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Molecular evidence of Anaplasma phagocytophilum infection in wild cervids and feeding Ixodes ricinus ticks from west-central Poland
10.1111/j.1469-0691.2008.02240.x
Molecular evidence of Anaplasma phagocytophilum infection in wild cervids and feeding Ixodes ricinus ticks from west-central Poland J. Michalik1, J. Stan´czak2, M. Racewicz2, S. Cieniuch2, B. Sikora1, A. Szubert-Kruszyn´ska1 and R. Grochowalska3 1
Department of Animal Morphology, Faculty of Biology, Adam Mickiewicz University Poznan, Poznan, Poland, 2Department of Tropical Parasitology, Institute of Maritime and Tropical Medicine, Medical University of Gdan´sk, Gdynia, Poland and 3Faculty of Biological Sciences, University of Zielona Go´ra, Zielona Go´ra, Poland
INTRODUCTION In Europe, Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA), is maintained in enzootic cycles involving the primary vector tick Ixodes ricinus and mammals, some of which are reservoirs for the bacterium. Identification of these host species in site-specific ecosystems is indispensable for understanding the epizootiology of A. phagocytophilum. In forest habitats, by hosting I. ricinus ticks, cervids are naturally exposed to A. phagocytophilum. Natural infections of these wild ruminants with the bacterium detected by PCR have been reported in several Central European countries [1,2]. In Poland, cervids are important game animals but their contribution as reservoirs for A. phagocytophilum, apart from a report concerning roe deer in north-western Poland [3], remains unknown. The objectives of the study were to determine the prevalence of A. phagocytophilum infection in three cervid species, the roe deer (Capreolus capreolus), red deer (Cervus elaphus) and fallow deer (Dama dama), as well as in deer-derived I. ricinus surveyed in west-central Poland. MATERIALS AND METHODS Sampling was performed at local tagging stations in 10 selected districts of the Wielkopolska province, in west-central Poland. EDTA-treated whole blood samples were collected from 298 animals harvested during the 2004 and 2005 hunting Corresponding author and reprint requests: J. Michalik, Department of Animal Morphology, Faculty of Biology, Adam Mickiewicz University Poznan, ul. Umultowska 89, 61-614 Poznan´, Poland E-mail: [email protected] No conflict of interest declared.
seasons. Roe deer were sampled during the summer and the fall in 10 districts (Czarnko´w, Krzykosy, Oborniki, Margonin, Miedzychod, Murowana-Goslina, Poniec, Sierako´w, Trzcianka, Wielen´), whereas samples from red and fallow deer were obtained only during the fall in nine (listed above with the exception of Krykosy) and two districts (Margonin, Murowana-Goslina), respectively. In total, 238 partially or fully engorged female and 63 non-engorged male I. ricinus ticks were obtained from 51 animals (20 fallow deer, 18 roe deer and 13 red deer). Total DNA was extracted from blood and I. ricinus ticks using the genomic Mini AX Blood (A&A Biotechnology, Gdynia, Poland) and Sherlock AX kit (A&A Biotechnology), respectively. Blood and tick samples were analysed for the presence of A. phagocytophilum DNA by PCR using three different primer sets: EHR521-EHR747, LA6-LA1 and HGE396-HGE921, based on the sequences of the 16S rDNA, ankA and hge-44 genes, respectively [4,5]. PCR products were considered positive when A. phagocytophilum DNA was detected by at least two primer sets. Additionally, selected PCR-positive samples tested with the primers LA6-LA1 specific for a 444-bp region of the ankA gene were sequenced with the ABI PRISM 3100 Genetic Analyser (Applied Biosystem 850, Foster City, CA, USA) according to the manufacturer’s protocol. Sequences were edited and compared with gene sequences deposited in the GenBank database using the NCBI BLAST program (U.S. National Institute of Health, Bethesda, Maryland).
RESULTS Anaplasma phagocytophilum DNA was identified in 9 (20.5%) of the 44 fallow deer, 9 (10.2%) of the 88 red deer, and in 16 (9.6%) of the 166 roe deer. Infection rates in host species varied depending on districts sampled (range: 17–25%, 0–33% and 0–25% for fallow, red and roe deer, respectively). The overall prevalence of A. phagocytophilum in female ticks (22.7%; range, 20–23.7%) was higher than in male I. ricinus (9.1%; range, 6.7–10.5%). Female and male ticks from PCR-positive animals were more frequently infected than ticks from PCR-negative hosts (35.7% vs. 7.3%, and 16.7% vs. 2.8%, respectively). These differences were
2009 The Authors Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 81–83
82 Clinical Microbiology and Infection, Volume 15, Supplement 2, December 2009
particularly evident in females from blood-positive and negative fallow deer (55.7% of 14 vs. 3.2% of 31). Sequence analysis of the ankA gene from three PCR-positive blood samples and seven infected ticks revealed 96–100% homology to each other and 99.2–100% homology to the other ankA sequences deposited in GenBank. Three of these sequences derived from one fallow deer (sample FD391-5), one female tick (391-8FD) detached from the same infected fallow deer, and from one red deer (RD392-8), were nearly or identical (99.7–100% similarity) to the reported sequences of the North American human pathogenic strains of A. phagocytophilum (Fig. 1). The other sequences from four ticks (samples 143-10FD, 143-7FD, 1432FD, 143-4FD) obtained from three fallow deer, two ticks (159-3RD, 160-1RD) from one red deer, and in one red deer-derived blood sample (RD211-3), were 98.5–99.5% identical to European sequences reported from a Polish roe deer, European HGA patients, a German horse, and infected German ticks. The sequences obtained in our study received GenBank accession numbers given in Fig. 1. CONCLUSIONS
Fig. 1. Phylogenetic relationships of 10 Anaplasma phagocytophilum sequences identified in this study (available in GenBank under acc. nos. EU779816 to EU779821, and EU781995) with the selected sequences from Europe and the United States based on the nucleotide sequences of the 444-bp fragment of the ankA gene. The sequence of sample 143-10FD was identical to the sequence of sample 143-7FD (EU779816). Two sequences of samples 159-3 ⁄ RD and RD2113 matched the sequence of sample 143-2 ⁄ FD (EU779817). The neighbour-joining algorithm method was used to construct the phylogenetic tree (Vector NTI Advance 10.3.0.).
Our results provide the evidence of A. phagocytophilum in populations of three wild cervids species and associated I. ricinus ticks in westcentral Poland. Among the sampled hosts, fallow deer appeared to be particularly susceptible to the infection and hosted the highest rate of infected female ticks on the positive individuals. The higher infection prevalence detected in female ticks than in non-engorged males as well as the higher infection level in ticks derived from PCR-positive animals strongly indicates acquisition of the agent from these hosts, which could serve as reservoirs. Sequence analysis of selected samples showed that cervids appear to participate in the maintenance of A. phagocytophilum strains potentially infective to humans. Further studies on the prevalence of the agent and identification of its genetic variants in various wild vertebrates are in progress. ACKNOWLEDGEMENTS This study was supported by the State Committee for Scientific Research (grant no. 2PO4C 111 29).
2009 The Authors Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 81–83
Michalik et al. Molecular evidence of Anaplasma phagocytophilum infection 83
REFERENCES 1. Petrovec M, Sixl W, Schweiger R et al. Infections of wild animals with Anaplasma phagocytophila in Austria and the Czech Republic. Ann N Y Acad Sci 2003; 990: 103–106. 2. Stefanidesova K, Kocianova E, Boldis V et al. Evidence of Anaplasma phagocytophilum and Rickettsia helvetica infection in free-ranging ungulates in central Slovakia. Eur J Wildl Res 2008; 54: 519–524. 3. Adamska M, Skotarczak B. Wild game as a reservoir of Anaplasma phagocytophilum in north-western Poland (in Polish). Wiad Parazytol 2007; 53: 103–107
4. Walls JJ, Caturegli P, Bakken JS, Asanovich KM, Dumler JS. Improved sensitivity of PCR using PCR for diagnosis of human granulocytic ehrlichiosis using epank1 genes of Ehrlichia phagocytophila-group ehrlichiae. J Clin Microbiol 2000; 38: 354–356. 5. IJdo JW, Meek JI, Cartter ML et al. The emergence of another tickborne infection in the 12-town area around Lyme, Connecticut: human granulocytic ehrlichiosis. J Infect Dis 2000; 181: 1388–1393.
2009 The Authors Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 81–83
Molecular evidence of Anaplasma phagocytophilum infection in wild cervids and feeding Ixodes ricinus ticks from west-central Poland J. Michalik1, J. Stan´czak2, M. Racewicz2, S. Cieniuch2, B. Sikora1, A. Szubert-Kruszyn´ska1 and R. Grochowalska3 1
Department of Animal Morphology, Faculty of Biology, Adam Mickiewicz University Poznan, Poznan, Poland, 2Department of Tropical Parasitology, Institute of Maritime and Tropical Medicine, Medical University of Gdan´sk, Gdynia, Poland and 3Faculty of Biological Sciences, University of Zielona Go´ra, Zielona Go´ra, Poland
INTRODUCTION In Europe, Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA), is maintained in enzootic cycles involving the primary vector tick Ixodes ricinus and mammals, some of which are reservoirs for the bacterium. Identification of these host species in site-specific ecosystems is indispensable for understanding the epizootiology of A. phagocytophilum. In forest habitats, by hosting I. ricinus ticks, cervids are naturally exposed to A. phagocytophilum. Natural infections of these wild ruminants with the bacterium detected by PCR have been reported in several Central European countries [1,2]. In Poland, cervids are important game animals but their contribution as reservoirs for A. phagocytophilum, apart from a report concerning roe deer in north-western Poland [3], remains unknown. The objectives of the study were to determine the prevalence of A. phagocytophilum infection in three cervid species, the roe deer (Capreolus capreolus), red deer (Cervus elaphus) and fallow deer (Dama dama), as well as in deer-derived I. ricinus surveyed in west-central Poland. MATERIALS AND METHODS Sampling was performed at local tagging stations in 10 selected districts of the Wielkopolska province, in west-central Poland. EDTA-treated whole blood samples were collected from 298 animals harvested during the 2004 and 2005 hunting Corresponding author and reprint requests: J. Michalik, Department of Animal Morphology, Faculty of Biology, Adam Mickiewicz University Poznan, ul. Umultowska 89, 61-614 Poznan´, Poland E-mail: [email protected] No conflict of interest declared.
seasons. Roe deer were sampled during the summer and the fall in 10 districts (Czarnko´w, Krzykosy, Oborniki, Margonin, Miedzychod, Murowana-Goslina, Poniec, Sierako´w, Trzcianka, Wielen´), whereas samples from red and fallow deer were obtained only during the fall in nine (listed above with the exception of Krykosy) and two districts (Margonin, Murowana-Goslina), respectively. In total, 238 partially or fully engorged female and 63 non-engorged male I. ricinus ticks were obtained from 51 animals (20 fallow deer, 18 roe deer and 13 red deer). Total DNA was extracted from blood and I. ricinus ticks using the genomic Mini AX Blood (A&A Biotechnology, Gdynia, Poland) and Sherlock AX kit (A&A Biotechnology), respectively. Blood and tick samples were analysed for the presence of A. phagocytophilum DNA by PCR using three different primer sets: EHR521-EHR747, LA6-LA1 and HGE396-HGE921, based on the sequences of the 16S rDNA, ankA and hge-44 genes, respectively [4,5]. PCR products were considered positive when A. phagocytophilum DNA was detected by at least two primer sets. Additionally, selected PCR-positive samples tested with the primers LA6-LA1 specific for a 444-bp region of the ankA gene were sequenced with the ABI PRISM 3100 Genetic Analyser (Applied Biosystem 850, Foster City, CA, USA) according to the manufacturer’s protocol. Sequences were edited and compared with gene sequences deposited in the GenBank database using the NCBI BLAST program (U.S. National Institute of Health, Bethesda, Maryland).
RESULTS Anaplasma phagocytophilum DNA was identified in 9 (20.5%) of the 44 fallow deer, 9 (10.2%) of the 88 red deer, and in 16 (9.6%) of the 166 roe deer. Infection rates in host species varied depending on districts sampled (range: 17–25%, 0–33% and 0–25% for fallow, red and roe deer, respectively). The overall prevalence of A. phagocytophilum in female ticks (22.7%; range, 20–23.7%) was higher than in male I. ricinus (9.1%; range, 6.7–10.5%). Female and male ticks from PCR-positive animals were more frequently infected than ticks from PCR-negative hosts (35.7% vs. 7.3%, and 16.7% vs. 2.8%, respectively). These differences were
2009 The Authors Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 81–83
82 Clinical Microbiology and Infection, Volume 15, Supplement 2, December 2009
particularly evident in females from blood-positive and negative fallow deer (55.7% of 14 vs. 3.2% of 31). Sequence analysis of the ankA gene from three PCR-positive blood samples and seven infected ticks revealed 96–100% homology to each other and 99.2–100% homology to the other ankA sequences deposited in GenBank. Three of these sequences derived from one fallow deer (sample FD391-5), one female tick (391-8FD) detached from the same infected fallow deer, and from one red deer (RD392-8), were nearly or identical (99.7–100% similarity) to the reported sequences of the North American human pathogenic strains of A. phagocytophilum (Fig. 1). The other sequences from four ticks (samples 143-10FD, 143-7FD, 1432FD, 143-4FD) obtained from three fallow deer, two ticks (159-3RD, 160-1RD) from one red deer, and in one red deer-derived blood sample (RD211-3), were 98.5–99.5% identical to European sequences reported from a Polish roe deer, European HGA patients, a German horse, and infected German ticks. The sequences obtained in our study received GenBank accession numbers given in Fig. 1. CONCLUSIONS
Fig. 1. Phylogenetic relationships of 10 Anaplasma phagocytophilum sequences identified in this study (available in GenBank under acc. nos. EU779816 to EU779821, and EU781995) with the selected sequences from Europe and the United States based on the nucleotide sequences of the 444-bp fragment of the ankA gene. The sequence of sample 143-10FD was identical to the sequence of sample 143-7FD (EU779816). Two sequences of samples 159-3 ⁄ RD and RD2113 matched the sequence of sample 143-2 ⁄ FD (EU779817). The neighbour-joining algorithm method was used to construct the phylogenetic tree (Vector NTI Advance 10.3.0.).
Our results provide the evidence of A. phagocytophilum in populations of three wild cervids species and associated I. ricinus ticks in westcentral Poland. Among the sampled hosts, fallow deer appeared to be particularly susceptible to the infection and hosted the highest rate of infected female ticks on the positive individuals. The higher infection prevalence detected in female ticks than in non-engorged males as well as the higher infection level in ticks derived from PCR-positive animals strongly indicates acquisition of the agent from these hosts, which could serve as reservoirs. Sequence analysis of selected samples showed that cervids appear to participate in the maintenance of A. phagocytophilum strains potentially infective to humans. Further studies on the prevalence of the agent and identification of its genetic variants in various wild vertebrates are in progress. ACKNOWLEDGEMENTS This study was supported by the State Committee for Scientific Research (grant no. 2PO4C 111 29).
2009 The Authors Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 81–83
Michalik et al. Molecular evidence of Anaplasma phagocytophilum infection 83
REFERENCES 1. Petrovec M, Sixl W, Schweiger R et al. Infections of wild animals with Anaplasma phagocytophila in Austria and the Czech Republic. Ann N Y Acad Sci 2003; 990: 103–106. 2. Stefanidesova K, Kocianova E, Boldis V et al. Evidence of Anaplasma phagocytophilum and Rickettsia helvetica infection in free-ranging ungulates in central Slovakia. Eur J Wildl Res 2008; 54: 519–524. 3. Adamska M, Skotarczak B. Wild game as a reservoir of Anaplasma phagocytophilum in north-western Poland (in Polish). Wiad Parazytol 2007; 53: 103–107
4. Walls JJ, Caturegli P, Bakken JS, Asanovich KM, Dumler JS. Improved sensitivity of PCR using PCR for diagnosis of human granulocytic ehrlichiosis using epank1 genes of Ehrlichia phagocytophila-group ehrlichiae. J Clin Microbiol 2000; 38: 354–356. 5. IJdo JW, Meek JI, Cartter ML et al. The emergence of another tickborne infection in the 12-town area around Lyme, Connecticut: human granulocytic ehrlichiosis. J Infect Dis 2000; 181: 1388–1393.
2009 The Authors Journal Compilation 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 81–83