Search

Background The latex from the medicinal plant Calotropis procera is often used in folk medicine against infectious and inflammatory diseases. Purpose In this study, we investigate a protein fraction with immunomodulatory properties, named LPPI, against experimental infections, in vitro and in vivo, with a virulent strain of Listeria monocytogenes. Study design LPPI was exposed to cultured macrophages or Swiss mice and then challenged with L. monocytogenes. Methods Peritoneal macrophages were obtained from Swiss mice, and cultured in 96-well microplates. Soluble latex proteins (LP) were subjected to fractionation by ion-exchange chromatography. The major peak (LPPI) was added into wells at 10 or 100 µg/ml. Albumin (100 µg/ml) was used for comparison between protein treatments. After incubation for 1 h at 5% CO2/ 37°C, the supernatant was discarded and 0.2 ml of L. monocytogenes overnight culture was added in the wells. Following 4 h and 24 h infection, the cytokine mRNA expression was evaluated as well as the number of intracellular colony forming units. Swiss mice (n = 16) were injected intraperitoneally (i.p.) with LPPI (5 and 10 mg/kg) while the control mice received albumin (10 mg/kg) or LP (10 mg/kg). After 24 h, all animal groups were challenged with L. monocytogenes (106 CFU/ ml), also by i.p. route. Results LPPI was not toxic to uninfected macrophages (pMO) and significantly increased mRNA expression of TNF-α, IL-6, IL-1β and iNOS. Following infection, cell viability was reduced by 50% in albumin-treated pMO (control); but only 17% in pMO treated with LPPI at 100 µg/ml. In this case, LPPI increased expression of TNF-α and IL-6 whereas the number of bacterial colony-forming units was reduced 100-fold in comparison to control groups. Swiss mice pretreated with LPPI showed dose-dependent survival rates that reached 80%, while mice that received albumin died 1–3 days after infection. After 24 h infection, leukocyte migration to the infectious foci was high in LPPI-treated mice whereas the number of viable bacteria in the peritoneal fluid, liver and bloodstream were significantly reduced. Conclusion We conclude that LPPI present immunomodulatory properties that are beneficial for prevention of systemic bacterial infections caused by the intracellular bacteria L. monocytogenes.

The Yersinia pestis capsular antigen F1 is widely used in plague laboratory diagnosis. Here, we describe the production of an F1 recombinant protein within reduced time and biosafety requirements. Its evaluation in hemagglutination tests indicated that the recombinant F1 can replace the conventional F1 protein for plague diagnosis.

A multiplex nested PCR method for detection of Vibrio cholerae O1 using a single tube was developed (MSTNPCR). Firstly, single-tube nested PCR (STNPCR) with primers directed to ctx A gene was standardized, and its detection limit was compared to simple PCR and two-step nested PCR. Secondly, primers directed to rfb N gene were added to the reaction. The detection limit of the multiplex reaction was determined using V. cholerae O1 DNA and V. cholerae O1 grown in alkaline peptone water (APW). STNPCR was shown to be approximately 100-fold more sensitive than simple PCR and 10 times less sensitive than two-step nested PCR. This drawback is compensated by a lower risk of cross-contamination. The addition of a second target did not impair the detection limit of STNPCR (as little as 1 pg of V. cholerae O1 DNA detected). MSTNPCR could specifically detect up to three V. cholerae O1 cells or colony forming units (cfu) directly from the APW growth. A diagnostic kit consisting of a set of microtubes having the inner primers fixed onto the inside of the tube cap and a set of tubes containing the reaction mixture was evaluated for stability, and it proved to be stable for five months at − 20 °C. Therefore, MSTNPCR would be useful in the detection of V. cholerae O1 directly from environmental waters in cholera endemic areas and in complementing the identification of toxigenic strains isolated by culture.