A typing scheme for Paenibacillus larvae, the causative agent of American Foul Brood

Barbara Morrisey, Food and Environment Research Agency (Fera) and the University of York with Ed Hayes, one of BDI's other sponsored PhD researchers

Barbara Morrisey writes:

"I am in the second year of my PhD at the University of York and Fera, funded by a BBSRC CASE partnership with Bee Disease Insurance (BDI). During this time I have created a typing scheme for identifying different strains of the bacteria Paenibacillus larvae the causative agent of American foulbrood (AFB).

American foulbrood is the most destructive brood disease globally, and it is found on every continent where honey bees are kept. It only affects the bee brood, larvae are fed the infective spores in contaminated larval food. The spores then germinate in the mid gut of the larvae before killing them. The dead larvae then dry onto the side of the broodcomb in a stage called scale, this scale is made up of millions of highly infective spores. Colonies that are infected with AFB will usually collapse if untreated. In many countries the best control method is thought to be burning infected hives. Antibiotics only mask the symptoms as they do not affect the highly resistant, disease spreading spores. Therefore, whether the colony is treated or not the bees are likely to die, leading to large losses in global apiculture. Monitoring and controlling AFB is costly to both the beekeepers and statutory bodies. The scheme I have developed will help us to understand the routes of transmission of this damaging and costly disease.

Outbreaks of infective diseases are often the result of exposure to a common source of the pathogen. This means that the organisms causing the outbreak will be clonally related and share genetic characteristics. The identification of strains of P.larvae causing AFB outbreaks can, therefore aid in the discovery of the routes of transmission in a disease outbreak. Identifying strains can also lead us to the source of infection, which means we could prevent further infection. Identifying strain types will also allow us to determine whether a site that is infected year after year is infected with the same strain which would suggest eradication has failed or different strains which would suggest reinfection from an outside source. It may also allow us to customise control methods depending on the strain type found in a colony.

In order to identify the strain types of a bacterial species you must use a typing scheme. A typing scheme is a kind of test used to distinguish different strains (or types) of a bacterial species from each other. There are various kinds of typing schemes for different bacterial species based on variances in characteristics such as the types of sugar an organism can metabolise, antibacterials which are produced or the types of proteins made by different strains. Recently methods based on the DNA sequence of bacteria have become more popular due to their precision and the decreasing cost of DNA sequencing.

The current scheme used to type P. larvae is known as the ERIC typing scheme. It only identifies four different strains, known as ERIC types (ERIC I to ERIC IV). ERIC types I and II are the most commonly found with ERIC I found throughout the world and ERIC II found mainly in Europe. ERIC III has been found recently in Chile but ERIC IV has not been found in the field in recent years. In terms of global apicultural losses ERICs I and II are the most important types, with ERIC I being the most important type in the UK. I wanted to create a scheme that still splits the bacteria up into their ERIC types but gives better resolution so that it can be used to study the population structure and evolutionary history of the bacterium. This new scheme identifies several ERIC I strain types and several ERIC II strain types.

Previous typing schemes for P. larvae did not identify many strain types of the bacterium, and some were difficult to use or did not give strains that could be recognised by different laboratories. I have created a scheme based on small DNA sequence differences within seven essential genes. When you combine the sequence differences from each gene you get the strain type (ST). This method is simple to use and easy to transfer between laboratories.

Last year I made a trip to the Bee research institute in Germany where I shared my scheme in exchange for gene sequences from P. larvae from around the world. The scheme was tested on around 300 samples from 31 different countries and it now characterises more than 20 different global isolates of P. larvae, several of which have been found here in the UK.

I am currently carrying out analyses on the global distributions of P.larvae types to give us some insight into the evolutionary history of the bacterium. The next step will be to study the distribution of these types around the UK in order to understand more about their spread and identify possible sources. I will regrow bacteria from Lateral flow devices (LFDs) that have been used by bee inspectors to verify infections in the field. P. larvae produces millions of very long lasting spores and because of this it is possible to regrow bacteria from LFDs that are several years old. This will give us a clearer picture of the causes of AFB outbreaks in the UK over the last few years.

My work is supervised by Dr Thorunn Helgason at the University of York, Dr Giles Budge at Fera (the Food and Environment Research Agency) and Mr Bernard Diaper at BDI (Bee Disease Insurance). The project is funded by a BBSRC CASE partnership with BDI. My trip to Germany was made possible by a grant from COLOSS under the COST grant system."

American Foulbrood photo - Courtesy The Food and Environment Research Agency (Fera), Crown Copyright