Effect of Two Strains of Probiotics on the Antioxidant Capacity , Oxidative Stress , and Immune Responses of Salmonella-Challenged Broilers

The present study aimed at evaluating the effect of two strains of probiotics (Bacillus subtilis PB6 and Bacillus Cereus var. toyoi) on selected indicators of the antioxidant status and immune responses of broilers before and after challenge with Salmonella enterica subsp. Typhimurium (S. typhimurium). Birds were distributed into the following five treatments: negative control (CTL−); negative control+S. typhimurium (CTL+); antibiotic+S. typhimurium, (AB); Toyocerin®+S. typhimurium (TOYO); and CloSTATTM+ S. typhimurium (CLOS). Birds in all treatments, except for CTL−, were orally challenged with 3 x109 CFUS. typhimurium at 16 days of age. The results showed that total antioxidant capacity (TAC), superoxide dismutase activity (SOD), and hydrogen peroxide (H2O2) values were similar among all groups. The antibody titers against S. typhimurium and Newcastle disease (ND) were not affected by the treatments (p>0.05). However, S. typhimurium and ND titers were influenced by the interaction between week and treatment (p<0.01, p<0.05, respectively) and by week (p<0.001, p<0.001, respectively). In conclusion, S. typhimurium challenge had an impact on anti-Salmonella and NC titers. Both probiotics did not affect the broilers’ antioxidant capacity.


IntRoduCtIon
Salmonella enterica serovar Typhimurium (S. typhimurium) is an enteric bacterium that can colonize chickens; it is a common serovar causing salmonellosis in broiler chickens (Ribeiro et al., 2007).
Probiotics have been reported to prevent the colonization of the gut by pathogenic bacteria like Clostridium perfringens and Salmonella through the mechanism of competitive exclusion (Teo and Tan, 2007).A Bacillus subtilis-based probiotic was capable of producing an antimicrobial factor against many bacteria (Yurong et al., 2005).Other reports showed that a probiotic based on Bacillus Cereus var.toyoi reduced the prevalence of Salmonella in poultry (Vilà et al., 2005).Sahin et al. (2004) reported Japanese quails submitted to stress presented higher oxidative stress and lower antioxidant status than non-stressed ones.Probiotic supplementation was shown to modulate the dynamics of oxidants and antioxidants in the body.For example, probiotics based on Bifidobacterium and Lactobacillus showed good antioxidant effect by inhibiting lipid peroxidation in vitro using intact cells and intracellular cell-free extracts (Lin and Yen, 1999).Similarly, probiotics have been shown to increase antioxidant levels and reduce glutathione concentrations and intestinal oxidative injury (Yadav et al., 2007).Sohail et al. (2011) demonstrated that a probiotic reduced the total antioxidant concentration.In another study, rats fed a probiotic showed increased SOD and glutathione peroxidase concentrations (D 'Souza et al., 2010).Zakaria HAH

Effect of Two Strains of Probiotics on the Antioxidant Capacity, Oxidative Stress, and Immune Responses of Salmonella-Challenged Broilers
Probiotics supplementation was reported to enhance disease resistance and improve the immune response of broiler chickens (Huang et al., 2004).A study by Haghighi et al. (2006)suggested that probiotics have an immune-boosting role due to enhanced induction of natural antibodies in chickens.
Therefore, the objective of the present study was to evaluate the effect of two strains of probiotics (Bacillus subtilis PB6 and Bacillus Cereus var.toyoi) on selected indicators of antioxidant status and immune responses of broilers before and after challenge with Salmonella enterica subsp.Typhimurium (S. typhimurium).

Animals and treatments
Two hundred day-old Cobb 500 broilers, vaccinated against Newcastle disease, infectious bronchitis and Marek's disease, were allotted to 50 experimental cages (four birds per cage) in a controlled-temperature environment, under 24-h lighting, and were offered feed and water ad libitum.The experiment was carried out from day one to day 42.

Measurements
Serum total antioxidant capacity (TAC), superoxide dismutase activity (SOD), and hydrogen peroxide (H 2 O 2 ) levels were measured using commercial kits (Cell Biolabs, Inc., San Diego, USA).For this purpose, blood samples were collected from 10randomly selected birds per treatment via brachial venipuncture and poured into plain tubes on day 42.Samples were centrifuged at 5 ºC and 3,000 rpm for 10 min, and then the serum was harvested.
Anti-S.typhimurium and Newcastle disease virus (ND) antibodies were titered in the serum from 10birds per treatment before (at 7 days of age) and after (at 21, 28 and 35 days)S.typhimurium challenge.Anti-S.typhimurium antibody titers were determined using a commercial ELISA kit (BioChek, London, UK).Microtiter plates pre-coated with inactivated S. typhimurium antigen were used.Serum samples were diluted (1:100) in diluent reagent and added to the wells of the microtiter plates to allow the formation of antigen-antibody complexes in the presence of anti-S.typhimurium antibodies.Anti-chicken IgG labelled with the enzyme alkaline phosphatase was then added to the wells.The chromogen added was para-nitrophenylphosphate (pNPP), and absorbance was read at 405 nm by using a microtiter plate reader.
A commercial Newcastle disease virus antibody test kit (IDEXX Laboratories, Inc., Westbrook, Maine, USA) was used for the detection of antibodies against ND.Serum samples were diluted (1:500) with diluent reagent and 100 µL of samples were added to the microtiter wells.Plates were incubated for 30 minutes at 18-26 C°, and then washed with 350 µL of distilled water, 100 µL of conjugate were dispensed into each well, and after incubation for 30 minutes at 18-26 C°, 100 µL of TMB substrate solution were dispensed into each well and 100 µL of stop solution were dispensed to stop the reaction.Absorbance was read at 650 nm.
On day 42, 10 birds per treatment were slaughtered, and their liver, bursa, spleen, and thymus were collected and weighed.Their weights relative to carcass weight were calculated and expressed as percentages.

Statistical Analysis
Data were analyzed according to a completely randomized block design, with five treatments of 10 replicates each.The general linear model procedure of SAS (version 9.1.SAS Institute, Cary, NC) was used.The effects of week of collection, treatment and week x treatment interaction were tested.Means showing significant differences in the analysis of variance were compared using the PDIFF option.The overall statistical significance level was set at p<0.05.All values were expressed as statistical means ± standard error of the mean (SEM).

ReSultS And dISCuSSIon
Table 2 shows the serum TAC, SOD and H 2 O 2 results of the birds submitted to the experimental treatments.Serum TAC, SOD, and H 2 O 2 values were similar (p>0.05)among all groups, suggesting that neither S. typhimurium challenge nor probiotics had an impact on total antioxidant capacity or oxidative stress.These results agree with Erdoğan et al. (2010),who concluded that the probiotics tested did not promote any significant improvement in the oxidant/antioxidant status in broilers.On the other hand, several other reports provided evidence that probiotics positively modulate the dynamics of oxidants and antioxidants in the body of chickens, Japanese quails, and rats (Lin and Yen, 1999;Sahin et al., 2004;Yadav et al., 2007;Sohail et al., 2011).This difference may be explained by several factors, such as probiotic strain and challenge type.Lin and Yen (1999) hypothesized that beneficial intestinal bacteria produce certain factors that have the ability to chelate free radicals, capturing ROS and inhibitingtheir cytotoxic activity.The mean corresponds to 10 birds per treatment.
Oxidative stress can be defined as a physiological disorder where there is an imbalance between concentrations of reactive oxygen species (ROS), such Zakaria HAH

Effect of Two Strains of Probiotics on the Antioxidant Capacity, Oxidative Stress, and Immune Responses of Salmonella-Challenged Broilers
as hydrogen peroxide (H 2 O 2 ), and an antioxidant.Sahin et al. (2002) reported that low endogenous antioxidant production is the causative factor of oxidative stress in birds.Excessive ROS accumulation leads to cell injury, including damage to the DNA, proteins, and lipid membranes.Generally, ROS are continually produced by aerobic cells and are instantly removed by endogenous antioxidant scavengers.It is established that changes in the activity of several antioxidant enzymes can be used to assess the level of oxidative stress and total antioxidant status in birds.TAC accurately reflects the antioxidative status of the organism.SOD catalyzes the dismutation of the superoxide anion into hydrogen peroxide, and molecular oxygen is considered as one of the most potent antioxidant.Hydrogen peroxide (H 2 O 2 ) is one of the most well-documented ROS produced under oxidative stress.It is a toxic product of normal aerobic metabolism and pathogenic ROS production involving oxidase and superoxide dismutase reactions.Under normal physiological conditions, cellular ROS generation is counter balanced by the action cellular antioxidant enzymes and redox molecules.
The results for anti-S.typhimurium responses in the different treatments are presented in Table 3.There was a significant treatment*week interaction (p<0.01).The S. typhimurium titer continued to increase in birds in treatments CTL+, AB, and TOYO from week one through week five.In the CTL− birds, S. typhimurium titer increased from week 1 to week 3, then decreased in week 4 and increased again in week 5 (0.6, 1.17, 1.02 and 2.38, for weeks 1, 3, 4, and 5, respectively).In the birds in the CLOS group, the titer increased until week 4, then decreased in week 5 (1.09, 1.42, 1.62 and 0.92, for weeks 1, 3, 4, and 5, respectively).On the other hand, week showed a significant effect on anti-S.typhimurium titer, which increased from week 1to week 5 (0.73, 1.85, respectively; p<0.001), while treatment had no significant effect on anti-S.typhimurium titers (p>0.05).The beneficial effects of probiotics of boosting the immune system of broilers is due to enhanced induction of natural antibodies, as previously reported (Haghighi et al., 2006).
The Newcastle disease (NC) titer results are shown in Table 4. Treatment showed no effect on NC titer; however, there was a significant treatment*week interaction (p<0.05) and a strong effect of week (p<0.001).Birds in all treatments presented lower NC titers after S. typhimurium challenge (week 3) as compared to week 1 (before the challenge).However, the response after week 3 varied according to the treatment.For example, the NC titer of birds in groups CTL−, CTL+, and TOYO continued to increase after week 3, but not in the birds fed AB and CLOS.Sadeghl et al. (2013) found that S. typhimurium-infected birds had higher antibody titers in response to NC vaccination when compared to uninfected birds.Holt et al. (1999) concluded that broilers exposed to Salmonella at a young age may remain infected upon maturity and do not develop significant immunity against Salmonella.Seo et al. (2000) evaluated the effects of the challenge with Salmonella Enteritidis, enrofloxacin and/or normal avian gut flora in layers.They found that antibodies titers (IgG) were similar among treatments at two days

HAH Effect of Two Strains of Probiotics on the Antioxidant Capacity, Oxidative Stress, and Immune Responses of Salmonella-Challenged Broilers
Abudabos et al. (2014)AT TM contains 2 x 10 7 CFU/g of Bacillus subtilis PB6 (CloSTAT, Kemin Industries Inc., Des Moines, IA).At day 16 of age, all birds except for those in the CTL−treatment were orally inoculated with 3 x10 9 CFU of Salmonella Typhimurium ATCC13311,according to the procedure described byAbudabos et al. (2014).

Table 2 -
Total antioxidant capacity and oxidative stress (SOD and H 2 O 2 levels) in serum of broiler chickens fed the experimental diets.

Table 3 -
Serum anti-Salmonella antibody titers according to treatment and weekMean results of 35 birds per treatments, and 50 birds per week.the same column with different superscripts differ significantly, **p<0.01,***p<0.001.
abcd Means in

Table 4 -
Serum anti-Newcastle disease titers according to treatment and week Mean results of 35 birds per treatments, and 50 birds per week.the same column with different superscripts differ significantly, **p<0.01,***p<0.001.
abcd Means in