Desenvolvimento e aplicação de Elisa indireto com peptídeo sintético p-p27 e ensaios moleculares para o diagnóstico da Leucemia viral felina (FeLV)

Abstract

Feline Leukemia Virus belongs to Retroviridae Family, gender Gammaretrovirus. The viral protein p27 is highly produced in the infected cells, and is found in the cytoplasm and corporal fluids. Although diagnostic tests are highly sensitive, is necessary to perform more than one test to confirm the infection, mainly serological ones, due to the variable characteristic of the progress of the infection. The aim of this work was to develop and standardize an indirect ELISA using a synthetic peptide p-p27, based on gag gene for the diagnosis of Feline Leukemia Virus (FeLV) and to evaluate coinfections. For the detection of FeLV antigen p27 and FIV antibodies, 80 samples were tested with a commercial kit (FIV/FeLV Test kit -AlereTM) and confirmed with ELISA (ELISA - SNAP® Combo FeLV/FIV). Stool samples were used for the detection of antigens for panleukopenia and feline coronavirus (Panleucopenia Felina Ag Test Kit and Coronavirose Felina Ag Test Kit (Alere TM). To confirm the presence of proviral DNA, a polymerase chain reaction was performed, for amplification of fragments of 450, 235 and 166 bp of FeLV gag gene. FeLV subtypes were detected through nestedPCR. Results showed that, of the 80 samples, 4 (5%) were positive for FeLV, 12 (15%) for FIV, 2 (2,5%) for Coronavirus and none for panleukopenia. The main clinical signs observed were: halitosis, intense salivation, gingivostomatitis, pharyngitis, oral bleeding, ulcerations on the dorsal and lateral portions of the tongue, glossopalatine arch lesions, cachexia, lethargy, diarrhea and co-infections (respiratory signs) (hydrosalpingitis and uterine congestion), nephropathy and liver disease. Nested-PCR revealed that the four FeLV positive samples amplified for subtypes A and B, demonstrating an AB combination and none for subtype C and / or ABC. Phylogenetic analysis identified a similarity (78% bootstrap) for FeLV-AB, to subtypes occurring in East Asia (Japan) and Southeast Asia (Malaysia), demonstrating that it is a highly genetically variable subtype, characterizing a circulating subtype with a differentiated genotype. The indirect ELISA was developed with a linear soluble peptide with 16 amino acid residues. p-p27 was efficient in detecting anti-FeLV antibodies in naturally infected felines and could be an auxiliary tool in the diagnosis.