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Anaplasma marginale is an economically important tick-borne pathogen of cattle that causes bovine anaplasmosis. A wide range of geographic strains of A. marginale have been isolated from cattle, several of which have been characterized using genomics and proteomics. While many of these strains have been propagated in tick lines, comparative analyses after propagation in tick cells have not been reported. The overall purpose of this research therefore was to compare the degree of conservation of selected genes after propagation in tick cell culture among A. marginale strains from the U.S. (the Virginia strain) and Brazil (UFMG1 and UFMG2 strains). The genes studied herein included those which encode the proteins HSP70 and SODB involved in heat shock and stress responses, respectively, and two genes that encode major surface proteins MSP4 and MSP5. Strain identities were first confirmed by sequencing the tandem repeats of the msp1a gene which encodes for the adhesin, MSP1a. The results of these studies demonstrated that the genes encoding for both stress response and heat shock proteins were highly conserved among the three A. marginale strains. Antibodies specific for MSP4, MSP5, SODB and HSP70 proteins were used to further characterize the A. marginale strains, and they reacted with all of these strains propagated in tick cell culture, providing further evidence for antigenic conservation. Although antigenic differences were not found among the three A. marginale strains, multi-locus sequence analysis (MLSA) performed with nucleotide sequences of these genes demonstrated that the A. marginale Brazilian and U.S. strains fall in different clades. These results showed that phylogenetically distant strains of A. marginale are antigenically conserved, even after several in vitro passages, supporting the use of some of the above conserved proteins as candidates for universal vaccines., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and will contribute to characterize gene regulation in ticks.

Anaplasma phagocytophilum, transmitted by ticks of the genus Ixodes, was first described in Scotland as the agent of tick-borne fever in sheep and more recently as the cause of human granulocytic anaplasmosis in the U.S. and Europe. We previously reported sheep as an experimental host for the human NY-18 isolate of A. phagocytophilum. While clinical signs were not observed and infected granulocytes were not seen in stained blood smears, these sheep served as a good host for infection of ticks. In this research we characterized tick feeding sites to better understand tick/host/pathogen interactions. Ixodes scapularis adults were allowed to feed for 2 and 4 days on experimentally infected sheep, after which biopsies were taken beneath tick feeding sites for histopathology, PCR and immunohistochemistry (IHC) studies. In addition, the expression of selected immune response genes was studied in blood and feeding site biopsies. While necrosis was too advanced in 4-day biopsies for accurate cell counts, higher numbers of eosinophils and neutrophils were found in 2-day biopsies from infected sheep as compared with the uninfected controls. An unexpected result was the documentation of higher dermal inflammation in infected sheep at sites without ticks. A. phagocytophilum infected granulocytes were localized by immunohistochemistry (IHC) in skin biopsies using rabbit antibodies against the recombinant A. phagocytophilum major surface protein 4 as the primary antibody for indirect peroxidase-anti-peroxidase and fluorescent antibody IHC. These infected cells are likely to be the source of infection for ticks. Sheep therefore served as good hosts for studying host/pathogen/tick interactions of this human strain of A. phagocytophilum, and provided a means of producing infected ticks for future studies on tick/pathogen developmental and transmission cycles., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Anaplasma and Mycobacterium species are among the most prevalent bacterial pathogens in European red deer (Cervus elaphus) in south-central Spain and are known to modify gene expression in ruminants. In this study, we used microarray hybridization and real-time RT-PCR analyses to characterize global gene expression profiles in red deer in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/Mycobacterium avium sub. paratuberculosis (MAP) infections, compare the expression of immune response genes between red deer infected with A. ovis, M. bovis and A. ovis/M. bovis/MAP, and characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and Anaplasma marginale. Global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer resulted in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes showed pathogen and host-specific signatures and the effect of infection with multiple pathogens on deer immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera produce similar genes expression patterns in infected ruminants. However, pathogen and host-specific differences could contribute to disease diagnosis and treatment in ruminants., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Abstract Background Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species. Results For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level. Conclusions These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.