Wilfried Vahjen

INDIGENOUS BACTERIA AND BACTERIAL METABOLIC PRODUCTS IN THE GASTROINTESTINAL TRACT OF BROILER CHICKENS

Abstract The gastrointestinal tract is a dynamic ecosystem containing a complex microbial community. In this paper, the indigenous intestinal bacteria and the microbial fermentation profile particularly short chain fatty acids (SCFA), lactate, and ammonia concentrations are reviewed. The intestinal bacterial composition changes with age. The bacterial density of the small intestine increases with age and comprises of lactobacilli, streptococci, enterobacteria, fusobacteria and eubacteria. Strict anaerobes (anaerobic gram-positive cocci, Eubacterium spp., Clostridium spp., Lactobacillus spp., Fusobacterium spp. and Bacteroides) are predominating caecal bacteria in young broilers. Data from culture-based studies showed that bifidobacteria could not be isolated from young birds, but were recovered from four-week-old broilers. Caecal lactobacilli accounted for 1.5 – 24% of the caecal bacteria. Gene sequencing of caecal DNA extracts showed that the majority of bacteria belonged to Clostridiaceae. Intestinal bacterial community is influenced by the dietary ingredients, nutrient levels and physical structure of feed. SCFA and other metabolic products are affected by diet formulation and age. Additional studies are required to know the bacterial metabolic activities together with the community analysis of the intestinal bacteria. Feed composition and processing have great potential to influence the activities of intestinal bacteria towards a desired direction in order to support animal health, well-being and microbial safety of broiler meat.

Influence of xylanase-supplemented feed on the development of selected bacterial groups in the intestinal tract of broiler chicks

The colonization of Lactobacillus spp., enterobacterian and facultatively anaerobic gram-positive cocci was monitored in intestinal samples of growing broiler chicks from 24 h to 28 days of age. Rapid bacterial growth occurred within the first week, followed by stabilization and decline of colony forming units (CFU). Xylanase supplementation led to significantly lowern CFU per gram of wet weight for total presumptive enterobacteria and total gram-positive coccin in luminal and tissue samples in the first 3 weeks. Lactobacillus spp. colony counts from tissue samples were higher for animals with the xylanase-supplemented diet, but luminal CFU were not.n The composition of dominant Lactobacillus spp. strains was different in duodenaln and jejunal tissues, but distribution of Lactobacillus spp. colony forms was unaffected by xylanase treatment. Mucosa-associated Enterococcus spp. displaced the dominant gram-positive cocci inn the jejunal samples. d - and l -lactic acid and acetic acid concentrations were significantly higher in ileal samples from the control group on days 7 and 14, while butyric acid concentrations were higher in the xylanase-treated group. It is concluded that the less viscous intestinal environment caused by the xylanase slowed proliferation of gram-positive cocci and presumptive enterobacteria in enzyme-supplemented animals in the first 3 weeks of life.

Bacterial Responses to Different Dietary Cereal Types and Xylanase Supplementation in the Intestine of Broiler Chicken

Several studies were carried out to investigate the influence of dietary cereals differing in soluble non starch polysaccharides (NSP) content and a xylanase preparation on selected bacterial parameters in the small intestine of broiler chicken. Compared to a maize diet colony forming units (CFU) of mucosa associated bacteria were higher in a wheat/rye diet, most notably for enterobacteria and enterococci. Xylanase supplementation to the wheat/rye diet generally led to lower CFU, especially in the first week of life. However, xylanase supplementation also displayed higher in vitro growth potentials for enterobacteria and enterococci. Bacterial growth of luminal samples in minimal media supplemented with selected NSP showed that the wheat/rye diet enhanced bacterial capacities to utilize NSP only in ileal samples. The xylanase application generally shifted respective maximum growth to the proximal part of the small intestine. The presence of soluble NSP from wheat or rye in the diet per se did not enhance bacterial NSP hydrolyzing enzyme activities in the small intestine, but xylanase supplementation resulted in higher 1,3-1,4- g - glucanase activity. Compared to a maize diet the activity of bacterial bile salt hydrolases in samples of the small intestine was not increased due to inclusion of wheat/rye or triticale to the diet. However, xylanase supplementation led to a reduction with a corresponding increase of lipase activity. It was concluded that dietary cereals producing high intestinal viscosities lead to increased overall bacterial activity in the small intestine. The supplementation of a xylanase to cereal based diets producing high intestinal viscosity, changes composition and metabolic potential of bacterial populations and may specifically influence fat absorption in young animals.

Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum.

The objective of this study was to follow the mycotoxin formation and changes in nutrient composition of wheat (cv. Ritmo) artificially inoculated with Fusarium culmorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on monitoring the progression of the contamination of the wheat kernels with deoxynivalenol (DON) and zearalenone (ZON). Both the uncontaminated control kernels and the contaminated kernels were examined also for the presence of zearalenone-4-beta-D-glucopyranoside and several trichothecenes at harvest. Furthermore, the impact of the Fusarium inoculation on some nutrients as starch, crude protein, amino acid composition, crude ash, non starch polysaccharides (NSP) as well as viscosity and thousand seed weight (TSW) was examined. Also proteolytic and amylolytic activity as well as the NSP-degrading enzyme activities of inoculated and control samples were analysed at the time of harvest. DON was detected in higher concentrations and in earlier stages, while ZON was found later and in smaller amounts. On average 7.79u2009mg/kg DM of DON and 100u2009μg/kg DM of ZON were found in the inoculated kernels at the time of harvest. Neither in the contaminated nor in the control samples glucose conjugates of ZON (Zearalenone-4-beta-D-glucopyranoside) were detected. Moreover, the infection with Fusarium culmorum had pronounced effects on some quality parameters. The crude protein content of the inoculated kernels showed significantly higher values over the whole period compared to the control kernels. The protein content of the inoculated kernels amounted 13.9% DM at harvest, while only a concentration of 12.5% DM was detected in the control samples. Similarly, in almost all stages of development the crude ash content of inoculated samples was higher than in control samples. These distinct differences in kernel composition resulted possibly from the changes of the thousand seed weight. In the present work the grain harvested from the control plots showed a significantly higher TSW (24.2u2009g) as compared to their inoculated counterparts (15.5u2009g). Despite lower extract viscosity of inoculated samples at time of harvest, the content of soluble NSP of inoculated plots was higher than in control samples at the same time. Moreover, inoculation resulted in markedly increased activities of protease, amylase and several NSP-degrading enzyme activities. This would suggest that the cell wall penetrating properties of the fungus itself and/or that the fungus induced alterations of the metabolic activity of the embryo or other constituents of the wheat kernel could be responsible.

Fermentable Fiber Ameliorates Fermentable Protein-Induced Changes in Microbial Ecology, but Not the Mucosal Response, in the Colon of Piglets

Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21-23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1β, IL10, TGFβ, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.

Response of performance characteristics and fecal consistency to long-lasting dietary supplementation with the probiotic strain Bacillus cereus var. toyoi to sows and piglets

Abstract As part of an interdisciplinary research project, we studied the performance response of sows and their litters to the probiotic strain Bacillus cereus var. toyoi as well as feces consistency of piglets. Gestating sows (n = 26) were randomly allotted into two groups. The probiotic B. cereus var. toyoi was administered by dietary supplementation to one group of sows and their respective litters (probiotic group) whereas the second group (control group) received no probiotic supplementation. The duration of the application was nearly 17 weeks for sows (day 90 ante partum until day 28 post partum) and six weeks for piglets (day 15–56). Piglets were weaned after 28 days. Body weight and feed consumption were recorded weekly and fecal consistency of weaned piglets was studied daily. B. cereus var. toyoi was recovered from feces of sows and piglets as well as from digesta of piglets in the probiotic group, while being absent from all samples of control animals. In addition, the probiotic was detected in piglet feces and digesta before pre-starter feed was offered, indicating a second route of uptake besides diet. Sows of the probiotic group nursed numerically more piglets and supported a higher sum of total nursing days of all piglets within each litter than control sows (p = 0.04). In turn, body weight (BW) up to day 35 was greater for control piglets (p < 0.01), while average daily gain and gain to feed ratio (G:F) in weeks six and eight postweaning was higher in the probiotic group (p < 0.05). The overall G:F of the total postweaning period was 680 g/kg and 628 g/kg in the probiotic group and control group, respectively (p = 0.009). During the trial a high prevalence of liquid feces with its maximum in the second week after weaning was observed. Probiotic supplementation led to a reduction in the incidence of liquid feces and postweaning diarrhea by 38% and 59%, respectively (p < 0.001).

Virulence factor gene profiles of Escherichia coli isolates from clinically healthy pigs.

ABSTRACT Nonpathogenic, intestinal Escherichia coli (commensal E. coli) supports the physiological intestinal balance of the host, whereas pathogenic E. coli with typical virulence factor gene profiles can cause severe outbreaks of diarrhea. In many reports, E. coli isolates from diarrheic animals were classified as putative pathogens. Here we describe a broad variety of virulence gene-positive E. coli isolates from swine with no clinical signs of intestinal disease. The isolation of E. coli from 34 pigs from the same population and the testing of 331 isolates for genes encoding heat-stable enterotoxins I and II, heat-labile enterotoxin I, Shiga toxin 2e, and F4, F5, F6, F18, and F41 fimbriae revealed that 68.6% of the isolates were positive for at least one virulence gene, with a total of 24 different virulence factor gene profiles, implying high rates of horizontal gene transfer in this E. coli population. Additionally, we traced the occurrence of hemolytic E. coli over a period of 1 year in this same pig population. Hemolytic isolates were differentiated into seven clones; only three were found to harbor virulence genes. Hemolytic E. coli isolates without virulence genes or with only the fedA gene were found to be nontypeable by slide agglutination tests with OK antisera intended for screening live cultures against common pathogenic E. coli serogroups. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations and that high numbers of E. coli cells harboring virulence genes and/or with hemolytic activity do not necessarily correlate with disease.

Increased dietary zinc oxide changes the bacterial core and enterobacterial composition in the ileum of piglets

This study was conducted to investigate the effects of increased dietary ZnO on the bacterial core and enterobacterial composition in the small intestine of piglets that were fed diets containing a total of 124 or 3,042 mg of Zn per kilogram of diet, respectively. Zinc was supplemented to the basal diet as ZnO. Bacterial 16S rRNA genes of ileal DNA extracts were PCR-amplified with 2 bar-coded primer sets and sequenced by 454 pyrosequencing. The bacterial core species were calculated from the relative abundances of reads present in 5 of 6 samples per group and at a minimum of 5 sequences per sample. The reference database SILVA was used to assign sequence reads at an alignment minimum of 200 bases and 100% identity. Lactic acid bacteria dominated the bacterial core, but showed diverse responses to dietary ZnO. Of the dominant Lactobacillus spp., Lactobacillus reuteri was reduced due to increased dietary ZnO (44.7 vs. 17.9%; P=0.042), but L. amylovorus was not influenced. However, the changes of relative abundances of other lactic acid bacteria were more noteworthy; Weissella cibaria (10.7 vs. 23.0%; P=0.006), W. confusa (10.0 vs. 22.4%; P=0.037), Leuconostoc citreum (6.5 vs. 14.8%; P=0.009), Streptococcus equinus (0.14 vs. 1.0%; P=0.044), and S. lutetiensis (0.01 vs. 0.11%; P=0.016) increased in relative abundance. Nonlactic acid bacteria that were influenced by increased dietary ZnO included the strict anaerobic species, Sarcina ventriculi, which showed a strong numerical decrease in relative abundance (14.6 vs. 5.1%). Species of the Enterobacteriaceae increased their relative abundance, as well as species diversity, in the high dietary ZnO experimental group. Bacterial diversity indices were increased due to increased dietary ZnO (P < 0.05), which was traced back to the increase of sequences from subdominant species. Increased dietary ZnO led to an increase of less prominent species and, thus, had a major impact on the bacterial composition and diversity in piglets. This effect may help to stabilize the intestinal microbiota in the sensitive postweaning period.

Bar-Coded Pyrosequencing of 16S rRNA Gene Amplicons Reveals Changes in Ileal Porcine Bacterial Communities Due to High Dietary Zinc Intake

ABSTRACT Feeding high levels of zinc oxide to piglets significantly increased the relative abundance of ileal Weissella spp., Leuconostoc spp., and Streptococcus spp., reduced the occurrence of Sarcina spp. and Neisseria spp., and led to numerical increases of all Gram-negative facultative anaerobic genera. High dietary zinc oxide intake has a major impact on the porcine ileal bacterial composition.

Growth behaviour of a spore forming probiotic strain in the gastrointestinal tract of broiler chicken and piglets

The growth behaviour of the probiotic strain Bacillus cereus var. toyoi in the gastrointestinal tract of broiler chicken and suckling piglets was evaluated. The strain germinated rapidly in intestinal samples from both animal species. Less than 10% of spores were recovered from the chicken crop and piglet stomach, respectively. Lumen samples and mucosal tissues from the hind gut of piglets displayed increasing colonization of the probiotic strain throughout the trial period. After oral administration of vegetative cells to broiler chicken and weaned piglets, sporulation was detected in all intestinal samples. The distribution of spore CFU indicated repeated germination and sporulation during the intestinal passage in piglets. B. cereus var. toyoi was not able to colonize the intestinal tract of both animal species. However, the probiotic strain was detected in suckling piglets before uptake of B. cereus var. toyoi supplemented feed. It is concluded that B. cereus var. toyoi germinates rapidly in broiler chicken and piglets, which is a necessary prerequisite for its possible probiotic effects. Germination and in vivo sporulation of vegetative cells indicated that the probiotic strain was metabolically active in the intestine of both animal species.