Photodynamic Effect of some Phthalocyanines on Enveloped and Naked Viruses.

Activity of three photosensitizing phthalocyanine derivatives was tested for photodynamic inactivation towards two coated and two non-enveloped viruses - bovine viral diarrhea virus (BVDV), influenza virus A(H3N2), poliovirus type 1 (PV-1) and human adenovirus type 5 (HAdV5). In the case of coated viruses, a combination of virucidal and irradiation effects was registered by octa-methylpyridyloxy-substituted Ga phthalocyanine (Ga8) toward BVDV, whereas tetra-methylpyridyloxy-substituted Ga phthalocyanine (Ga4) revealed a marked photoinactivation only. No such effect was observed towards influenza A virus. In contrast, the photoinactivating potential of Ga4 and Ga8 marked very high values on naked viruses, especially on HAdV5, at which both the virucidal as well as the irradiation effects became combined.

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.

Morphological variability and cell-wall deficiency in Mycobacterium tuberculosis 'heteroresistant' strains.

SETTING: Sofia State Hospital for Tuberculosis Treatment, Bulgaria. OBJECTIVE: To investigate the morphology of two 'heteroresistant' clinical isolates and one non-heteroresistant isolate, all isolated from newly diagnosed tuberculosis (TB) patients, as well as the reference strain H37Rv. Heteroresistant isolates contained clonally-related sensitive and drug-resistant organisms which could subsequently be separated using drug-containing primary cultures and had been isolated from patients originally diagnosed with susceptible TB by the 1% proportion method. Mycobacterial cultures were evaluated by transmission electron microscopy after 25 days of cultivation in Dubos broth. RESULTS: In contrast to H37Rv and the non-heteroresistant isolate, the bacterial populations in both heteroresistant isolates demonstrated distinct pleomorphic variability and coexistence of both classical and cell-wall deficient forms. Electron micrographs of mutants resistant to streptomycin and isoniazid showed predominance of atypical granular L-forms, which formed L-type colonies on Dubos agar. CONCLUSION: The L-form transformation processes, observed both in clinical heteroresistant isolates containing mixed populations of Mycobacterium tuberculosis organisms with different resistance gene genotypes and in the isolated resistant (mutant) clones, indicate a possible link between resistance and cell-wall deficient L-phase states and suggest one of the possible mechanisms by which resistant mutants are able to survive in vivo.

Intranasal application of lentinan enhances bactericidal activity of rat alveolar macrophages against Mycobacterium tuberculosis.

To study the in vivo interaction between lentinan-stimulated alveolar macrophages and Mycobacterium tuberculosis were used rats intranasally infected with 2 x 10(8) CFU. Before infection animals were treated intranasally with lentinan at a dose of 1 mg/kg (administrated three times at 2 days periods). Samples of broncho-alveolar lavage fluid were obtained from rats at different intervals -3, 24 and 72 h after infection. The process of phagocytosis in vivo was observed by light and electron microscopy, as well as acid phosphatase cytochemistry methods. Bactericidal activity of alveolar macrophages following the same intranasal installation of lentinan was assessed by in vitro "killing" ability test against M. tuberculosis. Nitrite production by lentinan activated alveolar macrophages was measured 24 h after ex vivo culture of these cells. It was demonstrated that lentinan induces high level of alveolar macrophage activation manifested through enhanced bactericidal effect against M. tuberculosis, which correlates with the induction of reactive nitrogen intermediates, increased activity of lysosomal enzymes (acid phosphatase), and with effective phagolysosomal fusion followed by destruction of Mycobacteria.

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.

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.

Proflavine-mediated inactivation of Salmonella dublin exposed to visible sunlight in natural fresh water.

The survival of Salmonella dublin exposed to visible sunlight, and heterotrophic bacteria in freshwater microcosms in the presence and absence of the photosensitizer proflavine, was studied. Enumeration of S. dublin and the heterotrophic bacteria showed that in both illuminated and nonilluminated systems (without proflavine) the bacteria remained viable and culturable for at least 6 days. The optimal proflavine concentration (no effect in the dark and a maximal photoinactivation of salmonellae after irradiation) was 2 mg l(-1). In contrast to S. dublin, the heterotrophic bacteria overcame the initial inhibitory effect of proflavine. The possible use of photosterilization against contamination with pathogenic bacteria in water model ecosystems, is discussed.

Interaction of alveolar macrophages with Staphylococcus aureus and induction of microbial L-forms during infection in rats.

In vivo cell interactions between Staphylococcus aureus and rat alveolar macrophages were investigated after intranasal inoculation during a 30-days period of examination. Some dynamic characteristics of microorganisms in the macrophages were examined by electron microscopy and acid phosphatase cytochemistry. It was found that at earlier infection intervals (days 3 and 7) the ingested cocci were sequestered in phagosomes and phagolysosomes and later many of the microbial cells were digested. An interesting finding was the intracellular appearance of cell wall-defective forms (L-forms) of S. aureus at later intervals (days 14 and 30 after challenge). Infection kinetics were evaluated by isolation and enumeration of colony-forming units of S. aureus from bronchoalveolar fluid and by assessment of blood and bronchoalveolar total and differential leukocyte counts. The results indicate that induction and survival of S. aureus L-forms may occur spontaneously in vivo. This phenomenon could explain some of the mechanisms, provoking the latent and relapsing lung infections.

Atypical behaviour and survival of Streptococcus pyogenes L forms during intraperitoneal infection in rats.

Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections.

Persistence and in vivo effects of Yersinia enterocolitica 0:3 endotoxin in rats.

In vivo effects of Yersinia enterocolitica 0:3 lipopolysaccharide (prepared from bacteria grown at 25 degrees C and 37 degrees C) were investigated after intraperitoneal (i.p.) and intraarticular (i.a.) injection in rats during 30 days of examination. The persistence of endotoxin in the peritoneal and the synovial cavities was demonstrated by the immunofluorescence technique. Peritoneal and synovial exudative cell infiltration, as well as changes in some parameters (glycolytic and acid phosphatase activity, and killing ability of peritoneal cells; lactate dehydrogenase concentration in synovial fluid) were studied. The results indicated that endotoxin could persist longer in the synovial than in the peritoneal cavity.

The fate of Yersinia pseudotuberculosis lipopolysaccharide (LPS) in intraperitoneally and intraarticularly infected rats.

Persistence and in vivo effects of Yersinia pseudotuberculosis serotype III LPS (prepared from bacteria grown at 25 degrees and 37 degrees C) in rats were investigated after intraperitoneal and intraarticular injection during the 30 day period of examination. Localization and persistence of LPS in the peritoneal and synovial cavities were demonstrated by using the immunofluorescence technique. Peritoneal and synovial exudative cell infiltration as well as changes in some parameters (glycolytic and acid phosphatase activities, killing ability of peritoneal cells, lactate-dehydrogenase concentration in synovial fluid) were studied. The results indicated that LPS expressed at 37 degrees C induced stronger peritoneal cell response (increased "killing" ability and elevation of glycolytic and acid phosphatase activities) in comparison to that synthesized at 25 degrees C. It was also found, that LPS persisted longer in the synovial rather than in the peritoneal cavity of rats and induced long-lasting synovial inflammation.

Killing of Pseudomonas pseudomallei by polymorphonuclear leukocytes and peritoneal macrophages from chicken, sheep, swine and rabbits.

Differences in the kinetics of Pseudomonas pseudomallei killing by peritoneal macrophages (PM) and polymorphonuclear leucocytes (PMNL) from chickens, sheep, swine and rabbits were found. P. pseudomallei was rapidly killed by porcine PM and PMNL. However the bacterial killing by ovine and lapine PM and PMNL proceeded at a slower rate. In contrast, chicken PM and PMNL ingested and killed the lowest number of P. pseudomallei bacteria. The differences in the bactericidal activity of PM and PMNL from different animal species correlated with the level of their acid phosphatase and glycolytic activity.

Cell wall-deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats.

Experimental infections were induced with different bacterial forms of Listeria monocytogenes: parental (S-forms), protoplastic (L-forms) and combined inoculum of both forms by i.p. injection of rats. The parental bacterial forms (S-forms) were isolated up to 7 days after challenge from the peritoneal cavity and the liver, while the L-forms were isolated up to 60 days from the peritoneal cavity. Continuous adhesion of L-forms on the peritoneal macrophage surface was found by scanning-electron microscopy. Erythrocyte and leucocyte count as well as some clinical chemistry parameters were measured during infections. They showed different dynamics in the three experimental groups. Histomorphological changes in the liver (microabscesses and mononuclear cellular granulomas) of infected animals were observed. They were less intensive and appeared later in rats infected with L-forms. The experiments demonstrated that infections caused by parental bacterial forms and by combined inoculum took an acute course, while the infection caused by L-forms could be distinguished as a prolonged and persistent one.

Influence of blood transfusion on bactericidal activity of human leukocytes and sera against Yersinia enterocolitica and Salmonella typhimurium.

Patients undergoing joint surgery and blood transfusion were studied. Serum and leukocyte bactericidal tests in vitro against Salmonella typhimurium and Yersinia enterocolitica were carried out preoperatively as well as on the 1st, 3rd and 7th days after the operation. The serum complement (C3 and C4) concentrations were determined at the same intervals. It was found that after blood transfusion the bactericidic activity of sera and the serum C3 complement concentrations were increased. In contrast the killing ability of leukocytes was suppressed.

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.

Experimental melioidosis in inbred mouse strains.

Experimental infection was induced in three inbred mouse strains (BALB/c, BDF1 hybrid and C57BL) by i. p. inoculation with Pseudomonas pseudomallei. The bacterial load in the viscera and the host response induced in different compartments (blood, peritoneal cavity and organs) were determined. Blood cell parameters and peritoneal exudative cell populations were evaluated during the infection with the aid of an automated haematology analyser Technicon H-1. It was found that all mouse strains produced a similar intraperitoneal inflammatory response with predominance of granulocytes at the early stage of infection and subsequent increase of macrophages especially in BDF1 hybrid and BALB/c mice. The highest bacterial count found in the liver and spleen of C57BL was associated with corresponding tissue damage (purulent pneumonia, abscesses in liver, karyorrhexis of hepatocytes and meningoencephalitis). The degree of bacterial load and histological changes found in BALB/c and BDF1 hybrid mice were lower than in C57BL mice. The results show that the variations in the infection magnitude among inbred mouse strains are host-dependent.

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.

Responsiveness of susceptible inbred mice to Yersinia pseudotuberculosis serovar III infection.

The susceptibility of BALB/c, C57BL and BDF1-hybrid mouse strains to Yersinia pseudotuberculosis serovar III infection was studied. The bacterial load in the viscera and brain and the host responses at different levels, i.e. blood, peritoneal cavity and organs were determined. Blood cell parameters and peritoneal exudate cell population were evaluated during the infection using the automated hematologic analyzer Technicon H-1. It was found that BDF1-hybrid mice produced an early peritoneal inflammatory response, while in BALB/c and C57BL mice it was not observed. The high susceptibility of C57BL was associated with a great number of microorganisms in the organs and with the corresponding histological changes. It was shown that the magnitude of the inflammation induced by Y. pseudotuberculosis varied among the host strains used. The variations of the susceptibility to Y. pseudotuberculosis among inbred mouse strains suggest the possible role of genetic factors regulating the host defence.

Dark interaction of haematoporphyrin and proflavine with Salmonella dublin cells.

The influence of haematoporphyrin and proflavine on the virulence, survivability, respiratory activity and cell wall ultrastructure of Salmonella dublin cells was studied. There was a decrease in all biological properties investigated, especially when haematoporphyrin was used for dark incubation in comparison with proflavine. The possible clinical use of negatively charged photosensitizers is discussed.

Experimental infection with Yersinia pseudotuberculosis of ground squirrels (Citellus citellus).

An experimental infection was induced in the European ground squirrel (Citellus citellus) by intraperitoneally injecting of 1 ml. of microbial suspension, containing 1 x 10(9) CFU (colony forming units) of Yersinia pseudotuberculosis, serotypes I and III. The infectious process was dynamically followed up from the 1st up to the 30th day, respectively. Account was rendered to the general condition, the internal body temperature and the mortality rate. The phagocytic activity of alveolar macrophages (aMa) and polymorphonuclear leucocytes (PMNL), isolated from the experimental animals, was also determined, as well as the degree of bacterial dissemination into the viscera and the histomorphological changes that found in the parenchymal organs. These investigations confirm the fact that the bacterial agent has a broad spectrum of alterations in the organism of the host--the European ground squirrel (Citellus citellus) in which the pseudotuberculosis develops as a generalized infection, especially pronounced in the animals that were infected with the serotype III.