Desenvolvimento de um RT-PCR RFLP para a detecção e diferenciação molecular das variantes vacinais e brasileiras do vírus da bronquite infecciosa das Galinhas

Abstract

The avian infectious bronchitis (AIB) is a disease caused by a single-stranded RNA virus pertaining to the coronaviridae family of the Gammacoronavirus genera. AIB is a disease of great economic significance to aviculture worldwide, not only because of the injury it causes to the breeders, but also because it is a differential diagnosis for several important pathologies that may affect poultry. The goals of the present research were to develop a RT-PCR RFLP technique capable of safely detecting and discriminating the strains of the IBV of the Massachusetts and BR serotypes from the trachea, kidney and intestines tissues in poultry farms of broilers and layers in the estate of Rio de Janeiro. In this study, 117 samples of intestines, kidneys and/or tracheae were collected from a total of 50 birds originating from 4 farms (three broiler farms and one layer farm) in the estate of Rio de Janeiro. The total RNA was extracted from all the samples and subjected to a reverse transcription process for the cDNA synthesis. The cDNA samples were subjected to the RT-qPCR technique. It was found that 92% of the birds (n= 46/50) were positive in at least one of the organs for the chicken bronchitis virus (vBIG). The RT-PCR RFLP technique developed in this work was able to detect a frequency of 94,64% (n= 53/56) of the samples belonging to the BR-I serotype, 3,7% (n= 2/56) of the samples showed fragments with patterns similar to what is expected from the Massachusetts Serotype and 1,78% (n= 1/56) showed patterns from both serotypes (Massachusetts and BR). There was no significant difference in the frequency of molecular detection of vBIG in the tissues of the trachea, kidney and intestines of the broiler and layer poultries from farms in the estate of Rio de Janeiro. The RT-PCR RFLP technique was able to safely detect and distinguish the Massachusetts and BR serotypes. The high frequency of the BR serotype demonstrates that, although vaccinated, the birds developed the disease, indicating that a new vaccine protocol must be addressed, since the majority of the strains found belong to a genetically distinct serotype.