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The qPCR to fight against the pathological risks of our bees

Until the end of the fifties, beekeeping was essentially a family beekeeping organized in small non-transhumant apiaries. The “flight hole” observation was the absolute reference to evaluate the health of a colony. The bees of autochthonous races in balance with their environment, which was then rich in biodiversity, were carriers of only a limited number of viruses (1 to 2), the main pathogens being rather bacterial (foulbrood) and fungal (nosemosis). Since the fifties, beekeeping practices have evolved and the mobility of the flocks has become the rule both in transhumance to ensure the harvest of honey and in the worldwide exchange of genetics.

Viral carriage has increased with no less than thirty-two viruses identified to date, with the same bee being able to harbor 7 to 8 different viruses. The dominant presence of Nosema apis, which is not very pathogenic, to the benefit of Nosema ceranae, which is much more deleterious, constitutes a new serious threat to the health of colonies.

What research has already shown:

The scientific literature on the multiple roles of pathogens is particularly abundant and we will limit ourselves to a few of the most decisive for beekeeping practices.

Nosema ceranae

Its presence in the gut can at any time cause the collapse of the colony:

– either by the development of the pathogenic fungus in case of dearth, especially of protein dearth or in case of indigestibility of the sugars offered to the bees

– or by collapse of the immune system in synergy with pesticides

– or by stimulation of the viral populations.

About viruses

Many facts must be kept in mind: the transmission of viruses can be vertical:

-all queens carry one or more viruses that they transmit to their offspring, the same observation is made about spermatozoa,

-the nurse bees excrete a lot of virus through their hypopharyngeal glands and thus contaminate the larvae,

– Varroa destructor disseminates a large number of viruses, either by passive carriage or as a multiplication host,

– Many viruses are not expressed directly but induce the development of other viruses present. Thus the virus of deformed wings may not produce alterations in the bees but may cause symptoms of sacciform brood because it has stimulated the multiplication of S.B.V. viruses.

– severe symptoms of contamination appear with a thousand times less virus particles if the penetration is done by the hemolymph route rather than by the oral route.

Efficiency of analytical means

The great revolution of the last few years has been the development of a new analytical method, qPCR (Polymerase Chain Reaction), which makes it possible to identify a pathogen by its DNA or RNA (RT-PCR).

The improvement of the different steps of the analysis and the multiplication of the primers allow to identify any living being in only a few hours.

Solu’Nature has partnered with the DNAucleis Laboratory to offer a PathoBEE 1 analytical pack that can routinely identify the presence of 10 pathogens.

Fungi Nosema apis and Nosema ceranae
Bacteria American foulbrood Paenibacillus larvae
APV (=ABPV) Acute paralysis virus
SBV Sacciform brood virus
CBPV Chronic paralysis virus = black disease
DWV Deformed wing virus phenotype A
VDV Varroa virus = DWV phenotype B
KBV Kashmir virus
SBPV Slow paralysis virus
BQCV Black royal cell virus

The analysis is carried out on 30 to 50 bees in a mixture because individual analyses do not always show the systematic carrying of pathogens per bee and the result is not representative in this case of the state of infection of their colony.

Thus, we can better understand the collapse of the densest colonies, those for which the observation “at the flight hole” is the most reassuring. Indeed, in case of overpopulation, the rubbing of bees against each other provokes micro lesions of the cuticle (in the same way as organic acids) which prove to be the most efficient entry door for the dissemination of viruses.

Be careful, the risk of collapse is important when going from a normal population to a colony with a large population. Thus, the removal of the supers may cause a sudden increase in the population density and initiate the development of various pathogens.

Dr Gilles Grosmond, veterinary expert, solu’nature

qPCR versus petri dish

qPCR versus petri dish

PCR versus petri dish

Real-time PCR is known to allow the isolation, identification and quantification of DNA from bacteria, viruses, parasites, allergens, GMOs in a very short time of about 1 to 3 hours in all fields: food hygiene, animal health and/or human health, cosmetic industries…

For a long time restricted for price reasons to the detection of a few pathogens in food hygiene or to short-lived products, the objective of Professor Michel Franck, CEO of ADNucleis, is to broaden the indications of this PCR technique to the identification and quantification of traditional hygiene indicators (Total Flora, E.coli, coliforms, Pseudomonas spp., Listeria spp., molds, yeasts…) and/or pathogens (Salmonella, Listeria monocytogenes, Cronobacter sakazakii, STEC such as E.coli O157H7, viruses, parasites…). This objective is now achieved thanks to a new “breakthrough” method at a price equivalent to the price of cultural methods.

Associated with the SEQUENCE PRO PCR robot – much less expensive than traditional pipetting machines – a compact robot (65cm x 65cm) that can be directly positioned on production lines, real-time PCR analysis can be performed by personnel not qualified in molecular biology, in situ and in real-time.

Thanks to its industrial partners such as Pasquier, ADNucleis continues to make real-time PCR an industrial standard, notably by simplifying and robotizing the entire analytical process.