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Journal of Clinical Microbiology, March 2005, p. 1309-1317, Vol. 43, No. 3
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.3.1309-1317.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Sequence Analysis of the msp4 Gene of Anaplasma phagocytophilum Strains

José de la Fuente,^1,2* Robert F. Massung,³ Susan J. Wong,⁴ Frederick K. Chu,⁵ Hans Lutz,⁶ Marina Meli,⁶ Friederike D. von Loewenich,⁷ Anna Grzeszczuk,⁸ Alessandra Torina,⁹ Santo Caracappa,⁹ Atilio J. Mangold,¹⁰ Victoria Naranjo,² Snorre Stuen,¹¹ and Katherine M. Kocan¹

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma,¹ Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia,³ Diagnostic Immunology Laboratory, Wadsworth Center,⁴ Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York,⁵ Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain,² Clinical Laboratory, Faculty of Veterinary Medicine, University of Zurich, Zurich, Switzerland,⁶ Institute of Medical Microbiology and Hygiene, Department of Medical Microbiology and Hygiene, University Clinic of Freiburg, Freiburg, Germany,⁷ Department of Infectious Diseases, Medical University of Bialystok, Bialystok, Poland,⁸ Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy,⁹ Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Rafaela, Santa Fe, Argentina,¹⁰ Department of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, Sandnes, Norway,¹¹

Received 22 January 2004/ Returned for modification 1 July 2004/ Accepted 7 November 2004

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The causative agent of human granulocytic ehrlichiosis was recently reclassified as Anaplasma phagocytophilum, unifying previously described bacteria that cause disease in humans, horses, dogs, and ruminants. For the characterization of genetic heterogeneity in this species, the homologue of Anaplasma marginale major surface protein 4 gene (msp4) was identified, and the coding region was PCR amplified and sequenced from a variety of sources, including 50 samples from the United States, Germany, Poland, Norway, Italy, and Switzerland and 4 samples of A. phagocytophilum-like organisms obtained from white-tailed deer in the United States. Sequence variation between strains of A. phagocytophilum (90 to 100% identity at the nucleotide level and 92 to 100% similarity at the protein level) was higher than in A. marginale. Phylogenetic analyses of msp4 sequences did not provide phylogeographic information but did differentiate strains of A. phagocytophilum obtained from ruminants from those obtained from humans, dogs, and horses. The sequence analysis of the recently discovered A. phagocytophilum msp2 gene corroborated these results. The results reported here suggest that although A. phagocytophilum-like organisms from white-tailed deer may be closely related to A. phagocytophilum, they could be more diverse. These results suggest that A. phagocytophilum strains from ruminants could share some common characteristics, including reservoirs and pathogenicity, which may be different from strains that infect humans.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), also known as the human granulocytic ehrlichiosis (HGE) agent, Ehrlichia equi, and Ehrlichia phagocytophila, is the causative agent of human granulocytic anaplasmosis, tick-borne fever of ruminants, and equine and canine granulocytic anaplasmosis (16). HGE was first described in 1994; since then, it has been reported in Europe, South America, and North America, where it is now the predominant form of anaplasmosis and among the most common tick-borne infections in the United States (28). In humans, the disease is characterized by fever, headache, myalgia, and malaise, leukopenia, thrombocytopenia, and evidence of hepatic injury (6). Tick-borne fever is reported as a febrile disease of goats, sheep, and cattle, with clinical signs varying from undetectable illness to severe febrile disease associated with opportunistic infections, hemorrhage, and abortions (16). Equine and canine diseases are characterized by fever, depression, anorexia, leukopenia, and thrombocytopenia; equine infection also frequently results in limb edema and ataxia and may lead to opportunistic infections (16).

The genera Anaplasma and Ehrlichia contain obligate intracellular bacteria that multiply within parasitophorous vacuoles in the cytoplasm. Within these vacuoles, the organisms undergo a developmental cycle involving reticulated and dense forms (6). Members of the Ixodes persulcatus complex transmit A. phagocytophilum, which is maintained, at least in part, in small and medium-sized mammals such as the white-footed mouse (Peromyscus leucopus), the raccoon (Procyon lotor), and the gray squirrel (Sciurus carolinensis) (21, 44). Some evidence suggests that subclinical persistent infections occur in domestic and wild ruminants, including deer (16). In cell culture, A. phagocytophilum infects and replicates in undifferentiated HL-60 human promyelocytic cells; in HL-60 cells differentiated into neutrophil-like cells, potential precursors of the myelomonocytic lineage (18); and in two tick cell lines (IDE8 and ISE6) that were derived originally from embryos of Ixodes scapularis (30-32).

A major protein antigen(s) is expressed on the outer membrane of A. phagocytophilum, and some of the immunodominant major surface proteins (MSPs) share sequence similarity with Anaplasma marginale MSP2 and MSP4 (22) and Ehrlichia ruminantium MAP1 (46). Although the biological function of A. marginale MSP4 is unknown, this MSP is probably involved in host-pathogen interactions and may evolve more rapidly than other nuclear gene proteins because of selective pressures exerted by host immune systems. Furthermore, the analysis of msp4 sequences provided phylogeographic patterns for A. marginale strains (11-14).

Although A. phagocytophilum has a broad geographic distribution, all strains identified thus far appear to have considerable serological cross-reactivity and a minor degree of variation in the nucleotide sequences of the 16S rRNA, groESL, gltA, ank, and msp2 genes, with the exception of some ank sequences from infected German ticks that are different from other ank sequences of human and animal strains (16, 22, 25, 26, 42, 47). However, the clinical and host tropism diversity of A. phagocytophilum suggests the presence of genetic differences among these bacteria that have not been characterized. The present study was undertaken to characterize variations in the sequence of an outer membrane protein gene, homologous to A. marginale msp4, in A. phagocytophilum strains obtained from different host species in the United States and Europe.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A. phagocytophilum samples. The A. phagocytophilum samples obtained from naturally infected humans, dogs, horses, donkeys, white-tailed deer (WTD), roe deer, sheep, European bison, and cows and from infected cultured human HL-60 and tick ISE6 cells (isolates) used in this study for the characterization of msp4 sequences are listed in Table 1. The geographic distribution of the samples covered the western and eastern United States and Germany, Poland, Norway, Italy, and Switzerland in Europe. Four samples of A. phagocytophilum-like organisms from WTD were also included in the study (Table 1). msp4 sequences for Anaplasma centrale (GenBank accession number AF428090), Anaplasma ovis (AF393742), and A. marginale (GenBank accession number AY010252, isolate from Wetumka, Okla.; GenBank accession number AF428083, isolate from Mexico) were used for outgroup comparisons. Analysis of the msp2 gene was done with sequences reported recently by Lin et al. (22) for human (HZ, LL, and MN-2), horse (MRK), and ovine (OS) strains; the sequences isolated in this study are from human blood samples gathered in New York and WI-H1-WI-H4, WI-C1, Webster, HGE2, NY18, Elsa, Olga, Ovine 5, and Ovine 7 strains of A. phagocytophilum (Table 1). For analysis of Ehrlichia spp. and Anaplasma spp., the Ehrlichia chaffeensis (Arkansas isolate) 28-kDa MSP (GenBank accession number AAO12929) (37), the E. ruminantium map1 (GenBank accession number AY028378) (1) and the Wolbachia spp. MSP (GenBank accession number AF020068) (5) sequences were also included in the analysis together with Anaplasma spp. msp4 sequences.

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TABLE 1. Samples used in the studya

msp4, msp2, and 16S rRNA PCR and sequencing. A sequence of 283 amino acids with 58% identity (BLAST score, E value = 1e^–99) to A. marginale (Oklahoma isolate) MSP4 (GenBank accession number AAG34718) was identified in silico in the A. phagocytophilum (HZ isolate) genome (TIGR 212042_contig: 366) by TBLASTN search of the preliminary sequence data obtained from The Institute for Genomic Research (http://www.tigr.org) at the National Center for Biotechnology Information (GenBank accession number NC_004351). Oligonucleotides (MAP4AP5 [5'-ATGAATTACAGAGAATTGCTTGTAGG-3'] and MSP4AP3[5'-TTAATTGAAAGCAAATCTTGCTCCTATG-3'], for upstream and downstream primers, respectively) were designed for amplification of the A. phagocytophilum msp4 849-bp coding region with the sequence of the HZ isolate. DNA was extracted as reported previously (3, 8, 13, 26). The msp4 gene was amplified from 1 µl (0.1 to 10 ng) of DNA by PCR with 10 pmol of each primer (MSP4AP5 and MSP4AP3) in a 50-µl volume (1.5 mM MgSO₄, 0.2 mM deoxynucleoside triphosphate, 1x AMV-Tfl 5x reaction buffer, 5 U of Tfl DNA polymerase) with the Access RT-PCR system (Promega, Madison, Wis.). Reactions were performed in an automated DNA thermal cycler (Mastercycler personal; Eppendorf, Westbury, N.Y.) for 35 cycles. After an initial denaturation step of 30 s at 94°C, each cycle consisted of a denaturing step of 30 s at 94°C and an annealing-extension step of 1 min at 68°C. The program ended by storing the reaction mixtures at 4°C. PCR products were electrophoresed on 1% agarose-ethidium bromide gels to check the size of amplified fragments in comparison with DNA size markers. Amplified fragments were resin purified (Wizard; Promega) and cloned into pGEM-T vector (Promega) or used directly for sequencing both strands by double-stranded dye-termination cycle sequencing (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University). When cloned, at least two clones were sequenced from each PCR.

Amplification and sequencing of msp2 genes was done as described above for msp4 genes but with the oligonucleotide primers described by Lin et al. (22) for the msp2 full-length DNA PCR (MSP25 [5'-TTATGATTAGGCCTTTGGGCATG-3'] and MSP23 [5'-TCAGAAAGATACACGTGCGCCC-3']) and amplification cycles consisting of a denaturing step of 30 s at 94°C, an annealing step of 30 s at 60°C, and an extension step of 1.5 min at 68°C.

A nested PCR for the amplification of a 546-bp amplicon of the 16S rRNA gene was done as described by Massung et al. (29).

Sequence alignment and phylogenetic analysis. The msp4 and msp2 gene coding regions and the MSP4 protein sequences were used for sequence alignment and phylogenetic analysis. Multiple sequence alignment was performed with the program AlignX (Vector NTI Suite, version 8.0; InforMax, North Bethesda, Md.) with an engine based on the CLUSTAL W algorithm (45). Nucleotides were coded as unordered, discrete characters with five possible character states: A, C, G, T, or N. Gaps were coded as missing data. Maximum parsimony (MP) analyses were conducted with equal weights for all characters and substitutions and heuristic searches with 10 random additions of input taxa. To examine the effect of the method of analysis on the resulting phylogeny, a phylogenetic tree was constructed based on the sequence distance method using the neighbor-joining (NJ) algorithm of Saitou and Nei (39) with the Kimura two-parameter model or Poisson corrections for nucleotide and amino acid sequences, respectively. Phylogenetic analyses were conducted with MEGA 2 (20). The stability or accuracy of the inferred topology(ies) was assessed via bootstrap analysis (17) of 1,000 iterations. Character state changes for A. phagocytophilum msp4 were polarized by designating A. marginale, A. centrale, and A. ovis as outgroups.

Nucleotide sequence accession numbers. The GenBank accession numbers for msp4 sequences of A. phagocytophilum HZ, WI-H, MRK, AP-V1, Elsa, Donkey, Horse 31, Roe deer, Ovine, and Bison strains are AY530194 to AY530198, AY702925, and AY706386 to AY706391; for msp2 sequences of Elsa and Ovine strains, the accession numbers are AY706392 and A706393.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The analysis of sequence variation in the msp4 coding region of A. phagocytophilum showed some heterogeneity in this locus at the nucleotide (90 to 100% identity) and protein (92 to 100% similarity) levels (Table 2). The 21 samples from infected human blood analyzed from New York during 1996 to 2000 had the same msp4 sequence, identical to the sequence of the HZ and NY18 New York isolates and to isolates HGE2 Webster (originally isolated from humans) and WI-C1 (originally isolated from a dog) (Table 1). The four strains from humans in Wisconsin (WI-H1 to WI-H4) (WI-H in Tables 2 and 3; Fig. 1 to 3) had identical msp4 sequences, differing only in one nucleotide at position 687 introducing an M-to-I amino acid change at position 229 with respect to the HZ reference sequence (Table 3). The MRK isolate, originally isolated from a horse in California and identified as E. equi, and the Italian strains from donkeys had identical msp4 sequences with a mutation at nucleotide position 648, resulting in an S-to-R amino acid change at position 216 with respect to the HZ sequence (Table 3). A strain from a horse in Germany had a sequence differing from the msp4 sequence of the MRK isolate, with 12 silent mutations with respect to the HZ sequence (Table 3). The most divergent strains of A. phagocytophilum were those derived from ruminants (Tables 2 and 3). The sequence of A. phagocytophilum variant 1 (AP-V1) from WTD differed in six nucleotide positions, producing two amino acid changes of D to A and I to T at positions 64 and 156, respectively, when compared to the sequence of the HZ isolate (Table 3). Swiss bovine Elsa and Olga samples and the Polish strain Bison 7 had 17 changes in the nucleotide sequence, resulting in two amino acid changes of E to D and I to V at positions 97 and 143, respectively, compared to the HZ sequence (Table 3). Polish Bison strains 12, 18, and 21 had 14 nucleotide changes, resulting in only one amino acid change identical to the E to D mutation at position 97 in Elsa, Olga, and Bison 7 strains when compared to the HZ sequence (Table 3). Polish Bison strains 15 and 16 had 18 changes at the nucleotide level when compared to the HZ sequence, resulting in the same two amino acid changes found in Elsa, Olga, and Bison 7 strains (Table 3). The Ovine strains from Norway had identical msp4 sequences, with 27 nucleotide mutations resulting in three amino acid changes when compared to the HZ sequence (Table 3). The sequence of the msp4 gene was identical in four A. phagocytophilum strains from German Roe deer strains, with 86 nucleotide mutations resulting in 23 amino acid changes, compared to the sequence of the HZ isolate (Table 3). The DNA samples from A. phagocytophilum-like organisms obtained from WTD in the United States (WTD-OK1, WTD-OK4, WTD-UGA1, and WTD-UGA2) did not produce any detectable signal in the msp4 PCR, but a PCR targeting a 546-bp amplicon of the 16S rRNA gene produced a positive signal (data not shown).

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TABLE 2. Percent identity among nucleotide (above diagonal formed by boldface entries) and percent similarity among deduced amino acid (below diagonal) sequences between Ehrlichia and Anaplasma speciesa

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TABLE 3. Differences in the msp4 nucleotide and amino acid sequences among A. phagocytophilum strainsa

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FIG. 1. Phylogenetic analysis of A. phagocytophilum strains based on the msp4 gene (A) and protein (B) sequences with NJ with Kimura two-parameter analysis or Poisson corrections and bootstrap analysis of 1,000 replicates. Numbers on the branches indicate percent support for each clade. Strains of A. phagocytophilum are described in Tables 1 and 2. Abbreviations: A.p., A. phagocytophilum; A.m., A. marginale.

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FIG. 3. Phylogenetic analysis of Anaplasma and Ehrlichia species based on MP analysis of msp4 sequence data and bootstrap analysis with 1,000 iterations. Numbers on the branches indicate percent support for each clade. Strains of A. phagocytophilum are described in Tables 1 and 2. Abbreviations: A.p., A. phagocytophilum; A.m., A. marginale.

The HGE2 isolate was analyzed following growth in both human HL-60 and tick ISE6 cells to evaluate if the msp4 sequence was conserved in bacteria grown in mammalian and tick cells; growth resulted in identical msp4 sequences.

Phylogenetic analysis of Anaplasma spp. msp4 gene and protein sequences did not provide phylogeographic information (Fig. 1). However, differences were observed in NJ and MP analyses of msp4 gene and protein sequences according to the original host of the strain, particularly differentiating the A. phagocytophilum strains obtained from bovine (Elsa and Olga), WTD (AP-V1), roe deer (Roe deer 2, 6, 8, and 9), European bison (Bison 7, 12, 15, 16, 18, and 21) and ovine (Ovine 5 and 7) ruminants (Fig. 1 and data not shown).

Phylogenetic analysis of A. phagocytophilum msp2 sequences confirmed this result (Fig. 2). The 21 samples from infected human blood analyzed from New York had the same msp2 sequence, identical to the sequence of the HZ and NY18 New York isolates. The msp2 sequences of isolates HGE2, WI-H1 to WI-H4, and WI-C1 were identical to the sequence of strain MN-2. The MRK and Webster isolates had identical msp2 sequences. The strains Elsa and Olga had identical msp2 sequences, different from the sequence of strains Ovine 2 and 5, which also had identical sequences.

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FIG. 2. Phylogenetic analysis of A. phagocytophilum strains based on the msp2 gene sequences with NJ with Kimura two-parameters correction and bootstrap analysis of 1,000 replicates. Numbers on the branches indicate percent support for each clade. Strains of A. phagocytophilum are described in Tables 1 and 2. A.p., A. phagocytophilum.

The phylogenetic analysis of Ehrlichia spp. and Anaplasma spp. was done using the msp4 data for A. phagocytophilum, A. marginale, A. centrale, and A. ovis; the E. chaffeensis 28-kDa MSP; and the E. ruminantium map1 and the Wolbachia spp. MSP sequences (Table 2). Anaplasma spp. were monophyletic with respect to Wolbachia and Ehrlichia spp. (Fig. 3). E. ruminantium and E. chaffeensis grouped together in a clade separated from Wollbachia and Anaplasma spp. (Fig. 3).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phylogenies of A. phagocytophilum have been largely inferred using ribosomal and housekeeping genes (16). The groESL genes have been used to differentiate North American from European strains and to compare A. phagocytophilum strains from roe deer and red deer in Slovenia (34, 42). Although ribosomal and housekeeping genes are powerful tools for determining relatedness at the genus and species level, their strong conservation gives little resolution of closely related species or strains. For this analysis, genes encoding structural or surface proteins like those involved in pathogen-host interactions are more appropriate. Massung et al. (26) used the ank gene encoding an A. phagocytophilum structural protein for phylogenetic analysis and were able to separate the North American strains into two clades, upper Midwest and Northeast, which were both distinct from the European strains. However, von Loewenich et al. (47) described ank variant sequences from infected ticks that are different from all other ank sequences and whose geographic distribution is still unknown. The homologue of A. marginale MSP4, which has been shown to provide phylogenetic and phylogeographic information about geographic isolates of A. marginale (11-14), was identified in silico in the A. phagocytophilum genome sequence, thus providing a potentially more informative genetic marker for the characterization of A. phagocytophilum strains.

The best characterized major antigenic protein of A. phagocytophilum, p44, is highly polymorphic in different populations of the organism derived from tick or mammalian host cells (3). For phylogenetic analyses, a genetic marker which is conserved through the life cycle of the bacterium is preferable. The msp4 sequence was stable in A. phagocytophilum (isolate HGE2) grown in human HL-60 and tick ISE6 cells, corroborating the results obtained for A. marginale MSPs in vitro and in vivo (4; unpublished results). The recently discovered msp2 gene, different from the antigenically variable p44 gene family of A. phagocytophilum (22), was also shown to be a candidate gene for phylogenetic studies of A. phagocytophilum strains. Future studies addressing the genetic stability of A. phagocytophilum msp2 and msp4 genes during the life cycle of the bacterium in vivo may further confirm the validity of the use of these genes in phylogenetic studies.

Strains of A. phagocytophilum generally showed a high degree of identity at the msp4 locus, similar to previous results using the 16S rRNA and groESL genes and confirmed by genome-wide analysis by pulsed-field electrophoresis (15, 29, 36, 42). However, the strain from European roe deer was unique in that its msp4 sequence was much more diverse than that for any other strain examined. Further studies to characterize additional genes and gene products of the roe deer strain are needed to determine if the diversity noted for the msp4 gene reflects genomic divergence or the specific evolution of the msp4 gene is under some selective pressure. With the exception of the roe deer strain, the msp4 sequences had a lower degree of diversity than ank gene sequences; but unlike results with the ank sequences, phylogenetic analysis of the msp4 sequences differentiated between strains of A. phagocytophilum from humans, dogs, and horses from those obtained from ruminants (26). As shown before for A. marginale strains in tick pathogen phylogenetic studies (13), the possibility of differentiating between A. phagocytophilum samples according to the mammalian host of origin suggested that msp4 sequences could be used for coevolutionary studies. The degree of msp4 sequence heterogeneity among strains of A. phagocytophilum was higher than among geographic isolates of A. marginale, with 99 to 100% identity (Table 2) (14). This higher degree of heterogeneity could be a result of the large number of reservoir hosts in which A. phagocytophilum strains have been identified, compared to the relatively limited host range of A. marginale, and may provide a higher level of resolution in coevolutionary studies involving the msp4 gene of A. phagocytophilum.

The DNA samples of A. phagocytophilum-like organisms obtained from WTD, WTD-OK1, WTD-OK4, WTD-UGA1, and WTD-UGA2, which did not produce any detectable signal in the msp4 PCR, were suitable for PCR analysis as revealed by the positive signal in the 16S rRNA targeting reaction. These results suggested that the sequence of the msp4 gene in these samples is more diverse than among strains of A. phagocytophilum. Alternatively, the sensitivity of the msp4 PCR was below the detection level for these samples (five copies of msp4 per nanogram of DNA). Nevertheless, although A. phagocytophilum-like organisms isolated from WTD may be closely related to A. phagocytophilum (33), they could prove to be a separate species after further analysis.

The A. phagocytophilum variant 1 (sample AP-V1) coexists with and is closely related to the human strains of A. phagocytophilum but has never been associated with human infection (9). AP-V1 has been found in I. scapularis ticks and WTD in Rhode Island, Connecticut, Maryland, Pennsylvania, and Wisconsin in the United States (9). Strains Elsa and Olga were obtained from infected cattle in Switzerland, where I. ricinus is the tick vector; these strains have not been associated with human infection (19, 36). Ovine strains of A. phagocytophilum produce a tick-borne fever in sheep with variable pathogenicity but have never been recovered from humans (40). A. phagocytophilum strains associated with human disease in Europe have been obtained from I. ricinus ticks but not from wild animals such as roe deer (23, 24, 34, 35, 47). These results suggested that A. phagocytophilum strains from ruminants could share some common characteristics, including reservoirs and pathogenicity, which may be different from strains that infect humans (27).

The organisms in the order Rickettsiales were recently reclassified based on biological characteristics and genetic analyses of 16S rRNA, groESL, and gltA genes (16). These phylogenetic analyses consistently supported the formation of four distinct genera within the family Anaplasmataceae: Anaplasma, Ehrlichia, Wolbachia, and Neorickettsia. On the basis of these analyses, Dumler et al. (16) proposed that E. equi, E. phagocytophila, and the HGE agent should be recognized as synonymous and be renamed as A. phagocytophilum. This reclassification has been supported by the analysis of other genetic markers (7, 26, 43). The results reported here using the surface protein gene msp4 also support this classification, although strains with different reservoirs and pathogenicity may exist.

 
This research was supported by project no. 1669 of the Oklahoma Agricultural Experiment Station; the Endowed Chair for Food Animal Research (K.M.K.), College of Veterinary Medicine, Oklahoma State University; the Forschungskommission of the Albert-Ludwigs-Unversität Freiburg, Germany (grant to F.V.L.); and the NIH Centers for Biomedical Research Excellence through a subcontract to J.D.L.F. from the Oklahoma Medical Research Foundation and the Oklahoma Center for the Advancement of Science and Technology, Applied Research Grants AR00(1)-001 and ARO21-37.

The Institute for Genomic Research and Yasuko Rikihisa (Ohio State University) are acknowledged for supplying the preliminary sequence data of A. phagocytophilum HZ isolate through the website at http://www.tigr.org. Anthony F. Barbet (University of Florida) is acknowledged for supplying DNA from HGE2 isolate grown in HL-60 and ISE6 cells. Janet J. Rogers (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University) is acknowledged for DNA sequencing.

 
* Corresponding author. Mailing address: Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078. Phone: (405) 744-0372. Fax: (405) 744-5275. E-mail: jose_delafuente{at}yahoo.com.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, March 2005, p. 1309-1317, Vol. 43, No. 3
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.3.1309-1317.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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