RESUMO
Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.
Assuntos
Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Mycoplasma hyopneumoniae/imunologia , Pneumonia Suína Micoplasmática/prevenção & controle , Adjuvantes Imunológicos , Administração Oral , Animais , Biópsia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/metabolismo , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imuno-Histoquímica , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Mycoplasma hyopneumoniae/classificação , Mycoplasma hyopneumoniae/genética , Pneumonia Suína Micoplasmática/microbiologia , Pneumonia Suína Micoplasmática/patologia , Reação em Cadeia da Polimerase em Tempo Real , Dióxido de Silício , Suínos , Resultado do Tratamento , Vacinação/métodosRESUMO
In bacteria, the biosynthesis of the cofactor flavin adenine dinucleotide (FAD), important in many physiological responses, is catalyzed by the bifunctional enzyme FAD synthase (FADSyn) which converts riboflavin into FAD by both kinase and adenylylation activity. The in silico 3D structure of a putative FADSyn from Mycoplasma hyopneumoniae (MhpFADSyn), the etiological agent of enzootic pneumonia was already reported, nevertheless, the in vitro functional characterization was not yet demonstrated. Our phylogenetic analysis revealed that MhpFADSyn is close related to the bifunctional FADSyn from Corynebacterium ammoniagenes. However, only the domain related to adenylylation was assigned by InterPro database. The activity of MhpFADSyn was evaluated through in vitro enzymatic assays using cell extracts from IPTG-inducible heterologous expression of MhpFADSyn in Escherichia coli. The flavoproteins were analyzed by HPLC and results showed that IPTG-induced cell lysate resulted in the formation of twofold increased amounts of FAD if compared to non IPTG-induced cells. Consumption of riboflavin substrate was also threefold greater in IPTG-induced lysate compared to non IPTG-induced cell extract. Thus, the recombinant MhpFADSyn protein could be associated to FAD biosynthesis. These findings contribute to expand the range of potential drug targets in diseases control and unveil metabolic pathways that could be attribute to mycoplasmas.
Assuntos
Mycoplasma hyopneumoniae/enzimologia , Nucleotidiltransferases/metabolismo , Escherichia coli/genética , Mycoplasma hyopneumoniae/classificação , Nucleotidiltransferases/genética , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Mycoplasma hyopneumoniae causes enzootic pneumonia (EP) in swine, a disease related to high economic losses in production systems. Epidemiological spread of M. hyopneumoniae clones was studied by multi-locus sequence typing (MLST) in several swine production regions but so far not in South America. Using MLST, we have therefore investigated M. hyopneumoniae clones circulating in farms from three main swine production regions in Brazil. Porcine lungs samples were collected between 2015 and 2016 in farms with EP outbreaks. Three geographically distant regions were selected, and 67 M. hyopneumoniae positive samples, each one from a different farm, were included in the study. The occurrence of five sequence types (ST) was demonstrated and the majority of the samples were identified as ST-69 (nâ¯=â¯60; 89.5%), followed by ST-70 (nâ¯=â¯3; 4.5%), ST-123 (nâ¯=â¯2; 3%), ST-124 (nâ¯=â¯1; 1.5%) and ST-127 (nâ¯=â¯1; 1.5%). There was no association of any specific ST with region or production system. The five STs were all new ones, probably representing unique Brazilian clones. ST-69 and ST-70 on one side and ST-123 and ST-124 on the other side are phylogenetically close, while ST-127 is singleton. In conclusion, our results showed a low variability and high clonality of M. hyopneumoniae genotypes from Brazilian farms affected by EP.
Assuntos
Mycoplasma hyopneumoniae/classificação , Mycoplasma hyopneumoniae/genética , Pneumonia Suína Micoplasmática/microbiologia , Animais , Brasil , Células Clonais , Fazendas , Variação Genética , Genótipo , Pulmão/microbiologia , Filogenia , Suínos/microbiologiaRESUMO
Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections.
Assuntos
Sistema Calicreína-Cinina , Mycoplasma hyopneumoniae/enzimologia , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Concentração de Íons de Hidrogênio , Cinética , Mycoplasma hyopneumoniae/classificação , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Filogenia , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Este trabalho teve como objetivo pesquisar a associação do PCV2 e M. Hyopneumoneae em amostras de pulmão de suínos. 32 fragmentos de pulmões, sendo 16 com lesões macroscópicas (CLM) e 16 sem lesões macroscópicas (SLM) foram analisados pela histopatologia, pelo teste de imuno-histoquímica (IHC) e pela reação de polimerase em cadeia em tempo real (qPCR). Não foi observada correlação positiva entre a histopatologia e a imuno-histoquímica. Não houve também, correlação positiva entre número de amostras com lesões características de pneumonia viral e a carga viral de PCV2 medida por qPCR. Analisando os dados obtidos da detecção dos agentes e lesões microscópicas associadas, constatou-se que em 4 amostras analisadas (12,5%) houve uma co-infecção entre o PCV2 e M. Hyopneumoneae. Este resultado demonstra uma possível associação deste dois agentes. Novos estudos deverão ser realizados para determinação de outros possíveis patógenos envolvidos na patogenia da PRDC na região da Zona da Mata Mineira.
The aim of this study was determine the association between PCV2 and M. Hyopneumoneae in pig lung samples. Thirty two lung fragments, from which 16 had macroscopic lesions (CLM) and the other 16 had no macroscopic lesions (SLM), were analyzed by histopathology, immunohistochemistry assays and polymerase chain reaction in real time (qPCR). No positive correlation was observed among the histology and the immunohistochemistry assay (IHC). There was also no positive correlation between the number of samples with viral pneumonia type of lesions and PCV2 viral load measured by qPCR. Analysis of the data obtained by detection of agents and associated microscopic findings showed that PCV2 and M. Hyopneumoneae co-infection was found in four samples (12,5%). This result reveals a possible association of these two agents. Further studies are required to determine additional potential pathogens involved in the pathogenesis of the PRDC in the Mata Mineira region, Brazil.
Assuntos
Coinfecção/classificação , Coinfecção/diagnóstico , Coinfecção/veterinária , Circovirus , Mycoplasma hyopneumoniae/classificação , Mycoplasma hyopneumoniae/virologiaRESUMO
Este trabalho teve como objetivo pesquisar a associação do PCV2 e M. Hyopneumoneae em amostras de pulmão de suínos. 32 fragmentos de pulmões, sendo 16 com lesões macroscópicas (CLM) e 16 sem lesões macroscópicas (SLM) foram analisados pela histopatologia, pelo teste de imuno-histoquímica (IHC) e pela reação de polimerase em cadeia em tempo real (qPCR). Não foi observada correlação positiva entre a histopatologia e a imuno-histoquímica. Não houve também, correlação positiva entre número de amostras com lesões características de pneumonia viral e a carga viral de PCV2 medida por qPCR. Analisando os dados obtidos da detecção dos agentes e lesões microscópicas associadas, constatou-se que em 4 amostras analisadas (12,5%) houve uma co-infecção entre o PCV2 e M. Hyopneumoneae. Este resultado demonstra uma possível associação deste dois agentes. Novos estudos deverão ser realizados para determinação de outros possíveis patógenos envolvidos na patogenia da PRDC na região da Zona da Mata Mineira.(AU)
The aim of this study was determine the association between PCV2 and M. Hyopneumoneae in pig lung samples. Thirty two lung fragments, from which 16 had macroscopic lesions (CLM) and the other 16 had no macroscopic lesions (SLM), were analyzed by histopathology, immunohistochemistry assays and polymerase chain reaction in real time (qPCR). No positive correlation was observed among the histology and the immunohistochemistry assay (IHC). There was also no positive correlation between the number of samples with viral pneumonia type of lesions and PCV2 viral load measured by qPCR. Analysis of the data obtained by detection of agents and associated microscopic findings showed that PCV2 and M. Hyopneumoneae co-infection was found in four samples (12,5%). This result reveals a possible association of these two agents. Further studies are required to determine additional potential pathogens involved in the pathogenesis of the PRDC in the Mata Mineira region, Brazil.(AU)