Oral supplementation of a cell preparation of Enterococcus faecalis strain EC-12 stimulates superoxide dismutase production in the livers of healthy and arthritis-induced mice.

Hepatitis, a major human chronic inflammation disease, has been linked to oxidative stress, which can be initiated by radicals produced during the oxidative metabolism. Oxidative damage has been also observed in arthritis-induced mice. Here we evaluated whether supplementation of a cell preparation of Enterococcus faecalis EC-12 could induce superoxide dismutase activity and/or damage in the livers of healthy mice or mice with arthritis. In Experiment 1, both healthy and arthritis-induced mice were orally given a saline solution, or a solution with a low (0.2 mg/mouse/day) or a high (2.0 mg/mouse/day) concentration of E. faecalis EC-12 for 49 consecutive days. Manganese superoxide dismutase activity increased in E. faecalis EC-12-supplemented mice but with no arthritis. In Experiment 2, mice received orally either a saline or an E. faecalis EC-12 suspension (10 mg/kg of body weight/day) for 28 consecutive days. No changes in tissues and levels of function markers and 8-hydroxy-2'-deoxyguanosine were observed in mouse livers, inferring that E. faecalis EC-12 supplementation caused no damage. While mRNA expression of copper/zinc superoxide dismutase remained unaltered, that of manganese superoxide dismutase increased in E. faecalis EC-12 administration mice. In conclusion, at least in healthy mice, E. faecalis EC-12 supplementation stimulated manganese superoxide dismutase activity in liver tissues with no side effects.

Evaluation of post-colostrum ingestion changes in the protein composition of peripheral blood of newborn piglets: A pilot study.

Putatively, colostral proteins are partly absorbed and transferred to blood circulation in newborn piglets, which suggests that colostrum ingestion alters the protein composition of their blood. Here, we conducted a pilot study to estimate the changes in the protein composition of piglet blood. Plasma collected from piglets pre- and post-ingestion of colostrum (PreC and PostC) was analyzed by shotgun proteomics. Proteins in colostrum were also analyzed. We identified 393 and 427 proteins in PreC and PostC plasma, respectively, and 596 colostral proteins. Whereas 202 unique proteins were identified in PostC, PreC and PostC commonly shared 225 proteins. By contrast, when compared with PreC, 54 proteins in PostC had their emPAI values increased >2-fold. Notably, using plasma samples collected from a separate experiment, the concentrations of growth differentiation factor 8 and haptoglobin were higher in PostC than in PreC, which was validated by ELISA. Approximately 60% of the uniquely identified or highly concentrated proteins in PostC were also found in colostrum, which were likely, at least partly, transferred from colostrum. The present study demonstrated that the protein composition of plasma of newborn piglets drastically changed post-colostrum ingestion, partly due to transfer of colostral proteins.

Highest concentration of breast-milk-derived exosomes in colostrum.

BACKGROUND: Exosomes are nanosized extracellular vesicles, that play important roles in intercellular immune regulation. They have potential therapeutic utility for neonatal diseases including necrotizing enterocolitis. Breast-milk-derived exosomes have recently shown beneficial effects on intestinal damage in vitro and in vivo. However, the chronological change in breast-milk-derived exosome concentrations after delivery are unclear. METHODS: In this prospective study, we enrolled 17 mothers who delivered premature infants admitted to a neonatal intensive care unit in Japan. We measured the consecutive concentrations of breast-milk-derived exosomes in the mothers for 48 weeks after delivery. RESULTS: The median concentration of breast-milk-derived exosomes was 1.62 × 108 particles/ml in colostrum, showing a significant decrease after 2 weeks (P < 0.01). There was no association between the exosome concentration in colostrum and maternal perinatal factors including parity, mode of delivery, maternal age, and gestational age at delivery. CONCLUSIONS: We concluded that breast-milk-derived exosomes were the richest in colostrum. Our basic data regarding breast-milk-derived exosomes are expected to aid in the clinical application of exosomes for treating neonatal diseases.

Composition and physiological functions of the porcine colostrum.

The first secretion, 24-h post parturition of the mammary glands of sows, known as colostrum, is high in protein and low in lactose and fat. As a consequence of an insufficient ingestion of colostrum, more than 50% of piglets fail to reach weaning and die. The composition and some functions of colostrum have been previously reported. For example, colostrum carbohydrates consist of mainly lactose. Lipids in the colostrum are mostly triacylglycerols, but <1% is fatty acids, which may act as homeostasis regulators. Similarly, proteins are found mostly as casein and whey, the latter being ≥80% immunoglobulins. Colostrum-derived immunoglobulins and bioactive proteins such as azurocidin help the immune system of the piglet fend off infections. In addition, leukocytes and exosomes are other minor but nonetheless equally crucial bioactive components in the porcine colostrum. Modern pig farming has achieved increases in pig productivity and litter size, but this has been accomplished in detriment of the health and the survival rate of piglets. Therefore, porcine colostrum is now even more important in pig farming. In the present review, we discuss the current knowledge on the composition and physiological functions of the porcine colostrum and briefly propose future research directions.

Preliminary investigation of the use of dietary supplementation with probiotic Bacillus subtilis strain QST713 shows that it attenuates antimicrobial-induced dysbiosis in weaned piglets.

Growth performance of pigs has been associated with healthy gut microbiota. To improve production, pigs are usually treated with antimicrobials. Nonetheless, while antimicrobials harm the gut-indigenous microbiota, probiotic supplementation seems to help keep it healthy. Here, using antimicrobials, we artificially induced dysbiosis in pigs and evaluated a possible preventive effect of probiotic supplementation. Three 6-week-old piglets were given a basal feed, and 3 more the feed supplemented with 2.0 × 106  CFU of Bacillus subtilis QST713/g of feed. After 14 days, antimicrobial enrofloxacin (5 mg/kg B.W.) was injected intramuscularly to all pigs on days 14-16. Feces were collected on days 14, 17, 19, 21, and 23. Total bacteria count was unaffected by enrofloxacin or QST713. However, Lactobacillus spp. and, in particular, Escherichia coli were affected by enrofloxacin, the latter not being observed in the feces on days 17 and 19. Interestingly, a reciprocal increase in E. coli was observed in control pigs on days 21 and 23, although in QST713-supplemented piglets, this increase was attenuated. While the gut microbiota composition did not return to initial levels in antimicrobial-administered piglets, it did in QST713-supplemented piglets. QST713 supplementation was likely crucial to keep the microbiota of piglets healthy.

Effects of dietary intake of heat-inactivated Lactobacillus gasseri CP2305 on stress-induced behavioral and molecular changes in a subchronic and mild social defeat stress mouse model.

The intestinal ecosystem is involved in the pathogenesis of mood disorders such as depression. Intestinal microbes can affect the central nervous system through the gut-brain axis, which raises the possibility of using probiotics for preventing depression. In this study, we examined the effect of heat-inactivated Lactobacillus gasseri CP2305 (CP2305) in a subchronic and mild social defeat stress (sCSDS) mouse model. sCSDS suppressed food intake. However, dietary CP2305 intake rescued it, suggesting that CP2305 improved the decreased appetite in sCSDS mice. sCSDS did not alter the gene expression of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3 in the hippocampus. However, dietary CP2305 provided following sCSDS increased the gene expression of these neurotrophins in the hippocampus. These findings suggest that CP2305 supplementation would aid in preventing psychosocial stress-induced disorders.

Poor Bifidobacterial Colonization Is Associated with Late Provision of Colostrum and Improved with Probiotic Supplementation in Low Birth Weight Infants.

This study aimed to evaluate the association between bifidobacterial colonization in low birth weight infants and perinatal factors, including the timing of initial colostrum and the effect of probiotics on this colonization. In this non-randomized controlled trial, we enrolled 98 low-birth-weight infants from a neonatal intensive care unit (NICU) in Japan. Infants were divided into three groups: group N (no intervention), group H (received non-live bifidobacteria), and group L (received live bifidobacteria). The number of bifidobacteria in the infants' stools at 1 month of age was measured using real-time polymerase chain reaction (PCR). We divided infants into "rich bifidobacteria" (≥104.8 cells/g feces) and "poor bifidobacteria" (<104.8 cells/g feces) subgroups. The ratio of "rich bifidobacteria" infants was 20/31, 34/36, and 30/30 in groups N, H, and L, respectively. In group N, the "rich bifidobacteria" group received first colostrum significantly earlier than the "poor bifidobacteria" group (1 day vs. 4 days, P < 0.05). Compared with the N group, both groups H and L had a significantly high proportion of "rich bifidobacteria" infants (P < 0.05). Bifidobacterial colonization was poor in premature infants at 1 month compared with term infants, and the level of colonization was associated with the timing of initial provision of colostrum. Providing probiotics to premature infants can improve bifidobacterial colonization.

Probiotic supplementation improves reproductive performance of unvaccinated farmed sows infected with porcine epidemic diarrhea virus.

We investigated if probiotic supplementation could improve the health and reproductive performance of unvaccinated lactating sows infected with porcine epidemic diarrhea (PED) virus. Twenty unvaccinated pregnant sows were equally allocated to probiotic-supplemented (P) and control (C) groups. For the experiment, 15 g/day of probiotic compound BIO-THREE PZ was given to P sows. Reproductive performance was checked daily. The number of neonates fostered by each sow was maintained at eight throughout the experiment. Individual milk production post-parturition was measured twice. Milk protein and fat ratios were determined by a milk analyzer. Total immunoglobulin (Ig) A and G concentrations were measured by ELISA. At day 7 post-parturition, the body weight of P sows was 10 kg higher than that of C sows, and at day 3 post-parturition, P sows produced more milk (+2 kg) and had a higher IgA concentration in whey than did C sows (p < .05). Finally, unlike C sows, P sows tended to return to estrus faster, and had larger piglets at birth with a lower mortality percentage during early days of suckling. In conclusion, probiotic compound BIO-THREE PZ helped strengthen the immune system of unvaccinated, PED-infected sows and improved their reproductive performance.

Fructo-oligosaccharide-Induced Transient Increases in Cecal Immunoglobulin A Concentrations in Rats Are Associated with Mucosal Inflammation in Response to Increased Gut Permeability.

Background: The mechanism underlying transient increases in immunoglobulin (Ig) A concentrations in the cecal contents of rats fed fructo-oligosaccharide (FOS) is unclear.Objective: This study was designed to test whether increased IgA concentrations represent one aspect of the inflammatory response to increased permeability induced by FOS in the cecum.Methods: Seven-week-old male Wistar rats were fed a fiber-free semipurified diet (FFP) with or without supplemental FOS (60 g/kg diet) for 9 or 58 d [experiment (expt.) 1], 7 d (expt. 2), or 7 or 56 d (expt. 3). In addition to measuring IgA concentrations in cecal content, we assessed gut permeability, inflammatory responses (expt. 1), the number of IgA plasma cells in the cecal lamina propria, polymeric Ig receptor (pIgR) expression in the cecal mucosa (expt. 2), and the condition of the cecal mucus layer (expt. 3).Results: The cecal IgA concentration in the FOS-fed rats was 15-fold higher than that of the rats fed FFP for 9 d (P < 0.05). Gut permeability estimated by urinary chromium-EDTA excretion, bacterial translocation to mesenteric lymph nodes, myeloperoxidase activity, and expression of inflammatory cytokine genes in the cecal mucosa was greater in the FOS-fed rats than in the rats fed FFP for 9 d. These effects were not observed in the rats fed FOS for 58 d (expt. 1). Accompanying the higher cecal IgA concentration, pIgR protein and the number of IgA plasma cells in the cecal mucosa were higher in the FOS-fed rats than in the rats fed FFP for 7 d (expt. 2). Destruction of the mucus layer on the epithelial surface, as evidenced by Alcian blue staining in the cecal sections, was evident in the rats fed FOS for 7 d, but the mucus layer appeared normal in the rats fed FOS for 56 d (expt. 3).Conclusions: These findings suggest that transient increases in cecal IgA concentrations induced by FOS in rats are associated with mucosal inflammation in response to increased gut permeability; these are presumably evoked by disruption of the cecal mucus barrier. The observed responses could contribute to the maturation of the gut immune system.

Improved porcine model for Shiga toxin-producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets.

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 1011  colony-forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum-deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.

Comparison of gene expression profiles of T cells in porcine colostrum and peripheral blood.

OBJECTIVE To compare gene expression patterns of T cells in porcine colostrum and peripheral blood. ANIMALS 10 multiparous sows. PROCEDURES Cytotoxic and CD4-CD8 double-positive T cells were separated from porcine colostrum and peripheral blood. Total RNA was extracted. The cDNA prepared from RNA was amplified, labeled, fragmented, and competitively hybridized to DNA microarray slides. The DNA microarray data were validated by use of a real-time reverse-transcription PCR assay, and expression of the genes FOS, NFKBI, IFNG, CXCR6, CCR5, ITGB2, CCR7, and SELL was assessed. Finally, DNA microarray data were validated at the protein level by use of flow cytometry via expression of c-Fos and integrin ß-2. RESULTS Evaluation of gene expression profiles indicated that in contrast to results for peripheral blood, numerous cell-signaling pathways might be activated in colostrum. Profile analysis also revealed that FOS and NFKBI (genes of transcription factors) were involved in most cell-signaling pathways and that expression of these genes was significantly higher in colostral T cells than in peripheral blood T cells. Furthermore, CCR7 and SELL (genes of T-cell differentiation markers) in colostral T cells had expression patterns extremely similar to those found in effector or effector memory T cells. CONCLUSIONS AND CLINICAL RELEVANCE All or most of the T cells in colostrum had an effector-like phenotype and thus were more activated than those in peripheral blood. This gene expression profile would enable T cells to migrate to mammary glands, be secreted in colostrum, and likely contribute to passive immunity provided by sows to newborn pigs.

Dietary administration of probiotics to sows and/or their neonates improves the reproductive performance, incidence of post-weaning diarrhea and histopathological parameters in the intestine of weaned piglets.

Probiotics have gained considerable attention with respect to their beneficial effects on livestock performance and health. The most significant effects of probiotics on the gut microbiota and the host animals take place when they are included in diets during particularly stressful periods such as weaning and/or at the beginning of the lactation period. The probiotics Bacillus mesentericus strain TO-A at 1 × 108 colony forming units (CFU)/g, Clostridium butyricum strain TO-A at 1 × 108 CFU/g and Enterococcus faecalis strain T-110 at 1 × 109 CFU/g were used. Litter weight at delivery and ratio of return to estrous improved significantly (17% and 24% improvement, respectively) by probiotic administration to sows (0.2% (w/w)). Furthermore, the feed intake of the probiotics-administered sows was greater than that of the control sows during the late lactation period. Post-weaning diarrheal incidence and growth performance was improved by probiotics administration to neonates (0.02% (w/w)), while the combined use of probiotics in sows and their neonates induced the enlargement of villous height and prevented muscle layer thinning in the small intestine of weaning piglets. The administration of probiotics of three species of live bacteria improved the porcine reproductive performance around stressful periods of sows (farrowing) and piglets (weaning). [Corrections added on 26 April 2016, after first online publication: 'Enterococcus faecalis strain T-100' has been corrected to 'Enterococcus faecalis strain T-110' in the above paragraph and in the 'Probiotics' section under the Materials and Methods heading.].

The effect of colostrum ingestion during the first 24 hours of life on early postnatal development of piglet immune systems.

It has been suggested that colostrum is important not only for direct protection from pathogens but also for proper development of immune systems in piglets. In this study, we focused on the effect of colostrum ingestion during the first 24 h of life on early postnatal development of piglet immune systems. Thirty-six piglets from five litters were divided into colostrum-fed (CoF) and colostrum-deprived (CoD) groups. The former group was allowed to suckle normally while formula milk was fed to the latter group during the first 24 h of life. At the weaning period, the concentrations of fecal immunoglobulin (Ig) A and plasma IgG as well as the number of blood leukocyte subsets were analyzed. Fecal IgA and plasma IgG concentrations in the CoF group were more than twice as high as those in the CoD group (P < 0.01). In addition, the number of blood B cells was significantly higher in the CoF group than that in the CoD group (P < 0.05). This study demonstrates that colostrum ingestion during the first 24 h plays a significant role in early postnatal development of both mucosal and systemic immunity of piglets.

Effect of monensin withdrawal on rumen fermentation, methanogenesis and microbial populations in cattle.

This study was designed to obtain information on the residual influence of dietary monensin on ruminant fermentation, methanogenesis and bacterial population. Three ruminally cannulated crossbreed heifers (14 months old, 363 ± 11 kg) were fed Italian ryegrass straw and concentrate supplemented with monensin for 21 days before sampling. Rumen fluid samples were collected for analysis of short chain fatty acid (SCFA) profiles, monensin concentration, methanogens and rumen bacterial density. Post-feeding rumen fluid was also collected to determine in vitro gas production. Monensin was eliminated from the rumen fluid within 3 days. The composition of SCFA varied after elimination of monensin, while total production of SCFA was 1.78 times higher than on the first day. Methane production increased 7 days after monensin administration ceased, whereas hydrogen production decreased. The methanogens and rumen bacterial copy numbers were unaffected by the withdrawal of monensin.

Effects of chronic mild food restriction on behavior and the hypothalamic malonyl-CoA signaling pathway.

Depression induces anorexia, leading to suppressed feeding behaviors and energy intake. Previously, we revealed that chronic social defeat induced a mild suppression of feeding in rats with elevated levels of hypothalamic malonyl-CoA which regulates feeding. Therefore, we attempted to elucidate the effects of chronic mild food restriction on behavior and on hypothalamic malonyl-CoA. The chronic mild food restricted rats were fed a restricted diet approximately 80% to 90% amount of diet compared to the control for 5 weeks. Ratios of restriction were adjusted with feed consumption in the chronic social defeat stressed rats. Chronic mild food restricted rats exhibited a suppression of body weight gain similar to that of the chronic social defeat stressed rats. Also these rats showed increased time spent in the center area of an open field (OF), prolonged immobility time in forced swim, increased phosphorylation of hypothalamic adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase and a decreased concentration of hypothalamic malonyl-CoA. Weight of the adrenal glands, locomotion in an OF, mitogen-activated protein kinase cascade and calcium/calmodulin-dependent protein kinases II in the hippocampus were not affected by chronic mild food restriction. Our findings suggest that chronic mild food restriction activates AMPK following a decreased hypothalamic malonyl-CoA.

Effects of chronic social defeat stress on peripheral leptin and its hypothalamic actions.

BACKGROUND: Suppression of body weight and symptom of anorexia are major symptoms of depression. Recently, we reported that chronic social defeat stress (CSDS) induced suppression of body weight gain and anorexic feeding behavior in rats. These abnormalities were the result of disrupted malonyl-coenzyme A (CoA) signaling pathway in the hypothalamus. However, the condition of peripheral leptin and its hypothalamic downstream signal molecules which regulate hypothalamic malonyl-CoA level in the CSDS-exposed rats (CSDS rats) is still unknown. RESULTS: CSDS rats showed suppressed body weight gain and food intake. The weight of the CSDS rats' epididymal white adipose tissues was decreased when compared to the control rats. The plasma cholesterol concentration was decreased significantly in the CSDS rats compared to the control rats (P < 0.05). The plasma glucose concentration was slightly decreased in the CSDS rats compared to the control rats (P < 0.1). The expression of leptin mRNA in epididymal white adipose tissues and the plasma leptin concentration were decreased in CSDS rats. Furthermore, the phosphorylation of the hypothalamic downstream signals of leptin, including extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3), was decreased in CSDS rats. CONCLUSIONS: Our results indicated that decreased peripheral leptin expression in CSDS rats could down-regulate the hypothalamic downstream signaling pathways of leptin while suppressed food intake. These data indicate that CSDS induces the down-regulation of hypothalamic AMPK following the elevation of hypothalamic malonyl-CoA levels and is independent of peripheral leptin and glucose.

Evaluation of secretion volume and immunoglobulin A and G concentrations in sow colostrum from anterior to posterior teats.

Among domestic animals, teat order is only observed in the pig. In order to achieve the healthy growth and weaning of piglets, it is important to elucidate if volume of colostrum secretion and immunoglobulin A (IgA) and IgG concentrations differ among the teats of a sow. Nine sows were used to evaluate the difference in colostrum secretion volume (CSV) and four of these sows were assessed for IgA and IgG concentrations from each teat. Samples were collected five times during 21 h following parturition. Teats were assigned anatomical locations of teat (1 to 7) from anterior to posterior. The CSV of anterior (locations 1 and 2) and middle teats (locations 3-5) was significantly higher than those of posterior teats (locations 6 and 7) throughout the experiment except for 18 h post-parturition (P < 0.05). The CSV of the teats at location 1 was significantly higher at most collection times than those at locations 6 and 7. A positive correlation of CSV was observed with IgA and IgG concentrations from 12 h and 6 h post-parturition, respectively (P < 0.05). The results suggest that anterior teats secrete greater volumes of colostrum and that these tend to contain higher IgA and IgG than posteriors teats.

Shotgun proteomic analysis of porcine colostrum and mature milk.

The epitheliochorial nature of the porcine placenta prevents the transfer of maternal immunity. Therefore, ingestion of the colostrum immediately after birth is crucial for neonatal piglets to acquire passive immunity from the sow. We performed a shotgun proteomic analysis of porcine milk to reveal in detail the protein composition of porcine milk. On the basis of the Swiss-Prot database, 113 and 118 proteins were identified in the porcine colostrum and mature milk, respectively, and 50 of these proteins were common to both samples. Some immune-related proteins, including interleukin-18 (IL-18), were unique to the colostrum. The IL-18 concentration in the colostrum and mature milk of four sows was measured to validate the proteomic analysis, and IL-18 was only detected in the colostrum (191.0 ± 53.9 pg/mL) and not in mature milk. In addition, some proteins involved in primary defense, such as azurocidin, which has never been detected in any other mammal's milk, were also identified in the colostrum.

Anorexic behavior and elevation of hypothalamic malonyl-CoA in socially defeated rats.

Suppression of body weight and eating disorders, such as anorexia, are one of the major symptoms of psychiatric disorders such as depression. However, the mechanisms of weight loss and reduced appetite in depressive patients and in animal models of depression are largely unknown. In this study, we characterized the mechanism of anorexia resulting from depression using socially defeated rats as an animal model of depression. Socially defeated rats showed suppressed body weight gain, enlarged adrenal glands, decreased home cage activity, decreased food intake, and increased immobility in the forced swim test. These results are representative of some of the core symptoms of depression. Simultaneously, we observed decreased levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-coenzyme A (CoA) carboxylase (ACC) and increased levels of malonyl-CoA in the hypothalamus of socially defeated rats. Hypothalamic malonyl-CoA controlled feeding behavior and elevation of malonyl-CoA in the hypothalamus induced inhibition of food intake. Our findings suggest that the suppression of body weight gain caused by social defeat stress is caused by anorexic feeding behavior via an increased concentration of malonyl-CoA in the hypothalamus.

Improvement of bone strength and dermal thickness due to dietary edible bird's nest extract in ovariectomized rats.

Oral administration of edible bird's nest extract (EBNE) improved bone strength and calcium concentration in the femur of ovariectomized rats. Dermal thickness was also increased by EBNE supplementation, whereas EBNE administration did not affect the serum estradiol concentration. These results suggest that EBNE is effective for the improvement of bone loss and skin aging in postmenopause all women.