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Poult Sci 2007. 86:2315-2321. doi:10.3382/ps.2007-00123
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IMMUNOLOGY, HEALTH, AND DISEASE

Effect of Probiotic Treatment in Broiler Chicks on Intestinal Macrophage Numbers and Phagocytosis of Salmonella Enteritidis by Abdominal Exudate Cells

S. E. Higgins, G. F. Erf, J. P. Higgins, S. N. Henderson, A. D. Wolfenden, G. Gaona-Ramirez and B. M. Hargis1

Center of Excellence in Poultry Science, University of Arkansas, Fayetteville 72701

1 Corresponding author: bhargis{at}uark.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Previous data have indicated that a Lactobacillus-based probiotic culture (FM-B11) is efficacious in reducing Salmonella Enteritidis colonization within 24 h when administered within 1 h of challenge. We hypothesized that the innate immune system, specifically macrophages, may play a role in the observed reduction of Salmonella Enteritidis colonization with probiotic treatment. Day-of-hatch chicks were challenged with Salmonella Enteritidis and then treated with the probiotic culture 1 h later. Three other treatment groups were not treated (negative control), challenged only, or treated with probiotic only. In all experiments, probiotic treatment on the day of hatch reduced (P < 0.05) cecal Salmonella Enteritidis recovery as compared with the control treatment. In experiments 1 and 2, immunohistochemistry was used to evaluate the presence of macrophages (KUL01+) in the ileum and cecum of 7 to 10 chicks per group at 24 h posttreatment. In experiment 1, the number of macrophages observed per 10,000 µm2 in the ileum of Salmonella Enteritidis-challenged chicks was higher (P < 0.05) than that of nonchallenged chicks (4.87 ± 0.31 vs. 3.05 ± 0.19). In the cecum, there were more (P < 0.05) macrophages per 10,000 µm2 in chicks receiving probiotic treatment without challenge than in negative control chicks (5.32 ± 0.41 vs. 3.66 ± 0.35). However, in experiment 2 we found no differences among treatments in the numbers of macrophages for both the ileum and cecum. Experiments 3 and 4 were performed to evaluate the ability of Sephadex-elicited abdominal exudate cells (AEC) from chicks to phagocytose Salmonella Enteritidis in vitro. Abdominal exudate cells were isolated from the abdominal cavity, maintained in tissue culture plates overnight, and then assayed for phagocytic activity by coincubating with Salmonella Enteritidis. In experiment 3, more (P < 0.05) Salmonella Enteritidis was recovered from AEC derived from probiotic-treated chicks than in any other treatment. However, in experiment 4, all treatments resulted in similar levels of elicited AEC, and phagocytosis of Salmonella Enteritidis was at low levels in all groups. Although not conclusive, the modest differences detected in experiments 1 and 3, and the fact that those differences were not repeatedly detectable, suggest that these macrophage-related changes were not solely responsible for the reductions of Salmonella Enteritidis following probiotic treatment.

Key Words: Salmonella • probiotic • macrophage • chick • phagocytosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Salmonella from poultry products has been associated with human foodborne illness (Kimura et al., 2004; Marcus et al., 2007). In the United States, it is estimated that 1.4 million humans contract salmonellosis and that the annual cost of this illness, including lost productivity, is $3 billion annually (World Health Organization, 2006). Competitive exclusion cultures and probiotic cultures consisting of live beneficial bacteria have been used to reduce levels of Salmonella in live poultry, with positive results (Nurmi and Rantala, 1973; Blankenship et al., 1993; Corrier et al., 1995; Waters et al., 2005). Additionally, improved performance has been reported with probiotic cultures (Huang et al., 2004; Higgins et al., 2005; Timmerman et al., 2006). The mechanisms behind these benefits are not well understood, although many studies have observed immunomodulatory effects from probiotic treatments.

Dalloul et al. (2005) described significantly higher levels of both interferon (IFN)-{gamma} and interleukin-2 in the intestine of probiotic-fed chickens 3 d following challenge with Eimeria acervulina. Another study by Yurong et al. (2005) reported increases in the number of Ig-producing cells (IgM, IgG) detected in Peyer’s patches and the cecal tonsils of chicks by d 7 and 10, respectively, following administration of a probiotic culture in the drinking water containing Bacillus subtilis, Candida utilis, and Lactobacillus acidophilus. The length of the cecal tonsils was also increased following 3 d of probiotic administration (Yurong et al., 2005). Koenen et al. (2004) found that oral administration of 2 probiotic Lactobacilli isolates to broiler chickens for 5 consecutive days caused increased phagocytosis by cells from the cecum and ileum, and also increased serum IgG and IgM.

Probiotics have previously been associated with activation of innate immunity through phagocytic cells. Recently, Farnell and coworkers (2006) reported that specific isolates of probiotic bacteria increased the oxidative burst capacity and degranulation of heterophils isolated from chicks treated 24 h following probiotic treatment, indicating that the innate immune system may also be activated through probiotic treatment. Olivares et al. (2006) reported an increase in both the number of circulating phagocytic cells and their activity in humans following consumption of either 2 lactic acid bacteria or a commercial yogurt. Additionally, Parra et al. (2004) observed that the consumption of fermented milk (containing lactic acid bacteria) by humans increased the oxidative burst capacity of monocytes and increased the tumoricidal capacity of natural killer cells. Sadeyen and coworkers (2004) found that inbred lines of chickens naturally susceptible to Salmonella infections had lower expression of IFN-{gamma} in the cecal tonsils at 3 wk of age compared with naturally resistant lines of chickens. Because IFN-{gamma} is a strong activator of macrophages, the production of this cytokine appears to be important to Salmonella resistance. Macrophages are present in most organs and possess effector functions such as phagocytosis, antigen processing and presentation, and cytokine secretion (Qureshi et al., 2000).

Because Salmonella spp. have a dynamic relationship with macrophages, we hypothesized that macrophages may play a role in the reduction of Salmonella following probiotic treatment. In the following experiments, we evaluated whether a Lactobacillus-based probiotic culture would increase the numbers of macrophages in the ileum and cecum. Additionally, we evaluated phagocytic activity in vitro by using Sephadex-elicited abdominal exudate cells (AEC) from chicks receiving the probiotic with or without Salmonella Enteritidis challenge.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Salmonella Enteritidis

A primary poultry isolate of Salmonella Enteritidis, bacteriophage type 13A (Salmonella Enteritidis), was obtained from the USDA National Veterinary Services Laboratory. This isolate was resistant to novobiocin (25 µg/mL, catalog no. N-1628, Sigma, St. Louis, MO) and was selected for resistance to naladixic acid (20 µg/mL, catalog no. N-1628, Sigma) in our laboratory. For these studies, 100 µL of Salmonella Enteritidis from a frozen aliquot was added to 10 mL of tryptic soy broth (catalog no. 211822, Becton Dickinson, Sparks, MD) and incubated at 37°C for 8 h. Then, 100 µL of culture was passed into 10 mL of fresh medium and incubated again for 8 h. This was repeated one more time for a total of 24 h. Bacterial cells were washed 3 times in sterile 0.9% saline by centrifugation at 1,864 x g, quantified with a spectrophotometer to a concentration of approximately 109 cfu/mL in sterile 0.9% saline, and diluted to inoculated concentrations as described below. Concentrations of Salmonella Enteritidis were determined retrospectively by spread-plating on xy-lose-Lys-deoxycholate (XLD) agar and enumeration for each experiment, and actual determined colony-forming units were reported.

Probiotic Culture

Eleven lactic acid bacterial isolates were previously selected and described (Higgins et al., 2005). These isolates, which were combined to form a probiotic culture that is now commercially available as FM-B11 (catalog no. 41069, IVS-Wynco LLC, Springdale, AR), were used for these experiments. The probiotic culture was diluted in reconstituted powdered skim milk to an expected concentration of 4 x 106 cfu/mL for oral gavage of chicks in these studies. Actual colony-forming units administered per chick from each experiment are reported below, which were determined retrospectively from spread-plating on de Man, Rogosa, Sharpe agar (catalog no. R1148, Sigma).

Experimental Design

These experiments were approved by the Institutional Animal Care and Use Committee of the University of Arkansas. Day-of-hatch broiler chicks were obtained from a local hatchery and were randomly assigned to 1 of 4 treatment groups (n = 25/group). Two groups were challenged by oral gavage with Salmonella Enteritidis in 0.25 mL of saline (experiments 1 and 2: 104 cfu/chick; experiment 3: 2.5 x 103 cfu/chick; experiment 4: 1.4 x 103 cfu/chick). The 2 remaining nonchallenged groups received the sterile saline vehicle by oral gavage (0.25 mL). All chicks were placed in a battery brooder unit (1 group per level) with unmedicated feed and water and were maintained at an age-appropriate temperature. One hour postchallenge, 1 challenged group and 1 unchallenged group received probiotic treatment by oral gavage (experiments 1 and 2: 3.0 x 106 cfu/chick; experiment 3: 4.1 x 106 cfu/chick; experiment 4: 5.7 x 106 cfu/chick), and the additional 2 groups received the skim milk vehicle by oral gavage.

Salmonella Enteritidis Recovery

In all experiments, Salmonella Enteritidis recovery from the cecal tonsils, to determine the efficacy of the probiotic culture, was performed as described below. Chicks were humanely killed by CO2 asphyxiation 24 h posttreatment. Cecal tonsils were aseptically removed and placed in tetrathionate broth (catalog no. 210420, Becton Dickinson) for Salmonella enrichment. The samples were incubated overnight at 37°C and then streaked for isolation on XLD agar plates (catalog no. 278820, Sigma-Aldrich, St. Louis, MO) containing novobiocin and naladixic acid. Plates were incubated overnight, and the presence or absence of Salmonella colonies was recorded.

Immunohistochemistry (Experiments 1 and 2)

At the same time the cecal tonsils were collected for Salmonella Enteritidis recovery, sections of the ileum and cecum were taken from 12 chicks per treatment group. Each sample was approximately 0.5 cm long and was immediately placed into a sterile sample bag and frozen in liquid nitrogen. The ileum sample was obtained from the midpoint between the end of the duodenal loop and Meckel’s diverticulum. Cecal tissues were taken from the midpoint of the cecum. Frozen samples were embedded in optimal cutting temperature medium (catalog no. 4583, Sakura Finetek, USA Inc., Torrance, CA) on blocks of dry ice and kept at –80°C until cut for immunohistochemistry.

Tissue samples were cut 5 µm thick with a cryostat, and sections were mounted on poly-L-Lys-coated slides and briefly fixed in acetone. Immunohistochemistry was performed in humidified chambers at room temperature, and blocking was performed with 10% horse serum in PBS overnight at room temperature. Slides were rinsed, and the KUL01 mouse antichicken monocyte-macrophage (catalog no. 8420-01, Southern Biotech, Birmingham, AL) primary antibody, at a dilution of 1:100, was applied to each tissue. Tissues were incubated for 30 min at room temperature. For each sample, an additional section was prepared and incubated with an antibody of irrelevant specificity to determine nonspecific binding of the primary antibody (isotype control, mouse IgG1, catalog no. M5909, Sigma-Aldrich). After 30 min, all tissues were rinsed with PBS, and horse antimouse IgG was applied at a 1:100 dilution for 30 min. All tissues were rinsed again and then incubated with avidin-enzyme-linked biotin complex (catalog no. PK-4002, Vector Laboratories Inc., Burlingame, CA) for 30 min. Incubation with avidin-enzyme-linked biotin complex was followed by several washes, after which diaminobenzidine substrate activated with 3% H2O2 was added for 5 min. All tissues were placed in methyl green stain for 30 min to counterstain, dehydrated in sequential baths, and mounted with a cover slip.

Quantification of Macrophages

Macrophages identified by immunohistochemistry were quantified by using a bright field microscope at 400x magnification. Three microscope fields were randomly selected on each sample. For each field, the area was determined and the number of macrophages enumerated. The number of macrophages (KUL01+ cells) per 10,000 µm2 was determined for each sample. The mean for individual tissues within a group was used to determine the mean for each treatment group.

Experiments 3 and 4

In these experiments, all chicks were injected in the abdominal cavity with 0.5 mL of a 3% Sephadex (catalog no. G5050, Sigma-Aldrich) suspension at the time of probiotic treatment, according to the method described by Qureshi et al. (1986). After 24 h, chicks were humanely killed by CO2 asphyxiation. Abdominal exudate cells were collected by making an incision in the abdominal cavity and washing the cavity with sterile PBS containing 0.5 U/mL of heparin. The washes were made by using 5-mL pipette tips, which were modified by the addition of 4 holes. The holes were made approximately 0.5 cm above the tip at equal intervals around the pipette tip by perforating the tip with an 18-gauge needle. Collected cells were added to sterile, siliconized glass 15-mL conical tubes and kept on ice for at least 10 min. After collection of AEC, the cecal tonsils were removed and processed as described above for Salmonella Enteritidis recovery.

After collection of the AEC suspension, debris was allowed to settle for 10 min, and the supernatant fluid containing the cells was poured into siliconized sterile 16 x 125 mm borosilicate glass tubes. Supernatants from 3 chicks were pooled into 1 tube, and 8 pools were assayed per treatment group. The AEC containing supernatants were centrifuged at 300 x g at 8°C for 15 min, and the pellet of cells was resuspended in 5 mL of RPMI medium (catalog no. 11835, Invitrogen, Grand Island, NY) with no phenol red, and 5% fetal bovine serum. The 5 mL of resuspended AEC was added on top of 5 mL of Histopaque-1077 (catalog no. 10771, Sigma-Aldrich) in sterile siliconized glass tubes. They were centrifuged for 30 min at 500 x g and then a Pasteur pipette was used to collect the mononuclear AEC at the interface. The cells were washed in PBS by centrifugation at 250 x g for 8 min, and the pellet was resuspended in 1.1 mL of RPMI medium. From each AEC pool, 10 µL of cells was combined with 10 µL of 10% Trypan blue for enumeration of mononuclear AEC and determination of cell viability. Each AEC suspension was found to consist of approximately 90 to 95% large viable mononuclear cells. The numbers of cells were recorded for each sample and were found to be between 105 and 106 cells/mL. Because of the minimal numbers of cells obtained from each pooled sample, AEC samples were immediately plated into 24-well tissue culture plates (0.5 mL/well, 2 wells per sample, catalog no. 3047, Becton Dickenson Co., Lincoln Park, NJ) and were not adjusted to equal numbers of cells per well. Cells were allowed to adhere for 2 h at 40°C in 5% CO2. Following adherence, the wells were carefully washed with sterile PBS 3 times, and 0.5 mL of RPMI was added. The cells were maintained in tissue culture plates in the incubator overnight.

Phagocytosis Assay (Experiments 3 and 4)

The phagocytosis assay was performed similarly to that described by Desiderio and Campbell (1983) and Kramer et al. (2001) for bovine and avian macrophages, respectively. The next day, AEC cultures were rinsed 1 time with sterile PBS, and 5 x 107 cfu of Salmonella Enteritidis diluted in 0.5 mL of RPMI medium was added to each well. Samples were incubated in the presence of Salmonella Enteritidis for 30 min at 40°C. After Salmonella Enteritidis incubation, 0.2 mL of supernatant was taken from each well, serially diluted, and spread-plated on XLD agar plates to determine the numbers of extracellular Salmonella Enteritidis. Wells were rinsed with sterile PBS 3 times, and 0.5 mL of RPMI containing 200 µg/mL of gentamicin was added to each well and incubated for 45 min at 37°C to kill any extracellular Salmonella Enteritidis. Wells were again rinsed 3 times with PBS, and 0.5 mL of PBS containing 1% Triton X was added to the wells for 10 min to lyse the AEC. A sample was taken from each well at this time to determine the numbers of intracellular Salmonella Enteritidis recovered. These aliquots were serially diluted and plated on XLD plates for enumeration of viable intracellular Salmonella Enteritidis (Desiderio and Campbell, 1983; Kramer et al., 2001). The numbers of cfu of Salmonella Enteritidis recovered from each well were then divided by the number of AEC originally counted in that sample, and data are reported as cfu of Salmonella Enteritidis per AEC.

Statistical Analysis

The incidence of Salmonella recovery within experiments was compared by using the chi-squared test of independence (Zar, 1984) to determine significant (P < 0.05) differences between control and treated groups. Data from enumerated macrophages in experiments 1 and 2 as well as Salmonella Enteritidis cfu in experiments 3 and 4 were analyzed by ANOVA and further separated by Duncan’s multiple range test (SAS Institute, 2004).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
To our knowledge, this is the first report of the numbers of macrophages present in intestinal tissue of 1-d-old chicks. In all experiments, we found significant reductions of Salmonella Enteritidis in probiotic-treated chicks compared with nontreated control chicks (Table 1Go). Using immunohistochemistry (Figure 1Go), we found higher numbers of ileal macrophages in the lamina propria of chicks in the Salmonella Enteritidis-challenged group of experiment 1 compared with the negative control chicks (4.87 ± 0.31 and 3.05 ± 0.19 macrophages/10,000 µm2, respectively, P < 0.05; Table 2Go). Similarly, in the cecum (Figure 2Go) we observed higher numbers of macrophages in the probiotic-treated chicks (5.32 ± 0.41 macrophages/10,000 µm2, P < 0.05) compared with negative control chicks (3.66 ± 0.35 macrophages/10,000 µm2). Chicks receiving the Salmonella Enteritidis challenge with or without probiotic treatment had intermediate numbers of cecal macrophages (4.38 ± 0.30 and 4.54 ± 0.35, respectively). In experiment 2, we observed no treatment differences in macro- phage numbers in either the ileum or cecum (Table 3Go). However, independent of treatment, the average numbers of macrophages present in the ileum and cecum of 24-h-old chicks were similar in both experiments.


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  		Table 1. Salmonella Enteritidis recovered from the cecal tonsils of chicks treated or not treated with a probiotic culture in experiments 1 through 4
 

Figure 1
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  		Figure 1. Macrophages in the ileum of a probiotic-treated 24-h-old chick. Macrophages in frozen tissue sections (5 µm) were identified by using an antichicken monocyte-macrophage mouse monoclonal antibody (KUL01) and an indirect immunoperoxidase staining procedure. Immunopositive cells are black.

 

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  		Table 2. The enumeration of macrophages in the cecae and upper ileum by immunohistochemistry1
 

Figure 2
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  		Figure 2. Macrophages in the cecum of a probiotic-treated 24-h-old chick. Macrophages in frozen tissue sections (5 µm) were identified by using an antichicken monocyte-macrophage mouse monoclonal antibody (KUL01) and an indirect immunoperoxidase staining procedure. Immunopositive cells are black.

 

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  		Table 3. Phagocytosis of Salmonella Enteritidis by abdominal exudate cells (AEC) obtained from chicks treated or not treated with a Lactobacillus-based probiotic1
 
In experiments 3 and 4, we recovered approximately 106 and 105 AEC/mL, respectively, in pooled samples from the abdominal cavity. For these assays, the cells were not diluted to maintain constant numbers in each well, but the Salmonella Enteritidis recovered was divided by the number of cells plated, so cfu per AEC were reported. In both experiments, there were no differences in the numbers of extracellular Salmonella Enteritidis recovered (data not shown). In experiment 3, we recovered significantly higher cfu of intracellular Salmonella Enteritidis from AEC collected from chicks receiving the probiotic and then challenged with Salmonella Enteritidis (approximately 4 cfu/AEC) compared with all other treatment groups (approximately 2 cfu/AEC; Table 3Go). The intracellular Salmonella Enteritidis recovered from AEC in experiment 4, however, did not differ between treatment groups. Our phagocytosis results (2 to 4 cfu of Salmonella Enteritidis per AEC) with AEC derived from 24-h-old chicks corresponded with those of Kramer et al. (2001), who reported that phagocytes isolated from the gut of 7-d-old chicks could phagocytose 2 Salmonella Enteritidis or less, but that phagocytes isolated from the spleen of those same chicks could phagocytose 13 to 33 Salmonella Enteritidis per cell.

The literature is limited on chicken intestinal macrophages and phagocytosis of Salmonella Enteritidis by chicken AEC, especially for very young chicks, and only a few investigators have attempted to enumerate phagocytosed Salmonella Enteritidis. The approach used here to enumerate Salmonella Enteritidis phagocytosed as cfu per AEC was based on the methods described by Desiderio and Campbell (1983) and Kramer et al. (2001) for bovine and avian macrophages, respectively. The fact that enumeration was based on phagocytosed Salmonella Enteritidis that are still alive in the macrophages prior to detection in this phagocytosis assay raises some questions; especially because survival in macrophages is one way that Salmonella spp. perpetuate infections in the host. Qureshi et al. (1986) reported that 80% of opsonized Salmonella typhimurium are killed within 15 min; hence, the presence of live Salmonella Enteritidis in AEC is a relative estimate of ongoing phagocytic activity, independent of downstream killing. Without knowing the downstream impact of surviving phagocytosed Salmonella Enteritidis, it is difficult to interpret an increase in cfu per AEC (e.g., experiment 3, Salmonella Enteritidis-challenged, probiotic-treated chicks) beyond enhanced phagocytic activity. However, with the same challenge model, recent data from our laboratory (Higgins et al., 2007)—showing that Salmonella Enteritidis-challenged, probiotic-treated chicks that did not completely clear Salmonella Enteritidis infection consistently had significantly lower counts of Salmonella Enteritidis in the cecal contents—support a positive effect of this probiotic treatment on Salmonella Enteritidis clearance.

In conclusion, these experiments evaluated the effect of a Lactobacillus-based probiotic culture on macrophages in neonatal chicks. This probiotic culture consistently reduced Salmonella Enteritidis recovery from the cecal tonsils within 24 h, as compared with nontreated controls. However, differences in the numbers of macrophages in situ were not consistent, and even the observed significant differences were not remarkable. Similarly, observed differences in phagocytosis in experiment 3 were not replicated in experiment 4. These data do not support the hypothesis that the observed reductions in Salmonella Enteritidis colonization from day-of-hatch chicks are due to the presence of macrophages or their ability to phagocytose Salmonella. However, these data do not preclude the possibility that macrophages are directly or indirectly participating in the consistently observed diminution of Salmonella colonization caused by administration of this probiotic.

Received for publication March 19, 2007. Accepted for publication June 26, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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B. Vila, A. Fontgibell, I. Badiola, E. Esteve-Garcia, G. Jimenez, M. Castillo, and J. Brufau
Reduction of Salmonella enterica var. Enteritidis colonization and invasion by Bacillus cereus var. toyoi inclusion in poultry feeds
Poult. Sci., May 1, 2009; 88(5): 975 - 979.
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