Caracterização patológica e molecular do vírus da laringotraqueíte infecciosa e diagnóstico diferencial em aves comerciais no estado de Minas Gerais

Abstract

Infectious laryngotracheitis (ILT) is a disease that affects the respiratory tract of chickens. In November 2010, its occurrence has been reported in southern Minas Gerais. For control of the disease, regular scheduled collections of tissues in 17 of the 27 farms in the area were carried out. In total, 159 laying hens of multiple ages were necropsied, and samples of sinuses and nasal turbinates, larynx, trachea, lungs, brain and conjunctiva were collected for further histological processing. For DNA extraction only trachea and trigeminal ganglion were collected, and then PCR was performed During the period of the project (2012-2013), three isolated episodes (at different times) suggestive of ILT, related to the introduction of unvaccinated birds occurred. The chickens (total of 18) were examined by veterinarians of Instituto Mineiro de Agropecuária (IMA), and samples were collected and sent to the pathology laboratory at UFMG. Five positive samples for ILT virus (GaHV -1), including a sample from previous studies (2011), had their genome fragments submitted to sequencing. Two fragments of ICP4 gene and a fragment of TK gene, which have been aligned to other strains, were deposited in GenBank. The chickens collected during the visits were also examined for the presence of histopathological lesions suggestive of other respiratory pathogens, and PCR was performed for Mycoplasma gallisepticum and Mycoplasma synoviae. Specific regions of the M. gallisepticum genome, a fragment of the intergenic region (IGSR) and a fragment of mgc2 gene, were sequenced and characterized. Eighteen egg-laying quails (Coturnix japonica) of the region were also evaluated for the presence of lesions for ILT by histopathology, as well as PCR for viral DNA. The chickens collected during scheduled visits to the farms had nonspecific lesions, and only four showed syncytial cells at histopathology, but without intranuclear inclusion bodies. On the other hand, chickens sent by IMA showed lesions compatible with ILT, which consisted of acute moderate diffuse fibrinous laryngitis and tracheitis associated with desquamation of epithelium and fusion of epithelial cells forming syncytia containing intranuclear inclusion bodies. The samples were sequenced and 100 % identity was observed among them, with no change in the fragments analyzed for three consecutive years (2011, 2012 and 2013). Furthermore, strains of Minas Gerais showed non-synonymous mutations compared with strains from an outbreak occurred in 2002, in São Paulo. The sequences obtained were not characterized as vaccine strains because no deletions were seen, in contrast to the sequence of the live attenuated vaccines (CEO and TCO). In the analysis of the TK gene fragment , mutation were not observed at position 252 of the gene for a threonine to methionine (related to the increased virulence of the strain). The epidemiological findings (low mortality and morbidity) associated with molecular analysis of the TK gene suggested less virulent strains. The vector vaccines for ILT (HVT and FPV) were implemented in the region in August 2011; however, as this alternative began to be tested for the first time in Brazil, it has become extremely important to monitor the birds from that area. The introduction of vector vaccines was effective in reducing viral infection, preventing clinical disease and reducing the presence of viral DNA, especially in the trigeminal ganglion. Among the co-infections observed, mycoplasmosis was the most prevalent, and from the sequencing of genomefragments, the M. gallisepticum was characterized with high nucleotide variation among the strains. No lesions to GaHV-1 were observed in tissue of quails by histopathology and in addition, all of them were negative by PCR, suggesting that they are not involved in the maintenance of the virus in the area. Although no other significant outbreaks of ILT was reported in the area, these results indicate that the virus is still present and circulating among birds and poultry farms. Biosecurity measures must be adopted with the aim to reduce or prevent chances of spreading the virus. Moreover, the vaccination program should be uniform in order to avoid infection of susceptible birds and the occurrence of clinical disease, and this should be extended not only to commercial poultry, but to backyard chickens as well.