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

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.

Objetivo: O objetivo do presente estudo foi avaliar morfologicamente os tecidos pulpar e periapical de molares de ratos com a polpa vital e expostos ao meio bucal durante o desenvolvimento radicular, além de detectar a localização do STRO-1 e do BMP4 nos mesmos. Métodos: Foram utilizados 24 ratos machos da linhagem Wistar com quatro semanas de idade. A fim de induzir a necrose, foi realizada a exposição pulpar dos primeiros molares inferiores esquerdos em condições não-assépticas e deixando a polpa exposta ao ambiente oral (grupo teste). Os molares inferiores do lado direito não sofreram intervenção e serviram como grupo controle. A eutanásia dos animais foi realizada nos tempos de 7, 10, 13 e 16 semanas de vida (o equivalente a 3, 6, 9 e 12 semanas após a intervenção). Após, as mandíbulas foram dissecadas e processadas para a análise histológica através de Hematoxilina-Eosina e Imunohistoquímica. Foram comparadas, entre os grupos, em cada um dos períodos, a reação inflamatória periapical, as características do tecido pulpar (células inflamatórias, vascularização, odontoblastos, degeneração, destruição tecidual), fechamento do forame e presença de reabsorções. Resultados: Todos os dentes do grupo teste apresentaram necrose pulpar e a interrupção do desenvolvimento radicular. Enquanto isso, os dentes do grupo controle apresentaram o desenvolvimento normal dos tecidos dentários. Quanto à imunohistoquímica, foi constatada a marcação pelo BMP-4 no tecido pulpar dos dentes do grupo controle. Não houve, no entanto, marcação nos dentes do grupo teste. Para o STRO-1, a marcação foi mais evidente na região dos vasos sanguíneos, tanto no grupo controle, quanto no grupo teste. Para nenhum dos anticorpos, foi constatada marcação na região da papila apical. Conclusão: No presente estudo, a técnica imunohistoquímica não foi suficiente para a avaliação da presença de células-tronco na região da papila de dentes com rizogênese incompleta e necrose pulpar. É possível que a presença de infecção e a reação inflamatória tenham bloqueado a expressão do STRO-1 e do BMP-4. / Objective: the aim of this study was to characterize the features of live and necrotic dental pulps of molar teeth in rats with developing roots, and to assess the expression of STRO-1 and BMP-4. Methods: twenty-four 4-month-old male Wistar rats were used. To induce necrosis, the pulps of the first lower left molars were exposed to the oral environment. The lower right molars served as the control non-operated group. Euthanasia of animals was carried out at 7, 10, 13 and 16 weeks and the jaws were dissected and processed for histological analysis by hematoxylin-eosin and immunohistochemistry. The periapical inflammatory reaction, the characteristics of the pulp tissue (inflammatory cells, vascularity, odontoblasts, degeneration, tissue destruction), closure of the foramen and presence of resorptions were described at each period. Results: all teeth from the test group presented pulp necrosis, occurring discontinuation of root development. Meanwhile, the teeth of the control group showed normal development of dental tissues. BMP-4-positive cells were detected in the pulp of teeth from the control group. However, there was no expression in teeth of the test group. For STRO-1, expression was more evident in the blood vessels, both for the control group and for the test group. Apical papilla evidenced no BMP-4 nor STRO-1 expression. Conclusion: Immunohistochemistry was not sufficient to evaluate the presence of stem cells in the apical papilla region of nonvital immature teeth. It is possible that the inflammatory process has blocked the expression of STRO-1 and BMP-4., Probably, infection has negatively modulated this expression.

Final thesis was worked out on theme Aplication of cryotherapy as a tool for plant viruses eradication from Prunus persica (L.) Batsch regenerants. Experiment was conducted on Faculty of Agronomy of the Mendel University in Brno and in the Crop Research Institute in Prague-Ruzyně. Literary research describes information about peach cultivation in vitro and plant pathogen eradication by cryotherapy. These information has been used in practical part of the final thesis. Plant material of the ´Redhaven´variety of peach was used for virus eradication by cryotherapy. Cryotherapy was applied on shoot tips. In the end of final thesis shoot tips viability and regeneration were evaluated.

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Necrotic enteritis (NE) is a major enteric disease in commercial poultry and manifests itself in clinical and subclinical forms. Despite years of research, NE is still among the leading diseases with the greatest economic impact on poultry production. Subclinical forms lead to poor performance (reduced feed intake, weight gain and eventually higher feed conversion ratio) but usually occurs with low mortality rates. The use of antibiotic growth promoters (AGPs) is proving to be an effective tool in maintaining gut health and modifying gut microbiota, thus improving broiler performance and reducing NE. Removal of AGPs has led to an increase in NE occurrence, particularly the subclinical forms. The lack of alternative strategies to control NE is mainly due to limited insight into the relationship between NE pathogenesis, the host microbiome and its immune responses. Therefore, key to overcoming NE is to define the cellular and molecular mechanisms that are involved in the progression of the disease, especially with regard to mucosal immune responses and gut microbiome. Also, assessing the impact of these changes on gut cell metabolism and function is of great importance. This information would be a valuable guide to prevent the onset or alleviate the negative impact of NE on bird's health and performance. In a series of experiments conducted for this project, the effect of single or multi-strain probiotics as well as multi-component additives were tested during NE challenge in order to define the cellular and molecular mechanisms that are involved in the progression of the disease. Results of these experiments revealed that challenging broilers with a 'naturally occurring' NE led to differential expression of tight junction (TJ) proteins in the jejunum compared to non-challenged birds. Supplementation of certain additives reduced NE lesion scores, improved performance and increased mRNA abundance of claudin-3, a key epithelial TJ protein. Multi-strain probiotics and multi-component additives (including a symbiotic and a product containing probiotics, prebiotics and essential oils) were more effective than single-strain probiotics or prebiotics. The aforementioned additives were also more effective in modulating immune responses to NE, especially by decreasing the mRNA abundance of IFN-γ and IL-10 in the jejunum. Furthermore, supplementation of these additives led to an increase in the expression of nutrient transporters (SGLT-1) and regulators of energy metabolism (PGC-1α, mTOR and AMPK); thus, improving nutrients absorption and metabolism. Microbial profiling using 16S rRNA sequencing showed that supplementation of each specific additive led to a signature-like microbiome in the ileal scrapings of broilers. However, supplementation of multi-component additives (including a symbiotic and a product containing probiotics, prebiotics and essential oils) modified the ileal microbiome in association with lower NE lesion scores, better performance and modulated immune responses. These additives reduced the relative abundance of bacteria such as ASF356, Bacteroides, Clostridium sensu stricto 1, Faecalibaculum, Lachnospiraceae UCG-001, Muribaculum, Oscillibacter, Parabacteroides, Rikenellaceae RC9 gut group, Ruminococcaceae UCG-014, and Ruminiclostridium 9 and increased the relative abundance of Lactobacillus compared to NE challenged birds. Collectively, these data indicate that during a subclinical naturally occurring NE, the use of multi-strain probiotics or multi-component additives, compared to single-strain probiotics or prebiotics, would be a more promising strategy in alleviating the effect of this enteric disease. / Doctor of Philosophy / Necrotic enteritis, an enteric disease, is one of the major diseases that negatively impacts the poultry industry and producers' profitability. The growing ban on the use of antibiotics that were used to prevent this disease has increased the number of necrotic enteritis outbreaks worldwide. Having a better understanding of the cellular and molecular mechanisms that are involved in the onset of this disease is of crucial importance and could lead to finding more effective ways to control this disease without drugs. The gut is the site of digestion and absorption of nutrients so any damage would lead to poor bird performance. In a series of experiments conducted for this project, several combinations of beneficial bacteria and nutrient sources that help bacterial growth in the gut (prebiotics) improved gut health leading to better performance during the grow-out period (days 0-42) when birds reach market age. These supplements protected the gut lining and reduced damages due to necrotic enteritis with less severe lesions. Barrier function of the gut was also improved by supplementing the diet with combination of beneficial bacteria and nutrients that help their growth in the gut. There are special types of proteins (called tight junctions) that seal up the space between intestinal cells (enterocytes) and prevent pathogens in the gut lumen from entering the body, thus preventing inflammation and disease. This helps the body to use the absorbed nutrients for growth rather than spending energy to fight pathogens, which collectively results in better growth performance. Concurrent supplementation of beneficial bacteria plus nutrients that help their growth balanced the immune responses in the gut by increasing the copy number of cytokines. Cytokines are proteins that orchestrate immune responses that the host mounts against pathogens. Certain cytokines regulate such responses by preventing the immune system from overreacting and mounting unnecessary reactions, thus preserving energy and nutrients for growth while reducing inflammation. Nutrient uptake from the gut lumen is facilitated by nutrient transporter proteins that reside on intestinal cells (enterocytes). Birds concurrently supplemented with beneficial bacteria and nutrients that help their growth in the gut increased the abundance of these proteins, resulting in improved nutrient uptake and performance compared to the control birds. Co-supplementation of beneficial bacteria and nutrient sources that help their growth modified the type and number of bacteria that are present in the gut lumen. The modified bacterial community were able to produce metabolites such as butyrate and propionate, which are beneficial for the health and growth of the intestinal cells, thus improving the bird's health and its performance. Overall, compared to beneficial bacteria alone, co-supplementation of beneficial bacteria with the nutrients that help their growth in the gut significantly reduced intestinal lesions and improved performance of broiler chickens during the production period. Moreover, dietary addition of these supplements improved gut barrier function by regulating the gene expression of tight junction proteins and gut mucosal immune responses as well as modifying the bacterial community of the gut. Therefore, such combination supplements hold promise in controlling necrotic enteritis in poultry and sustain good overall performance that translates into higher profitability to producers.

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.