Development of a hemi-nested PCR assay for the specific detection ofMelissococcus pluton

Abstract

A pair of oligonucleotide primers (MP1 and MP2) were used for the polymerase chain reaction (PCR) amplification of a 486 base pair (bp) fragment of the 16S rRNA gene of 26 geographically diverse Australian Melissococcus pluton (causative agent of European foulbrood) isolates. PCR primers spanning a region of the 16S rRNA gene from position 893-1377 failed to amplify a product when template DNA from a wide range of pathogenic and saprophytic bacteria were used including Paenibacillus larvae, Paenibacillus alve~ Enterococcus faecium and Spiroplasma melliferum. The PCR did, however; reliably amplify a 486 bp fragment (when the annealing temperature was lowered by 5°C) using template DNA isolated from the phylagenetically-related bacterium Enterococcus faecalis. PCR amplicons generated from E faecalis and M. pluton were readily distinguished by digestion with the restriction endonuclease Hinfl and electrophoresis in 1.5% agarose or by electrophoresis in 1% agarose containing bisbenzidene/polyethylene glycol. A hemi-nested PCR requiring a combination of primers MP1 and a third primer, MP3, which spanned 25 nucleotides from position 1168--1144 and internal to the 486 bp amplicon generated by primers MP1 and MP2 was developed. The hemi-nested PCR amplified a 276 bp M. plutonspecific product that was not amplified with E faecalis DNA. In sensitivity studies, the PCR assay could reliably detect approximately 1-10 organisms/mi. This level of sensitivity was achieved using crude DNA templates (boiled cell lysate) prepared using lnstagene matrix. The PCR assay could also detect M. pluton in brood with European foulbrood. Downloaded by [University of Birmingham] at 08

An in vitro evaluation of Cinnamon (Cinnamomum spp.) and Siwak (Salvadora persica) extracts for controlling the foulbrood pathogens of honeybee

“…While the bacterial colonies which appeared very slowly on Bailey's medium belonging to M. plutonius bacterium (Figure 6) had the description of a single colony, very small measuring 1 to 2 mm in diameter, biconvex, shape circular, had regular edges, white of color and sometimes grow deeply into the medium, and this description agreed with the study of Djordjevic et al (1998). Moreover, the used strains whether P. larvae or M. plutonius were catalase (-) and gram (+) (Figures 7 to 10) according to Djordjevic et al (1998) and Piccini and Zunino (2001). Table 1 that all used extracts of the two plants had antibacterial activities at their cleared concentrations against both of the two foulbrood bacterial growth on the culturing media (Figures 11 to 18) but with varying degrees and without inducing of a complete inhibition.…”

“…For confirming the isolates, some tests were performed such as description of the bacterial colonies characters according to Djordjevic et al (1998) and Del Hoyo et al (2001), catalase production, Gram stain test and the negative stain according to Shimanuki and Knox (2000).…”

The detection of Melissococcus pluton in honey bees (Apis mellifera) and their products using a hemi-nested PCR

“…Attempts by Djordjevic et al (1998a) to use this assay for the detection of M. pluton in honey and adult bees were unsuccessful (Djordjevic, unpublished results). More sensitive and robust methods using PCR for the detection of M. pluton in bees and bee products are important to better understand the ecology of M. pluton and for the certification of bee products free from M. pluton for export purposes.…”

“…Govan et al (1998) tested three bacterial species (Escherichia coli, Paenibacillus alvei and Staphylococcus aureus) to determine the specificity of their assay but did not determine the sensitivity of the assay. Djordjevic et al (1998a) described a hemi-nested PCR for which the amplification of the first generation product was shown to be specific for M. pluton when tested against 27 bacterial species which were closely phylogenetically related to M. pluton or commonly cultured from honey bee hives. The first generation PCR was capable of detecting between 1-10 M. pluton organisms/ml in serial titrations of an in vitro culture of M. pluton, a level of detection considerably more sensitive than direct culture.…”

“…Culture procedures, serological procedures, scanning electron microscopy and the preparation of smears of such material are time consuming and often ineffective or insensitive for such purposes. The aim of this study was to further develop the hemi-nested PCR (Djordjevic et al, 1998a) for the sensitive detection of M. pluton in honey, pollen, whole larvae and adult bee tissues, and also to determine the prevalence of M. pluton in Australian bulk honey samples using PCR compared to conventional culture techniques.…”