Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.

Isolamento de vibrios potencialmente patogênicos em moluscos bivalves

Neste estudo, 26 amostras de ostras (Crassostrea gigas) comercializadas na cidade de São Paulo e em alguns pontos do litoral de São Paulo, e 36 amostras de mexilhões (Perna perna) colhidas mensalmente em 3 pontos do litoral de Ubatuba - SP, foram submetidas à pesquisa de vibrios potencialmente patogênicos. As amostras desses moluscos eram submetidas a enriquecimento em água peptonada alcalina sem cloreto de sódio e com 1 por cento de cloreto de sódio, e GSTB. O isolamento foi realizado em ágar TCBS. Colônias sacarose positivas e negativas, sugestivas de espécies de Vibrio foram identificadas presuntivamente em meio de ágar ferro de Kligler, sendo confirmadas através de provas bioqufmicas complementares. Uma parte das amostras de vibrios potencialmente patogênicos isoladas foi submetida ao teste de Dean e teste de alça ligada em íleo de coelhos. Os vibrios potencialmente patogênicos encontrados em amostras de ostras foram V. alginolyticus (81 por cento ), V.parahaemolyticus (77 por cento ), V. cholerae não 0:1 (31 por cento ), V. fluvialis (27 por cento ), V. furnissii (19 por cento ), V. mimicus (12 por cento ) e V. vulnificus (12 por cento ) e em amostras de mexilhões foram V. alginolyticus(97 por cento ), V. parahaemolyticus(75 por cento ), V. fluvialis (47 por cento ), V. vulnificus (11 por cento ), V. cholerae não 0:1 (6 por cento ), V. furnissii (6 por cento ) e V. mimicus (6 por cento ). Observou-se acúmulo de fluido em alça ligada de íleo de coelho entre 0,25 e 0,49 ml/cm em 6,9 por cento das amostras, entre 0,5 e 0,99 ml/cm em 15,6 por cento e maior ou igual a 1 ml/cm em 15,1 por cento , e/ou intestino de camundongos lactentes (Teste de Dean) em 26,6 por cento das amostras testadas, confirmando o elevado potencial desses microrganismos em causar gastrenterite. Verificou-se ausência de variação sazonal e também, de correlação entre os vibrios potencialmente patogênicos isolados e os indicadores de contaminação fecal, confirmando que a presença desses microrganismos ocorre de forma autóctone e que, as condições climáticas foram favoráveis à sobrevivência dessas espécies em todas as épocas do ano. Considerando-se os resultados obtidos no presente estudo e o fato de que ostras e mexilhões são habitualmente ingeridos crus ou insuficientemente cozidos, pode-se concluir que sua ingestão constitui-se em um determinado grau de risco para a saúde do consumidor.

Riflessioni patologiche e pratiche sul cholera-morbus di Russia e delle Indie : precedute da un cenno geo-storiografico dell' invasione della malattia dall' Asia nella Russia europea /

Mode of access: Internet.

Enteritis in domestic animals: annotated bibliography, December 1953.

Mode of access: Internet.

Recherches anatomiques, pathologiques et thérapeutiques sur la maladie connue sous les noms de gastro-entérite, fièvre putride, adynamique, ataxique, typhoïde, etc., etc., comparée avec les maladies aigues les plus ordinaires /

Mode of access: Internet.

Wireless telemedicine for the delivery of specialist paediatric services to the bedside

A mobile interactive online health system was used to conduct virtual ward rounds at a regional hospital which had no specialist paediatrician. The system was wireless, which allowed telepaediatric services to be delivered direct to the bedside. Between December 2004 and May 2005, 43 virtual ward rounds were coordinated between specialists based in Brisbane and local staff at the Gladstone Hospital. Eighty-six consultations were provided for 64 patients. The most common conditions included asthma (27%), chest infections (12%), gastroenteritis (10%) and urinary tract infections (10%). In the majority of cases, there were partial (67%) or complete changes (11%) in the clinical management of patients. Specialist services were offered by a team of 13 clinicians at the Royal Children's Hospital: 10 general paediatricians, two physiotherapists and one registered nurse. Feedback from all consultants involved in the service and local staff in Gladstone was extremely positive. In 43 videoconference calls there were three technical problems, probably due to an intermittent mains power supply at the regional hospital. There appears to be potential for other rural and regional hospitals to adopt this model of service delivery.

An Incremental EM-based Learning Approach for On-Line Prediction of Hospital Resource Utilization

Objective: Inpatient length of stay (LOS) is an important measure of hospital activity, health care resource consumption, and patient acuity. This research work aims at developing an incremental expectation maximization (EM) based learning approach on mixture of experts (ME) system for on-line prediction of LOS. The use of a batchmode learning process in most existing artificial neural networks to predict LOS is unrealistic, as the data become available over time and their pattern change dynamically. In contrast, an on-line process is capable of providing an output whenever a new datum becomes available. This on-the-spot information is therefore more useful and practical for making decisions, especially when one deals with a tremendous amount of data. Methods and material: The proposed approach is illustrated using a real example of gastroenteritis LOS data. The data set was extracted from a retrospective cohort study on all infants born in 1995-1997 and their subsequent admissions for gastroenteritis. The total number of admissions in this data set was n = 692. Linked hospitalization records of the cohort were retrieved retrospectively to derive the outcome measure, patient demographics, and associated co-morbidities information. A comparative study of the incremental learning and the batch-mode learning algorithms is considered. The performances of the learning algorithms are compared based on the mean absolute difference (MAD) between the predictions and the actual LOS, and the proportion of predictions with MAD < 1 day (Prop(MAD < 1)). The significance of the comparison is assessed through a regression analysis. Results: The incremental learning algorithm provides better on-line prediction of LOS when the system has gained sufficient training from more examples (MAD = 1.77 days and Prop(MAD < 1) = 54.3%), compared to that using the batch-mode learning. The regression analysis indicates a significant decrease of MAD (p-value = 0.063) and a significant (p-value = 0.044) increase of Prop(MAD

Comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

Mini-dose glucagon rescue for mild hypoglycaemia in children with type 1 diabetes: The Brisbane experience

Objectives: To evaluate the use of small doses of glucagon using an insulin syringe in mild or impending hypoglycaemia in children with type 1 diabetes. Methods: Data were collected from patients attending the Paediatric Diabetes Clinic at the Queensland Diabetes Centre at the Mater Hospital, Brisbane in 2002-2004 following the institution of a new protocol for home management of mild or impending hypoglycaemia associated with inability or refusal to take oral carbohydrate. The protocol recommended the use of subcutaneous injections of glucagon using insulin syringes at a dose of two ' units ' (20 mu g) in children 2 years of age or younger, and for older children one unit per year of age up to a maximum of 15 units (150 mu g), with an additional doubled dose given if the blood glucose had not increased in 20 min. Results: Over a 2-year period, 25 children were treated with mini-dose glucagon on a total of 38 occasions. Additional doses were required for recurring hypoglycaemia on 20 (53%) occasions. The child could be managed at home on 32 (84%) of these 38 occasions, with only 6 (16%) children needing hospital treatment. Conclusions: Our study confirmed that small doses of glucagon given subcutaneously with an insulin syringe is a simple, practical and effective home treatment of mild or impending hypoglycaemia due to gastroenteritis or food refusal in children with type 1 diabetes.

Salmonella in companion animals

In an increasingly hygiene concerned society, a major barrier to pet ownership is the perceived role of companion animals in contributing to the risk of exposure to zoonotic bacterial pathogens, such as Salmonella. Manifestations of Salmonella can range from acute gastroenteritis to perfuse enteric fever, in both humans and dogs. Dogs are heavily associated with asymptomatic carriage of Salmonella as the microorganism can persist in the lower intestines of this host which can be then excreted into the environment. Studies in to the asymptomatic carriage of Salmonella in dogs are somewhat dated and there is limited UK data. The current UK carriage rate in dogs was investigated in a randomised dog population and it was revealed that the carriage rate in this population was very low with only one household dog positive for the carriage of Salmonella enterica arizonae (0.2%), out of 490 dogs sampled. Salmonella serotypes share phenotypic and genotypic similarities which are captured in epidemiological typing methods. Therefore, in parallel to the epidemiological investigations, a panel of clinical canine (VLA, UK) and human (Aston University, UK) Salmonella isolates were profiled based on their phenotypic and genotypic characteristics; using API 20E, Biolog Microbial ID System, antibiotic sensitivity testing and PFGE, respectively. Antibiotic sensitivity testing revealed a significant difference between the canine and human isolates with the canine group demonstrating a higher resistance to the panel of antibiotics tested. Further metabolic capabilities of the strains were tested using the Biolog Microbial ID System, which reveal no clear association between the two host groups. However, coupled with Principle Component Analysis two canine isolates were discriminated from the entire population on the basis of a high up-regulation of two carbohydrates. API 20E testing revealed no association between the two host groups. A PFGE harmonised protocol was used to genotypically profile the strains. A dendrogram depicting PFGE profiles of the panel of Salmonella isolates was performed where similarities were calculated by Dice coefficient and represented by UPGMA clustering. Clustering of the profiles from canine isolates and human isolates (HPA, UK) was diverse representing a natural heterogeneity of the genus, additionally, no clear clustering of the isolates was observed between host groups. Clustering was observed with isolates from the same serotype, independent of host origin. Host adaption is a common phenomenon in certain Salmonella serotypes, for example S. Typhi in humans and S. Dublin in cattle. It was of interest to investigate potential host adaptive or restricted strains for canine host by performing adhesion and invasion assays on Dog Intestinal Epithelial Cells (DIECs) (WALTHAM®, UK) and human CaCo-2 (HPA, UK) cell lines. Salmonella arizonae and Enteritidis from an asymptomatic dog and clinical isolate, respectively, demonstrated a significantly high proportion of invasion in DIEC in comparison to human CaCo-2 cells and other tested Salmonella serotypes. This may be suggestive of a potential host restrictive strain as their ability to invade the CaCo-2 cell line was significantly lower than the other serotypes. In conclusion to this thesis the investigations carried out suggest that asymptomatic carriage of Salmonella in UK dogs is low however the microorganism remains as a zoonotic and anthroponotic pathogen based on phenotypic and genotypic characterisation however there may be potential for particular serotype to become host restricted as observed in invasion assays

Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4,182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 µM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 µM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide and pyridazinone. Exploitation of analogues of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

Brote por Salmonella serovar Poona en una guardería

Introducción: se describe un brote de gastroenteritis causada por Salmonella poona en una guardería infantil en la ciudad de Valladolid (España) en los primeros tres meses del año 2011. Objetivos: describir las características epidemiológicas del brote, su relación con un brote supracomunitario declarado en España en 2010 y analizar el mecanismo de transmisión. Métodos: se realizó un estudio descriptivo bidireccional. Partiendo del caso índice, se elaboró una base de datos con la totalidad de niños asistentes a la guardería y se completó con la información recibida de los pediatras y con la información microbiológica. Se calcularon tasas de ataque por aulas y curva epidémica. Resultados: se encontraron 13 casos, de edades comprendidas entre los cinco meses y los cinco años, tres de los cuales fueron asintomáticos. La tasa de ataque global en la guardería fue del 28,2%, no encontrándose diferencias significativas entre las diferentes aulas. Todas las salmonelas aisladas excepto dos fueron enviadas al Centro Nacional de Microbiología (CNM) para su caracterización, identificándose todas ellas como Salmonella poona 13,22:z:1,6, idéntica a la aislada en el brote nacional. Conclusiones: parece evidente que el brote ocurrido en la guardería fue producido por el mismo microorganismo que el que causó el brote supracomunitario y que la fórmula láctea implicada en dicho brote fue el vehículo de transmisión que permitió la introducción del microorganismo en la guardería, propagándose por otras vías entre los alumnos de la misma.

Meningitis por Listeria monocytogenes en paciente inmunocompetente

Listeria monocytogenes es un patógeno de origen alimentario que suele producir gastroenteritis, procesos febriles, sepsis y meningitis. Afecta característicamente a neonatos, embarazadas, ancianos e inmunocomprometidos, con una epidemiología controvertida y poco conocida. Se presenta un caso de meningitis y síndrome de secreción inadecuada de hormona antidiurética secundario en paciente inmunocompetente.

Motivos de recurso ao Serviço de Urgência Pediátrica

Introdução: O recurso ao serviço de urgência (SU) hospitalar motivado por situações não urgentes é frequente e conduz a pior prestação de cuidados, insatisfação dos utentes e profissionais e aumento dos custos. Objectivos: Determinar os motivos para recurso a SU pediátrica hospitalar. Material e métodos: Estudo descritivo, transversal, entre 10/10 e 31/12/2013 em SU pediátrico hospitalar, através de análises de inquéritos preenchidos de forma anónima pelos acompanhantes e complementados com informação clínica pelo médico. Resultados: Foram analisados 481 questionários. O recurso ao SU ocorreu nas primeiras 24 horas de doença em 48% dos casos. Os principais motivos foram: noção de doença grave e urgente (33%), local de atendimento mais próximo (17%), preferência por avaliação por pediatra (17%). A maioria teve alta sem realização de exames complementares de diagnóstico ou tratamento (89%) e os principais diagnósticos de alta foram nasofaringite e gastroenterite agudas. Apenas 19,7% das idas ao SU poderiam ser consideradas como “justificadas” pelo cumprimento dos critérios de OMS para urgência hospitalar ou por orientação prévia por outra entidade de saúde. Não existem diferenças estatisticamente significativas entre as características das crianças que recorreram ao SU de forma “justificada” e “não justificada”. Discussão e conclusão: O reconhecimento de situações clínicas que justifiquem o recurso ao SU hospitalar não parece relacionado com habilitações literárias parentais ou atribuição de médico de família. A percepção de doença grave em situação benigna com recurso precoce e injustificado ao atendimento em contexto de serviço de urgência denota falta de educação para a saúde na população geral.

Effects of supplemental osteopontin on intestinal development and serum antibody responses to rotavirus vaccination in piglets

Milk contains numerous bioactive substances including immunoglobulins, cytokines, growth factors and components that exert antibiotic and prebiotic activity (Field, 2005). Little is known about the biological effects of individual milk bioactives, despite the fact that natural milk improves intestinal development and immune system functions in neonates (Donovan et al., 1994; Field, 2005) relative to milk formula. Characterization of the biological effects of such components is important for optimal production of infant milk formulas to be used when mother’s milk is not available. Milk components with preliminary evidence of positive effects on the intestinal growth and mucosal immunity include osteopontin (OPN). Osteopontin is a phosphorylated acidic glycoprotein expressed by a number of different immune and non-immune cells and tissues (Sodek et al., 2000). It is also present in body fluids including blood, bile and milk (Sodek et al., 2000). Osteopontin is a multifunctional protein that is implicated in a wide number of biological processes including cell survival, bone remodeling, and immune modulatory functions (Sodek et al., 2000). Furthermore, Schack and colleagues (2009) demonstrated that the concentration of OPN in human milk is considerably higher than in bovine milk and infant formulas. Taken together, it is likely that OPN plays a role in the early development of gastrointestinal tract and mucosal immune responses in infants. Since the neonatal pig shares anatomical, physiological, immunological, and metabolic similarities with the human infants (Moughan, et al., 1992), they were selected as the animal model in our studies. Our first aim was to investigate the effects of OPN on piglet intestinal development. Newborn, colostrum-deprived piglets (n=27) were randomized to receive three treatments: formula with bovine OPN (OPN; 140 mg/L); formula alone (FF); or sow reared (SR) for 21 days. Body weight, intestinal weight and length, mucosal protein and DNA content, disaccharidase activity, villus morphology, and crypt cell proliferation were measured. Statistical significance was assigned at P<0.05. No significant effects of OPN were observed for body weight, intestinal weight and length. Mucosal protein content of SR piglets was lower than FF and OPN piglets in the duodenum, but higher than FF and OPN piglets in the ileum. No significant effects of diet in mucosal DNA content were detected for the three regions of the small intestine. Lactase and sucrase activities of SR piglets were higher than the two formula-fed groups in the duodenum, lower in the ileum. No significant effects of diet on lactase and sucrase activities were noted between two formula-fed groups in the duodenum and ileum. Jejunal lactase activity of FF piglets was higher than SR piglets, whereas no significant effect of diet was observed in jejunal sucrase activity among the three groups. Duodenal and ileal villus height and villus area of SR piglets were lower than two formula-fed groups, while OPN piglets did not differ from FF piglets. There was a significant effect of diet (P<0.0001) on jejunal crypt cell proliferation, with proliferation in OPN piglets being intermediate between that of FF and SR. In summary, supplemental OPN increased jejunal crypt cell proliferation, independent of evident morphological growth, and had a minor impact on disaccharidase activity in the small intestine of neonatal piglets. Rotavirus (RV) is the most common viral cause of severe gastroenteritis in infants and young children worldwide (Parashar et al., 2006). Maeno et al. (2009) reported that OPN knockout (OPN-KO) suckling mice were more susceptible to RV infection compared to wild-type (WT) suckling mice. To detect the role of OPN in intestinal immune responses of neonates, the goal of the second study was to evaluate whether supplemental OPN influenced the serum antibody responses to RV vaccination in neonatal piglets. Newborn, colostrum-deprived piglets were randomized into two dietary groups: formula with bovine OPN (OPN; 140 mg/L) and formula alone (FF) for 35 days. On d7, piglets in each dietary group were further randomized to receive rotavirus (RV) vaccination (Rotarix®) (FF+RV and OPN+RV) or remained non-vaccinated (FF+NV and OPN+NV). Booster vaccination was provided on d14. Blood samples were collected on d7, 14, 21, 28 and 35. RV-specific serum immunoglobulin (Ig) G, IgA, IgM and total serum IgG, IgA, IgM were measured by ELISA. Statistical significance was assigned at P<0.05, with trends reported as P<0.10. Body weight gain was unaffected by diet and/or vaccination. No significant effect of oral OPN supplementation was observed for RV-specific antibody responses and total Igs levels. After the combination of dietary groups, RV piglets had significantly higher RV-specific IgM concentrations compared to NV piglets. Although there were higher means of RV-specific IgG and RV-specific IgA concentrations in RV group than their counterparts in NV group, the difference did not reach statistical significance. RV-specific IgM reached a peak at d7 post booster vaccination (PBV), whereas the RV-specific IgG and IgA peaked later at PBV 14 or 21. Total Igs were unaffected by RV vaccination but were significantly increased over time, following similar pattern as RV-specific Igs. In summary, neonatal piglets generated weak antibody responses to RV vaccination. Supplemental OPN did not enhance RV-specific serum antibody responses and total serum Igs levels in neonatal piglets with or without RV vaccination. In conclusion, we observed normal developmental changes in the small intestine and serum Igs levels in neonatal piglets over time. Oral OPN supplementation showed minimal impacts on intestinal development and no effect on serum Igs levels. The role of supplemental OPN on the growth and development of infants is still inconclusive. Future studies should measure other physiological and immunological parameters by using different models of vaccination or infection.