Performance of recombinant proteins in diagnosis and differentiation of canine visceral leishmaniasis infected and vaccinated dogs.

Control of canine visceral leishmaniasis (CVL), a major zoonotic disease in Brazil and many other tropical and subtropical countries, remains difficult as an accurate and reliable diagnosis is still missing. In endemic regions, infected dogs are the main parasitic reservoir host of human Visceral leishmaniasis (VL) infection. Vaccination of dogs against Leishmania infection constitutes an important strategy to prevent or to better control CVL, thus, a serological test that can discriminate between antibodies induced by immunization versus infection is highly desirable in order to improve and simplify diagnosis. Here, four recombinant proteins were evaluated for their ability to detect and differentiate between dogs that are infected with Leishmania or have been immunized with the anti-Leishmania vaccine Leish-Tec®. Receiver operating characteristic (ROC) curve analysis of the four Leishmania-specific IgG ELISA revealed superior performance of rK28, followed by rKLO8, rK39 and rLb6H. The rK28-based ELISA revealed not only the best accuracy against CVL, but also the lowest cross-reactivity with sera from Leish-Tec® immunized dogs. Our data show that the rK28-based ELISA is highly suitable for CVL screening as it shows high sensitivity with simultaneous low cross-reactivity. Further, the high specificity of the rKLO8 indicates its suitability for the confirmation of CVL diagnosis.

Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts.

This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves (ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.

Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts

ABSTRACT This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves (ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.