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Pioneer colonization of the gastrointestinal tract (GIT) by bacteria is thought to have major influence on neonatal tissue development. Previous studies have shown in ovo inoculation of embryos with saline (S), species of Citrobacter (C, C2), or lactic acid bacteria (L) resulted in an altered microbiome on day of the hatch (DOH). The present study investigated GIT proteomic changes at DOH in relation to different inoculations. Embryos were inoculated in ovo with S or ∼10 2  cfu of C, C2, or L at 18 embryonic days. On DOH, the GIT was collected, and tissue proteins were extracted for analysis via tandem mass spectrometry. A total of 493 proteins were identified for differential comparison with S at P ≤ 0.10. Different levels were noted in 107, 39, and 78 proteins in C, C2, and L groups, respectively, which were uploaded to Ingenuity Pathway Analysis to determine canonical pathways and biological functions related to these changes. Three members of the cytokine family (interleukin [IL]-1β, IL6, and Oncostatin M) were predicted to be activated in C2, indicated with Z-score ≥ 1.50, which suggested an overall proinflammatory GIT condition. This was consistent with the activation of the acute-phase response signaling pathway seen exclusively in C2 (Z-score = 2.00, P < 0.01). However, activation (Z-score = 2.00) of IL-13, upregulation of peroxiredoxin-1 and superoxide dismutase 1, in addition to activation of nitric oxide signaling in the cardiovascular system of the L treatment may predict a state of increased antioxidant capacity and decreased inflammatory status. The nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response (Z-score = 2.00, P < 0.01) was predicted to be upregulated in C which suggested that chicks were in an inflammatory state and associated oxidative stress, but the impact of these pathways differed from that of C2. These changes in the proteome suggest that pioneer colonizing microbiota may have a strong impact on pathways associated with GIT immune and cellular development., (Copyright © 2019. Published by Elsevier Inc.)

Coccidiosis has been a pervasive disease within the poultry industry, with test parameters used to measure effectiveness of treatment strategies often being subjective or influenced by non-disease-related activity. Four experiments were completed, which examined several test parameters of coccidiosis, including body weight gain (BWG), lesion scores, and oocysts per gram of feces (OPG). Each experiment included at least 2 parameters for measuring coccidial infection in chickens and turkeys. In experiment 1, an inoculated control was measured against 3 anticoccidial groups, whereas in experiments 2 to 4, noninoculated and inoculated controls were compared via BWG and OPG. Lesion scores were also included in experiments 1, 3, and 4. Experiment 4 resulted in high correlation, via Pearson correlation coefficient, between BWG and OPG (r = -0.69), very high correlation between OPG and lesion score (r = 0.86), and moderate correlation between BWG and lesion score (r = -0.49). Lesion scores proved to be effective in confirming Eimeria infection, although they did not correlate well with BWG or OPG. Each parameter tended to provide more useful information when lined up with the Eimeria life cycle. Incorporation of OPG, with BWG and lesion scores, as test parameters to measure coccidiosis intervention strategies, provides a global description of disease that may not otherwise be observed with the 2 latter measurements alone., (Copyright © 2019. Published by Elsevier Inc.)

Necrotic enteritis (NE) is a pervasive enteric disease responsible for large scale economic losses within the global poultry industry. The etiologic agent of NE is Clostridium perfringens (CP), an opportunistic pathogen that utilizes numerous extracellular toxins and glycoside hydrolases (GH) as key virulence and nutrient acquisition factors. Notably, some GH, mucinases, degrade components of mucin in the gastrointestinal tract as an energy source. Targeting this mechanism may serve to reduce the incidence of disease associated with CP. Two experiments were completed that evaluated mucinase vaccine targets sourced from conserved peptide sequences of carbohydrate binding module 32 of CP mucinases. In experiment 1, 37 antigen peptides were synthetically generated and used to produce hyper-immune sera, which was then evaluated for ability to obstruct CP growth in vitro. Total CFU of CP were measured at 4, 6, and 8 h incubation to determine growth rate. Peptides 4, 5, 22, 24, and 30 were selected for further in vivo testing based on conservation or the ability to inhibit CP growth by over 50% at 6 and 8 h. In experiment 2, the aforementioned peptides were conjugated to an agonistic, CD40-targetting antibody and evaluated in vivo. Broilers were given an Eimeria maxima and CP in order to induce NE and assess vaccine efficacy. Treatments included a non-vaccinated non-inoculated control, non-vaccinated inoculated control (NVIC), vaccination with peptide 4, 5, 22, 24, or 30 (VP4-VP30), or a combination of all 5 peptides (MC). There was a significant increase (P < 0.05) in the percent change in BWG relative to NVIC for vaccination with peptide 22 and MC of 18.54 and 17.43%, respectively. MC vaccinated group had the lowest lesions with a mean score of 0.63 ± 0.18. These results suggest the MC combination was the most successful in alleviating overall performance losses associated with NE-infected broilers and encourage future testing of MC in the development of an NE vaccine., (© 2019 Poultry Science Association Inc.)

Initial inoculation and colonization of the chicken gastrointestinal tract (GIT) by microbiota have been suggested to have a major influence on the growth performance and health of birds. Commercial practices in chicken production may alter or delay microbial colonization by pioneer colonizing bacteria that can have an impact on the development and maturation of the GIT and intestinal microflora. The objective of this study was to compare the impact of apathogenic Gram-negative isolates or lactic acid bacteria (LAB) as pioneer colonizers on the microbiome at the day of hatch (DOH) and evaluate the influence through 10 D of age on ceca. At 18 embryonic days (E), the amnion of embryos was inoculated with either saline (S), approximately 102 CFU of LAB (L), Citrobacter freundii (C), or Citrobacter species (C2). Once DNA was isolated from mucosal and digesta contents, samples underwent 2 × 300 paired-end Illumina MiSeq library preparation for microbiome analysis. An increased abundance of Lactobacillaceae family and Lactobacillus genus was observed in the L group at DOH (P < 0.05), whereas the abundance of Enterococcaceae and Enterococcus was numerically decreased. While Lactobacillus salivarius was one of the pioneer colonizers in the L group at 18E, the population decreased by 10 D (39.59 to 0.09%) and replaced with a population of undefined Lactobacillus (10.36%) and Lactobacillus reuteri (3.63%). Results suggest that L treatment may have accelerated a mature microbiota. Enterobacteriaceae was the dominant family (57.44%) in C group at DOH (P < 0.05). The C2 group only showed some abundance of the C2 species (7.92%) at DOH but had the highest overall abundance of undefined Lactobacillus in the ceca by 10 D (25.28%). Taken together, different isolates provided in ovo can have an impact on the initial microbiome of the GIT, and some of these differences in ceca remain notable at 10 D., (© 2019 Poultry Science Association Inc.)

Necrotic enteritis (NE) is a disease that has gained relevance in the poultry industry with both immediate and sustained effects on BW of broilers. The objective of the 3 experiments was to evaluate the impact of NE, induced by methods that reflect common broiler production systems, on the growth curve throughout the growth period. In addition, the impact of Eimeria maxima (EM) on NE, as well as the long-term impact of Clostridium perfringens (CP) on BW, were analyzed. In experiment 1, a dual infection model of EM and CP was compared to a non-challenged control, while experiment 2 evaluated 2 different strains of EM dual infection, as well as 6 CP-only groups. Similarly, experiment 3 tested dual infection and both high and low dose CP-only groups. Both NetB and non-NetB strains of CP were used to evaluate whether NetB toxin may potentially play a role in NE induction. In all 3 experiments, BW was measured immediately before infection on day 16, then weekly through the end of the test period. In all 3 experiments, a decrease (p < 0.05) in BW was observed immediately following the acute NE disease period of day 21 to day 23, with a negative impact also observed of BW gain during NE disease period (p < 0.05). A long-term effect on BW was most clearly detected in the EM + CP dual infection models, as well as when high levels of CP-only were administered. In these cases, BW was impacted long-term, with a requisite week or more to return to a BW similar to the non-challenged control. The separation in BW, though not significant, was nearly parallel with the non-challenged control throughout the growth period, indicating a shift in the growth curve. In addition to showing the long-term impact of various forms of NE on broiler growth, these shifts in the growth curve can be used to measure the effects of treatments on prevention and recovery of broilers impacted by NE., (© 2019 Poultry Science Association Inc.)

The impact of restrictive feeding programs on Salmonella and Campylobacter colonization and persistence after challenge was investigated for broiler breeder pullets housed in an experimental rearing facility. Pullet-chicks were placed on litter in 3 feeding program rooms and each room contained 2 replicate pens. The feeding programs were: (1) Skip-a-day in trough feeders (SAD); (2) Every-day in trough feeders (EDT); (3) Every-day on the pen litter (EDL). On d 1, an additional group of hatchmate chicks were housed in a separate room and gavaged with Salmonella Typhimurium, to later serve as seeder chicks. After seeders were confirmed Salmonella-positive at wk 4, at wk 5 seeders were placed into each feeding program pen to commingle with 135 penmates. At 7, 9, 11, 17, 18, and 20 wk the litter surface in each pen was sampled using intermittently stepped-on drag-swabs. At 8, 12, 16, and 20 wk of age the ceca were sampled from 10 penmates/pen and 2 pooled spleen samples/pen were collected. SAD litter remained Salmonella-positive through 20 wk of age while EDL and EDT pens had no detectible litter Salmonella recovery by 18 and 20 wk. EDL fed pens had no direct (<102 cfu/mL) litter Salmonella recovery during the entirety of the experiment. Salmonella prevalence for ceca from SAD pullets was significantly (P < 0.05) higher at 8 wk (70%) compared to EDT (40%) and EDL (30%). At wk 12, SAD pullets for both on and off-feed sampling days had significantly higher Salmonella recovery (40%), compared to EDT and EDL (both at 5% recovery). By 16 and 20 wk, only the SAD pullets on the on-feed day (48 h without feed) had recovery of Salmonella at 20%. Salmonella recovery in pooled spleen samples did not appear associated with feeding treatments (22% positive). The remaining pullets challenged with Campylobacter at 21 wk produced similar trends as was seen for Salmonella. SAD program pullets had significantly higher Campylobacter from ceca (80 to 100%) compared to pullets on EDL (30 to 60%) or EDT (40 to 95%). These results suggest that using a Skip-a-Day feeding program for broiler breeder pullets contributes to persistently higher Salmonella and Campylobacter ceca colonization and litter prevalence.

Aging, poor oxygenation, or improper storage temperature can lead to variable Eimeria oocyst viability, which is not readily assessed microscopically. Under fluorescent microscopy, some aged Eimeria maxima (EM) oocysts were strongly autofluorescent (AF) within the oocystoplasm and sporocysts, whereas others were distinctly non-fluorescent, leading to the hypothesis that non-viable oocysts may be detectible using this simple approach. Using accelerated aging conditions at 45°C, two experiments were conducted to evaluate variable percentages of autofluorescent EM oocysts on body weight gain (BWG), lesion scores (LS), and total oocyst shedding (OS) per bird. Through oral gavage, EM oocysts were administered on d10, whereas LS and BWG were determined d5 post-inoculation. Experiment 1 groups consisted of non-challenged controls (n = 15), or 25,000 EM exhibiting 12.8% (n = 14), 61.1% (n = 10), or 93.3% (n = 10) autofluorescence. BWG in 12.8% AF group was lower (P = 0.054) than non-challenged control. LS were higher (P < 0.05) in 61.1% and 12.8% AF groups as compared to non-challenged control and 93.3% AF groups. Experiment 2 groups consisted of non-challenged controls (LS n = 20/BWG n = 40), or 22,500 EM exhibiting 7% AF (LS n = 20/BWG n = 40), 80.6% AF (LS n = 19/BWG n = 39), or 99% AF (LS n = 19/BWG n = 39). BWG in 7% AF group was lower (P < 0.05) than non-challenged control and 99% AF groups. LS were higher (P < 0.05) in 80.6% and 7% AF groups as compared to non-challenged control and 99% AF groups. OS from d5-8 post-inoculation was determined for each of five replicates per group (n = 20/group; n = 4/replicate), with higher (P < 0.05) OS in 80.6% and 7% AF groups than in non-challenged control or 99% AF groups. Taken together, data indicate lower LS, higher BWG, and reduced OS with higher %AF oocysts, consistent with the hypothesis of lowered viable challenge with this EM isolate.

Three separate broiler Salmonella Typhimurium challenge experiments were conducted evaluating efficacy of formic and propionic acid feed supplements to suppress environmental and cecal Salmonella Typhimurium prevalence. In experiment 1, broilers were provided feed with 1 kg/ton formic acid or 5 kg/ton propionic acid feed additives or a basal control diet. At the day of placement, half of the pens were inoculated with seeder chicks orally challenged with a marker strain of Salmonella Typhimurium and to yield challenged and adjacent nonchallenged pens. No differences in weekly litter samples or cecal Salmonella prevalence at 3 or 6 wk among feeding treatments were detected. In experiment 2, treatments were: 2 kg/ton propionic acid in feed, 1.0 mL/L formic acid in water, both propionic acid in feed and formic acid in water, and a basal control. Every pen was challenged with seeder chicks inoculated with Salmonella Typhimurium. By 6 wk all pens maintained detectable litter Salmonella, and broilers provided both propionic acid in feed and formic acid in water had the lowest cecal recovery (35%), compared to the control (60%). In experiment 3, treatments were: formic acid at 4 or 6 kg/ton from wk 0 to 6 or for only the last wk, propionic acid at 5 or 10 kg/ton for only the last wk, and a basal control. Each pen was challenged with Salmonella Typhimurium inoculated seeder chicks. By 6 wk, broilers fed formic acid (4 kg/ton) for the entire growout had no Salmonella-positive ceca (0/30). All treatments that provided acid supplemented feed for only the last wk had 3-13% Salmonella-positive ceca. These experiments indicate that adding formic acid to broiler feed appears to prevent Salmonella colonization from challenge pens entering into the adjacent nonchallenge pens. Feeding formic acid (4 kg/ton) for 6 wk resulted in no recovery of Salmonella from ceca compared to the control prevalence of 17%., (Published by Oxford University Press on behalf of Poultry Science Association 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Broilers in the United States are typically electrically stunned using low voltage-high frequency (12-38 V, ≥400 Hz) DC or AC water bath stunners. In the European Union, however, broilers are required to be electrocuted using high voltage-low frequency (50-150 V, 50-350 Hz) AC. Low voltage stunned broilers regain consciousness in the absence of bleeding. In contrast, high voltage stunned broilers die due to induction of cardiac fibrillation. For birds stunned with low voltage systems, concerns have been raised regarding animal welfare during bleeding. This work evaluated the impact of extended DC stunning duration and alternative stunning methods (DC+AC combination) on the recovery of bird consciousness and meat quality. In the absence of bleeding, broilers that were DC stunned for extended times (60, 90, or 120 s), 63, 10, or 0% of broilers, respectively, were able to recover consciousness. Alternative stunning protocols included water bath stunning broilers at 15 or 25 V DC for 10 s followed by plate stunning at 100, 110, or 120 V AC for 5 s. Prior to shackling, live body weight and shank width were measured and during stunning, maximum mA for both DC and AC stuns were recorded. All of the alternative stunning protocols (DC+AC) resulted in non-recoverable stunning. The maximum mA recorded during both DC and AC stunning were moderately/strongly (r = 0.54-0.81) correlated to body weight and poorly/moderately (r = 0.27-0.74) correlated to shank width. No significant differences for carcass or meat quality characteristics (hemorrhages, red wing tips, broken clavicles, pH, cook loss, a* and b* color values, and MORS shear energy) were detected between control (15 or 25 V DC only) and treatment groups (DC+AC combination stunning). The only significant different meat quality parameter was L* values where the lowest voltage group (15 V DC) had the darkest fillets (53.27) and the 15 V DC+100 V AC group had the lightest fillets (55.61) with all other groups intermediate. These data indicate that stunning parameters combining DC and AC stunning may be viable protocols when a stun-to-death is desired., (Published by Oxford University Press on behalf of Poultry Science Association 2017.)

To estimate the potential for residual antimicrobial solution carryover, surface water accumulation and loss was measured on post-chill carcasses that were either dipped or sprayed with water. For all experiments, broilers were slaughtered, soft or hard scalded, defeathered, and eviscerated. Carcasses were immersion chilled, allowed to drip, and post-chill carcass weight (CW) recorded. For water dip treatment, carcasses were dipped for 0.5 min in water and hung by a wing (n = 33) or a leg (n = 30) and CW recorded at 0, 0.5, 1, 2, and 5 min post-dip. For water spray treatment, individual carcasses were hung by either the wings (n = 35) or legs (n = 34) from a shackle suspended from a scale. Water was sprayed at 80 psi and post-spray CW recorded. Initial water accumulation (0 min) for dipped carcasses was not significantly different (P > 0.05) for carcasses hung by the leg (101.0 g) or wing (108.8 g). Following the 5 min drip time, 31 g of water remained on the carcasses hung by the leg and only 10 g on carcasses hung by the wing (P < 0.05). When carcasses were sprayed with water, initial water accumulation (0 min) was 62 g for carcasses hung by the legs and 60 g for carcasses hung by the wings (P > 0.05). Following the 5 min drip time, 1 g or no water remained on the sprayed carcasses (P > 0.05). Carcasses that were dipped and hung by a leg for 5 min retained significantly more water (31 g) than carcasses that were dipped and hung by a wing (10 g) or sprayed carcasses hung either way (0.3 g) (P < 0.05). Post-chill water dip resulted in significantly higher initial carcass water accumulation than spraying (105 g vs. 61 g, P < 0.05). Carcass orientation during dripping only affected the amount of retained water for dipped carcasses. Dipped carcasses hung by a leg have the highest potential for residual carcass antimicrobial solution carryover and sprayed carcasses hung by either orientation have the lowest potential for residual antimicrobial solution carryover., (Published by Oxford University Press on behalf of Poultry Science Association 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Two experiments evaluated prebiotics added to feed on the recovery of Salmonella in broilers during grow-out and processing. In Experiment 1, "seeder" chicks were inoculated with Salmonella Typhimurium and placed with penmates. Treatments were: basal control diet, added 0.3% bamboo charcoal, 0.6% bamboo charcoal, or 0.12% Aromabiotic (medium chain fatty acids). The ceca from seeders and penmates were sampled to confirm Salmonella colonization at 3, 4, and 6 wk, and pen litter was sampled weekly. At 3 wk, charcoal fed chicks had significantly lower cecal recovery (37% lower) of Salmonella via direct plating but no differences at wk 4 or 6. At 6 wk, broilers fed Aromabiotic had no recovery of Salmonella from ceca with direct plating and significantly, 18%, lower recovery with enrichment. In Experiment 2, the treatments were: basal control diet, added 0.3% bamboo charcoal, 0.3% activated bamboo charcoal, or 0.3% pine charcoal. At placement, 2 seeders were challenged with Salmonella and commingled with penmates and ceca sampled at 1 and 2 wk, and ceca from 5 penmates/pen at 3 to 6 wk. Weekly, the pH of the crop and duodenum was measured from 1 penmate/pen and the litter surface sampled. At the end of grow-out broilers were processed. Results showed that penmates had colonized at 1 and 2 wk. Cecal Salmonella showed no differences except at 4 wk, when activated bamboo charcoal had a 18% lower recovery of Salmonella (enrichment) compared to the control (88%). Similar to Experiment 1, the recovery of Salmonella from the litter was not significantly different among treatments, however an overall decrease in recovery by 4 wk with direct plating reoccurred. The pH of the duodenum and the crop were not different among treatments. Crop pH (6.0) for all treatments were significantly higher at wk 1 compared to wk 2 to 6. Charcoals had minimal effect on Salmonella recovery in the ceca, but following defeathering, broilers fed charcoals had significantly lower Salmonella recovery from breast skin (charcoals 5+/60 compared to control 8+/20). While the addition of charcoals to broilers feed did not significantly affect Salmonella recovery during production (from litter or ceca samples) there was a lower Salmonella recovery from breast skin following scalding and defeathering., (Published by Oxford University Press on behalf of Poultry Science Association 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)