Chariya R. Brockelman

Opisthorchis viverrini: finding and recognition of the fish host by the cercariae.

The cercaria of Opisthorchis viverrini finds and recognizes its fish host by using at least four steps of behavioral patterns. (1) Dispersal and selection of plant-free water microhabitat are achieved by intermittent swimming behavior with positive phototactic orientation. (2) Attachment to the host is stimulated by water currents and a hydrophilic component of fish skin surface which has a molecular weight of more than 30,000. This component is sensitive to digestion with hyaluronidase and seems to be a glycosaminoglycan other than hyaluronic acid and chondroitin sulfate. (3) Remaining on the hosts surface is induced by an unknown chemical component of fish skin surface mucus. (4) Penetration into the host is triggered by a hydrophilic component of fish skin surface of a molecular weight of more than 30,000. This host signal has a proteinaceus character as it is sensitive to proteinase digestion but not to hyaluronidase and glycosidases. The requirement of O. viverrini cercariae for complete glycosaminoglycans and proteins as signals in host identification may have the advantage that the numerous small molecules in mud and decaying materials in the water cannot interfere with host-finding.

Finding and recognition of the bovine host by the cercariae of Schistosoma spindale

The cercaria ofSchistosoma spindale finds and identifies its bovine host with at least five behavioral phases. (1) Dispersal in and selection of midwater and water surface as the microhabitat are achieved by an intermittent swimming behavior with a weak geonegative but not photopositive orientation. (2) Attachments are stimulated by host-specific higher temperatures of the substrate but not by chemical host signals. (3) Remaining of the attached cercariae on the substrate is stimulated by host-specific higher temperatures of the substrate; chemical host signals have no effect. (4) The creeping of the cercariae is directed to the higher temperature in thermal gradients as weak as 0.07° C/mm. Chemical gradients had no effect on the creeping direction. This behavior may enable the cercariae to migrate along hairs to the hosts skin surface. (5) Penetrations are stimulated by the free fatty acid fraction of bovine skin-surface lipids. The characteristics of the stimulating fatty acids are the same as those identified for other schistosome species. Higher temperatures of the substrate alone do not stimulate penetrations.S. spindale cercariae do not use as many chemical host cues as stimuli for the identification of their host as doS. mansoni cercariae.S. spindale seems to be adapted to hairy hosts that are infected in shallow, muddy waters. The low host specificity of the cercarial host-finding behavior is compensated by an intimate parasite-snail intermediate host relationship, resulting in a high cercarial production of up to >7,000 cercariae per snail per day.

DEVELOPMENT OF AN IN VITRO MICROTEST TO ASSESS DRUG SUSCEPTIBILITY OF BABESIA BOVIS AND BABESIA BIGEMINA

Continuous cultivation of the bovine hemoparasites Babesia bovis and Babesia bigemina was developed as an in vitro microtest to assess parasite susceptibility to babesicidal compounds. Reproducibility of parasite multiplication rates was independent of culture size, making it possible to use a microscale of 100 microliters for each test sample. Inhibitory concentrations (IC50s) of a commonly used babesicide, quinuronium sulfate, evaluated by this in vitro method were found to be 5 x 10(-8) g/ml for B. bovis and 2 x 10(-9) g/ml for B. bigemina.

A specific DNA probe which identifies Babesia bovis in whole blood

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.

Carbohydrate reserves and hemolymph sugars of the African giant snail,Achatina fulica in relation to parasitic infection and starvation

In the snail,Achatina fulica, parasitized by the rat lungwormAngiostrongylus cantonensis, levels of carbohydrate in hemolymph and digestive glands were determined. The normal level of hemolymph glucose of 11.7 mg% dropped to 4.25 mg% in the infected snails, a significant difference after only one week of infection. The level of total reducing sugar in the hemolymph also decreased significantly (12.3 mg% to 3.6 mg%) in this period. Later on, the snails were capable of adapting themselves to the parasitic infection and the concentration of hemolymph sugars returned to the normal range. Starvation caused a decrease in the carbohydrate reserves of the digestive gland only when the starved snails had been previously infected.

CONTINUOUS CULTIVATION AND IMPROVED DRUG RESPONSIVENESS OF PLASMODIUM FALCIPARUM IN p-AMINOBENZOIC ACID-DEFICIENT MEDIUM

Long term cultivation of three culture lines of Plasmodium falciparum was achieved in Waymouth and RPMI media, both supplemented with 10% heat-inactivated human serum. Observations on parasite multiplication over a 6-mo period showed no difference in the rate of parasite population increase. Growth of asexual erythrocytic stages in Waymouth medium was as good as that in RPMI when measured by the rate of glucose utilization and extent of 3H-isoleucine and 3H-methionine incorporation. Responsiveness of the parasites to pyrimethamine and sulfadoxine was demonstrated to increase when tested in Waymouth medium because it contained no p-aminobenzoic acid to compete with the drugs.

Plasmodium falciparum: Variations in p-aminobenzoic acid requirements as related to sulfadoxine sensitivity

Plasmodium falciparum, Thai strain FCM-5S sensitive to sulfadoxine, was grown continuously for 1 year in a medium with a low concentration of p-aminobenzoic acid (PABA) without the emergence of a sulfadoxine-resistant line. The minimum PABA-requirement for growth of sulfadoxine-sensitive Thai strains in vitro was found to be 25.0 ng/ml, whereas resistant Burmese and Gambian strains completed their schizogony in the absence of PABA. Cultivation in dialyzed and nondialyzed human serum suggested that the sulfadoxine-resistant parasite might use another serum factor in place of PABA for folate biosynthesis, thus overcoming the effect of sulfadoxine, a PABA antagonist. The significantly lower rate of [14C]sulfadoxine incorporation into erythrocytes infected with sulfadoxine-resistant strains strongly supports this suggestion.

Effects of parasitism and stress on hemolymph protein of the African giant snail,Achatina fulica

SummaryEffects of parasitism and stress on the protein concentration of hemolymph have been investigated using the rat lungwormAngiostrongylus cantonensis in the snail hostAchatina fulica. The normal hemolymph protein concentration, averaging 1.77 g/100 ml in noninfected snails, did not show any reduction when the hosts were infected with third stage larvae. When the infected snails were bled repeatedly, protein concentration showed a significant decrease by 0.6 g/100 ml. Starved, infected snails were capable of maintaining their hemolymph protein level within the normal range. This treatment, however, in combination with frequent bleeding, caused much stress to the snails and reduced survival. The number of survivors depended on the frequency of bleeding and on the food level.

The influence of magnesium ion and ascorbic acid on the erythrocytic schizogony of Plasmodium vivax

Asexual erythrocytic stages of Plasmodium vivax have been cultivated for one schizogonic cycle to investigate parasite requirements for metal ions and vitamins. Waymouth and RPMI 1640 (GIBCO) media were used in varying proportions resulting in varying concentrations of organic salts, vitamins, and growth factors. A 1∶3 mixture gave the highest percentage (62.6%) of parasite development from the amoeboid forms to mature segmenters after 44 h of cultivation in a candle jar atmosphere at 38.5‡ C. Nevertheless, the total parasite count was significantly higher (P<0.50) in the mixture which had a Waymouth: RPMI ratio of 1∶2. Differentiation of schizonts to merozoites as well as parasite counts could be further enhanced by the addition of magnesium chloride to a final concentration of 1.8 mM magnesium ions. The minimal requirement for ascorbic acid which was studied in Science Mahidol (SCMI 612) medium appeared to vary among isolates. For example, all parasite population of four isolates tested declined proportionally with the decrease in concentration of ascorbic acid, the critical point being 3 Μg/ml medium. However, two isolates used in this study could no longer differentiate to segmenters when the ascorbic acid concentration of the medium was less than 6 Μg/ml.

Ultrastructural Study on the Interaction of β-Tha1assemicErythrocytes and Plasmodium falciparum

Summary β-thalassemic erythrocytes and normal red blood cells were experimentally infected in vitro with Plasmodium falciparum cultured for 4 days and studied by means of transmission electron microscopy. Conspicuous alterations of the parasites appear in the advanced stage of schizogony leading to the fact that only a small amount of malaria pigment is formed, which in general is not crystalline, but always enclosed in extremely large vacuoles. Furthermore some of the developing merozoites reveal features of cellular degeneration, thus these merozoites lost their ability to invade new erythrocytes. Despite these findings alterations of the host cells are induced by the parasites, which, however, are comparable to those found in infected normal red blood cells, e.g. knobs appeared on the surface of the erythrocytes and membrane-bounded clefts became apparent in the cytoplasm of the host cell.