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. 2005 Oct;12(10):1177–1183. doi: 10.1128/CDLI.12.10.1177-1183.2005

Serologic Cross-Reactivity between Anaplasma marginale and Anaplasma phagocytophilum

U M Dreher 1, J de la Fuente 2,3, R Hofmann-Lehmann 1, M L Meli 1, N Pusterla 4, K M Kocan 2, Z Woldehiwet 5, U Braun 6, G Regula 7, K D C Staerk 7, H Lutz 1,*
PMCID: PMC1247822  PMID: 16210480

Abstract

In the context of a serosurvey conducted on the Anaplasma marginale prevalence in Swiss cattle, we suspected that a serological cross-reactivity between A. marginale and A. phagocytophilum might exist. In the present study we demonstrate that cattle, sheep and horses experimentally infected with A. phagocytophilum not only develop antibodies to A. phagocytophilum (detected by immunofluorescent-antibody assay) but also to A. marginale (detected by a competitive enzyme-linked immunosorbent assay). Conversely, calves experimentally infected with A. marginale also developed antibodies to A. phagocytophilum using the same serological tests. The identity of 63% determined in silico within a 209-amino-acid sequence of major surface protein 5 of an isolate of A. marginale and one of A. phagocytophilum supported the observed immunological cross-reactivity. These observations have important consequences for the serotesting of both, A. marginale and A. phagocytophilum infection of several animal species. In view of these new findings, tests that have been considered specific for either infection must be interpreted carefully.

Anaplasma marginale is a rickettsial organism that causes bovine anaplasmosis in cattle. It invades the erythrocyte and leads to extravascular hemolysis (9). Cattle show clinical signs of anemia, icterus, fever and weakness, while several other domestic and wild ruminants are nonapparent carriers after infection (11). A. marginale is transmitted mechanically by lice, biting flies and fomites and biologically by various tick species. It is prevalent in many regions of the world with tropical and subtropical climates (for a review, see reference 9). In the context of a recent outbreak of bovine anaplasmosis in August 2002 in Switzerland (7), a study was initiated to determine the seroprevalence of A. marginale in cattle in Switzerland (4). The seroprevalence was likely to be zero with upper confidence limits lower than 2.5%. It was then realized that cattle seropositive for A. marginale or A. centrale in a USDA-approved test had a tendency to also be positive in an immunofluorescent antibody test for A. phagocytophilum (formerly known as Ehrlichia equi, E. phagocytophilum, and the agent of human granulocytic ehrlichiosis [5]).

Anaplasma phagocytophilum infects granulocytes and causes a variety of clinical signs, including human granulocytic anaplasmosis, fever and loss of milk yield in cattle, fever in small ruminants and fever, edemas, and petechia in horses (16, 18, 23, 25).

In view of the conserved nature of the msp5 gene of Anaplasma species (24) and the recent reorganization into the same genus, immunological similarities between A. marginale and A. phagocytophilum have to be expected. It was the goal of the present study to determine whether and to what degree antibodies against A. marginale would cross-react with A. phagocytophilum, whether cross-reactivity would interfere with the specificity of the serotesting, and to further investigate this cross-reactivity on the basis of the nucleotide and protein sequence of the target antigen of the enzyme-linked immunosorbent assay (ELISA) test, the major surface protein 5 (MSP5).

MATERIALS AND METHODS

Experimental design.

Serum samples were collected from cattle, sheep and horses before and after experimental infection with A. marginale (cattle) and A. phagocytophilum (cattle, sheep, and horses), respectively. To test for immunologic cross-reactivity, the samples were assayed with different serological methods detecting antibodies to either A. marginale, A. ovis, A. centrale, or A. phagocytophilum, respectively. Our hypothesis was that antibodies induced by A. phagocytophilum would react with A. marginale antigen and vice versa.

Experimental infection with A. marginale.

A total of five calves were included in the experiment. Experiments were carried out in Oklahoma. One calf (PA489) was infected intravenously (i.v.) with an A. marginale Okeechobee isolate blood stabilate, originating from a naturally infected North American bison (2). The other four calves were infected with an A. marginale Oklahoma isolate: two animals (PA 419 and PA 404) were injected i.v. with a blood stabilate from infected cows, and two animals (PA 387 and PA 412) were infected by feeding of Dermacentor variabilis and D. andersoni ticks, respectively. All calves were tested to be negative for A. marginale by a competitive ELISA (cELISA; VMRD, Pullman, WA) and by examination of Giemsa-stained blood smears prior to experimental infection.

Experimental infection with A. phagocytophilum.

Six clinically healthy Brown Swiss cattle, originating from tick-free regions of Switzerland and tested to be negative for A. phagocytophilum in an immunofluorescent antibody test (IFA, Ehrlichia equi slides, VMRD, Pullman, WA), were infected i.v. with 50 ml of whole blood collected from a cow with clinical anaplasmosis carrying a Swiss strain of A. phagocytophilum (17).

Five clinically healthy Cambridge sheep, aged 1 to 2 years, were infected i.v. with a British (Old Sourhope) strain of A. phagocytophilum by using 1 ml of a 10% dilution of blood stabilate with 10% dimethyl sulfoxide (20, 25, 26).

Four healthy 3- to 16-year-old geldings (two Thoroughbreds, one Warmbred, and one Hanoverian) and a 3- and a 17-year-old mare (one quarter horse and one standardbred), seronegative for A. phagocytophilum, were inoculated i.v. with a human isolate of A. phagocytophilum originating from Wisconsin (Webster strain [1]), cultured on HL60 cells. Each animal received 1 × 106 infected HL60 cells (18).

Serologic tests.

The cELISA (VMRD, Pullman, WA), licensed for the detection of antibodies to the MSP5 of A. marginale and to the respective surface proteins of A. centrale and A. ovis, was used (8, 22). In order to increase the reproducibility for individual serum samples, one positive and negative control sample each were tested together with six and three serum samples from experimental animals, respectively. This is in excess of the number of controls suggested by the manufacturer to be included per plate. The A. marginale cELISA has been approved by the U.S. Department of Agriculture for bovines but not for other species. In the present paper, it was used to evaluate the presence of antibodies in Anaplasma species in bovine, ovine and equine species. Based on the species differences, the absolute absorbance values measured in the different species cannot directly be compared. However, decreased absorbance values in individual animals during seroconversion induced by experimental infection were considered proof for induction of antibodies competing with the monoclonal antibody (MAb) present in the test kit. For the detection of antibodies to A. phagocytophilum, an IFA (Ehrlichia equi slides; VMRD, Pullman, WA) was employed. The tests were conducted according to the manufacturer's instructions. For the cELISA, the negative controls were always in the range between 0.4 and 2.1 optical densities (wavelength, 620 nm), and the positive controls showed an inhibition of more than 30%. Additionally, we required the coefficient of variation of the negative controls within a plate to be lower than 11%. For use in IFA, serum samples were serially diluted in twofold steps starting at 1:20.

For Western blot analysis, IDE8 tick cells infected with A. marginale (Virginia isolate) (14) were collected from various passages and pooled. Cells were lysed by repeated freezing and thawing on ice. As a control, material from an uninfected tick cell culture was processed identically. The protein concentration was determined in comparison to a bovine serum albumin standard by the Bradford method (Bio-Rad protein assay, Microassay Procedure for Microtiter Plates; Bio-Rad Laboratories, Hercules, California). The material was frozen in liquid nitrogen and lyophilized in a freeze drier (Benchtop, Ismatec, Glattbrugg, Switzerland). The pellet was solubilized in sample buffer (12) and adjusted to a protein concentration of 15 mg/ml. The antigens were homogenized by forcing the material through a 0.33-mm wide needle and by sonication (Sonoplus HD 2070 ultrasonic homogenizer, Bandelin electronic GmbH & Co. KG, Berlin, Germany; four cycles, 50 V, each cycle 0.2-s sonication and 0.8-s rest; 3-mm MS 73 probe; Bandelin). Preparative Western blot analysis was performed using the equivalent of 1 mg protein under conditions described previously (13). Molecular mass standards comprised 10- to 250-kDa proteins (Precision Plus protein dual-color standards, 161-0374; Bio-Rad Laboratories, Hercules, California). Strips of two to three millimeters width were cut from the membrane and incubated in serum diluted 1:100 in blocking solution (1% [wt/vol] liquid gelatin [Nordland], 50 mM trishydroxymethylaminomethan [Fluka 93349], 100 mM NaCl [Fluka 71380], 407 μM Tween 20 [Merck-Schuchardt, Hohenbrunn, Germany, pH, 7.4 adjusted with HCl]) at room temperature overnight. Antispecies antibodies conjugated to horseradish peroxidase (Cappel) were diluted 1:300 in blocking solution and strips were incubated for two hours at room temperature. For detection of MSP5, horseradish peroxidase conjugated MAbs removed from the cELISA test kit (100× Antibody Peroxidase Conjugate) were diluted 1:40 in blocking solution, and strips were incubated overnight. After thorough washing, the strips were incubated in substrate solution (2.8 mM 4-chloro-1-naphthol [Bio-Rad Laboratories, Hercules, California], 16.7%, [vol/vol] methanol, 41.65 mM trishydroxymethylaminomethan [Fluka 93349], 166 mM NaCl [Fluka 71380], 0.05% H2O2 [Fluka 95300]) for 10 min at room temperature. Strips were repeatedly dipped in aqua bidest and dried on Whatman paper (Fisher Scientific, Wohlen, Switzerland).

Statistical analysis.

Correlation of test results of the cELISA and the IFA was determined by the Spearman correlation coefficient. Results of repeated measures on individual animals at different time points were analyzed for statistically significant differences using the Wilcoxon signed rank test calculated by StatView software (version 2001; SAS Institute, Inc., Cary, NC). The hypothesis was that antibody levels after infection were higher than antibody levels at time zero (one-tailed P values). When more than two different time points were compared, the Friedman test (nonparametric repeated measures analysis of variance [ANOVA]) was used to assess whether significant differences over all time points exist before comparing individual time points with the Wilcoxon test.

The results in Fig. 1 through 4 are displayed in the form of box-plots where lower line of the box represents the 25th, the middle line the 50th and the upper line the 75th and the whiskers the 10th and 90th percentiles, respectively.

FIG. 1.

FIG. 1.

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Antibody detection in cELISA (A) and IFA (B) of five calves experimentally infected with A. marginale. Samples were taken from each animal prior to (week 0) and after infection (weeks 4 to 5.5). Antibody test results in samples collected after infection were significantly higher than in preinfection serum samples (P = 0.03) which is indicated.

FIG. 4.

FIG. 4.

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Antibody quantification by cELISA (A) and IFA (B) in sera from six horses experimentally infected with A. phagocytophilum. Samples were taken prior to (day 0) and after infection (day 12). Results of samples collected at day 0 were significantly different from those of samples collected at day 12, in both, the IFA and in the cELISA (both P = 0.02). The inhibitory values of cELISA shown in panel A were set to reach 0% inhibition in the noninfected horses.

Analysis of MSP5 protein sequences.

Multiple sequence alignment was performed using the program AlignX (Vector NTI Suite v.8.0; InforMax, North Bethesda, MD) with an engine based on the Clustal W algorithm (21). The phylogenetic tree was constructed with MSP5 protein sequences based on a sequence distance method utilizing the Neighbor Joining algorithm (19) as instrumented in AlignX. Protein topology was analyzed using TMpred and TMHMM v.2.0 algorithms for the prediction of transmembrane helices in proteins (6, 10).

RESULTS

Samples collected from experimentally infected cattle, sheep and horses were assayed with serologic tests specific for A. phagocytophilum and A. marginale, respectively, prior to and at different time points after infection.

Experimental infection with A. marginale.

Serum samples were collected from five calves experimentally infected with A. marginale. The increase in antibody concentration to A. marginale during the course of infection, detected by cELISA which is intended to detect antibodies to A. marginale, was paralleled by results obtained in the IFA which is intended to detect antibodies to A. phagocytophilum (Fig. 1). Test results from the cELISA and IFA showed strong correlation (Spearman's rho = 0.85, P = 0.002). Inhibition of the binding of MAbs to MSP5 in the cELISA and the antibody titers to A. phagocytophilum were significantly higher in serum samples collected after infection (P = 0.03 for both tests).

Experimental infection with A. phagocytophilum.

Sera were collected from six cows before and after experimental infection with a Swiss strain of A. phagocytophilum (Fig. 2). In the IFA which is based on A. phagicytophilum antigen, the overall increase of titer was significant (P = 0.004). High titers were already observed at week 2 after infection. Between week 2 and week 10, titer values did not change significantly. A significant overall difference among time points was not detected using the cELISA (P = 0.14). However, an increase in antibody titers was observed from week zero to week four (P = 0.02), with a subsequent decrease in week 10 (P = 0.06). Positive results (inhibition of 30% and higher compared to the negative control serum provided by the manufacturer) were obtained starting at week two after infection. The highest mean inhibitory values were observed by week four. By week 10 postinfection, antibody concentrations decreased. During the course of infection, each of the six cows became cELISA positive at least at one time point. Test results from IFA and cELISA were moderately correlated with each other (rho = 0.46, P = 0.02).

FIG. 2.

FIG. 2.

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Sera from six cows, prior to and after experimental infection with A. phagocytophilum. Antibodies measured in cELISA (A) to detect A. marginale and A. centrale antibodies and in IFA (B) designed to quantitate A. phagocytophilum antibodies in the same serum samples. The mean inhibitory value at week zero was set as baseline in panel A. Significant differences as indicated.

Antibodies were measured in five sheep before and after experimental infection with a British strain of A. phagocytophilum (Fig. 3). In the cELISA which is intended to detect antibodies to A. marginale antigen, overall inhibition values were increased significantly compared to preinfection samples starting two weeks after experimental infection until the end of the observation period (P = 0.01). Three of the infected sheep reached inhibitory values within the positive range for cattle (higher than 30% compared to the standardized negative bovine control serum of the cELISA test kit). A moderate correlation between the results of both tests existed (rho = 0.45, P = 0.03).

FIG. 3.

FIG. 3.

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Antibody values measured as inhibitory values in cELISA (A) and as titers in IFA (B) of five sheep experimentally infected with A. phagocytophilum. The mean inhibition before infection at week zero was set as baseline in panel A. Significant differences as indicated.

Sera were collected from six horses before and 12 days after experimental infection with a Wisconsin isolate of A. phagocytophilum (Fig. 4) and tested by IFA and cELISA. The results obtained with postinfection samples were significantly higher than in the pre-infection sera (P = 0.02) with both tests. The results of the cELISA and IFA tests were highly correlated (rho = 0.84, P < 0.001).

To confirm that antibodies produced against A. phagocytophilum after infection cross-react with MSP5 of A. marginale, Western blot analysis was done using A. marginale antigen from tick cell cultures (Fig. 5). Both, antibodies to A. marginale and to A. phagocytophilum recognized A. marginale MSP5.

FIG. 5.

FIG. 5.

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Western blot analysis. Antigen produced in tick cells (Ixodes scapularis embryonic cells, IDE8 cells, infected with A. marginale Virginia isolate). Ladders: 25, 20, 15, and 10 kDa. Lanes: 1, negative control serum included in the cELISA test; 2, positive control serum included in the cELISA test; 3, monoclonal antibody against MSP5; 4, serum of horse two, taken before infection; 5, serum of horse two, taken 12 days after infection with A. phagocytophilum. The band of 18 kDa (right arrow) detected by the positive control serum (2) and monoclonal antibody (3) is also detected in the horse after infection with A. phagocytophilum (5). Many bands are detected in both negative and positive control sera, presumably representing antibodies against tick cell antigens. The monoclonal antibody specific for A. marginale MSP5 recognized not only MSP5 but also a band of 15 kDa which was recognized by antibodies present in several sera collected from noninfected animals. No explanation was found for this MAb to bind to a band, which most likely is of tick cell origin.

Sequence analysis of MSP5.

A sequence of 209 amino acids with 63% identity (BLAST score E = 5.1e−69) to A. marginale (Florida isolate) MSP5 (A49213) was identified in silico in the A. phagocytophilum (HZ isolate) genome (TIGR contig: 513) 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 (NCBI).

Analysis of MSP5 sequences previously reported in the GenBank for A. marginale (strains Florida [A49213], Havana [AAS18265], and Brazil [AAO92930]) and A. centrale (strain Israel [AAL17671]) were compared to the A. phagocytophilum homologue identified in silico using as outgroups the E. canis (strain Oklahoma [AAD40619]), E. chaffeensis (strain Arkansas [AAD40620]), and E. ruminantium (strain Palm River [AAD40617]) sequences. The results showed that the A. phagocytophilum MSP5 has a 63% to 65% similarity to other Anaplasma sequences (Table 1; Fig. 6).

TABLE 1.

Percent similarity among deduced MSP5 amino acid sequences between Ehrlichia and Anaplasma speciesa

Strain A. marginale Brazil A. marginale Florida A. marginale Havana A. centrale Israel A. phagocytophilum HZ E. canis Oklahoma E. chaffeensis Arkansas E. ruminantium Palm River
A. marginale Brazil 100 98 97 92 63 50 49 47
A. marginale Florida 100 99 93 64 51 50 47
A. marginale Havana 100 93 64 51 50 47
A. centrale Israel 100 65 51 50 47
A. phagocytophilum HZ 100 54 49 52
E. canis Oklahoma 100 80 75
E. chaffeensis Arkansas 100 73
E. ruminantium Palm River 100
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a

Sequences were aligned and percent identity was determined using the program AlignX.

FIG. 6.

FIG. 6.

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Alignment of Anaplasma sp. MSP5 protein sequences. Conserved amino acids are shaded in gray and amino acid positions conserved in three or four of five sequences are shaded in black.

Analysis of protein topology suggested that Anaplasma sp. MSP5 contain a short N-terminal cytoplasmic domain with a single transmembrane region from amino acids 6 to 24. This model is consistent with the secondary and tertiary structures proposed for A. marginale MSP5 (15).

Sequence alignment showed the presence of regions of homology between Anaplasma and Ehrlichia sequences, including critical cysteine residues involved protein conformation (15) (Fig. 6). Furthermore, regions of sequence identity between Anaplasma MSP5 suggest the existence of cross-reactive epitopes between these proteins in the region involved in epitope recognition by monoclonal antibody ANAF16C1 used in the cELISA test (15).

DISCUSSION

After a recent A. marginale prevalence study in Swiss cattle (4), we suspected immunological cross-reactivity between A. marginale and A. phagocytophilum. We now report that animals experimentally infected by A. marginale or A. phagocytophilum produced antibodies that can be detected by both a cELISA using recombinant A. marginale antigen as well as an IFA using A. phagocytophilum morulae on the coated slides. Furthermore, the sequence comparison in silico supported our hypothesis of serologic cross-reactivity between these two agents.

The competitive ELISA used in our investigations is based on a monoclonal antibody directed against A. marginale MSP5. Therefore, it can be concluded that the epitope recognized by this antibody must also be present in the representatives of the group A. phagocytophilum comb. nov. used in the present study (isolates from Switzerland, Great Britain, and Wisconsin). We suspect that the immunological cross-reactivity is not restricted to MSP5 but might include also epitopes in other proteins of A. marginale and A. phagocytophilum. Sequence homologies have also been shown for the MSP4 gene of the two pathogens (3).

Alternatively to the postulated cross-reactivity, accidental cotransmission of A. marginale with A. phagocytophilum or preinfection of experimental animals had to be suspected. However, the experimentally infected cattle originated from and were kept in an A. phagocytophilum-free region (Oklahoma). In contrast to the northeastern and northwestern United States or Europe, in Oklahoma the early-stage tick vectors (Ixodes scapularis) feed on lizards and snakes that do not become infected with A. phagocytophilum.

The sheep examined in the present study originated from and were kept in Great Britain, free of A. marginale and A. ovis. And finally, the Brown Swiss cattle came from tick-free regions of Switzerland and samples collected from these animals tested A. marginale negative upon microscopical examination of blood smears before and after experimental infection. Therefore, the probability of concurrent A. marginale infection in the experimentally infected sheep and cattle seems low. Coinfection in horses is very unlikely, as A. marginale has never been reported to infect this species. Therefore, the observation that antibodies in horses induced by A. phagocytophilum infection react with the same band of 18 kDa as a MAb to A. marginale MSP5 further supports the occurrence of immunological cross-reactivity between the MSP5 proteins of these Anaplasma species. It cannot be completely excluded that antibodies to the 18-kDa protein were induced by the HL60 cells in which the A. phagocytophilum agent had been grown. However, although no control was done using serum from a horse injected with the uninfected HL60 cells, this appears to be unlikely, as the horses were infected by intravenous injection of 106 HL60 cells only. One million HL60 cells contain less than an estimated amount of 30 μg of protein and in the absence of an adjuvant this is hardly enough to induce antibodies against cellular components.

Our observations have important implications for the A. marginale and A. phagocytophilum serotesting. Positive results from serological A. marginale or A. phagocytophilum tests may result from infection with either the respective agent or a cross-reactive pathogen. This is especially important in domestic and wild ruminants that are susceptible to both agents, such as cattle, and especially in regions where both agents might be present. In these cases, we recommend very careful interpretation of the results obtained by the cELISA. A negative result suggests absence of A. marginale infection or a transient period with a very low A. marginale load. A positive result can be caused by A. marginale or A. phagocytophilum infection. Differentiation can be attempted by testing for antibodies to A. phagocytophilum. If the latter test is negative, the animal is most likely infected by A. marginale, A. centrale, or A. ovis. If the A. phagocytophilum test yields also a positive result, further differentiation should be attempted, e.g., by PCR testing of whole blood samples for the respective agents.

Knowledge of the reported cross-reactivity may prove useful for diagnostic of equine and canine samples. The cELISA could be considered for the detection of A. phagocytophilum infection in horses and dogs. However, first an evaluation needs be performed in order to define the species-specific baseline.

In view of the still limited information on nucleotide sequences and antigenic structures of many infectious agents and the ongoing/recent reorganization of the genus Anaplasma, the cross-reactivity observed between A. marginale and A. phagocytophilum may also be expected between other agents of this genus. This should be considered in any positive test where the nature of the infectious agent is unknown.

In conclusion, the immunologic cross-reactivity between A. marginale and A. phagocytophilum reported herein restricts the diagnostic efficacy of the A. marginale cELISA and the A. phagocytophilum IFA especially in species that are susceptible to both pathogens. Serodiagnosis may be considered indicative for a certain infection if only one of the two assays tests positive. If a sample tests positive in both assays, the nature of the infection must be further investigated, e.g., by PCR.

Acknowledgments

This study was funded by a grant from the Federal Veterinary Office. Laboratory work was performed using the logistics of the Center for Clinical Studies at the Vetsuisse Faculty of the University of Zurich. R.H.-L. is the recipient of a professorship by the Swiss National Science Foundation (PP00B-102866).

We are indebted to J. Schmidt for support. The expert technical support by E. Rogg, E. Rhiner, M. Nussbaumer, T. Meili-Prodan, and E. Gönczi is acknowledged. We thank C. Juhls, B. Willi, and M. A. Gomes Keller for helpful discussions. Further thanks go to M. Malanga, A. Metzler, A. Hehl, and A. Kipar.

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