High prevalence of IBV QX genotype in a densely populated poultry area of Northern Italy

Infectious bronchitis virus (IBV) is an highly infectious pathogen responsible for relevant economic losses all over the world, which causes diseases involving respiratory, uro-genital and, occasionally, gastroenteric systems. As other RNA viruses it is characterized by a remarkable genomic variability, due to both high substitution rate and recombination, triggering the frequent emergence of new genotypes, pathotypes and serotypes. In Italy, since the first description of the disease in 1956, several genotypes have been recognized though time with fluctuating prevalence. Considering the poor cross-protection between serotypes induced by both natural infections and vaccines, a constant monitoring of IBV epidemiology is fundamental to set up efficient control measures. A survey of IBV in 55 broiler, 10 breeder and 1 layer farms, located mainly in a densely populated poultry area of Northern Italy, performed from November 2012 to August 2013, is reported. Some farms were sampled more than once in different production cycles. A total of 83 samples were found positive for IBV by a commercial diagnostic real time RT-PCR kit targeting ORF1 sequence. A one step RT-PCR was performed on all positive samples using primer pair targeting the S1 protein gene. This protein, exposed on the viral surface, is responsible for viral attachment and, bearing several antigenic determinants, is subjected to strong immune derived selective pressure. As a consequence it is classically used for identification and genotyping of IBV variants. Samples which resulted negative to the second amplification were also tested with a second primer pair designed specifically for D1466 genotype. The PCR products were purified using the Wizard1 SV Gel and PCR Clean-Up System (Promega). Sequencing was performed at BMR Genomics (Padua, Italy) with the 3730xl DNA analyzer (Applied Biosystems, USA). Sixty-three strains were sequenced and based on phylogenetic analysis were classified in 6 different genotypes. The most prevalent one was the QX-like (47.61%) followed by the 793/B (38.09%). Even if our method could not discriminate between field and vaccine strains, it is noteworthy that almost all 793/B strains were closely related to two commercial vaccines. In particular 16 strains displayed 100% percentage of identity with 793/B based vaccines commercialized in Italy. Five Mass (7.93%), 3 Q1 (4.76%) and 1 D274 (1.58%) strains were also detected. Remarkably in layer/breeder farms QX-like genotype was never detected. In these poultry categories 793/B, Q1 and D274 genotypes were identified. No Italy02 genotype was detected in any of the samples analyzed. Our study confirmed the trend, observed in Europe, of an increasing prevalence of QX-like genotype that becomes the predominant one in Italy. A low level of circulation of the strain D274, recently reported in Italy, was also confirmed. Geographical distribution of genotypes and their association with clinical signs are discussed