Pseudomonas aeruginosa adhesion to normal and injured respiratory mucosa

Human nasal polyps outgrowth culture were used to study the adhesion of Pseudomonas aeruginosa to respiratory cells. By transmission electron microscopy, bacteria associated with ciliated cells were identified trapped at the extremities of cilia, usually as aggregates of several bacterial cells. They were never seen at the interciliary spaces or attached along cilia. Bacteria were also seen to adhere to migrating cells of the periphery of the outgrowth culture. Using a model of repair of wounded respiratory epithelial cells in culture, we observed that the adhesion of P. aeruginosa to migrating cells of the edges of the repairing wounds was significantly higher than the adhesion to non-migrating cells and that adherent bacteria were surrounded by a fibrocnectin-containing fibrillar material The secretion of extracellular matrix components is involved in the process of epithelium repair following injury. To investigate the molecular basis of P. aeruginosa adhesion to migrating cells, bacteria were treated with a fibronectin solution before their incubation with the respiratory cells. P. aeruginosa treatment by fibronectin significantly increased their adhesion to migrating cells. Accordingly, we hypothesize that during cell migration, fibronectin secreted by epithelial cells may favour P. aeruginosa adhesion by establishing a bridge between the bacteria and the epithelial cell receptors. Such a mechanism may represent a critical step for P. aeruginosa infection of healing injured epithelium.

The subclinical form of experimental visceral leishmaniasis in dogs

Pathological aspects of a subclinical form of experimental canine leishmaniasis is reported here for the first time. Fifteen mongrel dogs were used in the present study. Eight dogs were infected and seven were used as control. Four of the control dogs were inoculated with spleen cells from non-infected hamsters. The eight mongrel dogs inoculated intravenously with amastigotes forms of Leishmania chagasi envolved for periods as long as 25 months without any clinical characteristic sign of classical Visceral Leishmaniasis (VL). Most of the laboratory test results were compatible to those of the seven control animals but culture of bone marrow aspirated material and serologic testing (IIF) demonstrated or provided evidence that the animals were infected. The most important and predominant histopathological lesion in infected animals were epitheloid granulomas presented in the liver, spleen, adrenal gland and lung of some animals. Channels containing erythrocytes in some granulomas of the liver suggeste that these granulomas are formed inside sinusoidal capillaries. Despite the animals were proved to be infected and presented characteristic histologic lesions, they did not present external signs of disease. The granulomatous aspect of the lesions indicates a good immunologic reactivity and suggest that a host-parasite equilibrium does exist in the dog experimental model

Identification of Novel Membrane Structures in Plasmodium falciparum Infected Erythrocytes

Little is known about the molecular mechanisms underlying the release of merozoites from malaria infected erythrocytes. In this study membranous structures present in the culture medium at the time of merozoite release have been characterized. Biochemical and ultrastructural evidence indicate that membranous structures consist of the infected erythrocyte membrane, the parasitophorous vacuolar membrane and a residual body containing electron dense material. These are subcellular compartments expected in a structure that arises as a consequence of merozoite release from the infected cell. Ultrastructural studies show that a novel structure extends from the former parasite compartment to the surface membrane. Since these membrane modifications are detected only after merozoites have been released from the infected erythrocyte, it is proposed that they might play a role in the release of merozoites from the host cell

Trypanosoma cruzi: Maintenance in Culture Modify Gene and Antigenic Expression of Metacyclic Trypomastigotes

In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi

Sensitivity of a Vacuum Aspiratory Culture Technique for Diagnosis of Localized Cutaneous Leishmaniasis in an Endemic Area of Leishmania (Viannia) braziliensis Transmission

Sixty eight patients with localized cutaneous leishmaniasis from an area with Leishmania (Viannia) braziliensis transmission had cultures performed with a modified Marzochi´s vacuum aspiratory puncture technique to establish sensitivity and contamination rate with this new method. Overall sensitivity of three aspirates was 47.1%; (CI95% 39.4; 59.4) significantly greater than the sensitivity of a single one aspirate. Fungal contamination was observed in 6/204 (2.9%) inoculated culture tubes. We recommend that this useful technique should be adopted as routine for primary isolation of L. (V.) braziliensis from localized cutaneous ulcers.

A comparative study of preservation and storage of Haemophilus influenzae

The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20ºC and -70ºC. Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20ºC and -70ºC from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20ºC. Initial concentration did not influence survival after storage at -70ºC. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.

Oviposition Attractancy of Bacterial Culture Filtrates: response of Culex quinquefasciatus

Oviposition attractants could be used for monitoring as well as controlling mosquitoes by attracting them to lay eggs at chosen sites. In the present study, culture filtrates of seven bacterial species were tested for their attractancy against gravid females of Culex quinquefasciatus. When their oviposition active indices (OAI) were studied, the culture filtrates of Bacillus cereus and Pseudomonas fluorescens exhibited oviposition attractancy (OAI = >0.3) at 100 ppm and the OAI were respectively 0.70 and 0.47. Culture filtrates of B. thuringiensis var. israelensis (wild type), B. t. var. israelensis (mutant) and B. sphaericus showed attractancy at 2000 ppm with OAI of respectively 0.71, 0.59 and 0.68. However, the OAI of B. megaterium as well as Azospirillum brasilense was 0.13 (at 2000 ppm), which was less than 0.3 required to be considered them as attractants. When the oviposition attractancy of the bacterial culture filtrates were compared with that of a known oviposition attractant, p-cresol (at 10 ppm), the culture filtrates of B. t. var. israelensis (wild type) and B. cereus were found to be more active than p-cresol, respectively with 64.2 and 54.3% oviposition.

Standardization of bovine macrophage monolayers and isolation and culture of trypanosomes

We describe a method for culturing over 90% pure bovine macrophages from peripheral blood mononuclear cells separated with Nycoprep. The cells were cultured for 12 days and then stained with esterase and with anti CD14 to test for purity. The method is reproducible and ensures an adequate number of cells for immunological research. Additionally, we report the unexpected finding of Trypanosoma trypomastigotes in our macrophage cultures from bovines belonging to a geographic area from which no bovine trypanosomes had been reported before.

Techniques of DNA-studies on prehispanic ectoparasites (Pulex sp., Pulicidae, Siphonaptera) from animal mummies of the Chiribaya Culture, Southern Peru

During a paleoparasitological survey of several animal mummies (Cavia aperea f. porcellus and Canis familiaris) from Chiribaya Baja, an archaeological site in Southern Peru, an unexpected find was made. In the well preserved fur, large numbers of mummified fleas (Pulex simulans/irritans)that parasitized the animals during life were encountered. Due to the relative recent event of the host mummification and the outstanding preservation of the fleas, an attempt for the retrieval of DNA was made. A DNA extraction and sequencing protocol for archaeological ectoparasitic remains has been established, taking additional studies for tissue and protein preservation into account. Tissue preservation was assessed with transmission electron microscopy and the protein preservation was tested through the racemisation ratios of aspartic acid. Regions of the 28S rDNA gene were successfully amplified and sequenced. Further research perspectives are outlined.

Louse infestation of the Chiribaya Culture, Southern Peru: variation in prevalence by age and sex

In order to improve the interpretive potential of archaeoparasitology, it is important to demonstrate that the epidemiology of ancient parasites is comparable to that of modern parasites. Once this is demonstrated, then we can be secure that the evidence of ancient parasitism truly reflects the pathoecology of parasitic disease. Presented here is an analysis of the paleoepidemiology of Pediculus humanus infestation from 146 mummies from the Chiribaya culture 1000-1250 AD of Southern Peru. The study demonstrates the modern parasitological axiom that 10% of the population harbors 70% of the parasites holds true for ancient louse infestation. This is the first demonstration of the paleoepidemiology of prehistoric lice infestation.

Changes associated with laboratory rearing in antennal sensilla patterns of Triatoma infestans, Rhodnius prolixus, and Rhodnius pallescens (Hemiptera, Reduviidae, Triatominae)

We examined changes in the array of antennal sensilla of three species of Triatominae (Triatoma infestans, Rhodnius prolixus, and R. pallescens) following their establishment for different periods in laboratory culture. In each case, the laboratory colonies were compared with conspecific samples taken directly from the field, by quantitative analysis of the sensilla arrays on the three distal segments of the antenna in terms of the densities of three types of chemoreceptors (basiconics and thick and thin walled trichoids) and one type of mechanoreceptor (bristles). Sensilla densities were compared by ANOVA or non-parametric tests, and by multivariate discriminant analysis. Strains of the same species reared in different laboratories showed significant differences in their sensilla arrays, especially when compared to field-collected material from the same geographic origin. A Bolivian strain of T. infestans reared in the laboratory for 15 years and fed at monthly intervals, showed greatest differences from its conspecific wild forms, especially in terms of reductions in the number of chemoreceptors. By contrast, an Argentine strain of T. infestans reared for 25 years in the laboratory and fed weekly, showed a relative increase in the density of mechanoreceptors. A Colombian strain of R. prolixus reared for 20 years and fed weekly or fortnightly, showed only modest differences in the sensilla array when compared to its wild populations from the same area. However, a Colombian strain of R. pallescens reared for 12 years and fed fortnightly, did show highly significant reductions in one form of chemoreceptor compared to its conspecific wild populations. For all populations, multivariate analysis clearly discriminated between laboratory and field collected specimens, suggesting that artificial rearing can lead to modifications in the sensory array. This not only supports the idea of morphological plasticity in these species, but also suggests caution in the use of long-established laboratory material for experimental studies designed to extrapolate the natural behaviour and physiology of these species.

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

Primary culture of the region of the amebocyte-producing organ of the snail Biomphalaria glabrata, the intermediate host of Schistosoma mansoni

Biomphalaria glabrata snails are major hosts for the digenetic trematoda Schistosoma mansoni, the causative agent of human schistosomiasis. The success or failure of the infection will be dependent on the mobilization of the molluskan internal defense system, where a major role will be played by circulating hemocytes produced by the APO (amebocyte-producing organ) of the snail. In this report, the primary culture of the APO region of B. glabrata was obtained for the first time, as well as a control culture of the ovotestis. Three different cell populations migrated easily from the explants in culture, with no need of any dispersion agent. The cells grew in suspension at an incubation temperature of 15ºC and the cultures were maintained viable for up to two weeks. Two of these cell populations obtained resembled cell types known to be present in the hemolymph of Biomphalaria. The availability of APO cells in culture may contribute to a better understanding of the internal defense in mollusks, in general, as well as the specific response of B. glabrata to S. mansoni infection.