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PubMed2000

Uptake and interconversion of fluorescent lipid analogs in the protozoan parasite, Perkinsus marinus, of the oyster, Crassostrea virginica.

Uptake, distribution, and interconversion of fluorescent lipid analogs (phosphatidylcholine, PC; cholesteryl ester, CHE; phosphatidylethanolamine, PE; palmitic acid, C16; sphingomyelin, SM) by the two life stages, meront and prezoosporangium, of the oyster protozoan parasite, Perkinsus marinus, were investigated. Class composition of these two life stages and lipid contents in meront cells were also examined. Both meronts and prezoosporangia incorporated and modified fluorescent lipids from the medium, but their metabolic modes differ to some extent. Results revealed that among the tested analogs, neutral lipid components (CHE and C16) were incorporated to a greater degree than the phospholipids (PC, PE, and SM). HPLC analysis of meront lipids showed that while the majority of the incorporated PC, CHE, and PE remained as parent compounds, most of the incorporated C16 was in triacylglycerol (TAG) and SM was in ceramide and free fatty acids. The cellular distribution of fluorescent labels varied with lipid analogs and the extent of their metabolism by the parasite. Fluorescence distribution was primarily in cytoplasmic lipid droplets of both life stages after 24 h incubation with PC. After 24 h incubation with SM, fluorescence appeared in the membrane and cytosol. Total lipid contents in meront cultures increased during proliferation and TAG accounted for most of the increased total lipids. Since total lipid content per meront cell did not increase until the day of culture termination, the lipid increase in the meront culture was mainly a result of increased cell numbers. Both life stages contain relatively high levels of phospholipids, 53.8% in 8-day-old meronts and 39.4% in prezoosporangia. PC was the predominant phospholipid.

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PubMed2000

Immune parameters in carpet shell clams naturally infected with Perkinsus atlanticus.

Defence parameters of non-infected clams (Ruditapes decussatus) and clams heavily infected with Perkinsus atlanticus were assessed. Cellular (haemocyte density and phagocytic activity) and humoral (lysozyme and anti-bacterial activities, protein concentration and agglutination titre) parameters were measured in clams collected in an area enzootic for P. atlanticus. The infection intensity of each clam was assessed, and the immune parameters measured in the most infected clams were compared with those measured in the non-infected ones. Only the serum anti-bacterial activity and the agglutination titre were significantly different between infected and non-infected clams. The phagocytic rate, haemocyte density, lysozyme concentration and protein concentration were not statistically different but they showed the same trend in the two trials performed. Phagocytic rate, haemocyte concentration and anti-bacterial activity were higher in non-infected clams, while they had lower lysozyme concentration, serum protein concentration and agglutination titre than infected clams. Although infected and healthy clams were not different for every parameter measured, probably due to the high variability among individuals, P. atlanticus seems to affect the clam immune system, at least in advanced stages of the infection.

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PubMed2000

Modulation of the chemiluminescence response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes.

The influence of several factors on the chemiluminescence (CL) activity of haemocytes from the Mediterranean mussel (Mytilus galloprovincialis) was studied. Haemocytes were stimulated in vitro with different concentrations of zymosan, phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) (adding superoxide dismutase, SOD, to the zymosan-stimulated haemocytes in order to test the specificity of the reaction). The in vitro effect of the clam pathogens Vibrio tapetis (bacteria) and a Perkinsus atlanticus-like protozoan tentatively named Pseudoperkinsus taapetis on the mussel haemocytes CL response was also assessed. To study the in vivo stimulation of haemocytes, mussels were inoculated with zymosan and the CL response of their haemocytes was subsequently measured. Zymosan added in vitro produced the highest CL response, although PMA also enhanced the CL emission and, in addition, increased the zymosan-stimulated CL. LPS and V. tapetis did not activate haemocytes. SOD significantly decreased the CL emission in zymosan-stimulated haemocytes. P. tapetis cells, as well as their extracellular products, inhibited the CL response to zymosan. Haemocytes from mussels injected with zymosan showed lower levels of stimulation than in vitro treated cells, and CL increased with time after injection.

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PubMed1999

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.

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PubMed1997

Yeast as a model system to study drugs effective against apicomplexan proteins.

Biochemical and genetic analyses are required to identify potential drug targets in apicomplexan parasites, but these studies have proved difficult in most parasite systems. We have developed methods based on expression of parasite proteins in the budding yeast, Saccharomyces cerevisiae, to rapidly screen drugs directed against particular parasite targets, to study the structure and function of these target molecules, and to identify mutations in the parasite genes that alter enzyme specificity or drug sensitivity. In this paper we outline the parameters that need to be considered to design yeast strains that function efficiently to assay function of parasite proteins. Basic protocols and methods are included. We detail some problems that might be encountered in the engineering of these yeast strains and suggest possible solutions.

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PubMed2000

The cell cycle in protozoan parasites.

Research into cell cycle control in protozoan parasites, which are responsible for major public health problems in the developing world, has been hampered by the difficulties in performing classical genetic analysis with these organisms. Nevertheless, in a large part thanks to the data gathered in other eukaryotic systems and to the acquisition of the sequences of parasite genes homologous to cell cycle regulators, many molecular tools required for an in-depth study of the cell cycle in protozoan parasites have been collected over the past few years. Despite the considerable phylogenetic divergence between these organisms and other eukaryotes, and notwithstanding important specificities such as the apparent lack of checkpoints during cell cycle progression, available data indicate that the major families of cell cycle regulators appear to operate in protozoan parasites. Functional studies are now needed to define the precise role of these regulators in the life cycle of the parasites, and to possibly validate cell cycle control elements as potential targets for chemotherapy.

Animals↗
PubMed1981

Assessment of Theileria infections in Rhipicephalus appendiculatus ticks collected from the field.

Collections of adult Rhipicephalus appendiculatus ticks were made from bait cattle and vegetation at two field sites in areas of Kenya in which East Coast fever caused by Theileria parva is endemic. These ticks, together with two experimentally infected batches of ticks, were examined for infection with Theileria by four methods. Whole salivary glands were stained with methyl green pyronin or Feulgen's stain. Whole ticks were ground in medium, the suspensions were filtered and centrifuged and the treated material was examined microscopically and tested for infectivity by inoculation into cattle. All field collections and experimental batches of ticks were infected with Theileria and all four methods detected the infections. Approximately 1.5% of the ticks in the field collections were found to be infected with Theileria and the treated material from these ticks transmitted T. parva to cattle. It is considered that it will be feasible to survey field infection rates quantitatively by collecting ticks from bait cattle and vegetation for examination by a combination of salivary gland staining and preparation of tick suspensions for microscopy and infectivity tests.

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PubMed1976

An electron microscopic study of Babesia microti invading erythrocytes.

Intracellular sporozoan parasites invade the host cell through the invagination of the plasma membrane of the host and a vacuole is formed which accommodates the entering parasite. The vacuole may disappear and the invaginated membrane of the host then becomes closely apposed to that of the parasite's own membrane. As a result the parasite is covered by two membranes. Members of the class Piroplasmea differ from other Sporozoa in that their trophozoites are covered by a single membrane. By screening numerous sections of intraerythrocytic Babesia microti belonging to the class Piroplasmea, it was found that merozoites of Babesia enter the erythrocytes of hamsters in the same way as those of the other Sporozoa. When a merozoite touches the red blood cell with its anterior end it becomes attached to the membrane of the host, which starts to invaginate and a parasitophorous vacuole is formed. The vacuolar space disappears rapidly and the membrane of the vacuole and that of the parasite become closely adjacent. At this stage the parasite is surrounded by two plasma membranes. The outer membrane derived from the invaginated host membrane disintegrates quickly and the parasite is left with a single membrane throughout its life span.

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PubMed1979

Sporogenesis of a myxosporidan with motile spores.

Three types of cells comprise each Fabespora vermicola sporoblast: valvogenic (VAV), capsulogenic (CAP), and germinative (GEM). Walls, polar caps, and sutures are the main assemblages produced by the VAV cells. The unique polar cap organelle extends over the aperture region of the polar capsule component of the CAP cell. The VAV cell also assembles a wall located on the cytoplasmic side of the plasma membrane facing the sporoblast exterior. Bundles of 7 nm microfilaments develop within the extracellular space between the VAV and interior cells of the sporoblast. These microfilaments assemble late in sporogenesis when the spore acquires the capacity for locomotion. Polar filament construction takes place exclusively within the polar capsule primordium (PCP) by apparent self-assembly prior to the PCP being enveloped by membranes. The CAP and GEM cells accumulate considerable glycogen during sporogenesis. The first identifiable GEM cell is single, but has two unpaired nuclei. These GEM cell nuclei later form a paired structure which is sustained into the spore stage.

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