Four experiments evaluated the impact of probiotic administration on the immune response of broilers vaccinated with a live coccidiosis vaccine. Experiment one showed that probiotic administration increased heterophil and monocyte oxidative burst, and lymphocyte proliferation at multiple time points. In experiment two, probiotic + vaccine increased heterophil and monocyte oxidative burst on d 15 when compared with the negative controls. Overall, vaccine administration alone showed the highest response when compared to all other treatments. In the second trial, all birds were exposed to Eimeria oocysts in the litter and oral gavaged. The results showed that probiotic + vaccine resulted in greater heterophil and monocyte oxidative burst levels on d 14 and 28 when compared to the negative controls. Increases in lymphocyte proliferation were also seen in the probiotic + vaccine and probiotic alone broilers on d 14 among other treatments. In experiment three, heterophil oxidative burst was increased (p <= 0.05) in the vaccine alone group, vaccine with probiotic group, and the ionophore with probiotic group, when compared to the negative control. Monocyte oxidative burst was increased (p <= 0.05) in the vaccine with probiotic group on d 36 and 43, compared to the negative control. Lymphocyte proliferation was greater (p <= 0.05) on d 22 and 36 in the ionophore with probiotic group, when compared to the negative control. Experiment four showed that liver AVBD 2 gene expression elevated (p <= 0.05) in the probiotic + vaccine group relative to the probiotic alone group. Ileum AVBD 2 gene expression was not affected among any of the treatments was evaluated. Liver AVBD 9 was demonstrated to have higher (p <= 0.05) gene expression in the vaccine group when compared to controls. When AVBD 9 gene expression was evaluated in the ileum, a decrease (p <= 0.05) was observed in all treatments compared to the control group. These data suggest that simultaneous administration of probiotics during coccidiosis vaccination or ionophore treatment has the ability to modulate the immune response at varying time points.

Higher eukaryotes produce a vast range of antimicrobial peptides (AMPs) that play important roles in their defence against microbial infection. Beta defensins are small (3-5 kDa), cationic peptides that display broad, potent antimicrobial activity against a range of microbes and also act as chemoattractants of important immunomodulatory cells. To generate highly pure peptides for structural and functional studies, we developed a method to prepare recombinant human beta defensin-2 (HBD2). The HBD2 gene was synthesised by recursive PCR with codons optimised for expression in Escherichia coli. HBD2 was expressed as an insoluble fusion to a His-tagged ketosteroid isomerase. After cleavage from the fusion with cyanogen bromide, 1H NMR spectroscopy and mass spectrometry confirmed that the oxidised HBD2 was folded and possessed the correct b-defensin disulfide bond topology. The recombinant HBD2 was active against E. coli, P. aeruginosa, S. aureus and C. albicans and was also a chemoattractant against HEK293 cells expressing the chemokine receptor CCR6. 15N-labelled HBD2 was also prepared and was highly suitable for future structural studies. Since defensins are thought to interact with bacterial membranes we also tested the recombinant HBD2 in biophysical studies (surface plasmon resonance, SPR, Biacore). We observed different binding to artificial model membranes containing either E. coli Kdo2-lipid A or phospholipids. Bacterial resistance to AMPs has been linked to the covalent modification of the outer membrane lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N). This neutralises the charge of the LPS, thereby decreasing the electrostatic attraction of cationic peptides to the bacterial membrane. The pathogen Burkholderia cenocepacia displays extremely high resistance to AMPs and other antibiotics and the Ara4N pathway appears to be essential. To explore this further we expressed recombinant forms of two enzymes (ArnB and ArnG) from the B. cenocepacia Ara4N pathway. Purified ArnB is a pyridoxal 5’-phosphate (PLP)-dependent transaminase and we tested its ability to bind amino acid substrates. We investigated the binding of inhibitors L- and D-cycloserine to ArnB and tested their antibiotic activity against Burkholderia strains. We also studied the B. cenocepacia ArnG – a proposed membrane protein undecaprenyl-L-Ara4N flippase – and showed that the protein behaved as a dimer by non-denaturing gel analysis. The B. cenocepacia ArnG failed to complement E. coli knock-out strains encoding the equivalent flippase proteins ArnE and ArnF, suggesting that ArnG is a Burkholderia-specific protein.

Although inflammation is a crucial mechanism in response to injury and pathogen clearance, inappropriate or excessive induction of the inflammatory response in pregnancy can cause initiation of the labour cascade and subsequent preterm delivery. Host Defence Peptides (HDPs) have important anti-microbial properties but are also implicated as multifunctional modulators of immunity and infection. They are predominantly secreted by mucosal epithelial cells and released by leukocytes. The specific HDPs that are the focus of this thesis are Human beta-defensin 3 (hBD3) and Human Cathelicidin (hCAP-18/LL-37). The immunomodulatory effect of HDPs in reproductive tissues in response to infection/inflammation has not been well studied. In a pregnant state, the hypothesis of this thesis is that HDPs have a dual role in preventing ascending infection, but also preventing an exacerbated inflammatory response that can cause preterm birth by initiation of the labour cascade. To explore this I determine whether bacterial stimuli can regulate HDPs expression in pregnancy tissues. I also explore what interactions HDPs have on the production/induction of important cytokines that are vital to the inflammatory response. With the aid of HDP knockout mice, the role of these peptides in infection/inflammation and continuation of pregnancy is investigated in a mouse-model of induced preterm-labour. To understand how ascending infection might be controlled by HDPs in pregnancy, I explore how HDPs regulate commensal and pathogenic bacteria. This is achieved by interrogating the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene and next generation sequencing. Results Placental explants respond to Lipopolysaccharide (LPS) challenge by increasing production of pro-inflammatory cytokines. LL-37 but not hBD3 peptide was able to modulate this inflammation by inhibiting the release of these pro-inflammatory cytokines. To establish whether HDPs are critical in the continuation of pregnancy I use a LPS induced mouse–model of preterm labour in animals lacking the genes for the HDPs, Defb14 (Defb14-/-), or Camp (Camp-/-). Intrauterine injection of LPS induced preterm labour in wildtype mice. However, the Defb14-/- and Camp-/- mice do not have an increased rate of preterm labour. Key inflammatory mediators are increased in response to LPS-induced PTL. Camp-/- animals have a similar inflammatory response to wildtype mice when given LPS during pregnancy. To understand how ascending infection might be controlled by HDPs, I interrogated the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene. I established a workflow for 16S rRNA gene sequencing on next-generation sequencing platforms and a bioinformatic pipeline for data analysis. Using this approach I was able to show the mucosal microbiome of Camp-/- animals were significantly different to that of wildtype controls, showing increased diversity in the microbes present. In murine pregnancy, there were very little global cumulative or progressive shifts in bacteria, with the exception of Candidatus arthromitus, which significantly increases with gestation compared to non-pregnancy This thesis has demonstrated that Host Defence Peptides are expressed in pregnancy tissues and have anti-inflammatory properties in response to bacterial stimuli. It is not clear whether the HDPs, hBD3 and LL-37 are fundamental to the immune defence in pregnancy by preventing excessive inflammation, Although, I have shown LL-37 may have a role in modulation of the maternal microbiota.

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Differences in the expression of antimicrobial peptides in vivo have been proposed as underlying factors influencing susceptibility to infection. In this context, the role of avian b-defensins in inhibiting avian influenza infections is a study object in an ongoing collaboration with the Zoonotic Ecology and Epidemiology group at Lnu. In this report, an attempt to synthesize two variants of the peptide Anas Platyrhynchos AvBD-8, using Fmoc-based SPPS, is described. The length of AvBD-8 (43 aa) necessitated peptide synthesis in two segments to subsequently be ligated using native chemical ligation. The first component of a 19 aa segment was thus a Dbz-linker, which would allow to ligate this end with a second segment (24 aa). Halfway through the synthesis of this larger segment the batch was split into two pots, allowing the synthesis of two segments differing by one single amino acid (R for W). The composition of these segments were: Dbz-HDTSCTGGAQKCQVANNPA (Dbz-segment), SVVTRCCPIGQKCWGFARTNPPPC(boc) (W-segment), and  SVVTRCCPIGQKCRGFARTNPPPC(boc) (R-segment). Crude product yields were 284,5 mg; 67,6% (Dbz-segment), 137,6 mg; 52,3% (W-segment), and 166,3 mg; 64,2%. Preliminary mass spectrometric analysis on the crude products did not indicate the presence of the desired segments in major mass peaks. Further product purification is necessary in order to allow definite conclusions, but it appears as if the synthesis has not worked. Possible explanations are either impure or degraded reactant(-s), folding or shielding effects of the growing peptide chain at some point inhibiting synthesis, or experimental errors during one or more of the many steps involved in the synthesis.

No description available.

No description available.

Ferric ABC Transporters. Pathogenic bacteria have evolved specialised iron acquisition systems that allow them to effectively colonise a host. One of these systems is the ferric binding protein (Fbp) complex that is a member of the ATP-Binding Cassette (ABC) superfamily of small molecule transporters. The Fbp complex is made up of three-components (FbpABC) that transports ferric iron from the periplasm to the cytoplasm of many Gram negative bacteria. FbpA binds iron in the periplasm and transports it to the FbpB transporter complex that permeates the cytoplasmic membrane. Here the iron is actively transported by FbpB through the membrane that is powered by ATP hydrolysis catalysed by FbpC, the cytoplasmic ATPase. Burkholderia cenocepacia is an opportunist pathogen that colonises the lungs of cystic fibrosis patients and is particularly resistant to antibiotic treatment. In this study the iron uptake system of B. cenocepacia strain J2315 is investigated. A putative FbpA from B. cenocepacia J2315 was expressed in the periplasm of Escherichia coli cells and the recombinant FbpA B. cenocepacia protein purified. The structural and electrochemical properties of native FbpA B. cenocepacia were investigated using UV Visible spectroscopy, spectro-electrochemistry, mass spectrometry and crystallographic techniques. It appears that FbpA B. cenocepacia is a novel member of the FbpA superfamily that selectively utilises citrate as an exogenous anion in ferric iron co-ordination. This is the first instance that a recombinant ferric binding protein has been documented as preferentially utilising citrate in this manner. The putative ATPase from B. cenocepacia (FbpC B. cenocepacia) was also expressed in E. coli but it was found to be insoluble. A number of expression systems were tested but none were found to be successful in generating sufficient quantities of FbpC B. cenocepacia for structural studies. Human β-defensin 2. Despite daily contact with a range of microorganisms, mammals do not regularly succumb to pathogenic invasion. One reason is the presence of an important defence mechanism uses a reservoir of antimicrobial peptides (AMPs) that are expressed in eukaryotes as a means of innate immunity. The AMP superfamily is composed of over 900 members, displays broad structural and sequence diversity and is active against a wide range of bacteria, fungi and viruses. β-defensins are small (3-5 kDa), cationic peptides that display antimicrobial activity against a range of microbes and have also been shown to act as chemo-attractants (chemokines) within the adaptive immune system. In this study we obtained milligram amounts of pure human β-defensin 2 (HBD2) for functional studies by the development of a method for the rapid expression and purification of the recombinant peptide. A clone encoding a thioredoxin-HBD2 fusion protein was designed for the expression of soluble peptide in E. coli cells that was purified by simple affinity chromatography. The HBD2 peptide was cleaved from the fusion by an efficient protease step and further purified to yield pure HBD2. This recombinant HBD2 defensin was shown to be active against a Mycobacterium tuberculosis mutant strain.

Antimicrobial peptides are an ancient form of innate defense and is present in all ways of life. In humans they are present as cathelicidins and defensins. Both are important for the immune system and they exhibit activity against viruses, bacteria and fungi. Defensins exhibit less cytotoxicity and are better characterized and are thus more easily developed as therapeutic tools. Defensins are apt at doing a multitude of things, from inhibiting Herpes simplex virus replication and preventing anthrax’ lethality to helping with wound closure and acting as biomarkers for a variety of ailments. Defensins have consistently shown good results in a laboratory setting but have less than exemplary in vivo results. Defensins’ multifunctionality as well as the complex environment in living organisms makes characterizing why defensins are not performing as well in vivo difficult. They can also exhibit negative side-effects such as increasing the infectivity of the HIV and inhibiting anti-viral molecules of the innate immune system. Nevertheless, they exhibit big potential as complementary drugs, adjuvants, biomarkers, wound treatment and much more. Further characterization and development is absolutely necessary in these times of increasing antibiotic resistance.

A citricultura brasileira é a maior produtora e exportadora de citros e tem sido afetada por doenças que causam sérios prejuízos a produção e a qualidade dos frutos. No entanto, a cultura apresenta grandes problemas, entre eles, os fatores fitossanitários, que vem dizimando milhares de plantas e afetando a produtividade e a competitividade do setor. Atualmente, o huanglongbing (HLB), associado às bactérias de floema Candidatus Liberibacter spp., é considerado uma das mais destrutivas doenças de citros. A inexistência de cultivares de laranja doce resistentes ao HLB torna a transformação genética de citros uma ferramenta importante no controle desta doença. Para se defender do ataque de pragas e patógenos as plantas desenvolveram, durante o processo evolutivo, uma série de mecanismos de defesa, no qual pode-se incluir a produção de peptídeos com atividade antimicrobiana. As defensinas vegetais são peptídeos pequenos relacionadas à patogênese (PR), que possuem atividade antimicrobiana associada aos mecanismos de defesa das plantas. Assim, o objetivo deste trabalho foi a obtenção de plantas transgênicas de laranja doce (Citrus sinensis L.) cvs. \'Hamlin\', \'Natal\', \'Valência\' e \'Pera\', via Agrobacterium tumefaciens, superexpressando o gene codificador de defensina (def), isolado de Citrus sinensis cv. \'Valência\', dirigido pelo promotor com expressão preferencial no floema AtSUC2 (transportador de sacarose, clonado de Arabidopsis thaliana) ou pelo promotor constitutivo CaMV 35S (clonado do vírus do mosaico da couve-flor). Os explantes utilizados na transformação genética foram segmentos de epicótilo obtidos de plantas germinadas in vitro. A identificação das plantas transgênicas foi realizada por meio da análise da PCR, utilizando-se primers para a detecção do fragmento do gene de seleção nptII. As plantas PCR+ foram aclimatizadas e transferidas para casa-de-vegetação. A análise de Southern blot confirmou a integração do transgene em 36 plantas. Foram obtidas 7 plantas transgênicas da cultivar \'Hamlin\', 9 da cultivar \'Natal\', 1 da cultivar \'Pera\' e 9 da cultivar \'Valência\' contendo a construção gênica pC35S/def, e 3 plantas transgênicas da cultivar \'Hamlin\', 6 da cultivar \'Natal\' e 1 da cultivar \'Valência\' contendo a construção gênica pcAtSUC2/def. Os resultados obtidos neste trabalho serão importantes para futura avaliação e estudo visando o controle de Candidatus Liberibacter spp.. / The Brazilian citrus industry is the world\'s largest producer and exporter of citrus, however, it has been affected by diseases that cause serious production losses and damages to fruit quality. However, the culture faces problems, namely phytosanitary issues that have been damaging thousands of plants, affecting yield and competitiveness of the sector. Currently, Huanglongbing (HLB), associated with phloem bacteria Candidatus Liberibacter spp., is considered one of the most destructive citrus diseases. The lack of sweet orange cultivars resistant to HLB makes genetic transformation an important tool in the disease control. To defend from pest and pathogen attack, plants developed a series of defense mechanisms during the evolutionary process, which may include the production of peptides with antimicrobial activity. Plant defensins are small peptides related to pathogenesis (PR) which have antimicrobial activities, associated with plant defense mechanisms . The objective of this study was to obtain transgenic plants of sweet orange (Citrus sinensis L.) cultivars \'Hamlin\', \'Natal\', \'Valência\' and \'Pera\' with Agrobacterium tumefaciens overexpressing the defensin gene (def), isolated from Citrus sinensis cv. \'Valência\', controlled by the promoter with preferential expression in the phloem AtSUC2, (sucrose transporter, cloned from Arabidopsis thaliana) or by the constitutive promoter CaMV 35S (cloned from the mosaic virus of cauliflower). The explants used in genetic transformation were epicotyl segments obtained from germinated plants in vitro. The identification of transgenic plants was accomplished by PCR analysis using primers for the detection of nptII gene fragment. The PCR+ plants were acclimatized and transferred to greenhouse. The analysis of Southern blot confirmed the transgene integration in 36 plants. Seven transgenic plants were obtained for the cultivar \'Hamlin\', nine for \'Natal\', one for \'Pera\' and nine for \'Valência\' containing the gene construct pC35S/def and three transgenic plants for \'Hamlin\', six for \'Natal\' and one for \'Valência\' containing the gene construct pcAtSUC2/def. The results obtained in this work are important for future evaluation of the plants for resistance to Candidatus Liberibacter spp..