Lactobacillus sp. as a potential probiotic for the prevention of Paenibacillus larvae infection in honey bees

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Lactobacillus
sp. as a potential probiotic for the
prevention of
Paenibacillus larvae
infection in honey bees
Dagmar Mudroňová1*, Juraj Toporčák1, Radomíra Nemcová1, Soňa Gancarčíková1,
Vanda Hajdučková1 and Katarína Rumanovská1
1University of Veterinary Medicine, Komenskeho 73, 041 81, Košice, Slovak Republic.
Received 19 September 2011, accepted for publication 27 September 2011.
*Corresponding author: Email: mudronova@azet.sk
Keywords:
Lactobacillus
, probiotic, honey bee,
Paenibacillus larvae
Journal of Apicultural Research
50(4): 323-324 (2011) © IBRA 2011
DOI 10.3896/IBRA.1.50.4.11
American foulbrood (AFB) is amongst the most dangerous of bee
diseases, affecting many honey bee colonies worldwide each year.
This contagious disease is caused by sporulating Gram-positive
bacterium
Paenibacillus larvae
. In most countries veterinary legislation
requires that any bee colonies testing positive for AFB be destroyed,
with high financial costs. Elsewhere, AFB is fought radically by the
application of antibiotics and sulphonamides, but administration of
drugs has little beneficial effect, merely obscuring clinical symptoms
and risking the accumulation of drug residues in honey bee products.
One of the most progressive treatment possibilities without the use of
drugs is the application of probiotics or other natural substances.
Their beneficial biological influence has no side effects on the
organism or environmental risks. The use of probiotics in the
prevention and therapy of AFB is poorly studied, in particular the use
of autochtonous strains isolated from honey bees. The main aim of
this study was therefore to isolate lactobacilli from the honey bee
digestive tract and select them for potential probiotic use for the
prevention of
P. larvae
infection.
Forty digestive tracts (intestine, stomach and rectal sac) were
taken from healthy adult from three full sized honey bee colonies,
which belong to the University of Veterinary Medicine in Košice,
Slovakia. The digestive tracts were homogenized in 10 ml of sterile
physiological solution with 0.5% cysteine. Lactobacilli were isolated in
MRS broth resp. agar (Oxoid; Basingstoke, UK) for 24 and 48 h
respectively at 37°C in 80% CO2 and 20% N2 atmosphere. We
received 40 isolates of lactobacilli which were further selected for
probiotic use based on their ability to survive long term storage, anti-
bacterial activity against
P. larvae
,
growth properties, and production
of organic acids. From 20 isolates stored at -20°C for 5 months in
glycerine freezing medium 9 isolates (45%) were able to grow in MRS
broth and from 20 isolates stored at -20°C for 5 years 8 isolates
(40%) survived. All surviving isolates (17) were able to auto-
aggregate in MRS broth. Their antibacterial activity against a wild
strain of
P. larvae
, isolated from infected combs from an apiary in
Kožany, Slovakia, was tested by a modified paper disc assay
(Nowroozi
et al.,
2004). For each isolate of
Lactobacillus
an arithmetical
mean of zone diameters from three measurements was calculated.
Based on the calculated arithmetical means, the tested strains were
divided into non-inhibiting to weak inhibiting (< 10 mm), middle
inhibiting (10 mm ≤ zone ≤ 16 mm) and strong inhibiting strains (>
16 mm).
From 17 isolates tested, two were not able to inhibit the growth of
P. larvae
and the inhibitive activity of five strains was only weak.
Seven isolates showed middle inhibitive activity and three (22/C, 50/
C, 12/C) showed strong inhibition. These strains were characterized
based on their biochemical and growth properties and identification
was confirmed by rep-PCR method (Gevers
et al.
, 2001). Concentration
of organic acids in the bacterial cultures was determined by capillary
isotachophoresis (Nemcová
et al.
, 2007). Isolates 22/C and 12/C were
identified as the same strain of
L. brevis
and isolate 50/C as
L. plantarum
. The growth dynamic of both strains was similar
whereby no significant differences were found (Fig. 1). Decrease of
pH was significantly faster in
L. plantarum
from the 8th to 24th hour of
growth (p < 0.001) in comparison to
L. brevis
, in accordance with
significantly higher production of lactic acid by
L. plantarum
.
Homofermentative
L. plantarum
produced high concentration of lactic
acid (269.2 ± 14.30 mmol.l-1) and lower concentrations of acetic
(78.1 ± 4.83 mmol.l-1), acetoacetic (23.4 ± 2.08 mmol.l-1) and
propionic acid (17.9 ± 7.05 mmol.l-1). Heterofermentative
L. brevis
produced in the highest concentrations of acetic (73.9 ± 5.87 mmol.l-1),
lactic (42.9 ± 3.29 mmol.l-1), and acetoacetic acid (27.2 ± 1.69 mmol.l-1).
All these properties are good prerequisites for the use of both strains
for the prevention of
P. larvae
infection in honey bees, but further
studies are necessary.

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324 Mudroňová, Toporčák, Nemcová, Gancarčíková, Hajdučková, Rumanovská
Fig. 1.
The increase of absorbancy, numbers of lactobacilli and decrease of pH of the growing media during the growth of
L. brevis
and
L. plantarum
(
n
= 3).