Thursday, April 06, 2006
Ehrlichia chaffeensis: a Prototypical Emerging Pathogen
Christopher D. Paddock* and James E. Childs
Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Infectious Disease Pathology Activity, Mailstop G-32, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-1309. Fax: (404) 639-3043.
Ehrlichia chaffeensis is an obligately intracellular, tick-transmitted bacterium that is maintained in nature in a cycle involving at least one and perhaps several vertebrate reservoir hosts. The moderate to severe disease caused by E. chaffeensis in humans, first identified in 1986 and reported for more than 1,000 patients through 2000, represents a prototypical “emerging infection.” Knowledge of the biology and natural history of E. chaffeensis, and of the epidemiology, clinical features, and laboratory diagnosis of the zoonotic disease it causes (commonly referred to as human monocytic ehrlichiosis [HME]) has expanded considerably in the period since its discovery. In this review, we summarize briefly the current understanding of the microbiology, pathogenesis, and clinical manifestations associated with this pathogen but focus primarily on discussing various ecological factors responsible for the recent recognition of this important and potentially life-threatening tick-borne disease. Perhaps the most pivotal element in the emergence of HME has been the staggering increases in white-tailed deer populations in the eastern United States during the 20th century. This animal serves as a keystone host for all life stages of the principal tick vector (Amblyomma americanum) and is perhaps the most important vertebrate reservoir host for E. chaffeensis. The contributions of other components, including expansion of susceptible human populations, growth and broadening geographical distributions of other potential reservoir species and A. americanum, and improvements in confirmatory diagnostic methods, are also explored.
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.Microbiology
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).
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).Two antigen-expressing genes that contain repetitive elements have been identified. The 120-kDa protein gene contains a series of 240-bp serine-rich tandem repeat units; the number of repeat units varies among isolates. To date, three variants of the gene (represented by two, three, or four repeats) have been identified in DNA extracts of E. chaffeensis obtained from patients with HME and from infected ticks (255, 259, 287, 289). The 120-kDa gene encodes a heavily glycosylated, immunodominant surface protein that is preferentially expressed on dense-cored forms of E. chaffeensis and as a component of the intramorular fibrillary matrix (183, 219). This gene demonstrates interstrain variation, and p120 proteins expressed by different isolates of E. chaffeensis vary in molecular weight; however, immune sera from patients with HME react with p120 antigens from various strains regardless of variations in the number of repeat units (290). The VLPT gene demonstrates even greater interstrain diversity (209, 259). This gene is also characterized by a series of direct tandem repeats, whose number may vary among isolates. DNAs of VLPT genes amplified from cultured isolates of E. chaffeensis or from ticks or patient blood samples infected with this pathogen have shown two to six repeats. Qualitative differences in the nucleotide sequences of the imperfect 90-bp repeats results in at least seven different types of repeat units. Additional genetic diversity is produced by differences in the linear order of the individual repeats and by various deletions and substitutions along the length of the gene. Based on a relatively small number of DNAs evaluated, VLPT patterns of E. chaffeensis in the southeastern United States are most frequently represented by three or four repeats, and the six-repeat variant appears to be the rarest version of this gene (206, 255, 257, 259). The biological function of this gene has not yet been elucidated; however, VLPT sequences code for immunoreactive proteins with apparent molecular masses of 30 to 60 kDa (259). Collectively, the occurrence of genetic heterogeneity among several of the recognized genes of E. chaffeensis suggests that considerable molecular diversity exists within this bacterium: evaluation of 18 patient isolates by using genetic composites created by polymorphisms in the VLPT gene and the 120-kDa protein gene reveal eight distinct genotypes (255, 259). No distinct biological, clinical, or epidemiological correlates have been associated with a particular genotype, although future studies may be more revealing.
Isolate-dependent sequence polymorphisms have also been described for a locus of E. chaffeensis genes that encodes major outer membrane proteins (OMP), described as the omp cluster or the p28 multigene family (286). Detailed analysis of this locus in the Arkansas isolate of E. chaffeensis reveals 22 complete, paralogous genes from 813 to 900 bp distributed along a 27-kb segment of the genome (201). The p28 genes code for mature proteins with predicted molecular sizes of approximately 26 to 32 kDa; none of the proteins are identical, and the amino acid sequence identity varies from approximately 20 to 80% (286). Sequences of individual p28 genes also vary among different isolates of E. chaffeensis (171, 286, 291). At least 16 p28 alleles are actively transcribed, and it is likely that the antigenic diversity of E. chaffeensis results from differential expression within this gene family (171). Homologous immunodominant proteins encoded by multigene families have been identified in closely related bacteria, including E. canis, E. ruminantium, A. phagocytophila, and A. marginale (183, 201, 226).
Several major immunoreactive proteins of the Arkansas isolate of E. chaffeensis have been identified by using human antisera in immunoblot analyses. These include polypeptides with relative molecular masses of approximately 120, 66, 58, 55, 44, 29, 28, and 22- kDa (57, 59). Genetic correlates have been established for several of these antigens, including the 120-kDa protein, the GroEL protein (58 kDa), and the p28 proteins.
Variations in reactivity among different isolates of E. chaffeensis have been demonstrated by using monoclonal antibody (MAb) analyses. MAb 1A9 reacts with epitopes of various p28 proteins with different molecular sizes; however, it does not react with all isolates of E. chaffeensis (59, 285), reflecting heterogeneity in the antigenic composition among isolates created by the diversity of p28 proteins (286, 291). Isolate-specific reactivity is also demonstrated by MAb 6A1, which reacts with a surface-exposed, 30-kDa antigen of the Arkansas isolate but does not react with the 91HE17 isolate (56). Variation in sizes of apparently homologous proteins have also been detected by using MAbs (59) and immunoblot analyses demonstrating isolate-specific expression of the 120-kDa protein and VLPT repetitive-element gene products (56, 259). Biological correlates for these variably sized proteins to pathogen virulence or clinical disease in humans are incompletely characterized, although MAbs directed against specific epitopes of p28 OMPs can mediate the clearance of E. chaffeensis in a SCID mouse model (160).
Other than descriptions of the antigenic composition and immunolocalization of these proteins, relatively few phenotypic characteristics of E. chaffeensis have been identified. Experiments with a low-passage, culture-adapted isolate show that this strain can survive for at least 11 days in anticoagulated human whole blood and for as long as 21 days in cell culture media at 4 to 6°C (187).
Epidemiology and Ecology
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