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Anaplasma phagocytophilum and Borrelia burgdorferi in rabbits from southeastern China
Veterinary Parasitology 162 (2009) 354–356
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Anaplasma phagocytophilum and Borrelia burgdorferi in rabbits from southeastern China Lin Zhan a,1, Chen-Yi Chu a,1, Shu-Qing Zuo a, Xiao-Ming Wu a, J. Stephen Dumler b, Na Jia a, Bao-Gui Jiang a, Hong Yang a, Wu-Chun Cao a,* a
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dong-Da Street, Fengtai District, Beijing 100071, People’s Republic of China Division of Medical Microbiology and Immunology, Department of Pathology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 624, Baltimore, MD 21205, USA b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 September 2008 Received in revised form 18 February 2009 Accepted 2 March 2009
A total of 54 wild rabbits captured from southeastern China were examined for Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato by polymerase chain reaction (PCR) assays. One and three samples were positive for A. phagocytophilum and B. burgdorferi, respectively. Sequence analyses of PCR products identified a variant of A. phagocytophilum and a B. garinii genotype. This is the first detection of the two tick-borne agents in Chinese rabbits, the role of which in the maintenance of the agents deserve further investigations. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anaplasma phagocytophilum Borrelia burgdorferi Rabbit Host Gene variation
Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato (s.l.) are tick-borne pathogens with veterinary and public health significance (Dumler et al., 2005). The two agents are thought to be naturally maintained in small mammal–tick cycles, and mainly transmitted by Ixodes ticks, including I. scapularis and I. pacificus in the United States, I. ricinus in mainland Europe, and I. persulcatus in Asia (Swanson et al., 2006). Wild rodents have been implicated as natural reservoirs for the two tick-borne agents (Swanson et al., 2006). In Nantucket Island, A. phagocytophilum was detected in cottontail rabbits, which were considered as potential hosts of the agent (Goethert and Telford, 2003). Both A. phagocytophilum and B. burgdorferi s.l. were found in ticks as well as rodents collected in Zhejiang Province of southeastern China (Chu
* Corresponding author. Tel.: +86 10 63896082; fax: +86 10 63896082. E-mail address: [email protected] (W.-C. Cao). 1 These authors contributed equally to this work. 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.03.003
et al., 2008; Zhan et al., 2008). During the survey, wild rabbits were captured in the same area. The objectives of this study were to determine if the wild rabbits, the hosts of immature of ticks, were naturally infected by A. phagocytophilum and B. burgdorferi, and to characterize the two tick-borne agents using sequence analysis. The study In Januaries of 2006 and 2008, a total of 54 wild rabbits were captured from the forested rolling hills located at 298150 N latitude and 1218030 E longitude in Zhejiang Province of southeastern China, and all were recognized as Chinese hares (Lepus sinensis) by a zoologist. DNA was extracted from spleens and blood samples of the rabbits using Trizol (Invitrogen, Carlsband, CA, USA) as previously described (Cao et al., 2006). In order to improve sensitivity, nested PCR assays were performed to amplify the partial 16S rRNA gene of the A. phagocytophilum. Primers GE9f (50 -AACGGATTATTCTTTATAGCTTGCT-30 ) and GE10r
A A
G
T C C C C C C C C C C
A G G G G G G G G C
T
A A
T
A A
T
G
T A
G A A A A A A A A C C
T C C C C C C C C T
A A
G
C
T C
A
G
T A T
A
C T T T T T T T T T T A A C C A Japan South Africa Swedish USA USA
/, gap. a The same nucleotide as that of rabbit (this study) was not marked.
G C C
A A A
A A A A A
/ / A A A
G
G A A
A
G G G
G A A T T G
57 84 149 150 151 171 284 330 369 390 553 562 711 717 722 738 740 741 746 750
A A G
29 32 36
G G
27 28
T A
DQ458806 AF205140 AF227954 DQ458808 AB196721 AY570538 AY527241 AF172167 CP000235 Rabbit (this study) Tick Tick Rodent Deer/tick Dog Horse Horse Human
16
355
China
GenBank Nucleotide accession no. differencea Geographical Bio-origin origin
Table 1 Comparison of partial 16S rRNA gene sequences of Anaplasma phagocytophilum from different origins.
(50 -TTCCGTTAAGAAGGATCTAATCTCC-30 ) designed by Chen et al. (1994) were applied for the initial amplification. Two primer pairs, GE9f and GE2 (50 -GGCAGTATTAAAAGCAGCTCCAGG-30 ), and Ehr521 (50 -TGTAGGCGGTTCGGTAAGTTAAAG-30 ) and GE10r, were used in the nested reactions as previously described (Cao et al., 2000). After sequencing of the PCR products, a 919-bp fragment was obtained from the positive samples based on overlapping region of sequence. A nested PCR targeting a 253-bp fragment of conserved region of B. burgdorferi s.l. 5S–23S rRNA intergenic spacer was performed using the outer primers of 23S6 (50 -TCCTAGGCATTCACCATA-30 ) and 23S5 (50 -CTGCGAGTTCGCGGGAGA-30 ), and the inner primers of 23S3 (50 -CGACCTTCTTCGCCTTAAAGC-30 ) and 23Sa (50 TAAGCTGACTAATACTAATTACCC-30 ) as previously described (Chu et al., 2008). The PCR amplifications were conducted using a PerkinElmer model 2700 thermal cycler. PCR amplified products were purified by TIANgel Mini Purification Kit (Tiangen Biotech Co. Ltd., Beijing) and sequenced directly on both strands by automated dideoxynucleotide cycle sequencing (ABI PRISM 377, PerkinElmer, Inc.). Out of the 54 rabbits examined, one (1.86%) was positive for A. phagocytophilum and three (5.56%) were positive for B. burgdorferi. No coinfection was identified. Sequence analysis of 919-bp partial 16S rRNA gene indicated that the sequence of A. phagocytophilum (GenBank accession no. DQ458806) detected in this study was distinct from all known A. phagocytophilum sequence deposited in GenBank. The agent was genetically closed to the A. phagocytophilum variant detected in I. persulcatus ticks from Northeastern China (GenBank accession no. AF205140) with only 5-bp difference (Table 1). While the nucleotide sequence was different by 7-bp from that of an A. phagocytophilum variant (GenBank accession no. DQ458808) identified in rodents from the same area (Zhan et al., 2008), and by at least 8-bp from other known A. phagocytophilum strains detected in various animals (Table 1). It was reported that at least one A. phagocytophilum variants isolated from white tailed deer in the USA, which was characterized by its special host-tropism. For example, the ‘‘AP-Variant 1’’ was unable to establish infection in white-footed mice and SCID mice, but could infect goats by experimental inoculation (Massung et al., 2003a,b, 2005, 2006). The sheep infected with A. phagocytophilum variants could cause different clinical manifestations, hematological abnormalities, and serological responses (Stuen et al., 2003). Whether the A. phagocytophilum variant detected in Chinese hare is pathogenic to domestic animals and humans needs further investigations. A 253-bp product of B. burgdorferi 5S–23S ribosomal RNA intergenic spacer was obtained from each positive specimen and sequenced. The nucleotide sequences amplified from the three positive samples were identical to each other (GenBank accession no. FJ172758), and were identified as Borrelia garinii. Multiple sequence alignment revealed that the tested sequence in this study was 100% identity to that of B. garinii strain previously reported in southern China (GenBank accession no. EU160461), but different at least 1% from all known B. garinii sequences deposited in GenBank.
801 866 918
L. Zhan et al. / Veterinary Parasitology 162 (2009) 354–356
L. Zhan et al. / Veterinary Parasitology 162 (2009) 354–356
356
Lyme disease is the most common human tick-borne disease in the northern hemisphere (Haufs, 2003). Its causative agent, B. burgdorferi s.l., is subdivided into at least 11 genospecies. Four of which have been known to be pathogenic to human, including B. burgdorferi sensu stricto, B. afzelii, B. garinii, and B. spielmanii (Wilske et al., 2007). The pathogenicity of the detected B. garinii to human beings and animals remains to be determined. Our findings, together with the evidences previously accumulated (Chu et al., 2008; Zhan et al., 2008), imply that both tick-borne agents are circulating in southeastern China and indicate the wild rabbits may be involved in the maintenance of both pathogens. Further investigations are required to understand the wide geographic distribution and to determine the host range of A. phagocytophilum and B. garinii in China. Conflict of interest There are no potential conflicts of interest. Acknowledgments We are grateful to Dr. Xiao’ai Zhang for her technical assistance, Dr. Rong-Man Xu for identification of ticks. This study was supported by the National Science Fund for Distinguished Young Scholars (No. 30725032). References Cao, W.C., Zhan, L., He, J., Foley, J.E., De Vlas, S.J., Wu, X.M., Yang, H., Richardus, J.H., Habbema, J.D., 2006. Natural Anaplasma phagocytophilum infection of ticks and rodents from a forest area of Jilin Province. China. Am. J. Trop. Med. Hyg. 75, 664–668.
Cao, W.C., Zhao, Q.M., Zhang, P.H., Dumler, J.S., Zhang, X.T., Fang, L.Q., Yang, H., 2000. Granulocytic Ehrlichiae in Ixodes persulcatus ticks from an area in China where Lyme disease is endemic. J. Clin. Microbiol. 38, 4208–4210. Chen, S.M., Dumler, J.S., Bakken, J.S., Walker, D.H., 1994. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 32, 589–595. Chu, C.Y., Jiang, B.G., Liu, W., Zhao, Q.M., Wu, X.M., Zhang, P.H., Zhan, L., Yang, H., Cao, W.C., 2008. Presence of pathogenic Borrelia burgdorferi sensu lato in ticks and rodents in Zhejiang, south-east China. J. Med. Microbiol. 57, 980–985. Dumler, J.S., Choi, K.S., Garcia-Garcia, J.C., Barat, N.S., Scorpio, D.G., Garyu, J.W., Grab, D.J., Bakken, J.S., 2005. Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg. Infect. Dis. 11, 1828–1834. Goethert, H.K., Telford 3rd, S.R., 2003. Enzootic transmission of the agent of human granulocytic ehrlichiosis among cottontail rabbits. Am. J. Trop. Med. Hyg. 68, 633–637. Haufs, M.G., 2003. Lyme borreliosis. Lancet Infect. Dis. 3, 684. Massung, R.F., Courtney, J.W., Hiratzka, S.L., Pitzer, V.E., Smith, G., Dryden, R.L., 2005. Anaplasma phagocytophilum in white-tailed deer. Emerg. Infect. Dis. 11, 1604–1606. Massung, R.F., Mather, T.N., Levin, M.L., 2006. Reservoir competency of goats for the Ap-variant 1 strain of Anaplasma phagocytophilum. Infect. Immun. 74, 1373–1375. Massung, R.F., Mather, T.N., Priestley, R.A., Levin, M.L., 2003a. Transmission efficiency of the AP-variant 1 strain of Anaplasma phagocytophila. Ann. NY Acad. Sci. 990, 75–79. Massung, R.F., Priestley, R.A., Miller, N.J., Mather, T.N., Levin, M.L., 2003b. Inability of a variant strain of Anaplasma phagocytophilum to infect mice. J. Infect. Dis. 188, 1757–1763. Stuen, S., Bergstrom, K., Petrovec, M., Van de Pol, I., Schouls, L.M., 2003. Differences in clinical manifestations and hematological and serological responses after experimental infection with genetic variants of Anaplasma phagocytophilum in sheep. Clin. Diagn. Lab. Immunol. 10, 692–695. Swanson, S.J., Neitzel, D., Reed, K.D., Belongia, E.A., 2006. Coinfections acquired from Ixodes ticks. Clin. Microbiol. Rev. 19, 708–727. Wilske, B., Fingerle, V., Schulte-Spechtel, U., 2007. Microbiological and serological diagnosis of Lyme borreliosis. FEMS Immunol. Med. Microbiol. 49, 13–21. Zhan, L., Cao, W.C., De Vlas, S.J., Xie, S.Y., Zhang, P.H., Wu, X.M., Dumler, J.S., Yang, H., Richardus, J.H., Habbema, J.D., 2008. A newly discovered Anaplasma phagocytophilum variant in rodents from southeastern China. Vector Borne Zoonotic Dis. 8, 369–380.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Anaplasma phagocytophilum and Borrelia burgdorferi in rabbits from southeastern China Lin Zhan a,1, Chen-Yi Chu a,1, Shu-Qing Zuo a, Xiao-Ming Wu a, J. Stephen Dumler b, Na Jia a, Bao-Gui Jiang a, Hong Yang a, Wu-Chun Cao a,* a
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dong-Da Street, Fengtai District, Beijing 100071, People’s Republic of China Division of Medical Microbiology and Immunology, Department of Pathology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 624, Baltimore, MD 21205, USA b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 September 2008 Received in revised form 18 February 2009 Accepted 2 March 2009
A total of 54 wild rabbits captured from southeastern China were examined for Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato by polymerase chain reaction (PCR) assays. One and three samples were positive for A. phagocytophilum and B. burgdorferi, respectively. Sequence analyses of PCR products identified a variant of A. phagocytophilum and a B. garinii genotype. This is the first detection of the two tick-borne agents in Chinese rabbits, the role of which in the maintenance of the agents deserve further investigations. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anaplasma phagocytophilum Borrelia burgdorferi Rabbit Host Gene variation
Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato (s.l.) are tick-borne pathogens with veterinary and public health significance (Dumler et al., 2005). The two agents are thought to be naturally maintained in small mammal–tick cycles, and mainly transmitted by Ixodes ticks, including I. scapularis and I. pacificus in the United States, I. ricinus in mainland Europe, and I. persulcatus in Asia (Swanson et al., 2006). Wild rodents have been implicated as natural reservoirs for the two tick-borne agents (Swanson et al., 2006). In Nantucket Island, A. phagocytophilum was detected in cottontail rabbits, which were considered as potential hosts of the agent (Goethert and Telford, 2003). Both A. phagocytophilum and B. burgdorferi s.l. were found in ticks as well as rodents collected in Zhejiang Province of southeastern China (Chu
* Corresponding author. Tel.: +86 10 63896082; fax: +86 10 63896082. E-mail address: [email protected] (W.-C. Cao). 1 These authors contributed equally to this work. 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.03.003
et al., 2008; Zhan et al., 2008). During the survey, wild rabbits were captured in the same area. The objectives of this study were to determine if the wild rabbits, the hosts of immature of ticks, were naturally infected by A. phagocytophilum and B. burgdorferi, and to characterize the two tick-borne agents using sequence analysis. The study In Januaries of 2006 and 2008, a total of 54 wild rabbits were captured from the forested rolling hills located at 298150 N latitude and 1218030 E longitude in Zhejiang Province of southeastern China, and all were recognized as Chinese hares (Lepus sinensis) by a zoologist. DNA was extracted from spleens and blood samples of the rabbits using Trizol (Invitrogen, Carlsband, CA, USA) as previously described (Cao et al., 2006). In order to improve sensitivity, nested PCR assays were performed to amplify the partial 16S rRNA gene of the A. phagocytophilum. Primers GE9f (50 -AACGGATTATTCTTTATAGCTTGCT-30 ) and GE10r
A A
G
T C C C C C C C C C C
A G G G G G G G G C
T
A A
T
A A
T
G
T A
G A A A A A A A A C C
T C C C C C C C C T
A A
G
C
T C
A
G
T A T
A
C T T T T T T T T T T A A C C A Japan South Africa Swedish USA USA
/, gap. a The same nucleotide as that of rabbit (this study) was not marked.
G C C
A A A
A A A A A
/ / A A A
G
G A A
A
G G G
G A A T T G
57 84 149 150 151 171 284 330 369 390 553 562 711 717 722 738 740 741 746 750
A A G
29 32 36
G G
27 28
T A
DQ458806 AF205140 AF227954 DQ458808 AB196721 AY570538 AY527241 AF172167 CP000235 Rabbit (this study) Tick Tick Rodent Deer/tick Dog Horse Horse Human
16
355
China
GenBank Nucleotide accession no. differencea Geographical Bio-origin origin
Table 1 Comparison of partial 16S rRNA gene sequences of Anaplasma phagocytophilum from different origins.
(50 -TTCCGTTAAGAAGGATCTAATCTCC-30 ) designed by Chen et al. (1994) were applied for the initial amplification. Two primer pairs, GE9f and GE2 (50 -GGCAGTATTAAAAGCAGCTCCAGG-30 ), and Ehr521 (50 -TGTAGGCGGTTCGGTAAGTTAAAG-30 ) and GE10r, were used in the nested reactions as previously described (Cao et al., 2000). After sequencing of the PCR products, a 919-bp fragment was obtained from the positive samples based on overlapping region of sequence. A nested PCR targeting a 253-bp fragment of conserved region of B. burgdorferi s.l. 5S–23S rRNA intergenic spacer was performed using the outer primers of 23S6 (50 -TCCTAGGCATTCACCATA-30 ) and 23S5 (50 -CTGCGAGTTCGCGGGAGA-30 ), and the inner primers of 23S3 (50 -CGACCTTCTTCGCCTTAAAGC-30 ) and 23Sa (50 TAAGCTGACTAATACTAATTACCC-30 ) as previously described (Chu et al., 2008). The PCR amplifications were conducted using a PerkinElmer model 2700 thermal cycler. PCR amplified products were purified by TIANgel Mini Purification Kit (Tiangen Biotech Co. Ltd., Beijing) and sequenced directly on both strands by automated dideoxynucleotide cycle sequencing (ABI PRISM 377, PerkinElmer, Inc.). Out of the 54 rabbits examined, one (1.86%) was positive for A. phagocytophilum and three (5.56%) were positive for B. burgdorferi. No coinfection was identified. Sequence analysis of 919-bp partial 16S rRNA gene indicated that the sequence of A. phagocytophilum (GenBank accession no. DQ458806) detected in this study was distinct from all known A. phagocytophilum sequence deposited in GenBank. The agent was genetically closed to the A. phagocytophilum variant detected in I. persulcatus ticks from Northeastern China (GenBank accession no. AF205140) with only 5-bp difference (Table 1). While the nucleotide sequence was different by 7-bp from that of an A. phagocytophilum variant (GenBank accession no. DQ458808) identified in rodents from the same area (Zhan et al., 2008), and by at least 8-bp from other known A. phagocytophilum strains detected in various animals (Table 1). It was reported that at least one A. phagocytophilum variants isolated from white tailed deer in the USA, which was characterized by its special host-tropism. For example, the ‘‘AP-Variant 1’’ was unable to establish infection in white-footed mice and SCID mice, but could infect goats by experimental inoculation (Massung et al., 2003a,b, 2005, 2006). The sheep infected with A. phagocytophilum variants could cause different clinical manifestations, hematological abnormalities, and serological responses (Stuen et al., 2003). Whether the A. phagocytophilum variant detected in Chinese hare is pathogenic to domestic animals and humans needs further investigations. A 253-bp product of B. burgdorferi 5S–23S ribosomal RNA intergenic spacer was obtained from each positive specimen and sequenced. The nucleotide sequences amplified from the three positive samples were identical to each other (GenBank accession no. FJ172758), and were identified as Borrelia garinii. Multiple sequence alignment revealed that the tested sequence in this study was 100% identity to that of B. garinii strain previously reported in southern China (GenBank accession no. EU160461), but different at least 1% from all known B. garinii sequences deposited in GenBank.
801 866 918
L. Zhan et al. / Veterinary Parasitology 162 (2009) 354–356
L. Zhan et al. / Veterinary Parasitology 162 (2009) 354–356
356
Lyme disease is the most common human tick-borne disease in the northern hemisphere (Haufs, 2003). Its causative agent, B. burgdorferi s.l., is subdivided into at least 11 genospecies. Four of which have been known to be pathogenic to human, including B. burgdorferi sensu stricto, B. afzelii, B. garinii, and B. spielmanii (Wilske et al., 2007). The pathogenicity of the detected B. garinii to human beings and animals remains to be determined. Our findings, together with the evidences previously accumulated (Chu et al., 2008; Zhan et al., 2008), imply that both tick-borne agents are circulating in southeastern China and indicate the wild rabbits may be involved in the maintenance of both pathogens. Further investigations are required to understand the wide geographic distribution and to determine the host range of A. phagocytophilum and B. garinii in China. Conflict of interest There are no potential conflicts of interest. Acknowledgments We are grateful to Dr. Xiao’ai Zhang for her technical assistance, Dr. Rong-Man Xu for identification of ticks. This study was supported by the National Science Fund for Distinguished Young Scholars (No. 30725032). References Cao, W.C., Zhan, L., He, J., Foley, J.E., De Vlas, S.J., Wu, X.M., Yang, H., Richardus, J.H., Habbema, J.D., 2006. Natural Anaplasma phagocytophilum infection of ticks and rodents from a forest area of Jilin Province. China. Am. J. Trop. Med. Hyg. 75, 664–668.
Cao, W.C., Zhao, Q.M., Zhang, P.H., Dumler, J.S., Zhang, X.T., Fang, L.Q., Yang, H., 2000. Granulocytic Ehrlichiae in Ixodes persulcatus ticks from an area in China where Lyme disease is endemic. J. Clin. Microbiol. 38, 4208–4210. Chen, S.M., Dumler, J.S., Bakken, J.S., Walker, D.H., 1994. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 32, 589–595. Chu, C.Y., Jiang, B.G., Liu, W., Zhao, Q.M., Wu, X.M., Zhang, P.H., Zhan, L., Yang, H., Cao, W.C., 2008. Presence of pathogenic Borrelia burgdorferi sensu lato in ticks and rodents in Zhejiang, south-east China. J. Med. Microbiol. 57, 980–985. Dumler, J.S., Choi, K.S., Garcia-Garcia, J.C., Barat, N.S., Scorpio, D.G., Garyu, J.W., Grab, D.J., Bakken, J.S., 2005. Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg. Infect. Dis. 11, 1828–1834. Goethert, H.K., Telford 3rd, S.R., 2003. Enzootic transmission of the agent of human granulocytic ehrlichiosis among cottontail rabbits. Am. J. Trop. Med. Hyg. 68, 633–637. Haufs, M.G., 2003. Lyme borreliosis. Lancet Infect. Dis. 3, 684. Massung, R.F., Courtney, J.W., Hiratzka, S.L., Pitzer, V.E., Smith, G., Dryden, R.L., 2005. Anaplasma phagocytophilum in white-tailed deer. Emerg. Infect. Dis. 11, 1604–1606. Massung, R.F., Mather, T.N., Levin, M.L., 2006. Reservoir competency of goats for the Ap-variant 1 strain of Anaplasma phagocytophilum. Infect. Immun. 74, 1373–1375. Massung, R.F., Mather, T.N., Priestley, R.A., Levin, M.L., 2003a. Transmission efficiency of the AP-variant 1 strain of Anaplasma phagocytophila. Ann. NY Acad. Sci. 990, 75–79. Massung, R.F., Priestley, R.A., Miller, N.J., Mather, T.N., Levin, M.L., 2003b. Inability of a variant strain of Anaplasma phagocytophilum to infect mice. J. Infect. Dis. 188, 1757–1763. Stuen, S., Bergstrom, K., Petrovec, M., Van de Pol, I., Schouls, L.M., 2003. Differences in clinical manifestations and hematological and serological responses after experimental infection with genetic variants of Anaplasma phagocytophilum in sheep. Clin. Diagn. Lab. Immunol. 10, 692–695. Swanson, S.J., Neitzel, D., Reed, K.D., Belongia, E.A., 2006. Coinfections acquired from Ixodes ticks. Clin. Microbiol. Rev. 19, 708–727. Wilske, B., Fingerle, V., Schulte-Spechtel, U., 2007. Microbiological and serological diagnosis of Lyme borreliosis. FEMS Immunol. Med. Microbiol. 49, 13–21. Zhan, L., Cao, W.C., De Vlas, S.J., Xie, S.Y., Zhang, P.H., Wu, X.M., Dumler, J.S., Yang, H., Richardus, J.H., Habbema, J.D., 2008. A newly discovered Anaplasma phagocytophilum variant in rodents from southeastern China. Vector Borne Zoonotic Dis. 8, 369–380.