T. Ponnudurai

Infectivity of cultured Plasmodium falciparum gametocytes to mosquitoes.

Various factors that may influence routine and high levels of mosquito infection with cultured Plasmodium falciparum gametocytes are considered in this paper. One of the most important is the choice of an appropriate isolate, with facilities for cryopreservation and a good technique for initiation of cultures. The use of automated culture systems with strict adherence to detail and routine has eliminated much of the variability. The quality of the serum used for the culture of gametocytes and inclusion in the feed material for mosquitoes is of the highest importance. Blood collection for culture purposes must preferably involve alcohol as an antiseptic for cleaning donor skin or suitable receptacles. Mosquito blood meals should not include plasma with citrate phosphate dextrose or sera collected in microtainer tubes or from volunteers on proguanil-chloroquine prophylaxis. Sera of individuals on chloroquine alone do not influence transmission. Haematocrits of from 5 to 10% permit the culture of equally infective gametocytes. It was impossible to predict the outcome of an infection in mosquitoes based on the number of female gametocytes or gametes. Within any experiment, the oocyst load initially increased, followed by a decline with progressively lower numbers of gametocytes accompanied by a progressive increase in the efficiency of transmission. Some of the variability of mosquito infection within an experiment was due to individual differences in the speed of blood digestion of the mosquitoes. A new membrane feeder is described with three different sizes to accommodate a variety of goals.(ABSTRACT TRUNCATED AT 250 WORDS)

The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes

In vitro gametocytogenesis of Plasmodium falciparum was observed in all 22 isolates established in this laboratory. Gametocytes were produced in variable numbers--up to 3% of red cells--for a limited period of time after which this stage was seen only very sporadically. Complete maturation of microgametocytes in vitro was obtained in all 14 of the isolates that were tested for exflagellation. Up to 88.2% of membrane-fed Anopheles stephensi were infected from material produced in culture. It was also possible to infect A. gambiae and A. freeborni. Addition of fresh red cells and serum to culture material promoted infectivity of gametocytes. Gametocyte infectivity declined rapidly with time in the membrane feeders held at 38 degrees C.

Feeding behaviour and sporozoite ejection by infected Anopheles stephensi.

Anopheles stephensi mosquitoes infected with Plasmodium falciparum sporozoites were allowed to feed individually through fresh whole thickness mouse skin. More sporozoites were ejected into the skin in clusters than into the blood. Deposition of sporozoites in the blood was an infrequent occurrence and always coincided with ejection of these stages into the skin--perhaps a spill-over effect. The number of probes before feeding (median 4.5) was not correlated with the sporozoite inoculum (median 8), nor was the number of sporozoites in the glands (median 14,500). However, the number of sporozoite clusters in the skin (median 1) was positively correlated with the inoculum size. The median value of the sporozoite inoculum was 22, when only those mosquitoes that ejected sporozoites were included. When feeding was interrupted and recommended on a new membrane, sporozoite ejection occurred with equal frequency on both occasions. Sporozoites disappeared from the site of bites in living mice within 2 h of feeding. The epidemiological significance of these observations is discussed.

Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. 1. Static cultures.

A semi-automated cultivation apparatus for the in vitro culture of Plasmodium falciparum gametocytes is described. This apparatus has been designed to produce large numbers of fertile sexual stages for use in the development of a gamete vaccine or for the infection of suitable mosquitoes. These mosquitoes in turn may be used for the development of a possible sporozoite vaccine. Loss of red cells during medium change has been eliminated and the addition of warmed fresh medium simplified compared to similar systems described previously. Material harvested from this apparatus has been used for infecting mosquitoes. Up to 98% of Anopheles stephensi were infected with a mean oocyst count of 24 per positive gut (range one to 109). The importance of satisfactory presentation of gametocytes for mosquito infection is stressed. The possible presence of substances in normal human sera which inhibits exflagellation to a variable degree and reduces mosquito infectivity is also discussed.

Cloning and expression of the gene coding for the transmission blocking target antigen Pfs48/45 of Plasmodium falciparum

The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of Plasmodium falciparum has been cloned. The Pfs48/45 gene is a non-interrupted, single copy gene that codes for a hydrophobic, non-repetitive protein of 448 amino acid residues containing a putative signal peptide at the N-terminus, a hydrophobic C-terminus and 7 potential N-glycosylation sites. Antibodies directed against a Pfs48/45-glutathione-S-transferase fusion protein reacted with both the 45-kDa and 48-kDa proteins of gametocytes. When Pfs48/45 is expressed in the baculovirus-insect cell system the recombinant Pfs48/45 protein is targeted and exposed to the insect cell surface in such a configuration that it is recognized by transmission-blocking anti-45/48-kDa monoclonal antibodies.

Characterization of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures

Synchronised gametocyte cultures were used to study the biosynthesis of the sexual stage target antigens (Mr 230 000, 48 000 and 25 000) for anti gamete/zygote antibodies. These antigens were shown to be synthesized during gametocyte development from day 2-3 onwards until gametogenesis occurred. After gametogenesis a 25 kDa protein was predominantly synthesized, whereas synthesis of the other target proteins was hardly detectable. The 48, 45, and 25 kDa proteins appeared to be glycosylated, in addition the 25 kDa was also acylated in that it bound [3H]palmitic acid covalently. The iso-electric point (pI) of these proteins was assessed as being 6.0 +/- 0.1 (for both 48 and 45 kDa) and 5.6 +/- 0.1 (for 25 kDa).

Synchronization of Plasmodium falciparum gametocytes using an automated suspension culture system

An automated suspension culture system for the cultivation of Plasmodium falciparum is described which retains a degree of flexibility which is absent in other automated culture apparatuses. Not only does this system of cultivation promote rapid multiplication of asexual parasites but also permits the development and maturation of gametocytes. Using a combination of gelatin flotation and N-acetyl glucosamine treatment synchronous development of gametocytes was achieved. The total time for gametocyte maturation in vitro under the conditions provided was 7 days. Stages II and V required 48 h for development whilst I, III and IV needed 24 h each. Mature microgametocytes were relatively long lived in comparison with macrogametocytes. Electron microscopic study of the synchronized stages confirmed the observations of Sinden (1982) but, in addition, we noted the presence of Garnham bodies, a cytostome in all stages and dense spherules in stages I-III similar to the fenestrated buttons in sporozoites and exoerythrocytic forms. The relationship between the number of osmiophilic bodies in the mature gametocytes and their ability to escape from the red cell is reaffirmed.

Malaria sporozoite penetration: a new approach by double staining

To determine, whether a sporozoite is outside the hepatocyte membrane or internalized, a double staining test was carried out using, successively, antibody labeled with peroxidase and fluorescein. This test permits the quantification of sporozoite entry and outline sporozoite-hepatocyte interactions.

Plasmodium species: Flow cytometry and microfluorometry assessments of DNA content and synthesis

Fluorescence intensities were established by flow cytometry of different erythrocytic stages of Plasmodium berghei after staining of their DNA with Hoechst-33258 or Hoechst-33342. Parasites were obtained from highly synchronized infections or in vitro cultures. Most fluorescence measurements were performed using a low cost, clinical flow cytometer, equipped with a mercury arc lamp. Cells infected with P. berghei could be readily distinguished from uninfected cells on the basis of Hoechst-DNA fluorescence and single, double, and triple ring infected cells were separated clearly. The relative fluorescence intensities of different developmental stages (merozoites, ringforms, trophozoites, schizonts, and gametocytes) corresponded closely to the relative DNA contents of these stages as measured by microfluorometry. Flow cytometry appeared to be a sensitive and rapid method to measure DNA synthesis during asexual development; a C50 value of 5 microM of aphidicolin, a specific inhibitor of DNA synthesis, was established. Vital staining of parasites in culture was possible with both Hoechst dyes. After removal of Hoechst-33258, normal in vitro development of the stained parasites was observed. After Hoechst staining, the haploid ringforms of P. vivax showed slightly less fluorescence (15%) than ringforms of P. berghei and P. falciparum. No differences in fluorescence intensity were observed, however, by direct microfluorometry after Feulgen-pararosaniline staining, indicating that all three species have the same DNA content.

Plasmodium falciparum ookinetes migrate intercellularly through Anopheles stephensi midgut epithelium.

The migration ofPlasmodium falciparum andP. berghei ookinetes through the midgut epithelium inAnopheles stephensi was studied by transmission electron microscopy. With ruthenium red (RR) staining, the results of previous studies were confirmed:P. falciparum ookinetes take an intercellular route through the midgut epithelium. In the same mosquito species, the rodent parasiteP. berghei appeared to take an intracellular position, as previously suggested by other authors. The intra- or intercellular ookinete migration ofP. berghei orP. falciparum, respectively, can perhaps be related to the higher mortality ofP. berghei-infected mosquitoes within the first 2 days of infection. Evidence is presented that oocyst capsule formation begins as early as during the migration of the ookinete. After localization between the epithelial cells and the midgut basal lamina, the rapidly expanding oocyst stretches the overlying layer of the latter at the haemocoelic surface while a new basal lamina is generated between the oocyst and epithelial cell.