To identify novel antigens with immunoglobulin G2 (IgG2) specificity and immunostimulant properties for bovine Th1 cells, humoral and cellular responses were studied in cattle inoculated with initial bodies from a Mexican isolate of Anaplasma marginale and challenged with a heterologous strain. Analysis of post-immunization sera by ELISA and assaying of in vitro cellular responses in peripheral blood mononuclear cells (PBMCs) cultured in the presence of protein extracts from three Anaplasma marginale strains showed positive values of optical density ELISA readings and stimulation indices in the immunized but not control cattle. Post-immunization and post-challenge sera recognized in Western blots several proteins with molecular weights ranging from 15 to 209 kDa, twelve of which were recognized by IgG2 in the three Anaplasma marginale strains. Seven of these are novel and have not been previously reported for their IgG2 specificity; three are confirmed to be major surface proteins (MSP-1a, MSP-2, and MSP-5); and the others correspond to other well-studied MSPs but were not confirmed. Partially purified fractions of protein extracts of the Mex-17 strain were tested against PBMCs cultured in vitro. One out of the seven novel proteins induced detectable lymphoproliferation (LP) of PBMCs, and interferon-gamma was detected in supernatants of PBMC cultured in the presence of two protein fractions, including the one that caused LP. It is concluded that novel antigens, particularly the 28-kDa protein, played an additional role in the protection of immunized cattle and should be considered vaccine candidates after in vivo immunization experiments are concluded.
The current description of biological transmission of Anaplasma marginale by Rhipicephalus microplus ticks, includes of the biological intrastadial and transstadial transmission. Both transovarian transmission of Anaplasma from engorged ticks to their progeny and, transmission from infected unfed larvae to the mammalian host is controversial. In order to demonstrate vertical transmission of A. marginale by R. microplus ticks under experimental conditions, feed-acquisition infected engorged females were incubated at 18 °C or 28 °C for oviposition. Larvae hatched from these ticks were used to infest two steers for each incubation temperature. None of the four steers infested with either lot of larvae developed clinical disease, yet subclinical infection was observed in the steers infested with larvae from engorged ticks incubated at 28 °C for hatching. gDNA from, larvae used for the infection of the carrier tick donor, gDNA from larvae oviposited at 28 ºC, gDNA from blood of A. marginale-positive steers, were positive for amplification of msp5 and msp1α the variable region by PCR. All other DNA samples from the original stabilate, blood from the donor steer, larvae from ticks incubated at 28 °C and blood from steers infested with these same larvae were positive to both, msp5 and msp1α PCR. msp1α sequences of all PCR products were the same and are consistent with previously reported Tlapacoyan-2 sequence. The present evidence indicates that R. microplus is capable of passing A. marginale to its progeny and that these infected larvae can transmit the infection to susceptible hosts.
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