Biomedical subjects
Publications and source records attributed to C E Pedersen.
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.
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.
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.
The attenuated M-44 strain of Coxiella burneti was investigated for biological characteristics affecting its use as a live vaccine. After 25 serial passages in guinea pigs, the strain did not develop phase I properties detectable by the microagglutination test. Infectious organisms were not detected in untreated guinea pigs later than 12 weeks after inoculation; however, infection could be reactivated at later times by the production of stress (by pregnancy or drug treatment) in guinea pigs that had recovered. Evidence of reactivated infection was observed in recovered pregnant animals just before parturition at 46 weeks after infection, in methyl prednisolone-treated animals at 56 weeks, and in cyclophosphamide-treated guinea pigs at 96 weeks. A low rate of intercage and intracage infection was recorded.
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.
Envelope components were separated from Venezuelan, Eastern, and Western equine encephalomyelitis viruses after treatment of the virions with detergent. Vaccines prepared from the envelope component were capable of stimulating mice to produce humoral antibodies. Protective efficacy studies were performed using mono-, di-, and trivalent vaccine combinations. These elicited varying degrees of homologous protection, and Eastern and Venezuelan equine encephalomyelitis envelope products appeared to confer protection to mice challenged with Western equine encephalomyelitis virus.
Primary chicken embryo cell cultures were evaluated a s an alternate cell system for the production of attenuated Venezuelan equine encephalomyelitis (TC-83 strain) vaccine. The TC-83 strain virus was shown to remain stable during 10 serial passages in chicken embryo cell culture with regard to plaque size and morphology, virus yield, potency, and virulence for mice and hamsters.
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.
The effect of vaccination schedule on the immune response of Macaca mulatta to formalin-inactivated chicken embryo cell culture (CEC)-grown Rickettsia rickettsii vaccine was studied. Schedules consisted of inoculation on day 1 only, on days 1 and 15, on days 1 and 30, on days 1, 8, and 15, or on days 1, 15, and 45. Humoral antibody measured by microagglutination and indirect immunofluorescence and resistance to challenge with 10(4) plaque-forming units of yolk sac-grown R. rickettsii were assessed. Seroconversion was noted in all monkeys after the first dose of vaccine. A second dose administered 8 or 15 days after the primary infection, or a third given 7 or 30 days after the second, produced no long-term effect on antibody titer. Only monkeys given two doses of vaccine at a 30-day interval showed an increase in antibody titer during the period before challenge. Vaccination with one, two, or three doses of CEC vaccine prevented development of rash and rickettsemia after challenge. The two-dose schedules appeared to induce the highest degree of resistance to challenge, as indicated by unaltered hematological parameters and body temperature in monkeys. The one- and three-dose schedules were somewhat less effective, in that some challenged monkeys within each group displayed febrile and leukocyte responses associated with Rocky Mountain spotted fever infection. Our data suggest that administration of two doses of CEC vaccine at 15- or 30-day intervals is the immunization schedule of choice.
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Forty-seven male Macaca mulatta, 3 to 4 kg weight, were inoculated intravenously or subcutaneously with various doses of yolk sac-grown Rickettsia rickettsii. Thirty-four macaques became febrile and exhibited signs of infection ranging from transient illness with a few days of fever to severe illness with subsequent death. The rash appeared more frequently in the macaques inoculated subcutaneously. Febrile macaques that survived had leukocytosis, with concomitant neutrophilia. Febrile macaques that died had, in addition, marked terminal leukopenia and thrombocytopenia. Packed cell volume of all febrile macaques decreased. In almost all of the febrile macaques, there were increased serum urea nitrogen, glutamic-oxaloacetic transaminase, and lactate dehydrogenase and decreased total serum protein and amylase concentrations. A few febrile macaques had increased bilirubin values and decreased sodium, chloride, phosphorus, and alkaline phosphatase concentrations. Changes did not occur in serum glucose, potassium, calcium, and glutamic-pyruvic transaminase values. The experimental form of Rocky Mountain spotted fever in the macaque provides a subhuman primate model for studying the pathophysiology of this disease.
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.
Electron paramagnetic resonance observations were made on nitroxide spin- labeled molecules which were bound to the TC-83 vaccine strain of Venezuelan equine-encephalomyelitis virus. Paramagnetic resonance parameters derived from the observations and their dependence on sample temperature were similar but not identical to those which have been reported for these labels dissolved in lipid bilayer membranes of mammalian and bacterial origin. The data has a mechanical rigidity substantially greater than that of bilayers in cellular membranes. A model is presented which assumes the location of the lipid bilayer outside the nucleoprotein capsid and inside a spherical layer of envelope proteins. The model is in accord with Harrison's X-ray diffraction results for Sindbis virus. The model is discussed in terms of its implications with respects to the role played by lipid in viral maturation and infectivity.
Polyacrylamide gel electrophoretic examination of viruses selected from the Venezuelan equine encephalomyelitis (VEE) complex revealed distinct strain to strain differences in profiles of the two virion envelope proteins. The core protein was identical in all viruses tested. We detected five electrophoretic patterns into which the virus strains could be classified and these were designated alpha (alpha), beta (beta), gamma (gamma), delta (delta), and episolon (episolon). Isolates representing variant E of subtype I exhibited a profile characterized by only one apparent envelope band. The epizootic subtypes I-A, I-B, I-C and the sylvatic subtype II viruses contained at least two envelope proteins which differed in molecular weight according to virus strain but which were not necessarily specific for antigenic variety. These results generally, though not uniformly, support the serologic classification of the VEE virus complex and suggested that the usefulness of the classification scheme could be complemented by the inclusion of biochemical criteria.
Coxiella burneti phase I, purified from a formalin-inactivated yolk-sac vaccine, was separated into two bands of morphologically distinct cell types when subjected to sucrose gradient centrifugation. Recycling of the less dense, rod-shaped cells in unbuffered sucrose gradients (pH 5.5 to 6.0) resulted in the formation of bands having the location and appearance of the original two bands. Recycling of the denser band of larger ovoid-shaped cells yielded a single band, suggesting that the larger cell type arose from the smaller cell. In contrast to vaccine-derived rickettsiae, live, cell culture-propagated phase I organisms formed a single band in unbuffered sucrose gradients, at the same density as the upper band of the vaccine preparation. Centrifugation of cell culture-derived rickettsiae for 26 to 48 h in sucrose gradients of pH 5.5 resulted in the formation of a second band, at the same density as the lower band of the vaccine preparation. This did not occur in gradients of pH 7.0. Treatment of cell culture-propagated rickettsiae with formalin or germicidal ultraviolet radiation induced a total shift of the less dense cell population to a zone of higher density when centrifuged isopycnically in CsC1 gradients. This density change did not occur in sucrose gradients, suggesting a difference in the effect of these treatments on the permeability of the cell membrane to sucrose and CsC1.
Rocky Mountain spotted fever vaccine was produced from rickettsiae grown in chicken embryo cells in roller bottle cultures. The rickettsiae were concentrated and purified by passage through a sucrose gradient and inactivated with formalin. This vaccine satisfactorily passed preinactivation and final container testing and is believed to be superior to the presently available yolk sac vaccine.
Growth of Rocky Mountain spotted fever (RMSF) rickettsiae in duck embryo cell (DEC) cultures and chicken embryo cell (CEC) cultures was evaluated. Experimental lots of duck embryo cell- and chicken embryo cell-grown Rocky Mountain spotted fever vaccines and a commercial lot of yolk sac-grown vaccine were compared for protective efficacy in rhesus monkeys. Incidence and magnitude of antibody response, febrile response, and rickettsemia, as well as incidence of fatalities, suggested that both cell culture-derived vaccines were more immunogenic than the yolk sac-grown vaccine.
The development of a new diagnostic procedure for the identification of Venezvelan, eastern and western equine encephalomyelitis (VEE, EEE, WEE) viruses is described. The procedure utilizes virus precipitation with reference fluorescein-conjugated gamma globulin, followed by cellulose acetate electrophoresis. Clinical specimens containing varying concentrations of virus yielded, in primary duck embryo cell culture, sufficient virus for detection within 22 to 44 hours. Identification of VEE, EEE and WEE virus in specimens was accomplished by microprecipitation within this time. In contrast to conventional identification methods, our procedure eliminates the cost of utilizing laboratory animals and considerably reduces the time required for virus identification.