Towards in silico IBV vaccine design: defining the role of polymorphism in viral attenuation

The gammacoronavirus, Infectious Bronchitis Virus (IBV), is a respiratory pathogen of chickens. IBV is a constant threat to poultry production as established vaccines are often ineffective against emerging strains. This requires constant and rapid vaccine production by a process of viral attenuation by egg passage, but the essential forces leading to attenuation in the virus have not yet been characterised. Knowledge of these factors will lead to the development of more effective, rationally attenuated, live vaccines and reduction of the mortality and morbidity caused by this pathogen. M41 CK strain was egg passaged four times many years ago at Houghton Poultry Research Station and stored as M41-CK EP4 (stock virus at The Pirbright Institute since 1992). It was the first egg passage to have its genome pyrosequenced and was therefore used as the baseline reference. The overall aim of this project was to analyse deep sequence data obtained from four IBV isolates (called A, A1, C and D) each originating from the common M41-CK EP4 (ep4) and independently passaged multiple times in embryonated chicken eggs (figure 1.1). Highly polymorphic encoding regions of the IBV genome were then identified which are likely involved in the attenuation process through the formation of independent SNPs and/or SNP clusters. This was then used to direct targeted investigation of SNPs during the attenuation process of the four IBV passages. A previously generated deep sequence dataset was used as a preliminary map of attenuation for one virulent strain of IBV. This investigation showed the nucleocapsid and spike as two highly polymorphic encoding regions within the IBV genome with the highest proportion of SNPs compared to encoding region size. This analysis then led to more focussed studies of the nucleocapsid and spike encoding region with the ultimate aim of mapping key attenuating regions and nucleotide positions. The 454 pyrosequencing data and further investigation of nucleocapsid and spike encoding regions have identified the SNPs present at the same nucleotide positions within analysed A, A1, C and D isolates. These SNPs probably play a crucial role in viral attenuation and universal vaccine production but it is not clear if independent SNPs are also involved in loss of virulence. The majority of SNPs accumulated at different nucleotide positions without further continuation in Sanger sequenced egg passages presenting S2 subunit (spike) and nucleocapsid as polymorphic encoding regions which in nature remain highly conserved.

“Biomarkers in a One Health perspective: current knowledge of their ability to merge human and animal health

Biomarkers are nowadays essential tools to be one step ahead for fighting disease, enabling an enhanced focus on disease prevention and on the probability of its occurrence. Research in a multidisciplinary approach has been an important step towards the repeated discovery of new biomarkers. Biomarkers are defined as biochemical measurable indicators of the presence of disease or as indicators for monitoring disease progression. Currently, biomarkers have been used in several domains such as oncology, neurology, cardiovascular, inflammatory and respiratory disease, and several endocrinopathies. Bridging biomarkers in a One Health perspective has been proven useful in almost all of these domains. In oncology, humans and animals are found to be subject to the same environmental and genetic predisposing factors: examples include the existence of mutations in BR-CA1 gene predisposing to breast cancer, both in human and dogs, with increased prevalence in certain dog breeds and human ethnic groups. Also, breast feeding frequency and duration has been related to a decreased risk of breast cancer in women and bitches. When it comes to infectious diseases, this parallelism is prone to be even more important, for as much as 75% of all emerging diseases are believed to be zoonotic. Examples of successful use of biomarkers have been found in several zoonotic diseases such as Ebola, dengue, leptospirosis or West Nile virus infections. Acute Phase Proteins (APPs) have been used for quite some time as biomarkers of inflammatory conditions. These have been used in human health but also in the veterinary field such as in mastitis evaluation and PRRS (porcine respiratory and reproductive syndrome) diagnosis. Advantages rely on the fact that these biomarkers can be much easier to assess than other conventional disease diagnostic approaches (example: measured in easy to collect saliva samples). Another domain in which biomarkers have been essential is food safety: the possibility to measure exposure to chemical contaminants or other biohazards present in the food chain, which are sometimes analytical challenges due to their low bioavailability in body fluids, is nowadays a major breakthrough. Finally, biomarkers are considered the key to provide more personalized therapies, with more efficient outcomes and fewer side effects. This approach is expected to be the correct path to follow also in veterinary medicine, in the near future.

Estimation of climatological water deficit in an experimental watershed in the brazilian cerrado.

ABSTRACT: This study aimed to estimate the probability of climatological water deficit in an experimental watershed in the Cerrado biome, located in the central plateau of Brazil. For that, it was used a time series of 31 years (1982?2012). The probable climatological water deficit was calculated by the difference between rainfall and probable reference evapotranspiration, on a decennial scale. The reference evapotranspiration (ET0) was estimated by the standard FAO-56 Penman-Monteith method. To estimate water deficit, it was used gamma distribution, time series of rainfall and reference evapotranspiration. The adherence of the estimated probabilities to the observed data was verified by the Kolmogorov-Smirnov nonparametric test, with significance level (a-0.05), which presented a good adjustment to the distribution models. It was observed a climatological water deficit, in greater or lesser intensity, between the annual decennials 2 and 32.

Ingestive behavior of sheep fed Brazil nut cake in the diet.

Avaliou-se o comportamento ingestivo de ovinos alimentados com níveis de inclusão (0, 15, 30 e 60%) de torta de castanha-do-pará (TC) (Bertholletia excelsa Bonpl.), na dieta à base de silagem de milho. Foram utilizados 16 cordeiros, sem raça definida e peso médio de 33 ± 6.04 kg, distribuídos aleatoriamente nos quatro tratamentos. Os dados foram analisados quanto à normalidade, submetidos à análise de variância, ajustados em equações de regressão e ao teste de Williams, estimando-se o ponto W. Os consumos voluntários de matéria seca (MS) e fibra insolúvel em detergente neutro (FDN), reduziram linearmente (P=0,013 e P=0,002) em 5,0 g e 2,41 g dia-1, respectivamente, para cada 1% de TC inclusa na dieta. O tempo despendido em alimentação (288,75 min dia-1) não foi afetado significativamente, o tempo em ócio cresceu linearmente (P=0,002) em 3,10 minutos, e o tempo gasto em ruminação decresceu (P=0,001) em 2,62 minutos, para cada 1% de inclusão do coproduto. O número de bolos ruminados (NBR) sofreu efeito decrescente (P=0,004) em 4,61 nº dia-1. Quanto às mastigações merícicas, o tempo gasto por bolo (37,5 seg) e o número por bolo (56,14) não diferiram (P>0,05) entre os tratamentos, mas o tempo total de mastigação (TTM) reduziu linearmente (P=0,002) em 0,05 h dia-1 e o número de mastigações por dia apresentou efeito quadrático (P=0,008), com máximo estimado em 17,5% de TC. A eficiência de ruminação não diferiu entre os tratamentos (101,95 g MS h-1 e 36,76 g FDN h-1) e a eficiência de alimentação (EAL) sofreu redução linear (P=0,045) em 0,42 g FDN h-1, mas foi semelhante para g MS h-1 (172,5). Os consumos diários de MS e FDN apresentaram pontos W estimados em 51,96 e 30,67% de TC, respectivamente. As variáveis NBR, TTM e EAL (g FDN h-1) tiveram os pontos W estimados em 56,64, 56,19 e 56,33% de TC. A torta de castanha-do-pará, em níveis superiores a 56% da MS da dieta, afeta o comportamento ingestivo dos animais, principalmente em variáveis relacionadas à ruminação, sendo que a partir de 30,67% já passa a afetar o consumo de FDN, tendo como principais fatores o teor de extrato etéreo e a fonte de fibra das dietas.