| Summary: | 碩士 === 國立宜蘭大學 === 園藝學系碩士班 === 96 === American foulbrood (AFB) is a serious bacterial disease of honeybee (Apis mellifera L.) caused by the spores of Paenibacillus larvae. Since the disease signs of the early infection stage were difficult to find by naked eyes in bee colony, it is very important to detect the P. larvae spores for disease controls. This study collected honey samples from Taiwan and Thailand in 2005-2006 and detected P. larvae spores by plate counting methods. Results showed, in 2005, the rate of spore-positive samples from Taiwan is significant higher than from Thailand (18.8% vs. 9.8%, p < 0.05), and the highest (21.1%) was the apiary honey from Taiwan. Also in 2006 samples, the spore-positive rate from Taiwan is significant higher than Thailand (34.8% vs. 20.0%, p < 0.05), and apiary honey from Taiwan was up to 44.2%. These showed the AFB spores presented commonly in Taiwan.
Next, in order to confirm its accuracy of the plate counting method and to detect the other bee products, this study designed a nest PCR primer pair to detect P. larvae spores from bee products by polymerase chain reaction (PCR). Results showed a specific DNA fragment could be detected from infection adult bees, honey, bee pollen, and royal jelly by nest PCR. The detection limit is 0.125 CFU of the DNA template in primary PCR that equilibrated to 5 CFU per gram of bee products. Using this method, 80% (n = 15) of honey samples containing 0 ~ 10 CFU/15 g (n = 20) counted by plate counting methods were spore-positive. When tested the bee pollen samples (n = 36), 97% (n = 35) of them were spore-positive. These show that the proposed methods can succeed detecting the specific DNA fragment of P. larvae spores from bee pollen and honey at a lower spore density. When using this detection method to monitor the diseased levels of bee colony, royal jelly may be the suitable subject than honey or bee pollen.This study has demonstrated that the proposed nest PCR method can succeed detecting P. larvae spores from bee products. In addition, there are standard operation procedures for different bee products have been proposed to monitor AFB levels. Combing the well hive management, this system may pre-warn or prevent the emergence of American foulbrood without using antibiotics in bee colony.
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