Improved qPCR diagnosis of Ovavesicula popilliae (Microsporidia) infection of Japanese beetles (Coleoptera: Scarabaeidae) for monitoring pathogen establishment.

Based on reports of Japanese beetle (JB), Popillia japonica (Coleoptera: Scarabaeidae), population decline in Michigan following establishment of Ovavesicula popilliae Andreadis and Hanula (Microsporidia: Ovavesiculidae), USDA APHIS initiated a project to inoculate runway fields at major Midwest USA airports with O. popilliae, as part of a long-term plan to decrease JB populations at airports and thereby minimize its spread by cargo aircraft to quarantined states. The initial step is to evaluate the presence or absence of O. popilliae at major cargo airports. To accomplish this, improvements are needed in a previously validated qPCR diagnosis of O. popilliae infections of JB. In this study, the accuracy of qPCR diagnosis, which can be used for pooled samples of JB, is improved by minimizing PCR inhibition, then determining the Ct value for O. popilliae DNA that best distinguishes infected from healthy individuals based on microscopic diagnosis of the same individuals. This approach is validated in a second way by correlating the Ct value for O. popilliae DNA to the concentration of O. popilliae spore vesicles in the same samples of blended and filtered JB. Time efficiency of qPCR diagnosis is improved by combining rough homogenate from a pooled sample of 8 JB before DNA purification and qPCR analysis. Pooled samples can be used to accurately estimate percent infection at inoculated sites where less than 15% of the JB are infected. These new methods are used to detect O. popilliae infections of JB at a major airport in each of 8 different states.