Achados patológicos e aspectos moleculares do vírus da laringotraqueíte infecciosa em um surto em galinhas no sul de Minas Gerais

Abstract

Infectious laryngotracheitis (ILT) is a respiratory disease of chickens that has worldwide distribution resulting in severe economic losses. In November 2010, the Instituto Mineiro de Agropecuária (IMA) notified the MAPA (Ministério da Agricultura, Pecuária e Abastecimento) a suspicion of the disease in the southern state of Minas Gerais, which later was confirmed. The municipalities involved, included Itanhandu, Pouso Alto, Passa Quatro, Itamonte and Pedralva, belonging to the Highlands of Mantiqueira. Some epidemiological data were collected, necropsies of birds with respiratory clinical signs were performed and samples from larynx, trachea, lung, conjunctiva, turbinates and sinuses, trigeminal ganglia and brain of 78 chickens from laying hens farms in the area where the outbreak occurred were collected. Samples from the same tissues from 12 backyard chickens and laryngotracheal swabs from other 21 commercial chickens of this region were also collected. Additionally, tissue samples were collected from 18 chickens in other regions of the Minas Gerais state. Immunohistochemistry on 30 out 78 tissues from commercial laying hens was performed using a monoclonal antibody specific for the protein gJ. For DNA extraction, formalin fixed and paraffin embedded (FFPE) tissues from 78 commercial laying hens, eight backyard chickens and chickens from other regions of Minas Gerais state were used. Also, laryngotracheal swabs were made and used for DNA extraction. Later, polymerase chain reactions (PCR) were made using primers that amplify a product of 237 base pairs corresponding to a region of reversed diploid gene ICP4. Two FFPE and two swabs samples were sent for sequencing and aligned to other strains deposited in GenBank. Clinical signs were characterized by difficulty breathing, coughing, sneezing, nasal discharge, sometimes bloody, apathy and in some birds, swelling of the sinuses and conjunctiva. Mortality varied from 1% to 6% between different farms. At necropsy, some chickens had diffuse moderate acute fibrinous laryngitis and tracheitis; others had acute laryngitis and tracheitis fibrino-necrotic (diphtheric membrane) or formation and accumulation of yellowish necrotic material obstructing the larynx. Fibrinonecrotic lesions were also observed in the sinuses and conjunctiva. Histopathology revealed lesions characteristic of ILT in 25/78 (32%) larynx and trachea, characterized by hyperplasia and desquamation of the epithelium and fusion of epithelial cells forming syncytia containing intranuclear inclusion body. In 15/78 (19%) birds were observed syncytial cells with intranuclear inclusion body in the paranasal sinuses, and in 24/78 (30%) birds these cells were also observed in the conjunctiva. In the lung, 21/78 (26%) birds had ILT characteristic lesions. In the immunohistochemistry, positive marker was detected in the cytoplasm of syncytia cells, which inclusion body cells were visualized and also in some no syncytial cells of the four tested tissues. For PCR test, we observed a higher sensitivity for the diagnosis of ILT compared to histopathology. Samples sent for sequencing had 100% identity with United States (EU104910.1) strain, 99% with Australia (JN596963.1) strain and 91% with vaccine strains CEO (JN580316.1) and TCO (JN580315.1) strains. The tissue with more incidences of ILT characteristics lesions was the larynx/trachea. Nevertheless, lesions also were observed in the lungs and sinuses followed by conjunctiva. Validation of PCR on FFPE tissues offers the advantage of allowing the test tissue fixed by formalin, which enables retrospective and conservation alternative where there is no appropriate freezing. The sequence alignment of ILT BRASIL/2011/UFMG (KC182579) showed some differences in nucleotide that determined differences in the level of amino acid sequences (mutations) compared to the TCO and CEO vaccine strains. However, future sequencing of larger regions of the ICP4 gene should be made to determine if there is relation between the strain involved in the outbreak in southern LTI MG and other wild and vaccine strains.