Rapid Detection of Avian Eimeria Species Using Denaturing Gradient Gel Electrophoresis

A denaturing gradient gel electrophoresis (DGGE) assay was developed to rapidly discriminate species of avian Eimeria. Amplification by PCR of the small subunit ribosomal RNA gene (approximately 1,600 nucleotides) with Eimeria genus-specific primers followed by cloning and sequencing allowed us to carry out phylogenetic analyses and identify clone sequences to species level in most cases. Clones were subsequently used to amplify a smaller fragment (approximately 120 nucleotides) suitable for DGGE. The fragments were separated on denaturing gradient gel and bands with unique migration distances were mixed to obtain an identification ladder. The identification ladder and PCR products obtained from DNA extracted from fecal samples from several poultry farms were compared. Applying the DGGE method in this study allowed a rapid differentiation of Eimeria species present in fecal samples collected from poultry farms

Heparin modulates the 99-loop of factor IXa: Effects on reactivity with isolated Kunitz-type inhibitor domains

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (\u3e10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys98. Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys98 to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys98 and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite

Heparin modulates the 99-loop of factor IXa: Effects on reactivity with isolated Kunitz-type inhibitor domains

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (\u3e10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys98. Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys98 to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys98 and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite

Use of Pyrosequencing and Denaturing Gradient Gel Electrophoresis to Examine the Effects of Probiotics and Essential Oil Blends on Digestive Microflora in Broilers Under Mixed Eimeria Infection

A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre- and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in comparison to broilers not given the probiotics or the specific EO blends as feed supplements

Molecular methods to evaluate effects of feed additives and nutrients in poultry gut microflora Metodologias moleculares para avaliar efeitos de aditivos e nutrientes na microflora intestinal das aves

Intestines of each animal are the niche of a complex and dynamic ecosystem with important effects to the host. The members or final products of this ecosystem influence nutrient digestion, absorption, mucosa metabolism, general physiology, and local and systemic immunological responses of avian hosts. Better understanding of the avian gut microbial ecosystem may lead to improvements on poultry productivity, health, welfare, and reduction of food borne pathogens and the environmental impact of poultry production for a more sustainable industry. Molecular methods of microbial ecology are key tools to gain this knowledge. The objective of this presentation is to outline the basic concepts, applications, advantages, limitations, and evolution of these molecular methods used to study intestinal microbial ecology. The final goal is to stimulate their application in poultry applied research and development of new feed additives. Some practical examples in poultry research will be described to illustrate their relevance to advance in control methods for pathogens, avoid or manage disbiosis or subclinical intestinal diseases, reduce environmental impact, elucidate effects of nutrients in gut mucosa, microflora, and in general to improve poultry performance.<br>O intestino de cada animal é o nicho de um ecossistema complexo e dinâmico com efeitos importantes para o hospedeiro. As comunidades microbianas componentes deste ecossistema e/ou os produtos finais do metabolismo influenciam a digestão e absorção de nutrientes, o metabolismo das mucosas, a fisiologia geral e as respostas imunitárias locais e gerais da ave hospedeira. A melhor compreensão do ecossistema microbiano do intestino das aves pode levar a melhorias na produtividade, saúde, bem estar, e redução de agente patogênicos dos alimentos e do impacto ambiental da produção avícola para uma indústria mais sustentável. Os métodos moleculares da ecologia microbiana são ferramentas chaves para obter este conhecimento. O objetivo desta apresentação é descrever os conceitos básicos, aplicações, vantagens, limitações, e evolução destes métodos moleculares usados para estudar a ecologia microbiana do intestino. A meta final é estimular sua aplicação em pesquisa avícola aplicada e desenvolvimento de novos aditivos para alimentos. Alguns exemplos práticos em pesquisa avícola serão descritos para ilustrar sua relevância para avançar em métodos de controle de agentes patogênicos, evitar ou manejar a disbiose ou doenças intestinais sub-clínicas, reduzir o impacto ambiental, esclarecer os efeitos dos nutrientes na mucosa intestinal, a microflora e, em geral, melhorar o desempenho das aves