Both methods have high discriminatory power and are reproducible, allowing interlaboratory comparisons without the need for isolation and cultivation of the organism under biosafety level three conditions. The aim of this study was to identify C. The samples were not related to any outbreak, they were collected through routine diagnostic examinations. All tests were performed in accordance with all applicable institutional and national guidelines and regulations, approved by the ethics committees of the Institute for Veterinary Medical Research animal and the National Center for Epidemiology human and with the consent of the patient.
Map of Hungary showing the geographical distribution of C. ND: not determined. The blank map was downloaded from an open source [ 23 ]. To quantify the approximate bacterial load and to determine the cycle threshold Ct values, all samples were analysed by TaqMan based real-time polymerase chain reaction PCR assay targeting the multicopy IS insertion element of the C. MST consisted of amplification followed by sequencing of ten different spacer regions of the C. Primer sequences and reaction conditions were described earlier [ 17 ].
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Sequence types STs were determined using the MST database and previous publications [ 17 , 20 , 25 ]. Unfortunately, the allele codes for ST35 and ST36 are not publicly available therefore we were not able to include them in the phylogenetic analysis [ 25 ]. To determine the phylogenetic relationships of our samples to known MST genotypes [ 17 , 20 ] we used the sequence information of our three STs and constructed a phylogenetic tree with the polymorphisms and methods described in Hornstra et al.
MLVA was performed by single PCRs targeting six variable microsatellite markers as performed in other recent studies [ 18 , 27 — 30 ].
Ms27, Ms28 and Ms34 contain repeat units of six base pairs and Ms23, Ms24 and Ms33 contain repeat units of seven base pairs. The repeat numbers of each marker were determined by extrapolation using the obtained length of the sample fragments relative to the obtained fragment-length of the reference strain. A modified coding convention was used for Ms nine imperfect repeat units were counted in the Nine Mile strain G. Vergnaud, personal communication. Novel MLVA types were determined if the described allele combinations had not been previously described in any publications applying the same MLVA method [ 21 , 27 — 29 ].
Phylogenetic analysis according to Honstra et al. Parsimony tree showing the placement and phylogenetic relationships of the sequence types ST from this study with known STs [ 19 ] , rooted according to Pearson et al. Highlights indicate the placement of the STs of our samples; the red highlight denotes the newly proposed ST One hundred and twelve polymorphisms from Hornstra et al.
Tree length is and the homoplasy index excluding uninformative characters is 0.
Five MLVA genotypes were identified among the 11 cattle and ovine samples with complete genotypes, out of which two novel MLVA types were described in this study. In the case of the human sample only a partial genotype was obtained despite the relatively high DNA load Ct value was The identified allele combination in the human sample did not match any previously defined genotypes, implying the discovery of a novel MLVA type as well.
The partial MLVA genotype from the human sample showed high similarity to the novel genotype AG defined in sheep, as four out of the five microsatellites were the same. Host specificity of the genotypes was observed as all MLVA genotypes from ST20 were described from cattle only and the two other genotypes were detected in sheep exclusively.
Molecular characterisation of C. The main advantage of both MST and MLVA typing methods is their high discriminatory power and that the cultivation of the bacteria is unnecessary for their application. Three C.
Microvariants of the ST20 genotype occur as well and have been found elsewhere; MLVA genotypes I, J and M were described before from cow milk and milk products from several countries in Europe France, the Netherlands, Portugal, Spain and Switzerland and other parts of the world e. Qatar, Saudi Arabia [ 21 ]. ST20 was also detected in an outbreak among goats recorded from the United Kingdom [ 36 ].
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In conclusion, although ST20 is most often associated with cattle products, this genotype occasionally infects other species as well. ST28 samples have been collected from cows, sheep, Hyalomma sp. The comparison of the novel genotypes AG and AF with others described in Europe revealed similarities with genotypes AA and T, differing at 2 and 3 loci respectively and also associated with sheep, goats and human [ 28 , 29 ]. Again, that these genotypes are most commonly associated with sheep and goats provides further evidence for host preference [ 39 ].
The present study provides information about the genotypic diversity of C. From the examined 12 C. These genotypes cluster with host, suggesting host-specific evolutionary adaptations as shown by Pearson et al. The additional analyses of samples from a broader range of livestock species are needed to confirm this finding. Our results provide data for the European surveillance and help to increase information about Q fever in this Central European region.
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