Pasteurella multocida polyserositis in growing-finishing pigs.

Pasteurella multocida is the main secondary bacterium isolated from cases of swine pneumonia. Although highly pathogenic strains of P. multocida have been associated with primary septic lesions and polyserositis in pigs, studies on this pathological presentation in naturally occurring cases are limited. The aim of this work was to characterize the clinical, pathological and molecular findings in cases of P. multocida polyserositis in growing-finishing pigs in a commercial farm in Brazil. The mean age of 17 investigated pigs was 120 days. Clinically, the disease was acute (11/17), with clinical signs of dyspnoea and apathy. Sudden death occurred in some animals (6/17). The main gross findings included fibrinous serositis affecting the abdominal and thoracic cavities (17/17), fibrinous pericarditis (15/17), marked cranioventral pulmonary consolidation (17/17) and splenic infarcts (3/17). P. multocida was isolated in all cases from systemic sites, including the pericardial sac and abdominal exudate. Molecular typing of genus and species was performed on four isolates, and all were characterized as P. multocida type A. Another five isolates were positive for the pathogenicity marker gene pfhA by polymerase chain reaction. This study reinforces the role of P. multocida as a cause of polyserositis in growing-finishing pigs.

Re-Emergence of Salmonellosis in Hog Farms: Outbreak and Bacteriological Characterization.

Clinical salmonellosis has been increasing significantly in Brazil in recent years. A total of 130 outbreaks distributed among 10 swine-producing states were investigated. One representative Salmonella isolate from each outbreak was characterized through serotyping, antimicrobial resistance profiles, PFGE, and WGS. From 130 outbreaks: 50 were enteric, 48 were septicemic, 17 cases were characterized as hepato-biliary invasive, 13 as nodal and two were not classified. The most prevalent serovars were a monophasic variant of S. typhimurium (55/130), Choleraesuis (46/130), and Typhimurium (14/130). Most of the strains (86.92%) demonstrated a high rate of multi-drug resistance. The identification of a major Choleraesuis clonal group in several Brazilian states sharing the same resistance genes suggested that these strains were closely related. Six strains from this clonal group were sequenced, revealing the same ST-145 and 11 to 47 different SNPs. The detected plasmid type showed multiple marker genes as RepA_1_pKPC-CAV1321, the first to be reported in Salmonella. All AMR genes detected in the genomes were likely present on plasmids, and their phenotype was related to genotypic resistance genes. These findings reveal that salmonellosis is endemic in the most important pig-producing states in Brazil, emphasizing the need to make data available to aid in reducing its occurrence.

Survey of Campylobacter in foods implicated in foodborne diseases in Southern Brazil.

Campylobacter is not routinely tested in foodborne disease investigations in Brazil. Here, we studied the occurrence of Campylobacter among other food-related bacteria commonly found in foodborne disease outbreaks reported in Rio Grande do Sul State, Southern Brazil. Seventy-two food samples were analyzed by using culture-based detection methods during the 18-month investigation of 36 foodborne disease outbreaks. The sampled foods from the foodborne disease outbreaks were all negative for Campylobacter . However, at least one of other routinely investigated foodborne-related bacteria was detected in 29.17% (21/72) of the samples. Taken together, these results suggest the need to monitor Campylobacter in foodborne diseases to detect sporadic cases caused by Campylobacter that might go unnoticed in Rio Grande do Sul.

Detecting Infectious Bursal Disease Using a VP1 Gene-Based RT-qPCR Assay Compared to Standard Methods of Virus Isolation, ELISA, and Histopathology.

Infectious bursal disease (IBD) is an immunosuppressive viral disease of chickens, associated with severe economic losses and major threats to poultry production worldwide. Disease prevention programs rely on unequivocal identification of the pathogen, as well as vaccination programs. This study developed a sensitive, one-step, real-time, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay using a hydrolysis probe system for infectious bursal disease virus (IBDV, VP1 gene) detection and quantification, which was compared to other routinely used diagnostic methods. The assay successfully detected IBD reference viruses and field isolates. The absence of cross-reactivity was detected with negative samples or with other avian viruses in the analytical specificity test. The detection limit of this assay was 70 RNA copies. RT-qPCR was more sensitive in the detection of serially diluted IBDV isolates compared to virus isolation. For clinical samples, the sensitivity and specificity values of RT-qPCR compared to enzyme-linked immunosorbent assay (ELISA) were 97.5% and 100%, respectively, and compared to histopathology, these values were 100% and 93.94%, respectively. RT-qPCR can provide a simple and reliable assay for IBDV surveillance programs and for evaluation of control strategies.

Longitudinal study reveals persistent environmental Salmonella Heidelberg in Brazilian broiler farms.

The vast capacity for maintenance and dissemination in the environment are major challenges for the control of Salmonella spp. in poultry farms. The aim of this study was to assess environmental contamination by non-typhoidal Salmonella in successive broiler flocks in nine commercial broiler farms integrated with three companies in the south of Brazil, for a twelve-month production period. Recycled broiler litter, feed and swabs from the evaporative cooling system pads were analyzed, and the total enterobacteria count in the litter samples was ascertained. Positive broiler houses were identified in two of the three broiler companies studied, in which non-typhoidal Salmonella were detected for the first time in the first or second flock, and recurred in the recycled litter of subsequent flocks. Feed and evaporative cooling pad swab samples were also positive in at least one of the assessed flocks. The majority of the isolates (87.5%) originating from different flocks, broiler houses and companies that were sampled were identified as S. Heidelberg, with the prevalence of one single genotype. The total enterobacteria levels in the litter diminished as the flocks progressed, but the presence of Salmonella spp. was constant over the course of time, indicating that the litter management procedures were not capable of interrupting the cycle of residual contamination. The predominance of S. Heidelberg highlights its emergence and dissemination in this region, as well as its resistance and maintenance in the environment, and reinforces the need to improve prevention and recycled litter management measures.

Molecular serotyping of clinical strains of Haemophilus (Glaesserella) parasuis brings new insights regarding Glässer's disease outbreaks in Brazil.

Glässer's disease (GD) is an important infectious disease of swine caused by Haemophilus (Glaesserella) parasuis. Vaccination with inactivated whole cell vaccines is the major approach for prevention of H. parasuis infection worldwide, but the immunity induced is predominantly against the specific polysaccharide capsule. As a consequence, the available vaccines may not induce adequate protection against the field strains, when the capsules present in the vaccine strains are different from those in strains isolated from the farms. Therefore, it is crucial to map H. parasuis serovars associated with regional outbreaks so that appropriate bacterin vaccines can be developed and distributed for prevention of infection. In this study, 459 H. parasuis field strains isolated from different Glässer's disease outbreaks that occurred in 10 different Brazilian States were analyzed for serotype using PCR-based approaches. Surprisingly, non-typeable (NT) strains were the second most prevalent group of field strains and along with serovars 4, 5 and 1 comprised more than 70% of the isolates. A PCR-based approach designed to amplify the entire polysaccharide capsule locus revealed 9 different band patterns in the NT strains, and 75% of the NT strains belonged to three clusters, suggesting that a number of new serovars are responsible for a substantial proportion of disease. These results indicate that commercially available vaccines in Brazil do not cover the most prevalent H. parasuis serovars associated with GD.

Antimicrobial susceptibility profile of historical and recent Brazilian pig isolates of Pasteurella multocida / Perfil de suscetibilidade a antimicrobianos de isolados históricos e contemporâneos de Pasteurella multocida de suínos no Brasil

Pasteurella (P.) multocida is the causative agent of pneumonic pasteurellosis in swine, which is commonly associated with the final stages of enzootic pneumonia or porcine respiratory disease complex. Although this syndrome is one of the most common and important diseases of pigs, data on antimicrobial susceptibility of P. multocida isolates are uncommon in Brazil. Therefore, the present study was carried out to determine and to compare antimicrobial susceptibility profile of Brazilian P. multocida isolated from pigs with lesions of pneumonia or pleuritis during two-time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were used to determine the MIC of amoxicillin, enrofloxacin, florfenicol and tetracycline by microbroth dilution. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Multi-drug resistance (MDR) to amoxicillin/florfenicol/tetracycline was observed in 6% of recent isolates. There was a significant increase (p˂0.05) in resistance for amoxicillin and enrofloxacin in recent isolates compared with historic isolates (3.8% and 18%, respectively), most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. The results of this study showed an increase of isolates resistant to important drugs used in treatment of P. multocida infections in pigs, demonstrating the need for the implementation of rational use of antimicrobials in Brazilian swine industry.(AU)

Pasteurella (P.) multocida é o agente da pasteurelose pneumônica em suínos, a qual é comumente associada com o estágio final da pneumonia enzoótica suína ou complexo das doenças respiratórias dos suínos. Apesar de ser uma das doenças mais comuns e importantes na suinocultura, dados sobre suscetibilidade antimicrobiana de isolados de P. multocida são raros no Brasil. Dessa forma, o presente estudo foi realizado para determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. Isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) foram usados para determinar a concentração inibitória mínima (CIM) de amoxicilina, enrofloxacina, florfenicol e tetraciclina através do teste de microdiluição em caldo. Florfenicol apresentou o menor nível de resistência para ambos os isolados históricos e contemporâneos (0% e 6%, respectivamente), enquanto que tetraciclina apresentou o maior nível de resistência (20.5% e 34%, respectivamente). Resistência a múltiplos antimicrobianos (amoxicilina, florfenicol e tetraciclina) foi observada em 6% dos isolados recentes. Foi observado aumento significativo (p˂0.05) na resistência a amoxicilina e enrofloxacina em isolados recentes comparado com isolados históricos (3.8% e 18%, respectivamente), provavelmente devido à pressão de seleção de antimicrobianos usados no tratamento e prevenção de infecções causadas por P. multocida. Os resultados deste trabalho demostraram o aumento de isolados resistentes a importantes drogas utilizadas no tratamento de infecções causadas por P. multocida em suínos, evidenciando a necessidade da implementação do uso racional de antimicrobianos na suinocultura brasileira.(AU)

Impact of treatments for recycled broiler litter on the viability and infectivity of microorganisms.

The microbiological risk of recycled litter depends on the efficacy of the management system applied to inactivate residual microorganisms and preserve the health of the successive broiler flock. This study aimed to assess the viability and infectivity of the Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV) and Salmonella Heidelberg in recycled litter exposed to different treatments. The litter was contaminated with microorganisms and submitted to the treatments (T): T1: shallow fermentation; T2: quicklime (calcium oxide); T3: shallow fermentation followed by addition of quicklime; T4: no treatment. Sentinel chicks housed on the treated litter showed that T1 and T3 inactivated residual IBDV. Analysis of the litter subjected to T1 also showed reduced levels of total enterobacteria. T2 was not able to reduce the microorganisms assessed and its association with T1 (T3) failed to enhance the effect of the treatment. NDV did not survive in the broiler litter, regardless of the treatment applied, and it was also not detected in the sentinel chicks. S. Heidelberg remained viable in the litter submitted to all studied treatments, being isolated from the sentinel chicks of all the experimental groups. The antimicrobial activity of T1 and T3 was associated to higher ammonia contents in the broiler litter. The results indicate that the shallow fermentation treatment is efficient for controlling residual IBDV and total enterobacteria in the recycled litter.

Pasteurella multocida type A as the primary agent of pneumonia and septicaemia in pigs / 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...

Para entender melhor os aspectos patológicos e disseminação de Pasteurella multocida tipo A como causa primária de pneumonia em suínos foi realizado um experimento com inoculação intranasal de diferentes concentrações de inóculos [Grupo (G1): 108 Unidades Formadoras de Colônias (UFC)/ml; G2: 107 UFC/ml; G3: 106 UFC/ml e G4: 105 UFC/ml], usando dois suínos por grupo. Esses suínos foram obtidos de um rebanho de alto status sanitário. Os animais foram monitorados clinicamente por quarto dias e subsequentemente necropsiados. Todos os suínos apresentaram sinais clínicos e lesões associadas com doença respiratória. Dispneia e hipertermia foram os principais sinais clínicos observados. Broncopneumonia cranioventral supurativa, em alguns casos associados com pleuropneumonia necrossupurativa, pleurites e pericardite fibrinosa foram mais frequentes. A doença evoluiu com septicemia, caracterizada por infartos sépticos no fígado e baço, com detecção de P. multocida. Neste estudo, P. multocida tipo A isolado 11246 foi agente primário de pleurite fibrinosa e broncopneumonia cranioventral supurativa, pericardite fibrinosa e septicemia em suínos. Todas as concentrações de inóculo utilizado (105-108 UFC/ml) foram capazes de produzir sinais clínicos e patológicos de alterações de pneumonia, pleurites, pericardites e septicemia nos animais...

Aspectos patológicos e microbiológicos das doenças respiratórias em suínos de terminação no Brasil / Pathological and microbiological aspects of respiratory disease in fattening pigs in Brazil

Para avaliação dos aspectos patológicos e microbiológicos de casos clínicos de doenças respiratórias em suínos de terminação foram analisados 75 suínos doentes oriundos de 36 lotes. Suínos que apresentavam sinais clínicos respiratórios evidentes foram necropsiados para avaliação macroscópica e colheita de amostras para análise histopatológica e microbiológica. Foram realizados testes de isolamento bacteriano para as principais bactérias do sistema respiratório dos suínos, PCR para Mycoplasma hyorhinis, imuno-histoquímica para Influenza A, Circovirus suíno tipo 2 e Mycoplasma hyopneumoniae. A sensibilidade antimicrobiana de 24 amostras de Pasteurella multocida tipo A foi avaliada por testes de concentração inibitória mínima para os principais antimicrobianos utilizados em suinocultura. Mycoplasma hyopneumoniae e Pasteurella multocida tipo A foram os agentes infecciosos mais prevalentes. Broncopneumonia supurativa e pleurite foram as principais lesões respiratórias encontradas. Pasteurella multocida tipo A, quando presente, aumentou a extensão das lesões pulmonares. Todas as amostras de Pasteurella multocida testadas foram sensíveis aos antimicrobianos Doxiciclina, Enrofloxacina e Tilmicosina. Em 58% das amostras foi identificado mais de um agente infeccioso, evidenciando a alta prevalência da associação de agentes nas doenças respiratórias de suínos em terminação...

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...

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.

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.