RESUMO
This study attempted to determine whether one multiplex polymerase chain reaction (PCR) is an effective adjunct method for diagnosing Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infection, and whether M. hyorhinis should be considered as an enzootic pneumonia or porcine respiratory disease complex pathogen in Taiwan. To our knowledge, this study is the first to isolate and identify M. hyorhinis as a porcine pathogen in Taiwan. A novel isolation method and a multiplex PCR test were applied to detect and isolate M. hyorhinis. The correlation of M. hyorhinis with swine pneumonia was also examined using a challenge test. Based on weight, 18 pigs were assigned to three groups and housed throughout the study in a specific-pathogen-free (SPF) facility and provided with aseptic feed and water. Groups 1 (n=6) and 2 (n=6) were challenged with 5mL M. hyorhinis culture via tracheal intubation on day 1. The M. hyorhinis strains ATIT-1, -3, and-7 were used to infect group 1 and the strain ATCC 27717 was used for group 2. Culture medium was replaced by phosphate-buffered saline in group 3 (n=6). All pigs were slaughtered on day 28, and their lungs were removed for examination of lesions. Of the six pigs in group 1 challenged with wild-type strains, two had typical mycoplasma pneumonia lesions. No gross lung lesions were observed in groups 2 and 3. Although further examination is necessary to confirm that wild-type strains can cause pneumonia, it appears that M. hyopneumoniae is no longer the only mycoplasma pathogen implicated in the diagnosis of swine enzootic pneumonia (SEP).
Assuntos
Mycoplasma/isolamento & purificação , Mycoplasma/patogenicidade , Pneumonia por Mycoplasma/veterinária , Reação em Cadeia da Polimerase/veterinária , Doenças dos Suínos/diagnóstico , Animais , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Diagnóstico Diferencial , Pulmão/microbiologia , Pulmão/patologia , Mycoplasma/classificação , Mycoplasma/genética , Mycoplasma hyopneumoniae/isolamento & purificação , Mycoplasma hyopneumoniae/patogenicidade , Pneumonia Suína Micoplasmática/diagnóstico , Pneumonia Suína Micoplasmática/microbiologia , Pneumonia por Mycoplasma/diagnóstico , Pneumonia por Mycoplasma/epidemiologia , Pneumonia por Mycoplasma/microbiologia , Reação em Cadeia da Polimerase/métodos , Distribuição Aleatória , Sensibilidade e Especificidade , Organismos Livres de Patógenos Específicos , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/microbiologia , Taiwan/epidemiologiaRESUMO
Mutations in the fimH gene of Salmonella typhimurium result in a non-fimbriate, non-adhesive phenotype. This phenotype was shown to be due to the lack of both fimH and fimF expression since disruption of the fimH gene by insertion of a DNA cassette into this determinant results in mutants that are complemented by plasmids carrying both fimH and fimF. Deletion mutations within the S. typhimurium fimH gene carried on a recombinant plasmid can be used to complement the mutant, and these transformants are non-adhesive but fully fimbriate, consistent with the role of FimH as being necessary for fimbrial adhesin expression. Adherence to erythrocytes, HeLa, and Hep-2 cells is associated with expression of the FimH polypeptide, and fimbriate strains that cannot synthesize FimH are non-adhesive. Discrete differences in the amino acid sequences of the adhesive type 1 and the non-hemagglutinating type 2 FimH polypeptides were detected, and are most likely responsible for the differences in hemagglutinating activity.
Assuntos
Adesinas Bacterianas/genética , Adesinas de Escherichia coli , Aderência Bacteriana/genética , Proteínas de Fímbrias , Fímbrias Bacterianas/química , Salmonella typhimurium/genética , Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/química , Sequência de Aminoácidos , Aderência Bacteriana/fisiologia , Sequência de Bases , Southern Blotting , Linhagem Celular , DNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica , Células HeLa , Testes de Hemaglutinação , Humanos , Dados de Sequência Molecular , Família Multigênica/genética , Mutagênese Insercional , Fenótipo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/química , Salmonella typhimurium/ultraestrutura , Análise de Sequência de DNARESUMO
This study evaluated the suitability of fimY gene amplification by PCR as an effective means of detecting Salmonella species. Although fimY gene of Salmonella typhimurium is involved in regulating type 1 fimbrial expression, the amino acid sequence of FimY shares very little homology with other known prokaryotic proteins in the GenBank database. Therefore, fimY is a promising target gene to detect the presence of Salmonella species. Herein, two primers internal to the fimY gene of S. typhimurium are used to investigate the distribution of the fimY homologous sequence among 45 Salmonella serovars and 20 non-Salmonella species by using PCR. Experimental results indicated that only Salmonella species possessed the fimY homologous sequence, subsequently generating the specific 526-bp DNA fragments. The sensitivity of the fmY-specific primer set was demonstrated on a Salmonella-free swab sample from a pork carcass surface, which was then artificially contaminated with different concentrations of S. typhimurium. A combining of pre-enrichment step in buffered peptone water and PCR amplification of fimY allowed the detection of S. typhimurium at the concentration of 3.4 x 10(0) CFU/ml from the swab sample. With an additional enrichment step in Rappaport-Vassiliadis (RV) broth, this procedure can also detect pork carcass surface naturally contaminated with Salmonella species in a slaughterhouse. Results in this study demonstrate that fimY is unique to Salmonella species and is an appropriate PCR target for detecting these microorganisms.
Assuntos
Proteínas de Bactérias/análise , DNA Bacteriano/análise , Proteínas de Ligação a DNA , Microbiologia de Alimentos , Reação em Cadeia da Polimerase/métodos , Salmonella typhimurium/genética , Fatores de Transcrição/análise , Amplificação de Genes , Filogenia , Salmonella typhimurium/classificação , Salmonella typhimurium/isolamento & purificação , Homologia de Sequência do Ácido NucleicoRESUMO
The efficacy of Mycoplasma hyopneumoniae oral vaccine was investigated in microsphere dosage form. A co-spray drying process was used to apply an encapsulating material, Eudragit L30 D-55, to microspheres containing Mycoplasma hyopneumoniae antigens. The microspheres were generally effective (>93%) with protein release at pH 7.4, but almost none were released at pH 1.2, for 3 hr in an in vitro dissolution test. An SPF-swine model was used to evaluate the effectiveness of the microspheres as an oral vaccine, and the related immune responses. The serum's systemic IgG against M. hyopneumoniae was evoked by ELISA analysis, after a 2nd immunization of all pigs. The vaccinated groups' mean lesion score was significantly lower after the Mycoplasma hyopneumoniae challenge than that of the nonvaccinated/challenged groups (P<0.05). This study strongly suggests that the oral microspheres vaccine prepared by a co-spray drying method can provide effective protection against M. hyopneumoniae infection in pigs.
Assuntos
Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Infecções por Mycoplasma/prevenção & controle , Infecções por Mycoplasma/veterinária , Mycoplasma/imunologia , Doenças dos Suínos/prevenção & controle , Vacinação/veterinária , Administração Oral , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Formas de Dosagem , Composição de Medicamentos , Pulmão/microbiologia , Pulmão/patologia , Microesferas , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/patologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/patologia , Fatores de TempoRESUMO
The pH 6 antigen of Yersinia pestis is a virulence protein whose gene, psaA, is positively regulated at the transcriptional level by low pH, mammalian temperature, and an upstream locus, psaE. Low pH appears to be required for initial psaA transcription, although increased temperature is necessary for full expression of the gene. In addition, psaA is monocistronic and its transcript has a relatively long 5' nontranslated region.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Transcrição Gênica , Yersinia pestis/genética , Proteínas de Bactérias/imunologia , Sequência de Bases , Northern Blotting , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Yersinia pestis/imunologiaRESUMO
The Salmonella typhimurium fimA gene is controlled by several ancillary fim genes. One of these genes, fimZ, appears to be involved in increasing the expression of fimA. A fimZ mutant of S. typhimurium was constructed by allelic exchange, and this mutant was found to be nonfimbriate. The fimZ mutant demonstrated decreased levels of fimA expression compared with the parental strain when both were grown under conditions favoring fimbrial expression. An examination of the predicted amino acid sequence, deduced from the nucleotide sequence of fimZ, indicated that the FimZ polypeptide possessed a DNA binding motif. Bacterial lysates, derived from strains transformed with recombinant plasmids possessing a fimZ gene, demonstrated DNA binding activity with a fragment containing the fimA promoter. Lysates without a FimZ polypeptide did not exhibit any binding activity. These data are consistent with FimZ being a transcriptional activator of fimA, and FimZ acts by binding to the promoter region.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias , Mutação , Salmonella typhimurium/genética , Proteínas de Bactérias/biossíntese , Bacteriólise , Sítios de Ligação , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Pili Sexual , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Salmonella typhimurium/classificação , Sorotipagem , Especificidade da EspécieRESUMO
Bacteria in a nonfimbriate phase because of continuous aeration of liquid cultures produce FimA in amounts similar to those produced by fimbriate bacteria. However, relatively low FimA production was observed in nonfimbriate-phase cultures obtained by growth on solid media or by anaerobic incubation. Regardless of the fimbrial phase of Salmonella typhimurium, the fimA promoter region was always oriented in the direction that might allow fimA transcription.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Fímbrias , Regulação Bacteriana da Expressão Gênica/genética , Pili Sexual/fisiologia , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Anaerobiose , Proteínas de Bactérias/genética , Sequência de Bases , Inversão Cromossômica , DNA Bacteriano/genética , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas/genética , Salmonella typhimurium/genéticaRESUMO
Using formalin inactivated Actinobacillus pleuropneumoniae antigens and aqueous ethylcellulose dispersions, microspheres of oral vaccines were developed by a co-spray drying process. The present study attempted to determine whether the dosage formulations of microspheres could form enteric matrices. To assess the enteric characteristics, an in vitro dissolution test was performed with the AQ6-AP microspheres; 95% of the A. pleuropneumoniae protein was released within 3 h at pH 7, but there was no release at pH 1.5. The scanning microscopy revealed that the surface structure of AQ6-AP microspheres became porous at neutral pH. The SDS-PAGE analysis showed that the release rate of proteins from the microspheres was pH dependent not only for the AQ6-AP formulation but also when antigens of A. pleuropneumoniae were replaced with porcine serum. The results suggest that the A. pleuropneumoniae antigens were entrapped in the AQ6 microspheres under the acidic conditions. In a mouse model, oral immunization with AQ6-AP microspheres containing A. pleuropneumoniae evoked systemic IgG and mucosal IgA responses against A. pleuropneumoniae antigens. Thus, the present method may further provide an opportunity to develop oral vaccines and mucosal immunity.