Effect of oral supplementation of probiotic strains of Lactobacillus rhamnosus and Enterococcus faecium on the composition of the faecal microbiota of foals.

Effects of probiotics on the intestinal microbiota of foals are yet insufficiently studied. The aim of this study was to investigate whether supplementation of Lactobacillus rhamnosus (DSM 7133) and Enterococcus faecium (DSM 7134) influences the bacterial composition of the faecal microbiota of foals. A total of 34 newborn foals were randomly assigned to the placebo group (PG, n = 16) and the treatment group (TG, n = 18). From day 1 to day 14 of life, foals orally received 3 ml of either a probiotic preparation (1.05 × 109 CFU E. faecium and 4.50 × 108 CFU L. rhamnosus) or placebo (carrier) once a day. Faeces were collected directly from the rectum immediately after birth (meconium) and at day 14 and day 56 of life. Samples of 12 foals per group were selected for microbiological analysis. DNA was extracted and used for polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR. No DNA or amplicons were obtained from meconium. There were no differences in richness of bands and Shannon index of diversity regarding the Clostridium cluster XIVa between groups. Cluster analysis and principal coordinate analysis of DGGE data showed a clear effect of age. Band-based similarity of bacterial clusters (Dice coefficient) decreased from day 14 to day 56 of life (p < 0.001) in PG foals only resulting in lower similarity in PG versus TG foals when 2 month old (p < 0.01). Five of thirty re-amplified bands were identified on species level. Others were assigned either to family (mainly Lachnospiraceae) or genus level (Akkermansia). The bands related to Akkermansia muciniphila or Akkermansia spp. appeared almost in all DGGE profiles. Two-week supplementation of the probiotic preparation to foals had no significant impact on the composition of the faecal microbiota but it appears to have prevented the reduction of bacterial similarity between 2 and 8 weeks of age observed in not treated foals.

Effects of oral supplementation of probiotic strains of Lactobacillus rhamnosus and Enterococcus faecium on diarrhoea events of foals in their first weeks of life.

Foal first diarrhoea is one of the most prominent problems in the early life of horses. Probiotics might have the potency to prevent or at least diminish neonatal diarrhoea. We hypothesised that the treatment of foals with probiotic strains of Lactobacillus rhamnosus and Enterococcus faecium starting early after birth and then daily over 2 weeks would prevent or mitigate foal heat diarrhoea. The influence of this probiotic treatment on diarrhoea incidence and growth and health performance of young foals was investigated. Thirty-four foals were randomly allocated to two groups. From day 1 to 14 of life, the foals received either placebo (PG, n = 16) or the probiotic treatment (TG, n = 18). Clinical examination was performed, and the faeces consistency score (FCS, 1-5; with diarrhoea defined by ≤3) was recorded once per day in weeks 1 and 2 and once weekly in weeks 3-8 of life (WL). The body height was measured at birth and after two and eight WL. Diarrhoea occurred in the 1st WL in 19% and 61% of PG and TG foals respectively. In the 1st WL, diarrhoea lasted 0.3 ± 0.8 and 1.6 ± 1.4 days in PG and TG foals respectively. In the 2nd WL, diarrhoea occurred in 94% and 84% of PG and TG foals, respectively, and lasted for 3.0 ± 1.5 and 3.7 ± 1.6 days respectively. At least two periods of diarrhoea developed in 33% and 65% of PG and TG foals respectively. The TG foals grew slightly slower than the PG foals. The results indicated that the probiotic treatment of neonatal foals as performed in this study was not suitable to reduce diarrhoea within the first two WL, because contrary to the hypothesis, the TG foals suffered more frequently and for longer periods from diarrhoea than the PG foals.

New insights into the role of the porcine intestinal yeast, Kazachstania slooffiae, in intestinal environment of weaned piglets.

Kazachstania slooffiae is a porcine intestinal yeast whose role in the intestinal environment is largely unexplored. Therefore, the impact of K. slooffiae on growth performance, intestinal microbial metabolites and the microbiota of weaned piglets was investigated in this study. Forty-eight German Landrace pigs were weaned at day 27 or 28 of life and grouped into one control and three treatment groups. During the 5-week experiment, piglets had ad libitum access to feed and water. On days 5, 6 and 7 post weaning, pigs were orally supplemented with either placebo or K. slooffiae cells once a day. Faecal samples collected on days 5-8, 14, 21 and 28 post weaning were used for microbiological and chemical analyses. Between groups, there were no significant differences in the incidence of diarrhoea, pH and growth performance. Total yeasts and K. slooffiae correlated positively with total short-chain fatty acids, acetic, propionic, n-butyric, i-valeric and valeric acids, and negatively with pH. Pyrosequencing of the bacterial intestinal community revealed that K. slooffiae significantly affected the composition of the microbiota. The results of this study suggest that K. slooffiae may play an important role in the porcine digestive system, especially in the critical weaning period.

Development and Evaluation of qPCR Assay for Quantitation of Kazachstania slooffiae and Total Yeasts Occurring in the Porcine Gut.

Kazachstania slooffiae is the dominating yeast in pig's gut. No methods others than cultivation were applied for enumeration of yeasts within this ecosystem. Therefore, the aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay to quantitate total yeasts and K. slooffiae in the porcine gut. This work demonstrated that the copy numbers in gDNA can be determined by qPCR using PCR amplicons as a calibrator and one-point calibration method. The gDNA were then used as a calibrator for further analysis. The values of quantitation cycle and PCR amplification efficiency of gDNA calibrator were highly reproducible. DNA was extracted from feces and from 10 different cultured yeasts found in pigs' intestine. The qPCR results using primers NL1/LS2 encoding 26S rDNA correlated (r = 0.984, P < 0.0001) with cultivation results. From two primer sets developed, one set encoding act1 gene was suitable for quantitation of K. slooffiae. The copy numbers of K. slooffiae could be determined by 40% analyzed animals, amounting to about 70% of total yeasts. The application of this method in next studies will help to get more information about K. slooffiae and total yeasts in the gut of pigs.

Establishment of intestinal microbiota with focus on yeasts of unweaned and weaned piglets kept under different farm conditions.

This study aimed to characterize the intestinal yeasts in weaning piglets and to establish their possible relationships with main bacterial groups. German Landrace piglets were weaned (WP, n=32) at 28 days of age or kept with the dams until day 39 without creep feed (UP, n=32). The experiment was performed at an experimental and a commercial farm (CF). Faeces were collected from the piglets, sows and pen floors on days 28, 33 and 39 for isolation of DNA and cultivation for enumeration of yeasts, enterobacteria, enterococci and lactobacilli. Fragments of the D1 domain of 26S rRNA gene were amplified and separated by denaturing gradient gel electrophoresis (DGGE). No yeasts could be cultured from water and feed samples. No or only low numbers of yeasts were detected among all UP. In WP at CF, yeasts correlated with lactobacilli (r=0.456; P=0.009) and enterobacteria (r=-0.407; P=0.021). Kazachstania slooffiae dominated among the cultured yeasts. It was the only yeast species detected by PCR-DGGE. Yeasts, especially K. slooffiae, established in the porcine gastrointestinal tract after consumption of grain-based feed and may interrelate with the intestinal microbiota. The study provides data indicating importance of K. slooffiae for the development of balanced porcine gut microbiota.

Biological diversity of yeasts in the gastrointestinal tract of weaned piglets kept under different farm conditions.

The study was conducted to determine yeasts present in the gastrointestinal tract (GIT) of piglets kept under experimental farm (EF) and commercial farm (CF) conditions. Ninety five German Landrace full- and half-sibling piglets were sacrificed at 39 days of age. Sixty eight piglets were weaned at 28th day of life, when they were offered one diet ad libitum. Twenty seven piglets remained unweaned by their dams. None of the piglets received any creep feed before weaning. Digesta samples were collected from 1/3 distal small intestine (SI), caecum and proximal colon. One hundred seventy three colonies of isolated yeasts were characterized by sequence analysis of the PCR-amplified D1/D2 domain of the 26S rRNA gene with following alignment of the recovered sequences to GenBank entries. From the 17 phylotypes found, isolates most closely related to Galactomyces geotrichum, Kazachstania slooffiae and Candida catenulata dominated in the GIT of CF piglets. Kazachstania slooffiae and Candida glabrata dominated in GIT of EF piglets. Sørenson and Morisita-Horn similarity indices between farms were low (0.44 and 0.54 respectively) and the Simpson diversity index was higher for EF (7.58) than for CF (4.34). The study brings new data on yeasts composition in the pig GIT and shows differences in yeasts biodiversity between farms operated at different hygiene conditions.