Rapidly Quantitative Detection of in Honeybees Using Ultra-rapid Real-time Quantitative PCR

Overview

Journal J Vet Sci

Specialty Veterinary Medicine

Date 2021 May 31

PMID 34056881

Citations 4

Authors

A Tai Truong

Sedat Sevin

Seonmi Kim

Mi Sun Yoo

Yun Sang Cho

Byoungsu Yoon

Affiliations

Soon will be listed here.

Abstract

Background: The microsporidian parasite is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.

Objectives: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of in infected bees.

Methods: A procedure for UR-qPCR detection of was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.

Results: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with with 2.74 × 10⁴ copies of DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.

Conclusions: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

Citing Articles

Prevalence of honey bee pathogens and parasites in South Korea: A five-year surveillance study from 2017 to 2021.

Truong A, Yoo M, Seo S, Hwang T, Yoon S, Cho Y Heliyon. 2023; 9(2):e13494.

PMID: 36816323 PMC: 9929316. DOI: 10.1016/j.heliyon.2023.e13494.

Beneficial Bacteria and Plant Extracts Promote Honey Bee Health and Reduce Nosema ceranae Infection.

Garrido P, Porrini M, Alberoni D, Baffoni L, Scott D, Mifsud D Probiotics Antimicrob Proteins. 2023; 16(1):259-274.

PMID: 36637793 PMC: 10850026. DOI: 10.1007/s12602-022-10025-7.

Insights into the Role of Natural Products in the Control of the Honey Bee Gut Parasite ( spp.).

El-Seedi H, El-Wahed A, Al Naggar Y, Saeed A, Xiao J, Ullah H Animals (Basel). 2022; 12(21).

PMID: 36359186 PMC: 9656094. DOI: 10.3390/ani12213062.

Molecular Detection and Differentiation of Arthropod, Fungal, Protozoan, Bacterial and Viral Pathogens of Honeybees.

Lannutti L, Gonzales F, Dus Santos M, Florin-Christensen M, Schnittger L Vet Sci. 2022; 9(5).

PMID: 35622749 PMC: 9145064. DOI: 10.3390/vetsci9050221.

References

LARKIN M, Blackshields G, Brown N, Chenna R, McGettigan P, McWilliam H . Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23(21):2947-8. DOI: 10.1093/bioinformatics/btm404. View

Rubanov A, Russell K, Rothman J, Nieh J, McFrederick Q . Intensity of Nosema ceranae infection is associated with specific honey bee gut bacteria and weakly associated with gut microbiome structure. Sci Rep. 2019; 9(1):3820. PMC: 6405881. DOI: 10.1038/s41598-019-40347-6. View

Erler S, Lommatzsch S, Lattorff H . Comparative analysis of detection limits and specificity of molecular diagnostic markers for three pathogens (Microsporidia, Nosema spp.) in the key pollinators Apis mellifera and Bombus terrestris. Parasitol Res. 2011; 110(4):1403-10. DOI: 10.1007/s00436-011-2640-9. View

Glenny W, Cavigli I, Daughenbaugh K, Radford R, Kegley S, Flenniken M . Honey bee (Apis mellifera) colony health and pathogen composition in migratory beekeeping operations involved in California almond pollination. PLoS One. 2017; 12(8):e0182814. PMC: 5560708. DOI: 10.1371/journal.pone.0182814. View

Chen Y, Evans J, Murphy C, Gutell R, Zuker M, Gundensen-Rindal D . Morphological, molecular, and phylogenetic characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera. J Eukaryot Microbiol. 2009; 56(2):142-7. PMC: 2779023. DOI: 10.1111/j.1550-7408.2008.00374.x. View

Hamiduzzaman M, Guzman-Novoa E, Goodwin P . A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera). J Invertebr Pathol. 2010; 105(2):151-5. DOI: 10.1016/j.jip.2010.06.001. View

Riviere M, Ribiere M, Chauzat M . Recent molecular biology methods for foulbrood and nosemosis diagnosis. Rev Sci Tech. 2014; 32(3):885-92. DOI: 10.20506/rst.32.2.2207. View

Fu Z, He X, Cai S, Liu H, He X, Li M . Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae. J Microbiol Methods. 2015; 120:72-8. DOI: 10.1016/j.mimet.2015.12.003. View

Bourgeois A, Rinderer T, Beaman L, Danka R . Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee. J Invertebr Pathol. 2009; 103(1):53-8. DOI: 10.1016/j.jip.2009.10.009. View

10.

Fries I . Nosema ceranae in European honey bees (Apis mellifera). J Invertebr Pathol. 2009; 103 Suppl 1:S73-9. DOI: 10.1016/j.jip.2009.06.017. View

11.

Klee J, Besana A, Genersch E, Gisder S, Nanetti A, Tam D . Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera. J Invertebr Pathol. 2007; 96(1):1-10. DOI: 10.1016/j.jip.2007.02.014. View

12.

Forsgren E, Fries I . Comparative virulence of Nosema ceranae and Nosema apis in individual European honey bees. Vet Parasitol. 2010; 170(3-4):212-7. DOI: 10.1016/j.vetpar.2010.02.010. View

13.

Lawyer F, Stoffel S, Saiki R, Chang S, Landre P, Abramson R . High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity. PCR Methods Appl. 1993; 2(4):275-87. DOI: 10.1101/gr.2.4.275. View

14.

Ptaszynska A, Borsuk G, Wozniakowski G, Gnat S, Malek W . Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees. FEMS Microbiol Lett. 2014; 357(1):40-8. DOI: 10.1111/1574-6968.12521. View

15.

Williams G, Shafer A, Rogers R, Shutler D, Stewart D . First detection of Nosema ceranae, a microsporidian parasite of European honey bees (Apis mellifera), in Canada and central USA. J Invertebr Pathol. 2007; 97(2):189-92. DOI: 10.1016/j.jip.2007.08.005. View

16.

Higes M, Martin-Hernandez R, Botias C, Bailon E, Gonzalez-Porto A, Barrios L . How natural infection by Nosema ceranae causes honeybee colony collapse. Environ Microbiol. 2008; 10(10):2659-69. DOI: 10.1111/j.1462-2920.2008.01687.x. View

17.

Refardt D, Ebert D . Quantitative PCR to detect, discriminate and quantify intracellular parasites in their host: an example from three microsporidians in Daphnia. Parasitology. 2006; 133(Pt 1):11-8. DOI: 10.1017/S0031182006000060. View

18.

Gray F, Cali A, Briggs J . Intracellular stages in the life cycle of the microsporidian Nosema apis. J Invertebr Pathol. 1969; 14(3):391-4. DOI: 10.1016/0022-2011(69)90167-0. View

19.

Higes M, Martin R, Meana A . Nosema ceranae, a new microsporidian parasite in honeybees in Europe. J Invertebr Pathol. 2006; 92(2):93-5. DOI: 10.1016/j.jip.2006.02.005. View

20.

Martin-Hernandez R, Meana A, Prieto L, Salvador A, Garrido-Bailon E, Higes M . Outcome of colonization of Apis mellifera by Nosema ceranae. Appl Environ Microbiol. 2007; 73(20):6331-8. PMC: 2075036. DOI: 10.1128/AEM.00270-07. View