Antonio Francisco de Souza Filho

Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Immune response and protective profile elicited by a multi-epitope chimeric protein derived from Leptospira interrogans

INTRODUCTION Pathogenic Leptospira is the causative agent of leptospirosis, a widely disseminated disease of human and veterinary concern. The development of vaccines that elicit cross-protective immunity through multiple leptospiral serovars has long been pursued. The aim of this study was to develop a novel chimeric multi-epitope fusion antigen, containing sequences of previously studied outer membrane proteins (OMPs) of Leptospira. METHODS The chimeric protein was designed based on the amino acid sequences of the LigA, Mce, Lsa45, OmpL1, and LipL41 proteins, cloned into pAE vector, the protein expressed in Escherichia coli, and its immune response evaluated in the hamster infection model. RESULTS The recombinant chimeric protein (rChi) was recognized by antibodies present in serum samples of confirmed cases of human leptospirosis and experimentally infected hamsters, demonstrating that the rChi protein participates in the immune response activation during infection. However, despite high antibody titers achieved when the rChi protein was administered with either Alhydrogel or Bordetella pertussis monophosphoryl lipid A (MPLA), only 50% of the hamsters were protected against infection. CONCLUSIONS Although a complete characterization of the immune response elicited by rChi/adjuvant in hamsters is required, it is believed that the construction of chimeric genes is an important attempt towards the generation of an effective vaccine against leptospirosis.

Decrease in antithrombin III and prothrombin serum levels contribute to coagulation disorders during leptospirosis.

Pathogenic bacteria of the genus Leptospira are the causative agent of leptospirosis, an emergent infectious disease that affects humans and animals worldwide. Severe forms of the disease in humans include jaundice, multiple organ failure and intense haemorrhage. Up to now, mechanisms associated with the haemorrhage foci are poorly understood. We report in this work that, despite the low levels of antithrombin III in convalescent human serum samples, virulent, culture-attenuated and saprophyte strains of Leptospira are unable to bind and/or degrade this thrombin inhibitor, suggesting an indirect mechanism of pathogenesis. Lower levels of prothrombin were found in serum samples at the onset and convalescent phase of the disease when compared to normal human sera. The concomitant decreased levels of antithrombin III and prothrombin suggest a process of stimulated coagulation, which is corroborated by the increase of prothrombin fragment F1+2 in the serum samples. Data obtained with hamsters experimentally infected with virulent Leptospira interrogans serovars Kennewicki and Canicola strongly point out that haemorrhage is correlated with decreased levels of thrombin inhibitors and prothrombin. Activated coagulation might lead to an overconsumption of coagulation factors ultimately leading to bleeding and organ failure.

Molecular characterization and antimicrobial susceptibility pattern of Streptococcus agalactiae isolated from clinical mastitis in dairy cattle

The objectives of this study were to: (a) genotypically characterize Streptococcus agalactiae isolates recovered from clinical mastitis (CM) cases in dairy cows and, (b) determine the association of antimicrobial susceptibility (AMS) and genotypes of Strep. agalactiae clustered according to the genetic similarity. A total of 89 Strep. agalactiae isolates recovered from bovine CM were genotyped using random amplified polymorphic DNA (RAPD) analysis. In addition, the AMS of the isolates was determined using a commercial broth microdilution test composed of 10 antimicrobials (penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, penicillin/novobiocin, erythromycin, pirlimycin, tetracycline, and sulfadimethoxine). Descriptive analysis was used to report the frequency of RAPD-types and genotypic clusters within herd, housing system, season and CM severity scores. The minimal antimicrobial concentrations that inhibited 50% (MIC50) and 90% (MIC90) of the isolates were calculated and survival analysis was completed to verify the differences of AMS among genotypic clusters. Results of RAPD showed a great genotypic diversity of Strep. agalactiae (45 RAPD-types) and three clusters (Ia, Ib and II) were created based on the genetic similarity among genotypes. After clustering, a high genetic similarity was observed within and between herds. Overall, Strep. agalactiae showed high susceptibility to most antimicrobials, except to tetracycline and erythromycin. Differences in the AMS among clusters were observed for ampicillin, ceftiofur, erythromycin, pirlimycin, sulfadimethoxine and tetracycline. In conclusion, Strep. agalactiae is still highly susceptible to most antimicrobials, although differences in susceptibility to certain antimicrobials were observed among genotypic clusters.

Prospective study of canine leptospirosis in shelter and stray dog populations: Identification of chronic carriers and different Leptospira species infecting dogs

Dogs are highly susceptible to the leptospiral infection, notably stray and sheltered dogs. Unsanitary conditions often observed in dog shelters may predispose the introduction and spread of leptospires among sheltered populations, potentially increasing the chances for the inadvertent adoption of asymptomatically infected animals. The present work describes a longitudinal study using a multidisciplinary approach for the identification of chronically infected dogs and the characterization of potentially pathogenic strains circulating among stray and sheltered dog populations in São Paulo, Brazil. A total of 123 dogs from three populations were included. The initial evaluation consisted of blood and urine quantitative PCR testing (qPCR), the detection of specific antibodies by microscopic agglutination test (MAT), physical examination and hematological and serum biochemistry analyses. The qPCR-positive dogs were prospectively examined, and reevaluations also included culture from urine samples. Positive qPCR samples were subjected to 16S rRNA and secY gene phylogenetic analysis. The recovered strains were characterized by Multilocus Sequence Typing, polyclonal serogroup identification and virulence determination. Leptospiruria was detected in all populations studied (13/123), and phylogenetic analysis revealed that 10 dogs had L. interrogans infection. Three dogs (3/13) had L. santarosai infection. The secY phylogenetic analysis revealed that the L. santarosai sequences clustered separately from those obtained from other hosts. Ten leptospiruric dogs were reevaluated, and three dogs presented persistent leptospiruria, allowing culturing from two dogs. The strains were characterized as L. interrogans serogroup Canicola (virulent) and L. santarosai serogroup Sejroe (not virulent). Serum samples were retested by MAT using the DU92 and DU114 strains as antigens, and no increased seroreactivity was detected. Asymptomatic L. santarosai infection was observed in all populations studied, suggesting a possible role of dogs in the chain of transmission of this leptospiral species. The results suggest a genetic distinction between lineages of Brazilian L. santarosai maintained by dogs and other animal hosts. Our findings revealed that dogs could act as maintenance hosts for distinct pathogenic Leptospira, highlighting also that asymptomatically infected dogs can be inadvertently admitted and adopted in dog shelters, potentially increasing the risks of zoonotic transmission.

Evaluation of Lsa46 and Lsa77 Leptospiral Proteins for Their Immunoprotective Activities in Hamster Model of Leptospirosis

Leptospirosis is a neglected tropical disease caused by pathogenic Leptospira spp. The lack of an effective vaccine favors the increase of the disease. Currently, surface-exposed proteins are the main targets for the search of vaccine candidates. In this study, we examined whether the surface Lsa46 and Lsa77 proteins, previously identified as laminin and plasminogen binding proteins, have the capacity of inducing protection and sterilizing immunity against challenge with virulent Leptospira in hamster model. Animals were subcutaneously immunized with Lsa46, Lsa77, or a combination of both in Alum adjuvant and challenged intraperitoneally with L. interrogans serovar Kennewicki strain Pomona Fromm. Hamster immunization with Lsa46 or Lsa77 or both promoted a strong IgG response. Th2- and Th1-biased immune responses were observed when Lsa46 and Lsa77 were individually administered, respectively, as detected by the IgG1/IgG2/3 ratio. Immunized hamsters with the combined proteins induced a Th1-biased immune response. Although the immunization with Lsa46 and Lsa77 stimulated protective immunity with reduction of bacterial burden, when compared to animals individually immunized with the proteins, the data was not statistically significant. Thus, although promising, more studies are needed before the role of these proteins in stimulating sterilizing immunity in mammals is conclusively determined.

Binding of human plasminogen by the lipoprotein LipL46 of Leptospira interrogans

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira. Bacteria disseminate via the bloodstream and colonize the renal tubules of reservoir hosts. Leptospiral surface-exposed proteins are important targets, because due to their location they can elicit immune response and mediate adhesion and invasion processes. LipL46 has been previously reported to be located at the leptospiral outer membrane and recognized by antibodies present in serum of infected hamsters. In this study, we have confirmed the cellular location of this protein by immunofluorescence and FACS. We have cloned and expressed the recombinant protein LipL46 in its soluble form. LipL46 was recognized by confirmed leptospirosis human serum, suggesting its expression during infection. Binding screening of LipL46 with extracellular matrix (ECM) and plasma components showed that this protein interacts with plasminogen. The binding is dose-dependent on protein concentration, but saturation was not reached with the range of protein concentration used. Kringle domains of plasminogen and lysine residues of the recombinant protein are involved in the binding because the lysine analog, amino caproic acid (ACA) almost totally inhibited the reaction. The interaction of LipL46 with plasminogen generates plasmin in the presence of plasminogen activator uPA. Because plasmin generated at the leptospiral surface can degrade ECM molecules and decrease opsonophagocytosis, we tentatively infer that Lip46 has a role in helping the invasion process of pathogenic Leptospira.

Complete Genome Sequencing of Mycobacterium bovis SP38 and Comparative Genomics of Mycobacterium bovis and M. tuberculosis Strains

Mycobacterium bovis causes bovine tuberculosis and is the main organism responsible for zoonotic tuberculosis in humans. We performed the sequencing, assembly and annotation of a Brazilian strain of M. bovis named SP38, and performed comparative genomics of M. bovis genomes deposited in GenBank. M. bovis SP38 has a traditional tuberculous mycobacterium genome of 4,347,648 bp, with 65.5% GC, and 4,216 genes. The majority of CDSs (2,805, 69.3%) have predictive function, while 1,206 (30.07%) are hypothetical. For comparative analysis, 31 M. bovis, 32 M. bovis BCG, and 23 Mycobacterium tuberculosis genomes available in GenBank were selected. M. bovis RDs (regions of difference) and Clonal Complexes (CC) were identified in silico. Genome dynamics of bacterial groups were analyzed by gene orthology and polymorphic sites identification. M. bovis polymorphic sites were used to construct a phylogenetic tree. Our RD analyses resulted in the exclusion of three genomes, mistakenly annotated as virulent M. bovis. M. bovis SP38 along with strain 35 represent the first report of CC European 2 in Brazil, whereas two other M. bovis strains failed to be classified within current CC. Results of M. bovis orthologous genes analysis suggest a process of genome remodeling through genomic decay and gene duplication. Quantification, pairwise comparisons and distribution analyses of polymorphic sites demonstrate greater genetic variability of M. tuberculosis when compared to M. bovis and M. bovis BCG (p ≤ 0.05), indicating that currently defined M. tuberculosis lineages are more genetically diverse than M. bovis CC and animal-adapted MTC (M. tuberculosis Complex) species. As expected, polymorphic sites annotation shows that M. bovis BCG are subjected to different evolutionary pressures when compared to virulent mycobacteria. Lastly, M. bovis phylogeny indicates that polymorphic sites may be used as markers of M. bovis lineages in association with CC. Our findings highlight the need to better understand host-pathogen co-evolution in genetically homogeneous and/or diverse host populations, considering the fact that M. bovis has a broader host range when compared to M. tuberculosis. Also, the identification of M. bovis genomes not classified within CC indicates that the diversity of M. bovis lineages may be larger than previously thought or that current classification should be reviewed.