Infection of endothelial cells with Anaplasma marginale and A. phagocytophilum

Anaplasma marginale and A. phagocytophilum are obligate intracellular, tick-borne pathogens that target erythrocytes and neutrophil granulocytes, respectively. Because ticks do not directly tap blood vessels, an intermediate tissue may mediate infection of blood cells. We considered that vascular endothelium interacts with circulating blood cells in vivo, and could be involved in pathogenesis and dissemination of the organisms. We used light and electron microscopy and immune labeling to show that A. phagocytophilum invaded rhesus (RF/6A), human (HMEC-1, MVEC), as well as bovine (BCE C/D-1b) endothelial cell lines, whereas A. marginale infected rhesus and bovine endothelial cells. A. marginale formed large intracellular inclusions that appeared smooth and solid at first, and subsequently coalesced into discrete granules. A. phagocytophilum formed numerous smaller inclusions in each cell. Within 1-3 weeks, the monolayers were destroyed, and lysed cultures were diluted onto fresh monolayers. Electron microscopy demonstrated uneven distribution of A. marginale inside large inclusions, with reticulated forms grouped more tightly than denser cells, whereas in A. phagocytophilum individual organisms appeared more evenly spaced. Specific polyclonal and monoclonal antibodies both labeled A. marginale and A. phagocytophilum in endothelial cells, and oligonucleotide primers complimentary to either A. marginale or A. phagocytophilum amplified their expected target from these cultures. In conclusion, we demonstrate that relevant microvascular endothelium is susceptible to anaplasmas in vitro and may present a link that could explain development of the immune response and persistent infection.     

Investigation of endothelial cells as an in vivo nidus of Anaplasma marginale infection in cattle

Continuous culture of Anaplasma marginale in endothelial cells and the potential implications for vaccine development heightened interest in determining the importance of endothelial cells in the A. marginale life cycle. A. marginale-infection trials were performed to determine if endothelial cells are an in vivo host cell in cattle and if A. marginale from in vitro endothelial cells were infective to cattle. Adult, immunocompetent steers were infected by tick-feeding transmission and were euthanized at different points in the parasitemic cycle. Based on quantitative PCR, the tissue distribution of A. marginale DNA during peak and trough parasitemia was variable with higher quantities observed in spleen, lung, hemal nodes, and abomasum. A. marginale was not conclusively identified in tissue endothelial cells from the steers' tick-bitten dermis or post-mortem tissues using three microscopy techniques (dual indirect immunofluorescence, transmission electron microscopy, and in situ DNA target-primed rolling-circle amplification of a padlock probe). Intravenous inoculation of spleen-intact or splenectomized calves with endothelial cell culture-derived VA isolate A. marginale did not cause seroconversion or clinical anaplasmosis regardless of whether the endothelial culture-derived bacteria were inoculated as host cell-free organisms or within endothelial cells and regardless of the type of endothelial cell culture used - RF/6A primate endothelial cells or primary bovine testicular vein endothelial cells. Data presented here suggest that endothelial cells are likely not a pivotal component of the A. marginale life cycle in vivo.     

Applications of a cell culture system for studying the interaction of Anaplasma marginale with tick cells

A cell culture system for the tick-borne rickettsia Anaplasma marginale offers new opportunities for research on this economically important pathogen of cattle. A. marginale multiplies in membrane-bound inclusions in host cells. Whereas erythrocytes appear to be the only site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks and transmission occurs via the salivary glands during feeding. We recently developed a cell culture system for A. marginale using a cell line derived from embryos of Ixodes scapularis. Here we review the use of this cell culture system for studying the interaction of A. marginale with tick cells. Several assays were developed using the A. marginale/tick cell system. An adhesion assay was developed for the identification of proteins required by A. marginale for adhesion to tick cells. The effect of antibodies against selected major surface proteins in inhibiting A. marginale infection 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 for A. marginale was 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 on A. marginale infection. The tick cell culture system has proved to be a good model for studying A. 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.     

Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells

Major surface protein 1 (MSP1) of the cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is a complex of two proteins, MSP1a and MSP1b. Previous studies demonstrated that MSP1a and MSP1b are adhesins for bovine erythrocytes, while only MSP1a proved to be an adhesin for tick cells. In this study, a tick cell culture system for propagation of A. marginale was used to develop an infection inhibition assay for testing the ability of antisera to block infection of A. marginale for cultured tick cells. A. marginale derived from cell culture was incubated with various antisera prior to inoculation onto cell monolayers. The monolayers were harvested 7 days post-inoculation and A. marginale in the cultures was quantified using an antigen detection ELISA. Antisera tested in the infection inhibition assay were derived from persistently infected cattle, from cattle immunized with A. marginale purified from bovine erythrocytes, and from rabbits and cattle that were immunized with the recombinant MSP1a, MSP1b and MSP1 complex. Antibodies from cattle persistently infected with A. marginale, cattle immunized with A. marginale from bovine erythrocytes or cattle immunized with the recombinant MSP1 complex did not inhibit the infectivity of A. marginale for tick cells. Antiserum from rabbits immunized with MSP1a and MSP1b (individually or combined) reduced infection of both the Virginia and Oklahoma isolates of A. marginale for tick cells by 25-70%. Likewise, antisera from cattle immunized with recombinant MSP1a or MSP1b inhibited infection of tick cells by 26-37%. These results further confirm the role of MSP1 complex proteins in infection of tick cells. Lack of inhibition of infection by antisera from naturally infected cattle or cattle immunized with whole organisms suggests that the bovine immune response is not directed toward blocking infection of A. marginale for tick cells and may contribute to the continued infectivity of the pathogen for ticks.     

Antigenic analysis of Anaplasma marginale grown in bovine erythrocytes co-cultured with bovine endothelial cells

Two monoclonal antibodies (mAbs) for A. marginale were used to test the antigenic integrity of A. marginale grown in vitro in bovine erythrocytes co-cultured with endothelial cells. Both the mAbs reacted in the indirect immunofluorescent antibody test with A. marginale grown in vitro and also detected the antigens in Western immunoblots of SDS-PAGE separated antigens made from A. marginale infected erythrocytes from the cultures. Furthermore, active replication was evident as [35S]-methionine is incorporated by A. marginale present in the second passage of a culture maintained for six weeks as shown by immunoprecipitation of labeled antigens by the mAbs. This indicates that A. marginale grown in the in vitro culture system described previously [Waghela et al., Vet. Parasitol. 73 (1997) 43] maintain antigenic character, and with further development the system can be used for preparing immunogens or diagnostic antigens.     

Susceptibility of Bos indicus and Bos taurus to Anaplasma marginale and Babesia bigemina infections

The reaction of Bos taurus and pure-bred Bos indicus heifers to infection with the intraerythrocytic parasites Anaplasma marginale and Babesia bigemina was studied. B. bigemina infection at 18 months and A. marginale infection at 13 or 24 months resulted in slightly less severe reactions in pure-bred Bos indicus cattle than in Bos taurus. In both breeds, the reaction to A. marginale infection was more severe in older cattle. The severity of B. bigemina infection was not affected by a previous infection with A. marginale.     

A case of apparent suppression of Anaplasma marginale infection by eperythrozoonosis (Eperythrozoon teganodes)

The apparent suppression of Anaplasma marginale infection by Eperythrozoon teganodes in a splenectomized calf has been reported. A splenectomized calf, inoculated with 500 ml of blood having 23% erythrocytes infected with A. marginale, developed eperythrozoonosis on the fourth day post inoculation. A. marginale parasitaemia remained very low during the patent eperythrozoonosis. A. marginale parasites started to increase in number only after E. teganodes infection had been controlled with neoarsphenamine. A splenectomized calf treated identically, but not showing E. teganodes parasites in the peripheral blood, developed clinical anaplasmosis and fulminant parasitaemia within 3-4 days post inoculation.     

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.     

Mapping of B-cell epitopes in the N-terminal repeated peptides of Anaplasma marginale major surface protein 1a and characterization of the humoral immune response of cattle immunized with recombinant and whole organism antigens

Major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) together with MSP1b forms the MSP1 complex. MSP1a has been shown to be involved in adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle. A differential antibody response to MSP1a and MSP1b was observed in cattle immunized with A. marginale derived from bovine erythrocytes (anti-MSP1a response) or cultured tick cells (anti-MSP1b response). In this study, we further characterized the MSP1a antibody response of cattle using several immunogens, including recombinant MSP1a (rMSP1a) protein, erythrocyte- or tick cell culture-derived A. marginale, or a combination of tick cell culture-derived A. marginale and rMSP1a. The MSP1a antibody response to all these immunogens was directed primarily against the N-terminal region of MSP1a that contains tandemly repeated peptides, whereas low antibody levels were detected against the C-terminal portion. Linear B-cell epitopes of MSP1a were mapped using synthetic peptides representing the entire sequence of the protein that were prepared by SPOT synthesis technology. Only two peptides in the N-terminal repeats were recognized by sera from immunized cattle. These peptides shared the sequence SSAGGQQQESS, which is likely to contain the linear B-cell epitope that was recognized by the pools of bovine sera. The average differential of antibody titers against MSP1a minus those against MSP1b correlated with lower percent reductions in PCV. A preferential antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived, cell culture-derived plus rMSP1a or rMSP1a alone, and the percent reduction PCV was significantly lower in these cattle as compared with the other immunization groups. These results provide insight into the bovine antibody response against A. marginale and the role of MSP1a in protection of cattle against A. marginale infection.     

Characterization of the functional domain of major surface protein 1a involved in adhesion of the rickettsia Anaplasma marginale to host cells

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.     

Adhesion of outer membrane proteins containing tandem repeats of Anaplasma and Ehrlichia species (Rickettsiales: Anaplasmataceae) to tick cells

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.     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

Adaptive immunity to Anaplasma pathogens and immune dysregulation: implications for bacterial persistence

Anaplasma marginale is an obligate intraerythrocytic bacterium that infects ruminants, and notably causes severe economic losses in cattle worldwide. Anaplasma phagocytophilum infects neutrophils and causes disease in many mammals, including ruminants, dogs, cats, horses, and humans. Both bacteria cause persistent infection - infected cattle never clear A. marginale and A. phagocytophilum can also cause persistent infection in ruminants and other animals for several years. This review describes correlates of the protective immune response to these two pathogens as well as subversion and dysregulation of the immune response following infection that likely contribute to long-term persistence. I also compare the immune dysfunction observed with intraerythrocytic A. marginale to that observed in other models of chronic infection resulting in high antigen loads, including malaria, a disease caused by another intraerythrocytic pathogen.     

Evaluation of sequential coinfection with Anaplasma phagocytophilum and Anaplasma marginale in cattle

OBJECTIVE: To determine whether sequelae of infection differed among single versus double infection with Anaplasma phagocytophilum or Anaplasma marginale, with and without tick salivary extract, in cattle. ANIMALS: Eighteen 13-month old steers. PROCEDURES: Treatment groups of 3 cattle each included A marginale inoculated ID followed on day 35 by A phagocytophilum without tick saliva, A phagocytophilum followed on day 10 by A marginale without tick saliva, A marginale followed on day 35 by A phagocytophilum with tick saliva, A phagocytophilum followed on day 10 by A marginale with tick saliva, tissue culture control injection, and tick saliva control injection. Infection was monitored via clinical observations, CBC, serologic testing, and PCR analysis of blood and tissues. RESULTS: Infected cattle had significantly reduced weight gain. Anemia occurred 25 to 32 days after A marginale infection, which was attenuated by tick saliva. Parasitism was greater if cattle had not previously been inoculated with A phagocytophilum. Nine of the 12 treated cattle had positive results of PCR analysis for A phagocytophilum from at least 1 blood sample. Five tissue samples had positive results of PCR analysis for A phagocytophilum; PCR results for A marginale were positive in spleen, lung, lymph node, heart, and ear skin of infected cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva in disease kinetics and severity in cattle, which may be important for interpretation of diagnostic tests and management of disease in areas where both pathogens occur.     

Targeting the tick/pathogen interface for developing new anaplasmosis vaccine strategies

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.     

Co-feeding studies of ticks infected with Anaplasma marginale

Ticks often cluster at preferred feeding sites on hosts, and the co-feeding of ticks at the same site has been shown to increase feeding success and the transmission of some pathogens. While the major route of infection of ticks with pathogens is via the bloodmeal during feeding on a parasitemic host, non-systemic transmission of viruses and spirochetes has been shown to occur from infected to uninfected ticks at common feeding sites on uninfected hosts. In this research, two separate studies were done using the tick-borne rickettsial pathogen of cattle, Anaplasma marginale. In one study we tested whether A. marginale could be transmitted non-systemically from infected to uninfected Dermacentor variabilis males while co-feeding on rabbits. Infection of ticks was determined by allowing them to transmission feed on susceptible cattle and by DNA probe and microscopy studies on salivary glands. In the second study, we tested whether the co-feeding of male and female ticks on parasitemic cattle would increase the acquisition and development of A. marginale in males. A. marginale infections in salivary glands were determined by quantitative PCR after the ticks were allowed to transmission feed on susceptible cattle. Non-systemic transmission of A. marginale did not occur from infected and uninfected ticks that fed at the same site on rabbits and, therefore, does not appear to be a means of A. marginale transmission. A. marginale infections in male ticks were not increased while co-feeding with females. Thus, co-feeding of adult Dermacentor spp. does not appear to influence the dynamics of A. marginale transmission.     

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

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

High-yield preparation of purified Anaplasma marginale from infected bovine red blood cells

Purification of Anaplasma marginale from infected bovine RBC was achieved through enzyme treatment and density-gradient centrifugation. A relative yield of 41.6% was obtained by dividing the number of organisms in the final purified preparation by the number of A marginale-infected RBC. Purified parasites were verified as A marginale by light microscopy, electron microscopy, and immunologic tests. The purified parasites reacted positively with calf and rabbit anti-A marginale sera in interfacial and slide agglutination tests. Anti-bovine RBC serum did not agglutinate purified A marginale, indicating absence of any contaminating RBC stroma. Anaplasma marginale was antigenic, but did not cause infection when the preparation was inoculated into a susceptible calf. The density of A marginale was determined to be 1.19 g/ml and cell diameters ranged from 0.25 to 0.63 micron. This method provided procedures for obtaining A marginale free of bovine RBC antigens for accurate biochemical assays and vaccine production.     

Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection

The immunity induced by frozen and fresh Anaplasma centrale vaccines against anaplasmosis caused by A. marginale was tested in 12-month old Friesian steers. A. centrale parasitaemia occurred in all cattle inoculated with both types of vaccine. The average maximal decrease in PCV for the frozen and fresh vaccines was 41.0 and 40.3% respectively. All cattle recovered spontaneously. Vaccinated and control steers of the same age were challenged six months later with doses of 10(6), 10(7) or 10(8) A. marginale organisms. Vaccinated cattle showed average maximal A. marginale parasitemia of 1.2-4.0 versus 10.3-12.0% in control cattle. The average maximal decrease in packed cell volume (PCV) was 33.1 and 30.0% for steers vaccinated with frozen or fresh vaccine, respectively, and 57.4% for the non-vaccinated steers. All vaccinated cattle recovered spontaneously from the A. marginale infection while 7 out of 8 control steers required specific treatment. It thus appears that both frozen and fresh A. centrale vaccines are equally capable of inducing partial protection against infection with A. marginale and of preventing severe red blood cell destruction.