Multispacer sequence typing of Coxiella burnetii from milk and hard tick samples from ruminant farms in Lebanon

Supplementary Files

Table I
Table II
Table III
Table IV


Coxiella burnetii

How to Cite

Dabaja, M. F., Greco, G., Blanda, V., Tempesta, M., Bayan, A., Torina, A., Vesco, G., D’Agostino, R., Lelli, R., Ezzedine, M., Mortada, H., Raoult, D., Fournier, P. E., & Mortada, M. (2020). Multispacer sequence typing of Coxiella burnetii from milk and hard tick samples from ruminant farms in Lebanon. Veterinaria Italiana, 56(4), 289-296.


his 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.


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 burnetiiin bulk tank milk samples from commercial dairies in Indiana. BMC Veterinary Research, 11, 186-174.Angelakis E. & Raoult D. 2010. Q fever. Vet Microbiol, 140, 297-309.

Arricau-Bouvery N. & Rodolakis A. 2005. Is Q Fever an emerging or re-emerging zoonosis? Vet Res, 36, 327-349.Astobiza I., Tilburg J.J., Piinero A., Hurtado A., Garcia-Pérez A.L., Nabuurs Franssen M.H. & Klaassen C.H.V. 2012. Genotyping of Coxiella burnetii from domestic ruminants in northern Spain. BMC Vet Res, 8, 241.

Bernasconi M.V., Casati S., Péter O. & Piffaretti J.C. 2002. Rhipicephalus ticks infected with Rickettsia and Coxiellain southern Switzerland Canton Ticino. Infection, Genetics and Evolution, 2, 111-120.

Berri M., Souriau A., Crosby M., Crochet D., Lechopier P. & Rodolakis A. 2001. Relationships between the shedding of Coxiella burnetii, clinical signs and serological responses of 34 sheep. Vet Rec, 148 (16), 502-505.

Bottieau E., Raeve D.H., Colebunders R., Van den Ende J., Vervoort T. & Van Marck E. 2000. Q fever after a journey in Syria: a diagnosis suggested by bone marrow biopsy. Acta Clinica Belgica, 55, 30-33.

Cantas H., Muwonge A., Sareyyupoglu B., Yardimci H. & Skjerve E. 2011. Q fever abortions in ruminants and associated on-farm risk factors in northern Cyprus. BMC Veterinary Research, 7, 13.

Capin G.A., Emere Z., Canpolat S., Vatansever Y. & Düzgün A. 2013. Detection of Coxiella burnetii from ticks by polymerase chain reaction and restriction gragment length polymorphism. Ankara Üniv vet Fak Derg, 60, 263-268.

Cetinkaya B., Kalender H., Ertas H.B., Muz A., Arslan N., Ongor H. & Gurçay M. 2000. Seroprevalence of coxiellosis in cattle, sheep and people in the east of Turkey. Vet Rec, 146 (5), 131-136.Pérez-Eid C. 2009. Les tiques identification, biologie, importance médicale et vétérinaire, 2nd ed. Paris: 187-260.

Cutler S.J., Bouzid M. & Cutler R.R. 2007. Q fever. Journal of Infection, 54 (4), 313-318.Dabaja M.F., Tempesta M., Bayan A., Vesco G., Greco G., Torina A., Blanda V., La Russa F. Scimeca S., Ezzedine M., Mortada H., Raoult D., Fournier P.E. & Mortada M. 2017. Diversity and distribution of ticks from domestic ruminants in Lebanon. Vet Ital, 53 (2), 147-155. doi:10.12834/VetIt.1171.6503.2.

Dabaja M.F., Greco G., Villari S., Vesco G., Bayane A., El Bazzal B., Ibrahim E., Gargano V., Sciacca C., Lelli R., Ezzedine M., Mortada H., Tempesta M. & Mortada M. 2019. Occurrence and risk factors of Coxiella burnetii in domestic ruminants inLebanon. Comparative Immunology. Microbiology and Infectious Diseases, 64, 109-116. doi: 10.1016/j.cimid.2019.03.003. Epub 2019 Mar 8.

Di Domenico M., Curini V., De Massis F., Di Provvido A., Scacchia M. & Cammà C. 2014. Coxiella burnetii in central Italy: novel genotypes are circulating in cattle and goats. Vector Borne Zoonotic Dis, 14 (10), 710-715.

Dorko E., Rimárová K. & Pilipčinec E. 2012. Influence of the environment and occupational exposure on the occurrence of Q fever. Cent Eur J Public Health, 20 (3), 208-214.Duron O. 2015. The IS1111 insertion sequence used for detection of Coxiella burnetii is widespread in Coxiella-like endosymbionts of ticks. FEMS Microbiology Letters, 362(17), fnv132.

Elsa J., Duron O., Séverine B., Daniel González-Acuña D. & Sidi-Boumedine K. 2015. Molecular methods routinely used to detect Coxiella burnetii in ticks cross-react with Coxiella-like bacteria. Infection Ecology & Epidemiology, 5, 29230.

Esmaeili S., Naddaf S.R., Pourhossein B., Shahraki A.H., Amiri F.B., Gouya M.M. & Mostafavi E. 2016. Seroprevalence of brucellosis, leptospirosis, and Q Fever among butchers and slaughterhouse workers in South-Eastern Iran. PLOS ONE, 11 (1), 0144953.

Evstigneeva A.S., UI'Yanova T.Yu. & Tarasevich I.V. 2007. The survival of Coxiella burnetii in soil. Eurasian soil science, 40 (5), 565-568.

Faix D.J., Harrison D.J., Riddle M.S., Vaughn A.F., Yingst S.L., Earhart K. & Thibault G. 2008. Outbreak of Q fever among US military in western Iraq, June-July 2005. Clinical Infectious Diseases, 46, 65-68.

Fernández-Aguilar X., Cabezón Ò., Colom-Cadena A., Lavin S. & López-Olvera J.R. 2016. Serological survey of Coxiella burnetii at the wildlife-livestock interface in the Eastern Pyresees, Spain. Acta Vet Scand, 58, 26.

Fournier P.E., Marrie T.J. & Raoult D. 1998. Diagnosis of Q Fever. J Clin Microbiol, 36 (7),1823-1834.

Fuad M.M., Aldomy A.J., Wilsmore L. & Safi H. 1998. Q Fever and abortion in sheep and goats in Jordan. Pakistan Vet, 18 (1), 43.

Glazunova O., Roux V., Freylikman O., Sekeyova Z., Fournous G., Tyczka J., Tokarevich N., Kovacava E., Marrie T.J. &, Raoult D. 2005. Coxiella burnetii genotyping. Emerg Infect Dis, 11 (8), 1211-1217.

Hackstadt T. & Williams J.C. 1981. Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc Natl Acad Sci USA, 78, 3240-3244.

Hervé T.D., Amadei M.A., Nezri M. & Raoult D. 2004. Wind in November, Q fever in December. Emerg Infect Dis, 10 (7), 1264-1269.

Hoogstraal H. & Kaiser M.N. 1959. Observations on Egyptian Hyalomma ticks (Ixodoidea, Ixodidae). 5. Biological notes and differences in identity of H. anatolicumand its subspecies anatolicum Koch and excavatumKoch among Russian and other workers. Identity of H. lusitanicum. Ann Entomol Soc Am, 52 (3), 243-261.

Hoogstraal H. & Tatchell R.J. 1985. Ticks parasitizing livestock. In Ticks and tick-borne disease control. A practical field manual. Tick Control, 1, 1-73.IHU – Méditerranée Infection. 2020.

Multi spacers Typing: Coxiella database. (http://ifr48. burnetii/ accessed on 17 June 2020).

Klee S.R., Tyczka J., Ellerbrok H., Franz T., Linke S., Baljer G. & Appel B. 2006. Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii. BMC Microbiology,6, 2.

Marrie T.J. 1990. Q fever - A review. Can Vet J, 31, 555-563.Maurin M. & Raoult D. 1999. Q fever. Clin Microbiol Rev, 12, 518-553.

Mediannikov O., Fenollar F., Socolovschi C., Diatta G., Bassene H., Molez J.F., Sokna C., Trape J.F. & Raoult D. 2010. Coxiella burnetii in humans and ticks in rural Senegal. Plos Negleted Tropical Diseases, 4 (4), e654.

National Association of State Public Health Veterinarians (NASPHV) and National Assembly of State Animal Health Officials (NASAHO). 2013. Prevention and control of Coxiella burnetii infection among humans and animals: guidance for a coordinated public health and animal health response, 2013. 30 pp. ( accessed on 17 June 2020).

Nourollahi Fard S.R. & Khalili M. 2011. PCR-Detection of Coxiella burnetii in ticks collected from sheep and goats in Southeast Iran. Iran J Arthropod Borne Dis, 5 (1), 1-6.

Pearson T., Hornstra H.M., Hilsabeck R., Gates L.T., Olivas S.M., Birdsell D.M., Hall C.M., German S., Cook J.M., Seymour M.L., Priestley R.A., Kondas A.V., Friedman C.L.C., Price E. P., Schupp J.M., Liu C.M., Price L.B., Massung R.F., Kersh G.J. & Keim P. 2014. High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk. BMC Microbiology, 14, 41-49.

Péter O., Dupuis G., Bee D., Lüthy R., Nicolet J. & Burgdorfer W. 1988. Enzyme-linked immunosorbent assay for diagnosis of chronic Q fever. J Clin Microbiol, 26 (10), 1978-1982.

Psaroulaki A., Ragiadakou D., Kouris G., Papadopoulos B., Chaniotis B. & Teselentis Y. 2006. Ticks, tick-borne Rickettsiae, and Coxiella burnetii in the Greek Island of Cephalonia. Ann N Y Acad Sci, 1078, 389-399.

Reichel R., Mearns R., Brunton L., Jones R., Horigan M., Vipond R., Vincent G. & Evans S. 2012. Description of a Coxiella burnetii abortion outbreak in a dairy goat herd, and associated serology, PCR and genotyping results. Res Vet Sci, 93 (3), 1217-1224.

Roest H.I., Ruuls R.C., Tilburg J.J.H.C., Nabuurs-Franssen M.H., Klaassen C.H.W., Vellema P., van den Brom R., Dercksen D., Wouda W., Spierenburg M.A.H., van der Spek A.N., Buijs R., de Boer A.G., Willemsen P.T.J. & van Zijderveld F.G. 2011. Molecular epidemiology of Coxiella burnetii from ruminants in Q fever outbreak, the Netherlands. Emerg Infect Dis, 17 (4), 668-675.

Satta G., Chisu V., Cabras P., Fois F. & Masala G. 2011.Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick-transmitted micro-organisms in Sardinia, Italy. Journal of Medical Microbiology, 60, 63-68.

Seshadri R., Paulsen I.T., Eisen J.A., Read T.D., Nelson K.E., Nelson W.C., Ward N.L., Tettelin H., Davidsen T.M., Beanan M.J., Deboy R.T., Daugherty S.C., Brinkac L.M., Madupu R., Dodson R.J., Khouri H.M., Lee K.H., Carty H.A., Scanlan D., Heinzen R.A., Thompson H.A., Samuel J.E., Fraser C.M. & Heidelberg J.F. 2003. Complete genome sequence of the Q-fever pathogen Coxiella burnetii. Proc Natl Acad Sci USA, 100 (9), 5455-5460.

Spyridaki I., Psaroulaki A., Loukaides F., Antoniou M., Hadjichristdolou C. & Tselentis Y. 2002. Isolation of Coxiella burnetii by a centrifugation shell-vial assay from ticks collected in Cyprus: detection by nested polymerase chain reaction (PCR) and by PCR- restriction fragment length polymorphism analyses. Am J Med Hyg, 66 (1), 86-90.

Sulyok K.M., Kreizinger Z., Hornstra H.M., Pearson T., Szigeti A., Dán Á., Balla E., Keim P.S. & Gyuranecz M. 2014. Genotyping of Coxiella burnetii from domestic ruminants and human in Hungary: indication of various genotypes. BMC Vet Res, 10, 107-112.

Szymańska-Czerwińska M., Galińska E.M., Niemczuk K. & Zasepa M. 2013. Prevalence of Coxiella infection in foresters and ticks in south-eastern Poland and comparison of diagnostic methods. Ann Agric Environ Med, 20 (4), 699-704.

Tilburg J.J., Roest H.J., Buffet S., Nabuurs-Franssen M.H., Horrevorts A.M., Raoult D. & Klaassen C.H.V. 2012. Epidemic genotype of Coxiella burnetii among goats, sheep, and humans in the Netherlands. Emerg Infect Dis, 18, 887-889.

Vincent G., Stenos J., Latham J., Fenwick S. & Graves S. 2016. Novel genotypes of Coxiella burnetii identified in isolates from Australian Q fever patients. Int J Med Microbiol, 306 (6), 463-470.

Walker A.R., Bouattour A., Camicas J.L., Estrada-Peña A., Horak I.G., Latif A.A., Pegram R.G. & Preston P.M. 2014. Ticks of domestic animals in Africa: a guide to identification of species, 2nd ed. Bioscience Reports, Edinburgh, Scotland U.K, 74-217.