Chemical composition and physical characteristics of faeces in horses with and without free faecal liquid - two case-control studies.

BACKGROUND: Free faecal liquid (FFL) is a condition in horses characterised by two-phase (one solid and one liquid) separation of faeces. Causes of the condition are unknown, but disturbed hindgut fermentation has been suggested as it may alter biochemical composition and appearance of faeces in equines. However, information on faecal composition in horses with FFL is scarce. Faecal chemical composition (dry matter, osmolality, ash, macro minerals, short-chain fatty acids (SCFA) and pH) and physical characteristics (free liquid, sand, water holding capacity and particle size distribution) were compared in horses with (case) and without (control) FFL in two sub-studies. In sub-study I, faeces from 50 case-control horse pairs in Sweden and Norway were sampled in three sampling periods (SP1-SP3). In sub-study II, faeces from 32 case-control horse pairs in Germany were sampled on one occasion. RESULTS: In sub-study I, faecal concentration and proportion of lactic acid (of total short-chain fatty acids, SCFA) and water holding capacity was lower in case compared to control horses. Other variables (content of dry matter, ash, sodium, calcium, phosphorous, magnesium, sulphur, and concentrations of i-butyric, n-valeric and total SCFA, ammonia-N as proportion of total N, and pH) were similar in faeces from case and control horses. In sub-study II, all analysed variables were similar in faecal samples from case and control horses. Faecal particle size distribution was similar in case and control horses, but the proportion of larger particles (2 and 1 mm) were lower and proportion of smaller particles (< 1 mm) was higher in sub-study I compared to in sub-study II. CONCLUSIONS: To the authors' knowledge, this is the first study to investigate faecal chemical composition and physical characteristics in horses with FFL. Case and control horses had similar total SCFA, pH and osmolality, indicating that hindgut fermentation was similar. However, small differences in concentration and proportion (of total SCFA) of lactic acid and water holding capacity of faeces were shown and are of interest for further studies of horses with FFL.

Microbiota of bovine milk, teat skin, and teat canal: Similarity and variation due to sampling technique and milk fraction.

The aim of this study was to evaluate the effect of sampling technique and milk fraction on bovine milk microbiota data and to compare the microbiota in milk to microbiota on the teat end and in the teat canal. Representative milk samples are highly important for assessment of bacteriological findings and microbiota in milk. Samples were obtained from 5 healthy lactating dairy cows at udder quarter level during 1 milking. Swab samples from the teat end and teat canal, and milk samples collected using different techniques and in different milk fractions were included. Milk was collected by hand stripping and through a teat canal cannula before and after machine milking, through a trans-teat wall needle aspirate after milking, and from udder quarter composite milk. The microbiota of the samples was analyzed with sequencing of the V1-V3 region of the 16S rRNA gene. In addition, somatic cell counts and bacterial cultivability were analyzed in the milk samples. Microbiota data were analyzed using multivariate methods, and differences between samples were tested using analysis of similarity (ANOSIM). Differences between samples were further explored via individual studies of the 10 most abundant genera. The microbiota on the teat end, in the teat canal, and in udder quarter composite milk, collected using a milking machine, differed in composition from the microbiota in milk collected directly from the udder quarter. No differences in milk microbiota composition were detected between hand-stripped milk samples, milk samples taken through a teat canal cannula, or milk samples taken as a trans-teat wall needle aspirate before or after milking. We conclude that for aseptic milk samples collected directly from the lactating udder quarter, sampling technique or milk fraction has minor effect on the microbiota composition.

Composition of the mucosa-associated microbiota along the entire gastrointestinal tract of human individuals.

Background: Homeostasis of the gastrointestinal tract depends on a healthy bacterial microbiota, with alterations in microbiota composition suggested to contribute to diseases. To unravel bacterial contribution to disease pathology, a thorough understanding of the microbiota of the complete gastrointestinal tract is essential. To date, most microbial analyses have either focused on faecal samples, or on the microbial constitution of one gastrointestinal location instead of different locations within one individual. Objective: We aimed to analyse the mucosal microbiome along the entire gastrointestinal tract within the same individuals. Methods: Mucosal biopsies were taken from nine different sites in 14 individuals undergoing antegrade and subsequent retrograde double-balloon enteroscopy. The bacterial composition was characterised using 16 S rRNA sequencing with Illumina Miseq. Results: At double-balloon enteroscopy, one individual had a caecal adenocarcinoma and one individual had Peutz-Jeghers polyps. The composition of the microbiota distinctively changed along the gastrointestinal tract with larger bacterial load, diversity and abundance of Firmicutes and Bacteroidetes in the lower gastrointestinal tract than the upper gastrointestinal tract, which was predominated by Proteobacteria and Firmicutes. Conclusions: We show that gastrointestinal location is a larger determinant of mucosal microbial diversity than inter-person differences. These data provide a baseline for further studies investigating gastrointestinal microbiota-related disease.

Distribution of bacteria between different milk fractions, investigated using culture-dependent methods and molecular-based and fluorescent microscopy approaches.

AIMS: To develop a protocol for DNA extraction using whole milk and further, to investigate how the use of whole instead of skimmed milk during DNA extraction affected the resulting microbial composition. METHODS AND RESULTS: In a model study, three selected bacterial species (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 11775 and Lactobacillus reuteri PTA 4659) were added to raw milk and their distribution between different milk fractions was examined by cultivation on selective agar plates. Quantitative real-time PCR (qPCR) assays and Illumina amplicon sequencing were conducted after DNA extraction of whole milk and skimmed milk. In addition, fluorescent microscopy was used to visualize the distribution of Lactobacillus reuteri R2LC mCherry in milk samples with different fat contents. Depending on spike-in bacterial species, recovery rates of 7·4-26·5% of added bacteria were obtained in the fat fraction in culture-based enumeration. qPCR showed a 7-9 fold increase in recovery of spike-in bacteria when the milk fat fraction was combined with the pellet during the DNA extraction step. In the Illumina 16S amplicon approach, relative abundance of six of the top 11 operational taxonomic units identified differed significantly when comparing skimmed milk and whole milk as starting material. Fluorescent microscopy images demonstrated that L. reuteri R2LC mCherry was associated with fat globules in whole milk samples. CONCLUSIONS: This study demonstrates that milk fat harbours bacterial species that might be underestimated when skimmed milk, rather than whole milk, is used for DNA extraction. SIGNIFICANCE AND IMPACT OF THE STUDY: This study emphasizes the importance of using whole instead of skimmed milk for DNA extraction. A protocol for extracting DNA from whole milk is suggested.

Dietary live yeast and increased water temperature influence the gut microbiota of rainbow trout.

AIMS: The objective was to determine the effects of dietary substitution of fishmeal (FM) with live yeast and increasing water temperature on the diversity and composition of gut microbiota in rainbow trout. METHODS AND RESULTS: Fish were fed either FM or yeast (Saccharomyces cerevisiae) and reared in water temperatures of either 11°C (cold) or 18°C (warm) for 6 weeks. Luminal content and mucosa were collected from the distal gut and the load, diversity and species abundance of yeast and bacteria were analysed using agar plating, MALDI-TOF and rRNA gene amplicon sequencing. Yeast in the gut of fish fed FM were represented by S. cerevisiae, Rhodotorula spp. and Debaryomyces hansenii, while fish fed yeast contained 4-5 log higher CFU per g of yeast that were entirely represented by S. cerevisiae. For gut bacteria, sequencing of 16S rRNA gene amplicons using Illumina MiSeq showed lower bacterial diversity and abundance of lactic acid bacteria, especially Lactobacillus, in fish reared in warm rather than cold water. Fish fed yeast had similar bacterial diversity and lower abundance of Leuconostocaceae and Photobacterium compared with fish fed FM. CONCLUSIONS: Feeding live yeast mainly increased yeast load in the gut, while increased water temperature significantly altered the gut microbiota of rainbow trout in terms of bacterial diversity and abundance. SIGNIFICANCE AND IMPACT OF THE STUDY: Live yeast can replace 40% of FM without disrupting bacteria communities in the gut of rainbow trout, while increased water temperature due to seasonal fluctuations and/or climate change may result in a gut dysbiosis that may jeopardize the health of farmed fish.

Composition of human faecal microbiota in resistance to Campylobacter infection.

In mice, specific species composition of gut microbiota enhances susceptibility to Campylobacter jejuni but little is known about the specific composition of the human gut microbiota in providing protection from infections caused by enteropathogens. Healthy adult individuals, who travelled in groups from Sweden to destinations with an estimated high risk for acquisition of Campylobacter infection, were enrolled. Faecal samples, collected before travelling and after returning home, were cultured for bacterial enteropathogens, and analysed for Campylobacter by PCR and for the species composition of the microbiota by 16S amplicon massive parallel sequencing. The microbiota compositions were compared between persons who became infected during their travel and those who did not. A total of 63 participants completed the study; 14 became infected with Campylobacter, two with Salmonella and 47 remained negative for the enteropathogens tested. After exclusion of samples taken after antimicrobial treatment, 49 individuals were included in the final analyses. Intra-individual stability of the microbiota was demonstrated for samples taken before travelling. The original diversity of the faecal microbiota was significantly lower among individuals who later became infected compared with those who remained uninfected. The relative abundances of bacteria belonging to the family Lachnospiraceae, and more specifically its two genera Dorea and Coprococcus, were significantly higher among those who remained uninfected. The travel-related infection did not significantly modify the faecal microbiota composition. Species composition of human gut microbiota is important for colonization resistance to Campylobacter infection. Especially individuals with a lower diversity are more susceptible to Campylobacter infection.

Impact of chicory inclusion in a cereal-based diet on digestibility, organ size and faecal microbiota in growing pigs.

A total of 30 7-week-old pigs were used to evaluate the effects of chicory inclusion on digestibility, digestive organ size and faecal microbiota. Five diets were formulated: a cereal-based control diet and four diets with inclusion of 80 and 160 g/kg chicory forage (CF80 and CF160), 80 g/kg chicory root (CR80) and a mix of 80 g/kg forage and 80 g/kg chicory root (CFR). Generally, the pigs showed a high growth rate and feed intake, and no differences between the different diets were observed. The coefficients of total tract apparent digestibility (CTTAD) of energy, organic matter and CP did not differ between the control and CF80, whereas they were impaired in diet CF160. The CTTAD of non-starch polysaccharides and especially the uronic acids were higher (P < 0.05) with chicory inclusion, with highest (P < 0.05) values for diet CF160. Coliform counts were lower and lactobacilli : coliform ratio was higher (P < 0.05) in diet CFR than in the control. Global microbial composition was investigated by terminal restriction fragment length polymorphism combined with cloning and sequencing. Analysis of gut microbiota pattern revealed two major clusters where diet CF160 differed from the control and CR80 diet. Chicory forage diets were correlated with an increased relative abundance of one species related to Prevotella and decreased abundance of two other species related to Prevotella. For diet CFR, the relative abundance of Lactobacillus johnsonii was higher than in the other diets. This study shows that both chicory forage and root can be used as fibre sources in pig nutrition and that they modulate the composition of the gut microbiota differently.

Expression of heat shock protein 27 in gut tissue of growing pigs fed diets without and with inclusion of chicory fiber.

The physiological expression of cytoprotective heat shock protein 27 (Hsp27) in the gut was investigated in eighteen 7-wk-old pigs fed one of 3 fiber-rich diets for 18 d. The diets were a cereal-based control diet and a cereal-based diet with inclusion of either 80 g/kg chicory forage (CF80) or chicory root (CR80). Immunohistochemical staining showed that Hsp27 was expressed in all the samples from ileum and colon. The expression was most intensive in the apical intestinal epitheliums in close contact with luminal contents and lighter in crypt cells. The ileal Peyer's patches showed a strong expression of Hsp27, which was highly correlated with Hsp27 expression in the ileal epithelial cells (P = 0.003). The frequency of ileal Hsp27 expression with the most intensive staining was distributed higher in pigs fed chicory forage diet (CF80, 25%) followed by chicory root diet (CR80, 16.7%) and the control (11.1%). In proximal colon, the frequency of expression showed a similar pattern for the different diets. The intestinal microbiota profile was characterized with the intention to find correlations to heat shock protein (Hsp) expression in pig gastrointestinal (GI) tract and showed that the distal ileum and proximal colon encompass its own unique microbial profile. However, no significant relationship was found between gut microbiota diversity and Hsp27 expression. These indicate that Hsp27 expression in the porcine gut could be associated with specific dietary fiber components but not the overall microbiota diversity.