Búsqueda de candidatos proteicos para el desarrollo de vacunas frente a las infecciones por streptococcus suis en cerdos

Streptococcus suis es microorganismo de enorme repercusión económica para la industria porcina y una marcada importancia sanitaria, al ser considerado un agente zoonósico emergente. En base al polisacárido capsular, se reconoce la existencia de 35 serotipos, siendo los serotipos del 1 al 10, 14, 15, 16, 1/2 y 1/14 los que se asocian a la mayoría de los brotes clínicos en el cerdo y el hombre. El descubrimiento de una vacuna eficaz, elaborada con antígenos comunes y conservados entre serotipos se ha convertido en un objetivo prioritario mundial. Actualmente, la atención se ha desplazado hacia las proteínas de superficie, especialmente aquellas ancladas a la pared celular, que pueden ser detectadas con técnicas proteómicas. Hemos analizado mediante proteómica una colección de 39 cepas pertenecientes a 19 serotipos, utilizando la técnica del shaving, basada en la digestión de células vivas con proteasas y el posterior análisis de los péptidos generados por LC/MS/MS. Esto nos ha permitido definir el pan-surfoma de este grupo de aislados, identificado un total de 113 proteínas (33 lipoproteínas; 17 proteínas de la pared celular; 9 proteínas secretadas, 28 proteínas de membrana con un dominio transmembrana y 26 proteínas multitransmembrana). De todas las proteínas identificadas, tres proteínas de pared celular cell envelope proteinase (SSU0757), 5'-nucleotidase (SSU0860) y surface-anchored DNA nuclease o SsnA (SSU1760), podrían ser buenos candidatos vacunales, al estar presentes en más del 70 por ciento de los serotipos y estar más expuestas en la superficie. Seleccionamos la proteína “Surface-anchored DNA nuclease” (SsnA) para valorar su potencial como candidato vacunal, mediante ensayos de protección in vivo de la proteína recombinante rSsnA. Realizamos un estudio preliminar para seleccionar un adyuvante eficaz para su uso en formulaciones vacunales frente a las infecciones por S. suis en el modelo murino, utilizando un protocolo basado en...Streptococcus suis is a microorganism of enormous economic impact to the swine industry with a public health concern, considered an emerging zoonotic agent. Based on the capsular polysaccharide, the existence of 35 serotypes are recognized, serotypes 1 through 10, 14, 15, 16, 1/2 and 1/14 are associated with the majority of clinical outbreaks in pigs and human. The discovery of an effective vaccine, made with common and conserved antigens between serotypes has become a global priority. Currently, attention has shifted to the surface proteins, especially those anchored to the cell wall, which can be detected with proteomic techniques. We analyzed using proteomic techniques a collection of 39 strains belonging to 19 serotypes, using the technique of shaving, based on digestion of live cells with protease and subsequent analysis of the generated peptides by LC/MS/MS. We define the pan-surfoma this group of isolates, and identify a total of 113 proteins (33 lipoproteins, 17 cell wall proteins, 9 secreted proteins, membrane proteins, 28 with a transmembrane domain and 26 multitransmembrane proteins). Of all the identified proteins, three proteins of cell wall cell envelope proteinase (SSU0757), 5'-nucleotidase (SSU0860) and surface-anchored DNA nuclease or SsnA (SSU1760) could be good vaccine candidates, because they are present in more than 70 percent of the serotypes and more exposed on the surface. We select the protein Surface-anchored DNA nuclease (SsnA) to evaluate their potential as a vaccine candidate using in vivo protection assays with the recombinant protein rSsnA. We conducted a preliminary study to select an effective adjuvant for use in vaccine formulations against infections by S. suis in the mouse model,..

Métodos de enriquecimiento de glicoproteínas bacterianas para su posterior análisis y caracterización mediante espectrometría de masas

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El proceso de una auditoría de cuentas anuales: basado en las prácticas en empresa

El trabajo desarrolla de una forma teórica-práctica el desarrollo completo de una auditoría de cuentas anuales, comenzando desde la aceptación del encargo y finalizando con la emisión de un informe, en el que consta la opinión del experto independiente sobre las cuentas anuales de una compañía basándose en todo el trabajo realizado durante el proceso. En el trabajo se aplican todos los conocimientos adquiridos durante mis prácticas en una auditoría a través del análisis de varias áreas contables seleccionadas en las que se explican los procedimientos de auditoría aplicados.<br /

Identification of Potential New Protein Vaccine Candidates through Pan-Surfomic Analysis of Pneumococcal Clinical Isolates from Adults

Purified polysaccharide and conjugate vaccines are widely used for preventing infections in adults and in children against the Gram-positive bacterium Streptococcus pneumoniae, a pathogen responsible for high morbidity and mortality rates, especially in developing countries. However, these polysaccharide-based vaccines have some important limitations, such as being serotype-dependent, being subjected to losing efficacy because of serotype replacement and high manufacturing complexity and cost. It is expected that protein-based vaccines will overcome these issues by conferring a broad coverage independent of serotype and lowering production costs. In this study, we have applied the ‘‘shaving’’ proteomic approach, consisting of the LC/MS/MS analysis of peptides generated by protease treatment of live cells, to a collection of 16 pneumococcal clinical isolates from adults, representing the most prevalent strains circulating in Spain during the last years. The set of unique proteins identified in all the isolates, called ‘‘pan-surfome’’, consisted of 254 proteins, which included most of the protective protein antigens reported so far. In search of new candidates with vaccine potential, we identified 32 that were present in at least 50% of the clinical isolates analyzed. We selected four of them (Spr0012, Spr0328, Spr0561 and SP670_2141), whose protection capacity has not yet been tested, for assaying immunogenicity in human sera. All of them induced the production of IgM antibodies in infected patients, thus indicating that they could enter the pipeline for vaccine studies. The pan-surfomic approach shows its utility in the discovery of new proteins that can elicit protection against infectious microorganisms

Optimization of digestion of living cells of streptococcus pneumoniae for searching protein vaccine candidates

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Pre-test probability and likelihood ratios for clinical findings in canine leishmaniasis

CanineleishmaniasisisaparasiticzoonosismainlycausedbyL.infantum;anobligateintracellularprotozoantransmittedbyhaematophagousinsectsofthegenusPhleboto-mus,whichaffectsdogsandwildcanids.Theclinicalimplicationsofthisdiseasearehighlyvariable,sinceinfectedanimalsmayremainasymptomatic(absenceofobserv-ableclinicalsigns)orpresentawidespectrumofclinicalalterationsanddegreesofseverity, including the death of the animal. Symptoms such as lymphadenomegaly,alopecia,weightloss,keratoconjunctivitisandonychogryphosisareusuallythefirstdiagnosticreferenceavailable.Theobjectivesofthisstudyaretoevaluatethevalidity(sensitivity,specificityandlikelihoodratios)anddiagnosticutility(pre-testprobability)oftheclinicalsignscommonlyassociatedwithcanineleishmaniasisbasedonthepreva-lenceintheareaandtoexplorethecombinationofsymptomsthatbestpredictsthediagnosisofcanineleishmaniasis.Itisamatchedcase-controlstudyinthecaninepopu-lationofsouthernSpainbasedonthecomparisonofthefindingscollectedintheclinicalhistoryandtheresultsoftheLeisSCANquantitativeELISA.Atotalof39casesand78controlswereanalysed.Approximately80%oftheinfectedanimalsshowedsignscompatiblewiththedisease.Themostfrequentalterationswerecutaneous(64.1%),systemic(51.3%)andoculo-nasal(30.7%).Themostusefulsignstosupportthisdiagno-siswerealopeciaandepistaxis(LR+6.69and6.0,respectively)(pre-testleishmaniasisprobabilityis≥70%forprevalence≥28%whenalopeciaorepistaxisispresent),fol-lowedbylameness(LR+5.0).Thecombinationsofsignsthatshowedgreatervaliditywere alopecia with hyperkeratosis of the snout and alopecia with onychogryphosis(LR+>10).Noneoftheobservedsignsortheircombinationsresultedusefultoruleoutthediagnosis(LR–0.55to1.15).Theresultsfoundshownotabledifferencesinthediagnosticvalueoftheclinicalsigns,individuallyandincombination,sowebelievethatmedicaldecisionsshouldbebasedontheirdiagnosticvalidity(LR+)andtheestimationofthepre-testandpost-testprobability

Search of Potential Vaccine Candidates against Trueperella pyogenes Infections through Proteomic and Bioinformatic Analysis

Trueperella pyogenes is an opportunistic pathogen, responsible for important infections in pigs and significant economic losses in swine production. To date, there are no available commercial vaccines to control diseases caused by this bacterium. In this work, we performed a comparative proteomic analysis of 15 T. pyogenes clinical isolates, by “shaving” live cells, followed by LC-MS/MS, aiming at the identification of the whole set of surface proteins (i.e., the “pan-surfome”) as a source of antigens to be tested in further studies as putative vaccine candidates, or used in diagnostic tools. A total of 140 surface proteins were detected, comprising 25 cell wall proteins, 10 secreted proteins, 23 lipoproteins and 82 membrane proteins. After describing the “pan-surfome”, the identified proteins were ranked in three different groups based on the following criteria: to be (i) surface-exposed, (ii) highly conserved and (iii) widely distributed among different isolates. Two cell wall proteins, three lipoproteins, four secreted and seven membrane proteins were identified in more than 70% of the studied strains, were highly expressed and highly conserved. These proteins are potential candidates, alone or in combination, to obtain effective vaccines against T. pyogenes or to be used in the diagnosis of this pathogen

Risk factors associated with the antimicrobial resistance of staphylococci in canine pyoderma

This study reports the susceptibility to antimicrobial agents of staphylococci 4 (n=105) isolated from dogs, and the factors associated with this resistance. The study 5 animals were 23 healthy dogs (group A), 24 with first-time pyoderma (group B), and 27 6 with recurrent pyoderma that had undergone long-term antibiotic treatment (group C). 7 Staphylococci were more commonly isolated from the pyoderma-affected than the 8 healthy dogs (p<0.0001). 9 10 Some 78% of the isolates were resistant to at least one antimicrobial agent. 11 Resistance to amoxicillin-clavulanate,cephalosporins (OR 4.29, 95% CI [1.15, 16.3] 12 respectively), enrofloxacin (OR 9.47, 95% CI [1.53, 58.5]) and ciprofloxacin (OR 79.7 13 95% CI [3.26, 1947.4]) was more common among group C isolates. Some 32% of all 14 the isolates were multiresistant (MR) and 10.4% were methicillin-resistant (MRS). The 15 probability of isolating MRS staphylococci in group C increased by a factor of four 16 (95% CI [1.18, 17.9]) compared to A plus B. Multi-resistant (MR) isolates were 17 obtained more commonly from urban than rural dogs (OR 3.79, 95% CI [1.09, 13.17]). 18 All the MRS staphylococci encountered were obtained from urban dogs and more 19 commonly from male dogs (p= 0.07). 20 21 This study shows that dogs bred in urban habitat, with a history of antibiotic 22 therapy in the past year represents significant risk of being carriers of isolates resistant 23 to methicillin (MRS) and other antimicrobials. These factors should be considered 24 before applying an antimicrobial treatment in veterinary clinics

Identification of new immunoprotective surface protein vaccine candidates against streptococcus suis infection by proteomics

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The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation