Orientador: Ruben Pablo Schocken-Iturrino / Banca: Maria Luiza Poiatti / Banca: Alessandra Aparecida Medeiros / Banca: Hélio José Montassier / Banca: Antônio Carlos Monteiro / Resumo: Entre as espécies de Clostridium de importância em patologia animal, destaca-se o Clostridium perfringens, o Clostridium botulinum, o Clostridium chauvoei. O objetivo desta pesquisa foi verificar a presença de bactérias anaeróbias esporuladas (Clostridium sp), assim como, identificar as espécies de Clostridium patogênicos para a saúde animal, principalmente de bovinos, no solo de pastagem da micro-região de Jaboticabal, SP. Foram coletadas 250 amostras de solo e realizadas contagens de bactérias esporuladas do gênero Clostridium e identificação das espécies patogênicas presentes. Os resultados permitiram demonstrar a contagem de UFC de Closrtidium sp com média em log 10 igual a 2,79, sendo que os valores mínimo e máximo obtido foram 2,15 e 3,68 respectivamente. Para caracterização e identificação, os resultados permitiram identificar a presença de bactérias anaeróbias esporuladas em 233 amostras (93,2%), entre estas 180 eram do gênero Clostridium...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The species of Clostridium of major importance to animal pathology are Clostridium perfringens, Clostridium botulinum, and C. chauvoei. Considering this, the objective of this research was to verify the presence of anareobic sporulate bacteria (Clostridium sp), and also identify the species of pathogenic Clostridium for the animal health, mostly to bovine, in pasture soil of Jaboticabal-SP. A total of 250 samples were collected and used to determine the number of sporulated bacteria from Clostridium genderand identify the pathogenic species present. The results demonstrated that the average number of CFU in log 10 of Closrtidium sp was 2,79, and the minimum and maximum values obtained were 2,15 and 3,68 respectively. After characterization and isolation and identification, the results showed the presence of 233 samples (93,2%) of sporulated bacteria, of these 180 were of Clostridium gender. The biochemical tests were identified in 42 samples, being 23 samples (9,2%) of Clostridium perfringens, 13 samples (5,2%) of Clostridium botulinum and 6 samples (2,4%) of C. chauvoei ...(Complete abstract, click electronic access below) / Doutor

Orientador: Ruben Pablo Schocken-Iturrino / Banca: Clovis Wesley Oliveira de Souza / Banca: Caroline Petters Pigato de Nardi / Banca: Luiz Augusto do Amaral / Banca: Antonio Carlos Paulillo / Resumo: O gênero Clostridium é amplamente distribuído na natureza, está presente no solo e conteúdo intestinal dos animais, produzem toxinas que são capazes de provocar doenças e intoxicações. O Clostridium perfringens está envolvido com prejuízos na avicultura, pois a doença (Enterite Necrotica) na maioria das vezes é subclínica, e o produtor só observa na hora do abate que a ave não ganhou o peso esperado. O controle dessa e de outras enfermidades e agentes patogênicos na avicultura de corte tem grande importância uma vez que o Brasil está entre os principais países exportadores de carne. O objetivo deste trabalho primeiramente foi pesquisar Clostridium perfringens em frangos de corte sadios, provenientes de dois grandes frigoríficos com Inspeção federal, um no Estado de São Paulo e outro no Estado de Minas Gerais. Foram obtidos 300 raspados intestinais, os quais foram semeados em Agar SPS e incubados em anaerobiose. A identificação dos tipos A, B, C, D e E de C. perfringens foi realizada mediante o emprego da PCR multiplex por amplificação dos genes codificadores das toxinas alfa, beta, épsilon e iota do mesmo. A partir das amostras positivas na PCR (37), procedeu-se o isolamento do agente. Depois de muitas tentativas, verificou-se por meio do sequenciamento da região 16S rDNA, que as colônias sulfito redutoras que estavam sendo isoladas eram Enterococcus spp, microrganismo coexistente na microbiota intestinal de frango de corte, que compete com o C. perfringens pelo crescimento das colônias no mesmo meio de cultura, ainda que este seja seletivo e diferencial para clostrídios sulfito redutores. Dessa forma, notou-se que o crescimento de colônias de enterococos interferia muito na identificação e enumeração do C. perfringens e ainda apresentavam colônias muito semelhantes às de C. perfringens nos oito meios de cultura testados. Visto isto, foram feitos alguns testes para avaliar o comportamento dos... / Abstract: Clostridium genus is widely distributed in nature and is present in soil and intestinal contents of animals, produce toxins which are capable of causing diseases and intoxication. Clostridium perfringens is involved with losses in the poultry industry, because the disease is most often subclinical, and producer only observed at the time of slaughter the bird has not gained the expected weight. The control of this and other diseases and pathogens in poultry production is very important since Brazil is among the major meat exporting countries. The aim of this study was to evaluate the presence of Clostridium perfringens in poultry coming from two large slaughter-house, one from São Paulo state and another from Minas Gerais. Were obtained 300 intestinal scraped and cecal contents, which were sown onto SPS agar and incubated anaerobically. The identification of C. perfringens types A, B, C, D and E was performed by the use of multiplex PCR. After identification of the presence of C. perfringens in 37 of 300 samples by amplification of the cpa gene, alpha toxin encoder, the isolation of pure colonies was proceeded. However, through the sequencing of 16S rDNA region, it was also found the presence of Enterococcus spp. that lives in the intestinal microbiota of poultry and disputes in growth on the same culture medium, even been selective and differential for clostridia. Thus, it was observed that colony morphology of enterococci is very similar to C. perfringens in this medium what make isolation of pure colonies difficult. Having on mind the difficulty of C. perfringens isolation under these circumstances, the PCR is an rapid and secure alternative for detection of C. perfringens and its different types, being effective for diagnostics / Doutor

abstract: Necrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium perfringens, leading to an estimated 2 billion dollar global economic loss in the poultry industry annually. Traditionally, NE has been effectively controlled by antibiotics added to the diet of poultry. Concerns about increasing antibiotic resistance of poultry and human based pathogens have led to the consideration of alternative approaches for controlling disease, such as vaccination. NE causing strains of C. perfringens produce two major toxins, α-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. We have developed a fusion protein combining a non-toxic carboxy-terminal domain of the α-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for Nicotiana benthamiana to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system. The fusion protein was purified by metal affinity chromatography and used to immunize broiler birds. Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. However, the PlcC-NetB fusion had antibody titers four times that of the bacterially produced toxoids alone. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent C. perfringens when treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB is a promising vaccine candidate for controlling NE in poultry. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2018

No description available.

Necrotic enteritis (NE) caused by Clostridium perfringens type A is an important bacterial enteric disease of global broiler production. However, the dynamic interactions of NE and its predisposing factors are not fully presented by current studies. By using the System Dynamics (SD) Model, the epidemiological changes in susceptible-infected-removed models of NE and avian coccidiosis and their interactions in one or multiple grow-out cycles was established; meanwhile, the growth performance was measured by the average weights of infected and non-infected populations at harvest were estimated. The SD model provided direct and persuasive outcomes of the epidemiology and ecology of NE compared with models using statistical methodology. With interventions on certain predisposing factors of management practices and medication, effects which decreased disease incidence and growth performance were observed; moreover, the leverage points obtained from interventions on certain management practices provided quantitative results which were applicable and useful for improving the broiler production.

Clostridium perfringens (C. perfringens) causes diseases such as necrotic enteritis in poultry. Current methods for early detection of C. perfringens, including culture, and molecular techniques (PCR and qPCR), have logistical limitations in detecting pathogens on-site because they require specialized equipment and long sample preparation time. This study describes developing a highly sensitive on-site LAMP assay combined with a simple DNA extraction method to detect C. perfringens. Results showed that the LAMP assay was highly specific and sensitive with the optimal reaction conditions: 63°C for 30 mins with primer concentration of 1.2 mM FIP/BIP and 0.2 mM F3/B3. The HotSHOT method was the most convenient DNA extraction method for the on-site LAMP assay with respect to the processing time, cost, and minimal equipment requirement. Therefore, the HotSHOT DNA extraction method followed by the LAMP assay could serve as a rapid on-site molecular test for C. perfringens in the field.

No description available.

Clostridium perfringens is a significant human pathogen, causing 250,000 cases of food poisoning in addition to several thousand potentially lethal cases of gas gangrene each year in the United States. Historically, work in this field has centered around toxin production, as C. perfringens can produce over 13 toxins. This work expands the knowledge of the starvation-response of C. perfringens, which includes several potential virulence factors, sporulation, motility and biofilm formation. Sporulation protects cells from a variety of stresses, including starvation. Efficient sporulation requires the transcriptional regulator CcpA, mediator of catabolite repression. Sporulation is repressed by glucose, but, surprisingly, in a CcpA-independent fashion. C. perfringens cells in a biofilm are resistant to a number of environmental stresses, including oxygen and antibiotics. Biofilm formation is repressed by glucose, and other carbohydrates, independently of CcpA. Gliding motility, a type four pili (TFP)-dependent phenomenon, affords C. perfringens with a mechanism for moving across a solid surface in response to carbohydrate starvation, while carbohydrates supplementation at high levels delay the initiation of the motility response. CcpA is required for the proper initiation of motility, a ccpA^-C. perfringens strain showed a considerable increase in the time to initiation of motility on lactose and galactose, and was unable to move at all in the presence of glucose. Gliding motility represents the most significant finding of this work. TFP were previously undescribed in any Gram-positive bacterial species, and this work produced genetic evidence suggesting their presence in all members of the clostridia, and physical evidence for TFP-dependent gliding motility in a second species, C. beijerinckii. / Ph. D.

Clostridium perfringens is a spore-forming anaerobic Gram-positive rod which has gliding motility through type IV Pili (TFP). Since the discovery of TFP in Gram-positive bacteria is relatively new, more studies are required to understand the mechanism and interaction of the proteins of this machinery. Moreover, the similarities between TFP and type 2 secretion system (T2SS) suggest that C. perfringens has also a T2SS. We studied the localization of TFP ATPases, PilB1, PilB2 and PilT in Bacillus subtilis to compare the localization in an organism other than C. perfringens and which lacks any known genes similar to TFP. Unlike the case in C. perfringens, PilB1 in B. subtilis localized to the poles in the absence of PilT, with some central foci at the future division sites. Colocalization of PilB1 was also studied with PilT and the results suggested that PilB1 needs PilT to migrate from the poles to the center. Localization of PilB2 in B. subtilis, was similar to the results in C. perfringens and to the localization of PilB1 in B. subtilis. We have not been able to co-express PilB2 with PilT yet. Succeeding in this study will help us better understand the interactions between PilB proteins and PilT. In another project, we studied a von Willebrand factor Type A-Domain Containing protein (vWA) which is secreted from C. perfringens strain 13. We overexpressed and purified this protein and tested the effects on mammalian cells. We found that the vWA is probably not a toxin but since it seems to bind to macrophage membranes, we propose that the vWA could be part of a toxin complex, probably the subunit of the complex that binds to the host cells. / Master of Science

Clostridium perfringens is the causative agent of gas gangrene. A lethal infection in mice requires a large inoculum suggesting that the immune system is involved in inhibiting disease. Human monocytic cells and neutrophils killed C. perfringens in vitro when complement was present. Macrophages and neutrophils co-localized with C. perfringens in vivo when bacterial numbers were low. Depletion of neutrophils and monocytes in mice revealed that monocytic cells play a role in inhibiting C. perfringens gas gangrene in mice infected with an intermediate dose. C. perfringens can persist in the tissues and this could be mediated by persistence within macrophages. To examine if the toxin perfringolysin O (PFO) could mediate this, less active variants of PFO were used to examine what occurs between phagosomal escape and cell lysis. The mutant forms of PFO did mediate phagosomal escape in macrophages and were found within macrophages at higher numbers than wild-type C. perfringens. Our data were preliminary but may indicate that less active PFO mediates intracellular persistence. To investigate the role of sialidase in C. perfringens gas gangrene we made nanI-, nanJ-, and nanI-/nanJ- mutants. We observed that NanI is responsible for the majority of sialidase activity of C. perfringens strain 13, that NanJ is an extracellular sialidase, and that these genes are transcriptionally regulated by sialic acid. Murine infection trials revealed that these sialidases may be protective for mice during infection. In conclusion, murine monocytes inhibit disease onset and C. perfringens sialidase enhances mouse survival. However, the toxin PFO if less active promotes the survival of C. perfringens with macrophages. / Master of Science