Cytolytic CD8+ T Cells Recognizing CFP10 Are Recruited to the Lung after Mycobacterium tuberculosis Infection

Optimum immunity against Mycobacterium tuberculosis requires both CD4+ and CD8+ T cells. In contrast with CD4+ T cells, few antigens are known that elicit CD8+ T cells during infection. CD8+ T cells specific for culture filtrate protein-10 (CFP10) are found in purified protein derivative positive donors, suggesting that CFP10 primes CD8+ T cells in vivo. Using T cells from M. tuberculosis–infected mice, we identified CFP10 epitopes recognized by CD8+ T cells and CD4+ T cells. CFP10-specific T cells were detected as early as week 3 after infection and at their peak accounted for up to 30% of CD8+ T cells in the lung. IFNγ-producing CD8+ and CD4+ T cells recognizing CFP10 epitopes were preferentially recruited to the lungs of M. tuberculosis–infected mice. In vivo cytolytic activity of CD8+ T cells specific for CFP10 and TB10.3/10.4 proteins was detected in the spleen, pulmonary lymph nodes, and lungs of infected mice. The cytolytic activity persisted long term and could be detected 260 d after infection. This paper highlights the cytolytic function of antigen-specific CD8+ T cells elicited by M. tuberculosis infection and demonstrates that large numbers of CFP10-specific cytolytic CD8+ T cells are recruited to the lung after M. tuberculosis infection

Evaluating the removal of pigs from a group and subsequent floor space allowance on the growth performance of heavy-weight finishing pigs

Citation: Flohr, J. R., Tokach, M. D., DeRouchey, J. M., Woodworth, J. C., Goodband, R. D., & Dritz, S. S. (2016). Evaluating the removal of pigs from a group and subsequent floor space allowance on the growth performance of heavy-weight finishing pigs. Journal of Animal Science, 94(10), 4388-4400. doi:10.2527/jas2016-0407A total of 1,092 finishing pigs (initially 36.3 kg) were used in a 117-d study to evaluate the impact of initial floor space allowance and removal strategy on the growth of pigs up to 140 kg BW. There were 4 experimental treatments with 14 pens per treatment. The first treatment provided 0.91 m(2) per pig (15 pigs/pen). The other 3 treatments initially provided 0.65 m(2) per pig (21 pigs/pen) with 3 different removal strategies. The second treatment (2:2:2) removed the 2 heaviest pigs from pens on d 64, 76, and 95 when floor space allowance was predicted to be limiting. Treatment 3 (2:4) removed the 2 heaviest pigs on d 76 and the 4 heaviest pigs on d 105. Treatment 4 (6) removed the heaviest 6 pigs on d 105. All pigs remaining in pens after removals were fed to d 117. Overall (d 0 to 117), pigs initially provided 0.91 m(2) of floor space had increased (P < 0.05) ADG compared to pigs in pens on the 2: 4 or 6 removal strategy, but ADG was not different compared with pigs on the 2:2:2 removal strategy. Total BW gain per pen was greater (P < 0.05) for pens initially stocked at 0.65 m(2) compared to pens initially stocked at 0.91 m(2). Feed usage per pen was less (P < 0.05) for pens initially stocked at 0.91 m(2) compared to pens initially providing 0.65 m(2) of floor space and on removal strategies; however, feed usage per pig was greater (P < 0.05) for pigs initially stocked at 0.91 m(2) compared to pigs initially stocked at 0.65 m(2) and on removal strategies. Feed usage, on a pig or pen basis, was less (P < 0.05) for pigs on the 2: 2: 2 removal strategy compared to pigs on the 2:4 or the 6 removal strategy. Income over feed and facility cost (IOFFC) was less (P < 0.05) for pigs initially provided 0.91 m(2) compared to pigs initially provided 0.65 m(2) and on removal strategies. Also, IOFFC was less (P < 0.05) for pigs on the 2:2:2 compared to the 2:4 and 6 removal strategies. In conclusion, increasing the floor space allowance or the time points at which pigs are removed from the pen improved the growth of pigs remaining in the pen; however, IOFFC may be reduced because fewer pigs are marketed from each pen (pigs stocked at 0.91 m(2) throughout the study) or from reducing total weight produced (2:2:2 removal strategy)

Evaluating the impact of maternal vitamin D supplementation: I. Sow performance, serum vitamin metabolites, and neonatal muscle characteristics

Citation: Flohr, J. R., Woodworth, J. C., Bergstrom, J. R., Tokach, M. D., Dritz, S. S., Goodband, R. D., & DeRouchey, J. M. (2016). Evaluating the impact of maternal vitamin D supplementation: I. Sow performance, serum vitamin metabolites, and neonatal muscle characteristics. Journal of Animal Science, 94(11), 4629-4642. doi:10.2527/jas2016-0409In Exp. 1, 56 gestating sows (PIC 1050; 35 d postinsemination) were used in a 30-d trial to determine serum 25(OH)D-3 response to increasing concentrations of dietary vitamin D3. Sows were randomly allotted to 1 of 7 dietary D3 treatments (200, 800, 1,600, 3,200, 6,400, 12,800, or 25,600 IU of added D3 per kilogram of complete diet) with 8 sows per treatment. Increasing D-3 increased (quadratic; P < 0.001) serum 25(OH)D-3 with the response depicted by the prediction equation: serum 25(OH) D3, ng/mL = 35.1746 + (0.002353 x dietary D-3, IU/d)- (0.0000000156 x dietary D3, IU/d(2)). In Exp. 2, 112 sows and their litters were used to determine the effects of dietary vitamin D regimen on sow performance, subsequent preweaning pig performance, neonatal bone and muscle characteristics, and serum vitamin metabolites. Sows were allotted to 1 of 4 dietary treatments 3 to 5 d following breeding: 800, 2,000, or 9,600 IU of D-3 per kilogram of the diet or 50 mu g of 25(OH) D-3 (2,000 IU of D-3 equivalent from Hy- D, DSM Nutritional Products, Parsippany, NJ) per kilogram of diet. There were 25 to 27 sows per treatment. Increasing dietary D-3 increased (linear, P = 0.001) serum 25(OH) D-3 of sows on d 100 of gestation, at farrowing, and at weaning. Increasing D-3 in sow diets increased piglet serum 25(OH)D-3 at birth (linear, P = 0.001) and weaning (quadratic, P = 0.033). Sows fed 50 mu g of 25(OH)D-3/kg had intermediate (P < 0.004) serum 25(OH)D-3 concentrations on d 100 of gestation, at farrowing, and at weaning compared with sows fed 2,000 IU of D-3/kg and sows fed 9,600 IU of D3/kg. Pigs from sows fed 50 mu g of 25(OH) D3/ kg had greater serum 25(OH)D-3 compared with pigs from sows fed 2,000 IU of D-3/kg, but at weaning, serum 25(OH)D-3 concentrations were similar. Also, pigs from sows fed 9,600 IU of D-3/kg had greater (P = 0.011) serum 25(OH) D3 at birth and weaning compared with pigs from sows fed 50 mu g of 25(OH) D-3/kg. Maternal performance, litter characteristics, neonatal bone ash content, and neonatal muscle fiber characteristics were largely unaffected by the dietary vitamin D treatments. Overall, D3 and 25(OH) D3 are both useful at increasing serum 25(OH)D-3 concentrations, but more D3 (on an equivalent IU basis) is needed to achieve similar serum 25(OH)D-3 responses compared with feeding 25(OH)D-3. Concentration of maternal vitamin D supplementation in lactation impacted milk transfer of the vitamin more so than the form of the vitamin, as evidence by the weaned pig serum 25(OH)D-3 concentrations

Evaluating the impact of maternal vitamin D supplementation on sow performance: II. Subsequent growth performance and carcass characteristics of growing pigs

Citation: Flohr, J. R., Woodworth, J. C., Bergstrom, J. R., Tokach, M. D., Dritz, S. S., Goodband, R. D., & DeRouchey, J. M. (2016). Evaluating the impact of maternal vitamin D supplementation on sow performance: II. Subsequent growth performance and carcass characteristics of growing pigs. Journal of Animal Science, 94(11), 4643-4653. doi:10.2527/jas2016-0410A of subsample of 448 growing pigs (PIC 327 x 1050) weaned from 52 sows fed varying dietary vitamin D regimens were used in a split-plot design to determine the effects of maternal and nursery dietary vitamin D on growth performance. Sows were previously administered diets containing vitamin D as vitamin D-3 (800, 2,000, or 9,600 IU/kg) or as 25(OH) D3 (50 mu g [or 2,000 IU vitamin D equivalent]/kg from HyD; DSM Nutritional Products, Parsippany, NJ). Once weaned, pigs were allotted to pens on the basis of previous maternal vitamin D treatment, and then pens were randomly assigned to 1 of 2 nursery vitamin D dietary regimens (2,000 IU of vitamin D-3/ kg or 50 mu g 25(OH) D-3/kg). Pigs remained on nursery vitamin D treatments for 35 d, and then they were provided common finishing diets until market (135 kg). Growing pig serum 25(OH)D-3 suggested that maternal dietary vitamin D influenced (P < 0.001 at weaning) serum concentrations early after weaning, but nursery vitamin D regimen had a larger impact (P < 0.001) on d 17 and 35 postweaning. Overall growth performance was not influenced by nursery vitamin D dietary treatments. From d 0 to 35 in the nursery, pigs from sows fed increasing vitamin D3 had increased (quadratic, P < 0.003) ADG and ADFI, but G:F was similar regardless of maternal vitamin D regimen. Also, pigs from sows fed 50 mu g/kg of 25(OH) D-3 had increased (P = 0.002) ADG compared with pigs weaned from sows fed 800 IU of vitamin D3. Throughout finishing (d 35 postweaning until 135 kg), ADG was increased (quadratic, P = 0.005) and G: F was improved (quadratic, P = 0.049) with increasing maternal dietary vitamin D-3. Also, pigs from sows fed 50 mu g/kg of 25(OH)D-3 had increased (P = 0.002) ADG compared with pigs weaned from sows fed 800 IU of vitamin D-3. Carcass data were collected from a subsample population separate from that used for the growth performance portion of the study, and a total of 642 carcasses from progeny of sows fed the varying dietary vitamin D treatments were used. Live BW of pigs at marketing and HCW were heavier (P < 0.030) for pigs from sows previously fed 25(OH)D-3 compared with pigs from sows fed 9,600 IU of vitamin D-3. Overall, pigs from sows fed 2,000 IU of vitamin D-3 grew faster after weaning compared with pigs from sows fed 800 or 9,600 IU of vitamin D-3. Pigs from sows fed 25(OH)D-3 hag greater ADG compared with pigs from sows fed 800 IU of vitamin D-3, and they had increased final BW and HCW compared with pigs from sows fed 9,600 IU of vitamin D-3

Lipocalin-2 Functions as Inhibitor of Innate Resistance to Mycobacterium tuberculosis

Lipocalin-2 is a constituent of the neutrophil secondary granules and is expressed de novo by macrophages and epithelium in response to inflammation. Lipocalin-2 acts in a bacteriostatic fashion by binding iron-loaded siderophores required for bacterial growth. Mycobacterium tuberculosis (M.tb) produces siderophores that can be bound by lipocalin-2. The impact of lipocalin-2 in the innate immune response toward extracellular bacteria has been established whereas the effect on intracellular bacteria, such as M.tb, is less well-described. Here we show that lipocalin-2 surprisingly confers a growth advantage on M.tb in the early stages of infection (3 weeks post-challenge). Using mixed bone marrow chimeras, we demonstrate that lipocalin-2 derived from granulocytes, but not from epithelia and macrophages, leads to increased susceptibility to M.tb infection. In contrast, lipocalin-2 is not observed to promote mycobacterial growth at later stages of M.tb infection. We demonstrate co-localization of granulocytes and mycobacteria within the nascent granulomas at week 3 post-challenge, but not in the consolidated granulomas at week 5. We hypothesize that neutrophil-derived lipocalin-2 acts to supply a source of iron to M.tb in infected macrophages within the immature granuloma, thereby facilitating mycobacterial growth

EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in Mycobacterium tuberculosis by Affecting Bacterial Cell Wall Integrity

Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall

Estimation of unconfined aquifer hydraulic properties using gravity and drawdown data

2011 Fall.Includes bibliographical references.An unconfined aquifer test using temporal gravity measurements was conducted in shallow alluvium near Fort Collins, Colorado on September 26-27, 2009. Drawdown was recorded in four monitoring wells at distances of 6.34, 15.4, 30.7, and 60.2 m from the pumping well. Continuous gravity measurements were recorded with a Scintrex® CG-5 gravimeter near the closest well, at 6.3 m, over several multi-hour intervals during the 27 hour pumping test. Type-curve matching of the drawdown data performed assuming Neuman's solution yields transmissivity T, specific yield Sy, and elastic component of storativity S estimates of 0.018 m2s-1, 0.041, and 0.0093. The gravitational response to dewatering was modeled assuming drawdown cone geometries described by the Neuman drawdown solution using combinations of T, Sy, and S. The best fitting gravity model based on minimization of the root mean square error between the modeled and observed gravity change during drawdown resulted from the parameters T =0.0033 m2s-1, Sy =0.45, and S =0.0052. Conservative precision estimates in the gravity data widen these estimates to T =0.002-0.006 m2s-1, Sy =0.25-0.65, and S =0-0.2. Drawdown conforming to the Neuman solution was forward modeled using combinations of T, Sy, and S. Minimization of the root mean square misfit between these forward models and observed drawdown in the monitoring wells results in T =0.0080 m2s-1, Sy =0.26, and S =0.000004. Discrepancy between type-curve matching results, gravity analysis results, and drawdown modeling is attributed to heterogeneity and anisotropy within the aquifer, and a relatively large amount drawdown compared to initial saturated thickness, conditions which fail the Neuman solution assumptions. In this aquifer test, gravity was most sensitive to transmissivity, less sensitive to specific yield, and insensitive to the specific storage-saturated thickness quotient. Simultaneous deployment of multiple gravity stations during similar tests should better constrain gravity analysis aquifer property estimates of transmissivity and specific yield