Microsporidian Enterocytozoon hepatopenaei (EHP) spores are inactivated in 1 min at 75 °C

Abstract

The microsporidian Enterocytozoon hepatopenaei (EHP) is a major threat for shrimp culture. We hypothesized that commercial feeds processed at 75 °C or higher would present no risk for EHP transmission, even if they test positive for EHP by PCR. To test this hypothesis, the purified EHP spores (1 × 106 spores) were incubated at 25 °C (non-heated) or 75 °C (heated) for 45–60 s prior to determining viability by polar tube extrusion assay. Heat at 75 °C prevented extrusion when compared to spores incubated at 25 °C, confirming inactivation even though PCR detection of EHP was still positive. Further tests were carried out using oral injection of heated and non-heated spores. At day 14 post injection, SYBR Green-qPCR based on the EHP spore wall protein (SWP) sequence revealed 1063–36,449 copies of EHP per ng DNA in the hepatopancreatic tissues of 3 from 4 shrimp given non-heated spores while shrimp given spores heated at 75 °C for 45 s or 60 s showed no detectable copies. In another experiment, shrimp were fed EHP-PCR-positive, commercial feed samples (CS1 and CS2) containing the equivalent of 2.2 × 106 and 4.8 × 105 EHP copies/day for 21 days. The positive control consisted of EHP-PCR-negative feed supplemented with active EHP spores (2.0 × 105 spores/day) fed for 14 days followed by feeding with un-supplemented, EHP-PCR-negative feed from day 15 to 21. The negative control consisted of EHP-PCR-negative, commercial feed. After 21 days, all shrimp in the positive control group showed EHP infection by SYBR Green-qPCR and in situ hybridization. In contrast, shrimp fed with the negative control feed and the two PCR-positive commercial feed samples (CS1 and CS2) showed no signs of EHP infection. The results supported our hypothesis that CS1 and CS2 feeds contained inactive EHP DNA. Considering the heat exposure during manufacture (equal to or exceeding 75 °C for 60 s), such feeds should be regarded as posing no risk for EHP transmission.