INTRODUCTION

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Anaplasmosis is a tick-borne disease of cattle caused by the rickettsia Anaplasma marginale. Following transmission to cattle, animals develop an intraerythrocytic rickettsemia accompanied by severe anemia, weight loss, abortion, and sometimes death (1). Recovered cattle maintain low to undetectable levels of infection and become carriers of the disease, thus acting as reservoirs of infection for transmission to other animals (32). The disease is devastating to the production, movement, and utilization of cattle worldwide, including many areas of the United States (17).

Control measures for anaplasmosis have included various combinations of vector control, vaccination, and administration of antibiotics for treatment or disease prevention. Each method has associated drawbacks which have limited their use, and consequently, severe losses continue (24). Vaccination with live or killed organisms has been practiced for more than 75 years but has relied on infected bovine erythrocytes as a source of A. marginale antigen. Blood-derived vaccines have been expensive due to requirements for disease-free cattle and the difficulty of purifying A. marginale from erythrocyte components to avoid induction of isoantibodies (9). In addition, vaccine failures have been reported on heterologous challenge with various geographic isolates (24).

In bovine erythrocytes, A. marginale occurs within small membrane-bound inclusions containing four to six rickettsiae. In contrast, large colonies that contain many organisms develop in naturally infected ticks. A developmental cycle begins in midgut cells, with subsequent infection of a variety of tick tissues including the salivary glands, from where the rickettsiae are transmitted to cattle. Within each site of development in ticks, A. marginale undergoes a development cycle involving two stages. A reticulated or vegetative form that divides by binary fission is observed first within the colonies and subsequently changes into the infective dense form (14-16).

Recently, A. marginale has been grown in continuous culture in a cell line, IDE8, derived from embryos of the tick Ixodes scapularis (20). The culture-derived A. marginale remained infective for ticks and cattle after 3 years of continuous passage in the IDE8 cells. Colonies of A. marginale in tick cell culture were morphologically similar to those observed in ticks (8, 20). A. marginale electron-dense forms in the inoculum adhered to and infected cultured tick cells and transformed into reticulated forms that divided by binary fission. After formation of large colonies of organisms, the rickettsiae transformed into the dense form, which was released and was infective for other cells. Because the cell culture-derived A. marginale organisms were morphologically similar to organisms in naturally infected ticks, it was important to determine which surface antigens, if any, were conserved among the cell culture-, bovine erythrocyte-, and tick salivary-gland-derived A. marginale organisms in order to evaluate the potential of using cultured A. marginale for future research and for development of improved vaccines and diagnostic tests.

Surface antigens major surface protein 1 (MSP1) through MSP5 of erythrocytic-stage A. marginale have been extensively characterized in recent years, and the gene sequences, recombinant protein analogs, monospecific and monoclonal antibodies (MAbs), information on isolate variability, and potential value in diagnostic assays and vaccines are available (3-6, 10, 13, 18, 21, 22, 25, 28, 31, 34). A previous investigation suggested that antibodies against A. marginale from midgut of the tick Dermacentor andersoni recognize MSP1, MSP2, and MSP3 of erythrocyte-stage A. marginale, indicating that some epitopes are shared (29). MSP5 is present on both tick salivary-gland and bovine erythrocyte stages of A. marginale (13). In this study, we directly compared MSP1 through MSP5 of the erythrocyte stage with homologous proteins present on cell culture- and tick salivary-gland-derived A. marginale.

	MATERIALS AND METHODS

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Sources of rickettsiae. (i) Cell culture-derived A. marginale. The Virginia isolate of A. marginale was cultivated in the IDE8 cell line isolated from embryonated eggs of I. scapularis, as described previously (20). Cultures infected with A. marginale and containing >90% infected cells were harvested and frozen at 70°C in phosphate-buffered saline. Thawed cultures were solubilized in sample buffer for gel analysis. Uninfected IDE8 cells were processed similarly and used as negative controls in immunoblots.

(ii) Erythrocyte-derived A. marginale. Erythrocytic A. marginale organisms (Florida and Virginia isolates) were purified from blood (30) collected at ascending or peak rickettsemia in a splenectomized calf that was experimentally infected with cryopreserved A. marginale.

(iii) Tick salivary-gland-derived A. marginale. Infected tick salivary glands were collected from male D. andersoni ticks that were infected with the Virginia isolate of A. marginale as adults as described previously (15, 16). Tick salivary glands were dissected from infected ticks that were allowed to feed on susceptible calves in order to cause development of A. marginale in tick salivary glands. Salivary-gland infections were confirmed by electron microscopy and by use of an Anaplasma-specific DNA probe. Infected glands were stored at 70°C in phosphate-buffered saline until they were thawed and solubilized in sample buffer for gel electrophoresis. Uninfected salivary glands were dissected from uninfected, fed ticks and processed in a similar manner as the infected ones and used as negative controls in immunoblots.

MAbs and polyclonal sera. MAb 22B1 (anti-MSP1a [26]), rabbit 874 (monospecific antibody to affinity-purified MSP1 [7]), rabbit 883 and 884 (monospecific antibodies to affinity-purified MSP2 [31]), rabbit 955 (monospecific antibody to affinity-purified MSP3 [19]), MAb 75C2 (anti-MSP3 [19]), rabbit C (monospecific antibody to affinity-purified MSP4 [22]), MAb ANAF16C1 (anti-MSP5 [13]), and rabbit 821 (antibody to purified erythrocytic-stage A. marginale [30]) were all prepared previously during characterization of MSPs of bovine erythrocyte-stage A. marginale. All these antibodies were prepared against MSPs of the Florida isolate of A. marginale. Antiserum was also prepared from a spleen-intact cow infected with a cryopreserved blood stabilate of the Virginia isolate of A. marginale (PA188). Negative control antibodies included normal rabbit serum and preimmune serum, normal bovine serum, and MAbs Tryp2B1 and Tryp1E1 with specificity for a variable surface glycoprotein of Trypanosoma brucei.

Protein analysis. A. marginale-infected tick cells from in vitro culture or salivary glands, partially purified erythrocytic-stage rickettsiae, or the respective uninfected control preparations were solubilized in sample buffer for one (2% sodium dodecyl sulfate [SDS], 2.5% 2-mercaptoethanol, 25 mM Tris [pH 6.8], 15% glycerol, 0.002% bromophenol blue)- or two (9 M urea, 1% Triton X-100, 2.5% 2-mercaptoethanol, 0.8% 5/7 ampholyte, 0.2% 3/10 ampholyte)-dimensional gel electrophoresis. Proteins were separated on 7.5 to 17.5% polyacrylamide-SDS slab gels (one dimension) or on isoelectrofocusing tube gels followed by 7.5 to 17.5% polyacrylamide-SDS slab gels (two dimensions [2]) and immunoblotted to nitrocellulose. Nitrocellulose membranes were reacted with the above-described primary antisera, and reactive proteins were visualized by reaction with horseradish peroxidase-conjugated protein G and SuperSignal or SuperSignal Ultra substrate (Pierce, Rockford, Ill.) or alkaline phosphatase-conjugated secondary antibody and CDP-Star substrate (Tropix, Bedford, Mass.) and chemiluminescence as described by the manufacturers.

	RESULTS

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Initial immunoblots suggested that MSPs previously identified and characterized on erythrocyte-stage A. marginale were also present on A. marginale cultured in vitro. In Fig. 1, a positive reaction was obtained on cultured A. marginale with specific antibodies against MSP1a, MSP2, and MSP5 and also with an antiserum raised against purified erythrocyte-stage rickettsiae. No reactivity was observed with negative control antibodies or with MSP-specific antibodies against the uninfected tick cell line. Subsequently, the presence of each MSP was examined in more detail.

View larger version (59K): [in this window] [in a new window]   FIG. 1.   Antibodies to A. marginale erythrocyte-stage surface proteins react with A. marginale grown in vitro in tick cell culture. Proteins of A. marginale-infected (A) or uninfected (B) tick cells were separated by SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose membranes, and reacted with specific antibodies, and reactions were visualized by chemiluminescence. Antibodies are as follows: anti-MSP1a, R874 (1:4,000) and MAb 22B1 (10 µg/ml); anti-MSP2, R883 (1:10,000) and R884 (1:10,000); anti-MSP5, MAb F16C1 (10 µg/ml); anti-purified erythrocyte-stage A. marginale (A.m.), R821 (1:10,000); negative antibody (Ab) controls, Tryp2B1 (10 µg/ml), Tryp1E1 (10 µg/ml), and normal rabbit serum (1:4,000). Numbers to the right of each panel indicate molecular mass in kilodaltons.

MSP1 has been shown previously to confer partial protection on immunized animals against A. marginale challenge and to contain an epitope recognized by MAbs capable of in vitro neutralization of isolated rickettsiae (25, 26). This epitope is present on the MSP1a component of a dimeric complex of MSP1a and MSP1b (7, 33). The reaction of MAb 22B1, which recognizes this neutralization-sensitive epitope of MSP1a, is shown in Fig. 2. MSP1a of the Virginia isolate (erythrocyte stage) of A. marginale has an apparent molecular weight of 70,000 compared to 105,000 for MSP1a of the Florida isolate (erythrocyte stage) of A. marginale (Fig. 2A), agreeing with previous data. MSP1a is present on the in vitro-cultured Virginia isolate of A. marginale and is the same apparent size as MSP1a of Virginia erythrocyte stages (Fig. 2B). MSP1a of the same size is also present on Virginia isolate salivary-gland stages (Fig. 2C). Thus, despite the considerable variation in erythrocyte-stage MSP1a known to occur between geographic isolates of A. marginale (23), no size variation was observed between the different stages of a single isolate or on the same isolate of A. marginale grown in culture.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Antibody to MSP1a detects a 70,000-molecular-weight band in the Virginia isolate of A. marginale from erythrocytes, tick salivary glands, and in vitro culture but a 105,000-molecular-weight band in the Florida isolate of A. marginale. A. marginale organisms purified from infected bovine erythrocytes (er.) or total proteins of infected tick cell cultures (t.c.) or infected (inf.) or uninfected (uninf.) salivary glands of D. andersoni (s.g.) were separated and blotted with anti-MSP1a (MAb 22B1) or negative control (Tryp1E1) antibody. The figure shows a comparison of MSP1a in Florida and Virginia erythrocyte stages (A), in Virginia erythrocyte and cultured stages (B), and in Virginia cultured and salivary-gland stages (C). Numbers at right indicate molecular mass in kilodaltons.

MSP2 and MSP3 are the dominant polypeptides recognized on immunoblots by diluted sera from cattle infected with A. marginale (2, 27) (see also Fig. 4). Figures 3 and 4 show that MSP2- and MSP3-related polypeptides are present in erythrocyte, salivary-gland, and cultured stages of A. marginale. As in erythrocyte stages (2), MSP2 and MSP3 polypeptides appear to be the major polypeptide antigens in cultured and in salivary-gland stages recognized by sera from infected cattle (Fig. 4). The same two-dimensional blot pattern was obtained for MSP2 and MSP3 polypeptides no matter whether A. marginale was examined after 9, 13, 17, or 21 passages in culture (Fig. 5). However, this two-dimensional pattern was different from that of A. marginale in D. andersoni salivary glands (Fig. 6), where greater diversity in MSP2 polypeptide spots was observed.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Antibody to MSP2 detects a 36,000-molecular-weight band in Florida and Virginia-isolate, erythrocyte-stage (er.) A. marginale and in A. marginale from tick salivary glands (s.g.) or grown in vitro (t.c.). Antibodies are as follows: anti-MSP2, R883 (1:10,000); control, normal rabbit serum (1:10,000). Numbers at right indicate molecular mass in kilodaltons.

View larger version (38K): [in this window] [in a new window]   FIG. 4.   MSP2 and MSP3 are the major polypeptides recognized by bovine infection serum in A. marginale from bovine erythrocytes, tick salivary glands, and in vitro culture. (A) Virginia isolate, erythrocyte-stage A. marginale proteins were separated and reacted with a bovine infection serum (PA188, 1:300), specific antibodies to MSPs, or control antibodies. (B) PA188 (1:300) was reacted with separated proteins derived from Florida or Virginia isolate, erythrocyte-stage A. marginale (er.) or with infected or uninfected (unif.) tick salivary glands (s.g.) and in vitro cultures (t.c.). (C) As for panel B, except that the dilution of PA188 is 1:1,000. Antibodies are as follows: bovine infection serum PA188, anti-MSP2 R883 (1:10,000), anti-MSP3 R955 (1:10,000), control normal rabbit serum (1:10,000), anti-MSP1a MAb 22B1 (2 µg/ml), anti-MSP3 MAb 75C2 (2 µg/ml), and control MAb Tryp1E1 (2 µg/ml). Numbers at left indicate molecular mass in kilodaltons.

View larger version (40K): [in this window] [in a new window]   FIG. 5.   Conservation of MSP2 and MSP3 polypeptides in A. marginale grown in culture for varying times. Two-dimensional electrophoresis and immunoblotting with bovine infection serum PA188 (1:300) of A. marginale analyzed after passage 9 (30 January 1995) (A), passage 13 (6 June 1996) (B), passage 17 (13 November 1996) (C), and passage 21 (24 January 1997) (D). The migration positions of molecular weight standards (lysozyme, 14,300; trypsin inhibitor, 21,500; carbonic anhydrase, 30,000; ovalbumin, 46,000; bovine serum albumin, 66,000; phosphorylase b, 97,400; and myosin, 220,000) are shown on the left of each panel, and positions of isoelectric point standards (conalbumin, 6.3; carbonic anhydrase, 5.9; bovine serum albumin, 5.5; actin, 5.1; and trypsin inhibitor, 4.5) are shown at the bottom of each panel.

View larger version (56K): [in this window] [in a new window]   FIG. 6.   Diversity in MSP2 and MSP3 polypeptides in A. marginale (A.m.) from salivary glands of D. andersoni (D.a.). Two-dimensional electrophoresis and immunoblotting of infected tick salivary glands with bovine infection serum PA188 (1:300) are shown. The migration positions of standards are illustrated as described for Fig. 5.

MSP4 and MSP5 are conserved proteins of 31,000 and 19,000 in apparent molecular weight, respectively, found in erythrocyte stages of all isolates of A. marginale examined (22, 34). MSP4 and MSP5 are also present in all stages examined of A. marginale, including cultured organisms (Fig. 7 and 8).

View larger version (35K): [in this window] [in a new window]   FIG. 7.   Antibody to MSP4 detects a 31,000-molecular-weight band in A. marginale (A.m.)-infected but not in uninfected tick cells grown in vitro. Proteins were separated, blotted as described for Fig. 1, and reacted with anti-MSP4 antibody (rabbit C; 1:1,000) or preimmune serum (1:1,000), and reactions were visualized by chemiluminescence. Numbers to the right of each panel indicate molecular mass in kilodaltons.

View larger version (47K): [in this window] [in a new window]   FIG. 8.   Antibody to MSP5 detects a 19,000-molecular-weight band in Florida and Virginia-isolate, erythrocyte-stage (er.) A. marginale and in A. marginale from tick salivary glands (s.g.) or grown in vitro (t.c.). Antibodies are anti-MSP5 MAb F16C1 (10 µg/ml) (A) and control Tryp1E1 (10 µg/ml) (B). Numbers at right indicate molecular mass in kilodaltons.

	DISCUSSION

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All MSPs previously identified and characterized on erythrocyte-derived A. marginale were also found to be present on organisms grown in the IDE8 cultured tick cells. MSP1, MSP2, MSP4, and MSP5 show potential value in the development of serodiagnostic tests and vaccines for anaplasmosis. MSP1a varies in size in different geographic isolates of A. marginale, but a neutralization-sensitive epitope is conserved (23). Sequencing of the MSP1a gene of different isolates and characterization of this epitope with synthetic peptides showed that the neutralization-sensitive epitope was located on a tandem 28- or 29-amino-acid repeat in MSP1a. The number of tandem repeats varied between isolates of A. marginale, two being present in a Virginia isolate and eight being present in a Florida isolate (4). The lack of size variation between MSP1a of erythrocyte stages and that of cultured stages of the Virginia isolate suggested that gross isolate identity was maintained in the cultured rickettsiae.

Variation in MSP2 and MSP3 has also been observed previously in erythrocyte stages of A. marginale. Both polypeptides are encoded by polymorphic, multigene families (3, 28), and although sometimes visualized as single bands at around 36,000 (MSP2) or 90,000 (MSP3) in molecular weight on single-dimension gels, each can be resolved into multiple spots on two-dimensional gels (2). There is evidence that MSP2 is a protective antigen (31) but also that both MSP2 and MSP3 may be involved in antigenic or strain variation of A. marginale (2, 10, 11). More than three antigenic types of MSP2 were present in one rickettsemia cycle in A. marginale prepared from infected carrier cattle (11). Different antigenic types of MSP2 and MSP3 have been distinguished previously by polyacrylamide gel electrophoresis and immunoblotting (2, 10). Our two-dimensional blots of A. marginale cultured for varying times revealed no structural variation in MSP2 or MSP3 polypeptides, suggesting that antigenic stability may be maintained in culture. This was not the case when A. marginale was passaged through ticks, where the expressed MSP2 polypeptides appeared to be those found in culture in addition to others of similar size but different pI. Diverse structural types of MSP2 may be expressed in salivary glands before rickettsiae enter the mammalian host. Further confirmation of antigen stability in culture and diversity on passage between culture and different hosts will be required with different cultured isolates of A. marginale, since at this time only one isolate (Virginia) has been cultured.

MSP4 and MSP5 are also present on cultured A. marginale. MSP4 has some sequence homology with MSP2 polypeptides and therefore may be considered a related member of this protein family (28). MSP4 appears to be conserved and is expressed by all isolates tested (22). MSP5 is encoded by a single gene copy and is also conserved between different isolates. Infected cattle commonly develop antibodies to MSP5. The reaction between MAb F16C1 and recombinant MSP5 was inhibited by 34 of 35 known positive cattle serum samples, by sera from cattle infected up to 6 years previously, and not by sera from anaplasmosis-free cattle (13). MSP5 is, therefore, a strong candidate for a diagnostic test antigen.

The presence of these erythrocyte-stage MSPs on cultured, animal-infective rickettsiae suggests that the cell culture-derived A. marginale may serve as a source of A. marginale of great potential value for basic and applied research. Cultures might be used, for example, to assess antigenic stability and diversity during passage of A. marginale through cattle and ticks, to determine whether antigens of biological relevance are expressed selectively on A. marginale derived from ticks, to develop transformation systems and gene knockouts to discover rickettsial gene function, for screening of novel therapeutic agents, and for development of improved diagnostic reagents and vaccines. Use of cell culture-derived A. marginale may considerably reduce the need to use cattle as a source of rickettsiae.