Antimicrobial and toxicological evaluation of Origanum vulgare: an in vivo study

Abstract Origanum vulgare has been of great interest in academia and pharma industry due to its antioxidant, antifungal and antitumor properties. The present study aimed to find the anti-MRSA potential and in vivo toxicity assessments of O. vulgare. O. vulgare extract was used to monitor anti-MRSA activity in mice. Following MRSA established infection in mice (Mus musculus), treatment with O. vulgare was continued for 7 days. Autopsies were performed and re-isolation, gross lesion scoring and bacterial load in various organs were measured. Additionally, blood sample was analysed for hematological assays. Toxicity assessment of O. vulgare potential as medicine was done at 200 mg/kg and 400 mg/kg by evaluating liver and kidney functions. Bacterial load and gross lesion in lungs and heart were significantly low compared to positive control following O. vulgare treatment. Likewise, O. vulgare treated groups had hematological, neutrophil and TLC values similar to control groups. Increased AST, ALP and total bilirubin alongwith marked hepatocellular degeneration and distortion around the central vein, inflammatory cell infiltration, and cytoplasmic vacuolization of hepatic cells was observed at higher dose. It is concluded that crude extract of O. vulgare may contain beneficial secondary metabolites and in future may be explored for curing infectious diseases.

Resumo Origanum vulgare tem despertado grande interesse na academia e na indústria farmacêutica devido às suas propriedades antioxidantes, antifúngicas e antitumorais. O presente estudo teve como objetivo encontrar o potencial anti-MRSA e avaliações de toxicidade in vivo de O. vulgare. O extrato de O. vulgare foi usado para monitorar a atividade anti-MRSA em camundongos. Após infecção estabelecida por MRSA em camundongos (Mus musculus), o tratamento com O. vulgare foi continuado por 7 dias. As autópsias foram realizadas e o reisolamento, pontuação das lesões grosseiras e carga bacteriana em vários órgãos foram medidos. Além disso, a amostra de sangue foi analisada para ensaios hematológicos. A avaliação da toxicidade do potencial de O. vulgare como medicamento foi feita com 200 mg / kg e 400 mg / kg, avaliando as funções hepática e renal. A carga bacteriana e as lesões graves nos pulmões e no coração foram significativamente baixas em comparação com o controle positivo após o tratamento com O. vulgare. Da mesma forma, os grupos tratados com O. vulgare apresentaram valores hematológicos, de neutrófilos e de TLC semelhantes aos grupos de controle. Aumento de AST, ALP e bilirrubina total juntamente com degeneração hepatocelular marcada e distorção ao redor da veia central, infiltração de células inflamatórias e vacuolização citoplasmática de células hepáticas foram observados em doses mais altas. Conclui-se que o extrato bruto de O. vulgare pode conter metabólitos secundários benéficos e, no futuro, pode ser explorado para a cura de doenças infecciosas.

Antimicrobial and toxicological evaluation of Origanum vulgare: an in vivo study / Avaliação antimicrobiana e toxicológica de Origanum vulgare: um estudo in vivo

Abstract Origanum vulgare has been of great interest in academia and pharma industry due to its antioxidant, antifungal and antitumor properties. The present study aimed to find the anti-MRSA potential and in vivo toxicity assessments of O. vulgare. O. vulgare extract was used to monitor anti-MRSA activity in mice. Following MRSA established infection in mice (Mus musculus), treatment with O. vulgare was continued for 7 days. Autopsies were performed and re-isolation, gross lesion scoring and bacterial load in various organs were measured. Additionally, blood sample was analysed for hematological assays. Toxicity assessment of O. vulgare potential as medicine was done at 200 mg/kg and 400 mg/kg by evaluating liver and kidney functions. Bacterial load and gross lesion in lungs and heart were significantly low compared to positive control following O. vulgare treatment. Likewise, O. vulgare treated groups had hematological, neutrophil and TLC values similar to control groups. Increased AST, ALP and total bilirubin alongwith marked hepatocellular degeneration and distortion around the central vein, inflammatory cell infiltration, and cytoplasmic vacuolization of hepatic cells was observed at higher dose. It is concluded that crude extract of O. vulgare may contain beneficial secondary metabolites and in future may be explored for curing infectious diseases.

Resumo Origanum vulgare tem despertado grande interesse na academia e na indústria farmacêutica devido às suas propriedades antioxidantes, antifúngicas e antitumorais. O presente estudo teve como objetivo encontrar o potencial anti-MRSA e avaliações de toxicidade in vivo de O. vulgare. O extrato de O. vulgare foi usado para monitorar a atividade anti-MRSA em camundongos. Após infecção estabelecida por MRSA em camundongos (Mus musculus), o tratamento com O. vulgare foi continuado por 7 dias. As autópsias foram realizadas e o reisolamento, pontuação das lesões grosseiras e carga bacteriana em vários órgãos foram medidos. Além disso, a amostra de sangue foi analisada para ensaios hematológicos. A avaliação da toxicidade do potencial de O. vulgare como medicamento foi feita com 200 mg / kg e 400 mg / kg, avaliando as funções hepática e renal. A carga bacteriana e as lesões graves nos pulmões e no coração foram significativamente baixas em comparação com o controle positivo após o tratamento com O. vulgare. Da mesma forma, os grupos tratados com O. vulgare apresentaram valores hematológicos, de neutrófilos e de TLC semelhantes aos grupos de controle. Aumento de AST, ALP e bilirrubina total juntamente com degeneração hepatocelular marcada e distorção ao redor da veia central, infiltração de células inflamatórias e vacuolização citoplasmática de células hepáticas foram observados em doses mais altas. Conclui-se que o extrato bruto de O. vulgare pode conter metabólitos secundários benéficos e, no futuro, pode ser explorado para a cura de doenças infecciosas.

Antimicrobial and toxicological evaluation of Origanum vulgare: an in vivo study / Avaliação antimicrobiana e toxicológica de Origanum vulgare: um estudo in vivo

Origanum vulgare has been of great interest in academia and pharma industry due to its antioxidant, antifungal and antitumor properties. The present study aimed to find the anti-MRSA potential and in vivo toxicity assessments of O. vulgare. O. vulgare extract was used to monitor anti-MRSA activity in mice. Following MRSA established infection in mice (Mus musculus), treatment with O. vulgare was continued for 7 days. Autopsies were performed and re-isolation, gross lesion scoring and bacterial load in various organs were measured. Additionally, blood sample was analysed for hematological assays. Toxicity assessment of O. vulgare potential as medicine was done at 200 mg/kg and 400 mg/kg by evaluating liver and kidney functions. Bacterial load and gross lesion in lungs and heart were significantly low compared to positive control following O. vulgare treatment. Likewise, O. vulgare treated groups had hematological, neutrophil and TLC values similar to control groups. Increased AST, ALP and total bilirubin along with marked hepatocellular degeneration and distortion around the central vein, inflammatory cell infiltration, and cytoplasmic vacuolization of hepatic cells was observed at higher dose. It is concluded that crude extract of O. vulgare may contain beneficial secondary metabolites and in future may be explored for curing infectious diseases.

Origanum vulgare tem despertado grande interesse na academia e na indústria farmacêutica devido às suas propriedades antioxidantes, antifúngicas e antitumorais. O presente estudo teve como objetivo encontrar o potencial anti-MRSA e avaliações de toxicidade in vivo de O. vulgare. O extrato de O. vulgare foi usado para monitorar a atividade anti-MRSA em camundongos. Após infecção estabelecida por MRSA em camundongos (Mus musculus), o tratamento com O. vulgare foi continuado por 7 dias. As autópsias foram realizadas e o reisolamento, pontuação das lesões grosseiras e carga bacteriana em vários órgãos foram medidos. Além disso, a amostra de sangue foi analisada para ensaios hematológicos. A avaliação da toxicidade do potencial de O. vulgare como medicamento foi feita com 200 mg / kg e 400 mg / kg, avaliando as funções hepática e renal. A carga bacteriana e as lesões graves nos pulmões e no coração foram significativamente baixas em comparação com o controle positivo após o tratamento com O. vulgare. Da mesma forma, os grupos tratados com O. vulgare apresentaram valores hematológicos, de neutrófilos e de TLC semelhantes aos grupos de controle. Aumento de AST, ALP e bilirrubina total juntamente com degeneração hepatocelular marcada e distorção ao redor da veia central, infiltração de células inflamatórias e vacuolização citoplasmática de células hepáticas foram observados em doses mais altas. Conclui-se que o extrato bruto de O. vulgare pode conter metabólitos secundários benéficos e, no futuro, pode ser explorado para a cura de doenças infecciosas.

Antimicrobial and toxicological evaluation of Origanum vulgare: an in vivo study / Avaliação antimicrobiana e toxicológica de Origanum vulgare: um estudo in vivo

Origanum vulgare has been of great interest in academia and pharma industry due to its antioxidant, antifungal and antitumor properties. The present study aimed to find the anti-MRSA potential and in vivo toxicity assessments of O. vulgare. O. vulgare extract was used to monitor anti-MRSA activity in mice. Following MRSA established infection in mice (Mus musculus), treatment with O. vulgare was continued for 7 days. Autopsies were performed and re-isolation, gross lesion scoring and bacterial load in various organs were measured. Additionally, blood sample was analysed for hematological assays. Toxicity assessment of O. vulgare potential as medicine was done at 200 mg/kg and 400 mg/kg by evaluating liver and kidney functions. Bacterial load and gross lesion in lungs and heart were significantly low compared to positive control following O. vulgare treatment. Likewise, O. vulgare treated groups had hematological, neutrophil and TLC values similar to control groups. Increased AST, ALP and total bilirubin along with marked hepatocellular degeneration and distortion around the central vein, inflammatory cell infiltration, and cytoplasmic vacuolization of hepatic cells was observed at higher dose. It is concluded that crude extract of O. vulgare may contain beneficial secondary metabolites and in future may be explored for curing infectious diseases.(AU)

Origanum vulgare tem despertado grande interesse na academia e na indústria farmacêutica devido às suas propriedades antioxidantes, antifúngicas e antitumorais. O presente estudo teve como objetivo encontrar o potencial anti-MRSA e avaliações de toxicidade in vivo de O. vulgare. O extrato de O. vulgare foi usado para monitorar a atividade anti-MRSA em camundongos. Após infecção estabelecida por MRSA em camundongos (Mus musculus), o tratamento com O. vulgare foi continuado por 7 dias. As autópsias foram realizadas e o reisolamento, pontuação das lesões grosseiras e carga bacteriana em vários órgãos foram medidos. Além disso, a amostra de sangue foi analisada para ensaios hematológicos. A avaliação da toxicidade do potencial de O. vulgare como medicamento foi feita com 200 mg / kg e 400 mg / kg, avaliando as funções hepática e renal. A carga bacteriana e as lesões graves nos pulmões e no coração foram significativamente baixas em comparação com o controle positivo após o tratamento com O. vulgare. Da mesma forma, os grupos tratados com O. vulgare apresentaram valores hematológicos, de neutrófilos e de TLC semelhantes aos grupos de controle. Aumento de AST, ALP e bilirrubina total juntamente com degeneração hepatocelular marcada e distorção ao redor da veia central, infiltração de células inflamatórias e vacuolização citoplasmática de células hepáticas foram observados em doses mais altas. Conclui-se que o extrato bruto de O. vulgare pode conter metabólitos secundários benéficos e, no futuro, pode ser explorado para a cura de doenças infecciosas.(AU)

Isolation of Saprolegnia aenigmatica oomycetes and protocol for experimental infection of pacu (Piaractus mesopotamicus) / Isolamento do oomiceto Saprolegnia aenigmatica e protocolo de infecção experimental em pacu (Piaractus mesopotamicus)

Successful disease treatment depends on molecular studies under indoor conditions with experimental infection protocols that facilitate understanding the disease and the drug`s efficacy. The internal transcribed spacer (ITS) region was sequenced from three isolates, which were identified as Saprolegnia aenigmatica. Subsequently, healthy fish were immunosuppressed with dexamethasone (1.2 mg kg-1) and descaled to the skin using a sharp scalpel. These individuals were isolated in individual aquariums maintained at 22°C. Individuals in one group were subcutaneously inoculated with 9,000 zoospores (DDZ treatment), a second group was exposed to oomycetes in water with three colonized baits (DDB), a third group was maintained in water without zoospores (DD), and a control group (C) consisted of healthy animals. After 48 and 96 hours, two animals from each group were euthanized for fungal reisolation. The fish from groups DD and C did not show clinical signs, and no oomycetes were isolated. The animals from the DDZ and DDB groups showed cotton-wool-like masses on the skin, and S. aenigmatica was re-isolated. Thus, for infection using zoospores or baits parasitized by S. aenigmatica, an immunosuppressor (dexamethasone) and a sharp scalpel can be used effectively to establish an experimental infection in P. mesopotamicus.

O sucesso do tratamento de uma enfermidade depende do estudo de moléculas em condições de laboratório por meio de protocolos de infecção experimental que viabilizam a compreensão da doença e da eficácia dos fármacos. Pela sequência ITS foram identificadas três cepas de Saprolegnia aenigmatica. Dessa forma, pacus sadios foram submetidos à imunossupressão com dexametasona (1,2 mg kg-1), esfoliados com auxílio de bisturi e distribuídos em aquários a 22ºC. Após este procedimento, um grupo de animais foi inoculado com 9.000 zoósporos/peixe subcutâneo (DEZ), a outro foram adicionadas três iscas colonizadas com o oomiceto na água (DEI), um terceiro grupo foi mantido sem contato com o oomiceto (DE) e um quarto grupo, de animais sadios, representaram o controle (C). Após 48 e 96h deste procedimento, foram eutanaziados animais de cada grupo para reisolamento. Os animais do grupo DE e C não apresentaram sinais clínicos e não foi reisolado o oomiceto. Porém, tanto os animais do grupo DEZ quanto como os animais do grupo DEI apresentaram micélio branco na pele e foi reisolado Saprolegnia aenigmatica. Assim, a infecção com zoósporos ou com iscas colonizadas por S. aenigmatica, com o uso de dexametasona e abrasivo epitelial são formas eficazes de infecção experimental em P. mesopotamicus.

Isolation of Saprolegnia aenigmatica oomycetes and protocol for experimental infection of pacu (Piaractus mesopotamicus) / Isolamento do oomiceto Saprolegnia aenigmatica e protocolo de infecção experimental em pacu (Piaractus mesopotamicus)

Successful disease treatment depends on molecular studies under indoor conditions with experimental infection protocols that facilitate understanding the disease and the drug`s efficacy. The internal transcribed spacer (ITS) region was sequenced from three isolates, which were identified as Saprolegnia aenigmatica. Subsequently, healthy fish were immunosuppressed with dexamethasone (1.2 mg kg-1) and descaled to the skin using a sharp scalpel. These individuals were isolated in individual aquariums maintained at 22°C. Individuals in one group were subcutaneously inoculated with 9,000 zoospores (DDZ treatment), a second group was exposed to oomycetes in water with three colonized baits (DDB), a third group was maintained in water without zoospores (DD), and a control group (C) consisted of healthy animals. After 48 and 96 hours, two animals from each group were euthanized for fungal reisolation. The fish from groups DD and C did not show clinical signs, and no oomycetes were isolated. The animals from the DDZ and DDB groups showed cotton-wool-like masses on the skin, and S. aenigmatica was re-isolated. Thus, for infection using zoospores or baits parasitized by S. aenigmatica, an immunosuppressor (dexamethasone) and a sharp scalpel can be used effectively to establish an experimental infection in P. mesopotamicus.(AU)

O sucesso do tratamento de uma enfermidade depende do estudo de moléculas em condições de laboratório por meio de protocolos de infecção experimental que viabilizam a compreensão da doença e da eficácia dos fármacos. Pela sequência ITS foram identificadas três cepas de Saprolegnia aenigmatica. Dessa forma, pacus sadios foram submetidos à imunossupressão com dexametasona (1,2 mg kg-1), esfoliados com auxílio de bisturi e distribuídos em aquários a 22ºC. Após este procedimento, um grupo de animais foi inoculado com 9.000 zoósporos/peixe subcutâneo (DEZ), a outro foram adicionadas três iscas colonizadas com o oomiceto na água (DEI), um terceiro grupo foi mantido sem contato com o oomiceto (DE) e um quarto grupo, de animais sadios, representaram o controle (C). Após 48 e 96h deste procedimento, foram eutanaziados animais de cada grupo para reisolamento. Os animais do grupo DE e C não apresentaram sinais clínicos e não foi reisolado o oomiceto. Porém, tanto os animais do grupo DEZ quanto como os animais do grupo DEI apresentaram micélio branco na pele e foi reisolado Saprolegnia aenigmatica. Assim, a infecção com zoósporos ou com iscas colonizadas por S. aenigmatica, com o uso de dexametasona e abrasivo epitelial são formas eficazes de infecção experimental em P. mesopotamicus.(AU)

Establishment of a Pathogenicity Index for One-day-old Broilers to Pasteurella multocida Strains Isolated from Clinical Cases in Poultry and Swine

Although Pasteurella multocida is a member of the respiratory microbiota, under some circumstances, it is a primary agent of diseases , such as fowl cholera (FC), that cause significant economic losses. Experimental inoculations can be employed to evaluate the pathogenicity of strains, but the results are usually subjective and knowledge on the pathogenesis of this agent is still limited. The objective of this study was to establish a new methodology for classifying the pathogenicity of P. multocida by formulating a standard index. Strains isolated from FC cases and from swine with respiratory problems were selected. One hundred mL of a bacterial culture of each strain, containing 106 CFU, was inoculated in 10 one-day-old broilers. Mortality after inoculation, time of death (TD), and the presence of six macroscopic lesions were evaluated over a period of seven days post-inoculation (dpi). A Pathogenicity Index Per Bird (IPI), ranging 0 to 10, was calculated. Liver and heart fragments were collected to reisolate the bacteria. Blood was collected from the surviving birds, and an ELISA test was carried out to detect specific antibodies. The median of the pathogenicity indices, the number of lesions and the rate of bacteria reisolation were significantly different (p 0.05) among the origins of the isolates (p 0.05). The pathogenicity index developed in this study allows the classification of Pasteurella multocida pathogenicity and may be an alternative to the pathogenicity models currently used for screening.

Establishment of a Pathogenicity Index for One-day-old Broilers to Pasteurella multocida Strains Isolated from Clinical Cases in Poultry and Swine

Although Pasteurella multocida is a member of the respiratory microbiota, under some circumstances, it is a primary agent of diseases , such as fowl cholera (FC), that cause significant economic losses. Experimental inoculations can be employed to evaluate the pathogenicity of strains, but the results are usually subjective and knowledge on the pathogenesis of this agent is still limited. The objective of this study was to establish a new methodology for classifying the pathogenicity of P. multocida by formulating a standard index. Strains isolated from FC cases and from swine with respiratory problems were selected. One hundred mL of a bacterial culture of each strain, containing 106 CFU, was inoculated in 10 one-day-old broilers. Mortality after inoculation, time of death (TD), and the presence of six macroscopic lesions were evaluated over a period of seven days post-inoculation (dpi). A Pathogenicity Index Per Bird (IPI), ranging 0 to 10, was calculated. Liver and heart fragments were collected to reisolate the bacteria. Blood was collected from the surviving birds, and an ELISA test was carried out to detect specific antibodies. The median of the pathogenicity indices, the number of lesions and the rate of bacteria reisolation were significantly different (p 0.05) among the origins of the isolates (p 0.05). The pathogenicity index developed in this study allows the classification of Pasteurella multocida pathogenicity and may be an alternative to the pathogenicity models currently used for screening.(AU)

Análise da evolução do gene msp1 de Anaplasma marginale em bezerros e carrapatos e Propagação e caracterização de um isolado de Anaplasma marginale de Portugal em células IDE8

Este trabalho é composto de dois capítulos: O primeiro capítulo visou a avaliação da diversidade das sequências repetidas da proteína MSP1a de A. marginale em bovinos experimentalmente infectados e em R. microplus durante o período de 1 ano e realizou-se o reisolamento da estirpe AmRio 1 de A. marginale a partir de leucócitos. O segundo capítulo objetivou o isolamento, propagação e caracterização de uma estirpe de A. marginale de Portugal em células IDE8. Para a avaliação da diversidade de A. marginale, os bovinos foram infestados com larvas de R. microplus e posteriormente as estirpes AmRio 1 e AmRio 2 mantidas em cultivo celular foram inoculadas respectivamente no bovino 1 e no bovino 2. Posteriormente amostras de sangue foram colhidas durante 1 ano para realização de esfregaços, hematócrito, proteínas plasmáticas e a realização de seminested PCR para os genes msp5 e msp1. A partir de amostras de sangue também foram realizados isolamentos de A. marginale a partir de leucócitos. Carrapatos foram colhidos durante todo o período de estudo e suas glândulas salivares, intestinos e saliva foram submetidos à seminested PCR para o gene msp5 e msp1. Ambos os animais apresentaram estabilidade dos parâmetros avaliados e ausência de sinais clínicos, revelando uma persistência da infecção. Inclusões foram observadas em leucócitos em um esfregaço do animal 1, e foi realizado reisolamento de A. marginale nas quatro tentativas utilizando-se leucócitos para o inóculo. O animal 1 se tornou positivo para o gene msp5 a partir do dia 12 após a infecção, enquanto o animal 2 não se tornou positivo para a cepa inoculada durante todo o período de estudo analisado. As repetições em tandem avaliadas revelaram positividade para a estirpe AmRio 1 no animal 1. De 58 amostras de órgãos de carrapatos, 6 foram positivas para o gene msp5 no animal 1. No animal 2, de 20 amostras analisadas, 4 foram positivas para o gene msp5. No segundo capítulo utilizou-se eritrócitos de um bovino naturalmente infectado da região do Alentejo, Portugal, para a realização do isolamento de uma estirpe de A. marginale em células IDE8. O animal doador do inóculo foi considerado persistentemente infectado com 0,25% de rickettsemia. Observou-se inclusões características da infecção trinta e cinco dias após a inoculação em células IDE8.

This thesis consists of two chapters: The purpose of the first chapter was to evaluate the tandem repeat diversity in the major surface protein MSP1a of Anaplasma marginale in experimentally infected cattle and in Rhipicephalus microplus over the span of 1 year, and re-isolation of the A. marginale AmRio1 strain from cattle leukocytes was performed. The second chapter was aimed at isolating, propagating, and characterizing a novel Anaplasma sp. genotype from Portugal in IDE8 cells. To evaluate A. marginale genetic diversity, cattle were infested with R. microplus larvae and subsequently infected by A. marginale from tick cell cultures. The strains AmRio1 and AmRio2 were inoculated in bovine 1 and bovine 2, respectively. Subsequently, blood samples were collected over 1 year for blood smears, hematocrit, plasma proteins, and semi-nested polymerase chain reaction (PCR) for the msp5 and msp1 genes. A. marginale was also isolated from the cattle leukocytes. Ticks were collected and their salivary gland and gut DNA were tested using semi-nested PCR for the msp5 and msp1 genes. Both animals showed stability in the evaluated parameters and absence of clinical signs, showing that they had persistent infection. A blood smear from Animal 1 showed inclusions inside the leukocytes, and four attempts were made to isolate Anaplasma sp. from leukocytes for the inoculum. Animal 1 became positive for the msp5 gene on day 12 after infection, while positivity was detected in Animal 2 only 77 days after second exposure to the agent. The tandem repeats were stable when the blood and tick samples of both animals were evaluated, demonstrating the presence of the strains AmRio1 in Animal 1 and AmRio2 in Animal 2. In the second chapter, erythrocytes from a naturally infected bovine from Portugal were used for isolating Anaplasma sp. in IDE8 cells. Suggestive inclusions of infection were observed in the IDE8 cells 35 days after inoculation. The Anaplasmataceae family 16S rRNA gene from the cultures was sequenced, and showed that the isolated rickettsia was close to the A. platys species in the phylogenetic tree.

Recovery of Salmonella Gallinarum in the Organs of Experimentally-Inoculated Japanese Quails (Coturnix coturnix)

Salmonellosis is an infection caused by specific or non specific serotypes of theSalmonella genus, responsible for losses in the poultry industry. Fowl typhoid, caused by S. Gallinarum (SG) is important because it causes elevated mortality in adult birds, leading to economic losses in the poultry industry. This study aimed at quantifying the number of viable SG cells in the liver, spleen, lung, cecum, and reproductive tract (ovary and testicles) of experimentally inoculated Japanese quails (Coturnix coturnix), as well as SG shedding in their feces. One hundred and two Japanese quails, with four months of age at the beginning of the experiment, were used. The birds were inoculated with three bacterial cultures containing different concentrations (6x104CFU/0.1mL, 2x105 CFU/0.4mL, or 5x106CFU/0.2mL) of SG resistant to nalidixic acid. On days 1, 4, 7, and 14 after the inoculation (dpi) individual cloacal swabs were collected from six birds per group, which were subsequently sacrificed for organ sampling. The swab samples were streaked directly on plates containing brilliant green agar and nalidixic acid (VBNal). Samples that were negative after 24h, were streaked again. The collected organs were individually macerated and transferred to buffered peptone water at 0.1%. The solutions were immediately diluted serially for CFU counting in VBNal. SG was successfully recovered from one quail, which was inoculated with 2x105 CFU/0.4mL, and from five quails of the group inoculated with 5x106CFU/0.2mL inoculum. All of the analyzed cloacal swab samples were negative. Therefore, this study demonstrated it was difficult to isolate SG from the analyzed organs and that it was not possible to recover thepathogen in the cloacal swabs collected from inoculated quails. These results may be explained by the absence of flagella in SG, inducing weak intestinal immune response in the beginning of the infection and preventing its isolation in cloacal swab samples. The low positivity rate of the analyzed organs may be due to the immune status of the euthanized birds, since the SG dissemination in the animal organism occurs mostly close to death, which was observed in the birds found dead during the experiment.(AU)

Recovery of Salmonella Gallinarum in the Organs of Experimentally-Inoculated Japanese Quails (Coturnix coturnix)

Salmonellosis is an infection caused by specific or non specific serotypes of theSalmonella genus, responsible for losses in the poultry industry. Fowl typhoid, caused by S. Gallinarum (SG) is important because it causes elevated mortality in adult birds, leading to economic losses in the poultry industry. This study aimed at quantifying the number of viable SG cells in the liver, spleen, lung, cecum, and reproductive tract (ovary and testicles) of experimentally inoculated Japanese quails (Coturnix coturnix), as well as SG shedding in their feces. One hundred and two Japanese quails, with four months of age at the beginning of the experiment, were used. The birds were inoculated with three bacterial cultures containing different concentrations (6x104CFU/0.1mL, 2x105 CFU/0.4mL, or 5x106CFU/0.2mL) of SG resistant to nalidixic acid. On days 1, 4, 7, and 14 after the inoculation (dpi) individual cloacal swabs were collected from six birds per group, which were subsequently sacrificed for organ sampling. The swab samples were streaked directly on plates containing brilliant green agar and nalidixic acid (VBNal). Samples that were negative after 24h, were streaked again. The collected organs were individually macerated and transferred to buffered peptone water at 0.1%. The solutions were immediately diluted serially for CFU counting in VBNal. SG was successfully recovered from one quail, which was inoculated with 2x105 CFU/0.4mL, and from five quails of the group inoculated with 5x106CFU/0.2mL inoculum. All of the analyzed cloacal swab samples were negative. Therefore, this study demonstrated it was difficult to isolate SG from the analyzed organs and that it was not possible to recover thepathogen in the cloacal swabs collected from inoculated quails. These results may be explained by the absence of flagella in SG, inducing weak intestinal immune response in the beginning of the infection and preventing its isolation in cloacal swab samples. The low positivity rate of the analyzed organs may be due to the immune status of the euthanized birds, since the SG dissemination in the animal organism occurs mostly close to death, which was observed in the birds found dead during the experiment.

Deforming mandibular osteomyelitis in a cow caused by Trueperella pyogenes / Osteomielite mandibular deformante em vaca causada por Trueperella pyogenes

This study reports an unusual case of deforming mandibular osteomyelitis in a cow caused by Trueperella (Arcanobacterium) pyogenes, on the face of the ventrolateral caudal portion of the right branch of the mandible. Fragment aspired of lesion by fine needle allowed cytological characterization, isolation and identification of T. pyogenes. Radiographic examination showed marked periosteal reaction in the right mandible, numerous lytic areas and cortical bone destruction. Despite of treatment based on in vitro antimicrobial sensitivity test, it was recommended the euthanasia due to progressive worsening of the cow's condition. Multiple abscesses were observed in the mandibular region at necropsy. Pyogranuloma was characterized in histological exam. Sampled material collected from the lesion after necropsy resulted in microbiological reisolation of T. pyogenes.(AU)

Relata-se caso incomum de osteomielite mandibular deformante em vaca, causada por Trueperella (Arcanobacterium) pyogenes, na face ventro-lateral da porção caudal do ramo direito da mandíbula. A punção aspirativa de fragmento da lesão permitiu a caracterização citológica, o isolamento microbiano e identificação de T. pyogenes. Exame radiográfico mostrou acentuada reação periodontal na mandíbula direita, com predomínio de áreas líticas e destruição da cortical óssea. Apesar da instituição do tratamento baseado no teste de sensibilidade microbiana in vitro, foi recomendada a eutanásia, em virtude da piora progressiva do estado geral do animal. No exame post-mortem foram observados múltiplos abscessos na lesão que, histologicamente, foi caracterizada como piogranuloma. A colheita de material da região mandibular afetada, após a necropsia, resultou no reisolamento microbiológico de T. pyogenes.(AU)

Salmonella Gallinarum virulence in experimentally-infected Japanese quails (Coturnix japonica)

Salmonella Gallinarum is the agent of fowl typhoid in poultry and infects mainly adult galliforms, causing significant economic losses in poultry production. Because quails are susceptible to this disease and quail production is becoming increasingly important in Brazil, this study was carried out to evaluate the virulence of Salmonella Gallinarum strain to quails. The inoculum was prepared from S. Gallinarum strain resistant to nalidixic acid. Forty eight 16-week-old Japanese quails were randomly distributed in three groups. Before the experiment, cloacal swabs were collected from all birds in order to confirm they were free from Salmonella spp. Cloacal swabs and fecal samples were collected on days 03, 06, 09, 12, and 15 post-inoculation. Birds that died during the experiment were submitted to post-mortem examination, and had their organs aseptically collected for bacteriological examination. All eggs produced during the experiment were also examined. The mortality rate recorded during the experiment was 43.75% (21/48). S. Gallinarum was recovered from the organs of the birds that naturally died during the experiment, but the agent was not isolated from the organs of sacrificed birds. No egg sample was positive for Salmonella Gallinarum. It was concluded that S. Gallinarum may be recovered from the organs of experimentally-infected Japanese quails.

Salmonella Gallinarum virulence in experimentally-infected Japanese quails (Coturnix japonica)

Salmonella Gallinarum is the agent of fowl typhoid in poultry and infects mainly adult galliforms, causing significant economic losses in poultry production. Because quails are susceptible to this disease and quail production is becoming increasingly important in Brazil, this study was carried out to evaluate the virulence of Salmonella Gallinarum strain to quails. The inoculum was prepared from S. Gallinarum strain resistant to nalidixic acid. Forty eight 16-week-old Japanese quails were randomly distributed in three groups. Before the experiment, cloacal swabs were collected from all birds in order to confirm they were free from Salmonella spp. Cloacal swabs and fecal samples were collected on days 03, 06, 09, 12, and 15 post-inoculation. Birds that died during the experiment were submitted to post-mortem examination, and had their organs aseptically collected for bacteriological examination. All eggs produced during the experiment were also examined. The mortality rate recorded during the experiment was 43.75% (21/48). S. Gallinarum was recovered from the organs of the birds that naturally died during the experiment, but the agent was not isolated from the organs of sacrificed birds. No egg sample was positive for Salmonella Gallinarum. It was concluded that S. Gallinarum may be recovered from the organs of experimentally-infected Japanese quails.

Salmonella Gallinarum virulence in experimentally-infected Japanese quails (Coturnix japonica)

Salmonella Gallinarum is the agent of fowl typhoid in poultry and infects mainly adult galliforms, causing significant economic losses in poultry production. Because quails are susceptible to this disease and quail production is becoming increasingly important in Brazil, this study was carried out to evaluate the virulence of Salmonella Gallinarum strain to quails. The inoculum was prepared from S. Gallinarum strain resistant to nalidixic acid. Forty eight 16-week-old Japanese quails were randomly distributed in three groups. Before the experiment, cloacal swabs were collected from all birds in order to confirm they were free from Salmonella spp. Cloacal swabs and fecal samples were collected on days 03, 06, 09, 12, and 15 post-inoculation. Birds that died during the experiment were submitted to post-mortem examination, and had their organs aseptically collected for bacteriological examination. All eggs produced during the experiment were also examined. The mortality rate recorded during the experiment was 43.75% (21/48). S. Gallinarum was recovered from the organs of the birds that naturally died during the experiment, but the agent was not isolated from the organs of sacrificed birds. No egg sample was positive for Salmonella Gallinarum. It was concluded that S. Gallinarum may be recovered from the organs of experimentally-infected Japanese quails.(AU)

EFICÁCIA TERAPÊUTICA, SEGURANÇA CLÍNICA E ECOTOXICOLÓGICO DA FOSFOMICINA EM TILÁPIAS, Oreochromis niloticus, COM AEROMONOSE E ESTREPTOCOCOSE

O presente estudo teve por objetivos avaliar a eficácia terapêutica a segurança clínica e ecotoxicológica do antimicrobiano fosfomicina para o tratamento de aeromonose e streptococose em tilápia do Nilo (Oreochromis niloticus). Para tal, a segurança clínica foi avaliada com as doses de 10, 20 e 40 mg de fosfomicina.kg-1; os estudos de eficácia com a dose terapêutica de 10 mg de fosfomicina.kg-1, foram conduzidos com peixes desafiados com Aeromonas hydrophila e Streptococcus agalactiae; os ensaios de ecotoxicidade foram feitos para a determinação de CL50 em peixes (O. niloticus) e plantas aquáticas (Lemna minor) e de CE50 para crustáceos (Daphnia magna) e moluscos (Pomacea canaliculata). Nos estudos foram utilizadas tilápias de 112g ± 5,6g, acondicionadas em tanques de 400 L (n=10). Ambos os estudos de eficácia terapêutica com 10 mg.kg-1 demonstraram a eficácia para A. hydrophila e S. agalactiae, pois nas análises de reisolamento destes microrganismos não ocorreu crescimento dos mesmos em amostras de fígado, rim, baço e cérebro dos peixes tratados quando comparados aos grupos infectados e não tratados nos quais o número de unidades formadoras de colônias (UFC) foi de 7,6 ± 2,4 por placa. Na infecção por A. hydrophila apresentaram microcitose e linfopenia, assim como, trombocitose na fase inicial da infecção seguida de trombocitopenia. Lesões nos tecidos hepáticos e renais corroboraram as alterações da atividade enzimática sérica de ALT, AST, FA e nos níveis de creatinina, colesterol e triglicerídeos. Na infecção por S. agalactiae apresentaram diminuição do número de eritrócitos e dos valores percentuais de hematócrito, caracterizando quadro de hemólise, associado à linfopenia, trombocitopenia e neutrofilia na fase inicial da infecção. Verificou-se correlação entre as lesões nos tecidos hepáticos e renais aos valores séricos de ALT, AST, FA e nos níveis de creatinina. Nos ensaios ecotoxicológicos para peixe (O. niloticus), molusco (P. canaliculata), microcrustáceo (D. magna) e macrófitas (L. minor e A. caroliniana), os resultados demonstraram segurança ecotoxicológica da fosfomicina sendo considerada praticamente não tóxica para estes organismos (CL e CE50 > 100,0 mg.L-1). A formulação Fosfomicin C® atenuou as alterações patológicas em peixes infectados quando comparado aos não infectados. Porém essa mesma formulação causou alterações no estudo de segurança clínica. Os estudos demonstram a viabilidade do uso da fosfomicina na dose 10 mg.kg-1 para basal da estreptococose e aeromonose na tilapicultura, porém existe a necessidade de testar o antibiótico puro, para verificar se as alterações observadas foram causadas pelo antibiótico ou por algum outro componente.

The present study aimed to evaluate the therapeutic efficacy of clinical and ecotoxicological safety of antimicrobial fosfomycin for the treatment of aeromonose and streptococcosis in Nile tilapia (Oreochromis niloticus). For this, clinical safety was evaluated with doses of 10, 20 and 40 mg of fosfomycin.kg-1; Efficacy studies with the therapeutic dose of 10 mg of fosfomycin.kg-1 were conducted with fish challenged with Aeromonas hydrophila and Streptococcus agalactiae; The ecotoxicity assays were performed for the determination of LC50 in fish (O. niloticus) and aquatic plants (Lemna minor) and EC50 for crustaceans (Daphnia magna) and molluscs (Pomacea canaliculata). In the studies, tilapia of 112 g ± 5.6 g were used, packed in 400 L tanks (n = 10). Both therapeutic efficacy studies with 10 mg.kg-1 demonstrated efficacy for A. hydrophila and S. agalactiae, because in the reisolation analyzes of these microorganisms there was no growth in liver, kidney, spleen and brain samples of treated fish , While in the infected and untreated groups, mean growth was greater than ten colony forming units (CFU) per plaque. In the A. hydrophila infection they presented microcytosis and lymphopenia, as well as thrombocytosis in the initial phase of infection followed by thrombocytopenia. Liver and renal tissue damage corroborated the changes in serum enzyme activity of ALT, AST, FA and creatinine, cholesterol and triglyceride levels. In S. agalactiae infection, there was a decrease in the number of erythrocytes and percentage values of hematocrit, characterizing hemolysis, associated with lymphopenia, thrombocytopenia and neutrophilia in the initial phase of infection. There was a correlation between hepatic and renal tissue damage in serum ALT, AST, AF, and creatinine levels. In the ecotoxicological assays for fish (O. niloticus), mollusc (P. canaliculata), microcrustaceans (D. magna) and macrophytes (L. minor and A. caroliniana), the results demonstrated ecotoxicological safety of fosfomycin being considered practically non-toxic for these Organisms (CL and EC 50> 100.0 mg.L-1). The Fosfomicin C® formulation attenuated pathological changes in infected fish compared to uninfected fish. However, the same formulation caused changes in the clinical safety study. The studies demonstrate the feasibility of using fosfomycin at a dose of 10 mg.kg-1 for streptococcal and aeromonose control in tilapicultura, but there is a need to test the pure antibiotic to verify if the observed changes were caused by the antibiotic or some other component.

Avaliação da morfologia, virulência e atividades proteolítica e lipolítica de isolados de Beauveria bassiana s.l. antes e após reisolamento em Rhipicephalus microplus

Beauveria bassiana sensu lato (s.l.) é um fungo cosmopolita de grande importância agrícola. Seu uso na pecuária como biocontrolador de Rhipicephalus microplus apresenta bons resultados. Dentro deste contexto, estudos que associem a análise do desenvolvimento e do metabolismo fúngico permitem selecionar isolados de melhor potencial artropodopagênico. O presente estudo avaliou a morfologia, virulência e atividades enzimáticas de dez isolados brasileiros de B. bassiana s.l. antes (grupo Micoteca) e após reisolamento (grupo Reisolado) através de um ciclo de crescimento sobre R. microplus. Para a morfologia, foram realizadas análises macromorfológicas, através de ponto único de inóculo; micromorfológicas, a partir do cultivo entre lâmina e lamínula, e avaliação da produção de conídios. A virulência foi avaliada a partir da observação dos parâmetros biológicos de fêmeas ingurgitadas de R. microplus após imersão em suspensões aquosas de B. bassiana s.l. (108 e 107 conídios/mL). As atividades proteolítica (semi-quantitativa, total, Pr1 e Pr2) e lipolítica (-nitrofenil palmitato) foram realizadas após 72 horas de crescimento dos fungos em meio mínimo. A estatística dos experimentos foi realizada comparando os dados antes e após reisolamento para determinar os isolados de maior potencial patogênico, além de observar o efeito de uma passagem sobre um hospedeiro em potencial. Os dados paramétricos foram avaliados pela análise de variância (ANOVA) seguida do teste de Tukey e os não paramétricos pelo teste de Kruskal Wallis seguido de Student-Newman-Keuls (SNK) com nível de significância de 5%. Quando comparados entre si antes do reisolamento, foram observadas diferenças morfológicas, de virulência e atividades enzimáticas entre os isolados. CG 481 e CG 206 apresentaram os maiores percentuais de controle (65,75% e 61,65%, respectivamente), embora não tenham demonstrado alta atividade enzimática. Quanto ao reisolamento fúngico, o processo alterou em pequena escala a morfologia e a virulência. Em relação à atividade enzimática, a maioria dos isolados elevaram a atividade proteolítica específica Pr2 e a atividade lipolítica. Isolados como Bb 02 e CG 500 foram aqueles que mais sofreram influência após um ciclo de crescimento. Bb 02 aumentou em 1,9 vezes o percentual de controle (15,8% para 30,6%), elevando a produção de conídios e tornando as colônias mais pulverulentas. Além do mais, foi evidenciado também o aumento de todas as atividades enzimáticas (6,3 vezes para Pr1; 2,4 vezes para Pr2 e 9 vezes para atividade lipásica). Em contrapartida, CG 500 reduziu em 4,8 vezes o percentual de controle, além de alterar macromorfologicamente as colônias. As atividades enzimáticas também foram reduzidas em 3,6 vezes para Pr1; 1,2 vezes para Pr2 e 1,6 vezes para a atividade lipásica. Portanto, conclui-se que um ciclo de crescimento de B. bassiana s.l. sobre R. microplus foi capaz de alterar alguns dos isolados avaliados, embora mais passagens pelo hospedeiro sejam necessárias para propiciar resultados mais relevantes. Além disto, o estudo de outras enzimas como as quitinases, a produção de toxinas e a hidrofobicidade dos conídios devem ser considerados na determinação da eficácia fúngica.

Beauveria bassiana sensu lato (s.l.) is a cosmopolitan fungus of great importance in agriculture. Good results are obtained when this microrganism is used as a biocontrol of Rhipicephalus microplus. In this context, studies involving the analysis of the development and fungal metabolism enable to select arthropodopatogenic isolates with better potencial. The aim of this study was to evaluate the morphology, virulence and enzymatic activities of ten B. bassiana s.l. isolates before (Micoteca group) and after reisolation (Reisolado group) through one passage (one cycle) on R. microplus. For morphology was analyzed: macromorphology, performed using the single point of inoculum; micromorphology, with cultivation between slide and cover slip and conidia production. The virulence before and after reisolation was evaluated by observing the biological parameters of R. microplus engorged females after immersion in B. bassiana s.l. aqueous suspensions (108 and 107 conidia mL-1). The proteolytic (semi-quantitative, total, Pr1 and Pr2) and lipolytic activities (-nitrophenyl palmitate) were performed after 72 hours of fungal growth on minimal medium. The statistical analysis of the experiments was performed by comparing the data before and after reisolation for determining isolates with greater pathogenic potential and observing the effect of one passage to the potential host. Parametric data were evaluated by analysis of variance (ANOVA) followed by Tukey test and nonparametric data by Kruskal-Wallis test followed by Student-Newman-Keuls test (SNK) with 5% significance. When compared to each other before reisolation, morphological, virulence and enzymatic activities differences were observed among the isolates. CG 481 and CG 206 showed the highest control percentage (65.75% and 61.65%, respectively), although do not show the highest enzyme activity. After fungal reisolation, the process changed in small-scale the morphology and virulence. In enzymatic activity, most isolates increased the Pr2 and the lipase activities. Isolates as Bb 02 and CG 500 were those who suffered most influence after growth cycle. Bb 02 increased 1.9 times the control percentage (15.8% to 30.6%), incrementing the conidia production and making colonies that are more powdery. For this isolate was also evidenced an increase in all the enzyme activities (6.3 times to Pr1; 2.4 times to Pr2 and 9 times to lipase activity). Differently, CG 500 reduced 4.8 times the control percentage and changed the macromorphology. The enzymatic activities were also reduced by 3.6 times to Pr1; 1.2 times to Pr2 and 1.6 times to lipase activity. Therefore, the study concluded that one B. bassiana s.l. passage on R. microplus was able to change some of the isolates, although more passes by the host are necessary to provide relevant results with other isolates. Moreover, the study of enzymes such as chitinases, production of toxins and hydrophobicity of conidia should be considered to determine the fungal effectiveness.

Hematologia e histopatologia de tilápias (Oreochromis niloticus) vacinadas e desafiadas com Aeromonas hydrophila e Streptoccocus agalactiae

A intensificação dos sistemas aquicolas tem contribuído para o aumento da ocorrência das doenças infecciosas. Assim, o objetivo deste estudo foi analisar as alterações hematológicas e histopatológicas de tilápias (Oreochromis niloticus) vacinadas e desafiadas com Aeromonas hydrophila e Streptococcus agalactiae. Para tanto, peixes com pelo menos 15 gramas foram vacinados com 0,05 mL por peixe, na cavidade celomática entre as nadadeiras ventrais e foram mantidos por 21 dias para o desenvolvimento da imunidade. A indução da infecção foi realizada em períodos diferentes para cada bactéria, sendo após a vacinação, com 60 dias para Aeromonas hydrophila e 90 dias para Streptococcus agalactiae, com as respectivas concentrações de DL50. Após sete dias da inoculação foram coletados, de forma asséptica, fígado, rim, baço e cérebro dos peixes, a realização das provas bioquímicas e confirmação da presença das bactérias Aeromonas sp. e Streptococcus sp. e coleta do sangue para a contagem total dos leucócitos e trombócitos e diferencial dos leucócitos, da série vermelha foram analisados hematócrito, eritrócitos, VCM (Volume Corpuscular Médio), HCM (Hemoglobina Corpuscular Média) e CHCM (Concentração Hemoglobina Corpuscular Média), além dos testes bioquímicos de proteína total (PPT), colesterol, albumina, uréia, triglicérides, lactato e glicose e também foi realizado o reisolamento das bactérias. Ao término do período experimental ocorreram aumento nas variáveis hematológicas e bioquímicos do CHCM, globulinas e colesterol e a diminuição no hematócrito, VCM, leucócitos e lactato de tilápias infectadas por S. agalactiae e aumento nos parâmetros de leucócitos, globulinas e a diminuição dos trombócitos e colesterol de tilápias infectadas por A. hydrophila além de alterações histopatológicas em...

The intensification of aquaculture systems has contributed to the increased occurrence of infectious diseases. The objective of this study was to analyze the hematological and histopathological tilapia (Oreochromis niloticus) vaccinated and challenged with Aeromonas hydrophila and Streptococcus agalactiae To that end, with at least 15 fish were vaccinated with 0.05 grams mL per fish in between the coelomic cavity and ventral fins were maintained for 21 days for development of immunity. Induction of Infection was performed at different times for each bacteria, which after vaccination, 60 days and 90 days A. hydrophila to S. agalactiae, with respective concentrations of LD50. After seven days of inoculation were collected aseptically, liver, kidney, spleen and brain of fish carrying out the biochemical and confirmation of the presence of the bacteria Aeromonas sp. and Streptococcus sp. and blood collection for the total count of leukocytes and thrombocytes and differential leukocytes, red series were analyzed hematocrit, erythrocytes, MCV (Mean Corpuscular Volume) , MCH (Mean Corpuscular Hemoglobin) and MCHC (Mean Corpuscular Hemoglobin Concentration), in addition to biochemical assays of total protein (PPT), cholesterol, albumin, urea, triglycerides, and glucose and lactate was also performed reisolation of bacteria. At the end of the experimental period there was an increase in haematological and biochemical variables of MCHC, globulin and cholesterol and decrease in hematocrit, MCV and lactate tilapia infected with S. agalactiae and an increase in leukocyte parameters, globulin and the decrease of platelets and cholesterol tilapia infected with A. hydrophila as well as histopathological changes in skin, liver and kidney of...

Imunodetecção de antígenos de Escherichia coli em infecção experimental em aves / Imunodetection of Escherichia coli antigens in experimental infection in broiler flocks

A colibacilose é uma enfermidade importante na avicultura por causar prejuízos econômicos e danos à carcaça. Escherichia coli é um micro-organismo oportunista e a complexidade da infecção se deve a diversidade de sorotipos, variados fatores de virulência e baixa capacidade de estimular a imunidade cruzada. Assim, a compreensão dos mecanismos de virulência que ocorrem no hospedeiro, pode resultar no desenvolvimento de métodos diagnósticos e profiláticos. Neste estudo, 85 pintinhos foram divididos em quatro grupos, sendo três infectados com diferentes sorogrupos de Escherichia coli (O2, O78, O119) e um vacinado com Poulvac® E.coli (Pfizer®). As lesões sugestivas de colibacilose foram avaliadas e qualificadas aos 35 dias de idade, e em seguida realizada a colheita de sangue para a obtenção do soro. Foram obtidas proteínas in vitro dos sorogrupos de E. coli utilizados na infecção experimental e da vacina (mutante de Escherichia coli O78). Estas foram separadas por eletroforese unidimensional em gel de acrilamida utilizando dodecil sulfato de sódio (SDS) e foram submetidas a reação de western blot com a mistura de soros de cada grupo estudado e também com o pool de soros de aves livres de patógenos específicos (SPF), usadas como controle negativo. O perfil eletroforético revelou a presença de várias proteínas dos diferentes sorogrupos de E. coli quando cultivadas no meio casaminoácido e levedura. Houve diferenças entre os sorotipos, mas também proteínas comuns. Foi possível estabelecer uma relação entre virulência, quantidades de proteínas específicas e taxa de reisolamento

The colibacillosis is a serious disease in poultry industry to cause economic losses and damage to carcass. Escherichia coli is an opportunistic microorganism and the complexity of infection is due to the diversity of serotypes, several virulence factors and low ability to stimulate cross-immunity. Thus understanding of virulence mechanisms that occur in the host, may result in development of diagnostic and prophylactic methods. In this study, 85 chicks were divided into four groups being infected with three different serogroups of E. coli (O2, O78, O119) and vaccinated with Poulvac E. coli ® (Pfizer ®). The lesions suggestive of colibacillosis were evaluated at 35 days of age, and then the blood sample are collected to obtain the serum. Proteins were obtained in vitro from serogroups of E. coli used in experimental infection and vaccine (mutant of Escherichia coli O78). These were separated by one-dimensional electrophoresis on acrylamide gels using sodium dodecyl sulfate (SDS) and then reacted by western blot with the pool of sera from each group and also with the pool of sera from specific pathogen free poultry used as negative control. The electrophoretic pattern showed the presence of many proteins of different serogroups of E. coli when grown in the broth casaminoacid and yeast. There were differences among serotypes, but also common proteins. It was possible to establish a relationship between virulence and reisolation rate

Imunodetecção de antígenos de Escherichia coli em infecção experimental em aves / Imunodetection of Escherichia coli antigens in experimental infection in broiler flocks

A colibacilose é uma enfermidade importante na avicultura por causar prejuízos econômicos e danos à carcaça. Escherichia coli é um micro-organismo oportunista e a complexidade da infecção se deve a diversidade de sorotipos, variados fatores de virulência e baixa capacidade de estimular a imunidade cruzada. Assim, a compreensão dos mecanismos de virulência que ocorrem no hospedeiro, pode resultar no desenvolvimento de métodos diagnósticos e profiláticos. Neste estudo, 85 pintinhos foram divididos em quatro grupos, sendo três infectados com diferentes sorogrupos de Escherichia coli (O2, O78, O119) e um vacinado com Poulvac® E.coli (Pfizer®). As lesões sugestivas de colibacilose foram avaliadas e qualificadas aos 35 dias de idade, e em seguida realizada a colheita de sangue para a obtenção do soro. Foram obtidas proteínas in vitro dos sorogrupos de E. coli utilizados na infecção experimental e da vacina (mutante de Escherichia coli O78). Estas foram separadas por eletroforese unidimensional em gel de acrilamida utilizando dodecil sulfato de sódio (SDS) e foram submetidas a reação de western blot com a mistura de soros de cada grupo estudado e também com o pool de soros de aves livres de patógenos específicos (SPF), usadas como controle negativo. O perfil eletroforético revelou a presença de várias proteínas dos diferentes sorogrupos de E. coli quando cultivadas no meio casaminoácido e levedura. Houve diferenças entre os sorotipos, mas também proteínas comuns. Foi possível estabelecer uma relação entre virulência, quantidades de proteínas específicas e taxa de reisolamento (AU)

The colibacillosis is a serious disease in poultry industry to cause economic losses and damage to carcass. Escherichia coli is an opportunistic microorganism and the complexity of infection is due to the diversity of serotypes, several virulence factors and low ability to stimulate cross-immunity. Thus understanding of virulence mechanisms that occur in the host, may result in development of diagnostic and prophylactic methods. In this study, 85 chicks were divided into four groups being infected with three different serogroups of E. coli (O2, O78, O119) and vaccinated with Poulvac E. coli ® (Pfizer ®). The lesions suggestive of colibacillosis were evaluated at 35 days of age, and then the blood sample are collected to obtain the serum. Proteins were obtained in vitro from serogroups of E. coli used in experimental infection and vaccine (mutant of Escherichia coli O78). These were separated by one-dimensional electrophoresis on acrylamide gels using sodium dodecyl sulfate (SDS) and then reacted by western blot with the pool of sera from each group and also with the pool of sera from specific pathogen free poultry used as negative control. The electrophoretic pattern showed the presence of many proteins of different serogroups of E. coli when grown in the broth casaminoacid and yeast. There were differences among serotypes, but also common proteins. It was possible to establish a relationship between virulence and reisolation rate (AU)