Retrospective review and current knowledge on the occurrence of West Nile virus in mosquito vectors, reservoirs and hosts in Slovakia (Central Europe).

West Nile virus (WNV) is a mosquito-borne flavivirus (the genus Flavivirus) representing a medical and veterinary public health concern. Birds are the most important reservoirs. Culicine mosquitoes transmit WNV to vertebrate hosts (including horses and humans) and migratory birds play role in its long-distance transport. Slovakia is geographically localised at the crossroad of migration routes connecting South Europe and Africa with breeding localities in the Western, Northern, Central and Eastern Europe and Siberia. This review summarizes historical and present knowledge on WNV in Slovakia during a period of more than fifty years. Five European mosquito species capable to transmit WNV are native in Slovakia. Based on recent research results, the major role in the WNV transmission is attributed to Culex mosquitoes, which are also the most abundant species. Virus isolates from birds that succumbed to WNV infection are genetically close to Central European strains. Historical and recent results point out, that WNV circulates in the population of vectors, reservoirs and hosts for decades. Although West Nile fever epidemics in Slovakia were not reported yet, virus isolation, molecular detection and serological findings in reservoirs and hosts confirm that sporadic cases occur. Furthermore, the first autochthonous human case may indicate favourable conditions for WNV transmission to humans. The climate change and precipitation anomalies may favour to increase vector abundance, hence increase the chance of WNV epidemics. This review highlights an urgent need of a countrywide surveillance program aimed on the WNV occurrence in vectors and reservoirs in Slovakia. Keywords: West Nile virus; flavivirus; vector-borne pathogen; arbovirus.

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.

Histo-FISH protocol to detect bacterial compositions and biofilms formation in vivo.

The study of biofilm function in vivo in various niches of the gastrointestinal tract (GIT) is rather limited. It is more frequently used in in vitro approaches, as an alternative to the studies focused on formation mechanisms and function of biofilms, which do not represent the actual in vivo complexity of microbial structures. Additionally, in vitro tests can sometimes lead to unreliable results. The goal of this study was to develop a simple approach to detect bacterial populations, particularly Lactobacillus and Bifidobacterium in biofilms, in vivo by the fluorescent in situ hybridisation (FISH) method. We standardised a new Histo-FISH method based on specific fluorochrome labelling probes which are able to detect Lactobacillus spp. and Bifidobacterium spp. within biofilms on the mucosal surface of the GIT embedded in paraffin in histological slices. This method is also suitable for visualisation of bacterial populations in the GIT internal content. Depending on the labelling probes, the Histo-FISH method has the potential to detect other probiotic strains or pathogenic bacteria. This original approach permits us to analyse bacterial colonisation processes as well as biofilm formation in stomach and caecum of BALB/c and germ-free mice.

Prevalence of porcine circovirus 2 infection in pig population in Slovakia.

The prevalence of porcine circovirus 2 (PCV-2) infection in the pig population in Slovakia was investigated. Sera from pigs suspected for post-weaning multisystemic wasting syndrome (PMWS) as well as clinically healthy pigs were tested for viral DNA and specific IgM and IgG antibodies. Pigs (n = 198) were categorized to weaning, grower and fattening ones and sows. The results showed that PCV-2 antibodies were present in 53.4% of PMWS-suspects, in 50.0% of healthy pigs and in 69.0% of sows. In PMWS-suspect grower pigs, 40.7% were positive for IgM+IgG antibodies and 22.2% for viral DNA. In PMWS-suspect fattening pigs, 50.0% were positive for IgM+IgG antibodies and 25.0% for viral DNA. In healthy fattening pigs, almost 90.0% were positive for IgG antibodies and 38.5% for viral DNA. The highest proportion of PMWS-suspects was in grower pigs and specific antibodies were increasing with the age of pigs. A combination of positivities for IgG+IgM antibodies and viral DNA was a highly significant marker of PMWS. Viral DNA was detected in seropositive as well as seronegative PMWS-suspects. Overall, in all categories of pigs tested, specific antibodies and viral DNA were detected in 54.0% and 35.5%, respectively.

Induction of micronuclei in rat bone marrow after chronic exposure to lead acetate trihydrate.

Lead increasingly contributes to pollution of the environment and may play a role in the development of adverse effects in the human and animal body. Data concerning its mutagenic, clastogenic, and carcinogenic properties have been conflicting. In this study, we evaluated the frequency of micronuclei in bone marrow erythrocytes of rats treated with lead acetate trihydrate. Outbred Wistar rats were exposed to a daily dose of 100 mg/L drinking water for 125 days. The mean value of the total number of micronuclei observed in polychromatic erythrocytes of female rats was significantly higher than that found in the control group (13.375 +/- 2.722 against 9.625 +/- 3.204 micronuclei/1000 cells; P = 0.024 in ANOVA). In exposed female animals, no significant reduction of the ratio of polychromatic to normochromatic erythrocytes was observed (0.990 +/- 0.228 against 1.208 +/- 0.195; P = 0.060 in ANOVA). The effects of lead acetate trihydrate in male rats are both cytotoxic and genotoxic because of a decrease in ratio of polychromatic to normochromatic erythrocytes (0.715 +/- 0.431 against 1.343 +/- 0.306; P = 0.023, ANOVA followed by Tukey test) and an increase in frequency of micronucleated polychromatic erythrocytes (24.167 +/- 7.859 against 4.0 +/- 4.528 micronuclei/1000 cells; P < or = 0.001, ANOVA followed by Tukey test), respectively.

In vitro effect of pesticides (dichlofluanid, endosulfan, simazine, tolylfluanid and triallate) on proliferative activity of animal derived cell cultures.

In this study pesticides with different chemical structures (dichlofluanid, endosulfan, simazine, tolylfluanid and triallate) were examined for their potential cytotoxic effect on proliferative activity of cell cultures of mammalian origin. Cell lines Madin-Darby Bovine Kidney (MDBK), Rabbit Kidney (RK13), Porcine Kidney (PK15), and semicontinual line of Bovine Embryonic Pulmonary Cells (BEPC) were used in the study. From these cell cultures cell proliferative activity was suppressed most intensively in PK15 culture by endosulfan (10(-1) - 10(-6) M). The least effect on cell proliferation in all cell cultures tested, with the exception PK 15 (10(-1) - 10(-2) M), was recorded after simazine exposure. On the basis of IC50 values the cytotoxic effect was: dichlofluanid (IC50 = 10(-3.94) M) > tolylfluanid (IC50 = 10(-3.69) M) > endosulfan (IC50 = 10(-3.24) M) > triallate (IC50 = 10(-3.12) M) > simazine (IC50 = 10(-1.78) M). The comparison of average IC50 values of cell cultures revealed that the most sensitive cell lines were PK15 (IC50 = 10(-3.27) M) and RK13 (IC50 = 10(-3.21) M), whereas MDBK (IC50 = 10(-2.55) M) and BEPC (IC50 = 10(-2.52) M) were less sensitive to pesticide exposure.

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.

Effect of endosulfan on peripheral sheep leukocytes in vitro.

The immunotoxic and genotoxic effect of endosulfan, an organochlorine insecticide, on sheep peripheral blood leukocytes was examined in in vitro conditions. The immunotoxic effect was evaluated by assays of the metabolic activity of phagocytes and assays for lymphocyte activation--the leucocyte migration-inhibition assay (LMIA) and lympho-proliferation. The significant inhibitory effect of endosulfan on metabolic activity of peripheral blood phagocytes was registered at the actual concentrations of 10(-3)- 10(-4) M. At 10(-3) M the migration of leukocytes was inhibited, both in activated and non-activated with phytohemagglutinin (PHA) leukocyte suspensions (p < 0.01) in LMIA. This indicated the direct cytotoxic effect of endosulfan on the polymorphonuclears and monocytes of which the intensity of migration is an indicator of lymphocyte activation with mitogen. At the concentration of 10(-4) M an immunotoxic effect, ie significant decrease of lymphocyte activation with mitogen was recorded in LMIA. Lympho-proliferation test showed the significant inhibition of proliferation for PHA-stimulated lymphocytes at 10(-3) M and 10(-4) M. Micronucleus assay evaluated the genotoxic potential of endosulfan. Higher concentrations of insecticide (10(-5) M, 10(-6) M) resulted in a significant dose dependent increase in the number of micronuclei.

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.

The importance of lymphocyte micronucleus test for the detection of genotoxic events.

DNA lesion induced by xenobiotics is implemented either through direct interaction of the chemical with DNA through covalent binding or intercalation, or indirectly, through interference with the processes of DNA replication and repair, interaction with proteins, nuclease release from lysosomes etc. The present study emphasizes that the assessment of genotoxic risk arising from xenogenous substances demands the development of sensitive, direct and more exact genotoxic testing methods. One of them is micronucleus assay. It is a simple and quick test for investigating of cytogenic damage with the advantage that exposure to both clastogens and aneugens may be detected.

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.

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.

The effect of p-chloroaniline on leucocytes of sheep peripheral blood under the migration-inhibition test conditions.

Toxic and immunotoxic effects of p-chloroaniline--a metabolite of herbicide monolinuron--were investigated in peripheral blood leucocytic suspensions of five sheep using a migration-inhibition test. The toxic effect of p-chloroaniline was recorded at concentrations 1.0 to 0.1 mg.ml-1 and the immunotoxic one at concentrations 0.01-0.001 mg.l-1. The toxic effect was demonstrated by total inhibition of leucocyte migration. The immunotoxic effect, determined as mitogenic activation of leucocytes by phytohemagglutinin, concanavalin A and lipopolysaccharide, was detected at 10 to 100-fold lower concentrations of p-chloroaniline than those which resulted in toxic effects.

The influence of heavy metal emissions and Fasciola hepatica infestation on the immunogenicity of a Listeria vaccine.

The effect of oral dosing with emissions containing heavy metals (mercury, lead, copper, cadmium, zinc and chromium) on immune status was tested in 3 groups of sheep. Two groups (Groups I and II) were given emissions for 15 d. One of these groups (Group I) was then also infested with Fasciola hepatica metacercaria on day 16. These 2 groups (Groups I and II) and a control group (Group C) were immunized with a Listeria vaccine on days 8 and 22. A decreased index of metabolic response (IMR) of phagocytes and reduced responses of lymphocytes to mitogenic activation with phytohemagglutinin and L monocytogenes antigen stimulation in the leukocyte migration-inhibition test were recorded. Decreased agglutination titers of serum antibodies against L monocytogenes were observed. The F hepatica infestation had no significant effect on the migration index or IMR.

Effect of vitamin E on some leucocytic parameters and functions in transported calves.

The objective of this investigation was to determine the effect of vitamin E administration on leucocyte parameters and some of their functions in transported calves. In the study 8 calves, aged approx. 10 days, divided into two groups, were used. 20 mg of tocopheryl-acetate per kg body weight were administered orally to each of the 4 experimental calves 24 hours before loading. The calves were transported by road for 3 hours. Blood samples collected before and after the transportation were examined for total and differential leucocyte counts, T-lymphocyte subpopulation, phagocytic activity, leucocyte migration, serum immunoglobulin levels, and for plasma vitamin E and cortisol levels. The animals showed a leucocytosis with neutrophilia and lymphopenia after transportation. The administration of vitamin E led to a decrease of cortisol level in 24 hours. There was no difference between groups in cortisol reaction due to transportation stress. Leucocyte migration has been less inhibited in the control group after unloading. A mild decline in phagocytic activity was observed 3 hours after transportation. Serum immunoglobulins were unaffected by both vitamin E administration and transportation. Vitamin E had no significant effect on leucocyte function and cortisol levels in present study.

The induction of specific protection against Actinobacillus pleuropneumoniae infection by specific DLE.

Immunoprophylactic and immunotherapeutic effects of specific actinobacillus DLE (dialysable leukocyte extract) were studied in model pigs experimentally infected with Actinobacillus pleuropneumoniae. Specific DLE used for immunoprophylactic purposes was administered intramuscularly to pigs either in two doses in combination with one dose of actinobacillus vaccine (1st group) or in a single dose (2nd group). All animals of the two experimental groups survived the intranasal A. pleuropneumoniae challenge (2 x 2 ml 2-5 x 10(10) c.f.u.) while 3 of 5 animals of the control group died of haemorrhagic-necrotic fibrinous pneumonia. DLE administered to pigs for immunotherapeutic purposes was applied intravenously in two doses 1 and 5 h after the actinobacillus challenge (1st group), and in a single dose 5 h following the challenge (2nd group). All animals of the first group survived the challenge while 3 out of 5 animals of the second group and 4 out of 5 animals of the control group died during the experiment.