Effect of Enterococcus faecium on immune system of chickens challenged against Salmonella Minnesota

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1 Effect of Enterococcus faecium on immune system of chickens challenged against Salmonella Minnesota Published on: 09/10/2012 Author/s : Leandro NagaeKuritza, Leonardo BozziMiglino, Mariana Camargo Lourenço, Larissa Pickler, Antônio Leonardo Kraieski, Elizabeth Santin (Department of Veterinary Medicine, Laboratory of Microbiology and Ornitopathology, Federal University of Parana, Curitiba, Parana, Brazil) Summary The emergence of new outbreaks of Salmonella sp. leads to concern about new outbreaks of food borne disease, especially from chicken meat and by-products. Aiming to reduce this contamination, some alternatives are being tested, including probiotics. Enterococcus faecium(ef) is a lactic acid bacteria, considered as a probiotic, with the ability to reduce Salmonella in broilers. This experiment was designed, to evaluate the effect of Enterococcus faecium on immune system of chickens and control of Salmonella Minnesota. Broiler chicks were divided in three treatments. In treatment 1, birds were not inoculated and not fed with probiotic. In treatment 2, birds were inoculated with Salmonella Minnesota, acting as positive control and in treatment 3, birds were inoculated with Salmonella Minnesota and fed with diet containing EF. Before challenge, birds fed with EF presented an increase in goblet cell number in ileum and cecum and reduced CD8 + T lymphocytes in the ileum and increased CD4 + T lymphocytes in the cecum compared to negative control. After challenge, birds fed with EF showed a reduction in the number of Salmonella from cloacal swabs 48h post challenge and on cecum and litter at 35 days (15 days after challenge) of age and reduced CD4 + T cells in the ileum and CD8 + T cells in the cecum compared to birds challenged that did not received EF. In conclusion, EF on diet affected the immune cell population on intestinal mucosa. KEYWORDS:probiotic, T-lymphocyte, goblet cell, Immunohistochemistry INTRODUCTION Salmonellosis account for a severe problem of public health both in developing and developed countries and several controlling mechanisms of such pathogen have been applied to poultry production (Cardoso and Carvalho, 2006). The inhibition effect produced by probiotics on the population of pathogenic enterobacteria such as Salmonella through the competitive exclusion (CE) mechanism is widely documented in the literature (Reid and Friendship, 2002; Hariharan et al., 2004; Dahiyaet at., 2006; Callaway et al., 2008).

2 Enterococcus faecium(ef) is a lactic-acid producing bacterium which present reported inhibition effects against Escherichia coli and Salmonella spp (Lewenstein et al., 1979). Its probiotic effect has been demonstrated as it improves piglets feed conversion and performance (Mallo et al., 2010) and rats immunity modulator (Peng et al., 2010). Therefore, this study is intended to assess the efficiency of an EF-based probiotic in the infiltration by immune cells into the intestinal mucosa of chickens challenged with Salmonellaentericaserovar Minnesota (SM). MATERIALS AND METHODS Sixty male broiler chickens, Cobb 500 line, were used from the 1 st to 35 days of age, divided equally into 3 treatments. Treatment 1 (T1) Negative control, no SM inoculation; Treatment 2 (T2) Positive control, SM inoculation; Treatment 3 (T3) 50 g/ton of Enterococcus faecium (EF) Probiotic in feed (CYLACTIN ME20 plus, DSM Nutritional Products, Basel, Switzerland)and SM inoculation. Birds of each treatment were housed in separate but identical rooms, side by side, under negative pressure, which were previously cleaned, disinfected, using a sterilized wood shaving litter. Upon the animal s arrival, 5 animals underwent euthanasia and necropsy for the collection of liver and cecum and analysis as for the presence/absence of Salmonella. Animals were held at optimum comfort temperature as applicable to their ages, and supply of water and feed adlibitum, and fed balanced diets at levels equal to or higher than those recommended by NRC (1994) and pellet feed. At 15 days of age, treatment 2 and 3 animals were inoculated, per oral, with a Salmonellaentericaserovar Minnesota solution at 10 8 CFU/ml concentration. At 35 days of age, collections of fragments of ileum and cecum from 5 animals per treatment were conducted and processed as previous described (Smirnov et al., 2004) for the analysis of goblet cells and also for further analysis of CD4+ and CD8+ cells according to described earlier (Pickler et al., 2012). The histologic analysis and the quantification of CD4+ e CD8+ cells from the intestinal epithelium were conducted under light microscopy, in an image analyzing system (Motic Image Plus 2.0 Motic China Group Co. 2006) attached to the microscope (Olympus America INC., NY, USA). The quantification of the goblet cells and CD4+ and CD8+ lymphocytes were conducted per field (100X magnification) on ileum and cecum fragments, where 10 regions of interest of each tissue section were scanned. All the data were analyzed by using the statistical software Statistix for Windows Copyright (C) The ANOVA was used and Fischer test at 5% probability was used as post hoc test. The tested probiotic strain was Enterococcus faeciumncimb 10415, supplied by the DSM Nutritional Products (Basel, Switzerland). Triplicate samples of the feed before and after pelletization were assessed for the amount of probiotic present, and it was found out the probiotic-added feed had average 1x10 9 cfu/kg (data not shown).

3 RESULTS AND DISCUSSION At 7 days old, it is observed that birds fed with diets containing EF based probiotic show significant increases in goblet cells in the ileum and cecum compared the birds of the control group (Table 3). Colonization of different portions of the gastrointestinal tract by certain bacteria can occur by association with the mucus layer and immunoglobulins in the immune process described as inclusion / exclusion (Everett et al., 2004). This adhesion serves as the first line of defense against harmful microorganisms and against toxins (Nousiainen et al., 2005) which may also justify the reduction of SM isolated in birds fed with this probiotic. In CD4+ and CD8+, in the ileum, there was no statistical difference (P> 0.05) between the treatments, but in cecum was observed higher counts of CD4+ cells in the group treated with EF compared to the untreated group. For the CD4:CD8 ratio was observed in the cecum amount significantly (P <0.05) lower of CD4+ than CD8+ cells in control group compared to group treated with EF, while the ileum no difference was observed (Table 3). The boost produced by the colonization of probiotics are essential for the development of functional immune system and balanced, including the presence of T and B lymphocytes in the lamina propria and the expansion and maturation of IgA and also induction of tolerance by the present antigens (Borchers et al., 2009). Table 3 Count of goblet cells, CD4+, CD8+ cells and CD4+:CD8+ cell ratio (CD4/CD8) per field in ileum and cecum of broiler chickens at 7 days of age. (100X magnification). At 35 days of age, the results of cellular dynamics in the mucosa of the ileum and cecum of chickens show no significant differences between groups for goblet cells. However there are fewer CD4+ cells in ileum and CD8+ cells in cecum in the group treated with EF in the diet compared to the challenged group untreated with EF. For the CD4:CD8 ratio it was observed in ileum significantly (P 0.05) lower number of CD4+ than CD8+ cells in the group fed with EF in diet compared with the groups challenged and unchallenged with SM that was not receiving probiotics in diet. In cecum, the group challenged with SM showed a higher amount of mucosal CD8+ cells than not challenged group, however, the group fed with EF based probiotic was statistically similar to both groups (Table 4). Table 4 Count of goblet cells, CD4+, CD8+ cell and CD4+:CD8+ cell ratio per field in ileum and cecum of broiler chickens at 35 days of age. (100X magnification).

4 According to Van Immerseel (2009), the meeting of specialized epithelial cells with microorganisms rapidly stimulates the release of proinflammatorychemokines that attract innate immune cells such as granulocytes and macrophages, can trigger a wide range of new immune reactions, such as the appearance of T-helper cells (CD4+). CONCLUSION The use of probiotic-based Enterococcus faecium in broilers challenged by SalmonellaMinnesota, increased nonspecific immune response at 7 and 35 days of age, providing changes in the infiltration dynamics of ileal and caecal mucosa immune cells. REFERENCES CARDOSO, T.G., AND V.M. CARVALHO. (2006) Toxinfecçãopor Salmonella spp. RevistadoInstituto de Ciências da Saude24(2): REID, G., FRIENDSHIP, R. (2002) Alternative to antibiotic use: probiotics for the gut. Animal Biotechnology13: HARIHARAN, H., MURPHY, G.A., KEMPF, I. (2004) Campylobacter jejuni: Public health hazards and potential control methods in poultry: a review. Vet Med Czech.49: DAHIYA, J.P., WILKIE, D.C., VAN KESSEL, A.G., DREW, M.D. (2006) Potential strategies for controlling necrotic enteritis in broiler chickens in post-antibiotic era. Animal Feed and Science Technology129: CALLAWAY, T.R., EDRINGTON, T.S., ANDERSON, R.C., HARVEY, R.B., GENOVESE, K.J., KENNEDY, C.N., VENN, D.W., NISBET, D.J. (2008) Probiotics, prebiotics and competitive exclusion for prophylaxis against bacterial disease. Animal Health Research Reviews9: MALLO, J.J., RIOPEREZ, J., HONRUBIA, P. (2010) The addition of Enterococcus faecium to diet improves piglet s intestinal microbiota and performance. Livestock Science133:

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