Establishment of Lactobacillus plantarum strain in honey bee digestive tract monitored using gfp fluorescence.

Lactic acid bacteria are symbiotic bacteria that naturally reside in the gastrointestinal tract of honey bees. They serve a multitude of functions and are considered beneficial and completely harmless. In our experiments Lactobacillus plantarum strain B35, isolated from honey bee digestive tract, was modified using pAD43-25 plasmid carrying a functional GFP gene sequence (gfpmut3a) and used as a model for monitoring and optimisation of the mode of application. The establishment of this strain in honey bee digestive tract was monitored using GFP fluorescence. Three different modes of oral application of this strain were tested: water suspension of lyophilised bacteria, aerosol application of these bacteria and consumption of sugar honey paste containing the lyophilised lactobacilli. Two days after administration the L. plantarum B35-gfp was present throughout the honey bee digestive tract with 104-105 cfu/bee with highest count observed for aerosol application.

Toxic effects of cadmium on testis of birds and mammals: a review.

In humans and other mammals, cadmium (Cd) causes various damages to different organs and tissues of the body. This review presents a comprehensive overview on the effect of Cd on the structure of seminiferous tubules, Leydig cells and blood vessels in the testis. The main observation of the effect of Cd is destruction of the seminiferous tubules with severe necrotic areas. Damage is to all stages of developing germ cells by inducing their structural changes and the apoptotic cell death. Sertoli supporting cells are considered the most vulnerable cells. Their damage results in cytoplasmic rearrangement and disruption of inter-Sertoli tight junctions resulting in increased permeability of the blood-testis barrier, structural changes in the Leydig cells and decreased testosterone secretion. After long time of Cd exposure an increase of the amount of interstitial connective tissue occurs. In blood vessels Cd exposure causes various morphological and physiological changes in vascular endothelial cells and smooth muscle cells. In humans and other mammals, the range of effect depends on the dose, route, ways, and duration of exposure. After necrosis of the sensitive cells Cd produced lesions in surrounding tissue and activate free cells. Atrophy of the seminiferous tubules is followed by Leydig cell regeneration and interstitial revascularization. In birds, spermatogenic cells underwent irreversible degeneration or atrophy of seminiferous tubules in the absence of significant vascular lesions.

Limited genetic diversity of Aerococcus viridans strains isolated from clinical and subclinical cases of bovine mastitis in Slovakia.

The Aerococcus viridans isolates from bovine mastitis in Slovakia were isolated and characterized by classical microbiological and biochemical, and molecular techniques including IGS-PCR and rep-PCR, ARDRA and 16S rDNA gene sequencing. The substantial variability of antibiotic resistance patterns was observed. The majority of strains were resistant to beta-lactam antibiotics, the resistance to tetracycline was observed in 3 tested strains, resistance to lincomycin was found in 4 strains and practically all tested strains were sensitive to neomycin and ciprofloxacin. While variable at a phenotypic level, no significant genetic variability among A. viridans isolates was detected by molecular DNA based methods. The data obtained suggest that a few A. viridans strains spread among cow's population in Slovak farms.

The retention of cadmium and selenium influence in fowl and chickens of F1 generation.

The retention of cadmium and selenium influence on Cd retention in the muscle, liver and kidneys of hens, chickens and in eggs was studied. Cadmium (Cd) as cadmium chloride (CdCl(2)) and selenium (Se) as sodium selenite (Na(2)SeO(3)) were added to feed at dosages: group 0-control, group 1-20 mg/kg Cd, group 2-30 mg/kg Cd + 4 mg/kg Se. The birds were exposed to Cd for 8 weeks. Cadmium level in hens and cocks was found highest in the kidneys, followed by the liver and muscle. Se supplementation resulted in Cd increase in the muscle tissue and in the reduction of Cd content in the liver and in significant decrease in the kidneys (p < 0.05). A higher Cd level in the yolk and lower in the white was noted in both experimental groups. Nonsignificant increase of Cd in eggs was noted in experimental groups with Se supplementation. Level of cadmium in organs of 7-day-old chicks hatched from Cd-treated hens in both experimental groups was low but the tendency to accumulate preferentially the Cd in the liver and kidneys was recorded. Supplementation of selenium in hens and cocks was not reflected in the decrease of Cd in these two organs of F(1) chickens but was reflected in increase in the muscle. In spite of relatively high Cd levels in the organs of layers no layer-egg-chickens transfer was observed. It was confirm that kidneys and liver are organs more attacked by dietary cadmium than muscle. Supplementation of low dose of Se resulted in decrease of cadmium deposition in analyzed organs.

The effect of herbicide BASTA 15 on the development of mouse preimplantation embryos in vivo and in vitro.

The aim of this study was to evaluate the possible effect of maternal poisoning by BASTA-15 on developmental capacities and quality of preimplantation embryos in a mouse model. During in vivo tests, fertilized mice were fed with various doses of BASTA-15 for several days. During in vitro tests, isolated embryos were cultured in a medium with the addition of herbicide or its main compound glufosinate ammonium. Stereomicroscopic evaluation of embryonic pools obtained from treated dams showed that BASTA-15 at dose 58 µl/kg bw negatively affected their ability to reach the blastocyst stage. Moreover, as shown by morphological evaluation, based on cell counting and cell death assay, even the application of herbicide at the lowest dose (approx. 1/100 LD50) had a negative effect on obtained embryo quality. In vitro tests proved the direct ability of BASTA-15 to negatively affect embryo growth and quality. On the other hand, the addition of glufosinate ammonium at equivalent concentrations (from 0.015 to 15 µg/ml) had almost no damaging effect on embryos. It was harmful only at very high doses. Results show that maternal intoxication with BASTA-15 might affect the development of preimplantation embryos and suggest that the responsibility for this effect lies probably not solely with glufosinate ammonium, but in combination with the herbicide's secondary compounds.

Changes in the peritubular tissue of rat testis after cadmium treatment.

The present study demonstrates histological and immunohistochemical changes in the peritubular testicular tissue of rat testis after application of cadmium chloride. After 5-day cadmium exposure, advanced deterioration of the boundary testicular tissue, mainly oedema, disarrangement of collagen fibres and peritubular cells, dilatation and thrombosis of blood vessels were observed. Changes in the boundary tissue were accompanied with desquamation of the germinal epithelium. Immunohistochemically, positive reaction for alpha-smooth muscle actin and desmin in tunica media of large testicular blood vessels basically was not affected. No reaction for vimentin was seen in endothelial cells of blood capillaries, whereas positive reaction presented only these cells in large blood vessels. The myofibroblasts positively reacting for desmin and alpha-smooth muscle actin form a single incomplete layer in the lamina propria of seminiferous tubules. Vimentin reactivity in the myofibroblasts and in the supporting Sertoli cells as well as Leydig cells in damaged testicular tissue was not observed. An increase in fibroblasts and free inflammatory cells positive for vimentin in the peritubular space on the peripheric area of the testis was observed.

The alteration of glutathione peroxidase activity in rat organs after lead exposure.

The activity response of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR) and the contents of thiobarbituric reactive substances (TBARS) were investigated in rats exposed to lead. The enzyme activities were determined in the liver, kidney and heart of male and female rats which were received 100 mg and 1000 mg of lead acetate per liter water for 18 weeks. The statistical analyses indicated the differences related to the organs and to the sex of animals. Administration of lead evoked decrease of GPx activity in the kidney of both male and female rats. On the contrary, GPx activity increased in the heart of female rats, while in the male rats the higher dose of lead evoked a decrease in activity. In the kidneys of male rats and in the heart of female rats thiobarbituric acid reactive substances (TBARS), an indicators of oxidative stress, significantly increased in rats which were given the high lead dose. Most likely the observed changes could be a compensatory response to different lead accumulation in the male and female organs and also the possible distinct mechanisms in ROS elimination.

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.

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.

Effect of chloridazone on the animal organism.

The acute toxic effect of the herbicide chloridazone and mitochondrial respiration were investigated and typical clinical signs of intoxication were described in rats (Wistar), pheasants (Phasianus colchicus) and sheep (Slovak Merino). The LD50 of chloridazone was calculated to be for rats 800 mg/kg bw (range 552 to 1160 mg/kg bw) and for pheasants 3684 mg/kg bw (range 1768 to 7677 mg/kg bw). According to WHO chloridazone is moderately toxic for rats and slightly toxic for pheasants. The LD50 for sheep is 161 mg/kg bw (range 76 to 340 mg/kg bw). Chloridazone thus presents an acute risk for ruminants, which is in coincidence with the WHO classification characterising it as a very toxic compound. The following clinical features of intoxication were observed after p.o. administration of chloridazone: apathy, dyspnoea, hyperventilation, hypersalivation (sheep - foam hypersalivation), paralysis, tonic-clonic convulsions and death in clonic convulsions. Very quick rigor mortis. Chloridazone interfered with mitochondrial respiration in the liver of rats yet its mode of action was different from that of succinate substrate or glutamate-malate. Succinate dependent respiration was significantly decreased in both states (3 and 4) of respiration. Glutamate-malate respiration was not changed in state 4, though it significantly increased in state 3 after ADP administration. RCP (respiration control proportion) value was increased on using either of the substances.

Antioxidant and detoxifying enzymes in the liver and kidney of pheasants after intoxication by herbicides MCPA and ANITEN I.

The activity of antioxidant and detoxifying enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase, glutathione-S-transferase (GST), the contents of thiobarbituric acid reactive substances, and the superoxide dismutase and glutathione-S-transferase isoenzyme patterns, were determined in the liver and kidney of pheasants after acute intoxication by herbicides MCPA and ANITEN I. In the liver, the activity of antioxidant enzymes was significantly decreased in the group given ANITEN I. New superoxide dismutase isoforms (pI 6.30, 6.85, 7.00) and higher intensity of isoform with pI 6.60 were observed after isoelectrofocusing in all experimental groups. In the kidney, the activity of superoxide dismutase was significantly decreased, and a higher intensity of superoxide dismutase isoforms (pI 6.00 and 6.60) was observed in all experimental groups. The contents of thiobarbituric acid reactive substances were significantly increased in the group with ANITEN I. The glutathione-S-transferase isoenzyme pattern was studied by using subunit-specific substrates and by Western blotting. The activity of glutathione-S-transferase with ethacrynic acid and cross-reactivity with rat subunit 7 was lower in all experimental groups in the kidney and liver, except in the liver of the group given a higher dose of ANITEN I. In this group, we have found a 2.10-fold higher activity to ethacrynic acid and a strong induction of subunit 7.

Acute oral toxicity of the herbicide BUREX EKO in pheasants.

The aim of this study was to determine the acute LD50, clinical symptoms and pathological changes of acute BUREX EKO intoxication in pheasants according to OECD No 205. Medium lethal dose (LD50) of BUREX EKO in pheasant is 3.84 ml/kg body weight with the upper level of reliability 4.50 ml and lower level of reliability 3.27 ml/kg body weight. As far as the calculation to the effective substance is concerned it is 1077 mg of chloridazone per kg body weight with the interval of reliability from 919 to 1263 mg/kg body weight. Calculated the effective substance of chloridazone (3.84 ml is LD50 of BUREX EKO which contains 1077 mg of chloridazone) BUREX EKO can be classified as the moderately toxic substance to pheasants. There were following clinical symptoms of the BUREX EKO intoxication in pheasants: apathy, drowsiness, incapability to move, ruffled feathers, slight diarrhoea, strenuous respiration, tonico-clonical cramps before death, decease with the head expressively bent rearwards. There was a relatively fast beginning of rigor mortis in dead pheasants. Pathologico-anatomical dissection of the pheasants obtained under conditions of acute intoxication did not reveal any changes on the organs of both experimental and control pheasants which would be immediately connected with the effect of the administered substance. Hyperaemia was recorded by histologico-pathological investigation of the liver and kidneys. No changes on the brain and intestine wall were recorded.

The effect of small doses of mercury on the level of selected lysosomal enzymes in the plasma and lymphocytes of sheep.

1. The 12-month-old ewes, Slovak Merinos breed, were given 40 mg of mercury daily for 28 days in the form of HgCl2. 2. Administration of mercury had a significant influence on the activity of the investigated lysosomal enzymes in the plasma and lymphocytes of sheep.

[Complications during myelographic examination in dogs]. / Výskyt komplikácií pri myelografickom vysetrení psov.

The occurrence of spinal disease in dogs is quite frequent. Clinical examination and survey radiography do not allow to make an exact diagnosis in many cases. Therefore the radiographic method-myelograhy is used to make an exact diagnosis and to locate the pathological process in the spinal cord. Myelography can be accompanied by some complications caused by a contrast medium of anaesthesia. In this study, the occurrence of complications was evaluated when the contrast medium Dimer X and three types of general anaesthesia were used: xylazine-ketamine, chlorpromazine-piritramide, chlorpromazine-pentobarbital. Certain relations between the origin of complications, dog weight and total time of anaesthesia are indicated. The results obtained show that the highest number of cases with complicated regeneration was observed after the use of the combination xylazine-ketamine when the average duration of anesthesia was 31.1 minutes comparing to the combination chlorpromazine-pentobarbitale with average duration of anaesthesia was 127.7 minutes with the lowest number of complications. We also refer to certain relations between weight and the occurrence of complications where the highest correlation (r = 0.59) was recorded for xylazine-ketamine anaesthesia. The correlation coefficient (r = 0.27) for chlorpromazine-pentobarbital documents very low correlations of the followed values.

[The effect of emissions from a mercury-processing metallurgy plant on the intensity of experimental Fasciola hepatica infection in sheep]. / Vplyv imisií z kovohút na výrobu ortuti na intenzitu experimentálnej infekcie Fasciola hepatica u oviec.

A group of 14 improved Valashka lambs, aged 4 months, body weight 14-16 kg, were used in the experiment studying the effect of subchronic heavy metal intoxication on the course of experimental fasciolosis (Fasciola hepatica). The animals were divided into two groups, each of 7 lambs, 3 lambs--ewes and 4 lambs--rams. Each animal in group I was given orally for 27 days gelatinous capsules containing heavy metal emissions of the following composition: 4.5 mg Hg, 2.9 mg Pb, 147.8 mg Cu, 9.8 mg Zn, 0.9 mg Cr and 0.04 mg Cd per animal and day. Group II served as control. On Day 27, when the administration of heavy metals ceased, each animal in both groups received 250 F. hepatica metacercariae. The animals were sacrificed on Day 110 post infection, their liver necropsied and parenchymatous organs and muscles taken to examine the content of heavy metals. The heavy metal concentrations are presented in Tab. I. In the emission-intoxicated group, the maximum permissible reference level of mercury in the kidneys, liver and muscles was exceeded on the average by 1.94, 0.87 and 0.020 mg Hg/kg, respectively. Cu exceeded the reference level only in the liver, with 261.3 mg Cu/kg. In the kidneys and muscles, Cu was below the reference levels. An analogous situation was with Zn, Cr, and Cd. The F. hepatica infection mean intensity in this animal group was 38.3 +/- 3.01 specimens (min. 34 - max. 46 specimens). In control group, the heavy metal levels were several times lower than the reference values and the infection mean intensity was 27 +/- 5.4 (min. 19 - max. 35 specimens). In subchronically intoxicated animals, the reference values for mercury were exceeded and so were partially those of copper in the parenchymatous organs and muscles. The F. hepatica infection mean intensity also increased, compared with control. The results have proved the negative effect of the metal emissions on the animal immune system, which was manifested by the increased infection intensity.

[Heavy metal poisoning in relation to glucose-6-phosphate dehydrogenase activity in sheep erythrocytes]. / Intoxikácia tazkými kovmi vo vztahu k aktivite glukóza-6-fosfát dehydrogenázy v erytrocytoch oviec.

Peroxidative lesions of cells and production of free radicals from endo- and exogenous reasons, eg. due to air pollution, can result in severe lesions of cells and subsequent pathological processes (Rieger, 1992; Robbins and Cotran, 1988). A pentose cycle plays an important role in the system of antioxidative protection: glucose-6-phosphate dehydrogenase (G-6-PD; EC 1.1.1.49) is its first enzyme. G-6-PD activity was evaluated in the erythrocytes of sheep kept in the region contaminated by heavy metals with mercury dominating among them, and in the same animals after administration of a feed mixture containing Hg, Pb Cd, Zn Cr, Cu, Fe and As (Fig. 1). Boehringer Mannheim test was used to determine the G-6-PD activity. There was no significant differences in the enzyme activity in the sheep from a contaminated region and in the animals outside the air-pollution region (control animals) before the applications of heavy metals started. The average value of G-6-PD activity was 13.96 +/- 0.94 mU.10 (-9) Ec in control animals and 14.39 +/- 1.49 mU.10 (-9) Ec in the animals from a contaminated region. After eight-day applications of heavy metals the G-6-PD activity increased statistically significantly to 18.71 +/- 2.45 mU.10 (-9) Ec; P < 0.01, and to 23.55 +/- 1.87 mU 10 (-9) Ec after 16 days of application; P < 0.001 (Fig. 2). An increase in G-6-PD activity after heavy metal applications is probably a compensation mechanism in the system of erythrocyte antioxidative protection due to higher peroxidation. The long term increased intake of heavy metals from polluted air did not lead to any rise of G-6-PD activity probably due to the lower dose of heavy metals and/or to adaptation of animal organisms to long run emission exposure. The results demonstrate that G-6-PD can be one of the biochemical indicators at organism load by heavy metals with mercury dominating among them.

[The effect of supermethrin, an insecticide, on health status indicators in sheep during subchronic poisoning]. / Vplyv insekticídu supermetrín na niektoré ukazovatele zdravotného stavu oviec v podmienkach subchronickej intoxikácie.

The effect of supermethrin on the overall health with respect to weight gains, diet intake, triad values (body temperature, pulse rate and breathing rate) and potential intoxication signs was investigated in sheep of the Slovak Mertino breed (age of 8 months, males and females) during 6-week feeding of the insecticide supermethrin (Research Institute of Chemical Technology, Bratislava). This insecticide supermethrin contains a cyanide group in its molecule and can be included in the group of type II pyrethroids. It is an analog of cypermethrin and it has a different proportion of cis- and trans-isomers. Supermethrin mixed with molasses feed M was administered daily at a dose of 50 mg/kg (about 1/70 of LD50) to five sheep of experimental group I, at a dose of 200 mg/kg (about 1/15 of LD50) to five sheep of experimental group II, and the dose increased from 200 mg to 300 mg/kg l.w. (about 1/20 of LD50) since the fourth week of trial. The main signs of its toxic action involved depressive effects on weight gains (Fig. 4) Over the whole period of trial, the live weight rose by 5.44 +/- 1.94 kg in control group, by 2.66 +/- 1.48 kg in experimental group I, which equates a significant decrease by 51.10% and only 0.34 +/- 0.95 kg in experimental group II, which equates a decrease in weight gains by up to 93.75% against the control. We do not believe that the growth depression can be related to diet intake. There were no larger differences in feed intake between the experimental groups and the control. The growth depression was caused by incessant diarrhea. The patho-morphological examination did not reveal hyperemia and/or intestinal inflammation, the histological examination did not show any lesions of epithelium in the intestinal mucosa. An increase in supermethrin dose from 200 to 300 mg/kg l.w. resulted in signs coming from the CNS. Hypersensibility manifested by moderate unrest, head and neck shaking after auditory, and especially after touch stimuli was observed. This tremor was increasing to became spontaneous 3-4 days before trial termination. The above findings clearly suggest that supermethrin administration at lower doses has harmful effects primarily on the digestive tract, but at higher doses these effects are more intensive accompanied by the effects on the CNS. No negative effects on pulse rate (Fig. 1), breathing rate (Fig. 2) and internal body temperature (Fig. 3) were recorded.

Chronic influence of pyrethroid supermethrin on some reproductive features in Japanese quail (140 days long avian reproduction test).

We observed the influence of chronic effect of pyrethroid supermethrin on some reproduction features in Japanese quails. They received 3 different doses (P1 10.7 mg.kg-1.day-1; P2 21.4 mg.kg-1.day-1 and P3 35.7 mg.kg-1.day-1) of the tested substance during 20 weeks. The total health status, reproduction of Japanese quails, the damage rate (degree) of the organs and tissues as well as the determination of residue in the eggs and muscles of the experimental birds showed that pyrethroid insecticide supermethrin can be considered a safe pesticide.

[Biochemical aspects of the toxic effects of Supermethrin in a 140-day avian reproductive test]. / Biochemické aspekty toxického úcinku supermetrínu v podmienkach chronického 140-dnového aviárneho reprodukcného testu.

Chronic toxic effects of supermethrin on some biochemical parameters (AST, ALT, LDH, creatinine and total proteins) were investigated in 84 individuals of Japanese quail divided into four groups (control-K, experimental group I-P1, experimental group II-P2, experimental group III-P3) in the conditions of 140-day avian reproductive test. The three experimental groups received the tested substance at these doses: P1-10.7 mg/kg l.w./day, P2-21.4 mg/kg l.w./day, P3-35.7 mg/kg l.w./day. The results of observation of the enzyme activities AST and ALT show that only the AST activity (in the course of 140-day avian reproductive test) significantly increased to 1.225 mu kat/l in the females of experimental group P1, to 1.053 mu kat/l in P2 and to 1.014 mu kat/l in P3 against the control, in which the AST activity was 0.670 mu kat/l. The values of AST activity in the males were 1.143 mu kat/l in P1, 1.117 mu kat/l in P2 and 1.090 mu kat/l in P3 against the control 0.8395 mu kat/l. The investigation of variations in total LDH activity in Japanese quail after 140-day avian reproductive test has shown an increase in the LDH activity in the males (11.193 mu kat/l in P1, 11.269 mu kat/l in P2, 8.245 mu kat/l in P3 and 7.362 mu kat/l in K) as well as in the females (10.91 mu kat/l in P1, 12.023 mu kat/l in P2, 10.196 mu kat/l in P3 and 7.055 mu kat/l in K).(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance of ruminal microorganisms to supermethrin.

The effect of the pyrethroid insecticide supermethrin on the growth and cyanogenic activity of 8 ruminal bacterial species (S bovis A0 24/85, S xylosus 310, E faecium 2, L plantarum, M elsdenii 4MJ, S ruminantium A17, B succinogenes 16J and B ruminicola 3/3) in pure culture was examined. Bacteria grown in 0.66 and 6.6 mg supermethrin/ml had similar growth rates and resistance to supermethrin. Production of cyanide from supermethrin occurred by all examined strains of ruminal bacteria, but the enzyme activity varied considerably with species and with the supermethrin concentrations.