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Detection of T Lymphocytes in Intestine of Broiler Chicks Treated with Lactobacillus spp. and Challenged with Salmonella enterica Serovar Enteritidis

Department of Clinical Veterinary Medicine, College of Veterinary Medicine and Animal Science (FMVZ), Sao Paulo State University (UNESP), Botucatu, Sao Paulo, Brazil 18618-000

¹ Corresponding author: andreatti{at}fmvz.unesp.br

	ABSTRACT

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The expression of immune response in the form of leukocytic infiltrate by CD3+, CD4+, and CD8+ cells in the epithelium and in the intestinal lamina propria of chicks was studied in the present work by means of immunohistochemical reaction. The chicks were treated with Lactobacillus spp. or cecal microflora (CM) and experimentally challenged or not with Salmonella enterica serovar Enteritidis. The 320 birds utilized were divided into 4 groups containing 80 chicks each and submitted to treatments with Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus acidophilus, and CM. Each group was subdivided into 4 subgroups of 20 birds each and classified into a subgroup that did not receive treatment (negative control), subgroup treated, subgroup treated and challenged with Salmonella Enteritidis, and subgroup only challenged with Salmonella Enteritidis (positive control). The results obtained show that the treatment with L. reuteri, L. salivarius, L. acidophilus, or CM and challenged or not with Salmonella Enteritidis determine immune response in the form of leukocytic infiltrate by CD3+ and CD8+ lymphocytes followed by CD4+ in the epithelium and in the lamina propria of the duodenum, jejunum, and cecum of chicks up to 12 d of age. The quantity of CD3+ lymphocytes was significantly higher (P < 0.05) in the intestine of chicks treated with L. acidophilus or CM and challenged or not with Salmonella Enteritidis; however, the higher quantity of CD8+ lymphocytes was in the intestine of chicks treated with CM and challenged with Salmonella Enteritidis. The duodenum was the segment in which the immune response by T cells (CD3+, CD4+, and CD8+) was stimulated with the greatest intensity, followed by, respectively, the jejunum and cecum. The quantity of CD3+ lymphocytes present in the duodenum, jejunum, and cecum increases with the age of chicks, independent of the stimulus determined by treatments or challenge.

Key Words: chicken • probiotic • Salmonella • CD3+ • CD4+ and CD8+ lymphocytes • immunohistochemistry

	INTRODUCTION

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Major risk factors for food poisoning caused by Salmonella are associated with chickens, such as eating raw or undercooked eggs (Molbak and Neimann, 2002). Salmonella enterica serovar Enteritidis is the second most common serotype in humans and identified most commonly from clinical and nonclinical chicken sources in the United States (Centers for Disease Control and Prevention, 2005). This serovar can cause infection of chickens in the absence of a clinical disease (Gast and Beard, 1990). Several measures to control Salmonella have been used, among them the use of antimicrobial drugs. However, concerns about drug-resistant bacteria and the appearance of drug residues in food animals have stimulated interest in alternative treatments such as the use of probiotics, competitive exclusion products, and bacteriophages (Andreatti Filho et al., 2003, 2007; Joerger, 2003).

Lilly and Stillwell (1965) were the first to utilize the term probiotic and the work by Nurmi and Rantala (1973) was the initial mark of studies involving the concept of competitive exclusion. The principle of competitive exclusion is particularly interesting because it provides some protection to birds against the entrance of pathogens in the first days of life, although at this age the birds do not possess plenary intestinal microflora, and the immune system still is not found completely developed (Day, 1992).

Intestinal microflora is composed of various bacterial species, prominent among which is the genus Lactobacillus, whose most common species including Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus animalis, and Lactobacillus acidophilus have demonstrated great importance to intestinal health in chickens (Ramesh et al., 2000).

Treatment with Lactobacillus spp. has demonstrated immunostimulant effects on the intestinal mucosa (Simon et al., 2001). In chickens the presence of Lactobacillus spp. in the intestine stimulates synthesis of IgA by the immune system through release of short-chain peptides and increasing the resistance to diseases (Pulverer et al., 1990). Furthermore, Lactobacillus also are related to beneficial effects in therapy against tumors, exercising antimutagenic effects and modulating immune response, including T cells, probably due to the action of cytocines on CD4+ cells in humans (Schifrin et al., 1997; Pelto et al., 1998). In this manner, the presence of Lactobacillus is fundamental to regulating the composition of intestinal microflora, developing immunity of the intestine, and also promoting the health of chickens (Muir et al., 2000).

This study was designed to assess the capacity to stimulate the immune system in the form of leukocytic infiltrate by the CD3+, CD4+, and CD8+ lymphocytes in the intestinal epithelium and in the intestinal lamina propria of chicks treated with L. reuteri, L. salivarius, L. acidophilus, or cecal microflora (CM) isolated from breeders, and challenged or not with Salmonella enterica serovar Enteritidis.

	MATERIALS AND METHODS

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Chickens

Three hundred twenty day-of-hatch broiler chicks from a local hatchery were used. The chicks were housed in metal cages, maintained under heating for 12 d, receiving water and commercial ration ad libitum, without addition of antibiotics. Sixteen extra birds were euthanized by cervical dislocation, and cecal tonsils and liver were aseptically collected, placed individually in tubes containing 10 mL of tetrathionate broth (catalog number 210420, Becton Dickinson, Sparks, MD) and selenite cystine broth (catalog number 298251, Becton Dickinson), and incubate overnight at 37°C. After that, 1 loop from each tube was streaked on brilliant green agar (BGA; catalog number 228530, Becton Dickinson) and xylose lysine deoxycholate agar (catalog number 278850, Becton Dickinson), and the plates were incubated overnight at 37°C. Each plate was evaluated for the presence or absence of lactose negative colonies (Nagajara et al., 1991). Only chicks free of Salmonella were utilized.

Experimental Delineation

The 320 birds utilized were divided into 4 groups containing 80 chicks each and submitted to treatments with L. reuteri, L. salivarius, L. acidophilus, and CM. Each group was subdivided into 4 subgroups of 20 birds each and classified into a subgroup which did not receive treatment (negative control), subgroup treated, subgroup treated and challenged with Salmonella Enteritidis, and subgroup only challenged with Salmonella Enteritidis (positive control). At the ages of 2, 4, 8, and 12 d, 5 birds from each subgroup were euthanized by cervical dislocation, and fragments of duodenum, jejunum, and cecum were removed aseptically and fixed in Bouin 10% for 24 h.

Preparation of Cultures of Cecal Microflora and Lactobacillus

The CM was obtained from three 35-wk-old breeders, euthanized by cervical dislocation, and necropsied aseptically for removal of the cecum, which were cultivated in 10 mL of fluid thioglycolate medium (catalog number 0256-17, Becton Dickinson) in an anaerobic jar containing the Anaerobac (Probac do Brasil Ltda., Sao Paulo, Brazil) system, for 24 h at 40°C. Concomitantly, this material also was tested to verify the presence of Salmonella spp. (Nagajara et al., 1991). After incubation, determination of the number of colony-forming units (cfu) of intestinal bacteria was realized and, for this, decimal dilution series were performed in PBS solution with pH 7.2. From these decimal dilutions, 0.1 mL was inoculated in Petri dishes containing fluid thioglycolate medium added in 1.5% Bacto agar (catalog number 214010, Becton Dickinson) and cultivated in anaerobiose, for 24 h at 40°C. Day-of-hatch chicks received by oral gavage 7.0 x 10⁷ cfu/chick (0.5 mL) of CM utilizing graduated pipette. In treatments by oral gavage with Lactobacillus the following species were utilized: L. acidophilus (7.8 x 10⁷ cfu/chick), L. reuteri (7.5 x 10⁷ cfu/chick), or L. salivarius (7.8 x 10⁷ cfu/chick), isolated from the cecum of 35-wk-old breeders, identified biochemically and stored in nutrient broth containing 10% glycerol, and frozen in liquid nitrogen.

Biochemical identification was used by means of fermentation test of carbohydrates, in which samples of Lactobacillus were propagated in 3 mL of DeMan-RogosaSharpe (MRS; catalog number 38944, Sigma-Aldrich Co., St. Louis, MO) broth free from meat extract and glucose, added to 0.002% bromocresol purple as indicator and individually, 0.01% of the following carbohydrates: arabinose, fructose, galactose, glucose, mannitol, mannose, maltose, sucrose, salicyl, and sorbitol. The tubes were incubated at 37°C for 48 h (Kandler and Weiss, 1986).

Each cryotube containing 1.5 mL of culture of Lactobacillus spp. was thawed, ressuspended in MRS broth, and incubated in anaerobiose for 24 h at 40°C before determination of inoculum cfu, which was done by means of decimal dilution series of MRS broth in PBS and plating of 0.1 mL of these dilutions in Petri dishes containing MRS (catalog number 30912, Sigma-Aldrich Co.) agar, incubated in anaerobioses for 48 h at 40°C.

Preparation of Inoculum of Salmonella Enteritidis

For the challenge, a strain of Salmonella Enteritidis was utilized, resistant to nalidixic acid (catalog number N8878, Sigma-Aldrich Co.) and to rifampicin (catalog number R3501, Sigma-Aldrich Co.) and isolated from the liver of chickens. This resistance was developed through successive culture in BGA, containing 100 µg/mL of nalidixic acid and rifampicin (Weinack et al., 1982). The inoculum was obtained through culture in brain heart infusion (catalog number 237500, Becton Dickinson) and incubated at 40°C for 12 h. The challenge inoculum was quantified by the cfu number per chick, as described previously. For determination of cfu number, these dilutions were plated in BGA increased from antibiotics in a quantity of 100 µg/mL. After a period of incubation of 24 h at 40°C, reading was performed for determination of cfu. On the third day of life, the chicks received a challenge of 1.4 x 10⁶ cfu/chick of Salmonella Enteritidis by oral gavage (0.5mL).

Immunohistochemistry

The immunohistochemical reactions were done according to Hsu et al. (1981) for cuts conserved in paraffin. All intestinal fragments had been fixed in Bouin 10% for 24 h and embedded in paraffin. Sections were cut to a thickness of 3 µm, deparaffinized in xilene, rehydrated in graded ethanol, and rinsed in distilled water. Endogenous peroxidases were neutralized with hydrogen peroxide 3% for 15 min followed by rinsing for 5 min in distilled water. Antigen retrieval was achieved by incubating slides in EDTA (catalog number 433802, Sigma-Aldrich Co.) solution (ethylenediaminetetraacetic acid disodium salt), pH 8.0, in a steamer at 96°C for 30 min.

Nonspecific immunoglobulin was blocked by incubation of slides for 60 min with 5% BSA (catalog number A8531, Sigma-Aldrich Co.) before application of the primary antibody. Slides were incubated for 18 h at 4°C with a polyclonal rabbit anti-human pan T-lymphocytes (CD3+; catalog number A0452, Dakocytomation, Glostrup, Denmark) at dilution of 1:100, monoclonal anti-chicken T-lymphocytes (CD4+ UNLB; catalog number EW-13089-66, Southern Biotechnology Associates Inc., Vernon Hills, IL) at dilution of 1:25 and monoclonal anti-chicken T-lymphocytes CD8+/144B (catalog number M7103, Dakocytomation) at dilution of 1:100. A streptavi-din-immunoperoxidase staining procedure (catalog number K0690, Dakocytomation) was used for immunolabeling CD3+ and CD8+ positive cells. The catalyzed signal amplification system (catalog number K1500, Dakocytomation) procedure was used for immunolabeling CD4+ positive cells. The immunoreactions were observed with 3' 3-diaminobenzidine substrate (catalog number D8001, Sigma-Aldrich Co.). Sections were counterstained with Mayer’s hematoxylin.

The negatives controls were done with rabbit Ig (for the polyclonal antibodies) and mouse Ig (for the monoclonal antibody); 1 slide with only secondary antibody and 1 with only streptoavidine and peroxidase. All of these slides were negative.

After the immunohistochemical reactions, the counts of marked cells were completed in an optical microscope (JENAMED 2 – Carlzeiss Jena) utilizing an objective of 40x. The CD3+, CD4+, and CD8+ lymphocytes were counted in ten random fields in each of the slides in the epithelium and in the lamina propria from each segment (duodenum, jejunum, and cecum) present.

Statistical Analysis

The count of T cells (CD3+, CD4+, and CD8+) within experiments was compared using analysis of variances (Zar, 1996) in an entirely random delineation to determine significant (P < 0.05) differences between control and treated groups. The GLM procedure of SAS with a sig-nificance level of P < 0.05 was used.

	RESULTS

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Means for quantity of CD3+ lymphocytes in the intestine of chicks, obtained after treatments with Lactobacillus spp. or CM and challenged or not with Salmonella Enteritidis related to different ages of chicks, are demonstrated in Table 1, in which it is possible to observe that at the 4 moments analyzed for all the treatments, there was a difference (P < 0.05) from the negative control group and the groups treated with L. reuteri, L salivarius, L acidophilus, or CM and challenged or not with Salmonella Enteritidis. There was no difference (P < 0.05) from the challenge with Salmonella Enteritidis, the treatments with L. reuteri, L. acidophilus and L. reuteri challenged with Salmonella Enteritidis in 2-d-old chicks and from the challenged with Salmonella Enteritidis and the treatment with L. reuteri in chicks aged 4 and 12 d. All the groups challenged with Salmonella Enteritidis and treated with Lactobacillus spp. or CM presented difference (P < 0.05) from chicks only challenged with Salmonella Enteritidis and not treated, except in 2-d-old chicks treated with L. reuteri and challenged with Salmonella Enteritidis. The treatment with CM or L. acidophilus without challenged with Salmonella Enteritidis presented the most quantity of CD3+ lymphocytes in 12-days-old chicks. The groups treated with L. acidophilus or CM presented a difference (P < 0.05) among chick ages, with increasing means in quantity of CD3+ lymphocytes, concomitantly with the development of the birds. The means observed in these same treatments were greater than the mean quantities of lymphocytes in the challenge with Salmonella Enteritidis in chicks aged 4, 8, and 12 d. Furthermore, another result observed was that the negative control treatment presented a difference (P < 0.05) only among the first 3 moments (2, 4, and 8 d) and the final chicks age observed (12 d).

	DISCUSSION

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A similar result was obtained by Vervelde et al. (1998) who identified, also by immunohistochemistry, a great quantity of leukocytic infiltrate by CD3+ lymphocytes constituted, principally, of CD4+ and CD8+ cells in the epithelium and in the lamina propria of chick intestine, 7 d after the treatment realized with a mixture of recombinant antigen of Eimeria and choleric toxin. These results also are in accord with the affirmation of Elwood et al. (1997), in which in humans, swine, and rodents, T cells are deposited as much in the intestinal epithelium as in the intestinal lamina propria. Besides the identification of these same structures, another marked similarity between the study by Vervelde et al. (1998) and the present study was that, in both, the quantity of CD8+ lymphocytes was greater than that of CD4+.

McSorley et al. (2000) demonstrated that a significant fraction of Salmonella-specific CD4+ T cells respond to the flagellar filament protein, FliC, and that this Ag has the capacity to protect mice from lethal Salmonella infection. Choi et al. (1999) described the alterations of subpopulations of T cells, among them the CD4+, CD8+, TCR1, and TCR2 lymphocytes, as well as the transcription of IFN- and TGF-β4 mRNA in the intestine of chicks, after oral inoculation of Eimeria acervulina. According to Songserm et al. (2002), the influx of T cells to the intestinal epithelium is related to the presence of bacteria in the intestinal lumen. For these authors, intestinal immunity mediated by cells is directly related to the elimination of enteropathogens such as Pasteurella multocida and Eimeira from the intestine of chicks. Thus the results obtained by these researchers are in accord with the present work because an increase was proven in the population of the CD4+ and CD8+ lymphocytes, 4 d after the realization of treatments.

Yet according to Songserm et al. (2002), in addition to bacterial stimulus, the increase in chick age is responsible for the influx of lymphocytes into the intestinal epithelium of chicks. A study done by Lillehoj and Chung (1992) in broiler chickens verified that the percentage of CD3+ lymphocytes increased 24.4% by the age of 1 wk, 42% by the second week, and 62.6% by the fourth week. These results agree with the present work because in the latter there was also an increase in the quantity of CD3+ lymphocytes as chick age increased. In relation to variation in the number of CD4+ lymphocytes with the increase in chick age, the percentages obtained in the present work also agree with the affirmations made by Lillehoj and Chung (1992), in which a diminution in quantity of CD4+ lymphocytes was observed as the chicks aged. Yet in relation to the quantity of CD8+ lymphocytes, the numbers obtained by Lillehoj and Chung (1992) diverge from the present study because they show a gradual and discrete increase of these cells as the birds aged.

Our results suggest that the treatments with L. reuteri, L. salivarius, L. acidophilus, or CM and challenged or not with Salmonella Enteritidis, determine immune response in the form of leukocytic infiltrate by CD3+, CD8+, followed by CD4+ lymphocytes in the intestine of chicks in the period from 2 to 12 d of age. The quantity of CD3+ lymphocytes was significantly higher in the intestine of chicks treated with L. acidophilus or CM and challenged or not with Salmonella Enteritidis; however, the higher quantity of CD8+ lymphocytes was in the intestine of chicks treated with CM and challenged with Salmonella Enteritidis. The duodenum was the segment in which the immune response by T, CD3+, CD4+, and CD8+ cells was stimulated with the greatest intensity, followed by, respectively, the jejunum and cecum. The quantity of CD3+ lymphocytes present in the duodenum, jejunum, and cecum increases with the age of chicks, independent of the stimulus determined by treatments or challenge.

Received for publication November 23, 2007. Accepted for publication January 22, 2008.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Noujaim, J. C. Articles by Neto, R. T. Search for Related Content PubMed PubMed Citation Articles by Noujaim, J. C. Articles by Neto, R. T.