Risk factors associated with dog endoparasites infection spread in East Slovak Lowland.

This study aimed to determine the prevalence of endoparasites in dog feces from public places, investigate the occurrence of endoparasites in soil, and identify potential risk factors associated with the dog endoparasites infection spread in East Slovak Lowland - the region near the EU border with Ukraine. In a one-year study, a total of 803 dog fecal samples and 148 soil samples from public places were examined for the presence of endoparasite developmental stages. In general, 43.59 % of dog excrements were positive. Six different species of intestinal parasites were detected: Toxocara canis (22.65 %), Trichuris vulpis (14.07 %), family Ancylostomatidae (10.09 %), Capillaria aerophilla (5.60 %), Ascaris spp. (1.49 %). Altogether, 52.03 % of soil samples were positive for at least one endoparasites species. The occurrence of parasitic eggs in the soil was as follows: Ascaris spp. (37.16 %), Toxocara spp. (29.05 %), Trichuris spp. (29.05 %) %), family Ancylostomatidae (2.03 %) and Toxascaris leonina (2.03 %). The occurrence of endoparasites between the towns and villages varied significantly. The distribution of endoparasites in dogs and soil was affected by the population density, the minority people living in the studied territory, the number of people living in poor hygienic conditions with limited access to drinking water and the usage of sewerage. Persisting endoparasitic contamination of the environment in East Slovak Lowland forms a reservoir with zoonotic potential representing public and environmental health problems.

Alveolar Echinococcosis of the Liver with a Rare Infiltration of the Adrenal Gland.

Human alveolar echinococcosis (AE) is a silently-progressing disorder that has become a threat in many countries. Since 2000, when the first case was recorded, the number of human AE patients in Slovakia is on continuous raise. The article presents a rare case of alveolar echinococcosis with infiltration in the adrenal gland and discusses the problems associated with differential diagnosis of the disease. In 2016, abdominal ultrasound performed due abdominal pain complaint showed the presence of cystic lesions in the right liver lobe of 54-year old female patient. During surgery, another lesion in the right adrenal gland was found, and neoplastic processes or echinococcosis were considered in the differential diagnosis. Due to unclear correlation between radiology, serology and histopathology results and endemic situation in Slovakia, molecular examination was recommended. Subsequently E. multilocularis was confirmed as etiological agent of infection. Alveolar echinococcosis is considered as a rare disease, with very few patients referred to clinicians or hospitals that sometimes have almost none existing experience with the diagnosis and treatment of the disease. Therefore, the establishment of networks or reference centres specialized on management of the disease would be suitable way to provide the patients with the best care and improve the disease diagnosis, treatment and prognosis.

A Pilot Comparative Study Between Serological and Genetic Investigations in Relationship to Clinical Outcomes on Patients with Cystic Echinococcosis.

The aim of this study was to investigate whether Enzyme-Linked Immunosorbent Assays (ELISA) and Western Blotting (WB) methods could contribute to the assessment of clinical outcomes in genotype-defined cystic echinococcosis (CE) patients. Twenty-nine human isolates and blood samples have been taken from patients who underwent surgery or percutaneous aspiration (PAIR) for therapeutic purposes at Ege University and Manisa Celal Bayar University Hospitals. All sera of patients were screened for the presence of E. granulosus IgG antibodies using in-house approved ELISA and WB methods. According to the ELISA results, five patients had high, thirteen patients had medium and eight patients had low specific antibody level response which ranged 1/640 -1/5000. Despite confirmed WB positivity three patients were found to be negative by ELISA. Immunoblot analysis of EgAg showed many protein bands with size of 8, 12, 20, 22, 24, 36, 75 and 90 kDa. Among of them, 8 - 12 kDa bands (90 %), 20 - 22 kDa and 36 kDa bands presented strong reactivity against human serum specimens. No serum samples from healthy control reacted with EgAg. Phylogenetic analysis of resulting COX1 and NAD1sequences has revealed that all patients in our study were infected with the E. granulosus G1-G3 genotype. There was no consistent correlation between results of ELISA and WB, the number or size of cysts and genotype. Our study brings a unique contribution in terms of relationship between serological investigation, disease genotypes and clinical outcomes.

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.

Selective recruitment of T-cell subsets to the udder during staphylococcal and streptococcal mastitis: analysis of lymphocyte subsets and adhesion molecule expression.

During bacterial infection of the bovine mammary gland, large numbers of leukocytes migrate into the udder, resulting in the establishment of a host response against the pathogen. Currently, the specific leukocyte populations mediating this immune response are not well defined. In the studies described here, we analyzed blood and milk from healthy cows and cows with naturally occurring mastitis to determine if distinct alphabeta and gammadelta T-lymphocyte subsets were involved in the response of the udder to a mastitis pathogen and if the type of mastitis pathogen influenced the subset composition of these responding leukocytes. Although blood samples from cows with confirmed staphylococcal and streptococcal mastitis were characterized by increased numbers of gammadelta T cells, the most dramatic changes in leukocyte distributions occurred in milk samples from these cows, with a 75% increase in alphabeta T-cell levels and a 100% increase in gammadelta T-cell levels relative to the levels in milk samples from healthy animals. Interestingly, the increase in alphabeta T-cell numbers observed in milk from cows with staphylococcal mastitis was primarily due to increased numbers of CD4(+) T cells, while the increase in alphabeta T-cell numbers observed in cows with streptococcal mastitis was due to a parallel increase in both CD4(+) and CD8(+) T-cell numbers. The increased numbers of gammadelta T cells in milk from cows with staphylococcal and streptococcal mastitis were due to a selective recruitment of a distinct gammadelta T-cell subset (GD3.1(+)), while no change in the numbers of GD197(+) gammadelta T cells was observed. We also analyzed adhesion protein expression on blood and milk leukocytes and found that, in comparison to the situation for healthy cows, L-selectin was down-regulated and CD18 was up-regulated on leukocytes from cows with mastitis. Thus, shedding of L-selectin and up-regulation of CD18 by neutrophils may provide a sensitive indicator of early inflammatory responses during bovine mastitis. Overall, these studies suggest that distinct alphabeta and gammadelta T-cell subsets are involved in the host defense of the udder against mastitis infection and that selective recruitment of these T-cell subsets depends on the infectious agent involved.

[The treatment of acute lower limb ischemia]. / Leczenie ostrego niedokrwienia konczyny.

In 1988-1997 297 patients were operated on acute limb ischemia. In 131 patients ischemia was caused by arterial embolism, in 144 patients by acute arterial thrombosis, in 20 patients by arterial trauma and in 2 patients acute aortic dissection. Acute limb ischemia causes 12.8% risk of death especially in elderly and when the reason for ischemia is acute occlusion of aortic bifurcation. The majority of patients with acute arterial thrombosis requires arterial reconstruction.

Isolation of bovine neutrophils with biomagnetic beads: comparison with standard Percoll density gradient isolation methods.

A prerequisite for studies on bovine neutrophils is a reliable method of neutrophil isolation from blood to obtain highly purified cell populations that are functionally active. Since current techniques of neutrophil isolation fall short of these requirements, we have developed a newer and more effective technique for isolation of bovine neutrophils that utilizes biomagnetic beads coated with a monoclonal antibody that recognizes an abundant surface antigen on bovine neutrophils to purify these cells. Comparison of the purity and viability of bovine neutrophils isolated by a conventional method (continuous Percoll density gradient) with this new method showed that neutrophils isolated with biomagnetic beads were higher in purity and had an increased yield. In addition, cells isolated with biomagnetic beads demonstrated normal or even improved function in assays of chemotaxis, phagocytosis, degranulation, and respiratory burst activity. Finally, bovine neutrophils isolated using this method showed an overall lower level of spontaneous apoptosis, which correlates well with the high level of viability observed in the purified cell preparations. Thus, this method represents a significant advance over current methods for isolating bovine neutrophils and would be widely applicable to labs studying the biochemistry and signal transduction pathways in these cells.

Cellular immune responses in mice infected with the intestinal nematode Trichuris muris.

CBA and B10.BR mice show variation in immune response to the intestinal nematode Trichuris muris. CBA mice develop strong resistance, eliminating worms from the intestine; B10BR mice are permissive and develop chronic infections. It is already known that resistance and permissiveness reflect differential T helper responses. The data reported here show that resistant CBA mice express good antigen-specific lymphocyte proliferative responses to infection, whereas cells from B10.BR mice are relatively anergic, although still responsive to Concanavalin A (ConA). The possibility that the altered proliferative responsiveness seen in infected B10.BR mice reflected quantitative or qualitative changes in T helper cells was examined by comparing cytokine production and expression of cell surface markers (CD4, CD8, and CD28) in mesenteric lymph node cells and spleen cells from both strains and comparing these with the characteristics of cells from resistant CBA mice and from CBA mice that had been rendered permissive to infection by a combination of irradiation and corticosteroid treatment. As expected, cells from B10.BR mice produced high levels of interferon-gamma (IFN-gamma), whereas those from CBA mice released high levels of IL-5, whether stimulated with adult worm somatic antigens, excretory/secretory antigens, or ConA. Immunosuppressed CBA mice produced high levels of both IFN-gamma and IL-5 throughout the experiment. FACS analysis revealed a decrease of CD4+ and an initial increase in CD8+ cells in all infected mice. No major changes occurred in the relative proportion of CD28(+) cells. Further evaluation of the CD28 costimulatory molecule, measured as mean fluorescence intensity, displayed down-regulation in permissive and immunosuppressed mice. The data obtained suggest that lymphocyte unresponsiveness and permissiveness to T. muris infection may be associated with a down-regulation or an initially reduced expression of costimulatory CD28 molecules.

Modulation of endotoxin- and enterotoxin-induced cytokine release by in vivo treatment with beta-(1,6)-branched beta-(1,3)-glucan.

Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, beta-(1,6)-branched beta-(1,3)-glucan (soluble beta-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble beta-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-alpha), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-gamma) and suppressed production of IL-2 and TNF-alpha compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble beta-glucan-treated mice with LPS also resulted in suppressed TNF-alpha production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-alpha production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble beta-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-alpha. Taken together, our results suggest that treatment with soluble beta-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality.

Long-term reinforced fixed provisional restorations.

Extensive prosthodontic treatment often requires fabrication of long-term provisional restorations. Numerous materials and techniques have been described for prolonged insertion of interim restorations. This article describes a procedure for fabrication of long-term reinforced heat-processed provisional restorations based on a diagnostic wax-up. Reinforced heat-processed provisional restorations reduced flexure, which minimizes progressive loss of cement and diminished the possibility of recurrent decay. Occlusal stability and vertical dimension were maintained because of greater wear resistance. Occlusion, tooth contours, and pontic design developed in the provisional restoration were duplicated in the definitive restoration. The use of a matrix from a diagnostic wax-up facilitated fabrication of the prosthesis, and made the procedure less time-consuming and more predictable.

Development and morphological variability of Echinococcus granulosus.

Characteristics are presented of Echinococcus granulosus strobila from dogs experimentally infected with protoscolices from a single cyst isolated from the liver of a domestic pig in a central Slovakian district. Adult cestodes are characterized mainly by 28-32 hooks highly variable in shape and size; 26-51 testes situated mostly throughout the proglottis (1 or 3 rows behind the vitelline gland); a lobate ovary; the shape of the uterus, which in 10% of proglottides is more or less spherical, in 62% is saccular with lateral sacculations of variable size, in 27% is saccular without lateral sacculations and in 1% is more or less tubular or strongly ramified; and an intermediately rapid rate of development lasting until between 40 and 44 days p.i. The present report analyzes the high degree of variability of the clone studied, referring to the knowledge on the characteristics of the pig, sheep, and cattle strains of E. granulosus and E. multilocularis species, with which some taxonomic characters of the clone representatives overlap.

Effects of concurrently administered copper and mercury on phagocytic cell activity and antibody levels in guinea pigs with experimental ascariasis.

The subchronic effect of copper and mercury on selected immunological parameters in guinea pigs with experimental ascariasis was studied. Cupric sulphate and mercuric chloride administered for 28 days did not suppress the levels of specific circulating antibody in a subsequent Ascaris suum infection. Intoxication of animals significantly inhibited the phagocytic activity of peritoneal macrophages throughout the experiment. Ascaris suum infection elevated macrophage phagocytic activity but values remained significantly lower in comparison with infected non-intoxicated animals. Both the concurrently administered metal compounds changed the phagocytic ability of polymorphonuclear leucocytes in the blood. The intensity of infection assessed by the number of Ascaris larvae migrating in the lungs of animals currently intoxicated with both the metal compounds increased by 28.6%, compared with controls.

Effect of glucan immunomodulator on the immune response and larval burdens in mice with experimental toxocarosis.

Glucan immunomodulator, combined with immunoglobulin and zinc (GI), was tested in mice infected with Toxocara canis for its effects on the immune response and parasite recovery. Infection with 2 500 T. canis eggs per mouse induced a short-term depression of the proliferative response of T cells to phytohemaglutinin from Day 35 to a Day 49 post infection (p.i.). GI given in two doses at the start of the experiment markedly stimulated and restored cell proliferation at Days 21-63 p.i. Infection resulted in significant increase in specific circulating antibody level at Days 21 and 35 p.i. A striking reduction in the number of T. canis larvae, after GI administration was observed in the muscles of the GI-treated mice, compared with the untreated animals.

[The effect of copper on immune responses in guinea pigs with experimental ascariasis]. / Vplyv medi na imunitné prejavy morciat pri experimentálnej askarióze.

A subchronic effect of copper on selected immunological parameters was studied in guinea pigs with experimental ascariosis (Ascaris suum). CuSO4 given orally for 28 days moderately increased T and B cells in the animal lymphoid organs. CH50 and AH50 complement levels remained unchanged during CuSO4 administration. A subsequent infection of subchronically intoxicated guinea pigs caused a repeated insignificant stimulation of B cell values while the percentage of T cells was not increased. On the contrary, the complement levels increased significantly in intoxicated infected animals, namely CH50 levels from day 5 to day 15 and AH50 levels from day 1 to day 7 post infection. No statistically significant differences were observed in comparison with infected non-intoxicated guinea pigs. The lean intensity of infection evoked by A. suum larvae migrating in the lungs of intoxicated animals was 29% lower than in control.

Effect of mercury on the immune response and mean intensity of Ascaris suum infection in guinea pigs.

The subchronic effect of mercury on selected immunological parameters was studied in guinea pigs with experimental Ascaris suum infection. HgC1(2) given for 28 days reduced significantly T- and B-cell populations in the lymphoid organs and the phagocytic ability of peritoneal macrophages. The subsequent infection of HgC1(2)-intoxicated animals elevated the studied immunological parameters, but in comparison with infected non-intoxicated guinea pigs they remained significantly suppressed. The mercury compound in infection stressed animals caused a slight alteration of the complement CH50 and AH50 activity. The specific circulating antibody level in infected and HgC1(2) treated animals rose a little by day 12 p.i. and then again decreased significantly, compared with untreated guinea pigs. The mean intensity of infection with migrating Ascaris larvae in HgC1(2)-treated animals increased by 15%, compared with controls.

Immunorestorative effect of glucan immunomodulator on guinea pigs with experimental ascariosis.

The immunorestorative effect of glucan immunomodulator, combined with porcine immunoglobulin and zinc (GI) on T- and B-lymphocytes and peritoneal macrophage phagocytic ability was studied in guinea pigs with experimental ascariosis (Ascaris suum) after a cyclophosphamide (CY)-evoked immunosuppression. During the migration phase of A. suum infection GI exerted a significant restorative effect on the CY-reduced percentage occurrence of T- and B-cell populations in the mesenteric, mediastinal and hepatic lymph nodes and spleen of A. suum hosts. On the contrary, it did not influence the CY-suppressed phagocytic activity and index of phagocytic activity of the peritoneal macrophages. The protective effect of the GI evaluated by the reduction in the number of migrating ascarid larvae in the lungs of guinea pigs after immunosuppression with CY and administration of GI was 14.46% higher, compared with the suppressed and infected group without administration of GI.

Effect of heavy metal emission on the immune response of sheep with experimental fasciolosis.

The effect of industrial heavy metal emission on immune reactions was studied in lambs subsequently infected with Fasciola hepatica. The heavy metal emission (Cu, Hg, Pb, Cd, Cr, Zn) was collected from grass at 5 km distance from a mercury plant. Administration of the emission for 28 days prior to infection of the animals reduced the percent circulating T- and B-cell populations when compared with untreated animals and animals infected with F. hepatica only. The administration of emission had no suppressive effects on the levels of circulating antibodies to F. hepatica following infection. The intensity of liver infection with mature F. hepatica in the emission-fed and infected lamb group increased by 21.5%. This difference in comparison with control infected animals was not significant.

Effects of heavy metals imission on the cellular immunity of guinea pigs with experimental ascariosis.

A study was performed on the short-term effects of industrial heavy metals imission (Hg, Cu, Pb, Zn), collected at 5 km from a mercury-producing plant, on T- and B-lymphocytes and peritoneal macrophages of guinea pigs with experimental ascariosis (Ascaris suum). Administration of the imission for 5 days showed a significant decrease in T- and B-cell population and in macrophage phagocytic ability during the migration phase of Ascaris suum when compared with an infected but untreated group of animals. This was also manifested by an eight-fold increase in the density of migrating ascaris larvae in the lungs. Macrophage metabolic activity in animals given imission was significantly suppressed only until Day 7 post-infection. The most striking decrease in the immunological responses was observed in guinea pigs that were given imission but were not infected.

[Immunostimulatory effects of certain substances in experimental ascaridiasis in pigs]. / Imunostimulacné úcinky niektorých látok pri experimentálnej askaridóze prasiat.

The effects were studied of four immunomodulative substances of biological origin (purified glucan, glucan with porcine immunoglobulin and zinc, transfer factor and vitamin A) on T and B lymphocytes, along with their protective effects in pigs with experimental ascariasis. These substances activated T and B lymphocytes with different intensity. The nonspecific stimulation of these cells in the blood of pigs with the immunomodulative substances intensified the protective mechanisms; this resulted in a decreased number of migrating ascaris larvae in the lungs of pigs by 17% to 65%. Of the four immunomodulative substances studied, transfer factor and glucan-based substances with porcine immunoglobulin and zinc proved to be the most effective. Both the substances exerted a significant stimulative effect on T- and B-cell populations even in the first days after infection, reaching the maximum on days 7-9 p. i. (Figs. 1 and 2). The protective effect, assessed by the reduction of migrating ascaris larvae in the lungs of pigs, was 62% and 65% using transfer factor and glucan preparation with homologous immunoglobulin and zinc, respectively. The percentage of occurrence of T and B lymphocytes, obtained by the use of purified glucan and vitamin A, was not in correlation with their protective effect (purified glucan 17%, vitamin A 50%). The immunomodulative effects of little effective purified glucan increased when combined with porcine immunoglobulin and zinc. The results are significant from the aspect of the nonspecific stimulation of immunity in ascariasis and in some other helminthoses.

Effect of glucan preparation on immunocompetent cells and phagocytic ability of blood leucocytes in experimental ascariosis of pigs.

In experimental ascariosis (Ascaris suum) of pigs a preparation of glucan combined with pig immunoglobulin and zinc was found to significantly elevate the levels of both T and B lymphocytes in the blood, and to significantly stimulate the phagocytic ability of monocytes and neutrophilic granulocytes in experimental pigs. The protective effect of the preparation, assessed by the reduction in the number of migrating ascarid larvae in the lungs of pigs was 65%.