Desenvolvimento e padronização de um ELISA indireto para o diagnóstico sorológico de peste suína clássica

Um ensaio imunoenzimático do tipo ELISA indireto (ELISA-I) foi desenvolvido e padronizado para o diagnóstico sorológico de peste suína clássica. Na comparação foram utilizadas novecentas e trinta e sete amostras de soros suínos, as quais foram testadas pelo teste de soroneutralização seguido de revelação por imunoperoxidase (NPLA), tomado como padrão, resultando em 223 amostras positivas e 714 negativas. Em relação ao NPLA, o ELISA-I apresentou sensibilidade de 98,21%, especificidade de 92,86%, valor preditivo positivo de 81,11%, valor preditivo negativo de 99,4% e precis ão de 94,1%. A análise estatística dos resultados revelou uma correlação muito forte (r=0,94) entre os dois testes. Quando comparado com um kit de ELISA disponível comercialmente, a performance de ambos em relação ao NPLA foi similar. Concluiu-se que o ELISA-I é um teste apropriado para triagem em larga escala de soros para a detecção de anticorpos contra o Vírus da Peste Suína Clássica (VPSC), embora não seja capaz de diferenciar entre anticorpos induzidos pelo VPSC ou outros pestivírus.An indirect enzyme linked immunoassay (ELISA-I) was developed and standardized for the serological diagnosis of classical swine fever (CSF). For the comparison, nine hundred and thirty-seven swine serum samples were tested by serum neutralization followed by immunoperoxidase staining (NPLA), considered as the standard. Of these, 223 were positive and 714 negative for neutralizing antibodies to classical swine fever virus (CSFV). In relation to the NPLA, the ELISA-I presented a 98.2% sensitivity; 92.86% specificity, 81.11% positive predictive value, 99.4% negative predictive value and a 94.1% precision. Statistical analysis showed a very strong correlation (r=0,94) between both tests. When compared to a commercially available ELISA kit, the performance of both, in relation to the NPLA, was similar. It was concluded that the ELISA-I is suitable for large scale screening of antibodies to classical swine fever virus, although it does not distinguish antibodies to classical swine fever virus from those induced by other pestiviruses

Early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles

<div><p>Avian infectious bronchitis virus (IBV) primarily replicates in epithelial cells of the upper respiratory tract of chickens, inducing both morphological and immune modulatory changes. However, the association between the local immune responses induced by IBV and the mechanisms of pathogenesis has not yet been completely elucidated. This study compared the expression profile of genes related to immune responses in tracheal samples after challenge with two Brazilian field isolates (A and B) of IBV from the same genotype, associating these responses with viral replication and with pathological changes in trachea and kidney. We detected a suppressive effect on the early activation of TLR7 pathway, followed by lower expression levels of inflammatory related genes induced by challenge with the IBV B isolate when compared to the challenge with to the IBV A isolate. Cell-mediated immune (CMI) related genes presented also lower levels of expression in tracheal samples from birds challenged with B isolate at 1dpi. Increased viral load and a higher percentage of birds with relevant lesions were observed in both tracheal and renal samples from chickens exposed to challenge with IBV B isolate. This differential pattern of early immune responses developed after challenge with IBV B isolate, related to the downregulation of TLR7, leading to insufficient pro-inflammatory response and lower CMI responses, seem to have an association with a most severe renal lesion and an enhanced capability of replication of this isolate in chicken.</p></div

Log10 of IBV RNA copies in tracheas and kidneys from experimentally infected chickens or mock infected.

<p>(A) Log10 of IBV RNA copies in tracheas from chickens challenged with IBV A isolate and IBV B isolate, collected at 1dpi, 5dpi and 8dpi. (B) Log10 of IBV RNA copies in kidneys from chickens challenged with IBV A isolate and IBV B isolate, collected at 5dpi and 8dpi. Medians followed by different letters differ significantly (p≤0.05).</p

Relative expression of cell-mediated immune response related genes in tracheal samples from chickens experimentally infected with IBV or mock infected.

<p>Relative expression of cell-mediated immune response related genes measured by RT-qPCR: CD3 (A), CD4 (B), CD8β (C), IFNγ (D) and Granzyme homolog A (E), in tracheal samples collected 1 day-post infection (dpi), 5dpi and 8dpi from chickens experimentally challenged at 28 days of age with Brazilian field isolates A or B of infectious bronchitis virus, or mock infected (NC). Medians followed by different letters differ significantly (p≤0.05).</p