Screened the remaining suspected P. larvae isolates from Dawn D. Created crude genomic lysates of each isolate and performed diagnositc PCR using the AFB primers.
Results:
Not the best image, but the bands (or lack of bands) can be clearly seen. This is actually a photo of a photo of a photo due a corrupt saved file of the image on my USB drive. So, this image is of the printed out picture of the gel.
Top Row
| |||||||||
HiLo Marker
|
18
|
19
|
20
|
21
|
22
|
23
|
24
|
25
|
Positive
|
Bottom Row
| |||||||||
HiLo Marker
|
26
|
27
|
28
|
29
|
30
|
31
|
Neg1
|
Neg2
|
Positive control was P. larvae ATCC 9545. Neg1 was the E. coli strain pMarA ran in this experiment, and Neg2 was the E. coli strain ran in the previous experiment.
| Lab Code | EW Code |
| dd1-6a | 18 |
| dd1-8a | 19 |
| dd5-J | 20 |
| dd5-K | 21 |
| dd5-L | 22 |
| dd5-M | 23 |
| dd5-M | 24 |
| dd5-N | 25 |
| dd5-Q | 26 |
| dd8-55a | 27 |
| dd8-63a | 28 |
| dd8-86a | 29 |
| dd11-2a | 30 |
| dd12-22a | 31 |
| DH5a | Neg1 |
| pMarA | Neg2 |
Discussion:
Of all the isolates from Dawn D. that had been screened using the diagnostic PCR AFB primers, only one did not test positive. That one isolate was from the previous study on 12-22-14. The negative result was from isolate dd1-7a (EW code 02). All other isolates were confirmed as being Paenibacillus, specifically P. larvae because the AFB primers amplified a conserved region of the 16S rRNA only found in P. larvae. A complete list will be compiled of all the confirmed isolates and provided to Dawn D. for future studies. It is my understanding that freezer stocks will be made of the positive isolates, at -80C, to be used in future studies.
It was surprising to find that so many honey providers in the area have P. larvae present in their honey. However, I am uncertain on exactly how many isolates were initially screened by Dawn D. that proved to be both Gram positive and Oxidase positive before they were given to me for diagnostic PCR analysis. Also, there wasn't a relatively large presence of the bacteria in any honey source, and it could be that there is just always a low presence of the bacteria in any bee hive. Perhaps that is a route for a future study.
Of all the isolates from Dawn D. that had been screened using the diagnostic PCR AFB primers, only one did not test positive. That one isolate was from the previous study on 12-22-14. The negative result was from isolate dd1-7a (EW code 02). All other isolates were confirmed as being Paenibacillus, specifically P. larvae because the AFB primers amplified a conserved region of the 16S rRNA only found in P. larvae. A complete list will be compiled of all the confirmed isolates and provided to Dawn D. for future studies. It is my understanding that freezer stocks will be made of the positive isolates, at -80C, to be used in future studies.
It was surprising to find that so many honey providers in the area have P. larvae present in their honey. However, I am uncertain on exactly how many isolates were initially screened by Dawn D. that proved to be both Gram positive and Oxidase positive before they were given to me for diagnostic PCR analysis. Also, there wasn't a relatively large presence of the bacteria in any honey source, and it could be that there is just always a low presence of the bacteria in any bee hive. Perhaps that is a route for a future study.
//EWW
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