Effects of probiotics Pediococcus acidilactici strain MA18/5M and Saccharomyces cerevisiae subsp. boulardii strain SB-CNCM I-1079 on fecal and intestinal microbiota of nursing and weanling piglets.

In this study, the influence of (PA) and subsp. (SCB) on fecal and intestinal microbiota of piglets during lactation and after weaning was monitored. Forty sows and their litters were used and allocated to the following dietary treatments: 1) PA, 2) SCB, 3) a mixture of the 2 probiotics (PA+SCB), 4) antibiotics (ATB), and 5) control (CTRL). Four weeks before parturition, probiotic-treated sows started receiving a daily probiotic dose of at least 2.5 × 10 cfu mixed in 500 g of feed until the end of lactation. The other groups were fed a diet without probiotics and ATB. Two days after birth, piglets received, daily, 1 × 10 cfu of the same probiotics as their mother. At weaning (d 21), these piglets were fed a basal diet enriched with the same probiotics whereas piglets from untreated litters were fed the basal diet with or without ATB. Two piglets per litter were randomly chosen to evaluate the influence of treatments on fecal microbial composition (d 10 and 28) and on ileum and colon microbiota at d 37. The microbiota was characterized by culture on selective media and by 16S rRNA gene diversity assessment using the terminal RFLP technique and clone library analysis to evaluate diversity index and phylum affiliation. Terminal RFLP profiles were also analyzed to determine differences in microbial composition between animals receiving different treatments and to identify diet-specific terminal restriction fragments (TRF) using pairwise multiresponse permutation procedures (MRPP) and indicator species analysis. Before weaning, administration of probiotics to sows and piglets had minor effect on fecal microbiota of piglets. Most modulatory effects of probiotics on ileum and colon microbiota were observed on d 37. Results revealed that PA or ATB treatments reduced ileal microbiota diversity compared with the CTRL ( < 0.05) and promoted the establishment of Firmicutes whereas SCB consumption positively influenced the establishment of the Porphyromonadaceae and Ruminococcaceae bacterial families in the colon. Moreover, pairwise MRPP analysis indicated that ileum bacterial communities of pigs treated with PA or ATB differed from those of CTRL pigs ( < 0.05). In conclusion, PA and SCB supplements, respectively, influenced, in a strain-dependent manner, the ileum and colon microbiota of weaned piglets. Results also suggest that PA and SCB have the potential as feed additives to modulate bacterial populations associated with gut health.

Utilization of lactoferrin to fight antibiotic-resistant mammary gland pathogens.

The widespread use of antibiotics has lead to the increased presence of pathogens that are less susceptible to their antibacterial effect. Lactoferrin (Lf) is naturally produced by the mammary gland. Lactoferrin is the main whey protein in human milk and is also present in cow's milk but at a much lower concentration than in human milk. This protein appears to have many biological functions, including antibacterial and antiinflammatory activities. The best-known effect of Lf is to bind iron that is essential for bacterial growth. However, the cationic nature of this protein also appears to be important for the antimicrobial activity of this protein. Lactoferrin has a weak antibacterial effect when used alone, but interestingly, Lf appears much more effective when used at low concentration in combination with several antibiotics. The most striking observation is that Lf increases the inhibitory activity of penicillin up to 4-fold in most penicillin-susceptible Staphylococcus aureus strains, whereas this increase was 4- to 16-fold in penicillin-resistant strains. Indeed, Lf reduces beta-lactamase activity in S. aureus strains producing this enzyme. Transcription of beta-lactamase gene is dramatically repressed in the presence of Lf. We evaluated the efficacy of intramammary treatments containing penicillin G or bovine Lf (bLf), or both, to cure chronic mastitis caused by a clinical isolate of S. aureus highly resistant to beta-lactam antibiotics. In a first trial, mastitis was induced in lactating cows by injecting a low dose of S. aureus through the teat canal of all quarters. Bacterial cure rate was null for control quarters, 11.1% for bLf, 9.1% for penicillin, and 45.5% for the combination of bLf and penicillin. A second trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. Quarters were treated with 100,000 IU of penicillin G with or without 250 mg of bLf for 7 d. Bacterial cure rate was greater for the bLf + penicillin combination (33.3%) compared with penicillin alone (12.5%). In conclusion, bLf added to penicillin is an effective combination for the treatment of stable S. aureus infections resistant to beta-lactam antibiotics.

Efficacy of a lactoferrin-penicillin combination to treat {beta}-lactam-resistant Staphylococcus aureus mastitis.

The efficacy of intramammary (IM) treatments containing penicillin G (PG) alone or a combination of PG and bovine lactoferrin (bLF) was evaluated using a model of experimentally induced chronic bovine mastitis caused by a clinical isolate of Staphylococcus aureus highly resistant to beta-lactam antibiotics. First, we confirmed that this strain could cause mastitis and infection could not be cured with PG alone. In a second trial, chronic mastitis was induced in 19 late-lactating cows by injecting a low dose of Staph. aureus through the teat canal of all quarters. After 15 d, cows with stable infections in their 4 quarters had their mammary quarters randomly assigned, within cow, to 1 of 4 IM treatments as follows: 1) citrate buffer, 2) 100,000 IU of PG, (3) 1 g of bLF, or 4) 1 g of bLF + 100,000 IU of PG. Treatments were repeated twice a day for 5 d. A third trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. One month before dry-off, 20 gravid cows regrouped by dates of calving were infected in their 4 quarters. Once infections were established, cows were dried off abruptly. After calving, aseptic milk samples were collected separately from all quarters for 4 wk to monitor infection. Mammary quarters from enrolled cows were then randomly assigned, within cow, to 1 of 2 treatments as follows: 1) 100,000 IU of PG or 2) 250 mg of bLF + 100,000 IU of PG. Treatments were administered IM twice a day for 7 d. For all trials, milk samples were taken to monitor bacterial concentration and somatic cell count. Bacteriological cure rate was determined using milk samples taken 3 and 4 wk after initiation of treatments. For the second trial, cure rate was null for control quarters, 11.1% for bLF, 9.1% for PG, and 45.5% for the bLF + PG combination. For cows infected in their previous lactation, cure rate was higher for the bLF + PG combination (33.3%) compared with PG alone (12.5%). In conclusion, bLF added to PG is an effective combination (i.e., 3- to 5-times higher cure rate) for the treatment of stable Staph. aureu infections highly resistant to beta-lactam antibiotics.

Deferoxamine reduces tissue damage during endotoxin-induced mastitis in dairy cows.

The protective effects of 3 antioxidants on polymorphonuclear neutrophil-induced damage to mammary cells were evaluated in vivo using an endotoxin-induced mastitis model. Fifteen healthy, midlactation cows with no history of clinical Escherichia coli mastitis were randomly assigned to 1 of the 3 treatment groups corresponding to each modulator to be evaluated, that is, deferoxamine, catechin, and glutathione ethyl ester. Each cow had 1 quarter infused with saline and 1 quarter infused with the selected modulator; a third quarter was infused with lipopolysaccharides (LPS), whereas the fourth quarter received a combination of LPS and the modulator. Infusion of LPS caused acute mastitis as determined by visual observations and by large increases in milk somatic cell count, BSA, and proteolytic activity. These parameters were not affected by antioxidant administration. The extent of cell damage was evaluated by measuring milk levels of lactate dehydrogenase and N-acetyl-beta-D-glucosaminidase activity. Levels of these parameters were several times higher after LPS administration. Intramammary infusions of catechin or glutathione ethyl ester did not exert any protective effect, whereas infusion of deferoxamine, a chelator of iron, decreased milk lactate dehydrogenase and NA-Gase activity, suggesting a protective effect against neutrophil-induced damage. The protective effect of deferoxamine was also evidenced by a lower milk level of haptoglobin. The proteolytic activity of mastitic milk was not influenced by the presence of deferoxamine. Overall, our results suggest that local infusion of deferoxamine may be an effective tool to protect mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.

Antioxidants to prevent bovine neutrophil-induced mammary epithelial cell damage.

Activated neutrophils are able to produce a large quantity of bactericidal molecules such as reactive oxygen species that have been associated with tissue damage in several inflammation models. The protective effects of antioxidants in a context of neutrophil-induced damage to mammary epithelial cells were first evaluated in vitro using a coculture model of activated bovine neutrophils and a bovine mammary epithelial cell line (MAC-T cells). Cell damage was determined by quantifying the release of lactate dehydrogenase by MAC-T cells in culture medium. Morphological observation of cells stained with acridine orange was used to visualize the extent of cell damage. When incubated with neutrophils activated by lipopolysaccharides and phorbol 12-myristate 13-acetate, MAC-T cells released large amounts of lactate dehydrogenase indicating significant cell damage. The addition of dimethylthiourea or bathocuproine disulfonic acid did not reduce the damage whereas catechin, deferoxamine or glutathione ethyl ester significantly reduced neutrophil-induced cytotoxicity in a dose-dependent manner. The effect of deferoxamine, an iron chelator, on the growth of Escherichia coli and the ability of bovine neutrophils to phagocytose these bacteria were then assessed in vitro. Our data showed that deferoxamine did not interfere with the phagocytic activity of neutrophils but inhibited growth of the bacteria. Overall, our results suggest that antioxidants may be effective tools for protecting mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.

Proteases involved in mammary tissue damage during endotoxin-induced mastitis in dairy cows.

During and after diapedesis, milk polymorphonu-clear neutrophils (PMN) release many proteases that have the potential of degrading extracellular matrix proteins and milk proteins. However, the kinetics of milk proteolysis during inflammation and the underlying mechanisms are poorly defined. The enzymes involved in bovine mammary tissue destruction were investigated in this study using an endotoxin-induced mastitis model. Using zymography techniques, the proteolytic activity of milk and mammary tissue during mastitis was examined. Mastitic milk produced 6 caseolysis bands, 4 of which differed from the ones produced by plasmin. Peak proteolytic activity, bovine serum albumin contents, and mammary tissue damage occurred between 6 and 12 h postchallenge. Mastitic milk proteases hydrolyzed casein, gelatin, collagen, hemoglobin, mammary gland membrane proteins, and lactoferrin. These results confirm that mastitic milk proteases have a broad spectrum of activity. The hydrolytic activity of mastitic milk was partially inhibited by aprotinin, EDTA, 1,10-phenanthroline, leupeptin, and pefabloc. When cocultured with normal mammary tissue, mastitic milk, but not normal milk, caused mammary tissue degradation. In situ zymography of mammary gland showed increased proteolytic activity in mastitic tissue compared with normal tissue. The similarity of zymograms of mastitic milk, blood PMN, milk somatic cells, and PMN strongly suggests that proteases in mastitic milk mainly originate from milk PMN. These results suggest that proteases released by PMN are actively involved in udder tissue damage during mastitis.

Pancreatic inflammation, apoptosis, and growth: sequential events after partial pancreatectomy in pigs.

Positive signs of pancreas regeneration were observed in rats after induced pancreatitis and partial pancreatectomy (1,2). Although the human pancreas did not regenerate after partial anatomic resection (3), the pig pancreas exhibited growth responses to bombesin after partial pancreatectomy (4). This study was undertaken to establish the time course of pancreatic inflammation, apoptosis, and hypertrophy and/or hyperplasia after partial pancreatectomy in pigs.