Protocolo para aumentar a segurança no diagnóstico da Anemia Infecciosa Equina

Abstract

Equine infectious anemia (EIA) is a contagious viral disease that affects equids worldwide and has been recorded in Brazil since the 1960s. It is caused by the Equine Infectious Anemia Virus (EIAV), a lentivirus that integrates into the host genome, making the infection permanent. Due to the high genetic variability of the virus, there is no effective treatment or vaccine, making diagnosis and control challenging. Transmission occurs primarily through contact with infected blood, either via mechanical vectors or contaminated materials, and although more common in humid regions, its spread is global. The infection may present in acute, chronic, or subclinical forms, with cycles of fever, anemia, and weight loss. Diagnosis is based on antibody detection, with agar gel immunodiffusion (AGID) as the gold standard and ELISA as a complementary test. PCR can detect proviral DNA but is limited by the genetic diversity of viral isolates. Control measures include serological surveillance and restrictions on the trade of seropositive animals. In Brazil, positive animals must be euthanized (sanitary slaughter), except in endemic areas such as the Pantanal. However, current diagnostic protocols are vulnerable to fraud, including sample substitution, especially during retesting. Genetic identification of tested animals through DNA extraction from serum or clot samples could minimize fraud and improve diagnostic reliability. Moreover, molecular detection of the virus could help identify acute- phase infections, contributing to better EIA control. This study aimed to enable genomic DNA (gDNA) extraction from serum and clot samples for equine genotyping and proviral DNA detection, in order to establish a reliable diagnostic protocol for EIA. Different conditions for gDNA extraction were evaluated for equine genotyping and EIAV detection. Whole blood samples collected with four anticoagulants (EDTA, heparin, citrate, and fluoride) were subjected to gDNA extraction and PCR at different storage times (3, 30, 60, and 90 days) and temperatures (4 °C and -20 °C), using an equine genotyping-specific primer. Serum and clot samples were also analyzed, assessing the influence of tube type (with or without gel separator) and serum preparation method (centrifugation or decantation) on gDNA and proviral DNA extraction and amplification. The results showed that anticoagulants, time, and temperature did not negatively affect PCR performance. Serum proved to be a viable matrix, although results were influenced by the extraction and preparation methods. The clot samples yielded the most consistent results, with successful amplification under all tested conditions, and also allowed for molecular detection of EIAV in samples from seropositive animals from different regions of Brazil.