Yanez A.J.,Institute Bioquimica |
Valenzuela K.,Institute Bioquimica |
Silva H.,Institute Bioquimica |
Retamales J.,Andrés Bello University |
And 7 more authors.
Diseases of Aquatic Organisms | Year: 2012
Piscirickettsiosis or salmonid rickettsial septicaemia (SRS) caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. Since the first reports of the disease, it has been successfully isolated and maintained in eukaryotic cellculture systems, but these systems are time-consuming, the media are costly, and eliminating heavily contaminated host cell debris is difficult. In this report, we describe a marine-based broth supplemented with L-cysteine, named AUSTRAL-SRS broth, that facilitates superior growth of P. salmonis strains. Strains reached an optical density of approximately 1.8 when absorbance was measured at 600 nm after 6 d incubation at 18°C. Several passages (n = 6) did not alter the culture kinetics. We report for the first time the purification of DNA, lipopolysaccharide (LPS) and whole membrane protein obtained from P. salmonis grown in this liquid medium, and thus provide a suitable platform to simplify the preparation of P. salmonis cells for genetic and serological studies. Moreover, the results of the cytopathic effect test showed that P. salmonis grown in AUSTRALSRS broth maintained their virulence properties, inducing apoptosis after 3 d. This makes the medium a good candidate for the successful growth of P. salmonis and an excellent basis for the development of low cost vaccines. © Inter-Research 2012.
De Almeida J.P.L.,Institute Bioquimica |
De Almeida J.P.L.,University of Lisbon |
Freitas-Santos T.,University of Lisbon |
Saldanha C.,Institute Bioquimica |
Saldanha C.,University of Lisbon
Clinical Hemorheology and Microcirculation | Year: 2012
Recent evidence has shown that plasma fibrinogen, a major cardiovascular risk factor, interacts with the erythrocyte membrane and acts to influence blood flow via erythrocyte nitric oxide (NO) modulation. In the present in-vitro study, whole blood samples were harvested from healthy subjects and aliquots were incubated in the absence (control aliquots) and presence of fibrinogen at different degrees of band 3 phosphorylation, and the erythrocyte deformability was determined. The present study shows that in the presence of higher fibrinogen concentrations, similar to those found in inflammatory conditions, erythrocyte deformability is increased only when band 3 is dephosphorylated by the presence of syk inhibitor and at low shear stress. On the contrary, no changes were verified in the presence of fibrinogen when band 3 is allowed to be phosphorylated by inhibiting the phosphotyrosine phosphatase enzyme activity with calpeptin. We also observed that the presence of fibrinogen at higher concentration does not induce changes in erythrocyte deformability in the absence of modulators of the band 3 phosphorylation degree. However, the mechanisms by which fibrinogen signalling modulates erythrocyte function remain to be clarified and are currently under study. © 2012 IOS Press and the authors. All rights reserved.
Tapia-Cammas D.,Andrés Bello University |
Yanez A.,Institute Bioquimica |
Arancibia G.,Andrés Bello University |
Toranzo A.E.,University of Santiago de Compostela |
Avendano-Herrera R.,Andrés Bello University
Diseases of Aquatic Organisms | Year: 2011
A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg μl -1 for V. anguillarum, 500 fg μl -1 for P. salmonis, and 5 pg μl -1 for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 × 10 5 CFU ml -1 of V. anguillarum, 1.26 × 10 4 CFU ml -1 of S. phocae, and 5.33 × 10 4 CFU ml -1 of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 ± 0.54 × 10 7 CFU g -1 for V. anguillarum, 9.03 ± 1.84 × 10 5 CFU g -1 for S. phocae, 3.8 ± 0.78 × 10 3 CFU mg -1 for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of ~1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simul taneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms. © Inter-Research 2011.