Identification of Novel Coxiella burnetii Genotypes from Ethiopian Ticks

Background Coxiella burnetii, the etiologic agent of Q fever, is a highly infectious zoonotic bacterium. Genetic information about the strains of this worldwide distributed agent circulating on the African continent is limited. The aim of the present study was the genetic characterization of C. burnetii DNA samples detected in ticks collected from Ethiopian cattle and their comparison with other genotypes found previously in other parts of the world. Methodology/Principal Findings A total of 296 tick samples were screened by real-time PCR targeting the IS1111 region of C. burnetii genome and from the 32 positive samples, 8 cases with sufficient C. burnetii DNA load (Amblyomma cohaerens, n = 6; A. variegatum, n = 2) were characterized by multispacer sequence typing (MST) and multiple-locus variable-number tandem repeat analysis (MLVA). One novel sequence type (ST), the proposed ST52, was identified by MST. The MLVA-6 discriminated the proposed ST52 into two newly identified MLVA genotypes: type 24 or AH was detected in both Amblyomma species while type 26 or AI was found only in A. cohaerens. Conclusions/Significance Both the MST and MLVA genotypes of the present work are closely related to previously described genotypes found primarily in cattle samples from different parts of the globe. This finding is congruent with the source hosts of the analyzed Ethiopian ticks, as these were also collected from cattle. The present study provides genotype information of C. burnetii from this seldom studied East-African region as well as further evidence for the presumed host-specific adaptation of this agent.


Introduction
Coxiella burnetii, the causative agent of Q fever, is a zoonotic Gram-negative bacterium with a worldwide distribution. Q fever is typically an acute febrile illness with nonspecific clinical signs in humans, but hepatitis and atypical pneumonia are seen in severe cases, and a small proportion of infected people develop chronic infection with life-threatening valvular endocarditis [1,2]. Domestic ruminants are considered the primary hosts of C. burnetii, but it has been described in a wide range of other animal species including mammals, birds and ticks [3]. Domestic ruminants are often asymptomatic carriers of the bacterium, but the agent may cause abortion in these animals [1]. C. burnetii has been detected in about 40 different tick species which may act as reservoirs in nature. Transmission of C. burnetii by tick bite to animals has also been suggested [4].
In Ethiopia, Philip et al. [5] reported the first evidence of the existence of C. burnetii in ticks found on cattle. The seroprevalence of C. burnetii among slaughterhouse workers in an abattoir in Addis Ababa was reported to be 6.5% by complement fixation test [6]. According to recently published data, seroprevalence rates of C. burnetii determined by enzyme-linked immunosorbent assay (ELISA) were 31.6% in cattle, 90.0% in camels and 54.2% in goats. These values are higher than values reported in other parts of Africa [7].
Multispacer sequence typing (MST) [8] and multi-locus variable-number tandem repeat analysis (MLVA) [9,10] are two PCR-based C. burnetii genotyping methods with high discriminatory power. These techniques do not require previous cultivation of C. burnetii under biosafety level 3 conditions and can be implemented directly on clinical and environmental samples e.g. for routine diagnostic purpose or during epidemic investigations.
The aim of the present study was to genetically characterize C. burnetii DNA samples originating from Ethiopian ticks and compare them to previously identified genotypes in order to obtain genotypic information from this poorly studied region.

Materials and Methods
A total of 296 ticks (Amblyomma variegatum, n5118; A. cohaerens, n5100; A. lepidum, n52; Rhipicephalus decoloratus, n550; R. evertsi, n517; R. praetextatus, n58 and Hyalomma rufipes, n51) were collected from 109 cattle belonging to 18 different herds (zebu breed) in Didessa valley, south-western Ethiopia (09˚059N, 36˚339E -7˚409N, 36˚50E) in 2012 ( Figure 1) [11]. Because sampling was part of the regular veterinary care and the field studies did not involve endangered or protected species, no specific permissions were required for these activities. Ticks were identified on the basis of their morphology by microscopy [12], and the DNA of each tick was extracted using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the tissue protocol in the manufacturers' instructions. In order to detect C. burnetii DNA and to estimate the amount of C.
For the MST analysis ten selected spacer regions of the C. burnetii genome were amplified and sequenced according to the description of Glazunova et al [8]. The obtained sequences were trimmed, concatenated and aligned with genotypes from the MST database and previous publications using BioEdit Sequence Alignment Editor 7.1.11 [8,[14][15][16]. A Neighbor-joining phylogenetic tree was constructed using the maximum composite likelihood model and 1000 bootstraps with the Mega 5.05 software [17]. Sequence types (STs) were determined using data from the MST database and previous publications [8,14,15,18,19]. Unfortunately, the allele codes or nucleotide sequences of STs 35-51 are not publicly available therefore we were not able to include them in the phylogenetic analysis [18,19].
MLVA was performed by single PCRs targeting six variable microsatellite markers. Ms27, Ms28 and Ms34 containing repeat units of six base pairs and Ms23, Ms24 and Ms33 containing repeat units of seven base pairs. The 39-end-  labelled forward and reverse primer sequences, and PCR conditions were applied as described before [20,21]. The amplification products were run on an ABI 3100 Genetic Analyser and electropherograms were evaluated with the Peak Scanner Software 2.0 (Applied Biosystems Inc., Foster City, CA). DNA of the Nine Mile strain (RSA 493, Coxevac, Ceva Inc., Budapest, Hungary) was used as a reference. 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. The updated coding convention was used for Ms33: nine imperfect repeat units were counted in the Nine Mile strain [22]. Novel MLVA types were determined if the described allele combinations had not been previously published in the Coxiella MLVA database nor described in any publication applying the same MLVA method [21][22][23][24][25]. Novel MLVA types were denominated in accordance with the nomenclature of the Coxiella MLVA database and previous publications [21][22][23][24][25].
In the case of three A. cohaerens samples only partial MLVA genotypes were obtained, probably because of the insufficient DNA load (high Ct values). The combinations of the identified alleles in samples HSE-167 and HSE-170 were identical to the complete MLVA genotypes 26/AI, suggesting that they belong to the same genotype, while sample HSE-210 could belong to either of the newly determined MLVA types (26/AI or 24/AH).

Discussion
Both typing methods revealed novel C. burnetii genotypes when compared to genotypes with publicly available genetic data [8,14,15,[21][22][23][24][25]. However, since several MST genotypes (STs 35-51) were determined and denominated by previous publications without presenting the allele codes or nucleotide sequences [18,19], we cannot rule out the possibility that the novel ST documented in the present study may eventually be identical with one of them. The lack of data harmonization between the MST and different MLVA systems presents a further challenge.
Information on the genotypic characteristics of C. burnetii isolates originating from Africa is restricted. Up to now ST2 and ST16 from Central Africa, ST30 from Namibia and ST6, ST19 and two partial STs (ST 35 and 36) from Senegal are the only described C. burnetii genotypes from Africa, identified in ticks and human samples [8,19,26]. The newly discovered ST52 is closely related to the widely distributed ST20 (Figure 2). ST20 has been reported from Germany, France, Hungary, the Netherlands, Spain, the United Kingdom and the United States. This genotype is most often associated with cattle samples, but it occasionally infects other species as well [8,15,25,27]. ST52 is also related to ST19, described earlier from human clinical samples from Senegal [8]. ST52 was further discriminated into two newly identified MLVA types (24/AH and 26/AI). The comparison of the two novel genotypes with others described elsewhere revealed high similarity with genotypes originating from cattle. For instance the novel type 24/AH and type 25/L previously identified from Slovakian cow milk differs only in one out of the six loci (12 versus 13 copies at locus Ms24) [23].
Both typing methods revealed that these novel C. burnetii genotypes are closely related to genotypes previously described from cow samples from other parts of the globe. This finding is congruent with the source hosts of the Ethiopian ticks analyzed in the present study, as these were also collected from cattle. Therefore, our results provide additional evidence of the presumed host-specific evolutionary adaptation of C. burnetii [15,28].
In conclusion, the present study provides genetic information about C. burnetii from the poorly studied African continent. One novel MST type (proposed ST52) and two novel MLVA types (24/AH and 26/AI) have been identified among the eight examined C. burnetii DNA samples of Ethiopian ticks collected from cattle. Our results are bringing to light some missing parts of C. burnetii phylogeny and they also provide further evidence for the presumed host-specific adaptation of this agent.