Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-30Bitstream added on 2014-06-13T20:16:27Z : No. of bitstreams: 1 ferrarezi_mc_me_araca.pdf: 120865 bytes, checksum: 0f7d463195e9facb001a073816cb3dfd (MD5) / Universidade Estadual Paulista (UNESP) / A diarréia neonatal é uma das principais causas de perdas na bovinocultura. O Clostridium perfringens é um enteropatógeno amplamente distribuído na natureza e responsável por várias doenças nos animais, dentre elas a diarréia neonatal. Foram examinadas 141 amostras fecais de bezerros com diarréia e 129 amostras de animais sadios, com até 28 dias de idade e pertencentes a três rebanhos distintos. Do cultivo bacteriológico em anaerobiose foi possível isolar 36,2% e 30,2% amostras suspeitas de Clostridium perfringens dos animais enfermos e dos animais sadios, respectivamente. A genotipagem bacteriana foi efetuada empregando-se a técnica de PCR multiplex com os primers dos genes codificadores das toxinas alfa (cpa), beta (cpb), épsilon (etx), iota (itxA), enterotoxina (cpe) e toxina beta2 (cpb2). Dentre as amostras isoladas, 17/51 (33,3%) e 17/39 (43,6%) dos animais com diarréia e sadios, respectivamente, amplificaram um ou mais genes codificadores das toxinas de C. perfringens. Dos bezerros com diarréia, quatorze apresentaram somente o gene cpa (tipo A), um apresentou o cpa e cpb2 (tipo A beta2 positivo), um amplificou o cpa, itxA, e cpb2 (tipo E, beta2 positivo) e um amplificou o cpa, etx, itxA e cpb2 (tipo D e E, um ou ambos cpb2 positivo). Dentre os bezerros sadios, 10 eram exclusivamente tipo A, um era tipo A cpb2 positivo, dois eram tipo E, três eram tipo E cpb2 positivo e um era tipo D e E cpb2 positivo. Não houve correlação entre a genotipagem dos genes codificadores das toxinas de Clostridium perfringens e a presença de diarréia neonatal nos bezerros. / Neonatal diarrhea is one of the main causes of losses in cattle herds. Clostridium perfringens is a widespread enteropathogen, and is responsible for many animal diseases such as bovine neonatal diarrhea. Fecal samples from 141 diarrheic calves and 129 healthy calves, aged up to 28 days and belonging to three herds were examined. Rates of culture positivity were 36.2% and 30.2% for diarrheic and nondiarrheic calves, respectively. Multiple isolates from primary isolation plates were subjected to simultaneous genotyping by multiplex PCR, with primers amplifying fragments of alpha (cpa), beta (cpb), epsilon (etx), iota (itxA), enterotoxin (cpe) and beta2 (cpb2) toxinencoding genes. Only 17/51 (33.3%) and 17/39 (43.6%) of these mixtures from diarrheic and nondiarrheic calves, respectively, yielded genotype information, suggesting that this may not be a viable approach to genotyping of isolates. Fourteen isolate mixtures from animals with diarrhea had only cpa (type A), one had cpa and cpb2 (type A beta2 positive), one with cpa, itxA, and cpb2 (type E, beta2 positive), and one with cpa, etx, itxA, and cpb2 (Types D and E, one or both cpb2 positive). Among 17 isolate mixtures from healthy calves, 10 were exclusively type A, one was type A cpb2 positive, two were type E, three were type E cpb2 positive, and one was types D and E cpb2 positive. There was no correlation between isolation of a given toxin type and the presence of diarrhea.

Made available in DSpace on 2014-06-11T19:23:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-18Bitstream added on 2014-06-13T20:29:52Z : No. of bitstreams: 1 000721435.pdf: 591654 bytes, checksum: 7b7b1e9b77dc7c172c5d9fbb988ca07b (MD5) / toxina Épsilon (ETX) produzida pelo Clostridium perfringens tipos B e D é uma das mais potentes toxinas clostridiais superada apenas pelas neurotoxinas botulínica e tetânica. É responsável por quadros fatais de enterotoxemia em ovinos, caprinos e ocasionalmente em outros animais, caracterizados por edema em vários órgãos e aumento da permeabilidade vascular. Nos estudos “in vitro”, a linhagem de células “Madin-Darby canine kidney” (MDCK) é susceptível à ação da ETX, que se heptameriza nas membranas celulares formando um poro complexo que evolui para a lise celular. No presente estudo, foram avaliadas a morfologia e a viabilidade celular, a despolarização da membrana mitocondrial e a expressão de mediadores de morte celular programada (Bax e Bcl-2), após a exposição das células MDCK, com a ETX, a cada 24 horas, durante intervalo de 1 a 5 horas. Verificou-se o aparecimento de vacúolos no interior do citoplasma celular associados à perda de viabilidade celular, que evoluíram de forma progressiva, nos períodos de 1 a 5 horas pós-exposição. Foram realizadas análises por citometria de fluxo acústica para obtenção de uma visão mais aprofundada da patogenia causada pela ETX. Utilizando a citometria de fluxo acústica, considerada altamente sensível, as células MDCK expostas à ação da ETX, nos períodos de 1 a 5 horas, revelaram uma diminuição do potencial de membrana mitocondrial, seguido da expressão das proteínas Bax (25,48 %) e Bcl-2 (45,45 %) na fase de formação do pré-poro (1 hora pós exposição). Estes resultados, juntamente com alta citotoxicidade e visualização de vacúolos celulares, demonstra que a análise por citometria de fluxo acústico representa potencialmente uma ferramenta eficaz para estudar a patogênese da ETX / Epsilon toxin (ETX) produced by Clostridium perfringens types B and D is one of the most powerful clostridial toxins surpassed only by neurotoxins botulinum and tetanus. Studies blame the ETX by developing a fatal enterotoxemia in sheep, goats and occasionally in other animals, characterized by edema in multiple organs and increased vascular permeability. In in vitro toxicoinfection studies the Madin-Darby canine kidney (MDCK) cell line is susceptible to the action of ETX, which forms a heptamer in the cell membranes forming a pore complex that progresses to cell lysis. In the present study, we assessed cell viability and morphology, mitochondrial membrane depolarization and expression of programmed cell death mediators (Bax and Bcl-2) after exposure of MDCK cells with the ETX every 24 hours for range of 1 to 5 hours. Our results shows the appearance of vacuoles within the cytoplasm associated with loss of cell viability, which evolved gradually, in periods 1-5 hours after exposure. Analyzes were performed by acoustic flow cytometry to obtain further insight into the pathogenesis caused by ETX. Using acoustic flow cytometry, considered highly sensitive, MDCK cells exposed to the action of ETX, during 1 to 5 hours showed a decrease in mitochondrial membrane potential followed by the expression of Bax (25.48%) and Bcl-2 (45.45%) proteins at the pre-pore stage (1 hour post exposure). These results along with high cytotoxicity and visualization of cellular vacuoles, demonstrates that the flow cytometry analysis acoustic represents a potentially powerful tool for studying the pathogenesis of ETX

Submitted by TAÍS RAMALHO DOS ANJOS null (tranjos.vet@gmail.com) on 2017-04-24T12:41:20Z No. of bitstreams: 1 TAIS_RAMALHO_DOS_ANJOS (2).pdf: 1545211 bytes, checksum: 549d837c68e7609438e83800c2a4db56 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-25T20:34:24Z (GMT) No. of bitstreams: 1 anjos_tr_me_jabo.pdf: 1545211 bytes, checksum: 549d837c68e7609438e83800c2a4db56 (MD5) / Made available in DSpace on 2017-04-25T20:34:24Z (GMT). No. of bitstreams: 1 anjos_tr_me_jabo.pdf: 1545211 bytes, checksum: 549d837c68e7609438e83800c2a4db56 (MD5) Previous issue date: 2017-04-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O tratamento térmico por UAT elimina formas vegetativas dos microrganismos, porém, formas esporuladas altamente resistentes ao calor podem permanecer nos produtos e após a germinação dos esporos são capazes de liberar toxinas causadoras de doenças transmitidas por alimentos (DTAs). No presente estudo, objetivou-se avaliar a qualidade microbiológica de produtos tratados por UAT, por meio da contagem de microrganismos heterotróficos aeróbios mesófilos, isolamento e contagem de bactérias do grupo do Bacillus cereus e de Clostridium perfringens, comparar lotes dos três produtos quanto a tais microrganismos, identificar a espécie dos isolados e a presença dos fatores de virulência de bactérias do grupo do B. cereus. Para isso, foram obtidas 75 amostras, sendo 25 de cada produto (de leite de cabra UAT, alimento com soja UAT e bebida de leite de cabra e soja UAT), de 5 diferentes lotes, no comércio do interior do Estado de São Paulo. Os resultados evidenciaram possíveis falhas de processamento e utilização de matéria-prima inadequada, uma vez que altas populações (>104UFC/mL) de microrganismos heterotróficos aeróbios mesófilos foram verificadas em 100% dos lotes de alimento com soja e bebida de leite de cabra e soja e 80% dos lotes de leite de cabra. Microrganismos do grupo do B. cereus foram isolados de 80% dos lotes de alimento com soja, e 60% dos lotes de bebida de leite de cabra e soja e leite de cabra, com populações superiores a 1018 UFC/mL. C. perfringens foi isolado de 20% dos lotes de alimento com soja, com populações superiores a 103 UFC/mL. Na comparação entre os lotes, a data de validade não influenciou nas populações dos microrganismos analisados. Dos isolados de bactérias do grupo do B. cereus, 100% (29/29) apresentaram amplificação para o gene do complexo hbl, 96,5% (28/29) para o gene do complexo nhe e 75,8% (22/29) para o gene bceT. Tais resultados devem servir de alerta às autoridades sanitárias, pois leite de cabra e produtos à base de soja são normalmente consumidos por crianças e alérgicos, e o leite de cabra adicionado de soja apresentou maiores cargas de microrganismos patogênicos potencialmente causadores de intoxicações alimentares. / Heat treatment by UHT (ultra-high temperature) eliminates the vegetative forms of microorganisms, however, sporulated forms highly resistant to heat can remain in the products and following spore germination are capable of releasing toxins that cause foodborne diseases (FD´s). The objective of this study is to evaluate the microbiological quality of products submitted to UHT, namely goat milk, soy food, and goat and soy milk drinks, by the counting of heterotrophic aerobic mesophilic microorganisms, the isolation and counting of bacteria from the Bacillus cereus groups and Clostridium perfringens, comparing batches of the three products for such microorganisms, to identify the species of the isolated and the presence of the virulence factors of bacteria of the B. cereus group. For this, 75 samples were obtained, 25 of each product (from UAT goat milk, food with UAT soy and drink from goat milk and UAT soy) from 5 different lots, in the interior trade of the State of São Paulo. The results demonstrated possible failures in the processing and use of inadequate raw material since high population (>104 ) of heterotrophic aerobic mesophilic microorganisms were found in 100% of the lots of soy food and goat and soy milk drinks and 80% of the lots of goat milk. Microorganisms from the B. cereus group were isolated from 80% of the lots of soy food and 60% of the lots of goat and soy milk drinks, with population above 1018 . The C. perfringens microorganisms were isolated from 20% of the soy food lots, with population above 103 . In the comparison between the lots, the expiration date did not influence the population of the analyzed microorganisms, therefore, they were conveyed by the raw material used. Of the B. cereus isolated, 100% (29/29) showed amplification for the hbl complex gene, 96,5% (28/29) for the nhe complex gene and 75,8% (22/29) for the bceT gene. These results should serve as a warning to the health authorities because goat milk and soy-based products are generally consumed by children and individuals with allergies and goat milk added to soy was capable of carrying even higher doses of pathogenic microorganisms that may potentially cause food poisoning.

Made available in DSpace on 2014-06-11T19:30:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-19Bitstream added on 2014-06-13T20:07:37Z : No. of bitstreams: 1 boarini_l_me_jabo.pdf: 330765 bytes, checksum: 5a2e1936a8b0a2fe8933d469fc9b46a7 (MD5) / A suinocultura vem ganhando espaço no mercado brasileiro, atualmente o interesse por alimentos saudáveis trouxe a carne suína como aliada e não mais como vilã. A partir disso, novas tecnologias como sistemas de confinamento, foram adotadas a fim de obter melhorias que visam principalmente à higiene do processo, alimentação balanceada dos animais e instalações adequadas. Nesse enquadramento, doenças infecciosas entéricas representam um problema importante na suinocultura, e estão envolvidas com grandes perdas econômicas e contaminação da carcaça comercializada. Considerando estes aspectos, o trabalho objetivou identificar Clostridium perfringens e Salmonella spp., a fim de alertar o setor suinícola sobre os riscos ainda disseminados que causam enfermidades nos suínos e contaminam os produtos que serão comercializados; e correlacionar a presença de Clostridium perfringens com interferências no ganho de peso dos animais. Foram realizadas contagens bacterianas para Clostridium spp. que apresentaram médias de 1,2x105 UFC/mL na primeira coleta e 7,88x102 UFC/mL na segunda coleta do confinamento, e nas amostras do frigorífico contou-se 1,8x104 UFC/mL. Os resultados obtidos por PCR foram positivos apenas para a toxina alfa (cpa), caracterizando uniformidade dos positivos (25%) para Clostridium perfringens tipo A do confinamento e 46,4% do frigorífico. Para Salmonella spp. com o gene invA foram detectados 9,5% de positivos nos animais do confinamento e 21,4% das amostras do frigorífico. E Salmonella Typhimurium com o gene fliC, foram positivas 3,57% no confinamento e 8,33% no frigorífico. Foi possível concluir que Clostridium perfringens e Salmonella spp. foram... / Swine production is becoming more popular in the Brazilian market, currently interest in healthy foods brought swine as an ally and not as a villain. From this, new technologies such as confinement systems were adopted in order to achieve improvements aimed primarily to process hygiene, a balanced diet of animals and adequate facilities. In this framework, enteric infectious diseases represent a important problem in the swine production, and are involved with large economic losses and contamination of carcass marketed. Considering these aspects, the study aimed to identify Clostridium perfringens and Salmonella spp. in order to alert the swine sector still scattered about the risks that cause diseases in swine and contaminate the products to be marketed; and correlating the presence of Clostridium perfringens with interference on weight gains of animals. Bacterial counts were performed for Clostridium spp presenting averages of 1.2 x105 CFU/ mL in the first test and 7.88 x102 CFU / mL in the second collection of confinement, and the samples of slaughterhouse told by 1.8 x104 CFU/ mL. The results obtained by PCR were positive only for the alpha-toxin (cpa), featuring uniformity of positive samples (25%) for Clostridium perfringens type A of confinement and 46.4% of the slaughterhouse. For Salmonella spp. with the invA gene were detected 9.5% of positive animals in confinement and 21.4% of samples from the slaughterhouse. And Salmonella Typhimurium with the gene fliC, were positive 3.57% in the confinament and 8.33% in the slaughterhouse. It was concluded that Clostridium perfringens and Salmonella spp. were... (Complete abstract click electronic access below)

Necrotic enteritis (NE), a devastating enteric disease caused by Clostridium perfringens type A, contributes to the losses of 6 billion dollars worldwide per year and is currently being considered as a major global threat to the poultry industry. In past decades, it has been well-controlled by ineed antimicrobial growth promoters (AGPs). The withdrawal of AGPs due to antibiotic-resistance concerns resulted in a spike in NE incidence and led to the re-emergence of NE in the modern broiler production system. To unveil the association of toxin genes of C. perfringens, particularly for netB, with clinical NE, a self-designed qPCR primer set targeting netB was developed to qualify and quantify netB in NE-producing and non-NE-producing isolates. The netB was demonstrated to exist in the majority of C. perfringens type A isolates. The presence and the amount of netB were not significantly different between two types of isolate, indicating that those indicators are insufficient to predict an association with the pathogenicity of NE. The virulence of netB is suggested to be expressed or triggered under certain conditions, further promoting NE. A side by side trial was implemented with different combinations of netB-positive C. perfringens (CP1) and two predisposing factors to assess their role in NE development. Both CP1 and predisposing factor(s) are required for consistent NE reproduction, and particularly, Eimeria exerts significant effects on NE induction. The use of CP1 without a predisposing factor failed to induce NE. The severity and incidence of NE were positively correlated with the number of predisposing factors given in the NE induction. Analyzing gut microbiota in chickens challenged with CP1 and/or Eimeria by metagenomic sequencing, significant overgrowth of Clostridium sensu stricto 1, the genus contains C. perfringens, was associated with NE. Eimeria infection precedent to CP1 challenge had a synergistic effect on the overrepresentation. In addition to C. perfringens, the other member under Clostridium sensu stricto 1 was found to participate in NE development. Given supplementary dose of 0.4 kg/ton in feed, lauric acid neither exerted the inhibitory effect against proliferation of Clostridium sensu stricto 1 and C. perfringens nor reduced the incidence and severity of NE.

Necrotic enteritis (NE) is a disease that negatively affects broiler performance and has increasingly become a problem with the reduced use of antibiotics. There is increased interest to utilize alternative methods including coccidial vaccines and direct fed microbials (DFM) to reduce or prevent NE. Additionally, it has been demonstrated that reduced dietary Ca can reduce NE mortality. The objective of the first study was to examine the effects of Bacillus subtilis (DFM) in combination with attenuated (AV) and non-attenuated (NV) coccidial vaccines in broilers with NE. The objective of the second study was to determine the effects of DFM, antibiotic (BMD) or negative control (NC) with standard dietary Ca (0.9%) or below standard Ca (0.75%, 0.6%) in broilers with NE. To induce a natural NE occurrence, birds were vaccinated with the appropriate coccidial vaccine and then placed on reused litter from a previous flock exhibiting NE lesions and mortality. In the first trial DFM reduced BW and BW gain (P ≤ 0.05) during the starter phase (0 to 14 d). The addition of a DFM in the AV group resulted in decreased (P ≤ 0.05) BW gain and BW at 42 d. The DFM in the NV group resulted in improved (P ≤ 0.05) mortality corrected feed conversion ratio from 0 to 42 d. Overall, with a natural NE occurrence, the effect of DFM was variable depending on what type of coccidial preventative was utilized and was beneficial when used with NV. In the second trial BW and BW gain were increased in broilers fed 0.60% dietary Ca and DFM compared to 0.90% Ca and NC (P ≤ 0.05). Broilers fed 0.60% Ca had reduced NE mortality compared to broilers fed 0.90% Ca (P ≤ 0.05). Reducing dietary Ca to below industry standards was able to lessen the severity of NE though the addition of DFM was needed to improve BW. Overall, with a natural NE occurrence, the DFM was beneficial when used with NV and in diets with 0.6% dietary Ca. / Doctor of Philosophy / Necrotic enteritis (NE) is caused by the bacterium Clostridium perfringens and is a disease that reduces growth of broiler chicks and has increasingly become a problem in the chicken industry with the reduced use of antibiotics. Coccidiosis is a protozoal disease that causes intestinal damage and predisposes broilers to developing NE. Prevention of coccidiosis can be through medication or coccidial vaccines. Probiotics or direct fed microbials (DFM) have demonstrated the potential to reduce both coccidiosis and NE symptoms. Additionally, it has been shown that reducing the amount of calcium (Ca) in the diet can reduce chick death from NE. The first study examined the effects of a beneficial bacterium, Bacillus subtilis (DFM) in combination with two types of coccidial vaccines, attenuated (AV) and non-attenuated (NV). The second study investigated DFM with standard dietary Ca (0.9%) or below standard Ca levels (0.75%, 0.6%). To induce a "natural" NE disease occurrence, birds were vaccinated with the appropriate coccidial vaccine and then placed into pens. Pens contained reused litter from a previous flock of birds with NE (contained spores of Clostridium perfringens). In the first trial DFM reduced body weight of broilers during the first two weeks (P ≤ 0.05). The DFM decreased (P ≤ 0.05) BW gain and BW in the AV group but improved feed efficiency in the NV group (P ≤ 0.05). In the second trial BW and BW gain were increased in broilers fed DFM and lower Ca diets (0.6%) compared to those fed the standard (0.90%) Ca diets without DFM (P ≤ 0.05). Dead birds were reduced when broilers were fed lower Ca diets (0.6%) compared to broilers fed standard (0.90%) Ca diets (P ≤ 0.05). In conclusion, the DFM was beneficial when used with NV and in diets with lower Ca (0.6%) during a natural NE occurrence.

Researchers of <i>Clostridium perfringens</i>, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the <i>E. coli</i> reporter GusA, we characterized its induction in three different strains of <i>C. perfringens</i>. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when <i>C. perfringens</i> was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in <i>Bacillus subtilis</i>. PilT's typical localization in <i>Bacillus subtilis</i> was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins. / Master of Science

Thèse ou mémoire avec insertion d'articles. / Thèse en cotutelle : « Université Laval, Québec, Canada, Philosophiæ doctor (Ph. D.) et Universidad de La Rioja Logrono, Espagne » / Les antibiotiques ont été largement utilisés comme promoteurs de croissance et prophylactiques pour prévenir les maladies dans l'élevage de volailles. En raison de l'urgence de la prévention de la propagation des bactéries multirésistantes, de nombreux gouvernements ont interdit leur utilisation comme facteurs de croissance. Malheureusement, ces mesures ne sont pas universellement appliquées et, comme effet secondaire, l'incidence des infections à Clostridium perfringens associées à l'entérite nécrotique (EN) a augmenté dans les pays où elles ont été interdites. En outre, des C. perfringens résistants apparaissent, mais comme ce microorganisme ne fait pas l'objet de programmes de surveillance, davantage d'informations sont nécessaires pour comprendre pleinement son profil de résistance. Tout cela souligne la nécessité de rechercher et de mettre en œuvre de nouvelles approches pour éviter l'utilisation d'antibiotiques chez les volailles. Les bactéries productrices de bactériocines (BAC+), capables d'inhiber la croissance de C. perfringens, constituent une bonne approche. Les entérocoques se caractérisent par la production de bactériocines (entérocines) et peuvent donc être utilisés à cette fin. Cependant, ces genres contiennent souvent des facteurs de virulence et des mécanismes de résistance aux antibiotiques. Par conséquent, en raison de leur dualité en tant que pathogènes commensaux et opportunistes, une caractérisation plus approfondie de ces bactéries est nécessaire. C'est pourquoi l'utilisation de leurs entérocines pourrait être une approche encore meilleure et plus réaliste. Cette thèse tente de répondre à cette question en développant trois objectifs principaux. Le premier objectif était de caractériser au niveau génomique une collection d'isolats de C. perfringens provenant de volailles touchées par la NE. À cette fin, vingt isolats ont été caractérisés par séquençage du génome entier (WGS) et les données relatives à leur résistome, virulome, plasmidome, gènes de toxines et typage de séquences multilocus ont été analysés. Les résultats ont montré que les gènes tet (associés à la résistance à la tétracycline) étaient les gènes de résistance les plus fréquemment détectés et, fait intéressant, deux isolats portaient le gène erm (T) associé à la résistance à l'érythromycine, qui n'a été signalé que chez d'autres bactéries Gram-positifs. Douze des isolats ont été toxinotypés comme étant de type A et sept comme étant de type G. D'autres facteurs de virulence codant pour des hyaluronases et des sialidases, ainsi que des plasmides, ont été fréquemment détectés. Les types de séquences ont révélé une grande variabilité des isolats et de nouvelles combinaisons alléliques ont été trouvées. Parmi les isolats, C. perfringens MLG7307 présentait des caractéristiques uniques, même en l'absence du gène conservateur colA, ce qui suggère que cet isolat pourrait appartenir à une nouvelle classification. Dans l'ensemble, les résultats obtenus permettent de mieux comprendre les caractéristiques génomiques de C. perfringens et de mieux appréhender ce pathogène. Le deuxième objectif était de cribler et de caractériser les souches d'entérocoques d'origine avicole ayant une activité antimicrobienne contre C. perfringens. À cette fin, une collection de 251 entérocoques provenant de volailles a été criblée pour son activité antimicrobienne contre la collection de C. perfringens et des BAC+ ont été sélectionnées pour effectuer une analyse WGS en termes de profil de résistance aux antimicrobiens, de virulence, de plasmidome et de typage de séquences multilocus. Selon les résultats obtenus, des entérocoques potentiellement inoffensifs ont été sélectionnés pour tester la survie digestive dans les conditions de la volaille. Parmi tous les entérocoques, E. faecium X2893 et X2906 étaient les candidats les plus prometteurs pour des études ultérieures en tant que cultures protectrices pour l'élevage de volailles. Les deux souches appartiennent au type de séquence ST722, portent les gènes codant pour l'entérocine A et l'entérocine B, n'ont pas de gènes de résistance acquis, ne portent pas de plasmides, portent le gène acm impliqué dans la colonisation de l'hôte et ont montré des taux de survie élevés dans des conditions digestives in vitro chez la volaille. Ils sont donc de bons candidats pour être utilisés comme cultures protectrices dans des études à venir. Le dernier objectif était de produire et de purifier des entérocines ayant une activité contre la collection de C. perfringens et d'autres bactéries pathogènes pour la volaille. Les entérocines A, B, P, SEK4 et L50 ont été obtenues par synthèse peptidique en phase solide assistée par micro-ondes et leur activité antimicrobienne a été évaluée contre la collection de C. perfringens et d'autres bactéries pertinentes. Des combinaisons de ces entérocines, en fonction de leurs mécanismes d'action, ont été évaluées pour obtenir une synergie. Les résultats ont montré que les deux peptides de L50 étaient les plus actifs contre C. perfringens, L50A étant plus actif. Ces peptides ont également montré le spectre le plus large, étant actifs même contre Campylobacter coli ATCC 33559 et Pseudomonas aeruginosa ATCC 27855. Toutes les combinaisons testées ont montré une synergie ou une synergie partielle. Cette étude renforce l'idée d'utiliser les entérocines seules et en combinaisons synergiques pour inhiber la croissance de C. perfringens et d'autres agents pathogènes comme une alternative prometteuse aux antibiotiques dans le secteur de la volaille. En conclusion, l'utilisation d'entérocoques BAC+, et en particulier de leurs entérocines, représente une alternative très intéressante aux antibiotiques dans le secteur de la volaille. D'autres perspectives sur ce sujet pourraient inclure l'optimisation de la production d'entérocines et des études plus détaillées (toxicité, dégradation enzymatique, etc.), afin de s'assurer que leur utilisation est sûre. Les prochaines étapes pourraient également inclure des modèles d'infection in vivo afin d'évaluer leur efficacité dans la prévention de l'infection par C. perfringens. / Antibiotics have been widely used in therapeutic, prophylaxis and growth promotion in poultry farming. Due to the urgency of preventing the spread of multi-drug resistant bacteria, many governments have banned the use of antibiotics for growth promotion and are calling for a general reduction of these agents in food production, requiring alternatives to preserve human and animal health. Clostridium perfringens infections associated with necrotic enteritis (NE) are one of the major threats of the poultry sector. Antibiotic resistance in C. perfringens is emerging, but as this microorganism is not subject to surveillance programmes, more information is needed to fully understand its antibiotic resistance profile. All this underlines the need to deepen in the control of NE associated to C. perfringens in the poultry sector reducing the use of antibiotics. Bacteriocin-producing (BAC+) bacteria, capable of inhibiting the growth of C. perfringens provide a good approach. Enterococci are characterized by the production of bacteriocins (enterocins) and can therefore be used for this purpose. However, these genera often contain virulence factors and antibiotic resistance mechanisms. Hence, due to their duality as commensal and opportunistic pathogen, a deep characterisation of these BAC+ enterococci is required. For this reason, the use of their enterocins instead could be an even better and more realistic approach. This thesis attempts to address this issue through the development of three main objectives. The first objective was to characterise at the genomic level a collection of C. perfringens isolates from poultry affected by NE. To this end, twenty isolates were characterised by whole genome sequencing (WGS) and data on their resistome, virulome, plasmidome, toxin genes and multilocus sequence typing were analysed. The results showed that the tet genes (associated with tetracycline resistance) were the most common resistance genes detected and, interestingly, two isolates carried the erm(T) gene associated with erythromycin resistance, which has only been reported in other Gram-positive bacteria. Twelve of the isolates were toxinotyped as type A and seven as type G. Other virulence factors encoding hyaluronases and sialidases and plasmids, were frequently detected. Identified sequence types revealed a high variability of the isolates and new allelic combinations were found. Among the isolates, C. perfringens MLG7307 showed unique characteristics, even lacking the housekeeping gene colA, suggesting that this isolate could belong to a newspecies/variant. Overall, the results obtained provide insights into the genomic characteristics of C. perfringens and a better understanding of this pathogen. The second objective was to screen and characterise for safety enterococcal strains of poultry origin with antimicrobial activity against C. perfringens. To this end, a collection of 251 enterococci from poultry was screened for antimicrobial activity against the C. perfringens collection and BAC+ strains were selected to perform WGS analysis in terms of the resistome, virulence, plasmidome and multilocus sequence typing. According to the results obtained, potentially harmless selected enterococci were also tested for digestive survival under poultry conditions. Among all enterococci, E. faecium X2893 and X2906 were the most promising candidates for further studies as protective cultures for poultry farming. Both strains belong to sequence type ST722, carry the genes encoding enterocin A and enterocin B, have no acquired resistance genes, do not carry plasmids, contain the acm gene involved in host colonisation and showed high survival rates under in vitro poultry digestive conditions. They are therefore good candidates for use as protective cultures in future studies. The last objective was to produce and purify enterocins with activity against the C. perfringens collection and other relevant bacterial poultry pathogens. Enterocins A, B, P, SEK4 and L50 were obtained by microwave- assisted solid-phase peptide synthesis and their antimicrobial activity was evaluated against the C. perfringens collection and other relevant bacteria. Combinations of these enterocins, according to their mechanisms of action, were evaluated to achieve synergy. The results showed that the two peptides from L50 were the most active against C. perfringens, with L50A being more active. These peptides also showed the broadest spectrum, being active even against Gram-negative Campylobacter coli ATCC 33559 and Pseudomonas aeruginosa ATCC 27855. All combinations tested showed synergy or partial synergy. This study strengthens the idea of using enterocins alone and in synergistic combinations to inhibit the growth of C. perfringens and other pathogens as a promising alternative to antibiotics in the poultry sector. In conclusion, the use of BAC+ enterococci, and especially their enterocins, represents a very attractive alternative to antibiotics in the poultry sector. Further perspectives on this topic could include the optimisation of enterocin production and more detailed studied (toxicity, enzymatic degradation, etc), to ensure that their use is safe. Also, next steps can includen vivo infection models to assess their efficacy in preventing C. perfringens infection in poultry production. / Los antibióticos se han utilizado ampliamente en terapéutica, profilaxis y como promotores del crecimiento en producción aviar. Dada la necesidad urgente de prevenir la propagación de bacterias multirresistentes, muchos gobiernos han prohibido su uso como promotores del crecimiento en producción animal, y urgen en la necesidad de reducir de forma general el uso de antibióticos en este sector, requiriendo alternativas para preservar la salud humana y animal. Las infecciones por Clostridium perfringens asociadas con enteritis necrótica (EN) han aumentado en los últimos años en el sector avicola y suponen un gran problema, que exige una vigilancia estrecha. La resistencia a antibióticos esta aumentando en C. perfringens, pero como este microorganismo no está sujeto a programas de vigilancia, se necesita más información para conocer a fondo su perfil de resistencia. Todo esto resalta la necesidad de investigar en el control de la EN asociada a C. perfringens, aplicando nuevos enfoques para reducir el uso de antibióticos en producción aviar. Las bacterias productoras de bacteriocinas (BAC+), capaces de inhibir el crecimiento de C. perfringens, suponen un buen enfoque. Los enterococos destacan por la producción de bacteriocinas (enterocinas) y, por tanto, pueden utilizarse con este fin. Sin embargo, con frecuencia los enterococos contienen factores de virulencia y mecanismos de resistencia a los antibióticos. De ahí que, debido a su dualidad como comensales y patógenos oportunistas, sea necesario caracterizarlos exhaustivamente. Así pues, el uso de sus enterocinas podría ser un enfoque aún mejor y más realista. Esta tesis intenta abordar esta problemática mediante el desarrollo de tres objetivos principales. El primer objetivo fue caracterizar a nivel genómico una colección de aislados de C. perfringens procedentes de aves de corral afectadas por NE. Para ello, se caracterizaron veinte cepas mediante secuenciación del genoma completo (WGS) y se analizaron datos sobre su resistoma, viruloma, plasmidoma, genes de toxinas y tipificación molecular. Los resultados mostraron que los genes tet (asociados a la resistencia a la tetraciclina) eran los más frecuentes y fue de interés la detección de dos aislados que portaban el gen erm(T), asociado a la resistencia a la eritromicina, que sólo se ha descrito previamente en otras bacterias Gram-positivas. Doce de los aislados fueron toxinotipados como tipo A y siete como tipo G. También se detectaron con frecuencia otros factores de virulencia que codifican hialuronasas y sialidasas, así como plásmidos. El tipado molecular reveló una gran variabilidad de los aislados, hallándose incluso nuevas combinaciones alélicas. Entre los aislados, C. perfringens MLG 7307 mostró características únicas, careciendo del gen « housekeeping » colA, lo que sugiere que podría pertenecer a una nueva especie/variedad. En su conjunto, los resultados obtenidos permiten conocer mejor las características genómicas de C. perfringens y profundizar en el estudio de este patógeno. El segundo objetivo fue caracterizar los enterococos de origen aviar con actividad antimicrobiana frente a C. perfringens para garantizar su seguridad. Para ello, se examinó la actividad antimicrobiana de una colección de 251 enterococos de origen aviar frente a la colección de C. perfringens y se seleccionaron cepas BAC+ para realizar análisis de secuenciación masiva (WGS) y determinar así su resistoma, viruloma, plasmidoma y realizar la tipificación molecular. Posteriormente, se analizó la supervivencia digestiva en condiciones aviares de los enterococos potencialmente inocuos seleccionados. De entre todos los enterococos, E. faecium X2893 y X2906 fueron los candidatos más prometedores para futuros estudios como cultivos protectores. Ambas cepas pertenecen al linaje ST722, son portadoras de los genes codificantes de la enterocina A y la enterocina B, no tienen genes de resistencia a antibióticos adquiridos, no portan plásmidos, poseen el gen acm implicado en la colonización del hospedador y mostraron elevadas tasas de supervivencia in vitro bajo condiciones digestivas aviares. Por lo tanto, son buenos candidatos para se utilizados como cultivos protectores en estudios posteriores. El último objetivo fue producir y purificar enterocinas con actividad antimicrobiana contra la colección de C. perfringens y otros patógenos relevantes. Las enterocinas A, B, P, SEK4 y L50 se obtuvieron mediante síntesis peptídica en fase sólida asistida por microondas y se evaluó su actividad antimicrobiana frente a la colección de C. perfringens y otros patógenos. Se evaluaron diferentes combinaciones de estas enterocinas, seleccionadas según sus mecanismos de acción, para lograr sinergias. Los resultados mostraron que los dos péptidos de la enterocina L50 eran los más activos contra C. perfringens, siendo L50A el más activo. Estos péptidos también mostraron el espectro más amplio, siendo activos incluso frente a las bacterias Gram-negativas Campylobacter coli ATCC 33559 y Pseudomonas aeruginosa ATCC 27855. Todas las combinaciones estudiadas mostraron sinergia o sinergia parcial. Este estudio consolida la idea de emplear enterocinas solas y en combinaciones sinérgicas para inhibir el crecimiento de C. perfringens y otros patógenos como alternativa prometedora a los antibióticos en el sector avícola. En conclusión, el uso de enterococos BAC+, y especialmente de sus enterocinas, representa una alternativa muy atractiva a los antibióticos en el sector avícola. Entre las perspectivas futuras sobre este tema podrían figurar la optimización de la producción de enterocinas y estudios más detallados (toxicidad, degradación enzimática, etc.), para garantizar que su uso sea seguro. Asimismo, los próximos pasos pueden incluir modelos de infección in vivo para evaluar su eficacia en la prevención de la infección por C. perfringens.

Chickens genetically selected for low (LA) or high (HA) antibody response to sheep red blood cells (SRBC) displayed a correlated change in major histocompatibility complex (MHC), so that LA chickens were 96% B¹³ and HA chickens were 96% B²¹. During a clinical outbreak of necrotic enteritis, B²¹B²¹ genotypes experienced significantly less mortality (6% vs. 13 %) compared to B¹³B¹³ genotypes. A study was carried out to assess immunological differences between LA and HA lines during exposure to Clostridium perfringens. In Experiment 1, chickens were orally gavaged with a low (10⁷ CFU/mL) or high (10⁹ CFU/mL) dose of C. perfringens. In Experiment 2, chickens were orally gavaged with live coccidia oocysts on experiment d 1, followed by 10⁷ CFU/mL C. perfringens on d 5. Unfortunately, establishment of necrotic enteritis infection was unsuccessful in both experiments as evidenced by lack of significant intestinal lesions, as well as no negative effect on bird performance. In an ex vivo study, peripheral blood mononuclear cells (PBMCs) were isolated from each genetic line, cultured, stimulated with LPS (4 h), and exposed to varying concentrations of C. perfringens α-toxin (1, 10, 100, 1000 U/L) for 2 and 4 h. Evaluation of cellular proliferation, percent cytotoxicity and immunological gene expression was carried out in a variety of experiments. Genetic lines were found to be highly divergent in all analyses. / Master of Science

Clostridium perfringens is a Gram-positive (Gr+) anaerobic pathogen that was found to contain Type IV pilus (T4P) system genes within the genomes of all its sequenced strains. T4P are widely used in Gram-negative organisms for aggregation, biofilm formation, adherence, and DNA uptake. Because few examples of T4P-utilizing Gram-positive bacteria are studied to date, we wanted to characterize the T4P system in this Gr+ bacterium. To understand the regulation of T4P genes and therefore better understand their expression, we employed the highly powerful next-generation sequencing tool RNA-seq in a variety of conditions. RNA-seq uncovered previously unknown regulatory mechanisms surrounding T4P genes as well as provided transcriptional information for most of the genes in the C. perfringens strain 13 genome. We also utilized reporter gene assays to look at post-transcriptional regulation of T4P promoters. The wealth of RNA-seq data acted as a jumping-off point for many smaller projects involving transcriptional regulators that may influence T4P expression. We investigated a novel small RNA in close proximity to the major T4P operon, as well as two little-characterized transcriptional regulators that function in the same conditions as T4P genes. RNA-seq also provided data to develop a method for protein purification from C. perfringens without induction. / Master of Science