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[Background]: Ticks are obligate hematophagous arthropods that synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) associated with the alpha-gal syndrome (AGS) or allergy to mammalian meat consumption., [Research design and methods]: In this study, we used a proteomics approach to characterize tick proteins in salivary glands (sialome SG), secreted saliva (sialome SA) and with α-Gal modification (alphagalactome SG and SA) in model tick species associated with the AGS in the United States (Amblyomma americanum) and Australia (Ixodes holocyclus). Selected proteins reactive to sera (IgE) from patients with AGS were identified to advance in the identification of possible proteins associated with the AGS. For comparative analysis, the α-Gal content was measured in various tick species., [Results]: The results confirmed that ticks produce proteins with α-Gal modifications and secreted into saliva during feeding. Proteins identified in tick alphagalactome SA by sera from patients with severe AGS symptomatology may constitute candidate disease biomarkers., [Conclusions]: The results support the presence of tick-derived proteins with α-Gal modifications in the saliva with potential implications in AGS and other disorders and protective capacity against tick infestations and pathogen infection. Future research should focus on the characterization of the function of tick glycoproteins with α-Gal in tick biology and AGS., This work was supported by the State Research Agency, Spain, project BIOGAL [PID2020-116761GB-I00], the Spanish Research Council Grant [2020AEP123] and Consejería de Educación, Cultura y Deportes, JCCM, Spain, project CCM17-PIC-036 (SBPLY/17/180501/000185). Ixodes holocyclus saliva and salivary gland collections was supported by the Australian Research Council Linkage Grant [LP120200836 (2013-2018)]. MV was supported by the University of Castilla La Mancha, UCLM, Spain, and the Fondo Europeo de Desarrollo Regional, FEDER, EU.

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., This research was supported by the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, Center for Veterinary Health Sciences, Research Advisory Committee grants to KMK, RCG, and EJR, Project BFU2011-23896 to JF, and the EU FP7 ANTIGONE project number 278976. N. Ayllón and R.C. Galindo were funded by MEC, Spain.

Diseases caused by arthropod-borne pathogens greatly impact on human and animal health. Recent research has provided evidence that tick protective antigens can be used for development of vaccines with the dual target of controlling arthropod infestations and reducing their vector capacity for pathogens. As reviewed herein, protective antigens such as subolesin/akirin, which are highly conserved across vector species, show promise for use in development of a universal vaccine for the control of arthropod infestations and the reduction of pathogen transmission. However, further research is needed in critical areas towards achieving this goal., This work was supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain (project FAU2008-00014-00-00) and the Consejería de Educación y Ciencia, JCCM, Spain (project PEII09-0118-8907). J.A. Moreno-Cid is a recipient of a JCCM fellowship. M. Canales was funded by the Wellcome Trust under the “Animal Health in the Developing World” initiative (project 0757990). M. Villar and Ruth C. Galindo were funded by the JAE-DOC program (CSIC-FSE) and MEC, Spain, respectively.

Q fever, a zoonotic disease, is caused by a gram-negative intracellular bacterium, Coxiella burnetii. Although normally transmitted during exposure to infectious aerosols, C. burnetii is also found in arthropod vectors. In the environment, ticks are thought to play a crucial role in bacterial maintenance and transmission by infecting various mammalian species. However, the nature of the pathogen–tick relationship is not well defined. To determine C. burnetii's interactions with a cultured tick cell line, we introduced purified C. burnetii NMII into Ixodes scapularis–derived IDE8 cells and assayed for bacterial presence, replication, gene expression, and subsequent infectivity for mammalian cells. Tick cells were harvested at 24 h, 72 h, 7 days, and 11 days postinfection (PI). C. burnetii uptake and subsequent replication was demonstrated by indirect immunofluorescence assay, electron microscopy, and real-time polymerase chain reaction (PCR). When a genome equivalent multiplicity of infection of 30 was used, 30%–40% of exposed cells were seen to have small, rounded, vacuoles at 72 h PI, whereas at 7 and 11 days PI, 60%–70% of cells contained enlarged vacuoles harboring large numbers of bacteria. Quantitative PCR analysis of total genomic DNA confirmed that C. burnetii genome numbers increased significantly from 24 h to 11 days PI. Expression of C. burnetii type four secretion system homologs at 7 days PI was demonstrated by reverse transcriptase PCR. Finally, indirect immunofluorescence assay demonstrated that C. burnetii propagated within IDE8 cells were infectious for mammalian cells. These studies demonstrate the utility of cultured tick cell lines as a model to investigate C. burnetii's molecular interactions with its arthropod vectors.

Anaplasma marginale is a tick-borne pathogen of cattle responsible for the disease anaplasmosis. Data suggest that Rhipicephalus (Boophilus) microplus and R. annulatus may be the major tick vectors of A. marginale in tropical and subtropical regions of the world. In this work we demonstrated the first infection and propagation of a Brazilian isolate of A. marginale (UFMG1) in the BME26 cell line derived originally from embryos of R. (Boophilus) microplus. The establishment of A. marginale infection in a cell line derived from R. (Boophilus) microplus is relevant for studying the A. marginale/tick interface., This work was supported by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo, Brazil), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), the Consejería de Educación y Ciencia, JCCM, Spain (project PAI06-0046-5285) and was facilitated through the Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3), financed by the International Cooperation Program of the European Union, coordination action project No. 510561.

Subolesin was recently shown in vaccine and RNA interference (RNAi) studies to protect against tick infestations and to affect tick feeding, reproduction, and development as well as infection of host cells by Anaplasma marginale and A. phagocytophilum. Recent experiments provided evidence that infection of both tick and vertebrate host cells with these two pathogens modified gene expression. We therefore hypothesized that infection of host cells with A. marginale and A. phagocytophilum affects expression of subolesin. Subolesin mRNA levels were determined by real-time reverse transcriptase (RT)-PCR in uninfected and A. marginale-infected Dermacentor variabilis guts and salivary glands and IDE8-cultured tick cells and in uninfected and A. phagocytophilum-infected Ixodes scapularis nymphs, ISE6-cultured tick cells, and the human cell line HL-60. In addition, the effect of subolesin on Anaplasma spp. infection/multiplication was characterized by RNAi in tick tissues and/or cultured tick and human cells. These experiments presented evidence of differential expression of subolesin in A. marginale- and A. phagocytophilum-infected cells. Subolesin was differentially expressed in A. marginale-infected ticks in a tissue-specific manner in which mRNA levels increased in response to A. marginale infection in tick salivary gland cells but not in the gut cells. Subolesin knockdown by RNAi reduced Anaplasma infection/multiplication only in cells in which infection increased subolesin expression, i.e., in A. marginale-infected D. variabilis salivary glands and IDE8 cells. The results reported herein further support the role of subolesin in Anaplasma-host interactions and suggest a putative role of subolesin in vaccines for the control of pathogen infection/multiplication in ticks.

Summary Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in South Africa and results in considerable economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle raised in communal and commercial farms in the north-eastern and south-western regions of the Free State Province, South Africa, that varied in rainfall and vegetation. Seroprevalence to A. marginale was determined in 755 cattle by an Anaplasma spp. competitive enzyme-linked immunosorbent assay and ranged from 44% to 98% and was similar in both regions. While Anaplasma centrale was not targeted in this study, A. marginale infections were identified by species-specific msp1α polymerase chain reaction in 129 of 215 of the samples studied. Similar genetic diversity of A. marginale strains was found in both the north-eastern and south-western regions. The sequences of 29 A. marginalemsp1α amplicons from South African strains revealed considerable genetic diversity providing 14 new repeat sequences. However, 42% of MSP1a repeat sequences were not unique to this region. These results indicated the presence of common genotypes between South African, American and European strains of A. marginale. Cattle movement between different parts of South Africa was suggested by the presence of identical A. marginale MSP1a genotypes in north-eastern and south-western regions of the Free State Province. Control strategies for anaplasmosis in South Africa should therefore be designed to be protective against genetically heterogeneous strains of A. marginale.

Human granulocytic anaplasmosis (HGA), an emerging disease of public health concern in many areas of the world, is caused by Anaplasma phagocytophilum. Small animal models of A phagocytophilum in laboratory mice have been developed and used to study the pathogenesis of HGA. In this study, we characterized the pathologic changes in acute infection of C3H/HeJ mice experimentally infected with the NY18 isolate of A phagocytophilum. Although no clinical signs were noted, acute infection was associated with gross splenomegaly, microscopic inflammatory lesions in the lung and liver, hyperplastic lesions on the spleen, and clinical pathology abnormalities including neutropenia and monocytosis. This study emphasizes the use of well-defined animal models as a valuable tool for the study of A phagocytophilum infections., This research was supported by the project No. 1669 of the Oklahoma Agricultural Experiment Station, the Endowed Chair for Food Animal Research (K. M. Kocan, College of Veterinary Medicine, Oklahoma State University), NIH Centers for Biomedical Research Excellence through a subcontract to J. de la Fuente from the Oklahoma Medical Research Foundation, and by the Georgia Department of Agriculture through a contract to the Athens Veterinary Diagnostic Laboratory, University of Georgia. C. Almazán is supported by grants-in-aid from the CONACYT and Promep (University of Tamaulipas), Mexico.

Antigens protective against Ixodes scapularis infestations were identified by cDNA expression library immunization (ELI) and analysis of expressed sequenced tags (EST). Three cDNAs protective against larval tick infestations, 4F8, with homology to a nucleotidase, and 4D8 and 4E6 of unknown function, were characterized and obtained as recombinant proteins for immunization studies. Vaccination trials with recombinant proteins demonstrated an effect of these antigens against I. scapularis larvae in a mouse model. Herein, we evaluated the effect of recombinant antigens on I. scapularis adult infestations on immunized sheep. Vaccination with recombinant 4D8, 4F8, 4E6 and the combination of all three antigens reduced adult tick infestations by 58, 12, 20, and 16%, respectively, when compared to the control group but was statistically significant for 4D8 and 4F8 only. Oviposition was reduced by 22–49% in all groups immunized with recombinant tick antigens (P < 0.05). The overall efficacy of vaccine formulations considering the effect on tick infestations and oviposition averaged 33–71%. These antigens, and especially 4D8, appear to be good candidates for continued development of a vaccine for control of tick infestations., This research was supported by Pfizer Animal Health, Kalamazoo, MI, USA, the Sitlington Endowed Chair (K.M. Kocan) and the Oklahoma Agricultural Experiment Station Project 1669. Consuelo Almazán was supported by Pfizer Animal Health, a grant-in-aid from the CONACYT and Promep (University of Tamaulipas), Mexico. We thank Drs. J.C. Garcia-Garcia (The Johns Hopkins University School of Medicine, Baltimore, MD, USA), Shalu Ayalew and Jeremiah T. Saliki (Veterinary Pathobiology, Oklahoma State University, OK, USA) for assistance with the purification of recombinant proteins and the ELISA with sheep sera, respectively.

Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important arthropod vector of pathogens in North America. Development of vaccines directed against tick proteins may effect reduction of tick infestations and transmission of tick-borne pathogens. The limiting step for the development of tick vaccines has been the identification of tick protective antigens. Reverse vaccinology approaches aimed at reducing animal experimentation while allowing for the rapid screening of pools of potential tick vaccine candidates would greatly facilitate progress towards the development of tick vaccines. Herein, we describe the screening of Ixodes scapularis cDNAs for identification of tick protective antigens using RNA interference (RNAi). The results of the RNAi screening were similar to those obtained previously using expression library immunization and demonstrated that RNAi could serve as a more rapid and cost-effective tool for vaccine antigen discovery in ticks and in other nonmodel organisms.

Summary Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) causes human, equine and canine granulocytic anaplasmosis and tick-borne fever of ruminants. The rickettsia parasitizes granulocytes and bone marrow progenitor cells, and can be propagated in human promyelocytic and tick cell lines. In this study, microarrays of synthetic polynucleotides of 21 329 human genes were used to identify genes that are differentially expressed in HL-60 human promyelocytic cells in response to infection with A. phagocytophilum. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) of selected genes confirmed the results of the microarray analysis. Six genes in the A. phagocytophilum-infected cells were found to be upregulated greater than 30-fold, while expression of downregulated genes most often did not change more than sixfold. Genes that were found to be differentially regulated in infected cells were those essential for cellular mechanisms including growth and differentiation, cell transport, signalling and communication and protective response against infection, some of which are most likely necessary for infection and multiplication of A. phagocytophilum in host cells. The differentially regulated genes described herein provide new information on the gene expression profiles in A. phagocytophilum-infected HL-60 cells, thus expanding in a global manner the existing information on the response of mammalian cells to A. phagocytophilum infection.

Infection of cells by tick-borne rickettsiae appears to be mediated by outer membrane proteins that allow pathogens to adhere to host cells. Major surface protein (MSP) 1a of Anaplasma marginale , the type species for the genus Anaplasma , was shown previously to be an adhesin for tick cells. The A. marginale MSP1a has a variable number of tandem 28 or 29 amino acid repeats located in the amino terminal region of the protein that contains an adhesion domain that is necessary and sufficient for infection of tick cells. The MSP1a studies demonstrated the importance of combining structural and functional characteristics for identification of adhesive proteins. In the present study other outer membrane proteins containing tandem repeats were selected from organisms of the family Anaplasmataceae and studied for their adhesive properties to tick cells. The adhesive properties and protein characteristics were then analyzed in order to provide a predictor of the adhesion function of proteins identified from genome sequences. Proteins selected included the A. marginale MSP1a, A. phagocytophilum 100 and 130 kDa, Ehrlichia chaffeensis 120 kDa, E. canis 140 kDa and E. ruminantium “mucin”, which were all cloned and expressed in Escherichia coli and then tested as adhesins for cultured IDE8 cells. Of the proteins studied, the A. marginale MSP1a and the E. ruminantium “mucin” were found to be adhesins for tick cells. Although all of these recombinant outer membrane proteins were glycosylated, the A. marginale MSP1a and E. ruminantium “mucin” adhesins shared a common feature of having a high Ser/Thr content in the tandem repeats. The results reported herein provide new information on the role of E. ruminantium “mucin” as an adhesin for tick cells and also suggest a role of glycans in adhesin molecules.

Identification of antigens that induce an immune response against tick infestations is required for the development of vaccines against these economically important ectoparasites. In order to identify protective antigens, we constructed a cDNA expression library from a continuous Ixodes scapularis cell line (IDE8) that was initially derived from tick embryos. cDNA clones were subjected to several rounds of screening in which mice were immunized with individual pools and then challenge-exposed by allowing I. scapularis larvae to feed on the immunized and control mice. Immunity against tick infestation was determined by the reduction in the ability of the larvae to feed to repletion and molt to the nymphal stage. Individual clones in pools that induced immunity to larval infestations were partially sequenced and grouped according to their putative protein function by comparison with sequence databases. The screening identified several individual antigens that induced a protective immune response against I. scapularis infestations. Our studies demonstrated for the first time that cDNA expression library immunization (ELI) combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in vaccines against ticks.

The major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been shown to mediate adhesion, infection and transmission of the organism, as well as to contribute to protective immunity in cattle. MSP1a contains a variable number of tandemly repeated peptides in the amino-terminal region, while the remainder of the protein is highly conserved among isolates. The number of repeats varies among geographic isolates of A. marginale but is constant within an isolate and has been used as a stable genetic marker of isolate identity. Because the sequence of the tandem repeats is the most variable part of the protein among isolates, this region of the protein is most likely to be involved in adhesion to host cells, a prerequisite to infection. The purpose of this study was to characterize the organization and function of the MSP1a tandem repeats of A. marginale in adhesion to host cells. We demonstrated by use of recombinant mutant proteins that the tandemly repeated region of MSP1a was necessary and sufficient to mediate adhesion of MSP1a to tick cells and bovine erythrocytes. Synthetic peptides representing the predominant sequences of individual repeats were tested for their adhesive capacity for tick cell extract (TCE). Peptides containing acidic amino acids D or E at position 20 bound to TCE, while peptides with a G as the 20th amino acid were not adhesive to TCE. Antibodies produced in rabbits against a synthetic repeat peptide neutralized A. marginale infection of cultured tick cells, and the neutralization observed was similar to that effected by antibodies produced against the whole MSP1a recombinant protein. Analysis of tandemly repeated MSP1a peptides of several geographic isolates of A. marginale revealed a complex relationship between the msp1alpha genotype and the tick-transmissible phenotype of the isolate and suggested that both the sequence and conformation of the repeated peptides influenced the adhesive properties of MSP1a. These studies demonstrated that the tandemly repeated region of the protein mediates the adhesive function of MSP1a.

A cell culture system for the tick-borne rickettsiaAnaplasma marginaleoffers new opportunities for research on this economically important pathogen of cattle.A. marginalemultiplies in membrane-bound inclusions in host cells. Whereas erythrocytes appear to be the only site of infection in cattle,A. marginaleundergoes a complex developmental cycle in ticks and transmission occurs via the salivary glands during feeding. We recently developed a cell culture system forA. marginaleusing a cell line derived from embryos ofIxodes scapularis. Here we review the use of this cell culture system for studying the interaction ofA. marginalewith tick cells. Several assays were developed using theA. marginale/tick cell system. An adhesion assay was developed for the identification of proteins required byA. marginalefor adhesion to tick cells. The effect of antibodies against selected major surface proteins in inhibitingA. marginaleinfection was tested in an assay that allowed further confirmation of the role of surface proteins in the infection of tick cells. A drug screening assay forA. marginalewas developed and provides a method of initial drug selection without the use of cattle. The culture system was used to test for enhancing effects of tick saliva and saliva components onA. marginaleinfection. The tick cell culture system has proved to be a good model for studyingA. marginale–tick interactions. Information gained from these studies may be applicable to other closely related tick-borne pathogens that have been propagated in the same tick cell line.

Anaplasmosis, a hemolytic disease of cattle caused by the tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been controlled using killed vaccines made with antigen harvested from infected bovine erythrocytes. We recently developed a cell culture system for propagation of A. marginale in a continuous tick cell line. In this study, we performed a cattle trial to compare the bovine response to vaccination with A. marginale harvested from tick cell culture or bovine erythrocytes. All immunized and control cattle were then challenge-exposed by allowing male Dermacentor variabilis infected with A. marginale to feed and transmit the pathogen. Nine yearling cattle (three per group) were used for this study and were immunized with cell culture-derived A. marginale, erythrocyte-derived A. marginale or received adjuvant only to serve as controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale and three immunizations were administered at weeks 1, 4 and 6. At week 8, cattle were challenge-exposed by allowing 60 D. variabilis male that were infected with A. marginale as adults to feed on the cattle. Antibody responses of cattle against major surface proteins (MSP) 1a, 1b and 5, as determined by ELISAs, peaked 2 weeks after the last immunization. Cattle immunized with infected IDE8 cell-derived antigens had a preferential recognition for MSP1b while cattle immunized with erythrocyte-derived antigens had a preferential recognition for MSP1a. Protection efficacy was evaluated using the percent infected erythrocytes (PPE), the packed cell volume (PCV), and the prepatent period. A. marginale-immunized cattle showed lower PPE and higher PCV values when compared to control animals and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.

Gene and protein sequences of major surface proteins (MSP) 1a and 4 of Anaplasma marginale (Rickettsiales: Anaplasmataceae) were used to infer phylogenetic relationships between New World isolates from Argentina, Brazil, Mexico and the United States. Seventeen isolates of A. marginale plus two outgroup taxa (A. centrale and A. ovis) were used for maximum-parsimony analysis of MSP4, while 20 isolates were used for phylogenetic analysis of MSP1a. msp4 analysis provided strong bootstrap support for a Latin American clade and, within this clade, support was detected for Mexican and South American clades. Isolates of A. marginale from the United States also grouped into two clades from the southern (isolates from Florida, Mississippi, and Virginia) and west-central (isolates from California, Idaho, Illinois, Oklahoma, and Texas) states. Although little phylogeographic resolution was detected within these higher clades, msp4 sequences appear to be a good genetic marker for inferring phylogeographic patterns of A. marginale isolates. In contrast to the phylogeographic resolution provided by msp4, MSP1a DNA and protein sequence were quite variable and did not provide phylogeographic resolution. Most variation in MSP1a sequences appeared unique to a given isolate and similar DNA sequence variation in msp1alpha was detected within isolates from Idaho and Florida and from Idaho and Argentina. The results of these studies demonstrated that msp4 provided phylogenetic information on the evolution of A. marginale isolates. In contrast MSP1a sequences appeared to be rapidly evolving and these sequences may provide phylogeographic information only when numerous isolate MSP1a sequences are analyzed from a geographic area.

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.

The major surface protein (MSP) 1a of the ehrlichial cattle pathogenAnaplasma marginale, encoded by the single-copy genemsp1α, has been shown to have a neutralization-sensitive epitope and to be an adhesin for bovine erythrocytes and tick cells.msp1αhas been found to be a stable genetic marker for the identification of geographic isolates ofA. marginalethroughout development in acutely and persistently infected cattle and in ticks. The molecular weight of MSP1a varies among geographic isolates ofA. marginalebecause of a varying number of tandemly repeated peptides of 28–29 amino acids. Variation in the sequence of the tandem repeats occurs within and among isolates, and may have resulted from evolutionary pressures exerted by ligand–receptor and host–parasite interactions. These repeated sequences include markers for tick transmissibility that may be important in the identification of ehrlichial pathogens because they may influence control strategies and the design of subunit vaccines.

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.

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 1 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-2or 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. Anaplasmosis in cattle is caused by Anaplasma marginale ,a member of the ehrlichial genogroup II within the order Rickettsiales, which is transmitted biologically by ixodid ticks and mechanically by biting flies. A. marginale is genetically very closely related to other tick-borne ehrlichiae, including the human pathogens Ehrlichia chaffeensis and the agent of human granulocytic ehrlichiosis (16, 40). These ehrlichiae induce disease typified by an acute onset which can rapidly progress to death. A. marginale replicates exclusively within bovine erythrocytes (RBC), which results in severe anemia during acute infection. Immunization of calves with A. marginale outer membranes can induce complete protection from detectable rickettsemia against a virulent homologous challenge (39). Furthermore

Serologic diagnosis of anaplasmosis is currently done by the complement-fixation, ELISA, and card agglutination tests. These tests have utilized A. marginale harvested from bovine erythrocytes as antigen which is often contaminated with erythrocyte stroma. We are currently testing A. marginale propagated in a Ixodes scapularis cell line as antigen for serologic tests. In this study, we report the use of the cell culture-derived A. marginale as antigen for development of a rapid, semi-automated latex agglutination test. Diluted serum and latex (polystyrene microspheres), sensitized with cell culture-derived A. marginale proteins, were dispensed into 96-well microtiter plates. An initial reading of light transmission was recorded by a computer-interfaced scanning autoreader. After 30 minutes, the plates were mixed and read a second time, recording the delta % light transmittance. The sensitized latex microspheres (latex) agglutinated in the presence of A. marginale antibodies, thus producing an increase in light transmittance. In preliminary tests, 724/977 of the sera were positive for A. marginale antibodies with an apparent agreement of 83.3% when compared with the complement-fixation test. Sensitization and sera dilution buffers were shown to have a marked effect on the sensitivity and specificity of this assay. Results will be presented on the optimization of buffers and the testing of sera from experimentally and field-infected cattle.

Anaplasma marginale was propagated in a continuous tick cell line and detergent-solubilized infected cells were used as antigen in a competitive ELISA (C-ELISA) for detection of Anaplasma-specific antibody in bovine sera. Positive control sera competed well (> or = 35% inhibition) with an A. marginale-specific monoclonal antibody for binding to this antigen, while negative sera failed to compete (< 35% inhibition). The C-ELISA was compared to the standard complement-fixation test (CFT) using 2,208 bovine sera. Overall, C-ELISA was more sensitive than CFT (24.9% versus 9.4%), mainly because CFT yielded "suspicious" or "anti-complementary" results in 10.5% of the sera and also failed to identify several vaccinated and carrier cattle that were C-ELISA-positive. The apparent agreement between CFT and C-ELISA was 89.6% and the kappa value was 0.6. These results show that this C-ELISA would be a suitable replacement of the CFT as the standard test for detection of A. marginale antibody.

In situ hybridization (ISH), which allows localization of pathogen nucleic acid in tissue sections, was used to detect Anaplasma Marginale, a rickettsia1 pathogen of cattle, in its tick vector. Dermacentor andersoni male ticks were experimentally infected with A. marginale, and one-half of each of 20 ticks was emedded in paraffin or LR White for ISH: Companion halves were embedded in DER resin for light microscopy (LM). Sections were digested with proteinase K and hybridized with a digoxigenin-labeled DNA probe. In both paraffin and LR White embedded sections, dark-blue color precipitates of hybridization signals were visualized in both salivary gland and gut cells. Anaplasma infections were also confirmed by LM in companion tick halves. Positive hybridization signals and A. marginale colonies were not seen in uninfected control tissues. Although ISH detected A. marginale in both paraffin and LR White embedded sections, LR White was found to be optimum for ISH of A. marginale because of improved m...

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Anaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts., This research was supported by grants BFU2008-01244/BMC and BFU2011-23896 and CSIC intramural project PA1002451 to J.D.L.F., EU FP7 ANTIGONE project number 278976, and Oklahoma Agricultural Experiment grant 1669 and the Walter R. Sitlington Endowed Chair for Food Animal Research to K.M.K. M. Villar was funded by the JAE-DOC program (CSIC-FSE), Spain. N. Ayllón and R. C. Galindo were funded by MEC, Spain.

[Background]: Tick Subolesin and its ortholog in insects and vertebrates, Akirin, have been suggested to play a role in the immune response through regulation of nuclear factor-kappa B (NF-kB)-dependent and independent gene expression via interaction with intermediate proteins that interact with NF-kB and other regulatory proteins, bind DNA or remodel chromatin to regulate gene expression. The objective of this study was to characterize the structure and regulation of subolesin in Ixodes scapularis. I. scapularis is a vector of emerging pathogens such as Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti that cause in humans Lyme disease, anaplasmosis and babesiosis, respectively. The genome of I. scapularis was recently sequenced, and this tick serves as a model organism for the study of vector-host-pathogen interactions. However, basic biological questions such as gene organization and regulation are largely unknown in ticks and other arthropod vectors. [Principal Findings]: The results presented here provide evidence that subolesin/akirin are evolutionarily conserved at several levels (primary sequence, gene organization and function), thus supporting their crucial biological function in metazoans. These results showed that NF-kB (Relish) is involved in the regulation of subolesin expression in ticks, suggesting that as in other organisms, different NF-kB integral subunits and/or unknown interacting proteins regulate the specificity of the NF-kB-mediated gene expression. These results suggested a regulatory network involving cross-regulation between NF-kB (Relish) and Subolesin and Subolesin auto-regulation with possible implications in tick immune response to bacterial infection. [Significance]: These results advance our understanding of gene organization and regulation in I. scapularis and have important implications for arthropod vectors genetics and immunology highlighting the possible role of NF-kB and Subolesin/Akirin in vector-pathogen interactions and for designing new strategies for the control of vector infestations and pathogen transmission., This research was supported by grants BFU2008-01244/BMC and BFU2011-23896 to JF, the Oklahoma Agricultural Experimental Grant 1669 and the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, and the EU FP7 ANTIGONE project number 278976. V. Naranjo was funded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007-2013), Spain. N. Ayllón was funded by Ministerio de Educación y Ciencia (MEC), Spain.

Diseases transmitted by arthropod vectors such as mosquitoes, ticks and sand flies greatly impact human and animal health, and therefore, their control is important for the eradication of vectorborne diseases (VBD). Vaccination is an environmentally friendly alternative for vector control that allows control of several VBD by targeting their common vector. Recent results have suggested that subolesin (SUB) and its orthologue in insects, akirin (AKR) are good candidate antigens for the control of arthropod vector infestations and pathogen infection. SUB was discovered as a tick-protective antigen in Ixodes scapularis. Vaccination trials with recombinant SUB/AKR demonstrated effective control of arthropod vector infestations in various hard and soft tick species, mosquitoes, sand flies, poultry red mites and sea lice by reducing their numbers, weight, oviposition, fertility and/or moulting. SUB/AKR vaccination also reduced tick infection with tickborne pathogens, Anaplasma phagocytophilum, A. marginale, Babesia bigemina and Borrelia burgdorferi. The effect of vaccination on different hosts, vector species, developmental stages and vectorborne pathogen infections demonstrated the feasibility of SUB/AKR universal vaccines for the control of multiple vector infestations and for reduction in VBD., This research was supported by Grants BFU2011-23896,FAU2008-00014-00-00 and PEII09-0118-8907 to JF, andthe Walter R. Sitlington Endowed Chair for Food Animal Research to KMK. M. Villar was funded by the JAE-DOCprogramme (CSIC-FSE), Spain. O. Merino is an Early Stage Researcher supported by the POSTICK ITN (Postgraduatetraining network for capacity building to control ticks andt ickborne diseases) within the FP7-PEOPLE – ITN pro-gramme (EU Grant No. 38511).

Anaplasma phagocytophilum, first identified as a pathogen of sheep in Europe, more recently has been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. Transmission of A. phagocytophilum is reported to be by ticks, primarily of the genus Ixodes. While mechanical and transplacental transmission of the type genus organism, A. marginale, occur in addition to tick transmission, these modes of transmission have not been considered for A. phagocytophilum. Recently, we developed a sheep model for studying host-tick-pathogen interactions of the human NY-18 A. phagocytophilum isolate. Sheep were susceptible to infection with this human isolate and served as a source of infection for I. scapularis ticks, but they did not display clinical signs of disease, and the pathogen was not apparent in stained blood smears. In the course of these experiments, one sheep unexpectedly gave birth to a lamb 5 weeks after being experimentally infected by inoculation with the pathogen propagated in HL-60 cells. The lamb was depressed and not feeding and was subsequently euthanized 18 h after birth. Tissues were collected at necropsy for microscopic examination and PCR to confirm A. phagocytophilum infection. At necropsy, the stomach contained colostrum, the spleen was moderately enlarged and thickened with conspicuous lymphoid follicles, and mesenteric lymph nodes were mildly enlarged and contained moderate infiltrates of eosinophils and neutrophils. Blood, spleen, heart, skin and cervical and mesenteric lymph nodes tested positive for A. phagocytophilum by PCR, and sequence analysis confirmed that the lamb was infected with the NY-18 isolate. Transplacental transmission should therefore be considered as a means of A. phagocytophilum transmission and may likely contribute to the epidemiology of tick-borne fever in sheep and other mammals, including humans., This research was supported by the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, Center for Veterinary Health Sciences, Research Advisory Committee grants to KMK, RCG and the Spanish Ministerio de Ciencia e Innovación (MICINN) project BFU2008-01244/BMC to JF.

Anaplasma phagocytophilum, first identified as a pathogen of ruminants in Europe, has more recently been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. . A. phagocytophilum is transmitted by . Ixodes spp., but the tick developmental cycle and pathogen/vector interactions have not been fully described. In this research, we report on the experimental infection of sheep with the human NY-18 isolate of . A. phagocytophilum which then served as a host for infection of . I. scapularis nymphs and adults. . A. phagocytophilum was propagated in the human promyelocytic cell line, HL-60, and the infected cell cultures were then used to infect sheep by intravenous inoculation. Infections in sheep were confirmed by PCR and an . Anaplasma-competitive ELISA. Clinical signs were not apparent in any of the infected sheep, and only limited hematologic and mild serum biochemical abnormalities were identified. While . A. phagocytophilum morulae were rarely seen in neutrophils, blood film evaluation revealed prominent large granular lymphocytes, occasional plasma cells, and rare macrophages. Upon necropsy, gross lesions were restricted to the lymphoid system. Mild splenomegaly and lymphadenomegaly with microscopic evidence of lymphoid hyperplasia was observed in all infected sheep. Female . I. scapularis that were allowed to feed and acquire infection on each of the 3 experimentally infected sheep became infected with . A. phagocytophilum as determined by PCR of guts (80-87%) and salivary glands (67-100%). Female . I. scapularis that acquired infection as nymphs on an experimentally infected sheep transmitted . A. phagocytophilum to a susceptible sheep, thus confirming transstadial transmission. Sheep proved to be a good host for the production of . I. scapularis infected with this human isolate of . A. phagocytophilum, which can be used as a model for future studies of the tick/pathogen interface., This research was supported by the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, a Center for Veterinary Health Sciences, Research Advisory Committee grant, and the Spanish Ministerio de Ciencia e Innovación (MICINN) project BFU2008-01244/BMC to JF.

Video Article: http://www.jove.com/video/2474, Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases and the most important vectors affecting cattle industry worldwide. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health. RNA interference (RNAi) is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications. RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable. The genetic characterization of ticks has been limited until the recent application of RNAi. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens., This video presentation was supported by the Associate Dean for Research and the Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University. The research was funded by the Ministerio de Ciencia e Innovación, Spain (project BFU2008-01244/BMC), the CSIC intramural project PA1002451 to JF, the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, CVHS 2009 RAC grant, OAES Animal Health Funds and USDA, National Research Initiative Competitive Grant, No. 2007-04613.

12 pages, 5 figures, 3 tables.-- et al., [Background]: Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF)., [Results]: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism., [Conclusions]: Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks., This research was supported by the Ministerio de Ciencia e Innovación, Spain (project BFU2008-01244/BMC), the INIA, Spain (project FAU2008-00014- 00-00), the Ministry of Health, Italy (project IZS SI 10-06), the CSIC intramural projects 200830I249 and PA1002025 to JF, the Oklahoma Agricultural Experiment Station (project 1669), the Walter R. Sitlington Endowed Chair for Food Animal Research (K. M. Kocan, Oklahoma State University) and facilitated by ICTTD-3, financed by the International Cooperation Program of the European Union, coordination action project No. 510561. M. Villar was funded by the JAE-DOC program (CSIC-FSE), Spain.

The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs from multigene families that undergo antigenic change and selection in cattle, thus contributing to maintenance of persistent infections. Many geographic strains of A. marginale have been identified worldwide, which vary in genotype, antigenic composition, morphology and infectivity for ticks. Isolates of A. marginale may be maintained by independent transmission events and a mechanism of infection/exclusion in cattle and ticks. The increasing numbers of A. marginale genotypes identified in some geographic regions most likely resulted from intensive cattle movement. However, concurrent A. marginale strain infections in cattle was reported, but these strains were more distantly related. Phylogenetic studies of selected geographic isolates of A. marginale, using msp4 and msp1α, provided information about the biogeography and evolution of A. marginale, and msp1α genotypes appear to have evolved under positive selection pressure. Live and killed vaccines have been used for control of anaplasmosis and both types of vaccines have advantages and disadvantages. Vaccines have effectively prevented clinical anaplasmosis in cattle but have failed to block A. marginale infection. Vaccines are needed that can prevent clinical disease and, simultaneously, prevent infection in cattle and ticks, thus eliminating these hosts as reservoirs of infection. Advances in genomics, proteomics, immunology and biochemical and molecular technologies during the last decade have been applied to research on A. marginale and related organisms, and the recent development of a cell culture system for A. marginale has provided a format for studying the pathogen/tick interface. Recent advancements and new research methodologies should provide additional opportunities for development of new strategies for control and prevention of bovine anaplasmosis., This research was supported by the project No. 1669 of the Oklahoma Agricultural Experiment Station, the Sitlington Endowed Chair for Food Animal Research (K.M. Kocan) and the Instituto de Ciencias de la Salud, Spain (ICS-JCCM) (project 03052-00).

Ticks are ectoparasites of animals and humans that serve as vectors of Anaplasma and other pathogens that affect humans and animals worldwide. Ticks and the pathogens that they transmit have coevolved molecular interactions involving genetic traits of both the tick and the pathogen that mediate their development and survival. In this paper, the expression of heat shock proteins (HSPs) and other stress response proteins (SRPs) was characterized in ticks and cultured tick cells by proteomics and transcriptomics analyses in response to Anaplasma spp. infection and heat shock. The results of these studies demonstrated that the stress response was activated in ticks and cultured tick cells after Anaplasma spp. infection and heat shock. However, in the natural vector-pathogen relationship, HSPs and other SRPs were not strongly activated, which likely resulted from tick-pathogen coevolution. These results also demonstrated pathogen- and tick-specific differences in the expression of HSPs and other SRPs in ticks and cultured tick cells infected with Anaplasma spp. and suggested the existence of post-transcriptional mechanisms induced by Anaplasma spp. to control tick response to infection. These results illustrated the complexity of the stress response in ticks and suggested a function for the HSPs and other SRPs during Anaplasma spp. infection., This research was supported by the Ministerio de Ciencia e Innovacion, Spain (Project no. BFU2008-01244/BMC), the CSIC intramural Project no. PA1002451 to J. Fuente, and the Walter R. Sitlington Endowed Chair for Food Animal Research to K. M. Kocan M. Villar and R. C. Galindo were funded by the JAE-DOC program (CSIC-FSE) and MEC, Spain, respectively.

Development of tick vaccines provides new opportunities for control of tick infestations and tick-borne diseases. Recently, the tick-protective protein, subolesin, was identified in a cell line derived from Ixodes scapularis by expression library immunization and a mouse model of tick infestations. While subolesin was conserved among ixodid tick species, the biological function of this gene is unknown. Subolesin expression in ticks was silenced by RNA interference (RNAi) to provide information on the gene's function, and silencing of subolesin profoundly impacted tick survival, feeding, and reproduction. In this research we used RNAi in the IDE8 tick cell line to further study the role of subolesin in development of cultured tick cells. The cells were incubated with subolesin double-stranded (ds)RNA and cell growth was monitored. Incorporation of dsRNA by tick cells was monitored with Cy3-labeled dsRNA. After 72 h cells were harvested for cell counts, morphology, and for confirmation of gene silencing by reverse transcriptase-PCR. While the expression of subolesin in treated cells was reduced 80 +/- 9% by RNAi as compared with mock-treated cells, cell growth did not appear to be affected over the 72-h period. This is the first report of the use of RNAi in tick cell culture. RNAi is a powerful tool for studying tick gene function and will likely contribute to our understanding of the role that tick genes play in cell development and infection with pathogens.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells., This research was supported by the Ministerio de Ciencia e Innovación, Spain (project BFU2008-01244/BMC), the CSIC intramural project 200830I249 and PA1002025 to JF, the Oklahoma Agricultural Experiment Station (project 1669), and the Sitlington Endowed Chair for Food Animal Research (K. M. Kocan, Oklahoma State University). V. Naranjo was founded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007-2013), Spain. Raúl Manzano-Roman was funded by Ministerio de Educación y Ciencia, Spain.

Cytauxzoon felis was transmitted to a domestic cat by Amblyomma americanum. The infection was produced by the bite of A. americanum adults that were acquistion fed as nymphs on a domestic cat that naturally survived infection of C. felis. Fever, inappetence, depression, and lethargy were first noted 11 days post-infestation (dpi). Pale mucus membranes, splenomegaly, icterus, and dyspnea were also observed during the course of the disease. The body temperature of the experimentally infected C. felis cat was subnormal from 16 dpi until 24 dpi when it returned to within normal limits. All clinical signs of cytauxzoonsis began to resolve by 23 dpi when the cat became subclinically infected with C. felis. The cat developed a marked, regenerative anemia beginning by 13 dpi and reached a nadir at 20 dpi before recovering. A moderate neutrophilia and marked lymphocytosis also developed between 18 and 26 dpi. Schizonts of C. felis were observed in spleen aspirates of the infected cat at 15 dpi. DNA of C. felis was amplified by real-time PCR starting 17 dpi and piroplasms of C. felis were first noted by light microscopy 18 dpi. Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus were also tested in a similar manner at the same time but did not transmit C. felis. Prior to the present study, only D. variabilis had been shown experimentally to transmit infection of C. felis. This is the first report of C. felis being transmitted by A. americanum. The transmission of C. felis infection from one domestic cat to another indicates that domestic cats subclinically infected with C. felis may be a reservoir of infection for naive domestic cats.

Four white-tailed deer were inoculated with either the Ap-V1 or NY-18 strain of Anaplasma phagocytophilum. Ixodes scapularis nymphs were then allowed to acquistion feed on the inoculated deer and molt to adults. Only an Ap-V1 infected deer was infected persistently and able to infect nymphal Ixodes scapularis. Molted adult ticks maintained Ap-V1 infection as demonstrated by PCR and microscopy. We report, for the first time, a morphologic description of A. phagocytophilum in I. scapularis., The present project was funded by the Center for Veteri-nary Health Sciences, Oklahoma State University, project No.1669 of the Oklahoma Agricultural Experiment Station, andthe Sitlington Endowed Chair for Food Animal Research (K.M. Kocan).

This article belongs to the Special Issue Biology of ticks., Ticks are ectoparasites of wild and domestic animals and humans that most notably impact global health by transmitting disease-causing pathogens. While information on the molecular interactions between ticks and pathogens that facilitate pathogen infection, development and transmission is limited, a comprehensive understanding of the tick-pathogen interface would be fundamental toward development of new and novel measures for control of both tick infestations and tick-borne pathogens. Recently, vaccine studies using key tick antigens and characterization of tick gene function by RNA interference (RNAi) have provided new information on genes that impact the tick-pathogen interface. In this review we summarize current research and prospects of tick vaccines and genetic manipulation of ticks targeted to the tick-pathogen interface. The knowledge gained from these collective studies will be fundamental toward understanding of tick-pathogen interactions and for formulation of control methods targeted at both ticks and tick-borne pathogens. Use of these molecular approaches will likely contribute to control measures that will notably reduce tick populations and tick-borne diseases in the future., This work was supported by the project No. 1669 of the Oklahoma Agricultural Experiment Station and the Endowed Chair for Food Animal Research (K.M. Kocan), the Instituto de Ciencias de la Salud, Spain (ICS-JCCM) (project 06036-00), the Consejería de Educación y Ciencia, JCCM, Spain (project PAI06-0046-5285), and the Wellcome Trust under the Animal Health in the Developing World initiative through project 0757990 entitled “Adapting recombinant anti-tick vaccines to livestock in Africa”.

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.

[Background] Subolesin is an evolutionary conserved protein that was discovered recently in Ixodes scapularis as a tick protective antigen and has a role in tick blood digestion, reproduction and development. In other organisms, subolesin orthologs may be involved in the control of developmental processes. Because of the profound effect of subolesin knockdown in ticks and other organisms, we hypothesized that subolesin plays a role in gene expression, and therefore affects multiple cellular processes. The objective of this study was to provide evidence for the role of subolesin in gene expression., [Results] Two subolesin-interacting proteins were identified and characterized by yeast two-hybrid screen, co-affinity purification and RNA interference (RNAi). The effect of subolesin knockdown on the tick gene expression pattern was characterized by microarray analysis and demonstrated that subolesin RNAi affects the expression of genes involved in multiple cellular pathways. The analysis of subolesin and interacting protein sequences identified regulatory motifs and predicted the presence of conserved protein kinase C (PKC) phosphorylation sites., [Conclusion] Collectively, these results provide evidence that subolesin plays a role in gene expression in ticks., We thank A. Taoufik (UCTD) for technical assistance. This research was supported by the Oklahoma Agricultural Experiment Station (project 1669), the Walter R. Sitlington Endowed Chair for Food Animal Research (K. M. Kocan, Oklahoma State University), Pfizer Animal Health, Kalamazoo, MI, USA, the Junta de Comunidades de Castilla-La Mancha, Spain (project 06036-00 ICS-JCCM), and the Wellcome Trust under the "Animal Health in the Developing World" initiative (project 0757990). V. Naranjo was funded by Consejería de Educación, JCCM, Spain. R.C. Galindo was funded by Ministerio de Educación y Ciencia, Spain.

Bovine anaplasmosis is a tick-borne hemolytic disease of cattle that occurs worldwide caused by the intraerythrocytic rickettsiae Anaplasma marginale. Control measures, including use of acaricides, administration of antibiotics and vaccines, have varied with geographic location. Our research is focused on the tick-pathogen interface for development of new vaccine strategies with the goal of reducing anaplasmosis, tick infestations and the vectorial capacity of ticks. Toward this approach, we have targeted (1) development of an A. marginale cell culture system to provide a non-bovine antigen source, (2) characterization of an A. marginale adhesion protein, and (3) identification of key tick protective antigens for reduction of tick infestations. A cell culture system for propagation of A. marginale was developed and provided a non-bovine source of A. marginale vaccine antigen. The A. marginale adhesion protein, MSP1a, was characterized and use of recombinant MSP1a in vaccine formulations reduced clinical anaplasmosis and infection levels in ticks that acquired infection on immunized cattle. Most recently, we identified a tick-protective antigen, subolesin, that reduced tick infestations, as well as the vectorial capacity of ticks for acquisition and transmission of A marginale. This integrated approach to vaccine development shows promise for developing new strategies for control of bovine anaplasmosis.

Over 40 cell lines are currently available from 13 ixodid and one argasid tick species. The successful isolation and propagation of several economically important tick-borne pathogens in tick cell lines has created a useful model to study interactions between tick cells and these viral and bacterial disease agents. Tick cell lines have already proved to be a useful tool in helping to define the complex nature of the host–vector–pathogen relationship. With the availability of genomics tools, tick cell lines will become increasingly important as a complement to tick and tick-borne disease research in vivo once genetic transformation and gene silencing using RNA interference become routine.

[Background] The tick-borne intracellular pathogen, Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) causes human granulocytic anaplasmosis after infection of polymorphonuclear leucocytes. The human Sp110 gene is a member of the nuclear body (NB) components that functions as a nuclear hormone receptor transcriptional coactivator and plays an important role in immunoprotective mechanisms against pathogens in humans. In this research, we hypothesized that Sp110 may be involved in the infection of human promyelocytic HL-60 cells with A. phagocytophilum., [Methods] The human Sp110 and A. phagocytophilum msp4 mRNA levels were evaluated by realtime RT-PCR in infected human HL-60 cells sampled at 0, 12, 24, 48, 72 and 96 hours postinfection. The effect of Sp110 expression on A. phagocytophilum infection was determined by RNA interference (RNAi). The expression of Sp110 was silenced in HL-60 cells by RNAi using predesigned siRNAs using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Gaithersburg, MD, USA). The A. phagocytophilum infection levels were evaluated in HL-60 cells after RNAi by realtime PCR of msp4 and normalizing against human Alu sequences., [Results] While Sp110 mRNA levels increased concurrently with A. phagocytophilum infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels., [Conclusion] These results demonstrated that Sp110 expression is required for A. phagocytophilum infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which A. phagocytophilum modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells., This work was supported by the Oklahoma Agricultural Experiment Station (project 1669), the Sitlington Endowed Chair for Food Animal Research to K.M.K. and the Ministry of Science and Education (MEC), Spain (project AGL2005-07401). Dr. Raúl Manzano-Roman was funded by Ministerio de Educación y Ciencia, Spain. V. Naranjo was founded by Consejería de Educación, JCCM, Spain.

Ticks transmit pathogens that cause diseases which greatly impact both human and animal health. Vaccines developed against Boophilus spp. using Bm86 and Bm95 tick gut antigens demonstrated the feasibility of using vaccines for control of tick infestations. These vaccines also reduced transmission of tick-borne pathogens by decreasing exposure of susceptible hosts to ticks. The recently discovered tick antigens, 64P putative cement protein and subolesin involved in the regulation of tick feeding and reproduction, were also shown to reduce tick infestations. These antigens, together with the TROSPA receptor for Burrelia burgdorferi OspA were effective against tick-borne pathogens by reducing the infection levels in ticks and/or the transmission of the pathogen. Development of a vaccine targeted at both the tick vector and pathogen would contribute greatly to the control of tick infestations and the transmission of tick-borne diseases. These results have demonstrated that tick vaccines can be developed for control tick infestations and show promise for the prevention of the transmission of tick-borne pathogens.

The coevolution of ticks and the pathogens that they transmit has ensured their mutual survival. In these studies, we used a functional genomics approach to characterize tick genes regulated in response to Anaplasma marginale infection. Differentially regulated genes/proteins were identified by suppression-subtractive hybridization and differential in-gel electrophoresis analyses of cultured IDE8 tick cells infected with A. marginale. Nine of 17 of these genes were confirmed by real-time RT-PCR to be differentially regulated in ticks and/or IDE8 tick cells in response to A. marginale infection. RNA interference was used for functional studies. Six genes, which encode putative selenoprotein W2a, hematopoietic stem/progenitor cells protein-like, proteasome 26S subunit, ferritin, GST, and subolesin control, were found to affect A. marginale infection in IDE8 tick cells. Four genes, which encode putative GST, salivary selenoprotein M, vATPase, and ubiquitin, affected A. marginale infection in different sites of development in ticks. The results of these studies demonstrated that a molecular mechanism occurs by which tick cell gene expression mediates the A. marginale developmental cycle and trafficking through ticks., This research was partially supported by the Oklahoma Agricultural Experiment Station (Project 1669), the Walter R. Sitlington Endowed Chair for Food Animal Research (K.M. Kocan, Oklahoma State University), Pfizer Animal Health (Kalamazoo, MI, USA), the Junta de Comunidades de Castilla-La Mancha, Spain (Project 06036-00 ICS-JCCM), the Ministry of Science and Education, Spain (Project AGL2005-07401), and the Wellcome Trust under the “Animal Health in the Developing World” initiative (Project 0757990).