Assessment of H-ß zeolite as an ochratoxin binder for poultry.

Most of the cereal-based ingredients used in poultry feed are contaminated with ochratoxin-A (OTA). We have investigated H-ß zeolite (HBZ) as a new OTA binder for poultry, along with widely used clay mineral-based product (CM), using in vitro and in vivo methods. In vitro binding experiment was carried out using a biphasic assay, consisting of adsorption at pH 3.2 and desorption at pH 6.8. High adsorption (>98%) with less desorption (<5%) was observed for HBZ, whereas CM showed high binding (>98%) and moderate desorption (48%). In the in vitro experiments with the different simulated gastro-intestinal pH buffers, HBZ did not desorb OTA at any of the pH. Desorption of OTA was observed with CM, as the pH increases. From the in vitro kinetic and chemisorption studies, faster, stronger, and higher adsorption was observed for HBZ. Thermodynamic studies showed positive entropy (22.76 KJ/mol K) for HBZ, signifying predominant hydrophobic interactions towards OTA, whereas CM exhibited negative entropy (-3.67 KJ/mol K). The in vivo binding efficacy of HBZ and CM was tested in 5-wk-old broiler chickens. The study consisted of 4 experimental groups, each with 6 replicates having 2 birds per replicate. The groups were control, negative control (no toxin binder), T1 (HBZ at 1 kg/ton of feed), and T2(CM at 1 kg/ton of feed). Except control, all the replicates received 20 µg of OTA in the feed. Excreta samples of T1, T2, and NC contained 11.57, 7.16, and 2.78 µg of OTA respectively, which was significantly different from each other (P < 0.05). A growth performance trial was conducted in broiler chickens for 35 D. A total of 288 one-day-old birds were randomly segregated to 3 treatment groups, each with 8 replicates of 12 birds each. Treatment groups consisted of control, T1, and T2, treated with no toxin binder, HBZ, and CM at 1 kg/ton of feed, respectively. None of the treatment groups including control, affected BW gain, and feed conversion ratio (P > 0.05).

Bacillus subtilis PB6 improves intestinal health of broiler chickens challenged with Clostridium perfringens-induced necrotic enteritis.

Necrotic enteritis (NE) is an enterotoxemic disease caused by Clostridium perfringens that results in significant economic losses, averaging damage of $0.05 per bird. The present study investigated the influence of a dietary supplement, Bacillus subtilis PB6, on performance, intestinal health, and gut integrity against C. perfringens-induced NE in broiler birds. Bacillus subtilis PB6 (ATCC-PTA 6737) is a natural strain isolated from healthy chicken gut that has been shown in in vitro to produce antimicrobial substances with broad activity against various strains of Campylobacter and Clostridium species. The animal study was conducted on broiler chickens (Cobb 400) for the period of 35 d using a completely randomized design. The experimental design included 3 treatments groups. Each treatment group contained 6 replicates, 3 male and 3 female, with 12 birds in each replicate. The 3 treatment groups were an uninfected control, an infected control, and an infected group supplemented with B. subtilis PB6 at 500 g/t of feed, containing 5 × 10(11) cfu/kg. Necrotic enteritis was induced in the broiler birds via oral inoculation of 30,000 oocysts of mixed strains of Eimeria species on d 14 followed by C. perfringens (10(8) cfu/mL) on d 19 through 21 of trial. The birds were analyzed for BW gain, mortality, feed conversion ratio (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. The infected control group showed markedly thickened mucosa, hemorrhages, intestinal lesions, and ballooning of intestine. The supplementation of B. subtilis PB6 reduced the FCR (P < 0.05) and intestinal C. perfringens counts significantly (P < 0.05) compared with the infected control group. It was also observed that B. subtilis PB6 improved villi length by 10.88 and 30.46% (P < 0.05) compared with uninfected and infected control groups, respectively. The group supplemented with B. subtilis PB6 significantly (P < 0.05) increased the villi length to crypt depth ratio by 49.11% compared with the infected group. In conclusion, the supplementation of B. subtilis PB6 not only controlled C. perfringens-induced NE, but also improved intestinal health in the broiler birds.

Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line.

Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC50 concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors.

Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation.

BACKGROUND: Neuropilin is a transmembrane receptor for vascular endothelial growth factor (VEGF) and is expressed in normal endothelial cells and upregulated in cancer cells. Neuropilin-1 (NRP-1) has been shown to promote tumour cell migration and survival in colon cancer in response to VEGF binding. The expression profiles of neuropilins, associated co-receptors and known ligands have been mapped in three colorectal cell lines: Caco-2, HCT116 & HT29. We have previously shown that butyrate, a naturally occurring histone deacetylase inhibitor (HDACi) produced by fermentation of fibre in the colon, causes apoptosis of colon cancer cell lines. RESULTS: Here we demonstrate that butyrate down-regulates NRP-1 and VEGF at the mRNA and protein level in colorectal cancer cell lines. NRP-1 is a known transcriptional target of Sp1, whose activity is regulated by acetylation. NRP-1 down-regulation by butyrate was associated with decreased binding affinity of Sp1 for canonical Sp-binding sites in the NRP-1 promoter. siRNA-mediated knock-down of Sp1 implied that Sp1 may have strong DNA binding activity but weak transactivation potential. CONCLUSION: The downregulation of the key apoptotic and angiogenesis regulator NRP-1 by butyrate suggests a novel contributory mechanism to the chemopreventive effect of dietary fibre.

Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination.

The location and function of recognized cortex-lytic enzymes of Bacillus subtilis have been explored, and the involvement in germination of a number of related proteins tested. The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during B. subtilis spore germination. Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument. The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction. Neither protein changes location or size as the spore germinates. The ypeB gene is the second gene in a bicistronic operon with sleB. The SleB protein is absent from ypeB mutant spores, suggesting that YpeB is required for its localization or stabilization. In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB - in both the inner membrane and the integument. As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzyme's hydrolytic specificity could not be defined. The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD(600) fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores. The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spore's ability to complete germination. Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of B. subtilis. Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally. The ykvT gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination: the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.

Complete spore-cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB.

The role of the sleB gene of Bacillus subtilis, which encodes a putative spore-cortex-lytic enzyme, and the downstream ypeB gene were investigated. Both SleB and YpeB were required for normal germination to occur. The corresponding mutants formed phase-bright, heat-resistant spores with no apparent defects in dormancy. However, mutant spore suspensions lost optical density slower than the wild-type and spores were phase-grey even 12 h after the triggering of germination. Since the loss of heat resistance and release of dipicolinic acid was similar to the wild-type, these mutants were blocked in the later stages of germination. The mutants were nevertheless capable of outgrowth on rich agar to form colonies, indicating that other spore components can compensate for their function sufficiently to allow outgrowth. The expression and regulation of the operon was examined using a lacZ transcriptional fusion. Expression of the operon began 2 h after the onset of sporulation and was under the control of RNA polymerase containing the forespore-specific sigma factor, sigmaG. The application of reverse phase HPLC revealed that the mutants do not have any structural defect in the dormant spore cortex and therefore these genes are not required for normal spore-cortex synthesis. The analysis of peptidoglycan dynamics during germination showed, however, that the cortex was only partially hydrolysed in both mutants. This analysis also revealed that the likely hydrolytic bond specificity of SleB is likely to be that of a lytic transglycosylase.