Rickettsial Diseases

Prévia do material em texto

Rickettsial
Diseases
edited by
Didier Raoult
Unité des Rickettsies
Université de la Méditerranée
Marseille, France
Philippe Parola
Unité des Rickettsies
Université de la Méditerranée
Marseille, France
New York London
To my wife Natacha, my children Magali, Sacha and Lola, and 
my granchild César
—Didier Raoult
To my wife, Géraldine, and my parents, Marie-Claire and Jean-Pierre
—Philippe Parola
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Preface
In the last 20 years, the rickettsial field has undergone significant evolution at the epidemiolog-
ical, microbiological, and molecular levels. Between 1984 and 2004 alone, at least nine additional
rickettsial species or subspecies were identified as causes of tick-borne rickettsioses around the
world. Of these agents, six were initially isolated from ticks, often years or decades before a
definitive association with human disease was established. Also, outbreaks of the louse-borne
epidemic typhus re-emerged in Africa, with the most recent outbreak (and the largest since
World War II) observed in Burundi in the 1990s, during their civil war. Fleas, which have been
historically associated with the transmission of murine typhus, have been involved in the cycle
of Rickettsia felis, an emerging pathogen belonging to the spotted-fever group of Rickettsia.
Furthermore, about one million cases of scrub typhus due to Orientia tsutsugamushi have been
estimated to occur each year around the world and as many as one billion people may be
exposed, particularly in rural locations.
Recent developments in molecular taxonomic methods have resulted in the reclassifica-
tion of the rickettsiae. For example, the Rochalimaea, united within the genus Bartonella, and
Coxiella were removed from the order Rickettsiales and the classification of this order contin-
ues to be modified as new data becomes available. Because Q fever caused by Coxiella burnetii
is still considered as a “rickettsiosis” and remains in the field of rickettsiologists, it will be largely
described in this book.
The rickettsial field has recently entered the genome area. In 2001, the first genome of a
tick-transmitted rickettsia (R. conorii strain Seven) was fully sequenced and revealed several
unique characteristics among bacterial genomes, including long palindromic repeat fragments
irregularly distributed throughout the genome. Further, comparison of the R. conorii genome
with that of R. prowazekii (the agent of epidemic typhus and included in the typhus group of
the genus Rickettsia), provided additional data on the evolution of rickettsial genomes, the lat-
ter appearing to be a subset of the first. Recently, the genomes of R. sibirica, R. rickettsii, R. akari,
R. felis, and R. typhi have been reported. Those of R. belli, R. massiliae, R. africae, and R. slovaca
are currently being sequenced. These data will certainly provide insights into the mechanism
of rickettsial pathogenicity and will provide new molecular diagnostic targets and new tools
for phylogenetic and taxonomic studies as well as new treatment measures.
We anticipate that this book will appeal to physicians specializing in infectious diseases,
dermatology, or even travel medicine, as well as clinical laboratory personnel and epidemiolo-
gists. Not only physicians and scientists in Europe and the United States will find this book
useful, but also those from tropical regions in Africa, Asia, Australia, and the Americas, where
numerous emerging diseases have been described in recent years and where rickettsial dis-
eases constitute a differential diagnosis in febrile patients.
Didier Raoult
Philippe Parola
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Contents
Preface . . . . v
Contributors. . . . ix
Section I: Rickettsia and Human Rickettsioses
1. Bacteriology, Taxonomy, and Phylogeny of Rickettsia 1
Pierre-Edouard Fournier and Didier Raoult
2. Pathogenesis, Immunity, Pathology, and Pathophysiology in Rickettsial Diseases 15
David H. Walker, Nahed Ismail, Juan P. Olano, Gustavo Valbuena, and Jere McBride
3. Arthropods and Rickettsiae 27
Sam R. Telford III and Philippe Parola
4. Murine Typhus 37
Yanis Tselentis and Achilleas Gikas
5. Louse-Borne Epidemic Typhus 51
Linda Houhamdi and Didier Raoult
6. Rickettsialpox 63
Christopher D. Paddock and Marina E. Eremeeva
7. Flea-Borne Spotted Fever 87
Abir Znazen and Didier Raoult
Section II: Tick-Borne Rickettsioses
8. Rocky Mountain Spotted Fever 97
James E. Childs and Christopher D. Paddock
9. African Tick-Bite Fever 117
Mogens Jensenius, Lucy Ndip, and Bjørn Myrvang
10. Rickettsia conorii Infections (Mediterranean Spotted Fever, Israeli Spotted Fever, 
Indian Tick Typhus, Astrakhan Fever) 125
Clarisse Rovery and Didier Raoult
11. Other Tick-Borne Rickettsioses 139
Oleg Mediannikov, Philippe Parola, and Didier Raoult
12. Other Rickettsiae of Possible or Undetermined Pathogenicity 163
Oleg Mediannikov, Christopher D. Paddock, and Philippe Parola
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viii Contents
Section III: Anaplasmataceae and Human Anaplasmosis and Ehrlichioses
13. Bacteriology and Phylogeny of Anaplasmataceae 179
Philippe Brouqui and Kotaro Matsumoto
14. Vectors and Reservoir Hosts of Anaplasmataceae 199
Hisashi Inokuma
15. Human Ehrlichioses 213
Juan P. Olano
16. Anaplasmosis in Humans 223
Anna Grzeszczuk, Nicole C. Barat, Johan S. Bakken, and J. Stephen Dumler
Section IV: Orientia tsutsugamushi and Scrub Typhus
17. Orientia tsutsugamushi and Scrub Typhus 237
George Watt and Pacharee Kantipong
Section V: Coxiella burnetii and Q Fever
18. Bacteriology of Coxiella 257
Katja Mertens and James E. Samuel
19. Immune Response to Q Fever 271
Jean-Louis Mege
20. Epidemiology of Q Fever 281
Thomas J. Marrie
21. Clinical Aspects, Diagnosis, and Treatment of Q Fever 291
Hervé Tissot-Dupont and Didier Raoult
Section VI: Wolbachia
22. Wolbachia and Filarial Nematode Diseases in Humans 303
Kelly L. Johnston and Mark J. Taylor
Section VII: Diagnostic Strategy of Rickettsial Diseases in Humans
23. Diagnostic Strategy of Rickettsioses and Ehrlichioses 315
Florence Fenollar, Pierre-Edouard Fournier, and Didier Raoult
Section VIII: Rickettsial Diseases of Domestic Animals
24. Rickettsial Diseases of Domestic Animals 331
Patrick J. Kelly
Section IX: Genomics of Rickettsial Agents
25. Genomics of Rickettsial Agents 345
Hiroyuki Ogata and Patricia Renesto
Section X: Antimicrobial Susceptibility of Rickettsial Agents
26. Antimicrobial Susceptibility of Rickettsial Agents 361
Jean-Marc Rolain
Index . . . . 371
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Contributors
Johan S. Bakken Department of Family Medicine, School of Medicine, University of 
Minnesota at Duluth and St. Luke’s Infectious Disease Associates, St. Luke’s Hospital, Duluth,
Minnesota, U.S.A.
Nicole C. Barat Division of Medical Microbiology, Department of Pathology, The Johns Hopkins
University School of Medicine, Baltimore, Maryland, U.S.A.
Philippe Brouqui Faculté de Médecine, Unité des Rickettsies, Université de la Méditerranée,
Marseille, France
James E. Childs Department of Epidemiology and Public Health, Yale University School of
Medicine, New Haven, Connecticut, U.S.A.
J. Stephen Dumler Division of Medical Microbiology, Department of Pathology, 
The Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.
Marina E. Eremeeva Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.
Florence Fenollar Faculté de Médecine, Unité des Rickettsies, Université de la 
Méditerranée, Marseille, France
Pierre-Edouard Fournier Faculté de Médecine, Unité des Rickettsies,Université de la
Méditerranée, Marseille, France
Achilleas Gikas Laboratory of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical
Medicine, University of Crete, Crete, Greece
Anna Grzeszczuk Department of Infectious Diseases, Medical University of Bialystok, 
Bialystok, Poland
Linda Houhamdi Faculté de Médecine, Unité des Rickettsies, Institut Fédératif de Recherche 48,
Centre National de Recherche Scientifique, Université de la Méditerranée, Marseille, France
Hisashi Inokuma Department of Clinical Veterinary Science, Obihiro University of Agriculture
and Veterinary Medicine, Obihiro, Japan
Nahed Ismail Department of Pathology, University of Texas Medical Branch, Galveston, 
Texas, U.S.A.
Mogens Jensenius Department of Infectious Diseases, Ullevål University Hospital, 
Oslo, Norway
Kelly L. Johnston Filariasis Research Laboratory, Molecular and Biochemical Parasitology,
Liverpool School of Tropical Medicine, Pembroke Place, U.K.
Pacharee Kantipong Department of Internal Medicine, Chiangrai Regional Hospital, 
Chiangrai, Thailand
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Patrick J. Kelly Ross University School of Veterinary Medicine, Basseterre, St. Kitts, West Indies
Thomas J. Marrie Faculty of Medicine and Dentistry, University of Alberta, Edmonton, 
Alberta, Canada
Kotaro Matsumoto Faculté de Médecine, Unité des Rickettsies, Université de la Méditerranée,
Marseille, France
Jere McBride Department of Pathology, University of Texas Medical Branch, Galveston,
Texas, U.S.A.
Oleg Mediannikov Unité des Rickettsies, Université de la Méditerranée, Marseille, France and
Laboratory of Rickettsial Ecology, Gamaleya Institute of Epidemiology and Microbiology,
Moscow, Russia
Jean-Louis Mege Unité des Rickettsies, Université de la Méditerranée, Marseille, France
Katja Mertens Department of Microbial and Molecular Pathogenesis, Texas A&M University
System Health Science Center, College Station, Texas, U.S.A.
Bjørn Myrvang Department of Infectious Diseases and Center for Imported and Tropical
Diseases, Ullevål University Hospital, Oslo, Norway 
Lucy Ndip Department of Biochemistry and Microbiology, University of Buea, Buea, Cameroon
Hiroyuki Ogata Structural and Genomic Information Laboratory, Parc Scientifique de Luminy,
Marseille, France
Juan P. Olano Department of Pathology, University of Texas Medical Branch, Galveston, 
Texas, U.S.A.
Christopher D. Paddock Infectious Disease Pathology Activity, Division of Viral and Rickettsial
Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.
Philippe Parola Faculté de Médecine, Unité des Rickettsies, Université de la Méditerranée,
Marseille, France
Didier Raoult Faculté de Médecine, Unité des Rickettsies, Université de la Méditerranée,
Marseille, France
Patricia Renesto Faculté de Médecine, Unité des Rickettsies, Marseille, France
Jean-Marc Rolain Faculté de Médecine et de Pharmacie, Unité des Rickettsies, Université 
de la Méditerranée, Marseille, France
Clarisse Rovery Faculté de Médecine, Unité des Rickettsies, Université de la Méditerranée, 
Marseille, France
James E. Samuel Department of Microbial and Molecular Pathogenesis, Texas A&M University
System Health Science Center, College Station, Texas, U.S.A.
Mark J. Taylor Filariasis Research Laboratory, Molecular and Biochemical Parasitology,
Liverpool School of Tropical Medicine, Pembroke Place, U.K.
Sam R. Telford III Division of Infectious Diseases, Cummings School of Veterinary Medicine,
Tufts University, North Grafton, Massachusetts, U.S.A.
Hervé Tissot-Dupont Faculté de Médecine, Unité des Rickettsies, Centre National de
Référence, Marseille, France
x Contributors 
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Yanis Tselentis Laboratory of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical
Medicine, University of Crete, Crete, Greece
Gustavo Valbuena Department of Pathology, University of Texas Medical Branch, Galveston,
Texas, U.S.A.
David H. Walker Department of Pathology, University of Texas Medical Branch, Galveston,
Texas, U.S.A.
George Watt Family Health International, Asia-Pacific Regional Office, Bangkok, Thailand
Abir Znazen Laboratoire de Microbiologie, Centre Hospitalo-Universitaire, Habib Bourguiba
Sfax, Tunisie
Contributors xi
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1 Bacteriology, Taxonomy, and Phylogeny
of Rickettsia
Pierre-Edouard Fournier and Didier Raoult
Faculté de Médecine, Unité des Rickettsies, Université de la Méditerranée, Marseille, France
INTRODUCTION
Traditional identification methods used in bacteriology cannot be routinely applied to 
rickettsiae because of the few phenotypic characters expressed by these strictly intracellular
organisms. As a consequence, “Rickettsia” has long been used as a generic term for many small
bacteria that could not be cultivated and were not otherwise identified. However, taxonomic
progress made over the last 35 years has deeply modified the definition of “rickettsia.” In par-
ticular, the introduction of molecular techniques has revolutionized the study of gene and
genome evolution and has allowed new approaches to phylogenetic and taxonomic inferences.
As a result of deep taxonomic changes, the term “rickettsia” currently only applies to arthro-
pod-borne bacteria belonging to the genus Rickettsia within the family Rickettsiaceae in the
order Rickettsiales, �-Proteobacteria. The Rickettsia genus is currently made of 24 recognized
species, and also contains several dozens of as-yet uncharacterized strains or tick amplicons.
Most of these bacteria are associated with ticks, which are their vectors and reservoirs, but
some are vectorized by lice, fleas, or mites. In contrast with louse- and flea-borne rickettsioses,
tick-borne rickettsioses have specific geographic distributions, directly depending on the dis-
tribution of their vectors. Rickettsia species cause rickettsioses, which are among the oldest
known arthropod-borne diseases (1). Currently, 16 rickettsioses are recognized. Among these,
several are caused by rickettsiae that were initially isolated from ticks and subsequently con-
sidered as nonpathogenic. A priori, it is difficult to predict which rickettsiae are potential
human pathogens. It should be considered that rickettsiae found in arthropods capable of
biting humans are potential human pathogens.
In this chapter, we will describe the bacteriology, taxonomy, and phylogeny of members
of the genus Rickettsia.
BACTERIOLOGY
Bacteria within the genus Rickettsia are obligate intracellular short rods, 0.3 to 0.5 � 0.8 to
2.0 �m2. The cytoplasm of these bacteria contains ribosomes and strands of DNA and is limited
by a typical Gram-negative trilamellar structure made of a bilayer inner membrane, a peptido-
glycan layer, and a bilayer outer membrane. Within host cells, rickettsiae are surrounded by an
electron-lucent slime layer. Rickettsiae are not stained by the Gram method, but retain basic
fuschin when stained using the Gimenez method (2). Using this method, they appear bright
red, whereas the background is stained in pale blue with the malachite green counterstain.
Members of the genus Rickettsia are divided into two main groups: the spotted fever
group (SFG) and typhus group (TG), depending on several characters: (i) SFG rickettsiae are
mainly associated with ticks, but also with fleas (Rickettsia felis) and mites (R. akari), have an
optimal growth temperature of 32°C, have a G � C content between 32 and 33, can polymer-
ize actin and thus move into the nuclei of host cells (3–5), and cause spotted fevers in humans;
(ii) TG rickettsiae are associated with human body lice (R. prowazekii) or fleas (R. typhi), have 
an optimal growth temperature of 35°C, have a G � C content of 29, cannot polymerize 
actin and thus cannot enter the nucleiof host cells and are only found in the cytoplasm of host
cells (3,5), and cause typhus in humans (Table 1). Within ticks, transovarial and trans-stadial
Section I: RICKETTSIA AND HUMAN RICKETTSIOSES
(Text continues on p. 6)
DK7611-Raoult-Chap 01_R2.qxd 3/20/07 2:18 PM Page 1
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DK7611-Raoult-Chap 01_R2.qxd 3/20/07 2:18 PM Page 5
transmissions are essential mechanisms for the maintenance of spotted fever rickettsiae. In con-
trast, flea- or louse-borne rickettsiae are not transmitted transovarially and thus these bacteria
have mammal reservoirs (humans for R. prowazekii, rodents for R. typhi, and cats for R. felis).
Rickettsiae are unstable when separated from host components, except for highly stable forms
found in the feces of arthropod hosts, as observed for R. prowazekii and R. typhi which are able
to survive within louse feces for several weeks (7,8). Rickettsiae are rapidly inactivated at 56°C.
Until 2001, the genome size of rickettsiae was estimated by pulse field gel electrophore-
sis and ranged from 1.1 to 1.6 Mb. Since 1998, genome sequences from four Rickettsia species
have been determined and published, including R. conorii (9), R. felis (10), R. prowazekii (11), and
R. typhi (12). The analysis of the obtained sequences has highlighted unique characteristics
among bacterial genomes: a genome size �1.4 Mb; the presence of a large number of 95- to 150-
nucleotide long palindromic repeat fragments (13), including some inserted into protein-
coding genes but compatible with the encoded protein’s three-dimensional fold and functions;
an ongoing degradation process of genes, from complete transcribed genes to split transcribed
genes and then to split untranscribed genes (9). In addition, genome sequencing provided the
first evidence of a conjugative plasmid in an intracellular bacterium, in R. felis (10). This find-
ing suggested that conjugation could play a role in the evolution of rickettsial genomes.
Rickettsiae grow in association with eukaryotic cells within which they live free and
divide by binary fission in the cytoplasm (4,14). As a consequence, rickettsiae must be cultivated
in tissue culture or yolk sac of developing chicken embryos. L929 and Vero cells are used most
frequently. Rickettsial growth in cell monolayers is monitored by the development of plaques
that represent the disruption of massively infected cells. SFG rickettsiae form plaques with a
diameter of 2 to 3 mm after five to eight days, whereas TG rickettsiae form smaller plaques
(1 mm) after 8 to 10 days. Rickettsiae have a membrane-bound adenosine diphosphate/adeno-
sine triphosphate (ADP/ATP) translocase that mediates exchange of ATP and ADP. Five copies
of this gene are present in R. conorii and R. prowazekii genomes (9,15). The exchange of extracel-
lular ATP for intracellular ADP is regulated by the concentration of phosphate in the host.
Rickettsiae possess genes encoding all enzymes of the tricarboxylic acid cycle. They do not uti-
lize glucose, but metabolize glutamate as their main source of energy. They do not synthesize
or degrade nucleoside monophosphates. They produce endotoxins whose role is uncompletely
understood.
Rickettsiae possess major antigens such as lipopolysaccharide, lipoprotein, outer mem-
brane proteins of the surface cell antigen (SCA) family, and heat shock proteins. The Weil-Felix
test, initially developed as a diagnostic test for rickettsioses, was based on the antigenic cross-
reactions among rickettsial antigens, mostly lipopolysaccharide (LPS), and Proteus vulgaris
strains OX19 and OX2, and Proteus mirabilis OXK (16). Other antigens have been characterized
in Rickettsia species, including a 17-kDa lipoprotein (17) and members of the autotransporter
protein family SCA. These include the 120-kDa S-layer protein (OmpB or Sca5) (18), OmpA,
present only in SFG rickettsiae (19), and Sca4 (20). Additional 14 genes putatively encoding
SCA proteins were identified in sequenced rickettsial genomes (21), one of which, sca1, was
present in all species (22).
PHYLOGENY
Initially, phylogenetic studies of rickettsiae, as for other prokaryotes, were based on the com-
parison of morphological, antigenic, and metabolic characters. The order Rickettsiales, within
which bacteria of the genus Rickettsia were classified, historically contained small, rod-shaped
Gram-negative organisms that retained basic fuschin when stained by the method of Gimenez
(2), divided by binary fission, could be cultivated in living tissues and could cause diseases in
invertebrate hosts (which acted as vectors and reservoirs) or vertebrate hosts which were
infected through arthropod bites (23). However, phylogenetic relationships based on these cri-
teria were highly unreliable. The advent of molecular methods allowed phylogenic relation-
ships among intracellular bacteria to be reliably estimated. The phylogenetic study based on
the comparison of 16S rRNA gene sequences showed that several of the bacteria classified in
the order Rickettsiales did not belong to the �-subclass of the Proteobacteria phylum. As a conse-
quence, Coxiella burnetii and Rickettsiella grylli were reclassified within Legionellaceae (24,25),
6 Fournier and Raoult
DK7611-Raoult-Chap 01_R2.qxd 3/20/07 2:18 PM Page 6
Eperythrozoon sp. and Haemobartonella sp. within Mycoplasmataceae (26), Wolbachia persica within
the �-subdivision of Proteobacteria close to Francisella sp. (25), Wolbachia melophagi within
Bartonellaceae (Birtles and Molyneux, GenBank Accession No. X89110), and Bartonella sp.,
Rochalimaea sp., and Grahamella sp. within Bartonellaceae (27,28). In addition, within the genus
Rickettsia, taxonomic changes also occurred. Rickettsia tsutsugamushi, the agent of scrub typhus,
was found to be distinct enough by 16S rRNA gene sequence comparison to warrant transfer
into the genus Orientia which includes a single species, Orientia tsutsugamushi (29).
Within the genus Rickettsia, priorto gene sequencing, polymerase chain reaction (PCR)
coupled with restriction fragment length polymorphism (RFLP) applied to the gltA and ompA
genes showed that R. canadensis and R. bellii occupied an intermediate position between the
typhus and SFGs (30), that three clusters were identified within the SFG (R. rickettsii and R. slo-
vaca; R. rhipicephali and R. montanensis; R. sibirica, R. honei, and R. conorii), and that the R. conorii
species was heterogeneous (31).
However, it was not until gene sequencing that phylogenic relationships among Rickettsia
species could reliably be estimated. The first gene to be used for phylogenic purposes was the
16S rDNA (32,33). A specific sequence was obtained for each serotype, but high sequence sim-
ilarity between 99.9 and 97.2 was observed. These studies confirmed the evolutionary unity of
the genus (Fig. 1), but as the sequences were almost identical, significant inferences about intra-
genus phylogeny were not possible. R. felis was closely related to R. akari and R. australis, but
not related to the TG as deduced from serologic criteria. R. canadensis was shown to be outside
the TG. Within the SFG, a cluster including R. massiliae, R. rhipicephali, R. amblyommii, and 
Bacteriology, Taxonomy, and Phylogeny of Rickettsia 7
FIGURE 1 Phylogenic organization of Rickettsia species based on the comparison of sequences of the 16S rDNA,
gltA, ompA, ompB, sca4, sca1, and sca2 genes, using the Maximum Parsimony method.
DK7611-Raoult-Chap 01_R2.qxd 3/20/07 2:18 PM Page 7
8 Fournier and Raoult
R. montanensis was described. It was proposed that R. bellii and R. canadensis diverged prior to
the schism between the SFG and the TG (33).
Subsequently, phylogenic studies were inferred from sequences from more divergent
genes that included gltA (34), the gene encoding the 17-kDa protein (35), and genes from the
autotransporter family sca: ompA (19), ompB (36), sca4 (20), sca1 (22), and sca2 (37). Phylogenetic
analysis inferred from sequences of the 17-kDa protein-encoding gene provided nonsignificant
bootstrap values for most of the nodes (35). Phylogenic relationships inferred from sequences
of the gltA gene were supported by significant bootstrap values for all the nodes except those
within groups in the SFG (Fig. 1) (34). The R. rickettsii group included: R. conorii, R. honei, 
R. rickettsii, R. africae, R. parkeri, R. sibirica, R. slovaca, and R. japonica. The R. massiliae group was
made of R. massiliae, R. rhipicephali, R. aeschlimannii, and R. montanensis. The coherence of the
latter group was reinforced by the resistance to erythromycin and rifampin of the species it con-
tained (38). R. canadensis, AB bacterium, and R. bellii clustered neither in the TG nor in the SFG
and were shown to be the most outlying rickettsiae. R. helvetica, R. akari, and R. australis clus-
tered between the R. massiliae group and the TG. Using ompA sequence comparison, the phylo-
genetic organization in the R. rickettsii group was well established (19). Different clusters were
determined. The R. conorii complex included the different strains closely related to R. conorii
(Malish, Moroccan, Indian, Israeli, and Astrakhan). A second cluster included R. sibirica, 
R. africae, and R. parkeri. R. rickettsii, R. slovaca, R. honei, and R. japonica were found alone in dif-
ferent branches. Significant bootstrap values were found for all the nodes except for R. honei.
However, the ompA gene could not be amplified from all Rickettsia species (19). Phylogenetic
analysis inferred from the sequences of the ompB gene confirmed the groups identified by ompA
sequencing (36) (Fig. 1). Bootstrap values were significant for most of the nodes with the excep-
tion of those inside the cluster including R. parkeri and R. africae. The phylogenetic positions of
R. helvetica, R. akari, R. australis, R. typhi, and R. prowazekii were the same that those determined
by gltA sequence comparison. Sca4 sequence comparison identified five well-supported
phylogenetic groups (Fig. 1). These included: the previously described R. massiliae and R. rick-
ettsii groups as well as the TG; the R. helvetica group that contained only R. helvetica; and the R.
akari group including R. australis, R. akari, and R. felis. Finally, the phylogenetic organization of
Rickettsia species inferred from the comparison of sequences of the sca1 and sca2 genes were
similar to those obtained from the analyses of ompA, ompB, and sca4 (22,37). In addition to the
previously described groups, it was proposed that R. bellii, R. canadensis, and Rickettsia sp.
strain AB bacterium, being the outlayers of the Rickettsia species, be grouped into an “ances-
tral” group (33). However, the consistency of this group has later been discussed on the basis
of genetic criteria (39).
TAXONOMY
Because phenotypic methods used to classify axenically cultivable bacteria were not applicable
to intracellular bacteria, most bacteria obligately associated with eukaryotic cells were initially
included in the order Rickettsiales. The order Rickettsiales was initially divided into the families
Rickettsiaceae, Bartonellaceae, and Anaplasmataceae. Within the family Rickettsiaceae, the tribe
Rickettsiae was composed of the genera Rickettsia, Coxiella, and Rochalimaea. The development of
PCR and nucleotide sequencing, particularly the study of 16S rRNA or rDNA, has considerably
modified the taxonomic classification of bacteria, in particular, intracellular bacteria that express
few phenotypic criteria commonly used for identification and classification. Following the
reclassification of several genera, as described above, the order Rickettsiales is currently com-
prised the genera Anaplasma, Ehrlichia, Neorickettsia, Orientia, Rickettsia, and Wolbachia (29,40).
Currently, a Rickettsia is a strictly intracellular bacillus of 0.3 to 0.5 mm in diameter and
0.8 to 2.0 mm in length, with a Gram-negative-type membrane, and Gimenez stain positive (2).
The target cells of Rickettsia sp. in humans are endothelial cells within which they multiply in
the cytoplasm. In addition, Rickettsia strains share a high degree of 16S rDNA nucleotide
sequence similarity (41) and are phylogenetically close. As a matter of fact, due to the lack of
official rules, defining a species within the Rickettsia genus has long been a matter of debate.
The guidelines established for extracellular bacteria do not fit well with the strictly intracellu-
lar nature of these bacteria. In particular, their initial differentiation relied on a combination of
DK7611-Raoult-Chap 01_R2.qxd 3/20/07 2:18 PM Page 8
Bacteriology, Taxonomy, and Phylogeny of Rickettsia 9
few phenotypic and genomic parameters. Rickettsial isolates have initially been classified
within three groups: the SFG, TG, and the scrub typhus group (STG), on the basis of the follow-
ing characteristics: (i) the intracellular position in the nucleus and cytoplasm for SFG rickettsiae
and R. canadensis, which are able to polymerize cellular actin (42), but only in the cytoplasm for
others which cannot polymerize actin; (ii) an optimal growth temperature of 32°C for the SFG
and 35°C for the TG and STG; and (iii) the cross-reaction of sera from a patient with rickettsial
infection with the somatic antigen of strains of Proteus vulgaris, OX19 for the TG and 
R. rickettsii, OX2 for the SFG, and OXK for the STG. Subsequently, the STG was deleted follow-
ing reclassification of R. tsutsugamushi within the genus Orientia (29). Other criteria have also
been used to describe Rickettsia species, including the geographical distribution of strains; their
arthropod vector; pathogenicity for humans, mice, and guinea pigs; size; optimal culture
temperature; time for plaque formation; hemolytic activity (23); cross-immunity and vaccine
protection tests in guinea pigs (43); complement fixation (44); and toxin neutralization (45).
However, since 1978, the immunofluorescentantibody assay with acute-phase mouse sera has
been used as a reference method for the identification of new SFG rickettsiae (46). This test
detects species-specific epitopes of the surface-exposed S-layer proteins (rOmpA and rOmpB),
as well as the Sca4 (PS-120) protein, of rickettsiae. Using this method, a species corresponds to
a serotype, with a rickettsial isolate being assumed to belong to a species if both strains exhibit
a specificity difference of �3 (46). Although useful, mouse immunization suffers drawbacks
such as a lack of reproducibility and the necessity to compare each new isolate to all previously
described species. Other phenotypic methods such as the use of monoclonal antibodies (47,48)
and sodium dodecyl sulfate–polyacrylamide gel electrophoresis did not bring any determinant
progress to rickettsial taxonomy. 
In addition, the official molecular criteria used for the identification of bacterial species,
that is, the DNA G � C content (32–33% for the SFG and 29% for the TG), and DNA–DNA reas-
sociation (49) [degrees of DNA–DNA hybridization of 94%, 74%, and 73% between R. rickettsii
and R. conorii, R. sibirica and R. montanensis, respectively (50), and of 70–77% between R.
prowazekii and R. typhi (51)] are not adequate for rickettsiae. Likewise, the average nucleotide
identity (ANI) method (52), designed as an alternative of DNA–DNA hybridization for the
delineation of bacterial species, is not suitable for rickettsiae as well. Using this criterion, R.
conorii, R. rickettsii, and R. sibirica, with ANI values of �94%, belong to the same species, as do
R. typhi and R. prowazekii. Pulsed field gel electrophoresis is useful for differentiating rick-
ettsiae, but it suffers from the absence of any database allowing the comparison of PFGE pro-
files, and the lack of reproducibility. Over the last 15 years, a number of genes including those
encoding 16S rRNA (16S rDNA), citrate synthase (gltA), the 17-kDa common antigen, surface-
exposed, high-molecular-weight antigenic proteins of the sca family (ompA, ompB, sca4, sca1,
and sca2) have been used to rapidly and reliably differentiate members of the genus Rickettsia
either by analysis of PCR–RFLP or by direct sequence determination (19,20,22,32,34–37). To
facilitate the classification of bacterial isolates as rickettsiae at the genus, group, and species
levels, genetic criteria based on a multi-locus sequence typing (MLST) method were proposed
(39). The MLST-based criteria used sequences of the 16SrDNA, gltA, ompA, ompB, and sca4
genes (Fig. 2), and were established using a panel of 20 uncontested Rickettsia species previ-
ously officially validated using mouse serotyping (R. prowazekii, R. typhi, R. rickettsii, R. conorii,
R. africae, R. sibirica, R. slovaca, R. honei, R. japonica, R. australis, R. akari, R. felis, R. aeschlimannii,
R. helvetica, R. massiliae, R. rhipicephali, R. montanensis, and R. parkeri). To incorporate these
genetic criteria into the definition of a Rickettsia species, an international committee of expert
rickettsiologists recently proposed guidelines to classify rickettsial isolates at various taxo-
nomic levels (Fig. 2) and to clarify the nomenclature within the genus Rickettsia (53). In addi-
tion, the subspecies taxonomic rank was created for members of the Rickettsia genus to classify
isolates of a species that exhibited specific phenotypic characteristics (54,55). The guidelines
recommended the use of a polyphasic approach that incorporated phenotypic, genotypic, and
phylogenic criteria (Fig. 2). This polyphasic approach enabled classification of rickettsial iso-
lates of uncertain taxonomic rank (R. mongolotimonae within the R. sibirica species; Rickettsia sp.
strain S within the R. africae species; Rickettsia sp. strain Bar29 within the R. massiliae species;
Rickettsia sp. strain BJ-90 within the R. sibirica species; Indian tick typhus rickettsia, Astrakhan
fever rickettsia, and Israeli spotted fever rickettsia within the R. conorii species). These criteria
DK7611-Raoult-Chap 01_R2.qxd 3/20/07 2:18 PM Page 9
10 Fournier and Raoult
also allowed the creation of the new species R. heilongjiangensis (13). Finally, a sensitive method
was developed to discriminate among isolates of a single Rickettsia species. This method,
named multi-spacer typing (MST), was based on the assumption that intergenic spacers, being
noncoding sequences, undergo less evolutionary pressure than coding sequences such as
genes, and thus are more variable between strains of a bacterium. MST identified 27 genotypes
among 39 R. conorii strains (56), four genotypes among 15 R. prowazekii strains (57), and thus
demonstrated to be valuable for the discrimination of rickettsiae at the strain level.
CONCLUSIONS
Owing to the improved diagnostic methods and increased interest, the number of representa-
tives of the genus Rickettsia has increased dramatically over the past 20 years, with 24 currently
SPD > 3 with respect 
to the type strain
AND
Specific epidemio- clinical 
characteristics
Yes
Belongs to a 
validated species
No
Other genus
Yes
SPD > 3 with respect 
to the type strain
AND
Specific epidemio- clinical 
characteristics
Subspecies
Describe phenotypic characters
(geographic distribution, vector, pathogenicity (if any), mouse 
serotype with respect to genetically closest validated species)
AND
Deposit of the type strain into 
two independent official culture collections
AND 
publication in Int. J. Syst. Evol. Microbiol. 
or
publication in another peer-reviewed journal and
in a Validation list in Int. J. Syst. Evol. Microbiol. 
Candidatus
rrs > 98.1%
AND
gltA > 86.5% 
with at least one
validated Rickettsia species 
First step
Is the bacterium a rickettsia ?
> 1 of these criteria
rrs > 99.8%
gltA > 99.9%
ompA > 98.8%
ompB > 99.2%
sca4 > 99.3% 
with a validated
Rickettsia species
Second step
Is the rickettsia a new species ?
Third step
Is the rickettsia isolated in pure culture ?
No
Potential 
new species
No Yes Yes No
Strain of
the
species 
NoYes
New species
FIGURE 2 Polyphasic taxonomic guidelines for classification of rickettsiae. Genes: rrs encodes the 16S rDNA;
gltA encodes the citrate synthase; ompA encodes rOmpA; ompB encodes rOmpB; sca4 encodes Sca4 (5PS-120).
Abbreviation: SPD, specificity difference in mouse serotyping.
DK7611-Raoult-Chap 01_R2.qxd 3/20/07 2:18 PM Page 10
validated species and several dozens of as-yet unclassified isolates or genotypes. These
arthropod-associated intracellular bacteria are now recognized in all parts of the world. The
comparison of the phylogenic organizations obtained from the study of several genes with
different functions provided basis to establish a reliable taxonomy of the bacteria included in the
genus Rickettsia. These data were incorporated into polyphasic consensus guidelines that
provide clear recommendations for the taxonomic classification and nomenclature of rickettsiae
and rickettsial diseases. These guidelines may later be updated by the introduction of additional
genetic or phenotypic characteristics and of new Rickettsia species.
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2 Pathogenesis, Immunity, Pathology, and
Pathophysiology in Rickettsial Diseases
David H. Walker, Nahed Ismail, Juan P. Olano, Gustavo Valbuena, and Jere McBride
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, U.S.A.
INTRODUCTION
The pathogenesis of diseases caused by Rickettsia, Orientia, Ehrlichia, Anaplasma, Neorickettsia,
and Coxiella differ greatly owing to differences in target cells, bacterial genomes, and cell wall
structure, and bacteria–host cell interactions including subcellular location, immune responses
stimulated during the infection, pathogenic mechanisms, and pathologic lesions (1,2). This
chapter focuses on infections with Rickettsia. The concept pathogenesis comprises three compo-
nents: the sequence of events from transmission until immune clearance of the agent, the
host–pathogen interaction ranging from the whole patient to the cellular level, and the patho-
genic mechanisms of cellular and tissue injury.
SEQUENCE OF EVENTS IN RICKETTSIAL INFECTIONS
Transmission
All Rickettsia have an arthropod host which serves as the biologic vector that transmits the
pathogens to humans. Tick- and mite-borne Rickettsia are inoculated into the skin of the host
from the arthropod’s saliva during its blood meal. Typhus group Rickettsia are mainly transmit-
ted in the feces of the louse (Pediculus humanus corporis) for epidemic typhus or flea for murine
typhus. It is widely believed that the infected insect feces are autoinoculated into the skin of
the patient by scratching the pruritic bite site. Interestingly, extracellular Rickettsia prowazekii in
louse feces and R. typhi in flea feces are stably infectious for months if not longer. The possibil-
ities of inoculation via rubbing the mucous membranes (e.g., conjunctiva) or via inhalation of
aerosols of infectious R. prowazekii or R. typhi organisms should not be discounted, and the lat-
ter has occurred in the research laboratory. A small fraction of cases of murine typhus are trans-
mitted by flea bite. Furthermore, some cases of tick-borne rickettsioses are transmitted by
transfer of rickettsiae to the conjunctiva by fingers contaminated with infectious tick
hemolymph or organs after crushing a tick that has been removed from a person or animal.
Aerosol transmission has been demonstrated experimentally to be very efficient, requiring
1000-fold fewer inhaled rickettsial organisms than anthrax spores.
Routes of Spread in the Body
The occurrence of prominent lymphadenopathy in the region draining the rickettsial portal of
entry at the eschar in R. africae, R. slovaca, and R. parkeri infections supports the hypothesis that
rickettsiae spread from the site of tick feeding to the regional lymph node via lymphatic ves-
sels. Dissemination throughout the body occurs via the bloodstream. Rickettsial infection
involves every organ, but spread beyond the lymphatic and blood vessels does not occur.
Target Cells and Organs
The initial target cells of Rickettsia at the site of inoculation in the skin have not been identified.
Because rickettsiae are obligately intracellular organisms and do not replicate extracellularly, it
is highly likely that rickettsiae rapidly enter cells in the skin. Rickettsia are capable of infecting
DK7611-Raoult-Chap 02_R2.qxd 3/20/07 11:41 AM Page 15
any type of nucleated cells; thus, their initial target is not necessarily endothelium. Among the
possible dermal target cells are fibroblasts, macrophages, dermal dendritic cells, and lymphat-
ic endothelium. It is proposed that infected dendritic cells migrate to regional lymph nodes
where innate and adaptive immunity are stimulated.
The main target cells of rickettsiosis are endothelial cells, most likely the result of vascu-
lar location and hematogenous dissemination of rickettsiae rather than interaction between a
rickettsial adhesin and a special receptor expressed only on endothelial cells.
Rickettsiae are also engulfed by fixed mononuclear phagocytes during their spread
through the blood stream. Thus, macrophages, especially in the spleen and liver, are minor tar-
get cells. Rickettsia rickettsii is the only organism in the genus that invades beyond the blood
vessel lining endothelium; they invade adjacent vascular smooth muscle cells, particularly in
arterioles.
Although Rickettsia infects all organs, the lungs and brain are the critical targets determin-
ing the lethality of rickettsioses. The events that are visible in the skin—silent seeding of the
dermal blood vessels, vasodilatation (macular rash), perivascular edema (maculopapular
rash), disruption of vascular integrity (petechial maculopapular rash), and resolution in sur-
vivors—occur throughout the organs of the body.
Injury Associated with Rickettsial Infection
Rickettsial infection is multifocal occurring in networks of contiguous cells, and organ and tissue
injury occurs in these foci. The hundreds of maculopapules in the skin of a patient with Rocky
Mountain spotted fever or epidemic louse-borne typhus are representative of only a small frac-
tion of the visceral involvement. When endothelial cells infected with R. prowazekii or R. typhi are
filled to capacity with replicating organisms, they burst by releasing the bacteria that infect other
endothelial cells. Death of the burst endothelial cells allowsred blood cells to hemorrhage into
the surrounding tissue and exposes the basement membrane to platelets and clotting factors.
Spotted fever group rickettsiae spread from cell to cell by host actin-based mobility, do
not accumulate in large numbers in endothelial cells, and injure the cells by damaging their
membranes. Infection of endothelial cells stimulates them to produce reactive oxygen species
that cause lipid peroxidation of the cellular membranes (3–5). Water leaks into the cells with
injured membranes and is sequestered in the endoplasmic reticulum. Injured endothelial cells
may die and/or detach and be swept away in the blood stream, allowing hemorrhage to occur,
or endothelium may be activated by the immune system to kill the intracellular rickettsiae (6).
The pathogenic mechanism of oxidative stress associated with R. rickettsii injury to
endothelial cells has been investigated extensively in cell culture, and oxidative stress occurs in
experimentally infected animals. However, it has not been demonstrated how important this
mechanism is in the pathogenesis of the disease in animals or humans (7). Other potential path-
ogenic mechanisms have not been investigated except for the determination that rickettsial
lipopolysaccharide does not have significant endotoxin activity. The potential role of oxidative
stress in the pathogenesis of typhus rickettsioses has not been investigated.
Rickettsial infection of human endothelial cells in vitro leads to the development of
interendothelial gaps indicative of discontinuities in the adherens junctions (8). Formation of
the gaps coincides with change in endothelial cells from small polygonal to large spindle shape
with development of stress fibers.
The pathophysiologic event that is most important in rickettsial infections is increased
permeability of the microcirculation, possibly due to the presence of gaps between infected
endothelial cells (8–10). Changes in junctional proteins after infection include p120 in adherens
junctions with �-catenin and occludin. Specifically, the location of p120 moves away from the
adherens junction after rickettsial infection of murine and human microvascular endothelial
cells, and �-catenin changes from a linear to a granular arrangement in infected cells. Likewise,
the location of occludin is altered in primary brain murine microvascular endothelial cells.
Immune Clearance of Rickettsiae
Recovery from rickettsial infection is associated with strong immunity against reinfection.
Studies of experimentally infected animals have revealed reduction of the rickettsial load
16 Walker et al.
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below the limits of detection in association with recovery from the infection. However, conva-
lescence from human infection with R. prowazekii is followed by asymptomatic latent carriage
of the rickettsiae in an unknown location in the body. When undefined host events presumed
to be related to altered immunity occur, there is reactivation of latent R. prowazekii infection, an
illness known as recrudescent typhus or Brill-Zinsser disease. Although reactivation of other
rickettsioses has not been observed, R. rickettsii has been isolated from lymph nodes of patients
who had recovered from Rocky Mountain spotted fever a year or more previously.
THE RICKETTSIA–HOST CELL INTERACTION
Entry of Rickettsiae into the Host Cell
In order to survive, obligately intracellular bacteria such as Rickettsia must enter a cell in the
vertebrate or arthropod host. Two major surface proteins of spotted fever group rickettsiae,
outer membrane proteins A (OmpA) and B (OmpB), are rickettsial ligands for host cells (11,12).
OmpB binds to Ku70, a host cell protein that spans the cell membrane (12,13). This adhesion
results in recruitment of more Ku70 to the cell membrane for binding to highly abundant
OmpB molecules and in recruitment of ubiquitin ligase to the nascent rickettsial entry site (12).
The Ku70 molecules are ubiquitinated, and signal transduction events lead to phagocytosis of
the adherent rickettsia associated with Arp2/3 complex recruitment to the entry focus. The
small GTPase, Cdc 42, protein tyrosine kinase, phosphoinositide 3-kinase, and Src-family
kinases activate the Arp2/3 complex to direct changes in the cytoskeletal actin at the entry site
resulting in focal-induced phagocytosis of the rickettsia by the so-called zipper mechanism
(14). Rickettsial entry into the host cell is rapid, generally occurring in about 15 minutes.
Rickettsial Escape from the Phagosome
Rickettsia are highly adapted to living in the host cell cytosol where they acquire the necessary
nutrients, adenosine triphosphate, and nucleotides and amino acids for replication. The phago-
some in which they enter is a potential death trap if lysosomal fusion occurs, but they escape
rapidly by lysing the phagosomal membrane. The rickettsial proteins that can digest the host
cell phagosomal membrane are phospholipase D and hemolysin C (TlyC) (15,16).
Spread of Rickettsiae to Other Cells
All investigated spotted fever group rickettsiae except R. peacockii activate polymerization of
host cell actin at one pole of the bacterium. The continuous conversion of globular actin to fil-
amentous actin propels the rickettsia forward through the cytosol until it collides with the host
cell membrane (17,18). Some rickettsia carom off the inner surface of the cell membrane like a
billiard ball. Other rickettsia deform the membrane, and a filopodium develops into which the
polymerized actin pushes the rickettsia. If the endothelial cell membrane at the site of the
filopodium abuts an adjacent endothelial cell and the membranes of both cells are breached,
the rickettsia enters the second endothelial cell without exposure to the constituents of the
extracellular fluid. If the rickettsia exits a luminal filopodium, it enters the blood stream. If 
R. rickettsii exits the basal endothelial cell membrane, it can spread to an adjacent vascular
smooth muscle cell.
Actin-based mobility of R. conorii and other spotted fever group rickettsiae is initiated by
a rickettsial protein RickA that is expressed on the rickettsial cell wall (19,20). The carboxy ter-
minal domain of RickA is similar to particular domains of WASP-family proteins that activate
Arp2/3, and indeed Arp2/3 is activated by RickA. The Arp2/3 complex is recruited to the rick-
ettsial surface where it acts as a nucleator of actin polymerization. Unlike actin tails that move
other organisms, the tails of Rickettsia do not contain Arp2/3. Consistent with the importance
of RickA in rickettsial actin-based mobility, R. prowazekii and R. typhi lack the gene rickA, and
rickA of R. peacockii is inactivated by transposon insertion. Thus, R. prowazekii and R. peacockii
do not have actin tails and do not spread from cell to cell. The burst release of R. prowazekii is
the result. R. typhi has erratic actin-based mobility that must rely upon a rickA-independent
molecular mechanism.
Pathogenesis, Immunity, Pathology, and Pathophysiology in Rickettsial Diseases 17
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HOST DEFENSES AGAINST RICKETTSIAE
The development of excellent models of disseminated rickettsial infections of endothelium 
in inbred strains of mice has enabled the identification of the effectors of protective immunity.
Whether these effector mechanisms are important in human rickettsioses still remains to 
be determined. The early immune events occurring in the skin and draining lymph nodes,
including the potential immunomodulatory effects of arthropod saliva, also remain to be 
elucidated.
Innate Immunity
Rickettsial infection activates natural killer cell activity, which dampens rickettsial establish-
ment of infection and rickettsial growth early in infection in association with the production of
gamma interferon (IFN-�) (21). Rickettsial infectionalso stimulates the production of type I
interferon (IFN-� and IFN-�), which independently has no protective effect. However, IFN-�
and IFN-� enhance activation of natural killer cells, maturation of dendritic cells, and produc-
tion of interleukin-12, which is a cytokine that favors T helper type 1 cellular immunity and is
produced early in rickettsial infection. Indeed, the levels of IL-12 are increased in the sera of
C3H/HeN mice early in the course of R. conorii infection.
Rickettsia-infected endothelial cells produce IL-6, IL-8, and MCP-1, and rickettsia-infected
macrophages secrete tumor necrosis factor (TNF-�) (22). Moreover, the concentration of 
IL-6 is elevated in the sera of C3H/HeN mice infected with R. conorii, and IL-1 and TNF-� are
increased in the sera of C57Bl/6 mice infected with R. australis. IL-1 and IL-6 are proinflamma-
tory cytokines that play a role in the activation of innate immune cells and induction of the 
specific acquired immune response. Rickettsial infection is associated with an acute-
phase response. However, the roles, if any, of acute-phase proteins such as C-reactive protein,
complement components, C3 and C4, and fibrinogen as host defenses against rickettsiae are
not known.
Macrophages and polymorphonuclear leukocytes are professional phagocytic cells that
are essential components of innate immunity against many bacteria. Rickettsiae avoid killing
by macrophages during primary rickettsial infection by escaping from phagosomes into the
cytosol, avoiding the rickettsicidal effect of phagolysosomal fusion. Neutrophils are an
insignificant population in the sites of rickettsial infections.
Rickettsial infection of endothelial cells also activates NF-�B, an important transcription
factor that mediates the production of proinflammatory cytokines and chemokines. NF-�B acti-
vation occurs downstream of rickettsial activation of the I�B kinase complex which phospho-
rylates I�Ba and I�Bb (inhibitors of NF-�B) leading to their subsequent degradation by the 26S
proteasome (23). The degradation of I�Ba and I�Bb activates NF-�B dimers, which are translo-
cated into the nucleus, bind to NF-�B enhancer sequences via the DNA-binding domain, and
regulate the transcription of specific cytokine and chemokine genes. Another effect of NF-�B
activation is inhibition of apoptosis of Rickettsia-infected target cells by preventing activation of
upstream caspases-8 and -9 and the effector caspase-3. The prevention of apoptosis enhances
survival of endothelial cells in which further rickettsial growth can occur. NF-�B alterations
have been documented in vitro. In vivo, this mechanism could conceivably play an important
role early in the infectious process when rickettsiae are invading their initial targets until the
infection is established. However, later in the infectious process when both innate and adaptive
immunity are fully activated, NF-�B signals are most likely overridden by the CD8 cytotoxic 
T-lymphocytes, which induce apoptosis of rickettsia-infected endothelial cells.
Adaptive Immunity
Role of Endothelial Cells and Other Target Cells in Killing Intracellular Rickettsiae
Activation of murine endothelial cells by IFN-� and TNF-� results in synthesis of rickettsicidal
nitric oxide (6). These cytokines act synergistically to stimulate expression of inducible nitric
oxide synthetase. Neutralization of IFN-� or TNF-� or gene knockout of IFN-� results in an
overwhelming infection after a low-dose rickettsial challenge (24,25). Cytokine-activated rick-
ettsial killing by endothelial cells is associated with autophagy and rickettsial digestion within
18 Walker et al.
DK7611-Raoult-Chap 02_R2.qxd 3/20/07 11:41 AM Page 18
an autophagolysosome (26). It is hypothesized that healthy rickettsiae have evolved mecha-
nisms to inhibit autophagy, the cell’s second line of defense, but injured or host defense-
inhibited rickettsiae lose this immune evasive activity.
Human endothelial cells activated by IFN-�, TNF-�, IL-1�, and RANTES (CCL5) also kill
intracellular rickettsiae (27). Some activated human endothelial cells have been demonstrated
to produce rickettsicidal nitric oxide in vitro. All cytokine-activated human endothelial cells are
capable of killing intracellular rickettsiae by producing hydrogen peroxide. The formation of
both nitric oxide and reactive oxygen species could lead to the formation of peroxynitrite that
also has potent rickettsicidal activity.
In human rickettsial diseases, macrophages are a minor target cell of rickettsial infection,
and hepatocytes are a suspected minor target based on pathologic lesions in hepatic biopsies
and observations early in the course of experimental animal infections (28). Human
macrophages activated by IFN-�, TNF-�, and IL-1� kill intracellular rickettsiae by production of
hydrogen peroxide and tryptophan starvation of rickettsiae via indoleamine-2,3-dioxygenase-
mediated degradation of tryptophan (27). In contrast, a human hepatocyte cell line (AKN-1)
kills intracellular R. rickettsii through synthesis of nitric oxide after activation by IFN-�, TNF-�,
IL-1�, and RANTES.
The mRNA of inducible nitric oxide synthase, IFN-�, CCL5, and indoleamine-2,3-dioxy-
genase is expressed in biopsies of skin lesions from patients with Mediterranean spotted fever.
These data suggest that nitric oxide production and tryptophan degradation occur at the sites
of immunity to rickettsiae in humans. The cells that express these rickettsicidal effector mech-
anisms and mediators are thought to be the endothelium, perivascular lymphocytes, dendritic
cells, and macrophages.
Roles of Lymphocytes in Immunity to Rickettsiae
Both CD4 and CD8 T-lymphocytes contribute to the control of rickettsial growth and killing and
to the host’s survival of rickettsial infection, most likely by secreting IFN-� and TNF-� (6).
C3H/HeN mice depleted of CD4 T-lymphocytes have the same course of illness and recovery
from experimental infection with R. conorii as sham-depleted mice. In contrast, mice depleted of
CD8 lymphocytes die or remain persistently infected when they are inoculated with the same
dose of R. conorii that results in the survival of wild-type mice. Cytotoxic activity of CD8 T-
lymphocytes is crucial to the clearance of rickettsial infection (25). Perforin gene knockout mice
are 1000-fold more susceptible to death from R. australis infection than wild-type C57BL/6 mice.
The LD50 for R. australis in major histocompatibility class (MHC) I gene knockout mice is 0.5
plaque-forming units; one organism is sufficient to initiate an infection that cannot be controlled
without MHC-I molecule presentation of R. australis antigen to CD8 cytotoxic T-lymphocytes.
Mechanisms of Homing of Natural Killer Cells, Immune CD4 and CD8 Lymphocytes,
and Macrophages to Sites of Rickettsia-Infected Endothelium
An important phenomenon is the interaction of Rickettsia-infected endothelium with cells of
the immune system. The histopathologically visible result is perivascular infiltration of CD4
and CD8 T-lymphocytes and macrophages around the networks of contiguous infected
endothelial cells of the microcirculation. It is presumed that the initial, and sometimes only,
interaction of the endothelium with circulating lymphocytes and monocytes is at the luminal
interface.
Among the chemokines that can play a role in directing T-lymphocytes to adhere to
endothelium, CXCL9 (Mig), CXCL10 (IP-10), and CX3CL1 (fractalkine) are expressed at high
levels in the endothelium of R. conorii-infected mice, and CXCL9 and CXCL10 are expressed in
endothelium of the brain in fatal human cases of Rocky Mountain spotted fever (29). However,
treatment of mice with antibodies to CXCL9 and CXCL10 does not alter the survival, and 
R. conorii infection of mice with gene knockout for the receptor of these chemokines does not
affect survival or T-lymphocyte infiltration (30). The only evidence that