RESUMO
Caseous lymphadenitis (CLA) is an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants and is characterized by the development of granulomas in the lymph nodes, spleen, liver, and lungs. Although little is known about the host-pathogen relationship of this bacterium, it was previously reported that the pathogen's lipids are important for its taxonomic classification and survival inside macrophages. However, there are no studies regarding the composition of these molecules. In this study, cell wall glycolipids from two C. pseudotuberculosis strains presenting different virulence profiles were purified and its composition was characterized. A difference was observed between the electrophoretic and chromatogram profiles for cell wall components from the two strains, mainly among molecules with low molecular weights. IgM from sheep with acute CLA recognized antigens with an estimated molecular weight of 11 kDa of the low-pathogenicity strain, while low-molecular weight antigens from the high-pathogenicity strain presented a lower recognition by these antibodies. Mass spectrometry analysis showed that the cell wall of the high-pathogenicity strain contained glycolipids with high amounts of unsaturated fatty acids and glycerophosphoinositols, which may contribute to the capacity of this strain to cause severe disease. In conclusion, it is indicated that cell wall non-protein antigens can play a key role in C. pseudotuberculosis virulence.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Parede Celular/química , Corynebacterium pseudotuberculosis/química , Glicolipídeos/imunologia , Linfadenite/veterinária , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/imunologia , Corynebacterium pseudotuberculosis/patogenicidade , Glicolipídeos/química , Doenças das Cabras/imunologia , Doenças das Cabras/microbiologia , Cabras/microbiologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Linfonodos/imunologia , Linfonodos/microbiologia , Linfadenite/imunologia , Linfadenite/microbiologia , Ovinos , Doenças dos Ovinos/microbiologia , VirulênciaRESUMO
Caseous lymphadenitis (CL), a contagious infectious disease caused by Corynebacterium pseudotuberculosis, stands out with great economic relevance for goat breeding in the country because it is one of the main causes of carcass condemnation and intense depreciation of the leather of small ruminants. The objective was to carry out a descriptive study of the clinical and epidemiological aspects of CL in goats in the state of Pernambuco and also to evaluate the detection of C. pseudotuberculosis in milk, blood, and stool samples by real-time PCR. Six hundred and forty-three goats were evaluated, of which 76 (11.8%) presented clinical signs suggestive of CL. And 88.9% (24/27) of the breeding was considered focus. Among symptomatic animals, 25 were selected and submitted to the collection of abscess content for bacteriological isolation. Milk, blood, and stool samples were analyzed for PLD gene detection by real-time polymerase chain reaction (PCR). Out of the 25 samples of abscess content, 72% (18/25) were positive. The PLD gene was detected in the frequencies of 8% (2/25) and 4% (1/25) in milk and blood samples, respectively. Caseous lymphadenitis is possibly widespread in the dairy herds of goats in the state of Pernambuco, especially in adult animals, with a higher occurrence of submandibular lymph node abscesses. The preliminary detection of C. pseudotuberculosis by mammary and(AU)
A linfadenite caseosa (LC), doença infectocontagiosa ocasionada pelo Corynebacterium pseudotuberculosis, se destaca com grande relevância econômica para a caprinocultura no país por ser umas das principais causas de condenação de carcaças e intensa depreciação do couro de pequenos ruminantes. Objetivou-se realizar um estudo descritivo dos aspectos clínico-epidemiológicos da LC em caprinos no estado de Pernambuco e, adicionalmente, avaliar a detecção do C. pseudotuberculosis em amostras de leite, sangue e fezes por PCR em tempo real. Foram avaliados 643 caprinos, dos quais 76 (11,8%) apresentaram sinais clínicos sugestivos de LC. E 88,9% (24/27) das propriedades foram consideradas foco. Dentre os animais sintomáticos, 25 foram selecionados e submetidos à colheita de conteúdo de abscesso para isolamento bacteriológico. Amostras de leite, sangue e fezes foram analisadas para detecção do gene PLD pela reação em cadeia da polimerase (PCR) em tempo real. Das 25 amostras de conteúdo de abscesso, 72% (18/25) resultaram positivas. O gene PLD foi detectado nas frequências de 8% (2/25) e 4% (1/25) em amostras de leite e sangue, respectivamente. A linfadenite caseosa possivelmente encontra-se disseminada nos rebanhos leiteiros de caprinos do estado de Pernambuco, sobretudo em animais adultos, com maior ocorrência de abscessos em linfonodos submandibulares. A detecção preliminar do C. pseud(AU)
Assuntos
Animais , Linfadenite/diagnóstico , Linfadenite/epidemiologia , Linfadenite/veterinária , Ruminantes/anormalidades , Corynebacterium pseudotuberculosis/patogenicidadeRESUMO
Caseous lymphadenitis (CL), a contagious infectious disease caused by Corynebacterium pseudotuberculosis, stands out with great economic relevance for goat breeding in the country because it is one of the main causes of carcass condemnation and intense depreciation of the leather of small ruminants. The objective was to carry out a descriptive study of the clinical and epidemiological aspects of CL in goats in the state of Pernambuco and also to evaluate the detection of C. pseudotuberculosis in milk, blood, and stool samples by real-time PCR. Six hundred and forty-three goats were evaluated, of which 76 (11.8%) presented clinical signs suggestive of CL. And 88.9% (24/27) of the breeding was considered focus. Among symptomatic animals, 25 were selected and submitted to the collection of abscess content for bacteriological isolation. Milk, blood, and stool samples were analyzed for PLD gene detection by real-time polymerase chain reaction (PCR). Out of the 25 samples of abscess content, 72% (18/25) were positive. The PLD gene was detected in the frequencies of 8% (2/25) and 4% (1/25) in milk and blood samples, respectively. Caseous lymphadenitis is possibly widespread in the dairy herds of goats in the state of Pernambuco, especially in adult animals, with a higher occurrence of submandibular lymph node abscesses. The preliminary detection of C. pseudotuberculosis by mammary and
A linfadenite caseosa (LC), doença infectocontagiosa ocasionada pelo Corynebacterium pseudotuberculosis, se destaca com grande relevância econômica para a caprinocultura no país por ser umas das principais causas de condenação de carcaças e intensa depreciação do couro de pequenos ruminantes. Objetivou-se realizar um estudo descritivo dos aspectos clínico-epidemiológicos da LC em caprinos no estado de Pernambuco e, adicionalmente, avaliar a detecção do C. pseudotuberculosis em amostras de leite, sangue e fezes por PCR em tempo real. Foram avaliados 643 caprinos, dos quais 76 (11,8%) apresentaram sinais clínicos sugestivos de LC. E 88,9% (24/27) das propriedades foram consideradas foco. Dentre os animais sintomáticos, 25 foram selecionados e submetidos à colheita de conteúdo de abscesso para isolamento bacteriológico. Amostras de leite, sangue e fezes foram analisadas para detecção do gene PLD pela reação em cadeia da polimerase (PCR) em tempo real. Das 25 amostras de conteúdo de abscesso, 72% (18/25) resultaram positivas. O gene PLD foi detectado nas frequências de 8% (2/25) e 4% (1/25) em amostras de leite e sangue, respectivamente. A linfadenite caseosa possivelmente encontra-se disseminada nos rebanhos leiteiros de caprinos do estado de Pernambuco, sobretudo em animais adultos, com maior ocorrência de abscessos em linfonodos submandibulares. A detecção preliminar do C. pseud
Assuntos
Animais , Linfadenite/diagnóstico , Linfadenite/epidemiologia , Linfadenite/veterinária , Ruminantes/anormalidades , Corynebacterium pseudotuberculosis/patogenicidadeRESUMO
Background: Lumpy jaw is disease effecting wallabies and kangaroos, particularly in Macropus rufus and Macropusgiganteus. In the most serious situations, additional tooth loss and fistulas follow, accompanied by a stench, weight loss,and eventually death due to sepsis or blood poisoning. Lumpy jaw disease has seriously affected the normal display andhealth of kangaroos, and cause a huge economic loss. There was an outbreak of jaw infection in kangaroos at the HongshanForest Zoo. Two Macropus giganteus and two Macropus rufus died of lumpy jaw. The main objective of the describingcase was to isolate pathogens, provide a basis for follow-up treatment, and serve to establish a disease prevention protocol.Case: Four grown-up kangaroos (two Macropus giganteus and two Macropus rufus) were raised in Hongshan Forest Zoo,which had obviously clinical symptoms, such as oral lesions of pus, necrotic tissue, rotting teeth, then died of lumpyjaw. Oral swab samples were collected from the lesion sites of the dying kangaroos. Mice experiments were conducted toexamine the pathogenicity of the strains. Tests of antimicrobial susceptibity were performed to prescribe with better drugtreatments for kangaroos. Corynebacterium pseudotuberculosis and Yersinia pseudotuberculosis were identified based onmorphology, culture characteristics and biochemical tests. Corynebacterium pseudotuberculosis (G+) in Sucrose, Mannitol,Lactose, Maltose, Glucose tubes were positive, that acids and gases both production, in Gelatin liquefaction, Indol test,MR were positive, that only acids production, others were negative; Yersinia pseudotuberculosis (G-) in Urea, MR werepositive, that only acids production, others were negative.The infected mice presented with gum erosion or ulcers whenthe two pathogens were injected subcutaneous at the oral regional by 2-3 point at 0.2 mL of individual strains 1.0×109CFU/mouse...(AU)
Assuntos
Animais , Macropodidae/microbiologia , Corynebacterium pseudotuberculosis/patogenicidade , Yersinia pseudotuberculosis/isolamento & purificação , Yersiniose/veterinária , Infecções por Corynebacterium/veterinária , Animais de Zoológico/microbiologia , Resistência a MedicamentosRESUMO
Background: Lumpy jaw is disease effecting wallabies and kangaroos, particularly in Macropus rufus and Macropusgiganteus. In the most serious situations, additional tooth loss and fistulas follow, accompanied by a stench, weight loss,and eventually death due to sepsis or blood poisoning. Lumpy jaw disease has seriously affected the normal display andhealth of kangaroos, and cause a huge economic loss. There was an outbreak of jaw infection in kangaroos at the HongshanForest Zoo. Two Macropus giganteus and two Macropus rufus died of lumpy jaw. The main objective of the describingcase was to isolate pathogens, provide a basis for follow-up treatment, and serve to establish a disease prevention protocol.Case: Four grown-up kangaroos (two Macropus giganteus and two Macropus rufus) were raised in Hongshan Forest Zoo,which had obviously clinical symptoms, such as oral lesions of pus, necrotic tissue, rotting teeth, then died of lumpyjaw. Oral swab samples were collected from the lesion sites of the dying kangaroos. Mice experiments were conducted toexamine the pathogenicity of the strains. Tests of antimicrobial susceptibity were performed to prescribe with better drugtreatments for kangaroos. Corynebacterium pseudotuberculosis and Yersinia pseudotuberculosis were identified based onmorphology, culture characteristics and biochemical tests. Corynebacterium pseudotuberculosis (G+) in Sucrose, Mannitol,Lactose, Maltose, Glucose tubes were positive, that acids and gases both production, in Gelatin liquefaction, Indol test,MR were positive, that only acids production, others were negative; Yersinia pseudotuberculosis (G-) in Urea, MR werepositive, that only acids production, others were negative.The infected mice presented with gum erosion or ulcers whenthe two pathogens were injected subcutaneous at the oral regional by 2-3 point at 0.2 mL of individual strains 1.0×109CFU/mouse...
Assuntos
Animais , Corynebacterium pseudotuberculosis/patogenicidade , Infecções por Corynebacterium/veterinária , Macropodidae/microbiologia , Yersinia pseudotuberculosis/isolamento & purificação , Yersiniose/veterinária , Animais de Zoológico/microbiologia , Resistência a MedicamentosRESUMO
Phylogenomics and genome scale positive selection analyses were performed on 29 Corynebacterium pseudotuberculosis genomes that were isolated from different hosts, including representatives of the Ovis and Equi biovars. A total of 27 genes were identified as undergoing adaptive changes. An analysis of the clades within this species and these biovars, the genes specific to each branch, and the genes responding to selective pressure show clear differences, indicating that adaptation and specialization is occurring in different clades. These changes are often correlated with the isolation host but could indicate responses to some undetermined factor in the respective niches. The fact that some of these more-rapidly evolving genes have homology to known virulence factors, antimicrobial resistance genes and drug targets shows that this type of analysis could be used to identify novel targets, and that these could be used as a way to control this pathogen.
Assuntos
Adaptação Fisiológica , Corynebacterium pseudotuberculosis , Farmacorresistência Bacteriana , Evolução Molecular , Fatores de Virulência , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/metabolismo , Corynebacterium pseudotuberculosis/patogenicidade , Deleção de Genes , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The pathogenic bacteria of Corynebacterium pseudotuberculosis caused a chronic contagious infectious disease of the caseous lymphadenitis or pseudotuberculosis. Globally, isolates obtained from different injuries that affect sheep and goats, have been identified by fully or partially gene sequencing. However, in Mexico there is no complete study to identify by molecular and phylogenetic techniques the circulating isolates as well as its virulence factors. Therefore, in the present study we reported the identification of 57 isolates of C. pseudotuberculosis by bacteriological tests and the amplification of 16S rRNA, rpoB and pld genes, as well as, genes involved in virulence and pathogenicity: Fag A, Fag B, Fag C, Fag D and hsp60. Phylogenetic analysis was performed based on the partial sequence of the rpoB gene. Genes involved in virulence and pathogenicity were identified in the 98.2% of the isolates. Regarding the phylogenetic analysis, were identified the species and subspecies to which they belong of all the tested isolates. The phenotypic and genotypic characterization will allow to establish preventive and prophylactic measures aimed to the creation of effective immunogens against Corynebacterium pseudotuberculosis.
Assuntos
Corynebacterium pseudotuberculosis/classificação , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/isolamento & purificação , Cabras/microbiologia , Filogenia , Ovinos/microbiologia , Abscesso/microbiologia , Abscesso/veterinária , Animais , Proteínas de Bactérias/genética , Chaperonina 60/genética , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/patogenicidade , RNA Polimerases Dirigidas por DNA/genética , Genes Bacterianos/genética , Doenças das Cabras/microbiologia , Linfadenite/microbiologia , Linfadenite/veterinária , México , RNA Ribossômico 16S/genética , Doenças dos Ovinos/microbiologia , Virulência , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. RESULTS: A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family. CONCLUSION: Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.
Assuntos
Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/veterinária , Animais , Antígenos de Bactérias/sangue , Bacteriófagos/genética , Sequência de Bases , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/patogenicidade , Bases de Dados de Ácidos Nucleicos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Genes Bacterianos/genética , Genoma Bacteriano , Doenças das Cabras/sangue , Doenças das Cabras/imunologia , Doenças das Cabras/microbiologia , Cabras , Linfadenite/imunologia , Linfadenite/microbiologia , Reação em Cadeia da Polimerase/veterinária , Ovinos , Doenças dos Ovinos/sangue , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Vacinas de DNA/uso terapêuticoRESUMO
PURPOSE: We tested the efficacy of the esterase encoded by cp1002_RS09720 from Corynebacteriumpseudotuberculosis in recombinant subunit and DNA caseous lymphadenitis (CLA) vaccines. This target was predicted as one of the best CLA vaccine candidates by mature epitope density analysis. METHODOLOGY: Gene cp1002_RS09720 was cloned into two different vectors (pAE for subunit vaccine and pTARGET for DNA vaccine). Four groups of 15 mice each were immunized with the recombinant esterase rCP09720 associated with aluminium hydroxide adjuvant (G1), pTARGET/cp09720 DNA vaccine (G2), a naked pTARGET (G3) or PBS as a negative control (G4). Immunization occurred in two doses intercalated by a 21 day interval. Twenty-one days after the last dose administration, animals were challenged with a virulent C. pseudotuberculosis MIC-6 strain. RESULTS: G1 showed high levels of IgG1 and IgG2a on days 21 and 42 post-immunization and a significant level of IFN-γ (P<0.05), suggesting a Th1 response. The protection levels obtained were 58.3 and 16.6â% for G1 and G2, respectively. CONCLUSION: The subunit vaccine composed of the recombinant esterase rCP09720 and Al(OH)3 is a promising antigenic formulation for use against CLA.
Assuntos
Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/imunologia , Esterases/genética , Linfadenite/prevenção & controle , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Animais , Infecções por Corynebacterium/imunologia , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/patogenicidade , Citocinas/metabolismo , Esterases/administração & dosagem , Esterases/imunologia , Imunoglobulina G/sangue , Interferon gama/imunologia , Linfadenite/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologia , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
BACKGROUND: Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage. RESULTS: The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen. CONCLUSIONS: This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.
Assuntos
Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/metabolismo , Corynebacterium pseudotuberculosis/patogenicidade , Proteômica/métodos , Virulência , Animais , Proteínas de Bactérias/análise , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteoma/genética , Proteoma/metabolismo , Baço/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as "pigeon fever" which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at California compared to the other strains. Additionally, high variability of resistance islands suggests gene acquisition through several events of horizontal gene transfer.
Assuntos
Infecções por Corynebacterium/genética , Corynebacterium pseudotuberculosis/genética , Genoma Bacteriano/genética , Doenças dos Cavalos/genética , Rhodococcus equi/genética , Animais , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/patogenicidade , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Doenças dos Cavalos/microbiologia , Cavalos/microbiologia , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Rhodococcus equi/patogenicidadeRESUMO
Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Artrópodes/toxicidade , Proteínas de Bactérias/toxicidade , Permeabilidade Capilar/efeitos dos fármacos , Corynebacterium pseudotuberculosis/química , Diester Fosfórico Hidrolases/toxicidade , Aranhas/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/patogenicidade , Eritrócitos/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hemólise/efeitos dos fármacos , Cavalos , Humanos , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Carneiro Doméstico , Pele/efeitos dos fármacos , Pele/patologia , Aranhas/enzimologia , Aranhas/patogenicidadeRESUMO
BACKGROUND: Corynebacterium pseudotuberculosis, a facultative intracellular bacterial pathogen, is the etiological agent of caseous lymphadenitis (CLA), an infectious disease that affects sheep and goats and it is responsible for significant economic losses. The disease is characterized mainly by bacteria-induced caseous necrosis in lymphatic glands. New vaccines are needed for reliable control and management of CLA. Thus, the putative virulence factors SpaC, SodC, NanH, and PknG from C. pseudotuberculosis FRC41 may represent new target proteins for vaccine development and pathogenicity studies. RESULTS: SpaC, PknG and NanH presented better vaccine potential than SodC after in silico analyses. A total of 136 B and T cell epitopes were predicted from the four putative virulence factors. A cluster analysis was performed to evaluate the redundancy degree among the sequences of the predicted epitopes; 57 clusters were formed, most of them (34) were single clusters. Two clusters from PknG and one from SpaC grouped epitopes for B and T-cell (MHC I and II). These epitopes can thus potentially stimulate a complete immune response (humoral and cellular) against C. pseudotuberculosis. Several other clusters, including two from NanH, grouped B-cell epitopes with either MHC I or II epitopes. The four target proteins were expressed in Escherichia coli. A purification protocol was developed for PknG expression. CONCLUSIONS: In silico analyses show that the putative virulence factors SpaC, PknG and NanH present good potential for CLA vaccine development. Target proteins were successfully expressed in E. coli. A protocol for PknG purification is described.
Assuntos
Vacinas Bacterianas/genética , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/patogenicidade , Expressão Gênica , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Sequência de Aminoácidos , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Análise por Conglomerados , Corynebacterium pseudotuberculosis/imunologia , Corynebacterium pseudotuberculosis/metabolismo , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Escherichia coli/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Fatores de Virulência/metabolismoRESUMO
Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused by Corynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp in C. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of the oppD gene sequence. As occurred to the wild-type, the ΔoppD strain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ΔoppD mutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice.
Assuntos
Proteínas de Bactérias/genética , Transporte Biológico/genética , Corynebacterium pseudotuberculosis/genética , Linfadenite/genética , Proteínas de Membrana Transportadoras/genética , Animais , Proteínas de Bactérias/metabolismo , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/patogenicidade , Humanos , Linfadenite/microbiologia , Linfadenite/patologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Mutação , Óperon/genéticaRESUMO
To verify the occurrence of caseous lymphadenitis in sheep and goats on farms of Pernambuco, Brazil, and in animals slaughtered in two Brazilian cities (Petrolina/PE and Juazeiro/BA), and to characterize the susceptibility profile of Corynebacterium pseudotuberculosis to disinfectants and antimicrobials, and its relationship with biofilm production were the objectives of this study. 398 samples were tested for sensitivity to antimicrobial drugs, disinfectants, and biofilm production. Among the 108 samples collected on the properties, 75% were positive for C. pseudotuberculosis. Slaughterhouse samples indicated an occurrence of caseous lymphadenitis in 15.66% and 6.31% for animals slaughtered in Petrolina and Juazeiro respectively. With respect to antimicrobials, the sensitivity obtained was 100% for florfenicol and tetracycline; 99.25% for enrofloxacin, ciprofloxacin and lincomycin; 98.99% for cephalothin; 98.74% for norfloxacin and sulfazotrim; 97.74% for gentamicin; 94.22% for ampicillin; 91.71% for amoxicillin; 91.21% for penicillin G; 89.19% for neomycin and 0% for novobiocin. In analyzes with disinfectants, the efficiency for chlorhexidine was 100%, 97.20% for quaternary ammonium, 87.40% for chlorine and 84.40% for iodine. 75% of the isolates were weak or non-biofilm producers. For the consolidated biofilm, found that iodine decreased biofilm formation in 13 isolates and quaternary ammonia in 11 isolates. The reduction of the biofilm formation was observed for iodine and quaternary ammonium in consolidated biofilm formation in 33% and 28% of the isolates, respectively. The results of this study highlight the importance of establishing measures to prevent and control the disease.
Verificar a ocorrência de linfadenite caseosa de ovinos e caprinos em fazendas de Pernambuco, no Brasil e em animais abatidos em duas cidades brasileiras (Petrolina/PE e Juazeiro/BA), caracterizar o perfil de suscetibilidade de Corynebacgerium pseudotuberculosis a desinfetantes e a antimicrobianos e analisar sua relação com a produção de biofilme foram os objetivos deste estudo. 398 amostras foram testadas para a sensibilidade aos medicamentos antimicrobianos, desinfetantes e produção de biofilme. Entre as 108 amostras coletadas nas propriedades, 75% foram positivas para C. pseudotuberculosis. Amostras do matadouro indicaram a ocorrência de linfadenite caseosa em 15,66% e 6,31% para animais abatidos em Petrolina (PE) e Juazeiro (BA), respectivamente. Com relação aos antimicrobianos, a sensibilidade obtida foi de 100% para florfenicol e tetraciclina, 99,25% para a enrofloxacina, ciprofloxacina e lincomicina; 98,99% para cefalotina; 98,74% para norfloxacina e sulfazotrim; 97,74% para gentamicina; 94,22% para ampicilina, 91,71% para amoxicilina, 91,21% para a penicilina G, 89,19% para a neomicina e 0% para novobiocina. Nas análises com desinfetantes, a eficiência para a clorexidina foi de 100%, 97,20% para amônia quaternária, 87,40% para o cloro e 84,40% para o iodo. 75% dos isolados foram fracos produtores de biofilme. Para o biofilme consolidado, observou-se que o iodo diminuiu a formação de biofilme em 13 isolados e a amônia quaternária em 11 isolados. A redução da formação de biofilme foi observada no biofilme consolidado, para iodo e amônia quaternária, em 33% e 28% das amostras, respectivamente. Os resultados deste estudo destacam a importância de se estabelecer medidas para prevenir e controlar a doença.
Assuntos
Animais , Biofilmes , Cabras/imunologia , Corynebacterium pseudotuberculosis/patogenicidade , Linfadenite/veterinária , Ovinos/imunologia , Anti-Infecciosos/efeitos adversos , Desinfetantes/efeitos adversosRESUMO
To verify the occurrence of caseous lymphadenitis in sheep and goats on farms of Pernambuco, Brazil, and in animals slaughtered in two Brazilian cities (Petrolina/PE and Juazeiro/BA), and to characterize the susceptibility profile of Corynebacterium pseudotuberculosis to disinfectants and antimicrobials, and its relationship with biofilm production were the objectives of this study. 398 samples were tested for sensitivity to antimicrobial drugs, disinfectants, and biofilm production. Among the 108 samples collected on the properties, 75% were positive for C. pseudotuberculosis. Slaughterhouse samples indicated an occurrence of caseous lymphadenitis in 15.66% and 6.31% for animals slaughtered in Petrolina and Juazeiro respectively. With respect to antimicrobials, the sensitivity obtained was 100% for florfenicol and tetracycline; 99.25% for enrofloxacin, ciprofloxacin and lincomycin; 98.99% for cephalothin; 98.74% for norfloxacin and sulfazotrim; 97.74% for gentamicin; 94.22% for ampicillin; 91.71% for amoxicillin; 91.21% for penicillin G; 89.19% for neomycin and 0% for novobiocin. In analyzes with disinfectants, the efficiency for chlorhexidine was 100%, 97.20% for quaternary ammonium, 87.40% for chlorine and 84.40% for iodine. 75% of the isolates were weak or non-biofilm producers. For the consolidated biofilm, found that iodine decreased biofilm formation in 13 isolates and quaternary ammonia in 11 isolates. The reduction of the biofilm formation was observed for iodine and quaternary ammonium in consolidated biofilm formation in 33% and 28% of the isolates, respectively. The results of this study highlight the importance of establishing measures to prevent and control the disease. (AU)
Verificar a ocorrência de linfadenite caseosa de ovinos e caprinos em fazendas de Pernambuco, no Brasil e em animais abatidos em duas cidades brasileiras (Petrolina/PE e Juazeiro/BA), caracterizar o perfil de suscetibilidade de Corynebacgerium pseudotuberculosis a desinfetantes e a antimicrobianos e analisar sua relação com a produção de biofilme foram os objetivos deste estudo. 398 amostras foram testadas para a sensibilidade aos medicamentos antimicrobianos, desinfetantes e produção de biofilme. Entre as 108 amostras coletadas nas propriedades, 75% foram positivas para C. pseudotuberculosis. Amostras do matadouro indicaram a ocorrência de linfadenite caseosa em 15,66% e 6,31% para animais abatidos em Petrolina (PE) e Juazeiro (BA), respectivamente. Com relação aos antimicrobianos, a sensibilidade obtida foi de 100% para florfenicol e tetraciclina, 99,25% para a enrofloxacina, ciprofloxacina e lincomicina; 98,99% para cefalotina; 98,74% para norfloxacina e sulfazotrim; 97,74% para gentamicina; 94,22% para ampicilina, 91,71% para amoxicilina, 91,21% para a penicilina G, 89,19% para a neomicina e 0% para novobiocina. Nas análises com desinfetantes, a eficiência para a clorexidina foi de 100%, 97,20% para amônia quaternária, 87,40% para o cloro e 84,40% para o iodo. 75% dos isolados foram fracos produtores de biofilme. Para o biofilme consolidado, observou-se que o iodo diminuiu a formação de biofilme em 13 isolados e a amônia quaternária em 11 isolados. A redução da formação de biofilme foi observada no biofilme consolidado, para iodo e amônia quaternária, em 33% e 28% das amostras, respectivamente. Os resultados deste estudo destacam a importância de se estabelecer medidas para prevenir e controlar a doença. (AU)
Assuntos
Animais , Cabras/imunologia , Ovinos/imunologia , Linfadenite/veterinária , Corynebacterium pseudotuberculosis/patogenicidade , Biofilmes , Desinfetantes/efeitos adversos , Anti-Infecciosos/efeitos adversosRESUMO
Este trabalho teve como objetivo determinar a ocorrência e os fatores de risco associados à infecção por Corynebacterium pseudotuberculosis em caprinos e ovinos do semiárido da Paraiba. De 640 animais examinados, 7,7% (49/640) apresentavam evidências clínicas de linfadenite caseosa. Em 59,2% (29/49) destes animais havia apenas as cicatrizes de abscessos anteriormente rompidos; em 40,8% (20/49) dos animais, os abscessos estavam intactos. Desses 20 animais, 13 (65%) caprinos apresentaram 14 abscessos, enquanto que sete (35%) ovinos apresentaram oito abscessos. Em ambas as espécies, o linfonodo pré-escapular foi o mais acometido. No exame microbiológico, constatou-se que C. pseudotuberculosis foi o agente mais frequentemente isolado, em 15 (68,2%) amostras; em uma (4,5%) foi isolado Staphylococcus coagulase negativa; uma (4,5%) Enterococcus sp.; uma (4,5%) o Proteus mirabilis e Pseudomonas aeruginosa; e em quatro (18,2%) amostras não houve crescimento bacteriano. O modelo final de regressão logística mostrou que animais provenientes de rebanhos em que seus proprietários deixavam os abscessos romperem naturalmente tiveram maior chance de apresentar linfadenite caseosa (odds ratio =8,19; IC 95% =1,75-38,25; p=0,008). Conclui-se que os caprinovinocultores da região devem adotar medidas profiláticas em seus rebanhos, como abertura e drenagem precoce dos abscessos superficiais e destino adequado do conteúdo. Tais medidas, associadas à inspeção periódica do rebanho, descarte de animais doentes e não introdução de animais infectados, contribuirão significativamente para o controle desta infecção.
This study aimed to determine the occurrence and risk factors associated with Cory nebacterium pseudotuberculosis infection in goats and sheep in semiarid region of Paraiba State, Northeastern Brazil. Of the 640 animals examined, 7.7% (49/640) had clinical evidence of caseous lymphadenitis. In 59.2% (29/49) of these animals there was only the scars of previously ruptured abscesses and in 40.8% (20/49) of the animals abscesses were intact. Of these 20 animals 13 (65%) goats had 14 abscesses, whereas seven (35%) sheep had eight abscesses. In both species, pre-scapular lymph node was the most involved. C. pseudotuberculosis was the agent most frequently isolated, in 15 (68.2%) samples, and in one (4.5%) coagulase-negative Staphylococcus was isolated, one (4.5%) Enterococ cus sp., one (4.5%) Proteus mirabilis and Pseudomonas aeruginosa, and in four (18.2%) samples there was no bacterial growth. The final logistic regression model showed that animals from herds where their owners let the abscesses break naturally presented larger odds of caseous lymphadenitis (odds ratio = 8.19, 95% CI = 1.75 - 38.25, p = 0.008). We conclude that goat/sheep owners of the region should adopt preventive measures in their herds, such as early opening and drainage of superficial abscesses, and appropriate destination of the content. Such measures, in addition to regular inspection of the flock, disposal of diseased animals and introduction of non-infected animals will contribute to the control of this infection.
Assuntos
Animais , Corynebacterium pseudotuberculosis/patogenicidade , Linfadenite/veterinária , Ovinos/microbiologia , Surtos de Doenças/veterinária , /prevenção & controle , Prevenção de Doenças , Fatores de RiscoRESUMO
Este trabalho teve como objetivo determinar a ocorrência e os fatores de risco associados à infecção por Corynebacterium pseudotuberculosis em caprinos e ovinos do semiárido da Paraiba. De 640 animais examinados, 7,7% (49/640) apresentavam evidências clínicas de linfadenite caseosa. Em 59,2% (29/49) destes animais havia apenas as cicatrizes de abscessos anteriormente rompidos; em 40,8% (20/49) dos animais, os abscessos estavam intactos. Desses 20 animais, 13 (65%) caprinos apresentaram 14 abscessos, enquanto que sete (35%) ovinos apresentaram oito abscessos. Em ambas as espécies, o linfonodo pré-escapular foi o mais acometido. No exame microbiológico, constatou-se que C. pseudotuberculosis foi o agente mais frequentemente isolado, em 15 (68,2%) amostras; em uma (4,5%) foi isolado Staphylococcus coagulase negativa; uma (4,5%) Enterococcus sp.; uma (4,5%) o Proteus mirabilis e Pseudomonas aeruginosa; e em quatro (18,2%) amostras não houve crescimento bacteriano. O modelo final de regressão logística mostrou que animais provenientes de rebanhos em que seus proprietários deixavam os abscessos romperem naturalmente tiveram maior chance de apresentar linfadenite caseosa (odds ratio =8,19; IC 95% =1,75-38,25; p=0,008). Conclui-se que os caprinovinocultores da região devem adotar medidas profiláticas em seus rebanhos, como abertura e drenagem precoce dos abscessos superficiais e destino adequado do conteúdo. Tais medidas, associadas à inspeção periódica do rebanho, descarte de animais doentes e não introdução de animais infectados, contribuirão significativamente para o controle desta infecção.(AU)
This study aimed to determine the occurrence and risk factors associated with Cory nebacterium pseudotuberculosis infection in goats and sheep in semiarid region of Paraiba State, Northeastern Brazil. Of the 640 animals examined, 7.7% (49/640) had clinical evidence of caseous lymphadenitis. In 59.2% (29/49) of these animals there was only the scars of previously ruptured abscesses and in 40.8% (20/49) of the animals abscesses were intact. Of these 20 animals 13 (65%) goats had 14 abscesses, whereas seven (35%) sheep had eight abscesses. In both species, pre-scapular lymph node was the most involved. C. pseudotuberculosis was the agent most frequently isolated, in 15 (68.2%) samples, and in one (4.5%) coagulase-negative Staphylococcus was isolated, one (4.5%) Enterococ cus sp., one (4.5%) Proteus mirabilis and Pseudomonas aeruginosa, and in four (18.2%) samples there was no bacterial growth. The final logistic regression model showed that animals from herds where their owners let the abscesses break naturally presented larger odds of caseous lymphadenitis (odds ratio = 8.19, 95% CI = 1.75 - 38.25, p = 0.008). We conclude that goat/sheep owners of the region should adopt preventive measures in their herds, such as early opening and drainage of superficial abscesses, and appropriate destination of the content. Such measures, in addition to regular inspection of the flock, disposal of diseased animals and introduction of non-infected animals will contribute to the control of this infection.(AU)
Assuntos
Animais , Ovinos/microbiologia , Corynebacterium pseudotuberculosis/patogenicidade , Surtos de Doenças/veterinária , Linfadenite/veterinária , Fatores de Risco , Prevenção de Doenças , /prevenção & controleRESUMO
Background: Caseous lymphadenitis (CL), an infectious disease of sheep and goats caused by Corynebacterium pseudotuberculosis, is characterized by pyogranulomas, white to greenish-yellow contents with aspect of rennet-coagulated cheese, and concentric laminations. Transmission occurs through direct contact with animals showing superficial lesions, via iatrogenic, or by respiratory route from aerosols. This paper reports the association of pyogranulomatous spondylitis with caseous lymphadenitis in two sheep, in Rio Grande do Sul State, southern Brazil. Cases: Two male sheep, 7- and 6-months-old, Texel (lamb 1) and mixed Texel (lamb 2), respectively, were submitted to necropsy. Affected sheep were reared under semi-intensive system in the same group, and had not been tail-docked. Both lambs showed weakness, diarrhea and paralysis of hind limbs. At necropsy, sheep had good body conditions, pale mucous membranes and large numbers of Haemonchus sp. worms within the abomasum. Lamb 1 showed serous fat atrophy and two abscesse-like lesions, one measuring 3.0 cm in diameter in the left apical lung lobe and other affecting the last lumbar vertebrae body. In lamb 2 there were three lesions like abscesses, a thoracic lesion involving the 13th vertebrae and the 1st lumbar vertebrae, a 3.0 cm in diameter lesion in the left axillary region, and a 1.5 cm lung lesion in the left apical lobe, apart of a calcified nodule with 0.5 cm in diameter in the diaphragmatic lobe. Histologically, these lesions were characterized by areas of caseous necrosis with foci of mineralization associated with peripheral infl ammatory infi ltrate rich in neutrophils surrounded by macrophages, epithelioid cells and occasional multinucleated giant cells and plasma cells and peripheral abundant fi brous connective tissue proliferation. In the vertebral bodies these lesions were partly surrounded by fibrous connective tissue, and in lamb 1 reached the dura mater, while in lamb 2, it extends to the spinal cord with pyogranulomatous myelitis, characterized by foci containing large amounts of neutrophils, surrounded by macrophages and epithelioid cells. There was thickening of blood vessel walls, with macrophages within or around them. Spinal cord injury affects both gray and white matter, with peripheral axonal spheroids. Corynebacterium pseudotuberculosis grew in pure culture upon microbiological tests from samples of lung and vertebral lesions from both cases. Discussion: This paper describes, originally, caseous lymphadenitis as a cause of hind limb paralysis in sheep in Rio Grande do Sul State, Brazil. The association of the macroscopic and histological findings with the isolation of C. pseudotuberculosis allowed us to relate pyogranulomatous spondylitis with caseous lymphadenitis. The spondylitis and spinal cord compression and/or myelitis may explain the hind limb paralysis. The spread of the pathogen was probably hematogenous to the lungs and the other parts of the body. The hematogenous osteomyelitis caused by bacterial infection affects often young animals in the area of vascularization of the growth plate and epiphyseal articular complex. The blood vessels of these sites allow the bacterial installation due to slow flow and the blood turbulence in the larger descending vascular branches, but also due to lowed phagocytic capacity and discontinuity between endothelial cells. These factors, in addition to the antiphagocytic action of Corynebacterium pseudotuberculosis in endothelial cells may explain the location at spinal lesions of caseous lymphadenitis in these sheep.
Assuntos
Animais , Pelve/lesões , Doenças dos Ovinos/diagnóstico , Espondilite/veterinária , Corynebacterium pseudotuberculosis/patogenicidade , Infecções por Corynebacterium/veterinária , Linfadenite/veterinária , OvinosRESUMO
In this work, we report the complete genome sequence of a Corynebacterium pseudotuberculosis PAT10 isolate, collected from a lung abscess in an Argentine sheep in Patagonia, whose pathogen also required an investigation of its pathogenesis. Thus, the analysis of the genome sequence offers a means to better understanding of the molecular and genetic basis of virulence of this bacterium.