Assessing vaccine-mediated protection in an ultra-low dose Mycobacterium tuberculosis murine model.

Despite widespread immunization with Bacille-Calmette-Guerin (BCG), the only currently licensed tuberculosis (TB) vaccine, TB remains a leading cause of mortality globally. There are many TB vaccine candidates in the developmental pipeline, but the lack of a robust animal model to assess vaccine efficacy has hindered our ability to prioritize candidates for human clinical trials. Here we use a murine ultra-low dose (ULD) Mycobacterium tuberculosis (Mtb) challenge model to assess protection conferred by BCG vaccination. We show that BCGconfers a reduction in lung bacterial burdens that is more durable than that observed afterconventional dose challenge, curbs Mtb dissemination to the contralateral lung, and, in a smallpercentage of mice, prevents detectable infection. These findings are consistent with the ability of human BCG vaccination to mediate protection, particularly against disseminated disease, in specific human populations and clinical settings. Overall, our findings demonstrate that the ultra-low dose Mtb infection model can measure distinct parameters of immune protection that cannot be assessed in conventional dose murine infection models and could provide an improved platform for TB vaccine testing.

Supplementation of xylanase and phospholipase to wheat-based diets for weaner pigs.

The effects of supplementing a wheat-based diet for weaner pigs with exogenous xylanase and phospholipase on ileal and faecal nutrient digestibilities and on the level of microbial metabolites in ileal digesta were examined. Fourteen piglets, weaned at 11 days, were fitted with a simple T-cannula at the distal ileum. The pigs were offered a control diet or diets supplemented with xylanase and phospholipase individually or in combination, in a two period crossover design. The combination of xylanase and phospholipase tended to increase the ileal recovery of the amino sugar galactosamine, whereas the concentration expressed in mg/kg dry matter intake of glucosamine was slightly decreased (p < 0.10). There was neither an effect of enzyme supplementation on ileal and faecal digestibility of the other nutrients and energy, nor was there an effect on pH and on the level of microbial metabolites in ileal digesta. However, an increase in ileal and faecal nutrient and energy digestibility with increasing age was observed. The ileal and faecal digestibility coefficients (except for ether extract) were significantly (p < 0.05) higher in experimental period I than in period II. These higher values may be attributed to a lower feed intake during period I. Since a lower level of feed intake is generally associated with a slower rate of passage and a longer retention time of digesta, a positive impact on digestion and absorption of nutrients can be assumed, which, on the other hand, limits the potential of additional enzyme effects.

Analysis of 4-hydroxyproline using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol derivatization and micellar electrokinetic chromatography combined with laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection was used for internal standard (pyrrolidinol) based quantification of 4-hydroxyproline (Hyp) in muscle hydrolysates. Hydrolysates were first derivatized with o-phthaldialdehyde to reduce primary amine interference and then 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD) to enable selective detection of secondary amines. This method allows for rapid and sensitive detection of hydroxyproline in dilute hydrolysates using commercially available capillary electrophoresis equipment.

Mucin output in ileal digesta of pigs fed a protein-free diet.

Daily outputs of mucin in ileal digesta were estimated in three barrows fed a protein-free diet while administered either saline (SAI) or a complete amino acid mixture (AAI) intravenously. The water soluble-ethanol precipitable fraction of ileal digesta (crude mucin; CM) was used to estimate the composition of mucin in ileal digesta. This fraction exhibited a carbohydrate composition characteristic of mucin and had a high threonine, serine and proline content (40 mol/100 mol). The proportions of soluble gastric and intestinal mucins, approximately 27 and 73%, respectively, were estimated from the N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) ratio in CM. The daily outputs of soluble mucin, 2.75 and 3.41 g/day from SAI and AAI pigs (p = 0.13), respectively, were determined from the GalNAc outputs in CM, assuming the above contributions of gastric and intestinal mucins. The estimated soluble mucin outputs accounted for more than 99% of the fucose, galactose, GalNAc and GlcNAc in CM. Total mucin outputs in ileal digesta, 5.32 and 5.65 g/day from SAI and AAI Pigs (p = 0.24), respectively, were determined from the total GalNAc output in digesta, assuming soluble and insoluble mucin had similar compositions. Based on these outputs, mucin represented approximately 30, 7 to 22, 15 and 11% of the endogenous threonine, proline, serine and protein, respectively, in ileal digesta. Approximately 74, 76, 100 and 53% of the fucose, galactose GalNAc and GlcNAc, respectively, in ileal digesta from pigs in this study was attributed to mucin. The results from this study demonstrate the importance of mucin as a source of some endogenous amino acids and carbohydrates.

Evaluation of the 15N-isotope dilution technique for determining the recovery of endogenous protein in ileal digesta of pigs: effect of the pattern of blood sampling, precursor pools, and isotope dilution technique.

The 15N-enrichments (atom percentage excess) were determined in the plasma free amino acids of blood samples taken at the time of feeding and in samples taken hourly and pooled over 12 h, as well as in ileal digesta, crude mucin, and bacteria collected at the distal ileum of pigs fed barley while continuously administered [15N]leucine intravenously. The branched-chain amino acids were the only indispensable amino acids to exhibit incorporation of 15N (P < .05). All dispensable amino acids exhibited some incorporation. Enrichments in free leucine and alanine were higher (P < .02) in blood samples taken at the time of feeding, compared to those in pooled blood samples, resulting in an underestimation of the endogenous ileal recoveries of these amino acids. Enrichments in amino acids in crude mucin were usually similar to those in pooled plasma samples, providing some support for the use of plasma free amino acids to estimate enrichments in endogenous amino acids in ileal digesta. Enrichments in bacteria were not different (P > .05) from those in ileal digesta. The recoveries of endogenous amino acids in ileal digesta determined with the [15N]leucine and 15N-amino acid dilution techniques demonstrate the overestimation of these criteria with the 15N-isotope dilution technique, applied in its current form, and suggest that modifications in the composition of endogenous protein can occur when pigs are fed protein-containing diets. These study supports the use of 15N-isotope dilution techniques, with modifications, for determining the recovery of endogenous protein in ileal digesta of pigs fed protein-containing diets.

Evaluation of the 15N-isotope dilution technique for determining the recovery of endogenous protein in ileal digestion of pigs: effect of dilution in the precursor pool for endogenous nitrogen secretion.

Three barrows, average initial BW of 35 kg, were fitted with a re-entrant cannula at the distal ileum and catheters in each of the external jugular veins. The barrows were fed twice daily a diet containing barley as the sole source of protein. A 1-d intravenous infusion of saline was followed by an 8-d continuous infusion of [15N]leucine at a rate of 30 mg/(kg BW x d). Blood samples, taken hourly for 9 d, were pooled over the 12-h period between feeding. Ileal digesta were collected hourly on d 3 and 8 of the [15N]leucine infusion and pooled over 12 and 24 h. Enrichments (atom percentage excess 15N) in nitrogen (N), leucine, isoleucine, valine and alanine were measured in the trichloroacetic acid (TCA)-soluble and -insoluble fractions of plasma and in ileal digesta. Although the enrichments in N were similar in all pools, enrichments in amino acids in the TCA-soluble fraction of plasma were at least twice that in the TCA-insoluble fraction and in ileal digesta. The estimated contributions of endogenous N and amino acids to total N and amino acids in ileal digesta were 82.3 +/- 9.38, 39.0 +/- 3.83, 43.8 +/- 6.50, 51.9 +/- 3.24, and 51.9 +/- 9.80 for N, leucine, isoleucine, valine and alanine, respectively. The results from this study indicate that enrichments in total N in the TCA-soluble fraction of plasma may not accurately reflect enrichments in endogenous N secreted into the intestinal lumen. This factor should be considered when using the 15N-isotope dilution technique to estimate the recovery of endogenous protein in ileal digesta of pigs fed protein-containing diets.

Ileal recovery of nutrients and mucin in humans fed total enteral formulas supplemented with soy fiber.

The objective of this study was to determine whether soy fiber supplementation of total enteral nutrition formulas affected small intestinal recovery of nitrogen, amino acids, and carbohydrates or mucin output in eight human subjects (four males, four females) with ileostomies. The subjects ingested five test diets to provide 1.0-16.5 g soy fiber/L for 2 consecutive days each. The five test diets, each with a different soy fiber content were formulated by varying the relative proportion (1:0, 0.75:0.25, 0.5:0.5, 0.25:0.75, and 0:1) of two commercially available formulas. Effluent dry matter increased with soy fiber intake as a result of the quantitative recovery of soy fiber nonstarch polysaccharide. Nitrogen and amino acid digestibilities were unchanged by the ingestion of soy fiber. Nutrients from the total enteral nutrition formulas were well digested in the small intestine with true nitrogen and amino acid digestibilities in excess of 90% and starch digestibilities approaching 100%. Ileal mucin output was higher in male subjects and was unaffected by soy fiber intake. In summary, soy fiber supplementation does not compromise protein and carbohydrate absorption from the small intestine of humans.

Determination of apparent ileal amino acid digestibility in pigs: effect of dietary amino acid level.

Studies were carried out to investigate the effect of dietary amino acid level on apparent ileal amino acid digestibility. Six barrows, average initial BW 35 kg, were fitted with a simple T-cannula at the distal ileum and fed six diets according to a 6 x 6 Latin square design. Six cornstarch-based diets containing six levels of CP from SBM (4, 8, 12, 16, 20, and 24% CP, respectively) were formulated. Chronic oxide was included as a digestibility marker. Each experimental period consisted of 8 d. After a 6-d adaptation period, ileal digesta were collected for 24 h during d 7 and 9 at 2-h intervals. The pigs were fed twice daily, equal amounts, at 0800 and 2000. The dietary allowance was 1,600 g/d during the first period and increased by 100 g each following period. There was a quadratic increase (P < .05) in apparent ileal amino acid digestibility as the dietary CP content was increased from 4 to 24%. Initially, the apparent ileal amino acid digestibilities increased sharply then gradually reached their plateaus, after which there were no further increases and the digestibility values became independent of the dietary amino acid levels. The lower end points of 95% confidence intervals of the plateau ileal digestibility values were defined to be the initial plateau digestibilities. The dietary CP and amino acid contents, corresponding to the initial plateau digestibility values, represent the dietary threshold levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion-pair high-performance liquid chromatography of diaminopimelic acid in hydrolysates of physiological samples.

A method is reported for the determination of diaminopimelic acid (DAPA) in physiological samples. DAPA is derivatized with an o-phthaldialdehyde reagent solution, subjected to reversed-phase high-performance liquid chromatography and detected spectrofluorometrically. The method is a significant advance over previous methods because it uses the ion-pairing agent hexadecyltrimethylammonium bromide (HTMA) to facilitate DAPA measurement. Ion-pairing with HTMA avoids interference with co-eluting derivatives to provide simultaneous, sensitive, reproducible measurement of both DAPA peaks (DD,LL-DAPA and DL-DAPA).

15N-leucine and 15N-isoleucine isotope dilution techniques versus the 15N-isotope dilution technique for determining the recovery of endogenous protein and amino acids in digesta collected from the distal ileum in pigs.

Twelve gilts, with an average initial BW of 38 kg, were fitted with simple T-cannulas at the distal ileum and two catheters in the external jugular veins. The animals were fed twice daily (0800 and 2000) 700 g of one of four diets containing either soybean meal, canola meal, wheat, or barley as the sole protein source. 15N-leucine was infused continuously for 9 d at a rate of 40 mg/kg of BW daily via one of the jugular catheters. Blood samples were taken during feeding. Ileal digesta samples were collected continuously for 24 h on d 7 and 9 of the infusion period. 15N-enrichment excess in leucine and isoleucine in the trichloroacetic acid (TCA)-soluble fraction of blood and in digesta were measured using single-ion monitoring gas chromatography-mass spectrometry. Assuming that the 15N-enrichment excess in leucine and isoleucine in the TCA-soluble fraction of the blood is similar to that in endogenous protein secreted into the digestive tract and that the amino acid composition of endogenous protein is constant, the contribution of endogenous to total protein in ileal digesta was calculated using the 15N-isotope dilution technique for leucine and isoleucine, respectively. These contributions were much smaller for the 15N-leucine than for the 15N-isoleucine isotope dilution technique: 7.1 to 11.0 vs 21.8 to 24.9 g of protein/kg of DM intake. The values obtained with the 15N-isoleucine isotope dilution technique were close to those obtained with the 15N-isotope dilution technique as described in a previous publication.