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V L Popov

Publications and source records attributed to V L Popov.

At least 19 recordsLinked to original sources
PubMed2000

Mechanically gated channel activity in cytoskeleton-deficient plasma membrane blebs and vesicles from Xenopus oocytes.

1. A novel technique involving hypertonic stress causes membrane 'blebbing' of the Xenopus oocyte and the shedding of plasma membrane vesicles (PMVs). 2. Confocal fluorescence microscopy, immunocytochemistry and electron microscopy indicate that blebs and PMVs lack cortical cytoskeleton and are deficient in cytoskeleton proteins and devoid of microvilli. 3. Patch recordings from PMVs consistently reveal mechanically gated (MG) channel activity. The MG channels display the same single-channel conductance as control recordings but differ in terms of reduced mechanosensitivity and adaptation to sustained stimulation. 4. Whole PMV recordings show rapid and reversible activation of mechanosensitive currents in response to pressure pulses. The maximal currents activated in PMVs are consistent with MG channel activity recorded in patches. 5. The discrepancy between MG channel activity recorded in whole PMVs and oocytes most probably reflects their different membrane geometry and ability to develop activating bilayer tensions. 6. We propose that membrane blebbing, which is known to occur under specific physiological and pathological conditions (e.g. mitosis and apoptosis), may increase mechanosensitivity independently of the intrinsic properties of membrane proteins.

Animals↗
PubMed2000

The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry.

The immunodominant 120 kDa protein (p120) of Ehrlichia chaffeensis was demonstrated to be exposed on the surface of purified whole ehrlichial cells examined by immunoelectron microscopy with a rabbit antibody against a portion of the domain containing tandem repeat units. In the intracellular location, the 120 kDa protein was detected by immunoelectron microscopy in the outer membrane of the cell wall of dense-core forms of the ehrlichiae in infected canine macrophage-like cells and as a component of the intramorular fibrillary matrix. No 120 kDa protein was detected in the cell wall of ehrlichial reticulate cells. Recombinant Escherichia coli with a plasmid containing the entire 120 kDa protein gene, but no bacteria with non-recombinant plasmid, attached to the surface of HeLa cells as visualized by electron microscopy. Some of the recombinant 120 kDa protein expressing E. coli invaded the HeLa cells as determined by gentamicin protection assays and by intravacuolar localization ultrastructurally.

Animals↗
PubMed1999

Role of Salmonella enterotoxin in overall virulence of the organism.

In this study, the Salmonella enterotoxin gene (stn) was mutated by marker exchange mutagenesis, and the overall virulence of the organism was evaluated. Salmonella marker exchange mutants evoked significantly less fluid secretion in mouse intestinal loops compared to that seen with wild-type S. typhimurium. Salmonella mutants were as invasive as wild-type bacteria for HeLa cells; however, their capacity to cause destruction of the intestinal mucosa was impaired, when compared with wild-type bacteria by electron microscopy. Upon oral challenge of mice, the LD(50)of the Salmonella mutants was greater than that for the wild-type bacteria. The fluid secretory potential, as well as a reduction in the LD(50)of these mutants was restored when the mutated stn gene was replaced by the native stn gene sequence. These mutations had no effect on the aerobic growth of these bacteria in minimal or complete medium; anaerobic growth was also not affected. With these studies, we demonstrated that the presence of an intact stn gene contributed significantly to the overall virulence of S. typhimurium in a murine model.

Animals↗
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

Isolation and genetic characterization of a hantavirus (Bunyaviridae: Hantavirus) from a rodent, Oligoryzomys microtis (Muridae), collected in northeastern Peru.

This paper describes the isolation and partial genetic characterization of a hantavirus from a pygmy rice rat, Oligoryzomys microtis, collected within the urban area of Iquitos, Loreto Department, Peru. The virus, designated HTN-007, exhibited the highest degree of genetic similarity to Rio Mamore virus, which was originally described from the same rodent species in eastern Bolivia. Comparison of small and medium segment nucleotide sequence data from HTN-007 and Rio Mamore virus revealed 87% and 85% sequence identity, respectively. Based on these analyses, HTN-007 appears to be a variant of Rio Mamore virus. As such, it represents the first successful isolation of Rio Mamore virus and the first evidence for the existence of a hantavirus in Peru. Serologic studies done by immunofluorescence on blood samples of 56 O. microtis trapped at the collection site indicated that 21.4% had antibodies to hantavirus. In view of the proximity of this rodent species to humans and the close phylogenetic relationship of Rio Mamore virus to hantaviruses that have been associated with human disease, Rio Mamore virus may be a hantavirus of some public health importance in tropical South America.

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

Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants.

Transposon and marker exchange mutagenesis were used to evaluate the role of Aeromonas cytotoxic enterotoxin (Act) in the pathogenesis of diarrheal diseases and deep wound infections. The transposon mutants were generated by random insertion of Tn5-751 in the chromosomal DNA of a diarrheal isolate SSU of Aeromonas hydrophila. Some of the transposon mutants had dramatically reduced hemolytic and cytotoxic activities, and such mutants exhibited reduced virulence in mice compared to wild-type Aeromonas when injected intraperitoneally (i.p.). Southern blot data indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene (act). The transcription of the act gene was affected drastically in the transposon mutants, as revealed by Northern blot analysis. The altered virulence of these transposon mutants was confirmed by developing isogenic mutants of the wild-type Aeromonas by using a suicide vector. In these mutants, the truncated act gene was integrated in place of a functionally active act gene. The culture filtrates from isogenic mutants were devoid of hemolytic, cytotoxic, and enterotoxic activities associated with Act. These filtrates caused no damage to mouse small intestinal epithelium, as determined by electron microscopy, whereas culture filtrates from wild-type Aeromonas caused complete destruction of the microvilli. The 50% lethal dose of these mutants in mice was 1.0 x 10(8) when injected i. p., compared to 3.0 x 10(5) for the wild-type Aeromonas. Reintegration of the native act gene in place of the truncated toxin gene in isogenic mutants resulted in complete restoration of Act's biological activity and virulence in mice. The animals injected with a sublethal dose of wild-type Aeromonas or the revertant, but not the isogenic mutant, had circulating toxin-specific neutralizing antibodies. Taken together, these studies clearly established a role for Act in the pathogenesis of Aeromonas-mediated infections.

Aeromonas hydrophila↗
PubMed1998

Anti-inflammatory and antisecretory potential of histidine in Salmonella-challenged mouse small intestine.

Challenge of mouse small intestinal loops with Salmonella typhimurium invoked the accumulation of luminal fluid, acute inflammation, and extensive structural damage to the small intestinal mucosa, as determined by histology and electron microscopy. Intraperitoneal and intestinal luminal injection of L-histidine, a known antioxidant, reduced the amount of fluid accumulating in the intestinal lumen and protected the intestinal tissue from S. typhimurium-induced damage. The reduction in S. typhimurium-induced fluid accumulation by L-histidine was specific for the L-enantiomer because D-histidine had no significant protective effect. Efficacy of L-histidine in protecting the infected intestinal tissue was attributed to the capacity of the imidazole ring to scavenge reactive oxygen species (ROS) generated by cells in the intestine during the acute inflammatory response. Glutathione levels were markedly reduced in S. typhimurium-challenged, inflamed intestinal tissues as a result of ROS generation. Importantly, after dosing the S. typhimurium-challenged mice with L-histidine, the glutathione content of the small intestinal tissue was not significantly different from mock-challenged controls. Further evidence favoring this mechanism included the capacity of L-histidine to scavenge ROS produced as a result of lipopolysaccharide (LPS) exposure of mononuclear cells (U937), as demonstrated with a redox-sensitive fluorescent dye (2'7'-dichlorodihydrofluorescein [DCF]). Addition of L-histidine, and to a lesser extent D-histidine, to the culture media of U937 cells before LPS exposure, resulted in a significant dose-dependent reduction in LPS-induced intracellular DCF fluorescence, as measured quantitatively by flow cytometry. The potential therapeutic value of anti-inflammatory drugs containing an L-histidine-like structure could protect infected mucosal tissues irrespective of microbial etiology.

Amino Acids↗
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

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

Hyperproduction, purification, and mechanism of action of the cytotoxic enterotoxin produced by Aeromonas hydrophila.

A gene encoding the cytotoxic enterotoxin (Act) from Aeromonas hydrophila was hyperexpressed with the pET, pTRX, and pGEX vector systems. Maximum toxin yield was obtained with the pTRX vector. Approximately 40 to 60% of Act was in a soluble form with the pTRX and pET vector systems. The toxin protein was purified to homogeneity by a combination of ammonium sulfate precipitation and fast protein liquid chromatography-based column chromatographies, including hydrophobic, anion-exchange, sizing, and hydroxylapatite chromatographies. Purified mature toxin migrated as a 52-kDa polypeptide on a sodium dodecyl sulfate (SDS)polyacrylamide gel that reacted with Act-specific antibodies in immunoblots. The minimal amount of toxin needed to cause fluid secretion in rat ileal loops was 200 ng, and the 50% lethal dose for mice was 27.5 ng when injected intravenously. Binding of the toxin to erythrocytes was temperature dependent, with no binding occurring at 4 degrees C. However, at 37 degrees C the toxin bound to erythrocytes within 1 to 2 min. It was determined that the mechanism of action of the toxin involved the formation of pores in erythrocyte membranes, and the diameter of the pores was estimated to be 1.14 to 2.8 nm, as determined by the use of saccharides of different sizes and by electron microscopy. Calcium chloride prevented lysis of erythrocytes by the toxin; however, it did not affect the binding and pore-forming capabilities of the toxin. A dose-dependent reduction in hemoglobin release from erythrocytes was observed when Act was preincubated with cholesterol, but not with myristylated cholesterol. With 14C-labeled cholesterol and gel filtration, the binding of cholesterol to Act was demonstrated. None of the other phospholipids and glycolipids tested reduced the hemolytic activity of Act. The toxin also appeared to undergo aggregation when preincubated with cholesterol, as determined by SDS-polyacrylamide gel electorphoresis. As a result of this aggregation, Act's capacity to form pores in the erythrocyte membrane was inhibited.

Aeromonas hydrophila↗
PubMed1997

Cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells.

In a murine model of rickettsial disease in which, as in human rickettsioses, endothelial cells are the major target of infection, depletion of IFN-gamma or TNF-alpha converts a sublethal infection into a uniformly fatal disease with overwhelming rickettsial growth and decreased nitric oxide (NO) synthesis. The kinetics of NO production and rickettsial survival and growth were examined on Days 1, 2, and 3 after inoculation of endothelial cells with Rickettsia conorii under four different experimental conditions: (a) no cytokine treatment, (b) treatment with IFN-gamma and TNF-alpha, (c) treatment with cytokines and NG monomethyl-L-arginine, a competitive inhibitor of NO synthesis, and (d) treatment with sodium nitroprusside, a source of NO. Endothelial cells were examined for the presence of inducible nitric oxide synthase mRNA by specific reverse transcriptase-PCR after stimulation with IFN-gamma and TNF-alpha. Cytokine-stimulated and unstimulated rickettsiae-infected endothelial cells were examined by electron microscopy to observe the cellular and rickettsial events. Transformed and diploid mouse endothelial cells stimulated by the combination of recombinant murine IFN-gamma and TNF-alpha killed intracellular Rickettsia conorii by a mechanism that required the synthesis of NO. The antirickettsial effect and NO synthesis were inhibited by treatment of endothelial cells with NG monomethyl-L-arginine. Addition of nitroprusside, which released NO, also exerted a strong antirickettsial effect in the absence of IFN-gamma and TNF-alpha. Endothelial inducible nitric oxide synthase mRNA was detected 4 hours after cytokine stimulation, increased substantially at 8 hours, and decreased to low levels by 72 hours. Ultrastructural evaluation revealed that endothelial cells effected rickettsial killing in association with autophagy. Double membranes of endothelial cell granular endoplasmic reticulum surrounded rickettsiae, which were also observed being destroyed within phagolysosomes. This study demonstrated for the first time that endothelial cells are capable of killing rickettsiae. When stimulated by the combination of IFN-gamma and TNF-alpha, mouse endothelial cells kill Rickettsia conorii by an NO-dependent mechanism. Within the endothelium, NO exerts a rickettsicidal effect.

Animals↗
PubMed1997

Electron microscopic analysis of Mycobacterium avium complex isolates exposed to ciprofloxacin, rifabutin, ethambutol and clarithromycin.

SETTING: Mycobacterium avium complex (MAC) isolates grown in the presence of clarithromycin, ciprofloxacin, ethambutol or rifabutin were examined by electron microscopy for drug-induced ultrastructural changes. OBJECTIVE: To further the understanding of the activity of clarithromycin, ciprofloxacin, ethambutol and rifabutin against MAC. DESIGN: Four MAC isolates, 1 control and 3 patient, were cultured for 24 or 72 hours in liquid medium containing one of two concentrations of an antimicrobial agent prior to examination by electron microscopy. RESULTS: One or more of the following ultrastructural changes was observed after exposure to the antimicrobial agent: disorganized nucleoid and cytoplasm, condensation of nucleoid, vacuolization, enlargement of the periplasmic space with or without vesicles, intracytoplasmic lipid-like inclusions, and cell lysis. Changes were seen after as little as 24 hours of exposure to the antimicrobial agent and were more evident with the higher of the two concentrations of the drug. Changes were present regardless of the minimum inhibitory concentration (MIC) of the antimicrobial agent against the isolates tested, but were more pronounced if the MIC was less than the concentration of drug tested. CONCLUSION: Consistent ultrastructural changes were observed following exposure to a given antimicrobial agent. No drug-specific changes were identified.

Antitubercular Agents↗
PubMed1996

Analysis and ultrastructural localization of Ehrlichia chaffeensis proteins with monoclonal antibodies.

Ehrlichia chaffeensis, an obligately intracellular bacterium with tropism for monocytes, is the etiologic agent of human monocytic ehrlichiosis. To determine the nature and ultrastructural location of E. chaffeensis antigens, monoclonal antibodies (MAbs) to E. chaffeensis were developed. The MAbs were used for immunofluorescence and Western immunoblotting analysis of the antigens of density gradient-purified ehrlichiae. Monoclonal antibody 6A1 recognized an epitope of a 30-kD protein. This antibody reacted with a strain-specific epitope of E. chaffeensis, Arkansas strain, and did not cross-react with any other ehrlichia tested. Monoclonal antibodies 3C7 and 7C1-B recognized Arkansas strain proteins of 30 and 29 kD and reacted with E. chaffeensis (strain 91HE17) proteins of 31 and 29 kD and an E. canis protein of 30 kD. Lack of reactivity of these two MAbs with E. sennetsu and E. risticii suggests that the epitope is group-specific. Monoclonal antibody 5D11 recognized a 58-kD protein of both strains of E. chaffeensis as well as E. canis, apparently a group-specific, conformation-independent epitope. Monoclonal antibody 7C1-C reacted with 58- and 88-kD proteins of both the Arkansas and 91HE17 strains. Trypsin treatment destroyed the reactivity of E. chaffeensis antigens with all the MAbs when tested by Western immunoblotting, indicating that these antigens are proteins with trypsin-sensitive epitopes. Immunoelectron microscopy of negatively stained intact E. chaffeensis organisms showed that the 30- and 29-kD proteins are present on the surface of the ehrlichial cell wall along with the previously localized 28-kD protein.

Animals↗
PubMed1995

Ultrastructural variation of cultured Ehrlichia chaffeensis.

The ultrastructure of Ehrlichia chaffeensis (Arkansas strain) was studied in non-irradiated and irradiated monolayers of mouse embryo, Vero, BGM and L929 cells, and in non-irradiated DH82 cells. Within the intracellular parasitophorous vacuoles (morulae), two types of ehrlichial cells were found regularly--those with uniformly dispersed nucleoid filaments and ribosomes (dense-cored cells), which represent the normal life cycle of ehrlichiae. In addition, large reticulate cells were observed, forming long projections of the cell wall, protrusions of cytoplasmic membrane into the periplasmic space, or budding of protoplast fragments (minute forms) into the periplasmic space. Ehrlichiae with abnormalities of protoplast fission were found, apparently leading to formation of giant, multilobular or elongated rod-like ehrlichiae. Morulae were usually surrounded by cisterns of granular endoplasmic reticulum and mitochondria and often contained vesicles, long tubules 25nm in diameter, probably originating from the ehrlichial cell wall, and fibrillar ehrlichial antigen apparently shed from the surface of the cell wall. Some cells contained, in addition to normal morulae, a whole morula that had become dense and contained degenerating ehrlichiae. These results indicate that as well as normal growth and reproduction, ehrlichiae exhibit pathological events: they can be remarkably damaged inside the host cell vacuoles, presumably phagolysosomes, or enter a process morphologically similar to bacterial L-transformation.

Animals↗
PubMed1995

Cultivation of Ehrlichia chaffeensis in mouse embryo, Vero, BGM, and L929 cells and study of Ehrlichia-induced cytopathic effect and plaque formation.

We successfully propagated Ehrlichia chaffeensis in mouse embryo, Vero, BGM, and L929 cells inoculated with host cell-free ehrlichiae, indicating that E. chaffeensis is capable of entry, survival, and growth in a relatively wide range of cell types derived from different species. We demonstrated rapid adaptation of E. chaffeensis in these cell lines, so that typical morulae could be detected as early as 5 days after inoculation. E. chaffeensis-induced cytopathic effect with different morphological characteristics in mouse embryo, Vero, and L929 cells. The earliest cytopathic effect appeared in untreated and irradiated mouse embryo cells at 4 days postinoculation. As the infected foci gradually expanded, the center of the foci showed necrotic cells with pyknotic nuclei and degraded morulae. E. chaffeensis caused cell lysis in untreated and irradiated L929 cells, with formation of distinct, round macroscopic plaques at 18 days postinoculation. In untreated and irradiated Vero cells, E. chaffeensis produced infected foci composed of loosely interwoven necrotic cells, spaces of detached cells, cells filled with morulae, and uninfected cells, resulting in characteristic reticular foci. Irradiated cells generally contained many large morulae and presented larger cytopathic foci. DH82 and BGM cells did not develop obvious cytopathic foci under the conditions employed. The findings reported herein offer the opportunity to study the pathogenic mechanism of cell injury by E. chaffeensis, the basis for quantification of infectious E. chaffeensis, improved approaches for recovery of ehrlichiae from human patients and tick hosts, and additional methods for cultivation of E. chaffeensis for molecular analysis.

Animals↗
PubMed1995

Isolation and characterization of a new strain of Ehrlichia chaffeensis from a patient with nearly fatal monocytic ehrlichiosis.

Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis, a disease that ranges in severity from asymptomatic infection to death. Only one isolate of E. chaffeensis has been made, the Arkansas strain, upon which all characterizations of the agent of human monocytic ehrlichiosis have been based. We report the isolation and characterization of a new strain of E. chaffeensis, the 91HE17 strain, which was cultivated from a patient with a nearly fatal illness. The new isolate grows best in culture with careful control of pH. The two isolates are nearly identical as determined by light and electron microscopy and have significant antigenic identity in fluorescent-antibody and immunoblot assays using polyclonal antisera and the E. chaffeensis-specific monoclonal antibody 1A9. Isolate 91HE17 had 99.9% nucleotide sequence identity with the Arkansas strain in the 16S rRNA gene. Parts of the Escherichia coli GroE operon homologs had identical restriction enzyme digestion patterns, and a 425-bp region of the GroEL gene had at least 99.8% sequence identity between the E. chaffeensis Arkansas and 91HE17 strains. Isolate 91HE17 lacked an epitope identified in E. chaffeensis Arkansas by the monoclonal antibody 6A1. This new E. chaffeensis isolate is very similar to the Arkansas strain and provides the opportunity to substantiate the existence of diversity among ehrlichiae which infect humans. Specific factors which differ among strains may then be compared to assess their potential contributions toward cellular pathogenicity and ultimately toward the development of disease in humans.

Aged↗