Zuzana Ševčíková

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.

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.

Effect of Enterococcus faecium EF 55 on morphometry and proliferative activity of intestinal mucosa in broilers infected with Salmonella Enteritidis

Abstract Introduction: The present study aimed to investigate the effect of Enterococcus faecium EF55 on chickens, as well as its influence on proliferative activity of epithelial intestinal cells after infection with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4). Moreover, the length and area of duodenal and jejunal villi of the birds were examined. Material and Methods: A pool of 80 birds was divided randomly into four groups. Probiotic group (EF) and Salmonella + probiotic group (EFSE) received E. faecium EF55 (109 CFU – 3 g per group/day) during 22 d. Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis PT4 (108 CFU in 0.2 mL PBS) in a single dose per os on day four of the experiment, whereas control birds (C group) received only 0.2 mL of PBS on that day. Samples were taken on the 4th and 18th day post infection. Results: Supplementation of feed with E. faecium EF 55 confirmed its selective antibacterial activity against SE PT4. The chickens infected with SE PT4 and fed E. faecium EF55 supplemented diet showed increased proliferative activity of enterocytes in the jejunum in both samplings. Applied probiotic strain demonstrated positive impact on intestinal morphometry in the jejunum of both non-infected groups and in Salmonella-infected chickens. In the latter group, the beneficial effect of E. faecium EF 55 was manifested by more efficient tissue turnover in the jejunum.

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.

Mast cells and eosinophils in rat mammary gland tumours induced by N-Nitroso-N-methylurea

Abstract The aim of the study was to evaluate the distribution and number of mast cells and eosinophils in rat mammary gland tumours induced by N-Nitroso-N-methylurea. The highest density of mast cells was found in cystic papillary adenocarcinomas of grade II. Eosinophils were detected only in the cystic papillary adenocarcinoma of grades I and II, in non-invasive cribriform adenocarcinoma and comedo-type carcinoma. Mast cell populations were observed perivascularly in the tumour stroma, in the host tumour interface, as well as in necrotic areas of neoplasms. Mast cells were observed to be intact according to their morphological changes, collectively referred to as degranulation. The obtained results indicate that mast cells and eosinophils play an important role in tumour micro-environment formation. The increased density of these cells in experimentally-induced rat mammary gland tumours suggests a poor prognosis in these cancers. Our results also confirmed that rat mammary gland tumours are good models for the study of breast cancers.