Culture and molecular identification of microorganisms from Digital Dermatitis lesions in dairy cattle: Leptospira, an unexpected finding / Cultivo e identificação molecular de microrganismos isolados de lesões de dermatite digital em bovinos leiteiros: Leptospira, um achado inesperado

Bovine digital dermatitis (BDD) is an infectious and contagious disease characterized by ulcerative and proliferative lesions affecting the skin on the bulbs of the heel or the interdigital cleft in dairy cattle, often associated with lameness. Evidences on the etiology of BDD indicate that it is multifactorial, involving environmental factors and multiple bacterial colonization. We isolated and identified microorganisms from BDD biopsy samples obtained from five Holstein Friesian and two Jersey cows by cultivation and molecular identification of bacterial isolates using 16S rRNA gene sequence analysis. We identified six bacterial species: Spirochetes as Treponema pedis and Leptospira broomi/L. fainei, L. licerasiae/L. wolffii; Corynebacterium appendicis, Cupriavidus gilardii and Enterococcus casseliflavus/E. gallinarum. It was quite surprising to have isolated and identified Leptospira species in three out of seven cultures, from different individual cows and two different farms. The species identified belong to the intermediate pathogenic clade, which is a group found to cause human and animal disease. Our findings indicate the need to further investigate the association of Leptospira of intermediate pathogenicity with BDD lesions and whether its presence would have any veterinary and medical significance both in Leptospirosis and with the pathogenesis of BDD lesions, especially in tropical countries.(AU)

Dermatite digital bovina (DDB) é uma doença infecciosa, contagiosa, caracterizada por lesões ulcerativas e proliferativas da região dos talões e/ou do espaço interdigital, frequentemente associada com claudicação. Evidências indicam que a etiologia da DDB é multifatorial, envolvendo fatores ambientais e colonização polimicrobiana. Relata-se aqui o isolamento e a identificação bacteriana em amostras de biópsias em lesões de DDB, obtidas de cinco vacas da raça Holandesa e duas da raça Jersey, por meio de cultivo e identificação molecular de isolados, com base na análise de sequências de genes 16S rRNA. São identificadas seis espécies bacterianas: as espiroquetas Treponema pedis e Leptospira broomi/L. fainei, L. licerasiae/L. wolffii; Corynebacterium appendicis, Cupriavidus gilardii e Enterococcus casseliflavus/E. gallinarum. O isolamento e a identificação de espécies de Leptospira surpreenderam, destacando-se sua presença em três dos sete cultivos obtidos em diferentes vacas, de duas fazendas distintas. As espécies identificadas pertencem ao grupo tipificado como de patogenicidade intermediária, causador de doenças em animais e no homem. Os resultados apresentados indicam a necessidade de maiores investigações sobre a associação entre Leptospira de patogenicidade intermediária e a patogênese das lesões DDB, investigando-se sua presença e significado nas medicinas veterinária e humana, especialmente em países tropicais.(AU)

Brazilian propolis: physicochemical properties, plant origin and antibacterial activity on periodontopathogens.

Propolis samples collected in the dry and rainy seasons, from an experimental apiary located in a cerrado vegetation region in Brazil were used in this study. Microscopic analysis showed the presence of 31 pollen types, secretory hairs (genus Baccharis) and fragments of plant epidermis. The oxidation rates and the wax content of the samples after physicochemical analyses were in agreement with the Cuban Guideline NRAG 870-88. A high performance liquid chromatography analysis showed a similar pattern of chromatograms, characterized by the presence of ten phenolic compounds. There was no significant difference in the pro fi le of phenolic compounds and also in the total flavonoid concentration in propolis samples collected in different seasons. Antibacterial assays were performed by the method of dilution of an ethanol extract of propolis (EEP) in agar (v/v%) and showed that all 16 A. actinomycetemcomitans strains tested were inhibited by propolis concentrations of 0.1% to 0.25%, and did not grow at all at 0.5%. The growth inhibition of six Fusobacterium spp. and 16 black-pigmented anaerobes was observed at concentrations of 0.05% to 0.1%, and no growth was observed at 0.25%. There was no effect of seasonality on the inhibitory activity of propolis. The antibiotics tetracycline and meropenem were used as positive controls.

Actinobacillus actinomycetemcomitans genetic heterogeneity: amplification of JP2-like ltx promoter pattern correlated with specific arbitrarily primed polymerase chain reaction (AP-PCR) genotypes from human but not marmoset Brazilian isolates.

Specific clonal types of Actinobacillus actinomycetemcomitans, a major human periodontal pathogen, may be responsible for clinical manifestations and the production of leukotoxin virulence factors. Leukotoxicity is associated with genetic polymorphism at the promoter region of the leukotoxin (lItx) gene. Here, we describe the use of arbitrarily primed polymerase chain reaction (AP-PCR) and ltx promoter PCR to molecularly characterise 35 A. actinomycetemcomitans Brazilian isolates: 21 of human origin and 14 from captive marmosets (Callitrix spp., primates commonly used as animal models for periodontal research). The discriminative capacity of each of 12 arbitrary primers was found to be variable, yielding between 3 and 24 PCR amplitypes. Combination of the results for all primers led to characterisation of 14 genotypes that grouped into four major clusters based on genetic similarity. Clusters 2, 3, and 4 were discriminative to host origin. A correlation with periodontal disease was suggested for strains belonging to clusters 3 and 4. The JP2-like PCR amplification pattern, associated with highly leukotoxic strains, was exclusive to human isolates and present in 29% of human isolates where it occurred in close relationship with AP genotypes L and J (cluster 3).

Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria.

Propolis collected from a cerrado area in Minas Gerais State, Brazil, was subjected to chromatography on silica gel column and to partition between immiscible solvents. Propolis aqueous-ethanolic extract and fractions obtained were tested for inhibitory activity against periodontitis-causing bacteria. All of the assayed bacterium species were susceptible to propolis extract. The two fractionation methodologies yielded fractions which were active against bacteria, with minimum inhibitory concentrations (MIC) ranging from 64 to 1024 microg/ml. TLC and HPLC analyses of the extract and of active fractions showed the presence of phenolic compounds of varied polarity. None of the assayed fractions was more active than the extract, suggesting that the antibacterial activity is probably due to the synergistic effect of several compounds.

In vitro activity of meglumine antimoniate, a pentavalent antimonial drug, on Leishmania promastigotes

The activity of a pentavalent antimonial drug glucantime on the growth of promastigote forms of Leishmania strains involved in South American cutaneous and mucocutaneous leishmaniasis was investigated. A marked difference in susceptibility to glucantime among four different strains and cloned lines obtained from a single strain was observed. For the sensitive strains (L.braziliensis M2903 and L. guyanensis M1176), the cell growth was reduced in a dose-dependent manner for drug concentrations at a range of 0.23 to 23 mM. However, despite the relative sensitivity of the assay, no significant increase of effect was observed in the presence of higher drug concentrations. For the resistant strains (L. amazonensis M10996 and L. braziliensis LYB259) a dose-response line is obtained at a higher concentration range (20 mM to mM). The influence of the drug on surface properties, respiratory activity and incorporation of radiolabelled leucine by a sensitive strain - L-guyanensis M1176-was studied as an approach to its site of action. Despite the increased intensity of self-aglutination for cells growing in the presence of glucantime, no significant change was observed in electrophoretic mobility or Concanavian A reactivity. Since the oxygen uptake of glucose-stimulated promastigotes was only slightly reduced in the presence of 23 mM glucantime at 28-C, the reduction was not sufficient to explain the total growth inhibition observed. A significant decrease of 14C-leucine incorporation into the cold TCA-insoluble fraction of drug-treated cells was observed within the same concentration range that reduces promastigote growth