Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Identification of Diagnostic PCR Markers for Honeybee Foulbrood Disease from Specific Genes of Paenibacillus larvae

부저병 원인균 Paenibacillus larvae 특이 유전자 분석을 통한 진단마커 발굴

  • Na, Han-Heom (Department of Biological Sciences, College of Natural Sciences, Kangwon National University) ;
  • Kim, Keun-Cheol (Department of Biological Sciences, College of Natural Sciences, Kangwon National University)
  • 나한흠 (강원대학교 자연과학대학 생명과학과) ;
  • 김근철 (강원대학교 자연과학대학 생명과학과)
  • Received : 2016.08.26
  • Accepted : 2016.12.22
  • Published : 2017.01.30
Download PDF
⟨ Previous Next ⟩

Abstract

Foulbrood disease is infected by Paenibacillus larvae on larval stage of honeybee, and is lethal disease to result in population death. This disease was manifested in 2008 in Korea, is still suffered by the secondary damages. In this study, we are to examine diagnostic PCR approaches to manage the Foulbrood disease. PCR amplification of 16S rRNA is generally using for microbial infection, but the specificity is little poor for the correct diagnosis. Therefore, we are to identify specific genes expressed in Paenibacillus larvae, and perform PCR analysis. We selected five distinct genes from literature references. Those genes are commonly known as toxic genes for host infection, and include Toxin1, Toxin2A & 2B, SplA, CBP49, and SevA&SevB. PCR amplification for these genes is difficult to detect at the first time. So, we performed the second PCR using the first PCR product as a template. This approach using the nested PCR was very useful for detecting large marker genes. When Paenibacillus larvae was cultured in the medium containing plant extracts, PCR amplification of the identified genes is correlated with the microbial growth inhibition. Therefore, these results suggest that the identified genes might be useful to study diagnostic PCR markers for honeybee Foulbrood disease.

부저병이란 Paenibacillus larvae감염에 의하여 꿀벌 유충의 괴사를 유도하는 질병이다. 우리나라에서는 2008년 봄, 국내에 처음으로 대량 발병 사례가 보고되었으며, 지속적인 2차 피해로 큰 후유증을 앓고 있다. 본 연구에서는 부저병을 효율적으로 관리할수 있는 진단방법을 조사하고자 하였다. 따라서 부저병의 원인균인 P. larvae에서 특이적으로 발현되고 있는 유전자들을 동정하고자 하였으며, 이 유전자들은 주로 부저병균의 독성을 유발하는 것으로 알려진 Toxin1, Toxin2A & 2B, SplA, CBP49, SevA&SevB 들이다. 이들은 1차 PCR 에서는 검출하기 어려웠지만, 2차 nested PCR방법을 이용하여 검출이 용이함을 알 수 있었다. 한편 여러가지 식물 추출물을 혼합한 배지에서 부저병균을 배양하였을 때, 부저병균의 성장저해와 일치하게 우리가 검증한 유전자들의 발현이 감소하는 것을 알 수 있었다. 이러한 결과들은 부저병 원인균의 특이 유전자들은 향후 PCR진단마커로서 활용 가능성이 있을 것으로 사료된다.

Keywords

References

  1. Alvarado, I., Elekonich, M. M., Abel-Santos, E. and Wing, H. J. 2015. Comparison of in vitro methods for the production of Paenibacillus larvae endospores. J. Microbiol. Methods 116, 30-32. https://doi.org/10.1016/j.mimet.2015.06.011
  2. Antunez, K., Harriet, J., Gende, L., Maggi, M., Eguaras, M. and Zunino, P. 2008. Efficacy of natural propolis extract in the control of American Foulbrood. Vet. Microbiol. 131, 324-331. https://doi.org/10.1016/j.vetmic.2008.04.011
  3. Arai, R., Miyoshi-Akiyama, T., Okumura, K., Morinaga, Y., Wu, M., Sugimura, Y., Yoshiyama, M., Okura, M., Kirikae, T. and Takamatsu, D. 2014. Development of duplex PCR assay for detection and differentiation of typical and atypical Melissococcus plutonius strains. J. Vet. Med. Sci. 76, 491-498. https://doi.org/10.1292/jvms.13-0386
  4. Chan, Q. W., Melathopoulos, A. P., Pernal, S. F. and Foster, L. J. 2009. The innate immune and systemic response in honey bees to a bacterial pathogen, Paenibacillus larvae. BMC Genomics 10, 387-2164-10-387. https://doi.org/10.1186/1471-2164-10-387
  5. Djukic, M., Brzuszkiewicz, E., Funfhaus, A., Voss, J., Gollnow, K., Poppinga, L., Liesegang, H., Garcia-Gonzalez, E., Genersch, E. and Daniel, R. 2014. How to kill the honey bee larva: genomic potential and virulence mechanisms of Paenibacillus larvae. PLoS One 9, e90914. https://doi.org/10.1371/journal.pone.0090914
  6. Ebeling, J., Knispel, H., Hertlein, G., Funfhaus, A. and Genersch, E. 2016. Biology of Paenibacillus larvae, a deadly pathogen of honey bee larvae. Appl. Microbiol. Biotechnol. 100, 7387-7395. https://doi.org/10.1007/s00253-016-7716-0
  7. Funfhaus, A., Poppinga, L. and Genersch, E. 2013. Identification and characterization of two novel toxins expressed by the lethal honey bee pathogen Paenibacillus larvae, the causative agent of American foulbrood. Environ. Microbiol. 15, 2951-2965.
  8. Han, S. H., Lee, D. B., Lee, D. W., Kim, E. H. and Yoon, B. S. 2008. Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB). J. Invertebr. Pathol. 99, 8-13. https://doi.org/10.1016/j.jip.2008.04.010
  9. Lugo, M. R., Ravulapalli, R., Dutta, D. and Rod Merrill, A. 2016. Structural variability of C3larvin toxin. Intrinsic dynamics of the alpha/beta fold of the C3-like group of mono-ADP-ribosyltransferase toxins. J. Biomol. Struct. Dyn. 1-24.
  10. Okolie, C. E., Wooldridge, K. G., Turner, D. P., Cockayne, A. and James, R. 2015. Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes. BMC Microbiol. 15, 157-015-0490-9.
  11. Poppinga, L., Janesch, B., Funfhaus, A., Sekot, G., Garcia-Gonzalez, E., Hertlein, G., Hedtke, K., Schaffer, C. and Genersch, E. 2012. Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees. PLoS Pathog. 8, e1002716. https://doi.org/10.1371/journal.ppat.1002716
  12. Riessberger-Galle, U., Hernandez-Lopez, J., Rechberger, G., Crailsheim, K. and Schuehly, W. 2016. Lysophosphatidylcholine acts in the constitutive immune defence against American foulbrood in adult honeybees. Sci. Rep. 6, 30699. https://doi.org/10.1038/srep30699
  13. Saraithong, P., Li, Y., Saenphet, K., Chen, Z. and Chantawannakul, P. 2015. Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing. Insect Sci. 22, 606-618. https://doi.org/10.1111/1744-7917.12155
  14. Takamatsu, D., Sato, M. and Yoshiyama, M. 2016. Infection of Melissococcus plutonius clonal complex 12 strain in European honeybee larvae is essentially confined to the digestive tract. J. Vet. Med. Sci. 78, 29-34. https://doi.org/10.1292/jvms.15-0405