Development of inactivated heartwater (Ehrlichia ruminantium) vaccine in South Africa.

Heartwater, Ehrlichia ruminantium infection in cattle, sheep, goats, and some wild ruminants, is an economically important disease in Africa characterized by high mortality rates in susceptible populations. In South Africa, the current commercial heartwater vaccine is an infection and treatment type of immunization using virulent live E. ruminantium organisms generated from blood of infected sheep with subsequent treatment of the animals with antibiotics at specific times during the course of infection. This vaccine has several inherent problems preventing its wide use as the vaccine must be administered intravenously and it does not protect against all the South African field isolates. A vaccine based on inactivation of Zimbabwean E. ruminantium Mbizi strain organisms produced in endothelial cell cultures can be a sustainable option because it will not require antibiotic treatment and will be safe as there is no potential for reversion to virulence. Previous data generated in laboratory trials and under natural field setting provides support for this vaccine approach. Four inactivated vaccine formulations using the E. ruminantium Mbizi strain were tested for their efficacy in Merino sheep compared to an unvaccinated control group (11 sheep per group). Two vaccines were prepared by beta-propiolactone (BPL) inactivation, and two were inactivated with binary ethylenimine (BEI) while purification was done with both percoll and polyethylene glycol (PEG). The four vaccine preparations were formulated with Montanide ISA 50V2 adjuvant and administered twice subcutaneously (2 ml per dose) at an interval of 4 weeks. All groups were challenged with a virulent homologous cell-cultured E. ruminantium inoculated via the intra-venous route on day 56. The primary variable of efficacy was measured by the percentage survival rate or mortality between the Controls and Vaccine Groups. Three vaccine formulations (BEI/Percoll (Group 3), BEI/PEG (Group 4), BPL/Percoll, (Group 1) had a significantly higher percent of animal surviving challenge compared to the unvaccinated control (p-values 0.001, 0.035, 0.030, respectively). The highest number of survivors was obtained in Group 3 BEI/Percoll; 10/11 (91%). Groups 4 (BEI/PEG) and Group 1 (BPL/Percoll) produced similar percentage of survivals of 64%. In contrast, the lowest survival rate of 50% was observed in Group 2 (BPL/PEG) which was numerically different but not significantly different from the unvaccinated control which had an 18% survival rate (2/11). The inactivated vaccine using BEI or BPL as inactivating agents blended with ISA 50 adjuvant induced protective immunity against challenge. The BEI/Percoll (Group 3) vaccination regimen was most efficacious against a lethal heartwater challenge as it significantly protected sheep against mortality which is the most important aspect of heartwater infections. Future work should be directed towards improvement of this vaccine formulation especially from the down-stream processing point of view as the percoll method is not scalable for commercialization purposes.

Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease, Ehrlichia ruminantium, from the Panola Mountain Ehrlichia.

Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick-borne disease of cattle, sheep and goats responsible for stock losses in sub-Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross-reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP-1B peptides for ER and PME, we tested the cross-reactivity of this assay using sera from infected livestock. The MAP-1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual-plex Taqman™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease-causing organism.

Point prevalence survey for tick-borne pathogens in military working dogs, shelter animals, and pet populations in northern Colombia.

BACKGROUND: Based on the high tick-borne pathogen results from a 2011 surveillance study in three Colombian cities, an in-depth point prevalence survey was conducted to determine the seroprevalence of tick-borne pathogens at a specific point in time in 70 working dogs, 101 shelter dogs, and 47 client-owned dogs in Barranquilla, Colombia. RESULTS: Of the 218 serum samples, 163 (74%) were positive for Ehrlichia canis and 116 (53%) for Anaplasma platys. Exposure to tick-borne pathogens was highest in shelter and working dogs where more than 90% of the samples were seropositive or positive on polymerase chain reaction for one or more organisms as compared to 51% in client-owned animals. CONCLUSION: Surveillance for exposure to tick-borne pathogens provides vital information necessary to protect and conserve the health of local humans and animals, deployed military service members, and working dogs in various parts of the world. This study and resultant data demonstrate the value of following a broad-based surveillance study with a more specific, focused analysis in an area of concern. This area?s high levels of exposure warrant emphasis by medical planners and advisors on precautionary measures for military dogs, Special Operations Forces personnel, and the local public.

Genome annotation of five Mycoplasma canis strains.

To understand its potential to cause invasive disease, the genome of Mycoplasma canis strain PG14(T) from a dog's throat was compared to those of isolates from the genital tract or brain of dogs. The average nucleotide identity between strain pairs is 98%, and their genome annotations are similar.

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.

Persistence mechanisms in tick-borne diseases.

The use of new, highly sensitive diagnostic methods has revealed persistent infections to be a common feature of different tick-borne diseases, such as babesiosis, anaplasmosis and heartwater. Antigenic variation can contribute to disease persistence through the continual elaboration of new surface structures, and we know in several instances how this is achieved. Known or suspected mechanisms of persistence in babesial parasites include cytoadhesion and rapid variation of the adhesive ligand in Babesia bovis and genetic diversity in several merozoite stage proteins of different Babesia spp. In Anaplasma, extensive variation in the pfam01617 gene family accompanies cycling of organism levels in chronic infection. One result from the pioneering research at Onderstepoort is the definition of a related polymorphic gene family that is likely involved in immunity against heartwater disease. We are beginning to understand the sizes of the antigenic repertoires and full definition is close, with the possibility of applying simultaneous high-throughput sequencing to the order of 1000 small genomes. We also, for the first time, can consider modifying these genomes and looking at effects on persistence and virulence. However, important biological questions remain unanswered; for example, why we are seeing a new emerging Anaplasma infection of humans and is infection of endothelial cells by Anaplasma significant to persistence in vivo.

In vivo endothelial cell infection by Anaplasma marginale.

Anaplasma marginale has recently been shown to infect endothelial cells in vitro, but it remains unknown as to whether endothelial infection also occurs in vivo. In this report, we demonstrate through dual fluorescence microscopy that A marginale, detected by the monoclonal antibody ANAF16C1, co-localizes with the endothelial cell marker, von Willebrand factor, in tissue sections from an experimentally inoculated calf. The results indicate that A marginale infection includes endothelial cells and has implications for both pathogenesis and immune mechanisms.

Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale.

Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

Development of improved vaccines for heartwater.

Heartwater is controlled by frequent application of acaricides, which is costly, creates endemic instability and has the potential of contaminating the environment. The live blood vaccine currently available has limitations because it is laborious and inconvenient to use, difficult to standardise and can transmit other blood-borne pathogens. The UF/USAID/SADC Heartwater Research Project has conducted research on the development of two types of vaccine for heartwater. The first-generation inactivated vaccine has been intensively tested in the laboratory and subsequently field tested in four southern African countries. It protects cattle, sheep and goats against mortality from heartwater challenge. It can be modified to incorporate any Ehrlichia ruminantium strain to provide protection from field challenge. The second-generation DNA vaccine containing genes encoding immunogenic E. ruminantium proteins has been developed and evaluated in the mouse model as well as in cattle and sheep. The use of improved vaccines against heartwater would have a positive impact on livestock farming in sub-Saharan Africa and the Caribbean and could be used to control the spread of heartwater if it were to be introduced into regions such as the United States.

Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

A subset of Cowdria ruminantium genes important for immune recognition and protection.

Cowdria ruminantium causes the tick-borne rickettsial disease of heartwater, which is devastating to livestock production in sub-Saharan Africa. Current diagnosis and control methods are inadequate. We have identified and sequenced a subset of genes encoding recombinant antigens recognized by antibody and peripheral blood mononuclear cells from immune ruminants. The identified genes include many with significant similarity to those of Rickettsia prowazekii, genes predicted to encode different outer membrane proteins and lipoproteins and a gene containing an unusual tandem repeat structure. Evidence is presented for immune protection by recombinant antigens in a mouse model of C. ruminantium infection. These data identify new recombinant antigens for evaluation in vaccines and diagnostic tests to control heartwater.

Reduction in mortality from heartwater in cattle, sheep and goats exposed to field challenge using an inactivated vaccine.

Inactivated vaccines for heartwater prepared with the commercially acceptable Montanide ISA 50 (ISA 50) adjuvant were field tested in Boer goats in Botswana, Angora goats in South Africa, and Merino sheep in Zambia and Zimbabwe. Two vaccines, one made using the Zimbabwean Mbizi isolate and the other using the respective local field isolate (Sunnyside in Botswana; Bathurst in South Africa; Lutale in Zambia), were tested at each site, except in Zimbabwe where only the Mbizi vaccine was tested. Compared with unvaccinated animals, the Mbizi vaccine significantly protected goats and sheep against field Amblyomma tick challenge in Botswana, Zambia and Zimbabwe (P = 0.018, 0.002 and 0.017, respectively), but failed to protect Angora goats in South Africa. However, in South Africa the vaccine prepared using the local field isolate Bathurst, induced significant protection (P=0.008). The vaccines containing the local isolates at all other sites were less protective than the Mbizi vaccine. The Mbizi inactivated vaccine also significantly protected 17 of 21 cattle (P = 0.05) against heartwater challenge from field ticks in Zimbabwe. Against the same challenge only 7 of 21 unvaccinated control cattle survived. This study demonstrates that heartwater is a major constraint to upgrading livestock in endemic areas, and caused an overall mortality of 77.6% in naive sheep and goats (97 of 125 died) and 67% in cattle (14 of 21 died). In contrast, the vaccine had a protective effect by reducing the overall mortality in sheep and goats to 54.3% (113 of 208 died) and to 19% in cattle (4 of 21 died).

Recombinant major antigenic protein 2 of Ehrlichia canis: a potential diagnostic tool.

The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples from E. canis immunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected with E. canis and were previously demonstrated to contain antibodies reactive with E. canis by indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other than E. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 of E. canis in the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 of E. canis could be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis.

Antigenic variation of Anaplasma marginale: major surface protein 2 diversity during cyclic transmission between ticks and cattle.

The rickettsial pathogen Anaplasma marginale expresses a variable immunodominant outer membrane protein, major surface protein 2 (MSP2), involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B-cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of A. marginale and defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of A. marginale infecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain A. marginale transmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2 hypervariable region. At least three different MSP2 variants were encoded in each A. marginale population. The major sequence variants did not change on passage of A. marginale between culture, acute erythrocyte stage infections, and tick salivary glands but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in humans, domestic animals, and reservoir hosts.

Antibody responses to MAP 1B and other Cowdria ruminantium antigens are down regulated in cattle challenged with tick-transmitted heartwater.

Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405-2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85-87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C. ruminantium antigens are down regulated in cattle despite repeated exposure to C. ruminantium via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of C. ruminantium exposure in cattle in areas of endemic heartwater infection.

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

A polarized Th1 type immune response to Cowdria ruminantium infection is detected in immune DBA/2 mice.

Immune responses to Cowdria ruminantium, an intracellular organism that causes heartwater in domestic ruminants, were characterized in a DBA/2 mouse model. Immunity induced by infection and treatment was adoptively transferable by splenocytes and could be abrogated by in vivo depletion of T cells but not by inhibition of nitric oxide synthase using NG-monomethyl-L-arginine. IgG2a and IgG2b C. ruminantium-specific responses were detected in immune mice. Culture supernatants of splenocytes from immune DBA/2 mice, which were stimulated with crude C. ruminantium antigens or recombinant major antigenic proteins 1 or 2, contained significant levels of interferon (IFN)-gamma and interleukin (IL)-6, but insignificant levels of IL-1alpha, IL-2, IL-4, IL-5, IL-10, IL-12, tumor necrosis factor-alpha (TNF), and nitric oxide. A similar response was detected during primary infection, although IFN-gamma levels decreased significantly during clinical illness and then increased following natural or antibiotic-aided recovery. These data support the conclusion that protective immunity to C. ruminantium in DBA/2 mice is mediated by T cells and is associated with a polarized T helper 1 type of immune response. This murine model could be utilized to screen for protective C. ruminantium antigens that provoke Th1 type immune responses and for evaluation of these antigens in recombinant vaccines against heartwater.

Expression of a gene encoding the major antigenic protein 2 homolog of ehrlichia chaffeensis and potential application for serodiagnosis.

The major antigenic protein 2 (MAP2) homolog of Ehrlichia chaffeensis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of human monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody. However, immune sera failed to react with the recombinant on immunoblots when the antigen was denatured by heat or reduced using beta-mercaptoethanol. The recombinant MAP2 (rMAP2) was used in an ELISA format with 60 blinded serum samples. Twenty of the serum samples were previously demonstrated to contain antibodies reactive with E. chaffeensis by indirect immunofluorescence assays (IFAs). The remaining 40 samples were seronegative. All samples negative by IFA were also found to be negative for antibodies against the rMAP2 of E. chaffeensis by using the ELISA. Only 1 of 20 IFA-positive samples tested negative in the rMAP2 ELISA. There was 100% agreement using IFA-negative samples and 95% agreement using IFA-positive samples, resulting in a 97.5% overall agreement between the two assays. These data suggest that the rMAP2 homolog of E. chaffeensis may have potential as a test antigen for the serodiagnosis of human monocytic ehrlichiosis. To our knowledge, this recombinant is unique because it is thus far the only E. chaffeensis recombinant antigen that has been shown to work in an ELISA format.

CD8(+) T cell knockout mice are less susceptible to Cowdria ruminantium infection than athymic, CD4(+) T cell knockout, and normal C57BL/6 mice.

The role of T cells in immunity to Cowdria ruminantium was investigated by studying the responses to infection of normal, athymic, CD4(+) T cell knock out (KO) and CD8(+) T cell KO C57BL/6 mice. Normal C57BL/6 mice could be immunized by infection and treatment, and immunity was adoptively transferable from immune to naive mice by splenocytes. Following infection, athymic mice died sooner than normal mice (P=0.0017), and could not be immunized by infection and treatment. CD4(+) T cell KO mice were as susceptible to infection as normal mice and could be immunized by infection and treatment. In contrast, CD8(+) T cell KO mice were less susceptible than normal and CD4(+) T cell KO mice and 43% self-cured, while those that died did so after a prolonged incubation period. Antibody responses to C. ruminantium were CD4(+) T cell dependent, because responses were detected in immune normal and CD8(+) T cell KO mice but not in immune CD4(+) KO mice (P=0.005). Since CD8(+) T cell KO mice were less susceptible to infection, and since CD4(+) T cell KO mice could be immunized, it can be concluded that immunity to C. ruminantium can be mediated by both CD4(+) and CD8(+) T cells.