Total IgA in spleen, bursa and intestine of chickens pretreated with E. faecium AL41 and challenged with Salmonella Enteritidis PT4

Abstract

The concentration of secretory IgA (sIgA) in intestine flush and solid organs (spleen, bursa) in chickens pretreated with probiotic strain Enterococcus faecium AL41 (EF) and challenged with Salmonella Enteritidis PT4 (SE) was assessed. Forty 1-day-old chickens Cobb 500 were divided into four groups: control, EFAL41, SE and combined EFAL41+SE. The increased concentration of sIgA was determined on day 4 after salmonella infection in the intestine flush in EFAL41group. In combined EFAL41+SE group significant increase of sIgA in the intestine flush was found 7 days post-infection. Pre-treatment of chickens with E. faecium AL41 also showed beneficial effect in bursa in EFAL41 and EFAL41+SE groups. In spleen, total IgA was increased only in SE group. The results demonstrated positive effect of E. faecium AL41 on IgA production in chickens' intestine and solid organs after S. Enteritidis PT4 challenge. Validity of method using for IgA evaluation in solid organ was proved.

Keywords:

• Salmonella Enteritidis
• E. faecium AL41
• immunoglobulin A
• mucosal immunity
• chickens

Introduction

Salmonella enterica serovar Enteritidis is a gram-negative facultative intracellular enteric pathogen that continues to be a significant source of food poisoning, causing severe gastroenteritis in humans (Howard, O'Bryan, Crandall, & Ricke, 2012) and considerable health costs (Cardoen et al., 2009).

Probiotics are non-pathogenic bacteria that exert a beneficial influence on health or physiology (or both) of the host (Rajput & Li, 2012). Moreover, they accelerate the establishment of a stable microflora in the gut of neonate chicks and create the barrier effect against problematic pathogens (Revajová et al., 2013). Lauková et al. (2012) supposed that Enterococci are ubiquitous microbiota constituting a large proportion of autochthonous microflora in animals. Samli, Senkoylu, Koc, Kanter, and Agma (2007) demonstrated that the addition of probiotics (Enterococcus faecium) into chicken feed improved the intestinal architecture and increased the intestinal surface area. Levkut et al. (2012) showed the influence of E. faecium EF55 on the dynamic of intestinal mucin production in birds. Probiotics can increase the levels of IgA-producing cells in the mucous (Levkut et al., 2014) and in the lamina propria (Ohland & MacNaughton, 2010). These beneficial bacteria promote secretory IgA (sIgA) production into the luminal mucous layer (Ohland & MacNaughton, 2010). sIgA protects the intestinal epithelium against colonisation and/or invasion by binding antigens on pathogens or commensals (Macpherson, McCoy, Johansen, & Brandtzaeg, 2008).

The goal of our paper was to evaluate sIgA in the intestinal flush and solid tissue (spleen and bursa of Fabricii) of the chickens preventively administered with E. faecium AL41 and challenged with S. enterica subsp. Enteritidis PT4. For that reason the modified flush method in the intestine and the method for disaggregation of the spleen and bursa of Fabricii were used.

Material and methods

Experimental design

A total of 40 one-day-old chickens of Cobb 500 breed were included in the 11-day experiment. The chickens were kept in the standard breeding conditions. Application of cleaning and feeding regimens prevented from cross-contamination effectively throughout the experiment. Chickens were randomly divided into four groups: control (C), E. faecium AL41 (EFAL41), S. Enteritidis PT4 (SE) and combined E. faecium AL41+S. Enteritidis PT4 (EFAL41+SE), each group contained 10 chicks. The probiotic strain E. faecium AL41 (provided by Andrea Lauková, IAP SAS, Košice, Slovakia) was administered individually per orally to EFAL41 and EFAL41+SE groups from 1 day to 7 days of experiment in dose 1 × 10⁹ colony-forming unit (CFU)/0.2 ml phosphate buffered saline (PBS). Experimental infection of SE and EFAL41+SE groups was carried out individually per orally using S. enterica serovar Enteritidis PT4 (provided by František Šišák, VRI, Brno, Czech Republic) in a single dose of 1 × 10⁸ CFU/0.2 ml PBS on day 4 of experiment. Samples for determination of IgA from intestine were taken at 4 days and 7 days post-salmonella infection [day post-infection (p.i.)], and from bursa of Fabricii and spleen at 7 days p.i.

Intestine samples and flush protocol

The flush protocol was assessed as described by Holt, Gast, Porter, and Stone (1999) with some modifications. Five randomly chosen chickens from each group were killed by cervical dislocation on days 4 (early phase of infection) and 7 (late phase of infection; p.i.) with salmonella. The ileum and terminal section of jejunum were excised, and 10 ml of warm flush solution (1 M tris/glycine buffer with 0.25% Tween 20, pH 7, Sigma Aldrich, USA) was injected into the intestine lumen with a 10-cc syringe and a 18-ga needle. The solution was aspirated and injected several times to flush the secretions from the intestine wall, and then the contents were collected into the syringe and dispelled into a tube. The intestine flushes were centrifuged for 5 min at 12,000×g (Hettich Rotina 420R Centrifuge, DJB Labcare, UK), and the supernatants from each sample were frozen at −20°C until the enzyme-linked immunosorbent assay (ELISA) procedure.

Isolation of splenic and bursal cells

The tissue in PBS was further disrupted with the flat end of a 10-ml syringe plunger and strained through a 70-µm nylon cell strainer (BD Falcon, USA) to obtain a single-cell suspension. The suspension was overlaid on to a Histopaque-1077 (Sigma Aldrich, USA) and centrifuged for 40 min at 1600×g (Hettich Rotina 420R Centrifuge, DJB Labcare, UK). Final ring-shaped aggregation of cells visible at the interface were collected and washed twice in PBS. Cells obtained from sediment of both organs were homogenised for 30 sec at 2000×g on MagNA Lyser (Roche, Germany).

ELISA

Chicken IgA ELISA kit (Kamiya Biomedical Company, USA) was used for detection of secretory IgA in intestinal flush and immune cell homogenate obtained from bursa and spleen. The samples were diluted at 1:5 in 1× diluent solution (component of ELISA kit) and in dose 100 µl added into pre-designated wells in duplicates. Ninety-six-well microtiter plates were coated with affinity-purified anti-chicken IgA antibody. After the incubation of microtiter plates (22°C 20 min), the contents of the wells were aspirated and washed three times with wash solution (component of ELISA kit). Then, 100 µl of diluted enzyme–antibody conjugate binding with horseradish peroxidase in stabilising buffer was applied into the plate wells, and incubated at 22°C for 20 (20 ± 2) min. The plate was washed as described above. One hundred microliter of 3,3′,5,5′-tetramethylbenzidine substrate solution was added into each well. The reaction was stopped with 100 µl of stop solution and absorbance was measured spectroscopically at 450 nm on microplate reader (Revelation Quicklink, Opsys MR, Dynex technologies, USA).

Interpretation was made using calibration curve prepared according to the manufacturer's protocol.

Statistical analysis

Statistical analysis of data was done by one-way analysis of variance (ANOVA) with the post hoc Tukey multiple comparison test using GraphPad Software (USA). Differences between the mean values for different treatment groups were considered statistically significant at P < 0.05. Values in table are means and standard deviation (SD).

Results

The concentration of sIgA (ng/mL) in the intestine flush was decreased in the group E. faecium AL41+S. enterica compared to C (P < 0.05) and EFAL41 group (P < 0.01) at 4 day p.i. in the intestine flush (Table 1).The highest values of sIgA were found in EFAL41+SE group compared to C, EF AL41 and SE group (P < 0.001) at 7 day p.i. Moreover, the changes in the concentration of sIgA were observed in the EFAL41 group compared to SE and C animals (P < 0.05). Finally, the increase of sIgA was seen in SE group compared to C (P < 0.05) in the intestine flush at 7 day p.i.

Table 1. IgA expression (ng/mL) in intestine flush, bursa of Fabricii and spleen in chickens pretreated with E. faecium AL41 and challenged with S. enterica serovar Enteritidis PT4.

Samples	Day p.i.	C	EFAL41	SE	EFAL41+SE
Intestine flush	4	68.65 ± 13.64*	77.08 ± 22.19**	51.15 ± 10.49	34.94 ± 13.34*
	7	125.99 ± 32.39***/*	281.75 ± 42.94***/*	205.34 ± 55.81***/*	449.92 ± 27.62*
Bursa of Fabricii	7	27.91 ± 17.08	61.37 ± 18.21	47.74 ± 10.19	68.02 ± 16.54
Spleen	7	13.75 ± 5.07**	20.22 ± 6.03*	51.14 ± 11.26*	18.48 ± 6.86*

Note: All values are expressed as mean ± SD. Means with different superscripts within a row are significantly different.

p.i., post-infection; C, control; EFAL41, E. faecium AL41; SE, S. enterica PT4; EFAL41+SE, E. faecium AL41+S. enterica PT4.

* P < 0.05; **P < 0.01; ***P < 0.001.

Bursa of Fabricii showed tendency to increase concentration of determined IgA in EFAL41+SE group without significance compared to other groups. Spleen demonstrated the increased concentration only in SE group as compared to C (P < 0.01), EFAL41 (P < 0.05) and EFAL41+SE groups (P < 0.05) at 7 day p.i. with S. Enteritidis PT4.

Discussion

Many studies have focused on the effects of probiotics on diverse aspects of the immune response. The interaction between probiotics and enterocytes is the key to initiation of the immunomodulation. Previous studies demonstrated that there is influence of E. faecium on the expression of IgA⁺ cells in mucous intestine of birds (Revajová et al., 2013). Moreover, Levkut et al. (2014) showed that administration of E. faecium EF55 in chicks infected with S. Enteritidis 147 caused the increase of IgA⁺ cell subpopulation in caecal intraepithelial lymphocytes in later phase of infection. To obtain more information about the effect of E. faecium on intestinal sIgA in chickens challenged with S. Enteritidis PT4, we examined sIgA in the intestinal flush and solid organs. Our experiment demonstrated the highest concentration of sIgA in the chickens pretreated with EFAL41 at early phase of S. Enteritidis PT4 infection in the intestine flush. This result showed that pre-treatment of chickens with E. faecium AL41 enhanced the effect on sIgA production. On the contrary, later phase of S. Enteritidis PT4 infection presented the increased concentration of total sIgA in the chickens pretreated with E. faecium AL41 and challenged with S. Enteritidis PT4. This finding suggests being the result of cumulative effect of both bacteria.

For study of total IgA concentration in bursa of Fabricii and spleen, we homogenised cells from solid organs as described in the materials and methods section. We supposed later IgA immune response in other organs as in the intestine therefore it was evaluated only 7 days p.i. Pre-treatment of chickens with E. faecium AL41 showed beneficial effect in bursa in both groups (EFAL41; EFAL41+SE). On the contrary, the increased concentration of total IgA in SE group in the spleen suggests early penetration of salmonella into this organ and developing of systemic immune response. The decreased splenic concentration of IgA in chickens pretreated with E. faecium could be connected with immune exclusion (Tsuji et al., 2008), or production of antimicrobial molecules (Lievin-Le Moal & Servin, 2006). It is known that E. faecium has genes for the production of enterocins A and P, and has shown inhibitory activity against many bacteria including S. enterica PT4 (Levkut et al., 2009). This consumption is consistent with the increased concentration of total sIgA in the intestine flush on 7 days p.i.

In conclusion, our results demonstrated positive effect of E. faecium AL41 on the production of total sIgA in the intestine of chickens challenged with S. Enteritidis PT4. The beneficial effect was also shown in bursa of Fabricii. Using of method for evaluation of IgA in solid organ was proved.

Acknowledgements

The authors would like to thank MVDr. Andrea Lauková, CSc. (IAP SAS, Košice, Slovakia) for providing probiotic strain E. faecium AL41, MVDr. František Šišák, CSc. (VRI, Brno, Czech Republic) for providing Salmonella Enteritidis PT4 and Ing. Reiterová, CSc. (IP SAS, Košice, Slovakia) for expert advices.

Additional information

Funding

This work was supported by the Grant Agency for Science of Slovak Republic under Grant [VEGA 1/0313/12], [VEGA 1/0886/11]; Slovak Research and Developmental Agency under Grant [APVV-0302-11].