Detection of P. larvae in Local Honey

Goal: Identify P. larvae isolates from local honey providers in North Dakota and the Minnesota area. These isolates will be used for future studies as wild type strains of P. larvae. In order to protect the providers of the honey samples, the businesses will not be identified.

Dawn D., lab technician of the Fisher lab, has acquired 12 honey samples (Lab code for honey providers is dd1 to dd12) from a number of states in the country. These honey samples were diluted and cultured on MYPGP agar. Suspected P. larvae isolates were further isolated onto MYPGP. Suspected isolates were then Gram stained and oxidase tested to help confirm their identity as possibly being Paenibacillus.

Of the many isolates recovered, a handful were confirmed to be Gram positive and oxidase positive indicating potential Paenibacillus larvae isolates. The lab code was developed by Dawn D, however my own code was created for simplicity:

Lab Code	EW Code
dd1-5a	01
dd1-7a	02
dd5-4a	03
dd8-55a	04
dd8-63a	05
dd8-67a	06
dd8-70a	07
dd8-78a	08
dd8-82a	09
dd8-83a	10
dd8-85a	11
dd8-85a	12
dd8-86a	13
dd8-91a	14
dd8-95a	15
dd8-99a	16
dd8-119a	17

The code dd# is referring to one of the 12 honey sources, and the last part is referring to a specific isolate that was identified that source. The above list is only a portion of the isolates confirmed to be potential Paenibacillus.

Confirmation of identification of the suspected isolates as being P. larvae was performed by using diagnostic primers developed according to Development of a fast and reliable diagnostic method for American foulbrood disease (Paenibacillus larvae subsp. larvae) using a 16S rRNA gene based PCR (Dobbelaere, 2001). Primer 1 and Primer 2 were used according to the paper (referred to as AFB primers). PCR parameters were as dictated by the paper as well.

AMRESCO Ready PCR Mix, 2x was used for the amplification reaction. A master mix was created and 2 uL of crude genomic lysates were added for each PCR reaction.

Results of PCR:

Top Row
HiLo Marker	01	02	03	04	05	06	07	08	Positive
Bottom Row
HiLo Marker	09	10	11	12	13	14	15	16	17

The bottom row amplicons were mixed with EZ vision loading dye, whereas the top row was not. This explains the difference in band intensities between the top and bottom rows. Positive control was Paenibacillus larvae ATCC 9545 and the negative control was E. coli pMarA, however I ran out of room on this gel to run the negative control. I will the negative control on the subsequent gel. HiLo DNA marker was used.

Interestingly, all the isolates, except for one, tested positive as being P. larvae. The only isolate that didn't amplify using the AFB primers was EW 02 (dd1-7a). I will continue to screen the remaining suspected Paenibacillus isolates.

//EWW