The Use of RIDA^®COUNT for Monitoring the American Foulbrood Pathogen

Stepan Ryba, Vaclav Kristufek, Dalibor Titera

Bee Research Institute at Dol, Libcice nad Vltavou, Czech Republic.
Biology Centre of the Academy of Sciences of the Czech Republic, v. v. i., Institute of Soil Biology, Ceske Budejovice, Czech Republic.
Faculty of Science, Charles University in Prague, Prague, Czech Republic.

DOI: 10.4236/ojvm.2012.24037                

Abstract

American Foulbrood (AFB) is currently one of the most dangerous diseases in honeybees due to its high virulence and worldwide spread. Quick evaluation of the diagnosis of this disease is crucial. Successful eradication in the area indicates a need to test all bee colonies, but this is expensive and time consuming. A new method of detecting Paenibacillus larvae using the RIDA^?COUNT test (R-Biopharm AG, Germany) was verified in the present study. The test is based on the principle of the cultivation test with MYPGPn medium, coloration of the bacteria with TTC (2,3,5-triphenyltetra-zoliumchloride) chromophore, and heat treatment of the sample. Using this new method, color-highlighted colonies of P. larvae can be established on the seventh day after inoculating the spores. An identical number of colonies grown with the classic cultivation test on Petri dishes containing MYPGPn medium or RIDA^?COUNT-P. larvae (RC-PL) sheets were verified.

Keywords

AFB; Apis mellifera L.; Bacterial Disease; Diagnosis; Diseases of Honeybees; MYPGPn Medium; Paenibacillus larvae

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	H. Hansen and C. J. Brodsgaard, “American Foulbrood: A Review of Its Biology, Diagnosis and Control,” Bee World, Vol. 80, No. 1, 1999, pp. 195-201.
[2]	D. C. de Graaf, A. M. Alippi, M. Brown, J. D. Evans, M. Feldlaufer, A. Gregorc, M. Hornitzky, S. F. Pernal, D. M. Schuch, D. Titera, V. Tomkies and W. Ritter, “Diagnosis of American Foulbrood in Honey Bees: A Synthesis and Proposed Analytical Protocols,” Letters in Applied Microbiology, Vol. 43, No. 6, 2006, pp. 583-590. doi:10.1111/j.1472-765X.2006.02057.x
[3]	D. C. de Graaf, P. De Vos, M. Heyndrickx, S. Van Trappen, N. Peiren and F. J. Jacobs, “Identification of Paenibacillus larvae to the Subspecies Level: An Obstacle for AFB Diagnosis,” Journal of Invertebrate Pathology, Vol. 91, No. 2, 2006, pp. 115-123. doi:10.1016/j.jip.2005.10.010
[4]	E. Genersch, E. Forsgren, J. Pentikainen, A. Ashiralieva, S. Rauch, J. Kilwinski and I. Fries, “Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without Subspecies Differentiation,” International Journal of Systematic and Evolutionary Microbiology, Vol. 56, No. 3, 2006, pp. 501-511. doi:10.1099/ijs.0.63928-0
[5]	U. Riessberger-Galle, W. von der Ohe and K. Crailsheim, “Adult Honeybee’s Resistance against Paenibacillus larvae larvae, the Causative Agent of the American Foulbrood,” Journal of Invertebrate Pathology, Vol. 77, No. 4, 2001, pp. 231-236. doi:10.1006/jipa.2001.5032
[6]	N. Bakhiet and D. P. Stahly, “Ultrastructure of Sporulating Bacillus larvae in a Broth Medium,” Applied and Environmental Microbiology, Vol. 50, No. 3, 1985, pp. 690-692.
[7]	D. Titera and M. Haklova, “Detection Method of Paenibacillus larvae Larvae from Beehive Winter Debris,” Apimondia, Vol. 38, 2003, pp. 131-133.
[8]	T. Bakonyi, I. Derakhshifar, E. Grabensteiner and N. Nowotny, “Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization,” Applied and Environmental Microbiology, Vol. 69, No. 3, 2003, pp. 1504-1510. doi:10.1128/AEM.69.3.1504-1510.2003
[9]	H. Morita, M. Ushiyama, S. Aoyama and M. Iwasaki, “Sensitivity and Specificity of the Sanita-Kun Aerobic Count: Internal Validation and Independent Laboratory Study,” Journal of AOAC International, Vol. 86, No. 2, 2003, pp. 355-366.
[10]	F. M. Lauro, M. Favaretto, L. Covolo, M. Rassu and G. Bertoloni, “Rapid Detection of Paenibacillus larvae from Honey and Hive Samples with a Novel Nested PCR pRotocol,” International Journal of Food Microbiology, Vol. 81, No. 3, 2003, pp. 195-201. doi:10.1016/S0168-1605(02)00257-X
[11]	V. A. Govan, M. H. Allsopp and S. Davison, “A PCR Detection Method for Rapid Identification of Paenibacillus larvae,” Applied and Environmental Microbiology, Vol. 65, No. 5, 1999, pp. 2243-2245.
[12]	S. Ryba, D. Titera, M. Haklova and P. Stopka, “A PCR Method of Detecting American Foulbrood (Paenibacillus larvae) in Winter Beehive Wax Debris,” Veterinary Microbiology, Vol. 139, No. 1-2, 2009, pp. 193-196. doi:10.1016/j.vetmic.2009.05.009
[13]	J. Mulec, V. Kristufek and A. Chronakova, “Comparative Microbial Sampling from Eutrophic Caves in Slovenia and Slovakia Using RIDA?COUNT Test Kits. Use of RIDA?COUNT in Caves,” International Journal of Speleology, Vol. 41, No. 1, 2012, pp. 1-8. doi:10.5038/1827-806X.41.1.1