J. L. Grimes

Role of Campylobacter jejuni Respiratory Oxidases and Reductases in Host Colonization

ABSTRACT Campylobacter jejuni is the leading cause of human food-borne bacterial gastroenteritis. The C. jejuni genome sequence predicts a branched electron transport chain capable of utilizing multiple electron acceptors. Mutants were constructed by disrupting the coding regions of the respiratory enzymes nitrate reductase (napA::Cm), nitrite reductase (nrfA::Cm), dimethyl sulfoxide, and trimethylamine N-oxide reductase (termed Cj0264::Cm) and the two terminal oxidases, a cyanide-insensitive oxidase (cydA::Cm) and cbb3-type oxidase (ccoN::Cm). Each strain was characterized for the loss of the associated enzymatic function in vitro. The strains were then inoculated into 1-week-old chicks, and the cecal contents were assayed for the presence of C. jejuni 2 weeks postinoculation. cydA::Cm and Cj0264c::Cm strains colonized as well as the wild type; napA::Cm and nrfA::Cm strains colonized at levels significantly lower than the wild type. The ccoN::Cm strain was unable to colonize the chicken; no colonies were recovered at the end of the experiment. While there appears to be a role for anaerobic respiration in host colonization, oxygen is the most important respiratory acceptor for C. jejuni in the chicken cecum.

The role of respiratory donor enzymes in Campylobacter jejuni host colonization and physiology

The human pathogen Campylobacter jejuni utilizes oxidative phosphorylation to meet all of its energy demands. The genome sequence of this bacterium encodes a number of respiratory enzymes in a branched electron transport chain that predicts the utilization of a number of electron transport chain donor and acceptor molecules. Three of these electron donor enzymes: hydrogenase, formate dehydrogenase, and 2-oxoglutarate:acceptor oxidoreductase (OOR), oxidize hydrogen, formate and alpha-ketoglutarate as electron donors, respectively. Mutations were created in these donor enzymes to isolate mutants in hydrogenase (HydB::CM), formate dehydrogenase (Fdh::CM), and OOR (OorB::CM), as well as a strain with insertions in both hydrogenase and formate dehydrogenase (Hyd::Fdh). These mutants are deficient in their respective enzyme activities and do not reduce the components of the electron transport chain when provided with their respective substrates. The presence of either hydrogen or formate in the media stimulated the growth of wild type (WT) C. jejuni (but not the associated mutant strains) and at least one of these alternative substrates is required for growth of the OOR mutant strain OorB::CM. Finally, the importance of hydrogenase, formate dehydrogenase and OOR as well as the complex I of C. jejuni are elucidated by chicken colonization assays, where the double mutant Hyd::Fdh, OorB::CM and nuo mutants are severely impaired in host colonization.

Organic trace minerals and 25-hydroxycholecalciferol affect performance characteristics, leg abnormalities, and biomechanical properties of leg bones of turkeys

Leg problems and resulting mortality can exceed 1% per week in turkey toms starting at approximately 15 wk of age. Dietary supplementation of organic trace minerals (MIN) and 25-hydroxycholecalciferol (HyD) may improve performance, decrease incidence of leg abnormalities, and increase bone strength. Nicholas 85X700 toms were assigned to 4 treatments consisting of a factorial arrangement of 2 concentrations of MIN (0 and 0.1% of Mintrex P(Se), which adds 40, 40, 20, and 0.3 mg/kg of Zn, Mn, Cu, and Se, respectively) and 2 concentrations of HyD (0 and 92 microg/kg of HyD). Diets were formulated to be equal in nutrient content and fed ad libitum as 8 feed phases. Feed intake and BW were measured at 6, 12, 15, 17, and 20 wk of age. Valgus, varus, and shaky leg defects were determined at 12, 15, 17, and 20 wk of age. Tibia and femur biomechanical properties were evaluated by torsion and bending tests at 17 wk of age. There were no treatment effects on BW. Only MIN significantly improved feed conversion ratio through to 20 wk of age. Cumulative mortality at 3 wk of age was greater among the MIN birds, but it was lower by 20 wk (P = 0.085). The MIN decreased the incidence of varus defects at 17 wk of age; shaky leg at 12, 15, and 17 wk of age; and valgus defects at 15, 17, and 20 wk of age. There were no MIN x HyD interaction effects on individual gait problems. Maximum load and the bending stress required for tibias to break in a 4-point assay were increased with MIN supplementation, especially when HyD was also added. Maximum shear stress at failure of femoral bones in a torsion assay was increased by supplementation with both MIN and HyD together. Dietary supplementation of MIN and HyD may improve biomechanical properties of bones. Dietary MIN supplementation may improve feed conversion of turkeys, likely by decreasing leg problems.

Effects of a Direct-Fed Microbial (Primalac) on Turkey Poult Performance and Susceptibility to Oral Salmonella Challenge

A study was conducted to determine 1) the effect of a dietary direct-fed microbial (DFM) on turkey poult performance, 2) the effect of a DFM on a Salmonella challenge, and 3) the effect of feed processing on the efficacy of the dietary DFM. Day-of-hatch Large White female poults were placed in 2 rooms in 2 Petersime batteries per room. Twelve pens of 7 birds each were used in each battery (24 pens per room, 336 birds total). One of 4 dietary feed treatments was assigned to each pen (6 pens per room for each diet). One room housed non-Salmonella-challenged poults, and the other room housed poults challenged with a 1-mL oral gavage of Salmonella (10(10) cfu/mL). A single batch of starter ration was split into 4 parts and used to provide 4 dietary treatments: 1) mash feed with no DFM (M), 2) mash feed with DFM (Primalac; 0.9 kg/tonne of feed, MD), 3) pelleted (20-s steam conditioning at 80 degrees C) and crumbled feed with no DFM (C), and 4) pelleted and crumbled feed with DFM (CD). Feed and deionized, distilled water were provided ad libitum. Data were collected and analyzed separately for each room. Mortality was recorded for each pen on a daily basis and totaled by week and for the 3-wk period. Individual BW and feed consumption, by pen, were measured weekly. Weekly and cumulative BW gains and feed to gain ratios (F:G) were calculated. Liver, spleen, total and lower intestinal tract weights, intestinal length, and most-probable-number Salmonella populations were determined for one randomly selected bird per pen. Feeding processed feed resulted in improved BW and F:G. Feeding the DFM improved 3-wk cumulative F:G in birds not gavaged and reduced relative intestinal weight in birds gavaged. Salmonella populations were reduced 1 log by feeding DFM. Dietary DFM improved bird performance, reduced Salmonella populations, and was not affected by feed processing.

Effects of Incubator Temperature and Oxygen Concentration During the Plateau Stage of Oxygen Consumption on Turkey Embryo Long Bone Development

Temperature (TEM) and O(2) concentrations during the plateau stage of oxygen consumption are known to affect yolk utilization, tissue development, and thyroid metabolism in turkey embryos. Three experiments were conducted to evaluate these incubation effects on long bone development. Fertile eggs of Nicholas turkeys were used. In each trial, standard incubation conditions were used to 24 d, when the eggs containing viable embryos were randomly divided into 4 groups. Four experimental cabinets provided 4 TEM (36, 37, 38, or 39 degrees C) or 4 O(2) concentrations (17, 19, 21, or 23% O(2)). In the third experiment, 2 temperatures (36 and 39 degrees C) and 2 O(2) concentrations (17 and 23%) were evaluated in a 2 x 2 factorial design. Body and residual yolk weights were obtained. Both legs were dissected, and shanks, femur, and tibia weights, length, and thickness were recorded. Relative asymmetry of each leg section was calculated. Chondrocyte density was evaluated in slides stained with hematoxylin and eosin. Immunofluorescence was used to evaluate the presence of collagen type X and transforming growth factor beta. Hot TEM caused reduction of tibia weights and increase of shank weight when compared with cool TEM. The lengths of femur, tibia, and shanks were reduced by 39 degrees C. The relative asymmetry of leg weights were increased at 38 and 39 degrees C. Poult body and part weights were not affected by O(2) concentrations, but poults on 23% O(2) had bigger shanks and heavier tibias than the ones on 17% O(2). High TEM depressed the fluorescence of collagen type X and transforming growth factor beta. The O(2) concentrations did not consistently affect the immunofluorescence of these proteins. The chondrocyte density was affected by TEM and O(2) in resting and hypertrophic zones. In the third experiment, high TEM depressed BW, leg muscle weights, and shank length. Low O(2) reduced tibia and shanks as a proportion of the whole body. We concluded that incubation conditions affect long bone development in turkeys.

The Effect of Quicklime (CaO) on Litter Condition and Broiler Performance

High levels of phosphorus and pathogens in runoff are 2 major concerns following manure applications to fields. Phosphorus losses from fields following manure applications have been linked to the solubility of phosphorus in manure; therefore, by decreasing manure phosphorus solubility, a decrease in phosphorus loss in runoff should be apparent. The objective of this research was to develop a process using quicklime that would result in reduced phosphorus solubility and bacteria counts in broiler litter. The 4 litter treatments evaluated were T1, new wood shavings without the addition of quicklime; T2, used, untreated broiler litter; T3, used litter with 10% quicklime (based on the weight of the litter); and T4, used litter with 15% quicklime (based on the weight of the litter). Body weight, cumulative feed consumption, and feed conversion (feed:BW) were determined on a weekly basis through 42 d of age. Mortality was recorded daily. Carcass weights and percentages of carcass yield without giblets were determined prechill. Litter pH, total phosphorus, nitrogen, soluble phosphorus, litter moisture (%), and total plate counts were measured for each litter treatment on d 7 and 42 after bird placement. No significant differences were found for BW, feed consumption, feed conversion, mortality, carcass weight, or carcass yield. No breast or footpad blisters were observed. On d 7, 15% quicklime had higher (P < 0.001) pH (11.2) when compared with the other treatments. Percentages of phosphorus and nitrogen were lower (P < 0.001) for new wood shavings in comparison with the used litter treatments. Soluble phosphorus (ppm) was lower (P < 0.001) for 15% quicklime (2.75) when compared with new wood shavings (42.2), untreated broiler litter (439.2), and 10% quicklime (35.0). Although not significant, 15% quicklime had lower total plate counts (cfu/g) in comparison with the other treatments on d 1 and 10 postmixing and at 7 d after bird placement. Litter conditions on d 42 after bird placement were similar. We concluded that the use of quicklime as a treatment for broiler litter would initially reduce nitrogen and soluble phosphorus and bacteria counts without negatively affecting bird productivity.

Effect of direct-fed microbials on performance and Clostridium perfringens colonization of turkey poults

Clostridium perfringens is recognized as an enteric pathogen in humans, domestic animals, and livestock. This organism is associated with necrotic enteritis, gangrenous dermatitis, clostridial dermatitis (turkeys), and gizzard erosions in poultry. This study was conducted to evaluate the effectiveness of a direct-fed microbial (DFM), Primalac (Star Labs, Clarksdale, MO), in preventing intestinal colonization of turkey poults with C. perfringens. One-day-old turkey poults (n = 128) were randomly divided into 4 treatments with 4 replicates (8 birds/pen). Treatments were as follows: 1) basal diet without DFM (C); 2) basal diet supplemented with Primalac (1.5 kg/ton; PM); 3) basal diet with poults gavaged with C. perfringens (CCP); and 4) basal diet supplemented with Primalac and poults gavaged with C. perfringens (PMCP). Feed and water were provided ad libitum throughout the trials, and birds were inoculated with C. perfringens (10(8)cfu/mL) on d 3 and 7. On d 21, 2 birds/pen were killed, spleen and bursa of Fabricius were collected and weighed, and cecal contents were used for C. perfringens enumeration. Feed consumption, BW, and feed conversion were calculated throughout the trial (weekly and cumulatively). Data were analyzed using GLM of SAS (SAS Institute, Cary, NC; P < 0.05). Among the inoculated groups, birds fed the DFM-supplemented diet had significantly lower cecal C. perfringens counts than the birds fed the diet without the DFM. The C. perfringens (log(10) cfu/g) in ceca were as follows: C, 5.88; CCP, 7.26; PM, 5.35; PMCP, 6.19 ± 0.36. No differences were observed for BW (814 ± 11 g), feed conversion (1.33 ± 0.03), organ weights, or relative organ weights. Further studies are needed to fully ascertain the potential of using DFM to reduce the numbers of C. perfringens in the gastrointestinal tract of turkey poults.

Chromium propionate in broilers: effect on insulin sensitivity

The objective of this study was to evaluate the effects of dietary chromium (Cr), as chromium propionate, on measures of insulin sensitivity. Liver and muscle glycogen, and plasma glucose and non-esterified fatty acid (NEFA) concentrations were used as indicators of insulin sensitivity. In total, 288 newly hatched male Ross broilers were divided into 4 dietary treatments consisting of 0 (control diet analyzed 0.43 to 0.45 mg Cr/kg), 0.2, 0.4, or 0.6 mg supplemental Cr/kg diet, resulting in 4 treatments with 9 replicate pens per treatment containing eight birds per pen. At d 21, 2 birds per cage were removed based on the greatest deviation from pen mean BW, resulting in each pen containing 6 birds for the final analyses. Final BW were taken on d 40, and on d 42 two birds from each pen were sampled for plasma NEFA, glucose, and muscle and liver glycogen determination at the initiation and termination of a 22 h fast. The remaining 2 fasted birds were sampled after a 30 min refeeding period. No differences were observed in feed intake, BW gain, or feed efficiency on d 21 or d 40. Liver glycogen tended (P=0.10) to be greater in Cr-supplemented chicks in the fed state, and muscle glycogen concentrations tended (P=0.07) to be greater in Cr-supplemented chicks compared with controls following fasting and refeeding. Plasma glucose concentrations were not affected by dietary Cr in the fed, fasted, or refed state. Plasma NEFA levels were not affected by treatment in fed or fasted birds. However, plasma NEFA concentrations were lower (P<0.01) in chicks supplemented with Cr than in controls following fasting and refeeding, suggesting that Cr increased insulin sensitivity. No differences were detected among birds supplemented with 0.2 or 0.4 mg Cr/kg, and among those receiving 0.4 or 0.6 mg Cr/kg. Results of this study indicate that Cr propionate supplementation of a control diet containing 0.43 to 0.45 mg Cr/kg enhanced insulin sensitivity.

Acidifier Dosage Impacts on Ammonia Concentrations and Emissions from Heavy-Broiler Houses

Broiler production results in the production of ammonia, and at high concentrations, ammonia can affect bird performance, and hence, productivity. When released into the environment through ventilation, ammonia can adversely affect public health and the environment. Because of its environmental and health impacts, ammonia emissions from animal feeding operations may be regulated by the EPA. Acidifying amendments have been shown to be effective in reducing ammonia emissions from poultry operations. The purpose of this study is to evaluate in-house ammonia concentrations and emissions from four commercial heavy-broiler houses in eastern North Carolina receiving four levels of PLT® (a commercial amendment): control (0.37 – 0.49 kg/m2 center brood area), low (0.37 – 0.49 kg/m2 whole house), medium (0.73 kg/m2 whole house), and high (>0.73 kg/m2 whole house). Ammonia concentrations were measured with acid scrubbers and ammonia emissions were calculated from exhaust ammonia concentrations and ventilation volumes. Based on monitoring of three flocks (September 2007 to May 2008), in-house ammonia-N concentrations decreased with increasing PLT application rates. A medium application rate was adequate for maintaining ammonia levels at or below 25 ppm for 9-wk grow-out period during the spring. Based on data from three flocks, ammonia emission factors (only grow-out) for the control, low, medium, and high treatments were 1.06, 1.12, 0.97, and 0.92 g/bird-d, respectively. These emission factors are mostly higher than those reported in the literature mainly because they represent heavier (>4 kg) and older (9-wk) birds fed a higher protein diet. Acid scrubbers proved to be suitable for measuring time-averaged ammonia concentrations for a wide range of values. Emissions data will be collected for four more flocks and PLT impacts on bird performance and energy use will also be quantified.

Effect of Dietary Exogenous Enzyme Supplementation on Enteric Mucosal Morphological Development and Adherent Mucin Thickness in Turkeys

Anti-nutritional factors (ANFs) in feed ingredients can challenge gut health and reduce nutrient utilization. Birds typically activate their innate immune system as a protective response against the adverse effects of ANF, which often involves the secretion of mucin. Although dietary supplementation of exogenous enzymes are commonly used to alleviate the adverse effects of ANF on apparent nutrient digestibility, little is known about how they affect gut health, particularly in relation to the morphological development and mucin secretion of enteric mucosa. We carried out two trials to examine the effect of dietary supplementation of different types of exogenous enzymes on gut health of by accessing the effect of jejunum morphological development and ileal enteric adherent mucin thickness layer in turkeys. Dietary β-mannanase supplementation reduced ileal adherent mucin thickness layer (804 vs 823 μg/g; p < 0.05), while a commercial blend of xylanase, amylase, and protease (XAP) reduced ileal adherent mucin layer thickness (589 vs 740 μg/g; p < 0.05); thus reducing the apparent endogenous loss of nutrients. Both enzyme supplements also affected gut morphological characteristics. In comparison to the control treatment, dietary β-mannanase supplementation improved the jejunum tip width (219 vs 161; p < 0.05), base width (367 vs 300; p < 0.05), surface area (509,870 vs 380, 157; p < 0.05) and villi height/crypt depth ratio (7.49 vs 5.70; p < 0.05), and XAP improved the crypt depth (p < 0.05). In conclusion, dietary supplementation of exogenous enzymes may help alleviate the adverse effects of ANF on nutrient utilization by directly or indirectly removing the mucosal irritation that stimulates enteric mucin secretion.