Angiostrongrylus cantonensis as a cause of cerebrospinal disease in a yellow-tailed black cockatoo (Calyptorhynchus funereus) and two tawny frogmouths (Podargus strigoides)

A captive yellow-tailed black cockatoo (Calyptorhynchus funereus) and 2 free-living tawny frogmouths (Podargus strigoides), both native Australian species, were presented with neurologic signs including depression and pelvic limb paresis and paralysis. Despite supportive treatment, all 3 birds died or were euthanatized. On histologic examination, sections of metastrongyloid nematode larvae were found in the central nervous system of all 3 birds, whereas intact larvae, identified as Angiostrongylus cantonensis, were recovered from the brain and spinal cord of 2 birds. Angiostrongylus cantonensis, the rat lungworm. has an obligatory migratory phase through the host's central nervous system, which can cause severe pathologic lesions. Natural infections in accidental hosts have been documented only in mammals, and to our knowledge, angiostrongyliasis in avian hosts has not been previously reported.

Site-directed mutagenesis and kinetic studies of the West Nile virus NS3 protease identify key enzyme-substrate interactions

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ae-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in VWNV NS3 protease at S1 (V115A/F, D129A/ E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.

Review of the evidence for prehospital administration of benzyl penicillin in meningoccocal septicaemia - experience in Queensland

Meningococcal disease is a rare but potential killer in both adults and children. Community acquired meningococcal disease is caused by a variety of serogroups of Neisseria meningitides. Of the five main subgroups, A, B, C, W135 and Y, serogroups, A and Y are rarely identified in Australia. Alternatively, Serogroup B accounts for the highest number of cases followed by serogroup C strains. Meningococcal infection causes two distinct clinical profiles, though dual presentations are not uncommon. The first, meningitis presenting alone, is the more common form of infection and requires urgent but not immediate medical treatment. Conversely the second presentation, meningococcal septicaemia, is considered a medical emergency. In Queensland, careful and detailed consideration of the evidence for introduction of benzyl penicillin for the prehospital treatment of meningococcal septicaemia has been conducted. Notwithstanding the seriousness of the septicaemic presentation, these reviews have resulted in the decision not to introduce this drug in the ambulance service at the time. This paper describes the reasoning behind these decisions.

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Attachment of Shiga toxigenic Escherichia coli to beef muscle and adipose tissue

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (S-r) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. S-r values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and S-r values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.

In vitro antibacterial activity of Australian native herb extracts against food-related bacteria

The antibacterial activities of water, ethanol and hexane extracts of five Australian herbs (Backhousia citriodora, Anetholea anisata, Eucalyptus staigerana, Eu. olida and Prostanthera incisa) against seven food-related bacteria (Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Enteritidis, Sal. Typhimurium and Staphylococcus aureus) were determined by the microtitre broth microdilution assay. The water extracts of all the herbs displayed no or low antimicrobial activity against all of the bacteria tested with the exception of S. aureus. Relatively high levels of activity (minimum inhibitory concentrations of 125-15.6 mu g ml(-1)) against this pathogen were present in water extracts from all herbs except P. incisa. The ethanol and hexane extracts of all herbs displayed some activity against a number of the bacteria tested, with no one particular herb displaying an obviously higher level or range of activity. Staphylococcus aureus proved to be the most sensitive of the bacteria tested against the solvent extracts with all extracts displaying activity ranging from 125 to 7.8 mu g ml(-1), while E. coli and L. monocytogenes, on the other hand, proved the least sensitive with only five of 15 herb/extract combinations displaying any activity against these pathogens. The extracts of the Australian native herbs examined in this study have potential for application in foods to increase shelf-life or promote safety. (c) 2005 Elsevier Ltd. All rights reserved.

An overview of eye-related photophobias

This chapter provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is the experience of discomfort affecting the eyes as a result of exposure to light. It has a variety of causes, including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for photophobia associated with these conditions: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

Eye-related photophobia

This article provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is an experience of discomfort affecting the eyes due to exposure to light. It has a variety of causes including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for eye-related photophobia: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

Trattamenti mediante tecnologie basate sul plasma di carote julienne

Prodotti di IV gamma a base di frutta e verdura minimamente trattati o pronti all'uso non possono essere considerati sicuri da un punto di vista microbiologico e sono stati spesso associati a casi di tossinfezione. Per tali episodi è stato evidenziato che la qualità dell'acqua utilizzata per il lavaggio è una fase critica. D'altra parte è noto che la disinfezione è una delle fasi di lavorazione più importanti per i prodotti minimamente trattati in quanto ha effetti diretti sulla qualità dei prodotti finiti, sulla sicurezza e la loro shelf-life. Tradizionalmente, l'industria di IV gamma ha impiegato i composti derivati del cloro per la fase di disinfezione in virtù della loro efficacia, semplicità d'uso e basso costo. Tuttavia vi è una diffusa tendenza ad eliminare i prodotti a base di cloro a causa soprattutto della preoccupazione in relazione ai rischi ambientali e sanitari per il consumatore associati alla formazione di sottoprodotti alogenati cancerogeni. Tra le varie tecnologie emergenti, proposte come alternative al cloro, il plasma ha presentato buone potenzialità in virtù delle specie chimiche che lo compongono, principalmente specie reattive dell’ossigeno e dell’azoto, che sembrano essere responsabili di stress ossidativo alle cellule microbiche, con conseguenti danni per DNA, proteine e lipidi. L’obiettivo generale di questo elaborato finale è stato quello di valutare la possibilità di utilizzare la tecnologia del plasma per la decontaminazione superficiale di carote julienne. In particolare si sono presi in considerazione trattamenti diretti in cui il vegetale è stato esposto al plasma per differenti tempi compresi tra 5 e 40 minuti. Inoltre, si è utilizzo il plasma per il trattamento di acqua che è stata successivamente impiegata per il lavaggio delle carote julienne. L'efficacia di entrambe le modalità di trattamento è stata verificata nei confronti sia della microflora naturalmente contaminante le carote, sia di alcuni microrganismi patogeni che possono essere associati a tale vegetale: Listeria monocytogenes, Salmonella Enteritidis ed Escherichia coli.

Évaluation de l'efficacité de la pasteurisation à la vapeur pour le contrôle de la contamination microbiologique des carcasses bovines

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Avaliação da Qualidade e Segurança Microbiológica de Hortofrutícolas

Frutas e vegetais frescos, embora sejam essenciais para uma dieta equilibrada e nutritiva, estão frequentemente associados a surtos de origem alimentar, pois este tipo de produto é muitas vezes consumidos cru, sem qualquer processamento térmico que elimine ou reduza para níveis aceitáveis a carga microbiana patogénica. Desta forma, e para que o consumo deste tipo de produtos continue a aumentar, torna-se importante aumentar o controlo em termos de segurança alimentar em todas as fases de produção. O presente trabalho de investigação teve por objetivos estudar a ocorrência de cinco microrganismos patogénicos: Salmonella sp.; Escherichia coli; Staplylococcus aureus; Listeria monocytogenes; HAV (vírus da Hepatite A), em framboesas e alface à venda no mercado português, otimizando uma técnica simples e rápida para deteção desses microrganismos nesses produtos. Também neste trabalho foi feito um estudo sobre os hábitos de consumo de produtos minimamente processados, framboesas e alface através de um inquérito por questionário. A escolha dos produtos e microrganismos a pesquisar teve por base um levantamento de quais as contaminações e os hortofrutícolas mais afetados nos últimos anos. A investigação iniciou-se com a pesquisa do HAV em framboesas e alface, onde após uma revisão bibliográfica, foram testados quatro protocolos distintos. No entanto devido à complexidade das técnicas para pesquisa de vírus em alimentos, à falta de meios e controlos para o desenvolvimento das respetivas técnicas, este objetivo foi abandonado. Numa segunda fase foi otimizado um método de deteção (PCR) para pesquisa das bactérias patogénicas, igualmente em framboesas e alface. Após otimização das condições de PCR, foram testados dois métodos de preparação da amostra: um com um passo de rebentamento de células baseado na fervura; e outro onde apenas se recolheu algum volume de cultura de pré-enriquecimento, tendo-se usado a mesma diretamente para a reação de PCR. Concluiu-se que apenas o primeiro método é eficiente, mas apenas para bactérias Gram-negativas (Salmonella sp. e E. coli) uma vez que estas possuem uma parede celular fina, capaz de ser destruída pela fervura, ao contrário das Gram-positivas (S. aureus e L. monocytogenes). Das amostras de framboesas e alface à venda no mercado português analisadas, concluiu-se que, apesar do método ainda necessitar de alguns ajustes, apenas a amostra de alface fresca parece não apresentar qualquer contaminação com Salmonella sp. e E. coli. Para S. aureus e L. monocytogenes não foi possível obter conclusões definitivas quanto à sua presença nas amostras analisadas, já que vários testes de confirmação deverão ser repetidos. Relativamente aos resultados obtidos acerca do inquérito de consumo, tem-se que dos 186 inquiridos, a grande maioria consome produtos hortofrutícolas minimamente processados 1-3 vezes/semana, tendo, a grande maioria, o hábito de os lavar com água antes do seu consumo, prática esta que é aconselhada. Quanto ao consumo de alface, a frequência aumenta para 3-5 vezes/semana, sendo esta adquirida maioritariamente inteira fresca e em superfícies comerciais. Por fim o consumo de framboesas ainda é escasso, pois a grande maioria dos inquiridos alega consumir este produto esporadicamente, sendo estas adquiridas embaladas inteiras frescas em superfícies comerciais.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

Bioengineering strategies to improve functional qualities of nisin

The abuse of antibiotics and the emergence of multi-drug resistant bacterial strains have created the need to explore alternative methods of controlling microbial pathogens. The bacteriocin family of antimicrobial peptides has been proposed as one such alternative to classic antibiotics. Nisin A belongs to the subgroup of bacteriocins called the lantibiotics, which contain several unusual amino acids as a consequence of enzyme-mediated post-translational modifications. As nisin is produced by generally regarded as safe (GRAS) microorganisms, it could potentially be applied in a clinical setting. However, as lantibiotics are naturally produced in such small quantities, this can hinder their industrial potential. In order to overcome this, several approaches can be utilised. For example, given the gene encoded nature of lantibiotics, genetic engineering approaches can be implemented in order to yield variants with enhanced properties. Here, the use of mutagenesis-based strategies was employed to obtain a derivative of nisin with enhanced bioactivity in vitro. Investigations with purified peptide highlighted the enhanced specific activity of this variant, nisin M21V, against food-borne Listeria monocytogenes strains. Furthermore, this specific enhanced bioactivity was evident in a mouse model of listeriosis. Reductions in bioluminescence and microbial counts in organs from infected mice were observed following treatment with nisin M21V compared to that of wild-type nisin A. Peptide bioengineering approaches were also implemented to obtain additional novel derivatives of nisin. The generation of “S5X” and “S33X” banks (representing a change of natural serines at positions 5 and 33 to all possible alternative residues) by a combination of site-saturation and site-directed mutagenesis led to the identification of several derivatives exhibiting improved stability. This allowed the rational design of variants with enhanced stability compared to that of wild type nisin. Another means of tackling issues associated with lantibiotic yield is to combine lantibiotics with other antimicrobials. This could circumvent the need for enhanced production while also reducing concentrations of the peptide antimicrobials. We observed that combinations of nisin variants and low levels of plant essential oils (thymol, carvacrol, trans-cinnamaldehyde) significantly controlled Gram negative foodborne pathogens in in vitro assays compared to nisin A-essential oil combinations. This enhanced control was also evident in model food systems. Nisin variants used in conjunction with carvacrol significantly reduced numbers of E. coli O157:H7 in apple juice while a commercial nisin preparation used in combination with citric acid significantly controlled C. sakazakii in infant milk formula. It is noteworthy that while nisin is generally associated with Gram positive targets, upon combination with plant essential oils the spectrum of inhibition was broadened to Gram negative targets.

Évaluation de l'efficacité de la pasteurisation à la vapeur pour le contrôle de la contamination microbiologique des carcasses bovines

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Potenzialità applicative delle alte pressioni di omogeneizzazione nel settore alimentare

Le alte pressioni di omogeneizzazione sono considerate una tecnologia non termica basata sull’applicazione di pressioni comprese tra 60 e 400 MPa ad alimenti fluidi o fluidificabili, con un tempo di trattamento di pochi millisecondi. Questa tecnologia permette di ottenere una serie di effetti sull’alimento che variano in rapporto all’entità del trattamento applicato e alla matrice fluida considerata. Pertanto, le alte pressioni di omogeneizzazione rappresentano una delle tecnologie maggiormente studiate in ragione delle loro buone opportunità applicative a livello industriale. Tale tecnologia viene comunemente applicata per modificare le proprietà funzionali di alcune macromolecole caratteristiche degli alimenti ed ha permesso l’ottenimento di prodotti di origine lattiero-casearia ed anche succhi di frutta caratterizzati da migliore texture, gusto, flavour e aumentata shelf-life. L’omogeneizzazione ad alta pressione, considerata come trattamento di sanitizzazione a freddo, è in grado di disattivare sia microrganismi patogeni che degradativi presenti in un determinato sistema, contribuendo a ridurre o contenere quindi lo sviluppo microbico nei prodotti alimentari. L’effetto di tale tecnologia, quando applicata a livelli compresi tra 60-200 MPa bar è stato valutato nei confronti di diversi patogeni quali Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus, Salmonella typhimurium microrganismi degradativi, come Bacillus subtilis e lieviti, deliberatamente inoculati in prodotti diversi.