Characterization and evaluation of lactic acid bacteria candidates for intestinal epithelial permeability and Salmonella Typhimurium colonization in neonatal turkey poults.

The present study evaluated the microbiological properties of three probiotic candidate strains of lactic acid bacteria (LAB) (128; 131; CE11_2), their effect on intestinal epithelial permeability, and their ability to reduce intestinal colonization of Salmonella Typhimurium (ST) individually or as a batch culture in neonatal turkey poults. Isolates were characterized morphologically and identified using 16S rRNA sequence analyses. Each isolate was evaluated for tolerance and resistance to acidic pH, high osmotic NaCl concentrations, and bile salts in broth medium. In vitro assessment of antimicrobial activity against different enteropathogenic bacteria was determined using an overlay technique. In vitro intestinal permeability was evaluated using a stressed Caco-2 cell culture assay treated with/without the probiotic candidates. The in vivo effect of the selected LAB strains on ST cecal colonization was determined in two independent trials with neonatal turkey poults. The results obtained in this study demonstrate the tolerance of LAB candidates to pH 3, a NaCl concentration of 6.5%, and high bile salts (0.6%). All strains evaluated exhibited in vitro antibacterial activity against Salmonella Enteritidis, ST, and Campylobacter jejuni. Candidates 128 and 131 exhibited a coccus morphology and were identified as Enterococcus faecium, and bacterial strain CE11_2 exhibited clusters of cocci-shaped cells and was identified as Pediococcus parvulus. All three candidate probiotics significantly (P < 0.05) increased transepithelial electrical resistance (TEER) in Caco-2 cells following a 3-h incubation period with hydrogen peroxide compared to control and blank groups. The combination of all three candidates as a batch culture exhibited significant efficacy in controlling intestinal colonization of ST in neonatal turkey poults. Evaluation of the combination of these selected LAB strains according to performance and intestinal health parameters of chickens and turkeys are currently in process.

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.

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.

Comparison of different ELISA protocols for the detection of IgA against influenza nucleoproteins in trachea of vaccinated chickens.

Vaccines targeting mucosal immunity are important for the control of infection by pathogens with mucosal portals of entry, such as avian influenza. However, reliable and effective methods for determining levels of mucosal IgA stimulated by vaccination are not well developed in poultry and are necessary for determining efficacy. The objective of the present study was to compare different ELISA protocols to evaluate levels of mucosal IgA against two different sequences of nucleoprotein (NP:), a highly conserved internal protein in avian influenza virus, in trachea. Positive control tracheas were obtained through hyperimmunization of birds with adjuvated NP1 and NP2 peptide conjugated with keyhole limpet hemocyanin administered both orally and parenterally; negative birds received no antigen. Trachea samples were homogenized, and supernatant fluid was collected to separate IgA. ELISA was performed on NP1- or NP2-positive trachea samples, negative trachea samples, and blank wells with different levels of NP1 and NP2 coating peptides (5 or 10 µg/mL) using two different secondary antibodies (Gene Tex, GT:, or Thermo Scientific, TS:), with or without an acetate wash, and using maximum, medium, or low binding ELISA plates. The TS antibody resulted in a higher background signal compared to GT. Furthermore, coating plate wells with NP2 resulted in very high background compared to NP1. An acetate buffer wash resulted in the muffling of signals, and medium and low binding plates used in the study resulted in better results than maximum binding plates. These results suggest that the selection of appropriate secondary antibodies, binding plates, and ELISA reagent protocols all play important roles in determining NP1- or NP2-specific IgA levels in trachea samples.

Fate of Salmonella Senftenberg in broiler chickens evaluated by challenge experiments.

Experimental and epidemiological evidence has indicated the respiratory route to be a potential portal of entry for salmonellas in poultry. The purpose of this study was to evaluate and compare the infectivity of Salmonella enterica serovar Senftenberg following oral gavage, intratracheal or intravenous challenge in chickens. Seven-day-old chicks were challenged with either 10(4) or 10(6) colony-forming units of S. Senftenberg per chick by oral gavage, intratracheal or intravenous challenge, respectively, in two independent trials. Chickens were humanely killed 24 h post challenge and S. Senftenberg was cultured and enumerated from caecal contents, caecal tissue-caecal tonsils and liver and spleen. In both trials, intratracheal delivery of S. Senftenberg was the only route that allowed colonization of the caeca of chickens when compared with oral gavage or intravenous challenge in a dose response fashion (P < 0.05). Liver and spleen samples yielded no S. Seftenberg after the lower dose challenge by the oral or intratracheal route and only low levels following the high-dose administration by these routes, whereas intravenous challenge resulted in recovery of the organisms after both doses. The results of the present study suggest that S. Senftenberg entering the blood is likely to be cleared and will not be able to colonize caeca to the same extent as compared with intratracheal challenge. Clarification of the potential importance of the respiratory tract for transmission of salmonellas under field conditions may be of critical importance to develop intervention strategies to reduce the transmission in poultry.

Evaluation of the respiratory route as a viable portal of entry for Salmonella in poultry via intratracheal challenge of Salmonella Enteritidis and Salmonella Typhimurium.

Experimental and epidemiological evidence suggests that primary infection of Salmonella is by the oral-fecal route for poultry. However, the airborne transmission of Salmonella and similar enteric zoonotic pathogens has been historically neglected. Increasing evidence of Salmonella bioaerosol generation in production facilities and studies suggesting the vulnerabilities of the avian respiratory architecture together have indicated the possibility of the respiratory system being a potential portal of entry for Salmonella in poultry. Presently, we evaluated this hypothesis through intratracheal (IT) administration of Salmonella Enteritidis and Salmonella Typhimurium, as separate challenges, in a total of 4 independent trials, followed by enumeration of cfu recovery in ceca-cecal tonsils and recovery incidence in liver and spleen. In all trials, both Salmonella Enteritidis and Salmonella Typhimurium, challenged IT colonized cecae to a similar or greater extent than oral administration at identical challenge levels. In most trials, chickens cultured for cfu enumeration from IT-challenged chicks at same dose as orally challenged, resulted in an increase of 1.5 log higher Salmonella Enteritidis from ceca-cecal tonsils and a much lower dose IT of Salmonella Enteritidis could colonize ceca to the same extent than a higher oral challenge. This trend of increased cecal colonization due to IT challenge was observed with all trails involving week-old birds (experiment 2 and 3), which are widely considered to be more difficult to infect via the oral route. Liver-spleen incidence data showed 33% of liver and spleen samples to be positive for Salmonella Enteritidis administered IT (10(6) cfu/chick), compared with 0% when administered orally (experiment 2, trial 1). Collectively, these data suggest that the respiratory tract may be a largely overlooked portal of entry for Salmonella infections in chickens.

Evaluation of selected direct-fed microbial candidates on live performance and Salmonella reduction in commercial turkey brooding houses.

As effective probiotic Bacillus isolates that can increase BW gain (BWG) are identified, they may offer advantages in terms of stability, cost, and feed application over probiotics limited to drinking water application. Additionally, an effective direct-fed microbial (DFM) may offer an effective alternative to antibiotic growth promoters. Previously, 4 Bacillus isolates were identified and evaluated in our laboratory as potential DFM candidates. These isolates were shown to significantly increase BWG as well as reduce recovery of Salmonella after experimental infection. In the first experiment, isolates PHL-MM65 (a Bacillus laterosporus) and PHL-NP122 (a Bacillus subtilis) were evaluated using poults raised under commercial conditions. After 7 d of conventional brooding, poults were tagged, weighed, and placed in 1 of 4 replicate pens for each treatment group [negative control, 0.019% nitarsone, PHL-MM65 (10(6) spores/g of feed), or PHL-NP122 (10(6) spores/g of feed)] within the commercial turkey barn. At 23 d, poults were weighed and BW was calculated. Treatment with PHL-NP122 (853 g) or nitarsone (852 g) increased BW (P ≤ 0.05) compared with control (784 g), whereas treatment with PHL-MM65 (794 g) did not significantly improve BW. Also on d 23 of the trial, ceca were aseptically removed from 10 poults per pen and cultured for recovery of Salmonella. Both Bacillus isolates PHL-NP122 and PHL-MM65 resulted in a significant reduction (P ≤ 0.05) in the frequency of Salmonella by more than 25% compared with the controls. In a second experiment on a different farm, isolates PHL-NP122, PHL-RW33 (a B. subtilis), and PHL-B1 (a Bacillus licheniformis) were evaluated. None of the candidate Bacillus DFM or the group fed nitarsone had significantly different BW or BWG than untreated control. These data suggest that isolate PHL-NP122, when added as a DFM to turkey diets, may increase BW gain as well as nitarsone during the brooding phase of commercial turkey production.

The role of an early Salmonella Typhimurium infection as a predisposing factor for necrotic enteritis in a laboratory challenge model.

Necrotic enteritis (NE) caused by Clostridium perfringens (CP) in poultry is an important bacterial disease in terms of economic implications. The disease is multifactorial and is invariably associated with predisposing factors. In the present experiments, we investigated the potential predisposing role of neonatal Salmonella Typhimurium (ST) infection for NE-associated mortality in a laboratory challenge model. In two experiments, day-of-hatch chicks were randomly assigned to four groups: Group 1, nonchallenged control; Group 2, chickens received Eimeria maxima (EM) and CP; Group 3, chickens received EM and CP and were also challenged with ST at day 1 of age; Group 4, chickens received EM and CP and were also challenged with ST at day 17 of age. Challenged groups received an oral dose of EM at 18 days of age and CP (10(8) colony-forming units/chick) at 22-23 days of age. When compared to EM and CP, chicks challenged with ST (day 1) had increased NE-associated mortality and CP-associated lesion scores (P < 0.05) in both experiments. Furthermore, body weight and body weight gain were lower (P < 0.05) in chicks infected with ST (day 1) in the first experiment, even though no differences (P > 0.05) were observed in weight gain in the second experiment. Chicks challenged with ST (day 17) were similar to the EM and CP group in all of the above-mentioned parameters, indicating that a paratyphoid infection in younger chicks remarkably alters the susceptibility to secondary bacterial infections. Based on this work, the authors suggest that an ST infection early in the age of a chick may be important for altering susceptibility to NE, an observation that may be useful from the perspective of experimental reproduction of this disease and, perhaps, as an economically important reason to address the problem of paratyphoid Salmonella infections in young chicks.

Evaluation of Bacillus species as potential candidates for direct-fed microbials in commercial poultry.

Increasing sociopolitical concerns with antibiotic use have led to investigations of potential alternatives for food safety and growth promotion. Direct-fed microbials (DFM) including spore-based probiotics are amenable to feed inclusion and are extremely stable. We isolated several Bacillus spp. from environmental and poultry sources and tested them for their ability to reduce Salmonella in vitro. In a preliminary in vivo trial, day-of-hatch chicks and poults were randomly assigned to the following treatments (24 birds/treatment): control and one of 8 DFM candidates at 10(6) spores/g of feed. Chicks and poults were tagged, weighed, and orally challenged with Salmonella Typhimurium (ST). Body weight gain and ST recovery were measured 11 d posthatch. Total percentages of ST-positive crop and ceca were significantly lower (P < 0.05) in at least 3 DFM candidates compared with control. Additionally, beneficial effects on BW gain were observed in at least 5 DFM candidates (P < 0.05) compared with control. In a second study, birds treated with NP122 (identified as Bacillus subtilis) had significantly lower (P < 0.05) cecal ST than control and benefitted BW gain irrespective of the presence or absence of a Salmonella challenge. In conclusion, NP122 markedly reduced ST recovery and increased BW gain in both chicks and poults. This provides preliminary evidence that this isolate may have potential use as a DFM in poultry.

Transcriptional profiling of cecal gene expression in probiotic- and Salmonella-challenged neonatal chicks.

Probiotics are currently used to improve health and reduce enteric pathogens in poultry. However, the mechanisms by which they reduce or prevent disease are not known. Salmonella are intracellular pathogens that cause acute gastroenteritis in humans, and infections by nontyphoid species of Salmonella also can result in diarrhea, dehydration, and depression in poultry. Frequently, however, no clinical signs of infection are apparent in poultry flocks. In this study, day-of-hatch chicks were challenged with Salmonella enterica serovar Enteritidis (SE) and treated 1 h later with a poultry-derived, Lactobacillus-based probiotic culture (FloraMax-B11, Pacific Vet Group USA Inc., Fayetteville, AR). Cecae were collected 12 and 24 h posttreatment for Salmonella detection and RNA isolation for microarray analysis of gene expression. At both 12 and 24 h, SE was significantly reduced in chicks treated with the probiotic as compared with the birds challenged with only SE (P < 0.05). Microarray analysis revealed gene expression differences among all treatment groups. At 12 h, 170 genes were expressed at significantly different levels (P < 0.05), with a minimum difference in expression of 1.2-fold. At 24 h, the number of differentially regulated genes with a minimum 1.2-fold change was 201. Pathway analysis revealed that at both time points, genes associated with the nuclear factor kappa B complex, as well as genes involved in apoptosis, were significantly regulated. Based on this analysis, probiotic-induced differential regulation of the genes growth arrest-specific 2 (GAS2) and cysteine-rich, angiogenic inducer, 61 (CYR61) may result in increased apoptosis in the cecae of chicks. Because Salmonella is an intracellular pathogen, we suggest that increased apoptosis may be a mechanism by which the probiotic culture reduces Salmonella infection.

Effect of lactic acid bacteria probiotic culture for the treatment of Salmonella enterica serovar Heidelberg in neonatal broiler chickens and turkey poults.

In the present study, a series of experiments was conducted to evaluate the ability of a commercial probiotic culture (FloraMax, IVS-Wynco LLC, Springdale, AR) to reduce Salmonella enterica serovar Heidelberg (SH) in chicks and turkey poults. In experiments 1 and 2, chicks were randomly assigned to treatment groups and then challenged via oral gavage with SH. Chicks were treated 1 h following SH challenge with the probiotic culture via oral gavage. At 24 and 72 h posttreatment, cecal tonsils and ceca were collected for recovery and enumeration of enteric Salmonella Heidelberg, respectively. In experiment 3, day-of-hatch turkeys were randomly assigned to treatment groups and then challenged via oral gavage with SH. Poults were treated 1 h following challenge with the probiotic via oral gavage. At 24 and 72 h post probiotic treatment, cecal tonsils and ceca were collected for recovery and enumeration of enteric SH, respectively. The probiotic culture significantly reduced the incidence of SH in cecal tonsils at both time points in chicks in both experiments (P < 0.05). These data demonstrate that administration of probiotic 1 h post SH challenge significantly reduced the incidence of SH recovery from cecal tonsils of chicks compared with controls 24 and 72 h following treatment. Similarly, probiotic treatment resulted in significant reductions in the concentrations of SH within the ceca in both experiments. Although similar significant results were observed at both 24 and 72 h in experiment 3, it was clear that poults were more susceptible to SH colonization than chicks. Overall, a Lactobacillus-based probiotic significantly reduced Salmonella enterica serovar Heidelberg in chicks and turkey poults.

Development and evaluation of candidate recombinant Salmonella-vectored Salmonella vaccines.

Attenuated Salmonella Enteriditis (ΔSE) recombinant vaccine vectors incorporating a Salmonella flagellar filament protein (fliC) subunit, a putative cell-mediated epitope, for expression of the lamB gene (encoding a maltose outer membrane porin), with or without co-expression of a putative immune-enhancing CD154 oligopeptide, were developed and compared with wild-type Salmonella Enteriditis (experiments 1 and 2) or the attenuated ΔSE empty vector (experiment 3) as initial vaccine candidates against Salmonella infection. A total of 3 experiments were performed to assess the infection and clearance rate of each of these constructs. Each construct or Salmonella Enteriditis was orally administered to broiler chicks at day of hatch by oral gavage (~10(8) cfu/chick). In experiments 1 to 3, liver-spleen and cecal tonsils were removed aseptically for recovery of wild-type Salmonella Enteriditis or ΔSE mutants. These experiments suggested that cell surface expression of fliC alone markedly increased the clearance rate of the vector at or before 21d postvaccination in all 3 experiments. In a fourth experiment, broilers were vaccinated with one of the vaccine constructs or the ΔSE empty vector and then challenged with wild-type Salmonella Typhimurium. At 19 d posthatch, 16 d postinfection, neither candidate protected against challenge significantly better than the ΔSE empty vector, although there was significantly less Salmonella recovered from vaccinated chickens as compared with nonvaccinated controls. These experiments indicate that these experimental vaccines did not protect against heterologous challenge or enhance clearance after Salmonella Typhimurium challenge; as such, their value as vaccines is limited. The increased clearance of the candidate vaccines, particularly the vector expressing fliC alone, may have value in that the fliC epitope may decrease the clearance time of other recombinant vectored Salmonella vaccines.

Effect of lactic acid bacteria probiotic culture treatment timing on Salmonella Enteritidis in neonatal broilers.

In the present study, a series of experiments were conducted to evaluate the ability of a combination of 3 ATCC lactobacilli (LAB3) or a commercially available probiotic culture (PROB) to reduce Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) in broiler chicks. Additionally, we varied the timing of PROB administration in relationship to Salmonella challenge and determined the influence on recovery of enteric Salmonella. In experiments 1 to 3, chicks were randomly assigned to treatment groups and were then challenged via oral gavage with Salmonella Enteritidis. Chicks were treated 1 h after Salmonella Enteritidis challenge with LAB3 or PROB. Twenty-four hours posttreatment, cecal tonsils were collected for recovery of enteric Salmonella. In experiments 4 to 7, day-of-hatch chicks were randomly assigned to treatment groups and were then treated with PROB via oral gavage and placed into pens. Chicks were challenged with Salmonella Enteritidis 24 h after treatment via oral gavage. At 24 h after Salmonella Enteritidis challenge, cecal tonsils were collected and recovery of enteric Salmonella was determined. In experiments 8 to 10, 1-d-old chicks were randomly assigned to treatment groups and were then challenged via oral gavage with Salmonella Enteritidis and placed into pens. Chicks were treated 24 h after challenge with PROB via oral gavage. Twenty-four hours post PROB treatment, cecal tonsils were collected and enriched as described above. It was found that PROB significantly reduced cecal Salmonella Enteritidis recovery 24 h after treatment as compared with controls or LAB3-treated chicks in experiments 1 to 3 (P<0.05). Administration of PROB 24 h before Salmonella Enteritidis challenge significantly reduced recovery of Salmonella Enteritidis in 2 out of 4 experiments and no reduction in cecal Salmonella Enteritidis was observed when chicks were challenged with Salmonella Enteritidis and treated 24 h later with PROB. These data demonstrate that PROB more effectively reduced Salmonella Enteritidis than LAB3, and the timing of PROB treatment affects Salmonella Enteritidis-associated reductions.

Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge.

Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10(6) to 10(8) cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, DeltaaroA-DeltahtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain DeltaaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, and DeltaaroA-DeltahtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with DeltaaroA-DeltahtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.

Comparison of beak-trimming methods on early broiler breeder performance.

Beak trimming is necessary in commercial broiler breeders to prevent or decrease trauma as they mature. Two common beak-trimming methods were evaluated by early performance comparison with nontrimmed chicks (NBT). The robotic electrocautery device (ECD) trims and cauterizes the beak tip. The robotic infrared beak-trimming device (IBT) applies an infrared light beam to destroy the live basal tissue while leaving the hard corneum intact for the first approximately 10 d. In 2 experiments, day-of-hatch Ross 708 by-product chicks were obtained from a local hatchery, where 1/3 of the chicks were trimmed using IBT. All chicks were then transported to another hatchery where 1/3 were trimmed using ECD and 1/3 were NBT. Personnel at each hatchery were highly experienced and skilled with their respective technique. All chicks were then transported to University of Arkansas facilities. Before placement in each experiment, chicks were individually neck-tagged and weighed, and in experiment 1, beaks were measured using a digital caliper. A small but significant transient reduction in BW gain was observed at 14 d due to ECD as compared with NBT controls, although ECD was not different than IBT in experiment 1. In experiment 2, IBT birds were significantly heavier at 11 d by 7.8 and 8.7 g than the NBT or ECD, respectively. However, at d 21 and 42, no significant differences in BW or BW gain were observed. When beak trimming was performed on day of hatch by skilled and experienced personnel, little measurable effect on early performance was observed during the first 6 wk of life. Decreased broiler performance is generally considered a sensitive indication of physical or psychogenic stress. Given the marked reduction in beak-inflicted trauma with beak trimming birds as they reach sexual maturity, these results suggest that when properly performed, neither of these beak-trimming methods causes sufficient physical or psychogenic stress to markedly affect early growth rate.

Evaluation of Salmonella-lytic properties of bacteriophages isolated from commercial broiler houses.

Because of recent interest in bacteriophage therapy in poultry, information regarding the interaction of bacteriophages and potential host bacteria in the environment should be collected. The present studies were initiated with a rather typical commercial broiler integrator within the south-central United States to examine environmental Salmonella levels in two broiler complexes, attempt to isolate Salmonella-lytic bacteriophages, and elucidate a possible reason for differing apparent Salmonella prevalence. Significantly (P < 0.05) less Salmonella was isolated from houses in complex 1 (15/44 [34%] Salmonella-positive drag swabs) as compared to houses in complex 2 (22/24 [92%]). A total of seven Salmonella-lytic bacteriophages were isolated from Salmonella-positive environments, and two bacteriophages were isolated from a single Salmonella-negative house. During the initial bacteriophage isolation, individual bacteriophages did not replicate in the Salmonella host isolated from the same environment, and lysis of additional Salmonella hosts relied on high numbers of bacteriophage to be present. This suggests that the presence of these bacteriophages in the environment of a commercial broiler house had little to no effect on the presence of Salmonella. This study highlights the need to find additional bacteriophage sources, more effective isolation methods, and more innovative approaches to using bacteriophages to treat enteric disease.

Evaluation of a Lactobacillus-based probiotic culture for the reduction of Salmonella enteritidis in neonatal broiler chicks.

We evaluated the effect of a Lactobacillus-based probiotic culture (FM-B11) for reduction of Salmonella Enteritidis in neonatal broiler chicks. In all experiments, chicks were challenged with approximately 10(4) cfu of Salmonella Enteritidis upon arrival at our laboratory, and the treatments were administered 1 h postchallenge. Cecal tonsil samples were obtained 24 h posttreatment and enriched for Salmonella Enteritidis recovery. The first experiment compared the effects of oral administration of doses of 10(4), 10(6), and 10(8) cfu/chick. In this experiment, doses of 10(6) and 10(8) both significantly reduced Salmonella Enteritidis recovery compared with controls (15 vs. 85% Salmonella Enteritidis positive), but 10(4) cfu did not significantly reduce Salmonella Enteritidis recovery. The second experiment compared the efficacy of oral administration of the live probiotic culture, with or without supernatant removed, to inactivated cultures or supernatant alone. Live probiotic organisms, with or without supernatant, significantly reduced Salmonella Enteritidis in this experiment, but inactivated or cell-free treatments did not reduce Salmonella Enteritidis. In the final 2 experiments, differing doses of probiotic culture were administered on the vent lips, where the treatment was taken into the lower gastrointestinal tract via cloacal drinking. Concentrations of probiotic culture from 10(2) to 10(7) cfu/chick significantly reduced Salmonella Enteritidis, and there was no difference in Salmonella Enteritidis recovery between treatment concentrations. These data suggest that this Lactobacillus-based probiotic culture may be efficacious for reduction of Salmonella Enteritidis in neonatal chicks.

Effect of probiotic treatment in broiler chicks on intestinal macrophage numbers and phagocytosis of Salmonella enteritidis by abdominal exudate cells.

Previous data have indicated that a Lactobacillus-based probiotic culture (FM-B11) is efficacious in reducing Salmonella Enteritidis colonization within 24 h when administered within 1 h of challenge. We hypothesized that the innate immune system, specifically macrophages, may play a role in the observed reduction of Salmonella Enteritidis colonization with probiotic treatment. Day-of-hatch chicks were challenged with Salmonella Enteritidis and then treated with the probiotic culture 1 h later. Three other treatment groups were not treated (negative control), challenged only, or treated with probiotic only. In all experiments, probiotic treatment on the day of hatch reduced (P < 0.05) cecal Salmonella Enteritidis recovery as compared with the control treatment. In experiments 1 and 2, immunohistochemistry was used to evaluate the presence of macrophages (KUL01+) in the ileum and cecum of 7 to 10 chicks per group at 24 h posttreatment. In experiment 1, the number of macrophages observed per 10,000 microm(2) in the ileum of Salmonella Enteritidis-challenged chicks was higher (P < 0.05) than that of nonchallenged chicks (4.87 +/- 0.31 vs. 3.05 +/- 0.19). In the cecum, there were more (P < 0.05) macrophages per 10,000 microm(2) in chicks receiving probiotic treatment without challenge than in negative control chicks (5.32 +/- 0.41 vs. 3.66 +/- 0.35). However, in experiment 2 we found no differences among treatments in the numbers of macrophages for both the ileum and cecum. Experiments 3 and 4 were performed to evaluate the ability of Sephadex-elicited abdominal exudate cells (AEC) from chicks to phagocytose Salmonella Enteritidis in vitro. Abdominal exudate cells were isolated from the abdominal cavity, maintained in tissue culture plates overnight, and then assayed for phagocytic activity by coincubating with Salmonella Enteritidis. In experiment 3, more (P < 0.05) Salmonella Enteritidis was recovered from AEC derived from probiotic-treated chicks than in any other treatment. However, in experiment 4, all treatments resulted in similar levels of elicited AEC, and phagocytosis of Salmonella Enteritidis was at low levels in all groups. Although not conclusive, the modest differences detected in experiments 1 and 3, and the fact that those differences were not repeatedly detectable, suggest that these macrophage-related changes were not solely responsible for the reductions of Salmonella Enteritidis following probiotic treatment.