Estabelecimento de um protocolo para detecção de Nosema apis e Nosema ceranae a partir de amostras de mel

Abstract

Honey bee (Apis mellifera) is the main species responsible by pollination and vegetable propagation. Aside pollination, several products used by human beings, i. e. honey, apitoxin, wax, royal jelly and propolis are provided by honey bees. However, pathologies like nosemosis, which is one of the main causes of Colony Collapse Disorder, may be responsible for severe colony losses and overall production decline, leading to economic losses. This work aims to standardize a technique for detection of Nosema apis and Nosema ceranae in honey samples, providing a fast and reliable alternative for the current methods, using as sample the most abundant product of honey bees. For this work, three DNA extraction protocols were tested and compared. One protocol used a microwave to disrupt and further extract DNA from cells and two other protocols used commercial kits: a kit for genomic DNA extraction from animal tissue and bacteria cell samples and a kit for plant genomic DNA extraction. Fifteen honey samples collected in Rio Grande do Sul State in 2007 and 2008 were tested. Nosema spp. spores were previously detected using optic microscopy in the samples, but no species were reliably determined. Considering the three tested methods, only the method using the plant genomic DNA extraction kit presented was able to obtain the expected PCR products. Fourteen out of the 15 honey samples tested were positive for N. apis and 11 out of the 15 samples were positive for N. ceranae. Standardization of a technique for early detection of Nosema species in honey samples was possible. The developed protocol proved to be fast and efficient, allowing extraction of genomic DNA in appropriate quality from honey samples and suitable for N. apis and N. ceranae detection.