Immediately post-hatch, broiler chicks are exposed to microbes that begin colonizing the gut, including environmental pathogens. One of the costliest enteric diseases in broiler production is necrotic enteritis (NE), caused by the ubiquitous opportunistic bacteria Clostridium perfringens (CP). With the worldwide reduction in antibiotic growth-promoters (AGPs), there is increased interest in natural alternatives to reduce disease and improve broiler health. The overall objective of the studies described herein was to apply probiotics or synbiotics to birds by in ovo application or orally before they leave the hatchery, then evaluate bird performance and various intestinal responses. Data were analyzed in JMP with LS Means to separate means with significance assigned at P ≤ 0.05 and trends at 0.05 < P ≤ 0.10. The first 21-day (D) study used 480 male Cobb 500 broilers randomly divided into one of four treatments using a 2x2 factorial design: a no-additive control (CTRL), a one-time oral application of synbiotic at the hatchery fed a basal diet (HS), an oral application of water at the hatchery with dietary synbiotics (DS), and a hatchery synbiotic plus dietary synbiotic (HSDS). Performance was measured on day-of-hatch (DOH), D3, D7, D14, and D21. mRNA abundance of various intestinal markers was measured at D7 and D21, including tight junction proteins ZO-1, ZO-2, and CLD-1; nutrient transporters SGLT1 and PepT1; and immune response markers TLR2, TLR4, and IL-10. HS lowered feed intake (FI) and feed conversion ratio (FCR) without lowering body weight (BW) from D14-21. There was greater abundance of PepT1 mRNA (P ≤ 0.1) and IL-10 mRNA (P ≤ 0.05) on D21 in HSDS. Second, a 21-day pilot study with 480 male and female Cobb 500 broilers was conducted to determine the optimum in ovo dosage level of a probiotic or synbiotic (PROB or SYNB) applied at embryonic day 18 (E18) with subsequent NE challenge using seven treatments: in ovo application of sterile water (CTRL), low (PROB-L or SYNB-L: 1x105 CFU), medium (PROB-M or SYNB-M: 1x106 CFU), or high (PROB-H or SYNB-H: 1x107 CFU) probiotic or synbiotic doses dissolved in sterile water. Performance measurements were taken on DOH, D4, D8, D14 and D21. On D8, NE lesion scores were not impacted by treatment. D8 ileal samples were taken for mRNA abundance of TLR4, IL-10, IL-1β, AvBD8, AvBD10, and AvBD13. SYNB-H had higher abundance of AvBD10 mRNA compared to CTRL (P ≤ 0.1), and higher IL-1β mRNA compared to SYNB-L (P ≤ 0.05). PROB-H and SYNB-H had better performance than the low and medium doses, but were not better than the CTRL. The high doses were chosen for subsequent studies. Third, a longer 42-day study using 1,630 Ross 708 male and female broilers was conducted consisting of the following six treatments. A negative control (NC): sterile water in ovo fed basal corn/soybean meal mash diet without NE challenge; antibiotic growth-promoter (AGP+): sterile water in ovo fed basal diet with virginiamycin (0.5 kg/MT) as an AGP with NE challenge; NC+: same as NC plus NE challenge; SI+: synbiotic in ovo fed the basal diet and NE challenged; SD+: sterile water in ovo fed basal diet supplemented with synbiotic (0.5 kg/MT feed) and NE challenged; and SID+: synbiotic in ovo fed basal diet with synbiotic (0.5 kg/MT feed) with NE challenge. Cumulatively, SID+ had lower FI and FCR than NC+, but no change in BW or BWG. The combination treatment (SID+) often had an additive effect compared to SD+ or SI+ alone on mRNA abundance and D7 cecal fatty acid profiles. SD+ and SID+ also had higher D42 lean:fat ratios compared to NC+. Last, a 42-day study was conducted using 1,630 male and female Ross 708 broilers and the in ovo application of probiotics and subsequent NE challenge with five treatments. NC: sterile water in ovo, fed basal corn/soybean meal mash diet without NE challenge; AGP+: sterile water in ovo, fed basal diet with virginiamycin (0.5 kg/MT of feed) as AGP with NE challenge; NC+: NC treatment, with NE challenge; PI+: probiotic in ovo, fed basal diet, with NE challenge; PD+: sterile water in ovo, fed basal diet supplemented with probiotic (1.3 kg/MT of feed), with NE challenge. The use of probiotics in this study had little effect on performance, lean:fat ratios, and cecal fatty acid profiles, but PD+ increased mRNA abundance of D14 TLR2, D14 TNF-α, and D42 LEAP2 in cecal tonsils compared to controls. PI+ increased mRNA abundance of D7 and D42 MUC2, D7 LEAP2, and D42 TNF-α in the ileum. PI+ increased mRNA abundance in the cecal tonsils of D7 TLR2 and D42 TNF-α. These studies yielded interesting results about probiotics and synbiotics during a NE challenge by evaluating performance, intestinal immune responses, and fatty acid profiles in the ceca of broilers. In conclusion, the probiotic in this study did not improve broiler health during a NE challenge, but synbiotic use in ovo and continuation in the feed showed improvement over in ovo or dietary application alone. Synbiotic improved FCR over a challenged control, and altered mRNA abundance in the small intestine. / Doctor of Philosophy / The poultry industry is one of the most popular animal protein sources worldwide. As with any livestock operation, industry goals include optimizing animal health and well-being, maximizing animal productivity, and producing quality products in the most cost effective manner. Improvements in genetics, nutrition, and management have increased productivity and cut costs. One important application was the low-level use of antibiotics in feed. These medications reduced the risk of disease outbreak in flocks, which led to healthier birds and improved growth rates. However, when global concern of antibiotic resistance in human medicine came to light, both the livestock industry and governing bodies implemented voluntary and mandatory reduction or elimination of antibiotics. Previously, these important antibiotics helped to control costly diseases. As they are removed, alternatives to antibiotics will be important in disease control and prevention. A major group of alternatives to antibiotics in poultry includes probiotics, prebiotics, and synbiotics. Probiotic bacteria are considered 'good bacteria' in the gut, and provide various health benefits to the host. Prebiotics are non-living substances that support the growth of healthy bacteria. A synbiotic is the combination of both probiotics and prebiotics in a single application method. The goal of this research project was to give probiotics or synbiotics to broiler chicks and evaluate their potential benefits and effects on bird performance and the immune response. Ideally, applying probiotic bacteria as early as possible might translate into early colonization of the gut with healthy bacteria. This included oral application of synbiotics at the hatchery, or by safely injecting them into part of the egg that is swallowed by the chick embryo before hatch. This egg application, or in ovo application, is a safe, effective, widely-practiced method of vaccinating chicks to jumpstart their defense against disease. By vaccinating them in ovo, they can start to prime the immune system before they even hatch. Applying probiotics in ovo may improve health after early gut colonization with beneficial microbes. Numerous studies on natural alternatives to antibiotics have been conducted, with varying results. Results of this research indicate that in ovo application of probiotics and synbiotics is safe. Birds that received probiotics in the feed often performed similar to those that received none. However, the in ovo use of synbiotics combined with the continued use in the feed after hatch improved efficiency in broilers during an intestinal disease challenge and improved various aspects of gut function. Overall, as antibiotics are phased out, using probiotics and synbiotics may improve poultry health, but continued research will help understand the optimum ways to use them.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Made available in DSpace on 2017-07-25T19:29:42Z (GMT). No. of bitstreams: 1 Hellen Aparecida Arantes.pdf: 691625 bytes, checksum: a3ecb17b706b52a2674dc29e0a5e82cc (MD5) Previous issue date: 2008-12-15 / The development and grain yield of the crop of wheat are influenced by several factors, among which is the interference caused by pathogens present in crops. Currently, the main tool for controlling these pathogens in crops is the use of constant application of fungicides. However, this practice is causing pathogens resistant to these groups of fungicides used, as well as encouraging the expression of the mutagenic character of these populations. In an attempt to provide more tools to control the diseases of wheat to the farmer, this study aimed to: a) assess the effect of timing of implementation and sources of phosphite and acibenzolar-S-methyl alone and combined with the fungicide pyraclostrobin + epoxiconazole in control the powdery mildew (Blumeria graminis f. sp. tritici) and the leaf rust (Puccinia triticina), b) assess the effect of different rates, sources of phosphite and acibenzolar-S-methyl isolated and associated to the fungicide pyraclostrobin + epoxiconazole in control of the spots brown (Bipolaris sorokiniana) and yellow (Drechslera tritici-repentis); c) and in vitro effect of different concentrations of phosphite and pyraclostrobin + epoxiconazole on the mycelial growth and germination of conidia of Bipolaris sorokiniana. About the control of powdery mildew and rust can be concluded that the phosphites have no effect on these pathogens and do not change their productivity. It is also clear that there is no difference between the different sources of phosphites to control the powdery mildew and leaf rust, which occurs on powdery mildew and leaf rust's only when the phosphite associated with the fungicide is applied. About the control of yellow and brown stains concluded that the dose of 1048 g i.a. ha-1 potassium phosphite reduces the severity of leaf spots and can be used as a tool in the management of leaf spots. In in vitro tests concluded that the phosphites have direct action on Bipolaris sorokiniana. / O desenvolvimento e o rendimento de grãos da cultura do trigo são influenciados por diversos fatores, entre os quais se encontram a interferência causada por patógenos presentes nas lavouras. Atualmente, a principal ferramenta de controle destes patógenos nas lavouras é o uso de constantes aplicações de fungicidas. Contudo, essa prática vem ocasionando resistência destes patógenos aos grupos de fungicidas utilizados, bem como favorecendo a expressão do caráter mutagênico destas populações. Na tentativa de disponibilizar mais uma ferramenta de controle às doenças do trigo ao agricultor, este trabalho objetivou: a) avaliar o efeito de épocas de aplicação e de fontes de fosfito e acibenzolar-S-metil isolados e combinados com o fungicida epoxiconazole + piraclostrobina no controle do oídio (Blumeria graminis f. sp. tritici) e da ferrugem da folha (Puccinia triticina); b) avaliar o efeito de diferentes doses, fontes de fosfito e acibenzolar-S-metil isolados e associados ao fungicida epoxiconazole + piraclostrobina no controle das manchas marrom (Bipolaris sorokiniana) e mancha amarela (Drechslera tritici-repentis); c) e efeito in vitro de diferentes concentrações de fosfito e epoxiconazole + piraclostrobina sobre o crescimento micelial e a germinação de conídios de Bipolaris sorokiniana. Sobre o controle de oídio e ferrugem pode-se constatar que os fosfitos não possuem efeito sobre estes patógenos e não alteram a produtividade. Concluiu-se ainda que não existe diferença entre as diferentes fontes de fosfitos no controle de oídio e ferrugem da folha, que ocorre efeito sobre o oídio e a ferrugem da folha apenas quando o fosfito é aplicado associado ao fungicida. Sobre o controle das manchas amarela e marrom conclui-se que a dose de 1048 g i.a. ha-1 de fosfito potássico reduz a severidade das manchas foliares, podendo ser utilizado como uma ferramenta no manejo das manchas foliares. Nos testes in vitro conclui-se que os fosfitos possuem ação direta sobre Bipolaris sorokiniana.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Travail en codirection avec M. Frédéric Raymond / Avec la présente augmentation de la résistance aux antibiotiques et l’inquiétude des consommateurs, de nouvelles alternatives sont nécessaires afin de contrôler l’entérite nécrotique (EN), une maladie causant des millions de dollars en pertes économiques pour l’industrie de la volaille mondialement. Les bactériocines sont des peptides antimicrobiens produits par une bactérie pour tuer ou inhiber la croissance d’autres compétiteurs bactériens. La perfrine est la seule bactériocine reportée associée aux souches pathogènes de Clostridium perfringens, l’agent causal de l’EN. Les objectifs de cette étude étaient de détecter les souches commensales de Clostridium perfringens possédant une activité antimicrobienne contre des souches pathogènes de C. perfringens et d’identifier et caractériser les bactériocines produites. Les souches commensales de C. perfringens ont été sélectionnées à partir de notre banque de souches. L’activité antimicrobienne de ces souches a été testée contre des souches pathogènes de C. perfringens en utilisant la méthode d’inhibition sur gélose. Une souche commensale active démontrant une activité antimicrobienne a été cultivée et ses bactériocines ont été partiellement purifiées grâce à la précipitation au sulfate d’ammonium et par chromatographie (HPLC). À la suite de chaque chromatographie, l’activité antimicrobienne des fractions a été vérifiée en utilisant la méthode décrite précédemment afin de choisir les fractions contenant les bactériocines. La susceptibilité enzymatique, la stabilité à la chaleur et au pH et le poids moléculaire estimé des bactériocines ont été caractérisés. Les bactériocines étudiées étaient sensibles à la protéinase K, thermolabiles, stables à pH entre 4 et 8 et leur poids moléculaire étaient supérieur à 30 kDa. Le génome de la souche CP1676 a été séquencé et des analyses bio-informatiques ont été réalisées. Nous avons trouvé 28 séquences de bactériocines potentielles, mais seulement 4 d’entre elles semblaient être prometteuses. Dans cette étude, des souches commensales de C. perfringens produisant des bactériocines actives contre des souches pathogènes ont été identifiées. Ces bactériocines pourraient devenir une alternative intéressante aux antibiotiques afin de contrôler l’entérite nécrotique, mais davantage d’information est nécessaire. / With the current increase in antimicrobial resistance and consumers’ concern, new alternatives are needed to control necrotic enteritis (NE), a disease that causes billions of dollars in economic losses to the poultry industry worldwide. Bacteriocins are antimicrobial peptides produced by bacteria to kill or inhibit the growth of other bacterial competitors. Perfrin is the only reported bacteriocin associated with pathogenic strains of Clostridium perfringens, the causal agent of NE. The aims of this study were to screen for commensal Clostridium perfringens strains with an antimicrobial activity against C. perfringens pathogenic strains and to identify and characterize the produced bacteriocins. Commensal C. perfringens strains were selected from our bacterial collection. Antimicrobial activity of those selected strains was tested against C. perfringens pathogenic strains using the agar spot test method. An active commensal strain showing antimicrobial activity was cultured and its bacteriocins were partially purified using the ammonium sulfate precipitation method and HPLC. After each chromatography, antimicrobial activity of fractions was tested using the method described above to choose fractions containing bacteriocins. Enzyme susceptibility, heat and pH stability and the estimated molecular weight of the bacteriocins were characterized. The studied bacteriocins were sensitive to proteinase K, thermolabile, stable at pH between 4 and 8 and their molecular weight higher than 30 kDa. CP1676 strain genome was sequenced and bioinformatics were performed. We found 28 potential bacteriocin sequences, but 4 of them seemed to be promising. In this study, commensal C. perfringens strains producing bacteriocins active against pathogenic strains have been identified. These bacteriocins could be an interesting alternative to antibiotics for the control of necrotic enteritis but further data is still needed.

Avian pathogenic Escherichia coli and Clostridium perfringens cause economic and welfare concerns to the broiler industry. The recent shift to no antibiotics ever (NAE) production has increased disease incidence. The objectives of this study were to determine the influence of season, age of flock, and sample type on E. coli prevalence and virulence and to identify C. perfringens prevalence and toxinotypes in NAE farms. Results indicated high prevalence of virulent E. coli; prevalence of virulent E. coli decreased from Spring to Summer. Virulent E. coli showed high resistance to antimicrobials. Serogroups O8 and O78 were most prevalent in virulent E. coli. C. perfringens prevalence was very low and all recovered isolates were toxinotype A with variation in netB, cpb2, and tpeL presence. In conclusion, NAE farms should have measures to control E. coli infections, especially in spring season. Further studies are required to confirm the lower prevalence of C. perfringens.

Syftet med denna masterstudie var att jämföra den smärtlindrande effekten av fullständig utrensning och desinfektion av rotkanalsystemet med enbart utrymning av pulpakavum, utan instrumentering av rotkanalerna, som akutbehandling hos tänder med symtomatisk apikal parodontit. Materialet utgjordes av 16 patienter insamlade på Tandvårdshögskolans jourmottagning. Patienter med svullnad och allmänpåverkan exkluderades. Patienterna valdes slumpmässigt ut till att genomgå någon av behandlingarna. Den preoperativa smärtnivån och ev. analgetikaintag registrerades. Tre till fem dagar efter utförd behandling kontaktades patienterna per telefon och fick då gradera sin postoperativa smärtnivå samt analgetika- och/eller antibiotikaintag. Resultatet visade att 100% av patienterna som behandlades med fullständig utrensning erhöll tillfredställande smärtlindring jämfört med 71% av de patienter som behandlades med enbart utrymning av pulpakavum. Skillnaden var inte statistiskt signifikant. 44% av patienterna som behandlades med fullständig utrensning hade tagit smärtstillande postoperativt medan 29% av patienterna i utrymningsgruppen hade gjort det. Skillnaden var inte signifikant. Ingen av patienterna oavsett behandling hade tagit antibiotika efter behandlingen. Ingen skillnad förelåg i andelen patienter som blev tillräckligt smärtlindrade mellan fullständig utrensning och enbart utrymning av pulpakavum hos tänder med symtomatisk apikal parodontit. Det var inte heller någon skillnad i smärtsänkning mellan de två behandlingarna. / The aim was to compare the pain relieving effect of complete chemo mechanical disinfection of the root canal system with removal of necrotic tissue in the pulp chamber without instrumentation of the root canals as emergency treatment in teeth with symptomatic apical periodontitis. The material consisted of 16 patients collected at the emergency clinic at Malmö University. Patients with swelling and/or systemic involvement were excluded. The patients were randomized to either treatment. The preoperative pain level and intake of analgesics was registered. Three till 5 days postoperatively the patients were contacted and asked to grade the current pain level and intake of analgesics and/or antibiotics. The results showed that 100% of the patients treated with complete chemo mechanical disinfection of the root canal system obtained satisfying pain relief compared to 71% for patients treated with removal of necrotic tissue in the pulp chamber. 44% of the patients treated with chemo mechanical disinfection of the root canal system had some kind of analgesics postoperatively compared to 29% for those treated with removal of necrotic tissue in the pulp chamber. No patients in either group reported use of antibiotics postoperatively. The conclusion was that both complete chemo mechanical disinfection and removal of necrotic tissue implied a significant pain relief as emergency treatment in teeth with symptomatic apical periodontitis. There was no difference between the two treatments concerning the number of patients who obtained sufficient pain relief or in pain relieving effect.

Clostridium perfringens causa enterite necrótica aviária devido à produção de toxinas que lesam o intestino. Neste estudo, de 94 amostras nove apresentaram C. perfringens, totalizando 22 isolados. Todos exceto um isolado, possuíram os genes nanI (95%) e/ou nanJ (81%), e 19/22, mostraram atividade neuraminidase em hemácias de frango. A atividade hemaglutinante foi observada em poucos isolados (26%). Todos os isolados foram plc positivos (toxina &alpha;), sendo classificados como tipo A. Sete isolados (31,8%) abrigaram o gene tpeL que codifica a toxina TpeL. Isolados tpeL+ mostraram efeito citotóxico característico da ação desta toxina. Alguns isolados mostraram capacidade de aderir e invadir células Vero. A maioria dos isolados foi resistente à sulfaquinoxalina (100%), cefalexina (95%) e eritromicina (95%) e sensíveis (100%) à cefoxitina, amoxicilina, enrofloxacina, amoxicilina-ácido clavulânico, penicilina-estreptomicina, cloranfenicol e metronidazol. Todos os isolados foram agrupados geneticamente em sete clusters, apresentando se como um grupo heterogêneo. / Clostridium perfringens cause avian necrotic enteritis due to production of toxins that damage the intestine. In this study, nine out of 94 samples had C. perfringens, totaling 22 isolates. All the isolates with exception of one, possessed the genes nanI (95 %) and/or nanJ (81 %), and 19/22 showed neuraminidase activity in chicken erythrocytes. The hemagglutinating activity was observed in a few isolates (26 %). All isolates were plc positive (toxin &alpha;) being classified as type A. Seven isolates (31.8%) harbored tpeL gene encoding the toxin TpeL. TpeL + isolates showed characteristic cytotoxic effect of the action of this toxin. Some isolates showed ability to adhere and invade Hep-2 cells. Most of the isolates were resistant sulphaquinoxaline (100%), cephalexin (95%) and erythromycin (95%) and sensitivity (100%) to cefoxitin, amoxicillin, enrofloxacin, amoxicillin - clavulanic acid, penicillin -streptomycin, chloramphenicol and metronidazole. All isolates were genetically grouped into seven clusters, presenting itself as a heterogeneous group.

The ƒÑ-toxin of Clostridium perfringens is a toxin involved in numerous diseases of humans and agriculturally important animals. One of these diseases is necrotic enteritis (NE), a sporadic enteric disease which affects avian species world-wide. This study involved the inactivation of alpha-toxin (ƒÑ-toxin) for use as a potential vaccine candidate to combat NE in chickens, and other diseases caused by C. perfringens type A. During the course of this research a number of ƒÑ-toxin recombinant proteins were developed through molecular inactivation of the ƒÑ-toxin gene, plc. Proteins plc316 and plc204 were developed by the deletion of the first three and seven ƒÑ-helices of the N-terminal domain respectively. These deletions resulted in proteins which were unstable in solution, constantly aggregated into insoluble masses and elicited lower overall antibody responses when administered to mice. A third protein, plcInv3 was developed from the deletion of part of the catalytic domain of the ƒÑ-toxin. PlcInv3 was highly soluble and upon immunisation of mice elicited a significant antibody response which was also capable of protecting mice against a live challenge of C. perfringens. The fourth and final protein developed was plc104. The smallest of the recombinant ƒÑ-toxin proteins, it consisted entirely of the C-terminal domain of ƒÑ-toxin. Its small size did not affect its ability to induce a strong antibody response when administered to mice, the antibodies of which were also protective during a challenge with C. perfringens. STM1, an attenuated strain of S. Typhimurium was used in the development of a vectored vaccine for the expression and oral delivery of plcInv3 and plc104 within the mouse host. The proteins were expressed within STM1 from expression plasmids containing the in vivo inducible promoters PhtrA and PpagC. A measurable humoral immune response against ƒÑ-toxin was absent following three oral vaccinations with the vectored vaccines, although, cytokine profiling of splenocytes from vaccinated mice revealed an increase in the number of interleukin-4 (IL-4)secreting cells and the lack of interferon-gamma (IFN-ƒ×) secreting cells. This indicated the stimulation of a T-helper type 2 (TH2) immune response which also lead to partial protection against a live C. perfringens challenge. This study demonstrates the feasibility of using STM1 as a carrier for the in vivo expression of the C. perfringens ƒÑ-toxin recombinant proteins plcInv3 and plc104. It is the first study to express C. perfringens antigens within an attenuated strain of S. Typhimurium, STM1.The partial protection of mice immunised with these vaccines indicates there is potential for this vectored vaccine system to be used in the protection of diseases caused by the ƒÑ-toxin of C. perfringens.

No description available.