Biology of Anaplasmataceae

INTRODUCTION — Bacteria in the family Anaplasmataceae, have been known to cause diseases of interest to veterinarians for over 100 years. However, their role as agents of human disease was not recognized until 1987. Since then, the number of genera and species recognized to cause human disease has markedly expanded, as has our knowledge about the epidemiology, clinical characteristics, and treatment of the diseases caused by these organisms.

The biology of pathogenic members of the family Anaplasmataceae will be reviewed in this topic. A discussion of the diseases caused by these bacteria is presented separately. (See "Human ehrlichiosis and anaplasmosis".)

TAXONOMY AND PHYLOGENY — The taxonomy of the Rickettsiales order and the two major families (Rickettsiaceae and Anaplasmataceae) underwent a radical change during the last two decades [1,2], based mostly on genomic data, but also on phenotypic features. A detailed discussion of Rickettsiaceae is presented separately. (See "Biology of Rickettsia rickettsii infection".)

The current configuration of the genus Anaplasmataceae provides for four to five clades that represent individual genera that contain known pathogens: Ehrlichia, Anaplasma, Neoehrlichia, Neorickettsia, and Wolbachia. These Anaplasmataceae can be divided into tick and non-tick transmitted bacteria:

●Tick-transmitted – All members of the genera Ehrlichia, Anaplasma, and Neoehrlichia are transmitted by the bites of ticks. These include:

•Ehrlichia: E. chaffeensis, E. canis, E. muris subsp muris, E. muris subsp eauclairensis, E. ruminantium, E. minasensis, and E. ewingii.

•Anaplasma: A. phagocytophilum, A. platys, A. marginale, A. centrale, A. ovis, and A. bovis.

•Neoehrlichia: Candidatus N. mikurensis.

●Non-tick transmitted – Species in the Neorickettsia and Wolbachia genera have distinct and complex transmission mechanisms that do not involve ticks. Members of these genera include:

•Neorickettsia: N. sennetsu, N. risticii, and N. helminthoeca.

•Wolbachia: W. pipientis and other unnamed species.

HOST SPECIFICITY — Individual species of Anaplasmataceae infect a wide range of hosts and cause disease in humans, dogs, cats, sheep, goats, cattle, horses, and other animals (table 1). The most important causes of human disease are members of the genera Ehrlichia and Anaplasma. The majority of recognized human infections are caused by A. phagocytophilum and E. chaffeensis (see "Human ehrlichiosis and anaplasmosis"):

●The white-tailed deer (Odocoileus virginianus) is the major reservoir host for E. chaffeensis. The natural cycle of E. chaffeensis involves lone star ticks (Amblyomma americanum), which may transmit to and acquire the organism from infected deer.

●The major reservoir host for human-infective strains of A. phagocytophilum is generally a small mammal, such as the white-footed mouse (Peromyscus leucopus). P. leucopus is also the host for Ixodes scapularis, the main tick vector in North America. Similar small mammal/tick associations are seen with the related vectors I. pacificus (North America), I. ricinus and I. persulcatus (Europe and Asia), and Haemaphysalis species (Asia). Some variant strains of A. phagocytophilum that infect deer do not infect mice or humans but may be transmitted by Ixodes species ticks. While ticks often have a preference for a specific host, many are promiscuous in their feeding behavior and readily bite humans.

Other species that cause human infection include E. ewingii (which has a natural life cycle and tick vector similar to E. chaffeensis); E. muris subsp eauclairensis; the Panola Mountain Ehrlichia; "A. capra"; A. ovis; Candidatus N. mikurensis (a cause of an emerging potentially severe febrile disease found in Europe and Asia); and Neorickettsia sennetsu. Although evidence exists for human disease related to Wolbachia endosymbionts in filariasis [3], direct infection of humans has not been definitively identified.

CHARACTERISTICS OF ANAPLASMATACEAE

General characteristics — Ehrlichia and Anaplasma spp are obligate intracellular bacteria that grow within membrane-bound vacuoles in human and animal leukocytes. In humans, either monocytes (human monocytic ehrlichiosis) or granulocytes (human granulocytic anaplasmosis and Ehrlichia ewingii ehrlichiosis) can be infected. (See 'Taxonomy and phylogeny' above and 'Cell specificity' below.)

Unlike rickettsiae, which escape the endocytic vacuole to live freely in the cell cytoplasm, Anaplasmataceae spp replicate within modified vacuoles in the cytoplasm of the host cell. A microcolony of these bacteria within a vacuole is called a morula (picture 1A-B). Similar to forms described for Chlamydia species, Anaplasmataceae bacteria within morulae differentiate into both replicative "reticulate" forms and infectious but nonreplicative "dense-core" forms. (See "Biology of Rickettsia rickettsii infection".)

The genomes of Ehrlichia and Anaplasma species lack complete biosynthetic pathways for peptidoglycan and lipopolysaccharide biosynthesis, as suggested by their very simple cell wall ultrastructure, but can still synthesize some components of peptidoglycan. Their genomes also encode paralogous genes for major surface antigenic proteins that are both ligands by which the bacteria bind to host cells before entry, or that contribute to antigenic variation and immune evasion [4]. Likewise, both genera possess structurally unusual but functional type IV secretion systems that translocate bacterial proteins into host cells. These bacterial effector proteins: (1) trigger signaling in the host cell to induce modifications of the cell cytoskeleton that permit pathogen endocytic entry; (2) alter intracellular trafficking of vacuoles to avoid lysosome fusion; or (3) travel to the cell nucleus and alter host cell transcriptional programs and functions [5,6].

Anaplasmataceae spp are difficult to detect by light microscopy as single bacteria within a leukocyte; however, morulae are easily visualized when present within white blood cells. These can be observed in Giemsa and Wright or other Macchiavello method-stained samples. Individual organisms are generally coccoid but can be pleomorphic when visualized in tissue culture cells [1].

Growth characteristics — Individual organisms induce endocytic entry into host cell vacuoles where they grow, divide, and within a few days form small, tightly packed clusters observable as intracellular inclusions (morulae). These bacterial cells have an open chromatin configuration and are not highly infective. A. phagocytophilum and E. chaffeensis cells with condensed chromatin (dense-core cells) appear several days later. These dense-core cells express unique proteins APH_1235 (A. phagocytophilum) or TRP120 (E. chaffeensis) that are important for infectivity [7,8].

Like rickettsiae, Anaplasmataceae spp will not grow in cell-free culture media. Thus far, they have only been cultured in eukaryotic cells. E. chaffeensis and E. canis can be propagated in canine blood monocytes and in monocyte, endothelial, macrophage, and fibroblast cell lines from murine, human, primate, and canine origin. A. phagocytophilum can be cultivated in vitro using human myeloid leukemia cell lines and some primate and human endothelial cell lines. All can be cultivated in tick embryo cell lines. Axenic growth has not been achieved for any species in the Anaplasmataceae family.

Growth in vitro can take one week to one month or longer to detect. The duration of the organism's viability in human-donated blood is unknown; transfusion-transmitted infection is known to occur, even with leuko-reduced blood products, and organisms can be reisolated from spiked EDTA-anticoagulated blood stored at 4°C for 11 days for E. chaffeensis and stored for 18 days for A. phagocytophilum [9,10]. Fatal infection after transfusion-transmission of A. phagocytophilum has been reported [11].

The metabolism of Anaplasmataceae spp is primarily aerobic. Genes required for glycolysis are absent, but the presence of some intact metabolic pathways in their genomic repertoires suggests that A. phagocytophilum and E. chaffeensis can use simple substrates such as glyceraldehyde 3-phosphate and phosphoenolpyruvate for phospholipid and nucleotide synthesis. Carbon sources are proline and glutamine only, and the majority of amino acids must be imported from the host by membrane transporters [4].

E. chaffeensis and A. phagocytophilum evade intracellular defenses by detoxifying superoxide anions or by preventing assembly of phagocyte oxidase and by delaying normal cellular apoptosis. Many of the altered cellular functions in infected hosts are mediated by direct interactions of cellular proteins with microbial-secreted effector molecules [12,13]. However, there is evidence that microbial molecules that bind host nuclear DNA have a role in reprogramming of host cell gene transcription, ultimately altering cell function [5,6,14-16].

Antigenic characteristics — Serologic cross-reactions between E. chaffeensis and A. phagocytophilum occur. In addition, antibodies in the convalescent sera of humans infected with E. ewingii react with antigens of both E. chaffeensis and E. canis.

Cross-reactivity between Anaplasmataceae spp antigens and rickettsial antigens does not occur. Also, Anaplasmataceae spp do not elicit cross-reacting antibodies with Proteus antigens (Weil-Felix agglutinins).

All strains of E. chaffeensis and A. phagocytophilum have modest antigenic differences among strains that result from genetic variation, post-translational modifications, and perhaps variation in expression of the omp1/p28 and msp2/p44 multifamily genes encoded in their genomes. Genetic diversity varies by geographical region, and as a result, there are greater serologic differences among strains from distinct geographical regions.

The A. phagocytophilum genome contains at least 100 genes encoding Msp2/p44. As each organism expresses only a single Msp2 protein at a time, a population of organisms can produce substantial antigenic diversity and evade immune response by switching expression to an antigenically distinct paralog after negative selection by host immune response. Likewise, there are 22 tandemly arranged omp1/p28 genes on the circular genome of E. chaffeensis that are differentially expressed or undergo a variety of post-translational modifications [17]. This likely contributes to immune evasion and persistence among natural coadapted hosts, such as ticks, small mammals, and deer [18], but this is unlikely to contribute significantly in humans, in whom infection does not persist. There is evidence that T cell exhaustion could also contribute to persistent infection; whether this contributes to aberrant immune pathology or protection is unclear [19].

Cell specificity — Depending upon the species, the Ehrlichia or Anaplasma that cause human disease infect circulating granulocytes, monocytes, or tissue macrophages [20-25]. Individual species of Ehrlichia characteristically have different, highly specific cellular tropisms in vivo. As examples:

●A. phagocytophilum – A. phagocytophilum infects mostly neutrophils. Binding and entry into host cells in vitro involves several surface-exposed molecules, including the multigene family major surface protein (Msp2/p44), OmpA, Asp14, and AipA, mostly through interactions with neutrophil surface glycans and glycoproteins [26,27]. This interaction permits introduction of A. phagocytophilum protein AnkA via the type IV secretion apparatus that bridges the bacterial and cell membranes. AnkA interacts with and is phosphorylated by Abl-1 after cell entry. This interaction is critical for pathogen entry into the neutrophil vacuole [28].

Vacuoles that sustain replication of A. phagocytophilum accumulate a range of bacterial-secreted proteins that probably play a role in intracellular survival, vacuole trafficking, and acquisition of nutrients. At least one A. phagocytophilum protein (AnkA), enters the nucleus where it alters gene expression programs [6,14], perhaps including those that govern respiratory burst, apoptosis, and proinflammatory response.

Although monocytes resist infection by A. phagocytophilum, it is likely that induction of an innate immune response and activation of the inflammasome within monocytes and macrophages together mediate the inflammatory response that is the clinical hallmark of human A. phagocytophilum infection, while altering immune responsiveness of T lymphocytes for adaptive immunity [29-32].

●E. chaffeensis – Individual species of Ehrlichia characteristically have different, highly specific cellular tropisms in vivo. E. chaffeensis infects mostly monocytes and macrophages in vivo. As with A. phagocytophilum, it expresses specific surface adhesion proteins (eg, TRP120, EtpE) that mediate binding to monocyte and macrophage selectins or to DNase X in regions associated with lipid rafts [33-35]. Once within the vacuole, E. chaffeensis secretes protein effectors via its type IV secretion apparatus into the host cell that in turn influence intracellular signaling, vacuolar trafficking, apoptosis, and regulation of transcriptional programs that favor survival long enough for acquisition in a subsequent tick bite [12].

Other species, such as Neorickettsia risticii, infect intestinal epithelial cells in horses; Candidatus N. mikurensis likely infects endothelial cells, and "Anaplasma capra" likely infects erythrocytes. In contrast, the host target cells for infection by the E. muris subsp eauclairensis and the Panola Mountain Ehrlichia have not been specifically identified.

MECHANISMS OF DISEASE — Laboratory-based investigation of pathogenetic mechanisms has primarily been focused on E. chaffeensis and A. phagocytophilum infections. Despite genetic, antigenic, and morphologic similarities, these studies suggest that disease mechanisms caused by these two pathogens are distinct. Thus, they are discussed separately below.

E. chaffeensis and human monocytic ehrlichiosis — After introduction into the skin through a tick bite, E. chaffeensis likely spreads via the lymphatics or blood to mononuclear phagocyte-enriched tissues, such as blood, bone marrow, liver, lymph nodes, and spleen. Human monocytic ehrlichiosis does not produce endothelial cell infection manifesting as vasculitis and thrombosis, which are the hallmarks of spotted fever rickettsial infections such as Rocky Mountain spotted fever. (See "Human ehrlichiosis and anaplasmosis" and "Clinical manifestations and diagnosis of Rocky Mountain spotted fever" and "Other spotted fever group rickettsial infections".)

Bone marrow aspirates in patients with E. chaffeensis infection demonstrate hypercellularity of all hematopoietic elements, despite clinical findings such as leukopenia, thrombocytopenia, and anemia [25]. Other histopathologic findings include: noncaseating granulomas or mononuclear phagocyte-rich infiltrates, sometimes with hemophagocytosis in bone marrow; focal hepatocellular apoptosis ranging from mild lobular hepatitis and cholestasis to severe hepatic lobular necrosis; hypercellularity and dissolution of follicular architecture in lymph nodes and spleen, including occasional regions of marked apoptosis or necrosis; and lymphohistiocytic inflammatory infiltrates in tissues including the meninges, brain, and pulmonary interstitium [25,36-39].

The discordance between the marked degree of histopathologic injury that can occur in human monocytic ehrlichiosis and the relatively small burden of bacteria in tissues in infected hosts suggests that human disease is secondary to immunopathologic mechanisms [40]. This is supported by studies in murine models of infection, where infection leads to marked increases in proinflammatory and anti-inflammatory cytokine production and diminished Th1 cytokine production [41]. In addition, TNFa produced by natural killer T (NKT) and CD8 T lymphocytes results in killing of CD4 T cells, and therefore reduced production of IFN-gamma, a key cytokine for control of intracellular E. chaffeensis infection [42,43]. E. chaffeensis may also directly modulate host defense genes of the infected macrophage, or trigger inflammasome activation and marked cellular injury via pathogen- or damage-associated molecular patterns [44,45]. An increasing number of severe infections meet the criteria for diagnosis of hemophagocytic lymphohistiocytosis [46-48]. (See "Clinical features and diagnosis of hemophagocytic lymphohistiocytosis".)

A. phagocytophilum and human granulocytic anaplasmosis — A. phagocytophilum gains access to neutrophils that are recruited to the site of the tick bite before entering the vasculature and disseminating hematogenously [49]. Thereafter, infected cells predominantly remain in the intravascular compartment.

A. phagocytophilum alters normal neutrophil cell functions and their transcriptional programs. As a result, infected neutrophils no longer bind to or emigrate effectively through endothelial barriers and have defects in phagocytosis and intracellular killing. However, infected neutrophils still become activated, degranulate, and produce proinflammatory responses and chemokines that recruit new inflammatory host cells to sustain infection [50].

The histopathologic findings seen in human granulocytic anaplasmosis (HGA) include: normocellular to hypercellular bone marrow despite leukopenia, thrombocytopenia, and anemia; periportal lymphohistiocytic inflammatory infiltrates with focal lobular hepatic lesions and hepatocyte apoptosis (explaining increased serum hepatic transaminase activities); increased splenic red pulp cellularity and focal splenic necrosis; mild to moderate interstitial pneumonitis; and pulmonary hemorrhage. Hemophagocytosis is occasionally observed in spleen, bone marrow, and liver sinusoids, but vasculitis, granulomas, and meningeal inflammation are rare or absent [51].

Some of the clinical findings (eg, fever, leukopenia, and thrombocytopenia) and histologic features (lymphohistiocytic infiltrates) seen in HGA are suggestive of the macrophage activation or hemophagocytic syndrome. As an example, a study of 29 patients with HGA showed that the severity of disease in infected patients was correlated with higher serum levels of triglycerides, ferritin, interleukin-12 p70, and interleukin-10:IFN-gamma ratios compared with matched, uninfected controls [52]. In addition, several patients with severe disease fulfilled the diagnostic criteria for macrophage activation syndrome. Defects in lymphocyte cytotoxicity (CD8 T cell, NK, and NKT cells) in mouse models suggest infection-related dysfunction of MCH class I antigen-presenting cells in vivo [29,31,32,50]. Detailed discussions of HGA and hemophagocytic syndrome are presented separately. (See "Human ehrlichiosis and anaplasmosis", section on 'Clinical manifestations' and "Clinical features and diagnosis of hemophagocytic lymphohistiocytosis".)

IMMUNE RESPONSES — Specific antibody responses are elicited following infection for all members of the Anaplasmataceae family. Pretreatment with convalescent antibodies protects mice with severe combined immunodeficiency from fatal experimental E. chaffeensis infection and partially protects wild-type mice from experimental infection with A. phagocytophilum [53,54].

However, for full protection and recovery from infection, a contribution of cellular immunity by CD4 T lymphocytes is also required [41,55]. For some infected hosts, antibodies may not coincide with the disappearance of organisms or protective immunity. As an example, dogs infected with E. canis, deer infected by E. chaffeensis, small mammals infected with A. phagocytophilum, and ruminants infected by A. marginale can continue to have bacteremia for months or years after acute infection and the appearance of pathogen-specific antibodies [56-59].

SUMMARY

●Ehrlichia and Anaplasma spp are obligate intracellular bacteria that grow within vacuoles in human and animal leukocytes. (See 'General characteristics' above.)

●Ehrlichia and Anaplasma spp are classified in the Rickettsiales order and Anaplasmataceae family and are among five genera (Ehrlichia, Anaplasma, Neoehrlichia, Neorickettsia, Wolbachia), for which individual species can cause or contribute to human disease (table 1). (See 'Taxonomy and phylogeny' above.)

●Individual species of the Anaplasmataceae family infect a wide range of hosts, and disease can be recognized in humans, dogs, cats, sheep, goats, cattle, and horses (table 1). The most important causes of human disease are members of the genus Ehrlichia and Anaplasma. The majority of recognized human infections are caused by A. phagocytophilum and E. chaffeensis. (See 'Host specificity' above.)

●Individual organisms enter host cells by inducing endocytosis after introducing bacterial proteins into the host cells. The organisms redirect the vacuole from lysosome fusion by modifying intracellular trafficking to allow microbial growth and division. Within a few days, small, tightly packed clusters of organisms are observable as intracellular inclusions mostly in leukocytes. (See 'Characteristics of Anaplasmataceae' above.)

●The major target cells of E. chaffeensis, which causes human monocytic ehrlichiosis, are monocytes and macrophages. The major target cell of A. phagocytophilum, which causes human granulocytic anaplasmosis, and for E. ewingii is the neutrophil. (See 'Cell specificity' above and 'Mechanisms of disease' above.)

●Disease severity in Ehrlichia and Anaplasma infections appears to be related to immune pathology and innate immune responses. (See 'Mechanisms of disease' above and 'Immune responses' above.)