Construction of chimeric phagosomes that shelter Mycobacterium avium and Coxiella burnetii (phase II) in doubly infected mouse macrophages: an ultrastructural study.

Dual infection of cells may divert pathogens to intracellular compartments different from those occupied in mono-infected cells. In the present studies, mouse bone marrow in vitro-derived macrophages were first infected with virulent Mycobacterium avium, which are normally singly lodged within tight phagosomes. These phagosomes do not mature; they undergo homotypic fusion with early endosomes and do not fuse with lysosomes. Seven days later, the cultures were superinfected with phase II (non-virulent) Coxiella burnetii, organisms sheltered in lysosome- (or prelysosome)-like, multi-occupancy phagosomes. The latter can attain large size and engage in efficient homo- and heterotypic fusion with other phagosomes. Cultures were fixed for transmission electron microscopy 6, 12, 24, and 48 h later. Other M. avium-infected cultures were superinfected with amastigotes of the trypanosomatid flagellate Leishmania amazonensis, which are also sheltered in lysosome- (or prelysosome)-like multi-occupancy vacuoles, and fixed at the same time periods. Chimeric phagosomes containing both M. avium and C. burnetii, were found already at 6 h and the proportion of M. avium that colocalized with C. burnetii in the same phagosomes reached over 90% after 48 h. In such phagosomes, both organisms were ultrastructurally well preserved. In contrast, colocalization of M. avium and L. amazonensis was rarely found. Speculative scenarios that could underlie the formation of chimeric phagosomes could involve delayed maturation of C. burnetii-containing phagosomes in presence of M. avium, which would allow for fusion of C. burnetii- and M. avium-containing phagosomes; the production, by C. burnetii, of molecules that upregulate the fusion of M. avium-containing phagosomes with those that contain C. burnetii; and the secretion of factors that could favour the survival of M. avium within chimeric vacuoles.

High incidence of Coxiella burnetii markers in a rural population in France.

Since Coxiella burnetii, the causative agent of Q fever, is often transmitted from goats and sheep to humans through aerosols, we examined the sera from 168 persons involved in goat breeding in the Centre region of France and 40 members of veterinary and medical staff from the same region for the presence of antibodies against C. burnetii. An immunofluorescence assay was used to detect the presence of antibodies of the IgG isotope against epitopes from phase II of C. burnetii, which are the first antibodies to appear in infected people, and from phase I, which reflect more chronic stages of the infection. Our serological survey showed that most of the tested sera were positive for C. burnetii markers, indicating at least an encounter with the bacterium. In the overall population of 208 subjects, 71% of the sera had antibodies against phase II epitopes (titres > or = 1:40). Among the goat farmers and their immediate families, 78% had antibodies against phase II and 33% against phase I (titres > or = 1:40). Considering only high titres (> or = 1:320), though, only 37% of the farmers had antibodies against phase II and 15% against phase I. Only 3 out of 12 veterinarians working in the field had high titres of antibodies against phase II and phase I, while none of 28 members of veterinary and medical laboratories had significant levels of antibodies. These results emphasize the need for closer surveillance of populations at risk for Q fever, to prevent the infection by C. burnetii from reaching chronic stages of the disease.

Entry and survival of Leishmania amazonensis amastigotes within phagolysosome-like vacuoles that shelter Coxiella burnetii in Chinese hamster ovary cells.

Coxiella burnetii, a rickettsia, and Leishmania amazonensis, a protozoan flagellate, lodge in their host cells within large phagolysosome-like vacuoles. In the present study, C. burnetii-infected Vero or CHO cells were superinfected with L. amazonensis amastigotes to determine if these parasites can home to and survive within heterologous vacuoles. Six hours after superinfection, Leishmania amastigotes were located almost exclusively within large Coxiella-containing vacuoles. Thereafter, the numbers of parasites in the vacuoles increased at the same rate as those in cells infected with L. amazonensis alone. Furthermore, in cultures shifted to 25 degrees C, some of the amastigotes transformed into promastigote-like forms that moved their flagella within the adoptive vacuoles. Thus, L. amazonensis amastigotes not only entered Coxiella vacuoles, most likely by fusion of donor and recipient vacuoles, but temporarily survived, differentiated, and replicated therein. This appears to be the first account of the temporary cohabitation of two living pathogens within the same vacuole in a mammalian cell.

pH and calcium dependence of hemolysis due to Rickettsia prowazekii: comparison with phospholipase activity.

Rickettsia prowazekii invades nucleated cells through phagocytosis and subsequently proliferates in the cytoplasm of the host cell. Hemolysis and a phospholipase A2 (PLA2) activity at neutral pHs have previously been reported; even though the phagosomal environment is most likely acidic. We here show that R. prowazekii and R. typhi also lyse erythrocytes at mildly acidic pHs, compatible with an early phagosomal compartment. For R. prowazekii, hemolysis at an acidic pH but not a neutral pH is enhanced by Ca2+, raising the possibility that more than one membranolytic factor may be produced by the rickettsiae. The rickettsiae alone display PLA2 activity, implying that the enzyme is of bacterial rather than erythrocyte or host cell origin. Moreover, the PLA2 activity requires divalent cations (Ca2+ or Mg2+), and, as with many extracellular PLA2s from other species, it has a preference for acidic over neutral phospholipids. The pH dependence of PLA2 is similar to that of the hemolysis without Ca2+, but in the presence of the hemolysis buffers (which contain Mg2+), there is no calcium-induced enhancement at acidic pHs. Thus, these rickettsiae are endowed with a membranolytic activity that could contribute to the escape of the bacteria from early phagosomal compartments, and it is likely that multiple toxins may be used for membrane lysis.

Fusion between large phagocytic vesicles: targeting of yeast and other particulates to phagolysosomes that shelter the bacterium Coxiella burnetii or the protozoan Leishmania amazonensis in Chinese hamster ovary cells.

This report examines the fusion of phagocytic vesicles with the large phagolysosome-like vacuoles induced in Chinese hamster ovary cells by the bacterium Coxiella burnetti or the Protozoan flagellate Leishmania amazonensis. Infection by these organisms is compatible with cell survival and multiplication. Fusion was inferred from the transfer of microscopically identifiable particles from donor to target vesicles. Donor vesicles contained heat-killed yeast, zymosan, beta-glucan or latex beads taken up by the host cells. Yeast and zymosan were also coated with Concanavalin A to increase their uptake by the cells (Goldman, R., Exp. Cell Res. 104, 325-334, 1977). Particle localization, routinely ascertained by phase-contrast microscopy, was confirmed by confocal laser fluorescence and by transmission electron microscopy. Coxiella vacuoles admitted all the particles tested and transfer took place whether the particles were given to the cells prior to or after infection. Transfer of uncoated or Concanavalin-A-coated yeast or zymosan was dependent on the number of particles ingested and on the incubation period (between 2 and 24 hours). Furthermore, the transfer step was quite efficient, since over 85% of the particles ingested entered Coxiella vacuoles at all particle to cell ratios examined. The fraction of uncoated or Concanavalin-A-coated yeast or zymosan transferred to Leishmania vacuoles was consistently lower and diminished at higher particle loads. In addition, only rarely did latex beads enter these vacuoles. The models proposed may be useful for the delineation of biochemical and molecular mechanisms involved in the fusion of large phagocytic vesicles and the modulation of the latter by cellular and pathogen-derived signals.

Post-therapeutic evolution of serum chlamydial antibody titers in women with acute salpingitis and tubal infertility.

OBJECTIVE: To determine if serologic data and, more particularly, antichlamydial immunoglobulin (Ig) M can be used for diagnosis of current chlamydial intrapelvic gynecologic infection. DESIGN: Forty-two women with acute salpingitis (group A), 131 women with tubal factor infertility (group B), and 98 pregnant women (control group C) were studied. SETTING: Hôpital Jean Rostand, Sèvres (patients), Laboratories Magenta and Eylau, Paris (serology), Institut Pasteur, Paris (cultures). INTERVENTIONS: Study groups: endocervical/urethral swabs, pelvic samples; serologic study before and after treatment. CONTROL GROUP: Serologic study. MAIN OUTCOME MEASURES: Serum samples were collected from each patient initially and 6 to 9 weeks later; additionally, two to five sequential sera were obtained from 22 (group A) and 25 (group B) patients with positive cultures, evolutive serology, or positive antichlamydial IgM. Sera were tested for antichlamydial IgG by a microimmunofluorescence assay using Chlamydia trachomatis elementary bodies and for IgA and IgM by whole inclusion-fluorescent assay. RESULTS: Before treatment, there was a correlation between the presence of antichlamydial IgM and positive cervical and/or intrapelvic chlamydia cultures. After treatment, antichlamydial IgM, when initially positive, rapidly disappeared in most subjects; its persistence after 4 months was significantly associated with tubal sequelae in group A patients and persistence of positive intrapelvic chlamydial cultures in group B women. CONCLUSION: Serologic analysis of women with acute salpingitis or tubal infertility, including antichlamydial IgM, may aid both in the before treatment diagnosis of chlamydial infection and in the follow-up evaluation.

K-tuple frequency in the human genome and polymerase chain reaction.

The frequency occurrences of K-tuple (overlapping sequences of defined length, K) were computed from the known human genome sequences. The significance of these frequencies for the whole human genome was tested by polymerase chain reaction (PCR). A computer programs based on these results was written to choose primers to amplify DNA target sequences, either of human genes or of human infectious agents. The software also gave nested primer sequences which were used to synthesize non radioactive probes by PCR. We applied these two methods, primer selection and non radioactive probes, to easily and quickly set up very efficient PCR sets to work in the human genome context.

Clinical presentation of louse-borne relapsing fever among Ethiopian refugees in northern Somalia.

Louse-borne relapsing fever (LBRF) is still endemic among Ethiopian populations. In order to assess the clinical presentation of LBRF in an Ethiopian refugee camp in northern Somalia, a referral system was organized for all pyrexias of unknown origin. Among the 134 patients referred, 37 showed Borrelia in fresh and stained blood smears. Common clinical features were: high fever (above 39 degrees C in 73% of the cases), headache and general body pain (88%), liver tenderness (62%), petechia (54%), nausea and vomiting (46%), chills and rigors (30%) and epistaxis (11%). Jaundice was absent. No fatalities were observed. The clinical picture was less severe than in previous studies on LBRF. This difference might be due to the fact that the present study was community-based as opposed to the previous studies which were hospital-based.

Lung alterations in guinea-pigs infected with influenza virus.

Guinea-pigs were infected intranasally with influenza A Hong Kong 68 (H3N2) virus. Infective particles were re-isolated from lung homogenates up to 3 days after inoculation and indicated local replication. The subsequent lung inflammatory stages were studied by light microscopy, scanning and transmission electron microscopy (TEM). Lung alterations appeared after 24 h and intensified up to 7 days after virus inoculation, progressively decreasing until 3 weeks thereafter. The damage was reversible and complete restoration of structure was obtained within 5 weeks. The lesions commenced with the infiltration of bronchiolar and alveolar walls by polymorphonuclear cells, histiocytes and macrophages. A purulent exudate was seen to occupy the bronchiolar lumen. Cilia disappeared from tracheal and bronchiolar epithelia. Tracheal epithelium desquamated in some animals. TEM examination showed deterioration in type I pneumocytes, an increase in type II pneumocytes and concomitant damage to alveolar capillaries. Alveolar oedema and fibrinous deposits were seen. The pleura presented slight modifications. These results show that infection of guinea-pigs with influenza virus is a useful model for the study of lung pathology associated with a non-lethal respiratory viral infection.

Auto-antibody dependent activation of the autologous classical complement pathway by guinea-pig red cells treated with influenza virus or neuraminidase: in vitro and in vivo study.

Guinea-pig erythrocytes that had been exposed to influenza A virus or Vibrio cholerae neuraminidase activated the classical complement pathway in autologous serum. Because all viral particles were eluted from the treated cells, activation was not dependent on anti-viral antibodies or on the particles themselves. After a threshold of 45-55% desialation, had been reached, the relative capacity of treated cells to activate complement increased very rapidly with desialation. Desialation unmasked sites on which natural auto-antibodies of the IgM class were fixed. Antibody fixation on the membrane led to C3b deposition on the cell membrane and activation of the classical complement sequence then cell lysis. The relevance of in vitro lysis of desialated cells to in vivo clearance of these cells is not certain because C4-deficient guinea-pigs were able to eliminate desialated cells from the blood stream as efficiently as did normal guinea-pigs. Nevertheless, membrane desialation occurring during myxovirus infection could lead to autoimmunity and tissue changes, as well as to recovery by eliminating virus-modified cells.

Guinea pig erythrocytes, after their contact with influenza virus, acquire the ability to activate the human alternative complement pathway through virus-induced desialation of the cells.

Guinea pig erythrocytes that had been exposed to influenza A virus activated the alternative complement pathway in whole human serum in the absence of natural antibodies. Because all virus particles were eluted from the treated cells, activation was not dependent on antiviral antibodies or on virus particles themselves. The relative capacity of treated erythrocytes to activate the alternative pathway was dependent on the amount of virus to which the cells had been exposed and was directly related to the amount of sialic acid removed from the erythrocyte membrane during incubation with either whole virus particles or purified viral sialidase. C3b bound to cells that had been treated with virus, and P-stabilized amplification convertase sites P,C3b,Bb formed on these cells, exhibited increased resistance to the action of the regulatory proteins beta-1H and C3b Ina compared with C3b and P,C3b,Bb on untreated, nonactivating cells. The acquired resistance of the cell-bound, P-stabilized amplification convertase to decay-dissociation by beta-1H was directly related to the activating capacity of the treated cells in whole serum (r = 0.95) and to the amount of sialic acid removed from the cells by the virus (r = 0.98). Desialation represents a specific alteration of the cell surface by which a nonimmune host, through activation of the alternative pathway, may deposit C3b on a target cell that had been exposed to influenza virus and may lyse virus virus-modified cells during orthomyxovirus infections.