Rozprawy doktorskie na temat „Necrotic enteritis”

Necrotic enteritis in poultry is caused by Clostridium perfringens type A, and is estimated to cost the worldwide poultry industry approximately $2 billion dollars a year, due to increased mortality and decreased feed conversion and weight gain. Very little is known about the pathogenesis of this disease due to the lack of a consistently reproducible experimental model. This dissertation outlines the development of an effective and consistent experimental model for necrotic enteritis in broiler chickens. It was also found that in vivo passage through the chicken's intestinal tract let to increased virulence; we increased the proportion of birds developing disease from 34.6% to 81.4%. Researchers have proposed that alpha toxin (CPA) is believed to be the critical virulence factor of the disease. All type A isolates have the potential to produce CPA, thus we challenged birds with numerous type A isolates that are virulent in other animal hosts. However, we found that they did not produce necrotic enteritis in broiler chickens. In addition, challenge with culture supernatant alone failed to produce gross lesions in the birds, although challenging with washed whole cell cultures did do so. Vaccinating birds with HIS-tagged recombinant CPA provided partial protection against disease; there was a 42.0% decrease in lesion development. The conclusion of this doctoral research is that CPA does have a role in the pathogenesis of necrotic enteritis in broiler chickens, but there are apparently other critical virulence factors involved in the development of disease.

Diet composition and nutrient balance can have a critical impact on intestinal integrity during exposure to enteric pathogens. Researchers have extensively reported benefits on nutrient availability and broiler performance as a consequence of the impact of phytase supplementation. However, the poultry industry has little information on the effects of phytase supplementation in disease settings. The objective of these studies was to evaluate phytase supplementation impact on bird performance, intestinal morphology and pH, nutrient digestibility and bone mineralization during necrotic enteritis (NE). In each experiment, Cobb 500 broilers were obtained from a commercial hatchery and housed in floor pens at the Virginia Tech Turkey Research Center. Birds were placed on used litter from a previous flock that had presented clinical signs of NE. Broilers were fed non-medicated diets formulated to meet NRC (1994) nutrient requirements, except for calcium and phosphorus. In the first experiment, birds began exhibiting clinical signs of NE on d 9, and elevated NE-associated mortality persisted until d 26. Mortality was influenced by the main effects of dietary Ca or phytase. Dietary Ca supplemented at 0.9% or 1000 FTU/kg of phytase increased mortality compared to 0.6% Ca or 0 FTU/kg phytase, respectively, from d 0 to 19. Feed intake (FI) and feed conversion (FC) were affected by Ca x P interaction. From d 0 to 19, birds fed 0.9% Ca and 0.3% available P (avP) had decreased FI and improved FC compared to birds fed 0.9% Ca and 0.45% avP, while FI and FC were similar in birds fed diets with 0.6% Ca, regardless of avP level. Calcium x P x phytase interaction influenced BW or BWG from d 0-12. In general, birds fed 0.9% Ca and 0.45% avP with phytase were heavier compared to birds fed 0.6% Ca, 0.45% avP, and phytase. Calcium at 0.9% increased gizzard (d 19) and jejunum (d 12) pH. Dietary Ca supplemented at 0.9%, avP supplemented at 0.45%, and 1,000 FTU/kg phytase significantly increased tibia ash weight compared to 0.6% Ca, 0.3% avP, and 0 FTU/kg phytase, respectively, on d 12. A 3-way interaction was observed on d 35 for tibia ash percentage; birds fed 0.9% Ca and 0.45% avP had a significant increase in tibia ash percentage, regardless of phytase supplementation. A 3-way interaction was also observed for Ca and P digestibility on d 35. Phytase supplementation significantly increased Ca digestibility regardless of Ca and P levels of the diets. In addition, diets containing 0.6% Ca and 1,000 FTU/Kg of phytase resulted in a significant increase in P digestibility, regardless of P levels. In the second experiment, birds also began exhibiting clinical signs of NE on d 9, and elevated NE-associated mortality persisted until the end of the trial (d 21). Mortality was significantly affected by an interaction between Ca source and Ca levels. Significantly higher mortality was observed when animals were fed 0.9% Ca diets formulated with calcified seaweed from d 0-21 compared to 0.6% Ca diets (regardless of Ca source). From d 0-7, birds fed 0.6% Ca in diets supplemented with phytase had heavier BW than the other treatments regardless of Ca source. From d 0-14 and 0-21, animals fed diets with calcified seaweed had significantly higher FC than animals fed diets with limestone. On d 21, the gizzard of birds fed 0.9% Ca was significantly less acidic than the gizzard of birds fed 0.6% Ca. In conclusion, reducing dietary levels of Ca associated with phytase supplementation improved bird performance and nutrient digestibility. In addition, these experiments indicate that Ca is an important dietary factor in the pathogenesis of NE.
Ph. D.

Necrotic enteritis (NE) is an important enteric disease in poultry production that has re-emerged as a major problem following an EU wide ban on the use of in-feed antimicrobials. Although the primary aetiological agent of disease is Clostridium perfringens type A, a commensal in the gastrointestinal tract (GIT) of chickens, numerous additional influential factors have been reported that can predispose chickens to NE. These precipitating factors mainly include diet, co-infection with other pathogens particularly coccidia, as well as environmental and management factors. Despite being first described almost more than 50 years ago, a reliable, consistently reproducible experimental model for NE induction is still lacking. Here, a series of experiments were conducted to investigate the importance of the various potential predisposing factors, in isolation and in combination, that are believed to play a role in sub-clinical NE development: feed withdrawal, dietary protein sources, co- infection with coccidia, C. perfringens dose and contact with reused litter. In addition, chicken breed sensitivity to NE was assessed, and last, but not least, blood and gut tissue samples were used to identify novel biochemical markers for sub-clinical NE. Chapter 3 reports an experiment showing that feed withdrawal up to 24 hrs in experimentally challenged birds did not result in NE specific lesions. Chapter 4 shows that replacing dietary soyabean meal with potato protein concentrate or canola meal or adding synthetic trypsin inhibitor to the soyabean meal control diet did not induce sub-clinical NE in birds housed on reused litter, a natural source of C. perfringens challenge. Chapter 5 describes that in vitro growth of C. perfringens on in vitro digested grower diets was prolonged following the addition of fishmeal, suggesting that the role of fish meal as a predisposing factor for in vivo sub-clinical NE cannot be excluded. All subsequent diets therefore contained high levels of fish meal. When this was used in combination with high dose of coccidial vaccine, a repeated in-feed challenge for three days at 102 colony forming units (cfu) C. perfringens per g feed did not result in sub-clinical NE, though at 109 cfu/g resulted in 10% of challenged birds (3 out of 30) showing NE-specific lesions (Chapter 6). Further study is needed to determine if the two Ross birds with gross NE lesions compared to the one Hubbard bird (out of 15 birds each) was due to a lower level of NE resistance. 3 The failure to significantly induce sub-clinical NE in the previous experiments suggests that challenging the birds with C. perfringens in the isolated presence of suspected predisposing factors may not provide a suitable experimental model. Indeed, when birds were dosed twice daily with 108 cfu C. perfringens for three days in the presence of high levels of fishmeal, canola meal as main protein source, coccidial and IBD vaccinations, and feed withdrawal prior to challenge, 40.6% of the challenged birds developed lesions of sub-clinical NE without inducing mortality (Chapter 7). This concurred with reduced growth performance relative to the sham-infected control birds, and thus is a successful model for induction of sub-clinical NE. Finally this work has, for the first time provided novel information on potential biomarkers (Chapter 8). Whilst challenge did not impact on the expression of genes previously shown to be differentially expressed upon C. perfringens toxin exposure, the serum ceruloplasmin concentration increased, suggesting that monitoring this acute phase protein may indicate the presence of C. perfringens infection in poultry. However, as such markers generally lack specificity, further research confirming its role in response to sub-clinical NE is needed to provide a fully effective diagnostic and prognostic marker for flock health and welfare, as well as ultimately helping to gain better understanding of the pathophysiology of sub-clinical NE. Improved knowledge of the effect of different dietary components on the growth of C. perfringens may help in the formulation of broiler diets to assist in further reducing the incidence of NE particularly in the absence of antimicrobial growth promoters. It is hoped that host responses in terms of acute phase proteins, and possibley gene expression, will also provide greater insight into the pathogenesis of NE. Provided that the developed experimental sub-clinical NE model is reproducible, this will benefit the understanding of this billion dollar disease and enable further investigation of various chemical and non-chemical interventions to reduce its severity and impact on poultry production.

Previous reports have shown that some bacteria utilize a dissimilatory nitrate reductase enzyme (NR) in anaerobic environments. This enzyme reduces nitrate to nitrite and also has been shown to co-metabolize chlorate to cytotoxic chlorite. A commercially available competitive exclusion (CE) product was evaluated for its nitrate reductase activity and therefore its experimental chlorate product (ECP) sensitivity. Of the 29 constituent bacteria of the CE culture, 11 had slight utilization of NR, 3 had moderate utilization of NR; the remaining were NR negative (with slight and moderate utilization: >0.1 to < 1.0 mM and > 1.0 mM nitrate utilized within 6 h, respectively). In vivo studies utilizing CE and ECP showed significant reductions in Salmonella. Although some of the bacteria were affected by ECP, the combined effect of the CE culture and ECP were effective in reducing Salmonella. Clostridium perfringens (CP) is a pathogen in the commercial poultry industry, which is the etiologic agent of necrotic enteritis (NE). Day-of-hatch broilers were fed a wheat diet and assigned to the following groups: control, commercial coccidia vaccine, commercial bursal disease vaccine, or the combination of the two, and challenged with CP in order to develop a disease model. Broilers in each treatment group had significant increases (P≤ 0.05) in lesion scores, mortality, and CP incidence. As pressure mounts for discontinuing the use of antibiotics in the agriculture industry, it is important to develop new strategies to combat these costly enteric pathogens. In vitro investigations evaluated a mixed gut culture with CP and the ECP at 5 mM or a 10 mM concentrations, over time. By 3 h there was a reduction (P≤ 0.05) in the 5 mM ECP and 10 mM ECP treatment groups. In vivo studies showed significant reductions in the incidence of CP and populations of intrinsic E. coli in all of the chickens provided ECP in the drinking water. Birds administered ECP in the feed showed significant reductions in lesion scores, incidence of CP and also had reduced E. coli log10 values. These results show that an ECP could provide the industry with a new management tool for controlling NE.

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 (10^7 CFU/mL) or high (10^9 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

The aim of this study was to develop a heat-stable and slow-release lysozyme-based product using radiant energy under vacuum (REV) technology by formulating lysozyme in a chitosan matrix so that the product could be used as a feed additive for more effective control of clostridial necrotic enteritis (NE) in broiler chickens. Lysozyme/chitosan was made into pastes with water at 25-45% solids content. These were dried with REV technology (300 watts microwave power under 4 KPa absolute pressure). Lysozyme retained its full activity during this process. A REV-treated sample produced with a paste containing 25% solids had a Tm of 124.6°C, which was substantially higher than that of spray-dried lysozyme (101.7°C). Thermal destruction curves constructed over a range of 100-130°C were used to determine D- and z-values of lysozyme and the REV-treated samples. REV products were consistently more heat-resistant than native lysozyme powder. A small scale feed pelleting study showed significantly better recovery of lysozyme in the chitosan protected REV product(P<0.05). The total porosity and pore size distribution of the REV products were analyzed with an Hg porosimeter. In the REV-treated samples, more than 80% of pore volume represented pore sizes ≥ 300 μm in radius, which were larger than those of native lysozyme powder. Surface structure analysis by scanning electron microscopy suggested the REV samples contained lysozyme evenly distributed inside the chitosan matrix. The REV samples released lysozyme more slowly in aqueous solution than lysozyme powder alone in water. A cage study was conducted to demonstrate the effect of Entegard™REV, the REV-treated lysozyme antimicrobial mixture, on the performance of broiler chickens and necrotic enteritis disease reduction of birds that were challenged with Eimeria maxima and Clostridium perfringens. Entegard™REV included in feed at 200 g/metric ton (MT) was very effective in reducing the negative health effects in the birds following NE challenge, and its ability to control the disease was not statistically different from a commonly used antibiotic growth promotant, bacitracin methylene disalicylate (BMD), at 55 g/MT.

Epsilon toxin (Etx) and necrotic enteritis toxin B (NetB) are two pore-forming toxins produced by C. perfringens. While Etx has been shown to be the key virulence factor for enterotoxemia in goats and sheep, NetB has been associated with the pathogenesis of avian necrotic enteritis (NE), a gastro-intestinal disease causing economic damage to the poultry industry worldwide. The crystal structure of Etx H149A (an Etx variant with 6x reduced toxicity relative to wild type toxin) was solved to 2.4 Å and showed that the H149A mutation in domain III does not affect organization of the receptor binding region in domain I. The Etx H149A structure also revealed a second putative glycan binding site in domain III. In addition, site-directed mutagenesis in domain I of Etx H149A affirmed the important role of tyrosine residues for toxin binding and demonstrated the capability of Etx H149A to be used as a platform for further receptor binding studies in the future. The crystal structure of the pore-form of NetB was solved to 3.9 Å and revealed high similarities to the Staphylococcus aureus α-hemolysin heptameric structure. However, in particular the region thought to interact with the target cell membrane showed some interesting divergence in amino acid composition. Site-directed mutagenesis within this domain significantly affected binding and toxicity of NetB to target cells. Mutagenesis within the β-sandwich domain of NetB revealed important amino acid residues for toxin oligomerisation and pore-formation. In order to test NetB toxoids as candidate vaccines, a NetB genetic toxoid and a formaldehyde NetB toxoid were used to immunise poultry in an in vivo NE disease model. Vaccination with any of the two antigens resulted in the induction of specific antibody responses against NetB and provided significant protection against disease.

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.

O setor avícola apresenta o maior crescimento em volume produzido dentre todos os setores cárneos no Brasil. A grande participação dos produtos avícolas na alimentação humana somada ao risco do desenvolvimento de resistência bacteriana, levaram a União Européia (UE) a abolir utilização de antimicrobianos como aditivos e de forma profilática na ração de animais. A remoção dos aditivos associada ao sistema de criação intensivo acabaram por fazer que doenças, até então consideradas controladas, se tornassem reemergentes. A enterite necrótica aviária (NE) é considerada um exemplo. De forma geral, condições estressoras predispõem ao desenvolvimento de doenças, sendo o calor um dos estressores mais comuns que ocorrem em granjas aviárias. Este estudo enfoca o efeito do estresse térmico por calor (35±1ºC) sobre o desenvolvimento da NE em frangos de corte. Para isso, 60 frangos de corte machos foram divididos em 6 grupos experimentais: 1 Grupo Controle; 2 Grupo Controle Estressado (C/HS35); 3 Grupo Tioglicolato (T); 4 Grupo Tioglicolato Estressado (T/HS35); 5 Grupo Infectado (I); 6 Grupo Infectado Estressado (I/HS35). A infecção experimental com Clostridium perfringens foi feita por via oral, com a bacteria em meio de cultura misturada a ração, do 15º ao 21º dia de vida nos grupos I e I/HS35. O estresse por calor (35±1º C) foi realizado do 14º ao 21º dia de vida das aves dos grupos estressados. Durante todo período experimental os animais foram mantidos em isoladores. Em relação aos animais não estressados, os animais submetidos ao estresse por calor apresentaram: 1- menor escore lesional macro e microscópico no intestino delgado; 2- maior concentração de IgA no lavado intestinal do duodeno; 3 - menor concentração de IgA no jejuno; 4 - redução dos níveis séricos de IgA e IgY; 5 - maior concentração sérica de IgM; 6 - diminuição qualitativa evidente dos heterofilos intestinais em relação aos animais infectados e estressados. Portanto, mostrou-se que o estresse por calor apresentou efeito imunomodulador importante, ao reduzir a inflamação intestinal. Este achado associa-se provavelmente à diminuição da imunidade inata por redução da migração de heterófilos para a mucosa intestinal, desta forma prevenindo a manifestação de um um quadro clínico mais grave de NE, fato associado à diminuição das lesôes desencadeadas pelo processo inflamatório heterofílico.
The poultry sector presented the highest growth in the volume of production among all meat sectors in Brazil. The great participation of poultry products on human diet together with the risk of food and environmental contamination by resistant bacteria led the European Union (EU) countries to abolish the use of antibiotics as feed additives in animal production. This fact associated with the intensive farming system are being reported as responsible for the re-emergence of some already controlled diseases. The avian necrotic enteritis (NE) exemplify such an effect. Generally, stressful conditions are predisponent factors for disease development; heat stress is one of the most common stressor in poultry farms. This study focuses on the effects of heat stress (35 ± 1 º C) on the development of NE in broilers. For this purpose, 60 male broilers were divided into 6 groups: 1 - control group, 2 - stressed control group (C/HS35) 3 - thioglycolate group (T) 4 - thioglycolate stressed group (T/HS35); 5 - infected group (I) 6 - infected stressed group (I/HS35). Experimental infection with Clostridium perfringens, grown in thioglycollate broth medium, was given through the feed to the birds of groups I and I/HS35 from the 15th to 21st days of life. The heat stress (35 ± 1 °C) was induced continuously from the 14th to the 21st day of life in birds of the stressed groups. Throughout the experimental period the animals were kept in isolators. Compared to non-stressed animals, broilers subjected to heat stress showed: lower gross and microscopic score lesions in the small intestine; increased concentrations of IgA in duodenal lavage and decreased IgA concentrations in the jejunum; smaller concentrations of serum IgA and IgY; increased concentration of serum IgM; reduction in gut number of heterophils in the thioglycolate treated and in the infected groups. Therefore, this experimental model showed that heat stress presented a significant immunomodulatory role on the induced NE, most probably because it reduced intestinal inflammation via decrease in heterophils migration to the intestinal mucosa, which in turn might had reduced tissue damage during infamation, hence preventing the development of a more severe form of NE.

A enterite necrótica causada por Clostridium perfringens é, comprovadamente, um grande problema para frangos de corte, seja sob a forma clínica ou subclínica , com elevados prejuízos produtivos. Em perus, não está claramente identificada a influência deste patógeno sobre os resultados produtivos, e provavelmente por razões econômicas, o volume de pesquisa nesta área é limitado. Em dois experimentos consecutivos, conduzidos em granjas experimentais diferentes, e sobre cama nova e reutilizada, perus de 14 dias, sem medicação com anticoccidianos, foram inoculados, a partir de uma amostra de campo patogênica, com uma superdosagem de aproximadamente 1 x 1010 UFC/ml de Clostridium perfringens, tipo A; bactéria anaeróbica Gram positiva, que compõe a microflora intestinal dos perus. Lesões necróticas severas, com grande destruição da mucosa intestinal, foram observadas após o 4º dia de inoculação. Nos dois experimentos, a resposta dos perus à infecção foi diferente da descrita em frangos de corte. A mortalidade foi muito baixa ou nula e a recuperação das aves, independente da medicação utilizada, foi rápida com pouco ou nenhum comprometimento zootécnico. Escores das lesões histológicas foram desenvolvidos, para tentar correlacionar as lesões macroscópicas com as lesões microscópicas, mas a correlação foi baixa. Frente ao alto desafio observado, não foi possível avaliar adequadamente a ação do antimicrobiano utilizado. As respostas frente aos desafios de Clostridium perfringens nos perus, aparentam ser diferentes em relação às observadas em frangos de corte, sendo necessária cautela ao extrapolar padrões de uma espécie para outra. O uso de critérios subjetivos nas avaliações podem comprometer a tomada de decisão em relação aos tratamentos e as respostas esperadas.
Necrotic enteritis caused by Clostridium perfringens represents a major challenge in broilers, causing clinical or sub clinical diseases, and results in important economic losses for the poultry industry. In turkeys, however, the importance of this pathogen is not clearly defined, and there are few studies assessing its effects on the performance of turkeys, most probably for economic reasons. In two consecutive trials, conducted in two experimental farms, 14 day old turkey poults were reared on new and reused wood shavings litter, without any anticoccidials in the feed, and were inoculated with an overdose of approximately 1x1010 CFU/ml of a pathogenic field sample of Clostridium perfringens type A. This Gram positive anaerobic bacterium is a normal inhabitant of the gut micro flora in the enteric tract of turkeys. Severe necrotic lesions, with major damage to the intestinal mucosa were observed on Day 4 after inoculation. In both trials, the response to the turkeys to the clostridial infection was clearly different from what is described in the literature for broilers. The mortality rate was very low or inexistent, and the birds in all treatments groups recovered very fast, with little or no impact on performance results. An attempt was made to establish a pattern to correlate histological lesion scores with macroscopic and microscopic lesions, but the resulting correlation was very low. Considering the high level of challenge induced in both trials, it was not possible to evaluate in activity of the antibiotic in proper terms. The response of turkeys to a high level of challenge with Clostridium perfringens seems to be different when compared to what is observed in broilers under similar experimental conditions, and caution should be applied when using the same diagnostic methods for different species. The use of subjective criteria to assess and describe the lesions can lead to erroneous treatments decisions and misinterpretation of the responses to treatment.

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.

Made available in DSpace on 2014-08-20T13:32:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-10-02
Avian Necrotic Enteritis (NE) is an acute enterotoxaemia caused by Clostridium perfringens A and C. The control of the disease is based on antibiotics added to animal feed. The ban of this practice by consumer markets, considered the biggest challenge to industrial aviculture, demanded the adoption of other alternatives for its control, among others, immunization with recombinant vaccines. The aim of this work was to produce and evaluate C. perfringens recombinant α toxin (rAT) vaccines adjuvanted with either Al(OH)3 (rAT+Al(OH)3 or recombinant B subunit of the heat-labile enterotoxin of Escherichia coli (rLTB) (rAT+rLTB), and a chimeric protein containing the α toxin fused to rLTB (rLTB-AT). The rAT+Al(OH)3 was innocuous and protected mice against a challenge with native α toxin (sT), and it was immunogenic and did not affect productivity parameters in broilers. The rAT+rLTB showed a dose-protection relationship in mice, while rLTB-AT did not protect mice against sT challenge. The rAT could be an alternative for controlling NE.
A Enterite Necrótica Aviar (ENA) é uma enterotoxemia aguda, causada pelos Clostridium perfringens A e C, cujo controle baseia-se na adição de antibióticos na ração. A restrição dessa prática pelo mercado consumidor, que tornou seu controle o maior desafio para o setor avícola, exigiu a adoção de novas estratégias para o controle, entre elas a imunização. Vacinas recombinantes vêm despertando grande interesse entre pesquisadores e empresas do setor. O objetivo deste trabalho foi elaborar vacinas de toxina α recombinante de C. perfringens (rAT) utilizando como adjuvantes Al(OH)3 (rAT+Al(OH)3) e subunidade B recombinante da enterotoxina termolábil de Escherichia coli (rLTB) (rAT+rLTB), e construir e avaliar uma proteína quimérica contendo rAT fusionada a rLTB (rLTB-AT). A rAT+Al(OH)3 foi inócua e protetora contra agressão de toxina α nativa (sT) em camundongos, e imunogênica em frangos de corte, sem afetar a produtividade. A rAT+rLTB demonstrou relação dose-proteção em camundongos, entanto a rLTB-AT não protegeu camundongos contra agressão de sT. A rAT demonstrou ser uma alternativa para controlar a ENA. Palavras-chave: Enterite Necrótica Aviar, Clostridium perfringens A, toxina α recombinante, vacinas.

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 α), 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 α) 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.

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

Necrotic enteritis (NE) is an economically important, but poorly understood, disease of poultry, typically caused by Clostridium perfringens Type A strains that carry the NetB toxin gene. The objective of the current research was to identify additional genes associated with NE-causing C. perfringens strains, and thus putatively involved in virulence. To identify novel NE-associated genes, the draft genome sequences of seven C. perfringens NE isolates and one isolate from a healthy chicken were compared against nine non-poultry genomes, and three highly-conserved NE-associated loci (NELoc-1 – 3) were identified. The largest locus (NELoc-1) encoded 37 putative proteins, including NetB, an internalin-like protein, a ricin-domain protein, two leukocidins, several cell-surface proteins and a cyclic-di-guanidine monophosphate (c-di-GMP) signaling system. NELoc-1 and -3 were both localized to separate plasmids that are both predicted to undergo conjugative transfer. These findings suggest that NE pathogenesis involves multiple virulence factors that are encoded on discrete pathogenicity loci, some of which are plasmid-borne. To further elucidate the genetic basis of NE pathogenicity, a microarray was developed based on two of the sequenced NE bird isolates, and used to assess the gene content of 54 isolates from chickens with and without NE. Variable genomic regions associated with netB-positive isolates were identified, including several chromosomal fitness-related loci, such as a carbohydrate ABC transporter, ferric-iron siderophore uptake system, and adhesion locus. Additional loci were related to plasmid maintenance. This study suggests that chromosomal background confers a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism and plasmid maintenance Finally, the relationship between netB presence, NetB production and host NE status was examined to assess the hypothesis that netB-positive isolates from healthy birds frequently do not express NetB toxin. The expression of NetB toxin was determined in 57 poultry isolates, demonstrating that NetB expression is closely correlated with the presence of netB, and independent of host disease status. In conclusion, these studies have identified a number of C. perfringens genes predicted to play a role in NE pathogenesis, and suggest that NE is a complex, multifactorial disease involving both host and plasmid-encoded virulence factors.

碩士
國立宜蘭大學
生物技術與動物科學系動物科學碩士班
106
Abstract This study investigated the effectiveness of antibacterial substances produced by solid fermentation of Bacillus licheniformis against Brachyspira hyodysenteriae and Clostridium perfringens. Three experiments were designed as follows: Test 1. To optimize the surfactin production by fermentation of Bacillus licheniformis. Molasses were used as carbon sources for reduction of fermentation costs. The results showed that the 5 grams of molasses can increase the production of surface by Bacillus licheniformis. Increasing the nitrogen source from the 25 grams to 50 grams also increased the production of surfactin. Test 2, To study the effect of surface elements on Helicobacter erythropolis and Clostridium perfringens. The test use the minimum inhibitory concentration, inhibition zone, conjugated focus microscope and electron microscope to observe the effect of surface. The change in the surface tension of the water can be used as a basis for inferring the concentration of surface elements. A 10 ppm surface element can inhibit the Helicobacter pylori, and the survival of 500 ppm Helicobacter pallidum is reduced to 23%. For Clostridium perfringens, 25 ppm has an inhibitory effect, and 500 ppm can kill 63% of Clostridium perfringens. The use of MIC in the zone of inhibition results in 50 ppm of the Helicobacter pallidum with a 0.3 mm zone of inhibition, 500 ppm with a 1.4 mm zone of inhibition; Clostridium perfringens has a zone of 0.3 mm at 7.8 ppm, 250 ppm has a 0.8 mm zone of inhibition. Confocal microscopy observation, using SYBR green (live cells), PI (dead cells) and Hoechest (background values) were stained separately, and it was observed that the erythropoietin had a 36% killing effect at 50 ppm for 30 minutes. Increased to 250 ppm up to 83%, up to 46% and 94% in one hour treatment; in addition, Clostridium perfringens has a 41% killing effect at 25 ppm and 48 ppm at 250 ppm. The treatment time was extended to one hour and increased to 46% and 84%, respectively. Finally, the scanning electron microscope (SEM) was used to observe the erythropoiesis. The results showed that the surface of the Helicobacter pallidum was damaged at 10 ppm. Under the treatment of 250 ppm, the cells could be completely broken, forming a broken and broken type. Maintaining its original spiral shape, this result is consistent in the half-hour and one-hour treatment groups, and it can be known that surface substances have the effect of inhibiting or killing both pathogenic bacteria. Test 3: A white broiler animal test was carried out using a fermentation product of Bacillus, and the effect on the growth performance of the chicken and the necrotic enteritis caused by Clostridium perfringens was tested. 1% and 2% fermented materials were added to the diet, and the number of spores of B. licheniformis was 1.2 × 107 CFU/g and 2.4 × 107 CFU/g. The test treatment group was the control group (no addition) and the positive control group (Add enromycin 0.5 kg / ton, 500 ppm), treatment group A (fermentation 1 kg / ton) and treatment group B (fermentation 2 kg / ton), each group of three replicates, each cage five total 60 broilers, the test schedule was 35 days, and 1 mL of Clostridium perfringens liquid was administered orally on the 17th, 18th and 19th days of feeding, and the dose was 1 x 108 CFU/mL. The results showed that the growth performance, intestinal flora and inflammatory index of the feed-added Bacillus licheniformis fermented group were better than the control group.

Necrotic enteritis (NE) is an economically important enteric disease of broiler chicken primarily caused by a-toxin (Cpa) secreted by C. perfringens type A. Mice immunized with recombinant C-terminal domain of Cpa (CpaCD) had transient and fewer localized lesions upon challenge with C. perfringens type A. These results demonstrate the usefulness of CpaCD as an immunogen for vaccine development against NE for chickens. Chicken CD40 (chCD40) is mainly expressed on the surface of chicken antigen-presenting cells (APCs), and the interaction of chCD40 and chCD40L (natural ligand for chCD40) provides crucial activation signals for chicken B-cells. A hypothesis was proposed that in ovo vaccination with an adenovirus-vectored CpaCD vaccine capable of targeting immunogen to APCs through the CD40 pathway will improve protection against NE in chickens. One agonistic monoclonal anti-chCD40 antibody (designated 2C5) was produced and characterized. 2C5 not only detected expression of chCD40 on chicken APCs, but also induced NO synthesis in chicken HD11 macrophages and enhanced proliferation of serum-starved chicken DT40 B-cells. This demonstrated substantial functional equivalence of 2C5 with chCD40L. The potential of 2C5 as an immunological adjuvant was further assessed by targeting a hapten to chicken APCs in hopes of enhancing an effective IgG response. Seven-week old chickens were immunized subcutaneously once with a complex consisting of 2C5 and peptide, and relative quantification of the peptide-specific IgG response showed that this complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. An agonistic anti-chCD40 single-chain variable fragment (designated DAG1) was combined with an adenoviral delivery system to create a vaccine, Ad-(DAG1-Cp aCD-FLAG), for in ovo administration. The efficacy of in ovo vaccination of broilers with Ad-(DAG1-Cp aCD-FLAG) in controlling NE was evaluated by C. perfringens type A challenge at 18 days post-hatch. Neither statistically significant IgA / IgG response nor protection against C. perfringens type A challenge was found in the vaccinated birds. These preliminary data suggest that a super-optimal dose of Ad-(DAG1-Cp aCD-FLAG) may be the main issue, because Cpa-specific B-cells may undergo apoptosis through the CD40 pathway.

Incorporation of full-fat flaxseed, and to a lesser extent, canola seed in diets to produce n-3-enriched products has attracted interest in the poultry industry. However, high amounts of nonstarch polysaccharides (NSP) in oilseeds compromise their nutritive value. The objectives of the current research were to develop enzyme supplements effective in cell wall depolymerization and viscosity reduction, particularly in flaxseed; to evaluate the effects of enzyme addition and feed processing on oil utilization and egg n-3 fatty acid deposition in broiler chickens and laying hens fed oilseed-containing diets; to characterize the NSP hydrolysis products and to investigate the effects of diet type and enzyme addition on growth performance and the incidence of necrotic enteritis (NE) in broiler chickens challenged with Clostridium perfringens. Results showed that diets containing high levels of flaxseed reduced egg production and shell quality in laying hens, and impaired final body weight and feed conversion ratio (FCR) in broiler chickens. Reducing flaxseed particle size via grinding did not improve the growth performance of broiler chickens, whereas diet pelleting showed more pronounced and beneficial effects in improving the nutritive value of flaxseed, particularly when intact seeds were used. Multicarbohydrase supplementation resulted in a significant depolymerization of cell wall polysaccharides in soybean, canola and flaxseed meals, which was followed by the production of water-soluble NSP hydrolysis products, and the reduction of flax mucilage viscosity in vitro was also evident. Enzyme addition to flaxseed-containing diets improved FCR of broiler chickens and egg production performance of laying hens, and facilitated egg n-3 fatty acid deposition. The C. perfringens challenge caused intestinal NE lesions and increased the mortality of broiler chickens with the highest NE mortality and intestinal C. perfringens counts observed in those fed flaxseed-containing diets. Enzyme supplementation to diets containing high levels of water-soluble NSP (wheat/barley- or wheat/barley/flaxseed-based) facilitated post-disease compensatory growth in pathogen challenged birds. This was accompanied by a numerical reduction of intestinal C. perfringens by 1.4 log10 cfu/g in birds fed the flaxseed-containing diets. Such findings indicated that enzyme addition may be used as a nutritional strategy to reduce the risk of NE development in broiler chickens.

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.

L’entérite nécrotique aviaire causée par Clostridium perfringens est une maladie économiquement dévastatrice et celle-ci est en émergence dans les troupeaux de poulets de chair éliminant l’usage des antibiotiques. À ce jour, aucune alternative en élevage ne permet de prévenir efficacement la maladie et un contrôle par une stratégie vaccinale serait des plus prisé. Une approche par génomique comparative jumelée à la vaccinologie réverse soustractive et comparative identifiant des protéines bactériennes de surface immunogènes figure parmi les approches méthodologiques des plus prometteuses pour le développement rapide d’un vaccin efficace. Une étude génomique comparative réalisée sur 48 souches de C. perfringens provenant de poulets de chair en santé ou affectés par l’entérite nécrotique a permis d’établir que les génomes analysés étaient composés de 155 700 protéines distinctes, où 13% étaient extracellulaires, 65% cytoplasmiques et 22% membranaires. L’évaluation du pouvoir immunogène de ces protéines à l’aide de l’outil de prédiction VaxiJen v.2.0 a permis d’identifier 4 catégories de scores pour les protéines identifiées, allant de 0,5 (seuil minimal recommandé) à 1,5. Les protéines présentant les scores les plus élevés ont été majoritairement associées à des localisations extracellulaires. La combinaison du score d’immunogénicité et de la localisation cellulaire des protéines analysées a mené à la sélection de 12 protéines candidates vaccinales, la plupart d’entre elles étant de fonction hypothétique. Une description plus approfondie de ces protéines permettra de mieux définir leur fonction, d’évaluer leur potentiel antigénique réel en caractérisant leur interaction avec le système immunitaire de la volaille et ultimement, d’évaluer leur rôle probable dans la pathogénie de l’entérite nécrotique.
Avian necrotic enteritis caused by Clostridium perfringens is a disease with a major economical impact, generating losses up to 6 billion dollars for the poultry industry worldwide. This disease appears in broiler chicken flocks that no longer employ the use of antibiotics. To date, no alternative method allows for the efficient prevention of necrotic enteritis (NE) and a control by a vaccinal strategy would be mostly prized. A comparative genomics approach as well as comparative and subtractive reverse vaccinology identifying immunogenic bacterial surface proteins is one of the most promising methodologies for the rapid development of an efficient vaccine. A comparative genomic study was performed on 48 C. perfringens strains isolated from healthy broiler chickens and from broilers affected by necrotic enteritis. From this study, it was established that the genomes analyzed were composed of 155 700 distinct proteins where 13% were predicted to have an extracellular expression, 65% at the cytoplasma level and 22% within the plasma membrane. The evaluation of the immunogenic potential of these proteins was established with the prediction software VaxiJen v2.0 for which a 0.5 threshold score allowed for the identification of four score categories among the identified proteins, from 0.5 to 1.5. For the most part, proteins with the highest scores were associated with an extracellular localisation. The combination of the immunogenicity score and localisation of the analysed proteins led to the selection of 12 vaccinal candidate proteins that were mostly identified as hypothetical. A more in-depth description of these proteins would allow the assessment of their function, the evaluation of their true immunogenic potential by characterizing their interaction with the avian immune system and ultimately, evaluate their probable role in the pathogenesis of necrotic enteritis.