Overcoming function annotation errors in the Gram-positive pathogen Streptococcus suis by a proteomics-driven approach

Background: Annotation of protein-coding genes is a key step in sequencing projects. Protein functions are mainly assigned on the basis of the amino acid sequence alone by searching of homologous proteins. However, fully automated annotation processes often lead to wrong prediction of protein functions, and therefore time-intensive manual curation is often essential. Here we describe a fast and reliable way to correct function annotation in sequencing projects, focusing on surface proteomes. We use a proteomics approach, previously proven to be very powerful for identifying new vaccine candidates against Gram-positive pathogens. It consists of shaving the surface of intact cells with two proteases, the specific cleavage-site trypsin and the unspecific proteinase K, followed by LC/MS/MS analysis of the resulting peptides. The identified proteins are contrasted by computational analysis and their sequences are inspected to correct possible errors in function prediction. Results: When applied to the zoonotic pathogen Streptococcus suis, of which two strains have been recently sequenced and annotated, we identified a set of surface proteins without cytoplasmic contamination: all the proteins identified had exporting or retention signals towards the outside and/or the cell surface, and viability of protease-treated cells was not affected. The combination of both experimental evidences and computational methods allowed us to determine that two of these proteins are putative extracellular new adhesins that had been previously attributed a wrong cytoplasmic function. One of them is a putative component of the pilus of this bacterium. Conclusion: We illustrate the complementary nature of laboratory-based and computational methods to examine in concert the localization of a set of proteins in the cell, and demonstrate the utility of this proteomics-based strategy to experimentally correct function annotation errors in sequencing projects. This approach also contributes to provide strong experimental evidences that can be used to annotate those proteins for which a Gene Ontology (GO) term has not been assigned so far. Function annotation correction would then improve the identification of surfaceassociated proteins in bacterial pathogens, thus accelerating the discovery of new vaccines in infectious disease research

Effects of indomethacin on ovarian leukocytes during the periovulatory period in the rat

We have investigated the effects of indomethacin (IM), a non-steroidal anti-inflammatory drug, and the role of prostaglandins on the accumulation of leukocytes in the rat ovary during the periovulatory period. Adult cycling rats were injected sc with 1 mg of IM in olive oil or vehicle on the morning of proestrus. Some animals were killed at 16:00 h in proestrus. On the evening (19:00 h) of proestrus, IM-treated rats were injected with 500 micrograms of prostaglandin E1 in saline or vehicle. Animals were killed at 01:30 and 09:00 h in estrus. There was an influx of macrophages, neutrophils, and eosinophils into the theca layers of preovulatory follicles, and of neutrophils and eosinophils into the ovarian medulla from 16:00 h in proestrus to 01:30 h in estrus. All these changes, except the accumulation of neutrophils in the theca layers of preovulatory follicles, were blocked by IM treatment. At 09:00 h in estrus, large clusters of neutrophils were observed in IM-treated rats, around abnormally ruptured follicles. The accumulation of leukocytes was not restored by prostaglandin supplementation, despite the inhibition of abnormal follicle rupture and restoration of ovulation in these animals. These results suggest that different mechanisms are involved in leukocyte accumulation in the ovary during the periovulatory period, and that the inhibitory effects of IM on the influx of leukocytes are not dependent on prostaglandin synthesis inhibition

Genetic and virulence-phenotype characterization of serotypes 2 and 9 of Streptococcus suis swine isolates

The aim of this study was to analyze the genetic characteristics and virulence phenotypes of Streptococcus suis, specifically, in clinical isolates of serotypes 2 and 9 (n = 195), obtained from diverse geographical areas across Spain. Pulsedfield gel electrophoresis (PFGE) typing identified 97 genetic profiles, 68% of which were represented by single isolates, indicative of a substantial genetic diversity among the S. suis isolates analyzed. Five PFGE profiles accounted for 33.3% of the isolates and were isolated from 38% of the herds in nine different provinces, indicative of the bacterium’s widespread distribution in the Spanish swine population. Representative isolates of the most prevalent PFGE profiles of both serotypes were subjected to multilocus sequence typing (MLST) analysis. The results indicated that serotypes 2 and 9 have distinct genetic backgrounds. Serotype 2 isolates belong to the ST1 complex, a highly successful clone that has spread over most European countries. In accordance with isolates of this complex, most serotype 2 isolates also expressed the phenotype MRP+EF+SLY+. Serotype 9 isolates belong to the ST61 complex, which is distantly related to the widespread European ST87 clone. Also, in contrast to most isolates of the European ST87 clone, which express the large variant MRP*, the majority of serotype 9 isolates (97.9%) did not express the protein. [Int Microbiol 2009; 12(3):161-166

Genetic and virulence-phenotype characterization of serotypes 2 and 9 of Streptococcus suis swine isolates

The aim of this study was to analyze the genetic characteristics and virulence phenotypes of Streptococcus suis, specifically, in clinical isolates of serotypes 2 and 9 (n = 195), obtained from diverse geographical areas across Spain. Pulsed-field gel electrophoresis (PFGE) typing identified 97 genetic profiles, 68% of which were represented by single isolates, indicative of a substantial genetic diversity among the S. suis isolates analyzed. Five PFGE profiles accounted for 33.3% of the isolates and were isolated from 38% of the herds in nine different provinces, indicative of the bacterium's widespread distribution in the Spanish swine population. Representative isolates of the most prevalent PFGE profiles of both serotypes were subjected to multilocus sequence typing (MLST) analysis. The results indicated that serotypes 2 and 9 have distinct genetic backgrounds. Serotype 2 isolates belong to the ST1 complex, a highly successful clone that has spread over most European countries. In accordance with isolates of this complex, most serotype 2 isolates also expressed the phenotype MRP(+)EF(+)SLY(+). Serotype 9 isolates belong to the ST61 complex, which is distantly related to the widespread European ST87 clone. Also, in contrast to most isolates of the European ST87 clone, which express the large variant MRP*, the majority of serotype 9 isolates (97.9%) did not express the protein

Combination of essential oils and antibiotics against Streptococcus suis: a preliminary study

Streptococcus suis is a major pathogen in the pig industry, associated with a wide variety of pigs diseases, such as meningitis, arthritis, bronchopneumonia, endocarditis, polyserositis and septicaemia. In addition, it is a zoonotic agent causing severe infections in people related with infected pigs or pork-derived products. The control of the disease is based on the antimicrobial therapy and sanitary measures, since there are not available commercial vaccines yet

Efficacy of biosecurity measures in the control of microorganisms associated to endometritis in sows. Preliminary study

Biosecurity can be defined as all the applied measurements that take as a target to minimize the sanitary risks in a stock farm, and include measurements related to the facilities and the management. The efficacy of these measurements must be reflected in a decrease of the microorganism in different productive phases. A study was carried out to evaluate if the set of applied measurements influences the microbial uterine contamination after farrowing of healthy sows. Two swine farms were been completed about biosecurity measurements was completed and a microbiological study of uterine swabs of sows after the farrowing was carried out. A total of 60 animals were studied, and 27 (45%, 95% CI [33.3%, 56.7%]) resulted positive. Significant differences between production and selection and multiplication farms were detected (OR = 3.44, IC 95%, 1.135-11.047). The colonization frequency was 65% CI [51.3%, 78.6%] and 35% CI [21%, 49%] in production and selection farm, respectively (P = 0.02). A total of 66 isolates were obtained, represented mainly by Staphylococcus spp. (33.33%) and Aerococcus spp. (27.27%), although other species included in the genus Streptococcus (9.09%), Enterococcus (6.06%) and Pseudomonas (4.55%), as well as different fungi species were also isolated. The frequency of isolation of different microorganisms was similar in both farms, with the exception of the genus Enterococcus that was not isolated in the production farm (P = 0.01). The questionnaire showed some differences in biosecurity measures in the selection and multiplication farm when it is compared to the production farm, which together with the increased uterine microbial contamination observed in the latter leads us to propose a preliminary hypothesis about the possible risk factors associated with this process, highlighting the absence of measures to avoid the presence of vectors and the establishment of strict protocols for cleaning and disinfectio

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

Seroprevalencia de las infecciones por el virus Diarrea Vírica Bovina en ganado bovino en Andalucía

Se ha realizado un estudio seroepidemiológico frente al virus de la Diarrea Vírica Bovina (vDVB) en la cabaña bovina andaluza, utilizando para ello un ELISA indirecto para la detección de anticuerpos frente a una proteína altamente conservada (p80). Después de eliminar los animales vacunados, la encuesta se realizó sobre 4.768 individuos pertenecientes a 227 colectivos no vacunados frente al vDVB, mediante muestreo estadístico para un nivel de confianza del 95 por ciento. La seropositividad obtenida ha sido del 42,3 por ciento de los individuos analizados, mientras que la prevalencia estimada de rebaños seropositivos alcanzó el 70,9 por ciento. La proporción de bovinos persistentemente infectados (IP) encontrada en la muestra (0,063 % de los individuos y 1,32 de los colectivos), ha sido más baja de la esperada en función de la alta seroprevalencia detectada, hecho que demuestra que la supervivencia de estos animales lógicamente está condicionada

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

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

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