Viera Revajová

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.

Antimicrobial activity of Enterococcus faecium ef 55 against Salmonella enteritidis in chicks.

The protective effect of Enterococcus faecium EF 55 against Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was studied in 1-day-old chicks. The EF 55 strain (isolated and characterised by the authors earlier) was applied daily (1.10(9) CFU/0.2 ml PBS) for 7 days. Oral inoculation of the SE PT4 strain was performed on day 8 in a single dose of 5.10(8) CFU/0.2 ml PBS. The experiment lasted for 21 days. Samples were collected on day 1 of the experiment to verify the absence of Salmonella, on day 8 to check colonisation of EF 55 and immunological status in experimental birds, and on days 2, 4, 6, 8 and 14 after SE PT4 infection of chicks. Strain EF 55 sufficiently colonised the digestive tract of chicks after 7 days of application. The highest numbers of EF 55 in the faeces of chicks were observed before SE infection and persisted to day 6 post infection (p.i.) in both the EF and EF+SE groups. PCR confirmed the identity of the EF 55 strain. The counts of SE PT4 strain in faeces of the EF+SE group were significantly reduced in comparison to those in the SE group on days 2 and 14 p.i. (P < 0.01). The significant reduction of salmonellae in the caecum was recorded at the end of the experiment (day 14 p.i.) in the EF+SE group in comparison to the SE group (P < 0.01). At day 4 p.i., colonies of S. Enteritidis PT4 were found in the liver of chicks of the SE group in a higher concentration than in chicks of the EF+SE group (P < 0.001). Salmonellae were isolated from the liver until days 8 and 6 p.i. in the SE and EF+SE groups, respectively. The mean values of actual lymphocyte subpopulations in the blood and the relative percentage of caecal intraepithelial lymphocyte subpopulations (CD4, CD8, CD44, TCR, MHC II and IgM) were not influenced at a statistically significant level by the application of the EF 55 and/or the SE PT4 strain. The results demonstrate the antimicrobial effect of E. faecium EF 55 against S. Enteritidis PT4.

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.

The Effect of Lactobacillus paracasei and Raftilose P95 Upon the Non-specific Immune Response of Piglets

This work was focused on the effects of administration of Lactobacillus paracasei and the fructooligosaccharide (FOS) Raftilose P95 upon the immune system of piglets 10 days after birth and 10 days after weaning under standard farming conditions. The piglets were divided into three groups of 30 animals: F - combined application of Lc. paracasei and Raftilose P95; L - application of Lc. paracasei only; C - control group without treatment. In the first phase of the experiment, group L revealed a significantly higher value in total absolute leukocyte (Le) counts ( P < 0.01) as well as in neutrophil counts (Ne), CD2 + , CD4 + T lymphocyte and B lymphocyte counts ( P < 0.05) compared to group F. Significant differences ( P < 0.05) were obtained in phagocyte activity of leukocytes (%PA Le) and in phagocyte activity of neutrophils (%PA Ne) when groups C and F were compared. At the time of weaning in comparison with newborn piglets a tendency to increase the parameters under observation was seen in group F with a simultaneous decrease of most indices in group L and in the control group. A significant difference ( P < 0.05) in favour of the combined treatment compared to simple lactobacilli application was observed in absolute numbers of CD4 + T lymphocytes and B lymphocytes. In the second phase of the experiment - application after weaning - the counts of total anaerobes (TAn) and total aerobes (TA) in the faeces of animals receiving the combined treatment (F) were significantly higher ( P < 0.05) when compared to the lactobacillus-only treated piglets (L) and to untreated piglets (C). In the case of total lactobacilli (TLc), the value in group F was significantly higher ( P < 0.05) compared to group L ( P < 0.01) and compared to the control group. Application of FOS Raftilose P95 seems to be a suitable method for fortification of the beneficial effects of probiotic bacteria especially during changes of the intestinal microflora composition, e.g. during weaning.

T lymphocyte subpopulations and B lymphocyte cellsin caecum and spleen of chicks infected with Salmonella enteritidis

The effect of low and high doses of Salmonella enteritidis PT4 (SE) on immunocompetent cells in caecum and spleen of one-day-old chicks was investigated. Subsets of T lymphocytes positive for CD3, CD4, CD8 and B lymphocytes (Bu1b-positive cells) were counted in the caecum after immunohistochemical staining and the relative percentage of these cells in the spleen was analysed using a FACScan cytometer on days 7, 10, 14, 21, and 27 post-inoculation (pi). In the low dose group, the number of CD3+ and CD4+ cells in the caecum had significantly increased at day 10 pi. Both CD8+ and Bu1b+ cells were significantly higher on day 14 pi in this group. In the high-dose group, the number of CD4+ cells had significantly increased at day 7 pi. CD3+, CD8+, and Bu1b+ cells showed prolonged proliferation at days 7 up to 21 pi. Splenic lymphocytes demonstrated significant changes only in the high dose group. The percentage of splenic CD4+ cells was decreased at day 7 pi. A decrease in CD3+ and CD8+ cells was found at day 14 pi in this group.

Larval toxocarosis in sheep: The immunohistochemical characterization of lesions in some affected organs

Morphological and immunohistochemical responses of lambs following oral infection with 10,000 infective Toxocara canis (T. canis) eggs were studied up to 28 days post-infection (pi). The small intestine, liver, lungs and brain of both infected and control lambs were examined using the routine histological methods for paraffin sections and immunohistochemical techniques for frozen tissue sections. Eosinophil-rich hepatic granulomas and diffuse T. canis-induced pulmonary inflammation were the most prominent pathological features. CD2+, CD4+, CD8+, IgM bearing cells, and macrophages were demonstrated in the liver granulomas. The observed histomorphologic changes were similar to other paratenic hosts. It was concluded that larval toxocarosis followed the classical migratory path in the infected lambs.

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.

Dynamic of apoptosis of cells in duodenal villi infected with Eimeria acervulina in broiler chickens

The aim of our study was to evaluate the parasite — host interactions at apoptosis level. We studied histopathological changes and time course of apoptosis in the duodenum during Eimeria acervulina infection. One-day-old broiler chicks were randomly allocated into two equal groups. At the age of two weeks the first group was experimentally infected with a pure suspension of sporulated E. acervulina oocysts. The second group served as a negative control. Tissue samples from the upper part of duodenum were obtained at 0.5, 1, 2, 3, 4, 5 and 6 days post infection. Biopsies of duodenum were studied immunohistochemically using DeadEnd™ Colometric TUNEL System for apoptosis detection in duodenal mucosa. Number of parasites in duodenal epithelium was also investigated. Our experimental results demonstrate: (i) macroscopic and histopathological changes in epithelium detected mainly in proximal segment of duodenum in infected groups; (ii) the number of developmental stages of E. acervulina (DSEA) during our trial increased, reaching the maximum 5 days post infection (dpi) (332.2 ± 16.12) (mean ± SEM), whereas the amount of DSEA declined significantly as late as 6 dpi (124.6 ± 3.91); (iii) the highest apoptosis level was recorded in initiatory 0.5 dpi (13.2 ± 1.02) and on the end of parasite development cycle after 5 dpi (12.6 ± 1.36). Finally, results showed that there was a period of inhibition of apoptosis during infection by E. acervulina.

Effect of probiotic bacteria on phagocytosis and respiratory burst activity of blood polymorphonuclear leukocytes (PMNL) in mice infected with Trichinella spiralis

This study focusses on the effect of probiotic (bacteriocinogenic) strains on parasite infection and innate immunity - phagocytosis and oxidative burst of blood monocytes and polymorphonuclear leukocytes (PMNL) in mice infected with Trichinella spiralis. Bacteriocinogenic and probiotic strains of different origin (Enterococcus faecium AL41=CCM8558, Enterococcus durans ED26E/7, Lactobacillus fermentum AD1=CCM7421, Lactobacillus plantarum 17L/1) were administered daily in dose of 109CFU/ml in 100μl and mice were infected with 400 larvae of T. spiralis on 7th day of treatment. Phagocytic activity of blood leukocytes was inhibited at week 3 and 4 post infection (p.i.), i.e. in the time of massive muscle invasion with larvae T. spiralis. Administration of bacterial strains to mice prior to T. spiralis infection elevated and prolonged phagocytic activity of blood leukocytes and their ingestion capability from week 1 to 3 of the infection and the phagocytosis was inhibited only at week 4 p.i. The highest stimulative effect on phagocytosis was induced by strains E. durans ED26E/7, L. fermentum AD1=CCM7421, and L. plantarum 17L/1. The percentage of cells with respiratory burst and their enzymatic activity was increased after T. spiralis infection with the exception of week 3 p.i. In contrast, in all mice treated with bacterial strains the enzymatic stimulation was observed after the infection, with the highest intensity caused by strains E. durans ED26E/7, L. fermentum AD1=CCM7421 and L. plantarum 17L/1. The administration of probiotic strains stimulated phagocytosis and respiratory burst of blood PMNL that could contribute to a decreased larval migration and a destruction of muscle larvae and then reduced parasite burden in the host. The protective effect against T. spiralis infection was induced by all strains, but the highest reduction was recorded by E. faecium AL41=CCM8558.

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.