Rickettsia Rickettsii Descriptive Essay

General Concepts

Rickettsiae

The rickettsiae are a diverse collection of obligately intracellular Gram-negative bacteria found in ticks, lice, fleas, mites, chiggers, and mammals. They include the genera Rickettsiae, Ehrlichia, Orientia, and Coxiella. These zoonotic pathogens cause infections that disseminate in the blood to many organs.

Rickettsia

Clinical Manifestations

Rickettsia species cause Rocky Mountain spotted fever, rickettsialpox, other spotted fevers, epidemic typhus, and murine typhus. Orientia (formerly Rickettsia) tsutsugamushi causes scrub typhus. Patients present with febrile exanthems and visceral involvement; symptoms may include nausea, vomiting, abdominal pain, encephalitis, hypotension, acute renal failure, and respiratory distress.

Structure, Classification, and Antigenic Types

Rickettsia species are small, Gram-negative bacilli that are obligate intracellular parasites of eukaryotic cells. This genus consists of two antigenically defined groups: spotted fever group and typhus group, which are related; scrub typhus rickettsiae differ in lacking lipopolysaccharide, peptidoglycan, and a slime layer, and belong in the separate, although related, genus Orientia.

Pathogenesis

Rickettsia and Orientia species are transmitted by the bite of infected ticks or mites or by the feces of infected lice or fleas. From the portal of entry in the skin, rickettsiae spread via the bloodstream to infect the endothelium and sometimes the vascular smooth muscle cells. Rickettsia species enter their target cells, multiply by binary fission in the cytosol, and damage heavily parasitized cells directly.

Host Defenses

T-lymphocyte-mediated immune mechanisms and cytokines, including gamma interferon and tumor necrosis factor alpha, play a more important role than antibodies.

Epidemiology

The geographic distribution of these zoonoses is determined by that of the infected arthropod, which for most rickettsial species is the reservoir host.

Diagnosis

Rickettsioses are difficult to diagnose both clinically and in the laboratory. Cultivation requires viable eukaryotic host cells, such as antibiotic-free cell cultures, embryonated eggs, and susceptible animals. Confirmation of the diagnosis requires comparison of acute- and convalescent-phase serum antibody titers.

Control

Rickettsia species are susceptible to the broad-spectrum antibiotics, doxycycline, tetracycline, and chloramphenicol. Prevention of exposure to infected arthropods offers some protection. A vaccine exists for epidemic typhus but is not readily available.

Ehrlichia

Clinical Manifestations

Ehrlichia species cause ehrlichioses that vary in severity from a life-threatening febrile disease that resembles Rocky Mountain spotted fever, except for less frequent rash, to an infectious mononucleosis-like syndrome.

Classification and Antigenic Types

Ehrlichia sennetsu, E chaffeensis, and the human granulocytic ehrlichia are genetically distinct and are easily distinguished antigenically.

Pathogenesis

A reservoir of E chaffeensis is deer, and for both human monocytic and granulocytic ehrlichiosis are transmitted when ticks bite human skin and inoculate organisms, which then spread by the bloodstream. Macrophages or neutrophils have cytoplasmic vacuoles that contain ehrlichiae dividing by binary fission in each of these ehrlichioses.

Host Defenses

Host defenses against E chaffeensis include cytokine-mediated restriction of iron supplies to the ehrlichiae.

Epidemiology

Sennetsu ehrlichiosis has been documented in Japan and Malaysia. Human infections with E chaffeensis- and E phagocytophila- like organisms have been found recently. Human monocytic ehrlichiosis originates in most of the Atlantic, southeastern, and south central states from New Jersey to Texas. Human granulocytic ehrlichiosis has been identified in the upper midwest and New England thus far.

Diagnosis

Clinical and laboratory clues must be confirmed serologically or by polymerase chain reaction detection of specific ehrlichial DNA.

Coxiella

Clinical Manifestations

Coxiella burnetii causes Q fever, which may present as an acute febrile illness with pneumonia or as a chronic infection with endocarditis.

Structure, Classification, and Antigenic Types

Coxiella burnetii varies in size and has an endospore-like form. This species has lipopolysaccharide and phage type diversity.

Pathogenesis

Coxiella burnetii organisms are transmitted to the human lungs by aerosol from heavily infected placentas of sheep and other mammals and disseminate in the bloodstream to the liver and bone marrow, where they are phagocytosed by macrophages. Growth within phagolysosomes is followed by formation of T-lymphocyte-mediated granulomas. In the few patients who develop serious chronic Q fever, heart valves contain organisms within macrophages.

Host Defenses

Host defense depends on T lymphocytes and gamma interferon.

Epidemiology

Q fever is found worldwide. It is associated mainly with exposure to infected placentas and birth fluids of sheep and other mammals.

Diagnosis

The disease is difficult to diagnose clinically, and cultivation poses a biohazard. Therefore, serology is the mainstay of laboratory diagnosis.

Control

Antibiotics are effective against acute Q fever. A vaccine containing killed phase I organism shows promise in protecting against infection.

Bartonella

Bartonella (Rochalimaea) quintana, the agent of trench fever, was formerly considered as a rickettsial agent. It can be cultured outside of eukaryotic cells and is transmitted to humans via lice. Trench fever was a significant medical problem during World War I and has reappeared among homeless and alcoholic persons. Recently, cat scratch disease and bacillary angiomatosis and peliosis were discovered to be caused in most cases by a related organism, B henselae. Bartonella bacilliformis has long been known to cause a sand fly-transmitted acute infection in South America that destroys the red blood cells and a chronic infection that causes a vascular tumor-like lesion similar to those of B henselae.

Introduction

Rickettsiae are small, Gram-negative bacilli that have evolved in such close association with arthropod hosts that they are adapted to survive within the host cells. They represent a rather diverse collection of bacteria, and therefore listing characteristics that apply to the entire group is difficult. The common threads that hold the rickettsiae into a group are their epidemiology, their obligate intracellular lifestyle, and the laboratory technology required to work with them. In the laboratory, rickettsiae cannot be cultivated on agar plates or in broth, but only in viable eukaryotic host cells (e.g., in cell culture, embryonated eggs, or susceptible animals). The exception, which shows the artificial nature of using obligate intracellular parasitism as a defining phenotypic characteristic, is Bartonella (Rochalimaea) quintana, which is cultivable axenically, but was traditionally considered as a rickettsia. The diversity of rickettsiae is demonstrated in the variety of specific intracellular locations where they live and the remarkable differences in their major outer membrane proteins and genetic relatedness (Table 38-1). An example of extreme adaptation is that the metabolic activity of Coxiella burnetii is greatly increased in the acidic environment of the phagolysosome, which is a harsh location for survival for most other organisms. Obligate intracellular parasitism among bacteria is not unique to rickettsiae. Chlamydiae also have evolved to occupy an intracellular niche, and numerous bacteria (e.g., Mycobacteria, Legionella, Salmonella, Shigella, Francisella, and Brucella) are facultative intracellular parasites. In contrast with chlamydiae, all rickettsiae can synthesize ATP. Coxiella burnetii is the only rickettsia that appears to have a developmental cycle.

Table 38-1

Properties of Selected Rickettsial Organisms.

Some organisms in the family Rickettsiaceae are closely related genetically (e.g., Rickettsia rickettsii, R akari, R prowazekii, and R typhi); others are related less closely to Rickettsia species (e.g., Ehrlichia and Bartonella); and others not related to Rickettsia species (e.g., C burnetii). The phenotypic traits of the medically important organism Orientia (Rickettsia) tsutsugamushi suggest that the species may be an example of convergent evolution in a similar ecologic niche.

Rickettsioses are zoonoses that, except for Q fever, are usually transmitted to humans by arthropods (tick, mite, flea, louse, or chigger) (Table 38-2). Therefore, their geographic distribution is determined by that of the infected arthropod, which for most rickettsial species is the reservoir host. Rickettsiae are important causes of human diseases in the United States (Rocky Mountain spotted fever, Q fever, murine typhus, sylvatic typhus, human monocytic ehrlichiosis, human granulocytic ehrlichiosis, and rickettsialpox) and around the world (Q fever, murine typhus, scrub typhus, epidemic typhus, boutonneuse fever, and other spotted fevers) (Table 38-2).

Table 38-2

Distinguishing Characteristics Of Rickettsial Diseases.

Rickettsiae of the Spotted Fever and Typhus Groups

The rickettsial diseases are arranged into several major categories (Table 38-2), the first two of which are the spotted fever and typhus fever groups.

Clinical Manifestations

Rocky Mountain Spotted Fever

Rocky Mountain spotted fever is among the most severe of human infectious diseases, with a mortality of 20 to 25 percent unless treated with an appropriate antibiotic. The severity and mortality are greater for men, elderly persons, and black men with glucose-6-phosphate dehydrogenase deficiency. Although, in theory, the disease is always curable by early, appropriate treatment, the case fatality rate is still 4 percent. The incidence of disease parallels the geographic distribution of infected Dermacentor variabilis ticks in the eastern United States and D andersoni in the Rocky Mountain states, where the infection was first recognized. Rocky Mountain spotted fever was subsequently recognized in the eastern United States. The incidence has declined in the Rocky Mountain states and increased dramatically in the southeastern United States and Oklahoma. Currently most cases actually occur in the Atlantic states from Maryland to Georgia, as well as in Oklahoma, Missouri, Kansas, Ohio, Tennessee, Arkansas, and Texas, although cases are reported in nearly every state. In the southeastern states, the disease occurs during the seasonal activity of D variabilis ticks (April through September) and affects children more frequently than adults. Significant changes in incidence do occur. From a low of 199 cases reported in 1959, the annual number of cases rose steadily to a peak of 1,192 cases in 1981, with a subsequent decline and plateau of approximately 700 cases since 1985. The reasons for these fluctuations are unclear.

The rickettsiae are maintained in nature principally by transovarial transmission from infected female ticks to infected ova that hatch into infected larval offspring (Fig. 38-1). A low rate of acquisition of rickettsiae by uninfected ticks occurs when the ticks feed upon small mammals with enough rickettsiae in their blood to establish tick infection. This effect replenishes lines of infected ticks that are occasionally killed by massive rickettsial overgrowth. A recently observed factor of potential importance in this balance of nature is the interference phenomenon, by which infection of ticks with nonpathogenic spotted fever group rickettsiae prevents the establishment of infection by R rickettsii.

Figure 38-1

Transovarian passage of R rickettsii in the tick vector is an important cycle in maintaining the infection in nature from one generation of tick to another. Horizontal transmission (i.e., acquisition of the bacteria by uninfected ticks feeding on infected (more...)

The clinical gravity of Rocky Mountain spotted fever is due to severe damage to blood vessels by R rickettsii. This organism is unusual among rickettsiae in its ability to spread and invade vascular smooth muscle cells as well as endothelium. Damage to the blood vessels in the skin in locations of the rash leads to visible hemorrhages in one-half of all infected persons (Fig. 38-2). Attempted plugging of vascular wall destruction consumes platelets, with consequent thrombocytopenia also affecting approximately one-half of the patients.

Figure 38-2

Common clinical manifestations of the rickettsial diseases. .

Rickettsialpox and Other Spotted Fevers

In the 1940s an epidemic of disease characterized by fever, rash, and cutaneous necrosis appeared in one area of New York City. The etiology was traced to R akari transmitted by the bite of mites (Liponyssoides sanguineus) that infested the numerous mice in an apartment house in this area. The disease was named rickettsialpox because many patients had blister-like rashes resembling those of chickenpox. Epidemics were diagnosed in other cities, and R akari has been isolated in other countries (e.g., the Ukraine). Perhaps because this nonfatal disease is seldom considered by physicians, or its incidence is truly low, the diagnosis is rarely made. Transovarial transmission in the mite and periodic documentation of cases assure us that the etiologic agent is still with us.

Boutonneuse fever, so called because of the papular rash in some cases, has many synonyms, reflecting different geographic regions of occurrence (e.g., Mediterranean spotted fever, Kenya tick typhus, and South African tick bite fever). Cases are observed in the United States in travelers returning from endemic areas. The agent, R conorii, is closely related to R. rickettsii. Severe disease resembling Rocky Mountain spotted fever can cause death in high-risk groups (e.g., elderly, alcoholic, and glucose-6-phosphate dehydrogenase-deficient patients). Cutaneous necrosis caused by rickettsial vascular infection at the tick bite site of inoculation, known as an eschar or tache noire, is observed in only half the patients with boutonneuse fever. The curiously high prevalence of antibodies reactive with R conorii in healthy populations in endemic regions might be explained by missed diagnosis of prior illness, subclinical infection, infection with an antigenically related but less pathogenic rickettsia, or nonspecificity of the laboratory test.

Other spotted fevers occur in geographic distributions of little concern to many physicians in the United States. North Asian tick typhus caused by R sibirica, Queensland tick typhus caused by R australis, and the recently discovered oriental spotted fever caused by R japonica demonstrate that spotted fever group rickettsiae occur worldwide.

Epidemic Typhus and Brill-Zinsser Disease

Epidemics of louse-borne typhus fever have had important effects on the course of history; for example, typhus in one army but not in the opposing force has determined the outcome of wars. Populations have been decimated by epidemic typhus. During and immediately after World War I, 30 million cases occurred, with 3 million deaths. Unsanitary, crowded conditions in the wake of war, famine, flood, and other disasters and in poor countries today encourage human louse infestation and transmission of R prowazekii. Epidemics usually occur in cold months in poor highland areas, such as the Andes, Himalayas, Mexico, Central America, and Africa. Lice live in clothing, attach to the human host several times daily to take a blood meal, and become infected with R prowazekii if the host has rickettsiae circulating in the blood. If the infected louse infests another person, rickettsiae are deposited on the skin via the louse feces or in the crushed body of a louse. Scratching inoculates rickettsiae into the skin.

Between epidemics R prowazekii persists as a latent human infection. Years later, when immunity is diminished, some persons suffer recrudescent typhus fever (Brill-Zinsser disease). These milder sporadic cases can ignite further epidemics in a susceptible louse-infested population. In the United States Brill-Zinsser disease is seen in immigrants who suffered typhus fever before entering the country. In the eastern United States, sporadic human cases of R prowazekii infection have been traced to a zoonotic cycle involving flying squirrels and their own species of lice and fleas.

Murine Typhus

Murine typhus is prevalent throughout the world, particularly in ports, countries with warm climates, and other locations where rat populations are high. Rickettsia typhi is associated with rats and fleas, particularly the oriental rat flea, although other ecologic cycles (e.g., opossums and cat fleas) have been implicated. Fleas are infected by transovarian transmission or by feeding on an animal with rickettsiae circulating in the blood. Rickettsiae are shed from fleas in the feces, from which humans acquire the infection through the skin, respiratory tract, or conjunctiva. During the 1940s more than 4,000 cases of murine typhus occurred annually in the United States. The incidence declined coincident with increased utilization of the insecticide DDT. Although the infection and clinical involvement affects the brain, lungs, and other visceral organs in addition to the skin, mortality in humans is less than 1 percent.

Structure, Classification, and Antigenic Types

Rickettsia species include two antigenically defined groups that are closely related genetically but differ in their surface-exposed protein and lipopolysaccharide antigens. These are the spotted fever and typhus groups. The organisms in these groups are smaller (0.3 μm by 1.0 μm) than most Gram-negative bacilli that live in the extracellular environment (Fig. 38-3). They are surrounded by a poorly characterized structure that is observed as an electron-lucent zone by transmission electron microscopy and is considered to represent a polysaccharide-rich slime layer or capsule. The cell wall contains lipopolysaccharides, a major component that differs antigenically between the typhus group and the spotted fever group. These rickettsiae also contain major outer membrane proteins with both cross-reactive antigens and surface-exposed epitopes that are species specific and easily denatured by temperatures above 54°C. The major outer membrane protein of typhus group rickettsiae has an apparent molecular mass of 120,000 Da. Spotted fever group rickettsiae generally have a pair of analogous proteins with some diversity of their molecular masses. Rickettsia prowazekii has a transport mechanism that exchanges ATP for ADP in its intracellular environment, thus providing a means to usurp host cell energy sources under favorable circumstances. Rickettsiae also are able to synthesize ATP via metabolism of glutamate. Adaptation to the intracellular environment is further evidenced in a variety of transport mechanisms to obtain crucial substances such as particular amino acids from cytoplasmic pools in the host cell. These adaptations and the presence of numerous independent metabolic activities demonstrate that rickettsiae are not degenerate forms of bacteria, but rather have evolved successfully for survival with an intracellular life-style.

Figure 38-3

(A) Organisms of Rickettsia conorii(r) in a cultured human endothelial cell are located free in the cytosol. One rickettsia is dividing by binary fission (arrowhead). (B) These rickettsiae can move inside the cytoplasm of the host cell because of the (more...)

Pathogenesis

Rickettsiae are transmitted to humans by the bite of infected ticks and mites and by the feces of infected lice and fleas. They enter via the skin and spread through the bloodstream to infect vascular endothelium in the skin, brain, lungs, heart, kidneys, liver, gastrointestinal tract, and other organs (Fig. 38-1). Rickettsial attachment to the endothelial cell membrane induces phagocytosis, soon followed by escape from the phagosome into the cytosol (Fig. 38-4). Rickettsiae divide inside the cell. Rickettsia prowazekii remains inside the apparently healthy host cell until massive quantities of intracellular rickettsiae accumulate and the host cell bursts, releasing the organisms. In contrast, R rickettsii leaves the host cell via long, thin cell projections (filopodia) after a few cycles of binary fission. Hence, relatively few R rickettsii organisms accumulate inside any particular cell, and rickettsial infection spreads rapidly to involve many other cells. Perhaps because of the numerous times the host cell membrane is traversed, there is an influx of water that is initially sequestered in cisternae of cytopathically dilated rough endoplasmic reticulum in the cells more heavily infected with R rickettsii.

Figure 38-4

Pathogenesis of the rickettsial agents illustrating unique aspects of their interactions with eukaryotic cells.

The bursting of endothelial cells infected with R prowazekii is a dramatic pathologic event. The mechanism is unclear, although phospholipase activity, possibly of rickettsial origin, has been suggested. Injury to endothelium and vascular smooth muscle cells infected by R rickettsii seems to be caused directly by the rickettsiae, possibly through the activity of a rickettsial phospholipase or rickettsial protease or through free-radical peroxidation of host cell membranes. Host immune, inflammatory, and coagulation systems are activated and appear to benefit the patient. Cytokines and inflammatory mediators account for an undefined part of the clinical signs. Rickettsial lipopolysaccharide is biologically relatively nontoxic and does not appear to cause the pathogenic effects of these rickettsial diseases.

The pathologic effects of these rickettsial diseases originate from the multifocal areas of endothelial injury with loss of intravascular fluid into tissue spaces (edema), resultant low blood volume, reduced perfusion of the organs, and disordered function of the tissues with damaged blood vessels (e.g., encephalitis, pneumonitis, and hemorrhagic rash).

Diagnosis

Diagnosis of rickettsial infections is often difficult. The clinical signs and symptoms (e.g., fever, headache, nausea, vomiting, and muscle aches) resemble many other diseases during the early stages when antibiotic treatment is most effective. A history of exposure to the appropriate vector tick, louse, flea, or mite is helpful but cannot be relied upon. Observation of a rash, which usually appears on or after day 3 of illness, should suggest the possibility of a rickettsial infection but, of course, may occur in many other diseases also. Knowledge of the seasonal and geographic epidemiology of rickettsioses is useful, but is inconclusive for the individual patient. Except for epidemic louse-borne typhus, rickettsial diseases strike mostly as isolated single cases in any particular neighborhood. Therefore, clinico-epidemiologic diagnosis is ultimately a matter of suspicion, empirical treatment, and later laboratory confirmation of the specific diagnosis.

Because rickettsiae are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification (Fig. 38-5). Some laboratories are able to identify rickettsiae by immunohistology in skin biopsies as a timely, acute diagnostic procedure, but to establish the diagnosis physicians usually rely on serologic demonstration of the development of antibodies to rickettsial antigens in serum collected after the patient has recovered. Currently, assays that demonstrate antibodies to rickettsial antigens themselves (e.g., the indirect fluorescence antibody test or latex agglutination) are preferable to the nonspecific, insensitive Weil-Felix test that is based on the cross-reactive antigens of OX-19 and OX-2 strains of Proteus vulgaris.

Figure 38-5

Laboratory methods used in confirming a diagnosis of rickettsial infection. These bacteria can be cultivated as indicated, but use of serology is more common.

Control

Although early treatment with doxycycline, tetracycline, or chloramphenicol is effective in controlling the infection in the individual patient, this action has no effect on rickettsiae in their natural ecologic niches (e.g., ticks). Human infections are prevented by control of the vector and reservoir hosts. Massive delousing with insecticide can abort an epidemic of typhus fever. Prevention of attachment of ticks and their removal before they have injected rickettsiae into the skin reduces the likelihood of a tick-borne spotted fever. Control of rodent populations and of the access of rats and mice to homes and other buildings may reduce human exposure to R typhi and R akari.

Vaccines against spotted fever and typhus group rickettsiae have been developed empirically by propagation of rickettsiae in ticks, lice, embryonated hen eggs, and cell culture. Vaccines containing killed organisms have provided incomplete protection. A live attenuated vaccine against epidemic typhus has proved successful, but is accompanied by a substantial incidence of side effects, including a mild form of typhus fever in some persons. The presence of strong immunity in convalescent subjects indicates that vaccine development is feasible, but it requires further study of rickettsial antigens and the effective anti-rickettsial immune response. T-lymphocyte-mediated immune mechanisms, including effects of the lymphokines, gamma interferon tumor necrosis factor, and interleukin-1, seem most important.

Orientia (Rickettsia) tsutsugamushi and Scrub Typhus

Although the agents of scrub typhus bear a single taxonomic name, Orientia (Rickettsia) tsutsugamushi, these interrelated organisms are somewhat heterogeneous and differ strikingly from Rickettsia species of the spotted fever and typhus groups.

Clinical Manifestations

Patients with scrub typhus often have only fever, headache, and swollen lymph nodes and in some cases myalgia, gastrointestinal complains, or cough beginning 6 to 21 days following exposure to the vector. Fewer than half of the patients have an eschar at the site where the larval mite fed and the classic rash. The mortality varies but averages 7 percent without anti-rickettsial treatment.

Structure, Classification, and Antigenic Types

Orientia (Rickettsia) tsutsugamushi is a very labile rickettsia that is particularly difficult to propagate and separate from the host cells in which it grows. In contrast with spotted fever group and typhus group rickettsiae, O tsutsugamushi does not seem to possess lipopolysaccharides, peptidoglycan, a slime layer, or other T-independent antigens. The rickettsial cell wall consists of proteins linked by disulfide bonds. Antigenically distinguishable strains represent only part of what seems to be a great antigenic mosaic. Immunity to infection with the homologous strain wanes within a few years; cross-protective immunity to heterologous strains disappears within a few months. The reasons for this lack of long-term immunity are unclear.

Pathogenesis

Orientia (Rickettsia) tsutsugamushi is injected into the skin during feeding by a larval trombiculid mite (chigger). An eschar often forms at this location. Rickettsiae spread via the bloodstream and damage the microcirculation of the skin (rash), lungs (pneumonitis), brain (encephalitis), and other organs. The generalized enlargement of lymph nodes is unique among rickettsial diseases. Orientia (Rickettsia) tsutsugamushi is phagocytosed by the host cell, escapes from the phagosome into the cytosol, divides by binary fission, and is released from projections of the cell membrane (Fig. 38-4). The pathogenic mechanism of O tsutsugamushi is not known.

Epidemiology

Scrub typhus occurs where chiggers infected with virulent rickettsial strains feed upon humans. Leptotrombidium deliense and other mites are found particularly in areas where regrowth of scrub vegetation harbors the Rattus species that are hosts for the mites. Some of these foci are quite small and have been referred to as mite islands. Because O tsutsugamushi is transmitted transovarially from one generation of mites to the next, these dangerous areas tend to persist for as long as the ecologic conditions, including scrub vegetation, persist. Truly one of the neglected diseases, scrub typhus occurs over a vast area, including Japan, China, the Philippines, New Guinea, Indonesia, other islands of the southwest Pacific Ocean, southeastern Asia, northern Australia, India, Sri Lanka, Pakistan, Russia, and Korea. Recognized in western countries mainly because of large numbers of infections of military personnel during World War II and the Vietnam War, scrub typhus perennially affects native populations. Reinfection and undiagnosed infections are highly prevalent. Mortality ranges from 0 to 35 percent and has not been correlated with any specific factor.

Diagnosis

Classic textbook cases with fever, headache, eschar, and rash are far outnumbered by cases that lack rash or eschar. Such cases are usually misdiagnosed. Laboratory diagnosis is unavailable in many areas where scrub typhus occurs. Isolation of rickettsiae requires inoculation of mice or cell culture. Serologic diagnosis is made by specific methods (indirect fluorescence antibody test or enzyme immunoassay) or by the older method of demonstrating cross-reactive antibodies that agglutinate the OXK strain of P mirabilis.

Control

Scrub typhus can be treated with doxycycline, tetracycline, or chloramphenicol. Chigger repellents may prevent exposure. Prophylaxis with weekly doses of doxycycline during and for 6 weeks after exposure protects against scrub typhus. Attempts to develop a safe, effective vaccine have failed.

Ehrlichia

According to the evolutionary scheme suggested by 16S rRNA sequence homology, ehrlichiae are genetically related to Rickettsia species. The genus Ehrlichia contains Gram-negative bacteria that reside in a cluster (morula) within membrane-bound cytoplasmic vacuoles of monocytes and macrophages, or polymorphonuclear leukocytes. Ehrlichiae have been implicated as the agents of diseases of horses (E risticii and E equi), dogs (E canis, E ewingii and E platys, a platelet pathogen), and other animals. Ehrlichia sennetsu causes a human disease in Japan resembling infectious mononucleosis. Ehrlichiae are unusual in their cell wall structure and they can establish persistent infections.

In 1987 the first case of human ehrlichiosis was reported in the United States. A severely ill man with multiorgan system involvement had morula inclusions demonstrated in peripheral blood leukocytes. Subsequently, cases of human monocytic ehrlichiosis have been documented mainly in eastern and southern states between New Jersey and Texas. The infection has varied from severe and sometimes fatal, mimicking Rocky Mountain spotted fever, to oligosymptomatic and asymptomatic forms. A history of tick bite and the seasonal and geographic occurrence correlate with the predominant tick vector, Amblyomma americanum. Illness is often accompanied by leukopenia, thrombocytopenia, and damage to the liver. Lesions include perivasculitis in the central nervous system, kidney, heart, and lungs and granulomas in the bone marrow and liver. Clinical diagnosis is difficult. Laboratory diagnosis by indirect fluorescence antibody assay or polymerase chain reaction is not widely available. Ehrlichia chaffeensis morulae are difficult to detect in peripheral blood leukocytes.

In 1994 another serious new infectious disease, human granulocytic ehrlichiosis, was reported. Ehrlichiae seen within morulae in neutrophils in smears of peripheral blood were identified as very closely related to E phagocytophila (a European tick-transmitted infection of sheep, cattle, goats, and deer) and E equi. The causative organism, like other granulocytic ehrlichiae, has never been cultivated. Human granulocytic ehrlichiosis has been associated with the deer tick, Ixodes scapularis, and thus is found as far north as Minnesota, Wisconsin, and New England. Laboratory diagnosis is practically achieved by visualizing morulae in neutrophils, as serology and polymerase chain reaction for the agent are presently research procedures. Sometimes fatal, human granulocytic ehrlichiosis, like E chaffeensis infection, can be treated effectively with doxycycline.

Coxiella burnetii and Q Fever

Coxiella burnetii is sufficiently different genetically from the other rickettsial agents that it is placed in a separate group. Unlike the other agents, it is very resistant to chemicals and dehydration. Additionally, its transmission to humans is by the aerosol route, although a tick vector is involved in spread of the bacteria among the reservoir animal hosts.

Clinical Manifestations

Q fever is a highly variable disease, ranging from asymptomatic infection to fatal chronic infective endocarditis (Fig. 38-6). Some patients develop an acute febrile disease that is a nonspecific influenza-like illness or an atypical pneumonia. Other patients are diagnosed after identification of granulomas in their liver or bone marrow. The most serious clinical conditions are chronic C burnetii infections, which may involve cardiac valves, the central nervous system, and bone.

Structure, Classification, and Antigenic Type

Coxiella burnetii is an obligately intracellular bacterium with some peculiar characteristics. It is small, generally 0.25 μm by 0.5 to 1.25 μm. However, there is considerable ultrastructural pleomorphism, including small- and large-cell variants and possible endospore-like forms, suggesting a hypothetical developmental cycle. Among rickettsiae, C burnetii is the most resistant to environmental conditions, is the only species that resides in the phagolysosome, is activated metabolically by low pH, and has a plasmid. The extensive metabolic capacity of C burnetii suggests that its obligate intracellular parasitism is a highly evolved state rather than a degenerate condition. The cell wall is typical of Gram-negative bacteria and contains peptidoglycan, proteins, and lipopolysaccharide. When propagated under laboratory conditions in embryonated eggs or cell culture, C burnetii undergoes phase variation analogous to the smooth to rough lipopolysaccharide variation of members of the Enterobacteriaceae. Phase I is the form found in nature and in human infections. The phase II variant contains truncated lipopolysaccharide, is avirulent, and is a poor vaccine.

Pathogenesis

Human Q fever follows inhalation of aerosol particles derived from heavily infected placentas of sheep, goats, cattle, and other mammals. Coxiella burnetii proliferates in the lungs, causing atypical pneumonia in some patients. Hematogenous spread occurs, particularly to the liver, bone marrow, and spleen. The disease varies widely in severity, including asymptomatic, acute, subacute, or chronic febrile disease, granulomatous liver disease, and chronic infection of the heart valves. The target cells are macrophages in the lungs, liver, bone marrow, spleen, heart valves, and other organs. Coxiella burnetii is phagocytosed by Kupffer cells and other macrophages and divides by binary fission within phagolysosomes (Fig. 38-3). Apparently it is minimally harmful to the infected macrophages. Different strains have genetic and phenotypic diversity. The lipopolysaccharides are relatively nonendotoxic. Host-mediated pathogenic mechanisms appear to be important, especially immune and inflammatory reactions, such as T-lymphocyte-mediated granuloma formation.

Epidemiology

Coxiella burnetii infects a wide variety of ticks, domestic livestock, and other wild and domestic mammals and birds throughout the world. Most human infections follow exposure to heavily infected birth products of sheep, goats, and cattle, as occurs on farms, in research laboratories, and in abattoirs. Coxiella burnetii is also shed in milk, urine, and feces of infected animals. Animals probably become infected by aerosol and by the bite of any of the 40 species of ticks that carry the organisms.

Diagnosis

Clinical diagnosis depends upon a high index of suspicion, careful evaluation of epidemiologic factors, and ultimately, confirmation by serologic testing. Although C burnetii can be isolated by inoculation of animals, embryonated hen eggs, and cell culture, very few laboratories undertake this biohazardous approach. Likewise, the diagnosis is seldom made by visualization of the organisms in infected tissues. Acute Q fever is diagnosed by demonstration of the development of antibodies to protein antigens of C burnetii phase II organisms. Chronic Q fever endocarditis is diagnosed by demonstration of a high titer of antibodies, particularly IgG and IgA, against the lipopolysaccharide antigens of C burnetii phase I organisms in patients with signs of endocarditis whose routine blood cultures contain no organisms.

Control

Antibiotic treatment is more successful in ameliorating acute, self-limited Q fever than in curing life-threatening chronic endocarditis. Reduction in exposure to these widespread organisms is difficult because some serologically screened animals that have no detectable antibodies to C burnetii still shed organisms at parturition. Persons with known occupational hazards (e.g., Australian abattoir workers) have benefitted from a vaccine composed of killed phase I organisms. This vaccine is not readily available, but offers promise for development of safe, effective immunization.

Bartonella

It has been recognized recently that organisms thought to be closely related to rickettsiae such as the louse-borne causative agent of trench fever, Bartonella (formerly Rochalimaea)quintana, in fact, belong in the genus Bartonella. These bacteria can be cultivated in cell-free medium and hence do not fit the criterion of definition of rickettsiae as obligately intracellular bacteria. Bartonella quintana infections were a serious medical problem during World War I. Soldiers in the trenches were infested with body lice that passed B quintana in their feces onto the skin. Individuals who have recovered from trench fever continue to have R quintana circulating in this stage of infection and may serve as sources of infection for lice, which can transmit the infection to others.

In association with the AIDS epidemic, another species B henselae (in addition to B quintana) has been discovered to be the cause of opportunistic infections often masquerading as hemangioma-like lesions of skin and visceral organs, bacillary angiomatosis. Bartonella henselae was recognized subsequently to be the long sought after cause of cat scratch disease, which usually manifested as a self-limited enlargement and inflammation of lymph nodes of several months duration in the regional drainage of a cat scratch or bite.

Bartonella bacilliformis transmitted by the sandfly in certain regions of Western South America invades human red blood cells, causing acute, often severe, hemolytic anemia. In chronic infections, there are skin lesions known as verruga peruana (Peruvian warts) that are similar to those of bacillary angiomatosis. A Peruvian medical student, Daniel Carrion, proved these lesions to be caused by an infectious agent in 1885 when he fatally inoculated himself with material from a verruga peruana. He died of the acute infectious hemolytic anemia known today as Oroya fever or, in his memory, Carrion's disease.

References

  1. Adal KA, Cockerell CJ, Petri WA. Cat scratch disease, bacillary angiomatosis, and other infections due to Rochalimaea. N Engl J Med. 1994;330:1509–1515. [PubMed: 8164704]

  2. Audy JR (ed): Red Mites and Typhus. University Press, New York, 1968 .

  3. Bakken JS, Dumler JS, Chen S-M. et al. Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? JAMA. 1994;272:212–218. [PubMed: 8022040]

  4. Brouqui P, Dumler JS, Raoult D. Immunohistologic demonstration of Coxiella burnetii in the valves of patients with Q fever endocarditis. Am J Med. 1994;97:451–453. [PubMed: 7977434]

  5. Drancourt M, Mainardi JL, Brouqui P. et al. Bartonella (Rochalimaea)quintana endocarditis in three homeless men. N Engl J Med. 1995;332:419–423. [PubMed: 7529894]

  6. Fishbein DB, Dawson JE, Robinson LE. Human ehrlichiosis in the United States, 1985 to 1990. Ann Int Med. 1994;120:736–743. [PubMed: 8147546]

  7. Hechemy KE, Paretsky D, Walker DH, Mallavia (eds): Rickettsiology: Current Issues and Perspectives. Vol 590. NY Acad Sci, New York, 1990 .

  8. Helmick CG, Bernard KW, D'Angelo LJ. Rocky Mountain spotted fever: clinical, laboratory and epidemiological features of 262 cases. J Infect Dis. 1984;150:480. [PubMed: 6491365]

  9. Kaplowitz LG, Fischer JJ, Sparling PF. Rocky Mountain spotted fever: a clinical dilemma. Curr Clin Top Infect Dis. 1981;2:89.

  10. Marrie TJ: Q Fever. Vol I. The Disease. CRC Press, Boca Raton, FL. 1990 .

  11. McDade JE. Natural history of Rickettsia rickettsii. Annu Rev Microbiol. 1986;40:287. [PubMed: 3096192]

  12. McDade JE, Shepard CC, Redus MA. et al. Evidence of Rickettsia prowazekii infections in the United States. Am J Trop Med Hyg. 1980;29:277. [PubMed: 6154428]

  13. Moulder JW (ed): Intracellular Parasitism. CRC Press, Boca Raton, FL. 1989 .

  14. Walker DH (ed): Biology of Rickettsial Disease. Vols I and II. CRC Press, Boca Raton, FL, 1988 .

  15. Williams WJ, Radulovic S, Dasch GA. et al. Identification of Rickettsia conorii infection by polymerase chain reaction in a soldier returning from Somalia. Clin Infec Dis. 1994;19:93–99. [PubMed: 7948564]

  16. Wolbach SB, Todd JL, Palfrey FW: Etiology and Pathology of Typhus. The League of Red Cross Societies Harvard Press, Cambridge, Mass, 1922 .

  17. Zinsser H (ed): Rats, Lice, and History: Little, Brown, New York, 1935 .

Molecular Typing of Isolates of Rickettsia rickettsii by Use of DNA Sequencing of Variable Intergenic Regions▿

ABSTRACT

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is found throughout the Americas, where it is associated with different animal reservoirs and tick vectors. No molecular typing system currently exists to allow for the robust differentiation of isolates of R. rickettsii. Analysis of eight completed genome sequences of rickettsial species revealed a high degree of sequence conservation within the coding regions of chromosomes in the genus. Intergenic regions between coding sequences should be under less selective pressure to maintain this conservation and thus should exhibit greater nucleotide polymorphisms. Utilizing these polymorphisms, we developed a molecular typing system that allows for the genetic differentiation of isolates of R. rickettsii. This typing system was applied to a collection of 38 different isolates collected from humans, animals, and tick vectors from different geographic locations. Serotypes 364D, from Dermacentor occidentalis ticks, and Hlp, from Haemaphysalis leporispalustris ticks, appear to be distinct genotypes that may not belong to the species R. rickettsii. We were also able to differentiate 36 historical isolates of R. rickettsii into three different phylogenetic clades containing seven different genotypes. This differentiation correlated well, but not perfectly, with the geographic origin and likely tick vectors associated with the isolates. The few apparent typing discrepancies found suggest that the molecular ecology of R. rickettsii needs more investigation.

In the late 1800s, reports first described a fatal febrile illness affecting settlers in the Bitterroot Valley of western Montana. Howard Taylor Ricketts showed 100 years ago that this disease, Rocky Mountain spotted fever (RMSF), was caused by a bacterium later named in his honor (41, 47). Despite the localized geographic association of its name, RMSF is found throughout the continental United States as well as in Central and South America (12, 13, 39, 42, 43). RMSF is the most commonly fatal tick-borne bacterial disease reported in the world, with the fatality rate for untreated cases approaching 20% (12, 13, 30). Common clinical features include fever, chills, headache, malaise, myalgia, and rash. Early diagnosis and antibiotic therapy are critical to avoid severe disease and to decrease the chance of a fatal outcome, with doxycycline being the drug of choice for treatment of RMSF (11, 12, 25).

The etiological agent of RMSF is the obligately intracellular bacterium Rickettsia rickettsii. The life cycle of this bacterium involves both vertebrate and invertebrate hosts, with hard (ixodid) ticks serving as the vector and both the ticks and their mammalian hosts serving as reservoirs to maintain the bacterium in nature (7). In the United States, RMSF is spread primarily by two different tick vectors, Dermacentor variabilis in the eastern and midwestern part of the country (5, 44) and Dermacentor andersoni in the northwestern part of the country (34, 35). The brown dog tick, Rhipicephalus sanguineus, was recently confirmed to be a competent vector for R. rickettsii (9, 10, 14), while the rabbit tick, Haemaphysalis leporispalustris, has been implicated as a vector for low-virulence strains of R. rickettsii (2, 3, 32, 35) known as serotype Hlp. In California, a limited number of spotted fever isolates of serotype 364D that are closely related to R. rickettsii have also been recovered from Dermacentor occidentalis ticks (27, 35). However, formal identification of the Hlp and 364D serotypes as R. rickettsii has not been completed. Dermacentor ticks are not found in Central and South America (19); in these regions, Amblyomma cajennense and Amblyomma aureolatum have been implicated in the transmission of RMSF (23, 37, 42).

A number of phenotypic and genetic differences have been observed previously among isolates of R. rickettsii. During his early work on the virulence of R. rickettsii, Price classified isolates recovered from D. andersoni ticks collected in Montana into four categories (R, S, T, and U) based on the different degrees of pathology observed in a guinea pig model of infection (38). An in vitro endothelial cell system for differentiating virulence properties of R. rickettsii has also been described (17). Anacker and colleagues also reported differences in the virulence of R. rickettsii isolates in the guinea pig model, grouping their isolates into three categories: isolates with highest virulence, lesser virulence, and lowest virulence (2, 3). They also observed differences in the one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of proteins recovered from whole-cell lysates of different isolates, but these differences allowed only for the differentiation of isolate Hlp (3). Philip et al. demonstrated antigenic differences between putative R. rickettsii isolates 364D and Hlp#2, but not other isolates, through the use of microimmunofluorescence with mouse immune sera (35). By AluI PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of almost identical nucleotide regions of rompA, Regnery et al. (40) (nucleotides 70 to 602) and Eremeeva et al. (18) (nucleotides 70 to 701) were able to differentiate Hlp#2 from a combined total of 17 R. rickettsii isolates. Both groups also used AluI PCR-RFLP analysis of the citrate synthase gene, gltA, and found that Montana isolates Bitterroot and Sheila Smith had slightly different banding patterns from those of R. rickettsii isolates from either the Central or Eastern United States or Central or South America (18, 40). Eremeeva et al. also reported that isolates Lost Horse Canyon and Morgan shared the same, less common gltA AluI PCR-RFLP banding pattern as isolates Bitterroot and Sheila Smith (18). In the same study, Eremeeva et al. also showed that isolate Hlp#2 contains a unique nine-base-pair insertion in the sequence of a fossil gene in comparison to other R. rickettsii isolates. Finally, Gilmore and Hackstadt (22) used HincII digestion of a 3.8-kb repeated region fragment of rompA to separate five R. rickettsii isolates into three groups.

In the public health setting, molecular typing of infectious agents is important for tracing their origin and spread in outbreak investigations, the detection of disseminated antibiotic-resistant strains in managed care facilities, the identification of hypervirulent strains, and monitoring failures in live vaccination programs (29). Molecular typing also allows for the study of bacterial population dynamics and may provide an improved understanding of the ecological niches occupied by specific pathogen types in the environment (24, 45). Molecular typing schemes based on the sequencing of intergenic regions (IGRs) have been developed for Rickettsia conorii and Rickettsia prowazekii (21, 49), and genetic typing of R. rickettsii based on variable-number-tandem-repeat loci was described recently (15, 18a, 48). In this work, we present an IGR typing scheme for R. rickettsii based on nucleotide polymorphisms found within six sites. This typing method was applied to a collection of 38 R. rickettsii isolates from human RMSF patients, animals, and ticks from different geographic locations.

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MATERIALS AND METHODS

R. rickettsii isolates and DNA preparation.R. rickettsii isolates (Table 1) were cultivated in Vero cells (strain C1008; green monkey kidney cells) as described elsewhere (17). DNAs for isolates Brazil-A, 84JG, Hlp#2-A, Bitterroot, Colombia, Lost Horse Canyon, Morgan, PriceT, and Sheila Smith were prepared by phenol-chloroform extraction from partially purified R. rickettsii cells as described previously (16). DNAs for the remaining isolates were isolated from infected cell cultures by using a QIAamp DNA Mini kit from QIAGEN (Valencia, CA). DNAs were eluted with AE buffer (QIAGEN) and stored at 4°C prior to analysis. Hlp#2-A and Hlp#2-B refer to two samples of isolate Hlp#2 with unique passage histories maintained by two different laboratories. Since the nucleotide sequences of Hlp#2-A and Hlp#2-B are identical for all loci tested, they are referred to jointly as isolate Hlp#2 for the remaining of this communication. Likewise, Brazil-A and Brazil-B are samples of isolate Brazil with differing passage histories. These samples are also identical and thus are referred to singularly as isolate Brazil. All 38 isolates had rOmpA 70p-602n gene fragments (15, 18, 40) compatible with their typing as R. rickettsii, although the nucleotide sequences of the Hlp#2 and 364D fragments each differ by three bases from the Sheila Smith sequence.

TABLE 1.

R. rickettsii isolates used in this study

PCR primer design.An automated preliminary annotation of the genomic sequence of R. rickettsii strain Sheila Smith (GenBank accession number AADJ00000000) was used to identify suitable IGRs. IGRs of 200 to 500 nucleotides were chosen for further studies, and the nucleotide sequences were compared to the homologous sequences of seven rickettsial species, including Rickettsia akari strain CWPP-Hartford, Rickettsia canadensis strain McKiel #2678, R. conorii strain Malish 7 (AE006914), Rickettsia felis strain California 2 (CP000053), R. prowazekii strain Madrid E (AJ235269), Rickettsia sibirica strain CWPP-246, and Rickettsia typhi strain Wilmington (AE017197), by using blastn (1). IGRs which showed nucleotide polymorphisms between the eight species of Rickettsia were then analyzed to assess if PCR primers could be designed to yield an amplicon that spanned the polymorphic region of the IGR and was between 200 and 500 base pairs in length (Table 2).

TABLE 2.

Primers used in this study

PCR amplification and sequencing.PCR amplification was carried out in 30-μl reaction mixes, using Taq PCR master mix kits from QIAGEN (Valencia, CA) according to the manufacturer's directions. Each reaction mix contained 2 μl of diluted template DNA and 20 picomoles of each primer. After a 5-min denaturation at 95°C, each reaction underwent 35 cycles of a 30-second denaturation at 95°C, a 30-second annealing incubation, and a 1-min extension at 68°C. This was followed by a final 10-min extension at 72°C. The PCR products were purified using a Wizard SV gel and PCR clean-up system (Promega, Madison, WI). One microliter of purified PCR product was sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions, using an ABI 3100 genetic analyzer (Applied Biosystems). Each PCR amplicon was sequenced in both directions.

DNA sequence manipulation and analysis.Sequencing reads were assembled using the SeqMerge program of the GCG software package (Accelrys, San Diego, CA). ClustalW alignments were created for each IGR by using MEGA3 (26). Prior to phylogenetic analysis, the polymorphic IGR sequences for each isolate were concatenated in the following order: spo0J-abcT1, RR0155-rpmB, RR0345-tolC, cspA-ksgA, RR1372-RR1373, and RR1240-tlc5. The nucleotide sequences of the homologous loci from R. conorii strain Malish 7 were obtained from the National Center for Biotechnology Information database and added to the analysis as an outgroup (GenBank accession no. AE006914) (28). PAUP*4.0 (46) was used to perform a maximum parsimony analysis of a ClustalW alignment of the concatenated sequences, and 1,000 bootstrap replicates were used to estimate the likelihood of the tree. TreeView was used to visualize the resulting phylogenetic tree (31).

Nucleotide sequence accession numbers.The nucleotide sequences for all IGR loci determined have been deposited in GenBank under the accession numbers listed in Table 2.

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RESULTS

An initial subset of five isolates was used to screen seven candidate variable IGRs for nucleotide polymorphisms. This subset consisted of isolates Bitterroot, Colombia, AZ-8, Hlp#2, and 364D. These isolates were chosen to provide samples from different geographic locations and vector associations to maximize the potential for detecting genetic diversity. Four loci (RR0155-rpmB, RR0345-tolC, cspA-ksgA, and RR1372-RR1373) exhibited genetic diversity during this screen and were chosen for further study. In addition, primer pairs designed by Fournier et al. for the molecular typing of R. conorii isolates (21) were analyzed for their usefulness in the typing of R. rickettsii isolates. These primers were used to amplify and sequence loci from eight isolates (AZ-8, Hlp#2, 364D, BSF Rab1, Hauke, Morgan, Panama2004, and PriceT), again chosen based on their geographic location, vector association, and the IGR sequence diversity detected using the initial primers. Of the 27 Fournier primers tested, two IGRs (spo0J-abcT1 and RR1240-tlc5) exhibited sufficient diversity during the sequencing screen to warrant further study, while amplicons were not obtained for all tested DNAs for six other IGR primer pairs (Table 2). Therefore, a total of 28 loci were screened (Table 2), of which 6 were chosen for sequencing from all 38 isolates and were included in the genotypic and phylogenetic analyses. Of the 22 IGR loci sequenced in the screen but not chosen for further study, 7 loci had no nucleotide differences among the tested isolates, 6 loci exhibited sequence differences only in both 364D and Hlp#2, 1 locus exhibited differences only in 364D, and 1 locus exhibited differences only in Hlp#2. The remaining seven loci tested showed various amounts of diversity, but the isolates grouped together as seen for the six chosen loci.

To ensure that no polymorphisms were due to mistakes in the reference genome sequence, all six selected loci were resequenced from isolate Sheila Smith; these sequences were identical to the Sheila Smith reference genome sequence in GenBank.

Analysis of spo0J-abcT1 IGR.Two single nucleotide polymorphisms (SNPs) and two types of insertions were identified in the amplicons generated with the spo0J-abcT1 primer pair (Fig. 1; Table 2). These genetic differences allowed for the separation of the isolates into four different genotypes (A1 to D1). Compared to the reference isolate Sheila Smith (genotype A1), all isolates except Sawtooth, Bitterroot, Lost Horse Canyon, and Morgan have a guanine-to-adenine transition at nucleotide 194 of the consensus amplicon, while isolate 364D (genotype C1) has a unique adenine-to-guanine transition at nucleotide 83 and a single base pair insertion at nucleotide 132. Isolate Hlp#2 (genotype D1) exhibited a unique seven-base-pair insertion (GTATAAA) at consensus nucleotides 132 to 138.

FIG. 1.

Genotypes of sequenced IGRs. Each table shows individual genotypes identified for each IGR. The numbers in parentheses indicate the number of isolates in each genotype. The total number of nucleotides in the consensus sequence of each IGR is shown under the table, and the nucleotide positions indicated are relative to the aligned consensus sequence. Deleted nucleotides are indicated with dashes.

Analysis of RR0155-rpmB IGR.Four SNPs and two insertion/deletion (indel) events, of two and seven nucleotides, were detected in the RR0155-rpmB amplicons (Fig. 1; Table 2), allowing for the identification of five distinct genotypes (A2 to E2). Compared to Sheila Smith (genotype A2), all isolates except Sawtooth, Bitterroot, Lost Horse Canyon, Morgan, Brazil, Colombia, Panama2004, and Costa Rica (genotypes A2 and B2) have an adenine-to-guanine transition at nucleotide 170. Only isolates Sheila Smith, Bitterroot, Lost Horse Canyon, Morgan, and Sawtooth (genotype A2) have a seven-nucleotide deletion (CCTTGTC; consensus nucleotides 81 to 87). Isolate 364D (genotype D2) has two unique SNPs, a cytosine-to-thymine transition at nucleotide 158 and a thymine-to-cytosine transition at nucleotide 192, and it shares a third SNP, a thymine-to-cytosine transition at nucleotide 216, with Hlp#2 (genotype E2). A deletion of nucleotides 65 and 66 is also only found in isolate 364D. All Central and South American isolates are identical to each other (genotype B2).

Analysis of RR0345-tolC IGR.The RR0345-tolC amplicon had only two SNPs (Fig. 1; Table 2), producing three genotypes (A3 to C3). The Hlp#2 amplicon (genotype C3) contains a thymine-to-guanine transversion at nucleotide 86, while isolate 364D has an adenosine-to-cytosine transversion at nucleotide 109 (genotype B3).

Analysis of the cspA-ksgA IGR.Four SNPs, a six-nucleotide indel, and a polymorphic polycytosine region were identified within the amplicons generated by the cspA-ksgA primer pair (Fig. 1; Table 2), resulting in the identification of genotypes A4 to G4. Compared to genotypes A4 to E4, the amplicon from Hlp#2 (genotype G4) has the following four SNPs: an adenosine-to-cytosine transversion of nucleotide 179, a thymine-to-cytosine transition of nucleotide 220, an adenosine-to-cytosine transversion of nucleotide 348, and a guanine-to-adenosine transition of nucleotide 357. The 364D amplicon (genotype F4) shares two of these SNPs with Hlp#2: the transversion of nucleotide 179 and the transition of nucleotide 220, but not the others. Amplicons from all isolates except Sheila Smith, Sawtooth, Bitterroot, Lost Horse Canyon, and Morgan (genotypes A4 and B4) have a six-nucleotide insertion (AATTAT; consensus nucleotides 255 to 260). A polycytosine region consisting of 8, 9, or 10 cytosine residues is found for consensus nucleotides 270 to 279 of this locus. Amplicons from genotypes B4 (Bitterroot, Lost Horse Canyon, and Morgan), C4 (Colombia and all eight Arizona isolates), and F4 (364D) have 8 cytosines, while amplicons from genotypes E4 (84JG, Hino, OSU 83-13-4, OSU 84-21c, 76RC, and Hauke) and G4 (Hlp#2) contain 10 consecutive cytosines. The remaining 19 amplicons (genotypes A4 and D4) all contain nine consecutive cytosines. Genotypes C4 and E4 were distinguished from genotype D4 (the largest group of isolates, which included all Central and South American strains and most Eastern and Midwestern U.S. strains) only by the number of cytosines in the polycytosine region.

Analysis of the RR1372-RR1373 IGR.Only two genotypes (A5 and B5) were present in the RR1372-RR1373 amplicons (Fig. 1; Table 2). Isolate 364D (genotype B5) contains a guanine-to-thymine transversion of nucleotide 36 and an adenosine-to-guanine transition of nucleotide 193.

Analysis of the RR1240-tlc5 IGR.Four SNPs and three indels were detected in the RR1240-tlc5 amplicons, resulting in genotypes A6 to E6 (Fig. 1; Table 2). The Hlp#2 amplicon (genotype E6) had a thymine-to-guanine transversion at nucleotide 88, while the 364D amplicon (genotype D6) had two unique SNPs, namely, cytosine-to-adenosine transversions at nucleotides 147 and 255. All isolates except those with genotype A6 (Sheila Smith, Sawtooth, Bitterroot, Lost Horse Canyon, and Morgan) have a three-nucleotide insertion at consensus nucleotides 69 to 71 (ATA). The isolates with genotype B6 (all Central and South American isolates examined) can be distinguished from the remaining isolates (genotype C6) by a guanine-to-thymine transversion of nucleotide 131 in genotype C6.

Phylogenetic analysis.When the sequences of all six loci were concatenated and compared, the 38 isolates could be separated into nine genotypes. Maximum parsimony analysis of the concatenated sequences revealed the presence of five phylogenetic clades. Isolates 364D (unique at six of six loci analyzed) and Hlp#2 (unique at five of six loci analyzed) have unique genotypes both with respect to each other and compared to the other R. rickettsii isolates and were thus separated with great reliability (Fig. 2). The largest clade, clade I, contains 27 isolates in three closely related genotypes, comprising the eight Arizona isolates, a small group of 6 isolates (84JG, Hino, OSU 83-13-4, OSU 84-21c, 76RC, and Hauke), and the remaining 13 isolates. Clade II contains two genotypes, comprised of isolates Sawtooth and Sheila Smith and isolates Bitterroot, Lost Horse Canyon, and Morgan. A related clade, clade III, is comprised of two genotypes from Central and South America, one with three isolates (Brazil, Costa Rica, and Panama2004) and the other with only one isolate (Colombia). The closely related genotypes in each of these well-separated clades were distinguished only by the number of cytosines in the polycytosine region of the cspA-ksgA amplicon.

FIG. 2.

Phylogenetic relationships of R. rickettsii isolates. Maximum parsimony phylogenetic relationships of R. rickettsii isolates are based on the concatenated sequences of all six intergenic regions. Numbers at the nodes are bootstrap values based on 1,000 bootstrap replicates. Only bootstrap values of >50 are shown. R. conorii Malish 7 represents the outgroup. The scale bar corresponds to the number of steps.

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DISCUSSION

Fournier et al. examined the sequences of 52 IGRs and found only 4 that showed polymorphisms among 38 isolates of R. conorii subsp. conorii (21). One of these IGRs failed to provide additional discriminatory power to the analysis and was excluded from the study. Utilizing the remaining three IGRs (15, 5, and 2 genotypes), Fournier and colleagues were able to identify 27 unique genotypes. Phylogenetic analysis of these sequences grouped the isolates into three clusters, two of which correlated well with the geographic distribution of the isolates. In 2004, Zhu et al. used the same technique to type isolates of R. prowazekii (49). After analyzing 25 IGRs, only 2 contained nucleotide differences at the isolate level. These two IGRs contained only two SNPs, and one isolate exhibited a single 81-nucleotide repeat not found in the other isolates. Using these differences, Zhu and colleagues were able to identify four genotypes in a collection of 15 isolates. Two clusters were formed by phylogenetic analysis; however, no correlation between the phylogenetic groupings and the epidemiological characteristics of the R. prowazekii isolates was reported. However, another genetic method providing robust geographic groupings for R. prowazekii was recently developed (13a).

In the present study, the genotypic analysis and the phylogenetic tree resulting from maximum parsimony analysis of the concatenated sequences of six IGRs divided the 38 isolates of R. rickettsii into nine unique genotypes and five phylogenetic clades. There is a strong correlation between the phylogenetic grouping of most of the isolates and their geographic origin (Fig. 2). The three largest clades with multiple isolates contain all 36 isolates classically identified as R. rickettsii by serotyping and genotyping of the GltA and rOmpA genes. The largest of these clades contains three genotypes: genotype 1 contains only those isolates collected during an RMSF outbreak associated with R. sanguineus ticks in Arizona, while genotypes 2 and 3 (except isolate PriceT, which is a D. andersoni isolate from Montana [38]) encompass isolates collected from the Midwestern and Eastern United States that are associated with D. variabilis. Genotype 2 and 3 isolates are not separated geographically, and both types include patient isolates from western states (Hino from Oklahoma and 80JC from Nebraska). The second clade includes two genotypes that include the Sheila Smith isolate and D. andersoni tick isolates originating from the Bitterroot Valley of Montana, as well as a patient isolate from North Carolina (Morgan). Central and South American isolates are found in two genotypes in clade III.

With two exceptions, the genotyping system developed appears to associate specific genotypes with the different tick vectors of R. rickettsii. Among the eight isolates collected during a recent outbreak of RMSF in southeastern Arizona (14), five were isolated directly from R. sanguineus ticks, including one (AZ-7) that was removed from a human, and the other three isolates were all recovered from clinical specimens taken from humans who suffered from RMSF and were associated epidemiologically with R. sanguineus (15). Of the 20 isolates placed into genotypes 2 and 3, 13 were isolated from human RMSF patients from Ohio, Virginia, North Carolina, Maryland, Georgia, Nebraska, and Oklahoma. Twelve of these are thought to be associated with the tick D. variabilis because of where the patients resided when they became infected. The records for one of these patients mention that one D. variabilis tick was removed from the patient in the hospital. The three R. rickettsii isolates collected from animals in Virginia (vole, opossum, and rabbit) also have the same association based on tick habitat ranges and the collection of D. variabilis ticks from these sites (14, 44). Three additional isolates of R. rickettsii in this cluster were isolated directly from D. variabilis ticks collected from three different counties in Ohio. Three of the five isolates found in the two genotypes of clade II were isolated from the primary vector of RMSF in the Northwestern United States, i.e., D. andersoni. However, D. andersoni is not found in North Carolina, where isolate Morgan was obtained (35). Similarly, the PriceT isolate recovered from D. andersoni in Montana (38) did not group with this cluster. A separate phylogenetic analysis of the same DNA stocks using analysis of variable-number-tandem-repeat loci also grouped PriceT and Morgan similarly to the IGR analysis (18a; Eremeeva, unpublished data). PCR-RFLP analysis of the GltA gene in 12 R. rickettsii isolates also grouped isolate Morgan with isolates Bitterroot, Sheila Smith, and Lost Horse Canyon (18). Thus, it appears that these clusters reflect the present fundamental genotypic characteristics of the isolate stocks available to us. At present, it cannot be excluded that isolates Morgan and PriceT may have been cross-contaminated with other isolates of R. rickettsii or mislabeled in the laboratory during their long passage and handling histories. It will be necessary to characterize additional new isolates of R. rickettsii, particularly from D. andersoni, to determine if other inconsistencies in vector-genotype associations exist. Clade III contains two samples with different passage histories of an isolate collected in Brazil prior to 1943, as well as isolates collected in Costa Rica, Colombia, and Panama. The Colombia and Panama2004 isolates are from patients, but the specific origins of the Costa Rica and Brazil isolates are less certain. D. andersoni and D. variabilis ticks are not found in Central and South America (19). It is thought that the most likely vector of R. rickettsii in these regions is a member of the genus Amblyomma, with most evidence pointing to A. cajennense or A. aureolatum (23, 37, 42).

Since the initial isolations of Hlp#2 and 364D, these isolates, particularly Hlp#2, have frequently been associated with the species R. rickettsii despite several known differences between them and prototypical R. rickettsii isolates (2, 3, 32, 35). In their original paper describing rickettsial isolates from the rabbit tick, Parker and coworkers recovered seven Hlp isolates, of which they refer to only two by name (group 2 and group 3) (32). They reported that all seven Hlp isolates acted similarly in guinea pigs. In subsequent studies, some authors refer only to using isolate Hlp obtained from Parker, while others refer to isolate Hlp#2. It is thought that most studies in fact used Hlp#2, but this cannot be confirmed. In their paper, Parker et al. noted that although the Hlp isolates appeared serologically identical and provided protective immunity to other R. rickettsii isolates, there was a marked difference in the virulence of Hlp types observed in the guinea pig infection model (32). Whereas an unidentified “laboratory strain of Rocky Mountain spotted fever rickettsiae” showed fever and scrotal pathology typical of R. rickettsii infection, the Hlp isolates showed increased incubation periods, increased duration and decreased degree of fever, and decreased scrotal involvement. Anacker et al. (3) obtained similar results and noted that Hlp was even less virulent in the guinea pig than established R. rickettsii isolates (Morgan and Simpson) with decreased virulence. In 2001, Eremeeva et al. examined the cytotoxic effects of rickettsial infection on human endothelial cells and found that Hlp#2 actually caused more cellular injury than did some typical isolates of R. rickettsii (17).

Limited information exists concerning the relationship between isolate 364D and other isolates of R. rickettsii. 364D was originally isolated from D. occidentalis in Ventura County, CA, in 1966 (35). Early serological work by Philip et al. determined that while it is closely related to R. rickettsii and Hlp#2, 364D is actually a different serotype from both (35). Later work showed that this isolate exhibits low virulence in a guinea pig model, has cytotoxicity in Vero cells, and kills chick embryos in 4 to 5 days after yolk sac inoculation (36). It has been suggested that 364D may also cause disease in humans, since analysis of convalescent-phase sera from patients in California thought to have RMSF showed specific antibodies to 364D serotype antigen (27).

Our data argue for a strong species or subspecies differentiation between Hlp#2 and 364D and between these two isolates and the other R. rickettsii isolates tested. Neither genotype has been isolated from patients manifesting symptoms of spotted fever. For five of the six loci analyzed for all isolates used in this study, Hlp#2 exhibits a genotype that is unique from all other isolates tested, while 364D exhibits a unique genotype for all six loci. There is high bootstrap support for an early divergence of both Hlp#2 and 364D from the other isolates (Fig. 2). When all 28 IGR loci were included, 364D had 15 unique sequences while Hlp#2 had 14 unique sequences. At three additional loci, 364D and Hlp#2 shared a sequence that was different from those of all classical R. rickettsii isolates tested. Additional genomic analysis of isolates of serotypes 364D and Hlp#2 is being done to determine if this strong differentiation of Hlp#2 and 364D isolates really indicates that they are novel subspecies lineages within the species R. rickettsii or if they should be reclassified as novel rickettsial species that are closely related to R. rickettsii (20). This distinction will be important because it will help to define whether these isolates are not isolates of R. rickettsii and do not need to be treated as select agents in the United States.

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ACKNOWLEDGMENTS

This research was supported in part by an appointment of S. Karpathy to the Emerging Infectious Diseases (EID) Fellowship Program administered by the Association of Public Health Laboratories (APHL) and funded by the Centers for Disease Control and Prevention (CDC).

The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

We thank the many investigators who isolated and contributed the isolates used in this investigation. We also thank Christopher Paddock for his critical review of the manuscript and his insightful comments.

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FOOTNOTES

    • Received 16 February 2007.
    • Accepted 11 May 2007.
  • ↵*Corresponding author. Mailing address: Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Mail Stop G-13, 1600 Clifton Rd. NE, Atlanta, GA 30333. Phone: (404) 639-4612. Fax: (404) 639-4436. E-mail: mge6{at}cdc.gov
  • ↵▿ Published ahead of print on 6 June 2007.

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