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First detection of Anaplasma phagocytophilum-like DNA in the French izard Rupricapra pyrenaica
10.1111/j.1469-0691.2008.02143.x
First detection of Anaplasma phagocytophilum-like DNA in the French izard Rupricapra pyrenaica E. Laloy1, E. Petit1, H.-J. Boulouis1, C. Lacroux2, F. Corbiere2, F. Schelcher2, S. Bonnet1 and R. Maillard1 1
UMR BIPAR, Maisons-Alfort and 2UMR INRA-ENVT, 1225, Toulouse, France
INTRODUCTION Various mammal species host Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila). This intracellular bacterium is responsible for tick-borne fever, also named granulocytic anaplasmosis, in domestic ruminants. Moreover, A. phagocytophilum is the agent of granulocytic anaplasmosis in humans. The hard tick Ixodes ricinus is known as the main vector of A. phagocytophilum throughout Europe. A. phagocytophilum infects domestic ruminants, e.g. cattle, sheep and goats, in several European countries, e.g. Spain and France. Wild ruminants such as roe deer (Capreolus capreolus) in many countries, and chamois (Rupricapra rupricapra) in Switzerland, [1] are reservoirs of A. phagocytophilum. The izard (Rupricapra pyrenaica), another wild ruminant, lives in the Pyrenees mountains. Owing to its grazing habits, it is likely to be exposed to infected ticks. However, to our knowledge, no study has been published on this wild ruminant. The purposes of the present study were to search for evidence of infection by A. phagocytophilum in French izards and to characterize isolates. MATERIALS AND METHODS In 2007, 450 izards were randomly selected, after hunting by professionals, in the French Pyrenees mountains (de´partements: Haute-Garonne, Arie`ge and Pyre´ne´es-Orientales). Spleens were collected from all animals, and 120 of them were randomly selected. DNA extraction was performed from 25-mg pieces from each selected spleen. Six distinct PCR assays were employed, with two steps. The 120 DNA extracts were first screened with broad-spectrum PCR primers targeting the 16S rDNA gene of Anaplasmataceae [2], and then with an A. phagocytophilum-specific PCR targeting the major surface protein 4 gene, msp4 [3]. Then, positive samples were subjected to confirmation by A. phagocytophilum-specific nested PCR on the 16S rDNA gene [4] and groEL heat shock operon, the msp2 gene, and the citrate synthase gene, gltA assays. Corresponding author and reprint requests: R. Maillard, UMR BIPAR, 23 av. du Gal De Gaulle, Maisons-Alfort, France E-mail: [email protected] No conflicts of interest declared.
PCR amplicons were sequenced. Nucleotide sequence homology searches were performed through the National Center for Biotechnology Information BLAST network service (http://blast.ncbi.nlm.nih.gov/Blast.cgi). After alignment with Anaplasmataceae 16S rDNA gene partial sequences previously mentioned [5], a phylogenetic tree was constructed using the neighbour-joining distance method and Kimura twoparameter analysis.
RESULTS After screening, three of 120 izards were positive for the msp4 gene. Only one of them (izard number 22) was also positive for the 16S rDNA gene by both broad-range and specific nested PCR. Two partial sequences of the 16S rDNA gene of 226 bp and 406 bp, respectively, were obtained from izard no. 22. The two sequences revealed 98–99% identity with the A. phagocytophilum sequence from a tick removed from cattle in India (DQ648489). These sequences were also very close to other Anaplasma spp. rRNA 16S sequences. As they were overlapping but not chimeric, the two sequences were used to make a 612-bp contig (Genbank access number: EU857675). The phylogenetic tree of the Anaplasmataceae based on the 16S rDNA gene partial sequence (Fig. 1) had similar topology to the tree published by Dumler et al. in 2001 [5], except for Anaplasma bovis. The izard agent was in a clade with Anaplasma marginale, Anaplasma ovis, and Anaplasma centrale. DISCUSSION In the USA, the white-tailed deer, another wild ruminant, hosts an Anaplasma sp.: ‘WTD agent’. It is detected by a PCR assay originally designed to detect A. phagocytophilum 16S RNA gene [3]. In contrast, the A. phagocytophilum-specific groEL PCR assay does not work on WTD agent, and neither did it work on the izard agent described here. Therefore, we may consider as a first hypothesis that the izard agent might be an Anaplasma sp. different from A. phagocytophilum, as appears to be the case for WTD agent.
Ó 2009 The Authors Journal Compilation Ó 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 26–27
Laloy et al.
Detection of anaplasma DNA in French izard 27
Fig. 1. Phylogenetic analysis of the Anaplasmataceae based on 16S rDNA partial sequences (612 bp) with bootstrap analysis of 1000 replicates. Blue numbers on the branches indicate percentage support for each clade. The tree was constructed using the neighbour-joining distance method and Kimura two-parameter analysis. Rtyphi, Rickettsia typhi (outgroup); Nhelmintho, Neorickettsia helminthoeca; Wpipientis, Wolbachia pipientis; Emuris, Ehrlichia muris; Ecanis, Ehrlichia canis; Eewingii, Ehrlichia ewingii; Echaffeens, Ehrlichia chaffeensis; Eruminanti, Ehrlichia ruminantium; WTDAgt_ctg, WTD agent; Aplatys, Anaplasma platys; Aphagocyto, Anaplasma phagocytophilum; Abovis_Cor, Anaplasma bovis variant from Korea; Abovis_Jap, A. bovis variant from Japan; Abovis_USA, A. bovis variant from the USA; IzardAgent, agent from izard no. 22; Ac_Japon, Anaplasma centrale variant from Japan; Ao_Mozambi, Anaplasma ovis variant from Mozambique; Ac_Vaccine, A. centrale Vaccine strain; Ao_Idaho, A. ovis variant from Idaho; Amarginale, Anaplasma marginale.
Phylogenetic analysis brought strong support for this hypothesis: the agent seemed to be closely related to intraerythrocytic Anaplasma spp. (A. marginale, A. ovis, and A. centrale). In our study, the izard agent gave a positive result with the A. phagocytophilum-specific msp4 PCR assay, whereas WTD agent does not [3]. This suggests a second hypothesis: the izard agent might be a new A. phagocytophilum variant. However, the msp4 PCR signal was weak and could have been a false-positive result. CONCLUSIONS Anaplasmataceae DNA was present in one of 120 izard spleens. It was more likely to be an Anaplasma sp. distinct from A. phagocytophilum than a new variant of this species. Similar analysis on DNA from the other 330 spleens will be performed in order to improve our epidemiological knowledge. Broad-range groESL and gltA amplification would be useful for further phylogenetic analysis.
REFERENCES 1. Liz N, Sumner JW, Pfister K, Brossard M. PCR detection and serological evidence of granulocytic ehrlichial infection in roe deer (Capreolus capreolus) and chamois (Rupicapra rupicapra). J Clin Microbiol 2002; 40: 892–897. 2. Parola P, Roux V, Camicas JL, Baradji I, Brouqui P, Raoult D. Detection of Ehrlichiae in African ticks by polymerase chain reaction. Trans R Soc Trop Med Hyg 2000; 94: 707–708. 3. De La Fuente J, Massung RF, Wong SJ et al. Sequence analysis of the msp4 gene of Anaplasma phagocytophilum strains. J Clin Microbiol 2005; 43: 1309–1317. 4. Massung RF, Slater K, Owens JH et al. Nested PCR assay for detection of granulocytic Ehrlichiae. J Clin Microbiol 1998; 36: 1090–1095. 5. Dumler JS, Barbet AF, Bekker CPJ et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001; 51: 2145–2165.
Ó 2009 The Authors Journal Compilation Ó 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 26–27
First detection of Anaplasma phagocytophilum-like DNA in the French izard Rupricapra pyrenaica E. Laloy1, E. Petit1, H.-J. Boulouis1, C. Lacroux2, F. Corbiere2, F. Schelcher2, S. Bonnet1 and R. Maillard1 1
UMR BIPAR, Maisons-Alfort and 2UMR INRA-ENVT, 1225, Toulouse, France
INTRODUCTION Various mammal species host Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila). This intracellular bacterium is responsible for tick-borne fever, also named granulocytic anaplasmosis, in domestic ruminants. Moreover, A. phagocytophilum is the agent of granulocytic anaplasmosis in humans. The hard tick Ixodes ricinus is known as the main vector of A. phagocytophilum throughout Europe. A. phagocytophilum infects domestic ruminants, e.g. cattle, sheep and goats, in several European countries, e.g. Spain and France. Wild ruminants such as roe deer (Capreolus capreolus) in many countries, and chamois (Rupricapra rupricapra) in Switzerland, [1] are reservoirs of A. phagocytophilum. The izard (Rupricapra pyrenaica), another wild ruminant, lives in the Pyrenees mountains. Owing to its grazing habits, it is likely to be exposed to infected ticks. However, to our knowledge, no study has been published on this wild ruminant. The purposes of the present study were to search for evidence of infection by A. phagocytophilum in French izards and to characterize isolates. MATERIALS AND METHODS In 2007, 450 izards were randomly selected, after hunting by professionals, in the French Pyrenees mountains (de´partements: Haute-Garonne, Arie`ge and Pyre´ne´es-Orientales). Spleens were collected from all animals, and 120 of them were randomly selected. DNA extraction was performed from 25-mg pieces from each selected spleen. Six distinct PCR assays were employed, with two steps. The 120 DNA extracts were first screened with broad-spectrum PCR primers targeting the 16S rDNA gene of Anaplasmataceae [2], and then with an A. phagocytophilum-specific PCR targeting the major surface protein 4 gene, msp4 [3]. Then, positive samples were subjected to confirmation by A. phagocytophilum-specific nested PCR on the 16S rDNA gene [4] and groEL heat shock operon, the msp2 gene, and the citrate synthase gene, gltA assays. Corresponding author and reprint requests: R. Maillard, UMR BIPAR, 23 av. du Gal De Gaulle, Maisons-Alfort, France E-mail: [email protected] No conflicts of interest declared.
PCR amplicons were sequenced. Nucleotide sequence homology searches were performed through the National Center for Biotechnology Information BLAST network service (http://blast.ncbi.nlm.nih.gov/Blast.cgi). After alignment with Anaplasmataceae 16S rDNA gene partial sequences previously mentioned [5], a phylogenetic tree was constructed using the neighbour-joining distance method and Kimura twoparameter analysis.
RESULTS After screening, three of 120 izards were positive for the msp4 gene. Only one of them (izard number 22) was also positive for the 16S rDNA gene by both broad-range and specific nested PCR. Two partial sequences of the 16S rDNA gene of 226 bp and 406 bp, respectively, were obtained from izard no. 22. The two sequences revealed 98–99% identity with the A. phagocytophilum sequence from a tick removed from cattle in India (DQ648489). These sequences were also very close to other Anaplasma spp. rRNA 16S sequences. As they were overlapping but not chimeric, the two sequences were used to make a 612-bp contig (Genbank access number: EU857675). The phylogenetic tree of the Anaplasmataceae based on the 16S rDNA gene partial sequence (Fig. 1) had similar topology to the tree published by Dumler et al. in 2001 [5], except for Anaplasma bovis. The izard agent was in a clade with Anaplasma marginale, Anaplasma ovis, and Anaplasma centrale. DISCUSSION In the USA, the white-tailed deer, another wild ruminant, hosts an Anaplasma sp.: ‘WTD agent’. It is detected by a PCR assay originally designed to detect A. phagocytophilum 16S RNA gene [3]. In contrast, the A. phagocytophilum-specific groEL PCR assay does not work on WTD agent, and neither did it work on the izard agent described here. Therefore, we may consider as a first hypothesis that the izard agent might be an Anaplasma sp. different from A. phagocytophilum, as appears to be the case for WTD agent.
Ó 2009 The Authors Journal Compilation Ó 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 26–27
Laloy et al.
Detection of anaplasma DNA in French izard 27
Fig. 1. Phylogenetic analysis of the Anaplasmataceae based on 16S rDNA partial sequences (612 bp) with bootstrap analysis of 1000 replicates. Blue numbers on the branches indicate percentage support for each clade. The tree was constructed using the neighbour-joining distance method and Kimura two-parameter analysis. Rtyphi, Rickettsia typhi (outgroup); Nhelmintho, Neorickettsia helminthoeca; Wpipientis, Wolbachia pipientis; Emuris, Ehrlichia muris; Ecanis, Ehrlichia canis; Eewingii, Ehrlichia ewingii; Echaffeens, Ehrlichia chaffeensis; Eruminanti, Ehrlichia ruminantium; WTDAgt_ctg, WTD agent; Aplatys, Anaplasma platys; Aphagocyto, Anaplasma phagocytophilum; Abovis_Cor, Anaplasma bovis variant from Korea; Abovis_Jap, A. bovis variant from Japan; Abovis_USA, A. bovis variant from the USA; IzardAgent, agent from izard no. 22; Ac_Japon, Anaplasma centrale variant from Japan; Ao_Mozambi, Anaplasma ovis variant from Mozambique; Ac_Vaccine, A. centrale Vaccine strain; Ao_Idaho, A. ovis variant from Idaho; Amarginale, Anaplasma marginale.
Phylogenetic analysis brought strong support for this hypothesis: the agent seemed to be closely related to intraerythrocytic Anaplasma spp. (A. marginale, A. ovis, and A. centrale). In our study, the izard agent gave a positive result with the A. phagocytophilum-specific msp4 PCR assay, whereas WTD agent does not [3]. This suggests a second hypothesis: the izard agent might be a new A. phagocytophilum variant. However, the msp4 PCR signal was weak and could have been a false-positive result. CONCLUSIONS Anaplasmataceae DNA was present in one of 120 izard spleens. It was more likely to be an Anaplasma sp. distinct from A. phagocytophilum than a new variant of this species. Similar analysis on DNA from the other 330 spleens will be performed in order to improve our epidemiological knowledge. Broad-range groESL and gltA amplification would be useful for further phylogenetic analysis.
REFERENCES 1. Liz N, Sumner JW, Pfister K, Brossard M. PCR detection and serological evidence of granulocytic ehrlichial infection in roe deer (Capreolus capreolus) and chamois (Rupicapra rupicapra). J Clin Microbiol 2002; 40: 892–897. 2. Parola P, Roux V, Camicas JL, Baradji I, Brouqui P, Raoult D. Detection of Ehrlichiae in African ticks by polymerase chain reaction. Trans R Soc Trop Med Hyg 2000; 94: 707–708. 3. De La Fuente J, Massung RF, Wong SJ et al. Sequence analysis of the msp4 gene of Anaplasma phagocytophilum strains. J Clin Microbiol 2005; 43: 1309–1317. 4. Massung RF, Slater K, Owens JH et al. Nested PCR assay for detection of granulocytic Ehrlichiae. J Clin Microbiol 1998; 36: 1090–1095. 5. Dumler JS, Barbet AF, Bekker CPJ et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001; 51: 2145–2165.
Ó 2009 The Authors Journal Compilation Ó 2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15 (Suppl. 2), 26–27