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PubMed2000

Strain diversity in major surface protein 2 expression during tick transmission of Anaplasma marginale.

Specific major surface protein 2 (MSP2) variants are expressed by Anaplasma marginale within the tick salivary gland and, following transmission, are expressed during acute rickettsemia. In previous work, we have shown that a restricted pattern of MSP2 variants is expressed in the salivary glands of Dermacentor andersoni ticks infected with the South Idaho strain of A. marginale. Now we demonstrate that the identical restriction does not apply to two other strains of A. marginale, and that different variants are also expressed when the same strain is transmitted by different Dermacentor spp. This indicates that antigenic diversity among strains is maintained in tick transmission and may be a significant constraint to MSP2 vaccine development.

Amino Acid Sequence↗
PubMed2006

Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin.

Subolesin was recently shown by both gene silencing and immunization with the recombinant protein to protect against tick infestations, and to cause reduced tick survival and degeneration of gut and salivary gland tissues. In this research, we extended these studies by testing whether targeting subolesin by RNAi or vaccination interfered with the ability of ticks to become infected with two tick-borne pathogens, Anaplasma marginale which causes bovine anaplasmosis and Anaplasma phagocytophilum, the causative agent of human granulocytin anaplasmosis. For the A. marginale studies, Dermacentor variabilis males were injected with subolesin dsRNA or saline and then were allowed to feed on cattle with ascending rickettsemias, while for the A. phagocytophilum studies, mice were immunized with the recombinant subolesin protein, infected with the pathogen and then infested with larval Ixodes scapularis. Tick infections were determined by quantitative polymerase chain reaction of gut and salivary gland tissues. In both experimental approaches, tick infections were significantly reduced. These results suggest that subolesin appears to be a candidate vaccine antigen that may contribute to control of multiple tick species and the reduction of tick-borne pathogens.

Anaplasma marginale↗
PubMed1996

Strategies to interrupt the development of Anaplasma marginale in its tick vector. The effect of bovine-derived antibodies.

Anaplasma marginale is a rickettsia transmitted by ticks that invades and multiplies in bovine erythrocytes causing the disease anaplasmosis. A complex developmental cycle occurs within ticks that begins in midgut cells, with subsequent infection in gut muscle cells. Final development occurs in salivary glands from where the rickettsia is transmitted to the vertebrate host. At each site of development, A. marginale multiplies within membrane-bound inclusions. Attempts to control anaplasmosis have focused on cattle and have included immunization and prophylactic treatment with tetracyclines. New strategies for control of anaplasmosis are being focused on the tick vector. Development of vaccines against hemoparasites in ticks may be feasible because vertebrate host immunoglobulins appear to cross the midgut epithelium of invertebrates and enter the hemolymph without breakdown. We tested the effect of A. marginale antibodies ingested by ticks with the bloodmeal on infections in ticks. Cattle were immunized with purified outer membrane proteins of erythrocytic-derived parasites. Infections in ticks exposed to the immunized cattle were determined using an Anaplasma-specific DNA probe, light and electron microscopy, and tick transmission studies. Vaccine-derived antibodies did not appear to affect the development and transmission of A. marginale in ticks. Further studies are needed to determine if bovine antibodies remain intact within ticks and whether the tick stage of A. marginale has unique surface antigens from the erythrocytic stage.

Anaplasma↗
PubMed1994

Characterization of hemagglutinating components on the Anaplasma marginale initial body surface and identification of possible adhesins.

Interaction of Anaplasma marginale initial bodies with the bovine erythrocyte surface was examined by a direct hemagglutination assay. Purified initial bodies were shown to specifically hemagglutinate bovine erythrocytes but not erythrocytes from nonhost animal species. Hemagglutination was inhibited by treatment of purified initial bodies with trypsin, alpha-chymotrypsin, or proteinase K but not by treatment with neuraminidase or sodium periodate. Treatment of bovine erythrocytes with alpha-chymotrypsin or neuraminidase partially inhibited hemagglutination of the treated cells by initial bodies. In contrast, no inhibition occurred after treatment of erythrocytes with trypsin, phospholipases, or sodium periodate or when monosaccharides and disaccharides were used as potential competitive inhibitors. Thus, the initial body receptor is probably a surface protein, whereas the bovine receptor may comprise both protein and carbohydrate. Hemagglutination was unaffected by treatment of initial bodies with monoclonal or polyclonal antibodies raised against the A. marginale 31-kDa (MSP4) major surface polypeptide or non-A. marginale proteins or by treatment with a monoclonal antibody to the A. marginale MSP1a neutralization-sensitive epitope. In contrast, antiserum raised against whole A. marginale initial bodies or monospecific antibodies raised against purified A. marginale major surface polypeptides with molecular sizes of 105 (MSP1a), 100 (MSP1b), 61, and 36 (MSP2) kDa completely or partially inhibited hemagglutination. These data confirm the proposed surface location of the proteins susceptible to inhibition and suggest that they mediate hemagglutination of bovine erythrocytes. We propose that these surface proteins are possible adhesins.

Anaplasma↗
PubMed1999

Comparison of surface proteins of Anaplasma marginale grown in tick cell culture, tick salivary glands, and cattle.

Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide. Recently, the Virginia isolate of A. marginale was propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis. Development of A. marginale in cell culture was morphologically similar to that described previously in ticks. In order to evaluate the potential of the cell culture-derived organisms for use in future research or as an antigen for serologic tests and vaccines, the extent of structural conservation of the major surface proteins (MSPs) between the cell culture-derived A. marginale and the bovine erythrocytic stage, currently the source of A. marginale antigen, was determined. Structural conservation on the tick salivary-gland stage was also examined. Monoclonal and monospecific antisera against MSPs 1 through 5, initially characterized against erythrocyte stages, also reacted with A. marginale from cell culture and tick salivary glands. MSP1a among geographic A. marginale isolates is variable in size because of different numbers of a tandemly repeated 28- or 29-amino-acid peptide. The cell culture-derived A. marginale maintained the same-size MSP1a as that found on the Virginia isolate of A. marginale in bovine erythrocytes and tick salivary glands. Although differences were observed in the polymorphic MSP2 antigen between culture and salivary-gland stages, MSP2 did not appear to vary, by two-dimensional gel electrophoresis, during continuous passage in culture. These data show that MSPs of erythrocyte-stage A. marginale are present on culture stages and may be structurally conserved during continuous culture. The presence of all current candidate diagnostic and vaccine antigens suggests that in vitro cultures are a valuable source of rickettsiae for basic research and for the development of improved diagnostic reagents and vaccines against anaplasmosis.

Anaplasma↗
PubMed1978

Liquid nitrogen storage of Anaplasma marginale complement-fixation antigen by a multiple small aliquot technique.

Anaplasma marginale complement fixation test antigens were frozen as drops in liquid nitrogen using an easily constructed apparatus. Economy in the use of antigen was achieved as the frozen drops were retrieved from storage in any required volume. The potency of the antigen was unaffected by the freezing and thawing technique. The technique is useful for storing other biological reagents.

Anaplasma↗
PubMed2005

Genetic diversity of Anaplasma marginale strains from cattle farms in the province of Palermo, Sicily.

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in Sicily and results in economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle in the Province of Palermo, Sicily. Seropositivity of cattle >or=1 year old for A. marginale in the study area ranged from 62% to 100%. The observed prevalence of A. marginale infections in cattle herds ranged from 25% to 100%. Two predominant A. marginale msp4 genotypes were found. A positive correlation was found between the prevalence of infection and the presence of Rhipicephalus (Boophilus) annulatus. Phylogenetic analysis of msp4 sequences of European strains of A. marginale did not provide phylogeographical information. These results suggest that development of farm husbandry systems and vaccines for genetically heterogeneous populations of A. marginale are needed for control of anaplasmosis in this region of Sicily.

Anaplasma marginale↗
PubMed2000

Expression of polymorphic msp1beta genes during acute anaplasma Marginale rickettsemia.

Immunization of cattle with native MSP1 induces protection against Anaplasma marginale. The native immunogen is composed of a single MSP1a protein and multiple, undefined MSP1b polypeptides. In addition to the originally sequenced gene, designated msp1beta(F1), we identified three complete msp1beta genes in the Florida strain: msp1beta(F2), msp1beta(F3), and msp1beta(F4). Each of these polymorphic genes encodes a structurally unique MSP1b protein, and unique transcripts can be identified during acute A. marginale rickettsemia. The structural polymorphism is clustered in discrete variable regions, and each MSP1b protein results from a unique mosaic of five variable regions. Although each of the MSP1b proteins in the Florida strain contains epitopes recognized by serum antibody induced by protective immunization with the native MSP1 complex, the variable regions also include epitopes expressed by some but not all of the MSP1b proteins. These data support testing recombinant vaccines composed of the multiple antigenically and structurally unique MSP1b proteins combined with MSP1a in order to mimic the efficacy of native MSP1 immunization.

Amino Acid Sequence↗
PubMed1988

Characteristics of an attenuated Anaplasma marginale of deer origin as an anaplasmosis vaccine.

A fully virulent field Anaplasma marginale isolate, passaged to a splenectomised white tailed deer, lost virulence for cattle after 18 months in the non-bovine host. This attenuated A. marginale produced a mild response in four 18-month-old Holstein steers characterised by an average 2.4% parasitaemia, a 13% drop in PCV and a positive serological response. All vaccinated steers were immune to challenge with a fully virulent, heterologous A. marginale when compared with non-vaccinated controls.

Anaplasma↗
PubMed1988

Molecular size variations in an immunoprotective protein complex among isolates of Anaplasma marginale.

A major surface protein complex from the Florida isolate of Anaplasma marginale has been previously shown to induce protection in immunized cattle and has been proposed as the basis of a subunit vaccine against anaplasmosis. This complex in the Florida isolate is composed of two noncovalently associated polypeptides with molecular masses of 105 and 100 kilodaltons (kDa). The analogous protein complex from four geographically different isolates of A. marginale was immunoprecipitated and compared with the protein complex of the Florida isolate. The polypeptides of the complex varied in apparent molecular mass among the isolates. By using antibodies recognizing epitopes on each polypeptide of the Florida isolate, the antigenic identity of the polypeptides in the analogous complexes was determined. The polypeptides recognized by the neutralizing monoclonal antibody 22B1, which recognizes a 105-kDa polypeptide in the Florida isolate, ranged from 70 to 100 kDa in the other isolates. Those polypeptides recognized by rabbit antiserum R911, which recognizes a 100-kDa polypeptide in the Florida isolate, ranged from 97 to 100 kDa. The surface-exposed peptides in the complexes were compared by limited enzymatic digestion to assess structural homology among isolates. Despite the marked variations in molecular weight, there were conserved peptides between the 22B1-reactive polypeptides and between the R911-reactive peptides. Determination of the role of the conserved peptides in inducing immunity will be critical in the application of these polypeptides as the basis of a subunit vaccine for bovine anaplasmosis.

Anaplasma↗
PubMed1978

Serum levels of conglutinin, complement, and immunoconglutinin in cattle infected with Anaplasma marginale.

Sera from 10 splenectomized yearling cattle given massive doses of Anaplasma marginale (peracute cases) were assayed for conglutinin and complement. Sera from 5 nonsplenectomized yearling cattle given smaller doses (acute cases) were assayed for conglutinin, immunoconglutinin, and complement. In the peracute cases, conglutinating activity disappeared within 6 days after the cattle inoculated. In the acute cases, conglutining began to disappear at 3 weeks after inoculation and had disappeared at peak parasitemia at 4 weeks. Conglutinin returned by 7 weeks after inoculation. In the same acute cases, immunoconglutinain appeared at 6 weeks after inoculation and then slowly disappeared. The complement levels in the peracute and acute cases did not change appreciably.

Agglutination Tests↗
PubMed2005

Distinctly different msp2 pseudogene repertoires in Anaplasma marginale strains that are capable of superinfection.

Lifelong persistent infection of cattle is a hallmark of the tick transmitted pathogen Anaplasma marginale. Antigenic variation of Major Surface Protein 2 (MSP2) plays an important role in evasion of the host immune response to allow persistence. Antigenic variation of MSP2 is achieved by gene conversion of pseudogenes into the single operon linked expression site and the diversity of variants is defined by the pseudogene repertoire. Once an animal is persistently infected with one strain of A. marginale, infection with a second strain (superinfection) is rare. However, we recently detected animals superinfected with different strains of A. marginale and hypothesized that the msp2 pseudogene repertoire would be distinct in these superinfecting strains, consistent with encoding different sets of surface variants. Five strains of A. marginale were selected in order to identify and compare msp2 pseudogene content; these included strains with similar and different msp1alpha genotypes, and genotypes that were representative of the strains that were found in the superinfected animals. Southern blot analysis of strains associated with superinfection revealed distinctly different msp2 banding patterns, in contrast to a pattern suggesting identical pseudogene content among related strains not associated with superinfection. Indeed, targeted sequence analysis of msp2 pseudogenes showed identical pseudogene repertoires in genotypically closely related strains and varying amounts of dissimilarity in the pseudogene repertoire in strains with distinctly different msp1alpha genotypes, but totally different msp2 pseudogene repertoires between the strains that were found in superinfected animals. This finding supports the hypothesis that the occurrence of superinfection reflects the differences in the msp2 repertoire and corresponding diversity of variants.

Amino Acid Sequence↗
PubMed1989

Seroepidemiology of Anaplasma marginale in white-tailed deer (Odocoileus virginianus) from Louisiana.

Antibodies to Anaplasma marginale were detected by the indirect fluorescent antibody test (IFA) in six of 331 (2%) serum samples of white-tailed deer (Odocoileus virginianus) from Louisiana. None of the serum samples were positive using the A. marginale modified rapid card agglutination test. Of the six IFA positive sera retested by the complement fixation test four sera gave anticomplementary and two gave seropositive reactions. The low A. marginale reactor rate in this white-tailed deer population was probably a reflection of the lack of cohabitation between cattle and deer and the fact that the primary arthropod vectors in Louisiana are tabanids. The validity of the indirect fluorescent antibody test for A. marginale antibodies in white-tailed deer should be evaluated.

Agglutination Tests↗
PubMed2004

Differential expression of the msp1alpha gene of Anaplasma marginale occurs in bovine erythrocytes and tick cells.

Major surface proteins (MSP) 1a and 1b of the tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) are conserved on A. marginale derived from bovine erythrocytes and tick cells. MSP1a and MSP1b form the MSP1 complex and are adhesins involved in infection of host cells. While both MSP1a and MSP1b are adhesins for bovine erythrocytes, only MSP1a is an adhesin for cultured and native tick cells. These studies were initiated because antibody responses to MSP1a and MSP1b differed in cattle immunized with killed A. marginale derived from bovine erythrocytes or cultured tick cells. A strong antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived A. marginale, whereas cattle immunized with tick cell culture-derived A. marginale produced antibodies preferentially to MSP1b. The molecular basis of this differential antibody response was then studied using Western blot, confocal microscopy and reverse transcriptase (RT)-PCR. Whereas expression of MSP1b by A. marginale derived from both bovine and tick host cells was similar at the protein and RNA levels, expression of MSP1a by A. marginale in these cells differed. Low levels of MSP1a were observed in cultured tick cells and tick salivary glands, but high expression of MSP1a occurred on A. marginale derived from bovine erythrocytes. The analysis of the expression of the msp1alpha gene by RT-PCR suggests that the differential expression of MSP1a is regulated at the transcriptional level and may influence the infectivity of A. marginale for host cells. Variation in the expression of MSP1a may also contribute to phenotypic and antigenic changes in the pathogen.

Anaplasma marginale↗
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

Detection of the Anaplasma centralevaccine strain and specific differentiation from Anaplasma marginale in vaccinated and infected cattle.

Bovine anaplasmosis caused by the intraerythrocytic rickettsia Anaplasma marginale is the most prevalent tick-borne disease of cattle worldwide. The most efficient method to control anaplasmosis is by vaccination using live Anaplasma centrale, a closely related species or subspecies of low pathogenicity that is capable of inducing significant protection against the more virulent A. marginale. In the present study, we applied PCR assays to detect and discriminate field infection with A. marginale from A. centrale persistently infected vaccinates. Direct and one-stage nested PCR were based on A. centrale mbp58 specific sequence, with the assay sensitivity level of 0.00001% for nested PCR performed in a single amplification step. Size polymorphism in the A. marginale msp1 alpha gene among strains was used to design a PCR capable of discriminating between the Israel T and NT strains of A. marginale and the encoded MSP1a size polymorphism was confirmed by immunoprecipitation. The detection of A. centrale in 72% of vaccinated field-grazing cattle clearly indicated that the majority of vaccinated cattle remain carriers. A. marginalewas detected in 64% of these vaccinated cattle, demonstrating that, as expected, natural transmission occurs within the endemic region. The lack of severe A. marginaleoutbreaks in this region, despite ongoing transmission, is consistent with protection being provided by widespread vaccination with A. centrale.

Anaplasma↗
PubMed1983

The use of tick transmission by Boophilus microplus to isolate pure strains of Babesia bovis, Babesia bigemina and Anaplasma marginale from cattle with mixed infections.

Pure strains of Babesia bovis, Babesia bigemina and Anaplasma marginale were isolated from cattle infected with all 3 species as well as Theileria sp. and Eperythrozoon teganodes, using only transmission by the tick, Boophilus microplus. Unengorged adult ticks transferred to susceptible cattle transmitted A. marginale, but not Babesia. Engorged adults gave rise to progeny that transmitted Babesia, B. bovis by larvae and B. bigemina by male ticks. The Theileria and E. teganodes were not transmitted by the ticks and thus did not appear in calves used for isolating the pure strains of Babesia and A. marginale.

Anaplasma↗