Coxiella burnetii in ticks and wild birds.

The study objective was to get more information on C. burnetii prevalence in wild birds and ticks feeding on them, and the potentialities of the pathogen dissemination over Europe by both. MATERIALS: Blood, blood sera, feces of wild birds and ticks removed from those birds or from vegetation were studied at two sites in Russia: the Curonian Spit (site KK), and the vicinity of St. Petersburg (site SPb), and at two sites in Bulgaria: the Atanasovsko Lake (site AL), and the vicinity of Sofia (site SR). METHODS: C. burnetii DNA was detected in blood, feces, and ticks by PCR (polymerase chain reaction). All positive results were confirmed by Sanger's sequencing of 16SrRNA gene target fragments. The antibodies to C. burnetii in sera were detected by CFR (complement fixation reaction). RESULTS: Eleven of 55 bird species captured at KK site hosted Ixodes ricinus. C. burnetii DNA was detected in three I. ricinus nymphs removed from one bird (Erithacus rubecula), and in adult ticks flagged from vegetation: 0.7% I. persulcatus (site SPb), 0.9% I. ricinus (site KK), 1.0% D. reticulatus (AL site). C. burnetii DNA was also detected in 1.4% of bird blood samples at SPb site, and in 0.5% of those at AL site. Antibodies to C. burnetii were found in 8.1% of bird sera (site SPb). C. burnetii DNA was revealed in feces of birds: 0.6% at AL site, and 13.7% at SR site. CONCLUSIONS: Both molecular-genetic and immunological methods were applied to confirm the role of birds as a natural reservoir of C. burnetii. The places of wild bird stopover in Russia (Baltic region) and in Bulgaria (Atanasovsko Lake and Sofia region) proved to be natural foci of C. burnetii infection. Migratory birds are likely to act as efficient "vehicles" in dispersal of C. burnetii -infested ixodid ticks.

Study on the substituents' effects of a series of synthetic chalcones against the yeast Candida albicans.

A large series of chalcones were synthesized and studied for activity against Candida albicans. The SAR analysis showed that the antifungal activity was highly dependent on the substitution pattern of the aryl rings and correlated to a large extent with the ability of compounds to interact with sulfhydryl groups. The most active were the hydroxylated chalcones as their activity related to the location of the phenolic group in the aryl ring B as follows: o-OH>p-OH approximately 3,4-di-OH>m-OH. These and other correlations obtained strongly contribute to the knowledge for design of anticandidal chalcones.

Modulation of complement activity in vitro and in vivo by Yersinia wild and mutant strains.

The ability of released proteins (Yops) and surface lipopolysaccharides (LPS) from the wild-type strain Yersinia enterocolitica 8081-L2, serotype 0:8 to influence the complement activity was determined. Yops and LPS from wild-type and mutant strains showed different ability to affect the classical pathway (CP) functional complement activity in vitro. The serum CP activity was inhibited during the infection induced with six Y. enterocolitica and three Y. pseudotuberculosis strains in rabbits. The changed complement activity might be of importance for the course of Yersinia infections.

Antimicrobial and cytotoxic activity of Ruta graveolens.

The methanol, petroleum ether, ethyl acetate and water-methanol extracts of Ruta graveolens were found to possess antimicrobial and cytotoxic activities.

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits.

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.

Chemical composition and biological activities of the Black Sea algae Polysiphonia denudata (Dillw.) Kutz. and Polysiphonia denudata f. fragilis (Sperk) Woronich.

The two investigated algae had almost identical sterol composition, but there were significant differences in the composition of the polar components and especially in the composition of the volatiles. P. denudata f. fragilis extracts possessed a stronger biological activity (antibacterial, antifungal and toxicity against Artemia salina). Despite the minute morphological differences between the two algae, we recommend P. denudata f. fragilis to be regarded as P. denudata subsp. fragilis.

Protective effect of Oxadin on experimental Yersinia enterocolitica infection in rats.

The effect of Oxadin (a new Bulgarian antimicrobial chemotherapeutic agent) on some parameters of non-specific immune response was investigated in a rat model of infection. After mimicking natural Yersinia enterocolitica systemic infection the number and functional activity of blood leucocytes and peritoneal macrophages were compared between groups of animals treated with Oxadin before and after infection. A significant immunostimulating effect of Oxadin was found in both experimental groups but was better expressed when administered before Yersinia infection. Bactericidal response of peritoneal macrophages (killing ability) and phagocytic activity of polymorphonuclear leucocytes from animals treated with Oxadin and thereafter infected with Yersinia enterocolitica were significantly activated during the first week of study. These findings correlated with the enhanced number of both types of phagocytic cells and the higher glycolytic activity of peritoneal macrophages.

Attenuation and preserved immunogenic potential of Yersinia pseudotuberculosis mutant strains evidenced in oral pig model.

Experimental oral infection of pigs with a parental Yersinia pseudotuberculosis strain pIB102, serotype O:3 and two mutant isogenic strains - pIB155,DeltayopK and pIB44,DeltaypkA has been carried out. Clinical findings, microbiological and immunological parameters were examined in dynamics from day 7 to day 60 post-infection (p.i.). All types of infections ran asymptomatically, without hyperthermia, loss of appetite, etc. Experiments on the blood parameters demonstrated a transient leucocytosis with lymphocytosis and monocytosis better expressed after yopK infection. Even though pig is usually known as a reservoir of yersiniae, bacterial colonization was found in mesenterial lymph nodes and tonsils on day 7, respectively 14 p.i. with parental strain, and only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of mutants to the bactericidal effect of leukocytes and blood sera is the characteristic feature of attenuation in their pathogenicity, compared to the parental strain. Comparative in vitro experiments on the immune response and immunostimulating capacity of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential, predominantly in case of yopK. Hyperplasia and strong activation of the lymph tissue of Peyer's patches, mesenterial lymph nodes, tonsils and spleen of pigs challenged with both mutant strains were proved as immunomorphological rearrangements. The results obtained give the reason to claim that the genetically constructed yopK null mutant strain is significantly attenuated but is still immunogenic and has the potential for a live vaccine carrier strain.

Cross-reaction between Yersinia outer membrane proteins and anti-Borrelia antibodies in sera of patients with Lyme disease.

Diagnosis of Yersinia infections accompanied by reactive arthritis could be complicated by cross-reaction with other arthritogenic bacteria. The possible cross-reaction between Yersinia antigens and anti-Borrelia antibodies in blood sera of patients with Lyme disease was studied. The occurrence of specific IgA, IgG and IgM antibodies was analyzed in serum samples from 30 patients with Yersinia-triggered reactive arthritis, 30 patients with Lyme disease and five samples from healthy blood donors. For anti-Borrelia IgG antibodies, cross-reaction was detected with YopH, YopB, V-ag, YopD, YopN, YopP and YopE, and for IgA with YopD. For IgM, no cross-reaction was detected. Owing to cross-reactivity with Borrelia, the diagnosis of Yersinia-triggered reactive arthritis should be based on a combination of serological and clinical findings.

PCR for detection of Mycobacterium tuberculosis in experimentally infected dogs.

The susceptibility of dogs to infection with Mycobacterium tuberculosis was studied through different ways of experimental infection. The examination shows, that in most cases the disease runs subclinically with pathological changes localized mainly in the lungs, lymph nodes, small intestines, liver, kidneys and spleen. Histological findings demonstrate granulomatous inflammation with caseosation and predominance of epitheloide macrophages and single lymphocytes. Tissue samples from internal organs of experimentally infected dogs as well as non-infected but contact animals were investigated by direct PCR. Specific PCR-products were obtained in 44 of 96 studied samples. Eighty-three (86.5%) of PCR results coincided with bacteriological finds, 82 (85.4%) with the pathological and 71 (74.0%) simultaneously with bacteriological and pathological results. The observed specific DNA products in tissue samples of infected and non-infected dogs demonstrate significant sensitivity of PCR method. It could be assumed that the transmission of M. tuberculosis infection is possible by close contact between ill and healthy dogs and that the naturally infected dogs or dogs suffering from tuberculosis may serve as a permanent source of infection to humans and other animals.

Experimental mixed infection of rabbits with Yersinia enterocolitica and Listeria monocytogenes.

Experimental mixed infection was reproduced in rabbits after per os infection with Yersinia enterocolitica serotype 0:3 cells. Four days later some of animals were re-infected orally with Listeria monocytogenes serotype 4b cells. A third group of healthy rabbits was also infected per os with Listeria monocytogenes. The infectious process was followed dynamically from days 1-28. The experimental animals were examined for clinical, paraclinical and morphological findings. Augmentation of body temperature and alveolar macrophage number, a decreased number of peritoneal macrophages, leucopenia as well as purulent meningoencephalitis, catarrhal pneumonia, lienitis, lymphadenitis and enteritis were detected after experimental mixed infection. Both types of macrophages demonstrated a weak bactericidal activity against Yersinia enterocolitica and a highly expressed killing effect against Listeria monocytogenes. Yersinia and Listeria cells were isolated from the viscera and brain. Both species of bacteria were established intracellularly in the macrophages by electron-microscopic examination. The data received showed that mixed Yersinia enterocolitica 0:3 and Listeria monocytogenes 4b infection of rabbits runs with transitory hyperthermia as a generalized infection and is similar to the Listeria mono-infection. The immunosuppressive effect induced by oral Yersinia enterocolitica infection of rabbits promotes the expression of listerious agents.

Efficient subtyping of pathogenic Yersinia enterocolitica strains by pulsed-field gel electrophoresis.

Yersinia enterocolitica is an enteropathogen that has recently and rapidly expanded over the world. There is a close correlation between the biotypes, serotypes, and phage types of the strains, making it virtually impossible to distinguish isolates of the same serotype with the classical phenotypic markers. In the present study, pulsed-field gel electrophoresis (PFGE) was used to compare the NotI genomic profile (i.e., pulsotype) of 20 strains each of serotypes O:3, O:9, and O:5. Eleven, 12, and 18 different pulsotypes were obtained, respectively, indicating that this technique is very efficient for subtyping pathogenic isolates of Y. enterocolitica. Within strains of serotype O:5, PFGE differentiated two subgroups that corresponded to two biotypes (biotypes 1A and 3). Comparison of the pulsotypes of three strains of biotype 3 and serotype O:3 (referred to as 3/O:3) with those of strains 4/O:3 and 3/O:5 suggested that the pulsotype is closer to the biotype than to the serotype. The pulsotypes of five pairs of strains isolated from the same patient or siblings were also analyzed. In four pairs, the two strains displayed identical pulsotypes, indicating that PFGE might be a powerful epidemiological tool. In the fifth pair, one restriction fragment differed, suggesting that genomic polymorphism may occur in vivo in Y. enterocolitica. Finally, the in vitro genomic stabilities of one strain each of Y. enterocolitica O:3, O:9, and O:5 were investigated. The pulsotypes of 10 isolated colonies were identical within each strain, indicating that in vitro, the genome of Y. enterocolitica is much more stable than that of Y. pestis.

Experimental Burkholderia pseudomallei infection of pigs.

Experimental infection with Burkholderia pseudomallei was successfully produced after a single intravenous challenge of 2-month-old pigs with a dose of 5.0 x 10(9) bacterial cells. Clinical, paraclinical and morphological findings of the infectious process and post-infectious immunity were examined up to day 30 post infection (p.i.). A transient and short hyperthermia accompanied by enhanced and longer demonstrated pulse frequency. An increased erythrocyte sedimentation rate and tachypnea were observed too after clinical examination. The infection starts with significant leucopenia, and a reduced number of alveolar and peritoneal macrophages which have been overcome in the latest intervals of infection. In contrast, the phagocytic activity of leucocytes was statistically increased during the course of infection and up to day 15 p.i. in the case of alveolar macrophages. Burkholderia pseudomallei was able to colonize the lungs during the whole experiment and was only present 3 days in the spleen and mesenterial lymph nodes (MLN). Significant antibody response was developed as early as day 7 p.i. Hyperaemia, haemorrhages and necrotic foci were found in the brain, liver spleen and MLN. Lung tissue was also hyperaemic, with formation of small abscesses and signs of catarrhal pneumonia. Data obtained in this study revealed that B. pseudomallei causes a chronic generalized infection in pigs, even after intravenous challenge.

Isolation of pathogenic yersiniae from wild animals in Bulgaria.

Pathogenic Yersinia strains were isolated between December 1998 and April 1999 from 37 wild animals: rabbit (Lepus europeus), boar (Sus scrofa scrofa), asiatic jackal (Canis aureus), red fox (Vulpes vulpes), mouflon (Ovis musimon), european river otter (Lutra lutra), beech marten (Martes foina), polecat (Musleta putorius) and wild cat (Felis silvestris). It was established that among the wild animals Y. enterocolitica strains of serotype 0:3 predominated, accompanied by Y. pseudotuberculosis strains of serotype 0:3. In one sample from asiatic jackal and one sample from rabbit, Y. enterocolitica serotype 0:8 was isolated. Yersinia enterocolitica and Y. pseudotuberculosis strains were isolated from tonsils and tongues as well as from the viscera--lung, liver, heart, spleen, kidney and lymph nodes, mainly in young animals (1-2 years of age). The results showed that wild animals are a possible natural reservoir for pathogenic Y. enterocolitica and Y. pseudotuberculosis and are included in the epidemiological chain of yersinioses.

Experimental melioidosis in hens.

Experimental intramuscular infection of hens with Pseudomonas pseudomallei, strain 2796 (1 x 10(9) CFU from a 24-h culture) was reproduced. Clinical, paraclinical and pathomorphological findings were followed from 1 to 30 days after challenge. Haemagglutinin titre, bacterial dissemination in the viscera, number of leucocytes, alveolar (aMa) and peritoneal (pMa) macrophages and their phagocytic activity in vitro were studied. During the course of infection a leucocytosis as well as an increased haemagglutinin titre (1:256) were established. The number of bacteria per gram tissue in the spleen and liver was highest at 1 day post-infection (p.i.). Melioidose bacteria from egg yolk were isolated at 15 and 30 days p.i. Leucocyte and pMa phagocytic activity was maximal at 3 days p.i. unlike the activity of aMa which increased gradually until the end of the study. Inflammatory-necrotic changes were found in the viscera and brain at 3 and 15 days p.i. The investigation of experimental melioidosis infection in hens showed that they are susceptible to P. pseudomallei and this disease takes a generalized subacute course.

Studies of Yersinia enterocolitica 0:3 experimental infection in pigs.

Y. enterocolitica (serotype 0:3, pYV+, biotype 4) infection of 20-day-old pigs challenged per os with a total dose of 5 x 10(10) CFU was studied. Clinical, paraclinical and morphological findings were examined in dynamics from 1st to 25th days post infection (p.i.). Augmentation of body temperature and erythrocyte sedimentation rate during the first days p.i. were established. The number of leucocytes, peritoneal (pMa) and alveolar (aMa) macrophages was increased significantly from 4th to 15th days p.i. Phagocytic activity of pMa and aMa examined in vitro was maximal on days 15 and 25 p.i. The enhanced phagocytic activity of macrophages was in correlation with the observed histological changes--purulent meningoencephalitis, necrotic tonsillitis, peribronchial lymphoid-leucocytic cell infiltration and catarrhal enteritis. Extensive colonization of internal organs was detected at necropsy till the end of trial. Analysis of the results shows that this orally caused infection runs slowly with dissemination and persistency of Y. enterocolitica 0:3 in the macroorganism, like a generalized infection.

Arthritis after experimental infection with Yersinia enterocolitica 0:3 in rabbits.

Arthritis in rabbits was caused after experimental oral infection with Yersinia enterocolitica (serotype 0:3, biotype 4, pYV+). Clinical and laboratory signs, bacterial dissemination to the viscera, immune response and morphological findings were studied from day 1 to day 40 post-infection (p.i.). Augmentation of body temperature and erythrocyte sedimentation rate occurred on day 1, and on day 8 p.i. was accompanied by leucopenia. The number of alveolar macrophages was increased up to the 15th day p.i., in contrast to peritoneal macrophage numbers. Extensive bacterial colonization of the internal organs was detected at necropsy until the end of the experiment. Analysis of the cell immune response revealed activation of B cells in peripheral blood, spleen and thymus as well as augmentation of T-cell number in the lymphoid organs examined on days 15, 28 and 40 p.i. Histological changes typical of a generalized infection, such as purulent meningoencephalitis, catarrhal pneumonia and lymphadenitis, were observed. Clinical and morphological manifestations of arthritis were also established. The results obtained show that Y. enterocolitica (serotype 0:3, pYV+) induces a generalized, non-lethal infection in Chinchilla rabbits, complicated by arthritis.

Haematoporphyrin and proflavine-sensitized photoinactivation of Salmonella dublin.

The photosensitive activity of haematoporphyrin (HP) and proflavine (PF) on some biological parameters of Salmonella dublin cells was assessed. The investigations showed a decreased respiratory activity of photosensitized PF bacterial cells, accompanied by lower virulence. HP-treatment and light irradiation of salmonellae did not influence their survival in vitro, which was in contrast to the PF-incubated and irradiated cells. Light irradiation of HP- and PF-treated bacteria did not change their phagocytosis from guinea pig alveolar macrophages. In the presence of visible light the PF-treatment considerably reduced the survival rate and multiplication in alveolar macrophages in comparison with HP-treated and light-exposed bacteria. Correlation was established between the degree of structural damage, as observed by electron microscopy and the level of diminution of the chosen biological parameters, which were more strongly expressed after PF-treatment. PF as a photosensitizer which influences the bacterial genomes and its possible practical use, is discussed.

Studies on the interactions of immunostimulated macrophages and Yersinia enterocolitica O:8.

Immunological and electron microscopy investigations of the phagocytic and killing activities of peritoneal macrophages from rats and mice against Yersinia enterocolitica serotype O:8 cells were performed. The effect of in vivo application of cytoplasmic membranes (CM) from the stable Escherichia coli WF+ L-form on macrophage activity was also studied. It was established that rat macrophages more actively phagocytosed the plasmidless pYV(-) Y. enterocolitica cells, compared to the plasmid-bearing pYV(+) Y. enterocolitica cells. The killing ability against both variants of the Y. enterocolitica strain was significantly enhanced in macrophages from CM-treated rats after 2 h, 4 h, and 24 h incubation. The CM treatment enhanced the phagocytic activity of the macrophages. The in vitro interaction of normal and immunostimulated rat macrophages with both pYV(+) and pYV(-) variants of Y. enterocolitica did not lead to any additional apoptotic and necrotic changes in macrophages compared to control macrophages, which were cultivated without Y. enterocolitica. Electron-microscopic investigation showed that mouse macrophages eliminated Y. enterocolitica pYV(+) cells in vivo after 24 h. No engulfed or digested bacterial cells were observed. Activation of cell surfaces and vacuolization of macrophage cytoplasm, both of CM-treated non-infected and infected mice, were observed. The experimental results showed that Y. enterocolitica pYV(+) cells could be eliminated by peritoneal macrophages.