Immunization with Anaplasma centrale Msp2 HVRs Is Less Effective than the Live A. centrale Vaccine against Anaplasmosis.

Bovine anaplasmosis, caused by Anaplasma marginale, is the most prevalent tick-transmitted pathogen of livestock globally. In many parts of the world, Anaplasma centrale, a related organism, is used as a live blood-borne vaccine as it causes either no or only a mild clinical disease. Anaplasma centrale does not prevent infection with A. marginale but does prevent acute disease. Anaplasma centrale is prohibited from being used in the U.S. due to the risk of transmitting emerging pathogens. Both of these organisms encode proteins known as major surface protein 2 (Msp2), which is the most immunodominant protein for the organism. Both organisms persist in their host by evading clearance, i.e., the adaptive immune response, by recombining the hypervariable region (HVR) of msp2 with pseudogene alleles. The study goal was to test whether the Msp2 HVRs encoded by A. centrale are a sufficient source of immune stimulation to provide the clinical protection exhibited by the blood-borne vaccine. Calves were inoculated with recombinantly expressed A. centrale HVRs. Control groups were inoculated with saponin or infected with the A. centrale live vaccine and compared with the test group. A Western blot analysis demonstrated that the HVR immunizations and A. centrale live vaccine stimulated an immune response. All animals in the study became infected upon challenge with A. marginale-infected ticks. The saponin-immunized control group had a high PPE (5.4%) and larger drops in PCVs (14.6%). As expected, the A. centrale-immunized animals were protected from acute disease with lower (0.6%) parasitemia and lower drops in PCV (8.6%). The HVR-immunized group had intermediate results that were not statistically significantly different from either the negative or positive controls. This suggests that the HVR immunogen does not fully recapitulate the protective capacity of the live vaccine.

Segmental Variation in a Duplicated msp2 Pseudogene Generates Anaplasma marginale Antigenic Variants.

Anaplasma marginale is a prototypical highly antigenically variant bacterial pathogen dependent on the sequential generation of major surface protein 2 (Msp2) outer membrane variants to establish persistent infection. Msp2 is encoded by a single expression site, and diversity is achieved by gene conversion of chromosomally encoded msp2 pseudogenes. Analysis of the full complement of msp2 pseudogenes in the St. Maries strain revealed identical sequences in different loci. The Florida strain shared the same locus structure, but in the loci where the St. Maries strain had two identical pseudogenes, the Florida strain had one whose sequence was identical to the St. Maries sequences, while the sequence of the second pseudogene differed. Consequently, we hypothesized that the msp2 pseudogene repertoire arose via gene duplication, allowing structural variation to occur in one copy but the utility of the other to be retained. Using comparative genomics, we first established that duplication of msp2 pseudogenes is common among A. marginale strains: all seven examined strains had at least one duplicate pair in which either the genes in the pair were maintained as identical copies or the genes contained segmental changes. We then demonstrated that a minimal segmental change in a duplicated pseudogene locus is sufficient for immune escape from the broad antibody response generated in a natural host, as is a completely divergent pseudogene sequence in an otherwise conserved locus. The results support a model in which a locus first duplicates, resulting in a second identical copy, and then progressively incorporates changes to generate an msp2 repertoire capable of generating sufficient antigenic variants to escape immunity and establish persistent infection.

Protective immunity induced by immunization with a live, cultured Anaplasma marginale strain.

Despite significant economic losses resulting from infection with Anaplasma marginale, a tick-transmitted rickettsial pathogen of cattle, available vaccines provide, at best, only partial protection against clinical disease. The green-fluorescent protein expressing mutant of the A. marginale St. Maries strain is a live, marked vaccine candidate (AmStM-GFP). To test whether AmStM-GFP is safe and provides clinical protection, a group of calves was vaccinated, and clinical parameters, including percent parasitized erythrocytes (PPE), packed cell volume (PCV) and days required to reach peak bacteremia, were measured following inoculation and following tick challenge with wild type St. Maries strain (AmStM). These clinical parameters were compared to those obtained during infection with the A. marginale subsp. centrale vaccine strain (A. centrale) or wild type AmStM. AmStM-GFP resulted in similar clinical parameters to A. centrale, but had a lower maximum PPE, smaller drop in PCV and took longer to reach peak bacteremia than wild type AmStM. AmStM-GFP provided clinical protection, yielding a stable PCV and low bacteremia following challenge, whereas A. centrale only afforded partial clinical protection.

Expression patterns of Anaplasma marginale Msp2 variants change in response to growth in cattle, and tick cells versus mammalian cells.

Antigenic variation of major surface proteins is considered an immune-evasive maneuver used by pathogens as divergent as bacteria and protozoa. Likewise, major surface protein 2 (Msp2) of the tick-borne pathogen, Anaplasma marginale, is thought to be involved in antigenic variation to evade the mammalian host immune response. However, this dynamic process also works in the tick vector in the absence of immune selection pressure. We examined Msp2 variants expressed during infection of four tick and two mammalian cell-lines to determine if the presence of certain variants correlated with specific host cell types. Anaplasma marginale colonies differed in their development and appearance in each of the cell lines (P<0.001). Using Western blots probed with two Msp2-monospecific and one Msp2-monoclonal antibodies, we detected expression of variants with differences in molecular weight. Immunofluorescence-assay revealed that specific antibodies bound from 25 to 60% of colonies, depending on the host cell-line (P<0.001). Molecular analysis of cloned variant-encoding genes demonstrated expression of different predominant variants in tick (V1) and mammalian (V2) cell-lines. Analysis of the putative secondary structure of the variants revealed a change in structure when A. marginale was transferred from one cell-type to another, suggesting that the expression of particular Msp2 variants depended on the cell-type (tick or mammalian) in which A. marginale developed. Similarly, analysis of the putative secondary structure of over 200 Msp2 variants from ticks, blood samples, and other mammalian cells available in GenBank showed the predominance of a specific structure during infection of a host type (tick versus blood sample), demonstrating that selection of a possible structure also occurred in vivo. The selection of a specific structure in surface proteins may indicate that Msp2 fulfils an important role in infection and adaptation to diverse host systems. Supplemental Abstract in Spanish (File S1) is provided.