Diversity of Coxiella-like and Francisella-like endosymbionts, and Rickettsia spp., Coxiella burnetii as pathogens in the tick populations of Slovakia, Central Europe.

Ticks are important vectors of pathogens affecting humans and animals worldwide. They do not only carry pathogens but diverse commensal and symbiotic microorganisms are also present in ticks. A molecular screening for tick-borne pathogens and endosymbionts was carried out in Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis inermis questing ticks collected in Slovakia. The presence of Rickettsia spp., Coxiella burnetii, Coxiella-like and Francisella-like microorganisms was evaluated by PCR in 605 individuals and by randomly sequencing 66 samples. Four species of rickettsiae (R. raoultii, R. slovaca, R. helvetica and R. monacensis) were identified and reported with an overall prevalence range between 0.4 and 50.3% (±8.0) depending on tick species, sex and locality. Partial sequencing of the gltA gene of 5 chosen samples in H. inermis showed 99% identity with Candidatus Rickettsia hungarica. The total prevalence of C. burnetii in ticks was 2.2 ±â€¯1.7%; bacteria were confirmed in I. ricinus and D. reticulatus ticks. The sequences from 2 D. reticulatus males and 1 I. ricinus female ticks were compared to GenBank submissions and a 99.8% match was obtained with the pathogenic C. burnetii. Coxiella-like endosymbionts were registered in all three species of ticks from all studied sites with an average prevalence of 32.7 ±â€¯3.7%. A phylogenetic analysis of this Coxiella sp. showed that it does not group with the pathogenic C. burnetii. The prevalence of Francisella-like microorganisms in questing ticks was 47.9 ±â€¯3.9%, however H. inermis (n = 108) were not infested. Obtained sequences were 98% identical with previously identified Francisella-like endosymbionts in D. reticulatus and I. ricinus. Coxiella-like and Francisella-like microorganisms were identified for the first time in Slovakia, they might be considered as a non-pathogenic endosymbiont of I. ricinus, D. reticulatus and H. inermis, and future investigations could aim to assess their role in these ticks. However, this work provided further data and broadened our knowledge on bacterial pathogens and endosymbionts present in ticks in Slovakia to help understanding co-infestations, combined treatments and public health issues linked to tick bites.

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.

Preparing and staining of Coxiella burnetii natural phase II antigen for the microagglutination reaction.

An improved method of preparing Coxiella burnetii natural phase II antigen of high purity enabled its staining for use in the microagglutination reaction (MAR). The antigen was as specific and sensitive for detection of phase II antibodies as the artificial phase II C. burnetii antigen prepared by periodate treatment from purified phase I cells of C. burnetii.

Phagocytosis of Coxiella burneti by macrophages.

Live or killed purified phase I and phase II Coxiella burneti organisms were phagocytized to a similar extent by mouse or guinea pig peritoneal macrophages and polymorphonuclear leukocytes (PMN); phase II was much more susceptible to phagocytosis than phase I. Phagocytosis of phase I was enhanced only by immune sera containing phase I antibodies. Increased in vivo phagocytosis of phase I organisms by macrophages from animals immunized with live phase I C. burneti was lost following their in vitro cultivation and washing.

Attempts at demonstration of lipopolysaccharide in phase II Coxiella burnetii.

Comparison of some properties of phase I and phase II Coxiella burnetii cells suggests the presence of lipopolysaccharide (LPS) also in the surface structures of phase II cells. Polysaccharide chains were released from them by mild acid hydrolysis and corpuscular residues resulting from such hydrolysis elicited in rabbits anti-lipid A-antibodies. Toxicity for adrenalectomized and actinomycin D-sensitized mice was demonstrated with phase I cells, but not with much higher concentrations of phase II cells. By contrast, the extent of protection against formation of ascitic tumour in mice with comparable concentrations of phase I and phase II cells was similar.

[Properties of phase I purified cellular antigen of Coxiella burnetii]. / Wlasciwosci oczyszczonego, komórkowego antygenu Coxiella burnetii fazy I.

An attempt was made to purify phase I cell suspension of Coxiella burnetii used as an antigen in diagnostic serological tests. Homogenised suspension of chick embryos infected with phase I Henzerling and "Z" strains, after preliminary purification from host cell contaminants of chick embryos was subjected to consecutive centrifugation in sucrose/uropoline gradient and to continuous 20-45% uropoline gradient. The fractions obtained from uropoline gradient centrifugation were applied as phase I antigen C. burnetii in the following tests: complement fixation and microagglutination. Only fractions containing protein were serologically active. They proved to be of similar specificity and sensitivity as the antigens obtained by standard method. Moreover, it was found that after formalin treatment of C. burnetii cells no soluble antigens are liberated which could be detected by complement fixation test.

[Current data on the polymorphism of Rickettsia prowazekii and burneti in cultured cells]. / Novye dannye o polimorfizme rikketsii Provacheka i Berneta pri kul'tivirovanii v kul'ture kletok

In cultivation of Rickettsia prowazeki (strains Breinl and E) in the cell cultures of guinea pig kidneys (GPK) and chick embryo fibroblasts (CEF) ultrastructure of rickettsia of unusual shape (filamentous, irregularpleomorphic and spheroplast-like) were revealed along with rickettsia of the usual shape and size. The polymorphism was less pronounced in the GPK and the CEF cells of Rickettsia burneti (strain M-44). It is supposed that rickettsial polymorphism was not associated with their developmental cycle and served as a morphological expression of the changes in the microorganism under the effect of unfavourable ecological conditions. The appearance of filamentous forms could be associated with disturbed cell division process; changed rigidity of the cell wall could serve as the cause of appearance of pleomorphic rickettsia. In difference from polymorphism, the cycle of rickettsial development is considered to be (in the basis of modern electron microscopic data) as a biological replacement of the vegetative (rod-like, bacillary) forms by those more stable in the external environment, resting (coccoid).

A proposed model to explain persistent infection of host cells with Coxiella burnetii.

L929 mouse fibroblast cells and J774 macrophage-like cells are both susceptible to persistent infection with the Q fever agent Coxiella burnetti. Previously this laboratory has shown that persistently infected cell populations multiply with unaltered generation times or cell cycle progression. It has also been reported by others and us that highly infected cells typically exhibit one large parasite-containing vacuole. We now report that lightly and heavily infected cells are capable of division and in the process segregate the parasite-containing vacuole into one of the emerging daughter cells; the companion daughter cell emerges parasite-free. This asymmetric division of infected cells, revealed via photomicrography of stained cells, accounts for the appearance of uninfected cells within persistently infected host cell populations that were previously 100% infected. Some of the persistently infected L929 populations were maintained in culture for over two years without the addition of normal cells.

Purification of large quantities of coxiella burnetii rickettsia by density gradient zonal centrifugation.

The purification of large quantities of inactivated, phase II Coxiella burnetii by isopycnic zonal centrifugation for use as diagnostic antigen and as a vaccine is described. The fractionation of egg yolk sac-derived C. burnetii vaccine resulted in the separation of two distinct populations of organisms, each devoid of microscopically and serologically recognizable components of egg yolk sac. One population of organisms, characterized by an equilibrium density of 1.240, was rod shaped (1.0 by 0.5 mumole) with a thick, densely strained wall and prominent central body. The second population, with an equilibrium density of 1.280, had a coccobacillary shape (approximately 1 mumole in diameter), granular, sometimes fibrillar cytoplasm, thin cellular walls, and lacked a prominent nucleoid.

Isolation and characterization of two cell types of Coxiella burneti phase I.

Two morphologically distinct cell types of Coxiella burneti phase I have been separated on the basis of unique buoyant densities. When centrifuged to equilibrium in cesium chloride or density gradients of sucrose or Renografin, the cells band in two zones. Electron micrographs of ultrathin sections of the two cesium chloride-separated cell types indicate a considerable number of morphological differences. The lower-density cells are small, compact, and rodshaped and have very dense nucleoids. The cell type of highest density is larger, rounded, and more pleomorphic, and the nucleoid filaments are more dispersed. The two cell types are nearly identical in sedimentation rates, and both infect chick yolk sac cells and are lethal to chick embryos. They convert to a mixture of cell types when cultured separately. Treatment with Formalin induces all cells to band at the same position when centrifuged to equilibrium in cesium chloride. The cell type variance was found to be independent of the antigenic phase phenomenon of C. burneti.

Ribosomes and ribonucleic acids of Coxiella burneti.

This report describes the direct isolation and characterization of rickettsial ribosomes. Ribosomes from the rickettsia Coxiella burneti were isolated and partially characterized. The ribosomes had a sedimentation constant of about 70S and could be dissociated into 50 and 30S subunits. Electron microscopy revealed ribosomal particles with dimensions similar to those reported for other procaryotic organisms. Ribonucleic acid (RNA) species (23 and 16S) were isolated from the ribosomal particles. The nucleotide compositions of the ribosomal RNAs were found to be similar to those reported for bacterial ribosomal RNA. In addition to the high-molecular-weight ribosomal RNA, 5S RNA was also extracted from the organism.

Experimental Q fever in sheep.

The aim of the present investigation is the complex study of experimental infection in pregnant ewes by means of clinical, serological, biological, histological and Electron microscopy methods. Four ewes, pregnant from the 2nd to 5th month, were infected by intravenous (in one case by intraperitoneal) routes with a C. burnetii strain at 10(6) ID 50/ml. The clinical illness in all of the animals was characterized by fever and two-phase temperature reaction on the 5th and 12th days. The clinical symptoms were as follows: torpidity, reduced appetite, thirst, conjuctivitis, rhinitis, rapid breathing. As a result of the developed latent infection, after the acute stage, the animals gave birth to three unviable lambs who died within 24 h. Another lamb was still-born. The lambs showed cachexia, arthritis, ataxia, wrinkled skin. The highest CF-titers (1:256-1:512) were reached on the 40th day, but serum antibodies (1:8-1:32) first appeared on the 8th day. The titers began to decrease on the 60th day. The pathomorphological changes testify to a latent infection characterized by placentitis, lymphocellular proliferation of the lamb's parenchymal organs and lymph nodes, multiple thromboses, interstitial pneumonia and plural proliferative changes. The EM exam showed rickettsiae in placentas mainly in the form of inclusions in cytoplasm of leukocytes and epithelial cells.

Q fever pneumonia.

Pneumonia is one of several clinical syndromes that results from inhalation of Coxiella burnetii. This microorganism, the etiologic agent of "Q" (query) fever, infects a wide range of animals and insects. Cattle, sheep, goats, and cats are the reservoirs whereby this agent is spread to humans. High concentrations of C burnetii are present in the placenta and at parturition, the organism is shed into the environment to be inhaled by humans. Following an incubation period that ranges from four to 30 days (mean 14 days), fever, headache, malaise, and cough ensue. The clinical presentation of pneumonia may range from a mild to a severe illness--the latter with the clinical picture of rapidly progressive pneumonia. There are no characteristic features of Q fever pneumonia but the severe headache and the epidemiological history should serve as clues. Treatment with tetracycline or rifampin for two weeks usually results in cure. Many cases of Q fever pneumonia remit without antibiotic therapy. The diagnosis is usually confirmed serologically using a complement fixation or microimmunofluorescence test.