Retention of fluorescent antigenicity of virus-infected cells on spotslides under various conditions of storage.

Ten well, epoxy-coated spotslides containing arbo- and arenavirus-infected cells were tested as determined by fluorescent methods for the retention of antigenicity after storage in a nitrogen atmosphere at various temperatures and times. In most cases antigen stability was maintained at -70 or -20 degrees C for periods exceeding 1-3 years. Antigen deterioration was greatest at ambient temperature, and less so at 4 degrees C.

Identification of Rickettsia rickettsii in formalin-fixed, paraffin-embedded tissues by immunofluorescence.

With slight modification of a trypsin digestion technique, Rickettsia rickettsii were demonstrated specifically by immunofluorescence staining in Formalin-fixed, paraffin-embedded tissue sections from a human, rhesus monkey, and guinea pig with Rocky Mountain spotted fever and in infected membranes from a chicken embryo. Tissues were cut at 4 micron and, using geltain as a tissue adhesive, were hydrated in a routine manner. Sections were then digested in refrigerated 0.1% trypsin for 16 h, washed, and stained specifically for R. rickettsii by direct or indirect immunofluorescence. Rickettsial organisms were localized in affected vessels of the mammalian species and within the yolk sac epithelium of the chicken embryo. Specificity was confirmed by adsorbing antibody conjugates with R. rickettsii organisms. Trypsin digestion probably decreased tissue proteins which interfered with immunochemical attachment of antibody to the rickettsiae. The technique is valuable in that a diagnosis of Rocky Mountain spotted fever can be confirmed from Formalin-fixed tissues processed in a routine manner.

Prompt confirmation of Rocky Mountain spotted fever: identification of rickettsiae in skin tissues.

Rickettsia rickettsii were identifiable by the immunofluorescence technique in skin specimens obtained by biopsy on days 4 and 8 of illness from patients with Rocky Mountain spotted fever. The immunofluorescence technique is regarded as a practical means of confirming the diagnosis during the early stages of illness before positive serologic reactions can be obtained.

Functional and morphologic changes during experimental Rocky Mountain spotted fever in guinea pigs.

Experimental Rocky Mountain spotted fever was studied in guinea pigs following intraperitoneal inoculation of 10(7) Rickettsia rickettsii. After a 2-day incubation period, animals developed fever, progressive emaciation, and scrotal swelling with necrosis. Vasculitis, with increased small vessel permeability for colloidal carbon, was evident in cremaster muscles as early as 1 day after inoculation. Inflammatory changes in vessels became progressively more severe as numbers of circulating rickettsiae increased. Thrombosis and vascular occlusion were first evident on day 4. Mild thrombocytopenia developed, coinciding with the development of vasculitis, and preceding the appearance of either fibrin-split products in blood or thrombi in vessels. Rickettsiae were first detected in blood on day 2; peak rickettsemia occurred on days 5 to 8. Rickettsiae were demonstrated in inflamed vessels on day 5 and later, but not at earlier stages. Serum lysozyme concentration was moderately elevated and hemolytic complement was moderately depressed throughout the illness. Agglutinating antibody was present in low titers on days 3 to 10. Antibody titers increased on days 12 to 16 after the rickettsiae were cleared from blood. These studies indicate that vasculitis seen early in the course of Rocky Mountain spotted fever is the result of rickettsial infection, but is not dependent on the presence of rickettsiae in endothelial cells or other blood vessel components.

Demonstration of Rickettsia rickettsii in the rhesus monkey by immune fluorescence microscopy.

Indirect immune fluorescence was used to detect rickettsiae in the tissues of a primate inoculated subcutaneously with Rickettsia rickettsii. Rickettsiae were identified by indirect immuno fluorescence predominantly in skin, skeletal muscle, scrotum, testicles, nares, heart, kidney, liver, brain, spleen, pancreas, and larynx. Cell culture assay confirmed the presence of infectious organisms in those specimens.

Antibody response to Rocky Mountain spotted fever.

Various techniques were compared to determine the most sensitive method for detection of rocky Mountain spotted fever antibody. A radiometabolic technique for detection of Rocky Mountain spotted fever antibody is also described. In infected monkeys, the fluorescent antibody technique yielded the earliest evidence of seroconversion; with some monkeys the microagglutination procedure was equally effective. The fluorescent antibody and microagglutination measurements showed higher titers than those for complement fixation, Weil-Felix, or the radiometabolic techniques.