Tissue tropism and vertical transmission of Coxiella in Rhipicephalus sanguineus and Rhipicephalus turanicus ticks.

Arthropod symbionts present tissue tropism that corresponds to the nature of the association and the mode of transmission between host generations. In ticks, however, our knowledge of symbiont tissue tropism and function is limited. Here, we quantified and localized previously described Coxiella-like symbionts in several organs of the tick Rhipicephalus turanicus. Quantitative polymerase chain reaction revealed high densities of Coxiella in the female gonads, and both male and female Malpighian tubules. Using fluorescence in situ hybridization and transmission electron microscopy, we further showed that in the gonads of both Rh. turanicus and Rh. sanguineus, Coxiella does not colonize the primary oocytes but is found later in young and mature oocytes in a specific distribution, suggesting controlled vertical transmission. This method revealed the presence Coxiella in the distal part of the Malpighian tubules, suggesting a possible role in nitrogen metabolism. While testing Rickettsia symbionts, no specific tissue tropism was found, but a slightly higher densities in the tick gut. The low density of Rickettsia in the female ovaries suggests competition between Rickettsia and Coxiella for vertical transmission. The described tissue distribution supports an obligatory role for Coxiella in ticks.

Highly prevalent Coxiella sp. bacterium in the tick vector Amblyomma americanum.

Laboratory-reared and field-collected Amblyomma americanum ticks were hosts of a Coxiella sp. and a Rickettsia sp. While the Coxiella sp. was detected in 50 of 50 field-collected ticks, the Rickettsia sp. was absent from 32% of ticks. The Coxiella sp. showed evidence of a reduced genome and may be an obligate endosymbiont.

Coinfection of fibroblasts with Coxiella burnetti and Toxoplasma gondii: to each their own.

Intracellular pathogens have evolved distinct strategies to subvert host cell defenses. At diametrically opposed ends of the spectrum with regard to the host endosomal/lysosomal defenses are the obligate intracellular protozoan Toxoplasma gondii and the bacterium Coxiella burnetti. While the intracellular replication of T. gondii requires complete avoidance of the host endocytic cascade, C. burnetti actively subverts it. This results in these organisms establishing and growing in very different vacuolar compartments. In this study we examined the potential interaction between these distinct compartments following coinfection of mammalian fibroblasts. When present within the same cell, these organisms exhibit minimal interaction with each other. Colocalization of T. gondii and C. burnetti within the same vacuole occurs at a low frequency in doubly infected cells. In such instances only one of the organisms appears to be replication competent, emphasizing the different requirements for survival and/or intracellular growth. The potential basis for both the lack of interaction between these distinct pathogen-containing compartments, and the mechanisms to address their low frequency of colocalization are discussed in the context of our understanding of the biology of the organisms and membrane traffic in eukaryotic cells.

IFN-gamma-mediated control of Coxiella burnetii survival in monocytes: the role of cell apoptosis and TNF.

The treatment of infectious diseases caused by intracellular bacteria, such as Q fever, may benefit from cytokines acting on macrophages. Monocytic THP-1 cells were infected with Coxiella burnetii, the etiological agent of Q fever, and then treated with IFN-gamma. While C. burnetii multiplied in untreated monocytes, IFN-gamma reduced bacterial viability after 24 h of treatment and reached maximum inhibition after 96 h. IFN-gamma also affected the viability of infected cells. Cell death resulted from apoptosis; occurring 24 h after the addition of IFN-gamma, it reached a maximum after 48 h and was followed by necrosis. Reactive oxygen intermediates were not required for C. burnetii killing, since monocytes from patients with chronic granulomatous disease were microbicidal in response to IFN-gamma. The role of cytokines was also investigated. IFN-gamma elicited a moderate release of IL-1beta in infected monocytes. Moreover, the IL-1 receptor antagonist did not affect C. burnetii survival, suggesting that IL-1beta was not involved in the bacterial killing induced by IFN-gamma. TNF was involved in IFN-gamma-induced killing of C. burnetii and cell death. IFN-gamma induced mRNA expression and sustained secretion of TNF. Neutralizing Abs to TNF as well as Abs directed against TNF receptors I and II, significantly prevented IFN-gamma-dependent killing of C. burnetii and cell death. These results suggest that IFN-gamma promotes the killing of C. burnetii in monocytes through an apoptotic mechanism mediated in part by TNF.

Some contributions of electron microscopy to the study of the rickettsiae.

Electron microscopy has provided valuable insights into the study of rickettsiae as intracellular parasites from several important perspectives. This tool has allowed researchers to delineate the fine structural features of these organisms and to show that they truly resemble free-living bacteria. Furthermore, it has been shown that there are subtle, but distinct differences in the outer envelope structure of some members of the genus Rickettsia that may explain reported differences in tinctorial properties and in their sensitivity to certain antibiotics. With Coxiella burnetii, electron microscopy has helped significantly in the characterization of the pleomorphic nature of the organism including formation of terminal bodies that resemble endospores of gram-positive bacteria. Electron microsxopy has also helped to define the relationship of the rickettsiae to their host cells. For example, ultrastructural analysis can reveal whether organisms exist free within the cytoplasm or nucleus (members of the genus Rickettsia), or whether they are bound by a phagosomal or phagolysosomal membrane (Ehrlichia and Coxiella). Finally, although all rickettsiae eventually destroy their host cell, it has been shown through transmission electron microscopy that this destruction might be mediated by different mechanisms that are specific for different rickettsial species.

Interaction between Dermacentor reticulatus cells and Coxiella burnetii in vivo.

By electron microscopy the distribution of Coxiella burnetii was followed in females of Dermacentor reticulatus injected intracoelomally in a dose of about 10(3) EID50 per tick. The heaviest infestation with coxiellae was noticed in the cells of haemolymph, fat body, Malpighian tubulus and tracheal complex. No rickettsiae were found in Gene's organ. Unexpected was the propagation of rickettsiae in muscle fibres. C. burnetii multiplied in all organs affected. Heavy infection resulted at the marked damage of cell components. Coxiellae were evident in the haemocytes; in organs they formed small and large cell variants; endospore formations were observed free in the haemolymph.

Titration of Coxiella burnetii in Buffalo green monkey (BGM) cell cultures.

Titration of Coxiella burnetii, strain Nine Mile, phase I, and strain Frankfurt, phase II, in Buffalo green monkey (BGM) cover slip cell cultures yielded reproducible infectivity titers after centrifugation of infected cell cultures (3000 g, 30 min, 37 degrees C) and a subsequent 4-day incubation at 37 degrees C in Minimal Essential Medium (MEM). Compared to other titration procedures (plaque assays, titration in embryonated chicken eggs), this technique proved to be fast, less laborious and economical.

Red plaque formation of Coxiella burnetii and reduction assay by monoclonal antibodies.

A red plaque technique for C. burnetii which utilizes primary chicken embryo cells, is described. Red plaques could be consistently detected as early as 6 days, usually 8 days post inoculation (p.i.), reflecting that C. burnetii proliferated within the phagolysosomes of host cells. Incubation with phase II monoclonal antibodies or inactivated immune sera containing phase I and phase II antibodies or phase II antibodies only, markedly reduced phase II C. burnetii red plaques. On the other hand, red plaques from phase I organisms increased several times when phase I cells were mixed with phase I monoclonal antibodies or inactivated immune sera containing phase I and phase II antibodies. By indirect red plaque reduction assay red plaque production by phase II cells could be reduced as well.

Q fever pneumonia.

Pneumonia is one of several clinical syndromes that results from inhalation of Coxiella burnetii. This microorganism, the etiologic agent of "Q" (query) fever, infects a wide range of animals and insects. Cattle, sheep, goats, and cats are the reservoirs whereby this agent is spread to humans. High concentrations of C burnetii are present in the placenta and at parturition, the organism is shed into the environment to be inhaled by humans. Following an incubation period that ranges from four to 30 days (mean 14 days), fever, headache, malaise, and cough ensue. The clinical presentation of pneumonia may range from a mild to a severe illness--the latter with the clinical picture of rapidly progressive pneumonia. There are no characteristic features of Q fever pneumonia but the severe headache and the epidemiological history should serve as clues. Treatment with tetracycline or rifampin for two weeks usually results in cure. Many cases of Q fever pneumonia remit without antibiotic therapy. The diagnosis is usually confirmed serologically using a complement fixation or microimmunofluorescence test.

Immunofluorescence serology. A tool for prognosis of Q-fever.

The indirect immunofluorescence antibody test is currently the method of choice for Q-fever laboratory diagnosis. It permits the detection of IgG-, IgM-, and IgA-specific antibodies against the two phases of Coxiella burnetii. Sera from 20 cases of C. burnetii infection have been examined. Only total IgG against phase II were detected in cryptic infections. In acute Q-fever cases, the appearance of total IgG antibodies against phase I was a sign of aggravation, while IgM titers remained low. In subacute cases of Q-fever, anti-phase-I IgG titers were equal to or higher than anti-phase-II titers, and IgM against both phases were produced over a long time. Particularly high IgM titers were found in cases of granulomatous hepatitis. IgA antibodies against phase I were found in cases of Q-fever endocarditis, although the two cases that died had few or no IgA antibodies, despite very high IgG and IgM titers.

Interferon-gamma inhibits growth of Coxiella burnetii in mouse fibroblasts.

We studied the effects of various mouse interferon preparations on the growth of Coxiella burnetii in mouse fibroblasts. The addition of both recombinant interferon-gamma and a crude lymphokine preparation that contained interferon-gamma to infected L929 cells inhibited the growth of C. burnetii, whereas the addition of a crude preparation of type I interferons did not. Cycloheximide suppressed the inhibitory effects of recombinant interferon-gamma and crude lymphokines.

Monoclonal antibodies distinguish phase variants of Coxiella burnetii.

Monoclonal antibodies (MAbs) directed against phase I and II variants of Coxiella burnetii were produced by fusing myeloma SP2/O-AG 14 cells with spleen cells from mice immunized with the chloroform-methanol extraction residue of phase I whole cells. Two hybridoma clones which distinguished the phase variants by microimmunofluorescence assay were isolated and characterized. The MAbs showing specificity for phase I cells (MAbI-1, immunoglobulin G, subclass 3 kappa) reacted with the hot phenol-water extract of phase I C. burnetii in immunodiffusion and enzyme-linked immunosorbent assays. MAbI-1 reacted with high-molecular-weight components from phase I phenol-water extract and whole cell in an immunoblot assay. Specificity of MAbI-1 for a carbohydrate epitope in the phenol-water extract was demonstrated by periodic acid inactivation of binding by a competitive enzyme-linked immunosorbent assay. Phase I antigenic sites were apparently well represented on the surface of cells as demonstrated by complete fluorescence and microagglutination. The MAb showing specificity for phase II cells (MAbII-1, immunoglobulin G, subclass 2b kappa) reacted with whole cells in the microimmunofluorescence assay, microagglutination test, complement fixation test, and the enzyme-linked immunosorbent assay. MAbII-1 reacted specifically with a 29,500-dalton surface protein as demonstrated by immunoprecipitation of 125I-surface-labeled cells. Although MAbII-1 reacted with detergent-solubilized protein, it did not react with sodium-dodecyl sulfate-denatured protein by immunoblot assay. This protein was not exposed on the surface of phase I cells, but chloroform-methanol extraction of phase I cells exposed the phase II epitope.

The role of the house fly (Musca domestica L.) in the transmission of Coxiella burnetii.

The flies which are fed a single rickettsia suspension keep their infectivity throughout their life and can contaminate environment as long as that. Under conditions of experiment their life span is 32 days. Coxiella burnetii survives in the faeces of flies as long as 80 days, in the dead flies as long as 90 days.

Comparison of the ultrastructure of small dense forms of chlamydiae and Coxiella burnetii.

In the course of passaging of Coxiella burnetii (C.b.) in Alveonasus lahorensis ticks, the haemocytes contained cell forms with electrondense cytoplasm, intracytoplasmic lamellar membranes, and a peculiar limiting membrane--25 to 30 nm thick "envelope complex". Similar small forms occurred when C.b. had been cultured in the yolk sack of chick embryos. The dense forms of C.b. were similar to those of Rickettsiella cells. Dense forms (elementary bodies) surrounded by an "envelope complex" were found also in some chlamydiae cultured in yolk sacs of chick embryos.