Research Note: Immunocompetent cells in blood and intestine after administration of Lacto-Immuno-Vital in drinking water of broiler chickens.

The understanding of the synbiotics´ impact on the host is incomplete. To improve the knowledge, we study the effect of Lacto-Immuno-Vital synbiotic preparation in chickens on local and systemic immune response by evaluation of immunocompetent cells in the peripheral blood and jejunal mucosa. Hematological method was used for determination of white blood cell count, and flow cytometry for measurement the functions of phagocytes and subpopulation of lymphocytes (CD3, CD4, CD8, IgM, and IgA). Cell Qest programme (Germany) was used for analysing of data obtained from flow cytometer and GraphPad Prism version 4.0 for comparison by paired t test between control and experimental groups. The experiment was conducted in a commercial broiler chicken fattening farm, the birds were handled and sacrificed in a humane manner. A flock of 64,400 one-day-old Hybrid ROSS 308 chickens were included in the 42-d experiment. The chickens were randomly divided into 2 equal groups, experimental and control, and each group of chickens was housed in a different hall while maintaining the same conditions. The chickens in the experimental group (Lactovital) received 500 g of Lacto-Immuno-Vital (Hajduvet Kft., Hungary) in 1,000 L of drinking water. Lacto-Immuno-Vital was administered daily from the first day (D1) to D7 of the experiment. From D 7 to D 22 it was given in a pulsed manner (every third day) at a dose of 300 g in 1,000 L of drinking water. Control group received only the standard diet. For immune analyses 6 randomly chosen chickens from experimental and control group were taken from the halls. The sampling days were set at D 8 and D 22 of the experiment. Samples of peripheral blood were collected from vena subclavia. The chickens were euthanized and whole jejunum was taken during necropsy into Hanks ice solution (pH 7.2-7.3). Administration of Lacto-Immuno-Vital in drinking water of nonstressed broilers during fattening period in commercial production increased phagocytic activity and phagocytic index. The number of IgA+ and CD8+ cells in lamina propria of intestine was decreased in chickens fed diet supplemented with Lacto-Immuno-Vital in drinking water. We suggest that increased phagocytic activity and decreased number of immunocompetent cells in mucosa of intestine was caused by improved systemic and local immune system function.

Influence of Lacto-Immuno-Vital on growth performance and gene expression of IgA, MUC-2, and growth factor IGF-2 in the jejunum of broiler chickens.

The effects of Lacto-Immuno-Vital synbiotic preparation on gene expression of IgA, MUC-2, and growth factor IGF-2 in the jejunum and on BW gain in broiler chickens were studied. A flock of 64,400 1-day-old Hybrid ROSS 308 chickens was inducted in the 42-day experiment. The chickens were divided into 2 equally size groups in separate halls. The chickens in the experimental (E) group received 500 g of Lacto-Immuno-Vital in 1,000 L of drinking water. The preparation was administered daily from the first day (day 1) to day 7 of the experiment. From day 7 to day 22, it was given in pulsed manner (every third day) at a dose of 300 g in 1,000 L of drinking water. The broiler chickens in the E group gained more weight (P < 0.001) compared with control from day 10 to day 42. Death of animals during feeding period was 1,078 chickens in the E group compared with 1,115 dead chickens in the control group. Feed conversion ratio was 1.61 kg of supplemented diet/kg of BW in the E group compare with 1.67 kg of nonsupplemented diet/kg of BW in control. The relative expression of IgA gene in the jejunum was upregulated on day 22 in the E group compared with control (P < 0.05), whereas relative expression of MUC-2 gene was upregulated in the E group compared with control on day 8 and day 22 (P < 0.05; P < 0.001). Similarly, relative expression of IGF-2 gene was upregulated in the E group compared with control on both samplings (P < 0.01). The composition of Lacto-Immuno-Vital synbiotic preparation showed beneficial effects on growth performance, feed conversion ratio, morbidity, mortality, and selected parameters of mucosal immunity in the chicken jejunum.

The Influence of Chestnut Wood and Flubendazole on Morphology of Small Intestine and Lymphocytes of Peripheral Blood, Spleen and Jejunum in Broiler Chickens.

The study examined subpopulations of lymphocytes in peripheral blood, spleen, and jejunum including morphology of that segment in broiler chicken farm after treatment with flubendazole (Flimabend) and natural extract from chestnut wood (Farmatan). A total of 24 forty-day-old Kalimero-Super Master hybrid chickens were divided into 4 groups (n=6): the Fli group received Flimabend per os, 100 mg/g suspension in 1.43 mg of active substance/kg body weight during 7 day of experiment, Far group received Farmatan per os at 0.2 % concentration for 6 hours per day during 5 day (experimental days - from 3 to 7); the Far+Fli group received a combination of doses administered in the same way as for the first two groups; and control -C group with no active substance administration. The results demonstrated mild increase of leukocytes, lymphocytes, monocytes, leucocyte common antigen CD45, IgM+ and IgA+ cells in peripheral blood after administration of Flimabend. Similarly, subpopulations of followed lymphocytes (CD3+, CD4+, CD8+, IgM+) were increased in the jejunum after application of that drug. On the other hand, administration of Farmatan revealed opposite effect on determined immunocompetent cells what proves anti-inflammatory effect. Morphology of villi was also negatively influenced by administration of Flimabend. Administration of Farmatan suggests also its preventive administration in chickens. This tanin-containing drug as plant natural product may be used due to its antibacterial activity and as promising alternative to conventional drug with possible antihelminthic effect.

Muscle characteristics in chicks challenged with Salmonella Enteritidis and the effect of preventive application of the probiotic Enterococcus faecium.

The present study was conducted to assess the effects of the probiotic Enterococcus faecium AL41 (EF) and of the enteric pathogen Salmonella Enteritidis PT4 (SE) on the development of posthatch pectoralis major muscle (PM) of broiler chicks. The four experimental groups were control (CON), EF, SE, and EF+SE (EFSE). EF and SE were given per os from days 1 to 7 and at day 4 posthatch, respectively. Muscle samples from 6 chicks per group were taken at day 8 (D8) and day 11 (D11) to evaluate PM myofiber growth, capillarization, DNA, RNA, and protein content, as well as enzyme activities (isocitrate dehydrogenase, lactate dehydrogenase, creatine kinase). PM growth rate was 7.45 ± 2.7 g/d in non-SE groups (CON, EF) and 5.10 ± 1.82 g/d in SE-infected groups (P < 0.02). Compared with group CON, application of bacteria (groups EF and SE) reduced the fiber cross-sectional area (246 and 262 vs. 347 ± 19 µm2) and the number of myonuclei per fiber (0.66 and 0.64 vs. 0.79 ± 0.03). At D11, hypertrophic myofiber growth normalized in the EF group, but negative effects persisted in SE and EFSE birds contributing to lower daily PM gain. In addition, SE infection strongly disturbed PM capillarization. Negative effects on capillary cross-sectional area and on the area (%) covered by capillaries persisted until D11 in the SE group, whereas pre-feeding of EF restored capillarization in the EFSE group to control levels. We conclude that supplementation of the probiotic bacteria EF AL41 had positive effects on PM capillarization and, thus, on delivery of O2, supply of nutrients, and removal of metabolites. Supplementation of probiotic bacteria might therefore reduce energetic stress and improve muscle health and meat quality during SE infection.

Systemic immune response of gnotobiotic mice infected with porcine circovirus type 2 after administration of Lactobacillus reuteri L26 Biocenol™.

In our previous study we confirmed an antiviral activity of probiotic Lactobacillus reuteri L26 which was mediated by stimulation of local intestinal immunity. The aim of this paper was to evaluate the influence of L. reuteri L26 on the systemic immune response in gnotobiotic mice infected with porcine circovirus type 2 (PCV2). A total of 30 germ-free mice were divided into 3 groups and animals in noninfected and infected control groups (NC and IC; n=10) received sterile de Man-Rogosa-Sharpe broth for 7 days and animals in experimental group L+PCV (n=10) were inoculated with L. reuteri L26. Subsequently, mice in L+PCV and IC groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). The results showed an increase of percentage of cytotoxic cells (CD8+ and CD49b+CD8-) and oxidative burst of phagocytes, up-regulation of the gene expression of RANTES, granulocyte-macrophage colony-stimulating factor, interferon-γ and immunoglobulin A in blood above all in the later phase of infection (14 dpi) in L+PCV group accompanied by higher load of PCV2 in the serum. These findings indicate that L. reuteri L26 has a potential to induce systemic immune reaction, but in gnotobiotic mice immune stimulation can increase virus replication.

Influence of Lactobacillus reuteri L26 Biocenol™ on immune response against porcine circovirus type 2 infection in germ-free mice.

Probiotic bacteria are frequently used for prevention of bacterial infections of the gastrointestinal tract, but there are only limited studies on their efficacy against viral gut infections in animals. The aim of this study was to investigate the effect of probiotic Lactobacillus reuteri L26 BiocenolTM on the innate and adaptive immune responses in germ-free Balb/c mice, experimentally infected by porcine circovirus type 2 (PCV2), which confers immunosuppressive effect. A total of 30 six-week-old female mice were divided into 3 groups and animals in experimental group LPCV (n=10) were inoculated with L. reuteri L26, animals in the control group (C; n=10) and experimental group PCV (n=10) received sterile De Man-Rogosa-Sharpe broth for 7 days. Subsequently, mice from both experimental groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). Virus load in faeces, ileum and mesenteric lymph nodes (MLN); as well as gene expression of selected cytokines, immunoglobulin A (IgA) and polymeric Ig receptor (PIgR) in the ileum, and percentage of CD8+, CD19+ and CD49b+CD8- cells in the MLN were evaluated. Our results showed that L. reuteri significantly decreased the amount of PCV2 in faeces and in the ileum, and up-regulated the gene expression of chemokines, interferon (IFN)-γ, IgA and PIgR in the ileum. Increased IFN-γ mRNA level was accompanied by higher proportion of natural killer cells and up-regulated IgA and PIgR gene expressions were in accordance with significantly higher percentage of CD19+ lymphocytes in the MLN. These findings indicate that probiotic L. reuteri has an antiviral effect on PCV2 in the intestine which is mediated by stimulation of local gut immune response.

Leukocytic responses of broilers following dietary contamination with deoxynivalenol and/or treatment by dietary selenium supplementation.

1. This experiment was to investigate the effects of natural dietary contamination with a mycotoxin product (deoxynivalenol: DON) and/or with dietary selenised yeast (Se-yeast), on respiratory burst and phagocytic activity of granulocytes and the frequency of B- and T-lymphocytes in peripheral blood of broilers. 2. Sixty one-day-old chicks of both sexes were divided into 4 groups, each of 15 birds, fed on a control diet that contained 0.2 mg DON/kg and 0.4 mg Se/kg (CON group), a diet supplemented with 1 mg Se-yeast/kg (Se-yeast group), a diet contaminated with 3 mg DON/kg (DON group) or a diet contaminated with DON and supplemented with Se-yeast (DON plus Se-yeast group). 3. Blood samples collected from the birds at the age of 4 weeks showed that neither B- and T-cell numbers nor granulocytic respiratory burst were influenced by 3 mg DON/kg. Blood granulocyte phagocytic activity was not reduced by DON but numbers of heterophils were increased. In the DON plus Se yeast group phagocytic activity was the same as in the CON group. The Se-yeast and DON plus Se-yeast groups had increased numbers of CD3(+), CD4(+), and CD8(+) T-cells as well as IgM(+) B-cells in their blood compared to both CON and DON-groups. 4. The results show there is no significant effect of dietary DON up to 3 mg/kg on leukocytes apart from the compromised blood granulocytes phagocytic activity and increased numbers of heterophils. The increased numbers of B- and T-lymphocytes in blood of birds fed on diets with supplementation of organic Se indicates some positive effects of this essential microelement on poultry lymphoid cells.

Diagnosis and manifestation of encephalitozoonosis in mice after experimental infection with different species and application of dexamethasone.

Twenty-eight BALB/c mice were infected with different strains of Encephalitozoon species (Encephalitozoon cuniculi II - mouse type, E. cuniculi III - dog type, Encephalitozoon hellem, Encephalitozoon intestinalis). Five of them were infected with E. cuniculi II (mouse type) and simultaneously immunosuppressed with dexamethasone. Clinical signs of encephalitozoonosis were not remarkable. Ascites was found in two mice of dexamethasone-treated group 14 days post-infection (p.i.). The histopathological changes were found mainly in spleen and liver in the form of lymphoepithelioid granuloma. Spores were found in faeces since day 14 p.i. and visualized by Calcoflour White M2R. After cultivation on cellular cultures (VERO E6 - monkey kidney cells, RK-13 - rabbit kidney fibroblasts), the species differentiation was performed by PCR using panmicrosporidial primers (PMP1, PMP2) and specific primers (ECUN-F, ECUN-R, V1, SI-500). The differences were recorded in the immune response of immunocompetent and immunosuppressed mice. At day 60 p.i., the titres of specific antibodies measured by indirect immunofluorescence antibody test were lower (1:4096) in dexamethasone-treated mice when compared with non-immunosuppressed animals (1:8196). The significant increases of antibody titres were recorded in particular infected groups within the experiment (P < 0.01 between day 14 p.i. and day 30 p.i., P < 0.001 between day 14 p.i. and day 60 p.i.). Experimental encephalitozoonosis in non-immunosuppressed and immunosuppressed mice provides a useful model for the study of immune response and lesions associated with these protozoans.

Spreading of Salmonella enteritidis in the cecum of chickens.

Adhesion and colonization of high (2 x 10(8) CFU) and low doses (2 x 10(2) CFU) of Salmonella enteritidis (phage type 4) was determined in the ceca collected 6 h-4 weeks after inoculation (pi), of 1-d-old White Plymouth Rock orally-inoculated chickens. S. enteritidis was associated with the epithelial surface of the villi in the low-dose group 18 h-7 d pi, the penetration in the cecal lamina propria was observed on day 1 and 10 pi. In the high-dose group, adhesion and colonization was observed in all birds killed 6 h-14 d pi; penetration of the bacteria into the cecal lamina propria was seen 1-21 d pi. Large numbers of macrophage-like cells containing S. enteritidis were observed in the cecal lamina propria on days 3-21 pi. Colonization and migration by S. enteritidis in the intestinal tract of chickens was shown to be dose dependent.

Immune response of E. cuniculi infected mice to aflatoxin B1.

The immunomodulatory effects of aflatoxin B1 (AFB1) on mice experimentally infected with Encephalitozoon cuniculi (E. cuniculi) were studied. Mice inoculated intraperitoneally with spores of E. cuniculi drank a daily solution of AFB1 (0.2 mg kg(-1) of body weight) for 27 days. Application of AFB1 to mice demonstrated a decrease of immunocompetent cells. On the other hand, the mice infected with E. cuniculi and given AFB1 showed significant increased number of both monocytes and CD8+ T cells, and tendency to a decrease in CD4+ T cells. Aflatoxin B1 revealed to merely modulate systemic immune response of E. cuniculi infected mice.

Effect of aflatoxin B1 on CD3 T cells and alkaline phosphatase in the intestine of mice.

The aim of the study was to determine the effect of repeated applications of aflatoxin B1 (AFB1) on immunocompetent cells (CD3 T cells) and alkaline phosphatase in the intestinal mucosa. Mice were orally treated with AFB1 for 24 days. The mucosa of the intestine showed a significant decrease in the number of CD3 T cells and a significantly lower level activity of alkaline phosphatase on day 24 in AFB1 treated mice. Similarly, with changes in the small intestine, qualitative haematological parameters were modified in systemic immunity as lymphopenia, and neutropenia, monocytopenia. AFB1 treated animals showed reduction in body weight gain and increased liver weight. We supposed that changes found in the small intestine are secondary to primary systemic haematological lesions. The decrease in CD3 T cells suggests a connection with the decrease in the host's resistance to infectious diseases.

Influencing the immune parameters in germ-free piglets by administration of seal oil with increased content of omega-3 PUFA.

Oral administration of oil with an increased content of omega-3 polyunsaturated fatty acids to germ-free piglets resulted in a significant increase in the total values of CD4, CD8 lymphocytes, B lymphocytes, and monocytes, in peripheral blood in comparison with the controls. The metabolic activities of phagocytes as well as the polyclonal activation of lymphocytes were not significantly influenced. The level of growth factor was significantly higher, as determined on the basis of somatomedin in the blood serum. Biochemical indices showed a significant increase in the level of eicosapentaenoic and docosahexaenoic acids in blood serum and the decrease in the level of arachidonic acid at the same time.

Effect of low and high doses of Salmonella enteritidis PT4 on experimentally infected chicks.

Chicks (1-d-old, three groups, each containing 50 chicks) were inoculated with 2 x 10(2) and 2 x 10(8) CFU of Salmonella enteritidis; the third group were kept as uninoculated control. Five birds from each group were euthanized at intervals from 6 h to 4 weeks post-inoculation (pi). In the low-dose group S. enteritidis was isolated from 60% cecal samples at 18 h pi, and from 20% of livers at 3 d pi. Individual variation in the frequency of S. enteritidis recovery was observed in this group. The clearance of salmonella from the organs was faster in the low-dose group, and salmonella was not isolated from the liver and cecum at 21 and at 27 d pi, respectively. However, in the high-dose group, S. enteritidis was isolated from all ceca and 80% of liver 6 h pi, and salmonella was detected in the cecum and liver throughout the experiment. Serous typhlitis and unabsorbed yolk sac were the most prevalent lesions in both groups. Granulomatous nodules in the cecum were found occasionally in some cases in both inoculated groups, which can play a role as reservoirs in carrier chicks.

Cellular immunological responses of pheasant during endogenous development of Eimeria colchici.

We examined the time course and histological localisation of the developmental stages of Eimeria colchici. The prepatent period in the caeca of pheasants was 6 days. The patent period began on day 7 post-infection (p.i.) and ended on day 11 p.i. with peak production of oocysts on days 8-9. The peripheral blood lymphocytes of pheasant chicks showed a significant increase in proliferation to E. colchici antigen from day 5 p.i., with peak on day 14 p.i. The metabolic activity (respiratory burst) of heterophils increased on days 3, 4 and 14 p.i. The total number of peripheral blood leukocytes and lymphocytes in the infected pheasant chicks had increased by day 2 p.i. and reached a maximum on day 4 of the experiment. Days 5 and 6 p.i. were characterised by a drop in the number of these cells.

Study of immune response in pigs fed with high vitamin E-dosages.

The effects of long-term vitamin E-supplementation on phagocytic cells, lymphocyte sub-populations, and SWC3+ cell count were studied in pigs. Eighteen weaned pigs were divided into three groups: 1) 100 mg DL-alpha tocopheryl acetate/kg diet, 2) 200 mg DL-alpha tocopheryl acetate/kg diet, 3) control group (basic feed with 10 mg DL-alpha tocopheryl acetate/kg diet). The examination of the immune indices was performed on day 120 of feeding the supplemented diets. The higher dietary levels of vitamin E resulted in increased serum concentration of alpha-tocopherol for both experimental groups (p < 0.05) and there were no significant differences in counts of CD2+, CD4+, CD8+, B lymphocytes nor SWC3+ cells among the groups. Similarly, vitamin E supplementation did not affect the functions of phagocytic cells tested.

Effect of oil containing n-3 polyunsaturated fatty acids (PUFA) on the immune response and growth factors in piglets.

Oral administration of n-3 polyunsaturated fatty acids (PUFA) to piglets slightly enhances the immune response. As compared to the control, in the experimental piglets the absolute values of monocytes in the peripheral blood were significantly increased (P < 0.05), while the metabolic activity of phagocytes and the number of lymphocytes within the individual subpopulations were slightly higher. The level of growth factors, determined on the basis of somatomedin in the blood serum, was significantly higher in the experimental group (P < 0.05). n-3 PUFA interfere with the synthesis of prostaglandins and influence the metabolism of fatty acids. This finding may contribute to the therapy of inflammatory processes influencing immune and growth factors in piglets.

Immune response to Encephalitozoon cuniculi infection in laboratory mice.

The study was performed to determine the immune response to Encephalitozoon cuniculi infection in immunocompetent mice during 120 days of experiment. Mice infected with E. cuniculi had an increased number of CD4+ T cells up to Day 20 post infection (p.i.), but counts of CD8+ T lymphocytes were significantly lower up to Day 90 p.i. in peripheral blood. Blood monocytes were significantly increased on the Day 60 and Day 120 of infection. A lack of significant decrease of CD4+ T cells may be considered as an important event in the immune response to E. cuniculi infection in immunocompetent mice.

Morphological and functional development of the rumen in the calf: influence of the time of weaning. 1. Morphological development of rumen mucosa.

The objective of this study was to determine whether the nutritional regimen of rearing calves would influence the morphometric and histological development of rumen mucosa. Twelve male Holstein calves 7 d of age were assigned to three groups of 4 animals each: milk group (I), early weaned (6 weeks) group (II) and late weaned (9 weeks) group (III). All animals received additional solid feed. Animals of group I were slaughtered after 6 weeks of age, whereas those in groups II and III were slaughtered after 9 weeks of age. At slaughter, the ruminal digesta amounted to 2035 g (milk group), 3092 g (late weaned group) and 5374 g (early weaned group). The differences in the ruminal molar percentage of SCFA were not significant. There was a trend for lower pH and higher SCFA concentrations in the order late weaned, early weaned and milk fed animals (pH: 6.4, 6.6 and 6.7, respectively; SCFA: 96, 87 and 77 mmol/l, respectively). The mean length (1.07 mm in milk group, 1.45 mm in late weaned group and 1.87 mm in early weaned group), width (0.43, 0.58 and 0.71 mm, respectively) and surface of papillae (190, 232 and 241 mm2/cm2 mucosa, respectively) increased with both the age of the animals and the elevated intake of solid feed, whereas the number of papillae (210, 140 and 92 per cm2 mucosa, respectively) decreased. In both milk-fed groups type A and B corneal cells were present in the Stratum corneum, whereas in the earlier weaned calves type C-cells could be also seen. These findings indicate a more advanced stage of development of the rumen epithelium in the earlier weaned calves fed higher amounts of concentrate and hay.

Influence of dietary concentrate to forage ratio on the development of rumen mucosa in calves.

Effects of structural and non-structural carbohydrates on the development of rumen fermentation and ruminal mucosa in calves were examined during the weaning period. Barley/soybean meal (SBM) group was fed a concentrate starting from 2 weeks of age, whereas alfalfa group received a mixture of concentrate and alfalfa hay in which the proportion of the latter was gradually increased from 20% to 70% between weeks 2 and 9 of age. The total volatile fatty acid concentration in rumen fluid of calves increased with age, but at 9 weeks there were no significant differences between the two diets (barley/SBM group 153 mmol/l, alfalfa group 150 mmol/l). Rumen papillae at 9 weeks of age, as compared to 6 weeks of age, were longer and fewer in number per square centimetre mucosa, with larger cut surface. This resulted in a higher surface of papillae per square centimetre mucosa at 9 weeks (barley/SBM group 286 mm2/cm2, alfalfa group 245 mm2/cm2) than at 6 weeks of age (barley/SBM group 217 mm2/cm2, alfalfa group 198 mm2/cm2). At 9 weeks of age, the pH (barley/SBM 5.0, alfalfa 5.7), the acetate to propionate ratio (barley/SBM 2.2, alfalfa 3.2) as well as the length of the papillae in the ventral ruminal sac (barley/SBM 1.96 mm, alfalfa 2.37 mm) were increased in the alfalfa group when compared to the barley/SBM group (P < 0.1). In the former group, the proportion of butyrate revealed significantly increased values at 4 and 6 weeks of age. In animals of the barley/SBM group at 9 weeks of age, characteristic protrusions with proliferated thick epithelium occurred on the papillae and increased the surface for absorption. On the epithelium (Stratum corneum) desquamating cells with parakeratosis could be observed. In the alfalfa group the papillae of the ventral ruminal sac were longer, without protrusions. The morphotypes of the adhering rumen microflora differed between the groups. It can be concluded that feeding greater amounts of non-structural carbohydrates increases the surface for absorption of the rumen epithelium in calves. The absence of hyperkeratosis and rumenitis in the barley/SBM group indicated that there is no reason to limit high starch diets in the early weaning period of calves.