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Rickettsiae in ticks from wild ungulates of Sierra Nevada and Doñana national parks (Spain)
10.1111/j.1469-0691.2008.02148.x
Rickettsiae in ticks from wild ungulates of Sierra Nevada and Don˜ana national parks (Spain) F. J. Ma´rquez Dpto. Biologı´a Animal, Biologı´a Vegetal y Ecologı´a, Univ. Jae´n, Jae´n, Spain
In recent years there have been several reports about the presence of new spotted fever group (SFG) Rickettsia in a geographic range [1,2]. The specific identification of the tick rickettsiae is established by polymerase chain reaction (PCR) amplification and DNA sequence of relevant rickettsial genes, particularly the outer membrane proteins genes (ompA and ompB), the citrate synthase (gltA) and the 16S rRNA genes [3,4]. The epidemiology of rickettsiae and rickettsial diseases in Andalusia (south Iberian Peninsula) is not well known. The purpose of this study was to identify and characterise SFG rickettsiae present in ticks from wild ungulates in two significant natural parks and surrounding areas situated in Andalusia (P.N. Sierra Nevada and Don˜ana). The presence of Rickettsia in hard ticks was evaluated using molecular techniques (PCR amplification and sequencing) over a sample of 180 ticks of eight species (Ixodes ricinus Linnaeus, 1746, Haemaphysalis sulcata Canestrini and Fanzago, 1877, H. punctata Canestrini and Fanzago, 1877, Hyalomma lusitanicum Koch, 1844, Rh. turanicus Pomerantzev, 1940, Rh. bursa Canestrini and Fanzago, 1877, Rh. (Boophilus) annulatus (Say, 1821) and Dermacentor marginatus (Sulzer, 1776)) collected on 23 red deer, 5 fallow deer, 23 Iberian ibex and 32 wild boar during population control programmes in each national park during the years 2002–2003 (Table 1). D. marginatus, I. ricinus, Hy lusitanicum and Rh. bursa appeared in both natural spaces whereas Rh. annulatus and H. sulcata were restricted, respectively, to Don˜ana and Sierra Nevada. DNA was extracted individually using the kit Nucleo Spin Tissue (MachereyCorresponding author and reprint requests: F. J. Ma´rquez, Dpto. Biologı´a Animal, Biologı´a Vegetal y Ecologı´a, Univ. Jae´n, Campus Las Lagunillas s ⁄ n, Jae´n, 23071 – Jae´n, Spain E-mail: [email protected] No conflicts of interest declared.
Nagel, Du¨ren, Germany) and specific rickettsial sequences were detected by using PCR primers that amplify a portion of glta, ompA and ompB genes, respectively [3]. Positive PCR products were sequenced in a CEQ 2000XL capillary DNA sequencer (Beckman Coulter Life, Brea, CA, USA) according to the manufacturer’s instructions. Sequences were manually aligned and analysed with Bioedit and identified using the BLAST feature of GenBank. Overall, rickettsial DNA was detected in 51 (28.3%) of the examined ticks. Sequence analysis revealed that I. ricinus, captured on red deer, Iberian ibex and wild boar, were infected with R. monacensis, including strain IRS3, and R. helvetica (prevalences of 50.0% and 4.3%, respectively), while D. marginatus, mainly from wild boar, presents R. slovaca (13.9%) and R. raoultii (38.9%). R. massiliae was found in one Rh. turanicus (prevalence of 20.0%) from Iberian ibex. H. sulcata, from Iberian ibex of Sierra Nevada, shows the presence (6 ⁄ 17, prevalence of 35.3%) of a still not described Rickettsia sp. that is closer to R. hoogstraalii. None of the H. punctata, Hy. lusitanicum, Rh. annulatus or Rh. bursa ticks studied contained rickettsiae. This study confirms the presence of several rickettsiae species in ticks affecting wild ungulates in the south of the Iberian Peninsula. Ticks infected with zoonotic Rickettsia species (R. slovaca, R. raoultii, R. helvetica and R. monacensis) could be maintained and dispersed by wild ungulates. The rickettsia detected in H. sulcata appears to have a distribution limited to mediumhigh Mediterranean mountain ranges. The high prevalence detected should be taken into consideration because elevated ungulate host and vector population densities will be related to an increase in the risk of transmission of these pathogens to the human population that lives, works or uses temporally these natural spaces.
2009 The Author Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 227–229
F
0 (4) 2 (6)
1 (4)
1
2
4
6
8
2
4 2
2
0 (2) 16 (25)
9 (18) 1 (1)
9
R. mon.
0 (6)
0 (2)
4
5
11
R. rao.
2
2
F
4
1
M
5
R. slo.
1 (3) 0 (16)
9 (11) 5 (16)
Pos. ⁄ ass.
23
S. Nevada
Iberian ibex
1
R. sp.
8
R. mon.
16
2 13
1
M
1
R. hel.
30
12
18
F
5
M 5 6 3 24 18 1 18 4 75
1
R. mas.
Total ticks
R. sp.
41 11 4 2 18 4 19 18 105
F
10
1 4
5
M
46 17 7 26 36 5 37 22 180
Total
36
2 12
11 11
F
Pos. ⁄ ass., positive vs. assayed; M, male, F, female; R. hel., R. helvetica; R. mas., R. massiliae; R. mon., R. monacensis; R. rao; R. raoultii; R. slo., R. slovaca; R. sp., undescribed rickettsiae.
Ixodes ricinus Hamaphysalis sulcata Hamaphysalis punctata Hyalomma lusitanicum Dermacentor marginatus Rhipicephalus turanicus Rhipicephalus bursa Rhipicephalus annulatus Total tick number
Pos. ⁄ ass.
M
Pos. ⁄ ass.
Tick species
R. rao.
24
S. Nevada
4 2 2 2 5 6 29
8
10 0 36
3 18 3
8
Host number
1
2
Don˜ana
R. mon.
1
National Park
(7) (20) (5) (2) (15) (6)
5
F
Wild boar
0 0 1 0 0 0
6 (10)
M
Host
Ixodes ricinus Haemaphysalis sulcata Haemaphysalis punctata Hyalomma lusitanicum Dermacentor marginatus Rhipicephalus turanicus Rhipicephalus bursa Rhipicephalus annulatus Total tick number
R. rao.
Pos. ⁄ ass.
R. hel.
Pos. ⁄ ass.
Tick species
R. mon.
5
23
Host number
Don˜ana
Don˜ana
National Park
Fallow deer
Red deer
Host
Table 1. Capture data (ticks and host) from Don˜ana and Sierra Nevada National Parks
228 Clinical Microbiology and Infection, Volume 15, Supplement 2, December 2009
2009 The Author Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 227–229
Ma´rquez
ACKNOWLEDGEMENTS This study has been supported by grants from the Andalusia Government (Research Group BIO-294). The authors thank Celia Sa´nchez and Jose´ E. Granados for help in sampling and Dr Ricardo Oya of the Research Services of Jae´n University for his technical assistance in sequencing. The experiments comply with the current laws of the European Union.
Rickettsiae in ticks from wild ungulates 229
2. Brouqui P, Parola P, Fournier PE, Raoult D. Spotted fever rickettsioses in southern and eastern Europe. FEMS Immunol Med Microbiol 2007; 49: 2–12. 3. Ma´rquez FJ, Muniain MA, Soriguer RC et al. Genotypic identification of an undescribed spotted fever group Rickettsia in Ixodes ricinus from southwestern Spain. Am J Trop Med 1998; 58: 570–577. 4. Rydkina E, Roux V, Fetisova N et al. New Rickettsiae in ticks collected in territories of the former Soviet Union. Emerg Infect Dis 1999; 5: 811–814.
REFERENCES 1. Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world, emerging diseases challenging old concepts. Clin Microbiol Rev 2005; 18: 719–756.
2009 The Author Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 227–229
Rickettsiae in ticks from wild ungulates of Sierra Nevada and Don˜ana national parks (Spain) F. J. Ma´rquez Dpto. Biologı´a Animal, Biologı´a Vegetal y Ecologı´a, Univ. Jae´n, Jae´n, Spain
In recent years there have been several reports about the presence of new spotted fever group (SFG) Rickettsia in a geographic range [1,2]. The specific identification of the tick rickettsiae is established by polymerase chain reaction (PCR) amplification and DNA sequence of relevant rickettsial genes, particularly the outer membrane proteins genes (ompA and ompB), the citrate synthase (gltA) and the 16S rRNA genes [3,4]. The epidemiology of rickettsiae and rickettsial diseases in Andalusia (south Iberian Peninsula) is not well known. The purpose of this study was to identify and characterise SFG rickettsiae present in ticks from wild ungulates in two significant natural parks and surrounding areas situated in Andalusia (P.N. Sierra Nevada and Don˜ana). The presence of Rickettsia in hard ticks was evaluated using molecular techniques (PCR amplification and sequencing) over a sample of 180 ticks of eight species (Ixodes ricinus Linnaeus, 1746, Haemaphysalis sulcata Canestrini and Fanzago, 1877, H. punctata Canestrini and Fanzago, 1877, Hyalomma lusitanicum Koch, 1844, Rh. turanicus Pomerantzev, 1940, Rh. bursa Canestrini and Fanzago, 1877, Rh. (Boophilus) annulatus (Say, 1821) and Dermacentor marginatus (Sulzer, 1776)) collected on 23 red deer, 5 fallow deer, 23 Iberian ibex and 32 wild boar during population control programmes in each national park during the years 2002–2003 (Table 1). D. marginatus, I. ricinus, Hy lusitanicum and Rh. bursa appeared in both natural spaces whereas Rh. annulatus and H. sulcata were restricted, respectively, to Don˜ana and Sierra Nevada. DNA was extracted individually using the kit Nucleo Spin Tissue (MachereyCorresponding author and reprint requests: F. J. Ma´rquez, Dpto. Biologı´a Animal, Biologı´a Vegetal y Ecologı´a, Univ. Jae´n, Campus Las Lagunillas s ⁄ n, Jae´n, 23071 – Jae´n, Spain E-mail: [email protected] No conflicts of interest declared.
Nagel, Du¨ren, Germany) and specific rickettsial sequences were detected by using PCR primers that amplify a portion of glta, ompA and ompB genes, respectively [3]. Positive PCR products were sequenced in a CEQ 2000XL capillary DNA sequencer (Beckman Coulter Life, Brea, CA, USA) according to the manufacturer’s instructions. Sequences were manually aligned and analysed with Bioedit and identified using the BLAST feature of GenBank. Overall, rickettsial DNA was detected in 51 (28.3%) of the examined ticks. Sequence analysis revealed that I. ricinus, captured on red deer, Iberian ibex and wild boar, were infected with R. monacensis, including strain IRS3, and R. helvetica (prevalences of 50.0% and 4.3%, respectively), while D. marginatus, mainly from wild boar, presents R. slovaca (13.9%) and R. raoultii (38.9%). R. massiliae was found in one Rh. turanicus (prevalence of 20.0%) from Iberian ibex. H. sulcata, from Iberian ibex of Sierra Nevada, shows the presence (6 ⁄ 17, prevalence of 35.3%) of a still not described Rickettsia sp. that is closer to R. hoogstraalii. None of the H. punctata, Hy. lusitanicum, Rh. annulatus or Rh. bursa ticks studied contained rickettsiae. This study confirms the presence of several rickettsiae species in ticks affecting wild ungulates in the south of the Iberian Peninsula. Ticks infected with zoonotic Rickettsia species (R. slovaca, R. raoultii, R. helvetica and R. monacensis) could be maintained and dispersed by wild ungulates. The rickettsia detected in H. sulcata appears to have a distribution limited to mediumhigh Mediterranean mountain ranges. The high prevalence detected should be taken into consideration because elevated ungulate host and vector population densities will be related to an increase in the risk of transmission of these pathogens to the human population that lives, works or uses temporally these natural spaces.
2009 The Author Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 227–229
F
0 (4) 2 (6)
1 (4)
1
2
4
6
8
2
4 2
2
0 (2) 16 (25)
9 (18) 1 (1)
9
R. mon.
0 (6)
0 (2)
4
5
11
R. rao.
2
2
F
4
1
M
5
R. slo.
1 (3) 0 (16)
9 (11) 5 (16)
Pos. ⁄ ass.
23
S. Nevada
Iberian ibex
1
R. sp.
8
R. mon.
16
2 13
1
M
1
R. hel.
30
12
18
F
5
M 5 6 3 24 18 1 18 4 75
1
R. mas.
Total ticks
R. sp.
41 11 4 2 18 4 19 18 105
F
10
1 4
5
M
46 17 7 26 36 5 37 22 180
Total
36
2 12
11 11
F
Pos. ⁄ ass., positive vs. assayed; M, male, F, female; R. hel., R. helvetica; R. mas., R. massiliae; R. mon., R. monacensis; R. rao; R. raoultii; R. slo., R. slovaca; R. sp., undescribed rickettsiae.
Ixodes ricinus Hamaphysalis sulcata Hamaphysalis punctata Hyalomma lusitanicum Dermacentor marginatus Rhipicephalus turanicus Rhipicephalus bursa Rhipicephalus annulatus Total tick number
Pos. ⁄ ass.
M
Pos. ⁄ ass.
Tick species
R. rao.
24
S. Nevada
4 2 2 2 5 6 29
8
10 0 36
3 18 3
8
Host number
1
2
Don˜ana
R. mon.
1
National Park
(7) (20) (5) (2) (15) (6)
5
F
Wild boar
0 0 1 0 0 0
6 (10)
M
Host
Ixodes ricinus Haemaphysalis sulcata Haemaphysalis punctata Hyalomma lusitanicum Dermacentor marginatus Rhipicephalus turanicus Rhipicephalus bursa Rhipicephalus annulatus Total tick number
R. rao.
Pos. ⁄ ass.
R. hel.
Pos. ⁄ ass.
Tick species
R. mon.
5
23
Host number
Don˜ana
Don˜ana
National Park
Fallow deer
Red deer
Host
Table 1. Capture data (ticks and host) from Don˜ana and Sierra Nevada National Parks
228 Clinical Microbiology and Infection, Volume 15, Supplement 2, December 2009
2009 The Author Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 227–229
Ma´rquez
ACKNOWLEDGEMENTS This study has been supported by grants from the Andalusia Government (Research Group BIO-294). The authors thank Celia Sa´nchez and Jose´ E. Granados for help in sampling and Dr Ricardo Oya of the Research Services of Jae´n University for his technical assistance in sequencing. The experiments comply with the current laws of the European Union.
Rickettsiae in ticks from wild ungulates 229
2. Brouqui P, Parola P, Fournier PE, Raoult D. Spotted fever rickettsioses in southern and eastern Europe. FEMS Immunol Med Microbiol 2007; 49: 2–12. 3. Ma´rquez FJ, Muniain MA, Soriguer RC et al. Genotypic identification of an undescribed spotted fever group Rickettsia in Ixodes ricinus from southwestern Spain. Am J Trop Med 1998; 58: 570–577. 4. Rydkina E, Roux V, Fetisova N et al. New Rickettsiae in ticks collected in territories of the former Soviet Union. Emerg Infect Dis 1999; 5: 811–814.
REFERENCES 1. Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world, emerging diseases challenging old concepts. Clin Microbiol Rev 2005; 18: 719–756.
2009 The Author Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 227–229