Intracellular fate of phase I Coxiella burnetii in guniea pig peritoneal macrophages.

Cultivated guinea pig peritoneal macrophages were infected with radio-labeled phase I Coxiella burnetii in order to assess the intracellular distribution of ingested rickettsiae. Localization of organisms was determined by fractionation of macrophage homogenates by equilibrium density centrifugation on sucrose gradients. Macrophages isolated from either nonimmune or immune guinea pigs and infected with C burnetii opsonized with immune serum yielded equilibrium density distribution for rickettsiae similar to lysosomal enzymes, suggesting sequestration within macrophage lysosomes. To confirm these observations nonimmune or immune guinea pigs were injected with Triton WR-1339 prior to macrophage harvest to decrease the density of macrophage lysosomes. Triton-laden macrophages infected with opsonized rickettsiae resulted in equilibrium density distribution for lysosomal enzymes and organisms in less dense regions of the gradient. In contrast, when either nonimmune or immune macrophages were infected in the presence of normal guinea pig serum, the distribution of labeled rickettsiae in the gradient did not correspond with lysosomes. We conclude that in the absence of immune serum, ingested C burnetii are not sequestered within macrophage lysosomes. Phagolysomal fusion and subsequent degradation of rickettsiae within the lysosomes of the macrophages appear to occur only when C burnetii are opsonized with immune serum.

Aerosol Q fever infection of the nude mouse.

Athymic nude mice and euthymic littermate controls were exposed to 10(4) Coxiella burnetii organisms by small-particle aerosol. Antibody response with and without 2-mercaptoethanol treatment of serum was determined at various intervals after infection and serial kills were done to determine morphologic changes in both mouse phenotypes. Total antibody titers determined by the indirect fluorescent antibody technique to phase I and phase II C. burnetii were identical for both groups of mice. Microagglutinin titers determined on days 28 and 33 were abolished by 2-mercaptoethanol treatment of serum from both phenotypes, indicating that the antibody probably resided in the IgM fraction. Microscopically, the reaction to C. burnetii infection was similar in nude and euthymic mice on days 7 and 14. Later, the number and size of lesions attributable to Q fever diminished in euthymic mice. Infection was progressive in nude mice, with macrophage infiltration of most tissues, especially spleen and liver. Numerous rickettsiae were seen by immunofluorescence and electron microscopy in phagocytic vesicles of macrophages, many of which were dilated, giving the macrophage a vacuolated appearance. Results suggest that clearance of C. burnetii infection in mice is independent upon thymus-derived lymphocytes.

In vitro responses of guinea pig peritoneal macrophages to Legionella pneumophila.

Transmission and scanning electron microscopy were used to study the phagocytosis of virulent and avirulent strains of Legionella pneumophila. The interaction between L. pneumophila and peritoneal macrophages from normal guinea pigs or from animals that had survived infection was studied. The virulent strains survived and proliferated within the phagocyte after ingestion by either type of macrophage, whereas the avirulent strain of bacteria was killed by normal macrophages. Although the addition of immune serum enhanced phagocytosis, the outcome was the same as with normal serum.

Evaluation of a killed phase I Coxiella burnetii vaccine in cynomolgus monkeys (Macaca fascicularis).

The protective efficacy of a killed, purified, phase I Coxiella burnetii vaccine was tested in cynomolgus monkeys. Monkeys vaccinated once with 30 micrograms of the antigen were challenged 6 or 12 months later with virulent phase I rickettsiae administered in small-particle aerosols. The vaccine provided only partial protection, since some of the challenged monkeys developed clinical signs of illness. However, the vaccinated animals did not develop pneumonia as determined by radiographic evaluation nor any hematologic or chemical changes except for an increase in fibrinogen. Although rickettsiae were isolated from peripheral blood in vaccinated monkeys, the rickettsemia persisted for only 1-2 days; whereas, organisms were recovered from unvaccinated animals for 6-7 days. All vaccinated animals had circulating microagglutinating antibodies to phase I and phase II antigens 6 and 12 months after vaccination.

Prevalence of rickettsial antibody and cell-mediated reaction in cynomolgus monkeys (Macaca fascicularis).

Serologic studies on feral, colony-held cynomolgus monkeys indicated that 61% reacted to Coxiella burnetii antigens, and 36% reacted to Rickettsia conorii antigens. The results suggest that a high percentage of cynomolgus monkeys have been exposed to these organisms.

Mechanisms of protective immunogenicity of microbial vaccines: effects of cyclophosphamide pretreatment in Venezuelan encephalitis, Q fever and tularaemia.

Administration of high-dose (250 mg/kg) cyclophosphamide (CY) to guinea-pigs and mice 3 days prior to immunization with inactivated vaccine derived from Venezuelan encephalitis virus (VE), Coxiella burnetii and Francisella tularensis resulted in accentuated and prolonged delayed-type hypersensitivity (DTH) and in vitro cellular immunity (CMI) to specific antigen. Humoral antibody were either absent or significantly lower in CY-pretreated animals compared to immunized non-pretreated controls. CY pretreatments precluded protection in the VE virus model, suggesting that resistance is related to antibody. In the Q fever model, the protective immunogenicity of vaccine was preserved or increased by CY pretreatment suggesting that cell-mediated immunity is the important factor. In the tularaemia bacterial system, there was a complex effect of CY pretreatment on the low-grade protection afforded by killed vaccine against virulent infection. These findings suggest that the inability of killed vaccines to induce high-grade resistance against tularaemia and Q fever may be due in part to a suppressive B cell response which is eliminated by CY. These studies have given useful information on the relative significance of components of the specific immune response and may lead to an increased understanding of the mechanisms of action of vaccines and adjuvants.

In vitro studies of interaction of rickettsia and macrophages: effect of ultraviolet light on Coxiella burnetii inactivation and macrophage enzymes.

The inactivation of Coxiella burnetii in suspension or in cultures of guinea pig peritoneal macrophages by ultraviolet (UV) light was studied. The effect of UV treatment on the activity of macrophage organelle marker enzymes and their subsequent equilibration in linear sucrose gradients was also determined. It was shown that UV treatment of 600 mu W/cm2 for 15 s at a distance of 10 cm inactivated C. burnetti, either in suspension (10(8) organisms per ml) or within guinea pig peritoneal macrophages. Similar UV treatment had little effect on the activity or equilibration of macrophage organelle marker enzymes in linear sucrose gradients. However, longer exposure caused considerable inactivation of these enzymes.

Exposure of guinea pigs to Rickettsia rickettsii by aerosol, nasal, conjunctival, gastric, and subcutaneous routes and protection afforded by an experimental vaccine.

Guinea pigs were inoculated with Rocky Mountain spotted fever by the aerosol, conjunctival, subcutaneous, intragastric, and intranasal routes. Rickettsial infection was produced by all routes except intragastric. All animals with clinical signs of disease developed agglutinating antibody, and most developed a cell-mediated immune response. Disease produced by all experimental routes (except intragastric) was indistinguishable. The tissue culture-derived inactivated vaccine produced in this laboratory protected guinea pigs against an aerosol challenge.

In vitro interaction between normal cynolmolgus monkey alveolar macrophages and Legionnaires disease bacteria.

The interaction between normal cynomolgus monkey alveolar macrophages and Legionnaires disease bacteria was studied by transmission electron microscopy. After ingestion of Legionnaires disease bacteria, the organisms replicated within macrophages and destroyed the phagocytic cell.

Suppression of PHA-stimulated lymphocyte transformation in cynomolgus monkeys following infection with Coxiella burnetii.

Phytohemagglutin (PHA) induced blastogenesis of peripheral blood lymphocytes from cynomolgus monkeys infected with C. burnetii was suppressed between 14 and 28 days after infection. Lymphocytes became responsive to PHA again on day 35 with an increase in stimulation index when cultured with specific antigens. In contrast, production of specific humoral antibodies was not diminished during the acute and early convalescent stages of infection.

Cynomolgus monkey model for experimental Q fever infection.

A subhuman primate model was developed for study of the pathogenesis of infection with Coxiella burnetii. Cynomolgus monkeys (Macaca fascicularis) that were exposed to 10(5) mouse median infectious intraperitoneal doses of C. burnetii in a small-particle aerosol developed clinical signs of illness and pathologic changes characteristic of Q fever infection in humans. All monkeys had radiologic evidence of pneumonia by day 9. Antibodies to C. burnetii were detectable by the indirect fluorescent antibody test by day 7. These data indicate that the cynomolgus monkey is a suitable model for study of the pathogenesis of Q fever infection and may prove valuable in the evaluation of C. burnetii vaccines.

Recovery of alveolar macrophages from rhesus and cynomolgus macaques by lung lavage.

A lung lavage technique was developed to recover alveolar macrophages from rhesus and cynomolgus macaques. Sterile saline solution was injected through an endotracheal tube in anesthetized macaques; lung wash fluids containing leukocytes were withdrawn. The lung wash fluids from each animal routinely contained more than 16 x 10(6) leukocytes. The predominant cell type was the alveolar macrophage; lung wash fluids contained more than 53% and 80% alveolar macrophages from rhesus and cynomolgus macaques, respectively. Lung lavage was performed each week for 6 weeks in both species with no ill effects. This technique has many applications in the study of infection and of pulmonary defense mechanisms.

Experimental Coxiella burnetii infection of guinea pigs and mice.

The susceptibility of five inbred strains of mice, designated DBA/1J, DBA/2J, C57BL/6J, Balf/CJ, and AKR/J, as well as outbred Hartley and Moen-Chase guinea pigs to infection with Coxiella burnetii by several routes was studied. The DBA/2J mice were more susceptible to infection and had higher mortality rates than other strains of mice. Guinea pigs were more susceptible to infection than mice. Lesions observed in the infected animals were similar to those previously described in man and experimentally infected animals.

Experimental Q fever infection in congenitally athymic nude mice.

Congenitally athymic nude (nu/nu) mice and their phenotypically normal (nu/+) euthymic littermates were exposed to Coxiella burnetii administered as small-particle aerosols. After challenge, both strains of mice became infected, as characterized by rickettsemia, viable rickettsiae in the spleen, and serological conversion. The major difference noted was that euthymic animals had cleared rickettsiae from peripheral circulation and the spleen within 14 days. In contrast, rickettsiae were detected and isolated from spleen and blood of athymic mice through 60 days.

Cell-mediated immune responses of guinea pigs to an inactivated phase I Coxiella burnetii vaccine.

The ability of a killed phase I Coxiella burnetii vaccine to induce cell-mediated immune responses in guinea pigs was studied. Cell-mediated immune responses were assessed by the inhibition of macrophage migration and lymphocyte transformation assays. The macrophage migration response occurred rapidly and was detected at high levels, but was relatively short-lived. In contrast, the lymphocyte transformation response developed more slowly, and persisted for a longer period. The vaccine, given in a single dose or in two doses 1 week apart, protected guinea pigs from a subsequent virulent challenge.

In vitro guinea pig leukocyte reactions to Rickettsia rickettsii.

The presence of cell-mediated immunity in Rocky Mountain spotted fever-infected guinea pigs was determined by two in vitro assays: whole blood lymphocyte transformation (LT) and macrophage migration inhibition. Increased LT was detected as early as 1 week in guinea pigs infected with Rickettsia rickettsii and treated with oxytetracycline and was detected by two weeks in infected but untreated guinea pigs. Elevated LT was still detectable at 10 weeks postinfection. Guinea pigs vaccinated with killed rickettsiae failed to develop lymphocyte responsiveness; however, there was a rapid lymphocyte response after challenge with live organisms, suggesting potentiation by the vaccine. Vaccinated guinea pigs that were challenged and then treated with antibiotic failed to develop LT, suggesting that infection is necessary for the observed response. Macrophage migration inhibition was detected in both infected and vaccinated guinea pigs by 1 week after infection, but this response was no longer detected 4 to 5 weeks later. Antibody appeared at 2 to 3 weeks postinfection and was present at low levels through week 10. Antibody-treated rickettsiae were phagocytized and destroyed by guinea pig peritoneal macrophages, whereas normal serum-treated rickettsiae replicated and eventually destroyed the phagocytes.

Appearance of cellular and humoral immunity in guinea pigs after infection with Coxiella burnetii administered in small-particle aerosols.

The development of humoral and cell-mediated immune responses was studied in guinea pigs infected with Coxiella burnetti administered in small-particle aerosols. Direct macrophage migration inhibition was observed in cultured peritoneal exudate cells as early as 3 days after exposure. Maximum inhibition of macrophages cultured with phase I or II antigen occurred 14 to 21 days postexposure and persisted through 35 days. This inhibitory action was no longer detectable at 42 days. Serum antibody to the phase II antigen of C. burnetii was detected at 14 days, and serum antibody to phase I antigen was detected at 21 days, 18 days after the cell-mediated immune response.

Fat of Coxiella burnetti in macrophages from immune guinea pigs.

The interaction between Coxiella burnetii and peritoneal macrophages obtained from immune guinea pigs was studied by transmission electron microscopy. Phagocytosis and subsequent fate of ingested phase I and II rickettsiae were compared. Phase I rickettsiae were more resistant to phagocytosis than were phase II organisms. Macrophages from phase I- and II-immunized animals were equally capable of phagocytizing rickettsiae. Phase I and II rickettsiae previously treated with normal serum multiplied and destroyed macrophages from guinea pigs that had been immunized with phase II rickettsiae. Phase II organisms were initially suppressed in macrophages from phase I-immunized animals, but eventually multiplied in these cells. In contrast, only phase I organisms were destroyed by macrophages from phase I-immunized animals. Treatment of rickettsiae with immune serum enhanced ingestion by macrophages and potentiated the destruction of organisms by both types of macrophages. The macrophage migration inhibition assay was performed on peritoneal exudate cells from immune animals. Migration of peritoneal macrophages from phase I-immunized guinea pigs was inhibited, whereas macrophages from phase II-immunized animals migrated when cells were cultured in the presence of killed, intact phase I or II C. burnetii.