CICLO REPRODUCTIVO DE LA ESPECIE LUTJANUS PERU (NICHOLS Y MURPHY, 1922), ASPECTOS MICROSCÓPICOS DEL CRECIMIENTO Y DESARROLLO DE LOS OVOCITOS.

Snappers are edible fish of great commercial importance in Panama since their meat is highly appreciated for its excellent texture and taste; all species in the family are commercially exploited. Lutjanus peru (Nichols and Murphy, 1922) is an important species for the artisanal, industrial and recreational fisheries. The main objective was to describe at microscopic level the cycle of ripeness of the females of the red / silk snapper. Samples of histological cuts were prepared with the Hematoxilin-Eosin's dye routine. Due to the presence of oocytes at different stages of maturation throughout the year, this histological review allows us to deduce that Lutjanus peru has a spaced reproduction period that coincides with the rainy season, with a peak in June and most likely another spawning period towards November and December. Red snapper’s, has a reproductive period spread with a maximum gonadal maturation in June. As we find evidence that the total length (Lt) of catches has decreased and that most of the captured females are young specimens, this implies the need to improve management measures.Lutjanus peru (Nichols y Murphy, 1922) es una especie importante para la pesca artesanal, industrial y recreativa. El objetivo principal fue describir a nivel microscópico el ciclo de madurez de las hembras del pargo rojo/seda. Este análisis histológico nos permite deducir que Lutjanus peru tiene un período de reproducción espaciado que coincide con la temporada de lluvias, con un pico en junio. Las muestras de cortes histológicos fueron preparadas con la rutina de tinción de hematoxilina-eosina. Observamos que en una década (1990-1999) las longitudes totales promedio de la hembra del pargo de seda han disminuido, esto permite entender la necesidad de mejorar las medidas de manejo de la especie, con especial atención en la supervisión continua y cuidadosa de las capturas, del esfuerzo de pesca y de la selectividad para prevenir o controlar la captura de los especímenes menores a 40 cm. &nbsp

Antimicrobial Susceptibility and Characterization of Resistance Mechanisms of Corynebacterium urealyticum Clinical Isolates

Corynebacterium urealyticum is a non-diphtherial urease-producing clinically relevant corynebacterial, most frequently involved in urinary tract infections. Most of the C. urealyticum clinical isolates are frequently resistant to several antibiotics. We investigated the susceptibility of 40 C. urealyticum isolated in our institution during the period 2005-2017 to eight compounds representative of the main clinically relevant classes of antimicrobial agents. Antimicrobial susceptibility was determined by the Epsilometer test. Resistance genes were searched by PCR. All strains were susceptible to vancomycin whereas linezolid and rifampicin also showed good activity (MICs90 = 1 and 0.4 mg/L, respectively). Almost all isolates (39/40, 97.5%) were multidrug resistant. The highest resistance rate was observed for ampicillin (100%), followed by erythromycin (95%) and levofloxacin (95%). Ampicillin resistance was associated with the presence of the blaA gene, encoding a class A ?-lactamase. The two rifampicin-resistant strains showed point mutations driving amino acid replacements in conserved residues of RNA polymerase subunit ? (RpoB). Tetracycline resistance was due to an efflux-mediated mechanism. Thirty-nine PFGE patterns were identified among the 40 C. urealyticum, indicating that they were not clonally related, but producing sporadic infections. These findings raise the need of maintaining surveillance strategies among this multidrug resistant pathogen.This research was funded by Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI) D16/0016/0007 and RD16/0016/0008), and co-financed by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014-2020

Apoptosis of peritoneal leucocytes during early stages of Fasciola hepatica infections in sheep

Several immunomodulatory properties have been described in Fasciola hepatica infections. Apoptosis has been shown to be an effective mechanism to avoid the immune response in helminth infections. The aim of the present work was to study apoptosis in peritoneal leucocytes of sheep experimentally infected with F. hepatica during the early stages of infection. Five groups (n = 5) of sheep were used. Groups 2–5 were orally infected with 200 metacercariae (mc) and sacrificed at 1, 3, 9 and 18 days post-infection (dpi), respectively. Group 1 was used as the uninfected control (UC). Apoptosis was detected using three different methods 1) immunocytochemistry (ICC) with a polyclonal antibody anti-active caspase-3; 2) an annexin V flow cytometry assay using the Annexin V-FITC/propidium iodide (PI); and 3) transmission electron microscopy (TEM). The differential leucocyte count revealed that the majority of peritoneal granulocytes were eosinophils, which increased significantly at 9 and 18 dpi with respect to the uninfected controls. The ICC study revealed that the percentage of caspase-3+ apoptotic peritoneal leucocytes increased significantly from 3 dpi onwards with respect to the uninfected controls. The flow cytometry annexin V assay detected a very significant (P < 0.001) increase of apoptotic peritoneal macrophages, lymphocytes and granulocytes, which remained higher than in the UC until 18 dpi. Transmission electron microscopy studies also confirmed the presence of apoptosis in peritoneal eosinophils at 18 dpi. This is the first report of apoptosis induced by F. hepatica in the peritoneal leucocytes of sheep in vivo. The results of this work suggest the importance of apoptosis induction for the survival of the juvenile parasites in the peritoneal migratory stages of infection.This work was supported by EU grants (H2020-635408-PARAGONE) and the Spanish Ministry of Science grant AGL2015-67023-C2-1-R. The TEM studies were carried out by the Central Research Services (SCAI) of the University of CórdobaAccepted manuscriptVeterinari

Biliary PAH metabolites in European eel (Anguilla anguilla) from Mar Menor lagoon (SE Spain)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic contaminants present in marine sediments as a consequence of their continuous input from either land- or marine-based sources. Fishes have a higher capacity to metabolize and excrete PAHs than invertebrates such as mollusks and consequently the concentration of PAH metabolites in the bile fluid of fish can be used as an indirect indicator of exposure to PAH contamination. In this study the concentration and distribution of major PAHs metabolites in European eel (Anguilla anguilla) bile from the hypersaline Mar Menor coastal lagoon (SE Spain) were characterized for the first time. Eels of two different weight classes (350 g) were sampled from the northern and southern part of the lagoon using traditional fishing methods by local fishermen in spring 2014 and winter 2015. Bile samples were treated individually and maintained at -20ºC until analysis. 10 L of bile sample was diluted with water for liquid chromatography, incubated with β-glucuronidase/arylsulfatase for 2 hours at 37ºC, then the reaction was stopped with cold methanol and the sample centrifuged. The concentrations of PAHs metabolites (phenanthrol and pyrenol) in the supernatant were directly analysed by liquid chromatography with fluorescence detection using a standard solution for external calibration. Phenanthrol and pyrenol were found in all samples, with pyrenol being always the predominant one. The metabolite concentrations were higher in specimens sampled in spring 2014 than in winter 2015, suggesting a different seasonal pattern of PAH exposure to fish in the Mar Menor lagoon

Distribution of Foxp3+ T cells in the liver and hepatic lymph nodes of goats and sheep experimentally infected with Fasciola hepatica

Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and hepatic lymph nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n=6) and three groups of sheep (n=6) were used in this study. Goats in groups 1-2 and sheep in groups 4-5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin-biotin-complex method was used for the immunohistochemical study. At nine dpi in acute hepatic lesions, the number of both Foxp3+ and CD3+ T lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15-19wpi), the number of Foxp3+ and CD3+ T lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of hepatic lymph nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and hepatic lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.This work was supported by EU grants (H2020-635408-PARAGONE) and the Spanish Ministry of Science grant AGL2015-67023-C2-1-R. TM receives funding from the Scottish Government.Accepted manuscriptVeterinari

Aislados de Bacillus provenientes de la rizósfera de cactus incrementan la germinación y la floración en Mammillaria spp. (Cactaceae)

ResumenLas cactáceas son la vegetación característica de las zonas áridas en México, donde las lluvias son escasas, la evapotranspiración es elevada y la fertilidad de los suelos es baja. Las plantas han desarrollado estrategias fisiológicas como la asociación con microorganismos en la zona de la rizósfera para incrementar la captación de nutrientes. En el presente trabajo se obtuvieron 4 aislados bacterianos de la rizósfera de Mammillaria magnimamma y Coryphantha radians, los que fueron nombrados como QAP3, QAP19, QAP22 y QAP24 e identificados genéticamente como pertenecientes al género Bacillus. Estos aislados exhibieron in vitro propiedades bioquímicas como solubilización de fosfatos, producción de ácido indolacético y actividad ACC deaminasa, que se relacionan con la promoción del crecimiento de las plantas. Dicha promoción fue ensayada inoculando semillas de M. magnimamma y evaluando luego algunos parámetros. Se encontró que todos los aislados incrementaron la germinación desde un 17% hasta un 34,3% (con respecto a las semillas testigo sin inocular); el aislado QAP24 fue el que presentó el mayor efecto en este sentido y permitió la germinación de todas las semillas viables (84,7%) 3 días antes que en el testigo. La inoculación de este aislado en plantas de Mammillaria zeilmanniana mostró un efecto positivo sobre la floración: en 2 meses dentro del período de un año se detectó un incremento en el número de plantas en floración con respecto a las plantas testigo, de hasta el 31,0% en uno de ellos. Se concluye que los aislados de Bacillus spp. caracterizados poseen potencial para ser empleados en programas de conservación de especies vegetales de zonas áridas.AbstractCacti are the most representative vegetation of arid zones in Mexico where rainfall is scarce, evapotranspiration is high and soil fertility is low. Plants have developed physiological strategies such as the association with microorganisms in the rhizosphere zone to increase nutrient uptake. In the present work, four bacterial isolates from the rhizosphere of Mammillaria magnimamma and Coryphantha radians were obtained and named as QAP3, QAP19, QAP22 and QAP24, and were genetically identified as belonging to the genus Bacillus, exhibiting in vitro biochemical properties such as phosphate solubilization, indoleacetic acid production and ACC deaminase activity related to plant growth promotion, which was tested by inoculating M. magnimamma seeds. It was found that all isolates increased germination from 17 to 34.3% with respect to the uninoculated control seeds, being QAP24 the one having the greatest effect, accomplishing the germination of viable seeds (84.7%) three days before the control seeds. Subsequently, the inoculation of Mammillari zeilmanniana plants with this isolate showed a positive effect on bloom, registering during two months from a one year period, an increase of up to 31.0% in the number of flowering plants compared to control plants. The characterized Bacillus spp. isolates have potential to be used in conservation programs of plant species from arid zones

Comparative dynamics of peritoneal cell immunophenotypes in sheep during the early and late stages of the infection with Fasciola hepatica by flow cytometric analysis

Background: The peritoneal cell populations (PCP) are thought to play a crucial role during the early immune response in Fasciola hepatica infection while newly excysted juveniles (NEJ) are migrating in the peritoneal cavity (PC) towards the liver. In this study, we aimed to determine the immunophenotypes of the PCP and to analyse the dynamics of the recruitment of the PCP during the early and late stage of the infection in sheep infected with F. hepatica. Methods: Thirty-seven sheep were divided into three groups: Group 1 (n = 20) and 2 (n = 10) were challenged with F. hepatica, Group 3 (n = 7) was not infected and remained as uninfected control (UC). After the slaughtering, peritoneal lavages were carried out to isolate peritoneal cell populations at 1, 3, 9 and 18 days post-infection (dpi) for Group 1 and at 14 weeks post-infection (wpi) for Group 2 and 3. Flow cytometry was conducted to assess the dynamics of peritoneal cavity cell populations. Results: TCD4 cells showed a significant decrease at 1 and 18 dpi when compared to UC; no statistical differences were detected for TCD8 and WC1+ γδ during the early stage of the infection with respect to the UC. CD14 cells exhibited a decreasing trend, with a significant decrease at 9 and 18 dpi when compared to the UC. The dynamics of MHCII and CD83 cells showed a similar increasing pattern from 3 to 18 dpi. During the chronic stage, both TCD4 and TCD8 cells showed no significant differences when compared to the UC, although a slight but statistically significant higher level of WC1+ γδ cells was observed. A lower percentage of antigen-presenting cells (APCs) was detected with respect to the UC. Conclusions: The recruitment of the lymphocytes subsets did not show a significant increase during the course of the infection and only WC1+ γδ cells displayed a significant increase at the chronic stage. For the CD14, a decreasing trend was observed during the early stage, which was statistically significant at the chronic stage of the infection. Peritoneal CD83 and MHCII cells developed an increasing trend during the early stage of infection, and showed a significant decrease at the late stage of the infection.This study was funded by the European Union Grant H2020-635408- PARAGONE and by National Grant AGL2015-67023-C2-1-R. RPC was supported by an FPU grant of the Spanish Ministry of Education, Culture and Sport. Funding bodies were neither involved in the design of the study nor in analysis and interpretation of the dataVeterinari

Fasciola hepatica induces eosinophil apoptosis in the migratory and biliary stages of infection in sheep

The aim of the present work was to evaluate the number of apoptotic eosinophils in the livers of sheep experimentally infected with Fasciola hepatica during the migratory and biliary stages of infection. Four groups (n = 5) of sheep were used; groups 1–3 were orally infected with 200 metacercariae (mc) and sacrificed at 8 and 28 days post-infection (dpi), and 17 weeks post-infection (wpi), respectively. Group 4 was used as an uninfected control. Apoptosis was detected using immunohistochemistry with a polyclonal antibody against anti-active caspase-3, and transmission electron microscopy (TEM). Eosinophils were identified using the Hansel stain in serial sections for caspase-3, and by ultrastructural features using TEM. At 8 and 28 dpi, numerous caspase-3+ eosinophils were mainly found at the periphery of acute hepatic necrotic foci. The percentage of caspase -3+ apoptotic eosinophils in the periphery of necrotic foci was high (46.1–53.9) at 8 and 28 dpi, respectively, and decreased in granulomas found at 28 dpi (6%). Transmission electron microscopy confirmed the presence of apoptotic eosinophils in hepatic lesions at 8 and 28 dpi. At 17 wpi, apoptotic eosinophils were detected in the infiltrate surrounding some enlarged bile ducts containing adult flukes. This is the first report of apoptosis induced by F. hepatica in sheep and the first study reporting apoptosis in eosinophils in hepatic inflammatory infiltrates in vivo. The high number of apoptotic eosinophils in acute necrotic tracts during the migratory and biliary stages of infection suggests that eosinophil apoptosis may play a role in F. hepatica survival during different stages of infection.This work was supported by EU grants (FPVII-265862-PARAVAC, H2020-635408-PARAGONE) and the Spanish Ministry of Science grant AGL2009-08726. TEM studies were carried out by the Central Services for Research of the University of Córdoba (SCAI)Veterinari

Fused-filament 3D printing of drug products: Microstructure analysis and drug release characteristics of PVA-based caplets

Fused deposition modeling (FDM) 3–Dimensional (3D) printing is becoming an increasingly popular technology in the pharmaceutical field, since it allows the manufacture of personalized oral dosage forms by deposition of thin layers of material. Here, a filament extruder was used to obtain filaments of polyvinyl alcohol (PVA) containing paracetamol or caffeine appropriate for 3D printing. The filaments were used to manufacture caplets for oral administration by FDM 3D printing in order to evaluate the effect of the internal structure (micropore volume), drug loading and composition on drug dissolution behaviour. Micropore volume of the caplets was primarily determined by the presence of large pores due to gaps in the printed layers/net while printing, and the porosity of the caplets was 10 fold higher than the porosity of the extruded filament. Dynamic dissolution drug release tests on the caplets in biorelevant bicarbonate media revealed distinctive release profiles, which were dependent on drug solubility and drug loading. Porosity of the caplets did not help to predict the different drug release profiles. This study confirms the potential of 3D printing to fabricate caplets and helps to elucidate which factors influence drug release from this type of new dosage forms