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.     

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.     

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.     

A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep.

The DNA probe pCS20, which was cloned from the DNA of the Crystal Springs heartwater strain from Zimbabwe, cross-reacted with DNAs of heartwater strains from all endemic areas, including four heartwater strains from Zimbabwe, two strains from South Africa, one strain from Nigeria, and the Gardel strain from the Caribbean island of Guadeloupe. By nucleic acid hybridization, the pCS20 DNA probe detected Cowdria ruminantium DNA in all DNA preparations made from plasma samples from infected sheep before and during the febrile reaction. Synthetic oligonucleotides were prepared for amplification of specific C. ruminantium DNA sequences by the polymerase chain reaction (PCR). Amplification of two DNA products (181 and 279 bp) from pCS20 DNA and C. ruminantium genomic DNA of heartwater strains was demonstrated. In contrast, amplification of these products or any other products was not possible from genomic DNAs of Anaplasma marginale, Babesia bigemina, Trypanosoma brucei brucei, Escherichia coli, and bovine endothelial cells. The cross-reactivities of the 32P-labeled PCR products with genomic DNAs from several heartwater strains were similar to those with the pCS20 DNA probe. A nucleic acid-based test that uses hybridization assays and PCR provides a sensitive method for the detection of heartwater in both animals and ticks and has applications in epidemiological studies for the disease, which may allow for improved disease control.     

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.     

Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay.

Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Identification of an immunodominant 32-kilodalton membrane protein of Leishmania donovani infantum promastigotes suitable for specific diagnosis of Mediterranean visceral leishmaniasis.

Sera from 35 patients suffering from Mediterranean visceral leishmaniasis (caused by Leishmania donovani infantum) and 59 patients with various forms of cutaneous leishmaniasis prevalent in the sub-Mediterranean countries (caused by Leishmania major, L. donovani infantum, or Leishmania tropica) were tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA) with both membrane and soluble antigens prepared from L. donovani infantum parasites. Control sera were from healthy children (n = 41), adults with nonleishmanial diseases (n = 40), and patients with Chagas' disease (n = 12). A P32 antigen present in the membrane preparation from L. donovani infantum parasites was recognized by 95% of serum specimens from patients with Mediterranean visceral leishmaniasis but not by serum specimens from patients with cutaneous leishmaniasis or sera from control individuals. An ELISA with electroeluted P32 antigen was found to have a specificity and sensitivity of 94% in the serodiagnosis of Mediterranean visceral leishmaniasis. Healthy children with asymptomatic Leishmania infection were seronegative for the P32 antigen by ELISA. These results suggest that antibodies to P32 antigen develop only in patients with visceral leishmaniasis and that the P32 ELISA may be useful in areas where the disease is endemic for discriminating between patients with this disease and those with other clinical conditions.     

The immunodominant 90-kilodalton protein is localized on the terminal tip structure of Mycoplasma pneumoniae.

Immunoblot analysis of convalescent-phase sera of experimentally infected chimpanzees or monoclonal antibodies (MAbs) specific to the 90- and 40-kDa proteins of Mycoplasma pneumoniae indicated that both proteins were present in cytadsorbing, pathogenic strains PI-1428, M129, and FH but absent in noncytadsorbing, nonpathogenic strain M129-B176. Adsorption of convalescent-phase chimpanzee sera with virulent strain PI-1428 removed reactivity, whereas adsorption with avirulent strain M129-B176 did not remove reactivity to these two proteins. By using proteolysis and specific MAbs, we demonstrated that the 90- and 40-kDa proteins were surface exposed. Immunoelectron microscopy employing specific MAbs showed that the 90-kDa protein is localized on the terminal tip attachment apparatus. However, the MAb specific for the 40-kDa protein failed to indicate a similar localization. Nevertheless, these data, taken together, indicate that the immunodominant 90- and 40-kDa proteins are surface exposed, are localized on the terminal tip apparatus, and might be involved in the attachment mechanism.     

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 immunodominant 90-kilodalton Aspergillus fumigatus antigen is the subunit of a catalase.

We have identified, purified, and characterized structurally and functionally a 90-kDa immunodominant antigen associated with the water-soluble fraction of Aspergillus fumigatus. This antigen is recognized by 90.3% of serum samples from patients with aspergilloma and should be considered either by itself or better in combination with other purified antigens as a candidate for developing a standardized immunoassay for the detection of aspergilloma. p90 is a glycoprotein containing at least two two N-linked sugar chains of 2 and 5 kDa, respectively, which are not necessary for its reactivity with aspergilloma serum samples. Using specific anti-p90 rabbit serum, we have demonstrated that under native conditions, p90 exists in oligomeric form and has associated catalase activity. This activity is resistant to extreme temperatures (> 60 degrees C), reducing agents (40 mM dithiothreitol), high concentrations of denaturing agents such as 8 M urea and 8% sodium dodecyl sulfate, and treatments with ethanol-chloroform-water (5:3:10 [vol/vol]) mixtures.     

Diagnosis of systemic candidiasis by an enzyme-linked dot immunobinding assay for a circulating immunodominant 47-kilodalton antigen.

A dot immunobinding assay based on the detection of the immunodominant 47-kilodalton (kDa) antigen of Candida albicans is described for the serological diagnosis of systemic candidiasis. It was compared with a reverse passive latex agglutination test and a dot immunobinding assay with total unfractionated hyperimmune serum to C. albicans. Use of the 47-kDa antigen-specific probe increased both the sensitivity and specificity of the assay system. Patients with systemic candidiasis were detected earlier in the course of the infection. The rate of detection of systemic C. albicans infections in neutropenic patients was 77% compared with 55% with total antibody in the dot immunobinding assay and 29% with the latex test. All three assay systems were positive in over 73% of infected patients who were not neutropenic. The 47-kDa antigen-specific probe was relatively specific to C. albicans. Antibody probes to the immunodominant antigens of other yeasts might be incorporated in the same dot immunobinding assay to detect systemic candidiasis caused by other species of yeasts.     

A statistical method for the detection of false positives and false negatives in microtitre format PCR assays.

A method is described which can be used to determine whether a series of PCR reactions carried out in a microtitre plate are inherently unlikely to have occurred by chance, and hence to show 'false' results. The method is an extension of the familiar 'runs test' which can be used for tube-based PCRs. A Monte Carlo simulation program is discussed which can be used to generate expected probability distributions for either symmetric or asymmetric plate designs. In addition, systematic departures from a random pattern due to 'edge effects' can be detected.     

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.     

Reduction of false positives on the rectal tube in computer-aided detection for CT colonography

PURPOSE: To eliminate false-positive (FP) polyp detections on the rectal tube (RT) in CT colonography (CTC) computer-aided detection (CAD). METHODS: We use a three-stage approach to detect the RT: detect the RT shaft, track the tube to the tip and label all the voxels that belong to the RT. We applied our RT detection algorithm on a CTC dataset consisting of 80 datasets (40 patients scanned in both prone and supine positions). Two different types of RTs were present, characterized by differences in shaft/bulb diameters, wall intensities, and shape of tip. RESULTS: The algorithm detected 90% of RT shafts and completely tracked 72% of them. We labeled all the voxels belonging to the completely tracked RTs (72%) and in 11 out of 80 (14%) cases the RT voxels were partially labeled. We obtained a 9.2% reduction of the FPs in the initial polyp candidates' population, and a 7.9% reduction of the FPs generated by our CAD system. None of the true-positive detections were mislabeled. CONCLUSIONS: The algorithm detects the RTs with good accuracy, is robust with respect to the two different types of RT used in our study, and is effective at reducing the number of RT FPs reported by our CAD system.     

Identification of mycobacteria by PCR-based sequence determination of the 32-kilodalton protein gene.

In this study, a part of the nucleotide sequence of the mycobacterial 32-kDa protein gene was determined by PCR-based sequencing. A total of 24 mycobacterial strains, representing 10 species, were studied. Sequences of all tested members of the Mycobacterium tuberculosis complex were identical to each other and to the previously published sequence of M. tuberculosis H37Rv. The sequences of M. avium and M. intracellulare were different from each other. MAIX strains, identified with the Gen-Probe MAIX test, had sequences identical to each other but clearly different from those of M. avium and M. intracellulare. Each of the other mycobacterial species investigated, i.e., M. kansasii, M. gastri, M. gordonae, and M. malmoense, had a unique species-specific sequence. These results demonstrate that there is variation in the nucleotide sequence of the 32-kDa protein gene among different mycobacterial species. Thus, we propose that this gene can be used for PCR-based identification of mycobacteria.     

Serologic analysis of white-tailed deer sera for antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay and western immunoblotting.

White-tailed deer serum samples were collected in the Minneapolis-St. Paul, Minn., metropolitan area during the fall and winter months from 1989 to 1992 and analyzed for antibodies to Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. Ninety-eight percent of the serum samples were collected from regions where currently the vector tick, Ixodes dammini, is nonexistent. Antibodies to B. burgdorferi were detected in 2.2% of 508 samples by enzyme-linked immunosorbent assay, and their presence was confirmed by Western immunoblot analysis. Western immunoblotting yielded mean numbers of reactive bands of 0.1 and 6.0 for samples that were negative and positive for antibodies by enzyme-linked immunosorbent assay, respectively. The molecular weights of the antigens in many of the reactive bands from positive samples were similar to the molecular weights of antigens reactive with samples from humans with Lyme borreliosis. An antibody response to the major outer surface proteins A and B was not detected. Serologic analysis of deer sera may provide a valuable method for surveillance programs designed to monitor the spread of B. burgdorferi in nature.