Guillermo Tellez

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.

Effect of a Selected Lactobacillus spp.-Based Probiotic on Salmonella enterica Serovar Enteritidis-Infected Broiler Chicks

Abstract The effect of a Lactobacillus spp.–based probiotic (FM-B11TM) on Salmonella enterica serovar Enteritidis (SE) recovery was evaluated in liquid (Expt. 1) and lyophilized (Expt. 2) forms in two separate experiments with two trials each. For each trial, 80 broiler chicks were randomly allocated into two treatments: control and probiotic culture. All chicks were challenged with SE (∼104 colony-forming units [cfu]) upon arrival at our laboratory. In both experiments, probiotic culture was administered in the drinking water for 3 consecutive days at a final concentration of approximately 106 cfu/ml, beginning 1 hr after SE challenge. Cecal tonsils were aseptically removed at 24 and 72 hr postchallenge, followed by enrichment and plating on xylose lactose deoxycholate (XLD) agar for the presence or absence of Salmonella-typical colonies. In Expt. 1, a significant reduction (P < 0.05) in SE-positive samples was observed in both trials at 24 and 72 hr postchallenge. Additionally, in Expt. 2, the lyophilized probiotic decreased (P < 0.05) SE recovery at both 24 and 72 hr postchallenge compared with the control group in trial 1. In trial 2, SE evaluation was performed only at 72 hr after challenge and fewer (P < 0.001) treated samples were positive for SE. Results showed that application of either liquid or lyophilized probiotic culture in the drinking water for 3 consecutive days can help to reduce SE recovery from young birds, although further research is needed to elucidate the mechanism of this response.

Effect of a Commercial Competitive Exclusion Culture (Preempt@) on Mortality and Horizontal Transmission of Salmonella gallinarum in Broiler Chickens

A commercial competitive exclusion (CE) culture (PREEMPT) was evaluated for its ability to decrease mortality during the first 10-12 days posthatch resulting from the causative agent of fowl typhoid, Salmonella gallinarum. In the first experiment, chicks provided the CE culture on day of hatch and challenged with 10(5) S. gallinarum on day 3 had a significant decrease in mortality compared with non-CE-treated S. gallinarum-challenged chicks in all four replicates. Mortality for control chicks when averaged across all four replicates was 74% compared with 7.5% for the CE-treated chicks. A second experiment was performed that was designed to measure the efficacy of the CE culture in decreasing the horizontal transmission of this pathogen from infected to uninfected chicks when commingled. Day-of-hatch chicks that were directly infected (seeders) with 10(5) S. gallinarum and provided no CE culture averaged 86% S. gallinarum organ positive and 80% mortality during the first 12 days posthatch across four replicates. Untreated contact (not directly infected) chicks that were commingled with the seeder chicks averaged 84% S. gallinarum organ positive and 54% mortality, and the commingled CE-treated contact chicks (provided CE culture on day of hatch) average 35% S. gallinarum organ positive and 9% mortality during the same time period. These results are of importance to the poultry industry in geographic areas where poultry production is adversely affected by fowl typhoid and indicate that treating newly hatched chicks with this commercial CE culture may be a novel way of decreasing economic losses associated with this highly pathogenic organism.

Immunoprophylaxis of Salmonella enteritidis Infection by Lymphokines in Leghorn Chicks

Chickens were treated prophylactically with the soluble products from Con A-stimulated T-lymphocytes from Salmonella enteritidis-infected chickens in order to investigate the effect of such prophylactic treatment on organ invasion by S. enteritidis. At 18 days of age, chicks were injected intraperitoneally with one of the following: A) the Amicon YM 10 ultrafiltrate from immunized chickens, B) the Amicon YM 10 ultraretentate material from immunized chickens, or C) the Amicon YM 10 ultraretentate material from control nonimmune chickens. Thirty minutes after lymphokine injection, all birds were challenged per os with 10(8) colony-forming units of S. enteritidis. At both 1 and 6 days post-challenge, prophylactic treatment of chickens with the immune retentate resulted in a 51-60% reduction in S. enteritidis organ invasion. The reduction in S. enteritidis organ invasion was associated with a significant increase in lamina propria thickness based on morphometric analysis (P < 0.05). These results demonstrate that the prophylactic administration of S. enteritidis-immune lymphokines induces protection against S. enteritidis organ invasion, which is associated with a measurable microanatomical change in the cecal mucosa.

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.

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.

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.

Effect of Prolonged Administration of Dietary Capsaicin on Salmonella enteritidis Infection in Leghorn Chicks

The effect of 14 or 19 days of dietary capsaicin (18 ppm) on Salmonella enteritidis infection and histological, morphometric, and pH changes of the ceca was investigated. At day 13 or day 18, chicks were challenged with 10(8) colony-forming units of S. enteritidis. Chicks were killed and cultured 24 hours later. The total number of S. enteritidis-organ-culture-positive chicks was significantly lower among chicks fed capsaicin for either 14 or 19 days than among controls (P < 0.05). Subjective histological examination revealed a mild to moderate infiltration of mononuclear cells and heterophils in lamina propria of ceca, as well as epithelial cell proliferation in chicks following either 14 or 19 days of capsaicin administration. Using morphometric analysis, the mean lamina propria thickness and mean epithelial cell thickness in chickens fed capsaicin for 14 or 19 days were significantly greater than in controls (P < 0.05). Capsaicin significantly decreased luminal pH in both trials (P < 0.05). These data indicate that the observed capsaicin-induced resistance to S. enteritidis organ invasion is associated with measurable pH and morphological changes of the cecal mucosa.

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.

Evaluation of Salmonella-Lytic Properties of Bacteriophages Isolated from Commercial Broiler Houses

Abstract 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.