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D H Walker

Publications and source records attributed to D H Walker.

At least 55 records · Page 3Linked to original sources
PubMed1999

Genetic diversity of the 28-kilodalton outer membrane protein gene in human isolates of Ehrlichia chaffeensis.

The Ehrlichia chaffeensis 28-kDa outer membrane protein (p28) gene was sequenced completely by genomic walking with adapter PCR. The DNA sequence of the p28 gene was nearly identical to the previously reported sequence (N. Ohashi, N. Zhi, Y. Zhang, and Y. Rikihisa, Infect. Immun. 66:132-139, 1998), but analysis of a further 75 bp on the 5' end of the gene revealed DNA that encoded a 25-amino-acid signal sequence. The leader sequence was removed from the N terminus of a 30-kDa precursor to generate the mature p28 protein. A monoclonal antibody (MAb), 1A9, recognizing four outer membrane proteins of E. chaffeensis (Arkansas strain) including the 25-, 26-, 27-, and 29-kDa proteins (X.-J. Yu, P. Brouqui, J. S. Dumler, and D. Raoult, J. Clin. Microbiol. 31:3284-3288, 1993) reacted with the recombinant p28 protein. This result indicated that the four proteins recognized by MAb 1A9 were encoded by the multiple genes of the 28-kDa protein family. DNA sequence alignment analysis revealed divergence of p28 among all five human isolates of E. chaffeensis. The E. chaffeensis strains could be divided into three genetic groups on the basis of the p28 gene. The first group consisted of the Sapulpa and St. Vincent strains. They had predicted amino acid sequences identical to each other. The second group contained strain 91HE17 and strain Jax, which only showed 0.4% divergence from each other. The third group contained the Arkansas strain only. The amino acid sequences of p28 differed by 11% between the first two groups, by 13.3% between the first and third groups, and by 13.1% between the second and third groups. The presence of antigenic variants of p28 among the strains of E. chaffeensis and the presence of multiple copies of heterogeneous genes suggest a possible mechanism by which E. chaffeensis might evade the host immune defenses. Whether or not immunization with the p28 of one strain of E. chaffeensis would confer cross-protection against other strains needs to be investigated.

Amino Acid Sequence↗
PubMed1999

Comparison of Ehrlichia chaffeensis recombinant proteins for serologic diagnosis of human monocytotropic ehrlichiosis.

Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients' sera with the recombinant E. chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with E. chaffeensis. The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria. Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using E. chaffeensis antigen and by protein immunoblotting using recombinant E. chaffeensis proteins expressed in Escherichia coli. Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater. The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein. None of the recombinant antigens yielded false-positive results. All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein.

Amino Acid Sequence↗
PubMed1999

Serologic study of the prevalence of rickettsiosis in Yucatán: evidence for a prevalent spotted fever group rickettsiosis.

Because of the discovery of a spotted fever group rickettsiosis with signs and symptoms similar to dengue fever in Yucatan, Mexico, immunofluorescence assay (IFA) serology was performed on sera from 390 persons selected from a representative geographic distribution of rural Yucatan to detect antibodies reactive with Rickettsia rickettsii, R. akari, a Thai strain (TT-118) that is most closely related to a rickettsia identified in Amblyomma cajennense ticks in southern Texas, and R. typhi. The IFA antibodies at titers > or = 1:64 against R. akari were detected in 22 (5.6%) of the samples with the expected cross-reactivity against the other antigens of the spotted fever group. Immunoblotting with antigens of R. akari identified antibodies against antigens of spotted fever group lipopolysaccharides and not against rickettsial outer membrane proteins A and B, which contain the species-specific epitopes. A rickettsiosis most likely caused by a relative of R. akari appears to be both prevalent and widely distributed geographically in Yucatan.

Adolescent↗
PubMed1999

Monoclonal antibody-based immunohistochemical diagnosis of rickettsialpox: the macrophage is the principal target.

Cutaneous biopsies of five eschars and two rash lesions from five patients from New York City with documented rickettsialpox were examined by immunohistochemical methods with a monoclonal antibody directed against spotted fever group rickettsial lipopolysaccharide for the presence and cellular location of Rickettsia akari Rickettsiae were identified in all of the five patients, with good concordance of results for the same biopsy tissues with previously reported results by the direct immunofluorescence method. In contrast with immunofluorescence, which did not reveal the location of the organisms, immunohistochemical examination demonstrated R. akari to be in perivascular cells, morphologically resembling macrophages. Evaluation with double staining for rickettsiae and either CD68 or Factor VIII-related antigen revealed that the predominant infected cell type was CD68-positive macrophages, and only a rare rickettsia was detected in vascular endothelium, the major target cell for other rickettsioses. These results provide a diagnostic method for rickettsialpox and other spotted fever group rickettsioses and indicate that the elucidation of the pathogenesis of rickettsialpox must take into account that its target cell differs from that of Rocky Mountain spotted fever, boutonneuse fever, louse-borne typhus fever, and murine typhus.

Animals↗
PubMed1998

rOmpA is a critical protein for the adhesion of Rickettsia rickettsii to host cells.

rOmpA and rOmpB are immunodominant, surface-exposed proteins of Rickettsia rickettsii. Prior evidence suggests that adhesion of R. rickettsii to the host cell is mediated by a rickettsial protein. Five monoclonal antibodies to rOmpA, five to rOmpB, and one to the rickettsial lipopolysaccharide (LPS) were tested for inhibition of rickettsial attachment. All the monoclonal antibodies to rOmpA inhibited adhesion of rickettsiae to the L-929 cells with some inhibition rates as high as 90%. In contrast, monoclonal antibodies to rOmpB and LPS did not block attachment. When Fab fragments of monoclonal antibodies against rOmpA and rOmpB were used, similar results were observed as for the intact monoclonals, non-adhesion and adhesion, respectively. Purified rOmpA showed a competitive inhibitive effect on the attachment of R. rickettsii to host cells. Trypsin completely digested rOmpA but not rOmpB from the surface of intact R. rickettsii, resulting in loss of the ability of the rickettsiae to attach to the host cell. rOmpA appears to play an important role in the initial adhesion of R. rickettsii to the host cell.

Animals↗
PubMed1998

Detection of a spotted fever group rickettsia in Amblyomma cajennense (Acari: Ixodidae) in south Texas.

Thai tick typhus rickettsia (strain TT-118), a spotted fever group rickettsia of indeterminant pathogenicity, was isolated from a mixed pool of larval Ixodes and Rhipicephalus ticks collected from Thailand in 1962. Here we report the surprising finding of a spotted fever group rickettsia with closest sequence homology to the Thai tick typhus rickettsia in Amblyomma cajennense (F.) ticks from south Texas. Sequence analysis was performed on segments of 3 genes that differentiate rickettsial species; all 3 genes sequenced, the 17-kDa, glta, and rompA, when compared with those of other rickettsiae, showed the highest degree of similarity to the Thai tick typhus rickettsia with 99.5, 99.5, and 100% homology, respectively. This is the 1st finding of a rickettsial species in A. cajennense ticks in Texas and the United States. If this rickettsia is pathogenic, exposure to infected A. cajennense ticks may pose a previously unrecognized health risk to people who have been fed upon by these ticks.

Animals↗
PubMed1998

Ultrastructural differentiation of the genogroups in the genus Ehrlichia.

Ultrastructural characteristics of 15 strains and isolates of ehrlichiae belonging to three genogroups, or clades of genetically related organisms united in the genera Ehrlichia, Cowdria, Anaplasma, Neorickettsia and a strain of Wolbachia pipientis which represents a fourth genogroup in this cluster of species, were studied in continuous cell culture or in vivo: E. canis (Oklahoma strain and VHE isolate), E. muris (AS 145), E. chaffeensis (Arkansas, 91HE17 and Sapulpa), human granulocytic ehrlichiae (HGE)(BDS, 96HE27, 96HE37, #54, #55 and #72), E. equi (MRK), E. sennetsu (Miyayama), E. risticii (HRC-IL). Wolbachia pipientis was studied in the naturally infected Aedes albopictus mosquito cell line Aa23. All organisms were similar in the normal ultrastructure of individual cells and in the ability to form abnormal, pathological ehrlichial cells of the same type irrespective of the species. Normally all ehrlichiae studied in cell culture existed in two morphological forms - reticulate and dense-cored cells, both of which could divide by binary fission. Most alterations were related to their membranes, especially the cell wall. Differences in the structure of intravacuolar microcolonies (morulae) of ehrlichiae and their inter-relations with the host cells allowed differentiation of the genogroups: the E. canis-E. chaffeensis-E. muris genogroup formed large morulae, with many ehrlichiae, often suspended in a fibrillar matrix, and the host cell mitochondria and endoplasmic reticulum usually aggregated near the morulae and were in contact with the morula membrane; the E. phagocytophila-E. equi-HGE group morulae had no fibrillar matrix, no contacts with host cell mitochodria, and they did not aggregate around the morulae; E. sennetsu-E. risticii group usually developed in small individual vacuoles that did not fuse with each other and divided along with the ehrlichiae.

Animals↗
PubMed1998

Tick-transmitted infectious diseases in the United States.

The epidemiologic concept of tick-transmitted diseases has increased in importance with the recognition of the emerging infectious diseases, Lyme borreliosis, human monocytotropic and granulocytotropic ehrlichioses, and three different babesioses. Effective public health control of these diseases would depend upon critical knowledge of the vector biology of the ticks that transmit them. Rocky Mountain spotted fever and the human ehrlichioses are life-threatening yet treatable diseases. A major problem remains establishment of the diagnosis when treatment decisions are being made. Clinical manifestations, other than erythema migrans for Lyme borreliosis, do not provide strong diagnostic clues. Ehrlichiae or babesiae are often not detected in peripheral blood smears. Frequently there are no antibodies to these diverse agents at the time of presentation, and isolation does not yield sensitive and timely results. Polymerase chain reaction, still a research tool, promises the greatest sensitivity, specificity, and timeliness. Prevention by vaccines is not yet a reality, although OspA-based vaccines offer hope for the prevention of Lyme disease.

Animals↗
PubMed1998

Tick-borne diseases in Texas: a 10-year retrospective examination of cases.

The occurrence of Rocky Mountain spotted fever, human monocytotropic ehrlichiosis, tularemia, tick-borne relapsing fever, and a southern erythema migrans-like illness (Lyme/Lyme-like disease) is determined by the geographic distribution and seasonal activity of the particular vector tick(s). The flulike signs and symptoms early in the course of spotted fever rickettsiosis, ehrlichiosis, tularemia, and relapsing fever are nonspecific and do not readily suggest a particular diagnosis. Laboratory diagnosis, particularly during the acute stage of illness, often is elusive. Empiric treatment with doxycycline can be lifesaving for Rocky Mountain spotted fever and ehrlichiosis. This article provides insight into the significance of each of these tick-transmitted diseases in Texas.

Animals↗
PubMed1997

Sequence and characterization of an Ehrlichia chaffeensis gene encoding 314 amino acids highly homologous to the NAD A enzyme.

DNA sequence analysis of the nadA gene of Ehrlichia chaffeensis revealed a 942 bp open reading frame with the capacity to encode 314 amino acids. The amino acid sequence of the E. chaffeensis quinolinate synthetase A (NAD/A) has 53.6% identity and 82% similarity to the NAD A of the cyanelle of Cyanophora paradoxa. Portions of the homologous genes of E. canis and E. muris were also sequenced. The amino acid sequences of the NAD A of E. canis and E. muris have 89.2% and 93.2% homology, respectively, to the NAD A of E. chaffeensis. We propose that the nadA gene may be an excellent candidate for a genetic tool for the phylogenetic study of ehrlichiae.

Bacterial Proteins↗
PubMed1997

Role of T lymphocyte subsets in immunity to spotted fever group Rickettsiae.

To evaluate the roles of CD4 and CD8 T lymphocytes in immunity to disseminated endothelial infection with Rickettsia conorii (Malish 7 strain), these T cell subsets were depleted or adoptively transferred into subsequently infected C3H/HeN mice. CD4 T lymphocyte-depleted and sham-depleted mice underwent a similar course of illness with a sublethal rickettsial dose, cleared the infection by day 10, and recovered on days 10 to 11. In contrast, mice depleted of CD8 lymphocytes or CD4 and CD8 lymphocytes died or remained persistently infected through day 15 with the ordinarily sublethal dose. Endothelium was the major site of rickettsial persistence, including sites in the vital organs, brain, and lungs of CD8 lymphocyte-depleted mice. In nondepleted animals, CD8 T lymphocytes were observed in apposition to endothelial cells on day 10 at the time of rickettsial clearance. Adoptive transfer of immune CD4 or CD8 T lymphocytes protected mice against a lethal dose of R. conorii in the disseminated endothelial target model. Nonimmune CD4 or CD8 lymphocytes and immune lymphocytes that had passed through columns that depleted both CD4 and CD8 lymphocytes failed to protect mice against R. conorii. These studies represent the first analysis of the role of T lymphocyte subsets in immunity to spotted fever group rickettsiae and the first demonstration that clearance of spotted fever group rickettsiae from endothelial cells requires immune CD8 T lymphocytes.

Animals↗
PubMed1997

Cloning and sequencing of the gene for a 120-kDa immunodominant protein of Ehrlichia chaffeensis.

Ehrlichia chaffeensis is the tick-borne, obligately intracellular bacterium that causes human monocytic ehrlichiosis. A 120-kDa protein is one of the immunodominant proteins of E. chaffeensis that stimulates production of specific antibodies in infected humans. A genomic library of E. chaffeensis was constructed in a lambda ZAP II phage vector, and a clone expressing the 120-kDa protein of E. chaffeensis was identified using canine anti-E. chaffeensis serum. DNA sequence analysis of the cloned 120-kDa protein gene of E. chaffeensis identified a 1884-bp open reading frame with an ehrlichial promoter. Five identical 240-bp tandem repeat units were identified in the 120-kDa protein gene of E. chaffeensis, comprising 60% of the entire gene. Aside from the first repeat unit, all the other repeat units are identical. In the first repeat unit there are four nucleotides that are different from the other repeats. Hydropathy analysis of the deduced amino-acid sequence demonstrated that the repeat domain contains highly hydrophilic segments. The 120-kDa protein should be evaluated for a role in stimulating protective immunity.

Amino Acid Sequence↗
PubMed1997

Genetic and antigenic diversity of Ehrlichia chaffeensis: comparative analysis of a novel human strain from Oklahoma and previously isolated strains.

A new Ehrlichia strain, designated as Ehrlichia chaffeensis, Sapulpa strain, was isolated from a patient from Oklahoma with severe ehrlichiosis. Isolation of the ehrlichial pathogen was achieved by inoculating patient blood onto HEL cells and DH82 cells. Antigenic properties of the new isolate were characterized with monoclonal antibodies, homologous patient serum, and polyclonal rabbit serum by Western immunoblotting. The results showed antigenic differences and protein size variation of Sapulpa strain compared with the other 2 strains of E. chaffeensis. Sequencing of the 16S rRNA gene showed 100% identity to that of E. chaffeensis, strain 91HE17. Polymerase chain reaction and sequencing of DNA homologous to the 120-kDa protein gene of E. chaffeensis, Arkansas strain, showed that this gene of Sapulpa strain was smaller than that of Arkansas strain and contained a repeat region with three tandem repeat units.

Animals↗
PubMed1997

Rapid development of affinity matured monoclonal antibodies using RIMMS.

Affinity matured murine monoclonal antibody producing cell lines can now be rapidly generated using a novel repetitive, multiple site immunization strategy designated RIMMS. RIMMS capitalizes on rapid hypermutation and affinity maturation events which occur in B cell populations localized within secondary lymphatic tissue early in response to antigenic challenges. A murine myeloma cell line, P3XBcl-2-13, stably transfected with Bcl-2, enhances the outgrowth of hybridomas following somatic fusion with immune lymphocytes isolated from pooled peripheral lymph nodes (PLN) 8-14 days after the initial immunization. Immunizations somatic fusion, screening and isolation of affinity matured IgG secreting monoclonal antibody cell lines occur within a one month time period. By using RIMMS, we have been able to expedite the isolation of affinity matured monoclonal antibodies to numerous antigens, including a drug hapten.

Animals↗
PubMed1997

Western immunoblotting analysis of the antibody responses of patients with human monocytotropic ehrlichiosis to different strains of Ehrlichia chaffeensis and Ehrlichia canis.

In order to evaluate the relative sensitivity of the detection of antibodies against various antigenic proteins of Ehrlichia chaffeensis for the diagnosis of the emerging infectious disease human monocytotropic ehrlichiosis, Western immunoblotting was performed with 27 serum samples from convalescent patients with antibodies, as demonstrated by indirect immunofluorescence assay. Among 22 patients with antibodies reactive with the 120-kDa protein, 15 showed reactivity with the 29/28-kDa protein(s) and the proteins in the 44- to 88-kDa range. Two of the serum samples with this pattern reacted with the 29/28-kDa protein(s) of only the 91HE17 strain, and one sample reacted with only that of the Arkansas strain, indicating that the antibodies were stimulated by strain-specific epitopes. Overall, antibodies to the 29/28-kDa protein(s) were detected in only 16 patients' sera, suggesting that this protein is less sensitive than the 120-kDa protein. Two of 12 serum samples from healthy blood donors had antibodies reactive with the 120-kDa protein; one of these samples reacted also with the 29/28-kDa protein(s) of Ehrlichia canis, suggesting that unrecognized ehrlichial infection might have occurred, including human infection with E. canis. A high correlation between reactivity with the 120-kDa protein by Western immunoblotting and the recombinant 120-kDa protein by dot blot supports the potential usefulness of this recombinant antigen in diagnostic serology.

Adult↗