Clostridium perfringens is a versatile pathogen capable of causing a wide array of diseases, ranging from clostridial food poisoning to tissue infections such as gas gangrene. An important factor in virulence as well as in the distribution of C. perfringens is its ability to form an endospore. The symptoms of C. perfringens food poisoning are directly correlated to the release of an enterotoxin at the end of the sporulation process. The sporulation process in C. perfringens is subject to carbon catabolite repression (CCR) by sugars, especially glucose. CCR is a regulatory pathway that alters transcription based on carbon source availability. In Gram-positive bacteria, the HPr kinase/phosphatase is responsible for this nutritional sensing by phosphorylating or dephosphorylating the serine-46 residue of HPr. HPr-Ser-P then forms a complex with the transcriptional regulator CcpA to regulate transcription. We were able to show here that purified recombinant C. perfringens HPr kinase/phosphatase was able to phosphorylate the serine-46 residue of HPr. When the codon for this serine residue is mutated through PCR mutagenesis to encode alanine, phosphorylation could not take place. We have also shown that in gel retardation assays, CcpA and HPr-Ser-P were able to bind to two DNA fragments containing putative C. perfringens CRE-sites, sequences where CcpA binds to regulate transcription. The genome sequence of a food poisoning strain of C. perfringens was searched for potential CRE-sites using degenerate sequences designed to match those CRE-sites CcpA was shown to bind. DNA fragments containing these newly identified CRE-sites were then used in gel retardation assays to determine whether CcpA binds to these CRE-sites, making them candidates for CCR regulation. These results, combined with comparisons of metabolic characteristics of a ccpA- strain versus wild-type C. perfringens, provide evidence that CcpA participates in the regulation of carbon catabolite repression in the pathogenic bacterium C. perfringens / Master of Science

A proposta apresentada se baseia na introdução de um estágio intermediário de desinfecção previamento ao tratamento biológico visando intensificar os efeitos do estágio seguinte destinado à desinfecção convencional. Para estudo de caso foram aplicadas as técnicas de ozonização e radiação UV combinadas em instalações piloto que simulam duas condições seqüenciais de desinfecção. O desempenho do método proposto foi avaliado através de exames microbiológicos de amostras do efluente anaeróbio previa e posteriormente à desinfecção, utilizando indicadores de contaminação por bactérias (Escherichia coli e coliformes totais), vírus (colifagos) e protozoário (Clostridium perfringens). Os resultados obtidos no sistema combinado pré-desinfecção/desinfecção revelaram eficiência de inativação superior quando comparada ao procedimento convencional. Nas análises de E.coli, por exemplo, a aplicação de apenas 1 mg de ozônio/L ou 51 mW de radiação/'CM POT.2', na primeira etapa de desinfecção, foi suficiente para se alcançar 1 log acima do valor correspondente ao método convencional. Mesmo indicadores mais resistentes como C. perfringens apresentaram redução da fração N/No da ordem de 1 log em relação ao método proposto. Além disso, estes níveis de inativação foram alcançados mesmo sob a influência de elevada concentração de SST, SSV e DQO na entrada na unidade piloto destinada à pré-desinfecção. Entre as seqüências de experimentação investigadas ('O IND.3'/'O IND.3', 'O IND.3'/UV, UV/'O IND.3' e UV/UV) não foram observadas grandes variações. De modo semelhante, os resultados revelaram que a relação N/No, para os indivíduos submetidos ao sistema combinado, não foi afetada pelo aumento no tempo de exposição ao agente inativante ('O IND.3': 5, 7, 10 min; UV: 30, 60, 120s). Considerando as baixas dosagens de 'O IND.3' e UV aplicadas na primeira etapa, somada às condições limitadas de desempenho do sistema real examinado, os níveis de inativação alcançados sugerem grande potencialidade de utilização do método alternativo proposto, comprovando sua viabilidade técnica / The present proposition is based on the introduction of an intermediate disinfection stage before the biological treatment, in order to intensify the effects of the next stage employed in conventional disinfection. Studies were performed using combined ozonization and UV radiation techniques, in a model installation that simulates two sequential disinfection conditions. The performance of the method was evaluated using microbiological exams of samples taken from the anaerobic effluent before and after the disinfection. Bacterial (Escherichia coli and total coliforms), viral (coliphages) and protozoan (Clostridium perfringens contamination tracers were used in such exams. Results obtained by combining pre-disinfection and disinfection reveal superior inactivation efficiency as compared to the conventional procedure. For example, in the E. coli analysis the application of only 1 mg of ozone/L or 51 mW/'CM POT.2' of radiation in the first disinfection stage was enough for achieving 1 log above the convention method. Even more resistant tracers, such as C. perfringens, showed aproximatelly 1 log of reduction in the N/No fraction in the proposed method. Besides, these inactivation levels were achieved even for high concentrations of SST, SSV and DQO in the entrance of the pre-disinfection unit. No significant variations were observed among the disinfection sequences ('O IND.3'/'O IND.3','O IND.3'/UV,UV/'O IND.3', and UV/UV). Similarly, the results showed that the N/No relation, for individuals submitted to the combined system, was not affected by the increase of the exposition time to the inactivation agent ('O IND.3': 5, 7, 10 min; UV: 30, 60, 120 s). Taking into account the low dosages of 'O IND.3' and UV applied in the first stage and the limited performance conditions of the real system, the achieved inactivation levels suggest a great potential for the alternative method proposed, demonstrating its technical viability

Die im Biogasprozess anfallenden Gärreste werden oftmals als Wirtschaftsdünger verwendet. Krankheitserreger, die sich in den Gärresten befinden können über die Düngung in die Lebensmittelkette gelangen. Die Möglichkeit einer Vermehrung von Bakterien in den Biogasanlagen sowie deren Ausbreitung schürt die Bedenken der Öffentlichkeit. Das Ziel dieser Arbeit war es, Nachweismethoden für die Untersuchung von Proben aus Biogasanlagen zu etablieren, die Praxistauglichkeit dieser anhand von Proben aus Biogasanlagen zu überprüfen und die mikrobielle Belastung dieser Proben hinsichtlich ausgewählter Indikatorkeime zu erfassen. Bei den Indikatorkeimen handelte es sich um Clostridium perfringens, Clostridium botulinum, Enterokokken, Escherichia coli, ESBL-bildende Enterobaceriaceae und Salmonellen. Für die Etablierung der bakteriologischen Nachweismethoden wurde autoklavierter Gärrest mit einer definierten Keimmenge beimpft und auf verschiedene Nährmedien aufgebracht. Diese wurden bebrütet, ausgezählt und die KbE/ml berechnet. Mittels Probitanalyse wurde für jedes Medium die untere Grenze für den Nachweis aus beimpftem Gärrest bestimmt. Bei den Nährmedien handelte es sich um Brilliance™ Salmonella Agar, XLT4 Agar und XLD Agar für den Nachweis von Salmonella spp. Für E. coli wurden Tergitol 7 Lactose TCC Agar und Brilliance™ E. coli/Coliform Selektiv Agar verwendet. Der Nachweis von Enterokokken erfolgte mittels Slanetz Bartley Agar und Enterococcus Selektivagar. Für die ESBL-bildenden Enterobacteriaceae wurde der Brilliance™ ESBL Agar eingesetzt. Die getesteten Nährmedien zum Nachweis von C. perfringens waren Membran Clostridium Perfringens (mCP) Selektivnährboden sowie Tryptose Sulphite Cycloserine (TSC) Agar überschichtet mit TSC Agarbasis. Für C. botulinum erfolgte der Nachweis auf Eigelb Laktose Agar. Darüber hinaus wurde eine PCR zur C. perfringens Toxintyp-Bestimmung nach dem Protokoll von VAN ASTEN et al. (2009) etabliert. Zum Nachweis von C. botulinum wurde die PCR nach dem Protokoll von HILL et al. (2010) eingesetzt. Bei der Untersuchung der Praxistauglichkeit wurden Proben aus zehn Biogasanlagen des Freistaates Sachsen entnommen und untersucht. Hierbei handelte es sich um Proben aus Abschnitten vor, während und nach der Fermentation. Anhand der ermittelten Nachweisgrenze sowie der Handhabung wurden die folgenden Nährmedien für die Untersuchung der Biogasanlagen-Proben ausgewählt: Brilliance™ Salmonella Agar, XLT4 Agar, Brilliance™ E. coli/Coliform Selektiv Agar, Slanetz Bartley Agar, Brilliance™ ESBL Agar, TSC Agar überschichtet mit TSC Agarbasis und Eigelb Laktose Agar. Für die Anzucht anaerober Bakterien wurden die Proben vor der Beimpfung der Agarplatten erhitzt. Zudem erfolgte eine Anreicherung des zuvor erhitzten Probenmaterials in TPYG Bouillon. Diese wurde genutzt, um daraus aufgereinigte DNA mittels PCR auf C. botulinum und C. perfringens zu untersuchen. Die verwendeten Nährmedien wurden im Praxistest positiv evaluiert. Die Ergebnisse für die Proben aus den Biogasanlagen zeigten, dass, mit Ausnahme von C. perfringens, alle Indikatororganismen während des Biogasprozesses einer Reduktion unterlagen. Die durchschnittliche anaerobe Lebendkeimzahl belief sich auf 107 bis 108 KbE/g Probe. E. coli erfuhr eine Reduktion um bis zu vier Zehnerpotenzen. Enterokokken wurden um 1 bis 2 log10 Stufen reduziert. ESBL-bildende Enterobacteriaceae konnten in sechs der zehn Biogasanlagen nachgewiesen werden. Hierbei handelte es sich überwiegend um E. coli und Klebsiella spp. In keiner der Proben konnten Salmonellen oder C. botulinum nachgewiesen werden. Typ A war der am häufigsten nachgewiesene C. perfringens-Toxintyp. Das β2-Toxin-Gen wurde in 20 Fällen nachgewiesen. Einmal konnte C. perfringens Typ C, β2-Toxin-Gen-positiv detektiert werden. Der hygienische Status der Gärreste entsprach in etwa dem hygienischen Status von Gülle. In Abhängigkeit vom Indikatorkeim war eine Verbesserung des Status durch eine Reduktion der Keimzahl festzustellen.

A proposta apresentada se baseia na introdução de um estágio intermediário de desinfecção previamento ao tratamento biológico visando intensificar os efeitos do estágio seguinte destinado à desinfecção convencional. Para estudo de caso foram aplicadas as técnicas de ozonização e radiação UV combinadas em instalações piloto que simulam duas condições seqüenciais de desinfecção. O desempenho do método proposto foi avaliado através de exames microbiológicos de amostras do efluente anaeróbio previa e posteriormente à desinfecção, utilizando indicadores de contaminação por bactérias (Escherichia coli e coliformes totais), vírus (colifagos) e protozoário (Clostridium perfringens). Os resultados obtidos no sistema combinado pré-desinfecção/desinfecção revelaram eficiência de inativação superior quando comparada ao procedimento convencional. Nas análises de E.coli, por exemplo, a aplicação de apenas 1 mg de ozônio/L ou 51 mW de radiação/'CM POT.2', na primeira etapa de desinfecção, foi suficiente para se alcançar 1 log acima do valor correspondente ao método convencional. Mesmo indicadores mais resistentes como C. perfringens apresentaram redução da fração N/No da ordem de 1 log em relação ao método proposto. Além disso, estes níveis de inativação foram alcançados mesmo sob a influência de elevada concentração de SST, SSV e DQO na entrada na unidade piloto destinada à pré-desinfecção. Entre as seqüências de experimentação investigadas ('O IND.3'/'O IND.3', 'O IND.3'/UV, UV/'O IND.3' e UV/UV) não foram observadas grandes variações. De modo semelhante, os resultados revelaram que a relação N/No, para os indivíduos submetidos ao sistema combinado, não foi afetada pelo aumento no tempo de exposição ao agente inativante ('O IND.3': 5, 7, 10 min; UV: 30, 60, 120s). Considerando as baixas dosagens de 'O IND.3' e UV aplicadas na primeira etapa, somada às condições limitadas de desempenho do sistema real examinado, os níveis de inativação alcançados sugerem grande potencialidade de utilização do método alternativo proposto, comprovando sua viabilidade técnica / The present proposition is based on the introduction of an intermediate disinfection stage before the biological treatment, in order to intensify the effects of the next stage employed in conventional disinfection. Studies were performed using combined ozonization and UV radiation techniques, in a model installation that simulates two sequential disinfection conditions. The performance of the method was evaluated using microbiological exams of samples taken from the anaerobic effluent before and after the disinfection. Bacterial (Escherichia coli and total coliforms), viral (coliphages) and protozoan (Clostridium perfringens contamination tracers were used in such exams. Results obtained by combining pre-disinfection and disinfection reveal superior inactivation efficiency as compared to the conventional procedure. For example, in the E. coli analysis the application of only 1 mg of ozone/L or 51 mW/'CM POT.2' of radiation in the first disinfection stage was enough for achieving 1 log above the convention method. Even more resistant tracers, such as C. perfringens, showed aproximatelly 1 log of reduction in the N/No fraction in the proposed method. Besides, these inactivation levels were achieved even for high concentrations of SST, SSV and DQO in the entrance of the pre-disinfection unit. No significant variations were observed among the disinfection sequences ('O IND.3'/'O IND.3','O IND.3'/UV,UV/'O IND.3', and UV/UV). Similarly, the results showed that the N/No relation, for individuals submitted to the combined system, was not affected by the increase of the exposition time to the inactivation agent ('O IND.3': 5, 7, 10 min; UV: 30, 60, 120 s). Taking into account the low dosages of 'O IND.3' and UV applied in the first stage and the limited performance conditions of the real system, the achieved inactivation levels suggest a great potential for the alternative method proposed, demonstrating its technical viability

Die im Biogasprozess anfallenden Gärreste werden oftmals als Wirtschaftsdünger verwendet. Krankheitserreger, die sich in den Gärresten befinden können über die Düngung in die Lebensmittelkette gelangen. Die Möglichkeit einer Vermehrung von Bakterien in den Biogasanlagen sowie deren Ausbreitung schürt die Bedenken der Öffentlichkeit. Das Ziel dieser Arbeit war es, Nachweismethoden für die Untersuchung von Proben aus Biogasanlagen zu etablieren, die Praxistauglichkeit dieser anhand von Proben aus Biogasanlagen zu überprüfen und die mikrobielle Belastung dieser Proben hinsichtlich ausgewählter Indikatorkeime zu erfassen. Bei den Indikatorkeimen handelte es sich um Clostridium perfringens, Clostridium botulinum, Enterokokken, Escherichia coli, ESBL-bildende Enterobaceriaceae und Salmonellen. Für die Etablierung der bakteriologischen Nachweismethoden wurde autoklavierter Gärrest mit einer definierten Keimmenge beimpft und auf verschiedene Nährmedien aufgebracht. Diese wurden bebrütet, ausgezählt und die KbE/ml berechnet. Mittels Probitanalyse wurde für jedes Medium die untere Grenze für den Nachweis aus beimpftem Gärrest bestimmt. Bei den Nährmedien handelte es sich um Brilliance™ Salmonella Agar, XLT4 Agar und XLD Agar für den Nachweis von Salmonella spp. Für E. coli wurden Tergitol 7 Lactose TCC Agar und Brilliance™ E. coli/Coliform Selektiv Agar verwendet. Der Nachweis von Enterokokken erfolgte mittels Slanetz Bartley Agar und Enterococcus Selektivagar. Für die ESBL-bildenden Enterobacteriaceae wurde der Brilliance™ ESBL Agar eingesetzt. Die getesteten Nährmedien zum Nachweis von C. perfringens waren Membran Clostridium Perfringens (mCP) Selektivnährboden sowie Tryptose Sulphite Cycloserine (TSC) Agar überschichtet mit TSC Agarbasis. Für C. botulinum erfolgte der Nachweis auf Eigelb Laktose Agar. Darüber hinaus wurde eine PCR zur C. perfringens Toxintyp-Bestimmung nach dem Protokoll von VAN ASTEN et al. (2009) etabliert. Zum Nachweis von C. botulinum wurde die PCR nach dem Protokoll von HILL et al. (2010) eingesetzt. Bei der Untersuchung der Praxistauglichkeit wurden Proben aus zehn Biogasanlagen des Freistaates Sachsen entnommen und untersucht. Hierbei handelte es sich um Proben aus Abschnitten vor, während und nach der Fermentation. Anhand der ermittelten Nachweisgrenze sowie der Handhabung wurden die folgenden Nährmedien für die Untersuchung der Biogasanlagen-Proben ausgewählt: Brilliance™ Salmonella Agar, XLT4 Agar, Brilliance™ E. coli/Coliform Selektiv Agar, Slanetz Bartley Agar, Brilliance™ ESBL Agar, TSC Agar überschichtet mit TSC Agarbasis und Eigelb Laktose Agar. Für die Anzucht anaerober Bakterien wurden die Proben vor der Beimpfung der Agarplatten erhitzt. Zudem erfolgte eine Anreicherung des zuvor erhitzten Probenmaterials in TPYG Bouillon. Diese wurde genutzt, um daraus aufgereinigte DNA mittels PCR auf C. botulinum und C. perfringens zu untersuchen. Die verwendeten Nährmedien wurden im Praxistest positiv evaluiert. Die Ergebnisse für die Proben aus den Biogasanlagen zeigten, dass, mit Ausnahme von C. perfringens, alle Indikatororganismen während des Biogasprozesses einer Reduktion unterlagen. Die durchschnittliche anaerobe Lebendkeimzahl belief sich auf 107 bis 108 KbE/g Probe. E. coli erfuhr eine Reduktion um bis zu vier Zehnerpotenzen. Enterokokken wurden um 1 bis 2 log10 Stufen reduziert. ESBL-bildende Enterobacteriaceae konnten in sechs der zehn Biogasanlagen nachgewiesen werden. Hierbei handelte es sich überwiegend um E. coli und Klebsiella spp. In keiner der Proben konnten Salmonellen oder C. botulinum nachgewiesen werden. Typ A war der am häufigsten nachgewiesene C. perfringens-Toxintyp. Das β2-Toxin-Gen wurde in 20 Fällen nachgewiesen. Einmal konnte C. perfringens Typ C, β2-Toxin-Gen-positiv detektiert werden. Der hygienische Status der Gärreste entsprach in etwa dem hygienischen Status von Gülle. In Abhängigkeit vom Indikatorkeim war eine Verbesserung des Status durch eine Reduktion der Keimzahl festzustellen.

Clostridium perfringens est ubiquitaire dans l’environnement. Ce microorganisme peut être retrouvé dans la flore normale du tractus gastro-intestinal des mammifères et peut également causer une variété d’infections intestinales. Le phénotype de résistance à la bacitracine a déjà été rapporté chez C. perfringens mais les gènes associés n’ont pas été caractérisés. Dans cette étude, 24 des 99 isolats de C. perfringens aviaires testés ont démontré une résistance à la bacitracine. Les analyses ont révélé la présence d’un transporteur ABC ainsi que d’une undécaprénol kinase surproduite. Ces deux mécanismes semblent être codés par l’opéron bcrABDR. En amont et en aval des gènes bcr, un élément IS1216-like a été identifié, celui-ci pouvant jouer un rôle dans la dissémination de la résistance à la bacitracine. Des analyses d’hybridation sur ADN ont révélé que les gènes bcrABDR étaient localisés sur le chromosome. De plus, il a été démontré que les gènes bcr étaient exprimés en présence de bacitracine. Plusieurs études ont associé la tolérance aux antibiotiques et aux désinfectants à la formation de biofilm. Dans la littérature, peu d’informations sont disponibles sur le biofilm de C. perfringens. La majorité des isolats testés dans cette étude ont démontré la formation d’un biofilm. L’analyse de la matrice a démontré que celle-ci contenait des protéines, de l’ADN extracellulaire ainsi que des polysaccharides liés en bêta-1,4. Une meilleure survie des cellules en biofilm a été observée suite à une exposition à de fortes concentrations d’antibiotiques. Une exposition à de faibles doses de certains antibiotiques semblait diminuer le biofilm formé alors que pour d’autres, le biofilm semblait augmenter. Dans la présente étude, la susceptibilité des biofilms de C. perfringens à la désinfection a été également analysée. Les résultats ont démontré que la formation de biofilm protégeait les cellules de l’action du monopersulfate de potassium, des ammoniums quaternaires, du peroxyde d’hydrogène et du glutéraldéhyde. Toutefois, l’hypochlorite de sodium a été démontré comme étant efficace contre le biofilm de C. perfringens. Il a été démontré que les biofilms mixtes de C. perfringens cultivés en présence de Staphylococcus aureus ou d’Escherichia coli étaient plus résistants à la désinfection en comparaison aux biofilms simples de S. aureus ou d’E. coli. Toutefois, le biofilm simple de C. perfringens était plus résistant à la désinfection que les biofilms mixtes. Finalement, les profils de transcription entre les populations planctoniques et en biofilm ont été analysés par séquençage d’ARN. L’analyse transcriptomique du biofilm a identifié 238 gènes différentiellement exprimés entre les deux conditions. Les gènes négativement régulés sont impliqués dans la virulence, la production d’énergie, le métabolisme des sucres ainsi que dans la biosynthèse des acides gras et des acides aminés alors que les gènes induits sont impliqués dans la réponse au stress et au stress oxydatif, dans la biosynthèse d’acides gras et de phospholipides ainsi que dans la virulence. Cette étude décrit pour la première fois la découverte des gènes associés à la résistance à la bacitracine chez C. perfringens. Elle rapporte également de nouvelles données sur la matrice du biofilm, la tolérance aux antibiotiques et aux désinfectants ainsi que sur le transcriptome du biofilm de C. perfringens. / Clostridium perfringens is ubiquitous in the environment. This microorganism can be found in the intestinal tract of mammals as normal flora and can also cause many gastrointestinal infections. Phenotypic bacitracin resistance has been reported in the literature for C. perfringens but the genes responsible for this resistance have not yet been characterized. In this study, twenty-four of the 99 poultry isolates tested showed bacitracin resistance. Analysis revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase. These two mechanisms were shown to be both encoded by the putative bcrABDR operon. An IS1216-like element was found upstream and downstream from the bcr cluster, which may play a role in the dissemination of this resistance determinant. DNA hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Moreover, this gene cluster has been showed to be expressed under bacitracin stress. Many studies have associated tolerance to antibiotics and disinfectants to biofilm. In the literature, very little is known on the biofilm formation by C. perfringens. Most of the C. perfringens isolates tested in this study were able to form biofilms. Matrix composition analysis revealed the presence of proteins, extracellular DNA and beta-1,4 linked polysaccharides. Biofilm could also protect C. perfringens bacterial cells from an exposition to high concentrations of antibiotics. Exposition to low doses of antibiotics tended to lead to a diminution of the biofilm formed but for few isolates, the biofilm formation was increased. In the present study, susceptibilities of C. perfringens biofilms to disinfectants were also analysed. Results showed that biofilms can protect the bacterial cells from the action of potassium monopersulfate, quaternary ammonium chlorides, hydrogen peroxide and gluteraldehyde solutions. However, sodium hypochlorite solution was shown to be effective on C. perfringens biofilms. Our investigation of dual-species biofilms of C. perfringens with the addition of Staphylococcus aureus or Escherichia coli demonstrated that these dual-species biofilms were more tolerant to disinfection with sodium hypochlorite than the mono-species biofilms of S. aureus or E. coli. However, further disinfection studies using sodium hypochlorite suggest that the mono-species biofilms formed by C. perfringens is more tolerant to this disinfectant than the dual-species biofilms of C. perfringens with S. aureus or E. coli. Finally, the differential gene expression patterns between planktonic populations and biofilms of C. perfringens were investigated by RNA sequencing. The transcriptomic analysis identified 238 genes that were significantly differentially expressed between both conditions. Genes that were down-regulated in biofilm cells, relative to planktonic cells, included those involved in virulence, energy production, carbohydrate metabolism, fatty acids and amino acids biosynthesis. On the other hand, genes up-regulated in biofilm cells were involved in oxidative and stress responses, fatty acids and phospholipids biosynthesis and few genes were involved in virulence. This study reports on the discovery of genes associated to bacitracin resistance of C. perfringens. Our work brings also new data on matrix cohesion of the biofilm, tolerance to antibiotics and disinfectants, and on the transcriptome of the biofilm of C. perfringens.

Master of Science / Department of Animal Science / R. Scott Beyer / Daniel Y.C. Fung / Clostridium perfringens causes necrotic enteritis (NE), resulting in decreased feed efficiency and increased mortality, costing the poultry industry USD 2 billion a year worldwide. The objective of the first trial was to validate the Fung Double Tube (FDT) to detect and enumerate C. perfringens in chicken intestines. Two methods (FDT and petri plates) and three media (Shahidi Ferguson Perfringens [SFP] with egg supplement, polymyxin B [p], and kanamycin [k; E]; SFP with p and k [P]; and SFP with cycloserine [C]) were arranged in a 2 x 3 factorial, resulting in six treatments. The FDT with medium C (5.35 log CFU/g) had significantly (P<0.05) higher C. perfringens counts than any other media/method combination. The objective of the second and third trials was to determine the effect of diet type on the population of C. perfringens in broiler intestines using the FDT. Trial 2 tested: corn-soybean meal (SBM), low-crude protein (19.8%)/high synthetic amino acids (SAA), and barley (56%)-fishmeal (4%; BF). Diets in Trial 3 included: corn-SBM, barley (7.46%), fishmeal (4%), and BF. Diets in Trial 2 contained an antibiotic and a coccidiostat; diets in Trial 3 did not. After 21 days, birds in Trial 2 fed BF had significantly higher (P<0.05) counts (5.96 log CFU/g) of C. perfringens, as compared to all other diets. Both, corn-SBM and SAA diets resulted in 3.89 log CFU/g. In Trial 3, birds fed the corn-SBM diet (2.7 log CFU/g) had significantly lower (P<0.05) counts than broilers fed BF (4.15 log CFU/g). When broilers were fed fishmeal (3.583 log CFU/g) and barley (3.577 log CFU/g) separately, C. perfringens counts were numerically higher compared to the corn-SBM diet, but numerically lower than birds fed BF. Barley and fishmeal inclusion increased the incidence of C. perfringens, and their combination resulted in a cumulative effect. The FDT method is able to detect C. perfringens at higher levels than the conventional petri plate method (P<0.001) and it also proved to be an effective method to detect differences in C. perfringens counts from the intestines of chickens fed different diet.

Zum Studium des prophylaktischen Einflusses ausgewählter antibiotischer Leistungsförderer [Avilamycin (10 ppm), Avoparcin (15 ppm), Virginiamycin (20 ppm)] und ionophorer Antikokzidia [Monensin (100 ppm), Narasin (70 ppm)] sowie des metaphylaktischen bzw. therapeutischen Einsatzes von Tylosin [Tylan 0,5 g/l H2O] auf das Auftreten und die Ausprägung der Clostridium (Cl.) perfringens-Enterotoxämie (CPE) wurden Untersuchungen an insgesamt 33 Versuchsgruppen mit 825 Broilerküken durchgeführt. Die Erkrankung konnte mittels intraduodenaler Inokulation einer Vollkultur Cl. perfringens Typ A (ATCC 3624) sicher reproduziert werden. Die Morbiditätsrate betrug in allen infizierten Gruppen 100 %. An klinischer Symptomatik zeigte sich hauptsächlich profuser wässriger Durchfall. Schwerere Störungen, Apathie und Anorexie, waren selten und in allen beobachteten Fällen vom schnellen Tod des betreffenden Tieres begleitet. Allgemein fiel auf, daß die infizierten und nicht medikamentierten Tiere schneller und länger erkrankten. Bei infizierten und unmedikamentierten Tieren ergab sich eine Mortalitätsrate von 16 bis 36 %, in den medikamentierten Gruppen maximal 8 %. Tylosin zeigte eine sehr gute metaphylaktischen bzw. therapeutische Wirkung. Die Lebendmasseentwicklung betrachtend, konnte Avoparcin unter den Leistungsförderern beste Ergebnisse erzielen. Ähnliche Resultate wurden in den Kombinationsgruppen [Avilamycin plus Monensin oder Narasin] bzw. mittels Narasin erreicht. In absteigender Reihenfolge zeigten Avilamycin, Virginiamycin und Monensin eine geringere leistungsfördernde Wirkung. Die Bestimmung fäkaler bzw. ileozäkaler Clostridienkonzentrationen lebender, respektive verendeter Hühner erbrachte nur wenige und relativ unbedeutende statistisch gesicherte Korrelationen zu anderen Ergebnissen. Es konnten keine Zusammenhänge zwischen Erregerzahl und Lebendmassezunahme, bzw. Todesursache, aufgedeckt werden. Die Resultate aus allen Versuchen zusammenfassend, müssen den Kombinationen von Avilamycin mit Narasin bzw. Monensin beste Effekte hinsichtlich einer positiven Beeinflussung CPE-bedingter Morbidität, Mortalität und Lebendmasseverluste bescheinigt werden. Tylosin war in der Lage, die Verlustzahlen durch CPE rasch zu senken. Für die Ausprägung kompensatorischer Effekte hinsichtlich der Lebendmasseverluste unter der Infektion muß mit einer größeren Zeitspanne gerechnet werden. Die Polyether und auch Avilamycin sind als Futtermittelzusatzstoffe für die europäische Geflügelhaltung zugelassen. Durch die ständige Kokzidiosebedrohung in den Hühnerbeständen kann auf einen prophylaktischen Einsatz antikokzidieller Futtermittelzusatzstoffe momentan nicht verzichtet werden. Es ist zu vermuten, daß es durch den simultanen Einsatz von Polyether und Leistungsförderer zu einer positiven Beeinflussung der schädigenden Wechselwirkungen von Kokzidien und Cl. perfringens im Darm kommt. Bei vorhandener Empfindlichkeit der Eimerien sollte dies sowohl die Bekämpfung von CPE als auch von Kokzidiosen begünstigen. Der positive Eindruck von Avoparcin spielt, bedingt durch das europaweite Verbot, momentan für die Praxis keine Rolle. Die Entwicklungstendenzen auf dem Sektor antibiotisch wirksamer Futtermittelzusatzstoffe, eng verknüpft mit der bakteriellen Resistenzproblematik, werden in der Arbeit ausführlich diskutiert. / Investigations with 825 chickens in 33 trials were performed in order to find out the prophylactic effect of selected antibiotic growth promoters [avilamycin (10 ppm), avoparcin (15 ppm) virginiamycin (20 ppm)] and polyether ionophore antibiotics [monensin (100 ppm), narasin (70 ppm)] on the incidence of Clostridium (Cl.) perfringens enterotoxemia (CPE) in chickens as well as the therapeutic resp. metaphylactic influence of tylosin [Tylan 0,5 g/l H20]. The enterotoxemia could be reproduced regularly by intraduodenal infection with high numbers of vegetative cells of Cl. perfringens type A (ATCC 3624). The morbidity rate always reached 100 %. In spite of a profuse and watery diarrhoea the chickens normally showed no further considerable disturbances of the general status. Apathy or anorexia were rather rare and immediately followed by Exitus letalis of the related chickens. It was striking that the infected and non-medicated broilers contracted the disease more quickly and for a longer time. The mortality rate among the infected and non-medicated animals was 16 to 36 %, among the medicated groups max. 8 %. Tylosin showed a considerable metaphylactic effect in decreasing CPE mortality. The avoparcin group showed the best weight gain among the growth promoters, comparable to the results by means of the combinations [avilamycin + monensin or narasin] or narasin only. Decreasingly avilamycin, virginiamycin and monensin were less successful. Analysing the faecal resp. ileocecal quantities of Cl. perfringens adduced only a few statistically guaranteed correlation with other results. There was no causal connection between numbers of Cl. perfringens and life weight development. It was impossible to discover a numerical threshold of germs responsible for the death of the chickens. Summarising all the results of the entire attempts the combinations of avilamycin and narasin resp. monensin were the most effective concerning the reduction of morbidity, mortality and life weight losses by CPE. By application of tylosin it was possible to stop the mortality rate quickly. But it needs more time to achieve reductions of the CPE related weight losses. The two polyethers and also avilamycin are still admitted in the European Union. Currently an abandonment of anticoccidial feed supplements seems to be impossible due to the present danger of coccidiosis in poultry. By means of monensin/narasin plus avilamycin the adverse health effects of interactions of both pathogens should be reduced. Presupposing susceptibility of the coccida this should be a notable contribution to a better controlling and to the prevention of CPE and coccidiosis, too.

Made available in DSpace on 2014-07-29T15:01:41Z (GMT). No. of bitstreams: 1 Dissertacao Waleska Fernanda F Morgado.pdf: 1282396 bytes, checksum: 4ca186add1105f1358d9922c71f2b024 (MD5) Previous issue date: 2008-08-25 / Latin-American countries are facing serious problems related to waterborne diseases due to the lack of basic sanitation, affecting in particular those people living in small and rural communities. Solar radiation for water disinfection, SODIS, seems a promising process for small communities since it does not require electric energy and it has low cost and easy operation. This work aimed to evaluate the inactivation of the pathogens Escherichia coli and Clostridium perfringens by SODIS in the Center-West region of Brazil, Goiânia-GO. The Colilert® and multiple tubes (NMP/100 mL) were used to determine the Escherichia coli and the Clostridium perfringens bacterias, respectively. The inactivation and the re-growth of these bioindicators, the physico-chemical parameters of the raw and disinfected waters were the main focus of this work. Raw water was collected from a well located at the Civil Engineering School (EEC) of Federal University of Goiás (UFG) and it was inoculated apart using pre-determined concentrations of these bio-indicators. Samples were put in transparent PET bottles with capacity of 2L and left under sunlight exposure between 9am and 3pm. Samples were collected and analyzed in the laboratory after 0, 2, 4 and 6 hours of exposure. The work was divided into two phases: the first evaluate the effect of the use of two water volumes (1.5 L and 2 L) on the pathogen inactivation. In addition, the re-growth of these pathogens in the PET bottles after 3 storage days at ambient temperature was also investigated. In the second phase, PET bottles containing 1.5 L of contaminated water were exposed to sunlight radiation with and without solar reflectors. The results showed that there was a small difference (0,25-Log to Clostridium perfringens and 0,5-Log to Escherichia coli) in the inactivation of both bioindicators between the two volumes evaluated in the first phase. The use of the solar reflector did not improve the inactivation of the Clostridium perfringens and their re-growth was proportional to the final concentration after 6 hours of sunlight exposure. / Os países latino-americanos enfrentam sérios problemas com a alta incidência de doenças relacionadas com a falta de saneamento básico, sendo mais afetadas as populações que vivem em localidades pobres, periféricas e em zonas rurais. A utilização da radiação solar no processo de desinfecção de água, SODIS, é introduzida nesse contexto como uma alternativa de desinfecção da água independente de insumos, que funciona sem fornecimento de energia elétrica, baixo custo e de fácil operação em comunidades pequenas. Este trabalho teve por objetivo avaliar a inativação dos patógenos Escherichia coli e o Clostridium perfringens através da SODIS na região Centro-Oeste do Brasil, na cidade de Goiânia GO. Para a determinação das bactérias Escherichia coli e Clostridium perfringens foi utilizado os métodos Colilert® e tubos múltiplos (NMP/100 mL), respectivamente. A inativação e o recrescimento destes bioindicadores, a análise dos parâmetros físico-químicos da água bruta e da água desinfetada pela SODIS foram os enfoques principais deste estudo. Água bruta foi coletada de um poço raso da Escola de Engenharia Civil (EEC) da Universidade Federal de Goiás (UFG) e inoculada com concentrações determinadas dos bioindicadores, separadamente, acondicionadas em garrafas de PET e expostas ao sol pelo período das 09:00 às 15:00 horas. Amostras de água foram coletadas e analisadas em laboratório após os tempos de exposição de 0, 2, 4 e 6 horas. O trabalho experimental foi dividido em duas etapas, a primeira considerou a diferença de volumes nas garrafas, 1,5 L e 2 L, para investigar o efeito das concentrações de oxigênio, a verificação do recrescimento após 3 dias de armazenamento na própria garrafa de PET em temperatura ambiente e a avaliação da água bruta antes e após a SODIS. Na segunda etapa, garrafas com volume padrão de 1,5 L foram usadas em combinação com e sem concentrador solar. Os resultados mostraram que houve uma pequena diferença, (0,25-Log para Clostridium perfringens e 0,5-Log Escherichia coli) nas inativações bacterianas em relação aos diferentes volumes, sendo maiores nas garrafas de 1,5 L para ambos os bioindicadores na etapa 1. O uso do concentrador solar não apresentou melhoria no processo em relação ao Clostridium perfringens e o recrescimento foi proporcional à concentração final dos bioindicadores após as 6 horas de exposição.

246 samples of bulk and packaged spices from retail stores in the western, southeastern, southern, midwestern, and northeastern areas of the U.S. were examined for the presence of Clostridium -perfringens. Isolates were checked for the presence of the lecithinase gene (cpa) and enterotoxin genes (cpe) by PCR. Enterotoxin formation during sporulation was investigated using the Oxoid Toxin Detection Kit. Forty-three confirmed isolates (from 17% of total samples) were cpa-positive. Of those, 27 were cpe-positive. Together, levels of C. perfringens spores ranged from 3.6-2400/gm. The amount of enterotoxin in cell extracts ranged from 2-16 ng/ml. Some of the SEM images of isolated spore (# 78) and one plasmid-borne ent control (FD-153) showed an organized surface structure termed “candy-wrapper”. This extracellular structure remained after treatment with 0.1 % SDS for 1 hr, suggesting it was not composed of membrane debris from the mother cell. The D values of spores ranged from 1.19- 3.31 min. The addition of lysozyme in the plating medium elevated the recovery rate of heat-treated spores. The growth rate of a cocktail of spores from spices (# 31, # 32, # 45) between 4 to 5 hr after inoculation was determined with a doubling time of 6.82 min in hamburger. A cocktail of spores of plasmid-borne ent control showed an optimum growth rate between 5 to 8 hr after inoculation with doubling time of 15.98 min. However; spice isolate cocktail, plasmid-borne ent control cocktail (FD-5603 and FD-153), and a chromosome-borne ent control (NTCT 8239) were unable to germinate and outgrowth at 20oC. Inoculation in laboratory medium FTG indicated the same result as hamburger at 20oC. The ability of C. perfringens spores in spices to potentially survive cooking procedures can be followed by germination and growth of vegetative cells during improper cooling to levels associated with foodborne illness caused by this organism. Our results suggest that retail spices are potential vehicles of transmission of enterotoxin-positive C. perfringens.