Effect of 4-amino-6-methyl-3-phenylamino-1,2,4-triazin-5(4H)-one on the integrity of chloroplasts from barley leaves

4-Amino-6-methyl-3-phenylamino-1,2,4-triazin-5(4H)-one alters the integrity of chloroplasts in barley (Hordeum vulgare L. var. Hassan) leaves as judged by their sedimentation profiles in linear gradients of sucrose. This effect may be related to the inhibition caused by 4-amino-6-methyl-3-phenylamino-1,2,4-triazine-5(4H)-one of both ascorbate peroxidase and superoxide dismutase activities, and could thus be explained by an accumulation of toxic reduced-oxygen species in illuminated chloroplasts.     

Morphological and quantitative study of the Leydig cells of pigs fed with anabolic doses of clenbuterol

The effects of clenbuterol administered at anabolic doses on the testicular interstitium were studied in 30 pigs allocated to three experimental groups. The diet of two groups was supplemented with clenbuterol (Clb) (1 ppm), but whereas in the Clb+ group the treatment was given until slaughter (treatment period: 3 months), in the Clb- group the clenbuterol was withdrawn 2 weeks before slaughter (treatment period: 2-5 months); in the control group, the pigs were fed without clenbuterol. For histological procedures, a fractional sampling scheme was applied and routine techniques for light and transmission electron microscopy were used. The results of subjective morphology and morphometrics showed slight differences between the treated and the control groups. Conversely, the stereological results identified a prominent hyperplasia of the Leydig cells and ultrastructural analysis of these cells revealed a conspicuous increase in the organelles related to testosterone production, suggesting a functional activation of the interstitial cells in response to the clenbuterol treatment.     

Influence of pesticides on the activity and kinetics of invertase,urease and acid phosphatase enzymes

The effects of four pesticides (three herbicides, atrazine, paraquat, glyphosate, and an insecticide, carbaryl) on the activity and kinetics of an invertase (from yeast), a urease (from jack bean) and an acid phosphatase (from potato) were investigated. Glyphosate and paraquat showed a marked activation effect on invertase activity. From the dependence of V_max and K_m parameters on pesticide concentrations, a mixed-type activation mechanism was suggested. The kinetic behaviour of urease and acid phosphatase appeared to be uninfluenced by both pesticides. Methanol, used as solvent for both carbaryl and atrazine, inhibited invertase, urease and acid phosphatase with, respectively, competitive, non-competitive and uncompetitive mechanisms. The extent of the inhibition was different, as estimated by the values of the inhibition constant K₁. An additional inhibition effect of urease and invertase activities was achieved in the presence of carbaryl, whereas no influence was detected on the activity and kinetics of acid phosphatase. Finally, atrazine was shown to alter the kinetics of the enzymes only at higher concentrations.     

The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy

Peripheral blood mononuclear cell subsets, in vitro lymphoproliferative response to leishmanial antigen, and Leishmania-specific serum antibody levels were examined in 11 dogs, naturally infected with L. infantum, and 9 healthy control dogs. A decrease in the percentage of CD4+ T-cells and an increase in the proportion of gammadelta T-cells and sIgG+ B-cells were observed during canine visceral leishmaniasis (CVL). These changes may be responsible for the marked humoral response and the absence of in vitro lymphoproliferation to mitogen and specific parasite antigens. This possibility was supported by the analysis of these subsets after treatment with amphotericin B. One month after therapy, a significant increase in the percentage of CD4+ T-cells and a decrease of gammadelta T-cells and sIgG+ B-cells were observed. At the same time, the lymphocyte blastogenesis assay with leishmanial antigen was positive and the levels of specific antibodies to Leishmania were significantly lower than before the treatment. Five months after therapy, lymphocyte proliferative response to LSA disappeared, antibody and lymphocyte subsets levels returned to those observed during CVL. Therapeutic failure in CVL is associated with the inability of antileishmanial drugs to completely revert the profound immunodepression induced by the infection and prevent relapse.     

Humoral response (IgG) of goats experimentally infected with Fasciola hepatica against cysteine proteinases of adult fluke

The use of cysteine proteinases from Fasciola hepatica adult flukes for the serodiagnosis of caprine fasciolosis by means of an indirect ELISA test was studied. Two proteolytic fractions from adult fluke homogenates, with apparent molecular weights of 28 and 34 kDa (P28 and P34 respectively), were characterised as cysteine proteinases using azocasein assays and gelatin gel analysis. Both P28 and P34 fractions were electroluted and used as antigens in two different indirect ELISA tests. Serum IgG levels against P28 and P34 in goats given an experimental primary infection with 200 metacercariae or in goats given two experimental infections with 200 metacercariae were determined and compared with those observed in an uninfected control group. ELISA tests using both cysteine proteases showed a rapid and consistent detection of specific IgG in all experimentally infected goats. The IgG response to P28 was the first to be detected as early as 2-3 weeks post-infection and remained elevated throughout the experiment. The response to P34 was detected later (4-6 wpi) and disappeared in some animals at 18 wpi, while flukes were still present in the bile ducts. No significant differences were observed between the anti-P28 and anti-P34 IgG responses between animals receiving a primary or a challenge infection. The results of our study, although preliminary, are promising since the P28 ELISA described here may be a reliable method for the immunodiagnosis of F. hepatica infection in goats.     

Quantitative changes in the normal and apoptotic thymocytes of pigs treated with anabolic doses of the beta2 adrenergic agonist clenbuterol

Although the beta2 adrenergic agonists have been seen to have important effects on the mechanisms regulating the development and death of T-cells in the thymus, the side-effects on the immune system of anabolic treatments of these substances have hardly been considered. In order to evaluate the effects exerted by the beta2 adrenergic agonist clenbuterol on the thymocyte population, the thymus of eight pigs treated with anabolic doses of this substance was studied by morphometric methods, regarding apoptotic (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive) and normal (TUNEL-negative) cells. The thymus of another eight pigs fed without clenbuterol served as a control. The clenbuterol treatment had a clear effect on the thymocyte size, decreasing their mean nuclear area. The T-cell apoptosis index was also affected by the clenbuterol, significantly increasing the apoptosis percentage in the treated group with respect to the control. In the light of our results, the clenbuterol induced thymocyte apoptosis throughout the thymus and caused morphometric changes in the thymocyte population, which was in line with the immunosuppressive role attributed to other beta2 adrenergic agonists.     

Evaluation of a specific immunochemotherapy for the treatment of canine visceral leishmaniasis

The efficacy of specific immunochemotherapy against Leishmania infantum infection in dog was studied. The effects on transmission of the disease, as well as the cellular and humoral immune response were examined. The treated animals showed a significant reduction in the infection rates that were detected in Phlebotomus perniciosus females fed on the dog. The humoral immune response, assayed with an indirect immunofluorescence antibody test (IFAT), did not show significant variations under the influence of the therapy. The characterisation of the peripheral blood mononuclear cells (PBMC) using flow cytometry indicated a significant increase in the proportion of T lymphocytes, especially of CD4/TcR(alpha)(beta)(+) and CD4/CD45RA(+) cells, without showing evidence for modifications in the other leukocyte subsets. Cellular lymphoproliferation studies indicated a lack of a specific response to soluble leishmanial antigen (SLA), but the non-specific lymphoproliferative capacity assayed with phytohemagglutinin (PHA) was maintained.     

Isolation and characterization of immortalized porcine aortic endothelial cell lines

Primary porcine endothelial cells have a limited life span in culture. After four to five passages, they tend to de-differentiate and eventually reach senescence. The aim of this work was to establish immortalized porcine aortic endothelial cell lines (AOCs) to facilitate in vitro studies of different pathological process involving the endothelium. Primary porcine aortic endothelial cells (PAECs) were transfected with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. Flow cytometry analysis demonstrated uptake of acetylated low density lipoproteins (Ac-LDL) and constitutive expression of SLA class I, CD29, CD31, CD41/61, CD80/86, CD46, SWC3, and LAMP-1 antigens by all analyzed lines and showed little differences to primary cells. The functional similarity between primary and immortalized endothelial cells was demonstrated in a cytotoxicity assay using a human natural killer cell line (NKL) as effector. The AOCs cell lines should be valuable tools for in vitro study of the human immune response against pig endothelial cells. In addition, they would be very useful to gain insight in the pathogenesis of some viral haemorrhagic diseases of pig such as African swine fever (ASF) or classical swine fever (CSF).     

Evaluation of 65% permethrin spot-on for prevention of canine visceral leishmaniasis: effect on disease prevalence and the vectors (Diptera: Psychodidae) in a hyperendemic area

A study was designed to examine the effect of 65% permethrin spot-on on the prevalence of canine visceral leishmaniasis and the abundance of sand flies in two neighborhoods in Corumbá, Mato Grosso do Sul, Brazil known to have a high prevalence of visceral leishmaniasis. An enrollment survey was conducted to determine the prevalence of visceral leishmaniasis. Area residents were provided with information about the project; the study area was defined, and all dogs (160 in Cristo Redentor and 230 in Popular Velha) identified in the study area were enrolled. Three 65% permethrin spot-on treatments (June 15-25, July 13-15, and August 10-12) were administered to 150, 110, and 99 dogs, respectively, in Popular Velha, according to label recommendations. Dogs in Cristo Redentor were untreated controls. Visceral leishmaniasis was diagnosed periodically by indirect immunofluorescence assay. A reduction in canine visceral leishmaniasis prevalence was observed at the Popular Velha site. The infection rate for treated dogs 1 month following the final treatment was approximately 50% reduced from that observed before treatment(19.3% vs 9.6%). Conversely, the infection rate at the control site was more than 80% higher at the September sampling than that observed pretreatment (4.1% vs 7.4%). Similar numbers of sand flies were captured and identified from both sites throughout the study. The results suggest that regular use of 65% permethrin during months of high risk for canine visceral leishmaniasis can be a useful strategy for reducing the prevalence of this disease in hyperendemic areas. It should be stressed, however, that the success of this strategy depends not only on the efficacy of the product itself but also on the adoption of other control measures and on economic variables, considering the low purchasing power of the populations living in higher-risk neighborhoods.