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PubMed1996

Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale rickettsemia.

Antigenic variants of Anaplasma marginale major surface protein 2 (MSP-2), a target of protective immune responses, have been detected by use of copy-specific monoclonal antibodies reactive with some, but not all, organisms during acute rickettsemia. The presence of polymorphic msp-2 genes was confirmed by cloning and sequencing two gene copies, 11.2 and DF5, each of which encodes a full-length MSP-2 with a unique amino acid sequence. Transcription of msp-2 genes during acute rickettsemia was analyzed by use of cDNA cloning of hybrid-selected msp-2 mRNA. Sequencing of cDNA clones, designated AR1 to AR14, indicated that DF5 msp-2 was transcribed during acute rickettsemia. Two classes of variant msp-2 genes were also transcribed during acute rickettsemia. The first class of variant transcripts, typified by clones AR3, AR4, AR7, and AR14, each encoded a single or small number of amino acid substitutions relative to DF5. The second type, AR5, encoded a large region of amino acid polymorphism, including additions, deletions, and substitutions, as compared to DF5. Specific antibody directed against the AR5 polymorphic region bound a unique MSP-2 expressed on A. marginale that was not recognized by antibody generated against DF5. Similarly, anti-AR5 peptide antibody reacted with a different MSP-2 that was not bound by anti-DF5 antibody. This expression confirmed that variant msp-2 transcripts encode structurally distinct MSP-2 molecules which bear unique B-cell epitopes. These results support the hypothesis that the large msp-2 gene family, which constitutes a minimum of 1% of the genome, encodes antigenic variants critical to evasion of protective immune response directed against surface MSP-2 epitopes.

Acute Disease↗
PubMed1998

CD4(+) T-lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge.

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.

Anaplasma↗
PubMed1998

The repertoire of Anaplasma marginale antigens recognized by CD4(+) T-lymphocyte clones from protectively immunized cattle is diverse and includes major surface protein 2 (MSP-2) and MSP-3.

Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4(+) T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406-5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains of A. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains of A. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains of A. marginale for inclusion in a nucleic acid or recombinant protein vaccine.

Anaplasma↗
PubMed1998

Persistence of Anaplasma ovis infection and conservation of the msp-2 and msp-3 multigene families within the genus Anaplasma.

Goats which have recovered from acute Anaplasma ovis infection remain seropositive, although infected erythrocytes cannot be detected by microscopic examination. Persistence of A. ovis 17 to 21 months following experimental infection was demonstrated by PCR detection of the msp-5 gene. Quantitative analysis of persistent rickettsemia over time showed that all levels were below the limit of microscopic detection and ranged from a low of 10(2) organisms/ml to peaks of 10(6) organisms/ml. Two patterns of persistent rickettsemia were observed: the first was characterized by cyclic fluctuations at 6- to 9-week intervals, similar to the pattern described for A. marginale-infected cattle, while in the second pattern, repetitive cycles did not occur and the rickettsemia levels were relatively constant. The msp-2 and msp-3 multigene families, which provide the genetic capacity for outer membrane protein antigenic variation during persistent A. marginale rickettsemia, were identified in the A. ovis genome by Southern blot analysis, and expression of an MSP-2 homologue was confirmed by using immunoblots.

Anaplasma↗
PubMed1998

Expression of Anaplasma marginale major surface protein 2 variants during persistent cyclic rickettsemia.

Anaplasma marginale is an intraerythrocytic rickettsial pathogen of cattle in which infection persists for the life of the animal. Persistent A. marginale infection is characterized by repetitive rickettsemic cycles which we hypothesize reflect emergence of A. marginale antigenic variants. In this study, we determined whether variants of major surface protein 2 (MSP-2), a target of protective immunity encoded by a polymorphic multigene family, arise during persistent rickettsemia. By using a quantitative competitive PCR to identify rickettsemic cycles, msp-2 transcripts expressed in vivo were isolated from peak rickettsemia of sequential cycles. Cloning and sequencing of msp-2 cDNA revealed that genetic variants of MSP-2 emerge representing a minimum of four genetic variant types in each cycle during persistent infection. Two-color immunofluorescence using variant-specific antibody showed that emergence of MSP-2 variants resulted in expression of a minimum of three antigenic types of MSP-2 within one rickettsemic cycle. Therefore immune control of each cycle would require responses to an antigenically diverse A. marginale population. These findings demonstrate that polymorphic MSP-2 variants emerge during cyclic rickettsemia in persistent A. marginale infection and suggest that emergent variants play an important role in persistence.

Amino Acid Sequence↗
PubMed1999

Emergence of Anaplasma marginale antigenic variants during persistent rickettsemia.

Anaplasma marginale is an ehrlichial pathogen of cattle, in the order Rickettsiales, that establishes persistent cyclic rickettsemia in the infected host. Within each rickettsemic cycle, A. marginale expressing antigenically variant major surface protein 2 (MSP2) emerge. By cloning 17 full-length msp2 transcripts expressed during cyclic rickettsemia, we determined that emergent variants have a single, central hypervariable region encoding variant B-cell epitopes. The N- and C-terminal regions are highly conserved among the expressed A. marginale variants, and similar sequences define the MSP2 homologues in the agent of human granulocytic ehrlichiosis (HGE). This is in contrast to the MSP2 homologues in ehrlichial genogroup I pathogens, Ehrlichia chaffeensis, Ehrlichia canis, and Cowdria ruminantium, that have multiple hypervariable regions. By defining the variable and conserved regions, we were able to show that the single hypervariable region of A. marginale MSP2 encodes epitopes that are immunogenic and induce variant-specific antibody responses during persistent infection. These findings demonstrate that the MSP2 structural variants that emerge during each cycle of persistent rickettsemia are true antigenic variants, consistent with MSP2 antigenic variation as a mechanism of A. marginale persistence.

Amino Acid Sequence↗
PubMed2002

Major histocompatibility complex class II DR-restricted memory CD4(+) T lymphocytes recognize conserved immunodominant epitopes of Anaplasma marginale major surface protein 1a.

Native major surface protein 1 (MSP1) of Anaplasma marginale, composed of covalently associated MSP1a and MSP1b proteins, stimulates protective immunity in cattle against homologous and heterologous strain challenge. Protective immunity against pathogens in the family Anaplasmataceae involves both CD4(+) T cells and neutralizing immunoglobulin G. Thus, an effective vaccine should contain both CD4(+) T- and B-lymphocyte epitopes that will elicit strong memory responses upon infection with homologous and heterologous strains. Previous studies demonstrated that the predominant CD4(+) T-cell response in MSP1 vaccinates is directed against the MSP1a subunit. The present study was designed to identify conserved CD4(+) T-cell epitopes in MSP1a presented by a broadly represented subset of major histocompatibility complex (MHC) class II molecules that would be suitable for inclusion in a recombinant vaccine. Transmembrane protein prediction analysis of MSP1a from the Virginia strain revealed a large hydrophilic domain (HD), extending from amino acids (aa) 1 to 366, and a hydrophobic region extending from aa 367 to 593. The N terminus (aa 1 to 67) includes one 28-aa form A repeat and one 29-aa form B repeat, which each contain an antibody neutralization-sensitive epitope [Q(E)ASTSS]. In MSP1 vaccinates, recombinant MSP1a HD (aa 1 to 366) stimulated recall proliferative responses that were comparable to those against whole MSP1a excluding the repeat region (aa 68 to 593). Peptide mapping determined a minimum of five conserved epitopes in aa 151 to 359 that stimulated CD4(+) T cells from cattle expressing DR-DQ haplotypes common in Holstein-Friesian breeds. Peptides representing three epitopes (aa 231 to 266, aa 270 to 279, and aa 290 to 319) were stimulatory for CD4(+) T-cell clones and restricted by DR. A DQ-restricted CD4(+) T-cell epitope, present in the N-terminal form B repeat (VSSQSDQASTSSQLG), was also mapped using T-cell clones from one vaccinate. Although form B repeat-specific T cells did not recognize the form A repeat peptide (VSSQS_EASTSSQLG), induction of T-cell anergy by this peptide was ruled out. The presence of multiple CD4(+) T-cell epitopes in the MSP1a HD, in addition to the neutralization-sensitive epitope, supports the testing of this immunogen for induction of protective immunity against A. marginale challenge.

Amino Acid Sequence↗
PubMed2002

Expression of Anaplasma marginale major surface protein 2 operon-associated proteins during mammalian and arthropod infection.

The antigenically variant major surface protein 2 (MSP2) of Anaplasma marginale is expressed from a 3.5-kb operon that contains, in a 5'-to-3' direction, four open reading frames, opag3, opag2, opag1, and msp2. This operon structure was shown to be conserved among genotypically and phenotypically distinct A. marginale, A. ovis, and A. centrale strains. The individual OpAG amino acid sequences are highly conserved among A. marginale strains, with identities ranging from 95 to 99%. OpAG2 and OpAG3 were expressed by all examined A. marginale strains during the acute rickettsemia in the mammalian host and, like MSP2, localize to the bacterial surface. OpAG2 and OpAG3 were also expressed in an infected Ixodes scapularis tick cell line. In contrast, the same A. marginale strains expressed only OpAG2 in two different Dermacentor spp. during transmission feeding. OpAG1 expression was not detected in the infected mammalian host, the infected tick cell line, or within infected Dermacentor ticks. The differential expression of outer membrane proteins from within an operon is a novel finding in tick-transmitted bacteria, and the regulation of expression may be broadly applicable to understanding how the pathogen adapts to the mammalian host-tick vector transition.

Amino Acid Sequence↗
PubMed2003

Simultaneous variation of the immunodominant outer membrane proteins, MSP2 and MSP3, during anaplasma marginale persistence in vivo.

Vector-borne bacterial pathogens persist in the mammalian host by varying surface antigens to evade the existing immune response. To test whether the model of surface coat switching and immune evasion can be extended to a vector-borne bacterial pathogen with multiple immunodominant surface proteins, we examined Anaplasma marginale, a rickettsia with two highly immunogenic outer membrane proteins, major surface protein 2 (MSP2) and MSP3. The simultaneous clearance of variants of the two most immunodominant surface proteins of A. marginale followed by emergence of unique variants indicates that the switch rates and immune selection for MSP2 and MSP3 are sufficiently similar to explain the cyclic bacteremia observed during infection in the immunocompetent host.

Amino Acid Sequence↗
PubMed2004

CD4+ T lymphocytes from Anaplasma marginale major surface protein 2 (MSP2) vaccinees recognize naturally processed epitopes conserved in MSP3.

Major surface protein 2 (MSP2) and MSP3 of the persistent bovine ehrlichial pathogen Anaplasma marginale are immunodominant proteins that undergo antigenic variation. The recently completed sequence of MSP3 revealed blocks of amino acids in the N and C termini that are conserved with MSP2. This study tested the hypothesis that CD4+ T cells specific for MSP2 recognize naturally processed epitopes conserved in MSP3. At least one epitope in the N terminus and two in the C terminus of MSP2 were also processed from MSP3 and presented to CD4+ T lymphocytes from MSP2-immunized cattle. This T-lymphocyte response to conserved and partially conserved epitopes may contribute to the immunodominance of MSP2 and MSP3.

Amino Acid Sequence↗
PubMed1972

Deoxyribonucleic acid of Anaplasma marginale.

Deoxyribonucleic acid from isolated marginal bodies and calf erythrocytes infected with Anaplasma marginale is found to be double stranded and to contain 51 moles per cent guanine plus cytosine.

Anaplasma↗
PubMed1967

Evidence of lactate dehydrogenase in Anaplasma marginale.

Extracts from preparations of partially purified Anaplasma marginale revealed low levels of lactate dehydrogenase (LDH). Enzyme inhibition by immune sera further indicated that A. marginale possesses a protein moiety the same as that of the normal red blood cell (RBC), although data suggested an alteration of LDH(1) from that observed in normal RBC. Bimodal isozyme distribution was detected after electrophoresis of the extracts. One isozyme approached the cathode and the other the anode, and both appeared to be nicotinamide adenine dinucleotide-dependent. Heterogeneity of parasite and host cell isozymes was established on the basis of zone electrophoresis on cellulose acetate strips.

Anaplasma↗
PubMed1987

Detection of antibodies to Anaplasma marginale by an improved enzyme-linked immunosorbent assay with sodium dodecyl sulfate-disrupted antigen.

Sensitivities of two enzyme-linked immunosorbent assays (ELISAs) with particulate and sodium dodecyl sulfate (SDS)-disrupted Anaplasma marginale antigen were compared. The quotient of positive reference sera divided by the absorbance quotient of a negative reference serum at identical dilution was termed the signal-to-noise ratio. Optimal signal-to-noise ratios were dependent on both pretreatment of antigen and antigen concentration. SDS disruption of anaplasmal antigen resulted in a markedly improved signal-to-noise ratio of ELISA compared with ELISA with untreated antigen at identical antigen and serum dilutions. This represented higher sensitivity and lower background absorbance of the ELISA with disrupted antigen. SDS-disrupted A. marginale antigen was standardized by protein determination, and antigen, as well as precoated microtiter wells, was stored frozen without apparent loss of antigenic properties. ELISA results were in agreement with results of positive and negative control sera tested by the complement fixation test or by light microscopy Anaplasma diagnosis in Giemsa-stained blood films.

Anaplasma↗
PubMed1989

Cloned DNA probes identify Anaplasma ovis in goats and reveal a high prevalence of infection.

Anaplasma organisms are observed in erythrocytes from goats with anemia and weight loss in Kenya. Three anaplasmas have been isolated in nature, Anaplasma ovis, Anaplasma marginale, and Anaplasma centrale. The two recognized species, A. ovis and A. marginale, are known to infect goats. Since only A. ovis causes clinical disease in goats, the Anaplasma species in goats in Kenya were identified. To detect A. ovis, a 9.6-kilobase-pair section of genomic DNA was cloned into pBR322 (pAO12A) and was used in conjunction with an A. marginale DNA probe previously derived from a gene coding for a 105,000-molecular-weight surface protein (Am105L) of A. marginale. In Southern blots, pAO12A DNA hybridized to several at least partially homologous sequences that were present in A. ovis and A. marginale genomic DNAs. The pAO12A DNA did not hybridize to Babesia bovis genomic or goat leukocyte DNA. The Anaplasma species that infected goats was identified as A. ovis by (i) DNA hybridization with pAO12A, (ii) hybridization of the A. marginale DNA probe to A. centrale and A. marginale genomic DNAs and lack of hybridization to A. ovis genomic DNA from an isolate obtained in Idaho and Anaplasma DNA from infected goats in Kenya, (iii) the intraerythrocytic location of Anaplasma organisms in infected goat blood, and (iv) the host specificity of the Anaplasma organisms for goats but not for cattle. Also, by using the two Anaplasma DNA probes, the prevalence of A. ovis in goats from seven locations in Kenya was found to range from 22 to 87%. The pAO12A DNA probe detected a 0.0035% A. ovis parasitemia in infected blood, an improved sensitivity which is suitable for use in surveillance and epidemiological studies.

Anaplasma↗
PubMed1993

Impact of persistent Anaplasma marginale rickettsemia on tick infection and transmission.

Anaplasma marginale, an intraerythrocytic rickettsia of cattle, is transmitted biologically by ticks. Because of the brevity of acute A. marginale infection, transmission may rely on the tick's ability to acquire the organism from persistently infected cattle with low rickettsemia levels. By using a nucleic acid probe to quantitate low-level infection, we found that rickettsemia levels in persistently infected cattle fluctuated at approximately 5-week intervals during a 24-week period, from < 10(4) infected erythrocytes per ml of blood to high levels of approximately 10(7) infected erythrocytes per ml of blood. Cattle maintained very low rickettsemia levels (< 10(4.3) infected erythrocytes per ml of blood) for approximately 4 to 8 days of every 5-week cycle. The effect of fluctuations in rickettsemia in persistently infected cattle on acquisition by Dermacentor andersoni nymphal and adult male ticks was examined. A positive correlation was observed between rickettsemia levels in cattle and the resulting infection rates of ticks. At high rickettsemia levels, up to 80% of ticks acquired infection, but even at extremely low rickettsemia levels, 27% of adult male ticks became infected. Moreover, once ticks acquired infection, biological replication of the organism within the ticks appeared to make up for initial differences in the infecting dose. The high infection rates in adult males, combined with their intermittent feeding behavior and the observation that only a few infected ticks were required for transmission to a susceptible host, suggest that adult male D. andersoni ticks are epidemiologically important in A. marginale transmission. Because cattle with all levels of rickettsemia were capable of efficient transmission to ticks, population control efforts must include decreasing transmission from persistently infected individuals.

Anaplasma↗
PubMed1995

Detection of Anaplasma ovis infection in goats by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) based on a major surface protein 5 (MSP5) B-cell epitope conserved among Anaplasma species was used to detect goats infected with Anaplasma ovis. We examined strains of A. ovis isolated from goats in Kenya and demonstrated that MSP5 and the target B-cell epitope, bound by monoclonal antibody ANAF16C1, were conserved. Sera from 149 goats in four regions of Kenya and from 302 goats in six U.S. states were tested for the presence of epitope-specific antibodies with the MSP5 competitive inhibition ELISA. Evidence that the assay can be used to detect A. ovis-infected goats includes the following: (i) 53 goats raised in confinement with arthropod control were all seronegative; (ii) six goats experimentally infected with A. ovis seroconverted at the same time that they developed detectable rickettsemia; (iii) seroconverted goats remained seropositive, consistent with the persistence of A. ovis in goats and the presence of anti-MSP5 antibody in cattle persistently infected with Anaplasma marginale; and (iv) 119 of 127 known A. ovis-infected goats in Kenya were seropositive. A. ovis infection, as determined serologically and by demonstration of infected erythrocytes, in goats from the four regions in Kenya was highly prevalent. In contrast, despite the presence of A. ovis and competent arthropod vectors in the United States, the prevalence of infection appeared to be very low. The high prevalence in Kenya and the occurrence of anemia in persistently infected goats may be impediments to current efforts to increase milk yields on small farms.

Anaplasma↗
PubMed1998

Detection of cattle naturally infected with Anaplasma marginale in a region of endemicity by nested PCR and a competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5.

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.

Anaplasmosis↗