RESUMO
This work evaluated aspects of the immune response of BALB/c mice infected with Corynebacterium pseudotuberculosis (T1 and C57). The fifteen BALB/c mice were euthanized after 70 days of infection and morphologically evaluated, also analyzing the innate and adaptive immune responses. The C57 strain induced more pronounced morphological changes than the T1 strain. There was an increase in CD4+ and CD8+ T cells identified during infection with the C57 strain. Cytokines of the inflammatory profile IL-1α and IL-6 and regulatory IL-13 and IL-10 presented significant differences. Cytokines IL-2, IL-4, INF-γ, IL-22, IL-21, and IL-27 did not differ significantly between groups. The obtained results contribute to a better understanding of the type of response and the immunological mechanisms involved during infection with different strains of C. pseudotuberculosis.
Assuntos
Linfócitos T CD8-Positivos , Infecções por Corynebacterium , Corynebacterium pseudotuberculosis , Citocinas , Camundongos Endogâmicos BALB C , Animais , Corynebacterium pseudotuberculosis/imunologia , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Camundongos , Citocinas/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-10 , Imunidade Adaptativa , Imunidade Inata , Interleucina-6 , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Interleucina-1alfa/metabolismo , Interleucina-1alfa/imunologia , Interferon gama/metabolismo , Interleucina-4/metabolismo , Interleucinas , Interleucina-2/metabolismoRESUMO
Caseous Lymphadenitis (CLA) is a chronic disease that affects also small ruminants. CLA is caused by Corynebacterium pseudotuberculosis and is responsible for high economic losses due to the formation of superficial and visceral granulomas, the latter is considered as asymptomatic CLA causing high levels of dissemination. Several vaccination strategies, in which the use of synthetic peptides stands out. Thus, this work aimed to evaluate the protective potential of peptide vaccines designed to determine the immunodominant epitopes of CP40 against CLA in mice. The animals were divided into eight groups separated in controls (G1-PBS, G2-Saponin and G9-rCP40) and experimental (G3-pep1, G4- pep2, G5-pep3, G6-pep4, G7-pep5 and G8-pep6), these were vaccinated on days 0 and 15 by a subcutaneous route. 60 days after the first immunization, all animals were challenged with C. pseudotuberculosis. On days 0, 15, 60, and 120 after the first immunization, blood samples were taken to measure immunoglobulins. On the same day of the challenge, the splenocytes were isolated and assayed for the production of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17, and IL-10. After vaccinations, the animals were challenged and all of them were affected by the disease which led to their death. The G6 and G8 groups provided 10% protection and the G7 provided 20%. The G3 and G4 groups provided 30% and 40% protection respectively. The peptides showed the production of Total IgG antibodies and cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α), indicating a possible activation of the Th1 type response. However, groups G3, G5, G6, and G8 showed production of IL-17. None of the study groups showed IL-10 production. The immunogenicity of the peptides was not enough to protect these animals and it is believed that the use of adjuvants based on PAMPs may improve the immune response offered by these peptides.
Assuntos
Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/prevenção & controle , Desenvolvimento de Vacinas , Vacinas de Subunidades Antigênicas , Animais , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , CamundongosRESUMO
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 (CLA) is a small ruminant disease characterized by the development of granulomatous lesions in superficial and internal lymph nodes, as well as in some organs, and causes significant economic losses worldwide. The aetiological agent of CLA is the bacterium Corynebacterium pseudotuberculosis; however, the commercially available diagnostic tools present problems with regard to specificity, which can lead to false-negative results. This study aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins in goats and sheep using recombinant C. pseudotuberculosis PLD, CP40, PknG, DtxR and Grx proteins. For validation of the ELISAs, 130 goat serum samples and 160 sheep serum samples were used. The best ELISA for goats was developed using a combination of PLD and CP40 as antigens at a 1:1 ratio, which presented 96.9% sensitivity and 98.4% specificity. The most effective ELISA for sheep presented 91% sensitivity and 98.7% specificity when recombinant PLD alone was used as the antigen. These ELISAs can be used as highly accurate tools in epidemiological surveys and for the serodiagnosis of C. pseudotuberculosis infection in goats and sheep.
Assuntos
Anticorpos Antibacterianos/sangue , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/imunologia , Doenças das Cabras/diagnóstico , Linfadenite/veterinária , Testes Sorológicos/métodos , Doenças dos Ovinos/diagnóstico , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Infecções por Corynebacterium/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Cabras , Linfadenite/diagnóstico , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Ovinos , Medicina Veterinária/métodosRESUMO
Corynebacterium pseudotuberculosis is the etiologic agent of veterinary relevance diseases, such as caseous lymphadenitis, affecting different animal species causing damage to the global agribusiness. So far, there are no completely effective treatment methods to overcome the impacts caused by this pathogen. Several genomes of the species are deposited on public databases, allowing the execution of studies related to the pan-genomic approach. In this study, we used an integrated in silico workflow to prospect novel putative targets using the core genome, a set of shared genes among 65 C. pseudotuberculosis strains. Subsequently, through RNA-Seq data of the same abiotic stresses in two strains, we selected only induced genes to compose the reverse vaccinology workflow based in two different strategies. Our results predicted six probable antigens in both analysis, which indicates that they have a strong potential to be used in further studies as vaccine targets against this bacterium.
Assuntos
Vacinas Bacterianas/genética , Corynebacterium pseudotuberculosis/genética , Antígenos de Bactérias/genética , Simulação por Computador , Corynebacterium/genética , Corynebacterium pseudotuberculosis/imunologia , Corynebacterium pseudotuberculosis/metabolismo , Perfilação da Expressão Gênica , Genes Bacterianos , Genoma Bacteriano , Mapeamento de Interação de Proteínas , Análise de Sequência de RNA , VacinologiaRESUMO
For a long time, the scientific community has described the need for a continued update in practices that ensure the welfare of animals undergoing experimentation. In addition to approaches on principles of care and use of animals, there is a more current emerging concern: defining an appropriate end point in experiments that use animals for research, teaching and testing. The term "endpoint" is defined as the point at which an experimental animal's pain and/or distress is terminated, minimized, or reduced humanely. In the present study, we established an endpoint in Balb/C mice for caseous lymphadenitis vaccine trials, which can be considered as a highly important parameter since several studies are being developed to control the disease efficiently. Mice were monitored daily until the 30th day after infection with pathogenic strain of C. pseudotuberculosis MIC-6 using the most relevant parameters for the appearance of clinical signs of caseous lymphadenitis (CLA), such as abscesses, lethargy, and loss of weight and hair. The endpoint was found to be a weight loss of 0.2167 g after five days or 10% weight loss in less than five days. In conclusion, the findings reported here will help improve animal's well-being during vaccine trials for CLA and consequently represent significant contribution to animal's welfare.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Corynebacterium/prevenção & controle , Modelos Animais de Doenças , Determinação de Ponto Final/métodos , Linfadenite/prevenção & controle , Redução de Peso , Bem-Estar do Animal , Animais , Infecções por Corynebacterium/imunologia , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/microbiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLDâ¯+â¯rCP09720 (G3), and rPLDâ¯+â¯rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21â¯days after the last immunization. The animals were evaluated daily for 40â¯days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (pâ¯<â¯.05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (pâ¯<â¯.05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLDâ¯+â¯rCP09720), and 50% (rPLDâ¯+â¯rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/veterinária , Fosfolipase D/imunologia , Proteínas Recombinantes/imunologia , Fosfatase Ácida/administração & dosagem , Fosfatase Ácida/genética , Fosfatase Ácida/imunologia , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/química , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/genética , Esterases/administração & dosagem , Esterases/genética , Esterases/imunologia , Cabras/microbiologia , Imunidade Celular , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interferon gama/imunologia , Linfadenite/imunologia , Linfadenite/microbiologia , Linfadenite/prevenção & controle , Camundongos , Fosfolipase D/administração & dosagem , Fosfolipase D/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Ovinos/microbiologia , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/prevenção & controle , Células Th1/imunologia , Vacinação/veterinária , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
This study aimed to evaluate aspects of host immune response using an experimental infection model of Corynebacterium pseudotuberculosis (CP) in C57/Black6 wild-type and knockout for nitric oxide (KO-NO) mice. 28 mice were evaluated: 4 wild-type controls; 10 wild-type infected with CP; 4 KO-NO controls; 10 KO-NO infected with CP. Infection procedures were carried out by intraperitoneal inoculation using 107. Infected C57/Black6 KO-NO mice began to die after the 5° day post-inoculation, up until the 14º day. Neutrophils were found in increased numbers in the infiltrate of KO-NO murine peritoneal cavities. Examination of splenic tissue revealed an accumulation of lymphocytes, predominantly CD8 T-cells, in experimental animal groups. KO-NO animals were found to have a predominance of granulomas 7 days post-inoculation, primarily in the lymph nodes. In addition, greater amounts of bacteria were recovered from the mesenteric lymph nodes of KO-NO mice. There was no statistically significant difference in the levels of total IgG and its subclasses 14 days post-inoculation between KO-NO and wild groups. The results suggest the importance of nitric oxide in the process of controlling CP infection, as KO-NO animals were observed to be markedly more affected by infection with this bacterium.(AU)
O objetivo deste estudo foi avaliar os aspectos da resposta imune do hospedeiro, mediante o uso de um modelo experimental de infecção de Corynebacterium pseudotuberculosis (CP) em camundongos C57/Black6 do tipo selvagem e em C57/Black6 knockout para o óxido nítrico (KO-NO). Foram avaliados 28 camundongos: quatro controles de tipo selvagem; 10 do tipo selvagem infectados com CP; quatro controles KO-NO; e 10 KO-NO infectados com CP. A infecção foi realizada via intraperitoneal, usando-se 107. Os animais C57/Black6 KO-NO infectados começaram a vir a óbito no quinto dia pós-inoculação, o que aconteceu até o 14º dia. Um número maior de neutrófilos foi encontrado na sua cavidade peritoneal. O exame do baço revelou um acúmulo de linfócitos, predominantemente células T CD8, nos grupos de animais experimentais. Nos animais KO-NO, foi observada a presença de granulomas, sete dias pós-inoculação, principalmente nos gânglios linfáticos. Além disso, uma maior quantidade de bactérias foi detectada dos linfonodos mesentéricos desses animais. Não houve diferença estatisticamente significante nos níveis séricos IgG total e em suas subclasses aos 14 dias pós-inoculação nos grupos KO-NO e selvagem. Os resultados obtidos sugerem a importância do óxido nítrico no processo de controle da infecção por CP.(AU)
Assuntos
Animais , Camundongos , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/prevenção & controle , Camundongos Knockout/imunologia , Óxido Nítrico/uso terapêutico , Linfadenite/veterináriaRESUMO
This study aimed to evaluate aspects of host immune response using an experimental infection model of Corynebacterium pseudotuberculosis (CP) in C57/Black6 wild-type and knockout for nitric oxide (KO-NO) mice. 28 mice were evaluated: 4 wild-type controls; 10 wild-type infected with CP; 4 KO-NO controls; 10 KO-NO infected with CP. Infection procedures were carried out by intraperitoneal inoculation using 107. Infected C57/Black6 KO-NO mice began to die after the 5° day post-inoculation, up until the 14º day. Neutrophils were found in increased numbers in the infiltrate of KO-NO murine peritoneal cavities. Examination of splenic tissue revealed an accumulation of lymphocytes, predominantly CD8 T-cells, in experimental animal groups. KO-NO animals were found to have a predominance of granulomas 7 days post-inoculation, primarily in the lymph nodes. In addition, greater amounts of bacteria were recovered from the mesenteric lymph nodes of KO-NO mice. There was no statistically significant difference in the levels of total IgG and its subclasses 14 days post-inoculation between KO-NO and wild groups. The results suggest the importance of nitric oxide in the process of controlling CP infection, as KO-NO animals were observed to be markedly more affected by infection with this bacterium.(AU)
O objetivo deste estudo foi avaliar os aspectos da resposta imune do hospedeiro, mediante o uso de um modelo experimental de infecção de Corynebacterium pseudotuberculosis (CP) em camundongos C57/Black6 do tipo selvagem e em C57/Black6 knockout para o óxido nítrico (KO-NO). Foram avaliados 28 camundongos: quatro controles de tipo selvagem; 10 do tipo selvagem infectados com CP; quatro controles KO-NO; e 10 KO-NO infectados com CP. A infecção foi realizada via intraperitoneal, usando-se 107. Os animais C57/Black6 KO-NO infectados começaram a vir a óbito no quinto dia pós-inoculação, o que aconteceu até o 14º dia. Um número maior de neutrófilos foi encontrado na sua cavidade peritoneal. O exame do baço revelou um acúmulo de linfócitos, predominantemente células T CD8, nos grupos de animais experimentais. Nos animais KO-NO, foi observada a presença de granulomas, sete dias pós-inoculação, principalmente nos gânglios linfáticos. Além disso, uma maior quantidade de bactérias foi detectada dos linfonodos mesentéricos desses animais. Não houve diferença estatisticamente significante nos níveis séricos IgG total e em suas subclasses aos 14 dias pós-inoculação nos grupos KO-NO e selvagem. Os resultados obtidos sugerem a importância do óxido nítrico no processo de controle da infecção por CP.(AU)
Assuntos
Animais , Camundongos , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/prevenção & controle , Camundongos Knockout/imunologia , Óxido Nítrico/uso terapêutico , Linfadenite/veterináriaRESUMO
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, 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
BACKGROUND: Caseous lymphadenitis (CLA) is an infectious disease that affects small ruminants and is caused by Corynebacterium pseudotuberculosis. This disease is responsible for high economic losses due to condemnation and trim of infected carcasses, decreased leather and wool yield, loss of sales of breeding stock and deaths from internal involvement. Treatment is costly and ineffective; the most cost-effective strategy is timely immunisation. Various vaccine strategies have been tested, and recombinant vaccines are a promising alternative. Thus, in this study, different vaccine formulations using a recombinant protein (rCP40) and the CP09 live recombinant strain were evaluated. Five groups of 10 mice each were immunised with saline (G1), rCP40 (G2), CP09 (G3), a combination of CP09 and rCP40 (G4) and a heterologous prime-boost strategy (G5). Mice received two immunisations within 15 days. On day 30 after primary immunisation, all groups were challenged with a C. pseudotuberculosis virulent strain. Mice were monitored and mortality was recorded for 30 days after challenge. RESULTS: The G2, G4 and G5 groups showed high levels of IgG1 and IgG2a; G2 presented significant IgG2a production after virulent challenge in the absence of IgG1 and IgG3 induction. Thirty days after challenge, the mice survival rates were 20 (G1), 90 (G2), 50 (G3), 70 (G4) and 60% (G5). CONCLUSIONS: rCP40 is a promising target in the development of vaccines against caseous lymphadenitis.
Assuntos
Vacinas Bacterianas/uso terapêutico , Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/genética , Linfadenite/prevenção & controle , Vacinas Sintéticas/uso terapêutico , Animais , Linhagem Celular Tumoral , Clonagem Molecular , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/imunologia , DNA Bacteriano/genética , Linfadenite/microbiologia , Camundongos/imunologia , Camundongos/microbiologia , Camundongos Endogâmicos BALB C , Mutação/genética , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Caseous lymphadenitis (CL) is a contagious infectious disease of small ruminants caused by Corynebacterium pseudotuberculosis. Is characterized by the formation of abscesses in the lymph nodes and intestines of infected animals, induced by inflammatory cytokines. The production of cytokines, such as IL-10, TNF-α, IL-4 and IFN-γ, is regulated by mitogen-activated protein kinase (MAPK) pathway activation. The present study investigated the involvement of MAPK pathways (MAPK p38, ERK 1 and ERK 2) with respect to the production of cytokines induced by antigens secreted by C. pseudotuberculosis over a 60-day course of infection. CBA mice (n = 25) were divided into three groups and infected with 102 colony forming units (CFU) of attenuated strain T1, 102 CFU of virulent strain VD57 or sterile saline solution and euthanized after 30 or 60 days. Murine splenocytes were treated with specific inhibitors (MAPK p38 inhibitor, ERK 1/2 inhibitor or ERK 2 inhibitor) and cultured with secreted antigens obtained from pathogenic bacteria (SeT1 or SeVD57). RESULTS: The MAPK pathways evaluated were observed to be involved in the production of IL-10, under stimulation by secreted antigens, while the MAPK p38 and ERK 1 pathways were shown to be primarily involved in TNF-α production. By contrast, no involvement of the MAPK p38 and ERK 1 and 2 pathways was observed in IFN-γ production, while the ERK 2 pathway demonstrated involvement in IL-4 production only in the mouse splenocytes infected with VD57 under stimulation by SeT1. CONCLUSION: The authors hypothesize that MAPK p38 and ERK 1 pathways with respect to TNF-α production, as well as the MAPK p38 and ERK 1 and 2 pathways in relation to IL-10 production under infection by C. pseudotuberculosis are important regulators of cellular response. Additionally, the lack of the MAPK p38 and ERK 1/2 pathways in IFN-γ production in infected CBA murine cells stimulated with the two secreted/excreted antigens, in IL-4 production showing involvement only via the ERK 2 pathway under stimulation by SeT1 antigen during 60-day infection period with the virulent strain, suggests that these pathways regulated the production of pro-inflammatory and regulatory cytokines in the splenic cells of CBA mice.
Assuntos
Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/imunologia , Citocinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Animais , Antígenos de Bactérias/imunologia , Células Cultivadas , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/patologia , Feminino , Leucócitos Mononucleares/imunologia , Masculino , Camundongos Endogâmicos CBA , Baço/imunologiaRESUMO
BACKGROUND: Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a disease that affects small ruminants and is responsible for economic losses, including condemnation of carcasses and damaged hides. OBJECTIVE: The goal of this study was to determine if serum haptoglobin and plasma fibrinogen concentrations and peripheral blood leukocyte counts are biologic markers of CLA in sheep. METHODS: Blood from 38 clinically healthy Santa-Inês ewes selected and segregated from a commercial flock of 2500 sheep in an area endemic for C. pseudotuberculosis was collected every 30 days for 6 months. An indirect ELISA was used to detect IgM and IgG antibodies against C. pseudotuberculosis. Serum haptoglobin concentration was measured using a hemoglobin-binding assay and plasma fibrinogen concentration by refractometry following heat precipitation. Total leukocyte counts were determined using a hemocytometer, and differential leukocyte counts were performed on smears of peripheral blood. RESULTS: Twenty-one sheep were seropositive at the start of the study; 15 became seropositive during the study. Only 2 sheep were seronegative at the conclusion of the study. Haptoglobin and fibrinogen concentrations and WBC counts were not significantly different for seropositive and seronegative animals. Nine sheep, 5 that were seropositive positive at the start and 4 that became seropositive during the study period, developed abscesses in peripheral lymph nodes. There were 15 animals that became seropositive during the study, and their values did not differ significantly among the 3 phases--seronegative, acute (IgM+/IgG±), and chronic (IgM-/IgG+)--of infection. However, 11 of these sheep did not develop peripheral abscesses and had significantly higher haptoglobin concentrations and lower monocyte counts during the acute phase of the disease than did the 4 sheep that later developed abscesses. CONCLUSION: Serum haptoglobin concentration and monocyte counts may be potential markers for progression of CLA in sheep.
Assuntos
Biomarcadores/análise , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/imunologia , Leucócitos/citologia , Linfadenite/veterinária , Doenças dos Ovinos/diagnóstico , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/metabolismo , Brasil/epidemiologia , Infecções por Corynebacterium/diagnóstico , Infecções por Corynebacterium/epidemiologia , Infecções por Corynebacterium/microbiologia , Progressão da Doença , Feminino , Fibrinogênio/análise , Haptoglobinas/análise , Contagem de Leucócitos/veterinária , Linfadenite/diagnóstico , Linfadenite/epidemiologia , Linfadenite/microbiologia , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/microbiologiaRESUMO
The aim of this study was to evaluate an indirect specific ELISA developed for the detection of humoral immune response in vaccinated sheep and/or challenged with a Corynebacterium pseudotuberculosis strain. Healthy 4 month-old lambs were distributed into 4 groups: Group 1 immunized (G1, n = 5), Group 2 vaccinated/inoculated (G2, n = 8), Group 3 inoculated (G3, n = 2) and Group 4 control (G4, n = 2). Groups G1 and G2 received two doses of an experimental bacterin. Four weeks postvaccination, G2 and G3 groups were challenged with a C. pseudotuberculosis strain. Serological titers were studied by ELISA for 7 months and pathological studies were performed in groups G2, G3 and G4 by taking lung and lymph node samples for bacteriology and histopathology. The inoculated strain in G2 and G3 animals reproduced the macroscopic and microscopic lesions typical of caseous lymphadenitis (CL) and was isolated from the inoculation site, lymph nodes and/or lung in 7/8 animals from G2, and 2/2 animals of G3. The developed ELISA test had sensitivity and specificity of 98% and 100% respectively, detected significant differences between serological reactors of different experimental groups and allowed to establish a relationship with the type of treatment. We conclude that the developed ELISA may be a useful tool to identify infected animals with positive clinical CL.
Assuntos
Anticorpos Antibacterianos/análise , Vacinas Bacterianas/imunologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Linfadenite/veterinária , Doenças dos Ovinos/imunologia , Ovinos/imunologia , Vacinação/veterinária , Animais , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/prevenção & controle , Ensaio de Imunoadsorção Enzimática/métodos , Pulmão/imunologia , Linfonodos/imunologia , Linfadenite/imunologia , Linfadenite/microbiologia , Linfadenite/prevenção & controle , Distribuição Aleatória , Sensibilidade e Especificidade , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/prevenção & controleRESUMO
In the current study, the applicability of the quantification of gamma interferon (IFN-γ) levels for the detection of animals infected with Corynebacterium pseudotuberculosis and for determining caseous lymphadenitis (CLA) clinical status was evaluated. Peripheral blood leukocytes were collected from CLA nonendemic areas animals, from CLA seropositive animals without clinical signs of the disease, and from seropositive animals presenting CLA clinical signs. The leukocytes were stimulated with C. pseudotuberculosis-secreted antigens that were concentrated by the three-phase partitioning technique. An ovine IFN-γ enzyme-linked immunosorbent assay was used to quantify IFN-γ production. Goats and sheep with CLA had higher IFN-γ levels than uninfected seronegative animals. Leukocytes from sheep with CLA chronic abscesses produced higher IFN-γ levels when compared with seropositive sheep without CLA clinical signs, but this difference was not significant in goats. The sensitivity of the assay was 55.8% and 56%, whereas the specificity was 100% and 93%, for goats and sheep, respectively. In conclusion, IFN-γ is a potential marker for the determination of CLA infection status in small ruminants; however, further research is needed to improve assay sensitivity.
Assuntos
Corynebacterium pseudotuberculosis/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças das Cabras/microbiologia , Interferon gama/análise , Leucócitos/imunologia , Linfadenite/veterinária , Doenças dos Ovinos/microbiologia , Animais , Antígenos de Bactérias , Corynebacterium pseudotuberculosis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Doenças das Cabras/sangue , Doenças das Cabras/diagnóstico , Doenças das Cabras/imunologia , Cabras , Linfadenite/diagnóstico , Linfadenite/imunologia , Linfadenite/microbiologia , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Ovinos , Doenças dos Ovinos/sangue , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/imunologia , Estatísticas não ParamétricasRESUMO
El objetivo de este trabajo fue evaluar un ELISA indirecto desarrollado para medir la respuesta inmune humoral en carneros vacunados contra la linfoadenitis caseosa (LC) y/o desafiados con una cepa de Corynebacterium pseudotuberculosis homóloga. Se distribuyeron corderos de 4 meses clínicamente sanos en 4 grupos: grupo 1, corderos vacunados (G1, n = 5); grupo 2, corderos vacunados e inoculados (G2, n = 8); grupo 3, corderos inoculados (G3, n = 2); y grupo 4, control (G4, n = 2). Los animales del G1 y del G2 recibieron dos dosis de una bacterina experimental; los del G2 y del G3 fueron desafiados con una cepa de C. pseudotuberculosis cuatro semanas posvacunación. Se estudiaron por ELISA los títulos serológicos durante 7 meses y se efectuaron las necropsias en los grupos G2, G3 y G4. Se tomaron muestras de pulmón y linfonódulos para efectuar estudios bacteriológicos e histopatológicos. La cepa inoculada en los animales del G2 y del G3 reprodujo las lesiones macroscópicas y microscópicas típicas de la LC; ésta fue aislada del sitio de inoculación, de linfonódulos o de pulmón en 7/8 animales del G2 y en 2/2 animales del G3. La prueba de ELISA, con una sensibilidad del 98% y una especificidad del 100%, detectó diferencias significativas entre los serorreactores de los diferentes grupos experimentales y permitió establecer una relación con el tipo de tratamiento aplicado. Se concluye que el ELISA desarrollado puede ser una herramienta útil para identificar animales infectados y con clínica positiva a la LC.
The aim of this study was to evaluate an indirect specific ELISA developed for the detection of humoral immune response in vaccinated sheep and/or challenged with a Corynebacterium pseudotuberculosis strain. Healthy 4 month-old lambs were distributed into 4 groups: Group 1 immunized (G1, n = 5), Group 2 vaccinated/inoculated (G2, n = 8), Group 3 inoculated (G3, n = 2) and Group 4 control (G4, n = 2). Groups G1 and G2 received two doses of an experimental bacterin. Four weeks postvaccination, G2 and G3 groups were challenged with a C. pseudotuberculosis strain. Serological titers were studied by ELISA for 7 months and pathological studies were performed in groups G2, G3 and G4 by taking lung and lymph node samples for bacteriology and histopathology. The inoculated strain in G2 and G3 animals reproduced the macroscopic and microscopic lesions typical of caseous lymphadenitis (CL) and was isolated from the inoculation site, lymph nodes and/or lung in 7/8 animals from G2, and 2/2 animals of G3. The developed ELISA test had sensitivity and specificity of 98% and 100% respectively, detected significant differences between serological reactors of different experimental groups and allowed to establish a relationship with the type of treatment. We conclude that the developed ELISA may be a useful tool to identify infected animals with positive clinical CL.
Assuntos
Animais , Anticorpos Antibacterianos/análise , Vacinas Bacterianas/imunologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Linfadenite/veterinária , Doenças dos Ovinos/imunologia , Ovinos/imunologia , Vacinação/veterinária , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/prevenção & controle , Ensaio de Imunoadsorção Enzimática/métodos , Pulmão/imunologia , Linfonodos/imunologia , Linfadenite/imunologia , Linfadenite/microbiologia , Linfadenite/prevenção & controle , Distribuição Aleatória , Sensibilidade e Especificidade , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/prevenção & controleRESUMO
Background: Caseous lymphadenitis (CL) is considered one of the most important diseases, economically, in sheep and goats in the United States, Canada and Australia. Annual losses in wool production reaches 17 million dollars in Australia, the disease cause important impact in Europe, Africa, Middle East, Australia, and in the Americas. The CL is a chronic disease, which affects mainly sheep and goats. The etiologic agent is the Corynebacterium pseudotuberculosis. The presence of the agent in feces contributes to environmental contamination and increases the risk of infections in herds. For countries with international marketing of live animals, the CL is a serious health problem to livestock disease-free. The objective of this study was to report the occurrence of lesions caused by CL in sheep slaughtered under the official service of inspection of the state of Rio Grande do Sul and correlate it to the economic losses due to the lesions found. Materials, Methods & Results: Data concerning the fate of raw materials, the total number of animals slaughtered and injuries from the ante and post-mortem were provided by the Federal Inspection Service (SIFs do Ministério da Agricultura, Pecuária e Abastecimento no Rio Grande do Sul (SFA/MAPA-RS). Data from the slaughtered animals were generated by the information system management service inspection (SIGSIF), requested in official character, which included the daily slaughter maps, as well as monthly and annual reports. Were collected data from 932,547 sheep slaughtered under federal inspection, from January 2004 to August 2009. The number of cases recorded only in Federal Inspection Service was 348 and the number of animals condemned at slaughter was 86.[...](AU)
Assuntos
Animais , Linfadenite/veterinária , Inspeção Sanitária , Corynebacterium pseudotuberculosis/imunologia , Doenças dos Ovinos/prevenção & controleRESUMO
Background: Caseous lymphadenitis (CL) is considered one of the most important diseases, economically, in sheep and goats in the United States, Canada and Australia. Annual losses in wool production reaches 17 million dollars in Australia, the disease cause important impact in Europe, Africa, Middle East, Australia, and in the Americas. The CL is a chronic disease, which affects mainly sheep and goats. The etiologic agent is the Corynebacterium pseudotuberculosis. The presence of the agent in feces contributes to environmental contamination and increases the risk of infections in herds. For countries with international marketing of live animals, the CL is a serious health problem to livestock disease-free. The objective of this study was to report the occurrence of lesions caused by CL in sheep slaughtered under the official service of inspection of the state of Rio Grande do Sul and correlate it to the economic losses due to the lesions found. Materials, Methods & Results: Data concerning the fate of raw materials, the total number of animals slaughtered and injuries from the ante and post-mortem were provided by the Federal Inspection Service (SIFs do Ministério da Agricultura, Pecuária e Abastecimento no Rio Grande do Sul (SFA/MAPA-RS). Data from the slaughtered animals were generated by the information system management service inspection (SIGSIF), requested in official character, which included the daily slaughter maps, as well as monthly and annual reports. Were collected data from 932,547 sheep slaughtered under federal inspection, from January 2004 to August 2009. The number of cases recorded only in Federal Inspection Service was 348 and the number of animals condemned at slaughter was 86.[...]