1. Iran J Arthropod-Borne Dis, 2011, 5(1): 1–6 SR Nourollahi Fard and M Khalili: PCR-Detection of … 1 Original Article PCR-Detection of Coxiella burnetii in Ticks Collected from Sheep and Goats in Southeast Iran *SR Nourollahi Fard, M Khalili Department of Pathobiology, School of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran (Received 20 Sep 2010; accepted 24 May 2011) Abstract Background: There is a little data on Coxiella burnetii (Q fever agent) in Iran. Ticks may play a significant role in the transmission of C. burnetii among animals. The aim of this study was to use polymerase chain reaction for the detection of C. burnetii in ticks collected in Southeast Iran. Methods: One hundred and sixty ticks were collected from domestic animals in three localities of Kerman Province, Southeast Iran from November to June 2009. The collected ticks were divided into 35 pools and examined by Trans- PCR for C. burnetii. Results: Three pools, each consisting of five female of Hyalomma anatolicum anatolicum and one pool (6 ticks) of Rhipicephalus sanguineus ticks collected from goats and sheep were found to be positive by Trans-PCR. Conclusion: This paper documents the first molecular detection of C. burnetii in ticks, which shows their role as putative vectors and reservoirs for this pathogenic agent. Keywords: Coxiella burnetii, Ticks, Trans-PCR, Iran Introduction Ticks are well known for their impact on the health of both human and animals during in- festation (Noda et al. 1997, Vilcins et al. 2005). Ticks can transmit the widest range of patho- gens, including protozoa, bacteria, rickettsiae, spirochetes and viruses (Kim et al. 2006, Psaroulaki et al. 2006). The etiological agent of Q fever, Coxiella burnetii, has been iden- tified in over 40 tick species (Kazar 2005, Psaroulaki et al. 2006). Coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of acute and chronic Q fever in humans. Q fever has been described worldwide except in New Zealand. From 1999 to 2004, there were 18 reported outbreaks of Q fever from 12 differ- ent countries involving two to 289 people. Six outbreaks involved sheep; three involved goats; one resulted from exposure to goat manure; one from exposure to ovine manure; one involved exposure to wild animals; one involved exposure to cats and dogs; and in two outbreaks the source was unknown (Maurin and Raoult 1999). A few studies were conducted on Q fever in Iran some 50 years ago. Q fever cases have been reported from some countries neighbor- ing Iran, such as Turkey and Oman (Scrim- geour et al. 2003, Kennerman et al. 2008). Re- sults of a serosurvey undertaken on 42 sheep flocks in Turkey showed that 20% of sheep were seropositive (Kennerman et al. 2008). Recently, an outbreak of Q fever occurred with high morbidity in U.S. marines located in Iraq (Faix et al. 2008). The reservoirs are extensive but only partially known and include mammals, birds, and arthropods, mainly ticks. Farm animals, mainly cattle, sheep, and goats, are the primary reservoirs of infection. High numbers of C. *Corresponding author: Dr Mohammad Khalili, Email: mdkhalili1@yahoo.com Iran J Arthropod-Borne Dis, 2011, 5(1): 1–6 SR Nourollahi Fard and M Khalili: PCR-Detection of … 2 burnetii are present in the placenta of in- fected parturient animals and are shed in the environment following labor or abortion. Hu- mans acquire the infection mainly via inhala- tion of contaminated aerosolized particles or in- gestion of unpasteurized dairy products (Maurin and Raoult 1999). Clinical Q fever in human can present as two basic forms: acute or chronic. Acute Q fe- ver normally manifests as a self-limiting flu- like illness characterized by high-grade fever, peri-orbital headache, and myalgia. However, in some cases pneumonia occurrence requires hospitalization. Coxiella burnetii can estab- lish a persistent, latent infection that may re- activate months or years after initial exposure to the organism to cause chronic disease. Chro- nic Q fever is typically associated with patients who are immunocompromised and/or who have pre-existing heart valve defects and most com- monly presents as endocarditis (Maurin and Raoult 1999) It is likely that factors such as the route of infection and the inoculum size, affect the expression of C. burnetii infection. Indeed the respiratory route is associated with pneumo- nia and the intra peritoneal route with hepa- titis (Marrie et al. 1996). Coxiella burnetii may induce reproductive disorders such as abor- tion, stillbirth, and delivery of weak and non- viable neonates in ruminants (Lang et al. 1994). Ticks can serve as indicators of infection in nature. For example, 10 C. burnetii strains were isolated from Ixodes ricinus, Derma- centor reticulatus, D. marginatus, Haema- physalis concinna, and H. inermis ticks spe- cies collected in different habitats in Slovakia (Rehacek et al. 1991). The organism multi- ply in the gut cells of ticks and large num- bers of C. burnetii are shed in tick feces. Infected ticks are probably the most impor- tant agents in maintaining the whole cycle of C. burnetii. Ticks may play a significant role in the transmission of C. burnetii among the wild vertebrates, especially in rodents, lago- morphs, and wild birds. Also, experimental transmission of C. burnetii from infected to uninfected guinea pigs via tick bite has been performed with Ixodes holocyclus, Haema- physalis bispinosa and Rhipicephalus san- guineus (Angelakis and Raoult 2010). DNA-based methods have been success- fully used for detection of C. burnetii in ticks, fresh tissues, paraffin-embedded clinical sam- ples, frozen samples, formalin-fixed tissues, serum samples, and milk (Maurin and Raoult 1999). The isolation of the pathogen is a reli- able diagnostic method, but it remains time- consuming and hazardous and requires bio- safety level BL3 practices. Therefore, the di- agnosis of C. burnetii infection is usually done by PCR or serological examination. The PCR assay with primers targeting IS1111, the re- petitive, transposon-like element (Trans-PCR), has been found to be very specific and sensi- tive for the detection of C. burnetii in differ- ent clinical samples (Vaidya et al. 2008). The aim of our study was to use polymerase chain reaction for the detection of C. burnetii in ticks collected in Southeast Iran. Materials and Methods Standard strain of C. burnetii Phenol-killed, purified, and lyophilized cells of the C. burnetii Nine Mile, phase I, strain (RSA 493) were used for this study. Tick collection During November and June 2009, one hundred and sixty ticks were collected from domestic animals (goats, sheep) in Kerman and Bardsir cities, Kerman Province (south- east of Iran) and put them in 35 pools (each pool formed from 5-7 ticks). The collected ticks were properly noted and placed in properly-labeled bottles containing denatured alcohol (95% ethanol+4% methanol+1% pyri- dine). The ticks were transferred to the para- sitology laboratory of School of Veterinary Medicine, Shahid Bahonar University of Ker- man and adults were identified using a stereo- Iran J Arthropod-Borne Dis, 2011, 5(1): 1–6 SR Nourollahi Fard and M Khalili: PCR-Detection of … 3 microscope, according to general identification keys (Kaiser and Hoogstraal 1963, Mazlum 1968, Walker et al. 2003). DNA extraction Prior to DNA extraction, ticks were re- peatedly washed with 70% ethanol and al- lowed to air dry for 10 min on sterile paper. Ticks were divided into pools of 3-10, ac- cording to species, gender and locality, and were homogenized in minimum essential me- dium (Biochrom AG, Germany) supplemented with 4% fetal bovine serum. DNA was ex- tracted from 300 µ l of the homogenized sus- pension using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The extracted DNA stored at –20 °C until used. Trans-PCR A PCR assay targeting IS1111 fragment, a transposon-like repetitive region, was used to detect C. burnetii in clinical samples. In this study, trans-1 and trans-2 primers were used from the published data sequence of a trans- poson-like repetitive region of the C. burnetii genome (Hoover et al. 1992). The primers trans-1 (5′-TAT GTA TCC ACC GTA GCC AGT C-3′) and trans-2 (5′-CCC AAC AAC ACC TCC TTA TTC-3′) were synthesized by Copenhagen (Denmark). The trans-1 and trans-2 primers were designed to amplify a 687-bp fragment of the transposon-like re- petitive element. The trans-PCR assay was performed as described previously (Vaidya et al. 2008). The PCR mixture (25 µl) included 2.5 µl of 10× PCR buffer (100 mM Tris-HCl buffer, pH 8.3, 500 mM KCl, 15 mM MgCl2, and 0.01% gelatin), 200 µM deoxynucleo- side triphosphate mix, 2 µM of each primers, 0.3 U of Taq DNA polymerase, 3 µl of tem- plate DNA, and sterilized water to make up the reaction mixture volume. The DNA am- plification reaction was performed in a MG thermocycler (Eppendorf, Germany). The cy- cling conditions for PCR included an initial de- naturation of DNA at 95° C for 2 min, fol- lowed by five cycles at 94° C for 30 s, 66 to 61° C (the temperature was decreased by 1° C between consecutive steps) for 1 min, and 72° C for 1 min. These cycles were followed by 35 cycles consisting of 94° C for 30 s, 61° C for 30 s, and 72° C for 1 min and then a final extension step of 10 min at 72° C. Amplicons were visualized by agarose gel electrophoresis, stained with ethidium bromide at a final con- centration of 0.5 mg/mL, and photo documented. Results Of a total of one hundred and sixty ticks, three pools consisting of 5 female Hyalomma anatolicum anatolicum and one pool of three Rhipicephalus sanguineus ticks collected in June 2009 on goats and sheep were found positive using Trans-PCR (Fig.1). Fig. 1. Lanes 1–3, an example of positive samples, lane 4, DNA template from the standard C. burnetii Nine Mile, lane 5, 100-bp DNA ladder, and lane 6, Non Template Control (NTC) Discussion Serologic evidences indicate people and animals in Iran are exposed to C. burnetii (Khalili and Sakhaee 2009, Khalili et al. 2010, Sakhaee and Khalili 2010), but there is not information available regarding the presence of this agent in specific vectors. This report presents the first detection C. burnetii in ticks in Iran. Iran J Arthropod-Borne Dis, 2011, 5(1): 1–6 SR Nourollahi Fard and M Khalili: PCR-Detection of … 4 It is well known that ticks participate in the transmission of different pathogenic mi- cro-organisms to man and to animals. In na- ture, C. burnetii is found primarily in cycles involving ticks and vertebrates particularly ro- dents. C. burnetii is transmitted to domestic animals either by tick bites or through con- tact with infected excreta. Ticks are consid- ered as natural primary reservoirs of C. bur- netii and are responsible for transmission of the infection to wild animals and domestic animals (Norlander 2000). Ticks may play a significant role in the transmission of C. bur- netii among the wild vertebrates, especially in rodents, lagomorphs, and wild birds (An- gelakis and Raoult 2010). In ticks, C. bur- netii can multiply to very high titers, remains viable during their entire life, and can be trans- mitted transovarially to next generations. In the enzootic cycle, ticks and vertebrates such as rodents are important components (Aitken 1987). Also, experimental transmission of C. burnetii from infected to uninfected guinea pigs via tick bite has been demonstrated by Ixodes holocyclus, Haemaphysalis bispinosa, and Rhipicephalus sanguineus (Maurin and Raoult 1999). Ticks expel heavy loads of C. burnetii with their feces onto the skin of the animal host at the time of feeding. Appropri- ate tick control strategies and good hygiene practice can decrease environmental contami- nation (Angelakis and Raoult 2010). This study examined the occurrence of C. burnetii, the infectious agent of Q fever, in ticks in Iran, as a possible rout of infection in Q- fever outbreaks. We used pools of ticks for DNA extrac- tion, which offers the possibility of testing a large number of ticks collected in the field. We demonstrated the presence of C. burnetii by Trans-PCR in Hyalomma anatolicum ana- tolicum and Rhipicephalus sanguineus ticks collected in southeast Iran. Among the vari- ous species of hard ticks, Hyalomma ana- tolicum anatolicum is one of the most fre- quently found tick throughout Iran (Abbasian 1961). This paper documents the first mo- lecular detection of C. burnetii in ticks. This communication confirms the presence of C. burnetii in Rhipicephalus sanguineus and Hya- loma spp. as previously reported in Cyprus and Italy (Spyridaki et al. 2002, Satta et al. 2010). The preventive measures in animals should incorporate: the quarantine of imported ani- mals, routinely testing of animals for anti- bodies to C. burnetii, tick control and main- tenance of surrounding bushes. Furthermore, considering the lack of in- formation on the specific tick vector(s) of Q fever in the country, present preliminary data are meaningful. We are presently working on tick species identification, and hope to carry out similar studies covering other sites in the country. Investigations on C. burnetii using Trans-PCR are important method for diagno- sis and disease control of Q fever. In conclu- sion, this paper documents the first detection of C. burnetii in tick species in Iran. 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