Mária Levkutová

Leukocytic responses and intestinal mucin dynamics of broilers protected with Enterococcus faecium EF55 and challenged with Salmonella Enteritidis

The protective effect of Enterococcus faecium EF55 in chickens challenged with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was assessed. The antibacterial effect on the bacterial microflora in the small intestine in relation to white blood cell count, phenotyping of peripheral blood and intestinal lymphocytes, functional activity of lymphocytes and phagocytes and mucin quantitation were investigated. Day-old chicks (85) were randomly divided into four groups. The probiotic group (EF) and Salmonella+probiotic group (EFSE) received E. faecium EF55 (10(9) CFU - 3 g/group/day) for 21 days. The Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis (10(8) CFU in 0.2 ml PBS) in a single dose per os on day four of the experiment. The control group chicks (C) were fed a commercial diet without added bacteria. Supplementation of EF55 in the diet of the chickens in the EFSE group, challenged with S. Enteritidis, caused the density of the intestinal mucin layer to increase significantly in non-specific regions (duodenum and jejunum), but decrease significantly in target regions (caeca) for S. Enteritidis. Probiotic treatment also appeared to result in a significantly higher number of lymphocytes in peripheral blood and a tendency to increase CD3, CD4, CD8, and IgM positive cells 3 days post-infection with S. Enteritidis. The results demonstrated an antibacterial effect and suggested that EF55 had a moderating effect on intestinal mucin production and leukocytic response in the early phase of S. Enteritidis infection.

Interaction of TGF-β4 and IL-17 with IgA secretion in the intestine of chickens fed with E. faecium AL41 and challenged with S. Enteritidis.

The relative mRNA expression of IgA, TGF-β4, IL-17, and concentration of secretory IgA (sIgA) in small intestine of chickens pretreated with Enterococcus faecium AL41 and challenged with Salmonella Enteritidis PT4 were studied. Salmonella-free day-old chicks (40) Cobb 500 breed, were divided into four groups of 10 chicks each (n = 10): control (C), treated with E. faecium AL41 strain (EFAL41), challenged with Salmonella Enteritidis PT4 (SE), and combined (EFAL41+SE). Expression of IgA and sIgA concentration was upregulated in EFAL41 group in jejunum and ileum on 4 days post-Salmonella infection (dpi). Chicks in combined group demonstrated upregulation of cytokines and IgA expression, and increased sIgA concentration in the intestine flush on 7 dpi. The experiment demonstrated beneficial effect of E. faecium AL41 on IgA production and secretion in intestine. Findings also indicated that IgA played important role in decrease of S. Enteritidis in the intestine, and cytokines TGF-β4 and IL-17 contributed to the increased IgA secretion.

Influence of oregano and salvia extracts on lymphocyte subpopulation and functional activity of blood phagocytes and lymphocytes in chickens

Abstract Supplementation of oregano and sage extracts into the feed of experimental chickens a caused significant increase of mitogen-activated lymphocyte proliferation on the seventh week of feeding. On the other hand, fed diet supplemented with both extracts did not change number of CD3, CD4, CD8, IgM and IgG positive peripheral blood cells examined by flow cytometry. Similarly, no significant changes have been found in values, demonstrating the functional activity of phagocytes like phagocytic activity, the index of phagocytic activity and index of metabolic burst. Level of plasma immunoglobulins was and temporary on Week 4 of feeding, significantly decreased in chickens treated with oregano extract only. Lower levels of plasma immunoglobulin suggest a connection with antibacterial effect of carvacrol. Results showed no pro-inflammatory effects in studied parameters of systemic immune response. On the other hand, lymphocyte stimulation in vitro suggest a higher immune defence ability.

IgA gene expression and quantification of cecal IgA+, IgM+, and CD4+ cells in chickens treated with EFAL41 and infected with Salmonella Enteritidis.

IgA gene expression and quantification of mucous IgA+, IgM+ and CD4+ lymphocytes in the cecum of chicks was studied by qRT-PCR, immunohistochemistry and flow cytometry. A total of 220 1-day-old Salmonella-free chicks of Cobb 500 were divided into four groups (n=55). Group 1 served as control (C), group 2 was pretreated with probiotic bacterial strain Enterococus faecium AL41 (EFAL41), group 3 was infected with Salmonella Enteritidis PT4 (SE), and group 4 was pretreated with E. faecium AL41 and subsequently challenged with Salmonella Enteritidis PT4 (EFAL41+SE). The relative mRNA expression of IgA was upregulated in the EFAL41 group (P<0.05) when compared to control group at 4dpi. In comparison to the control, EFAL41 and SE group, the relative mRNA expression of IgA was also upregulated in EFAL41+SE group at 7dpi (P<0.001). Immunohistochemistry revealed, that the density of IgA+ cells was higher in EFAL41+SE group comparing to the controls and SE groups (P<0.001). Significantly more CD4+ cells were present in the SE group than in EFAL41 (P<0.05), and EFAL41+SE groups (P<0.001) at 4dpi. In contrast, higher density of CD4+cells at 7dpi was seen in EFAL41+SE group as compared with controls (P<0.05). Flow cytometry determined that relative percentage of intraepithelial lymphocytes (IEL) IgA+ cells was higher in EFAL41 than in SE and EFAL41+SE groups (P<0.05). Comparing to controls the number IgM+ cells increased in SE group (P<0.05) at 7dpi. The results demonstrated beneficial effect of E. faecium AL41 on the mRNA expression of IgA and number of IgA+ cells. Lamina propria lymphocytes (IgA+, IgM+) were not affected by EFAL41 intake or salmonella infection. Probiotic bacterial strain EFAL41 positively influenced the number of IEL during the first days of infection.

INFLUENCE OF OREGANO EXTRACT ON THE INTESTINE, SOME PLASMA PARAMETERS AND GROWTH PERFORMANCE IN CHICKENS

intestinal alkaline phosphatase (IAP) level, enterocyte proliferative ability, plasma proteins, and plasma minerals (calcium and magnesium) were studied in 1 day-old Ross 308 hybrid broiler chickens under commercial conditions during 42 experiment. Chickens fed oregano oil supplemented diet (0.707 g.kg -1 ) had significantly higher body weight gain (BWG) in the grower (19-29 d) and finisher (30-42 d) periods. Total plasma immunoglobulin levels were lower while mineral levels were higher during the finisher period. Significant decrease of IAP activity was demonstrated in animals fed oregano oil supplemented diet on 29, and 42 days of the experiment. Proliferative activity of enterocytes significantly increased in the finisher period along duodenal villi in animals treated with essential oil extracted from oregano. We suppose that higher body performance was probably the result of higher digestibility in the digestive tract. It is suggested that the antibacterial effect of carvacrol caused a lower number of intestinal bacteria, decrease of immunoglobulins and increase of investigated plasma proteins.

Expression of cytokines in chicken peripheral blood mononuclear cells after stimulation by probiotic bacteria and Campylobacter jejuni in vitro

The aim of this study was to delineate the ability of selected probiotic strains to reduce invasion by Campylobacter jejuni in peripheral blood mononuclear cells (PMBCs). PMBCs were isolated from chicken blood and exposed in vitro to probiotic strains Enterococcus faecium EF55, E. faecium AL41, E. faecium H31, Lactobacillus fermentum AD1, and infected with C. jejuni CCM6191. The mRNA expression levels for selected cytokines were determined at 24 and 48 h postinfection (pi) using reverse transcriptase-polymerase chain reaction. The level of expression of IL-1β, LITAF, and MIP1-β was upregulated (P < 0.05; P < 0.01; P < 0.001) in LAD + CJ (L. fermentum AD1 + C. jejuni) group when compared to control (C), CJ (C. jejuni), and other combined groups (EF55 + CJ, AL 41 + CJ, H31 + CJ) 24 h pi. Overall, L. fermentum AD1 and E. faecium AL41 demonstrated the highest immunomodulatory activity against campylobacter infection in vitro.

Caecal IEL, blood lymphocytes and intestinal mucin study in chickens after probiotic prevention and S. Enteritidis infection

Salmonella employs multiple, parallel systems of innate immune activation in order to effect a specific series of inflammatory changes. Hosts susceptible to Salmonella infection mobilize responses that are necessary for effective control of infection.

Effect of lignin supplementation of a diet contaminated with Fusarium mycotoxins on blood and intestinal lymphocyte subpopulations in chickens

The objective of the study was to investigate the effects of lignin supplementation of a diet contaminated with the Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) on peripheral blood leukocytes and duodenal immunocompetent cells in broiler chickens. From day 1 after hatching, all chickens were fed an identical control diet for two weeks. Then chickens of Group 1 continued to be fed the control diet, whereas Group 2 was fed the same diet supplemented with lignin at 0.5% level. Simultaneously, Group 3 started to receive a diet contaminated with DON (2.95 mg kg-1) and ZEA (1.59 mg kg-1), while Group 4 received an identical contaminated diet supplemented with 0.5% lignin for further two weeks. Samples of blood and duodenal tissue were collected from 6 birds of each group at 4 weeks of age. Neither counts of white blood cells nor phagocytic function in the peripheral blood were significantly affected in the mycotoxin- and/or lignin-treated birds. As compared to the control, increased numbers of IgM-bearing cells were found in the peripheral blood in Group 3 fed the contaminated diet (P < 0.05) and in Group 4 given the contaminated diet supplemented with lignin (P < 0.01). While the contaminated diet led to reduced numbers of duodenal CD4+ cells, in Group 2 treated only with lignin the number of duodenal CD4+ cells was increased. Lignin enrichment of the contaminated diet did not eliminate the mycotoxin-induced reduction in the number of duodenal CD4+ cells. The results suggest that dietary supplementation of lignin as an indigestible compound to poultry feed may increase the density of some intestinal immunocompetent cells without exerting effects on that in the peripheral blood. However, when added to a diet contaminated with Fusarium mycotoxins, lignin did not prevent the mycotoxin-induced changes in the numbers of blood and intestinal immunocompetent cells.

Leukocytic response and composition of enteral microbiota in chickens fed a sage extract supplemented diet and infected with Salmonella Enteritidis PT4

The effects of sage on the enteral microbiota and leukocytic response of chickens challenged with Salmonella Enteritidis (SE) PT4 were studied. Chickens were divided into four groups: control, sage (S), SE and sage+S. Enteritidis (SSE). Groups S and SSE were fed sage extract (8.2 g kg−1) for 21 days. Groups SE and SSE were challenged with SE (108 CFU) on day 4. The administration of the sage extract to the SSE group confirmed the selective antibacterial activity on SE, and resulted in an increased number of leukocytes, lymphocytes, heterophils and phagocytic activity index at day 3 post-infection. In the early phase of the infection, there was a tendency to increase the numbers of CD3+, CD8+ and IgM+ cells in the SSE group. The later phase led to a decrease of IgM+, CD3+ and CD4+ cells, but an increase in influx of macrophages, which suggested their movement into inflammatory regions.