Evaluation of the preservation of shrimp samples with Davidson’s AFA fixative for infectious myonecrosis virus (IMNV) in situ hybridization

The potential negative effect of prolonged storage of shrimp tissues in Davidson’s AFA fixative on in situ hybridization (ISH) signal was demonstrated previously for Taura syndrome virus (TSV), which has a single-stranded RNA genome. In this study we evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson’s AFA (Alcohol, Formaldehyde, Acetic acid) fixative will degrade its double-stranded RNA genome resulting in false negative ISH reactions. Twenty-one shrimp (3 g) specific-pathogen-free Litopenaeus vannamei were used in this study. Three shrimp were used as negative control and 18 shrimp were inoculated with a tissue homogenate prepared from frozen IMNV-infected L. vannamei obtained from Brazil in 2003 (positive control). Shrimp were collected at Day 12 post-injection and fixed in Davidson’s AFA for five different preservation times (1, 2, 4, 7 and 10 days). After the different fixation times, the Davidson’s AFA was replaced with several changes of 70% ethanol until the pH was stable. IMNV lesions were confirmed in all positive control shrimp by routine H & E histology and ISH. Myonecrosis lesions were strongly positive by ISH at all five preservation times evaluated. Hence, in the present report it was found that the length of time (up to 10 days) in Davidson’s AFA did not have a deleterious effect on the ISH reaction for IMNV.