Research on the Effect of Pediococcus pentosaceus on Salmonella enteritidis-Infected Chicken.
ABSTRACT: Salmonella enteritidis can cause significant morbidity and mortality in humans and economic loss in the animal industry. Improving the innate immunity is an effective method to prevent S. enteritidis infection. Pediococcus pentosaceus is a Gram-positive coccus which had probiotics properties. Numerous previously published studies reported that probiotics were beneficial to gut microbiota by changing the intestinal flora structure and inhibiting the harmful microbial growth to enhance the innate immunity. We investigated the immunological effects of P. pentosaceus on Salmonella-infected chickens by the following experiment. A total of 120 broilers from AA line were fed and divided into 2 groups (treated and control groups) for the experiment from day 1. The control group was fed with the basic diet, while the treated group was fed with the basic diet adding P. pentosaceus microcapsule with the bacterial concentration of 1?g/kg in the feed and bacterial counts 2.5 × 109?CFU/g. All the birds were given with 0.5?ml of S. enteritidis bacterial suspension (109?CFU/ml) through oral cavity at day 9. The number of dead birds was recorded and used in the analysis. The bacterial culture method and quantitative real-time PCR analysis were used to evaluate the effects of P. pentosaceus on chickens infected with S. enteritidis and to ascertain the mechanism of the effect. The results showed that the P. pentosaceus could restrain the pathogenicity of S. enteritidis and reduce the death rate from 44.4% to 23.3%. The flora in the caecum exhibited "rising-declining" trends, and the gene (TLR4, MyD88, TRAF6 NF-?B, IFN-?, TNF-a, IL6, and IL8) expression pattern was different between the experimental and control group. P. pentosaceus as a probiotic may competitively inhibit the growth of S. enteritidis and control the inflammatory response through regulating the gene expression which involved in the toll-like receptor pathway and inflammation pathway.
Project description:Restrictions of in-feed antibiotics use in poultry has pushed research toward finding appropriate alternatives such as Direct-Fed Microbials (DFM). In this study, previously tested Bacillus isolates (B. subtilis and B. amyloliquefaciens) were used to evaluate their therapeutic and prophylactic effects against Salmonella enterica serovar Enteritidis (S. Enteritidis) in broiler chickens. For this purpose, initial antibacterial activity of Bacillus-DFM (104 spores/g or 106 spores/g) against S. Enteritidis colonization in crop, proventriculus and intestine was investigated using an in vitro digestive model. Furthermore, to evaluate therapeutic and prophylactic effects of Bacillus-DFM (104 spores/g) against S. Enteritidis colonization, altogether 60 (n = 30/group) and 30 (n = 15/group) 1-day-old broiler chickens were randomly allocated to either DFM or control group (without Bacillus-DFM), respectively. Chickens were orally gavaged with 104 cfu of S. Enteritidis per chicken at 1-day old, and cecal tonsils (CT) and crop were collected 3 and 10 days later during the therapeutic study, whereas they were orally gavaged with 107 cfu of S. Enteritidis per chicken at 6-day-old, and CT and crop were collected 24 h later from two independent trials during the prophylactic study. Serum superoxide dismutase (SOD), FITC-d and intestinal IgA levels were reported for both chicken studies, in addition cecal microbiota analysis was performed during the therapeutic study. DFM significantly reduced S. Enteritidis concentration in the intestine compartment, and in both proventriculus and intestine compartments as compared to the control when used at 104 spores/g and 106 spores/g, respectively (p < 0.05). DFM significantly reduced FITC-d and IgA as well as SOD and IgA levels (p < 0.05) compared to the control in therapeutic and prophylactic studies, respectively. Interestingly, in the therapeutic study, there were significant differences in bacterial community structure and predicted metabolic pathways between DFM and control. Likewise, phylum Actinobacteria and the genera Bifidobacterium, Roseburia, Proteus, and cc_115 were decreased, while the genus Streptococcus was enriched significantly in the DFM group as compared to the control (MetagenomeSeq, p < 0.05). Thus, the overall results suggest that the Bacillus-DFM can reduce S. Enteritidis colonization and improve the intestinal health in chickens through mechanism(s) that might involve the modulation of gut microbiota and their metabolic pathways.
Project description:An eight-week feeding trial was conducted to evaluate the effects of different dietary probiotic supplements in juvenile whiteleg shrimp, Litopenaeus vannamei. A basal control diet without probiotics (CON), and five other diets by supplementing Bacillus subtilis at 107 CFU/g diet (BS7), B. subtilis (BS8), Pediococcus pentosaceus (PP8), and Lactococcus lactis (LL8) at 108 CFU/g diet, and oxytetracycline (OTC) at 4 g/kg diet were used. Whiteleg shrimp with initial body weights of 1.41 ± 0.05 g (mean ± SD) were fed with these diets. Growth of shrimp fed BS8 and LL8 diets was significantly higher than those of shrimp fed the CON diet (p < 0.05). Superoxide dismutase activity in shrimp fed PP8 and LL8 diets was significantly higher than that of shrimp fed the CON diet (p < 0.05). Lysozyme activity of shrimp fed probiotics and OTC diets significantly improved compared to those on the CON diet (p < 0.05). The intestinal histology showed healthier guts for shrimp fed the probiotic diets (p < 0.05). Immune-related gene expression in shrimp fed BS8, PP8 and LL8 diets was recorded as significantly higher than that of shrimp fed CON and OTC diets (p < 0.05). Also, results of the challenge test for 7 days and the digestive enzyme activity of shrimp fed BS8, PP8, and LL8 were significantly improved compared to those on the CON diet (p < 0.05). Therefore, these results indicated that L. lactis at 108 CFU/g could be an ideal probiotic for whiteleg shrimp, and also B. subtilis WB60 and P. pentosaceus at 108 CFU/g could improve the growth, immunity, histology, gene expression, digestive enzyme activity, and disease resistance, while replacing antibiotics.
Project description:The efficacies of trans-cinnamaldehyde (TC) and eugenol (EG) for reducing Salmonella enterica serovar Enteritidis colonization in broiler chickens were investigated. In three experiments for each compound, 1-day-old chicks (n = 75/experiment) were randomly assigned to five treatment groups (n = 15/treatment group): negative control (-ve S. Enteritidis, -ve TC, or EG), compound control (-ve S. Enteritidis, +ve 0.75% [vol/wt] TC or 1% [vol/wt] EG), positive control (+ve S. Enteritidis, -ve TC, or EG), low-dose treatment (+ve S. Enteritidis, +ve 0.5% TC, or 0.75% EG), and high-dose treatment (+ve S. Enteritidis, +ve 0.75% TC, or 1% EG). On day 0, birds were tested for the presence of any inherent Salmonella (n = 5/experiment). On day 8, birds were inoculated with ∼8.0 log(10) CFU S. Enteritidis, and cecal colonization by S. Enteritidis was ascertained (n = 10 chicks/experiment) after 24 h (day 9). Six birds from each treatment group were euthanized on days 7 and 10 after inoculation, and cecal S. Enteritidis numbers were determined. TC at 0.5 or 0.75% and EG at 0.75 or 1% consistently reduced (P < 0.05) S. Enteritidis in the cecum (≥3 log(10) CFU/g) after 10 days of infection in all experiments. Feed intake and body weight were not different for TC treatments (P > 0.05); however, EG supplementation led to significantly lower (P < 0.05) body weights. Follow-up in vitro experiments revealed that the subinhibitory concentrations (SICs, the concentrations that did not inhibit Salmonella growth) of TC and EG reduced the motility and invasive abilities of S. Enteritidis and downregulated expression of the motility genes flhC and motA and invasion genes hilA, hilD, and invF. The results suggest that supplementation with TC and EG through feed can reduce S. Enteritidis colonization in chickens.
Project description:This study was performed to investigate the differential expression of eight immunity genes and the bacterial profiles in the caecum of growing chickens challenged with Salmonella enterica serovar Enteritidis (SE) at 1 and 23 days post inoculation (dpi) in response to SE infection at 19 days of age and administration of the phytobiotic Intebio. Following infection, the genes CASP6 and IRF7 were upregulated by greater than twofold. Chicks fed Intebio showed at 1 dpi upregulation of AvBD10, IL6, IL8L2, CASP6 and IRF7. At 23 dpi, expression of AvBD11, IL6, IL8L2, CASP6 and IRF7 lowered in the experiment subgroups as compared with the control. Examination of the caecal contents at 1 dpi demonstrated a significant decrease in the microbial biodiversity in the infected subgroup fed normal diet. Bacterial content of Lactobacillus and Bacillus declined, while that of Enterobacteriaceae rose. In the infected subgroup fed Intebio, a pronounced change in composition of the microflora was not observed. In the early infection stages, the phytobiotic seemed to promote response to infection. Subsequently, an earlier suppression of the inflammatory reaction took place in chickens fed Intebio. Thus, use of Intebio as a drug with phytobiotic activity in chickens, including those infected with Salmonella, proved to be promising.
Project description:Salmonella Enteritidis is vertically transmitted to eggs from laying hens through infected ovaries and oviducts. S. Enteritidis can also penetrate the eggshell from contaminated feces. Reducing S. Enteritidis in laying hens is vital to provide safer eggs and minimize the spread of salmonellosis to humans. Antibiotics have been widely used to control bacterial diseases in broilers and laying hens. However, there is a major concern that the use of antibiotics leads to the development of antibiotic resistance and adverse effects on microbiota of the treated birds. Thus, there is an interest in developing alternatives to antibiotics, such as dietary prebiotics. In the present study, feed supplemented with the red seaweeds: Chondrus crispus (CC) or Sarcodiotheca gaudichaudii (SG), was offered to laying hens late in production to control S. Enteritidis. Diets contained one of the following; 2% or 4% Chondrus crispus (CC2, and CC4, respectively) or Sarcodiotheca gaudichaudii (SG2 and SG4, respectively). Chlortetracycline was used in the positive control diet. During week-4, 48 birds were orally challenged with 2 × 109 CFU/mL of S. Enteritidis. Eggs and fecal samples were collected 1, 3, 5, and 7 days' post inoculation. Birds were euthanized and organs (ceca, ovary, liver, and spleen) were sampled and analyzed for the presence of S. Enteritidis, 7 days' post inoculation. Results showed that seaweed reduced the negative effect on body weight and egg production in S. Enteritidis-challenged laying hens. Analysis of fecal samples showed that the antibiotic (CTC) reduced S. Enteritidis in the intestinal tract and fecal samples, 3 days' post inoculation. Fecal samples from Chlortetracycline and CC4 supplemented birds tested negative for S. Enteritidis on days 5 and 7 post inoculation (lowest detection limit = 10-1). S. Enteritidis colonization in the ceca was also significantly reduced in birds fed CC (4%) and Chlortetracycline. Blood serum profiles revealed that there were no significant differences in serum aspartate aminotransferase (AST) and sodium. However, the level of serum immunoglobulin (IgA) was higher in the CC4 treatment. The relative abundance of Lactobacillus acidophilus was significantly higher in CC4 while, the abundance of the pathogenic bacteria, Clostridium perfringens and Salmonella Enteritidis were reduced compared to control. Results indicate that feed supplemented with 4% CC is effective in providing protection against Salmonella Enteritidis colonization in laying hens.
Project description:The purpose of this study was to evaluate the antibacterial activity and safety of bacterias with probiotic potential isolated from free-ranging Tibetan yaks in high altitude regions of Tibet. For this purpose, one Leuconostoc pseudomesenteroides strain (named P1) and two Lactobacillus johnsonii and Lactobacillus mucosae strains (named LY1 and LY2), respectively, were isolated from fecal samples of Tibetan yaks. The antibacterial activity of the isolates was studied using Escherichia coli (E. coli ATCC 25922), Staphylococcus aureus (S. aureus ATCC 26112), and Salmonella enteritidis (S. enteritidis NCTC 13349) as indicator pathogens. The results showed that LY1 had high antibacterial efficacy against E. coli and S. enteritidis, while P1 had the most powerful bacteriostatic ability against S. aureus. PCR amplification showed that all the isolated strains were positive for Ent P2 (enterocin P-like bacteriocin) and exhibited a high tolerance to bile and low pH. Moreover, the safety of P1, LY1, and LY2 was determined through antibiotic resistance experiments, resistance gene testing, and hemolytic analysis while the antibacterial activity was assessed by in vitro and in vivo experiments. The LY2 strain was abandoned as a potential probiotic due to the detection of the vanA gene. The mice were fed from days 1 to 30 in six groups, the P1-1 (gavaged with P1 1 × 108 CFU/day), P1-2 (gavaged with P1 1 × 109 CFU/day), LY1-1 (gavaged with LY1 1 × 108 CFU/day), LY1-2 (gavaged with LY1 1 × 109 CFU/day), control (gavaged with an equal volume of vehicle), and blank control (gavaged with an equal volume of vehicle) groups. After 30 days, mice in the P1-1, P1-2, LY1-1, LY1-2, and control groups were intraperitoneal challenged with 1 × 108 CFU of E. coli (n = 10) in the abdomen. After 2 days of infection, the mice in the control group showed more severe damage in the liver, spleen and intestine than the mice in the P1-2 and LY1-2 groups. The mice in the P1-2 and LY1-2 groups had lower rates of diarrhea and mortality than other groups. In conclusion, bacteria with probiotic potential isolated from yaks may possibly be effective and safe antibacterial substances, providing a new treatment method to reduce the incidence of diarrhea associated with bacterial diseases in yaks.
Project description:Chicks in commercial production are highly sensitive to enteric infections and their resistance can be increased by administration of complex adult microbiota. However, it is not known which adult microbiota members are capable of colonising the caecum of newly hatched chicks. In this study, we therefore orally inoculated chicks with pure cultures of 76 different bacterial isolates originating from chicken caecum on day 1 of life and determined their ability to colonise seven days later. The caecum of newly hatched chickens could be colonised by bacteria belonging to phyla Bacteroidetes, Proteobacteria, Synergistetes, or Verrucomicrobia, and isolates from class Negativicutes (phylum Firmicutes). On the other hand, we did not record colonisation with isolates from phyla Actinobacteria and Firmicutes (except for Negativicutes), including isolates from families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Lactobacillaceae. Representatives of genera commonly used in probiotics such as Lactobacillus, Enterococcus, or Bacillus therefore did not colonise the chicken intestinal tract after a single dose administration. Following challenge with Salmonella enterica serovar Enteritidis, the best protecting isolates increased the chicken's resistance to S. Enteritidis only tenfold, which, however, means that none of the tested individual bacterial isolates on their own efficiently protected chicks against S. Enteritidis.
Project description:Toll-like receptors (TLRs) signaling pathways are the first lines in defense against Salmonella enteritidis (S. enteritidis) infection but the molecular mechanism underlying susceptibility to S. enteritidis infection in chicken remains unclear. SPF chickens injected with S. enteritidis were partitioned into two groups, one consisted of those from Salmonella-susceptible chickens (died within 5 d after injection, n = 6), the other consisted of six Salmonella-resistant chickens that survived for 15 d after injection. The present study shows that the bacterial load in susceptible chickens was significantly higher than that in resistant chickens and TLR4, TLR2-1 and TLR21 expression was strongly diminished in the leukocytes of susceptible chickens compared with those of resistant chickens. The induction of expression of pro-inflammatory cytokine genes, IL-6 and IFN-β, was greatly enhanced in the resistant but not in susceptible chickens. Contrasting with the reduced expression of TLR genes, those of the zinc finger protein 493 (ZNF493) gene and Toll-interacting protein (TOLLIP) gene were enhanced in the susceptible chickens. Finally, the expression of TLR4 in peripheral blood mononuclear cells (PBMCs) infected in vitro with S. enteritidis increased significantly as a result of treatment with 5-Aza-2-deoxycytidine (5-Aza-dc) while either 5-Aza-dc or trichostatin A was effective in up-regulating the expression of TLR21 and TLR2-1. DNA methylation, in the predicted promoter region of TLR4 and TLR21 genes, and an exonic CpG island of the TLR2-1 gene was significantly higher in the susceptible chickens than in resistant chickens. Taken together, the results demonstrate that ZNF493-related epigenetic modification in leukocytes probably accounts for increased susceptibility to S. enteritidis in chickens by diminishing the expression and response of TLR4, TLR21 and TLR2-1.
Project description:BACKGROUND:Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, are now accepted as suitable alternatives to antibiotics in the control of animal infections and improving animal production. Lactic acid bacteria (LAB) with remarkable functional properties have been evaluated in different studies as possible probiotic candidates. The purpose of this study was to isolate, characterize and assess the potentials of LAB from poultry gastrointestinal tract as potential poultry probiotics. RESULTS:Potential LAB probiotics were isolated from broilers, characterized and evaluated for probiotic properties including antagonistic activity (against Escherichia coli, E. coli O157: H7, Enterococcus faecalis, Salmonella Typhimurium, S. Enteritidis and Listeria monocytogenes), survivability in simulated gastric juice, tolerance to phenol and bile salts, adhesion to ileum epithelial cells, auto and co-aggregation, hydrophobicity, ?-glucosidase inhibitory activity, and antibiotic susceptibility tests. Most promising LAB strains with excellent probiotic potentials were identified by API 50 CHL and 16S rRNA sequencing as Lactobacillus reuteri I2, Pediococcus acidilactici I5, P. acidilactici I8, P. acidilactici c3, P. pentosaceus I13, and Enterococcus faecium c14. They inhibited all the pathogens tested with zones of inhibition ranging from 12.5?±?0.71 to 20?±?0?mm, and competitively excluded (P?<?0.05) the pathogens examined while adhering to ileum epithelial cells with viable counts of 3.0 to 6.0 Log CFU/ml. The selected LAB strains also showed significant (P?<?0.005) auto and co-aggregation abilities with ?-glucosidase inhibitory activity ranging from 12.5 to 92.0%. The antibiotic susceptibility test showed 100.00% resistance of the LAB strains to oxacillin, with multiple antibiotic resistance indices above 0.5. CONCLUSION:The selected LAB strains are ideal probiotic candidates which can be applied in the field for the improvement of poultry performance and control of pathogens in poultry, hence curtailing further transmission to humans.
Project description:Foodborne pathogens can cause foodborne illness. In reality, one food sample may carry more than one pathogen. A rapid, sensitive, and multiple target method for bacteria detection is crucial in food safety. For the simultaneous detection of Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella Enteritidis, multi-objective recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD) was developed in this study. The whole process, including amplification and reading, can be completed in 15 min at 37 °C. The detection limits were 2.6 × 101 CFU/mL for Staphylococcus aureus, 7.6 × 101 CFU/mL for Vibrio parahaemolyticus, and 1.29 × 101 CFU/mL for Salmonella Enteritidis. Moreover, colored signal intensities on test lines were measured by a test strip reader to achieve quantitative detection for Staphylococcus aureus (R2 = 0.9903), Vibrio parahaemolyticus (R2 = 0.9928), and Salmonella Enteritidis (R2 = 0.9945). In addition, the method demonstrated good recoveries (92.00%-107.95%) in the testing of spiked food samples. Therefore, the multiplex LFD-RPA assay is a feasible method for the rapid, sensitive, and quantitative detection of bacterial pathogens in seafood.