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Page 2 of 19 Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16

their genomes were compared to extant sequenced P. larvae phage genomes. We test the host ranges of the
bacteriophages and formulate cocktails to undertake in vitro testing on a set of representative bacterial strains.
These results form the basis of a promising solution for protecting honey bees in New Zealand from AFB.

Keywords: Apis mellifera, phage, environmental sampling, community science

INTRODUCTION
The European honey bee (Apis mellifera) is a valuable livestock animal globally . In New Zealand, this
[1]
value comes from their role in the pollination of horticultural and agricultural crops which contributes
,
roughly 8.7 billion dollars to New Zealand’s current GDP per annum, assuming this ratio has maintained
[2]
since 2013 . The export of apiculture products, including honey, beeswax and live bees, contributes a
further $483 million NZD p.a. . Since 2006, New Zealand has seen a steep increase in the number of
[3]
beekeepers and apiaries; with these rising numbers, there has also been a rising number of colony losses
[3]
observed .
Honey bees are under constant attack by abiotic and biotic factors, including but not limited to herbicides,
[4]
pesticides, parasites, viruses and bacteria . The two biggest biotic threats to honey bees today are the
parasitic Varroa mite (Varroa destructor) and American foulbrood (AFB), which is caused by the spore-
forming, bacterial pathogen Paenibacillus larvae (P. larvae). AFB is a serious and destructive disease that
attacks honey bees in their larval and pupal stages . AFB has detrimental consequences at both the larval
[5,6]
[7-9]
and colony level .
AFB has been present in New Zealand for at least 146 years after first being discovered in 1877 [10,11] . By 1887,
AFB had caused significant damage around the country and led to a 70% reduction in honey production .
[12]
The use of antibiotics to treat or mask an AFB infection in New Zealand is strictly prohibited [13,14] . Current
legislation stipulates that beekeepers must destroy hives infected with AFB within seven days of discovery,
using petrol fumes and incineration to ensure all traces of AFB are removed [13,14] . This method is costly to
both the beekeeping community and the New Zealand economy.

A potential solution to AFB infection in New Zealand is the prophylactic application of bacteriophages in a
phage cocktail. Bacteriophages, or phages informally, are self-propagating viruses that are only able to infect
and replicate within bacteria. Phages are ubiquitous and are the most numerous biological entity on Earth,
with at least 10 phages in existence globally at any point in time [15,16] .
31

Work undertaken overseas has shown it is possible to protect honey bee larvae from AFB infection through
[18]
the application of P. larvae phage cocktails both in vitro and in honeybee colonies in an at-risk apiary .
[17]
In the latter, phage protection appeared to remain intact for at least four months after application of the
cocktail. In addition, a recent genomic analysis was performed in which 48 completely sequenced P. larvae
phages were described and classified into four clusters and one “singleton” . These provide a rich resource
[19]
of information on phage diversity. However, New Zealand biosecurity laws prohibit us from importing
non-native phages for domestic release. Therefore, in order to create phage cocktails to protect honeybees in
our domestic honey production sector, it is necessary that we isolate P. larvae phages that are native to New
[20]
Zealand. While a collection of P. larvae bacterial isolates was previously reported , that collection was
subsequently destroyed (P. Lester personal communication). Discovering novel New-Zealand-based P. larvae
isolates was therefore necessary.