Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain
1 Laboratorio de Espiroquetas y Patógenos Especiales, Department of Bacteriology, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. de Pozuelo km 2.6, Majadahonda, Madrid, 28220, Spain
2 Microbiology Service and Infectious and Tropical Diseases Unit, Hospital Universitario Insular de Gran Canaria, Av Marítima del Sur, s/n, 35016, Las Palmas, Spain
3 Department of Production and Animal Health, NEIKER - Instituto Vasco de Investigación y Desarrollo Agrario, Berreaga Kalea, 1, Derio, Bizkaia, 48160, Spain
4 Department of Animal Health, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro, s/n, Madrid, 28040, Spain
5 Department of Animal Production, Universidad de Lleida, Pl. de Víctor Siurana, 1, Lleida, 25003, Spain
6 Internal Medicine Service, Hospital Comarcal de Laredo, Avda. de los Derechos Humanos, s/n, Laredo, Cantabria, 39770, Spain
7 Hospital Universitario Donostia and CIBERES, P° Doctor José Beguiristain s/n, Donostia-San Sebastián, Guipúzcoa, 20014, Spain
8 Department of Animal Medicine and Surgery, Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria, Campus Universitario de Arucas s/n, Arucas, Las Palmas, 35413, Spain
9 Present Address: Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, NY, 11794-5120, USA
BMC Microbiology 2012, 12:91 doi:10.1186/1471-2180-12-91Published: 1 June 2012
Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes.
To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples.
This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.