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Some isolates of Chlamydia pecorum from sheep feces failed to produce inclusions on passage in cycloheximide-treated monolayers, but chlamydiae could be recovered several weeks later. Chlamydia psittaci from sheep abortions did not show this phenomenon.

The effects of suspension media and rate of freezing on the survival of Chlamydia trachomatis LGV2 and Chlamydia pneumoniae after lyophilization were assessed. The highest loss in infectious elementary bodies (EBs) occurred during lyophilization. The survival was higher after freezing at a rate of 1 degree C min-1 and lyophilization than that after rapid freezing at -70 degrees C or -196 degrees C. The recovery (+/- 5%) was higher when fetal calf serum (FCS) containing glucose, saccharose or lactose were used as lyophilization media than that (0.5-3%) when yolk-sac, skimmed milk or phosphate buffer containing sucrose, glutamine and 10% FCS (SPG) were used. After lyophilization, the survival was not affected in the tested range from 10(4) to 5 x 10(6) inclusion-forming units (ifu) ml-1 prior to freezing. After storage for 4 months at 4 degrees C, the numbers of ifu of both Chlamydia serovars that were recovered were identical to the numbers of ifu immediately after lyophilization. It was concluded that chlamydias can be stored and transported in lyophilized form. However, a loss of 95% in infectious EBs should be taken into account.

Chlamydia is an obligate intracellular bacterial pathogen that severely challenges the patience and creativity of all its investigators--even to the point that some investigators have forsaken this field for more productive and fertile areas of research. The two principal difficulties that touch every aspect of chlamydial research are (a) that chlamydiae only grow within eukaryotic host cells and (b) there are limited genetic approaches available. Despite these technical difficulties, the fundamental underlying problem has been the expectation that chlamydiae are similar to other bacteria (or, historically, viruses) and amenable to study from this perspective. However, this has often turned out not to be the case. Chlamydiae have shown themselves to be unique at many levels and thus represent a formidable, yet enticing, research challenge.

Chlamydiae are Gram-negative bacteria with obligate intracellular reproduction and disability to synthesize high-energy compounds such as ATP. Their cycle of development is unique among the prokaryotes: the host cells, mainly epithelial cells, are infected by so-called elementary bodies (EB) which undergo reorganization to form metabolically active reticulate bodies (RB). These RB multiply by binary fission, and after transition into infectious EB they are released within 48-72 hours. Chlamydiae cause prolonged subclinical infections of the conjunctiva, lung, cervix, and urethra. Complications in newborns are inclusion conjunctivitis, nasopharyngitis and pneumonia; in females, salpingitis, infertility, and perihepatitis; in male patients, epididymitis and prostatitis; and in both sexes, Chlamydiae-induced arthritis. Identification of the pathogenic agent confirms clinical diagnosis; tissue culture identification remains the diagnostic method of choice. Therapeutical drugs are tetracycline, erythromycin, josamycin, and in certain cases quinolone derivatives.

To examine the possible role of lipids as adhesion receptors for infection, Chlamydia trachomatis and Chlamydia pneumoniae were labeled with 125I and layered on thin-layer chromatograms (tlc) of separated lipids isolated from target cells, and bound bacteria were detected by autoradiography. Elementary bodies from both species bound specifically and with high affinity to one lipid in HeLa 229 cells. Purification of this receptor by column chromatography on DEAE Sepharose followed by continuous preparative tlc, and structural analysis by 500-MHz 1H-NMR spectroscopy and fast atom bombardment mass spectrometry confirmed the HeLa cell chlamydial receptor to be phosphatidylethanolamine (PE). The chlamydiae also bound strongly to purified asialo-GM1 and asialo-GM2, but not to other neutral or acidic lipids tested. The relative binding of chlamydiae to human PE and asialo-GM1 was modified in the presence divalent cations, suggesting that chlamydiae have two interrelated receptor binding sites.

The obligately intracellular bacteria of the genus Chlamydia, which is only remotely related to other eubacterial genera, cause many diseases of humans, nonhuman mammals, and birds. Interaction of chlamydiae with host cells in vitro has been studied as a model of infection in natural hosts and as an example of the adaptation of an organism to an unusual environment, the inside of another living cell. Among the novel adaptations made by chlamydiae have been the substitution of disulfide-bond-cross-linked polypeptides for peptidoglycans and the use of host-generated nucleotide triphosphates as sources of metabolic energy. The effect of contact between chlamydiae and host cells in culture varies from no effect at all to rapid destruction of either chlamydiae or host cells. When successful infection occurs, it is usually followed by production of large numbers of progeny and destruction of host cells. However, host cells containing chlamydiae sometimes continue to divide, with or without overt signs of infection, and chlamydiae may persist indefinitely in cell cultures. Some of the many factors that influence the outcome of chlamydia-host cell interaction are kind of chlamydiae, kind of host cells, mode of chlamydial entry, nutritional adequacy of the culture medium, presence of antimicrobial agents, and presence of immune cells and soluble immune factors. General characteristics of chlamydial multiplication in cells of their natural hosts are reproduced in established cell lines, but reproduction in vitro of the subtle differences in chlamydial behavior responsible for the individuality of the different chlamydial diseases will require better in vitro models.

The means by which pathogens suppress, subvert, or elude host defenses and establish latent infections include microbially induced immunosuppression or antigenic variation, gaining access to sites of the body that are inaccessible to the immune system, and manipulating of the immune response to the advantage of the pathogen. Various risk factors of the host, such as immunosuppression, may be crucial in determining the frequency with which latency is the outcome of primary infection, as well as the likelihood that subsequent reactivation occurs. Mechanisms of reactivation of latent infections may also be triggered by disruption of anatomic or ecologic barriers, or through the cooperative efforts of a second pathogen. Although viruses, due to their unique ability to incorporate their genetic material into host genomes, are best known for their capacity for persistence, examples of latency can be found among all classes of microorganisms. The clinical and epidemiological importance of microbial latency is enormous, because such infections represent potential reservoirs from which dissemination of pathogens to new susceptibles can occur, because they may reactivate to cause acute or chronic progressive disorders in the original host, and because such infections might play a role in the origin of some human cancers. Few areas of basic research hold greater promise of substantially contributing to our understanding of infectious diseases and the eventual relief of human suffering.

Urine from a patient after kidney transplantation added to a human embryonic lung culture caused the appearance of dark spots shown to contain cells with phages resembling group A, as well as bodies resembling Chlamydiae.

Ultrastructural studies of the entry of Chlamydia strain TWAR into HeLa cells showed that the elementary bodies first attach to host cells by the pointed end, secure other binding sites on the host cells by forming cell wall protrusions, enter host cells by invaginating the host cell membrane, and form vacuolated endocytic vesicles. Differences were demonstrated between TWAR and other chlamydiae in the mode of attachment and endocytosis.

The genus Chlamydia consists of two species, Chlamydia trachomatis and C. psittaci. The former includes (a) the trachoma/inclusion conjunctivitis (TRIC) agents, subdivided into the serotypes A-K; (b) the lymphogranuloma venereum (LGV) agents, subdivided into the serogroups (L 1-3); and (c) the mouse penumonitis agent. The major characteristics differentiating the two species are sulfonamide susceptibility and the formation of distinct inclusion granula in host cells, in the case of C. trachomatis, whereas C. psittaci is resistant to sulfonamides and forms less dense inclusions. Chlamydiae are characterized by a special growth cycle. The infective form of Chlamydia is the elementary body (EB), which stimulates its own phagocytosis by the host cell, an event followed by the EB's transformation into the reticulate body (RB). The RB forms small buds which subdivide by simple fission to produce several RB within the host cell cytoplasm. The growth cycle is completed by transformation of the RB into the infective EB from before liberation of the latter from the host cell. The chlamydiae are deficient in independent energy metabolism. Thus, their supply of ATP and essential building blocks must be obtained from the host cell cytoplasm. The TRIC agents have neuraminidase localized to the surface structures. Receptors of the microbe are temperature-sensitive, whereas the host cell is trypsin-sensitive.

Living cells and their intracellular parasites show many of the characteristics ascribed to extreme environments and their dominant species. The diversity of species colonizing intracellular habitats is low, and successful inhabitants exhibit special fitness traits that often render them obligately dependent on residence within a host cell. However, the diversity-limiting factor in the extreme environment of the host cell interior is not abiotic, as it is in conventional extreme environments. It is biotic: the living cell itself and its many activities. Host cells bar the entrance to most would-be parasites, they destroy most of those that do manage to get inside, and they deny parasites free access to many components of their soluble metabolite pools. Successful intracellular parasites have evolved fitness traits that give them the capacity to survive in the face of diversity-limiting factors or to modify the intracellular habitat so that those factors no longer operate. Looking on the cell as an extreme habitat emphasizes its simultaneous roles as environment, antagonist, and competitor.

Chlamydiae are small bacteria that have a unique life cycle. There are two species, Chlamydia psittaci and C. trachomatis, which cause a wide spectrum of clinical disease, including neonatal conjunctivitis and pneumonia, sexually transmitted disease, psittacosis, and trachoma. The importance of chlamydial disease in public health is being increasingly recognized, and the incidence in developed countries seems to be increasing. An understanding of chlamydial disease, its prevention and treatment, is essential for the infection control practitioner, who can play a significant role in patient education.

Chlamydiales are bacteries showing a growth cycle unique among procaryotes. The two species Chlamydia trachomatis and Chlamydia psittaci are genetically very distant and their pathogenicity for man is very distinct. Human chlamydia infections by Chlamydia trachomatis are diseases chiefly sexually transmitted and their epidemiological importance is growing. The relationship between chlamydial infections, Reiter disease, and cat scratch disease are discussed. The various laboratory diagnostic procedures are reported, including the techniques and their indications; the method of choice is in the majority of cases the isolation of Chlamydia on cell culture.

During half a century, the agent of trachoma could be mainly demonstrated by inoculation to the conjunctiva of animals; by this mean the cycle of the agent could be revealed. There was a huge progress when T'ANG for these studies inoculated embryonated chicken eggs. However, experimentally infected animals are used at present time not only in trachome countries where do not exist laboratories: monkeys, guinea pigs, rabbits, rats and mice allow modern studies of chlamydial infection. Monkeys living in the countries where trachoma is endemic were selected because of their cheapness (orangoutan in Java, macaques in Northern Africa and in Taïwan, baboons in Africa). The monkeys selected by American workers are coming from South America. First pioneers (NICOLLE, CUENOD and NATAF, PAGES, JULIANELLE) have demonstrated the infectivity of animals and the place of the agent of trachoma on taxonomic point of view. As PAGES, we have demonstrated that infection could be regularly provoked when inoculating macaques; moreover a pannus could appear when adding hydrocortison drops or when infiltrating the cornea with tuberculin. Cultures of WEEKS bacilli were introduced in the eyes of trachomatous animals; we could observe an aggravation of the disease. If biology of trachoma is better known at present time, experimental trachoma is until now fundamentally important. It permits immunological studies especially for the purpose of vaccination; one can check terapeutical means for instance antibiotics; studies are performed to demonstrate cross immunizations or enhancement of defence (with levamisole). Experimental trachoma is hitherto and again for a long time commonly requested for the study of trachoma.

The effects of Chlamydia trachomatis (serovars E, K, and L2) on human neutrophil activation were examined with respect to the organisms both as primary agonists and as agents that modulate cell responses to a second stimulus. Unopsonized chlamydiae alone, at ratios of 1.5 to 100 organisms per cell, failed to elicit changes in intracellular calcium or membrane depolarization or to stimulate the respiratory burst or degranulation during 60 min of incubation. Each of these functions except the respiratory burst was also normally activated when chlamydia-infected neutrophils were subsequently stimulated with formylmethionyl leucine phenylalanine or phorbol myristate acetate; the respiratory burst was inhibited 30 to 65%. Inhibition was dependent on live organisms and was maximal within 5 min of incubation. The organisms had no effect on the superoxide (O2-) assay, and the site of chlamydial inhibition was determined at the level of the NADPH oxidase itself, not at an intermediate step in the activation cascade. The mechanism of enzyme inactivation could not be determined. These results show that unopsonized chlamydiae do not elicit responses from infected neutrophils and suggest that microbicidal mechanisms other than those dependent on elaboration of toxic oxygen-derived species are required to inactivate chlamydiae.