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.

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.

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.

Prevalence of antibodies to Encephalitozoon cuniculi (microsporidia) in angora goats--a potential risk of infection for breeders.

The presence of antibodies against Encephalitozoon cuniculi in Angora goats was detected by the method of indirect immunofluorescence (IFAT). The animals reacting at the titre 1: 64 and more were considered positive. Of the total number of 48 sera examined, 4 were positive at the titre 1: 32 and 2 were positive at the titre 1: 64. The occurrence of antibodies against E. cuniculi indicates that one of the causes of disorders in the reproductive cycle in Angora goats may be microsporidia Encephalitozoon cuniculi, and that these animals may be potential sources of infection for people.

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.

An unusual manifestation of encephalitozoonosis in chinchilla rabbits.

This disease occurred in 6 rabbits in which leukemic changes after infection with the bovine leukemia virus were being studied.

Subpopulations of lymphocytes in cattle naturally infected with papillomavirus.

Subpopulations of blood lymphocytes (CD2, CD4, CD8, WC1 and IgM-mu chain) were evaluated in clinically manifested bovine papillomatosis. Significantly lower numbers of CD2 (44.7%), CD4 (22.8%) and a lower ratio of CD4/CD8 (1.5) were found in animals with tumours compared to a group of cattle free of papillomas (62.3%, 34%, and 2.3, respectively). On the other hand, significantly higher numbers of gamma/delta+ T lymphocytes (9.6%) and of lymphocytes expressing IgM molecules (35%) were observed in the group of tumour-bearing cattle than in the papilloma-free group (4.8% and 22.1%, respectively). The animals came from a region characterised by elevated concentrations of copper, zinc, arsenic, cadmium and lead in the soil and in the organs of animals. It is supposed that environmental factors predisposed the animals to the development of papillomas.

Catecholamines and encephalitozoonosis in rabbits.

Twenty four rabbits (Oryctolagus cuniculus f. domestica) were used to detect specific anti-Encephalitozoon cuniculi antibodies. To identify microsporidian infection, a haemolytic test in agar gel was carried out. Blood samples of animals with and without spontaneous encephalitozoonosis were evaluated, and compared for the presence of epinephrine (EPI), norepinephrine (NE), and dopamine (DA). Rabbits infected spontaneously with E. cuniculi had significantly lower levels of catecholamines than healthy animals. This decrease in catecholamines is of special interest because of their role as factors modifying the immune response. These neuromediators also have different influences on the function of immune cells.

Surface analysis of IgM-expressing bovine lymphocytes in BLV-infected cattle by immuno-scanning electron microscopy.

Surface of IgM-expressing lymphocytes infected by bovine leukemia virus and with/without persistent lymphocytosis (BLV+PL-, BLV+PL+) in cattle is heterogenous. Three distinct morphological structures of B lymphocytes were found in these groups by immuno-scanning electron microscopy (ISEM). B lymphocytes with generally elongated and pleomorphic microvilli and sometimes short ruffles were observed in infected, and as well as in non-infected animals. A second morphological type endowed with a relatively small number of elongated villi-like veils, and a third morphological type characterized by a relatively small number of short microvilli or blunt stub-like microvilli covering the smooth surface were seen, mainly in BLV+PL+ and seldom in BLV+PL- Cells of the second type were more intensively labeled, than lymphocytes of the third type.

Tissue macrophages of calves after high doses of glucan.

Relatively high doses of the soluble form of fungal glucan (50 mg/kg of body weight) were administered to two clinically healthy calves. Hepatosplenomegaly was not observed. Quantitative and significant qualitative changes were seen in macrophages in the medulla of lymph nodes as compared to the control animals. Both the fibrillar and the granular components in the vacuoles of phagocytosing cells appeared to be positive in the periodic acid-Schiff reaction (PAS); an amorphous component was PAS negative. Quantitative changes occurred also in the expression of IgM molecules on the surface of B lymphocytes. The results indicate that glucan can be used in ruminants also in a single high dose.

Eosinophils in lymph nodes of cows infected by bovine leukaemia virus.

Prescapular lymph nodes from 109 animals positive to bovine leukaemia virus (BLV) were evaluated in this study. Lymph nodes of 81 animals (74.3%) showed an increased number of eosinophils (Eo) in a variety of pathological reactions. Eo counts in T-zonal hyperplasia (47 cases, 43.1%) and mixed hyperplasia (13 cases, 11.9%) were significantly higher (P < 0.05 and P < 0.01, respectively) than in the control group. Similarly, a significantly increased number of blood eosinophils (BEo) was observed in cows with diffuse infiltration by Eo in the lymph nodes as compared to the number of BEo in cows serologically positive to BLV but without morphological changes in the lymph nodes and in cows which were serologically negative (P < 0.05 and P < 0.01, respectively). The possible role of eosinophilic granulocytes in the regulation of immune response to enzootic bovine leukaemia (EBL) is discussed.

Monoclonal antibodies recognized bovine leukocyte antigens used in immuno-scanning electron microscopy.

Antibovine leukocyte monoclonal antibodies were tested in order to use them for scanning electron microscopy in backscattered electron imaging mode. The tests revealed that numerous antibovine leukocyte monoclonal antibodies still recognize lightly glutaraldehyde prefixed antigens and can be used to identify various blood cell types. The results of these tests are presented and discussed. Clearcut differences in the surface morphology exist among bovine peripheral blood normal lymphocytes. Expression of IgM and IgG molecules was observed first of all on the surface of B lymphocyte microvilli.

Monoclonal cytoplasmic immunoglobulin and pathomorphological reactions in lymph nodes in spontaneous bovine leukemia virus infection.

Prescapular lymph nodes from 109 cows seropositive to bovine leukemia virus (BLV) were examined for the presence of light chains of bovine immunoglobulin (Ig) kappa or lambda. A variety of reactions were present in prescapular lymph nodes, including B-zonal hyperplasia (23 cases, 21.1%), T-zonal hyperplasia (52 cases, 47.7%), mixed hyperplasia (18 cases, 16.5%), pulp proliferation (six cases, 5.5%), and atrophy (ten cases, 9.2%). In the case of pulp proliferation the immunohistological examination identified only Ig of a single light chain type lambda. The possible relationship of a monoclonal expression of Ig and development of tumorous proliferation is discussed in the study.

[The effect of primary BHV-1 infection on the dynamics of T and B lymphocytes in the peripheral blood and levels of specific serum antibodies in calves treated with glucan]. / Efekt primárnej BHV-1 infekcie na dynamiku T a B lymfocytov periférnej krvi a hladinu specifických sérových protilátok u teliat osetrených glukánom.

In the present study we investigated the dynamics of circulating T and B lymphocytes and serum specific antibodies in calves experimentally infected with IBR virus (I group) and in calves administered glucan (seven days before infection) and the infected (GI group). The percentages of T and B lymphocytes in the peripheral blood were determined from analyses by rosette methods; the titer of serum anti-IBR antibodies was determined by virus-neutralizing test on cell cultures. The dynamics of the percentage of circulating T lymphocytes showed a similar decreasing trend in both groups, with significant values on days 3 to 5 after infection (AI). In comparison with the calves of I group, the outset of T cell reduction was found to be less pronounced (within the first two days after infection), with a statistically significant difference on day 2 AI (P < 0.05), Fig. 1. A decrease in the percentage of T lymphocytes was related to an increase in the percentage of circulating B lymphocytes, with maximum on days 3 and 4 AI (P < 0.05), Fig. 1. All the calves before experiment beginning were free of serum anti-BHV 1 antibodies. They started responding to the experimental infection with IBR virus by production of serum antibodies between week 1 and 2 AI. The dynamics of serum anti-IBR antibodies showed an identical course in both experimental groups, with the more pronounced outset (P > 0.05) of immunological response in the calves of GI group (Fig. 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Pathomorphological and immunohistological changes in lymph nodes of cows spontaneously infected by bovine leukaemia virus.

Prescapular lymph nodes from 109 cows seropositive to bovine leukaemia virus (BLV) were subjected to histologic, cytochemical and in dubious cases also to immunohistologic examination for the presence of light chains of bovine immunoglobulin (Ig) kappa or lambda. By their morphological features, the histologically detected changes were divided into B-zonal hyperplasia (23 cases, 21.1%), T-zonal hyperplasia (52 cases, 47.7%), pulp proliferation (6 cases, 5.5%), hyperplasia of mixed type (18 cases, 16.5%), and atrophy (10 cases, 9.2%). Some changes resembled those reported in infections with human or feline immunodeficiency virus. Eosinophilic infiltration was a frequent feature. Immunohistochemical examination revealed only a lambda Ig chain in the cytoplasm of plasma cells or plasmacytoid cells and immunoblasts in pulp proliferation. Cytochemical examination showed a considerable number of cells with a diffuse positive reaction to acid phosphatase (AP).

[The effect of an insoluble fungal glucan on indicators of immunity in calves]. / Vplyv nerozpustného fungálneho glukánu na niektoré ukazovatele imunity teliat.

Immunomodulative effect of insoluble fungous glucan (IFG) was observed on nine accidentally chosen calves, which were in the age from 4.5-5.5 months. The following items were observed: T, B lymphocytes, white blood cell count, index and activity of phagocytosis, induction and determination of interferon from leukocytes. In the experimental group there was a decrease in B-rosseting cells observed. On the 28-th day and on the 42-th day of experiment duration a significant increase in B-rosseting cells in comparison with control group has been observed. Average values were significantly different on the levels P < 0.01 and P < 0.05. Percentages of T-rosseting cells were almost the same during the whole experiment. In the experimental group on the 7-th and on the 14-th day the decrease has been observed. By the Student's t-test a significant difference in favour of group, where IFG was applicated, has been observed. Percentage of white blood cell count was from 47 to 72 in both groups. When phagocytic activity was evaluated, there was the higher percentage of phagocytic cells in the 7th, 14-th, 21-st, 28-th and 42-th day of experiment when compared with the 0-day value. Concerning the phagocyte index a statistical difference was observed, when compared to the initial value in favour of the experimental group. The initial disbalanced values of interferon, which have been observed during the period from the 7-th to the 21-st day, increased on the 28-th and the 42-nd day of experiment which was statistically confirmed on the following levels: P < 0.05 and P < 0.01.

[The effect of Zindep on B-lymphocyte levels in pregnant cows]. / Vplyv prípravku Zindep na hladinu B-lymfocytov u gravidných kráv.

In the present study the immunomodulation effects of the Zindep preparation, based on elementary zinc and zinc oxide, were followed on the humoral response of gravid cows in the 8th and 9th months, a week after parturition and in calves immediately after parturition and on day 15 of their life. An increased level of B-lymphocytes was observed on days 2, 5, 8, 15, 30 and 50 of the experiment. The highest average values were recorded in the experimental group on days 8 and 15 of the experiment. The mentioned values were statistically significant (T-test) at the level 0.05 and 0.01. The average values of B-lymphocytes in cows after parturition reached 22.3% in the cows of the experimental group and 20.5% in the control group. No pronounced differences in the number of B-lymphocytes in the calves of both followed groups were recorded. The statistical evaluation of the values obtained in the cows after parturition as well as in the calves was not carried out due to a small number of samplings.