Vaccine prophylaxis of abattoir-associated Q fever: eight years' experience in Australian abattoirs.

During the period 1981-8 a clinical trial of a Q fever vaccine (Q-vax; Commonwealth Serum Laboratories, Melbourne) has been conducted in abattoir workers and other at-risk groups in South Australia. Volunteers in four abattoirs and visitors to the abattoirs were given one subcutaneous dose of 30 micrograms of a formalin-inactivated, highly-purified Coxiella burnetii cells, Henzerling strain, Phase 1 antigenic state, in a volume of 0.5 ml. During the period, over 4000 subjects have been vaccinated and the programme continues in the abattoirs and related groups. 'Common' reactions to the vaccine comprised tenderness and erythema, rarely oedema at the inoculation site and sometimes transient headache. Two more serious 'uncommon' reactions, immune abscess at the inoculation site, were observed in two subjects, and two others developed small subcutaneous lumps which gradually dispersed without intervention. Protective efficacy of the vaccine appeared to be absolute and to last for 5 years at least. Eight Q fever cases were observed in vaccinees, but all were in persons vaccinated during the incubation period of a natural attack of Q fever before vaccine-induced immunity had had time (greater than or equal to 13 days after vaccination) to develop. On the other hand, 97 Q fever cases were detected in persons working in, or visiting the same abattoir environments. Assays for antibody and cellular immunity showed an 80-82% seroconversion after vaccination, mostly IgM antibody to Phase 2 antigen, in the 3 months after vaccination. This fell to about 60%, mostly IgG antibody to Phase 1 antigen, after 20 months. On the other hand, 85-95% of vaccinees developed markers of cell mediated immunity as judged by lymphoproliferative responses with C. burnetii antigens; these rates remained elevated for at least 5 years. The Q fever vaccine, unlike other killed rickettsial vaccines, has the property of stimulating long-lasting T lymphocyte memory and this may account for its unusual protective efficacy as a killed vaccine.

Rickettsiae as organisms.

Although most pathogenic rickettsiae are obligate intracellular parasites, it is clear that they are bacteria. As such, form and function in rickettsiae are closely similar to form and function found in their free-living counterparts. This review of rickettsiae as bacteria portrays the broad similarities of rickettsiae and free-living bacteria, as well as the differences which distinquish one group from the other and one rickettsia from another. Growth characteristics and requirements, ecologic influences, special adaptations, antibiotic susceptibilities and host-parasite relationships will be considered in a broad survey of likenesses and differences displayed by rickettsiae pathogenic to man.

Vaccine prophylaxis of abattoir-associated Q fever.

Q fever is an important cause of morbidity in Australian meatworkers; recently there have been sharp outbreaks of Q fever in abattoirs in several states. In an attempt to control Q fever by vaccination, 924 nonimmune volunteers at two South Australian abattoirs were inoculated with one dose of a purified, formalin-inactivated, Coxiella burneti, Henzerling strain, phase 1 vaccine. Some 56% of workers in one abattoir, and 64% in the other, seroconverted after vaccination. In the 18 months after vaccination, no Q fever occurred in fully vaccinated subjects, whereas there were 34 cases in 1349 unvaccinated workers. Transient local reactions were noted in most vaccinated subjects; only a few had mild general reactions. No cases of vaccine-enhanced disease were observed. Vaccination of susceptible individuals with a purified C burneti phase 1 vaccine appears to be safe and effective in preventing Q fever in the abattoir.

A comparison of some biologic characteristics of isolates of the Legionnaires' disease bacterium.

The ability of three isolates of serogroup 1 and one isolate of serogroup 4 of Legionnaires' disease bacterium (LDB) to infect and cause fever and death in guinea pigs was studied, as well as their ability to produce plaques in cultured primary chick embryo cells. The serogroup 4 isolate originally was recovered from cord clot and placental tissue from a healthy mother following delivery of a normal child. The effects on LDB of prolonged cultivation on supplemented Mueller-Hinton (MH) agar medium and of subsequent cultivation in yolk sacs of chick embryos were examined. Prolonged cultivation of LDB on MH medium resulted in great loss of ability to produce plaques and to cause fever and death in guinea pigs. Subsequent passage in embryonated eggs of MH-adapted LDB tended to restore ability to produce plaques and to cause infection and illness in guinea pigs. Fatty acid composition profiles of the four strains were similar to each other.

Q fever endocarditis in the United States.

A patient with Q fever endocarditis, which is almost unknown in the United States, was followed for a total of 32 months; the study was begun 3 1/2 months before aortic valve replacement. Diagnosis was confirmed by serology, visualization of Coxiella burnetii in excised aortic valve tissue by direct and immunofluorescence staining, and isolation of C. burnetii from aortic valve tissue. Serum antibodies against phase I and phase II antigens of C. burnetii were identified. Almost all phase I and phase II antibodies were IgG. These findings are compared with those in an uncomplicated case of acute Q fever. New findings on the immune response to chronic Q fever are presented.

Antibody response in man following a small intradermal inoculation with Coxiella burnetii phase I vaccine.

A small inoculum (0.2 microgram) of phase I Coxiella burnetii vaccine given to individuals previously sensitized to CO burnetii elicited a positive skin reaction and a strong IgM phase I antibody response as determined by microagglutination, complement fixation and microimmunofluorescence tests. A similar inoculum administered to nonsensitized individuals did not elitic a skin reaction nor stimulate a recognizable antibody response. Serum from one of these sensitized and skin tested individuals was fractionated by gel filtration methods. The serum and serum fractions were titrated in a mouse seroprotection test using primary chicken embryo cell culture plaque technique as the assay procedure. Results of the mouse seroprotection test indicated that most of the protective activity of the serum was associated with the IgM fraction and that phase I IgM antibody suppressed the growth of C. burnetii in mouse spleen when mixed with the rickettsial suspension prior to inoculation.

Diagnostic specificity of immunoglobulin M (IgM) response in differentiation Legionnaires' disease from psittacosis.

Specific IgM and IgG antibody responses to Legionella pneumophila (LDB) and Chlamydia psittaci (PSI) in serum specimens from 22 cases of Legionnaires' Disease (LD) were examined by micro-immunofluorescence (IF) tests to explore the diagnostic significance of the IgM antibody response. Serial samples from 5 patients with LD showed greater than or equal to 4-fold changes in IgG antibody against LDB and PSI. All 5 patients possessed IgM antibodies against LDB but not against PSI. In single convalescent serum samples from 17 additional cases, 16 exhibited IgG and 15 showed IgM antibodies against LDB; all 17 exhibited IgG but not IgM antibodies against PSI. The IgM antibody response appears more specific than the corresponding IgG response in the serodiagnosis of LD, and may be valuable in differentiating LDB infections from those due to PSI.

Legionnaires' disease: antigenic peculiarities, strain differences, and antibiotic sensitivities of the agent.

Paired sera from victims of Legionnaires' disease showed, in many cases, significant rises in immunoglobulin G antibodies to both the causative agent (LA) of Legionnaires' disease and Chlamydia psittaci, but concurrent rises in immunoglobulin M antibodies only against LA. Guinea pigs experimentally infected with LA likewise responded with antibodies to both C. psittaci and LA. Guinea pigs infected with LA also reflected significant differences in antigenic makeup and in pathogenicity among four strains of LA examined. In antibiotic studies, rifampin was 200 times more effective than erythromycin and 17,000 times more effective than tetracycline in plaque reduction tests of LA in monolayer cultures of primary chick embryo cells. An isolate of LA recovered from a healthy person was compared with three isolates from persons with fatal infections.

Production of antibody in induced granulomas.

A method of producing antibodies in artificially induced granulomas with various microbial antigens is examined.

Microimmunofluorescence test for the serological study of rocky mountain spotted fever and typhus.

A microimmunofluorescence test was used to study antibody responses to various spotted fever group and typhus group rickettsiae during Rocky Mountain spotted fever (RMSF) and epidemic typhus (ET). Patients with RMSF reacted most strongly to Rickettsia rickettsii; those with ET reacted predominantly to R. prowazekii. The degree of cross-reaction to other rickettsial strains varied from patient to patient, but a particular pattern of cross-reaction was consistently observed in serial sera from the same patient. Fresh isolates from three Montana RMSF cases were indistinguishable from each other and from strain R of R. rickettsii used as a standard antigen in all tests. Immunoglobulin M (IgM) antibodies were usually present in high titer in early-convalescent-phase sera from RMSF, as well as ET, patients. After RMSF, IgM antibodies persisted for a few months and, in one instance, for as long as 10 months. IgM responses to laboratory-acquired infections were infrequent in persons previously vaccinated with antigens related to the infecting strain. Previous antigenic conditioning from infection or vaccination may have accounted partly for the apparent lack of IgM response in a few study participants.

Plaque assay of rickettsiae in a mammalian cell line.

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells.

A search for the epidemic typhus agent in Ethiopian ticks.

The presence of antibodies to Rickettsia prowazeki in domestic animals from several parts of Africa, and the isolation of this rickettsia from the blood of goats and sheep and from ticks off cattle or camels in Ethiopia, led to the hypothesis that R. prowazeki in nature may occur in an extrahuman cycle involving ticks and domestic animals. This study attempted to recover R. prowazeki from 2 624 ticks (4 genera, 10 species) collected in central and southern Ethiopia. The ticks were examined by the haemolymph test and by the injection of tissues into guineapigs. No strains of typhus rickettsia were received and there was no serologic evidence suggesting the presence of this agent in any of the ticks examined. One Amblyomma cohaerens contained an organism that reacted specifically with fluorescing antibodies against R. prowazeki; attempts to isolate and identify this agent failed. Fifty-seven (2.2%) Amblyomma ticks (26 A. gemma, 17 A. variegatum, 14 A. cohaerens) were infected with rickettsiae of the spotted fever group, and probably represented R. conori or closely related rickettsial agents.

DNA base composition of rickettsiae.

There is a small but distinct difference in DNA base composition between the typhus and spotted fever groups of rickettsiae. The molar percentages of guanine plus cytosine for Rickettsia prowazeki, R. typhi, and R. canada are approximately 30, for R. rickettsi, R. conori, and R. akari they are about 32.5. The percentage for trench fever rickettsia, Rochalimaea quintana, is 38.6.

Effects of various suspending media on plaque formation by rickettsiae in tissue culture.

Effects of some media used for suspending rickettsiae during purification, for metabolic studies, and in titrations of infectious rickettsiae were examined with respect to the plaque-forming ability of Rickettsia rickettsi and R. typhi in primary chicken embryo tissue cultures and the infectivity of R. typhi in mice. Brain heart infusion broth (BHI) was found superior to all other media tested in preventing both a significant decrease in plaque-forming units (PFU) and a delay in plaque formation. Skim milk, egg yolk, and some metabolic media were effective in maintaining PFU at 0 C, but did not prevent a significant delay in plaque formation. However, infectivity of R. typhi for tissue culture and mice was markedly decreased when suspended in metabolic media at 26 C. Addition of BHI to the routine tissue culture overlay reversed the deleterious effects of sucrose-phosphate solutions. The effects of Mg(2+), Mn(2+), K(+), Na(+), sucrose, and glutamate were also examined. No significant differences were observed between R. rickettsi and R. typhi in their responses to different media. The results of this study suggest the necessity for a reappraisal of previous studies of metabolism and infectivity of rickettsiae in these media.

Studies of the rickettsial plaque assay technique.

A plaque assay system for pathogenic rickettsiae, which utilizes primary chick embryo tissue cultures, is described. It proved to be a highly reproducible measure of infectiousness for Rickettsia rickettsi and R. typhi, which were employed in most studies; as well as for R. canada, R. prowazeki, R. sibirica, R. akari, R. conori, and Coxiella burneti. Plaque-forming units (PFU) were compared to direct rickettsial counts and to 50% infectious dose (ID(50)) values for embryonated eggs, mice, and guinea pigs. Plaque size, appearance, and number were influenced by diluent, incubation temperature after nutrient overlay, centrifugation of inoculated tissue cultures, and number of host cells planted initially in each flask. The most critical factors in plaque formation were diluent used in making rickettsial suspensions and incubation temperature (32 C) after nutrient overlay. Brain Heart Infusion was the only diluent capable of preventing significant delay in plaque formation and decreases in PFU and mouse ID(50). Plaque formation was unaffected by genetic background of host cells, volume of inoculum, temperature and length of incubation period before nutrient overlay, and rapid freezing and thawing of rickettsial seed. Centrifugation of inoculated cultures at 600 x g resulted in 100% irreversible absorption of rickettsiae to host cells within 5 min, whereas without centrifugation at least 4 hr was required to achieve the same effect.