Short Communication |
Corresponding author: Lubiane Guimarães-Cestaro ( lubi.guimaraes@gmail.com ) Academic editor: Jack Neff
© 2016 Lubiane Guimarães-Cestaro, José Eduardo Serrão, Dejair Message, Marta Fonseca Martins, Érica Weinstein Teixeira.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Guimarães-Cestaro L, Serrão JE, Message D, Martins MF, Teixeira EW (2016) Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products. Journal of Hymenoptera Research 49: 43-50. https://doi.org/10.3897/JHR.49.7061
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Honey bees are responsible for pollinating many native and cultivated plant species. These insects can be affected by many pathogens, including fungi and bacteria, both of which can form spores that are easily dispersed within the colony by means of the stored products, among other routes. The objective of this study was to develop a method to detect spores of the honey bee pathogens Nosema apis, Nosema ceranae, Ascosphaera apis and Paenibacillus larvae in samples of honey, bee pollen and royal jelly. The method was standardized for each product individually, and then analyzed by monoplex and multiplex PCR, which showed the same detection thresholds: 1.25 spores/mL of honey for N. ceranae; 7.5 spores/mL of honey for A. apis; and 0.4 spore/mL of honey for P. larvae, respectively. The standardized technique was effective and rapid for the detection of these pathogens in bee products and can be used for the establishment of official methods of sanitary control of bee products, considering the growing national and international trade of these products and the movement or migration of colonies between regions.
Apis mellifera , Bee pathogens, Honey, Pollen, Royal Jelly, Detection of spores
The honey bee, Apis mellifera, is essential to global agriculture by pollinating a wide range of food crops, besides supplying economically important products (
Within the colony, products like honey, pollen and royal jelly are susceptible to contamination by these pathogens, mainly through storage in honeycomb and trophallaxis (
Multiplex PCR is used if more than one pathogen is amplified simultaneously in a single reaction, resulting in considerable savings of both time and effort (
The aim of this study was to develop a method to detect spores of N. apis, N. ceranae, A. apis and P. larvae in honey, bee pollen and royal jelly samples.
Solutions containing spores of Nosema ceranae and Ascosphaera apis were prepared from naturally contaminated bees according the protocols described by
For detection of the pathogens, the samples were weighed (10 g for pollen, 5 g for royal jelly and 20 mL for honey) in sterile tubes and diluted in 40 mL of sterile distilled water. The samples of pollen were diluted in 45 mL of sterile distilled water. The samples were vigorously homogenized and centrifuged at 12,500 × g for 40 min. The supernatant was discarded and the pellet was resuspended in 1 mL of sterile distilled water, with subsequent homogenization and centrifugation at 10,000 × g for 20 min. The supernatant was again discarded and the pellet was submitted to DNA extraction using the Qiagen DNeasy® Plant Mini Kit, following the manufacturer’s instructions. In the case of bee pollen, after homogenization the samples were filtered with Whatman 1 filter paper using a vacuum pump and then the same procedure was followed as for the samples of honey and royal jelly.
Monoplex and multiplex PCR were carried out following
Decreasing spore concentrations were tested (2500, 250, 100, 50, 25 spores of Nosema ceranae; 3000, 300, 150, 75 spores of Ascosphaera apis; 1000, 100, 10, 8, 4, 1 spores of Paenibacillus larvae), until reaching the minimum detectable number, in order to identify the technique’s sensitivity. All the analyses were performed at the Honey bee Health Laboratory of São Paulo State Agribusiness Technology Agency (LASA/APTA), located in Pindamonhangaba, São Paulo.
Multiplex PCR allowed the simultaneous detection of N. ceranae, A. apis and P. larvae in different bee products, and the amplified product for each pathogen presented the expected fragment lengths (218 bp, 485 bp and 700 bp, respectively). Threshold values were the same for the monoplex and multiplex reactions (Figure
Agarose gel (2%) stained with SYBR® Safe of the monoplex and multiplex PCR products referring to the detection thresholds of the pathogens in honey bee products. M: 100 bp marker. 1 2.5 spores/g of pollen for Nosema ceranae 2 15 spores/g of pollen for Ascosphaera apis 3 0.8 spore/g of pollen for Paenibacillus larvae 4 multiplex PCR for N. ceranae (2.5 spores/g of pollen), A. apis (15 spores/g of pollen) and P. larvae (0.8 spore/g of pollen) 5 multiplex positive controls for N. ceranae (218 bp), N. apis (321 bp), A. apis (485 bp) and P. larvae (700 bp) 6 negative control. M: 100 bp marker 7 1.25 spores/mL of honey for N. ceranae 8 7.5 spores/mL of honey for A. apis 9 0.4 spore/mL of honey for P. larvae 10 multiplex PCR for N. ceranae (1.25 spores/mL of honey), A. apis (7.5 spores/mL of honey) and P. larvae (0.4 spore/mL of honey) 11 multiplex positive controls for N. ceranae (218 bp), N. apis (321 bp), A. apis (485 bp) and P. larvae (700 bp) 12 negative control. M: 100 bp marker 13 5 spores/g of royal jelly for N. ceranae 14 30 spores/g of royal jelly for A. apis 15 1.6 spores/g of royal jelly for P. larvae 16 multiplex PCR for N. ceranae (5 spores/g of royal jelly), A. apis (30 spores/g of royal jelly) and P. larvae (1.6 spores/g of royal jelly) 17 multiplex positive controls for N. ceranae (218 bp), N. apis (321 bp), A. apis (485 bp) and P. larvae (700 bp) 18 negative control.
Because N. apis occurs in very low prevalence (
Detection of Nosema apis, N. ceranae, Ascosphaera apis and Paenibacillus larvae in samples of bee products has been reported (
Some studies have presented detection limits that corroborate this idea.
Although the technique presented here is similar to those employed by other authors, our tests showed that the samples centrifuged below 12,000 × g did not undergo sedimentation, and consequently a considerable number of spores were still present in the supernatant.
To assure that no spores remained in the supernatant, besides performing a centrifugation at 12,000 × g for 40 min, we also submitted the resuspended pellet obtained to new centrifuging at 10,000 × g for 20 min, for subsequent extraction of DNA. This factor is likely responsible for the technique’s sensitivity.
For detection of the bacterium Paenibacillus larvae, various methods have been described, mainly using growth in culture medium and PCR (
According to the
Microbiological techniques have also been used to detect Paenibacillus larvae, but it is important to consider the time for analysis and efficacy in detecting this bacterium. According to the official technique specified in Brazil (
The protocol developed showed important results when used to analyze samples of bee products marketed in São Paulo state (in preparation), supporting the idea that PCR is a fast and reliable technique to diagnose pathogen infections (
The multiplex PCR method offers a significant cost-saving advantage, especially when large numbers of samples are analyzed (
The protocol presented here is useful to detect simultaneously Nosema apis, Nosema ceranae, Ascosphaera apis and Paenibacillus larvae in samples of honey, pollen and royal jelly and can support definition of official methods for surveillance actions.
This work was supported by the São Paulo State Research Foundation (FAPESP, EWT 2012/18802-3). We thank the Office to Improve University Personnel (CAPES) for the scholarship funding, the São Paulo State Agribusiness Technology Agency (APTA, SAA-SP) for the institutional support, and Maria Luisa T. M. F. Alves for her help with the analyses. MFM is a research fellow of the National Council for Scientific and Technological Development (CNPq). JES is a research fellow of the CNPq and Minas Gerais Research Foundation (FAPEMIG).