Introduction: the doubly labelled water (DLW) method has an accuracy of 1% and within-subject precision of 5-8%, depending on subject's age and environments issues. Energy intake assessment is prone to errors (>15-20%) depending in the method utilized. Objective: to quantify DLW methodology errors in four to five year olds that could affect the comparison with energy intake. Methods: energy expenditure (TEE, by DLW), was assessed during 14 days in 18 preschool children, who attended eight hours daily to day-care centres. Energy intake was determined by a combined method: food weighing during weekdays and recall after leaving the Centre (17h to sleep time) plus 24 h recall, during the weekend. Several assumptions affecting DLW total error were assessed to determine their influence in the comparison to energy intake (i.e. background variability, space ratio, proportion of water subject to fractionation, food quotient value). Results: the individual mean energy expenditure was 1 373 ± 177 kcal and the energy intake (1 409 ± 161 kcal). The overall difference between intake and expenditure was 42.9 kcal/day (limits of agreement + 259.1 to -112.3 kcal/day). TEE measurement error only explained a minor quantity (2.4%), between both measurements, and the observed mean isotope dilution space was 1.030 ± 0.010 confirming the value utilized in adults studies. Conclusions: energy expenditure data is similar to other studies in preschool children. The small difference found between energy intake and expenditure may be attributed to the applied energy intake methodology, the homogeneous diet at care centres during the week-days and the lower DLW methodology error.

Introducción: el método del agua doblemente marcada (ADM) tiene una precisión del 1% y en un mismo sujeto es de 5-8%, dependiendo de la edad y el entorno del sujeto. La evaluación de la ingesta energética es propensa a errores (>15-20%), dependiendo del método utilizado. Objetivo: cuantificar los errores metodológicos del ADM en niños de 4-5 años que podrían afectar la comparación con la ingesta de energía. Métodos: el gasto de energía (GTE, por ADM), se evaluó durante 14 días en 18 preescolares, asistentes a guarderías infantiles. La ingesta energética se determinó mediante un método combinado: pesaje de alimentos durante los días de la semana y registro después de salir del centro (17 horas en adelante), además de un recordatorio de 24 horas, durante un día del fin de semana. Resultados: el promedio individual del gasto energético total fue 1373 ± 177 kcal y la ingesta de energía (1409 ± 161 kcal). La diferencia global entre la ingesta y el gasto fue 42,9 kcal/día. El error de medición del GET explicó una variación del 2,4%, entre ambas mediciones, y el espacio de dilución de isótopos fue 1030 ± 0.010, confirmando el valor utilizado en los estudios de adultos. Conclusiones: los datos de GET fueron similares a otros estudios realizados en niños en edad preescolar. La pequeña diferencia encontrada entre la ingesta y el gasto energético se puede atribuir a la metodología de la ingesta de energía aplicada, la dieta homogénea en los centros de atención, durante los días de la semana, y el bajo error metodológico del ADM.

The highly pathogenic H5N1 avian influenza virus (AIV) has caused important economic losses and public health problems, mainly in Asia, Africa and Europe. Counteracting this virus by vaccination of poultry is crucial. In our study, recombinant antigens based on the fusion of the extracellular segments of both molecules, hemagglutinin H5 of AIV and the chicken CD154, were tested to enhance the immune response in chickens. For this purpose, chickens were immunized with recombinant adenoviral vectors carrying the nucleotide sequence of the hemagglutinin H5 alone (AdHA) or fused to the CD154 recombinant protein (AdHACD). Also, these two proteins (rHA and rHACD) were produced and assayed as antigens. Both, the adenoviral vector AdHACD and the recombinant protein rHACD, induced hemagglutination-inhibiting antibody titers significantly higher than their counterparts carrying HA alone. Similar results were observed when the cellular response was measured by real time PCR. AdHACD and rHACD significantly increased the production of IFN-? transcripts, which were lower when AdHA or rHA were used. To obtain a higher quality antigen, rHACD was purified by size exclusion chromatography, resulting in the preservation of its immunogenic properties. However, rHACD did not work as immunogen when an immunoaffinity chromatography was performed by eluting at acidic pH or with a chaotropic agent. These results demonstrate that chicken CD154 can significantly enhance humoral and cellular responses against hemagglutinin H5 of AIV when both molecules are administered as a fusion protein in chickens. Therefore, AdHACD or rHACD could become promising vaccine candidates against H5N1 virus.

El virus de la influenza aviar (AIV) H5N1 ha causado serios problemas económicos y de salud pública en Asia, África y Europa. Por este motivo, la protección de la masa avícola contra este virus es fundamental. En este estudio se desarrollaron dos antígenos basados en la fusión del segmento expuesto de la hemaglutinina H5 del AIV con el segmento expuesto del CD154 de pollo. Estos se inmunizaron como vectores adenovirales recombinantes que contenían la hemaglutinina H5 simple (AdHA) o la proteína de fusión (AdHACD), o como antígenos proteicos recombinantes rHA y rHACD. Tanto el AdHACD como la rHACD indujeron títulos de anticuerpos inhibidores de la hemaglutinación significativamente superiores a los obtenidos con el AdHA y la rHA. Al medir la respuesta celular por PCR en tiempo real, los resultados fueron similares. El AdHACD y la rHACD incrementaron significativamente la producción de transcritos de IFN-?. Estos fueron menores con el AdHA y la rHA. Para obtener un antígeno de mayor calidad, se purificó la rHACD por cromatografía de exclusión molecular. Este método mantuvo la inmunogenicidad de la rHACD. Sin embargo, cuando se realizó una cromatografía de inmunoafinidad mediante elución a pH ácido o con un agente caotrópico, la rHACD no funcionó como inmunógeno. Estos resultados demuestran que el CD154 de pollo puede incrementar significativamente la inmunidad humoral y celular contra la hemaglutinina H5 del AIV, cuando ambas moléculas se administran fusionadas en pollos. Por tanto, el AdHACD y la rHACD podrían convertirse en candidatos vacunales prometedores contra el virus H5N1.

In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking regions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen molecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2-4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.

Four epizootics caused by rabbit hemorrhagic disease virus (RHDV) have been recorded in Cuba from 1993 to 2005. Each time, thousands of animals have died or have been slaughtered to avoid the spread of the disease. Cell culture systems allowing the in vitro replication of RHDV are not available to date. Moreover, the amount of the recombinant capsid protein (VP60) obtained in heterologous expression systems does not commonly exceed a few tens of milligrams per liter of culture. In this paper, we report the expression of VP60 in two strains of the Pichia pastoris yeast with the highest expression levels obtained so far. VP60 was glycosylated, associated to the yeast cell disruption pellet at a concentration of 1.5 g/L in the first case, or soluble in the intracellular fraction at approximately 300 mg/L, following a different cloning strategy. These recombinant variants showed similar antigenic properties to those of the native protein, as determined by monoclonal antibodies. The soluble VP60 showed a higher number of protective epitopes, due to the formation of multimers that were similar in size and structure to the native RHDV capsids. Both antigens induced potent RHDV-specific immune responses in experimental animals. The antibodies produced were able to inhibit the in vitro hemagglutination of a viral strain isolated during the last outbreak in Cuba. A molecular and antigenic characterization of this strain was also carried out and led to its classification as a member of the highly pathogenic RHDVa subtype. Both recombinant antigens induced a specific, protective and long-lasting immune response against “classical” strains and also against the RHDVa subtype.