VECTOR-BORNE AND ZOONOTIC DISEASES Volume , Number , 2008 © Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2008.0070
Detection of Coxiella burnetii in Ticks Collected from Central Spain A. Toledo,1,2 I. Jado,2 A. S. Olmeda,1 M. A. Casado-Nistal,1 H. Gil,2 R. Escudero,2 and P. Anda2
Abstract
A total of 1482 adult ticks collected from vegetation and animals in central Spain in 2003–2005 were tested for the presence of Coxiella burnetii by polymerase chain reaction and subsequent reverse line blot hybridization (PCR-RLB). C. burnetii was identified in 7.7% of questing ticks (80/1039) and 3.4% of ticks collected from animals (15/443) belonging to four species: Hyalomma lusitanicum, Dermacentor marginatus, Rhiphicephalus sanguineus, and R. pusillus. These findings show an active role of ticks in maintaining C. burnetii in wild and peridomestic cycles in central Spain. Key Words: Coxiella burnetii; Ticks al. 2008), we have no data about the distribution of this agent in the environment. The aim of the present study was to detect the presence of C. burnetii in ticks collected both from vegetation and animals in this area in order to determine the intensity of the circulation of this pathogen in ticks, and to establish the role of some species of ixodids in the maintenance of this pathogen in nature. To do this, questing adult ticks were collected monthly by the blanket-dragging method in six locations within central Spain, five in the region of Madrid and one in the province of Toledo, during a two-year longitudinal study (2003–2005). At the same time, feeding ticks were removed from animals in adjacent areas, including wild and domestic mammals, birds, lagomorphs, and reptiles. For DNA extraction, 70%-ethanol-disinfected ticks were individually crushed (conical tissue grinder, ICN Biomedicals Inc., Irvine, CA) and processed with the QiAamp DNA mini kit (QIAGEN, Hilden, Germany) after treatment with a proteinase K solution (QIAGEN) at 56°C overnight. Of the DNA extracted, about 300 ng, as measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DE), were subjected to the PCR methodology described elsewhere (Berri et al. 2000) with biotinilated primers (Sigma-Genosys; Sigma-Aldrich Química, S.A. Tres Cantos, Madrid, Spain) and subsequent reverse line blotting (RLB). For the latter, PCR amplicons were hybridized to a DNA probe specific for C. burnetii transposase IS1111 as previously described (Barandika et al. 2007). The probe was synthesized with a C6 amino linker (Sigma-Genosys). Preparation of RLB membranes and hybridization were carried out as previously de-
Introduction
Q
FEVER IS A WORLDWIDE ZOONOSIS caused by Coxiella burnetii. Infection is mainly acquired through inhalation of infectious aerosols (Tissot-Dupont et al. 1999) through which C. burnetii is able to infect a broad spectrum of susceptible hosts, including wild and domestic mammals, and even nonmammalian species such as reptiles, fish, birds, and ticks (Woldehiwet 2004). Ticks may act as reservoirs of C. burnetii in nature as they transmit the agent vertically, i.e., transtadially and transovarially, to their progeny (Walker and Fishbein 1991). Although C. burnetii transmission by tick bite to animals has been suggested, this is not the main route of infection for livestock and is still controversial in humans. Over 40 tick species have been found to harbor the bacterium (Mantovani and Benazzi 1953), serving as indicators of its circulation in nature. In northern Spain (Basque Country), a hyperendemic area of Q fever, C. burnetii develops in a peridomestic cycle associated with infected flocks, rather than in a wild cycle, as assessed by examination of small mammals (Barandika et al. 2007) and ticks (Barandika et al. 2008). In these studies performed in the surroundings of livestock farms, only 3 of 253 small mammals and 1 of 691 questing ticks analyzed, mostly Ixodes ricinus, have been found to be positive by polymerase chain reaction (PCR). Central Spain is also an area where C. burnetii has an active role in human disease (de los RíosMartín et al. 2006). However, although studies were performed on livestock farms (Téllez et al. 1989, Carballedo et 1Departamento 2Centro
de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain. Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
1
2
TOLEDO ET AL.
FIG. 1. IS1111 polymerase chain reaction/reverse line blot (PCR/RLB) results for selected samples from the present study: lanes 1–6: specimens of H. lusitanicum; lanes 7–11: specimens of D. marginatum; lanes 12–13: specimens of R. sanguineus; lane 14: negative control for the extraction; lanes 15–18: positive controls (103, 102, 10, and 1 genome equivalents); lane 19: PCR negative control (water).
scribed by Gubbels et al. (1999), with the following adaptations: a 1:15 dilution of the PCR products in 2 SSPE-0.1% sodium dodecyl sulphate (SDS) was loaded onto the blotter; hybridization with the PCR products and subsequent incubation with a 1:32,000 dilution of streptavidin-POD conjugate (Roche Farma, S.A., Madrid, Spain) were carried out at 52°C for 60 minutes, and washing steps were performed at 48°C. After developing with Super Signal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL), PCR products were stripped from the membrane as previously described (Gubbels et al. 1999) and the membrane was rinsed and stored in 20 mM ethylene diamine tetraacetic acid (EDTA) (pH 8.0) at 4°C until the next hybridization, and reused a maximum of 8 times. The prevention of cross-contamination and false positive results was managed by using plugged tips and setting PCRs in a room separate from that used for DNA extraction. To exclude false positive results, negative controls (water) were included during DNA extraction and PCR amplification, and they were also subjected to RLB hybridization. Prevalences were analyzed according to independent variables such as sampling site, biological origin of the specimens (questing or feeding) and tick species, by the chisquare test or Fisher’s exact test using the SPSS statistical package (version 15.0; SPSS Ibérica, Madrid, Spain). Significance was set at a p value of 0.05. To assess the sensitivity of the test, 103, 102, 10, and 1 genome equivalents (GE) of the positive control (C. burnetii Nine Mile phase II) were subjected to the detection method, and it was calculated to be of 1 GE (Fig. 1). Specificity was
TABLE 1.
TICKS ANALYZED
AND
FOUND POSITIVE
FOR
tested against a comprehensive panel of bacterial species (Anaplasma phagocytophilum, Borrelia burgdorferi, Coxiella burnetii, Legionella pneumophila, Mycoplasma pneumoniae, Chlamidophila pneumoniae, Rickettsia conorii, and Orientia tsutsugamushi), and was found to be of 100% (data not shown). A total of 1039 questing adult ticks were collected, H. lusitanicum (701 specimens) and D. marginatus (265) being the most frequently found species (Table 1). In addition, 443 adult ticks were collected from livestock (horse, swine, sheep, and cattle), birds [the booted eagle (Hieraaetus pennatus), the common buzzard (Buteo buteo), and the red kite (Milvus milvus)], wild mammals (red deer, wild boars, foxes, hedgehogs, and beech martens), pets (dogs and cats), lagomorphs (hares and rabbits), and one imported python (Table 2), where H. lusitanicum and D. marginatus were the most abundant species as well. Overall, the infection rate was 6.4%, and C. burnetii was detected in 95 of 1482 specimens from four tick species: H. lusitanicum (67 of 795 specimens of this species: 8.4%), D. marginatus (25/348: 7.2%), R. sanguineus (2/146: 1.4%), and R. pusillus (1/108: 0.9%). No specimens of the rest of the species tested were found to be positive (Table 1). An example of the hybridization is shown in Figure 1. In ticks collected from vegetation, the overall infection rate was 7.7%, the bacterium being detected in 80 specimens from three tick species: H. lusitanicum (61/701: 8.7%), D. marginatus (18/265: 6.8%), and R. pusillus (1/47: 2.1%) (Table 1). The scarce number of tick species other than H. lusitanicum and D. marginatus made these figures statistically not significant (p 0.05)
COXIELLA
No.a
H. lusitanicum H. marginatum D. marginatus R. sanguineus R. pusillus R. bursa I. ricinus H. hispanica H. punctata A. latum Total
701 0 265 0 47 16 8 1 1 0 1039
aNumber
of ticks studied.
Positive (%) 61 0 18 0 1 0 0 0 0 0 80
(8.7) (6.8) (2.1)
(7.7)
SPECIES
AND
BIOLOGICAL ORIGIN
Ticks collected from animals
Questing ticks Tick species
BURNETII, BY
No. 94 13 83 146 61 40 0 5 0 1 443
Positive (%) 6 0 7 2 0 0 0 0 0 0 15
(6,4) (8,4) (1.4)
(3.4)
Total No. 795 13 348 146 108 56 8 6 1 1 1482
Positive (%) 67 0 25 2 1 0 0 0 0 0 95
(8.4) (7.2) (1.4) (0.9)
(6.4)
COXIELLA BURNETII IN TICKS IN SPAIN TABLE 2. Tick species H. lusitanicum H. marginatum D. marginatus R. sanguineus R. pusillus R. bursa H. hispanica A. latum Total
INFECTION RATE
3 OF
TICKS COLLECTED
FROM
ANIMALS SORTED
BY
HOST
Livestock
Birds
Wild mammals
Pets
Lagomorphs
Reptiles
Total
3/71 (4.2)a,b 0/12 7/60 (11.6)d 0/1 0 0 0 0 10/185 (5.4)
0/1 0/1 0 0 0/1 0 0 0 0/3
3/21 (14.2)c 0 0/23 2/38 (5.2)e 0/28 0 0 0 5/110 (4.5)
0/1 0 0 0/106 0/9 0 0 0 0/116
0 0 0 0/1 0/22 0 0/5 0 0/28
0 0 0 0 0 0 0 0/1 0/1
6/94 (6.4) 0/13 7/83 (8.4) 2/146 (1.4) 0/61 0/40 0/5 0/1 15/443 (3.4)
aPositives/studied;
numbers in parentheses indicate the percentage of positives. positives among 30 specimens collected from sheep (10%). cThree positives among 10 specimens collected from red deer (30%). dSeven positives among 50 specimens collected from horses (14%) eTwo positives among 14 specimens collected from foxes (14%). bThree
In ticks collected from animals, the overall infection rate was 3.4%, the highest being that of D. marginatus (7/83: 8.4%), followed by H. lusitanicum (6/94: 6.4%) and R. sanguineus (2/146: 1.4%) (Table 1). Significant differences were observed among tick species (p 0.05). All the positives removed from animals corresponded to ticks collected from livestock (horses and sheep: 10 specimens) and wild mammals (red deer and foxes: 5 specimens), that accounted for 3.4% of the ticks collected (15/443). In addition, H. lusitanicum showed the highest rate of infection in wild mammals (14.2%), whereas in livestock it was D. marginatus (11.6%) (Table 2). No significant differences between livestock and wild mammals were observed (p 0.05). The detection of C. burnetii in four tick species is not surprising because over 40 species have been found to be infected with the bacterium (Sˇpitalská and Kocianová 2003). The results in questing ticks showed a high infection rate in H. lusitanicum and D. marginatus that could be explained by transovarial and transtadial transmission, as has been previously reported in different tick species. These results suggest that H. lusitanicum and D. marginatus may act as reservoirs for the bacterium in central Spain. However, transmission experiments must be carried out with H. lusitanicum to elucidate the role of this species in the maintenance and transmission of the agent because this is, to our knowledge, the first mention of C. burnetii in H. lusitanicum. We also detected the presence of C. burnetii in questing R. pusillus, but its low incidence suggests that the role of this tick in the maintenance of the bacterium may be marginal. Overall, the presence of C. burnetii in ticks removed from animals was lower than in questing ticks (3.4% and 7.7%, respectively), and this difference was statistically significant (p 0.05). The two species that showed the highest infection rate in feeding ticks were D. marginatus and H. lusitanicum, as happened with questing ticks. Consequently, the difference in the overall percentage of positives between questing and feeding ticks was due to R. sanguineus, which was only found feeding on animals and was only rarely positive (1.4%). Interestingly, none of the specimens of R. sanguineus removed from dogs were infected, although dogs are competent hosts for C. burnetii and R. sanguineus is known to act as a vector for this host (Smith 1941). However, these spec-
imens were removed from dogs living in urban areas, where the presence of C. burnetii is less common. R. pusillus specimens were found in both vegetation and animals (47 and 108 specimens, respectively). Its low positivity (2.1% in questing ticks and no positives in feeding ticks) would further account for the lower overall percentage of positives detected in feeding ticks. The overall prevalence of infection in ticks from central Spain (6.4%) was higher than that reported from any other area of Spain [0.1% (1/691) in the Basque Country (Barandika et al. 2008)], Europe [0/1716 in Germany (Rehácek et al. 1993); 2.5% in Slovakia and Hungary (6/235) (S˘pitalská and Kocianová 2003), and 0.8% (3/390) in D. marginatus in Greece (Psaroulaki et al. 2006)], or other Mediterranean countries [2% (20/1019) in Egypt (Loftis et al. 2006)]. Tick species are probably the reason for these differences. It seems that H. lusitanicum and D. marginatus (the former species tested in the present study only, and the latter in central Spain as well as in Slovakia, Hungary, and Greece) may be considered as potential vectors. Also, I. ricinus, the most prevalent species in the Basque Country and Germany, does not seem to play an active role in C. burnetii ecology and, consequently, seems to be responsible for the low percentages of positive ticks found in these areas (Rehácek et al. 1993, Barandika et al. 2008). C. burnetii is known to be transmitted to humans mainly through a peridomestic cycle and can cause outbreaks in humans via aerosol transmission associated with parturient livestock. In addition, a wild cycle may be present in certain areas. The two cycles are interrelated when control measures are not well implemented. Previous serological studies performed on livestock in Madrid indicated that up to 76.6% of goats and 8.8% of cattle had antibodies against C. burnetii (Téllez et al. 1989). More recently, Carballedo et al. (2008) found rates of positivity of 31.5%, 22.4%, and 5.6% for sheep, goats, and cattle, respectively. In this latter study, 76% of livestock farms tested positive for at least one animal. In addition, in the Basque Country, where C. burnetii was not detected in ticks (Barandika et al. 2008), the percentage of seropositive sheep, goats and cattle was of 16.8%, 8.7% and 6.7%, respectively, with 57% of farms having at least one positive animal (A. L. García-Pérez, personal communication).
4 Consequently, taking into account the lack of a wild cycle for C. burnetii in the Basque Country, we can hypothesize that, without tick species that can act as competent vectors for Coxiella in the environment, its circulation in livestock appears to be much less intense. Recently, an outbreak associated with a farm used for school tours near Madrid City affected up to 22 patients, mostly children (de los Ríos-Martín et al. 2006). Although data about the incidence of Q fever in the area are not available, this outbreak highlights the intensity of the circulation of C. burnetii in our area. In the present study the detection of C. burnetii in questing ticks and ticks feeding on wild and domestic animals provides initial evidence of a potential wild cycle of C. burnetii in central Spain that could be feeding the perydomestic cycle in the area and put at risk further measures to control transmission to humans. Conclusions The presence of C. burnetii in ixodids in central Spain has been demonstrated by PCR-RLB in this study. Its detection in both questing ticks and ticks collected from animals indicates that C. burnetii develops in a transmission cycle of high intensity in central Spain, leading us to conclude that control measures should be implemented toward reducing the incidence of Q fever in this area. The high infection rate observed in H. lusitanicum and D. marginatus highlights the importance of these tick species as putative vectors and reservoirs for C. burnetii. Acknowledgments C. burnetii Nine Mile phase II purified DNA was kindly provided by Dr. Elena Kovác˘ová, of the Institute of Virology, Bratislava, Slovak Republic. The authors are grateful to Frank M. Hodgkins for reviewing the English version of this manuscript. Disclosure Statement Álvaro Toledo was supported by a fellowship from the Fondo de Investigación Sanitaria (FIS) PI051374. This work was also supported by FIS PI050901 and INIA FAU200600002-C04-02 and FAU2006-00002-C04-04. References Barandika, JF, Hurtado, A, García-Esteban, C, Gil, H, Escudero, R, Barral, M, Jado, I, Juste, RA, Anda, P, García-Pérez, AL. Tick-borne zoonotic bacteria in wild and domestic small mammals in northern Spain. Appl Environ Microbiol 2007; 73:6166–6171. Barandika JF, Hurtado A, García-Sanmartín J, Juste RA, et al. Prevalence of tick-borne zoonotic bacteria in questing adult ticks from northern Spain. Vector Borne Zoonotic Dis 2008 (in press). Berri, M, Laroucau, K, Rodolakis, A. The detection of Coxiella burnetii from ovine genital swabs, milk and fecal samples by
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