Pathogenic rickettsiae: An overview

▶The History of Discovery

The first description of pathogenic rickettsiae was made in 1910 by the American scientists H. Ricketts and R. Wilder, who revealed small oval-shaped bacteria both in blood of patients with Mexican typhus and in lice, inhabiting patient’s body. Later in 1913, the Czech microbiologist S. Prowazek discovered similar agents in blood of patients with typhus fever.

Finally, in 1916 the Brazilian pathologist H. da Rocha-Lima, who has been working in close collaboration with S. Prowazek, thoroughly investigated the newly discovered bacteriae, revised all collected data and finally established these organisms as the causative agents of epidemic typhus. He termed them Rickettsia provazekii in honor of H. Ricketts and S. Prowazek, who both died, investigating this life-threatening disease.

Further it was found that rickettsioses comprise a numerous group of arthropod-borne diseases. Today they are regarded as the emerging diseases because of 19 currently known rickettsioses more than 10 were discovered in last 20-25 years.

▶Modern Classification of Rickettsiae

Until quite recently the family Rickettsiaceae encompassed a great variety of pathogenic bacteria. Later many of them were organized into separate taxa, which became the members of new bacterial families and even classes.

According to current classification the order Rickettsiales comprises two families with pathogenic bacterial representatives: Rickettsiaceae and Anaplasmataceae.

The family Rickettsiaceae encompasses the genus Rickettsia (with human and animal pathogenic species R. prowazekii, R. typhi, R. conorii, R. sibirica, R. akari and many others) and genus Orientia (species O. tsutsugamushi).

The family Anaplasmataceae includes the genera Ehrlichia, Anaplasma, Neorickettsia, and Wolbachia.

Only the members of the first family are regarded now as the causative agents of rickettsioses.

Former representatives of Rickettsiales order, namely bartonellae and coxiellae, are placed now into the same name new families, Bartonellaceae and Coxiellaceae.

The main rickettsial pathogens and their diseases are listed in table

Pathogenic species from the family Anaplasmataceae pertain to genera Anaplasma and Erlichia.

Anaplasma phagocytophylum causes human granulocytic anaplasmosis; Erlichia chaffeensis – human monocytic ehrlichiosis, Erlichia ewingii – human granulocytic ehrlichiosis.

▶Structure and Properties of Rickettsiae

  • Morphology

Rickettsiae are pleomorphic gram-negative bacteria. Coccoid forms are about 0.5 μm in size. Rod-shaped rickettsiae also demonstrate substantial polymorphism; short organisms of 1 by 1.5 μm as well as long or curved thin rods 3-4 μm in size occur. The thread-like or filamentous forms are up to 40 μm in length.

Rickettsiae are non-motile bacteria; they don’t contain spores and capsules. R. provazekii may produce capsule-like substance.

These bacteria are visualized by Romanowsky-Giemsa stain, Gimenez stain (applies fuchsin and malachite green dye for counterstain) and by modified Ziehl-Neelsen stain (Zdrodovsky method).

Rickettsial genome is composed of small single circular chromosome 1,000-1,600 kb in size.

  • Cultivation

All rickettsiae are obilgate intracellular parasites.

Typhus group (TG) rickettsiae are localized exclusively in the cytoplasm of affected cells, while spotted fever group (SFG) rickettsiae can invade the cell nuclei, as they possess intracellular motility due to cellular actin polymerization.

For primary isolation various cell cultures both of tick and mammalian origin are used. Bacterial generation time is about 8-10 hours. Less fastidious is the cultivation of rickettsiae in yolk sacs of embryonated eggs. For animal inoculation guinea pigs, rats and mice are used.

  • Biochemical properties

Rickettsiae metabolism largely depends on cellular biochemical pathways, e.g., the bacteria can’t synthesize proteins. For energy gain they possess the enzymes ATP translocases that deliver ATP molecules directly from the infected cells. Also rickettsiae may acquire ATP by oxidative phosphorylation.

Rickettsiae are capable of producing gram-negative cell wall that is composed of peptidoglycan with muramic and diaminopimelic acids.

  • Antigenic structure

As an example, R. provazekii contains specific superficial protein antigens – outer membrane proteins OmpA and OmpB, and cell wall heat stable polysaccharide antigen, common for R. provazekii, R. typhi and certain strains of enterobacterial member, Proteus OХ-19.

  • Virulence factors

Rickettsiae contain polysaccharide heat stable endotoxin and heat labile protein toxic substance tightly associated with microbial body. The latter can be transformed into toxoid by formaldehyde treatment.

In the course of infection the main deleterious effects of rickettsiae are related with their active propagation inside the infected cells, followed by cell destruction and severe inflammatory response.

  • Resistance

In normal conditions rickettsiae can survive only in the body of infected host, vector or microbial reservoir. They rapidly lose viability in the natural environment. Dried bacteria usually stay viable for about 5-6 days.

Treatment with ordinary disinfectants and as well as heating at 80oС destroys rickettsiae within several minutes, heating at 100oС cause immediate microbial death.

  • Pathogenesis and Clinical Findings in Rickettsioses

Rickettsial diseases of the same clinical group show certain similarity in pathogenesis and clinical features. All rickettsioses are characterized by high fever, skin rashes, and generalized vasculitis.

Most severe disorder, epidemic typhus, is caused by R. provazekii and transmitted by body louse.

Epidemic typhus is the anthroponotic disease, which follows social disasters (wars, starvation, socioeconomic disorganization with substantial lack of hygienic conditions, etc.) It is believed that epidemic typhus has caused even more deaths than all the wars in human history.

Humans are the main sources of infection. The patients are contagious at fever period and within the week after. The persons, recovered from typhus, retain some viable bacteria for the whole life, thus maintaining the persistent infection.

Feeding louse becomes infected and within 4-5 days can spread the disease. Nevertheless, lice are lack of transovarial transmission of rickettsiae.

Incubation period of epidemic typhus is about 2 weeks (6 to 24 days) after primary inoculation.

Humans acquire infection rubbing louse excrements containing rickettsiae into injured skin after louse bite.

Rickettsiae multiply primarily in the site of penetration. Then they reach regional lymph nodes and enter the bloodstream, where the bacteria invade endothelial cells. Rickettsiae propagate within cytoplasm of endotheliocytes thereby causing their damage and lysis. The progressing endovasculitis is manifested by high fever, generalized roseolous-petechial skin rashes, myalgias, pneumonias and severe disorders of central nervous system with headaches, brain sinus thromboses and mental abnormalities (status typhosus). Disseminated intravascular coagulation (DIC) may occur.

When untreated, the disease fatality is about 10-30% at the peak of the infection.

During convalescence the growing specific antibodies eliminate bacteria. The disease confers long-lasting immunity.

However, rickettsiae can stay viable within phagocytes for many years, thus provoking the relapse of epidemic typhus (or Brill-Zinsser disease) in elderly persons.

Endemic or murine typhus is similar but much milder zoonotic rickettsial disease, caused by R. typhi. Ubiquitous rats and mice are the main reservoirs and sources of this infection in nature. The disease is transmitted by various arthropod vectors (several flea species, lice, mites, and ticks). In fleas transovarial transmission is possible.

Occasional disease acquisition by humans occurs via contamination of the injured skin, conjunctivae or respiratory tract by aerosols with infectious material, e.g. infected flea feces. Patients can develop fever, headaches, and rash.

The infection confers a relatively stable immunity, cross-reactive with epidemic typhus agent.

Scrub typhus is caused by Orientia tsutsugamishi, and transmitted by mites of genus Trombicula. It is found in Asia, including India and Japan, and in northern Australia.

The disease is generally similar with epidemic typhus, but the patients reveal the eschar in the primary site of mite bite followed by progressing lymphadenopathy and lymphocytosis.

Rickettsioses from the spotted fever group comprise a large number of fever diseases. Among them are Rocky Mountain spotted fever (RMSF), caused by R. rickettsii, Mediterranean spotted fever (MSF), produced by R. conorii, Siberian tick typhus (by R. sibirica), rickettsialpox (by R. akari) and several newly described diseases such as Japanese fever (by R. japonica), Astrakhan fever (Astrakhan fever rickettsia), African tick bite fever (R. africae), Israeli spotted fever (Israeli tick typhus rickettsia) and some others.

Spotted fevers are largely endemic diseases, transmitted by numerous arthropod species (ticks, mites, fleas, etc).

Rodents are the main sources of infection.

These disorders are generally characterized by fever, headache, rash, and eschar; the latter appears in the most of the diseases.

Particular diseases (e.g., RMSF) can cause large local outbreaks with high lethality (>30-40%).

▶Laboratory Diagnosis of Epidemic Typhus and Other Rickettsioses

Specimens are collected from patient’s blood, tissue biopsies and autopsy material. Serum is taken for serological tests.

Serological tests are most available for laboratory diagnosis of epidemic typhus and other rickettsioses. Immune fluorescent technique, complement fixation test and ELISA are commonly used. Diagnostic titer of antibodies in epidemic typhus determined by complement fixation test is 1/160 and higher.

By means of serological methods the differential diagnosis between primary epidemic typhus and relapsing Brill-Zinsser disease is possible. The reaction is made with two samples of titrated patient serum, where one sample was previously treated with potent reductive agent, e.g. cystein. Primary epidemic typhus is characterized by high levels of specific serum IgM that are noticeably susceptible to chemical reduction because of large cystin content. Fall of serum antibody titer verifies the presence of specific IgM and confirms the diagnosis of primary epidemic typhus whereas the lack of changes in antibody titers reveals the presence of IgG class antibodies testifying the diagnosis of epidemic typhus relapse, or Brill-Zinsser disease.

Direct detection of rickettsiae in biopsy specimens and arthropod material is possible by immunofluorescence or molecular tests, e.g. PCR.

For culture the specimens are inoculated into yolk sacs of embryonated eggs, various cell lines of tick or mammalian origin, or into susceptible animals, e.g. guinea pigs, mice, etc. Isolation is made only in reference laboratories with appropriate biosafety level.

Inoculation of male guinea pigs with patient’s blood makes it possible to discriminate endemic typhus from epidemic disease. Once infected with R. typhi, animals display specific periorchitis (the scrotal swelling) due to rickettsial propagation in the coats of guinea pig testis.

▶Specific Prophylaxis and Treatment of Epidemic Typhus and Other Rickettsioses

A single dose of 200 mg of doxycycline is effective for prevention of epidemic fever, thus any suspected case should be treated immediately. Fluoroquinolones are administered in spotted fevers. Treatment with chloramphenicol and macrolides is also possible.

Nevertheless, rickettsiae have intrinsic resistance to β-lactams and aminoglycosides and low sensitivity to sulfonamides.

Various live, formaldehyde-treated, and chemical vaccines are used for specific prophylaxis of epidemic typhus and other rickettsioses in the centres of disease outbreak or epidemic.