of Science were searched to retrieve relevant articles published up to 7 December 2021. Information on study design, characteristics of study population, pre-analytics (sample collection, processing, and storage), fecal miRNA extraction and quantification technologies, and diagnostic performance (including sensitivity, specificity, and area under the curve (AUC)) were summarized. Twenty studies reporting on 31 individual miRNAs and 16 miRNA panels (with 2–9 markers) for CRC diagnosis were identified. Substantial heterogeneity existed regarding stool sample collection, processing, storage, and miRNA extraction and normalization. For two individual miRNAs and one miRNA panel, values ≥ 80% were reported for both sensitivity and specificity; however, none of these results were either internally or externally validated. In a study among fecal immunochemical test-positive cases recruited from a true screening setting, better diagnostic performance was identified and internally validated for a combination panel including two miRNAs, fecal hemoglobin level, and patient age and sex, compared with fecal hemoglobin concentration alone. Fecal miRNAs or miRNA panels, possibly in combination with fecal hemoglobin test, may be promising candidates for noninvasive CRC early detection. However, large prospective and well-designed studies in CRC screening cohorts are required to validate promising miRNAs or miRNA panels.

1002. Activation of the Enterococcus faecalis Cell Envelope Stress Response through the Novel MadRS System Is Associated With Increased Size of Cardiac Microlesions

BackgroundEnterococci are opportunistic pathogens that can present a therapeutic challenge due to the acquisition of antibiotic resistance. Our previous work has shown the MadRS stress response system plays an important role in defending the enterococcal membrane against daptomycin and antimicrobial peptides (AMP) made by the innate immune system. Strains lacking the MadR response regulator show increased susceptibility to the cathelicidn LL-37 in vitro. A change from alanine to glutamate in the sensor kinase MadS (madSA202E) leads to activation of the system and impaired killing by AMPs. In this study, we evaluated the impact of MadRS function in vivo using a mouse peritonitis model of E. faecalis (Efs) infection.MethodsA laboratory strain Efs OG1RF and two derivatives, OG1RFΔmadR and OG1RFmadSA202E were included. Six mice per strain were inoculated via intraperitoneal injection of ~5x108 CFU/mL of bacteria in 50% sterile rat fecal extract, and followed for 96 hours post infection. Difference in survival between strains was determined by Mantel-Cox test. At the time of death, hearts were aseptically removed, fixed in formalin, and embedded in paraffin. Organs were bisected and sectioned, with every 4th section stained with hematoxylin and eosin (8 total sections per animal). Sections were imaged at 40x magnification, the number of lesions for each section was recorded, and lesion size was determined using imageJ.ResultsThere was no difference in median survival between animals infected with OG1RF and OG1RFΔmadR (22.5 v 21 hours, p=0.31), OG1RF and OG1RFmadSA202E (22.5 v 24 hours, p=0.29), or OG1RFΔmadR and OG1RFmadSA202E (21 v 24 hours, p=0.13). There was a significant difference in the number and size of cardiac lesions between the strains. Mice infected with OG1RFmadSA202E had a significantly higher number of cardiac microlesions as compared to those infected with OG1RFΔmadR (Fig 1). The size of the lesions in mice infected with OG1RFmadSA202E was also significantly larger than those in OG1RF wild type (Fig 1).Figure 1. Cardiac microlesions in a mouse peritonitis model of Enterococcus faecalis infection. Mouse hearts were removed at time of animal death, placed in formalin, and embedded in paraffin. Organs were bisected, then sectioned with every 4th section stained with hematoxylin and eosin (H&E, 8 sections per animal). H&E stained sections were imaged at 40x magnification, the number and size of lesions was determined for 48 sections per strain. Representative cardiac microlesions (arrow) are shown for A) E. faecalis OG1RF, B) OG1RFΔmadR and C) OG1RFmadSA202E, scale bar 20 μm. D) The number of microlesions observed in each section, and E) the area of the lesions for each strain are shown above, differences in means were determined with one way ANOVA using Tukey’s test for multiple comparisons. ns, not significant.ConclusionChanges in MadRS did not impact overall survival in mice, but did alter the number and size of cardiac microlesions. Further experiments are needed to determine if these changes could adversely affect therapy or rates of relapse.Disclosures William R. Miller, MD , Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD , Entasis (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Grant/Research Support Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

Functionalized Ferroferric Oxide Nanomagnetic Beads for Extraction of Nucleic Acid and Its Application in Early Screening of Colorectal Cancer.

Early screening is an important way to reduce the incidence and mortality of colorectal cancer (CRC). Fecal DNA testing stands out among many screening methods due to its high sensitivity. However, at this stage, researchers have not found a high-efficiency method for fecal DNA extraction. To this end, this work carried out a new round of exploration. Here, this experiment synthesized a kind of nanomagnetic beads (NH2-SiO2@Fe3O4) with good stability for nucleic acid extraction. A comparative study with the centrifugal adsorption column method revealed the significant advantages of the magnetic bead method in extracting fecal DNA. The DNA extracted by the magnetic bead method is of high purity, can also achieve high-throughput tests, and is more suitable for polymerase chain reaction detection, greatly simplifying the stool DNA detection process and providing a basis for the widespread promotion of early screening.

Reduced production of the major allergens Bla g 1 and Bla g 2 in Blattella germanica after antibiotic treatment.

Allergens present in the feces or frass of cockroaches can cause allergic sensitization in humans. The use of fecal and frass extracts for immunotherapy has been previously investigated but has not yet been fully standardized. Here, we treated cockroaches with ampicillin to produce extracts with reduced amounts of total bacteria. We performed targeted high-throughput sequencing of 16S rDNA to compare the microbiomes of ampicillin-treated and untreated (control) cockroaches. RNA-seq was performed to identify differentially expressed genes (DEGs) in ampicillin-treated cockroaches. Analysis of the microbiome revealed that alpha diversity was lower in the ampicillin-treated group than in the control group. Beta diversity analysis indicated that ampicillin treatment altered bacterial composition in the microbiome of cockroaches. Quantitative polymerase chain reaction revealed that almost all bacteria were removed from ampicillin-treated cockroaches. RNA-seq analysis revealed 1,236 DEGs in ampicillin-treated cockroaches (compared to untreated cockroaches). Unlike bacterial composition, the DEGs varied between the two groups. Among major allergens, the expression of Bla g 2 decreased significantly in ampicillin-treated cockroaches (compared to untreated group). In this study, the reduced level of allergens observed in cockroaches may be related to lower amounts of total bacteria caused by treatment with antibiotics. It is possible to make a protein extract with few bacteria for use in immunotherapy.

Induction of mucosal and systemic immune responses against the common O78 antigen of an oral inactivated ETEC vaccine in Bangladeshi children and infants

We tested an oral enterotoxigenic Escherichia coli (ETEC) vaccine, ETVAX, consisting of inactivated E. coli overexpressing the most prevalent ETEC colonization factors (CFs) and a toxoid (LCTBA), in Bangladeshi children for capacity to induce mucosal and plasma immune responses against O78 lipopolysaccharide (LPS) expressed on the vaccine strains. The vaccine was given ± double-mutant heat-labile toxin (dmLT) adjuvant. We evaluated the impact of dmLT on anti-O78 LPS immune responses and whether such responses can predict responses against the CFs as a marker for vaccine “take”. Two fractionated doses of ETVAX ± different amounts of dmLT were administered biweekly to groups of children 24–59 (n = 125), 12–23 (n = 97) and 6–11 (n = 158) months of age. Immune responses were evaluated in antibody in lymphocyte supernatants (ALS), fecal extracts and plasma. ALS IgA responses against O78 LPS were induced in 44–49% of the children aged 12–59 months. The magnitudes of the ALS responses were significantly higher in children receiving a half-dose (5 × 1010 bacteria) of ETVAX ± dmLT than in placebo recipients. <10% of the vaccinees aged 6–11 months mounted ALS responses against O78 LPS. However, 49% of the infants developed fecal secretory IgA responses which were significantly more frequent in those receiving a quarter-dose (2.5 × 1010 bacteria) of vaccine + dmLT (62%) compared to a quarter-dose alone (36%). Plasma IgA antibody responses were induced in 80% of older children and 36% of infants. The frequencies of O78 LPS responses in plasma and feces were comparable or higher than against the vaccine CFs in infants. Our findings show that ETVAX induced mucosal and systemic immune responses against O78 LPS in all age groups and that dmLT improved intestinal immune responses among infants. These observations may have implications for more successful use of other oral vaccines based on O antigens in children.

A beacon of dung: using lemming (Lemmus lemmus) winter nests and DNA analysis of faeces to further understand predator\u2013prey dynamics in Northern Sweden

The hypothesis that predation is the cause of the regular small rodent population oscillations observed in boreal and Arctic regions has long been debated. Within this hypothesis, it is proposed that the most likely predators to cause these destabilizing effects are sedentary specialists, with small mustelids being possible candidates. One such case would be the highly specialized least weasel (Mustela nivalis) driving the Norwegian lemming (Lemmus lemmus) cycle in Fennoscandia. These predators are often elusive and therefore distribution data can only be based on field signs, which is problematic when various mustelid species are sympatric, such as weasels and stoats (Mustela erminea). Here we present the results of using mustelid faeces in predated winter lemming nests to correctly identify the predator and thus discern which species exerts the strongest predation pressure on lemming winter populations. Samples were obtained during different phases in the lemming cycle, spanning 6 years, to account for different prey densities. Faecal mitochondrial DNA extraction and amplification of a 400-bp fragment was successful in 92/114 samples (81%); the sequencing of these samples proved that most predation occurrences (83%) could be attributed to the least weasel. These findings support the hypothesis that weasels in particular show high specificity in predation and could therefore be candidates to driving the lemming cycle in this area. We conclude that DNA analysis of faecal remains around predated nests can be a useful tool for further investigations concerning predator–prey interactions in the tundra.

High Prevalence and Factors Associated With the Distribution of the Integron intI1 and intI2 Genes in Scottish Cattle Herds.

Integrons are genetic elements that capture and express antimicrobial resistance genes within arrays, facilitating horizontal spread of multiple drug resistance in a range of bacterial species. The aim of this study was to estimate prevalence for class 1, 2, and 3 integrons in Scottish cattle and examine whether spatial, seasonal or herd management factors influenced integron herd status. We used fecal samples collected from 108 Scottish cattle herds in a national, cross-sectional survey between 2014 and 2015, and screened fecal DNA extracts by multiplex PCR for the integrase genes intI1, intI2, and intI3. Herd-level prevalence was estimated [95% confidence interval (CI)] for intI1 as 76.9% (67.8–84.0%) and intI2 as 82.4% (73.9–88.6%). We did not detect intI3 in any of the herd samples tested. A regional effect was observed for intI1, highest in the North East (OR 11.5, 95% CI: 1.0–130.9, P = 0.05) and South East (OR 8.7, 95% CI: 1.1–20.9, P = 0.04), lowest in the Highlands. A generalized linear mixed model was used to test for potential associations between herd status and cattle management, soil type and regional livestock density variables. Within the final multivariable model, factors associated with herd positivity for intI1 included spring season of the year (OR 6.3, 95% CI: 1.1–36.4, P = 0.04) and watering cattle from a natural spring source (OR 4.4, 95% CI: 1.3–14.8, P = 0.017), and cattle being housed at the time of sampling for intI2 (OR 75.0, 95% CI: 10.4–540.5, P < 0.001). This study provides baseline estimates for integron prevalence in Scottish cattle and identifies factors that may be associated with carriage that warrant future investigation.

Body fluids from the rat exposed to chlorpyrifos induce cytotoxicity against the corresponding tissue\u2212derived cells in vitro

BackgroundThis study aims to establish an in vitro monitoring approach to evaluate the pesticide exposures. We studied the in vitro cytotoxicity of three different body fluids of rats to the respective corresponding tissue-derived cells.MethodsWistar rats were orally administrated daily with three different doses of chlorpyrifos (1.30, 3.26, and 8.15 mg/kg body weight/day, which is equal to the doses of 1/125, 1/50, and 1/20 LD50, respectively) for consecutive 90 days. Blood samples as well as 24-hour urine and fecal samples were collected and processed. Then, urine, serum, and feces samples were used to treat the correspondent cell lines, i.e., T24 bladder cancer cells, Jurkat lymphocytes, and HT-29 colon cancer cells respectively, which derived from the correspondent tissues that could interact with the respective corresponding body fluids in organism. Cell viability was determined by using MTT or trypan blue staining.ResultsThe results showed that urine, serum, and feces extract of the rats exposed to chlorpyrifos displayed concentration- and time-dependent cytotoxicity to the cell lines. Furthermore, we found that the cytotoxicity of body fluids from the exposed animals was mainly due to the presence of 3, 4, 5-trichloropyrindinol, the major toxic metabolite of chlorpyrifos.ConclusionsThese findings indicated that urine, serum, and feces extraction, especially urine, combining with the corresponding tissue-derived cell lines as the in vitro cell models could be used to evaluate the animal exposure to pesticides even at the low dose with no apparent toxicological signs in the animals. Thus, this in vitro approach could be served as complementary methodology to the existing toolbox of biological monitoring of long-term and low-dose exposure to environmental pesticide residues in practice.

Assessment of Biphasic Extraction Methods of Mouse Fecal Metabolites for Liquid Chromatography-Mass Spectrometry-Based Metabolomic Studies.

With the increasing knowledge about the important roles of gut microbiota on the biological system, a systematic strategy to profile the fecal metabolome is urgently needed. Thus, an unbiased, efficient, and reproducible fecal metabolite extraction protocol needs to be established; however, the effect of biphasic extraction methods for the fecal samples remains unclear. In this study, five different methods were assessed in the extraction of polar and non-polar metabolites for the liquid chromatography-mass spectrometry (LC-MS)-based mouse fecal metabolomic study. First, the detection coverage of two extraction systems, the Bligh and Dyer extraction method (M1, chloroform/methanol/water, 2/2/1.8) and Matyash method (M2, methyl tert-butyl ether (MTBE)/methanol/water, 10/3/2.5), was compared; then, MTBE/methanol/water system with different solvent ratios (M3, 2.6/2.0/2.4; M4, 4.5/1/2.5; and M5, 3/2.5/2.5) were further evaluated. The results showed that M2 showed higher detection coverage than M1. For the MTBE/methanol/water system with different solvent ratios, M3 showed the largest detection coverage based on peak numbers and numbers of putatively annotated metabolites, while M4 presented the least overlap between two phases, higher peak intensities of metabolites, and superior reproducibility. Based on the above evidence, M4 was recommended for the biphasic extraction of fecal metabolites in the LC-MS-based mouse fecal metabolomic study.

The Effect of Common Viral Inactivation Techniques on 16S rRNA Amplicon-Based Analysis of the Gut Microbiota.

Research investigating the gut microbiome (GM) during a viral infection may necessitate inactivation of the fecal viral load. Here, we assess how common viral inactivation techniques affect 16S rRNA-based analysis of the gut microbiome. Five common viral inactivation methods were applied to cross-matched fecal samples from sixteen female CD-1 mice of the same GM background prior to fecal DNA extraction. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA. Treatment-dependent effects on DNA yield, genus-level taxonomic abundance, and alpha and beta diversity metrics were assessed. A sodium dodecyl sulfate (SDS)-based inactivation method and Holder pasteurization had no effect on measures of microbial richness, while two Buffer AVL-based inactivation methods resulted in a decrease in detected richness. SDS inactivation, Holder pasteurization, and the AVL-based inactivation methods had no effect on measures of alpha diversity within samples or beta diversity between samples. Fecal DNA extracted with TRIzol-treated samples failed to amplify and sequence, making it unsuitable for microbiome analysis. These results provide guidance in the 16S rRNA microbiome analysis of fecal samples requiring viral inactivation.

Contribution of cysteine and serine proteases to proteolytic digestion in an allergy-eliciting house dust mite

The digestive physiology of house dust mites (HDM) is of interest to understand their allergenicity towards humans since many of their allergens are digestive enzymes and/or are excreted into airborne fecal pellets. The aim of this study is to provide insight on the biochemical basis of proteolytic digestion in Dermatophagoides pteronyssinus, the most widespread HDM species. First, assays using non-specific protein substrates on purified fecal and body extracts determined that body-associated activity is almost exclusively dependent on cysteine proteases, and specifically on major allergen Der p 1. By contrast, cysteine and serine proteases contributed similarly to the activity estimated on fecal extracts. Second, the screening of group-specific peptide-based protease inhibitors followed by ingestion bioassays revealed that the human skin-derived cysteine protease inhibitor cystatin A produces a significant reduction in mite feeding (i.e. excreted guanine), and triggers the overproduction of Der p 1 (3-fold increase by ELISA). Noteworthy, the inhibition of cysteine proteases by cystatin A also resulted in a reduction in three non-target serine protease activities. Further incubation of these extracts with exogenous Der p 1, but not with other commercial cysteine proteases, restored trypsin (Der p 3) and chymotrypsin (Der p 6) activities, indicating that Der p 1 is responsible for their activation in vivo. Finally, the role of serine proteases on the mite's digestive physiology is discussed based on their remarkable activity in fecal extracts and the autocoprophagic behavior reported in mites in this study.

A field‐friendly method of measuring faecal glucocorticoid metabolite concentration as a simple stress checker in snow leopards

Abstract The hormonal analysis of excrement has been employed to non‐invasively assess the physiological conditions of domestic, experimental, zoo and wild animals to. However, conventional hormone analysis techniques require frozen and refrigerated reagents, and laboratory equipment; therefore, it is almost impossible to obtain the results on‐site. This study attempted to establish a method for the simple and rapid quantitative on‐site analysis of faecal glucocorticoid metabolite (fGM) concentrations; this method involved using hand‐shaking for faecal hormone extraction, and immunochromatography using test strips and a smartphone application. This study focused specifically on snow leopards and developed a new, simple fGM measurement method to evaluate their stress levels on‐site. First, the effects of ultraviolet exposure and bacterial activity on fGM concentration, including sample age, were evaluated using faeces from a captive snow leopard. Second, a field‐friendly hormone extraction method was evaluated; this method does not require storage of faeces in the field because the hormones are extracted by hand‐shaking of faeces in ethanol at the time of collection. In addition, to measure hormone concentrations on‐site, a simple immunochromatography measurement kit was used. The concentrations were measured by quantifying the colour reactions using a smartphone application. There was no consistent increase or decrease in fGM concentration with ultraviolet irradiation time. In addition, a high correlation was observed between concentrations extracted using the conventional method (using a vortex mixer and methanol extraction) and those from the field‐friendly method (r = 0.812, p &lt; 0.05). Changes in fGM concentration measured using immunochromatography were also highly consistent with those measured using the conventional method (enzyme immunoassay; r = 0.825, p &lt; 0.05). The analytical validation of immunochromatography also showed high accuracy, that is parallelism (ANCOVA: F = 0.597, df = 1, p = 0.445) and recovery tests (98.2% ± 5.1% and 107.5% ± 18.7%). Further, for biological validation, the change in fGM concentration was examined before and after the transportation of the snow leopards to another zoo. This field‐friendly hormone analysis method is expected to contribute as a simple and rapid stress evaluation tool to manage conservation and animal welfare by monitoring the stress levels.

Lactobacillus rhamnosus GG modifies the metabolome of pathobionts in gnotobiotic mice

BackgroundLactobacillus rhamnosus GG (LGG) is the most widely used probiotic, but the mechanisms underlying its beneficial effects remain unresolved. Previous studies typically inoculated LGG in hosts with established gut microbiota, limiting the understanding of specific impacts of LGG on host due to numerous interactions among LGG, commensal microbes, and the host. There has been a scarcity of studies that used gnotobiotic animals to elucidate LGG-host interaction, in particular for gaining specific insights about how it modifies the metabolome. To evaluate whether LGG affects the metabolite output of pathobionts, we inoculated with LGG gnotobiotic mice containing Propionibacterium acnes, Turicibacter sanguinis, and Staphylococcus aureus (PTS).Results16S rRNA sequencing of fecal samples by Ion Torrent and MinION platforms showed colonization of germ-free mice by PTS or by PTS plus LGG (LTS). Although the body weights and feeding rates of mice remained similar between PTS and LTS groups, co-associating LGG with PTS led to a pronounced reduction in abundance of P. acnes in the gut. Addition of LGG or its secretome inhibited P. acnes growth in culture. After optimizing procedures for fecal metabolite extraction and metabolomic liquid chromatography-mass spectrometry analysis, unsupervised and supervised multivariate analyses revealed a distinct separation among fecal metabolites of PTS, LTS, and germ-free groups. Variables-important-in-projection scores showed that LGG colonization robustly diminished guanine, ornitihine, and sorbitol while significantly elevating acetylated amino acids, ribitol, indolelactic acid, and histamine. In addition, carnitine, betaine, and glutamate increased while thymidine, quinic acid and biotin were reduced in both PTS and LTS groups. Furthermore, LGG association reduced intestinal mucosal expression levels of inflammatory cytokines, such as IL-1α, IL-1β and TNF-α.ConclusionsLGG co-association had a negative impact on colonization of P. acnes, and markedly altered the metabolic output and inflammatory response elicited by pathobionts.

Cross-amplification of ungulate microsatellite markers in the endemic Indian antelope or blackbuck (Antilope cervicapra) for population monitoring and conservation genetics studies in south Asia

The Indian antelope or blackbuck (Antilope cervicapra) is endemic to the Indian subcontinent, inhabiting scrublands and dry grasslands. Most of the blackbuck populations are small, isolated, and threatened by habitat fragmentation and degradation. Management of such disjunct populations requires genetic characterization, which is critical for assessing hazards of stochastic events and inbreeding. Addressing the scarcity of such information on the blackbuck, we describe a novel panel of microsatellite markers that could be used to monitor blackbuck demography and population genetic parameters using non-invasive faecal sampling. We screened microsatellites (n = 40) that had been reported to amplify in bovid and cervid species using faecal samples of the blackbuck collected from Kaimoor Wildlife Sanctuary, Uttar Pradesh, India and its vicinities. We selected 12 markers for amplification using faecal DNA extracts (n = 140) in three multiplex reactions. We observed a mean amplification success rate of 72.4% across loci (92.1-25.7%) with high allele diversity (mean number of alleles/locus = 8.67 ± 1.03). Mean genotyping error rates across the markers were low to moderate (allelic drop-out rate = 0.09; false allele rate = 0.11). The proportions of first- and second-order relatives in the study population were 0.69% and 6.21%, respectively. Based on amplification success, genotyping error rates and the probability of identity (PID), we suggest (i) a panel of five microsatellite markers (cumulative PID = 1.24 × 10-5) for individual identification and population monitoring and (ii) seven additional markers for conservation genetics studies. This study provides essential tools capable of augmenting blackbuck conservation strategies at the landscape level, integral to protecting the scrubland-grassland ecosystem.

Prolonged Suppression of Butyrate-Producing Bacteria Is Associated With Acute Gastrointestinal Graft-vs-Host Disease and Transplantation-Related Mortality After Allogeneic Stem Cell Transplantation.

Butyrogenic bacteria play an important role in gut microbiome homeostasis and intestinal epithelial integrity. Previous studies have demonstrated an association between administration of short-chain fatty acids like butyrate and protection from acute graft-vs-host disease (GvHD) after allogeneic stem cell transplantation (ASCT). In the current study, we examined the abundance and butyrogenic capacity of butyrate-producing bacteria in 28 healthy donors and 201 patients after ASCT. We prospectively collected serial stool samples and performed polymerase chain reaction analysis of the butyrate-producing bacterial enzyme butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) in fecal nucleic acid extracts. Our data demonstrate a strong and prolonged suppression of butyrogenic bacteria early in the course of ASCT. In a multivariable analysis, early use of broad-spectrum antibiotics before day 0 (day of transplantation) was identified as an independent factor associated with low BCoAT copy numbers (odds ratio, 0.370 [95% confidence interval, .175-.783]; P = .009). Diminished butyrogens correlated with other biomarkers of microbial diversity, such as low 3-indoxylsulfate levels, reduced abundance of Clostridiales and low inverse Simpson and effective Shannon indices (all P < .001). Low BCoAT copy numbers at GvHD-onset were correlated with GI-GvHD severity (P = .002) and associated with a significantly higher GvHD-associated mortality rate (P = .04). Furthermore, low BCoAT copy numbers at day 30 were associated with a significantly higher transplantation-related mortality rate (P = .02). Our results are consistent with the hypothesis that alterations in the microbiome play an important role in GvHD pathogenesis and that microbial parameters such as BCoAT might serve as biomarkers to identify patients at high risk of lethal GI-GvHD.