RESUMO
Epizootiologic outbreaks of disseminated neoplasia have been reported in association with massive mortalities of various bivalve species. In cockles, Cerastoderma edule, this pathological condition was described in Ireland and France. Since 1997, different populations affected by this pathology have been detected in Galicia (NW Spain). Transmission electron microscopy allowed the visualization of virus-like particles in neoplastic cells, resembling a retrovirus-like agent. To confirm this hypothesis, we used a commercial kit for detection and quantification of reverse transcriptase (RT) activity, based on the use of bromo-deoxyuridine triphosphate (BrdUTP) and a BrdU binding antibody conjugated to alkaline phosphatase. In addition, we developed a product-enhanced RT assay using RNA of hepatitis A virus as a template. These two assays showed positive RT activity in 90.9 and 81.8% of samples, respectively, from cockles displaying disseminated neoplasia as determined by light microscopy. These results strongly support the hypothesis of retroviral etiology for this pathological condition.
Assuntos
Doenças dos Animais/virologia , Cardiidae/virologia , Neoplasias/veterinária , Infecções por Retroviridae/veterinária , Retroviridae/patogenicidade , Doenças dos Animais/patologia , Animais , Surtos de Doenças , Transtornos do Desenvolvimento Sexual , Feminino , Gônadas/patologia , Gônadas/ultraestrutura , Gônadas/virologia , Corpos de Inclusão/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão/veterinária , Neoplasias/patologia , Neoplasias/virologia , Retroviridae/isolamento & purificação , Retroviridae/ultraestrutura , Infecções por Retroviridae/patologia , Infecções por Retroviridae/virologia , Espanha/epidemiologiaRESUMO
In the present study, the efficiency of several primer sets for the RT-PCR detection of hepatitis A virus (HAV) and astrovirus from both crude viral extracts and experimentally infected shellfish tissues was evaluated. Differences were observed depending on the primer set employed in the sensitivity of amplification of both viral types. For HAV primers, HAV240/HAV68 yielded the higher sensitivity: showing a detection limit of 0.02-0.1 infectious particles/microL or mg of tissue (either crude extracts or seeded mussel tissues). Regarding detection of AsV, a better performance was observed with primer set A1/A2 achieving a sensitivity of 0.1-1 PFU/microL or mg of tissue. The results obtained in this work strongly indicated that selection of primer sets to be employed for the routine detection of enteric viruses was a critical point in the design of the RT-PCR protocols.
