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Chlamydiae

Introduction: The Chlamydiales

The Chlamydiales are bacteria that, like the Rickettsiales, are obligate intracellular parasites of eukaryotic cells.  Unlike the Rickettsiae, the Chlamydiales have a  distinctive developmental cycle for their replication. Members of the Chlamydiales share greater than 80% sequence identity for the gene encoding their 16S ribosomal rRNA (ribosomal ribonucleic acid) and/or greater than 80% identity for the gene encoding their 23S rRNA [Everett et al., 1999]. They are found within the cells of vertebrates and amoebae, while similar particles have  been reported in invertebrate species including coelenterates, arthropods and molluscs. Members of the Chlamydiales are trivially referred to as chlamydiae. Many chlamydiae coexist in an apparently asymptomatic state within hosts which probably act as a natural reservoir for them.

Descriptions of a "chlamydia-like" disease of human eyes resembling the disease now known as trachoma (meaning  'rough eye') have been found in ancient Chinese and Egyptian manuscripts. In 1907, Halberstaedter and von Prowazek, working in Java, described the transmission of trachoma from man to orang-utans by inoculating their eyes with conjunctival scrapings [see pictures below]. 

Fig 1: Title page of the original Halberstaedter von Prowazek paper of 1907, the first to describe the chlamydiae. Double click on the image. 

In Giemsa-stained conjunctival epithelial cells, they found intracytoplasmic vacuoles (chlamydial inclusions) containing numerous minute particles (small chlamydial elementary bodies (EBs) and larger chlamydial reticulate bodies (RBs)) which they correctly inferred  represented the causal agent of trachoma.

Fig 2: Halberstaedter and von Prowazek's drawings of a normal conjunctival epithelial cell (left), an infected cell (centre) and free chlamydial particles (right). 1907.

Some of the small particles (the infectious EBs) were also observed lying outside the cells (see figure above), a triumph of observational science as the 0.3 µm chlamydial EBs are only just above the ~0.25 µm resolution limit of the conventional light microscope.

The newly discovered organisms were called the Chlamydozoa ( from the Greek khlamus, a mantle / cloak) because of the blue-staining matrix in which the particles were apparently embedded [See: Collier 1990]. This was not correct, as the chlamydiae are not "mantled protozoans". However, the Greek-derived stem remains as a tribute to these outstanding workers.

Similar inclusions were subsequently described in the conjunctival cells of babies with non-gonococcal ophthalmia neonatorum in the uterine cervix from some of their mothers and in the urethral epithelium from male patients with non-gonococcal urethritis. Thus trachoma, inclusion conjunctivitis of the newborn and infection of the adult genital tract were caused by similar infective agents (now Chlamydia trachomatis) all of which were capable of passing filters that otherwise generally retained bacteria. This property, coupled with inability of these agents to grow in artificial media. led to the erroneous belief that these agents were viruses.

In 1929-30, widespread outbreaks occurred of an atypical and often severe pneumonia, acquired from psittacine birds (budgerigars, parrots etc), which was termed psittacosis. These outbreaks stimulated research, and Levinthal Coles and Lillie independently described minute basophilic particles in Giemsa stained blood and tissue from the infected birds and human patients. Bedson and co-workers soon proved the aetiological relationship of these particles with psittacosis and went on to define the characteristic developmental cycle that now defines all members of the order Chlamydiales. Serendipitously, Bedson referred to this agent (subsequently improperly referred to as Bedsoniae) as "an obligate intracellular parasite with bacterial affinities", a concept of great insight that was not generally accepted for another 30 years. 

As early as 1934, Thygeson, an ophthalmologist, had drawn attention to the resemblances between the development and morphology of the inclusions seen in trachoma and inclusion conjunctivitis and of those found in psittacosis. The finding of a common complement-fixing antigen strengthened the idea that these agents and those of lymphogranuloma venereum (LGV) and of mouse pneumonitis were related within the same unique group. By 1935 the psittacosis agent had been grown in the chick embryo chorio-allantoic membrane while the the LGV agent was  propagated first, in monkey brain, in1931.

The trachoma "virus" was first isolated in the chick-embryo yolk sac in China in 1957 by T'ang and colleagues and reported in the Chinese Medical Journal to considerable occidental incredulity [for a review of the contribution of T'ang and colleagues see: Wang, 1999].

The aetiological relationship of this organism with trachoma was proved in 1958 by the inoculation of human volunteers. Their aetiological role in inclusion conjunctivitis, and also in genital tract infection was confirmed by inoculating the eyes of volunteers and baboons.

Names for these bacteria included ‘Bedsonia’, ‘Miyagawanella’, 'Halprowia',  ornithosis-, TRIC- and PLT-agents. The term ‘Chlamydia’ (Gr. Chlamus cloak) appeared in the literature in 1945. That chlamydiae were not viruses became evident in 1965 with the advent of tissue culture techniques and of electron microscopy, when evidence for bacterial rRNA, ribosomes and cell wall structures in chlamydiae finally became overwhelming.  However chlamydiae were grouped with Rickettsia until the genus Chlamydia was validated by Page in 1966. For years, Chlamydiales was the only bacterial order that had just one family and one genus (Chlamydiaceae and Chlamydia, respectively). In the 1990s, with the introduction of new diagnostic methods, chlamydiae were described as emerging disease agents. DNA-DNA reassociation studies and subsequently gene sequencing lead to two new species in the late 90's, Chlamydia pneumoniae and Chlamydia pecorum [both now placed into the genus Chlamydophila, see below].  New molecular diagnostic methods based on nucleic acid amplification led to chlamydiae being discovered in tissues and cells never before reported (joints, atherosclerotic plaques, brains, amoebae) and associated with diseases of previously unknown aetiology (arthritis, Alzheimer disease, coronary artery disease, etc.) More than 40 chlamydial strains were deposited in the American Type Culture Collection, including type strains for all species and families. New environmental chlamydiae were also discovered across a wide range of animal phyla. The new molecular knowledge led to a new taxonomy of the order Chlamydiales [Everett et al., 1999], which split the former family Chlamydiaceae into two genera, Chlamydia and Chlamydophila [ the name Chlamydophila, given by Hans Truper and Johannes Storz, means  'like chlamydia'], encompassing nine species, and added three new non Chlamydiaceae families, the Parachlamydiaceae, Waddliaceae and Simkaniaceae [see: diagram of old versus new classification]. 

Relics of the remarkable history of the chlamydiae are found in the names still used to identify its developmental stages. An ‘elementary body’ (EB, a term borrowed from virologists in the middle of the twentieth century) is the electron-dense, infectious chlamydial form, typically 0.2–0.6 µm in diameter. EBs that have been endocytosed by eukaryotic cells remain in vacuolar ‘inclusions’ (a virology term), where they transform into ‘reticulate bodies’ (RBs; the intracellular dividing and replicating stage; the contents of each RB were ‘reticulated’, i.e. homogeneous). RBs may be up to 1.5 µm in diameter, take up nutrients from the host cell and undergo multiple rounds of binary division. EBs and RBs both have inner and outer membranes and a variable periplasmic space. During the characteristic chlamydial developmental cycle, both EBs and RBs are present in the inclusion, their differing appearances being evident when viewed using electron microscopy.

Two or more days after host cell infection, the RBs transform back into metabolically inactive (but infectious) EBs, which are released through host cell rupture or, possibly, by fusion of the inclusion/and host cell plasma membranes. Conceptually, EBs can be considered as environmentally resistant structures, adapted to survive in  hostile (to chlamydiae) extracellular space in order to transmit infection to new host cells following their attachment and endocytosis. Chlamydiae are spread by aerosol or by contact and require no alternate vector, although flies may help transfer ocular infection in trachoma endemic areas. 

For the early historical literature references see Collier, L. H. (1990).

[KDE & MEW] November 2002

NEXT: Diagram of the old versus the new classification of the Chlamydiales.

Reference

Collier, L. H. (1990). Chlamydia. In: Topley and Wilson's Principles of Bacteriology, Virology and Immunity. 8th edition. Published Edward Arnold, London, pp 629 - 646.

Everett, K. D. E., Bush, R. M. & Andersen, A. A. (1999). Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. International Journal of Systematic and Evolutionary Microbiology 49, 415 - 440.  

Wang, Y. (1999). Etiology of trachoma: a great success in isolating and cultivating Chlamydia trachomatis. Chinese Medical Journal 112, 938 - 941. [In English].