Comparison of methods for detection of microsporidia species of the genus Nosema in honey bees (Apis mellifera)

Abstract

Two microsporidia species of the Nosema genus cause nosemosis in the adult honeybee: N. apis and N. ceranae. For diagnostic purposes and the determination of infection level various microscopic and molecular biological methods are used. The aim of this research was to compare the reliability of the traditional microscopic assessment and two PCR techniques: simplex- and duplex-PCR. Honey bee samples were taken from 150 colonies. Microscopic examination, performed according to the recommendations of the OIE, revealed Nosema spores in 68.67% samples analysed, whilst with the simplex-PCR method all samples (100.0%) proved positive. On the other hand, duplex-PCR method used for the identification of Nosema species resulted in 84.0% positive samples, all of which were N. ceranae. Our recommendation of the simplex-PCR method for the monitoring of honey bees in field conditions is based on its higher reliability than the microscopic assessment in the detection of low-level infections, as well as its potential for the detection of vegetative Nosema sp. stages; thus the early detection and timely prevention of Nosema infection would be possible. Nosema species identification is simplest and most cost-effective if performed with the duplex-PCR analysis. However, the simplex-PCR is more reliable, thus, it is suggested that samples that were negative when assessed with microscopy and duplex-PCR analysis undergo simplex-PCR.

Dve vrste mikrosporidija roda Nosema uzročnici su nozemoze kod odrasle medonosne pčele: N. apis i N. ceranae. Za postavljanje dijagnoze i utvrđivanje stepena infekcije koristi se nekoliko mikroskopskih i molekularno-bioloških metoda. Cilj našeg rada bilo je poređenje pouzdanosti tradicionalne mikroskopske metode i dve PCR metode: simplex- i duplex-PCR. Pregledano je ukupno 150 uzoraka pčela. Mikroskopskim pregledom, obavljenim prema preporukama OIE, prisustvo spora Nosema utvrđeno je u 68,7% uzoraka. Međutim, simplex-PCR metodom dobijeni su pozitivni rezultati u svih 150 uzorka (100,0%). Sa druge strane, primenom duplex-PCR metode infekcija je ustanovljena kod 84,0 %; u svim slučajevima determinisana je vrsta N. ceranae. Veća pouzdanost simplex-PCR metode u odnosu na mikroskopski pregled, kako u otkrivanju infekcije malog intenziteta, kao i mogućnost detekcije vegetativnih oblika nozeme, navodi nas da preporučimo uvođenje simplex-PCR metode kao obavezne za praćenje stanja pčelinjih društava na terenu; time bi se postigla rano utvrđivanje prisustva infekcije i blagovremena prevencija njenog širenja. Specijska identifikacija mikrosporidija roda Nosema najjednostavnija je i najisplativija metodom duplex-PCR. Međutim, simplex-PCR ima veću pouzdanost, te preporučujemo da se uzorci koji su negativni na osnovu mikroskopskog pregleda i duplex-PCR analize ispitaju i simplex-PCR metodom.