Size variation of the major immunodominant protein of Cowdria ruminantium.

An immunodominant response is made to a polypeptide of approximately 32 kDa in animals infected with the rickettsial pathogen Cowdria ruminantium. We show here using cultured strains of the rickettsia from different geographical areas that the apparent size of this polypeptide varies with strain origin. Changes in the primary structure between strains should be considered in the design of vaccines and diagnostic tests based on this antigen.     

Identification of an immunodominant antigenically conserved 32-kilodalton protein from Cowdria ruminantium.

Western blotting (immunoblotting) of Cowdria ruminantium antigens with goat or mouse antiserum identified a periodate-resistant, proteinase K-sensitive immunodominant antigen of 32,000 daltons. This protein, designated Cr32, could be demonstrated in goat choroid plexus infected with one of two different Cowdria stocks. Antisera against nine different Cowdria stocks from Africa and the Caribbean region recognized Cr32, which indicates that this protein contains conserved antigenic determinants.     

Sequence heterogeneity of the major antigenic protein 1 genes from Cowdria ruminantium isolates from different geographical areas.

The genes for the immunodominant major antigenic protein 1 (MAP1) of Cowdria ruminantium from four African and two Caribbean isolates were cloned, restriction mapped, and sequenced to identify conserved epitopes for development of serodiagnostic tools for heartwater. Restriction length polymorphisms were observed among the respective MAP1 genes analyzed and were confirmed by sequencing. The sequence data generated for these isolates were compared with data for the previously reported Senegal isolate MAP1 gene. These sequences were found to differ from each other by 0.6 to 14.0%. These differences translate into a 0.8 to 10.0% variation in the predicted protein sequence. In the entire coding sequence, several amino acid substitutions were identified in addition to deletions or insertions at three regions of the gene. These variable regions are referred to as variable regions I, II, and III. From the sequence data, an evolutionary distance tree was constructed; this tree suggested that at least two genetically distinct C. ruminantium strains exist in the Caribbean: the isolate from Antigua is similar to that from Senegal, while the isolate from Guadeloupe is closely related to that from Sudan.     

An immunoblotting diagnostic assay for heartwater based on the immunodominant 32-kilodalton protein of Cowdria ruminantium detects false positives in field sera.

Heartwater, a major constraint to improved livestock production in Zimbabwe, threatens to invade areas which have been previously unaffected. To monitor its spread in Zimbabwe, an immunoblotting diagnostic assay based on the responses of animals to the immunodominant, conserved 32-kDa protein of Cowdria ruminantium was evaluated. In this assay, no false reactions were detected with sera known to be positive and negative, but sera from some cattle, sheep, and goats from heartwater-free areas of Zimbabwe reacted strongly with the 32-kDa protein, suggesting that either these animals had previous exposure to heartwater or they were false positives. To investigate the possibility of previous exposure to heartwater, 11 immunoblot-positive and 6 immunoblot-negative sheep from heartwater-free areas of Zimbabwe were compared regarding their susceptibilities to challenge with C. ruminantium. Prior to challenge, C. ruminantium could not be detected in any sheep by transmission to Amblyomma hebraeum ticks or by the polymerase chain reaction (PCR) conducted with plasma samples. All sheep were equally susceptible to the challenge, and infection was confirmed by brain biopsy, necropsy, PCR, and transmission of C. ruminantium to ticks. Our data suggest that the immunoblot-positive reactions of sera from heartwater-free areas were due not to previous C. ruminantium infection but rather to antigenic cross-reactivity between C. ruminantium and another agent(s) such as Ehrlichia species. In conclusion, the immunodominant 32-kDa protein is not antigenically specific to C. ruminantium and its use in serological diagnosis of heartwater requires reevaluation.     

Validation of the Indirect MAP1-B Enzyme-Linked Immunosorbent Assay for Diagnosis of Experimental Cowdria ruminantium Infection in Small Ruminants

The major antigenic protein 1 fragment B (MAP1-B) enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Cowdria ruminantium infections was validated to determine cutoff values and evaluate its diagnostic performance with sheep and goat sera. Cowdria-infected populations consisted of 48 sheep and 44 goats, while the noninfected populations consisted of 64 sheep and 107 goats. Cutoff values were determined by two-graph receiver-operating characteristic (TG-ROC) curves. The cutoff value was set at 31 and 26.6% of the positive control reference samples for sheep and goat sera, respectively. The test’s diagnostic performance was evaluated with measurements of the area under the concentration-time curve (AUC) of the ROC curves and by the valid range proportion (VRP). The AUCs were 0.978 for sheep sera and 0.989 for goat sera. The VRP for both sheep and goat sera was approximately 1.0. The intermediate range (IR), which defines results that are neither positive nor negative, was 0 for goat sera and 2.81 for sheep sera. In an ideal test, the AUC and VRP would be 1.0 and the IR would be 0. In this study these parameters were close to those of an ideal test. It is concluded that the MAP1-B ELISA is a useful test for the diagnosis of C. ruminantium infection in small ruminants.     

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.     

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.     

Development of an in vitro cloning method for Cowdria ruminantium.

Cowdria ruminantium is a tick-borne rickettsia which causes severe disease in ruminants. All studies with C. ruminantium reported so far were carried out with stocks consisting of infective blood collected from reacting animals or from the same stocks propagated in vitro. Cloned isolates are needed to conduct studies on immune response of the host, on genetic diversity of the parasite, and on mechanisms of attenuation and the development of vaccines. A method of cloning based on the particular chlamydia life cycle of Cowdria was developed. Instead of cloning extracellular elementary bodies, it appeared more convenient to clone endothelial cells infected by one morula resulting from the infection of the cell by one elementary body of Cowdria. Two hundred and sixteen clones were obtained by limiting dilution of infected cells. The method was experimentally validated by comparing randomly amplified polymorphic DNA fingerprints from individual clones obtained from endothelial cell cultures coinfected with two different stocks of C. ruminantium.     

Cross-reactive epitope mimics in a fragmented-genome phage display library derived from the rickettsia, Cowdria ruminantium.

BACKGROUND: Epitopes can be mapped by comparing immunoaffinity-selected peptides from fragmented-gene display libraries with the target gene. With larger libraries derived from unsequenced genomes, this is not possible. Spurious epitope mimics may be created by expressing DNA in a variety of meaningless reading frames and orientations. OBJECTIVES: To determine empirically whether panning a large fragmented-genome phage display library with antibodies to MAP1, the major antigenic protein of the rickettsial parasite Cowdria ruminantium, would result in the selection of irrelevant, cross-reactive mimotopes. STUDY DESIGN: A gene III phage library displaying peptides derived from C. ruminantium was constructed using cloned DNA from a bacteriophage lambda genomic library. After in vivo excision, plasmids were cleaved with PvuII followed by PCR. Genes with a PvuII site, including MAP1 were therefore not amplified. DNA was sonicated, partially digested with DNase and cloned into the display vector fUSE2. Affinity-purified MAP1 antibodies were used for panning. Peptides expressed by panned phages were tested for recognition in Western blot and ELISA. Oligonucleotides representing antigenic sequences were used to locate their encoding DNA sequences in the original lambda library. The phage display library was also panned with two monoclonal antibodies (Mabs) against bluetongue virus (BTV). RESULTS: Five different peptide sequences were selected from the MAP1-deficient phage display library. None was identical to MAP1, but four peptides had regions that were similar, both to each other, and to the parasite protein. They produced strong signals in ELISA and Western blot. None could be located to any C. ruminantium open reading frame. Two BTV Mabs recognised a sequence similar to their authentic epitope. CONCLUSION: Large genome-targeted phage display libraries may be sufficiently diverse to allow the selection of peptides that mimic actual antigenic determinants. This diversity may be exploited in the search for useful epitopes.     

Heartwater (Cowdria ruminantium infection) as a cause of postrestocking mortality of goats in Mozambique.

A serological survey in Mozambique to detect antibodies to Cowdria ruminantium, the etiologic agent of heartwater, revealed a seroprevalence of 8.1% (n = 332) for goats in the northern province of Tete and of 65.6% (n = 326) for goats in the southern provinces. Translocation of 10 serologically negative goats from Tete to farms in the south resulted in two clinical cases of heartwater that were fatal. In addition, four goats seroconverted within the study period of 5 weeks. One goat showed no symptoms. Two goats died of other causes, whereas the remaining goat went missing after 1 week. Experimental needle infections of goats and sheep were conducted to confirm results and to isolate different strains of C. ruminantium. These data indicate that translocation of goats from the north to the south of Mozambique bears a high risk of C. ruminantium infection, which can cause fatal disease.     

Distribution of heartwater in the Caribbean determined on the basis of detection of antibodies to the conserved 32-kilodalton protein of Cowdria ruminantium.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect immunoglobulin G antibodies to the major 32-kDa protein of Cowdria ruminantium. A total of 1,804 serum samples collected from cattle on 19 islands in the eastern Caribbean Basin were tested by this cELISA. A total of 133 serum samples from 10 islands (Antigua, Dominica, Grenada, Guadeloupe, Martinique, Montserrat, St. Kitts, St. Lucia, St. Martin, and St. Vincent) were found to be positive. The presence of antibodies to C. ruminantium in cattle on these islands was confirmed by immunofluorescence and Western blotting (immunoblotting). In earlier studies, C. ruminantium has been demonstrated only on Guadeloupe, Antigua, and Marie Galante. This study shows that the causative agent of heartwater is now firmly established in the Caribbean.     

Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium.

Cowdria ruminatium, the causative agent of heartwater disease, expresses an immunodominant and conserved 32-kilodalton protein (MAP1; formerly called Cr32), which is currently in use for serodiagnosis of the disease. The gene encoding this protein, designated map1, was detected, cloned, and characterized. The gene is conserved between four different stocks of C. ruminantium originating from Senegal, Sudan, South Africa, and Zimbabwe. Homology searches revealed MAP1 to be homologous to the Anaplasma marginale surface protein MSP4, a potential protective antigen. The MAP1 protein, expressed in Escherichia coli fused with glutathione S-transferase, is specifically recognized by sera from animals infected with seven different stocks of C. ruminantium.     

Growth of Cowdria ruminantium, the causative agent of heartwater, in a tick cell line

The tick-borne rickettsia Cowdria ruminantium has been propagated continuously for over 500 days in the Ixodes scapularis tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells.     

Heartwater (Cowdria ruminantium Infection) as a Cause of Postrestocking Mortality of Goats in Mozambique

A serological survey in Mozambique to detect antibodies to Cowdria ruminantium, the etiologic agent of heartwater, revealed a seroprevalence of 8.1% (n = 332) for goats in the northern province of Tete and of 65.6% (n = 326) for goats in the southern provinces. Translocation of 10 serologically negative goats from Tete to farms in the south resulted in two clinical cases of heartwater that were fatal. In addition, four goats seroconverted within the study period of 5 weeks. One goat showed no symptoms. Two goats died of other causes, whereas the remaining goat went missing after 1 week. Experimental needle infections of goats and sheep were conducted to confirm results and to isolate different strains of C. ruminantium. These data indicate that translocation of goats from the north to the south of Mozambique bears a high risk of C. ruminantium infection, which can cause fatal disease.     

Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe.

The sensitivities of a PCR assay and a DNA probe assay were compared for the detection of Cowdria ruminantium in Amblyomma ticks that were fed on C. ruminantium-infected, clinically reacting, and recovered carrier animals. The PCR assay and DNA probe detected infection in 86.0 and 37.0%, respectively, of 100 ticks fed on a febrile animal. In 75 ticks fed on carrier animals, PCR and the DNA probe detected infection in 28.0 and 1.33% of ticks, respectively. This demonstrates that the DNA probe has poor sensitivity for the detection of low levels of infection in ticks and that PCR is necessary for this purpose. The PCR assay had a detection limit of between 1 and 10 C. ruminantium organisms and did not amplify DNA from Ehrlichia canis, which is phylogenetically closely related to C. ruminantium, Theileria parva, or uninfected Amblyomma hebraeum or A. variegatum. PCR detected infection in A. hebraeum and A. variegatum adult ticks infected with one of six geographically different C. ruminantium strains. Amplification was also possible from desiccated ticks and ticks fixed in 70% ethanol, 10% buffered formalin, or 2% glutaraldehyde. The PCR assay supersedes the DNA probe and older detection methods for the detection of C. ruminantium in ticks, particularly those fed on carrier animals, and is suitable for both prospective and retrospective studies which require accurate detection of C. ruminantium in individual ticks. Application of the PCR assay should significantly improve the understanding of heartwater epidemiology, particularly through the determination of field tick infection rates.     

Use of a recombinant envelope protein subunit antigen for specific serological diagnosis of West Nile virus infection

Serological diagnosis of West Nile virus (WNV) infection is complicated by extensive antigenic cross-reactivity with other closely related flaviviruses, such as St. Louis encephalitis virus. Here we describe a recombinant, bacterially expressed antigen equivalent to structural domain III of the WNV envelope protein that has allowed clear discrimination of antibody responses to WNV from those against other related flaviviruses in indirect enzyme-linked immunosorbent assays using standardized control antisera and field-collected samples.     

Conconavalin A-stimulated bovine T-cell supernatants inhibit growth of Cowdria ruminantium in bovine endothelial cells in vitro.

Conconavalin A-stimulated bovine T-cell supernatants inhibited the growth of Cowdria ruminantium in bovine endothelial cells in vitro but did not affect their entry. This finding represents one mechanism by which T cells may control C. ruminantium multiplication and hence affect the severity of disease.     

Use of cloned excretory/secretory low-molecular-weight proteins of Cooperia oncophora in a serological assay.

The potential of Cooperia oncophora excretory/secretory (ES) proteins as antigens in a serological assay which aims to establish exposure levels in cattle was assessed. ES proteins were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and immunoblotting. The N-terminal domains of two ES proteins were sequenced, and the corresponding cDNAs were cloned. Two cDNAs, designated CoES14.0 and CoES14.2, were expressed in Escherichia coli. The recombinant proteins were tested in an indirect enzyme-linked immunosorbent assay (ELISA) in which crude worm antigen (CWA) was used as a reference standard. In total, 67 reference serum samples were used: 27 negative serum samples, 29 C. oncophora-specific serum samples, 7 Dictyocaulus viviparus-specific serum samples, and 4 Ostertagia ostertagi-specific serum samples. This showed respective sensitivities and specificities of 17 and 84%, 0 and 100%, and 100 and 100% by the ELISAs with the three different types of proteins (CWA, CoES14.0, and CoES14.2, respectively). Since the CoES14.2 ELISA had the best sensitivity and specificity with reference sera, its specificity was further validated in an antigen inhibition ELISA. In this assay CoES14.2 and CWA preparations of C. oncophora, Cooperia curticei, O. ostertagi, Nematodirus helvetianus, Fasciola hepatica, D. viviparus, Haemonchus placei, and Trichostrongylus colubriformus were used as competitor antigens. This experiment showed that only the homologous antigens C. oncophora CWA and CoEs14.2 resulted in 100% inhibition. The CWA preparations of all other nematodes did not affect the ELISA, even if concentrations of 250 times the 50% inhibitory concentration of C. oncophora CWA were used. These results indicate that CoES14.2 does not share cross-reactive epitopes with heterologous CWAs. Finally, we tested the CoES14.2 ELISA with sequential serum samples from naturally infected groups of animals. The optical density values that were obtained correlated well with exposure levels based on cumulative egg excretion. Thus, the CoES14.2 ELISA seems to be a very sensitive tool for estimating exposure levels in cattle.     

Detection of the agent of heartwater, Cowdria ruminantium, in Amblyomma ticks by PCR: validation and application of the assay to field ticks

We have previously reported that the pCS20 PCR detection assay for Cowdria ruminantium, the causative agent of heartwater disease of ruminants, is more sensitive than xenodiagnosis and the pCS20 DNA probe for the detection of infection in the vector Amblyomma ticks. Here, we further assessed the reliability of the PCR assay and applied it to field ticks. The assay detected DNA of 37 isolates of C. ruminantium originating from sites throughout the distribution of heartwater and had a specificity of 98% when infected ticks were processed concurrently with uninfected ticks. The assay did not detect DNA of Ehrlichia chaffeensis, which is closely related to C. ruminantium. PCR sensitivity varied with tick infection intensity and was high (97 to 88%) with ticks bearing 10(7) to 10(4) organisms but dropped to 61 and 28%, respectively, with ticks bearing 10(3) and 10(2) organisms. The assay also detected C. ruminantium in collections of Amblyomma hebraeum and Amblyomma variegatum field ticks from 17 heartwater-endemic sites in four southern African countries. Attempts at tick transmission of infection to small ruminants failed with four of these collections. The pCS20 PCR assay is presently the most characterized and reliable test for C. ruminantium in ticks and thus is highly useful for field and laboratory epidemiological investigations of heartwater.     

Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection.

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.