Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48-72 hours

Fish photobacteriosis—The importance of rapid and accurate identification of Photobacterium damselae subsp. piscicida

MALDI-TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on-target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24-hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48-hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI-TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate-dependent

Evaluation of micronucleus and erythrocytic nuclear abnormalities in Balkan whip snake Hierophis gemonensis

The aim of the study was to evaluate the frequency of micronuclei (MN), nuclear abnormalities (NA) including vacuolated nuclei (VN) and cytoplasmic vacuoles (CV) in erythrocytes of Balkan whip snake Hierophis gemonensis and establish the level of spontaneous appearance during the annual cycle. Average frequency of NA was 10.89 ± 4.72 % while the MN (0.03 ± 0.03 %) and VN(0.04 ± 0.08 %) were seldom detected. NA significantly positively correlated with MN (r = 0.319 ; P < 0.05) and VN (r = 0.363 ; P < 0.05). Appearance of CV did not correlate with other measured parameters and average frequency was 11.06 ± 8.33 %. Significant seasonal variation was found in NA appearance with the lowest value in spring and the highest in winter. VN increase was observed in autumn. MN and CV levels varied between seasons but not significantly. Considering the biological cycle, frequency of NA, VN, MN and CV recorded in pre-hibernation/hibernation increased compared to the active phase, but only NA elevation was significant. Although the obtained results showed differences according to sex, statistical analysis of measured parameters showed the same pattern of seasonal variation in both sexes

Evaluation in situ of genotoxicity and stress in South American common toad Rhinella arenarum in environments related to fluorite mine

Little attention has been paid to the impact of wastewater generated by mining activities on fluoride. In this study, we evaluated the hematology responses of common South American toad Rhinella arenarum inhabiting natural and artificial environments associated with a fluorite mine from central Argentina. We analyzed three sampling stations associated with the fluorite mine: (I) Los Cerros Negros stream (CN), which runs on granitic rock with a high fluorite content; (II) Los Vallecitos stream (LV), which runs on metamorphic rock with low fluorite content; and (III) artificial decantation ponds (DP) containing sediments produced by fluorite flotation process. We calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, and immature erythrocytes. In addition, we performed a differential leukocyte count and determined neutrophils/lymphocyte ratio as a stress response estimator. We found high micronucleus (MN) and erythrocyte nuclear abnormality (ENA) frequencies in DP and CN but low frequencies in LV. The neutrophil/lymphocyte ratio was different among sites, with a significant increase in individuals from DP. Values registered in DP could be caused by exposure to mixture of compounds registered in dams that hold wastewater, while high values registered in CN stream might be due to natural concentrations of fluoride. Our results suggest that blood is an effective and non-destructive sensitive indicator for monitoring genotoxic agents in freshwater ecosystems.Fil: Pollo, Favio Ezequiel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Ecología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Grenat, Pablo Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Ecología; ArgentinaFil: Salinas, Zulma Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Ecología; ArgentinaFil: Otero, Manuel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Ecología; ArgentinaFil: Salas, Nancy Edith. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Ecología; ArgentinaFil: Martino, Adolfo Ludovico. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Ecología; Argentin