Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
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. ijado@isciii.es
Isabel
I
Jado
Cristina
C
Carranza-Rodríguez
Jesús Félix
JF
Barandika
Álvaro
Á
Toledo
Cristina
C
García-Amil
Beatriz
B
Serrano
Margarita
M
Bolaños
Horacio
H
Gil
Raquel
R
Escudero
Ana L
AL
García-Pérez
A Sonia
AS
Olmeda
Ianire
I
Astobiza
Bruno
B
Lobo
Manuela
M
Rodríguez-Vargas
José Luis
JL
Pérez-Arellano
Fernando
F
López-Gatius
Francisco
F
Pascual-Velasco
Gustavo
G
Cilla
Noé F
NF
Rodríguez
Pedro
P
Anda
D000818:Animals; D002417:Cattle; D016997:Coxiella burnetii / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004783:Environmental Microbiology; D014644:Genetic Variation; D005838:Genotype; D006041:Goats; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods; D058889:Molecular Typing; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D011778:Q Fever / Q000382:microbiology* / Q000662:veterinary*; D051381:Rats; D012756:Sheep; D013030:Spain; D034421:Sus scrofa; D013987:Ticks
D015345:Oligonucleotide Probes