Draft genome of the Leptospira interrogans strains, Acegua, RCA, Prea, and Capivara, obtained from wildlife maintenance hosts and infected domestic animals.

In the present paper, we announce new draft genomes of four Leptospira interrogans strains named Acegua, RCA, Prea, and Capivara. These strains were isolated in the state of Rio Grande do Sul, Brazil, from cattle, dog, Brazilian guinea pig, and capybara, respectively.

Blocking ELISA using recombinant NcSRS2 protein for diagnosing bovine neosporosis.

Neospora caninum is the etiologic agent of neosporosis, which leads to economic impacts on cattle industry. The reference method for serodiagnosis of neosporosis is the indirect fluorescent antibody test (IFAT). However, IFAT is laborious, expensive, and is not practicable in high throughput screening. In order to facilitate the serological diagnosis of neosporosis, we developed a blocking enzyme-linked immunosorbent assay (b-ELISA) based on NcSRS2 recombinant protein (rNcSRS2) and polyclonal antibodies against rNcSRS2 (b-ELISA/rNcSRS2). Compared to IFAT, b-ELISA/rNcSRS2 showed 93.7 % accuracy (98.7 % sensitivity and 88.7 % specificity), suggesting its potential as diagnostic assay to detect N. caninum antibodies in cattle sera.

Phenotypic and Molecular Characterization of Leptospira interrogans Isolated from Canis familiaris in Southern Brazil.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes from the genus Leptospira, which includes 20 species and more than 300 serovars. Canines are important hosts of pathogenic leptospires and can transmit the pathogen to humans via infected urine. Here, we report the phenotypic and molecular characterization of Leptospira interrogans isolated from Canis familiaris in Southern Brazil. The isolated strain was characterized by variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, the isolate was recognized by antibodies from human and canine serum samples previously tested by microscopic agglutination test. Ultimately, the expression of membrane-associated antigens (LipL32 and leptospiral immunoglobulin-like proteins) from pathogenic leptospires using monoclonal antibodies was detected by indirect immunofluorescence assay. In conclusion, identification of new strains of Leptospira can help in the diagnosis and control of leptospirosis.

Lectin of Abelmoschus esculentus (okra) promotes selective antitumor effects in human breast cancer cells.

The anti-tumor effects of a newly-discovered lectin, isolated from okra, Abelmoschus esculentus (AEL), were investigated in human breast cancer (MCF7) and skin fibroblast (CCD-1059 sk) cells. AEL induced significant cell growth inhibition (63 %) in MCF7 cells. The expression of pro-apoptotic caspase-3, caspase-9, and p21 genes was increased in MCF7 cells treated with AEL, compared to those treated with controls. In addition, AEL treatment increased the Bax/Bcl-2 ratio in MCF7 cells. Flow cytometry also indicated that cell death (72 %) predominantly occurred through apoptosis. Thus, AEL in its native form promotes selective antitumor effects in human breast cancer cells and may represent a potential therapeutic to combat human breast cancer.

Serological analysis by enzyme-linked immunosorbent assay using recombinant antigen LipL32 for the diagnosis of swine leptospirosis.

Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14-60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys.

Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool.

Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.

Detection of virulence factors and molecular typing of pathogenic Leptospira from capybara (Hydrochaeris hydrochaeris).

Leptospirosis is a globally prevalent zoonosis caused by pathogenic Leptospira spp.; several serologic variants have reservoirs in synanthropic rodents. The capybara is the largest living rodent in the world, and it has a wide geographical distribution in Central and South America. This rodent is a significant source of Leptospira since the agent is shed via urine into the environment and is a potential public health threat. In this study, we isolated and identified by molecular techniques a pathogenic Leptospira from capybara in southern Brazil. The isolated strain was characterized by partial rpoB gene sequencing and variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, to confirm the expression of virulence factors, the bacterial immunoglobulin-like proteins A and B expression was detected by indirect immunofluorescence using leptospiral specific monoclonal antibodies. This report identifies capybaras as an important source of infection and provides insight into the epidemiology of leptospirosis.

Molecular characterization of virulent Leptospira interrogans serogroup icterohaemorrhagiae isolated from Cavia aperea.

Leptospirosis is a worldwide zoonotic infection caused by pathogenic Leptospira. Synanthropic rodents are recognized carriers of leptospires; however, the role of wild rodents in the epidemiology of the disease is still incipient. In this work, we describe Leptospira strain isolated from Cavia aperea (Brazilian guinea pig). The isolated strain was characterized by partial rpoB gene sequencing, variable-number tandem-repeats and histopathological analysis. The strain was identified as Leptospira interrogans, serogroup Icterohaemorrhagiae and caused clinical signs of leptospirosis in the hamster model, attesting to its virulence. In conclusion, these findings could be useful for elucidating the epidemiological role of C. aperea in leptospirosis.

Monoclonal antibodies against the leptospiral immunoglobulin-like proteins A and B conserved regions.

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira that affects humans and a wide variety of animals. Recently the genomes of Leptospira interrogans, Leptospira borgpetersenii and Leptospira biflexa species were sequenced allowing the identification of new virulence factors involved in survival and pathogenesis of bacteria. LigA and LigB are surface-exposed bacterial adhesins whose expression is correlated with the virulence of Leptospira strains. In this study, we produced and characterized five monoclonal antibodies (MAbs) against a recombinant fragment of LigB (rLigBrep) with approximately 54kDa that comprise the portions of LigA and LigB (domains 2-7). The 5 MAbs obtained were of the IgG1 (2) and IgG2b (3) isotypes and their affinity constants for rLigBrep ranged from 7×10(7) M(-1) to 4×10(8) M(-1). The MAbs were able to react with the native antigen on the L. interrogans, L. borgpetersenii and Leptospira noguchii surfaces by indirect immunofluorescence, immunoblotting and immunoelectron microscopy. These results demonstrate that the MAbs anti-rLigBrep can be useful to complement genetic studies and to aid studies aiming understanding the role of Lig proteins in Leptospira pathogenesis and the development of Lig-based vaccines and improved diagnostic tests for leptospirosis.

Testing different antigen capture ELISA formats for detection of Leptospira spp. in human blood serum.

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira. The illness is characterized by an acute bacteremic phase followed by an immune phase, in which specific antibodies are found in blood and leptospires are eliminated in urine. Novel diagnostic strategies for use in the acute phase of leptospirosis are needed since clinical manifestations in this phase mimic other feverish tropical diseases. In the present study, mAbs and polyclonal IgY were used in the standardization of three different antigen capture ELISA formats for direct detection of leptospires in human blood during the acute phase of the disease. Detection limit of leptospires in experimentally contaminated human sera ranged from 10(5) to 10(7) cells ml(-1) in the different formats. The ELISA format with the best performance was able to detect 10(5) leptospires ml(-1) in human sera using a mAb against LipL32, the major outer membrane protein of pathogenic leptospires, as capture antibody, and a biotinylated polyclonal IgY against a pathogenic serovar of L. interrogans Icterohamorrhagiae as detection antibody. By increasing the degree of IgY biotinylation this detection limit could be improved to make the assay clinically useful.

Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool

Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.