289 #These authors contributed equally to this work. 1Faculty of Veterinary Medicine, University of Bari, Bari, Italy. 2Lebanese University, Doctoral School of sciences and Technology, Beirut, Lebanon. 3Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Italy. 4URMITE, UM 63, CNRS 7278, IRD 198, Inserm 1095, Aix-Marseille University, Marseille, France. 5Lebanese Agricultural Research Institute, Fanar, Lebanon. 6Lebanese University, Faculty of Agriculture, Beirut, Lebanon. 7Lebanese University, Faculty of Sciences, Section I, Hadath, Lebanon. 8Istituto Zooprofilattico Sperimentale dell' Abruzzo e del Molise, Teramo, Italy. *Corresponding author at: Faculty of Veterinary Medicine, University of Bari, Bari, Italy Doctoral School of sciences and Technology (EDST ), Rafic Hariri University Campus, Hadath-Liban. Tel.: +961 (3)959739, Fax: +961-470941, e-mail: mayssaa.dabaja@gmail.com. Keywords Coxiella burnetii, MST, Lebanon, Ticks, Milk. Summary This study was carried out to detect and characterize Coxiella burnetii in ruminant milk samples and in different tick species from seropositive farms in four Lebanese regions. Milk and tick samples were screened for C. burnetii presence by quantitative real-time PCR (qPCR) targeting IS1111 region followed by multispacer sequence typing (MST). The overall positive percentages of 9.6% (27/282) and 95.45% (84/88) for C. burnetii were recorded in ruminant milk and tick samples, respectively. In detail, the C. burnetii DNA was recorded in 52/54 (96.3%) of Rhipicephalus annulatus, 20/21 (95.24%) of Rhipicephalus turanicus, 6/6 (100%) of Hyalomma anatolicum, 5/6 (83.3%) of Rhipicephalus sanguineus and 1/1 of Rhipicephalus bursa. After genotyping of some IS1111-positive samples (17/111), different MST genotypes were identified. Out of 15 positive ticks, 10 were infected with MST2 genotype, 4 were infected with MST7 genotype and 1 was infected with MST57. Moreover, genotypes MST20 and MST58 were found in one cow and one goat milk samples, respectively. The present study confirmed the high genetic diversity of C. burnetii in Lebanon. Mayssaa Fawaz Dabaja1,2,5*#, Grazia Greco1#, Valeria Blanda3, Maria Tempesta1, Ali Bayan7, Alessandra Torina3, Gesualdo Vesco3, Rosalia D'Agostino3, Rossella Lelli8, Mohamad Ezzedine2,7, Hussein Mortada6, Didier Raoult4, Pierre Edouard Fournier4 and Mohamad Mortada2,7 Multispacer sequence typing of Coxiella burnetii from milk and hard tick samples from ruminant farms in Lebanon Veterinaria Italiana 2020, 56 (4), 289-296. doi: 10.12834/VetIt.1799.13290.1 Accepted: 17.06.2020 | Available on line: 31.12.2020 is considered as potential weapon for bioterrorism (Alibek et al. 1999). C.  burnetii reservoirs include many wild and domestic mammals (Fernández-Aguilar et  al. 2016), with ruminants being the main source for humans (Berri et al. 2001, Fournier et al. 1998). The bacterium may also occasionally be detected in arthropods including ticks (Szymańska-Czerwińska et  al. 2013). Hard and soft ticks are one of the most important arthropods that are known to be naturally infected with C.  burnetii (Cutler et  al. 2007, Maurin et  al. 1999, Angelakis et  al. 2010). Ticks get infected with C.  burnetii during feeding on their animal host and Introduction Q Fever is a zoonotic disease caused by Coxiella burnetii worldwide distributed except in New Zealand (Cutler et  al. 2007). This pathogen is a small Gram negative, intracellular bacterium (Maurin et al. 1999, Péter et al. 1988) that multiplies in the phagolysosomes of eukaryotic host cells (Hackstadt et al. 1981, Arricau-Bouvery et al. 2005). This bacterium evolves in highly infective spore-like forms that are able to survive in the environment for several months (Evstigneeva et  al. 2007). C.  burnetii is classified as a category ‘B’ agent by the Centers for Disease Control (Atlanta, USA) and 290 Coxiella burnetii in Lebanon Dabaja et al. Veterinaria Italiana 2020, 56 (4), 289-296. doi: 10.12834/VetIt.1799.13290.1 qPCR targeted to the IS1111 region (Klee et  al. 2006) (Table I). Additionally, to investigate for C. burnetii genotypic diversity in Lebanon and to exclude cross-reactions with Coxiella-like endosymbionts (Duron et  al. 2015, Elsa et  al. 2015), some of IS1111 PCR-positive milk (n.  2) and tick (n. 15) samples from C.  burnetii seropositive animals (Dabaja et  al. 2017, Dabaja et  al. 2019) were investigated by using MST (Glazunova et al. 2005). Preparation of samples and PCR analysis Total DNA was extracted from milk and tick samples by using the Pure link Genomic DNA kit (Thermo Fisher™ Applied Biosystems™, Waltham, MA USA) as described by the manufacturer’s instruction. Briefly, in order to extract DNA, 200 μl of milk samples and/ or an hemilateral salivary gland of tick were mixed with 180 μl PureLink Genomic Digestion buffer and 20 μl proteinase K, followed by an incubation at 55 °C with occasional vortexing until lysis is complete over 30  minutes for milk sample and from 4 hours until overnight for tick samples . The detection of the IS1111 target (Klee et al. 2006) of C.  burnetii in milk and tick samples was carried out by using a high sensitive qPCR (Biorad CFX96 Real Time System). The IS1111 was selected as a target because it is present in multiple copies in the genome of this bacterium (Klee et al. 2006). The forward primer, Cox-F (5’-GT CTTA AGG TGG GCT GCG TG) and the reverse primer, Cox-R (5’-CCC CGA ATC TCA TTG ATC AGC) amplifies a 295 bp fragment that was revealed by a TaqMan probe (FAM-AGC GAACCA TTG GTA TCG GAC GTT TAT GG-TAMRA). The qPCR reactions were performed in a final volume of 25 µl using a mixture containing: 1X SsoAdvanced Universal Probe Supermix (Bio-rad), 0.4 µM of each primer, 0.5 µM of probe, 2 µl buffer of amplification internal control 10X (Applied biosystems by life Technologies), 0.5 µl internal control of DNA amplification 50X (Applied by life Technologies), 10 µl of DNA extract, and H 2 O to volume. PCR parameters were as follows: incubation at 50 °C for 2  min, incubation at 95  °C for 5 min, following 45  denaturation cycles at 95  °C for 15  sec then annealing and extension at 60  °C for 1 min. Each sample was determined in duplicate. The sample was considered as positive if the Ct was < 40. Genotyping of C. burnetii DNA detected in tick and milk samples A partial number of tick- milk- IS1111 positive samples were submitted to the genotyping step by using the MST assay (Glazunova et al. 2005). The limited sample size used in this step was due to the over 40 tick species can be naturally infected by this bacterium (Maurin et  al. 1999). C.  burnetii can multiply to very high titer levels in the mid-gut and stomach cells of the infected ticks, which can excrete bacteria via saliva and feces. Infected ticks can transmit C.  burnetii transtadially and transovarially (Dorko et al. 2012). The transmission of C. burnetii to mammal hosts might occur via tick bites or by feces contamination of their wool and skin (NASPHV and NASAHO 2013, Marrie et al. 1990). The Middle East is the epicenter of the disease, and outbreaks have being reported in Jordan (Fuad et  al. 1998), Syria (Bottieau et  al. 2000), Turkey (Cetinkaya et al. 2000), Iraq (Faix et al. 2005), Cyprus (Cantas et  al. 2011) and Iran (Esmaeili et  al. 2016). Lebanon has Mediterranean climate that makes it suitable environment for Q fever disease (Dabaja et  al. 2019) and arthropods (Dabaja et  al. 2017). Genotyping characterization of C.  burnetii strains detected in animals, humans and in ticks is useful for epidemiological purpose. Multispacer sequence typing (MST ) is a suitable tool to genotype C. burnetii strains because of its high discriminatory power (Glazunova et al. 2005, Walker et al. 2014). In the present study, the detection and the genotyping of C.  burnetii DNA in milk and hard tick samples from ruminant farms in Lebanon was performed, as no information is available in the area. Materials and methods Sampling The analyzed samples were collected under the frame of a previous cross-sectional study performed to evaluate both seroprevalence and via milk shedding of C.  burnetii in 1,633 animals from 429  ruminant farms distributed in 7 Lebanese provinces (Akkar, Baalback-ElHermel, Bekaa, Mount Lebanon, Nabatieh, North Lebanon and South-Lebanon) in 2014 (Dabaja et al. 2019). Breafly, in that study 39.86% of farms (95% CI: 35.23-44.56) and 17.27% (95% CI: 15.43-19.1) of ruminants resulted seropositive (Dabaja et al. 2019). Moreover, 27/282 (14.08%) milk samples from C.  burnetii seropositive animals were positive for the IS1111 target of C.  burnetii (Dabaja et  al. 2019). Furthermore, 219 adult hard ticks from 30 seropositive farms were collected in June 2014, as previously described (Dabaja et  al. 2017, Dabaja et  al. 2019). Collected ticks belonged to the genera Rhipicephalus and Hyalomma distributed in 5 species R. annulatus, R. turanicus, R. bursa and R. sanguineus and H. anatolicum (Dabaja et al. 2017). In the present study, 2 or 3 ticks were selected from each farm. A total of 88 out of the 219 collected ticks were individually investigated by using 291 Dabaja et al. Coxiella burnetii in Lebanon Veterinaria Italiana 2020, 56 (4), 289-296. doi: 10.12834/VetIt.1799.13290.1 Table I. Prevalence of C. burnetii in ticks collected in June 2014 from ruminants in Lebanon detected by real-time PCR. ID farm Locality town or village of origin Province Kind of farm ID and species of ticks Sex of ticks IS 1111 Cycle threshold: Ct 1 Barich *33°16'22''N **35°21'9''E ***358 m South Lebanon Bovine 1 H. a F 33.56 2 R. a F 26.6 3 R. a F 31.62 2 Qinarit *33°30'17''N **35°22'44''E ***233 m South Lebanon Bovine 10 R. a F 25.0 11 R. a F 32.7 12 R. a F 25.87 3 AynEdelbe *33°32'40.87''N **35°24'25.834''E ***41 m South Lebanon Bovine 13 R. a F 34.15 14 R. a F 22.32 15 R. a F 33.23 4 Maaroub *33°17'6''N **35°20'49.2''E ***270 m South Lebanon Ovine 16 R. t F 26.15 17 R. t F 36.26 18 R. t F 23.19 5 Zayta *33°30'29''N **35°23'03''E ***300 m South Lebanon Bovine 49 R. a F 28.08 50 R. a F 32.86 6 Bourghlieh *33°18'36''N **35°14'24''E ***19 m South Lebanon Bovine 56 R. a F 31.76 57 R. a F 37.18 58 R. a F 33.06 7 Hasbaya *33°23'N ***35°41'E ***750 m Nabatieh Bovine 4 R. a F 29.32 5 R. a F 30.66 6 R. a F 25.14 8 El Koulayaa *33°19'48''N **35°34'12''E ***650 m Nabatieh Caprine + Ovine 7 R.s F 37.19 8 R.b F 25.32 9 H.a F 34.43 9 Zawtar El Charkiyi *33°19' 33''N **35°28'34''E ***475 m Nabatieh Bovine 19 R. a F Negative 20 R. a F 21.12 21 R. a F 34.25 10 Mayfadoun *33°20'9.6''N **35°27'43.2''E ***470 m Nabatieh Bovine 22 R. a F 24.26 23 R. a F 30.86 24 R. a F 21.12 11 Marjiiyoun *33°30'N **35°30'E ***860 m Nabatieh Bovine 25 H. a M 30.3 26 H .a M 34.0 27 H. a M 35.0 12 Wata El Khiyam *33°19'37.8''N **35°36'40''E ***700 m Nabatieh Caprine 28 H.a M 26.35 29 R.s M 32.49 30 R.s M 27.29 13 Ibel El Saki *33°12'36''N **35°22'48''E ***800 m Nabatieh Ovine 31 R. t F 34.5 32 R. s F Negative 33 R. t F Negative 14 El Wazani *33°16'32''N **35°37'22''E ***297 m Nabatieh Bovine 34 R. a F 31.0 35 R. a F 32.8 36 R. a F 35.4 15 El Wazani *33°16'32''N **35°37'22''E ***279 m Nabatieh Bovine 37 R. a F 30.2 38 R. a F 27.48 39 R. a F 34.49 16 El Wazani *33°16'32''N **35°37'22''E ***279 m Nabatieh Caprine 40 R. t F 28.25 41 R. s F 37.7 42 R. t F 30.5 ID farm Locality town or village of origin Province Kind of farm ID and species of ticks Sex of ticks IS 1111 Cycle threshold: Ct 17 AynEbel *33°00'42''N **35°14'24''E ***800 m Nabatieh Bovine+ Ovine 43 R. t F 35.35 44 R. t F 30.32 45 R. t F 34.36 18 Kfarkila *33°10'12''N **35°19'48''E ***700 m Nabatieh Bovine 46 R. a F 33.2 47 R. a F 31.4 48 R. t F 35.0 19 Rmeich *33°00'54''N **35°24''E ***690 m Nabatieh Ovine 59 R. t F 32.82 60 R. t F 28.84 61 R. t M 33.78 20 AynEbel *33°00'42''N **35°14'24''E ***800 m Nabatieh Caprine 62 R. t F 30.85 63 R. t F 35.0 64 R. t F 30.8 21 AynEbel *33°00'42''N **35°14'24''E ***800 m Nabatieh Bovine 65 R. a F 35.86 66 R. a F 34.14 67 R. a F 37.63 22 Zahleh *33°50'48''N **35°4'07''E ***963 m Bekaa Ovine 51 R. t F 39.4 52 R. t F 30.7 23 Zahleh *33°50'48''N **35°54'07''E ***963 m Bekaa Ovine 53 R. t F 33.44 54 R. s F 30.0 55 R. t F 32.54 24 Machha *34°32'25''N **36°7'56''E ***349 m Bekaa Ovine 68 R. a F 30.89 69 R. a F 33.98 70 R. a F 31.6 25 Adbel *34°32'6''N **36°57'50.4''E ***300 m Akkar Bovine 71 R. a F Negative 72 R. a F 33.95 73 R. a F 30.93 26 Al Kantara *34°31'33.078''N **36°00'3.0711''E ***375 m Akkar Ovine 74 R. a F 36.24 75 R. a F 32.1 76 R. a F 36.55 27 Machha *34°32’25’’N **367'56''E ***349 m Akkar Bovine 77 R. a F 33.0 78 R. a F 35.2 79 R. a F 33.41 28 Michmich *34°21'24.0012''N **35°55'51''E ***1,100 m Akkar Bovine 80 R. a F 36.0 81 R. a F 31.47 82 R. a F 35.33 29 Bazbina *34°31' 0'' N **36°12'0''E ***955 m Akkar Bovine 83 R. a F 32.84 84 R. a F 36.23 85 R. a F 34.65 30 Sahel Halba *34°33'2'' N **36°4'41''E ***120 m Akkar Bovine 86 R. a F 34.37 87 R. a F 32.13 88 R. a F 33.41 Positivity percentage averall 84/88(+) (95.45%) CT Average: 32 *Latitude; **Longitude ***Altitude; F = Female; M = Male. R. a = Rhipicephalus annulatus; H. a = Hyalomma anatolicum; R. b = Rhipicephalus bursa; R. s = Rhipicephalus sanguineus; R. t = Rhipicephalus turanicus. 292 Coxiella burnetii in Lebanon Dabaja et al. Veterinaria Italiana 2020, 56 (4), 289-296. doi: 10.12834/VetIt.1799.13290.1 database containing C.  burnetii genotypes from countries in Europe and other parts of the world using BLAST; the new sequences were deposited in the available database on the website: http://ifr48. timone.univ-mrs.fr/MST_Coxiella/mst/group_detail. Results Arthropods IS1111 target was detected in 84 of the 88 (95.45%) investigated ticks from C. burnetii seropositive farms with Ct value being between 21.12 and 39. Positive samples included 52 of 54 (96.3%) R. annulatus, 20  of  21 (95.24%) R. turanicus, 6 of 6 (100%) H. anatolicum, 5 of 6 (83.3%) R. sanguineus and 1 of 1 R. bursa (Table III). Milk Among 282 milk samples from seropositive ruminants, IS1111 DNA had been detected in 9 of 86 (10.47%) cattle, in 6 of 93 (6.45%) sheep and in 12  of  103 (11.65%) goat specimens, as indicated in previous study (Dabaja et al. 2019). MST Because of the low amount of DNA, it was possible to perform MST genotyping in 84 IS111 positive ticks and 2 IS111 positive milk samples only (Tables III and IV). As result of this study, three previously described genotypes and two incomplete ones were identified (Tables III and IV). Of the 15 positive ticks, 10 hosted the MST 2 genotype, 4  the MST 7 genotype and 1 the MST 57 incomplete genotype. Of the 2 positive milk samples, one was infected with the known MST 20 and the other one with the incompletely characterized MST 58 (Tables III and IV). MST 2 genotype was widely found in different genera and species of ticks from South Lebanon and Nabatieh; MST 7 was identified in R. annulatum and R. turanicus from Bekaa and Akkar whereas MST 20 was detected in the cow milk samples in Bekaa region (Table III). MST 57 and MST 58 genotypes, incompletely characterized probably due to the low DNA concentration, were found in one tick and one goat milk sample from Nabatieh and Bekaa, respectively  (Table III). Discussion Almost all the collected ticks (95.45%) from C.  burnetii seropositive ruminant farms in Lebanon were positive for IS1111 target. Since IS1111-based low amount DNA remaining from each specimen to perform the MST. Multi-spacer typing was performed on IS1111 positive specimens using a set of primers targeting 10 variable spacers (Cox: 2; 5; 18; 20; 22; 37; 51; 56; 57; 61) according to previous study (Glazunova et al. 2005). Five µL of DNA preparation was amplified in a 50 µL reaction mixture containing 0.2 µM of each primer, 0.05 mM (each) dATP, dTTP, dCTP and dGTP; 1.25 U Taq Polymerase; MgCl 2 2.5 mM and 1X Taq buffer. DNA from Nine Mile strain of C.  burnetii was used as positive control. Amplifications were carried out using a 2720 thermal cycler (Applied Biosystems) according to the following conditions: an hot start step of 15 min at 95  °C, followed by 40  cycles of denaturation for 1 min at 95 °C, annealing for 30 sec at 59  °C, elongation for 1 min at 72  °C and final extension for 7 min at 72 °C. PCR amplicons were visualized by electrophoresis of 6 µL of the PCR product with 2 µL of blue loading buffer on 1.5% agarose gel (0.5xTBE) with SyberSafe under UV light. PCR products were purified via vacuum filtration through the NucleoFast 96 PCR Plate (Thomas Scientific, Dueren, Germany), as described by the manufacturer. Sequencing reactions were carried out using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems). Four µL of purified PCR were added to a 10 µL reaction containing 0.5 µL primers, 1.5 µL Big Dye buffer, and 1 µL Big Dye. The sequencing reaction was run in a thermal cycler as follows: an initial denaturation step of 1 min at 96  °C followed by 25 cycles of denaturation for 10 sec at 96  °C, annealing for 5 sec at 50 °C and elongation for 5 min at 60 °C, followed by a final step at 15 °C. Sequencing reactions were purified using Millipore Sephadex plates (Millipore, Billerica, Massachusetts) as per the manufacturer’s instructions, and stored at 4 °C until analyzed. Sequencing reactions were analyzed on an ABI 3130X Genetic Analyser (Applied Biosystems) and sequence assembly performed using the multisequence align software Chromaspro (v.2.1.1). The obtained sequences were further compared with the sequences included in the MST reference Table II. Percentage of ticks positive for IS1111 detection by real-time PCR. Tick species No. of positive ticks Percentage of positive ticks (95%CI) Rhipicephalus annulatus 52/54 96.3% (91.1-100) Rhipicephalus turanicus 20/21 95.3% (94.88-95.72) Rhipicephalus sanguineus 5/6 83 % (53-100) Rhipicephalus bursa 1/1 - Hyalomma anatolicum 6/6 100% CI = Confidence interval 293 Dabaja et al. Coxiella burnetii in Lebanon Veterinaria Italiana 2020, 56 (4), 289-296. doi: 10.12834/VetIt.1799.13290.1 2013), in R. annulatus in Senegal (Mediannikov et al. 2010), in R. sanguineus in Iran, Cyprus, Italy and Switzerland (Bernasconi et al. 2002, Nourollahi Fard et al. 2011, Satta et al. 2011, Spyridaki et al. 2002), in R. turanicus in Turkey, Italy, Switzerland and Greece (Capin et al. 2013, Bernasconi et al. 2002, Satta et al. 2011, Psaroulaki et  al. 2006), in H. anatolicum in Cyprus (Spyridaki et  al. 2002), thus supporting the evidence that C.  burnetii seems to be endemic in ticks in more areas. In our study several MST genotypes of C.  burnetii were found in tick and milk samples from ruminant farms in Lebanon. The most frequently detected MST 2 genotype was found in R. annulatus, R. bursa, R. turanicus and H. anatolicum ticks from the South Lebanon and Nabatieh regions. Indeed, the same MST 2 genotype had already been detected in blood samples of human beings affected with acute Q fever from France, Ukraine and Kyrgyzstan (Glazunova et  al. 2005). MST 7 genotype, detected in both R. annulatus and R. turanicus ticks from the Bekaa and the Akkar regions, had already been found in France and Russia in human blood samples and cardiac valves (Glazunova et al. 2005). Conversely, the sequence type detected in the cow milk sample from Bekaa region was similar to MST  20. This genotype had been found in animal, human and tick samples from Germany, France, Spain, Italy, Hungary, the United Kingdom, United States and Netherlands and central Africa. In PCR-assays designed to detect C.  burnetii cross react with Coxiella-like bacteria (Elsa et  al. 2015), the results of surveys carried out on ticks and based only on IS1111 PCR assay should be interpreted with caution since ticks can harbor either C. burnetii or Coxiella-like bacteria. In the present study, in order to exclude misinterpretations, some of the IS1111-positive samples were genotyped by using MST that is based on the characterization of a set of targets (Glazunowa et al. 2005). Based on results obtained using this combined approach, different MST genotypes in tick and a few milk samples from different C.  burnetii seropositive ruminant farms of different provinces of Lebanon were detected. C.  burnetii infection had already been recorded in R. bursa in Turkey (Capin et  al. Table III. Multispacer sequence typing (MST ) genotypes of C. burnetii in ticks and milk specimens from ruminant farms in Lebanon. Sample ID Source & host species Town or village of origin: GPS coordinates, elevation Province Date Cycle threshold: Ct MST genotype 2 R. a (F) Bovine Barich: *33°16'22''N; **35°21'9''E; ***358 m South Lebanon 6/2014 26.6 2 6 R. a (F) Bovine Hasbaya: *33°23'N;**35°41'E; ***750 m Nabatieh 6/2014 25.14 2 8 R. b (F) Ovine El Koulayaa: *33°19'48''N; **35°34'12''E; ***650 m Nabatieh 6/2014 25.32 2 10 R. a (F) Bovine Qinarit: *33°30'17''N;**35°22'44''E; ***233 m South Lebanon 6/2014 25 2 14 R. a (F) Bovine Ayn Eldeleb: *33°32'40.87''N; **35°24'25.834''E; ***41 m South Lebanon 6/2014 22.32 2 20 R. a (F) Bovine Mayfadoun: *33°20'9.6''N; **35°27'43.2''E; ***470 m Nabatieh 6/2014 21.12 2 24 R. a (F) Bovine ZawtarCharkiyi: *33°19'33''N; **35°28'34''E; *** 475 m Nabatieh 6/2014 21.12 2 28 Ha. a (M) Caprine WataElkhiyam: *33°19'37.8''N; **35°36'40''E; ***700 m Nabatieh 6/2014 26.35 2 38 R. a (F) Bovine El Wizani: *33°16'32''N; **35°37'22''E; ***279 m Nabatieh 6/2014 27.48 2 40 R. t (F) Caprine El Wizani: *33°16'32''N; **35°37'22''E; ***279 m Nabatieh 6/2014 28.25 2 44 R. t (F) Ovine Ayn Ebel: *33°00'42''N; **35°14'24''E; ***800 m Nabatieh 6/2014 30.2 57 52 R. t (F) Ovine Zahle: *33°50'48''N; **35°54'07''E; ***963 m Bekaa 6/2014 30.7 7 53 R. t (F) Ovine Zahle: *33°50'48''N; **35°54'07''E; ***963 m Bekaa 6/2014 33.44 7 70 R. a (F) Bovine Machha: *34°32'25''N; **36°7'56''E; ***349 m Akkar 6/2014 31.6 7 75 R. a (F) Ovine AlKantara: *34°31'33.078''N; **36°00'3.0711''E; ***375 m Akkar 6/2014 32.1 7 19 Milk Goat Rayak: *33°51'3''N; **36°00'42''E; ***929 m Bekaa 5/2014 37.74 58 3038 Milk Bovine Alkarak: *33°51'N; **35°55'35''E; ***1000 m Bekaa 3/2014 39 20 *Latitude; **Longitude ***Altitude; F = Female; M = Male. R. a = Rhipicephalus annulatus; H. a = Hyalomma anatolicum; R. b = Rhipicephalus bursa; R. s = Rhipicephalus sanguineus; R. t = Rhipicephalus turanicus. Table IV. Genotyping details of the detected strains according to Multispacer sequence typing (MST ). M ST ge no ty pe Co x2 Co x5 Co x1 8 Co x2 0 Co x2 2 Co x3 7 Co x5 1 Co x5 6 Co x5 7 Co x6 1 2 5 6 3 5 x 5 8 1 5 6 7 4 6 3 5 6 5 8 x 5 x 20 3 2 6 x 5 4 x 10 6 5 57 4 6 3 5 5 5 x x 5 6 58 4 8 2 x x x x x x x 294 Coxiella burnetii in Lebanon Dabaja et al. Veterinaria Italiana 2020, 56 (4), 289-296. doi: 10.12834/VetIt.1799.13290.1 Conclusions This study provides the first molecular evidence of C.  burnetii as well as a preliminary picture of the genetic diversity of the Q fever agent in different tick species and milk samples from ruminant farms in Lebanon. Although, based on the results of our study, there is no evidence of a role of ticks in the transmission of infection to the ruminants, appropriate biosecurity measures should be taken to prevent zoonotic risk since different genotypes were found in ticks and milk samples. Acknowledgments We thank the director general of the Lebanese Agricultural Research Institute (LARI) Dr. Michel Afram, and our many friends from the Ministry of Agriculture - Lebanon who collect us milk and ticks samples from different region of the country. particular, MST 20 was most frequently associated with cattle but rarely with goats. MST 20 was found in a single goat sample in the Netherlands (Tilburg et  al. 2012), in two milk samples in the United States (Pearson et al. 2014), and in a large goat herd with abortion problems in the United Kingdom (Reichel et  al. 2012). However, in most cases, the genotype MST 20 was associated with cattle and cow’s milk (Glazunova et  al. 2005, Astobiza et  al. 2012, Pearson et  al. 2014, Sulyok et  al. 2014, Mediannikov et  al. 2010). These findings provide further evidence for the presumed host-specific adaptation of this agent. Finally, although incompletely characterized for technical reasons, other two genotypes, MST 57 and MST 58 were detected in a R. turanicus tick and a goat milk sample, respectively, thus confirming the high genetic diversity of C. burnetii in Lebanon. Alibek K. 1999.The chilling true story of the largest covert biological weapons program in the world, Random House, NewYork, NY, USA. Bauer A.E., Olivas S., Cooper M., Hornstra H., Keim P., Pearson T. & Johnson A.J. 2015. Estimated herd prevalence and sequence types of Coxiella burnetii in bulk tank milk samples from commercial dairies in Indiana. BMC Veterinary Research, 11, 186-174. Angelakis E. & Raoult D. 2010. Q fever. 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