Effect of Probiotics on Serum Biochemical and Blood Constituents in Chicken Challenged with Salmonella enterica Subsp Typhimurium

Purpose: To examine the effects of two commercial probiotics (Toyocerin and CloSTAT) on serum enzyme activities, and hematological and biochemical indices of broiler chickens challenged with Salmonella enterica serovars Typhimurium (ST). Methods: The chicks received one of the following treatments at 0 day of age: T1 = control group, unmedicated diet, unchallenged birds, (negative control); T2 = unmedicated diet + bacterial challenge (positive control); T3 = medicated diet with neoxyval (0.05 g/kg diet) + bacterial challenge (NEOX); T4 = toyocerin (1 g/kg diet) + bacterial challenge (Toyocerin); and T5 = CloSTAT (1 g/kg diet) + bacterial challenge (COLS). Blood samples were withdrawn from 7 selected chicks in each treatment at 7, 21, 28, 35 and 42 days of age, and analyzed for total protein, albumin and globulin concentration, and the albumin: globulin ratio computed. Glutamate-oxaloacetate transaminase (GOT) and glutatamate pyruvate transaminase (GPT) levels in serum were measured on days 7 and 42. Results: The results revealed that albumin (p < 0.001), globulin (p < 0.001) and albumin: globulin ratio (p < 0.001) were influenced by the time of blood collection. Globulin increased significantly after ST challenge while albumin decreased significantly. Glutamate-oxaloacetate transaminase (GOT) after ST challenge was affected by treatment (p < 0.05). Higher levels were obtained from birds which had received NEOX or positive control, compared to negative control group. Conclusion: Salmonella challenge affects serum albumin, globulin and GOT enzyme. The results obtained suggest that the probiotic, Toyocerin, mitigates the negative effects of Salmonella challenge.


INTRODUCTION
Salmonella enterica serovars typhimurium (ST) is an enteric bacterium that can colonize chickens; it is a common serovar causing salmonellosis in broiler chickens [1,2]. The extent of Salmonella infection after oral inoculation can be systemic and often comprises infection of the liver [3].
After oral inoculation of adult Leghorn hens with doses of 109 cells of Salmonella enteritidis, it was recovered from 53 % of liver samples after 5-weeks post-inoculation [4].
A significant decline in growth performance was reported in broilers as a result of Salmonella [5,6]. The reduction in performance was ascribed to decrease in feed intake in broilers due to mucosal damage, diarrhea and systemic infection [7,8]. The liver is one of the main systemic organs affected by Salmonella and as a result of damaged liver cells, liver enzymes increase in the blood.
Currently, probiotics are used in the poultry industry as an alternative to antimicrobial growth promoters [9]. 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 [9,10]. Bacillus subtilis is capable of producing an antimicrobial factor against many bacteria [11]. Other reports showed that Bacillus cereus var. toyoi based probiotics reduced the prevalence of Salmonella in poultry [12]. There is scarcity of information on the effects of probiotics on serum hematological, biochemical indices and liver enzymes activity in broiler chickens challenged with oral salmonella. Therefore, in the present study, the objective was to evaluate the effect of two strains of probiotics (Bacillus subtilis PB6 and Bacillus cereus var. toyoi) on hematological and biochemical indices of ST challenged broiler chickens.

EXPERIMENTAL Animals, husbandry and treatment
The study was conducted under a protocol approved by King Saud University and complies with the current laws of Saudi Arabia. The experiment was carried out from 0 to 42 days of age by utilizing a total of two hundred, Cobb 500 broiler chicks which were allotted into 50 experimental cages with four chicks per cage. The chicks had been vaccinated for Marek's disease, Newcastle and infectious bronchitis. Feed and water were provided ad libitum and the birds were maintained at 24 h light schedule. A typical isocaloric and isonitrogenous starter (0 -14 d) and finisher (15 -42 d) diets based on corn-soybean meal diets were formulated in mashed form which met or exceed the recommendations in commercial practice (Tables  1, 2 and 3).

Challenge inoculum
At day 16 of age, chicks in treatments 2 to 5 were challenged with ST (3 x 10 9 CFU/ml). The strain used in this experiment was Salmonella typhimurium ATCC13311. Chicks were gavaged with 1 ml of cocktail containing 3 x 10 9 CFU/mL ST which was obtained commercially (MicroBiologics, Cloud, MN-U.S.A) according to the procedure described by [6].

Hematological and biochemical measurements
The following analyses were conducted by using enzymatic colorimetric kits: total protein (Biuret method), albumin (Bromoreesol green method), globulin concentration was calculated, thereafter, as the difference between total protein and albumin concentrations. Albumin: globulin ratio was calculated. Seven chicks per group were selected for blood collection via brachial venipuncture into plain tubes for serological analysis starting at days 7 (before bacterial challenge), 21, 28, 35 and 42 (after the challenge). Samples were centrifuged by using plain tubes at 5 ºC and 3,000 rpm for 10 min. Serum samples were stored in eppendorf tubes and stored at -80 ºC until further analysis, unless fresh sample is required for the analysis.
The activities of glutamate-oxaloacetate transaminase (GOT) and glutatamate pyruvate transaminase (GPT) in the serum were measured according to the colorimetric method by using GOT-GPT assay kits, Asan Pharmaceutical). For this purpose blood samples were withdrawn from 7 selected chicks of each treatment at days 7 (before bacterial challenge) and 42 (after the challenge).
At the end of the trial, blood samples were withdrawn from 5 selected chicks of each treatment via brachial venipuncture into EDTA tubes for hematological analysis and were placed inside an ice box and transferred to the laboratory. Within 1 h after collection, the hematological parameters were determined (Maxcom Auto Hematology Analyzer (MC-6200, China.

Statistical analysis
Data were analyzed by using the general linear model procedure of SAS [13]. Five treatments were arranged in 10 replications in a randomized complete block design. Means of measurements showing significant differences in analysis of variance (ANOVA) were tested using the difftype statement (PDIFF). Overall level for statistical significance was set at p < 0.05. All values are expressed as statistical means ± standard error of the mean (SEM).

RESULTS
The data related to serum biochemical indices are shown in Table 4. Total protein, albumin, globulin, and albumin : globulin ratio (A/G ratio) were similar among all groups (p > 0.05). The interaction term (treatment*week) was not significant for any parameters measured for serum biochemistry (p > 0.05).
On the other hand, albumin, globulin and their ratio (A/G ratio) were influenced by time of collection (week) (p < 0.001). Albumin concentration was the lowest when measured after the challenge (1.13 g/dl for week 3), then albumin concentration the uttermost at weeks five and six (1.94, 194 g/dl, respectively). In the contrary, globulin concentration increased right after the challenge (0.89 vs. 1.58 g/dl, for weeks one and three, respectively) and then started to decline after week four. On weeks five and six, the values were similar to the value before the challenge in week one (0.72, 0.75 and 0.89 g/dl, respectively). The A/G ratio dropped significantly after the challenge from 3.02 in week one to 0.87 in week three. In weeks five and six, A/G ratio was not different from week one.
The activities of glutamate-oxaloacetate transaminase (GOT) and glutatamate pyruvate transaminase in the serum are presented in Table 5. Treatment showed no effect on GOT or GPT in serum before the challenge (week one) (p > 0.05). However, after the challenge (week 6), serum from birds which had received NEOX or positive control had significantly higher GOT value as compared to negative control (274.4, 244.6 and 195.6 IU/L, for NEOX, positive and negative control, respectively) (p < 0.05). GOT for birds which had received CLOS was 230.6 IU/L. The lowest GOT value was obtained from birds which had received the negative control or TOYO (195.6 and 200.4 IU/L, respectively). On the other hand, week of collection showed a significant effect on GOT (p < 0.01), the mean for GOT in week six was 229 IU/L as compared to 199 IU/L in week one. In this study, the GPT values after the challenge were not affected by any treatments (p > 0.05). Table 6 shows the effect of treatment on hematological parameters of broiler blood profile. The values of the blood parameters measured in this trial were comparable and treatment had no effect on blood hematological values (p > 0.05).

DISCUSSION
The data related to serum biochemical indices indicated no differences between treatments in total protein, albumin, globulin, and A/G ratio among all groups. The findings of this trial are in agreement to the findings of [14] who reported no significant differences in serum biochemical indices examined as a result of probiotic supplementation.
The activities of glutamate-oxaloacetate transaminase (GOT) and glutatamate pyruvate transaminase in the serum were not affected by treatment before the bacterial challenge. However, after the challenge higher GOT values were found in birds which had received NEOX or positive control while probiotics lowered the GOT value especially for TOYO group. This finding agrees with [15] who reported that colonization of Salmonella in organs increases the liver enzyme activities in blood. Several studies have assessed the effects of Salmonella on the gene expression of chicken immune tissues or cells [16,17]. It was reported that the effects of Salmonella infection on the liver transcriptome profiles of broilers reflect a predominance of down regulation of genes with cell cycle and metabolic functions [18]. A clear sign of hepatic damage is the leaking of cellular enzymes into the plasma [19].
The values of the hematological parameters of broiler blood profile were comparable and treatment had no effect on blood hematological values. Similar to our findings, it was reported that probiotics did not show any harmful changes on blood hematological parameters and it could improve health condition by enhancing concentration of hemoglobin, hematocrit and red blood cell count in broilers [20]. Conversely, probiotics lowered lymphocytes and neutrophil values when compared to the control [21].

CONCLUSION
Neither challenge nor treatment has a significant impact on blood hematological values. Albumin, globulin and their ratio (A/G ratio) were influenced by time of collection. Albumin concentration was lowest when measured after the challenge. On the contrary, globulin concentration increased right after the challenge. The results from this study suggest that the probiotic, TOYO, alleviates the negative effects of Salmonella challenge.