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

Abstract

BackgroundCoxiella 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.ResultsTo 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.ConclusionsThis 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.

Coxiella burnetii total immunoglobulin G, phase I and phase II immunoglobulin G antibodies, and bacterial shedding in young dams in persistently infected dairy herds

“…9 The C. burnetii strain that circulates in Spain represents a low risk for Q fever transmission in zoonotic terms. 5,20,23 However, further studies designed to examine the cellular response are needed to clarify the pathogenesis of coxiellosis during acute infection in cattle.…”

Host and Environmental Factors Modulate the Exposure of Free-Ranging and Farmed Red Deer (Cervus elaphus) to Coxiella burnetii

“…Although the majority of human Q fever outbreaks are linked to the transmission of C. burnetii from domestic ruminants (4,5), the ability of C. burnetii to infect wild hosts (3,6) and its high environmental resistance (1) make wildlife species potential reservoirs of C. burnetii. Based on this hypothesis, wildlife could maintain C. burnetii and transmit it to wildlife (7), domestic animals (8), or humans (9). It is therefore of paramount relevance (i) to identify those potential wild reservoir species that could, through direct and indirect interactions, transmit C. burnetii to target species (domestic animals and humans) and (ii) to determine which environmental factors are the main drivers of C. burnetii within the most relevant wild reservoirs.…”

<b><i>Coxiella burnetii</i></b> - Pathogenic Agent of Q (Query) Fever

“…The most extensive characterization of Coxiella strains was performed by amplification and sequencing of 139 chromosomal and plasmidic open reading frames (ORFs), including the genes for LPS biosynthesis [120]. This method was used for characterization of the strains of the outbreak in the Netherlands [20] and the strains in Spain [126]. …”