The Family Rickettsiaceae

Abstract

Bacteria of the order Rickettsiales (Alphaproteobacteria) are gram-negative, small, rod-shaped, and coccoid, with all described species existing as obligate intracellular parasites of a wide range of eukaryotic organisms (Gillespie et al. 2012b). Before the DNA revolution, bacteria were assigned to Rickettsiales based primarily on chemical composition and morphology. Intraordinal classification employed a taxonomic system (i.e., generic characteristics) based on five major biological proper- ties: (1) human disease and geographic distribution, (2) natural vertebrate hosts and other animal vectors, (3) experimental infections and serology reactions and cross-reactions, (4) strain cultivation and stability, and (5) energy production and biosynthesis. This pioneering systematic work resulted in a Rickettsiales hierarchy of three families containing nine obligate and facultative intracellu- lar pathogenic genera: (1) Rickettsiaceae: genera Rickettsia, Coxiella, Rochalima, and Ehrlichia; (2) Bartonellaceae: genera Bartonella, Haemobartonella, Eperythrozoon, and Grahamella; and (3) Anaplasmataceae: genus Anaplasma. However, rickettsial classification has been substantially revised since the turn of the millennium, owing to the technological advances in molecular sequence genera- tion and the advent of several new fields of study, including molecular systematics, phylogenomics, and bioinformatics. Contemporary Rickettsiales taxonomy is radically different from the traditional system, with such tremendous changes as the reassignment of the Q-fever agent (Coxiella burnetii) to Gammaproteobacteria and the placement of the causative agents of several human diseases such as endocarditis, trench fever, and cat-scratch disease (Bartonella spp.) to the Order Rhizobiales.

Full text

547
31 The Family Rickettsiaceae
Magda Beier-Sexton, Timothy P. Driscoll,
Abdu F. Azad, and Joseph J. Gillespie
INTRODUCTION
Bacteria of the order Rickettsiales (Alphaproteobacteria) are gram-negative, small, rod-shaped, and
coccoid, with all described species existing as obligate intracellular parasites of a wide range of
eukaryotic organisms (Gillespie etal. 2012b). Before the DNA revolution, bacteria were assigned to
Rickettsiales based primarily on chemical composition and morphology. Intraordinal classication
employed a taxonomic system (i.e., generic characteristics) based on ve major biological proper-
ties: (1) human disease and geographic distribution, (2) natural vertebrate hosts and other animal
vectors, (3) experimental infections and serology reactions and cross-reactions, (4) strain cultivation
and stability, and (5) energy production and biosynthesis. This pioneering systematic work resulted
in a Rickettsiales hierarchy of three families containing nine obligate and facultative intracellu-
lar pathogenic genera: (1) Rickettsiaceae: genera Rickettsia, Coxiella, Rochalima, and Ehrlichia;
(2) Bartonellaceae: genera Bartonella, Haemobartonella, Eperythrozoon, and Grahamella; and (3)
Anaplasmataceae: genus Anaplasma. However, rickettsial classication has been substantially revised
since the turn of the millennium, owing to the technological advances in molecular sequence genera-
tion and the advent of several new elds of study, including molecular systematics, phylogenomics,
and bioinformatics. Contemporary Rickettsiales taxonomy is radically different from the traditional
system, with such tremendous changes as the reassignment of the Q-fever agent (Coxiella burnetii) to
Gammaproteobacteria and the placement of the causative agents of several human diseases such as
endocarditis, trench fever, and cat-scratch disease (Bartonella spp.) to the Order Rhizobiales.
AQ1
CONTENTS
Introduction ....................................................................................................................................547
Rickettsial Classication ................................................................................................................548
Rickettsiaceae ............................................................................................................................548
Bartonellaceae ........................................................................................................................... 549
Rickettsiella .......................................................................................................................... 549
Anaplasmataceae ....................................................................................................................... 551
Midichloriaceae ......................................................................................................................... 551
Holosporaceae ........................................................................................................................... 553
Rickettsial Genotypic and Phenotypic Characteristics .................................................................. 553
Rickettsial Virulence and Pathogenesis.......................................................................................... 558
Changing Ecology of Rickettsial Pathogens .................................................................................. 559
Classic Epidemic Typhus .......................................................................................................... 559
Endemic Typhus ........................................................................................................................560
Rocky Mountain Spotted Fever (RMSF) ..................................................................................560
Scrub Typhus ............................................................................................................................. 561
Rickettsial Pathogens as Biothreat Agents .....................................................................................562
Acknowledgments ..........................................................................................................................563
References ......................................................................................................................................563
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548 Practical Handbook of Microbiology
This chapter primarily focuses on the family Rickettsiaceae, which is currently comprised exclu-
sively of described species in the genera Rickettsia and Orientia. These two genera contain many
known and potential pathogens of great concern as the causative agents of emerging and reemerging
human diseases (Table 31.1). However, this work also reviews other families that are either currently
included in the order Rickettsiales (Holosporaceae, Anaplasmataceae and Midichloriaceae) or were
previously classied as Rickettsiales (e.g., Bartonellaceae). A summary of the current knowledge of
rickettsial phenotypic and genotypic characteristics is presented, as well as a synopsis of the factors
governing rickettsial virulence and pathogenicity. A perspective on the changing ecology of rickettsial
pathogens is provided, illustrating the emerging and reemerging properties of many pathogenic spe-
cies. Finally, a discussion is given on the utilization of rickettsial pathogens as agents of bioterrorism.
RICKETTSIAL CLASSIFICATION
Rickettsiaceae
Within Rickettsiaceae, only species of the genus Rickettsia, unique bacteria with highly reduced
genomes (Andersson et al. 1998, Andersson and Andersson 1999) and a close evolutionary rela-
tionship with the mitochondrial ancestor (Emelyanov 2003), remain from the original four genera.
Of particular note, one former species of Rickettsia, the scrub typhus agent R. tsutsugamushi, has
been split from Rickettsia and placed in a novel genus, Orientia (Tamura etal. 1995). ForCoxiella,
a monotypic genus originally considered a sister lineage to Rickettsia, reassignment to the
TABLE 31.1
Some Epidemiological Features of Rickettsial Diseases
Agent
Disease/Dominant
Symptoms Vector/Reservoir Host
Geographic
Distribution
Typhus fevers Rickettsia prowazekii Epidemic typhus Human body louse/humans Africa, Asia, America
Sylvatic typhus Flea and louse/ying squirrels United States (only)
Rickettsia typhi Murine (endemic)
typhus
Rodent and cat eas/rats, mice,
opossums (United States)
Worldwide
Transitional
Group
Rickettsia akari Rickettsial pox Mite/house mice Worldwide
Rickettsia felis Cat ea rickettsiosis Fleas/domestic cats, opossums
(United States)
Worldwide
Rickettsia australis Queensland tick typhus Tick/rodents Australia, Tasmania
Tick-transmitted
spotted fevers
Rickettsia rickettsii Rocky Mountain
spotted fever
Tick/rodents, rabbits North, Central and
South America
Rickettsia parkeri Mild spotted fever Tick/rodents? United States,
Brazil, Uruguay
Rickettsia conorii Mediterranean spotted
fever
Tick/rodents, hedgehogs Europe, Asia, Africa
Rickettsia sibirica North Asian tick typhus,
Siberian tick typhus
Tick/rodents Russia, China,
Mongolia, Europe
Rickettsia africae African tick-bite fever Tick/rodents Sub-Saharan Africa,
Caribbean
Rickettsia japonica Oriental spotted fever Tick/? Japan
Rickettsia slovaca Necrosis, erythema Tick/rodents and lagomorphs Europe
Rickettsia helvetica Aneruptive fever Tick/rodents Old World
Rickettsia honei Finders Island spotted
fever, Thai tick typhus
Ticks/? Australia, Thailand
Scrub typhus Orientia
tsutsugamushi
Scrub typhus Mites/rodents Indian subcontinent,
Asia, Australia
AQ2
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549The Family Rickettsiaceae
Gammaproteobacteria was suggested based on phylogenetic analysis of 16S ribosomal DNA
sequences (Weisburg etal. 1989). Robust phylogenomic analysis has conrmed the close relation-
ship of Coxiella spp. with Legionella within the Gammaproteobacteria (Williams etal. 2010).
Phylogenetic analysis of the genus Rochalima resulted in its unication with Bartonella in the
Bartonellaceae, with all species renamed as Bartonella spp. (Brenner etal. 1993). Finally, the genus
Ehrlichia has been moved to the Anaplasmataceae, as its current described species share greater
afnity with Anaplasma than Rickettsia (Dumler etal. 2001).
Species of the genus Rickettsia have traditionally been classied into either the typhus group
(TG) or the spotted fever group (SFG) rickettsiae. However, this classication is no longer valid for
several reasons. First, many uncharacterized species of Rickettsia, particularly those with unknown
pathogenicity and no known association with blood-feeding arthropods or vertebrates, are ancestral
to the more commonly known pathogenic strains (Perlman etal. 2006). Second, several character-
ized species of Rickettsia, such as R. bellii (Stothard etal. 1994), Rickettsia canadensis (Gillespie
etal. 2007), and R. helvetica (Driscoll etal. 2013), do not group within the TG or SFG based on
robust phylogenetic analysis. Finally, a monophyletic clade that lies between TG and SFG rickettsiae,
containing R. felis, R. akari, and R. australis, has been shown to share many traits similar to non-
SFG rickettsiae (Gillespie etal. 2007). This group was named transitional group (TRG) rickettsiae,
with all other early diverging Rickettsia lineages assigned to the ancestral group (AG) rickettsiae.
Including the Scrub Typhus Group rickettsiae (STG), which comprises O. tsutsugamushi and related
strains as sister to the Rickettsia lineages, this reclassication of Rickettsiaceae has been continu-
ally supported in various robust phylogenomic analyses (Gillespie etal. 2008, 2009, 2012a,b). Recent
advances in phylogenetic methods that accommodate base compositional biases in nucleotide and
amino acid sequences, which are critical to employ for the highly AT-rich genomes of Rickettsiales,
suggest that TRG and TG rickettsiae share a common origin relative to the other rickettsial lineages
(Driscoll etal. 2013) (Figure 31.1). The monophyly (common ancestry) of the reclassied Rickettsiaceae
is indisputable, thus providing a nal stability to the classication of this natural rickettsial group.
BaRtonellaceae
DNA hybridization studies and early DNA and RNA sequence comparisons revealed that many spe-
cies within the Bartonellaceae were more closely related to Brucella spp. in the order Rhizobiales
(Alphaproteobacteria) (Brenner etal. 1993). This work prompted the removal of Bartonellaceae from
the Rickettsiales, and the family remains in the Alphaproteobacteria as a lineage of Rhizobilaes, as
supported from phylogenomic analyses (Williams etal. 2007, Driscoll etal. 2013) (Table 31.2 and
Figure 31.1). As with species of Rochalima, species of Grahamella were united with other members
of the genus Bartonella (Birtles etal. 1995). The genera Haemobartonella and Eperythrozoon were
subsequently demonstrated to belong in the mollicutes (family Mycoplasmaceae), a very distantly
related gram-positive lineage of Firmicutes (Rikihisa etal. 1997). Thus, the Bartonellaceae classi-
cation has stabilized, with the contemporary composition consisting of only the genus Bartonella,
some unclassied and environmental specimens, and the incorrectly named Wolbachia melophagi.
Rickettsiella
The genus Rickettsiella, originally described as a member of Rickettsiales (Weiss etal. 1984), com-
prises a group of intracellular pathogens of diverse arthropod species. Based on characteristics
divergent from typical rickettsiae, such as unique cell morphology (Weiser and Zizka 1968), mode of
reproduction (Götz 1971, 1972), and an unusual morphogenic cycle most similar to Chlamydia spp.
(Federici 1980), the placement of Rickettsiella within Rickettsiales was always considered tenuous.
Phylogenetic analysis conrmed the erroneous taxonomic assignment of Rickettsiella (Roux etal.
1997), with an eventual reassignment to the Coxiellaceae (Gammaproteobacteria: Legionellales)
(Garrity etal. 2005). Robust phylogenomic analysis has conrmed the close relationship of Coxiella
spp. with Rickettsiella within the Gammaproteobacteria (Leclerque 2008, Williams etal. 2010).
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550 Practical Handbook of Microbiology
Candidatus Pelagibacter sp. IMCC9063
Candidatus Pelagibacter sp. HTCC7211
Candidatus Pelagibacter ubique HTCC1062
Candidatus Pelagibacter ubique HTCC1002
Methylobacterium sp. 4–46 [Methylobacteriaceae]
Hoeflea phototrophica DFL-43 [Phyllobacteriaceae]
Beijerinckiaceae (2)
Aurantimonadaceae (2)
Phyllobacteriaceae (2)
Rhizobiaceae (6)
Holosporaceae
Rhodospirillaceae (2), in part
Acetobacteraceae (6)
Candidatus Odyssella thessalonicensis L13
Rhodospirillales (8)
Azospirillum sp. B510 [Rhodospirillales; Rhodospirillaceae]
Rhizobiales (15)
Phyllobacteriaceae, in part
Rhizobiales; Hyphomicrobiaceae (2), in part
Parvibaculum lavamentivorans DS-1 [Rhizobiales; Phyllobacteriaceae]
Pelagibacterium halotolerans B2 [Rhizobiales; Hyphomicrobiaceae]
Pseudovibrio sp. FO-BEG1 [Rhodobacterales; Rhodobacteraceae]
Polymorphum gilvum SL003B-26A1 [unclassified]
Rhodobacterales (16)
Rhodobacteraceae, in part
Sphingomonadales (5)
SAR11 (5)
Parvularcula bermudensis HTCC2503 [Parvularculales]
Rhodobacterales; Hyphomonadaceae (3)
Caulobacterales (5)
Rhizobiales (12)
Alphaproteobacterium BAL199 [unclassified]
Candidatus Puniceispirillum marinum IMCC 1322 [unclassified]
Micavibrio aeruginosavorus ARL-13 [unclassified]
Mitochondria (12)
Neorickettsia spp. (2)
Wolbachia endosymbionts (9)
Ehrlichia ruminantium (3)
Ehrlichia canis Jake
Anaplasma marginale (12)
Anaplasmataceae
Rickettsia
Rickettsiaceae
0.5 sub/site
Rickettsiales
Reduced 50%
Orientia tsutsugamushi (2)
Anaplasma centrale lsrael
Culex quinquefasciatus Pel
D. willistoni TSC#14030-0811.24
sp. wRi
Drosophila simulans
Drosophila ananassae
Midichloriaceae
Rickettsiales endosymbiont (Trichoplax adhaerens)
Candidatus Midichloria mitochondrii lricVA
Typhus group
Spotted
fever
group
Transitional group
Ancestral group
Scrub typhus group
Rickettsia akari Hartford
Rickettsia rhipicephali 3-7-female 6-CWPP
Rickettsia peacockii Rustic
R. prowazekii (8)
Rickettsia rickettsii (8)
Rickettsia philipii 364D
Rickettsia sibirica (3)
Rickettsia africae ESF-5
Rickettsia parkeri Portsmouth
Rickettsia conorii (2)
Rickettsia slovaca (2)
Rickettsia heilongjiangensis 054
Rickettsia japonica YH
Rickettsia montanensis OSU 85-930
Rickettsia massiliae (2)
Candidatus Rickettsia amblyommii GAT-30V
Rickettsia endosymbiont (lxodes scapularis)
R. typhi (3)
Rickettsia australis Cutlack
Rickettsia felis URRWXCal2
Rickettsia helvetica C9P9
Rickettsia canadensis (2)
Rickettsia bellii (2)
Drosophila melanogaster
Muscidifurax uniraptor
str. TRS (Brugia malayi)
Culex quinquefasciatus JHB
Anaplasma phagocytophilum HZ
Ehrlichia chaffeensis (2)
Bradyrhizobiaceae (6)
Bartonellaceae (2)
Brucellaceae (2)
Xanthobacteraceae (3)
Alphaproteobacteria
Alphaproteobacterium HIMB114
FIGURE 31.1 Genome-based phylogeny estimated for 164 alphaproteobacterial taxa, 12 mitochondria, and
two outgroup taxa (not shown). The phylogenetic pipeline that entails orthologous protein group (OG) genera-
tion, OG alignment (and masking of less conserved positions), and concatenation of aligned OGs is described
elsewhere (Driscoll etal. 2013). Tree was estimated using the CAT-GTR model of substitution as implemented
in PhyloBayes v3.3 (Lartillot and Philippe 2004, 2006). Tree is a consensus of 1522 trees (post-burn-in) pooled
from two independent Markov chains that run in parallel. Branch support was measured via posterior prob-
abilities, which reect frequencies of clades among the pooled trees (all branches were recovered at 100%).
Rickettsiales is noted with a star. Mitochondria, boxed; rickettsial families Holosporaceae, Anaplasmataceae, and
Midichloriaceae boxed and shaded light gray; Rickettsiaceae, boxed and shaded dark gray. Classication scheme
for Rickettsia spp. follows previous studies (Gillespie etal. 2007, 2008). More details such as taxon names and
genome accession numbers are provided elsewhere. (From Driscoll, T. etal., Genome Biol. Evol., 5(4), 621, 2013.)
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551The Family Rickettsiaceae
anaplasmataceae
Molecular phylogenetic analysis revealed that the once monotypic Anaplasmataceae was comprised
of four lineages containing ve described genera: Anaplasma, Cowdria, Ehrlichia, Neorickettsia,
and Wolbachia. In a landmark study, Dumler etal. (2001) meticulously placed the majority of the
species in these ve genera into Neorickettsia, Wolbachia, Anaplasma, and Ehrlichia, with sub-
stantial revision of the latter two genera. Robust phylogeny estimations have supported the mono-
phyly of the contemporary Anaplasmataceae, and its sister relationship with the Rickettsiaceae
within Rickettsiales (Williams etal. 2007, Gillespie etal. 2012b, Driscoll etal. 2013). Neorickettsia
spp. are the most ancestral lineage within the Anaplasmataceae, followed by the Wolbachia endo-
symbionts and parasites, then the sister clade containing Anaplasma and Ehrlichia (Figure 31.1).
The latter two genera are much less divergent from one another than any other generic comparisons
within the Rickettsiales, suggesting their relatively recent divergence from one another (Driscoll
etal. 2013).
midichloRiaceae
A novel rickettsial family, Midichloriaceae, was recently proposed based on the discovery of a large
clade of species that is distinct from the Rickettsiaceae and Anaplasmataceae (Gillespie etal. 2012b,
Driscoll etal. 2013, Montagna etal. 2013). This family includes many species with diverse eukary-
otic hosts, which are predominantly aquatic (i.e., corals, sponges, placozoans, Hydra spp., and pro-
tists). Midichloriaceae was named after the tick symbiont Candidatus Midichloria mitochondrii,
which is known to invade and devour mitochondria (Sassera etal. 2006). Unique genomic traits
dene Candidatus M. mitochondrii, particularly genes encoding agella and cbb(3) oxidase (Sassera
etal. 2011). However, the phylogenetic position of the Midichloriaceae within the Rickettsiales is
still tenuous. Genome-based phylogenies support the sister relationship of Midichloriaceae with
Rickettsiaceae (Sassera et al. 2011, Driscoll et al. 2013); however, 16S rDNA-based phylogenies
suggest a closer relationship of Midichloriaceae with Anaplasmataceae (Gillespie et al. 2012b,
Driscoll etal. 2013). The lineages sampled within the analysis of 16S rDNA sequences outline the
extraordinary diversity within the Midichloriaceae and pave the way for future genome sequenc-
ing of additional taxa to better understand the proximate position of this natural group within the
Rickettsiales (Figure 31.2).
TABLE 31.2
Pathogens That Are No Longer Associated with Rickettsiales
Agent
Disease/Dominant
Symptoms Vector/Reservoir Host
Geographic
Distribution
Coxiella C. burnetii Q-fever Tick/goats, sheep, cattle,
domestic cats
Worldwide
Bartonella B. henselae Cat-scratch disease Cat ea/domestic cat Worldwide
B. quintana Trench fever Human body louse/humans Worldwide
B. bacilliformis Oroya fever Sand y/? Peru, Ecuador, Colombia
Ehrlichia E. chaffeensis Monocytic Ehrlichiosis Tick/mammals
(deer, rodents)
Worldwide
E. ewingii ? Tick/deer? United States
Neorickettsia N. sennetsu Sennetsu fever Snail/sh Japan, Malaysia
Anaplasma A. phagocytophilum Anaplasmosis Tick/rodents, other small
mammals
United States, Europe,
Asia, Africa
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552 Practical Handbook of Microbiology
Candidatus Caedibacter acanthamoebae (Acanthamoeba polyphaga AHN-3)
Caedibacter caryophila str. 221 (ciliate Paramecium caudatum)
Uncultured deep-sea bacterium clone Ucm1520 (Atlantic Ocean, Angola Basin) [ES]
Candidatus Paraholospora nucleivisitans (ciliate Paramecium sexaurelia)
Holospora obtusa (ciliate Paramecium caudatum)
UB clone HOCICi16 (drinking water distribution system simulator)
Candidatus Captivus acidiprotistae (unidentified acidophilic protist)
UB clone Oh3123O11E (prairie dog flea Oropsylla hirsuta)
Holosporaceae bacterium str. Serialkilleuse_9403403 (Acanthamoeba sp.)
Candidatus Odyssella thessalonicensis str. L13 (Acanthamoeba spp.)
URB clone PRTBB8516 (ocean water at 6000 m depth from Puerto Rico Trench) [ES]
Endosymbiont (Acanthamoeba sp. TUMK-23)
Candidatus Odyssella sp. 5-F (Sanwayao wastewater plant) [ES]
Candidatus Odyssella thessalonicensis str. L13 (Acanthamoeba spp.)
Endosymbiont (Acanthamoeba sp. KA/E23)
UB clone R2-Liz3 (euglenid protist Petalomonas sphagnophila) [ES]
UB clone R2-FM3 (euglenid protist Petalomonas sphagnophila) [ES]
Chara vulgaris (Streptophyta; Charophyceae)
Reclinomonas americana (Jakobida; Histionidae)
Phytophthora infestans (stramenopiles; Oomycetes)
Rhodomonas salina (Cryptophyta; Pyrenomonadales)
Malawimonas jakobiformis (Malawimonadidae)
Prototheca wickerhamii (Chlorophyta; Trebouxiophyceae)
Physcomitrella patens (Streptophyta; Embryophyta)
Chaetosphaeridium globosum (Streptophyta; Coleochaetophyceae)
Mesostigma viride (Streptophyta; Mesostigmatophyceae)
Nephroselmis olivacea (Chlorophyta; Prasinophyceae)
UB clone R1-FM1 (euglenid protist Petalomonas sphagnophila) [ES]
Orientia tsutsugamushi str. lkeda (mite Leptotrombidium sp.)
Rickettsia endosymbiont (deer tick Ixodes scapularis)
UB clone SHFG464 (coral Montastraea faveolata)
Rickettsia bellii str. RML369-C (tick Dermacentor variabillis)
URB clone EV221H2111601SAH71 (South Africa: Kalahari Shield, subsurface water) [ES]
UA clone SM1B06 [ES]
Rickettsiaceae endosymbiont (volvocalean green algae Pleodorina japonica)
Rickettsiaceae endosymbiont (volvocalean green algae Carteria cerasiformi s)
Bacterial symbiont (ciliate Diophrys appendiculata)
URB clone Ho(lakePohlsee)_4(epithelium of Hydra oligactis)
Secondary endosymbiont (weevil Curculio sikkimensis)
Rickettsia endosymbiont (leech Torix tagoi)
Rickettsia endosymbiont (water beetle Deronectes semirufus)
Secondary symbiont Hefei (aphid Sitobion miscanthi)
Candidatus Occidentia massiliensis str. Os18 (soft tick Ornithodoros sonrai)
Candidatus Cryptoprodotis polytropus (ciliate Pseudomicrothorax dubius)
UB clone R1-Liz1 (euglenid protist Petalomonas sphagnophila) [ES]
UB clone ELB16-030 (Antarctica: Southern Victoria Land, Lake Bonney) [ES]
Neorickettsia helminthoeca (trematode Nanophyetus salmincola)
Wolbachia endosymbiont clone KrWlbOkn1 (birch catkin bug kleidocerys resedae)
Wolbachia endosymbiont str. TRS (nematode Brugia malayi)
Ehrlichia ruminantium str. Gardel (tick Amblyomma variegatum)
Candidatus Neoanaplasma japonica(Ixodidae)
Anaplasma marginale str. St. Maries (Ixodidae)
Aegyptianella pullorum (soft tick Argas (Persicargus) persicus)
Candidatus Neoehrlichia mikurensis(Homo sapiens)
Endosymbiont (weevil Rhinocyllus conicus)
Uncultured neorickettsia sp.(garfish xenentodon cancila)
Neorickettsia risticii str. Illinois (trematode Acanthatrium oregonense)
Candidatus Xenohaliotis californiensis (abalone Haliotis cracherodii)
Candidatus Anadelfobacter veles (protist ciliate Euplotes harpa)
UP clone PEACE2006/237_P3 [ES]
UB clone Gven_P15 (coral Gorgonia ventalina)
UB clone Mfav_P11 (coral Montastraea faveolata)
UB clone Cc045 (marine sponge Cymbastela concentrica)
Rickettsiales endosymbiont (placozoan Trichoplax adhaerens)
UB clone RPR28 (drinking water)
UB clone RNA62799 (drinking water)
URB Ho_lab_2.5 (Hydra oligactis)
UA clone MD3.55 (coral Montastraea faveolata)
Candidatus Cyrtobacter comes (protist ciliate Euplotes harpa)
URB clone Hv(lakePohlsee)_25 (Hydra vulgaris)
URB Montezuma (ticks Ixodes persulcatus, Haemaphysalis concinnae)
Candidatus Lariskella arthropodarum clone AmLaKk1 (seed bug Arocat us melanostomus)
UB clone CF2 (cat flea Ctenocephalides felis)
Anaplasmataceae
sensu lato
Bootstrap
<.50
0.1
.51–.60
.61–.70
.71–.80
.81–.90
.91–1
Midichloriaceae Anaplasmataceae Holosporaceae
Mitochondria
Rickettsiaceae
UB clone XC1 (rat flea Xenopsylla cheopis)
Candidatus Lariskella arthropodarum clone DbLaKnz (Hairy Shieldbug D olycoris baccarum)
Candidatus Lariskella arthropodarum (seed bug Nysius plebeius isol ate NPTA1 bacteriome)
Candidatus Lariskella arthropodarum clone KrLaSpr (Birch Catkin bug Kleidocerys re sedae)
Rickettsiales bacterium str. Huangshan-1 (castor bear tick Ixodes (ovatus) ricinus)
Candidatus Midichloria mitochondrii str. IricVA (castor bean tick I. ricinus)
Candidatus Midichloria mitochondrii (castor bean tick Ixodes ricinus)
Candidatus Nicolleia massiliensis (castor bean tick Ixodes ricinus)
Candidatus Midichloria sp. Ixholo1 (paralysis tick Ixodes holocyclus)
UB clone Hw124 (tick Haemaphysalis wellingtoni from chicken)
URB clone ID25L(rainbow trout Oncorhynchus mykiss)
UA clone sw-xj63 (cold spring) [ES]
Endosymbiont (Acanthamoeba sp. UWC36)
UB clone Mfav_F04 (coral Montastraea faveolata)
UB clone Mfav_B15(coral Montastraea faveolata)
FIGURE 31.2 Phylogeny of SSU rDNA sequences estimated for 78 Rickettsiales taxa, 10 mitochondria, and
5 outgroup taxa (not shown). The phylogenetic pipeline that entails alignment and tree-building methods is
described elsewhere (Driscoll etal. 2013). Tree is nal optimization likelihood: (−22042.321923) using GTR
substitution model with GAMMA and proportion of invariant sites estimated. Brach support is from 1000
bootstrap pseudoreplications. For nodes represented by 2 bootstrap values, the left is from the analysis that
included 10 mitochondrial sequences, with the right from the analysis without the mitochondrial sequences.
All nodes with single bootstrap values had similar support in both analyses. Dashed branches are reduced
75% (mitochondria) or increased 50% (within Rickettsiaceae). Cladograms depict minimally resolved lin-
eages within the Midichloriaceae. For each taxon, associated hosts are within parentheses, with ES depicting
an environmental sample. Other abbreviations: UB, uncultured bacterium; UP, uncultured proteobacterium;
UA, uncultured alphaproteobacterium; URB, uncultured Rickettsiales bacterium. Taxa within black boxes
have available genome sequence data. More details such as taxon names and sequence accession numbers are
provided elsewhere. (From Driscoll, T. etal., Genome Biol. Evol., 5(4), 621, 2013.)
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553The Family Rickettsiaceae
holospoRaceae
Family Holosporaceae was rst added to the Rickettsiales in 2001 (Boone etal. 2001). This group is
comprised primarily of endosymbionts of protists, particularly amoebae, and was shown to form a
distinct lineage basal to the derived rickettsial lineages (Baker etal. 2003). Much of the knowledge
pertaining to these organisms comes from the described species of Holospora, particularly the par-
amecia-associated H. obtusa (Gromov and Ossipov 1981). Very recently, a draft genome sequence
has been generated for H. undulata (Dohra etal. 2013), though no studies have analyzed this infor-
mation within a phylogenomic context. Another member of Holosporaceae, Candidatus Odyssella
thessalonicensis, which is an obligate intracellular parasite of Acanthamoeba species (Birtles etal.
2000), is the only member of Holosporaceae previously included in phylogenomics studies. Initial
analysis of the Candidatus O. thessalonicensis genome suggested its removal from the Rickettsiales,
as phylogeny estimation grouped it among other basal lineages of Alphaproteobacteria (Georgiades
etal. 2011). However, robust phylogenomic analysis subsequently placed Candidatus O. thessa-
lonicensis as the most divergent lineage of Rickettsiales, branching off prior to the mitochondrial
ancestor and other derived rickettsial lineages (Driscoll etal. 2013) (Figure 31.1). This is consis-
tent with the phylogenetic analysis of 16S rDNA sequences, which captures a large monophyletic
group of mostly protist-associated species at the base of the rickettsial tree (Figure 31.2). Like
Midichloriaceae, the diversity encompassed within the Holosporaceae is extraordinary, necessitat-
ing the collection of more genome sequences from other species to bolster the taxonomic position at
the root of the rickettsial phylogeny. This information will prove invaluable for further stabilization
of rickettsial classication and will also shed light on the evolution of diverse strategies of obligate
intracellular lifestyle.
RICKETTSIAL GENOTYPIC AND PHENOTYPIC CHARACTERISTICS
The rst sequenced genome of a rickettsial species, R. prowazekii, revealed numerous pseudo-
genes and a highly reduced genome relative to facultative intracellular and free-living bacterial
species (Andersson etal. 1998). Since then, dozens of additional genomes from species within all
major genera have been sequenced, all being highly reductive yet encoding slightly different meta-
bolic capacities (Gillespie etal. 2012b). Regarding Rickettsiaceae, as of April 2014, three genome
sequences of STG rickettsiae are available, coupled with 59 genome sequences from species of
Rickettsia (Table 31.3). The genomes of two strains of O. tsutsugamushi possess a staggering prolif-
eration of mobile genetic elements (MGEs), on par with that of the human genome (Cho etal. 2007,
Nakayama etal. 2008). Still, these genomes contain several hundred core genes in common with
Rickettsia genomes, though with slightly fewer encoding metabolic enzymes (e.g., an incomplete
TCA cycle) (Nakayama etal. 2010). The third STG rickettsiae genome was recently generated from
an uncharacterized species, Rickettsiaceae bacterium Os18, which likely corresponds to a species
previously conditionally named Candidatus Occidentia massiliensis. This genome has not been
thoroughly analyzed within a phylogenomic context, but its phylogenetic position suggests it is
an intermediate between O. tsutsugamushi and Rickettsia spp. (Gillespie, unpublished data). The
potential for pathogenesis by Rickettsiaceae bacterium Os18 is unknown.
Importantly, the genome of Rickettsiaceae bacterium Os18 does not encode the proliferated
MGEs that characterize the O. tsutsugamushi genomes, suggesting the latter acquired these ele-
ments after diverging from the former. It is tempting to speculate that these proliferated MGEs in
O. tsutsugamushi genomes play a role in pathogenesis, possibly in providing the rickettsiae with a
dynamic arsenal of surface antigens for avoidance of the host immune response (Cho etal. 2007,
Gillespie et al. 2012a). Aside from several known host factors (i.e., bronectin, α5β1 integrins,
and syndecan-4 receptors), three proteins have been shown to mediate attachment and entry of
O.tsutsugamushi into host cells through a clathrin-dependent endosomal pathway (Ge and Rikihisa
2011). Two of these proteins, TSA (47 kDa surface protein) and surface antigen ScaC, are unknown
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554 Practical Handbook of Microbiology
TABLE 31.3
Characteristics of Rickettsiaceae Genome Sequencesa
Genome Contig %GC Length Genes Plasmids
Scrub Typhus Group (STG)
Rickettsiaceae bacterium str. Os18 301 31.25 1,469,247 1310 0
O. tsutsugamushi str. Ikeda 1 30.51 2,008,987 2197 0
O. tsutsugamushi str. Boryong 1 30.53 2,127,051 2364 0
Ancestral Group (AG)
Rickettsia sp. MEAM1 (Bemisia tabaci) 607 32.40 1,105,415 1036 0
R. bellii str. RML369-C 1 31.65 1,522,076 1612 0
R. bellii str. OSU 85–389 1 31.63 1,528,980 1657 0
R. canadensis str. CA410 1 31.01 1,150,228 1130 0
R. canadensis str. McKiel 1 31.05 1,159,772 1230 0
Incertae sedis
R. helvetica str. C9P9 1 32.20 1,369,827 1739 1
Transitional Group (TRG)
R. felis str. LSU-Lb 35 32.44 1,467,654 1831 2
R. felis str. URRWXCal2 1 32.45 1,485,148 1810 2
R. felis str. LSU 21 32.02 1,483,097 2194 1
R. akari str. Hartford 1 32.33 1,231,060 1437 0
R. australis str. Cutlack 1 32.25 1,296,670 1565 1
R. australis str. Phillips 45 31.88 1,274,508 1525 0
Typhus Group (TG)
R. typhi str. TH1527 1 28.92 1,112,372 875 0
R. typhi str. B9991CWPP 1 28.92 1,112,957 877 0
R. typhi str. Wilmington 1 28.92 1,111,496 892 0
R. prowazekii str. GvV257 1 28.99 1,111,969 902 0
R. prowazekii str. RpGvF24 1 28.99 1,112,101 897 0
R. prowazekii str. GvF12 10 27.94 1,109,257 966 0
R. prowazekii str. BuV67-CWPP 1 29.00 1,111,445 901 0
R. prowazekii str. Katsinyian 1 29.00 1,111,454 902 0
R. prowazekii str. NMRC Madrid E 1 29.00 1,111,520 926 0
R. prowazekii str. Madrid E 1 29.00 1,111,523 924 0
R. prowazekii str. Rp22 1 29.00 1,111,612 910 0
R. prowazekii str. Cairo 3 20 36.12 1,113,960 900 0
R. prowazekii str. Chernikova 1 29.01 1,109,804 892 0
R. prowazekii str. Dachau 1 29.01 1,108,946 907 0
R. prowazekii str. Breinl 1 29.01 1,109,301 914 0
Spotted Fever Group (SFG)
Rickettsia monacensis str. IrR/Munich 88 32.09 1,268,385 1684 1
Rickettsia endosymbiont of Ixodes scapularis 16 38.05 1,776,032 2404 4
R. montanensis str. OSU 85–930 1 32.57 1,279,798 1513 0
Candidatus Rickettsia amblyommii str. GAT-30V 1 32.43 1,407,796 1846 3
R. rhipicephali str. 3–7-female6-CWPP 1 32.41 1,290,368 1621 1
Rickettsia massiliae str. AZT80 1 32.61 1,263,659 1601 1
R. massiliae str. MTU5 1 32.54 1,360,898 1721 1
Candidatus Rickettsia gravesii str. BWI-1 28 32.58 1,327,625 1687 0
R. japonica str. YH 1 32.35 1,283,087 1575 0
Rickettsia heilongjiangensis str. 054 1 32.32 1,278,468 1562 0
(Continued )
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555The Family Rickettsiaceae
from Rickettsiaceae bacterium Os18. The other protein, HtrA (47 kDa surface protein), is highly
conserved across Rickettsiaceae genomes, with its function as a surface protein in O. tsutsugamushi
possibly a moonlighting role that needs to be determined for other rickettsial species. Collectively,
in spite of a lack of phenotypic data for Rickettsiaceae bacterium Os18, it is apparent that substan-
tial genotypic differences underlay the STG rickettsiae genomes, illustrating the need to employ
phylogenomics as a means to correlate pending characterized phenotypic traits with underlying
genotype.
Substantial variation in size and gene number is seen across the 59 sequenced Rickettsia genomes
(Table 31.3). The TG rickettsiae genomes are the smallest and encode the fewest number of genes,
consequences of being the most degraded and rapidly evolving genomes of Rickettsia (Blanc etal.
2007). The TG rickettsiae genomes also contain a slightly higher AT-bias within their genomes.
Outside of TG rickettsiae, such genome metrics do not consistently dene other monophyletic
groups. This is exemplied by the larger Rickettsia genomes, which are found in the AG (R. bellii),
TRG (R. felis), and SFG (REIS) rickettsiae. In fact, the larger Rickettsia genomes tend to have higher
numbers of transposases and other MGEs relative to the smaller genomes, illustrating the role of
lateral gene transfer in the evolution of Rickettsia species (Gillespie etal. 2012a). The presence of
plasmid(s) is also variable across species (and sometimes strains), yet it is likely that all species
are able to harbor plasmids given that R. prowazekii, which does not encode plasmids, can stably
maintain a transected SFG-like plasmid (Wood etal. 2012). Notwithstanding these variable features
across Rickettsia genomes, mapping of protein families over generated genome-based phylogenies
strongly supports the four-group classication scheme, with each group having a distinct genetic
prole that is predominantly of vertical descent (Gillespie etal. 2008, 2012b).
TABLE 31.3 (continued)
Characteristics of Rickettsiaceae Genome Sequencesa
Genome Contig %GC Length Genes Plasmids
R. honei str. RB 11 32.54 1,268,758 1614 0
R. peacockii str. Rustic 1 32.56 1,288,492 1558 1
Rickettsia philipii str. 364D 1 32.47 1,287,740 1570 0
R. rickettsii str. Hlp#2 1 32.47 1,270,751 1574 0
R. rickettsii str. Colombia 1 32.46 1,270,083 1560 0
R. rickettsii str. Brazil 1 32.45 1,255,681 1547 0
R. rickettsii str. Sheila Smith 1 32.47 1,257,710 1577 0
R. rickettsii str. Arizona 1 32.44 1,267,197 1580 0
R. rickettsii str. Hauke 1 32.45 1,269,721 1570 0
R. rickettsii str. Iowa 1 32.45 1,268,175 1595 0
R. rickettsii str. Hino 1 32.45 1,269,837 1593 1
R. slovaca str. D-CWPP 1 32.50 1,275,720 1598 0
R. slovaca 13-B 1 32.50 1,275,089 1611 0
R. parkeri str. Portsmouth 1 32.43 1,300,386 1604 0
R. africae str. ESF-5 1 32.40 1,278,530 1545 1
R. sibirica str. 246 1 32.47 1,250,020 1554 0
R. sibirica subsp. sibirica BJ-90 8 32.51 1,254,734 1588 0
R. sibirica subsp. mongolitimonae HA-91 21 31.92 1,252,337 1616 0
R. conorii str. Malish 7 1 32.44 1,268,755 1578 0
R. conorii subsp. indica ITTR 10 32.49 1,249,482 1601 0
R. conorii subsp. israelensis ISTT CDC1 33 31.83 1,252,815 1640 0
R. conorii subsp. caspia A-167 25 32.38 1,260,331 1657 0
a All sequences were annotated with RAST (Aziz etal. 2008).
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556 Practical Handbook of Microbiology
Phylogenomics has provided some clues for why certain species of Rickettsia do not t well
into the traditional TG and SFG rickettsiae. For instance, R. canadensis was previously associ-
ated with both TG and SFG rickettsiae until a robust phylogeny estimation suggested its closer
afliation with R. bellii, which together were united in the AG rickettsiae (Gillespie etal. 2007).
This result was consistent with only a few other reports (Stothard and Fuerst 1995, Vitorino
etal. 2007), as most other phylogenetic studies grouped R. canadensis erroneously based on the
molecular marker being employed for phylogeny estimation. This instability of R. canadensis
in analysis of different genes was clearly illustrated (Vitorino etal. 2007), and correlates with
phenotypic traits that do not clearly delineate this species into TG or SFG rickettsiae. For exam-
ple, like SFG rickettsiae, R. canadensis (1) infects ticks and is maintained transstadially and
transovarially (Burgdorfer 1968, Brinton and Burgdorfer 1971), (2) grows within the nuclei of
its host (Burgdorfer 1968), and (3) contains genes encoding both Sca0 and Sca5 surface antigens
(Dasch and Bourgeois 1981, Ching etal. 1990) (Figure 31.3). However, similar to TG rickett-
siae, R. canadensis grows abundantly in yolk sac, lyses red blood cells, is susceptible to eryth-
romycin, and forms smaller plaques relative to SFG rickettsiae (Myers and Wisseman 1981).
Genomic characteristics are just as ambiguous, as despite sharing roughly the same G+C% as
TG rickettsiae (Myers and Wisseman 1981, Eremeeva etal. 2005) and being more similar to
TG rickettsiae in the percentage of coding sequence per genome and number of predicted genes
(Table 31.3), R. canadensis shares more small repetitive elements with SFG rickettsiae than
TG rickettsiae (Eremeeva etal. 2005). Collectively, these attributes for R. canadensis illustrate
how a rigorous phylogenomic analysis is imperative for generating a robust systematic position
for rickettsial species that defy older paradigms for classication into TG and SFG rickettsiae
(Gillespie etal. 2007, 2009).
Phylogenomics has also been employed to characterize the TRG rickettsiae (Gillespie etal. 2007,
2008), as well as identify that R. helvetica does not currently t within the four-group classication
scheme (Driscoll etal. 2013). The emerging diversity of Rickettsia (Perlman etal. 2006, Weinert
etal. 2009, Gillespie etal. 2012b) suggests that additional groups of rickettsiae will likely be needed
to further stabilize Rickettsia classication, though such endeavors must employ rigorous phyloge-
nomic analysis. Additionally, phenotypic data is critical for understanding the underlining genetic
attributes of Rickettsia genomes. Without such data, the results of phylogenomics cannot be effec-
tively interpreted. For example, the analysis of 19 secreted proteins across 59 Rickettsia genomes
shows some patterns that dene the four groups of rickettsiae (Figure 31.3b). However, it is difcult
to associate phenotypic traits with these genotypic maps, for several reasons. First, thorough, rigor-
ously tested phenotypic traits are not available in the literature for all rickettsial species. Second,
presence alone of a gene suspected to contribute to a phenotype is not enough; expression and other
postgenomic data are necessary to determine if the gene is functional. Finally, and most concerning,
is the poor quality of genomes sequenced with the latest sequencing technologies. It is apparent that
most genomes sequenced since 2010 have not been manually curated for conrmation of automated
annotation, and many genome projects have not published an assembly. The WGS raw data may
contain areas of low sequence coverage that, unfortunately, result in misassembly or, even worse,
errors in gene prediction and annotation. This results in apparently truncated genes, split genes, or
pseudogenes; however, the probable full-length of these genes is masked by poor quality of data and
a lack of manual evaluation.
Collectively, a vast treasure trove of genomic data is amassing for species of Rickettsiaceae.
Phylogenomics is providing an invaluable tool for improving rickettsial classication and identify-
ing genotypic features that dene specic lineages of rickettsial species. Undoubtedly, the chal-
lenges highlighted previously will be met with the careful fusion of experimental data and genomic
data, with the latter manually processed to provide an accurate genotype for all species. Thus,
the framework for scaffolding pathogenicity factors over rickettsial lineages, correlating phenotype
with genotype, is set and provides an invaluable resource for future research.
AQ3
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557The Family Rickettsiaceae
(a)
RMSF Epidemic
typhus
Murine
typhus
Bouton-
neuse
fever
North
Asian tick
typhus
Rickettsial
pox
Murine
typhus
like?
Unknown
pathogenicity
AGAG
TickTickTickTickTick
Classification
Primary vector(s)
Transmission
Actin-based motility
Hemolytic activity
Intranuclear growth
Plaque formation
In vitro growth
V/H V V/H V/H
Y
Y Y
Y
Y
Y
Y
YY
Y
Y
Y
Y
Y
N
N N
N Min
NNY
Y
Y Y
Y
Y
Y
Y
Y C
C
CY
N Y
N
Y
Y
Y
Y
Y
Y
N
Y
V/H V/H V/H V/H V/H
FleaFlea
SFGSFGSFG TGTG
Louse/
flea
TRGTRG
Mite
R. rickettsii
R. prowazekii
R. typhi
R. conorii
R. sibirica
R. felis
R. bellii
R. canadensis
R. akari
(b)
D
nAbsent
Pseudogene
Truncated
T5SS Sec/
BAM Sec/
TolC
3
2
D
D
2
2
?
?
Sec
SS
Sca0
Sca1
Sca2
Sca3
Sca5
Sca4
Adr1
Adr2
RickA
RARP-1
RARP-2
VapC-d
VapC-1
Pat2
Pat1
PLD
TlyC
RalF
Multicopy
Rickettsia sp. MEAN1 (Bemisia tabaci)
R. akari Hartford
R. helvetica C9P9
R. canadensis [2 genomes]
R. felis [3 genomes]
R. typhi [3 genomes]
R. prowazekii [12 genomes]
Candidatus R. amblyommii GAT-30V
R. rhipicephali 3-7-female6-CWPP
REIS
R. australis [2 genomes]
R. sibirica [3 genomes]
R. africae ESF-5
R. parkeri Portsmouth
R. conorii [4 genomes]
R. slovaca [2 genomes]
R. rickettsii [8 genomes]
R. philipii 364D
R. peacockii Rustic
R. honei RB
R. heilongjiangensis 054
R. japonica YH
R. montanensis OSU 85-930
R. massiliae [2 genomes]
R. bellii [2 genomes]
Divergent
Full length
FIGURE 31.3 Rickettsia phenotypic and genotypic variability. (a) Variation in pathogenicity (decreasing
left to right) and several phenotypic traits for nine well-studied Rickettsia species. For transmission: V, verti-
cal (transovarial); H, horizontal (transstadial only). For R. typhi, min reects the small actin tails responsible
for erratic rickettsia movement (Teysseire and Raoult 1992, Heinzen etal. 1993, VanKirk etal. 2000). For
R. felis, C denotes contradictory data. (b) Conservation and distribution across 59 Rickettsia genomes of
genes encoding 18 secretory proteins. Phylogeny at left was estimated as previously described (Driscoll etal.
2013), with additional genomes annotated using RAST (Aziz etal. 2008). Classication scheme for Rickettsia
spp. follows previous studies: white, ancestral group; light gray, transitional group; gray, TG; dark gray, SFG
(Gillespie etal. 2007, 2008). R. helvetica is unclassied (incertae sedis) following recent recommendations
(Driscoll etal. 2013). Protein names are listed at top. Secretion pathway information is above protein names,
with checkmarks depicting proteins with NT Sec secretion signals (Ammerman etal. 2008). Inset: large black
circles, full-length proteins; medium-sized open circles, truncated proteins that may have additional frag-
ments encoded by separate genes; small black circles, probable pseudogenes with one or more fragments that
do not span the complete protein; Xs, absent genes with zero signicant matches using BLASTP. For Sca2,
D denotes divergent passenger domains fused to conserved Sca2 autotransporter domains. Numbers on other
circles depict proteins encoded by multiple genes.
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558 Practical Handbook of Microbiology
RICKETTSIAL VIRULENCE AND PATHOGENESIS
The majority of extant, described species of Rickettsia and Orientia are associated with arthropods
during some phase of their life cycle. Vector maintenance of rickettsiae involves either horizontal
(arthropod–vertebrate cycle) or vertical (transovarial) transmission, or both, as is the case for many
of the well-studied Rickettsia pathogens (Figure 31.3a). Aside from the spread of R. prowazekii
via lice-feeding among tightly associated communities, humans are considered a dead-end host
for rickettsiae, the result of accidental infection (Sahni etal. 2013). The rickettsiae transmitted via
blood-feeding arthropods are found within all ve phylogenetic groups (STG, AG, TRG, TG, SFG
rickettsiae) and include pathogens vectored by various species of ticks, eas, lice, and mites. These
rickettsial species have varying degrees of pathogenicity for eukaryotic hosts, including mammals
and arthropods. For example, R. prowazekii and R. rickettsii produce severe and often fatal disease
in humans, and their maintenance in their arthropod vectors (human body lice and tick, respec-
tively) is lethal to the host (Azad and Beard 1998). However, infection with R. typhi, while symp-
tomatic and even fatal in humans, has no effect on the vector eas. While both R. prowazekii and
R. typhi infection are lethal to human body lice, infection with two pathogenic SFG rickettsiae
(R.rickettsii and R. conorii) had no effect on louse tness. In contrast to all described species
of TG and TRG rickettsiae, not all species of SFG rickettsiae cause disease in vertebrate hosts;
for example, Rickettsia montanensis, R. peacockii, and Rickettsia rhipicephali are proposed to be
nonpathogenic for mammals and lack any apparent adverse effects on the survival and tness of
their tick hosts (Azad and Beard 1998). Furthermore, the pathogenicity of species of AG rickettsiae
remains unknown, as does the ability of other poorly described species not associated with blood-
feeding arthropod vectors (Gillespie etal. 2012b).
Over the past 15years, the application of molecular biology tools, especially recombinant DNA
technology for rickettsial diagnosis, resulted in the discovery of several new species of pathogenic
rickettsiae. Of paramount importance was the sequencing of over 60 genomes of the members of
Rickettsiaceae (Table 31.3). While this information revealed some intriguing information (e.g., close
phylogenetic relationship to mitochondria, extensive numbers of pseudogenes in varying states of
degradation, genome reduction, and loss of many genes needed for free living life style), it also con-
rmed many features of rickettsial pathogens that were already known (e.g., ATP/ADP transporter
system, absence of rickettsial bacteriophages, lack of genes for glycolysis, and the biosynthesis of
most amino acids, vitamins, and cofactors) (Gillespie etal. 2012b). Aside from evolutionary studies
and comparative metabolic proling, more recent studies utilizing genomic data tend to focus on
specic genes that potentially play a direct role in host pathogenicity. To this end, studies focusing
on genes that dene particular rickettsial groups are invaluable for understanding lineage-specic
pathogenicity factors (Ammerman etal. 2009, Rahman etal. 2010, 2013).
For Rickettsia spp., genomics has driven the characterization of genes encoding secreted pro-
teins involved in (or predicted to be involved in) adhesion, phagosomal escape, host actin polymer-
ization, toxin secretion, and hemolysis (Figure 31.3b). The secretion systems themselves (Rahman
etal. 2003, 2005, 2007, Gillespie etal. 2009, 2010, Kaur etal. 2012, Sears etal. 2012), as well as
the factors mediating protein secretion (Ammerman et al. 2008, 2009), are also becoming bet-
ter understood. Collectively, these components of the rickettsial secretome are highly targeted by
researchers interested in identifying the mechanisms of rickettsial pathogenicity. Although rickett-
sial invasion and destruction of eukaryotic host cells is considered the basis for rickettsial patho-
genicity, the precise role of presumed virulence factors requires further elucidation. The question
that remains to be answered is: what are the underlying mechanisms that make some strains of
Rickettsia so virulent and others avirulent? While the relevance of these ndings to virulence is
still an open question, a blend of (1) advances in genetic manipulation, (2) postgenomic approaches,
and (3) phylogenomics will undoubtedly provide insights into the underlying molecular basis of
rickettsial virulence. It is only a matter of time before the genetic basis of rickettsial pathogenicity
is illuminated.
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559The Family Rickettsiaceae
CHANGING ECOLOGY OF RICKETTSIAL PATHOGENS
Throughout modern history, the morbidity and mortality associated with rickettsial infections has
been underestimated, primarily due to misdiagnosis. This unfortunate truth remains at present,
with the signicance and impact of rickettsioses greatly underappreciated worldwide, including
in the United States. Fatalities associated with rickettsial infections continue to decline world-
wide due to the effectiveness of antibiotics and physician awareness and improved rapid diagno-
sis. However, the u-like symptoms presented by patients during early infection often delay the
administration of appropriate antibiotics. Furthermore, the long incubation time (often greater
than 1 week) allows rickettsiae to grow to large numbers and disseminate throughout the vascular
system, compromising vascular integrity and setting the stages for more serious complications,
including noncardiogenic pulmonary edema, acute respiratory distress, compromised central ner-
vous system (CNS), and failure of multiple organ systems (Sahni et al. 2013). However, perhaps
most important to the underestimation of rickettsial diseases is the growing number of emerging
pathogens that were previously considered nonpathogenic, such as R. massiliae, R. felis, R. parkeri,
R. aeschlimanii, R. slovaca, and R. helvetica. This occurrence, in conjunction with reemerging
pathogens in novel focal areas, which in some cases are vectored by previously unrecognized
arthropod species (Dumler and Walker 2005), presents a dire need to better understand the eco-
logical landscapes of rickettsial pathogens and their ability to adapt to changing environments and
maintain their virulence.
In recent decades, the improvement of molecular diagnostics and the importance of selected
rickettsial pathogens as biothreat agents have rekindled interest in rickettsioses. Recent serosur-
veys have demonstrated a high prevalence of rickettsial diseases worldwide, particularly in warm
and humid climates. Additionally, new molecular diagnostic technologies helped to narrow the
gap of the continental divide in rickettsial distributions. For example, several rickettsial species
that were known only in the United States are now reported in Central and South America. The
substantial diversity of rickettsial pathogens throughout the world, coupled with variable clinical
manifestations, provides a daunting task to review the ecology of all rickettsial diseases. Thus, fur-
ther description given in the following text is limited to four important rickettsial pathogens: (1) the
louse-borne epidemic typhus agent R. prowazekii, (2) the ea-borne endemic typhus agent R. typhi,
(3) the tick-borne Rocky Mountain Spotted Fever (RMSF) agent R. rickettsii, and (4) the mite-borne
scrub typhus agent, O. tsutsugamushi.
classic epidemic typhus
Epidemic typhus is one of the most virulent diseases known to humanity. Symptoms appear
about 10days after an infected body louse has bitten a person, and include a high fever of about
42°C, extreme pain in the muscle and joints, stiffness, and cerebral impairment. During the
second week, the patient may become delirious with neurological symptoms and may experi-
ence stupor. Gangrene and necrosis may occur due to thrombosis of the small vessels in the
extremities. Mortality rate in untreated patients is approximately 20%. In severe epidemics, the
mortality rate is often as high as 40%. The human body louse, Pediculus humanus corporis,
is the principal vector for R. prowazekii. Although the head louse, P. h. capitis, is capable of
maintaining R. prowazekii experimentally, its role in the transmission of this rickettsiosis is
not well established. Body lice feed only on humans, although the laboratory-adapted colony
can be maintained on rabbit or articially fed on debrinated blood, and all three stages of the
louse life cycle (i.e., eggs, nymphs, and adults) may occur on the same host. The lice prefer a
lower temperature (20°C) and are normally found in the folds of clothing. The body louse will
abandon a patient with a temperature greater than 40°C to seek another host. This attribute is a
major factor in the transmission of typhus within the susceptible population. Humans serve as
host to R. prowazekii and lice and are reservoirs of the rickettsiae. In addition, humans serve
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560 Practical Handbook of Microbiology
as a mobile component of the louse-borne typhus cycle, such that behavior inuences the pat-
tern of typhus transmission. The conditions that allow for the coexistence of body lice and a
susceptible population could be the starting point for an epidemic of R. prowazekii to are in
refugee camps.
The louse-borne epidemic typhus is still endemic in highlands and cold areas of Africa, Asia,
Central and South America, and in parts of Eastern Europe. Another face of typhus, hardly
studied in depth, is recrudescent typhus (Brill–Zinsser disease), in which the symptoms are less
pronounced and the mortality rate is less than 1%. However, these patients could serve as a long-
range source of R. prowazekii, permitting transmission of rickettsiae to occur months to years
after the primary infection. In the United Sates, R. prowazekii is maintained in the sylvatic form
involving the ying squirrel and its eas and lice. Fortunately, immunity to typhus rickettsiae
develops after recovery from infection. Furthermore, successful treatment with tetracycline and
doxycycline is the approach of choice to reduce morbidity and eliminate mortality in susceptible
populations. Although vaccination against R. prowazekii has been partially achieved with inocu-
lation of inactivated rickettsiae or attenuated strains (e.g., str. Madrid E), unfortunately, these
vaccine approaches have been accompanied with undesirable toxic reactions and difculties in
standardization.
endemic typhus
In the United States, a major concern is the changing ecology of endemic typhus, hereafter referred
to as murine typhus. For instance, in both south Texas and southern California, the classic cycle
of R. typhi, which involves commensal rats and primarily the rat ea (Xensopsylla cheopis), has
been replaced by the Virginia opossum (Didelphis virginiana)/cat ea (Ctenocephalides felis)
cycle. Curiously, however, infected rats and their eas are difcult to document within Texas’ and
California’s murine typhus foci (Azad etal. 1997, Azad and Beard 1998). Similarly, the association
of 33 cases of locally acquired murine typhus in Los Angeles county with seropositive domestic cats
and opossums was also conrmed. Additionally, based on serological surveys, R. typhi infections
also occur in inland cities in Oklahoma, Kansas, and Kentucky, where urban and rural-dwelling
opossums thrive. Thus, the maintenance of R. typhi in the cat ea/opossum cycle is of potential
public health importance and a major health risk considering the distribution of opossum, which
spans the United States, Mexico, Central America, and Canada. A previous search for the rural res-
ervoir of murine typhus also resulted in the discovery of R. felis, the second ea-borne-associated
rickettsial species in opossums (Azad etal. 1997, Azad and Beard 1998). Urban rat/ea populations
are still the main reservoir of R. typhi worldwide and particularly in many cities where urban set-
tings provide a constellation of factors for the perpetuation of the R. typhi cycle, including declining
infrastructures, increased immunocompromised populations, homelessness, and high population
density of rats and eas.
Rocky mountain spotted FeveR (RmsF)
RMSF is one of the most virulent human infections in the United States. R. rickettsii, the causative
agent of RMSF, is a true zoonotic bacterium that cycles between ixodid ticks and wildlife popula-
tions, not only in the United States but also in Mexico and in Central and South America. RMSF,
like all rickettsial infections, is classied as a zoonosis requiring a biological vector such as tick
to be transmitted between animal hosts (and accidentally to humans). Human infection occurs via
the bite of an infected tick. Initial signs and symptoms of the disease include fever, headache, and
muscle pain. This is followed by rash and organ-specic symptoms such as nausea, vomiting, and
abdominal pain. Delayed treatment results in hospitalization and sequelae, such as amputation,
deafness, and permanent learning impairment. The disease can be difcult to diagnose in the early
AQ4
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561The Family Rickettsiaceae
stages due to nonspecic presentations and, unfortunately, in the absence of prompt and appropriate
treatment, it often kills the infected patient. Mortality of up to 75% had been reported before anti-
biotic discovery, and even today 5%–10% of children and adults die from RMSF, with many more
requiring intensive care. RMSF is a reportable disease in the United States, although the number of
reported cases annually ranges between 250 and 1200.
All SFG rickettsiae are transmitted by ixodid ticks (Azad and Beard 1998, Parola etal. 2005).
Inaddition to R. rickettsii, the etiologic agent of RMSF, several other tick-borne rickettsial species
are also human pathogens (Table 31.1). In the United States, at least ve other SFG rickettsiae,
namely, R. amblyommii, R. montanensis, R. peackockii, Rickettsia endosymbiont of Ixodes scapu-
laris, and R. rhipicephali, are routinely isolated only from ticks; yet, they cause limited or no known
pathogenicity to humans or certain laboratory animals. R. parkeri, which was originally considered
nonpathogenic, was recently identied as a human pathogen (Paddock etal. 2004); thus, the other
ve rickettsial species could also be etiologic agents of as-yet-undiscovered, less severe rickettsio-
ses. However, the genome sequences of R. peackockii (Felsheim etal. 2009) and REIS (Gillespie
etal. 2012a) reveal the proliferation of mobile elements that have interrupted many genes, some
of which are candidate virulence factors, suggesting that these species may be restricted to their
arthropod vectors.
Distribution of SFG rickettsiae is limited to that of their tick vectors. In the United States, a
high prevalence of SFG species in ticks cannot be explained without the extensive contributions
of transovarial transmission. The transovarial and transstadial passage of SFG rickettsiae within
tick vectors in nature ensures rickettsial survival without requiring the complexity inherent in an
obligate multihost reservoir system (Azad and Beard 1998). Although many genera and species of
ixodid ticks are naturally infected with rickettsiae, Dermacentor andersoni (Rocky Mountain wood
tick) and D. variabilis (American dog tick or Wood tick) are the major vectors of R. rickettsii in the
United States.
scRuB typhus
Scrub typhus is an acute, febrile, infectious illness caused by O. tsutsugamushi (formerly
Rickettsia tsutsugamushi, see previous text). Long considered a monotypic genus, it is now
apparent that substantial diversity exists within the sister clade to Rickettsia (Figure 31.2), with
a recently described novel species of Orientia, O. chuto (Izzard etal. 2010). Additionally, novel
strains of O. tsutsugamushi continue to be discovered in geographically isolated regions world-
wide (Odorico etal. 1998, Kelly etal. 2009, Yang etal. 2012, Jiang etal. 2013). Like Rickettsia
spp., O. tsutsugamushi is an obligate intracellular gram-negative bacterium. However, as com-
pared to Rickettsia spp., O. tsutsugamushi possesses a different cell wall structure, lacking pep-
tidoglycan and lipopolysaccharide, and also has a slightly different composition of metabolic
genes (Gillespie etal. 2012b). Humans acquire scrub typhus via the bite of infected larval stages
of numerous species of trombiculid mites (Leptotrombidium spp.). Scrub typhus is endemic in
regions of eastern Asia and the southwestern Pacic (Korea to Australia), and also from Japan to
India and Pakistan. While no signicant morbidity or mortality occurs in patients who receive
appropriate treatment, pneumonia, myocarditis, disseminated intravascular coagulation, and
death can occur in 0%–30% of untreated patients or those infected with antibiotic-resistant
O.tsutsugamushi strains.
The contemporary urban and rural cycles of rickettsial pathogens present a potential for future
outbreaks amid the drastic increase in housing construction, urban sprawl, and related develop-
mental expansion, all of which provide mammalian reservoirs and their blood-sucking ectopara-
sites ample harborage and proximity to human habitations. Considering the existing trends in
global population expansion, rickettsial agents will continue to be introduced into human popula-
tions, with susceptible populations the primary targets for emerging and reemerging pathogens.
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562 Practical Handbook of Microbiology
To this end, the advancements in classication and diagnostics brought about by the molecular
technologies described earlier have a broader impact, reshaping the knowledge of the ecodynamics
and compositional patterns of rickettsial species worldwide.
RICKETTSIAL PATHOGENS AS BIOTHREAT AGENTS
Throughout modern history, the epidemics of louse-borne typhus have been important in the mold-
ing of human destiny, being credited with causing more deaths than all the wars in history (Azad
and Radulovic 2003, Azad 2007). For example, more than 30 million cases of louse-borne typhus
occurred during and immediately after World War I, causing an estimated three million deaths. In
the wake of war, famine, ood, and other disasters, the explosive spread of the brutal epidemic of
louse-borne typhus within crowded human populations made a deep impression on the commanders
of the Russian Red Army, and by 1928, R. prowazekii was transformed into a battleeld weapon.
Many years later, the Japanese Army successfully tested biobombs containing R. prowazekii, caus-
ing outbreaks of typhus. Thus, the precedent exists for utilizing pathogenic rickettsiae in warfare,
and recent increased risk of misuse of bacterial pathogens as weapons of terror is no longer ction
(Azad and Radulovic 2003, Azad 2007).
The rickettsial diseases vary from mild to very severe clinical presentations, with case fatality
ranging from 2% to over 30% (Table 31.4). The severity of rickettsioses has been associated with
age, delayed diagnosis, hepatic and renal dysfunction, CNS abnormalities, and pulmonary compro-
mise. Despite the variability in clinical presentations, pathogenic rickettsiae cause debilitating dis-
eases and sometimes death, and several rickettsial pathogens could be used as a biological weapon.
Realistically, only R. prowazekii and R. rickettsii pose serious problems, especially when some
salient features of pathogenic rickettsial species are compared with those from several category A
agents (Table 31.4). Despite the fact that effective chemotherapy is available and effective control
measures are known, rickettsial diseases continue to be a problem in the United States and many
other parts of the world.
TABLE 31.4
Comparison of Selected Rickettsial Pathogens to Examples of Biological Warfare Bacteria
Bacteria Disease/Incubationa
Natural
Hosts
Treatment/
Vaccine
Rapid
Diagnosis Transmissionb
Mortality
Ratec (%)
Bacillus
anthracis
Anthrax/variable + +/+ + Inh, Ing 5–80
Clostridium
botulinum
Botulism/12–36h − +/− + Ing 70
Yersinia pestis Plague/2–7days + +/±+ V, Inh 30–90
Burkholderia
mallei
Glanders/3–6days + +/− + Inh, C 95
Francisella
tularensis
Tularemia/2–10days + +/− + C, V 5
Coxiella burnetti Q-fever/7–14days + +/− + Inh, V 2–3
R. prowazekii Epidemic Typhus/6–14days ++/− +V, Inh 30
R. rickettsii RMSF/3–15days ++/− + V, Inh 20–25
R. typhi Endemic typhus/6–14days + +/− + V, Inh 4
a Incubation period: Dependent upon bacterial dose and mode of exposure.
b Mode of exposure: C, cutaneous; Inh, inhalation (aerosol); Ing, ingestion; V, vector borne.
c Fatality rate (dependent upon the mode of exposure).
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563The Family Rickettsiaceae
ACKNOWLEDGMENTS
The work presented in this chapter has been supported with funds from the National Institute of
Health/National Institute of Allergy and Infectious Diseases grants R01AI017828 and R01AI59118
awarded to AFA. TPD acknowledges support from NIAID Contract HHSN266200400035C
awarded to Bruno Sobral (Virginia Bioinformatics Institute at Virginia Tech). The content is solely
the responsibility of the authors and does not necessarily represent the ofcial views of the funding
agencies. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
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AUTHOR QUERIES
[AQ1] Please check if identied heading levels are okay.
[AQ2] Please provide appropriate text instead of question marks (?) in Tables 31.1 and 31.2.
[AQ3] Please specify “a” or “b” for Gillespie etal. (2009).
[AQ4] Please check if edit to sentence starting “Curiously, however...” is okay.
[AQ5] Please provide page range for Dohra etal. (2013).
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