Coxiella burnetii and Coinfections in Ixodes ricinus Ticks in Central Germany

Introduction

I xodes ricinus is the most abundant tick species in Germany and the main vector of Lyme borreliosis spirochetes in Central Europe. Additionally, this tick species transmits several emerging pathogens e.g. Anaplasma phagocytophilum, spotted fever group rickettsiae, Coxiella burnetii, Francisella tularensis, and Babesia spp. (Stanek 2009). C. burnetii is the causative agent of Q fever, a zoonosis that occurs worldwide and infects a variety of different animals, including domestic mammals such as cattle and sheep. In humans, Q fever presents mainly as a self-limited respiratory infection, sometimes complicated by pneumonia, hepatitis, or myopericarditis. Only 30% of the infected individuals develop clinical signs. In a few cases, infection leads to chronic Q fever presenting as culture-negative endocarditis (Maurin and Raoult 1999). The reservoirs are extensive but only partially known and include mammals, birds, and arthropods, mainly ticks. More than 40 tick species can be naturally infected with C. burnetii worldwide (Maurin and Raoult 1999). In Central Europe, the sheep tick Dermacentor marginatus is the most important vector. The organism multiplies in the gut cells of ticks and large numbers of bacteria are shed in tick feces (Liebisch 1977).

Material and Methods

A total of 1000 ticks were collected in 2006 and 2007 (430 nymphs, 293 female adults, 277 male adults) by blanket-dragging. The investigation area, located in Thuringia in Central Germany, consists of brushwood, foliage, and grassland along a small stream and is frequently used for hiking, jogging, and biking. Ticks were assigned to the species I. ricinus and developmental stage using a standard key for morphological identification (Hillyard 1996). DNA extraction was performed directly from the ticks using the GenElute^TM Bacterial Genomic DNA Kit (Sigma-Aldrich). As negative control, a 1.5-mL tube with phosphate-buffered saline was processed in parallel for each 10 tick preparations. To avoid cross contaminations, all steps were performed in separate rooms under a laminar air flow bench. DNA of the positive control was pipetted in a final step after all other samples. Detection and quantification of C. burnetii were performed with TaqMan-based real-time polymerase chain reaction (PCR) assays targeting the transposase element IS1111 and isocitrate dehydrogenase (icd) gene (Klee et al. 2006). Quantification of C. burnetii was done with 10-fold serial dilutions of plasmids with cloned icd fragments ranging from 1 × 10⁰ to 1 × 10⁵ plasmid copy numbers.

For detection of coinfections, conventional PCRs were performed using the following specific targets: Borrelia spp., ospA (Michel et al. 2003); Rickettsia spp., citrate synthase gene gltA (Nilsson et al. 1999); and Babesia spp., 18S rRNA gene (Hartelt et al. 2004). To identify Borrelia species, all positive amplicons were separately digested with the restriction enzymes Kpn2I, BglII, SspI, HindIII, XbaI (MBI Fermentas), and SfuI (Roche Diagnostics) (Michel et al. 2003, Lenčáková et al. 2006). Rickettsia spp.- and Babesia spp.-positive PCR products were purified by means of the Agarose Gel Extraction Kit (Jena Bioscience GmbH) for sequence analysis. Sequencing reactions were performed using the DYEnamic^TM ET Dye Terminator Cycle Sequencing Kit (GE Healthcare), followed by preparation for running through the ABI PRISM 310 genetic analyzer (Applied Biosystems). The obtained sequences were aligned with the program ClustalW (www.ebi.ac.uk/Tools/clustalw2/index.html) and compared with the data stored in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.).

Results

C. burnetii was detected in 1.9% (19/1000) I. ricinus ticks (10 nymphs, 4 female adults, 5 male adults). Moreover, C. burnetii-specific DNA was quantified with serial dilutions of plasmids with cloned icd fragments (Table 1). Based on the DNA extraction volume of 100 μL and the quantification of singular icd, we found 4 × 10⁵ genomic equivalents per tick, varying only slightly in a range of 2 × 10⁵ and 7 × 10⁵ genomic equivalents. Interestingly, coinfections with a broad spectrum of other tick-transmitted pathogens were present in 10 C. burnetii-positive ticks: Borrelia afzelii (2 nymphs), Borrelia garinii OspA type 6 (1 male adult), Bo. garinii OspA type 7 (1 male adult), Borrelia burgdorferi (2 nymphs), Borrelia valaisiana (1 male adult), Babesia microti (1 female adult), Borellia garinii OspA type 6 and Borrelia burgdorferi (1 nymph), Borrelia burgdorferi and Rickettsia helvetica (1 nymph) (Table 1). Overall, Borrelia spp., Rickettsia spp., and Babesia spp. were present in 27.0%, 14.7%, and 5.0% of the investigated I. ricinus ticks. We detected a broad heterogeneity of Borrelia species and OspA types including Bo. burgdorferi, Bo. afzelii, Bo. garinii OspA types 3, 5, 6, 7, and 8, Bo. bavariensis (formerly Bo. garinii OspA type 4), Bo. valaisiana subtypes I and II, Bo. spielmanii, and Bo. lusitaniae. Fourteen ticks harbored mixed infection with several Borrelia species. Detailed information about the infection rates of these pathogens with respect to species differentiation and seasonal aspects had been published (Hildebrandt et al. 2010a, 2010b).

Discussion

Q fever is a notifiable disease in West Germany from 1962 and in the former German Democratic Republic (GDR, East Germany) from 1979 onwards (Hellenbrand et al. 2001). A lot of regions notify sporadic cases or outbreaks regularly or even every year, for example, in Jena (Thuringia), Göppingen (Baden Württemberg), and Aschaffenburg (Bavaria) (www.rki.de). In areas where D. marginatus is not endemic but Q fever incidence is relatively high, I. ricinus might play a role in the transmission cycle of C. burnetii, although evidence is lacking. In Germany, the bacterium has been isolated sporadically from I. ricinus ticks in Baden (Hengel et al. 1950), Northrhine-Westphalia (Schaaf 1961), Hesse (Thoms 1996), and Thuringia (Kramer 1990). Our study is the first to report on C. burnetii in I. ricinus ticks from a landscape conservation area in Central Germany (Thuringia), which is frequently used for hiking, jogging, and biking. Moreover, we got an interesting insight into a broad spectrum of coinfections. To our knowledge, no data on coinfections of ticks with C. burnetii and other tick-borne pathogens exist so far.

In the last years, the biggest human outbreaks of Q fever in Germany, in Jena and Soest, were due to direct or indirect contact with infected animals. In both areas, D. marginatus is not present. Previous veterinary serological studies in Thuringia identified seropositive cattle and sheep and even sporadic seropositive reactions in roe deer, red deer, and wild boars (Kramer 1991, Lange and Klaus 1992). Transovarial and transstadial transmission of C. burnetii in ticks was observed, and through highly infectious tick feces, domestic animals can be a source of human infection (Liebisch 1977). The broad spectrum of copathogens indicates interactions in transmission cycles not only for Borrelia spp., but also for emerging pathogens such as Babesia spp. or Rickettsia spp.

The main source of human infection is the inhalation of contaminated aerosols. Interestingly, the main potential reservoir hosts in the investigated area are small mammals, roe deer, and red deer, whereas sheep are absent according to the responsible forestry office. The coexistence of C. burnetii in I. ricinus ticks together with other pathogens indicates the possibility of coinfections in humans in areas where people are in close contact with infected ticks and domestic animals.