Biochemistry of rickettsiae and their infected hosts, with special reference to Coxiella burneti.
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BACKGROUND: Lone Star ticks (Amblyomma americanum) have been suggested as a vector of the agent of Lyme disease (Borrelia burgdorferi sensu lato) in the USA, based on associations with an infection manifesting mainly as erythema migrans. In laboratory experiments, however, they failed to transmit B. burgdorferi sensu stricto. METHODS: In this study, carried out from 1994 to 1996, we determined the seroprevalences of B. burgdorferi (1.2%), Ehrlichia chaffeensis (7%), E. phagocytophila (0%), Rickettsia rickettsii (0%), R. typhi (0%), Coxiella burneti (0%), Francisella tularensis (0%), and Babesia microti (0%) by standard serological methods for 325 residents (97% of the total population) of Gibson Island, coastal Maryland, USA, where 15% of the residents reported having had Lyme disease within a recent 5-year span. FINDINGS: Of the 167 seronegative individuals who were followed up prospectively for 235 person-years of observation, only 2 (0.85%) seroconverted for B. burgdorferi. Of 1556 ticks submitted from residents, 95% were identified as Lone Star ticks; only 3% were deer ticks (Ixodes dammini), the main American vector of Lyme disease. B. burgdorferi s.s. infected 20% of host-seeking immature deer ticks, and borreliae ("B. lonestari") were detected in 1-2% of Lone Star ticks. Erythema migrans was noted in 65% of self-reports of Lyme disease, but many such reports indicated that the rash was present while the tick was still attached, suggesting a reaction to the bite itself rather than true Lyme disease. Sera from individuals reporting Lyme disease generally failed to react to B. burgdorferi or any other pathogen antigens. CONCLUSION: The residents of Gibson Island had an exaggerated perception of the risk of Lyme disease because they were intensely infested with an aggressively human-biting and irritating nonvector tick. In addition, a Lyme disease mimic of undescribed etiology (named Masters' disease) seems to be associated with Lone Star ticks, and may confound Lyme disease surveillance. The epidemiological and entomological approach used in this study might fruitfully be applied wherever newly emergent tickborne zoonoses have been discovered.
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Rickettsiae of the spotted (SF) group were found in a selected locality of the Tribec Mountains in southern Slovakia in 1974. The rickettsiae were found in 10.8% of females and 6.1% of males of Haemaphysalis inermis and 7.3% of females and 4.8% of males of Ixodes ricinus. No significant differences in the incidence were observed during the year. Coxiella burneti was found in 1 male and 4 females of H. inermis and 1 male of I. ricinus in ad adjacent locality. Antibodies against rickettsiae of the SF group were detected in 22.5% and against C. burneti in 1.9% of small mammals in other adjacent localities. Trypanosomes and Nosema slovaca were recovered from I. ricinus ticks; both records are the first for Europe.
Microcinematography, cytological and cytochemical studies revealed new features of D. sibericus, D. murinus, and R. tsutsugamushi biology, mainly a very active movement in the cell. In contrast to D. sibericus, development of D. murinus and R. tsutsugamushi in cells is characterized by severe irritation of the infected cells early in the infection, a greater speed of movement of rickettsiae and their active release from the affected cells, as well as the development of necrobiotic changes in the latter. The mitotic activity of the cells increased early in cultures infected with Cox. burneti, R. prowazeki, D. sibericus, and T. tsutsugamushi. Subsequently, cell nuclei shrink and different cytotic changes develop in various rickettsia. Studies on the metabolism of the affected cells revealed activation of nucleic metabolism and redox enzymes associated with mitochondria in the course of the first 3 days followed by a low activity in 5 postinfection days. The lysosomal apparatus of the cells underwent drastic changes and the activity of acid phosphatase increased markedly with further elevation in the permeability of lysosomal membranes.
A rickettsia isolated from a parrot was found by use of electron microscopic and serological technic to be a Coxiella burneti. In cell culture one can see a dramatic polymorphism and a Gram negative-like structure of the cell wall. They divide by binary fission with no appearance of a complexe cycle of development like the one described for Chlamydia psittaci. Many myelinic fibrils and altered Coxiella suggest that the host cell react very deaply.
The authors studied a possibility of using the antigen neutralization test with dry immunoglobulin typhus erythrocytic diagnostic agent for the purpose of detection of Rickettsia prowazeki antibodies. Blood sera of 315 healthy persons, 24 patients with sporadic typhus, and 18 laboratory animals immunized with R. sibirica and R. burneti, as well as with Proteus OX19 were examined. The results obtained pointed to the high specificity and sensitivity of the given serological test. A possibility of its use for antibody detection both in the typhus patients and in persons who sustained this infection in the past was demonstrated. In difference from the complement fixation test it permits to study anticomplementary sera.
Based on a long-term study of many years, a report is given on the behaviour of Coxiella burneti in the argasid tick, Ornithodoros moubata. Recent observations were made on ticks infected on mice and subsequently maintained separately. Particular emphasis was placed upon the localization of the ricksettsiae in certain organs of the ticks, its excretion -also with regard to routes of transmission - and transovarial passage. C. burneti invade primarily the gut epithelial cells of the tick and these cells remain infested with the causative agent throughout the entire life of the vector. After a certain infection period, other organs of the tick may become also infected. Thus, infection of the coxal organ, of salivary glands, rectal ampullae and ovaries has been confirmed through the presence of the rickettsiae in the coxal fluid, saliva and excreta or through transovarial passage of the agent with the saliva during feeding transmission to a new host takes place. Infection of the various organs of the tick and excretion or passage of the agent is not necessarily the rule, but may be even considered as an exception. This is especially the case for transovarial passage. On the other side, the multiplication of rickettsiae in the tick can be so intensive that the haemolymph and all internal organs become flooded with the organisms. These observations have been made in moribund and dead ticks. One of the most remarkable results was that the behaviour of C. burneti in its tick host varied considerably not only within the same series of experiments but more frequently in one and the same individual tick as well. Thus, e.g. excretion of the agent - with the saliva or coxal fluid - could be interrupted and resumed again later on. Evidently, these variations indicate a shift in the host-parasite interrelationship in which the multiplication of the rickettsiae is greatly enhanced by a decrease in the host's immune response, whereas an increase in its defence reaction will cause suppression of multiplication. It is not known, however, if the relevant impulse is primarily given by the tick or the rickettsial organism. The behaviour of C. burneti in O. moubata which does not follow a definite pattern coincides well with the high adaptability and variability characterizing this microorganism in other aspects as well.
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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.
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The study was made on reservoirs of infections (rodent, sheep, goat, dromedary), vectors (louse) and human-receptors. Examinations were made on smears examined with staining and indirect-fluorescente techniques and on sera examined by complement fixation test. Chlamydia psittaci appears very often in rodent, sheep, goat, dromedary and man. Antibodies against Coxiella burneti were found present in sheep, goat but mainly in dromedary and less than 16 years old boys. Two lice were carrier of Rickettsia prowazeki; Rickettsia mooseri was detected on one louse and one mouse.
The role played by ixodes ticks in the epizootiology and epidemiology of Q fever was studied by following up the transphasal and transovarial passing of Rickettsia burnetii in ticks of the Rhipicephalus bursa and Hyalomma detritum species. Use was made of the 269th passage of the rickettsial strain BP, isolated in this country from sheep. The infection was followed up from larva Ist generation to larva IInd generation of the two ticks. In 10 out of the eleven samples of ticks studied there were rickettsiae in all developmental stages of the ticks, and this pointed to the phase transmission of strain BP via the metamorphosis. Of all 16 samples of eggs and second generation larvae of infected ticks, three egg samples were positive of both species of ticks, and one samples of larvae was positive of H. detritum ticks. Results showed that the infection may be passed transovarially, however, in a comparatively low per cent.