PCR identification and prevalence of Eimeria species in commercial turkey flocks of the Midwestern United States.

The present study used a PCR approach to characterize prevalence of coccidial species in fecal samples obtained from 40 individual Midwestern turkey flocks to characterize distribution of species in commercial flocks. Each sample was screened for 6 prominent Eimeria species using species-specific primers and was supplemented with a primary nested-PCR approach for amplification of mitochondrial cytochrome c oxidase subunit gene I where initial sample DNA concentrations were low. All samples were positive for at least one species of Eimeria, while most presented 2 (20/40) or 3 (14/40) species in total. Prevalence across farms was primarily dominated by E. meleagrimitis (97.50%), E. adenoeides (95%), and E. gallopavonis (40%). Of the samples positive for E. adenoeides and E. meleagrimitis, almost half (17/40) contained additional species. Data presented here offer insight into Eimeria species currently challenging the Midwestern US turkey industry and potential need to evaluate flocks for species prior to implementing vaccination programs.

Enteric permeability and inflammation associated with day of hatch Enterobacteriaceae inoculation.

Early exposure to Enterobacteriaceae may result in inappropriate microbial colonization of the gastrointestinal (GI) tract, induce mild GI inflammation, alter immune system development, and predispose poultry to opportunistic infection. Four experiments were conducted to test Enterobacteriaceae isolates Escherichia coli LG strain (LG), E. coli Huff strain (Huff), Salmonella Enteritidis LB (SE) and Salmonella Typhimurium (ST) on ability to induce GI inflammation. All 4 experiments included a noninoculated control, and day of hatch (DOH) oral inoculation of LG, Huff, SE and ST in experiment 1, LG and SE in experiment 2, and LG, Huff, SE, and ST in experiment 3. Experiment 4 included LG, Huff, a noninoculated control (NIC), and Clostridium perfringens only (NCP) wherein birds received oral C. perfringens challenge on d15-16 to induce necrotic enteritis. Body weight was measured, yolk sacs and spleens were collected, and blood was obtained for serum fluorescein isothiocyanate dextran (FITC-d) recovery and alpha-1-acid glycoprotein (A1GP) concentrations. Samples were taken weekly through 2 wk of age in experiments 1 and 2, or 4 wk of age in experiments 3 and 4. Increased FITC-d recovery was observed for LG and SE on d13 in experiment 2 (P < 0.05), and C. perfringens only birds on d27 in experiment 4 (P < 0.05) as compared to noninoculated controls. Each experiment resulted in notable differences in A1GP serum concentrations over time, with fluctuations in A1GP patterns through d14 based on DOH inoculation (P < 0.05). Over time, A1GP was increased for DOH inoculated birds from d 22 to 29, the fourth wk of life, and d 2-29, the entire experiment, vs. noninoculated controls in experiment 3 (P < 0.05). Similarly, NCP and LGCP showed increased A1GP from d 20 to 27 and d 6 to 27, vs. NIC in experiment 4 (P < 0.05). In experiment 4, C. perfringens challenge resulted in earlier A1GP response in DOH inoculated birds, d 17-20, as compared to NCP birds, d 20-27 (P < 0.05). These results suggest early Enterobacteriaceae exposure may influence early inflammatory state in the GI tract and may also alter patterns of inflammation and responsiveness to pathogens.

Evaluation of day of hatch exposure to various Enterobacteriaceae on inducing gastrointestinal inflammation in chicks through two weeks of age.

Inappropriate microbial colonization can induce gastrointestinal (GI) inflammation may predispose poultry to opportunistic infections and reduce growth performance. Four independent experiments were completed to test ability of select Enterobacteriaceae isolates to induce GI inflammation. Experiments 1 and 2 included a non-inoculated control (NC), and a low (L), medium (M), or high (H) day of hatch (DOH) oral inoculation level. In experiment 1, birds in L1, M1, and H1 received 102 to 104 CFU of a mixed dose of 2 species of Citrobacter and Salmonella Enteritidis LB (SE). In experiment 2, birds in L2, M2, and H2 received 103 to 105 CFU of E. coli LG (LG) and included NC. Body weight was recorded on d 0, 7, and 14, with blood collected for chicken serum alpha-1-acid glycoprotein (A1GP) measurements on d14. Neither experiment resulted in differences in BWG, however, A1GP was increased (P < 0.05) on d 14 when DOH inoculation dose 103 CFU/chick was used compared to NC. This observed increase in A1GP resulted in selection of 103 CFU/chick for DOH inoculation in experiments 3 and 4. Experiment 3 consisted of NC, E. coli Huff (Huff), and SE. On d 0, 7 and 15, BW was measured, with blood collected on d 15 for A1GP. Both d 15 A1GP and BWG from d 7 to 15 were reduced in inoculated chicks, Huff and SE, in experiment 3 (P < 0.05). Experiment 4 evaluated NC and LG with BW measured on d 0, 2, 7 and 14. Yolk sacs were evaluated for retention and bacterial enumeration, and blood for serum A1GP were collected on d 2 and 14. Experiment 4 resulted in no differences in yolk sac parameters or A1GP, whereas there was an increase in BWG for LG from d 0 to 14 (P < 0.05). When evaluated over time, serum A1GP increased between d 2 and d 14 by nearly 46% in LG, compared to negligible changes in NC (P = 0.111). Mild GI inflammation induced by early Enterobacteriaceae exposure may not drastically impact growth or inflammation parameters but may increase susceptibility to opportunistic infection necessitating further study of this model.

Effect of dietary synbiotic supplementation on performance parameters in turkey poults administered a mixed Eimeria species inoculation I.

This study examined the effects a synbiotic feed additive (PoultryStar meUS) on performance and intestinal health parameters in turkey poults administered a mixed Eimeria inoculation. The synbiotic feed additive consisted of Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, Pediococcus acidilactici and a fructo-oligosaccharide prebiotic. Dietary treatments began on day of hatch, and poults were placed on a normal starter, starter containing Clinacox, or starter containing PoultryStar until the conclusion of the experiment on day 42. In addition, on day of hatch, all poults, with exception of the negative control, were orally inoculated with Salmonella enterica Enteritidis. On day 16, poults in inoculated treatment groups received an oral dose of Eimeria adenoides and Eimeria meleagrimitis oocysts resulting in a 2 × 3 factorial arrangement of treatments. BW were measured at weekly intervals after challenge, and fecal samples were collected from all pens during day 21 to day 33 to monitor fecal shedding and calculate oocyst per gram of feces. Five day after Eimeria inoculation, inoculated PoultryStar-fed (I-PS) and inoculated Clinacox-fed (I-CL) poults, on average, weighed and gained significantly more weight (P < 0.05) than inoculated controls (I-CON) and were similar to uninoculated treatments. Between day 21 and day 28, I-PS and I-CL poults showed a 23% improvement (P < 0.001) in percent change in BW gained relative to I-CON, and overall weight gain as a percentage was similar to the uninoculated control. Overall incidence of macroscopic intestinal lesions on day 21 and day 28 was low, but I-PS and I-CL poults were generally less positive than I-CON, and no oocysts were detected in the feces of any group except I-CON which cycled as expected. From this study, it can be concluded that incorporating PoultryStar into the diet of poults reared to 6 wk ameliorates and prevents aspects of performance loss and negative impacts on gut health seen with mixed Eimeria inoculation.

Research Note: Effect of synbiotic supplementation on caecal Clostridium perfringens load in broiler chickens with different necrotic enteritis challenge models.

Studies were conducted to determine the efficacy of synbiotic applications to combat the negative effects of necrotic enteritis (NE). An in vitro study was conducted to test the effect of probiotics species supernatants to decrease Clostridium perfringens (CP) proliferation. Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, and Pediococcus acidilactici culture supernatants decreased the proliferation of CP at 1:1 supernatant-to-pathogen dilution in vitro. Two in vivo studies were conducted to determine the in vivo response of synbiotic supplementation containing the aforementioned probiotic strains on broiler production performance and caecal CP load in broilers induced with NE infection. In experiment 1, 75 broiler chicks were randomly allotted to 3 treatment groups, control (basal diet), ionophore (Salinomycin), and synbiotic (PoultryStar me), from day of hatch, and NE was induced in all birds. There were no significant treatment effects on BW, feed consumption, and feed gain ratio. However, at 35 D, ionophore or synbiotic supplementation increased (P < 0.05) villi height and decreased interleukin (IL)-1 mRNA abundance, while synbiotic supplementation increased (P < 0.05) IL-10 mRNA abundance compared with the control group, respectively. In experiment 2, 360 broiler chicks were randomly allotted to 3 treatments, an unchallenged negative control (control; basal diet), challenged positive control (NE; basal diet), or NE + synbiotic group (synbiotic). At both 21 and 42 D of age, NE birds had decreased (P < 0.05) BW, feed conversion, and jejunal villi height compared with control, while NE + synbiotic birds were not different from control groups. At 42 D of age, NE birds had 2.2 log/g increased CP in the ceca contents compared with control, while synbiotic birds had CP load that was not different than that of the control group. NE + synbiotic birds had significantly greater amounts of bile anti-CP IgA than the control and NE groups. It can be concluded that synbiotic supplementation decreased CP proliferation in vitro and caecal CP load in vivo while improving production parameters during an NE infection in broilers.

Impact of in ovo administered pioneer colonizers on intestinal proteome on day of hatch.

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 ∼102 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]-1ß, 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.

Research Note: Evaluating fecal shedding of oocysts in relation to body weight gain and lesion scores during Eimeria infection.

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.

Preliminary studies on development of a novel subunit vaccine targeting Clostridium perfringens mucolytic enzymes for the control of necrotic enteritis in broilers.

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.

Evaluation of the impact of in ovo administered bacteria on microbiome of chicks through 10 days of age.

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.

Comparison of multiple methods for induction of necrotic enteritis in broilers: II. Impact on the growth curve.

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.

Evaluation of autofluorescent Eimeria maxima oocysts as a potential indicator of non-viability when enumerating oocysts.

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.

Impact of Enteric Health and Mucosal Permeability on Skeletal Health and Lameness in Poultry.

Intestinal barrier leakage and/or altered gut microbial composition has been shown to markedly impact both osteoblast and osteoclast activities, systemically through circulation of gut immune cells and cytokines and locally by causing inflammation of extraintestinal organs such as the liver and bone marrow. Mild cases of heightened intestinal inflammation can cause bone loss in male mice in the absence of any overt nutritional deficiencies or weight loss, which has also been shown in chickens that have been infected with Salmonella. For poultry, ingredients selected for feed formulation have also a significant impact on gut health, intestinal microbiota, bone quality, and performance parameters. Consumption of diets with a high content of soluble non-starch polysaccharides (NSP) can affect bone quality parameters by reducing the amount of conjugated bile acids in the intestine, therefore diminishing the absorption of fat-soluble vitamins such as vitamin D and minerals like calcium and phosphorus. Recent enteric inflammation studies have shown that high NSP-containing diets have effects on intestinal viscosity, bone mineral content, and breaking strength, along with increased fluorescein isothiocyanate-dextran (FITC-d) leakage. Other skeletal diseases, such as bacterial chondronecrosis with osteomyelitis and enterococcal spondylitis, have a microbial component that is associated with increased mucosal permeability of the gut. Probiotics targeted toward control of enteric inflammation, either created through infectious disease or poor diet, may serve as a strategy for control of predisposing factors that lead to bone disorders.

In ovo evaluation of FloraMax®-B11 on Marek's disease HVT vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis infection in broiler chickens.

Three experiments were conducted to evaluate the effect of in ovo administration of FloraMax®-B11 (FM) on Marek's disease (MD) herpesvirus of turkeys (HVT) vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis (SE) infection in chickens. Experiment 1 consisted of 3 trials. In trials 1 and 2, d 18 White Leghorn 15I5x71 embryos were randomly distributed in 4 groups: 1) HVT vaccinated in ovo and no Marek's disease virus (MDV) challenge; 2), HVT + FM vaccinated in ovo and no MDV challenge; 3) HVT vaccinated in ovo and challenge with virulent MDV (vMDV; strain 583A); and 4), HVT + FM vaccinated in ovo and challenge with vMDV. Trial 3 was designed exactly the same as Experiment 1 but chicks were challenged with very virulent MDV (vvMDV; strains Md5 and 612). Birds were monitored until 8 wk of age, and tested for MD incidence. Experiment 2 consisted of 3 trials. In each trial, d 18 broiler embryos were injected in ovo with either saline or FM to measure hatchability and gastrointestinal bacterial composition. In Experiment 3, d 18 broiler embryos were injected in ovo with either saline or FM. All chickens that hatched were orally gavaged with SE at hatch and kept for 7 d to monitor post-hatch BW. No significant difference (P > 0.05) between MD percentage in birds vaccinated with HVT alone or HVT + FM were observed in Experiment 1. In Experiment 2, probiotic did not negatively affect hatchability, but did reduce lactose positive Gram-negative bacteria. Further, increase in BW was associated with higher villi surface area in the ileum in chickens that received the probiotic as well as a significant reduction in the SE incidence in Experiment 3. These results suggest that in ovo administration of FM does not negatively impact the ability of HVT to protect against MD or hatchability of chickens, but improves BW during the first 7 d of life and decreases SE recovery in chickens.

Effects of In Ovo feeding of dextrin-iodinated casein in broilers: I. Hatch weights and early growth performance.

This study was conducted to determine the effect of in ovo feeding of dextrin (Dext) and iodinated casein (IC) on hatch and early growth in broilers. Three experiments were conducted at a commercial hatchery using a commercial Inovoject™ system with treatments occurring in conjunction with vaccination at transfer from incubator to hatcher units (18.5 to 19 d embryonic development). In all 3 experiments, approximately 15,000 eggs (2,500 eggs per group) were treated and transferred to a single hatcher unit. Treatments in Exp. 1 consisted of buffered saline solution alone (Control, Cont) or a dextrin solution (Dext, 18% maltodextrin, 10% potato starch dextrin) containing zero, 80, 240, 720, or 2,160 µg IC/mL. The results of this initial experiment indicated that broiler chicks at hatch that received 240 and 720 µg IC/mL in Dext were heavier (P < 0.05) compared to the other treatment groups; there were no differences in hatchability between groups. Based on these findings, subsequent studies used treatments of zero, 240, and 480 µg/mL IC in Dext or Cont. In Exp. 2, hatch weights in all treatment groups were higher (P < 0.05) compared to those receiving Cont. In Exp. 3, chicks given Dext alone or 240 and 480 µg/mL in saline weighed less at hatch compared to the other treatment groups. However, chicks provided Dext alone in Exp. 3 had less weight loss after a 24-hour holding period compared to the other groups. All treatment groups exhibited greater weight gain from one to 10 d compared to the Cont group. The results indicate that in ovo feeding of broiler embryos with Dext containing 240 and 480 µg IC/mL may have beneficial effects on broiler hatch weights and early growth rate.

Effect of in ovo feeding of dextrin-iodinated casein in broilers: II. Hatch window and growth performance.

Studies were conducted using a commercial InovojectTM system to determine effects of in ovo feeding of dextrin and iodinated casein (IC) on hatch and posthatch growth in broilers. At ∼18.5 d embryonic development, eggs were treated with 0, 240, or 480 µg IC/mL in saline (Cont, IC240, and IC480) or dextrin (Dext, DextIC240 and DextIC480). The Dext solution consisted of 18% maltodextrin and 10% potato starch dextrin; saline was the vehicle used by the company for in ovo vaccination. The volume for all in ovo treatments was 50 µL/injection. Eggs in Experiment 1 were transferred to a commercial hatcher unit whereas eggs in Experiments 2 and 3 were transferred to a research hatcher unit to assess effects of treatments on timing of hatch. At hatch, chicks were randomly selected and placed in floor pens and grown to 6 wk. In Experiment 1, there were no differences in hatch weights, but broilers provided Dext IC240 in ovo were heavier (P < 0.05) at 6 wk compared to other treatments with the exception of the Dext IC240 group. In Experiment 2, hatch weights were heavier (P < 0.05) in chicks receiving IC240 and DexIC480 treatments compared to Controls. At 6 wk, broilers in all treatments were heavier (P < 0.05) than Cont with the exception of IC480. In Experiment 3, hatch was stimulated by IC240 (in saline), but was delayed by Dext IC240. Serum analysis of ß-hydroxybutyrate (µM/mL), as an indicator of ketone accumulation from fat metabolism of chicks held in chick boxes for 24 h posthatch (to simulate delay in placement after hatch), indicated that chicks in the IC240 group (that hatched earlier) had higher blood ketones compared to chicks that received Dext or DextIC240 in ovo (that hatched later). We conclude dextrin and iodinated casein (240 µg/mL) provided in ovo (∼18.5 d of embryonic development) has the potential to improve chick quality and posthatch body weight by delaying or narrowing hatch window.

Evaluation of changes in serum chemistry in association with feed withdrawal or high dose oral gavage with dextran sodium sulfate- (DSS-) induced gut leakage in broiler chickens.

Dextran sodium sulfate ( DSS: ) has been shown to be effective at inducing enteric inflammation in broiler chickens, resulting in increased leakage of orally administered fluorescein isothiocyanate dextran to circulation. In a previous study, 2 doses of DSS (0.45 g/dose) administered as oral gavage resulted in increased mucosal permeability. The main objective of the present study was to compare serum turbidity in control and DSS treated birds plus with feed restriction ( FR: ), and evaluate the associated serum chemistry. Three independent experiments were conducted with different combinations of treatment groups. In Experiment 1, control full-fed ( CON: ) and DSS full-fed ( FFD: ) with n = 15 birds/group were evaluated, Experiment 2 had groups (n = 15/group) CON, FFD, feed restriction ( FRS: for 34 h), and DSS with feed restriction ( FRD: ), and Experiment 3 (n = 15/group) had CON, FFD, and FRS (29 h FRS). All DSS treated birds received one or 2 doses of DSS by oral gavage (0.45 g/dose/bird). Results showed that, compared to CON group, there was an increase (P < 0.05) in serum turbidity in FFD birds, even though the difference between FRS and FRD was not apparent (P > 0.05). Administration of DSS did not result in increase of serum enzymes such as alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase ( LDH: ), nonetheless, the FFD showed lower (P < 0.05) LDH level compared to CON in Experiment 2. Among the various serum chemistry parameters evaluated triglycerides had the highest positive correlation (r2 = 0.85; P < 0.05) with serum turbidity. DSS administration resulted in decreased serum protein levels, especially albumin. These results suggest that oral gavage with DSS in broiler chicks could result in changes to serum chemistry parameters which could be developed as potential marker/s for gut leakage.

Evaluation of a Bacillus direct-fed microbial candidate on digesta viscosity, bacterial translocation, microbiota composition and bone mineralisation in broiler chickens fed on a rye-based diet.

1. The effects of the dietary inclusion of a Bacillus-based direct-fed microbial (DFM) candidate on digesta viscosity, bacterial translocation, microbiota composition and bone mineralisation were evaluated in broilers consuming rye-based diets. 2. In the present study, control mash rye-based diets (CON) or Bacillus-DFM supplemented diets (TRT) were administered ad libitum to male broilers in three independent experiments. 3. In Experiments 1 and 2 (n = 25/group), liver samples were taken to evaluate bacterial translocation, digesta samples were used for viscosity measurements and the intestinal microbial flora was evaluated from different intestinal sections to enumerate total recovered gram-negative bacteria (TGB), lactic acid bacteria (LAB) and anaerobic bacteria (TAB). Additionally, both tibias were removed for assessment of bone quality. 4. In Experiment 3, each experimental group had 8 replicates of 20 chickens (n = 160/group). Weekly, body weight (BW), feed intake (FI) and feed conversion ratio (FCR) were evaluated. At d 28-of-age, samples were taken to determine bacterial translocation, digesta viscosity and bone quality characteristics. 5. In all experiments, consumption of Bacillus-DFM reduced bacterial translocation to the liver and digesta viscosity. Additionally, DFM supplementation improved BW, bone quality measurements and FCR. Moreover, chickens fed on the Bacillus-DFM diet in Experiments 1 and 2 showed a significant reduction in the number of gram-negative and anaerobic bacteria in the duodenal content compared to control. 6. In summary, chickens fed on a rye-based diet without DFM inclusion showed an increase in bacterial translocation and digesta viscosity, accompanied by reduced performance and bone quality variables relative to the Bacillus-DFM candidate group. Hence, incorporation into the feed of a selected DFM ameliorated the adverse anti-nutritional effects related to utilisation of rye-based diets in broilers chickens.

Effect of dexamethasone in feed on intestinal permeability, differential white blood cell counts, and immune organs in broiler chicks.

We have previously shown that intestinal barrier function can be adversely affected by poorly digested diets or feed restriction, resulting in increased intestinal inflammation-associated permeability. Three experiments were conducted in broilers to evaluate the effect of dexamethasone (DEX) treatment on systemic fluorescein isothiocyanate-dextran (FITC-D; 3-5 kDa) levels, indicative of increased gut epithelial leakage. Experiment 1 compared DEX injections of 1 mg/kg, once per day on d 3, 5, and 9, with feed administration at 0.57, 1.7, or 5.1 ppm d 4 to 10, with FITC-D serum concentrations 2.5 h after gavage with 4.16 mg/kg FITC-D. All DEX treatments resulted in marked (2 to 6X; P<0.05) increased serum FITC-D levels. Feed DEX administration resulted in greater (P<0.05) gut permeability than injection at any dose, with numerically optimal effects at the lowest dose tested. In experiments 2 and 3, chicks were randomly assigned to a starter ration containing either control (CON) or DEX treated feed (0.57 ppm/kg; d 3 to 10 experiment 2, d 4 to 10 experiment 3). At d 10, all chicks were treated by oral gavage with FITC-D and serum samples were obtained as described above. Samples of the liver were aseptically collected, homogenized, diluted 1:4 wt/vol in sterile saline, and serial dilutions were plated on tryptic soy agar to evaluate total numbers of aerobic bacteria in the liver as an index of bacterial translocation (BT). In both experiments, FITC-D absorption was significantly enhanced (P<0.05) in DEX-treated chicks, again indicating increased paracellular leakage across the gut epithelium associated with dissolution of tight junctions. Experiment 2 differential cell counts showed an increased heterophil/lymphocyte ratio, and immune organ (spleen and bursa of Fabricius) weights for experiments 2 and 3 were decreased (P<0.05) from controls. In experiments 2 and 3, dietary DEX administration resulted in numerically (experiment 2) or significantly (P<0.05) increased enteric BT to the liver, supporting the observation that dietary DEX causes a stress-like inflammatory GI response, which may contribute to subclinical or clinical disease, and may be a useful model for ongoing disease mitigation research related to stress-related diseases of GIT origin.

Poultry enteric inflammation model with dextran sodium sulfate mediated chemical induction and feed restriction in broilers.

Gut inflammation is a cardinal event occurring in various gastrointestinal diseases regardless of etiology. A potential mechanism of action for antibiotic growth promoters and probiotics is alleviation or attenuation of such inflammation. In vivo inflammation models and markers to quantify changes in inflammation, such as paracellular leakage and tight junction function, are necessary tools in the search for methods to reduce enteric inflammation. Dextran sodium sulfate (DSS) and feed restriction (FRS), and fluorescein isothiocyanate dextran (FITC-d; 3 to 5 kDa) marker were evaluated for induction and assessment of enteric inflammation in broilers. Three independent experiments were conducted where birds received an inflammation inducer treatment and an oral gavage of FITC-d (2.2 mg/bird) 2.5 h before killing on d 4, followed by measurement of serum FITC-d levels and release of FITC-d from different regions of gastrointestinal tract (GIT) to evaluate tight junction function. Experiment 1 tested control (CON) and DSS; Experiments 2 and 3 evaluated CON, DSS, and FRS. In all experiments DSS, as well as FRS in Experiments 2 and 3, showed higher (P<0.05) leakage of FITC-d into serum than CON, but FRS was not different from DSS. The amount of FITC-d retained in duodenal and cecal tissue was affected (P<0.05) by FRS in Experiments 2 and 3, and DSS affected FITC-d retention in duodenum only, suggesting differences in gut passage or absorption/adsorption. In conclusion, DSS oral gavage and FRS could induce leaky gut, with changes in serum FITC-d and migration of FITC-d from GIT.

Dose titration of FITC-D for optimal measurement of enteric inflammation in broiler chicks.

Traditionally, antibiotic growth promoters (AGP) have been used in foodstock animals to reduce enteric inflammation and maintain intestinal homeostasis, thus improving growth and performance. Due to increasing restrictions regarding the use of AGP however, precise and high throughput enteric inflammation models and markers to search for effective alternatives are urgently needed. In this paper, oral administration of fluorescein isothiocyanate dextran (FITC-d, 3-5 kDa) and its passage into blood was used as a marker for tight junction permeability. In experiement 1, broilers were assigned to a control group, a group which received 24 h feed restriction (FR), or a group which received dextran sodium sulfate (DSS) (0.75% in water for 5 d), and each group then underwent an oral gavage of FITC-d 2.5 h before sample collection on d10. FITC-d in serum and intestinal samples (duodenum and ceca) were found to be higher (P<0.05) after FR than in the DSS and control groups. In experiment 2, FR was evaluated for its effect on mucosal leakage and an oral dose of FITC-d of 0.5, 1.1, or 2.2 mg/chick was used to measure the gastrointestinal tract (GIT) permeability at 6 d of age. The amount of FITC-d remaining in the duodenal tissue of the control birds increased with dose, only the 1.1 mg FITC-d/chick dose resulted in differences (P<0.05) between the control and FR groups. No differences were noted between the control and FR groups, regardless of FITC-d dosage in cecal recovery of FITC-d. Additionally, FR increased FITC-d serum levels when compared to the control group and in a dose-dependent manner. Experiment 3 compared serum levels after administration of 0.55 and 1.1 mg/chick doses of FITC-d in birds treated with FR, rye-based diet (RBD), and DSS. Intestinal sections were collected for FITC-d recovery in the 1.1 mg dosage group. All inflammation treatments significantly increased serum FITC-d levels at both doses. Only FR resulted in increased (P<0.05) FITC-d recovery from duodenum, ileum, and ceca. In conclusion, FR, DSS, and RBD affected GIT tight junction integrity, suggesting their value for enteric inflammation models, and FITC-d may be a good indicator of permeability.