A reliable method for quantification of non-viable microbe-based nutritional and zootechnical additives introduced into feed is essential in order to ensure regulatory compliance, feed safety and product authenticity in industrial applications. In the present work, we developed a novel real-time quantitative polymerase chain reaction (qPCR) -based analysis protocol for monitoring two microbial additives in feed matrices. To evaluate the applicability of the method, pelleted wheat- and maize-based broiler chicken diets containing a non-viable phytase-producing strain of Aspergillus niger produced in solid state fermentation (150 or 300 g/t) and a non-viable selenium-enriched Saccharomyces cerevisiae (100 or 200 g/t) as model feed ingredients, were manufactured and subjected to analysis. Power analysis of the qPCR results indicated that 2 to 6 replicate feed samples were required to distinguish the product doses applied, which confirms that the microbial DNA was efficiently recovered and that potential PCR inhibitors present in the feed material were successfully removed in DNA extraction. The analysis concept described here was shown to be an accurate and sensitive tool for monitoring the inclusion levels of non-viable, unculturable microbial supplements in animal diets.
ABSTRACT In the 1990s, Salmonella enterica subsp. enterica serovar Enteritidis has caused 15 outbreaks in Finland; 12 of them were caused by phage type 1 (PT1) and PT4. Thus far, there has been no clear evidence as to the source of these Salmonella Enteritidis PT1 and PT4 strains, so it was necessary to try to characterize them further. Salmonella Enteritidis PT1 ( n = 57) and PT4 ( n = 43) isolates from different sources were analyzed by genomic pulsed-field gel electrophoresis (PFGE), plasmid profiling, and antimicrobial resistance testing to investigate the distribution of their subtypes in Finland. It was also hoped that this investigation would help in identifying the sources of the infections, especially the sources of the outbreaks caused by PT1 and PT4 in the 1990s. The results showed that both PFGE and plasmid profiling, but not antimicrobial susceptibility testing, were capable of differentiating isolates of Salmonella Enteritidis PT1 and PT4. By genotypic methods, it was possible to divide both PT1 and PT4 isolates into 12 subtypes. It could also be shown that all PT1 outbreak isolates were identical and, at least with this collection of isolates, that the outbreaks did not originate from the Baltic countries or from Russia, where this phage type predominates. It was also established that the outbreaks caused by PT4 all had different origins. Valuable information for future investigations was gained on the distribution of molecular subtypes of strains that originated from the tourist resorts that are popular among Finns and of strains that were isolated from livestock.
To investigate the correlations between self-reported symptoms of irritable bowel syndrome (IBS) and the gastrointestinal (GI) microbiota composition.Fecal samples were collected from a total of 44 subjects diagnosed with IBS. Their symptoms were monitored with a validated inflammatory bowel disease questionnaire adjusted for IBS patients. Thirteen quantitative real-time polymerase chain reaction assays were applied to evaluate the GI microbiota composition. Eubacteria and GI bacterial genera (Bifidobacterium, Lactobacillus and Veillonella), groups (Clostridium coccoides/Eubacterium rectale, Desulfovibrio desulfuricans) and distinct bacterial phylotypes [closest 16S rDNA sequence resemblance to species Bifidobacterium catenulatum, Clostridium cocleatum, Collinsella aerofaciens (C. aerofaciens), Coprococcus eutactus (C. eutactus), Ruminococcus torques and Streptococcus bovis] with a suspected association with IBS were quantified. Correlations between quantities or presence/absence data of selected bacterial groups or phylotypes and various IBS-related symptoms were investigated.Associations were observed between subjects' self-reported symptoms and the presence or quantities of certain GI bacteria. A Ruminococcus torques (R. torques)-like (94% similarity in 16S rRNA gene sequence) phylotype was associated with severity of bowel symptoms. Furthermore, among IBS subjects with R. torques 94% detected, the amounts of C. cocleatum 88%, C. aerofaciens-like and C. eutactus 97% phylotypes were significantly reduced. Interesting observations were also made concerning the effect of a subject's weight on GI microbiota with regard to C. aerofaciens-like phylotype, Bifidobacterium spp. and Lactobacillus spp.Bacteria seemingly affecting the symptom scores are unlikely to be the underlying cause or cure of IBS, but they may serve as biomarkers of the condition.
Stimbiotics are a new category of feed additives that can increase fibre fermentability by stimulating fibre-degrading microbiota in the gut. The aim of this study was to test,
The objective of this study was to determine the response of broilers to the combination of multi-enzymes and direct-fed microbial (DFM) under commercial production settings. A total of 7,000 1-day-old male broilers (Ross 308) were distributed over 10 pens (700 broilers/pen). Two dietary treatments were tested using complete randomized design, including a control diet and a test diet with addition of multi-enzymes (xylanase, amylase, and protease (XAP)] and DFM (a combination of spores from 3 strains ofBacillus amyloliquefaciens). Pelleted diets were offered ad libitum in 3 phases and water was freely available. During starter and grower phases (0 to 21 d), the enzyme and DFM combination resulted in improved FE (P < 0.05). During the finisher phase, higher feed intake and BW gain (P < 0.05) were observed for the test group. Overall, there were significantly higher feed intake, BW gain, and lower water-to-feed ratio in test group compared to the control group. This was related to improved (P < 0.05) modified production efficiency factor which was calculated based on final BW, survival rate, feeding period, and mortality-weight-corrected FCR. The test group had improved litter quality and a reduced foot-pad lesion score compared to the control. In addition, there was a tendency (P < 0.1) of reducingClostridium perfringens population in cecal digesta and higher lactic acid content in the ileal digesta, when expressed on an as-is basis, in the test group. In this study, we demonstrated that using a multi-enzymes and DFM combination in the diet for broilers can result in improved FE in starter/grower phases and animal welfare parameters, and lead to improved production efficiency under commercial settings.
Aquamin is a calcium-rich multi-mineral supplement derived from the red marine algae, Lithothamnion species. Calcium supplementation has been shown to exert a prebiotic-like effect on the gut microbiota and has been associated with distinct changes in lactate and short chain fatty acid (SCFA) production. Irritable bowel syndrome (IBS) subtype is associated with changes in SCFA levels compared with healthy controls. Using an ex vivo simulation model, and a fecal inoculum from a patient diagnosed with IBS, we evaluated the effects of Aquamin (at 6 and 30 mg/mL) on SCFAs and lactate production, pH and gas production, and human microbiota composition. Our results demonstrate that Aquamin increased SCFA production (acetate and propionate by 8% and 24%, respectively, at 30 mg/mL dose), significantly decreased lactate production (30 mg/mL), and increased colonic fluid pH without inducing changes in colonic gas production or gastrointestinal (GI) microbiota composition. These results indicate that Aquamin may play a role in optimizing GI microbial function in an ex vivo setting.
