Isolation and characterization of Ehrlichia chaffeensis strains from patients with fatal ehrlichiosis

Abstract

Two new isolates of Ehrlichia chaffeensis (designated Jax and St. Vincent) were obtained from patients with fatal ehrlichial infections. Patients developed characteristic manifestations of severe disease due to E. chaffeensis, including marked thrombocytopenia, pulmonary insufficiency, and encephalopathy. Primary isolation was achieved in DH82 cells; the Jax and St. Vincent isolates were detected within 19 and 8 days postinoculation, respectively. The isolates were characterized by molecular evaluation of the 16S rRNA gene, the groESL heat shock operon, a 120-kDa immunodominant protein gene, and an incompletely characterized repetitive-motif sequence (variable-length PCR target [VLPT]). The sequences were compared with those of the corresponding molecular regions in the type isolate (Arkansas). St. Vincent contained one fewer repeat unit in both the 120-kDa protein gene and the VLPT compared with corresponding sequences of the Jax and Arkansas isolates. 16S rRNA gene sequences from the two new isolates had 100% identity to the corresponding sequences of the 91HE17 and Sapulpa isolates of E. chaffeensis, and to the corrected 16S rRNA gene sequence of the Arkansas isolate. The Jax isolate grew more slowly than the St. Vincent isolate in DH82 cells, and both of the new isolates grew more slowly than the extensively passaged Arkansas isolate. Although specific associations between ehrlichial pathogenicity and genotype were not identified from these comparisons, recovery of this organism from a spectrum of clinical presentations remains an integral step in understanding mechanisms of disease caused by E. chaffeensis.

Survival of Ehrlichia chaffeensis in refrigerated, ADSOL‐treated RBCs

“…The conclusions drawn from this current study on the survival of ehrlichiae are complemented and supported by our experience in isolating E. chaffeensis from clinical samples. We have successfully isolated E. chaffeensis from EDTA blood obtained from clinically ill patients and refrigerated at 4°C for as long as 7 days 11 . Our single attempt at isolating this pathogen from a PCR‐positive sample more than 11 days old was unsuccessful.…”

“…The initial cell culture used to grow E. chaffeensis for experimental use was a canine monocytic cell line (DH82), which is the standard cell line used to grow and isolate this bacterium 16 . E. chaffeensis (St. Vincent isolate) was maintained on a semiconfluent monolayer of DH82 cells in 25‐cm 2 polystyrene tissue‐culture flasks, as previously described in detail 11 . The culture was incubated at 37°C in a 5‐percent CO 2 ‐in‐air atmosphere.…”

“…Ficoll‐isopaque preparations were obtained from samples at each time interval by a previously described method 11 . Briefly, 3.0 mL was removed from each Vacutainer tube and processed for the recovery of the WBC fraction.…”

“…Although there are no documented cases of transfusion‐associated infection with E. chaffeensis , persons receiving units of infected blood may be at particular risk for developing severe disease because of their underlying illness. The ehrlichioses appear to be most severe in elderly or immunocompromised patients 11 . The diagnosis of ehrlichiosis in hospitalized patients receiving blood components for other conditions may be problematic, as the disease would occur outside the normal epidemiologic setting for acquiring a tick‐borne infection.…”

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

“…Human ehrlichiosis is characterized by fever, headache, myalgia, thrombocytopenia, leucopenia, and elevated liver enzyme levels (Perez et al, 1996(Perez et al, , 2006Buller et al, 1999;Paddock and Childs, 2003;Martinez et al, 2008;Pritt et al, 2011). Complications, such as pulmonary insufficiency, renal failure, encephalopathy, and disseminated intravascular coagulation can occur in fatal human cases of E. chaffeensis infection (Paddock et al, 1997). Human ehrlichiosis have been reported from the United States, Europe, Asia, Africa, and South America, although most of the case reports have originated in the United States.…”