Our last blog (From Hive to Report: A Flowchart) described the steps we follow to analyze the samples that we receive in the lab for the National Honey Bee Disease Survey (NHBDS) each year. We also touched on how we generate the corresponding reports and send them to the beekeepers. One question beekeepers often ask is why they may receive two different results from their sample when detecting the Nosema. The quick answer is “the difference in results depends on the process of the detection,” because we actually use two different methodologies for detecting Nosema in the lab—microscopy and real-time polymerase chain reactions (PCR).
Microscopy method: Using this method, we crush the bee (actually 100 bees), add water, and place a small drop of the mixture on a special type of glass slide called a hemocytometer, which helps us count the Nosema spores when viewed under a high-magnification microscope. Under the microscope, we can only count the spores that occur after bees have been infected for a long time (what can be called the dormant or spore stage). For this reason, we suggest catching older bees if we need to check hives for Nosema only.
Molecular (PCR) method: Detecting Nosema using this method is based on analyzing the RNA that we extract from each sample (USDA APHIS National Honey Bee Survey Viral Sample Processing). The extracted RNA undergoes a procedure called PCR, which detects and calculates the amount of genomic material coming from Nosema. The PCR test detects the Nosema during its vegetative growth, meaning the Nosema infections are new—as we would expect in young bees.
According to our 2011-2012 National Honey Bee Pests and Diseases Survey Report, “The molecular techniques employed in this survey are based on analysis of the RNA extracted from each sample. Therefore, our molecular identification focuses on detection of actively reproducing Nosema (vegetative stage,) not dormant (spore stage) Nosema. Subsequently, it is possible that the samples examined by microscopy had detectable levels of Nosema (spores) while the molecular analysis quantifies active infection. This accounts for the difference in the PCR and microscopic detection of Nosema in these samples”.
2018 Samples Microscopic and PCR results:
In 2018, we analyzed 849 samples using both methods. There were 57 samples that were positive for microscopic method but negative for PCR and 251 samples that were negative for microscopic method but positive for PCR. There were 215 samples that were negative for both methods and 326 samples that were positive for both methods.
2019 Samples Microscopic and PCR Results:
During the 2019 survey year, we analyzed 795 samples using both methods. There were 48 samples that were positive for microscopic method but negative for PCR and 193 samples that were negative for microscopic method but positive for PCR. There were 189 and 365 samples that were negative and positive respectively for both methods, the same pattern that was present in the 2018 survey results.
Knowing that the two different methods are best at detecting Nosema in different stages, it is not surprising that we get a positive result in PCR method and negative result in microscopic method, or vice versa. Understanding the two methods of detection also may help better inform beekeepers about their colonies’ Nosema infections. Presence of Nosema in PCR results but none in microscope results could mean a newer infection. Presence of Nosema spores detected by microscope suggests an older infection, regardless of vegetative growth.