Catia Silene Klein

Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products

Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see.

Mycoplasma hyopneumoniae type I signal peptidase: Expression and evaluation of its diagnostic potential

Type I signal peptidase (SPase I) is a membrane-anchored protease of the general secretory pathway, which is encoded by the sipS gene in Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia (PEP). In this study, the expression of the M. hyopneumoniae SPase I (MhSPase I) was analyzed in virulent and avirulent strains, and the recombinant protein (rMhSPase I), expressed in Escherichia coli, was evaluated regarding its potential as an immunodiagnostic antigen. It was demonstrated that the sipS coding DNA sequence (CDS) is most likely part of an operon, being co-transcribed along with four other CDSs. Quantitative reverse transcriptase PCR and immunoblot assays showed that MhSPase I is expressed by all three strains analyzed, with no transcriptional difference, but with evidence of a higher protein level in a pathogenic strain (7422), in comparison to another pathogenic (7448) and a non-pathogenic (J) strain. rMhSPase I was strongly immunogenic for mice, and the MhSPase I antigenicity was confirmed. Polyclonal serum anti-rMhSPase I presented no detectable cross-reaction with Mycoplasma flocculare and Mycoplasma hyorhinis. Moreover, phylogenetic analysis demonstrated a low conservation between MhSPase I and orthologous proteins from other porcine respiratory disease complex-related bacteria, Firmicutes and other Mycoplasma species. The potential of an rMhSPase I-based ELISA for PEP immunodiagnosis was demonstrated. Overall, we investigated the expression of sipS and the encoded MhSPase I in three M. hyopneumoniae strains and showed that this protein is a good antigen for use in PEP serodiagnosis and possibly vaccination, as well as a potential target for antibiotic development.

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.

First partial proteome of the poultry pathogen Mycoplasma synoviae

Mycoplasma synoviae is responsible for respiratory tract disease and synovitis in chickens and turkeys. In an attempt to identify the most prominent proteins expressed by this microorganism, a proteome map of M. synoviae was developed by using two-dimensional gel electrophoresis in combination with mass spectrometry. Based on the genome sequence of M. synoviae, a total of 30 different coding DNA sequences, including one hypothetical and one conserved-hypothetical protein, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The identified proteins were assigned according to the Clusters of Orthologous Groups of proteins functional classification. M. synoviae has 694 predicted CDSs. Overall, in this work 416 proteins spots were resolved in Coomassie Blue stained 2DE gels and were analyzed by mass spectrometry (MS). Altogether, we have achieved by MS the identification of 78 protein spots, corresponding to 30 different proteins. This is the first proteome map to be described in M. synoviae, and it is expected to be useful as a reference for comparative analysis.

Comparação de métodos de isolamento de bactérias NAD-dependentes do trato respiratório superior de suínos sadios

Secrecoes nasais, tonsilares e tecido tonsilar foram coletados de 67 leitoes de 9 a 15 semanas de idade, provenientes de tres rebanhos positivos para Actinobacillus pleuropneumoniae (App), e de 50 leitoes provenientes de dois rebanhos negativos. Foram classificados como positivos aqueles rebanhos com isolamento previo de sorotipos 3, 5 e 7 e rebanhos negativos aqueles submetidos a controle veterinario, sem notificacao de sintomas clinicos, lesoes de pleuropneumonia suina e sem isolamento do agente. O material coletado foi submetido a tres diferentes metodos de cultivo: 1- semeadura direta em meio de cultivo solido seletivo; 2- diluicao em caldo seletivo seguido de subsemeadura em meio de cultivo solido seletivo; 3- diluicao em caldo seletivo seguido de subsemeadura em agar sangue. Entre as amostras NAD-dependentes recuperadas 86 foram classificadas como App, 13 como grupo minor e 21 como grupo taxon (C, D, E e F). Dos rebanhos positivos foram recuperadas quatro amostras de App (sorotipos 3, 7 e 12) e 51 nao sorotipificaveis. Dos rebanhos negativos foram recuperadas 31 amostras de App nao sorotipificaveis, indicando que o App faz parte da flora normal do trato respiratorio superior dos suinos. O melhor metodo de isolamento de amostras NAD-dependentes de leitoes portadores foi da biopsia de tecido tonsilar semeado diretamente em meio solido seletivo (PPLO agar adicionado de 2m g de cristal violeta, 10m g NAD, 1m g de lincomicina, 1,4m g de bacitracina por ml).

DETECTION OF MYCOPLASMA HYOPNEUMONIAE BY POLYMERASE CHAIN REACTION IN SWINE PRESENTING RESPIRATORY PROBLEMS

Since Mycoplasma hyopneumoniae isolation in appropriate media is a difficult task and impractical for daily routine diagnostics, Nested-PCR (N-PCR) techniques are currently used to improve the direct diagnostic sensitivity of Swine Enzootic Pneumonia. In a first experiment, this paper describes a N-PCR technique optimization based on three variables: different sampling sites, sample transport media, and DNA extraction methods, using eight pigs. Based on the optimization results, a second experiment was conducted for testing validity using 40 animals. In conclusion, the obtained results of the N-PCR optimization and validation allow us to recommend this test as a routine monitoring diagnostic method for Mycoplasma hyopneumoniae infection in swine herds.

Pasteurella multocida tipo A como agente primário de pneumonia e septicemia em suínos

In order to understand better the pathological aspects and spread of Pasteurella multocida type A as the primary cause of pneumonia in pigs, was made an experiment with intranasal inoculation of different concentrations of inocula [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml], using two pigs per group. The pigs were obtained from a high health status herd. Pigs were monitored clinically for 4 days and subsequently necropsied. All pigs had clinical signs and lesions associated with respiratory disease. Dyspnoea and hyperthermia were the main clinical signs observed. Suppurative cranioventral bronchopneumonia, in some cases associated with necrosuppurative pleuropneumonia, fibrinous pericarditis and pleuritic, were the most frequent types of lesion found. The disease evolved with septicaemia, characterized by septic infarctions in the liver and spleen, with the detection of P. multocida type A. In this study, P. multocida type A strain #11246 was the primary agent of fibrinous pleuritis and suppurative cranioventral bronchopneumonia, pericarditis and septicaemia in the pigs. All concentrations of inoculum used (105-108 CFU/ml) were able to produce clinical and pathological changes of pneumonia, pleuritis, pericarditis and septicemia in challenged animals.

Comparison of methods for the isolation of NAD-dependent bacteria from the upper respiratory tract of healthy pigs.

Secrecoes nasais, tonsilares e tecido tonsilar foram coletados de 67 leitoes de 9 a 15 semanas de idade, provenientes de tres rebanhos positivos para Actinobacillus pleuropneumoniae (App), e de 50 leitoes provenientes de dois rebanhos negativos. Foram classificados como positivos aqueles rebanhos com isolamento previo de sorotipos 3, 5 e 7 e rebanhos negativos aqueles submetidos a controle veterinario, sem notificacao de sintomas clinicos, lesoes de pleuropneumonia suina e sem isolamento do agente. O material coletado foi submetido a tres diferentes metodos de cultivo: 1- semeadura direta em meio de cultivo solido seletivo; 2- diluicao em caldo seletivo seguido de subsemeadura em meio de cultivo solido seletivo; 3- diluicao em caldo seletivo seguido de subsemeadura em agar sangue. Entre as amostras NAD-dependentes recuperadas 86 foram classificadas como App, 13 como grupo minor e 21 como grupo taxon (C, D, E e F). Dos rebanhos positivos foram recuperadas quatro amostras de App (sorotipos 3, 7 e 12) e 51 nao sorotipificaveis. Dos rebanhos negativos foram recuperadas 31 amostras de App nao sorotipificaveis, indicando que o App faz parte da flora normal do trato respiratorio superior dos suinos. O melhor metodo de isolamento de amostras NAD-dependentes de leitoes portadores foi da biopsia de tecido tonsilar semeado diretamente em meio solido seletivo (PPLO agar adicionado de 2m g de cristal violeta, 10m g NAD, 1m g de lincomicina, 1,4m g de bacitracina por ml).

Aspectos patológicos e microbiológicos das doenças respiratórias em suínos de terminação no Brasil

For pathological and microbiological evaluation of porcine respiratory disease in fattening pigs, seventy five animals showing respiratory distress, fever and/or cough were analyzed. These pigs were necropsied and samples were collected for histological and microbiological analysis. Bacterial isolation procedures were performed aiming to detect major swine bacterial respiratory pathogens. Also, PCR for Mycoplasma hyorhinis, and immunohistochemistry for Influenza A, porcine circovirus type 2, and Mycoplasma hyopneumoniae were carried out. Mycoplasma hyopneumoniae and Pasteurella multocida type A were the most prevalent infectious agents. The antimicrobial sensitivity of 24 samples of P. multocida type A was evaluated by minimum inhibitory concentration tests and all these samples were sensitive to doxycycline, tilmicosin and enrofloxacin. Suppurative bronchopneumonia and pleuritis were main respiratory lesions found. When P. multocida type A was present, the extension of lung lesions was increased. In 58% of the samples more than one infectious agent was identified, suggesting a high prevalence of infectious agents associations in porcine respiratory disease in Brazil.

Extracellular Proteins of Mycoplasma synoviae

Mycoplasma synoviae is a Gram positive bacteria lacking of cell wall that affects chickens and turkeys causing infection in the upper respiratory tract and in some cases arthritis, with economical impact to broiler breeders. Treatment and prevention of avian synovitis depend on knowledge of the infectious process. Secreted or surface-exposed proteins play a critical role in disease because they often mediate interactions between host and pathogen. In the present work, we sought to identify possible M. synoviae secreted proteins by cultivating the bacteria in a modified protein-free Frey medium. Using this approach, we were able to detect in the cell-free fraction a number of proteins that have been shown in other organisms to be secreted, suggesting that they may also be secreted by M. synoviae.