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Cell-Immunomodulation Against Salmonella enteritidis in Herbal Extract-Treated Broilers
Elie K. Barbour†
Vatche K. Sagherian†
Rabih S. Talhouk‡
Salma N. Talhouk*
†Department of Animal Science, American University of Beirut, Beirut, Lebanon
‡Department of Biology, American University of Beirut, Beirut, Lebanon
*Department of Plant Science, American University of Beirut, Beirut, Lebanon
KEY WORDS: Cell-immunomodulation, Salmonella enteritidis, herbal extracts, broilers
Water extracts of Calendula officinalis (1.5 X 104 µg/mL) and Melissa officinalis (3.0 Ą 104 µg/mL) were administered orally, in defined volumes, to respective groups of chicken broilers between 10 to 21 days of age, while control broilers were administered a similar volume of plain drinking water. At 41 days of age, a volume of 0.1 mL of inactivated Salmonella enteritidis (SE) culture, adjusted to 3% transmittance at a wave length of 540 nm, was injected intradermally in the right footpad of each assigned bird in the three treatments, while the left footpad was left as control, injected with 0.1 mL of plain sterile saline. Twenty-four hours after SE administration, four slides, each carrying 6 tissue sections (3 µm thickness), were prepared of each footpad. Immunohistochemistry technique, based on mouse monoclonal biotinylated antibodies against CD3-T cells, CD4-T cells, and IgM-marked B cells, was used to count these cells in the first three slides, and H & E staining was applied on the fourth slide to count the neutrophils. The C. officinalis-treated birds had the highest mean CD3-cell count (P < 0.05) and the lowest mean neutrophil count (P < 0.05) among the three treatments. No chemotactic presence of CD4 or IgM-marked B cells was noticed at the SE-administered right footpad of all individual birds of the three treatments. In addition, the left control footpad of all birds, injected with plain saline, showed a complete absence of the four cell types. This work concludes that cell-immunomodulation against SE was present only in C. officinalis–treated birds, a finding worthy of future investigation directed toward control of zoonoses caused by SE.
The increase in the frequency of human Salmonella enterica serotype enteritidis (SE) outbreaks in the United States,1 the United Kingdom,2 and in Western Europe3 was associated with consumption of poultry products contaminated with SE.4 Immunomodulation targeting the control of this zoonoses was focused mainly on vaccines5 and other chemical substances, including thymulin and zinc.6 The alarmingly high incidence of antimicrobial resistance in bacteria caused many governments in Europe to ban the use of many antimicrobials in animals, encouraging alternative approaches such as the use of herbal immuno modulators that might have significance in organic farming7,8 with a clinical value in controlling the emergence of resistant microbial strains.9
The immunomodulation concept can be traced back to observation recorded by workers more than a century ago.10 Later, another report showed that immuno active substances could produce either suppression or enhancement of immunologic activities.11 The available improved experimental techniques and the greater understanding of the immune system made it possible to classify a large number of physiologic, microbial synthetic substances and natural herbal extracts as immuno modulators.12–14
Melissa officinalis extracts were found to contain Rosmarinus acid substances responsible for immunomodulation of several complement-dependent inflammatory processes with a major effect on the C5 convertase enzyme.15
However, reports related to Calendula officinalis indicate that the immunomodulation by this plant is mainly caused by a high-molecular-weight polysaccharide that stimulates the immune system.16 Others have documented an immunomodulation potential in C. officinalis extract to control the human immunodeficiency virus (HIV).17
To our knowledge, this is the first report that evaluates immunomodulation of CD3, CD4, IgM-marked B-cells, and neutrophils against S. enteritidis antigens in herbal extract-treated broilers.
Materials and Methods
Preparation of Plant Extracts
A total of 90 g of fresh-cut flowers and leaves of C. officinalis were immersed for 15 minutes in 1 L of boiled distilled water; the extract was filtered through Watman No. 1 filter paper and made up with distilled water to 6 L volume. The final weight of C. officinalis to volume of water solvent was 1.5 X 104 µg/mL. Moreover, an amount of 180 g of fresh-cut leaves of Melissa officinalis were immersed in one liter of boiled distilled water for 15 minutes, filtered through Watman No. 1 filter paper, and the filtrate was made with distilled water to a 6-L volume. The final weight of M. officinalis to volume of water solvent was 3.0 Ą 104 µg/mL.
Birds and Experimental Design
Three groups of day-old broiler chicks, each composed of 105 birds, were put in 9 pens (3 pens per treatment, with 35 birds per pen). All birds were fed similarly according to formulations recommended by the National Research Council.18 Each of the three divided groups was treated differently; group one received C. officinalis extract, group two received M. officinalis extract, and group three was the control, receiving plain drinking water. The extracts and the drinking water were administered orally to each individual bird, using a disposable pipet, in an amount of 2.8 mL/bird/d between the ages of 10 and 15 days, and in daily amounts of 4.4 mL/bird between the ages of 16 and 21 days, when the immune system of birds is completely mature. Besides this treatment, the drinking water was made available ad libitum. The standardized feed and water availability and the proper space of 10 birds per meter square comply with the “Ethical principles and guidelines for scientific experiments on animals,” as instructed by the Swiss Academy of Medical Sciences.
Salmonella Enteritidis preparation and administration: Pure culture of SE was grown in tryptose phosphate broth (TPB) at 37˚C for 24 hours. The culture was inactivated by formalin with a concentration in TPB culture of 0.3%. Inactivation was confirmed by subculturing formalized TPB-SE culture onto brilliant green agar. The inactivated SE was pelleted from TPB by centrifugation at 1265.4 Ą g. The recovered SE pellets were washed three times with sterile saline. The washed pellets were reconstituted in sterile saline to form a suspension density equivalent to 3% transmittance at a wavelength of 540 nm.
Ten birds 41 days of age were randomly selected from each of the three treatments marked with leg bands, and individually administered a 0.1 mL of the 0.3% inactivated SE cells intradermally in the right footpad. The left footpad of the selected birds was injected with 0.1 mL sterile saline, serving as a control to each bird’s case. Immunohistochemistry and histopathology procedures were applied on the skin organ collected from the footpads, 24 hours after the inactivated SE-delivery.
Immunohistochemistry and Histopathology
The birds were killed 24 hours after inactivated SE delivery. Skin of the footpads at the site of injection was cut and fixed in 10% formalin solution. Embedding in paraffin wax and microtome sectioning to 3 µm thickness was performed according to a previously described procedure.19 Six sections of each footpad were placed on each of four microscopic slides for immunohistochemistry and histopathology procedures.
The immunohistochemistry procedure was performed according to a previously described protocol.20 Briefly, the first three slides of each footpad were assigned for marking the CD3, CD4, and IgM-mature B cells, respectively. The three slides were deparafinized in xylene and bleached in 3% methanol peroxide to quench the endogenous peroxide. Next, 0.2% bovine serum albumen was used for blocking. The three slides from each footpad were treated respectively with 1:200 dilution of mouse monoclonal biotinylated antibodies against markers on CD3-T cells, CD4-T cells, and IgM-mature B cells (Southern Biotechnology Associates, Inc., Birmingham, AL, U.S.A.). Strepavidin-peroxidase conjugate (Zymed Laboratories Inc, San Francisco, CA, U.S.A.) and DAB substrate supplemented with H2O2 were used in sequence to color the specific targeted cells in the footpad tissues.
The CD3, CD4, and IgM-mature B cells were counted on the 3 respective slides. Five microscopic fields at magnification of 400X were randomly selected for counts on each of the 6 tissue sections of each footpad. The mean of a cell count is reported per one field.
The histopathology procedure based on H & E staining21 was applied on the fourth slide of each footpad, carrying six tissue sections. The neutrophils were counted at magnification of 1000 X, in which five microscopic fields were randomly selected for counts on each of the 6 tissue sections of each footpad.
The analysis of variance (ANOVA) of the mean count of CD3, CD4, IgM-mature B cells, and neutrophils was carried using SPS version 9.0. The experimental layout was a randomized complete block design. Means were then separated by Dunkan’s Multiple Range Test (? = 0.05).
Figure 1 shows the presence of neutrophils in right footpads, injected with inactivated-SE cells of birds treated with M. officinalis (Figure 1B); however, the left footpad of the same bird (Figure 1B), injected with sterile saline, showed a complete absence of neutrophils. In addition, the right footpad of C. officinalis–treated broilers, injected with SE, showed presence of CD3-T cells (Figure 2B), and the left footpad of the same bird (Figure 2A), injected with sterile saline, showed a complete absence of CD3-T cells.
Table 2 shows the comparison of the mean count of neutrophils, CD3, CD4, and IgM-B cells in right footpads injected with SE cells among the three broiler treatments. These were controls deprived of herbal extracts and broilers administered C. officinalis or M. officinalis extracts. A complete absence of the 4 types of cells was seen in the left footpad (sterile saline) of broilers in the three treatments, as indicated in the third footnote of Table 1.
Broilers treated with C. officinalis showed cell immunomodulation resulting in a significant increase in CD3 cells (P < 0.05) in the right footpads receiving inactivated SE cells compared with the right footpads of M. officinalis extract-treated birds, and with controls deprived of herbal extracts (Table 1). The magnitude of this increase in CD3 cell count in C. officinalis-treated broilers was about 10 times that of controls and about 18 times that of M. officinalis-treated birds.
On the contrary, the C. officinalis birds had a significant reduction (P < 0.05) in the number of neutrophils in SE-injected right footpads compared with the other two treatments (Table 1). The magnitude of this reduction in neutrophil cell count of C. officinalis-treated broilers was about 2.0 times less than that of M. officinalis-treated birds and about 2.5 times less than that of controls. No chemotactic presence of CD4 and IgM-B cells was noticed in SE-injected footpads of all individual birds of the three treatments (P > 0.05).
The herbal extracts did not induce a higher infiltration of neutrophils to the site of inactivated SE cells in the footpad of broilers compared with controls not given extracts (Table 1). On the contrary, the C. officinalis –treated birds had a significant drop in neutrophil count (P < 0.05) compared with controls, and the M. officinalis-treated birds showed an insignificant drop in neutrophil compared with controls (P > 0.05).
Previous research documented the presence of rosmarinic acid, a naturally occurring active ingredient in water extracts of M. officinalis that proved to inhibit several complement-dependent inflammatory processes15 In addition, the significant reduction in neutrophil count in C. officinalis–treated birds could be caused by immunomodulation by anti-inflammatory components present in the extract. Previous workers showed the presence of an anti-inflammatory effect in C. officinalis extract.22 Others reported that tannins in C. officinalis water extracts had inhibitory effects on neutrophils chemotaxis.23 This was in agreement with reports from other researchers proving the anti-phlogistic activity of tannins in C. officinalis extracts.24 Practically, herbal infusion, tinctures, and ointments of C. officinalis are now used to treat skin and mucous membrane inflammation such as pharyngitis, dermatitis, leg ulcers, conjunctivitis, superficial wounds, sores, and burns.25
A significant positive immunomodulation of CD3-T cells was present in the C. officinalis–treated broilers (Figure 2 and Table 1), resulting in 18 and 10 times increase in mean count of CD3 cells compared with that in M. officinalis–treated birds and in controls, respectively (P < 0.05). A previous work noted that C. officinalis extract induced significant stimulation of T cell activation.26 The intradermal introduction of inactivated SE cells in the footpad clearly did not induce a CD4 cell response (TD cell) in all treatment groups. However, it did induce a significant CD3 response in C. officinalis–treated birds and, to a less extent, in M. officinalis–treated birds and in controls. This absence of CD4 cells is indicative of absence of a delayed hypersensitivity reaction to killed SE cells. This is in agreement with previous workers agreeing that CD4 T-cells are not required for this early host response to Salmonella sp.27-29 In addition, the absence of IgM-B cells at SE site indicates the absence of a presensitized B-cell clone to SE antigens in the experimental birds.
Conversely, the C. officinalis-treated birds showed a significant reduction (P < 0.05) in the number of neutrophils in SE-injected right footpads, compared with the other treatments (Table 1). The magnitude of this reduction in neutrophil cell count of C. officinalis-treated broilers was about 2 times less than that of M. officinalis-treated birds and about 2.5 times less than that of controls (untreated by herbal extracts).
No chemotactic presence of CD4 and IgM-B cells was noticed in the SE-injected footpads of individual birds in the three treatment groups (P > 0.05).
The significant rise in CD3 cells (T cytolytic cells) could have a positive impact on more production of macrophage activation factor such as interferon-gamma, which is crucial for protection against intracellular microbial pathogens, such as SE.30 Other workers have emphasized the role of CD3 cells (TC) in mediation of cytolysis of Salmonella–infected target cells in an interferon–dependent mechanism.31 The cytolysis of host cells by TC cells to clear the intracellular infection will definitely need a tissue regeneration that could be provided by the C. officinalis extract itself, an ability that is proved to be present in such extract leading to tissue regeneration and epithelial tissue development.32 The ability in C. officinalis to regenerate tissues forms the basis in marketing ointments of such extract for treatment of slow-healing tissue cuts.25
Future investigation should target understanding the impact of C. officinalis extract on the clearance of intracellular SE cells by immunomodulated CD3 cells in chickens in an attempt to reduce the threat of transmission of the etiologic agent of this major zoonoses to human consumers of poultry products.
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Figure 1. (A) Presence of neutrophils (arrow) in right footpad of Melissa officinalis–treated bird, injected with inactivated-SE cells.
Figure 1. (B) Absence of neutrophils in left footpad of the same bird, injected with sterile saline.
Figure 2. (A) Presence of CD3-T cells (arrow) in right footpad of Calendula officinalis–treated bird, injected with inactivated-SE cells.
Figure 2. (B) Absence of CD3-T cells in left footpad of the same bird, injected with sterile saline.
Table 1. Mean Count* of CD3, CD4, IgM-mature B cell, and neutrophils after 24 hours of Inactivated SE Injection† in right footpads of herbal extract-treated broilers
Treatment Mean count of different immune cells
Neutrophils CD3 CD4 IgM-B cells
Control‡ 7.59a 1.91b 0.0a 0.0a
Calendula officinalis 2.98b 20.17a 0.0a 0.0a
Melissa officinalis 5.83a 1.13b 0.0a 0.0a
SEM§ 0.779 5.134 0.0a 0.0a
* Mean count at microscopic magnification of 1000 Ą (neutrophils) and 400 Ą (CD3, CD4, and IgM-B cells) in which five microscopic fields were randomly selected on each of the 6 tissue sections of each footpad. The mean is count/field/footpad.
† Injection of 0.1 mL of 3% transmission inactivated SE cells at a wavelength of 540 nm.
‡ Count in right footpad injected with SE cells in control birds deprived of herbal extracts. The count of the 4 types of cells in the left footpads of birds in the 3 treatments was zero.
§ SEM, standard error of means.
a–b Means with different alphabet superscripts in a column are significantly different (P < 0.05).
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