Epidemic typhus infection in cynomolgus monkeys (Macaca fascicularis).

A nonhuman primate model of clinical Rickettsia prowazekii infections was developed in cynomolgus monkeys (Macaca fascicularis). Monkeys infected intravenously with 10(7) plaque-forming units developed clinical signs of illness and pathological changes characteristic of epidemic typhus infection in humans. Increases in total leukocyte counts, serum alkaline phosphatase, blood urea nitrogen, and serum glutamic pyruvate transaminase values were observed. Microscopic examination revealed typical typhus nodules in the brains of two monkeys that died. These data indicated that the cynomolgus monkey is a suitable model for study of the pathogenesis of epidemic typhus infection and may prove valuable in the evaluation of candidate R. prowazekii vaccines.

Immune responses to Rickettsia akari infection in congenitally athymic nude mice.

Athymic BALB/c nude mice and euthymic BALB/c mice were infected with Rickettsia akari by the intraperitoneal route. The rickettsialpox infection was terminated in euthymic mice with only two intraperitoneal injections of the antibiotic oxytetracycline, whereas prolonged treatment was necessary to terminate the infection in athymic mice. Both athymic and euthymic mice produced specific antibody, but athymic mice were still susceptible to reinfection. Killed R. akari served as a protective immunogen in euthymic, but no in athymic, mice. When spleen cells from convalescent euthymic mice were transferred to syngeneic athymic mice, recipients showed protection against challenge. This suggests that a T-cell-dependent step is generally necessary to terminate the rickettsialpox infection.

Evaluation of a killed Rocky Mountain spotted fever vaccine in cynomolgus monkeys.

A nonhuman primate model of Rocky Mountain spotted fever infection was developed in cynomolgus monkeys (Macaca fascicularis) infected by the subcutaneous route or by aerosol. Clinical responses, hematology and serum chemistry values, and pathological findings were similar to those found in humans ill with Rocky Mountain spotted fever. The clinical model was then used to test the efficacy of a killed Rocky Mountain spotted fever vaccine grown in chicken embryo cells. Monkeys were immunized with varying dilutions of the vaccine with a two-dose schedule and then challenged at 2 months with virulent Rickettsia rickettsii by the subcutaneous route or by aerosol. The undiluted vaccine totally protected monkeys against both challenges, even at extremely high doses.

Breast gross cystic disease fluid analysis. I. Isolation and radioimmunoassay for a major component protein.

Human breast gross cystic disease (GCD) fluid was analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis, and four major proteins (GCDFP-70), GCDFP-44, GCDFP-24, and GCDFP-15) were identified. By fractionation techniques, these proteins were separated from one another. The GCDFP-70 was immunologically identical to human albumin and was present in GCD fluid at approximately a 100-fold lower concentration than in plasma. The GCDFP-44 was immunologically identical to human plasma Zn-alpha2-glycoprotein; however, it was present in GCD fluid at an approximately 50-fold higher concentration than in plasma. The GCDFP-24 was the major component protein of GCD fluid. It had progesterone binding activity, and immunologically it was identical to a component of human plasma; however, antisera that identified 30 separate components of plasma failed to identify the GCDFP-24 as one of these plasma proteins. The GCDFP-24 concentration in GCD fluid was approximately 100-fold higher than the plasma analog. The GCDFP-15 component was immunologically distinct from any plasma components, as judged by Ouchterlony analysis. It was, however, immunologically identical with a component of both human milk and saliva. As revealed by radioimmunoassay, plasma levels in normal subjects were 7-85 ng/ml. In patients with metastatic breast carcinoma, markedly plasma levels (150-30,000 ng/ml) of this protein were detected. Short-term tissue cultures of breast carcinoma explants released this protein into the culture medium.

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.

Suppression of cellular immune responses in guinea pigs infected with spotted fever group rickettsiae.

Using a guinea pig model, we demonstrated that infections with pathogenic species of spotted fever group rickettsiae transiently and nonspecifically suppress established cellular immune responses as measured by in vitro lymphocyte transformation and in vivo delayed cutaneous hypersensitivity responses to unrelated, nonrickettsial antigens. The correlation of the duration of this immunosuppression with the virulence of the infecting rickettsial species suggests that this suppression is a pathological effect of the rickettsial infection. Although we did not specifically study the mechanism of this suppression, it is not associated with either lymphocytopenia or leukocytosis.

Enhancement of the immunogenicity of phase I antigen of Coxiella burnetii.

The immunogenicity of the soluble phase I antigen of Coxiella burnetii for guinea pigs was enhanced by a nuclease-resistant complex of polyriboinosinic-polyribocytidylic acid, poly-L-lysine, and carboxymethyl cellulose.

Initial clinical evaluation of a new Rocky Mountain spotted fever vaccine of tissue culture origin.

Currently available Rocky Mountain spotted fever (RMSF) vaccines are relatively ineffective in preventing infections in humans and contain considerable amounts of contaminating egg protein. A new formalin-inactivated vaccine was prepared by sucrose density gradient centrifugation of the Sheila Smith strain of Rickettsia rickettsii grown in chick embryo cell tissue culture. The new product has greater protective immunogenicity in rheusus monkeys and guinea pigs than commercial vaccines. Six volunteers without immunologic evidence of prior exposure to RMSF received from one to three inoculations of the vaccine diluted 1:10, and there were two benign local reactions. Titers of antibody (determined by microagglutination and indirect fluorescence techniques) increased in all recipients as did lymphocyte tranformation responses to specific rickettsial antigen. Ten volunteers were immunized twice with vaccine diluted 1:3; there were no local reactions, and immunologic responses were similar to those in the six volunteers in the first group. The proper dosage and immunization schedule for the vaccine must be determined in further studies.

Alterations of body fluid compartments and distribution of tissue water and electrolytes in rhesus monkeys with rocky mountain spotted fever.

Chair-restrained rhesus monkeys (Macaca mulatta) were inoculated subcutaneously with 10(2)--10(3) plaque-forming units of virulent Rickettsia rickettsii. The latent period for fever and rickettsemia was three to four days; death occurred six to eight days after infection. Total circulatory electrolyte levels and fluid volumes, including plasma, red blood cell, true circulatory blood, and extracellular fluid, increased. The expansion of the extracellular and plasma volumes resembled findings reported during severe Rocky Mountain spotted fever in humans, guinea pigs, and rabbits. Total water content of the liver also increased. Intracellular concentrations of K+, as well as total Na+ and K+, decreased in the diaphragm. Both the lung and medulla oblongata showed increased levels of intracellular Na+ and water with simultaneously decreased levels of extracellular Na+ and water. Such an intracellular overhydration of the medulla oblongata could contribute to death as a result of depression of the cardiovascular and respiratory centers. On the basis of the findings in monkeys, the intravenous infusion of fluids and electrolytes during clinical therapy of severe rickettsial infections should be considered extremely dangerous.

Prophylactic treatment of Rocky Mountain spotted fever.

Prophylactic treatment of Rocky Mountain spotted fever with a single dose of oxytetracycline was investigated in guinea pigs. Disease was prevented when treatment was administered shortly before expected onset. Relapses occurred when treatment preceded expected onset by 48 h or more.

Plaque formation by strains of spotted fever rickettsiae in monolayer cultures of various cell types.

Some strains of spotted fever rickettsiae could be distinguished by their ability or inability to form plaques in monolayer cultures of various mammalian and avian cell types.

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.

Laboratory-acquired Rocky Mountain spotted fever. The hazard of aerosol transmission.

Nine patients with laboratory-acquired Rocky Mountain spotted fever were seen during the period 1971 to 1976. Investigation of each case revealed either definite or probable exposure to an aerosol containing infectious rickettsiae; in no case was there evidence of parenteral exposure either by accidental self-inoculation or by tick bite. These illnesses are believed to represent infection acquired via the respiratory route. This report emphasizes the aerosol hazard of Rickettsia rickettsii in the laboratory and discusses the possibility of respiratory transmission of Rocky Mountain spotted fever in nature. The illness occurred only in personnel who had received either no vaccination or the primary series of the commercial (Lederie) vaccine against this infection. Other personnel who had received the primary series with multiple booster vaccinations demonstrated increased immunity as measured by humoral antibody titers and rickettsial antigen-induced lymphocyte transformation; no cases of clinical disease developed in these multiply-vaccinated personnel.

Lesions in guinea pigs infected with Coxiella burnetii strain M-44.

Guinea pigs inoculated with the live M-44 vaccine strain of Coxiella burnetii were examined grossly and microscopically for the presence of Q fever-related lesions. Mild myocarditis was observed in 38% of the infected animals but in none of the control animals. Livers showed significant incidence of hepatitis, necrosis, and granuloma formation, especially during the first eight days of the infection. A much lower incidence of splenitis was also found but was considered to be of borderline significance. Generally, lesions were mild in nature, and none seemed to endanger the life of the animal or to cause observable distress.

Studies on Macaca mulatta infected with Rocky Mountain spotted fever.

Acid-base alterations and changes in other selected serum constituents (free fatty acids, triglycerides, cholesterol, copper, cortisol, alpha1-acid glycoprotein, haptoglobin, and albumin) were measured during a study of Rocky Mountain spotted fever in 16 male rhesus macaques. Blood samples were taken from nonanesthetized macaques conditioned to repeated handling. Arterial pH increased and PCO2 decreased during the febrile period. Free fatty acids, triglycerides, copper, cortisol, alpha1-acid glycoprotein, and haptoglobin increased, whereas albumin decreased during the disease. Significant changes were not observed in arterial PO2. Cholesterol remained unchanged. The increase in arterial pH and decrease in PaCO2 indicated that respiratory alkalosis was present in macaques acutely affected with Rocky Mountain spotted fever.

Scanning and transmission electron microscopy of Rickettsia rickettsii propagated in cell culture.

Scanning electron microscopy utilizing critical-point drying and transmission electron microscopy employing air-dried agar pseudoreplicas and critical-point dried carbon replicas were used to study the surface of Rickettsia rickettsii propagated in cell culture.

Susceptibility of laboratory animals to infection by spotted fever group rickettsiae.

An attempt was made to find a suitable animal model for studies of spotted fever group rickettsiae. Inbred and outbred mice, the guinea pig, ferret, gerbil, hamster, wild rabbit, cotton rat, sheep, and miniature swine were tested. Of these, only certain strains of the mouse [Mai:(S) and BALB/cJ] and the guinea pig [Hla:(HA)] exhibited, overtly, the desired characteristics of disease. Other laboratory animals (such as sheep or rabbits) can be used for the production of antiserum against the spotted fever group of rickettsiae; however, these rickettsiae apparently have little or no effect on several other animal species. The lack of overt disease might explain the role of these animals or related genera as reservoirs for the tick-borne spotted fever rickettsiae.