Bacterial Infections

In April 1986, a medical intern scanning the peripheral blood smear of a severely ill man with an unexplained illness observed peculiar intracytoplasmic inclusions in several of the patient's monocytes. The patient described multiple tick bites sustained approximately 2 weeks earlier during a visit to a rural area in northern Arkansas, and a presumptive diagnosis of Rocky Mountain spotted fever had been made (104, 174). Clinicians and scientists subsequently identified these inclusions as clusters of bacteria belonging to the genus Ehrlichia, previously known in the United States solely as veterinary pathogens (174). Within the next 5 years, the organism was isolated in cell culture, characterized by molecular techniques, and formally named Ehrlichia chaffeensis (9, 73). During this interval, surveillance efforts identified several hundred cases of moderate to severe, and occasionally fatal, ehrlichiosis in patients with unexplained illnesses following tick exposures (97, 106, 107, 125, 233, 263). These findings indicated that ehrlichiosis was a widespread and significant public health problem of increasing but undefined magnitude.

During the 1990s, two additional Ehrlichia spp., Anaplasma (formerly Ehrlichia) phagocytophila (the agent of human granulocytic ehrlichiosis [HGE]) and E. ewingii (a cause of granulocytic ehrlichiosis in dogs), were identified as human pathogens, and these reports greatly expanded the geographic region and the size of the human population at risk for acquiring one of these potentially lethal infections (19, 42). While most of the cases of ehrlichiosis caused by E. chaffeensis were being identified in the southeastern and south central United States, within a relatively few years of the initial recognition of HGE the number of cases of human ehrlichiosis identified in the northeastern and north cental states surpassed other regional totals (188).
Although the term “emerging infection” has become almost hackneyed, the Ehrlichia spp. that cause human disease in the United States epitomize the intended application of this designation (156). Not only are these pathogens new to science, but their maintenance in nature requires the complex interactions of tick vectors and vertebrate hosts that are sensitive to environmental influences that can drive epidemics (6). Changes in host susceptibility within a population can be a critical factor in disease emergence (193). Ehrlichiosis caused by E. chaffeensis has increasingly been identified in population segments immunosuppressed through aging, infectious causes, malignancy, or medical therapy (206). Reports of severe and fatal ehrlichioses in these population segments will increase as an unavoidable consequence of environmental forces that increase the risk of exposure to these pathogens, coupled with dramatic changes in human demography and the geographic distribution of AIDS cases (63, 154). This entire process has been fueled by technical developments and the application of sensitive and versatile diagnostic methods, particularly PCR, and a renewed interest in tick-borne and other zoonotic diseases (156, 212, 272).

Several reviews have been written on the microbiology and molecular biology of ehrlichiae and the clinical characteristics of the human ehrlichioses (86, 87, 93, 111, 186, 205, 227, 228). This review of E. chaffeensis summarizes much of this material but focuses primarily on the ecological and epidemiological factors that have contributed to its recognition as an agent of human disease. Although disease caused by E. chaffeensis has been termed human monocytic ehrlichiosis or human monocytotropic ehrlichiosis (i.e., HME), designations of ehrlichioses based on cell tropism may become less useful monikers as additional ehrlichial pathogens are recognized. However, this nomenclature is firmly established in the literature, and to avoid confusion in this review, the acronym HME is used to designate disease caused by E. chaffeensis.

Taxonomy and Phylogenetic

PlacementE. chaffeensis is an obligately intracellular bacterium in the family Anaplasmataceae and is a member of the α subdivision of the Proteobacteria. Until 2001, the genus Ehrlichia was composed of a heterogeneous collection of several recognized species (e.g., E. canis, E. phagocytophila, E. sennetsu, E. equi, E. risticii, E. chaffeensis, E. ewingii, and E. muris) and various other taxa that do not have current standing in bacterial nomenclature. This assemblage of species demonstrates considerable molecular diversity based on phylogenetic analyses of 16S rRNA genes, surface protein genes, and groESL heat shock protein operon sequences. On the basis of these differences, Ehrlichia spp. were until recently segregated into three informal “genogroups” (86). A contemporary taxonomic revision reassigned several of these species to other genera (E. sennetsu and E. risticii to Neorickettsia and E. phagocytophila and E. equi to Anaplasma) and emended the genus Ehrlichia to include Cowdria ruminantium, a closely related tick-borne pathogen that causes severe disease (“heartwater”) in ruminants in Africa and the Caribbean. In this classification, all members of the tribe Ehrlichieae were reassigned to the family Anaplasmataceae (88). The bacteria that cause human “ehrlichioses” are now represented by three genera rather than the single genus Ehrlichia; they include Neorickettsia sennetsu (the agent of sennetsu fever) Anaplasma phagocytophila, E. ewingii, and E. chaffeensis.

The emended genus Ehrlichia includes E. canis, E. chaffeensis, E. ewingii, E. muris, and E. ruminantium. These ehrlichiae share various genetic, morphologic, clinical, and ecological features: all are at least 97.7% similar in 16S rRNA gene sequences, all reside and multiply in cytoplasmic vacuoles of host cells (the principal cell types include mononuclear and polymorphonuclear leukocytes and endothelial cells, depending on the particular species); all cause disease in animals, humans, or both; and all are transmitted by hard-tick vectors (88).

Morphology

Light microscopic and ultrastructural descriptions of E. chaffeensis have been based on observations of the pathogen in human leukocytes and tissues and in various cell lines of mammalian origin. In these habitats, these small, nonmotile bacteria reside and grow in cytoplasmic vacuoles derived from an early endosome, forming loose to condensed aggregates of bacteria termed morulae. By light microscopy, these morulae appear as mulberry-like, bosselated intracytoplasmic inclusions that stain dark blue to purple with Romanovsky-type stains (Fig. 1) (227).

By electron microscopy, two distinct morphologic cell types are identified: coccoid and coccobacillary forms with ribosomes and nucleoid DNA fibrils uniformly dispersed throughout the cytoplasm (reticulate cells) (Fig. 2), and predominantly coccoid bacteria with centrally condensed nucleoid DNA and ribosomes (dense-cored cells). Reticulate cells measure 0.4 to 0.6 μm by 0.7 to 1.9 μm, and dense-cored cells measure 0.4 to 0.6 μm in diameter. Both cell types replicate by binary fission, and both demonstrate a gram-negative-type cell wall, characterized by a smooth-contoured cytoplasmic membrane and a generally ruffled outer membrane, separated by a periplasmic space. Members of the genus Ehrlichia do not appear to contain significant amounts of peptidoglycan (227). Both cell types have been demonstrated in clinical samples (209), although the microbiological significance of these distinct morphological forms is unknown. Morulae range from 1.0 to 6.0 μm in width and contain 1 to >40 organisms of uniform or mixed cell types (218, 228). The intramorular space may contain a fine, striated fibrillar matrix and intramorular tubules 25 nm in diameter and as long as 1.5 μm, which originate from the outer membrane of reticulate cells. In cell culture and infected human cells, host cell mitochondria are frequently apposed to the margins of morulae (209, 218).

Isolates of E. chaffeensis

At least 21 isolates of E. chaffeensis have been obtained from patients with HME, infected in Arkansas (73, 90), Oklahoma (59), Florida and Georgia (209, 259), Tennessee (206, 255), and Maryland (262). Isolates of E. chaffeensis from sources other than human tissues are few and include five from white-tailed deer (169) and one from a domestic goat (85), each obtained in Georgia.
Described isolates have been obtained in primary culture by using a continuous canine histiocytoma cell line (DH82 cells) and less frequently, human embryonic lung fibroblasts (HEL 299 cells) (59, 73, 85, 90, 169, 206, 209, 255).

In vitro, E. chaffeensis has been adapted to grow in various other cell lines, including human microvascular endothelial cells (HMEC-1 cells), African green monkey kidney cells (Vero cells), human cervical epithelioid carcinoma cells (HeLa cells), human monocytic leukemia cells (THP-1 cells), HEL 299 cells, mouse embryo cells, buffalo green monkey cells, and murine fibroblasts (25, 38, 58, 128, 187).

Genetic, Antigenic, and Phenotypic Characteristics

The genome size of E. chaffeensis is approximately 1250 kb (239). Among the nucleotide sequences that have been characterized are the 16S rRNA gene (9), various genes coding for immunoreactive proteins including the variable-length PCR target (VLPT) (259) and the 120-kDa (289), 106-kDa, and 37-kDa (290) protein genes, the groESL heat shock operon (260), a quinolate synthetase A gene (292), and a locus that contains 22 homologous but not identical genes (the p28 multigene family) (201, 286).

# posted by Pat O'Connor @ 6:08 AM