Probiotics & Antimicro. Prot. DOI 10.1007/s12602-014-9175-1
Immune Response of Salmonella Challenged Broiler Chickens Fed Diets Containing GalliproÒ, a Bacillus subtilis Probiotic Ali Asghar Sadeghi • Parvin Shawrang Shirin Shakorzadeh
•
Ó Springer Science+Business Media New York 2014
Abstract This study was conducted to investigate the effect of feeding a probiotic, Bacillus subtilis, on antibody titers against Newcastle and infectious bursal viruses in broiler chickens challenged with Salmonella enterica serotype Enteritidis. One hundred and sixty 1-day-old broiler chicks were randomly assigned to four treatments in a completely randomized design. The treatments were negative control, probiotic-treated group, challenged group, and challenged probiotic treated group. Salmonella challenging decreased (P \ 0.05) the relative weights of spleen and bursa. Inclusion of probiotic to diet of challenged chickens increased the relative weight of spleen, but had no effect on the relative weight of bursa. There were no differences for the antibody titers of chickens between negative control and probiotic-treated group. Salmonella challenging decreased (P \ 0.05) antibody titers against Newcastle and infectious bursal viruses. Improvements in the antibody titers were observed by the addition of probiotic to diet of these chickens. The results showed that dietary inclusion of probiotic had no significant effect on immune parameters of chickens at non-contaminated environment, display a greater efficacy at environment contaminated with pathogen and can improve immune responses of infected chickens.
Introduction Antibiotics are supplemented in the broiler rations worldwide to prevent poultry disease and as growth promoters to enhance health status and improve meat production. However, the use of antibiotics has resulted in problems such as drug residues in meat, development of drug-resistant bacteria, and imbalance of normal microflora [1]. As a consequence, it has become necessary to develop alternatives such as beneficial microorganisms termed probiotics. The use of probiotics has been shown to modulate the systemic antibody response to antigens in chickens [2, 3], piglets [4], and fish [5] and can result in the secretion of cytokines and enhancement of immunity [6, 7]. Despite the fact that several studies have shown immune enhancement resulting from oral administration of probiotics, there is a dearth of information regarding the effects of probiotics on the immune response of broiler chickens challenged with Salmonella enterica Enteritidis. The present study was carried out to determine the effect of the probiotic GalliproÒ, Bacillus subtilis, on immune organ weights and on antibody titers against Newcastle and infectious bursal viruses in broilers, and the potential protection it might provide to the birds against a Salmonella challenge.
Keywords Probiotics Bacillus subtilis Immune organ Pathogen challenge Immune cells profile Materials and Methods A. A. Sadeghi (&) S. Shakorzadeh Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, Iran e-mail: [email protected] P. Shawrang Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute, AEOI, Karaj, Iran
Experimental Birds and Treatments One hundred and sixty 1-day-old broiler chicks (Ross 308) were obtained from a commercial hatchery. Using a completely randomized design, the birds were randomly divided into four groups and housed in the separated pens of
123
Probiotics & Antimicro. Prot.
identical size (1.3 9 1.2 m) in a litter system. Each group had 4 replicates with 10 birds per each. The treatments included a negative control (basal diet without probiotic supplementation and challenging), probiotic-treated group (basal diet supplemented with 0.02 % probiotic, without pathogen challenge), challenged group (basal diet, with pathogen challenge at day 7 of age), and a challenged probiotic-treated group (basal diet supplemented with probiotic and pathogen challenge at day 7 of age). Probiotic sample, GalliproÒ, which contains a B. subtilis was prepared from Biochem Co. (Zusatzstoffe GmbH, Germany) and included in the diets at its catalog recommendation. The birds had free access to water and feed. Environmental temperature in the first week of life was 33 °C and decreased to 20 °C until the end of the experiment. During the first week, 22 h of light was provided with a reduction to 20 h thereafter. Salmonella Culturing and Challenging Salmonella enterica serotype Enteritidis (PTCC 1709) was obtained as freeze-dried from the Persian Type Culture Collection (IROST, Tehran, Iran) isolated from the liver of chickens. Briefly, freeze-dried inoculum was grown in tryptic soy broth (Acumedia Manufacturers Inc., Baltimore, MD, USA) at 37 °C for 8 h and passed to fresh tryptic soy broth for three-incubation periods. Determination of the number of colony-forming units (cfu) through decimal dilution series was performed in sterile buffered 0.9 % peptone water with pH 7.2. For this, 0.1 ml of diluted medium was inoculated in Petri dishes containing Shigella–Salmonella agar and cultivated for 24 h at 37 °C, and then, cfu per ml was calculated. Day 7 of age, chicks in challenged groups received by oral gavage 1.0 9 105 cfu per chick (0.2 ml) of S. enterica Enteritidis suspension. Unchallenged birds received the same amount of sterile buffered peptone water by oral gavage. Vaccination and Serology The immunization program included vaccination against Newcastle disease (B1, day 10, eye drop; Lasota days 19 and 32 drinking) and infectious bursal disease (Gumboro, D78, days 12 and 24, eye drop). Blood samples (3.0 ml) of eight birds per treatment (two birds per pen) were collected from the wing veins, using sterile syringes, on days 21 and 42 of age. Immediately after collection, 900 ll of blood was transferred to micro-tube containing 100 ll sodium citrate solutions (3.85 mg/100 ll) and were immediately mixed. The tubes were transferred to Veterinary Laboratory of Mabna (Karaj, Iran) for counting total and differential white blood cells according to Wright’s stain method. Remainder of blood samples (2.1 ml) was
123
Table 1 The relative weights of spleen and bursa of chickens at days 21 and 42 of age (as g/kg body weight) Treatments
Negative control Probiotic treated
Day 21 of age
Day 42 of age
Spleen
Bursa
Spleen
Bursa
1.19
2.53
1.03a,b
1.47a
1.22
2.61
a
1.48a
b
1.07
Salmonella challenged Salmonella challenged and probiotic treated
1.10 1.16
2.13 2.19
0.90 1.00a,b
1.35b 1.36b
SEM
0.102
0.101
0.052
0.086
a, b
Within the same column, means with different superscripts are significantly differed (P \ 0.05)
transferred to clear glass tubes, kept at room temperature for 2 h, then overnight at 4 °C in refrigerator and centrifuged at 1,5009g for 15 min. Serum was obtained, and complement inactivation was done at 56 °C for 30 min and stored at -20 °C to conduct antibody titration. The titers of antibody against Newcastle virus were measured by the hemagglutination–inhibition test. The titers of antibody against infectious bursal virus were measured using a commercial ELISA kit (IDEXX Lab., CA, USA). Immune Organ Weights On days 21 and 42 of age, a total of 32 birds (eight per treatment; two per pen) were randomly selected, individually weighed, stunned, killed by cervical dislocation, and were plucked in the slaughterhouse. The carcasses were opened, and then, the spleen and bursa were removed and weighed. The relative weights of the spleen and bursa were then calculated as [organ weight (g)/body weight (kg)]. Statistical Analysis Statistical analyses were conducted with the general linear model procedure of SAS for Windows version 9.1 (SAS Institute Inc., Cary, NC) to determine whether variables differed among groups. The Kolmogorov–Smirnov test was used to test the normal distribution of the data before statistical analysis was performed. Group comparisons were made using the Tukey test. Probability values of P \ 0.05 were considered significant.
Results Immune Tissue Weights As shown in Table 1, the relative weights of the spleen and bursa at day 21 of age were not affected by treatments. At
Probiotics & Antimicro. Prot. Table 2 Effects of experimental treatments on total and differential counts of white blood cells at day 21 of age Treatments
WBC 9 103
L (%)
H (%)
H/L
E (%)
M (%)
Negative control
23.4d
76.0a
22.0c
0.29b
0
2
Probiotic treated
c
24.8
a
74.0
c
22.0
0.30b
2
2
Salmonella challenged
27.9a
68.0b
29.0a
0.42a
0
3
b
a
b
Salmonella challenged and probiotic treated SEM
26.4
0.61
72.0
1.82
25.0
1.08
a,b
0.35
1
2
0.011
0.7
0.9
WBC white blood cells count, L lymphocyte, H heterophil, H/L heterophil to lymphocyte ratio, E eosinophils, and M monocytes a, b, c, d
Within the same column, means with different superscripts are significantly differed (P \ 0.05)
Table 3 Effects of experimental treatments on total and differential counts of white blood cells at day 42 of age Treatments
WBC 9 103 21.8
c
Probiotic treated
22.9
b
Salmonella challenged
23.4a,b
Salmonella challenged and probiotic treated
Negative control
SEM
L (%) a
H (%)
H/L
E (%)
M (%)
c
0
4
c
19.0
c
75.0
21.0
b,c
0.27
1
3
71.0c
24.0a
0.33a
2
3
24.3a
74.0b
22.0a,b
0.29b
1
3
0.52
0.72
0.91
0.009
0.9
1.3
77.0
b
0.25
WBC white blood cells count, L lymphocyte, H heterophil, H/L heterophil to lymphocyte ratio, E eosinophils, and M monocytes a, b, c
Within the same column, means with different superscripts are significantly differed (P \ 0.05)
this time period, Salmonella challenging resulted in numerical decreases in the relative weights of the spleen and bursa, in comparison with the negative control. Probiotic supplementation increased the relative weights of the immune organs in the probiotic-treated chickens when compared to the challenged chickens. At day 42 of age, differences were present among the relative weights of the spleen or bursa of chickens for the different treatments. Dietary inclusion of the probiotic increased the relative weights of spleen, but had no effect on the relative weight of bursa. At this time period, challenging the birds with Salmonella significantly decreased the relative weights of the spleen and bursa by 12 and 7 % compared with the negative control, respectively. Addition of the probiotic to the diet of these chickens increased the relative weight of the spleen by 10 % in comparison with the spleens of the chickens in the challenged group, but it had no effect on the relative weight of the bursa. Effect on Immune Cells The results of total and differential counts of white blood cells at day 21 of age are reported in Table 2. At this period, there was a significant difference in total white blood cells among treatments (P \ 0.05). The highest mean of total white blood cells was related to the chickens in both Salmonella challenged groups, and the lowest one was for negative control. White blood cells counts
decreased in challenged probiotic-treated chicken by 5 %, compared with challenged chickens. Challenged chickens had the lowest lymphocyte percentage compared with other treatments. There were no significant differences in lymphocyte percentage among the negative control and the chickens fed the diet containing the probiotic with or without challenging. In contrast to the lymphocyte data, the chickens in the positive control group had the highest heterophil percentage and H/L ratio in comparison with the other treatments. The addition of the probiotic to the diet of the challenged chickens reduced heterophil percentage and the H/L ratio, in comparison with the chickens in the challenged group by 16 %, respectively. There were no significant differences for eosinophils and monocytes among treatments. Total and differential counts of white blood cells at day 42 of age are reported in Table 3. At that time period, the lowest and highest total white blood cells counts were observed in the negative control and in the challenged chickens fed the diet containing the probiotic, respectively. Salmonella challenging and probiotic supplementation increased total white blood cells, especially when both of them were applied. The highest lymphocyte percentage and lowest heterophil percentage were found in the probiotictreated chickens, and vice versa, for the challenged chickens. Salmonella challenging resulted in an increased H/L ratio, but the addition of the probiotic to the diet decreased it significantly.
123
Probiotics & Antimicro. Prot.
Fig. 1 Antibody titer (log 2) against Newcastle virus in the unchallenged and Salmonella challenged chickens (Ross308 broilers) fed diets containing probiotic at days 21 (up) and 42 (down) of age
Fig. 2 Antibody titer (log 2) against Infectious Bursal virus in the unchallenged and Salmonella challenged chickens (Ross308 broilers) fed diets containing probiotic at days 21 (up) and 42 (down) of age
Effect on Antibody Titers
chickens at the both periods. At day 21 of age, Salmonella challenging decreased the antibody titers against Newcastle and infectious bursal viruses by 25 and 27 %, respectively, compared with levels observed in the negative control. At that same period, the antibody titer against Newcastle virus in the challenged probiotic-treated chickens increased (18 %) significantly, in comparison with the levels observed in the challenged chickens, but no change was observed for the antibody titer against infectious bursal virus. At day 42 of age, antibody titers against Newcastle and infectious bursal viruses in challenged probiotic-treated chickens increased significantly by 21 and 14 %,
The geometric means of antibody titers against Newcastle and infectious bursal viruses at days 21 and 42 of age are depicted in Figs. 1 and 2. At both time periods and for both antigen levels, there were no differences for antibody titers between the negative control and probiotic-treated chickens, although addition of the probiotic to the diet did appear to increase the numerically antibody titers slightly, but not significantly. Challenging with Salmonella decreased (P \ 0.05) the antibody titers against both antigens, as the lowest means were observed in challenged
123
Probiotics & Antimicro. Prot.
respectively, in comparison with the levels observed in the challenged chickens.
Discussion The main purpose of present study was to examine the effect of dietary inclusion of the probiotic GalliproÒ on the immune responses of Salmonella challenged broiler chickens when vaccinated with Newcastle and infectious bursal viruses. Effects of these treatments on white blood cell counts and the relative weights of the immune functions were also measured. There were no differences in the antibody titers against Newcastle and infectious bursal viruses between the negative control and the unchallenged probiotic-treated chickens, but numerically the latter had slightly but not significantly higher titers than the former. Our results are in agreement with several studies [8, 9], in which the addition of a probiotic to the diet of unchallenged chickens had no significant effect on antibody titers against Newcastle virus and sheep red blood cells when used as antigens. In contrast to our results, the beneficial effects of probiotics treatment on immunity have been reported in number of other studies using probiotics in broilers [6, 10, 11]. Differences among these studies can be related to different age groups used, the different probiotics administered, and the different dose of the probiotics used. Recently, Elangovan et al. [12] reported that probiotic supplementation (500 g, GalliproÒ, B. subtilis) had no effect on cell-mediated immune response, whereas it significantly increased the humoral immune response of broiler chickens. These discrepant findings might be associated with the dose of probiotic used and the class of the antigen to which the antibodies were directed. The addition of the probiotic GalliproÒ to the diet of unchallenged chickens significantly increased the white blood cell in comparison with what was observed in the negative control, but it had no effect on the heterophil to lymphocyte ratio. Our results are in agreement with those of Rahimi and Khaksefidi [13], in which supplementation of the diet with probiotic significantly increased the white blood cell count in comparison with the count found in the control group. Results of this study showed that Salmonella challenge results in decreased antibody titers against Newcastle and infectious bursal viruses, a lower lymphocyte count, and depressed immune organ weights. The exact mechanisms by which the immunomodulatory activities of probiotics are mediated are not clear. However, it has been shown that Salmonella stimulate different subsets of immune system cells to produce cytokines, especially interleukin-1b [14], which in turn play a role in the induction and regulation of
the immune response [15]. The lower humoral immune response of challenged broilers can be explained by increase in production of interleukin-1. Interleukin-1 stimulates the hypothalamus, leukocytes, or both to produce the corticotropin-releasing factor, which stimulates the production of adrenocorticotropic hormone (ACTH) by the anterior pituitary, leukocytes, or both. ACTH then stimulates corticosterone production from the adrenal gland [15]. Corticosterone has been found to be immunosuppressive [16], inhibiting the production and actions of antibodies [17], increasing the heterophil to lymphocyte ratio [18] and depressing immune organ growth [19]. The results in Tables 2 and 3 are consistent with the mentioned events. White blood cell counts and the heterophil to lymphocyte ratio led us to conclude that challenged chickens were in physiological stress status. The heterophil to lymphocyte ratio has been accepted as a reliable index for determining stress in poultry [20]. Heterophils are parts of natural immunity and cellular defense against microbial infections, and lymphocytes are cells that produce antibodies. The increases in heterophil to lymphocyte ratio in challenged chickens may be attributed to increased corticosterone secretion [18], which finally results in a decreased antibody titer. Although the addition of the probiotic GalliproÒ to the diet had no effect on immune response of unchallenged chickens; when Salmonella challenge was administered, significant positive effects were observed. We found no study in the literature in which the effects of probiotic on antibody titers against Newcastle and infectious bursal viruses of Salmonella challenged chickens were evaluated. The present results support the findings of Higgins et al. [21] and suggest that performance-related benefits of some probiotics and probiotic cultures may be most apparent when low-level enteric challenge, such as Salmonella infection, is present. The two most likely mechanisms by which probiotic reduce the negative effects of Salmonella on antibody titers involve competitive exclusion through competition for receptor sites and in the production of volatile fatty acids that are inhibitory of certain enteric pathogens, production of bacteriocins, or competition with pathogens and native flora for limiting nutrients or stimulation of a host innate immune response [22]. The B. subtilis could reduce Salmonella counts in intestines through a number of these inhibitory mechanisms. Recently, an interesting study [23] in humans revealed that the oral administration of B. subtilis could reduce pro-inflammatory cytokines (TNF-a, interleukin-1b, interleukin-6, and Interferon-c) and increase anti-inflammatory cytokine (interleukin-10 and TGF-b) in the plasma of patients with inflammatory bowel disease. These events may have reduced the negative effects of Salmonella on immune response and hematological parameters of the chickens in this study.
123
Probiotics & Antimicro. Prot.
In the present study, white blood cells were increased by Salmonella challenging and were decreased in similar chickens fed a diet containing the probiotic GalliproÒ. An increased level of white blood cells is indicative of an inflammation process or infection. It has been shown that cytokines (especially interleukin 1-b), whose synthesis is increased by infection, are important factors in the inflammatory response [14, 15]. Several in vitro and in vivo studies have established an inflammatory immune response to Salmonella in chickens by demonstrating an up-regulation in the expression of the inflammatory cytokine interleukin-1 [14, 24]. Inflammatory factors stimulate the hypothalamic production of corticotrophin-releasing factor [16]. As mentioned previously, this factor can increase ACTH concentration, thereby stimulating corticosterone production. Corticosterone can inhibit lymphocyte functions and, thereby, decrease the population of leukocytes. Immune tissue development is the basis of immune system functionality. At day 42 of age, the relative organ weights of bursa and spleen were influenced negatively by Salmonella challenging. Stress-induced bursal atrophy has been suggested to be caused by an increased corticosteroid production [25]. Low bursa weight could be interpreted as an indicator of low immune activity because it is a major lymphoid organ in poultry. The decrease of immune tissue weight produces an effect on immune cell phenotypes, immune cell proliferation, and antibody production. As mentioned previously, challenging could induce the production of interleukin-1, which finally can increase the level of serum corticosterone concentration [14]. It was concluded [19] that under this condition, retardation of immune organs occurs. The addition of the probiotic GalliproÒ to the diet of the challenged chickens had no effect on the relative weight of the bursa, but increased the relative weight of the spleen. This same situation was observed in the unchallenged probiotic-treated chickens in comparison with what was observed in their negative control. We cannot find the reason for these events. The results of this study indicated that Salmonella challenging had a negative effect on the immune response of broiler chickens through a retardation of the growth of the immune organs, a change in the profile of the immune cells produced and due to a decrease in antibody production. Dietary inclusion of the probiotic GalliproÒ had no significant effect on the immune parameters of chickens in a non-contaminated environment, but it displayed a greater efficacy when the chickens were in an environment contaminated with a pathogen, and it can improve the efficacy of vaccination and health of infected chickens. Acknowledgments The authors are grateful to the Islamic Azad University for research funding support, to the Agricultural, Medical
123
and Industrial Research School, NSTRI, for supplying the basal diet and allowing the use of their poultry unit, and to the Biochem Company (Zusatzstoffe GmbH, Germany) for providing a sample of the probiotic GalliproÒ. We also thank all of the staff members at the poultry unit for the assistance in the care and feeding of the chickens used in this research. Conflict of interest Ali Asghar Sadeghi, Parvin Shawrang and Shirin Shakorzadeh declare that there is no conflict of interest.
References 1. Sorum H, Sunde M (2001) Resistance to antibiotics in the normal flora of animals. Vet Res 32:227–241 2. Koenen ME, Kramer J, Van Der Hulst R et al (2004) Immunomodulation by probiotic lactobacilli in layer- and meat-type chickens. Brit Poult Sci 45:355–366 3. Allan WH, Gough LE (1974) A standard haemaglutination inhibition test for Newcastle disease: a comparison of macro and micro-methods. Vet Rec 95:120–123 4. Schierack P, Wieler LH, Taras D et al (2007) Bacillus cereus var. toyoi enhanced systemic immune response in piglets. Vet Immunol Immunopathol 118:1–11 5. Panigrahi A, Kiron V, Kobayashi T et al (2004) Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136. Vet Immunol Immunopathol 102:379–388 6. Haghighi HR, Gong J, Gyles CL et al (2005) Modulation of antibody-mediated immune response by probiotics in chickens. Clin Diag Lab Immunol 12:1387–1392 7. Kim DH, Austin B (2006) Cytokine expression in leucocytes and gut cells of rainbow trout, Oncorhynchus mykiss Walbaum, induced by probiotics. Vet Immunol Immunopathol 114:297–304 8. Panda AK, Ramarao SV, Reddy MR et al (1999) Effect of dietary inclusion of probiotic on growth, carcass traits and immune response in broilers. Indian J Poult Sci 34:343–346 9. Seifert S, Fritz C, Carlini N et al (2011) Modulation of innate and adaptive immunity by the probiotic Bifidobacterium longum PCB133 in turkeys. Poult Sci 90:2275–2280 10. Khaksefidi A, Ghoorchi T (2006) Effect of probiotic on performance and immunocompetence in broiler chicks. J Poult Sci 43:296–300 11. Li SP, Zhao XJ, Wang JY (2009) Synergy of Astragalus polysaccharides and probiotics (Lactobacillus and Bacillus cereus) on immunity and intestinal microbiota in chicks. Poult Sci 88:519–525 12. Elangovan AV, Mandal AB, Shrivastav AK, Yadhav AS (2011) Supplementing probiotics (GalliPro) to broiler chicken on growth performance, immunity and gut microbial population. Anim Nut Feed Technol 11:169–176 13. Rahimi S, Khaksefidi A (2010) A comparison between the effects of a probiotic (Bioplus 2B) and an antibiotic (virginiamycin) on the performance of broiler chickens under heat stress condition. Iran J Vet Res 7:23–28 14. Okamura M, Lillehoj HS, Raybourne RB et al (2004) Cellmediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-c production. Comp Immunol Microbiol Infect Dis 27:255–272 15. Maassen CB, Van Holten-Neelen C, Balk F et al (2000) Straindependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains. Vaccine 18:2613–2623 16. Post J, Rebel JMJ, Huurne AAHM (2003) Physiological effects of elevated plasma corticosterone concentrations in broiler chickens.
Probiotics & Antimicro. Prot.
17.
18.
19. 20. 21.
An alternative means by which to assess the physiological effects of stress. Poult Sci 82:1313–1318 Gross WB (1992) Effect of short-term exposure of chickens to corticosterone on resistance to challenge exposure with Escherichia coli and antibody response to sheep erythrocytes. Am J Vet Res 27:972–979 Vleck CM, Vertalino N, Vleck D et al (2000) Stress, corticosterone, and heterophil to lymphocyte ratios in free-living ade´lie penguins. The Condor 102:392–400 Khansari DN, Murgo AJ, Faith RE (1990) Effects of stress on the immune system. Immunol Today 11:170–175 Maxwell MH, Robertson GW (1998) The avian heterophil leukocyte: a review. World’s Poult Sci J 54:155–178 Higgins SE, Torres-Rodriguez A, Vicente JL et al (2005) Evaluation of intervention strategies for idiopathic diarrhea in commercial turkey brooding houses. J Appl Poult Res 14:345–348
22. Gaggia F, Mattarelli P, Biavati B (2010) Probiotics and prebiotics in animal feeding for safe food production. Int J Food Microbiol 141:S15–S28 23. Selvam R, Maheswari P, Kavitha P et al (2009) Effect of Bacillus subtilis PB6, a natural probiotic on colon mucosal inflammation and plasma cytokines levels in inflammatory bowel disease. Indian J Biochem Biophys 46:79–85 24. Swaggerty CL, Kaiser P, Rothwell L et al (2006) Heterophil cytokine mRNA profiles from genetically distinct lines of chickens with differential heterophil-mediated innate immune responses. Avian Pathol 35:102–108 25. Riddell C (1987) Page 10 in: avian histopathology. American Association of Avian Pathologists, Kennett Square
123
Immune Response of Salmonella Challenged Broiler Chickens Fed Diets Containing GalliproÒ, a Bacillus subtilis Probiotic Ali Asghar Sadeghi • Parvin Shawrang Shirin Shakorzadeh
•
Ó Springer Science+Business Media New York 2014
Abstract This study was conducted to investigate the effect of feeding a probiotic, Bacillus subtilis, on antibody titers against Newcastle and infectious bursal viruses in broiler chickens challenged with Salmonella enterica serotype Enteritidis. One hundred and sixty 1-day-old broiler chicks were randomly assigned to four treatments in a completely randomized design. The treatments were negative control, probiotic-treated group, challenged group, and challenged probiotic treated group. Salmonella challenging decreased (P \ 0.05) the relative weights of spleen and bursa. Inclusion of probiotic to diet of challenged chickens increased the relative weight of spleen, but had no effect on the relative weight of bursa. There were no differences for the antibody titers of chickens between negative control and probiotic-treated group. Salmonella challenging decreased (P \ 0.05) antibody titers against Newcastle and infectious bursal viruses. Improvements in the antibody titers were observed by the addition of probiotic to diet of these chickens. The results showed that dietary inclusion of probiotic had no significant effect on immune parameters of chickens at non-contaminated environment, display a greater efficacy at environment contaminated with pathogen and can improve immune responses of infected chickens.
Introduction Antibiotics are supplemented in the broiler rations worldwide to prevent poultry disease and as growth promoters to enhance health status and improve meat production. However, the use of antibiotics has resulted in problems such as drug residues in meat, development of drug-resistant bacteria, and imbalance of normal microflora [1]. As a consequence, it has become necessary to develop alternatives such as beneficial microorganisms termed probiotics. The use of probiotics has been shown to modulate the systemic antibody response to antigens in chickens [2, 3], piglets [4], and fish [5] and can result in the secretion of cytokines and enhancement of immunity [6, 7]. Despite the fact that several studies have shown immune enhancement resulting from oral administration of probiotics, there is a dearth of information regarding the effects of probiotics on the immune response of broiler chickens challenged with Salmonella enterica Enteritidis. The present study was carried out to determine the effect of the probiotic GalliproÒ, Bacillus subtilis, on immune organ weights and on antibody titers against Newcastle and infectious bursal viruses in broilers, and the potential protection it might provide to the birds against a Salmonella challenge.
Keywords Probiotics Bacillus subtilis Immune organ Pathogen challenge Immune cells profile Materials and Methods A. A. Sadeghi (&) S. Shakorzadeh Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, Iran e-mail: [email protected] P. Shawrang Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute, AEOI, Karaj, Iran
Experimental Birds and Treatments One hundred and sixty 1-day-old broiler chicks (Ross 308) were obtained from a commercial hatchery. Using a completely randomized design, the birds were randomly divided into four groups and housed in the separated pens of
123
Probiotics & Antimicro. Prot.
identical size (1.3 9 1.2 m) in a litter system. Each group had 4 replicates with 10 birds per each. The treatments included a negative control (basal diet without probiotic supplementation and challenging), probiotic-treated group (basal diet supplemented with 0.02 % probiotic, without pathogen challenge), challenged group (basal diet, with pathogen challenge at day 7 of age), and a challenged probiotic-treated group (basal diet supplemented with probiotic and pathogen challenge at day 7 of age). Probiotic sample, GalliproÒ, which contains a B. subtilis was prepared from Biochem Co. (Zusatzstoffe GmbH, Germany) and included in the diets at its catalog recommendation. The birds had free access to water and feed. Environmental temperature in the first week of life was 33 °C and decreased to 20 °C until the end of the experiment. During the first week, 22 h of light was provided with a reduction to 20 h thereafter. Salmonella Culturing and Challenging Salmonella enterica serotype Enteritidis (PTCC 1709) was obtained as freeze-dried from the Persian Type Culture Collection (IROST, Tehran, Iran) isolated from the liver of chickens. Briefly, freeze-dried inoculum was grown in tryptic soy broth (Acumedia Manufacturers Inc., Baltimore, MD, USA) at 37 °C for 8 h and passed to fresh tryptic soy broth for three-incubation periods. Determination of the number of colony-forming units (cfu) through decimal dilution series was performed in sterile buffered 0.9 % peptone water with pH 7.2. For this, 0.1 ml of diluted medium was inoculated in Petri dishes containing Shigella–Salmonella agar and cultivated for 24 h at 37 °C, and then, cfu per ml was calculated. Day 7 of age, chicks in challenged groups received by oral gavage 1.0 9 105 cfu per chick (0.2 ml) of S. enterica Enteritidis suspension. Unchallenged birds received the same amount of sterile buffered peptone water by oral gavage. Vaccination and Serology The immunization program included vaccination against Newcastle disease (B1, day 10, eye drop; Lasota days 19 and 32 drinking) and infectious bursal disease (Gumboro, D78, days 12 and 24, eye drop). Blood samples (3.0 ml) of eight birds per treatment (two birds per pen) were collected from the wing veins, using sterile syringes, on days 21 and 42 of age. Immediately after collection, 900 ll of blood was transferred to micro-tube containing 100 ll sodium citrate solutions (3.85 mg/100 ll) and were immediately mixed. The tubes were transferred to Veterinary Laboratory of Mabna (Karaj, Iran) for counting total and differential white blood cells according to Wright’s stain method. Remainder of blood samples (2.1 ml) was
123
Table 1 The relative weights of spleen and bursa of chickens at days 21 and 42 of age (as g/kg body weight) Treatments
Negative control Probiotic treated
Day 21 of age
Day 42 of age
Spleen
Bursa
Spleen
Bursa
1.19
2.53
1.03a,b
1.47a
1.22
2.61
a
1.48a
b
1.07
Salmonella challenged Salmonella challenged and probiotic treated
1.10 1.16
2.13 2.19
0.90 1.00a,b
1.35b 1.36b
SEM
0.102
0.101
0.052
0.086
a, b
Within the same column, means with different superscripts are significantly differed (P \ 0.05)
transferred to clear glass tubes, kept at room temperature for 2 h, then overnight at 4 °C in refrigerator and centrifuged at 1,5009g for 15 min. Serum was obtained, and complement inactivation was done at 56 °C for 30 min and stored at -20 °C to conduct antibody titration. The titers of antibody against Newcastle virus were measured by the hemagglutination–inhibition test. The titers of antibody against infectious bursal virus were measured using a commercial ELISA kit (IDEXX Lab., CA, USA). Immune Organ Weights On days 21 and 42 of age, a total of 32 birds (eight per treatment; two per pen) were randomly selected, individually weighed, stunned, killed by cervical dislocation, and were plucked in the slaughterhouse. The carcasses were opened, and then, the spleen and bursa were removed and weighed. The relative weights of the spleen and bursa were then calculated as [organ weight (g)/body weight (kg)]. Statistical Analysis Statistical analyses were conducted with the general linear model procedure of SAS for Windows version 9.1 (SAS Institute Inc., Cary, NC) to determine whether variables differed among groups. The Kolmogorov–Smirnov test was used to test the normal distribution of the data before statistical analysis was performed. Group comparisons were made using the Tukey test. Probability values of P \ 0.05 were considered significant.
Results Immune Tissue Weights As shown in Table 1, the relative weights of the spleen and bursa at day 21 of age were not affected by treatments. At
Probiotics & Antimicro. Prot. Table 2 Effects of experimental treatments on total and differential counts of white blood cells at day 21 of age Treatments
WBC 9 103
L (%)
H (%)
H/L
E (%)
M (%)
Negative control
23.4d
76.0a
22.0c
0.29b
0
2
Probiotic treated
c
24.8
a
74.0
c
22.0
0.30b
2
2
Salmonella challenged
27.9a
68.0b
29.0a
0.42a
0
3
b
a
b
Salmonella challenged and probiotic treated SEM
26.4
0.61
72.0
1.82
25.0
1.08
a,b
0.35
1
2
0.011
0.7
0.9
WBC white blood cells count, L lymphocyte, H heterophil, H/L heterophil to lymphocyte ratio, E eosinophils, and M monocytes a, b, c, d
Within the same column, means with different superscripts are significantly differed (P \ 0.05)
Table 3 Effects of experimental treatments on total and differential counts of white blood cells at day 42 of age Treatments
WBC 9 103 21.8
c
Probiotic treated
22.9
b
Salmonella challenged
23.4a,b
Salmonella challenged and probiotic treated
Negative control
SEM
L (%) a
H (%)
H/L
E (%)
M (%)
c
0
4
c
19.0
c
75.0
21.0
b,c
0.27
1
3
71.0c
24.0a
0.33a
2
3
24.3a
74.0b
22.0a,b
0.29b
1
3
0.52
0.72
0.91
0.009
0.9
1.3
77.0
b
0.25
WBC white blood cells count, L lymphocyte, H heterophil, H/L heterophil to lymphocyte ratio, E eosinophils, and M monocytes a, b, c
Within the same column, means with different superscripts are significantly differed (P \ 0.05)
this time period, Salmonella challenging resulted in numerical decreases in the relative weights of the spleen and bursa, in comparison with the negative control. Probiotic supplementation increased the relative weights of the immune organs in the probiotic-treated chickens when compared to the challenged chickens. At day 42 of age, differences were present among the relative weights of the spleen or bursa of chickens for the different treatments. Dietary inclusion of the probiotic increased the relative weights of spleen, but had no effect on the relative weight of bursa. At this time period, challenging the birds with Salmonella significantly decreased the relative weights of the spleen and bursa by 12 and 7 % compared with the negative control, respectively. Addition of the probiotic to the diet of these chickens increased the relative weight of the spleen by 10 % in comparison with the spleens of the chickens in the challenged group, but it had no effect on the relative weight of the bursa. Effect on Immune Cells The results of total and differential counts of white blood cells at day 21 of age are reported in Table 2. At this period, there was a significant difference in total white blood cells among treatments (P \ 0.05). The highest mean of total white blood cells was related to the chickens in both Salmonella challenged groups, and the lowest one was for negative control. White blood cells counts
decreased in challenged probiotic-treated chicken by 5 %, compared with challenged chickens. Challenged chickens had the lowest lymphocyte percentage compared with other treatments. There were no significant differences in lymphocyte percentage among the negative control and the chickens fed the diet containing the probiotic with or without challenging. In contrast to the lymphocyte data, the chickens in the positive control group had the highest heterophil percentage and H/L ratio in comparison with the other treatments. The addition of the probiotic to the diet of the challenged chickens reduced heterophil percentage and the H/L ratio, in comparison with the chickens in the challenged group by 16 %, respectively. There were no significant differences for eosinophils and monocytes among treatments. Total and differential counts of white blood cells at day 42 of age are reported in Table 3. At that time period, the lowest and highest total white blood cells counts were observed in the negative control and in the challenged chickens fed the diet containing the probiotic, respectively. Salmonella challenging and probiotic supplementation increased total white blood cells, especially when both of them were applied. The highest lymphocyte percentage and lowest heterophil percentage were found in the probiotictreated chickens, and vice versa, for the challenged chickens. Salmonella challenging resulted in an increased H/L ratio, but the addition of the probiotic to the diet decreased it significantly.
123
Probiotics & Antimicro. Prot.
Fig. 1 Antibody titer (log 2) against Newcastle virus in the unchallenged and Salmonella challenged chickens (Ross308 broilers) fed diets containing probiotic at days 21 (up) and 42 (down) of age
Fig. 2 Antibody titer (log 2) against Infectious Bursal virus in the unchallenged and Salmonella challenged chickens (Ross308 broilers) fed diets containing probiotic at days 21 (up) and 42 (down) of age
Effect on Antibody Titers
chickens at the both periods. At day 21 of age, Salmonella challenging decreased the antibody titers against Newcastle and infectious bursal viruses by 25 and 27 %, respectively, compared with levels observed in the negative control. At that same period, the antibody titer against Newcastle virus in the challenged probiotic-treated chickens increased (18 %) significantly, in comparison with the levels observed in the challenged chickens, but no change was observed for the antibody titer against infectious bursal virus. At day 42 of age, antibody titers against Newcastle and infectious bursal viruses in challenged probiotic-treated chickens increased significantly by 21 and 14 %,
The geometric means of antibody titers against Newcastle and infectious bursal viruses at days 21 and 42 of age are depicted in Figs. 1 and 2. At both time periods and for both antigen levels, there were no differences for antibody titers between the negative control and probiotic-treated chickens, although addition of the probiotic to the diet did appear to increase the numerically antibody titers slightly, but not significantly. Challenging with Salmonella decreased (P \ 0.05) the antibody titers against both antigens, as the lowest means were observed in challenged
123
Probiotics & Antimicro. Prot.
respectively, in comparison with the levels observed in the challenged chickens.
Discussion The main purpose of present study was to examine the effect of dietary inclusion of the probiotic GalliproÒ on the immune responses of Salmonella challenged broiler chickens when vaccinated with Newcastle and infectious bursal viruses. Effects of these treatments on white blood cell counts and the relative weights of the immune functions were also measured. There were no differences in the antibody titers against Newcastle and infectious bursal viruses between the negative control and the unchallenged probiotic-treated chickens, but numerically the latter had slightly but not significantly higher titers than the former. Our results are in agreement with several studies [8, 9], in which the addition of a probiotic to the diet of unchallenged chickens had no significant effect on antibody titers against Newcastle virus and sheep red blood cells when used as antigens. In contrast to our results, the beneficial effects of probiotics treatment on immunity have been reported in number of other studies using probiotics in broilers [6, 10, 11]. Differences among these studies can be related to different age groups used, the different probiotics administered, and the different dose of the probiotics used. Recently, Elangovan et al. [12] reported that probiotic supplementation (500 g, GalliproÒ, B. subtilis) had no effect on cell-mediated immune response, whereas it significantly increased the humoral immune response of broiler chickens. These discrepant findings might be associated with the dose of probiotic used and the class of the antigen to which the antibodies were directed. The addition of the probiotic GalliproÒ to the diet of unchallenged chickens significantly increased the white blood cell in comparison with what was observed in the negative control, but it had no effect on the heterophil to lymphocyte ratio. Our results are in agreement with those of Rahimi and Khaksefidi [13], in which supplementation of the diet with probiotic significantly increased the white blood cell count in comparison with the count found in the control group. Results of this study showed that Salmonella challenge results in decreased antibody titers against Newcastle and infectious bursal viruses, a lower lymphocyte count, and depressed immune organ weights. The exact mechanisms by which the immunomodulatory activities of probiotics are mediated are not clear. However, it has been shown that Salmonella stimulate different subsets of immune system cells to produce cytokines, especially interleukin-1b [14], which in turn play a role in the induction and regulation of
the immune response [15]. The lower humoral immune response of challenged broilers can be explained by increase in production of interleukin-1. Interleukin-1 stimulates the hypothalamus, leukocytes, or both to produce the corticotropin-releasing factor, which stimulates the production of adrenocorticotropic hormone (ACTH) by the anterior pituitary, leukocytes, or both. ACTH then stimulates corticosterone production from the adrenal gland [15]. Corticosterone has been found to be immunosuppressive [16], inhibiting the production and actions of antibodies [17], increasing the heterophil to lymphocyte ratio [18] and depressing immune organ growth [19]. The results in Tables 2 and 3 are consistent with the mentioned events. White blood cell counts and the heterophil to lymphocyte ratio led us to conclude that challenged chickens were in physiological stress status. The heterophil to lymphocyte ratio has been accepted as a reliable index for determining stress in poultry [20]. Heterophils are parts of natural immunity and cellular defense against microbial infections, and lymphocytes are cells that produce antibodies. The increases in heterophil to lymphocyte ratio in challenged chickens may be attributed to increased corticosterone secretion [18], which finally results in a decreased antibody titer. Although the addition of the probiotic GalliproÒ to the diet had no effect on immune response of unchallenged chickens; when Salmonella challenge was administered, significant positive effects were observed. We found no study in the literature in which the effects of probiotic on antibody titers against Newcastle and infectious bursal viruses of Salmonella challenged chickens were evaluated. The present results support the findings of Higgins et al. [21] and suggest that performance-related benefits of some probiotics and probiotic cultures may be most apparent when low-level enteric challenge, such as Salmonella infection, is present. The two most likely mechanisms by which probiotic reduce the negative effects of Salmonella on antibody titers involve competitive exclusion through competition for receptor sites and in the production of volatile fatty acids that are inhibitory of certain enteric pathogens, production of bacteriocins, or competition with pathogens and native flora for limiting nutrients or stimulation of a host innate immune response [22]. The B. subtilis could reduce Salmonella counts in intestines through a number of these inhibitory mechanisms. Recently, an interesting study [23] in humans revealed that the oral administration of B. subtilis could reduce pro-inflammatory cytokines (TNF-a, interleukin-1b, interleukin-6, and Interferon-c) and increase anti-inflammatory cytokine (interleukin-10 and TGF-b) in the plasma of patients with inflammatory bowel disease. These events may have reduced the negative effects of Salmonella on immune response and hematological parameters of the chickens in this study.
123
Probiotics & Antimicro. Prot.
In the present study, white blood cells were increased by Salmonella challenging and were decreased in similar chickens fed a diet containing the probiotic GalliproÒ. An increased level of white blood cells is indicative of an inflammation process or infection. It has been shown that cytokines (especially interleukin 1-b), whose synthesis is increased by infection, are important factors in the inflammatory response [14, 15]. Several in vitro and in vivo studies have established an inflammatory immune response to Salmonella in chickens by demonstrating an up-regulation in the expression of the inflammatory cytokine interleukin-1 [14, 24]. Inflammatory factors stimulate the hypothalamic production of corticotrophin-releasing factor [16]. As mentioned previously, this factor can increase ACTH concentration, thereby stimulating corticosterone production. Corticosterone can inhibit lymphocyte functions and, thereby, decrease the population of leukocytes. Immune tissue development is the basis of immune system functionality. At day 42 of age, the relative organ weights of bursa and spleen were influenced negatively by Salmonella challenging. Stress-induced bursal atrophy has been suggested to be caused by an increased corticosteroid production [25]. Low bursa weight could be interpreted as an indicator of low immune activity because it is a major lymphoid organ in poultry. The decrease of immune tissue weight produces an effect on immune cell phenotypes, immune cell proliferation, and antibody production. As mentioned previously, challenging could induce the production of interleukin-1, which finally can increase the level of serum corticosterone concentration [14]. It was concluded [19] that under this condition, retardation of immune organs occurs. The addition of the probiotic GalliproÒ to the diet of the challenged chickens had no effect on the relative weight of the bursa, but increased the relative weight of the spleen. This same situation was observed in the unchallenged probiotic-treated chickens in comparison with what was observed in their negative control. We cannot find the reason for these events. The results of this study indicated that Salmonella challenging had a negative effect on the immune response of broiler chickens through a retardation of the growth of the immune organs, a change in the profile of the immune cells produced and due to a decrease in antibody production. Dietary inclusion of the probiotic GalliproÒ had no significant effect on the immune parameters of chickens in a non-contaminated environment, but it displayed a greater efficacy when the chickens were in an environment contaminated with a pathogen, and it can improve the efficacy of vaccination and health of infected chickens. Acknowledgments The authors are grateful to the Islamic Azad University for research funding support, to the Agricultural, Medical
123
and Industrial Research School, NSTRI, for supplying the basal diet and allowing the use of their poultry unit, and to the Biochem Company (Zusatzstoffe GmbH, Germany) for providing a sample of the probiotic GalliproÒ. We also thank all of the staff members at the poultry unit for the assistance in the care and feeding of the chickens used in this research. Conflict of interest Ali Asghar Sadeghi, Parvin Shawrang and Shirin Shakorzadeh declare that there is no conflict of interest.
References 1. Sorum H, Sunde M (2001) Resistance to antibiotics in the normal flora of animals. Vet Res 32:227–241 2. Koenen ME, Kramer J, Van Der Hulst R et al (2004) Immunomodulation by probiotic lactobacilli in layer- and meat-type chickens. Brit Poult Sci 45:355–366 3. Allan WH, Gough LE (1974) A standard haemaglutination inhibition test for Newcastle disease: a comparison of macro and micro-methods. Vet Rec 95:120–123 4. Schierack P, Wieler LH, Taras D et al (2007) Bacillus cereus var. toyoi enhanced systemic immune response in piglets. Vet Immunol Immunopathol 118:1–11 5. Panigrahi A, Kiron V, Kobayashi T et al (2004) Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136. Vet Immunol Immunopathol 102:379–388 6. Haghighi HR, Gong J, Gyles CL et al (2005) Modulation of antibody-mediated immune response by probiotics in chickens. Clin Diag Lab Immunol 12:1387–1392 7. Kim DH, Austin B (2006) Cytokine expression in leucocytes and gut cells of rainbow trout, Oncorhynchus mykiss Walbaum, induced by probiotics. Vet Immunol Immunopathol 114:297–304 8. Panda AK, Ramarao SV, Reddy MR et al (1999) Effect of dietary inclusion of probiotic on growth, carcass traits and immune response in broilers. Indian J Poult Sci 34:343–346 9. Seifert S, Fritz C, Carlini N et al (2011) Modulation of innate and adaptive immunity by the probiotic Bifidobacterium longum PCB133 in turkeys. Poult Sci 90:2275–2280 10. Khaksefidi A, Ghoorchi T (2006) Effect of probiotic on performance and immunocompetence in broiler chicks. J Poult Sci 43:296–300 11. Li SP, Zhao XJ, Wang JY (2009) Synergy of Astragalus polysaccharides and probiotics (Lactobacillus and Bacillus cereus) on immunity and intestinal microbiota in chicks. Poult Sci 88:519–525 12. Elangovan AV, Mandal AB, Shrivastav AK, Yadhav AS (2011) Supplementing probiotics (GalliPro) to broiler chicken on growth performance, immunity and gut microbial population. Anim Nut Feed Technol 11:169–176 13. Rahimi S, Khaksefidi A (2010) A comparison between the effects of a probiotic (Bioplus 2B) and an antibiotic (virginiamycin) on the performance of broiler chickens under heat stress condition. Iran J Vet Res 7:23–28 14. Okamura M, Lillehoj HS, Raybourne RB et al (2004) Cellmediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-c production. Comp Immunol Microbiol Infect Dis 27:255–272 15. Maassen CB, Van Holten-Neelen C, Balk F et al (2000) Straindependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains. Vaccine 18:2613–2623 16. Post J, Rebel JMJ, Huurne AAHM (2003) Physiological effects of elevated plasma corticosterone concentrations in broiler chickens.
Probiotics & Antimicro. Prot.
17.
18.
19. 20. 21.
An alternative means by which to assess the physiological effects of stress. Poult Sci 82:1313–1318 Gross WB (1992) Effect of short-term exposure of chickens to corticosterone on resistance to challenge exposure with Escherichia coli and antibody response to sheep erythrocytes. Am J Vet Res 27:972–979 Vleck CM, Vertalino N, Vleck D et al (2000) Stress, corticosterone, and heterophil to lymphocyte ratios in free-living ade´lie penguins. The Condor 102:392–400 Khansari DN, Murgo AJ, Faith RE (1990) Effects of stress on the immune system. Immunol Today 11:170–175 Maxwell MH, Robertson GW (1998) The avian heterophil leukocyte: a review. World’s Poult Sci J 54:155–178 Higgins SE, Torres-Rodriguez A, Vicente JL et al (2005) Evaluation of intervention strategies for idiopathic diarrhea in commercial turkey brooding houses. J Appl Poult Res 14:345–348
22. Gaggia F, Mattarelli P, Biavati B (2010) Probiotics and prebiotics in animal feeding for safe food production. Int J Food Microbiol 141:S15–S28 23. Selvam R, Maheswari P, Kavitha P et al (2009) Effect of Bacillus subtilis PB6, a natural probiotic on colon mucosal inflammation and plasma cytokines levels in inflammatory bowel disease. Indian J Biochem Biophys 46:79–85 24. Swaggerty CL, Kaiser P, Rothwell L et al (2006) Heterophil cytokine mRNA profiles from genetically distinct lines of chickens with differential heterophil-mediated innate immune responses. Avian Pathol 35:102–108 25. Riddell C (1987) Page 10 in: avian histopathology. American Association of Avian Pathologists, Kennett Square
123
