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Clin Diagn Lab Immunol. May 2002; 9(3): 658–668.
PMCID: PMC119989

Infection of Tick Cells and Bovine Erythrocytes with One Genotype of the Intracellular Ehrlichia Anaplasma marginale Excludes Infection with Other Genotypes

Abstract

Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, is endemic in several areas of the United States. Many geographic isolates of A. marginale that occur in the United States are characterized by the major surface protein 1a, which varies in sequence and molecular weight due to different numbers of tandem repeats of 28 or 29 amino acids. Recent studies (G. H. Palmer, F. R. Rurangirwa, and T. F. McElwain, J. Clin. Microbiol. 39:631-635, 2001) of an A. marginale-infected herd of cattle in an area of endemicity demonstrated that multiple msp1α genotypes were present but that only one genotype was found per individual bovine. These findings suggested that infection of cattle with other genotypes was excluded. The present study was undertaken to confirm the phenomenon of infection exclusion of A. marginale genotypes in infected bovine erythrocytes and cultured tick cells. Two tick-transmissible isolates of A. marginale, one from Virginia and one from Oklahoma, were used for these studies. In two separate trials, cattle inoculated with equal doses of the two isolates developed infection with only one genotype. Tick cell cultures inoculated with equal doses of the two isolates became infected with only the Virginia isolate of A. marginale. When cultures were inoculated with different ratios of the Oklahoma and Virginia isolates of A. marginale, the isolate inoculated in the higher ratio became established and excluded infection with the other. When cultures with established infections of one isolate were subsequently infected with the other, only the established isolate was detected. We documented infection exclusion during initial infection in cell culture by labeling each isolate with a different fluorescent dye. After 2 days in culture, only a single isolate was detected per cell by fluorescence microscopy. Finally, when Anaplasma ovis infections were established in cultures that were subsequently inoculated with the Virginia or Oklahoma isolate of A. marginale, A. marginale infection was excluded. These studies confirm that infection exclusion occurs with A. marginale in bovine erythrocytes and tick cells, resulting in the establishment of only one genotype, and appears to be the first report of infection exclusion for Anaplasma and Ehrlichia species.

Anaplasmosis, a tick-borne disease of cattle caused by the obligate intraerythrocytic bacterium Anaplasma marginale (Rickettsiales: Anaplasmataceae) is endemic in many tropical and subtropical areas, including several areas of the United States. Several geographic isolates of A. marginale, which differ in biology, morphology, sequence, and antigenic characteristics, have been identified in the United States (1, 11, 12, 37, 40). Feeding ticks effect biological transmission of A. marginale, whereas mechanical transmission occurs when infected blood is transferred to susceptible animals by biting flies or by blood-contaminated fomites. Cattle that recover from acute infection remain persistently infected and serve as reservoirs for mechanical transmission and infection of ticks (reviewed in reference 15).

A. marginale multiplies only within membrane-bound inclusions in the cytoplasms of host cells. The only known site of development of A. marginale in cattle is within erythrocytes (35), whereas A. marginale develops in several tissues in ticks and undergoes a development cycle that is complex and coordinated with the tick feeding cycle (16, 20-22).

MSP1a is one of six major surface proteins (MSPs) that have been described for A. marginale from bovine erythrocytes. MSP1a is encoded by a single gene, msp1α, which is conserved during the multiplication of the parasite in cattle and ticks, therefore resulting in a stable genetic marker of A. marginale geographic isolates (2, 3, 7). The molecular weight of MSP1a differs among geographic isolates because of a variable number of tandem repeats of 28 or 29 amino acids (1, 11).

Recently, A. marginale was propagated in continuous culture in a cell line, IDE8, derived from embryos of the tick Ixodes scapularis (6, 27). A. marginale organisms harvested from cell culture were infective for both cattle and ticks (6, 27). The MSPs characterized for A. marginale from bovine erythrocytes were found to be conserved on the cell culture-derived organisms, and the antigenic composition of A. marginale remained the same after successive passage in cell culture (3) or after passage through ticks (3, 4). The A. marginale isolate antigenic identity, as determined by the sequence and molecular weight of MSP1a, was retained in culture (3, 6, 7, 27).

A recent report by Palmer et al. (32) documented genetic heterogeneity in the structure of msp1α sequences recovered from infected animals in a herd of cattle in eastern Oregon, where A. marginale is endemic. However, single msp1α genotypes were identified in individual cattle that were naturally or experimentally infected and sampled at different stages of infection (32). These findings suggest that exclusion of other A. marginale isolates may occur in infected cattle (14).

Infection (superinfection) exclusion or homologous interference is a phenomenon that was first described to occur in bacteriophages (29, 38) and later confirmed in animal viruses (9, 18, 24). Bacterial, vertebrate, or invertebrate cells infected with one virus did not become productively infected with the same or a closely related virus (18). Infection exclusion has been documented for Rickettsia spp. (8). The mechanism of infection exclusion is not well understood, and it has been suggested that a number of factors, including competition for host cell receptors or intracellular host factors, the production of interferon or interferon-like substances by the infected host cell, the production of defective interfering viral genomes from the first infecting virus, and the production of transacting protease by the first virus, have been posited to contribute to this phenomenon (18).

In this report, we used two tick-transmissible A. marginale isolates from Virginia and Oklahoma to test the hypothesis that exclusion of more than one genotype of A. marginale occurs in bovine erythrocytes and cultured tick cells. We also used a second species of Anaplasma in tick cell culture, Anaplasma ovis, to test whether infection exclusion occurs between two different Anaplasma species.

MATERIALS AND METHODS

Isolates of Anaplasma spp.

The Virginia isolate of A. marginale used for these studies was originally obtained in 1978 from the USDA Animal Disease Research Laboratory, Beltsville, Md., and has been used for tick transmission and cell culture studies in our laboratory (5, 20-22, 27). The Oklahoma isolate of A. marginale was obtained from a naturally infected bovine from Wetumka, Okla., in 1997 (6, 11). Each isolate was inoculated into a splenectomized calf, and blood samples collected at the peak of infection were prepared as stabilates and stored in liquid nitrogen or used to inoculate tick cell monolayers. The Idaho strain of A. ovis (28, 31) was obtained from Guy Palmer (Washington State University, Pullman) and used to infect a splenectomized sheep. Blood samples collected at the peak of infection were also used for preparation of blood stabilates and for inoculation and propagation of A. ovis in cultured IDE8 tick cells.

Coinfection studies in cattle. (i) Cattle.

Four mixed-breed cattle (8 to 10 months old) determined to be free of infection by an A. marginale-specific enzyme-linked immunosorbent assay (ELISA) (36) were used for the coinfection studies. Cattle were housed in the Anaplasmosis Research Barn and cared for by the Oklahoma State University Laboratory Animal Research Unit with the approval of the Institutional Animal Care and Use Committee. Calves experimentally infected with the A. marginale isolates were monitored three times a week by examination of stained blood smears and determination of the packed cell volume. Thin smears were made on glass slides from cattle blood collected by venopuncture in EDTA-treated Vacutainer tubes and stained with Protocol Hema 3 stain (Biochemical Sciences, Inc., Sweedesburg, N.J.). The percentage of infected erythrocytes out of 500 was determined and reported as the percentage of parasitized erythrocytes (PPE). Once infection was detected in blood smears, the calves were monitored daily.

(ii) Trial 1: coinfection of A. marginale in cattle.

Bovine PA444 was inoculated intravenously (i.v.) with 8 ml of the Oklahoma A. marginale stabilate made from blood collected from bovine PA417 with a PPE of 9.3% and 8 ml of the Virginia A. marginale stabilate made from blood collected from bovine PA432 with a PPE of 9.8%. Blood samples (1 ml each) were collected daily during patent acute infection and stored at −70°C until used for PCR analysis for identification of the A. marginale isolate.

(iii) Trial 2: coinfection of A. marginale in cattle.

Three cattle (PA465, PA466, and PA467) were used for trial 2. PA465 was inoculated i.v. with 4 ml of the Virginia A. marginale-infected blood stabilate (PPE, 25.9%) made from blood collected from PA432. PA466 was inoculated i.v. with 4 ml of the Oklahoma A. marginale-infected blood stabilate (PPE, 31.6%) made from blood collected from PA417. PA467 was inoculated i.v. with 4 ml of each of the same Oklahoma and Virginia A. marginale-infected blood stabilates used for inoculation of PA465 and PA466. Blood samples (1 ml each) were collected daily from all cattle during patent infection and stored at −70°C until used for PCR analysis for identification of the A. marginale isolate.

Coinfection studies of A. marginale and A. ovis in cultured tick IDE8 cells. (i) Maintenance of infected and uninfected tick cells.

Monolayers of IDE8 (ATCC CRL 1973) cells, originally derived from embryonic I. scapularis ticks, were maintained at 31°C in L-15B medium supplemented with 5% fetal bovine serum, tryptose phosphate broth, and bovine lipoprotein concentrate (ICN, Irvine, Calif.) as described by Munderloh et al. (26). Monolayers were subcultured at ratios of 1:5 to 1:10 (vol/vol) when monolayers reached a density of approximately 5 × 106 cells/cm2. A. marginale- and A. ovis-infected I. scapularis cells were maintained at 34°C in L-15B medium as described previously (27). The medium was replaced weekly until the desired infection levels were reached. Cultures were routinely harvested when infection levels reached 90 to 100% and detachment of infected host cells was apparent (terminal culture). Culture material was subinoculated onto uninfected tick cells at ratios of 1:5 to 1:10 (vol/vol) in 25-cm2 flasks. The bacterial concentration was determined by comparison with an erythrocyte-based A. marginale standard in an antigen capture ELISA (36). Colony counts in infected tick cell monolayers were made by pelleting a sample of infected cells on a glass slide, staining the samples with Giemsa stain, and counting the number of colony-containing cells by light microscopy.

(ii) Simultaneous infection of tick cells with the Virginia and Oklahoma A. marginale isolates.

In the first trial, a confluent monolayer of IDE8 cells was simultaneously inoculated with 1.4 × 1010 organisms each of the Virginia and Oklahoma isolates of A. marginale. Separate IDE8 monolayers received 1.4 × 1010 organisms of either the Virginia or Oklahoma isolate of A. marginale. Cultures received fresh L-15B medium and were maintained at 34°C as described above. At day 10 postinfection (p.i.), samples were collected from each flask for genotype identification and from flasks receiving one isolate only for determination of bacterial concentration by antigen capture ELISA (36). Infected material from the dual-infection flask was passaged onto an uninfected IDE8 monolayer and maintained as described above. A sample was collected from this flask at day 7 p.i. for genotype identification. In a second trial, IDE8 monolayers were simultaneously inoculated with the cultured Virginia and Oklahoma isolates of A. marginale at different ratios. Monolayers were inoculated, per flask, with 1.4 × 1010 A. marginale organisms of either the Virginia or the Oklahoma isolate alone. Additional monolayers received the same infection dose but of both the Virginia and Oklahoma isolates at ratios of 1:3, 2:2, and 3:1. Samples were collected from each flask at day 13 p.i. for isolate identification. Each experimental treatment was done in duplicate.

(iii) Superinfection of tick cells with the Virginia and Oklahoma isolates of A. marginale, followed by heterologous infection.

Monolayers of IDE8 tick cells were inoculated with either Virginia (flask 1) or Oklahoma (flask 2) isolate at a greatly increased density of 1.4 × 1011 organisms per flask. After a 60-min incubation period, the inoculum was removed and the monolayers were washed with fresh L-15B medium. At 3 days p.i., flask 1 was inoculated with the Oklahoma isolate at a dose of 1.4 × 1010 organisms. At 5 days p.i., flask 2 was inoculated with a similar dose of the Virginia isolate. Samples were collected from flasks 1 and 2 prior to infection with the second isolate of A. marginale, and colony counts were done by light microscopy. Flasks were maintained as described above until monolayers began detaching, and samples were collected for isolate identification. Each experimental treatment was done in duplicate.

(iv) Differential live staining of the Virginia and Oklahoma A. marginale isolates in tick cells.

IDE8 monolayers were infected with 1.4 × 1011 organisms of either the Virginia or the Oklahoma isolate and exposed to the inoculum for 60 min. Inoculum was removed, and the monolayers were washed in L-15B medium. After 24 h, fresh medium containing a 50-μg/ml concentration of CellTracker Green BODIPY dye (catalog number C-2102; Molecular Probes, Eugene, Oreg.) was added to the culture of the Virginia A. marginale isolate. Medium containing a 1-μg/ml concentration of CellTracker Orange CMTMR dye (catalog number C-2927; Molecular Probes) was added to the Oklahoma isolate culture. After 24 h incubation samples were collected from both cultures to verify the staining by each dye and the level of infection. Cell suspensions were dried on glass slides, washed three times in phosphate-buffered saline, fixed for 15 min in 2% glutaraldehyde (in 0.1 M sodium cacodylate buffer), and rinsed in phosphate-buffered saline. Samples were mounted with Mowiol-glycerol-1,4-diazabicyclo-(2,2,2)-octane (DAPCO; Sigma, St. Louis, Mo.) as the mounting medium (17) and with glass coverslips and examined under a Leica DM LB fluorescence microscope attached to a Spotcam (Diagnostic Inc., Sterling Heights, Mich.) ISA-bus 12-bit/channel RGB Peltier-cooled charge-coupled device camera. Cells labeled with CellTracker Green BODIPY and CellTracker Orange CMTMR were identified by using an A filter (450 to 490 nm band pass, long pass at 515 nm; Leica I3) or B filter (515 to 560 nm band pass, dichromatic at 580 nm, long pass at 590 nm; Leica N21), respectively. The stained cultures of the Virginia and Oklahoma isolates of A. marginale were then harvested and combined, and tick cells were mechanically disrupted by vigorously pipetting with a 6-ml syringe and a 19-gauge needle to release bacteria. Culture material containing the labeled Virginia and Oklahoma isolates was held for 15 min to allow intact host cells to settle, and 5 ml of supernatant was added to an uninfected monolayer of IDE8 cells. Samples were collected at 48 h p.i. and processed for examination by fluorescence microscopy as previously described.

(v) Infection of tick cells with A. ovis followed by infection with the Virginia and Oklahoma isolates of A. marginale.

Monolayers of IDE8 cells were infected with terminal cultures of A. ovis from one T-25 flask and exposed to the inoculum for 60 min. Inoculum was removed, and monolayers were washed in L-15B medium. At day 1 p.i., samples were collected for A. ovis colony counts by light microscopy and flasks were then inoculated with 2.7 × 1010 organisms of either the Virginia or the Oklahoma isolate of A. marginale. At day 10 p.i. with A. ovis, cultures were harvested for isolate identification. Each experimental treatment was done in duplicate.

Analysis of Anaplasma isolate identity. (i) PCR.

A. marginale cells from bovine erythrocytes or infected tick cell cultures were characterized by PvuII restriction analysis of the msp1α gene amplified by PCR. A. marginale DNA was extracted from 0.5-ml stored blood samples containing infected bovine erythrocytes collected during different stages of acute infection or from infected IDE8 cells by using 500 μl of Tri Reagent (Sigma) as described in the manufacturer's recommendations. The msp1α gene, coding for MSP1a and containing 5′ and 3′ regulatory sequences, was amplified from 100 ng of DNA by PCR (13) with 10 pmol of each primer, MSP1aP (5′-GCATTACAACGCAACGCTTGAG-3′) and MSP1a3 (5′-GCTTTACGCCGCCGCCTGCGCC-3′), in a 50-μl-volume mixture (1.5 mM MgSO4, 0.2 mM deoxynucleoside triphosphate, 1× avian myeloblastosis virus reverse transcriptase-Thermus flavus reaction buffer, 5 U of Thermus flavus DNA polymerase) using the Access reverse transcriptase-PCR system(Promega, Madison, Wis.. Reactions were performed in an automated DNA thermocycler (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 2.5 min at 68°C. The program ended with the reaction mixtures being stored at 4°C. PCR products were electrophoresed on 1% agarose gels to check the sizes of the amplified fragments. Control samples with no DNA added were included in the experiments. Ten microliters of the PCR product were mixed with 0.5 μl (5 U) of PvuII (Gibco BRL, Gaithersburg, Md.) and incubated for 30 min at 37°C. The reaction mixture was analyzed in a 1% ethidium bromide (EtBr)-stained agarose gel. The gene coding for MSP1a in A. ovis has not been cloned, but the msp4 gene has a sequence distinguishable from that of A. marginale isolates (J. de la Fuente, unpublished results). Therefore, in experiments with A. ovis, the msp4 gene was amplified by PCR and sequenced as previously reported (11).

(ii) Western blotting.

Expression of MSP1a in infected IDE8 cells was analyzed by Western blotting. Total proteins were extracted from infected cells with 500 μl of Tri Reagent (Sigma) in accordance with the manufacturer's recommendations after DNA precipitation. Proteins (10 μg/well) were separated by sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis (23) and transferred to a nitrocellulose membrane for 1 h at a 60-mA constant current in a Hoefer TE 70 (Amersham Pharmacia) semidry transfer unit. The membrane was blocked with 5% skim milk for 1 h at room temperature. The anti-MSP1a mouse monoclonal antibody Ana15D2 (25) was used. Antibodies were diluted to a concentration of 10 μg/ml in 3% bovine serum albumin in Tris-buffered saline (TBS). The membrane was incubated with the antibodies for 1 h at room temperature and washed three times with TBS. The membrane was then incubated with a goat anti-mouse alkaline phosphatase conjugate (KPL, Gaithersburg, Md.) diluted to 1:5,000 in TBS. The membrane was washed three times with TBS and finally developed with BCIP-NBT (5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium; Sigma) for 20 min.

(iii) Light microscopy studies.

Cultured tick cells were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and pelleted in Eppendorf tubes with a microcentrifuge at 10,000 × g for 5 min. The supernatant was removed, replaced with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), and repelleted. The cell pellets were then postfixed in 2% cacodylate-buffered osmium tetroxide (pH 7.4), dehydrated in a graded series of ethanol, and embedded in epoxy resin by the method of Kocan et al. (19). Thick sections (1.0 μm) were cut and stained with Mallory's stain (33) for observation with a light microscope. Light micrograph images were recorded with a Leica DM LB microscope with the Spotcam camera system described above.

Nucleotide sequence accession number.

The A. ovis msp4 coding sequence was deposited in the GenBank under accession number AF393742.

RESULTS

Validation of the assay system.

We based our identification of the A. marginale isolate in blood and tick cell culture samples on the sequence of the msp1α gene, which varies among different geographic isolates (11). The msp1α gene of the Virginia isolate has two tandem repeats, whereas the gene of the Oklahoma isolate has three tandem repeats (11) and varies in the number of PvuII sites (positions 69, 876, and 712 with respect to the translation initiation codon) (11). PvuII digestion of the msp1α gene PCR therefore resulted in distinguishable patterns for the Virginia and Oklahoma isolates (Fig. (Fig.1).1). The Virginia isolate gave two bands, of 1,071 and 997 bp, while the Oklahoma isolate gave three bands, of 997, 849, and 313 bp (Fig. (Fig.1).1). We demonstrated that both the Virginia and Oklahoma A. marginale isolates could be identified simultaneously by identification of the Virginia and Oklahoma isolate msp1α genes after PCR and PvuII digestion of amplification products in samples in which bovine blood or cultured tick cells infected with the two isolates were mixed at different ratios (4:0, 3:1, and 2:2) prior to DNA extraction and analysis (Fig. (Fig.11 and results not shown). These experiments confirmed that the two isolates could be detected by our procedure if they were present in the sample.

FIG. 1.
Validation of the assay system. Identification of the msp1α genotype of the Virginia (VaAM) and Oklahoma (OkAM) A. marginale isolates after PCR and PvuII digestion of amplification products in samples in which cultured tick cells infected with ...

Exclusion of A. marginale isolates in cattle.

When cattle were infected with equal amounts of erythrocytes infected with either the Virginia or Oklahoma isolate, only one of the isolates established infection (Fig. (Fig.22 and and3B).3B). In the first trial, the A. marginale Virginia isolate was the only isolate detected in bovine PA444 at the early stages of infection (1.6% infected erythrocytes) and thereafter until the end of the acute phase of infection (Fig. 2A and B). The A. marginale isolate from Virginia was also the only isolate detected in salivary glands of ticks that acquired infection after feeding on PA444 during acute infection (data not shown). In the second trial, the A. marginale isolate from Oklahoma was the only isolate detected in bovine PA467 (Fig. (Fig.3B)3B) that was inoculated with equal amounts of blood stabilate infected with the Virginia or Oklahoma isolate. The Oklahoma isolate that established infection was detected at very early stages of infection (0.6% infected erythrocytes) (Fig. (Fig.3).3). In this second trial, blood stabilates of each isolate caused infection when inoculated into separate susceptible cattle (bovine PA465 for the Virginia isolate and bovine PA466 for the Oklahoma isolate) (Fig. (Fig.3A3A).

FIG. 2.
Trial 1: exclusion of A. marginale isolates in cattle. Only the Virginia A. marginale isolate was present in bovine PA444 inoculated with equal amounts of erythrocytes infected with the Oklahoma or Virginia A. marginale isolate. DNA was extracted, and ...
FIG. 3.
Trial 2: exclusion of A. marginale isolates in cattle. (A) Bovines PA465, PA466, and PA467 were inoculated with erythrocytes infected with the Virginia (VaAM) or Oklahoma (OkAM) isolate or both (VaAM+OkAM). (B) DNA was extracted, and the msp1α ...

Exclusion of A. marginale isolates in tick cells.

Tick cell cultures that were infected simultaneously with 1.4 × 1010 A. marginale organisms of each of the Virginia and Oklahoma isolates and analyzed 10 days p.i. contained only the Virginia isolate, and this was the only isolate detected 7 days after subpassage of the cultures (Fig. (Fig.4B).4B). When cultures were infected with each individual isolate, the culture containing the Virginia isolate contained twice as many organisms as the Oklahoma isolate-infected culture, as detected by antigen detection ELISA (Fig. (Fig.4A4A).

FIG. 4.
Exclusion of A. marginale isolates in tick cells infected with equal amounts of the Virginia (VaAM) and Oklahoma (OkAM) A. marginale isolates. Only the Virginia A. marginale isolate was present in IDE8 cells inoculated with 1.4 × 1010 organisms ...

Tick cell cultures were inoculated with different infection ratios of the Virginia and Oklahoma A. marginale isolates. When infected cultures were analyzed for msp1α DNA by PCR, the Virginia isolate established the predominant infection and excluded the Oklahoma isolate at infection ratios of 3:1 and 2:2 (Virginia/Oklahoma A. marginale isolates), whereas at a 1:3 infection ratio of the Virginia/Oklahoma isolates, the Oklahoma isolate established the predominant infection and excluded the Virginia isolate (Fig. (Fig.5A).5A). When the same cultures were analyzed for MSP1a protein by Western blotting, the Virginia and Oklahoma isolates established the predominant infection at infection ratios of 3:1 and 1:3, respectively (Fig. (Fig.5B).5B). However, at a 2:2 infection ratio, bands corresponding in size to the Virginia and Oklahoma isolates were observed (Fig. (Fig.5B).5B). Although the results were essentially similar at the DNA and protein levels, the relative amounts of the Virginia and Oklahoma isolates in the sample infected with equal amounts of both isolates could have differed as the result of differences in the DNA and protein half-lives.

FIG. 5.
Exclusion of A. marginale isolates in tick cells infected with different ratios of the Virginia (VaAM) and Oklahoma (OkAM) isolates. IDE8 cells were inoculated with 1.4 × 1010 organisms per flask and cultured for 13 days. The Virginia isolate ...

In a third series of experiments, when cell cultures that were first allowed to develop established infections with either the Virginia or Oklahoma isolate(Fig. isolate(Fig.6A6A and B) were subsequently infected with the heterologous isolate and tested at 10 to 12 days following the second infection, each culture was infected with only the isolate used for establishing the initial infection (Fig. (Fig.6C6C).

FIG. 6.FIG. 6.
Exclusion of A. marginale isolates in tick cells infected with the Virginia or Oklahoma A. marginale isolates. Only the isolate used for primary infection was present in IDE8 tick cells infected with 1.4 × 1011 organisms per flask of either the ...

The Virginia and Oklahoma isolates, labeled with the green and orange fluorescent CellTracker dyes (Molecular Probes C-2102 and C-2927, respectively), were used to coinfect cultured tick cells in order to document initial infection events (Fig. (Fig.7A7A through C). At 2 days p.i., cells infected with individual isolates were seen (Fig. (Fig.7D7D through F), although at an approximately 2.5-fold higher level for cells infected with the Virginia isolate of A. marginale. In monolayers infected with both labeled isolates, very few (<1%) cells with possible dual infections were seen (Fig. (Fig.7G7G through I).

FIG. 7.
Infection of IDE8 tick cells with differentially labeled Virginia (VaAM) and Oklahoma (OkAM) A. marginale isolates in parasites. The Virginia and Oklahoma isolates were labeled with CellTracker Green BODIPY and Orange CMTMR dyes (Molecular Probes), respectively. ...

Exclusion of A. marginale by A. ovis infection in tick cells.

To assess the capacity of a second Anaplasma species, A. ovis, to exclude A. marginale infection, tick cell cultures with established infections of A. ovis (Fig. (Fig.8A8A and B) were infected with the Virginia or Oklahoma isolate of A. marginale. After 12 days of infection with the Virginia or Oklahoma isolate, only A. ovis was detected in cell monolayers, as demonstrated by the sequence of the amplified msp1α gene (Fig. (Fig.8C8C).

FIG. 8.FIG. 8.
Exclusion of A. marginale by A. ovis infection in tick cells. Only the sequence corresponding to A. ovis msp4 was present in IDE8 cells infected with A. ovis and then reinfected with the Virginia or Oklahoma isolate. (A and B) Light micrographs of 1-μm-thick ...

DISCUSSION

These studies were undertaken to test the hypothesis that a mechanism of infection exclusion occurs in host cells infected with A. marginale that results in the establishment of a single genotype and that excludes infection with other genotypes. In two trials, we demonstrated that cattle experimentally inoculated with two A. marginale isolates became infected with only one isolate. We then extended these studies and demonstrated that infection exclusion of A. marginale also occurs in cultured tick cells, resulting in the establishment of only one isolate per culture. When cell cultures were inoculated simultaneously with equal numbers of two A. marginale isolates, a single isolate (the Virginia isolate) became established in the culture. The Virginia A. marginale isolate may have become established in these cultures because it multiplied more rapidly in tick cells than did the Oklahoma isolate; the Virginia isolate grew twice as fast in control cultures as the Oklahoma isolate. However, when cell cultures were infected with various ratios of the Virginia and Oklahoma A. marginale isolates, the isolate inoculated at the higher ratio became established and excluded infection with the other. Infection exclusion occurred in cultures with established infections of one isolate that were subsequently inoculated with the heterologous isolate. Therefore, the results of these studies provided evidence that infection of bovine erythrocytes and cultured tick cells with one A. marginale genotype excluded infection with a second genotype.

We subsequently demonstrated that infection exclusion also occurred with a second Anaplasma species, A. ovis. While A. ovis infects different hosts (sheep and goats), it is genetically and antigenically related to A. marginale (28, 31). A. ovis and A. marginale MSP4s have 94% amino acid identity and serve for isolate identification (de la Fuente, unpublished). However, A. ovis MSP1a appears to have little homology to A. marginale MSP1a, because we were unable to amplify the msp1α gene in A. ovis by PCR using primers designed for the A. marginale gene (de la Fuente, unpublished). A. ovis-infected tick cultures proved to exclude subsequent infection by the Virginia or Oklahoma A. marginale isolates. These data suggest that the mechanism of infection exclusion is conserved among Anaplasma species.

Infection of tick IDE8 cells with fluorescence-labeled Virginia or Oklahoma A. marginale isolate cells allowed us to document early infections of the two isolates, and we demonstrated individual cells in the same culture that were infected with one or the other genotype. We cannot guarantee that all these cells were infected de novo, because infected cells may have been introduced with the inoculum. However, although both labeled isolates were detected in initial samples, at 2 days postinoculation with both isolates, most cells were infected with the Virginia isolate. Therefore, while individual cells in the culture may become infected with either of the genotypes, the mechanism of infection exclusion appears to occur early in infection, resulting in the establishment of only one genotype. Similar observations have been reported for animal viruses (18).

Based on these results and the results of Palmer et al. (32), tick vectors would not be expected to become infected with more than one A. marginale genotype simply because only one genotype would occur in the bovine used for tick acquisition feeding. However, infection exclusion may also occur in ticks, allowing for infection of individual ticks with only a single genotype. Although multiple genotype infections per tick are not likely to occur in nature because of the lack of a blood source infected with multiple isolates, it would be interesting to test whether the infection exclusion mechanism also occurs in ticks by allowing ticks to feed on a pool of blood containing equal amounts of two A. marginale isolates in an artificial tick feeding system as described by Waladde et al. (39).

The findings reported herein were similar to the results of Palmer et al. (32) from studies on a persistently infected reservoir herd within a region in Oregon where A. marginale is endemic. In herds in an area of A. marginale endemicity where more than one A. marginale genotype was detected, individual cattle were found to be infected with a single genotype. This mechanism of infection exclusion, which allows for infection of individual cattle with one genotype, would reduce competition and favor the survival of individual A. marginale genotypes.

Infection exclusion has been reported previously for bacteria of the genus Rickettsia only (8). Burgdorfer et al. documented exclusion of Rickettsia rickettsii from Dermacentor andersoni ovaries by the nonpathogenic Rickettsia peacockii (8). In a different study, Ridderhof and Barnes (34) demonstrated that infection exclusion did not occur with the intracellular bacterium Chlamydia trachomatis. In their experiments, more than 88% of HeLa cells were coinfected with two different C. trachomatis serovars (34). Phylogenetic studies have demonstrated that Chlamydiae are not closely related to Anaplasmataceae and may have evolved different mechanisms of survival that are host or host cell associated, whereas Rickettsieae are closely related to Anaplasmataceae (10, 30).

The epidemiology of anaplasmosis has been poorly understood. The mechanism of infection exclusion in A. marginale resulting in one genotype per animal would contribute to our understanding of anaplasmosis epidemiology. This mechanism would contribute to the geographic localization of A. marginale isolates with distinctive genotypes and antigenic characteristics (11). If cattle infected with another genotype were introduced into a herd in which A. marginale infection was endemic, these genotypes would be maintained and most likely also become endemic if they were transmitted to susceptible cattle. Both persistently infected cattle and ticks could serve as reservoirs of the introduced genotype.

Infection exclusion may contribute to the success of the live Anaplasma centrale vaccine used in Australia, Israel, Latin America, and Africa in which development of persistent infections with this less pathogenic strain prevents clinical anaplasmosis. If infection exclusion between Anaplasma spp. occurs, the A. centrale infections would prevent cattle from being infected with A. marginale, as well as provide immunoprotection due to persistent infection. The protection afforded to cattle that are persistently infected with A. marginale, therefore, may be due to infection exclusion as well as to immunologic mechanisms.

The mechanism of infection exclusion which results in one genotype infection per animal may constrain the mobility and establishment of multiple A. marginale geographic isolates per geographic area and supports the need for novel vaccine preparations that are cross protective against multiple genotypes that may be introduced from other areas via shipment of cattle.

Acknowledgments

This research was supported by project no. 1669 of the Oklahoma Agricultural Experiment Station, the Endowed Chair for Food Animal Research (College of Veterinary Medicine, Oklahoma State University; to K. M. Kocan), NIH Centers for Biomedical Research Excellence through a subcontract to J. de la Fuente from the Oklahoma Medical Research Foundation, and the Oklahoma Center for the Advancement of Science and Technology, Applied Research Grant AR00(1)-001.

We acknowledge G. H. Palmer (Washington State University, Pullman) for providing the A. ovis isolate, A. F. Barbet (University of Florida) for providing the anti-MSP1a mouse monoclonal antibody Ana15D2, Dollie Clawson and Brian McEwen (Department of Veterinary Pathobiology, Oklahoma State University) for technical assistance, and Sue Ann Hudiburg and Janet J. Rogers (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University) for oligonucleotide synthesis and DNA sequencing, respectively.

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