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)

Influence of different media, incubation times, and temperatures for determining the MICs of seven antifungal agents against Paracoccidioides brasiliensis by microdilution.

MIC assays with Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, had been conducted with variable protocols, employing both macrodilution and microdilution tests and including differences in inoculum preparation, media used, incubation periods, and temperatures. Twenty-one clinical and environmental isolates of Paracoccidioides were tested using amphotericin B, itraconazole, ketoconazole, fluconazole, sulfamethoxazole, sulfamethoxazole-trimethoprim, and terbinafine, according to the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, document M27-A2, 2002), with modifications such as three medium formulations (RPMI 1640 medium, McVeigh and Morton [MVM] medium, and modified Mueller-Hinton [MMH] medium), two incubation temperatures (room temperature [25 to 28 °C] and 37 °C), and three incubation periods (7, 10, and 15 days). The antifungal activities were also classified as fungicidal or fungistatic. The best results were obtained after 15 days of incubation, which was chosen as the standard incubation time. The MICs for most individual isolates grown for the same length of time at the same temperature varied with the different media used (P < 0.05). Of the isolates, 81% showed transition from the yeast to the mycelial form in RPMI 1640 medium at 37 °C, independent of the presence of antifungals. MMH medium appears to be a suitable medium for susceptibility testing of antifungal drugs with P. brasiliensis, except for sulfamethoxazole and the combination of sulfamethoxazole-trimethoprim, for which the MVM medium yielded better results. The incubation temperature influenced the MICs, with, in general, higher MICs at 25 °C (mycelial form) than at 37 °C (P < 0.05). Based on our results, we tentatively propose a microdilution assay protocol for susceptibility testing of antifungal drugs against Paracoccidioides.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii.

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, <0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 µg/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 µg/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 µg/mL). The quantitation of cytokines revealed a predominance of IFN-γ and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.

LyeTx I, a potent antimicrobial peptide from the venom of the spider Lycosa erythrognatha.

LyeTx I, an antimicrobial peptide isolated from the venom of Lycosa erythrognatha, known as wolf spider, has been synthesised and its structural profile studied by using the CD and NMR techniques. LyeTx I has shown to be active against bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Candida krusei and Cryptococcus neoformans) and able to alter the permeabilisation of L: -alpha-phosphatidylcholine-liposomes (POPC) in a dose-dependent manner. In POPC containing cholesterol or ergosterol, permeabilisation has either decreased about five times or remained unchanged, respectively. These results, along with the observed low haemolytic activity, indicated that antimicrobial membranes, rather than vertebrate membranes seem to be the preferential targets. However, the complexity of biological membranes compared to liposomes must be taken in account. Besides, other membrane components, such as proteins and even specific lipids, cannot be discarded to be important to the preferential action of the LyeTx I to the tested microorganisms. The secondary structure of LyeTx I shows a small random-coil region at the N-terminus followed by an alpha-helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction. The high activity against bacteria together with the moderate activity against fungi and the low haemolytic activity have indicated LyeTx I as a good prototype for developing new antibiotic peptides.

PTX3 function as an opsonin for the dectin-1-dependent internalization of zymosan by macrophages.

Pentraxin 3 (PTX3) is a tumor necrosis factor and interleukin-1beta-stimulated gene that encodes a long PTX with proinflammatory activity. Here, we show that peritoneal macrophages derived from PTX3 transgenic (Tg) mice express higher levels of PTX3 mRNA than macrophages from wild-type (WT) mice, at basal level as well as upon stimulation with zymosan (Zy). Macrophages from Tg mice also showed improved opsonin-independent phagocytosis of Zy particles and the yeast form of the fungus Paracoccidioides brasiliensis. In the case of P. brasiliensis, an enhanced microbicidal activity accompanied by higher production of nitric oxide was also observed in macrophages from Tg mice. Using fluorescein-activated cell sorter analysis and reverse transcriptase-polymerase chain reaction, we demonstrated that basal level of Toll-like receptor-6 and Zy-induced dectin-1 expression was slightly but consistently higher in macrophages from Tg mice than in macrophages from WT mice. Recombinant (r)PTX3 protein binds to Zy particles as well as to yeast cells of P. brasiliensis and addition of rPTX3, to a culture of WT-derived macrophages containing Zy leads to an increase in the phagocytic index, which parallels that of Tg-derived macrophages, demonstrating the opsonin-like activity of PTX3. It is important that blockade of dectin-1 receptor inhibited the phagocytosis of Zy particles by WT and PTX3 Tg macrophages, pointing out the relevant role of dectin-1 as the main receptor involved in Zy uptake. Our results provide evidence for a role of PTX3 as an important component of the innate-immune response and as part of the host mechanisms that control fungal recognition and phagocytosis.

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).

In vitro granuloma formation, NO production and cytokines profile from human mononuclear cells induced by fractionated antigens of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the main mode of protection against this fungal infection. We investigated in vitro the response of peripheral blood mononuclear cells (PBMC) from paracoccidioidomycosis (PCM) patients presenting different clinical forms to antigenic fractions from P. brasiliensis yeast cell lysate (PbAg). These fractions designated F0 to FV were obtained using anion-exchange chromatography on a FPLC system. Our studies showed variation in the cellular responses induced by different antigenic fractions. The fraction F0 caused significant decrease in cellular proliferation, granuloma formation, accompanied by significant elevation in the production of IL-10. The fractions FII and FIII increased in vitro granuloma formation associated with high production of TNF-alpha. Besides that, FII and FIII evoked decrease in NO production but not F0 that induced very high levels, among patients with PCM from acute form. The findings suggest that P. brasiliensis antigenic components participate in the modulation or activation of PBMC response in PCM, and IL-10 and NO could be important in the regulation of in vitro granuloma formation.

Polymorphism in the gene coding for the immunodominant antigen gp43 from the pathogenic fungus Paracoccidioides brasiliensis.

The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM). It is protective against murine PCM and is a putative virulence factor. The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp. Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points. In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P. brasiliensis isolates after sequencing two PCR products from each fungal sample. The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp. Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein. The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms. The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots. The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine.

PCR for diagnosis of paracoccidioidomycosis.

A PCR assay based on oligonucleotide primers derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis DNA in sputa. In the standardized conditions, it could detect 10 cells/ml of sputum, providing sufficient accuracy to be useful for diagnosis of paracoccidioidomycosis.

In vitro human immune reactivity of fast protein liquid chromatography fractionated Paracoccidioides brasiliensis soluble antigens.

Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were fractionated in a fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that could elicit cell reactivity and antibody recognition in human paracoccidioidomycosis (PCM). PbAg fractions were eluted by 20 mM Tris-HCl solution (pH 9.6) with an increasing gradient up to 1 M NaCl. The FPLC system was able to resolve 7 fractions, enumerated from 0 to VI, according to the elution on the NaCl gradient. The analysis of each fraction on SDS-PAGE showed that fractions 0 to V were constituted by multiple protein bands with molecular mass ranging from 18 to 114 kDa. Large amounts of nucleic acids were evidenced in fraction VI, as revealed by agarose gel stained with ethidium bromide. Sera from PCM patients presenting different clinical forms contained antibodies that recognized antigens in all fractions with the exception of fraction VI as detected by ELISA. Further studies were designed to investigate the capacity of these fractions to induce cell proliferation. It was demonstrated that fractions III and V (200 and 450 mM NaCl, respectively) stimulated a significant proliferative response of peripheral blood mononuclear cells, while fraction 0 induced the lowest proliferative response among patients with PCM, in either acute, acute treated, or chronic forms.

Electrophoretic karyotypes and genome sizing of the pathogenic fungus Paracoccidioides brasiliensis.

Here we present the karyotype analysis and genome sizing of Paracoccidioides brasiliensis, a pathogen refractory to conventional genetic analysis. We have established pulsed-field gel electrophoresis (PFGE) conditions to resolve the high-molecular-weight chromosomal bands of two clinical isolates of P. brasiliensis. Both isolates showed four megabase-sized bands, ranging from 2.0 to 10.0 Mbp. Significant differences in chromosome sizes and in the chromosomal location of genes for the gp43 antigen and chitin synthase were found. Different technical approaches were employed to estimate the DNA content and to define the ploidy of P. brasiliensis. An estimated genome size in the range of 45.7 to 60.9 Mbp was provided by the analysis of data generated by measuring the amplitude of fluorescence intensity of DAPI (4',6-diamidino-2-phenylindole)-stained nuclei (by confocal microscopy). The nuclear genome size estimated by confocal microscopy is twice that estimated by the average sum of the molecular weight of chromosome-sized DNA molecules by PFGE, suggesting that each separated P. brasiliensis chromosomal band is diploid.

Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis.

The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular responses in patients with paracoccidioidomycosis. In order to identify the peptide epitopes involved in the immunological reactivities of the gp43 and to obtain highly specific recombinant molecules for diagnosis of the infection, genomic and cDNA clones representing the entire coding region of the antigen were sequenced. The gp43 open reading frame was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides. The gene is present in very few copies per genome, as indicated by Southern blotting and chromosomal megarestriction analysis. A single transcript of 1.5 kilobase pairs was verified in the yeast phase. The gene encodes a polypeptide of 416 amino acids (Mr 45,947) with a leader peptide of 35 residues; the mature protein has a single N-glycosylation site. The deduced amino acid sequence showed similarities of 56-58% with exo-1,3- beta-D-glucanases from Saccharomyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not cross-react immunologically with the fungal glucanases. Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted with antibodies elicited against the native antigen.

[The microhemagglutination reaction for the routine serologic diagnosis of syphilis]. / A reação de microhemaglutinação para sífilis no diagnóstico sorológico de rotina.

The authors present the results of VDRL reactions (qualitative and quantitative) and microhaemagglutination assays (MIPHA) on 305 serum samples. The samples selected among the sera asked for syphilis serology at a clinical laboratory, were those which the results on VDRL reactions were observed to be reactive at low titles, not reactive or inconclusive. From these 305 samples, 87 (28.5 por 100) were also processed by the FTA-Abs test. The reactivity concordance between the tests was compared and the efficiency of the MIPHA as confirmatory diagnostic test was discussed. The conclusions prompt to the usefulness of the MIPHA on syphilis serodiagnosis routine as a function of its sensitivity, specificity, low costs and technical simplicity.