BIOFILM FORMATION OF Vibrio cholerae ON STAINLESS STEEL USED IN FOOD PROCESSING

Vibrio cholerae represents a significant threat to human health in developing countries. This pathogen forms biofilms which favors its attachment to surfaces and its survival and transmission by water or food. This work evaluated the in vitro biofilm formation of V. cholerae isolated from clinical and environmental sources on stainless steel of the type used in food processing by using the environmental scanning electron microscopy (ESEM). Results showed no cell adhesion at 4 h and scarce surface colonization at 24 h. Biofilms from the environmental strain were observed at 48 h with high cellular aggregations embedded in Vibrio exopolysaccharide (VPS), while less confluence and VPS production with microcolonies of elongated cells were observed in biofilms produced by the clinical strain. At 96 h the biofilms of the environmental strain were released from the surface leaving coccoid cells and residual structures, whereas biofilms of the clinical strain formed highly organized structures such as channels, mushroom-like and pillars. This is the first study that has shown the in vitro ability of V. cholerae to colonize and form biofilms on stainless steel used in food processing.

Aspectos interessantes observados na meiose de alguns hemípteros

Particular aspects of the meiosis of two species of Hemiptera, namely Megalotomus pallescens (Stal) (Coriscidae) and Jadera sanguinolenta (Fabr.); (Corizidae) are described and discussed in this paper. Megalotomus pallescens This species has primary spermatocytes provided with 7 autosomal tetrads plus a single sex chromosome. The X is smaller than the autosomes and may be found either in the periphery of the circle formed by the autosomal tetrads or in the center together with the m-tetrad which always occupies this position. The X chromosome - In the primary spermatocytes this element, which is tetradiform, orients itself parallelly to the spindle axis and divides transversely by its median constriction. In the secondary spermatocytes it passes undivided to one pole. The m-chromosomes - These chromosomes have been frequently found in close association with the sex chromosome in nuclei wich have passed the diffuse stage, a fact which was considered as affording some evidence in support of the idea /developed by the present writer in another paper with regard to the origin of the m-chromosomes from the sex chromosome. Formation of tetrads - Tetrads appear at first as irregular areas of reticular structure, becoming later more and more distinct. Then, two chromosomal strands very loose and irregular in outline, connected whit each other by several transverse filaments, begin to develop in each area. Growing progressively shorter, thicker and denser, these strands soon give origin to typical Hemiptera tetrads. Jadera sanguinolenta Spermatogonia of this species have 13 chromosomes, that is, 10 autosomes, 2 m-chromosomes and one sex chromosome, one pair of autosomes being much larger than the rest. Chromosomes move toward the poles with both ends looking to them. Primary spermatocytes show 6 tetrads and a single X. The sex chromossome in the first division of the spermatocytes divides as if it was a tetrad, passing undivided to one pole in the second division. In the latter it does not orient, being found anywhere in the cells. Its most common situation in anaphase corresponds therefore to precession. Tetrads are formed here in an entirely different way : the bivalents as they become distinct in the nuclei which came out. of the diffuse stage they appear in form of two thin threads united only at the extremities, an aspect which may better be analized in the larger bivalent. Up from this stage the formation of the tetrads is a mere process of shortening and thickening of both members of the pair. Due to the fact that the paired chromosomes are well separated from each other throughout their entire lenght, the author concluded that chiasmata, if present, are accumulated at the very ends of the bivalents. If no chiasmata have been at all formed, then, what holds together the corresponding extremities must be a strong attraction developed by the kinetochores. If one interprets the bivalents represented in the figures 17-21 as formed by four chromatids paired by one of the ends and united by the opposite one, then the question of the diffuse attachment becomes entirely disproved since it is exactly by the distal extremities that the tetrads later will be connected with the poles. In the opinion of the present writer the facts referred to above are one of the best demonstration at hand of the continuity of the paired threads and at the same time of the dicentricity of Hemiptera chromosomes. In view of the data hitherto collected by the author the behavior of the sex chromosome of the Hemiptera whose males are of the XO type may be summarized as follows: a) The sex chromosome in the primary metaphase appears longitudinally divided, without transverse constriction. It is oriented with the extremities in the plane of the equator and its chromatids separate by the plane of division. (Euryophthalmus, Protenor). In the second division the sex chromosome, provided as it is with an active kinetochore at each end, orients itself with its lenght parallelly to the spindle axis and passes undivided to one pole (Protenor?), or loses to the other pole a centric end (Euryophthalmus) In the latter case it has to become dicentric by means of a longitudinal spliting beginning at the kinetochore. b) The sex chromosome in the primary metaphase is tetradiform, that is, it is provided with a longitudinal split and a median transverse constriction. Orients with its length paral lelly to the spindle axis (what is probably due to the kinetochores being not yet divided) and divides transversely. (Corizas hyalinus, Megalotomus pallescens). in the secondary metaphase the sex chromosome which turned to be dicentric in consequence of a longitudinal spliting initiated in the kineto chore, orients perpendicularly to the equatorial plane and without losing anyone of its extremities passes undivided to one pole (Megalotomus). Or, distending between both poles passes to one side, in which case it loses one of its ends to the other side. (Corizas hyalinus). c) The very short sex chromosome in the first division of the spermatocytes orients in the same manner aa the tetrads and divides transversely. In the second division, due to the inactivity o the inetochore, it remains monocentric and motionless anywhere in the cell, finishing by being enclosed in the nearer nucleus. In the secondary telophase it recuperates its dicentricity at the same time as the autosomal chromatids. (Jadera sanguinolenta, Diactor bilineatus). d) The sex chromosome in the first division orients in the equador with its longitudinal axis parallelly to the spindle axis passing integrally to one pole or, distending itself between the anaphase plates, loses one of its ends to the opposite pole. In this case it becomes dicentric in the prometaphase of the second division, behaving in this division as the autossomes. It thus divides longitudnally. (Pachylis laticomis, Pachylis pharaonis).

Sobre o comportamento mitótico e meiótico de cromossomas policêntricos ou com ponto de inserção difuso

Material: Studies were made mainly with Ascaris megalocephála Cloq. univalens and bivalens, and also with Tityus bahiensis Perty. 1) Somatic pairing of heterochromatic regions. The heterochromatic ends of the somatic chromosomes in Ascaris show a very strong tendency for unspecifical somatic pairing which may occur between parts of different chromosomes (Figs. 1, 2, 3, 7, 10, 11, 12, 13, 14, 16, 18,), between the two ends of the same chromosome either directly (Figs. 4, 5, 7, 8, 11, 12, 13, 15, 16, 17, 18) or inversely (Fig. 8, in the arrow) and also within a same chromosomal arm (Fig. 6). 2) During the early first cleavage division the chomosomes are an isodiametric cylinder (Figs. 6, 9, 11, 13, 14). But in later metaphase the ends become club shaped (Figs. 1, 2, 3, 4, 5, 7, 10) which is interpreted as the beginning of migration of chromatic substance from the central euchromatic region towards the heterochromatic regions. This migration becomes more and accentuated in anaphase (Figs. 19, 22, 23) and in the vegetative cells where euchromatic region looses more and more staing power, especially in the intersititial zones between the individual small spherical chromosomes into which the euchromatic region desintegrates. The emigrated chromatin material is finally eliminated with the heterochromatic chromosome ends (Fig. 23 and 24). 3) It seems a general rule that during mitotic anaphase all chromosomes with diffuse or multiple spindle fiber attachement (Ascaris, Tityus, Luzula, Steatococcus, Homoptera and Heteroptera in general) move to the poles in the form of an U with precedence of the chromosomal ends. In Ascaris, the heterocromatic regions are pulled passively towards the poles and only the euchromatic central portion may be U-shaped (Fig. 19, 22, 25). While in the other species this U-shape is perfect since the beginning of anaphase, giving the impression that movement towards the poles begins at both ends of a chromosome simultaneously, this is not the case in Ascaris. There the euchromatic region is at first U-shaped, passing then to form a straight or zig-zag line and becoming again U-shaped during late anaphase. This is explained by the fact that the ends of the euchromatic regions have to pull the weight of the passive heterochromatic portions. 4) While it is generally accepted that, during first meio-tic division untill second anaphase, all attachement regions remain either undivided or at least united closely, this is not the case in chromosomes with diffused or multiple attachment. Here one clearly sees in all cases so far studied four parallel chromatids at first metaphase. In Luzula and Tityus (for Tityus all figs. 26 to 31) this division is allready quite clear in paraphase (pro-metaphase) and it cannot be said wether in other species the division in sister chromatids is allready present, but not visible at this stage. During first anaphase the sister chromatids of Titbits remain more or less in contact, while in Luzula and especially in Ascaris they are quite separated. Thus one can count in late anaphase or telophase of Ascaris megalocephala bivalens, nearly allways, four separate chromosomes near each pole, or a total of eight chromatids per division figure (Figs. 35, 36, 37, 38, 39, 40, 41).

Macrocheles muscaedomesticae (Acari, Macrochelidae) and a species of Uroseius (Acari, Polyaspididae) phoretic on Musca domestica (Diptera, Muscidae): effects on dispersal and colonization of poultry manure

Differences in the phoresy of the mites Macrocheles muscaedomesticae (Scopoli, 1972) (Macrochelidae) and Uroseius sp. (Polyaspidae) on the house fly, Musca domestica (Linnaeus, 1758) and the similarities in their phoretic dispersal and parasitism are discussed, altogether with the effects on predator-prey interactions. The prevalence and intensity of phoresy in the mite species were significantly related to the attachment site on the hosts. The phoresy of Uroseius sp. was correlated with temperature but not with rainfall and relative humidity. Selective pressure in the environment resulted in displacement and the emergence of local and regional populations. These results suggest that in each habitat the populations will use different resources and will show several relationships with other species, as well as a selection for morphological and behavioral types.

Feeding habits of six anuran (Amphibia: Anura) species in a rainforest fragment in Northeastern Brazil

Although Brazil encompasses one of the most abundant anuran faunas in the world, quantitative information on anuran ecology and diet are limited, especially in the Northeastern region. We analyzed the diet of six species: Hyla albomarginata, Hyla cf. branneri, Hyla minuta, Phyllomedusa aff. hypochondrialis (Hylidae), Leptodactylus natalensis, and Physalaemus cuvieri (Leptodactylidae) in a temporary pond in a rainforest remnant in Pernambuco, between 1999-2000. We analyzed diet composition, degree of food preference, and seasonal variations in diet. Leptodactylus natalensis and P. cuvieri showed higher diet diversity, whereas H. minuta consumed fewer food items. Insecta, Arachnida, and plants were preferential items for most species. Acari were consumed by all species; Hymenoptera, Odonata, and Coleoptera were also often consumed. A slight increase in diet diversity occurred in the rainy season. The species showed a generalist feeding behaviour, although P. cuvieri consumed Formicidae as major prey item.

Effect of cAMP on macromolecule synthesis in the pathogenic protozoa Trypanosoma cruzi

Macromolecule synthesis of Trypanosoma cruzi in culture was monitored using radioactive tracers. Cells of different days in culture displayed a preferential incorporation of precursors as follows: 1 day for (³H)-thymidine cells; 3 days for (³H)-uridine cells, and 4 days for (³H)-leucine cells. Autoradiographic studies showed that (³H)-thymidine was incorporated in the DNA of both kinetoplast and nucleus in this order. Shifts in the intracellular content of cAMP either by addition of dibutyryl-cAMP or by stimulation of the adenylcyclase by isoproterenol, caused an inhibition in the synthesis of DNA, RNA and proteins. Addition to the T. cruzi cultures of these agents which elevate the intracellular content ofcAMP provoked an interruption of cell proliferation as a result of the impairment of macromolecule synthesis. A discrimination was observed among the stereoisomers of isoproterenol, the L configuration showing to be most active.

Morphological evidence by scanning electron microscopy of excretion of metacyclic forms of Trypanosoma cruzi in vector's urine

Comparision by scanning electron microscopy (SEM) of Trypanosoma cruzi flagellates attached to the cuticle of the rectal gland of infected Dipetalogaster maxima nymphs, showed marked differences before amd after feeding. Before feeding numerous metacyclic trypomastigotes were observed among the abundant epimastigotes that formed the carpet of flagellates. On the other hand, in insects that were allowed to urinate for 24 hours after a meal, the metacyclics were scarce,indicating that they had been detached by the urine flow. An asymetric type of cell division, probably originating both an epi-and a trypomastigote, was occasionally observed. The occurrence of swellings at different levels of the flagella of epimastigotes suggests that secondary sites of attachment may be common.

Ultrastructure of Giardia duodenalis trophozoites group from hamster: some relevant aspects

Throphozoites of Giardia duodenalis group obtained from fragments or scratched of hamster's mucosa were examined by transission electron microscopy. The fine structure of the trophozoites are presented and comapred with those described for other animals. Some of the trophozoites present the cytoplasm full of glycogen, rough endoplasmic reticulum-like structures and homogeneous inclusions not enclosed by membranes, recognized as lipid drops, which had not been observed in Giardia from other animals. The adhesive disk is composed of a layer of microtubules, from which fibrous ribbons extend into the cytoplasm; these ribbons are linked by layer of crossbridge filaments that shows an intermediary dense band, described for the first time in this paper. The authors regard this band as the result of the cross-bridge filaments slinding in the medium region between adjacent fibrous ribbons, and suggest a contractile activity for them. The role of the adhesive disk on the trophozoite mechanism of attachment to host mucosa is also discussed.

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

Ultrastructure of the adults of Bunostomum phlebotomum (Nematoda: Ancylostomatidae)

The morphology of the male and female of Bunostomum phlebotomum are described based on scanning electron microscope (SEM) observations. The attachment of the worms to the small intestinal mucosa and during the copula were observed. Structures of the bucal capsule and genital organs were also studied.

Optimization and inhibition of the adherent ability of Plasmodium falciparum-infected erythrocytes

The vast majority of the 1-2 million malaria associated deaths that occur each year are due to anemia and cerebral malaria (the attachment of erythrocytes containing mature forms of Plasmodium falciparum to the endothelial cells that line the vascular beds of the brain). A "model" system"for the study of cerebral malaria employs amelanotic melanoma cells as the "target"cells in an vitro cytoadherence assay. Using this model system we determined that the optimum pH for adherence is 6.6 to 6.8, that high concentrations of Ca²* (50mM) result in increased levels of binding, and that the type of buffer used influences adherence (Bis Tris > MOPS > HEPES > PIPES). We also observed that the ability of infected erythrocytes to cytoadhere varied from (erythrocyte) donor to donor. We have produced murine monoclonal antibodies against P. falciparum-infected red cells which recognized modified forms of human band 3; these inhibit the adherence of infected erythrocytes to melanoma cells in a doso responsive fashion. Antimalarials (chloroquine, quinacrine, mefloquine, artemisinin), on the other hand, affected adherence in an indirect fashion i.e. since cytoadherence is due, in part to the presence of knobs on the surface of the infected erythrocyte, and knob formation is dependent on intracellular parasite growth, when plasmodial development is inhibited so is knob production, and consequently adherence is ablated.

Duplex Hemodinamic evaluation of hepatosplenic mansoni schistosomiasis

Conventional ultrasonography highly contributes to a non invasive diagnosis of HS schistosomiasis (Cerri et al., 1984). The introduction of Dopple allowed new advances in the knowledge of the portal dinamics of this disease (Taylor et al., 1985; Moriyasu et al., 1986). The aim of this paper was to analize the hemodinamic behavior of the portal vessels, considering caliper, maximum flow speed, direction of flow and preferential disposition of the collateral vessels. Thirty two patients with schistosomiasis mansoni with confirmed hepatosplenic form (HSSM), were analyzed. Fourteen patients with the intestinal form, have been analyzed as a control group. The results demonstrated that the maximum speed of the portal vein in the two groups has not been significantly diferent. Nevertheless, the diameter of the PV in the hepatosplenic group has been larger. The splenic vein presented speed and caliper larger than the superior mesenteric vein. The hepatic artery has been detectly in only 40% of the cases. The hepatic veins presented normal caliper and spectral pattern. The duplex proved to be an useful technich complementar and non-invasive, in the study of the HSSM.

Trypanosoma cruzi recognition by macrophages and muscle cells: perspectives after a 15-year study

Macrophages and muscle cells are the main targets for invasion of Trypanosoma cruzi. Ultrastructural studies of this phenomenon in vitro showed that invasion occurs by endocytosis, with attachment and internalization being mediated by different components capable of recognizing epi-or trypomastigotes (TRY). A parasitophorus vacuole was formed in both cell types, thereafter fusing with lysosomes. Then, the mechanism of T. cruzi invasion of host cells (HC) is essentially similar (during a primary infection in the abscence of a specific immune response), regardless of wether the target cell is a professional or a non-professional phagocytic cell. Using sugars, lectins, glycosidases, proteinases and proteinase inhibitors, we observed that the relative balance between exposed sialic acid and galactose/N-acetyl galactosamine (GAL) residues on the TRY surface, determines the parasite's capacity to invade HC, and that lectin-mediated phagocytosis with GAL specificity is important for internalization of T. cruzi into macrophages. On the other hand, GAL on the surface to heart muscle cells participate on TRY adhesion. TRY need to process proteolytically both the HC and their own surface, to expose the necessary ligands and receptors that allow binding to, and internalization in the host cell. The diverse range of molecular mechanisms which the parasite could use to invade the host cell may correspond to differences in the available "receptors"on the surface of each specific cell type. Acute phase components, with lectin or proteinase inhibitory activities (a-macroglobulins), may also be involved in T. cruzi-host cell interaction.

Development of trypanosomatids in the phytophagous insect Dysdercus peruvianus (Hemiptera: Pyrrochoridae): a light and eletron microscopic study

Experimental infections of the phytophagous Hemiptera Dysdercus peruvianus with different trypanosomatids were studied for up to 55 days by light microscopy while the course of infection with Leptomonas seymouri and the Leptomonas isolate 49/553G.O. was analyzed by electron microscopy. Rates of infection of D. peruvianus varied according to the infecting flagellate. The lower part of the midgut was found to be the preferential site of colonization where most flagellates were found isolated or arranged in clumps or rosettes. Specialized junctional structures with host cells were never observed. Flagellates could also be seen inside midgut cells within a parasitophorous vacuole. Infection of haemocoele and salivary glands was also observed.

Characterization of the hemagglutinin receptor specificity and neuraminidase substrate specificity of clinical isolates of human influenza A viruses

Six clinical isolates of influenza A viruses were examined for hemagglutinin receptor specificity and neuraminidase substrate specificity. All of the viral isolates minimally passaged in mammalian cells demonstrated preferential agglutination of human erythrocytes enzymatically modified to contain NeuAc alpha 2,6Gal sequences, with no agglutination of cells bearing NeuAc alpha 2,3Gal sequences. This finding is consistent with the hemagglutination receptor specificity previously demonstrated for laboratory strains of influenza A viruses. The neuraminidase substrate specificities of the clinical isolates examined were also identical to that described for the N2 neuraminidase of recent laboratory strains of human influenza viruses. The H3N2 viruses all displayed the ability to release sialic acid from both alpha 2, 3 and alpha 2, 6 linkages. In addition, two clinical isolates of H1N1 viruses also demonstrated this dual neuraminidase substrate specificity, a characteristic which has not been previously described for the N1 neuraminidase. These results demonstrate that complementary hemagglutinin and neuraminidase specificities are found in recent isolates of both H1N1 and H3N2 influenza viruses.