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.

Transmission blockade of Plasmodium falciparum: its variability with gametocyte numbers and concentration of antibody.

The transmission of Plasmodium falciparum through Anopheles stephensi was measured in the presence of anti-gamete (anti-45/48 kDa) and anti-zygote/ookinete (anti-25 kDa) antibodies. With lowering numbers of infectious gametocytes in the presence of the same concentration of antibodies, different results were obtained with the two types of antibodies. Transmission blocking appeared to weaken when using anti-45/48 kDa antibody, whilst, with the anti-25 kDa antibody, transmission inhibition was markedly strengthened with lower parasite numbers. Keeping the parasite numbers constant and progressively lowering the concentration of both types of antibodies resulted in a gradual loss of the latters' blocking ability. No enhancement of transmission occurred at low antibody concentrations.

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.

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.

Sporozoite load of mosquitoes infected with Plasmodium falciparum.

In the laboratory, mosquitoes given a second blood meal 5-11 d after an infective one have more sporozoites in their salivary glands than do those given a single infective blood meal only. The presence of specific anti-sporozoite antibody in the second blood meal does not reduce the number of sporozoites in salivary glands. On the contrary, the presence of the raised immunoglobulin levels--even non-specific ones--may result in higher gland infections. Oocyst maturation is extremely asynchronous in mosquitoes given a single blood meal, the maturation time being 10-22 d or more. The explanation for the increased density of sporozoites in salivary glands in mosquitoes having a second blood meal may be acceleration of oocyst maturation. Multiple blood meals are a normal event for infectious mosquitoes in nature, and therefore have no special epidemiological significance. However, in the laboratory a second blood meal could be a simple procedure for increasing the efficiency of sporozoite production.

A scanning electron microscopic study of the sporogonic development of Plasmodium falciparum in Anopheles stephensi.

The full development of Plasmodium falciparum in Anopheles stephensi mosquitoes was studied by scanning electron microscopy. Ookinetic development was described from in vitro cultures. Growing oocysts beneath the basal lamina of the midgut wall mechanically stretch this lamina until it is torn and displaced by day 7. In young oocysts the wall appears smooth. In older oocysts wrinkles in the wall are visible after routine fixation. Osmium tetroxide postfixation greatly reduced the occurrence of these wrinkles. Intracapsular development of sporozoites was visualized after mechanical manipulation of the oocysts during sample preparation. In contrast to P. berghei, no ectopic development was seen in P. falciparum in the mosquito midgut. The mechanism of sporozoite escape from the oocyst appears to be similar to that described for rodent malaria. Fracturing of salivary glands provided the first view by scanning electron microscopy of sporozoites located in proximal and distal gland cells and in the draining duct.

Transmission-reducing immunity is inversely related to age in Plasmodium falciparum gametocyte carriers.

Immunity to the sexual stages of Plasmodium falciparum is induced during natural infections and can significantly reduce the transmission of parasites to mosquitoes (transmission reducing activity; TRA) but little is known about how these responses develop with increasing age/exposure to malaria. Routinely TRA is measured in the standard membrane feeding assay (SMFA). Sera were collected from a total of 199 gametocyte carriers (median age 4 years, quartiles 2 and 9 years) near Ifakara, Tanzania; 128 samples were tested in the SMFA and generated TRA data classified as a reduction of > 50% and > 90% of transmission. TRA of > 50% was highest in young children (aged 1-2) with a significant decline with age (chi(2) trend = 5.79, P = 0.016) and in logistic regression was associated with prevalence of antibodies to both Pfs230 and Pfs48/45 (OR 4.03, P = 0.011 and OR 2.43 P = 0.059, respectively). A TRA of > 90% reduction in transmission was not age related but was associated with antibodies to Pfs48/45 (OR 2.36, P = 0.055). Our data confirm that antibodies are an important component of naturally induced TRA. However, whilst a similar but small proportion of individuals at all ages have TRA > 90%, the gradual deterioration of TRA > 50% with age suggests decreased antibody concentration or affinity. This may be due to decreased exposure to gametocytes, probably as a result of increased asexual and/or gametocyte specific immunity.

Infectivity of malarial parasites to mosquitoes: 'the interdependent roles of parasite, vector and host'.

It has been possible to infect mosquitoes routinely with cultured gametocytes of Plasmodium falciparum since 1980. This has enabled the development of a reliable bio-assay for potential transmission-blocking vaccines and research on the role of specific antibodies from the host on the parasitic stages in the mosquito midgut. After some development and fine-tuning of the assay, it became apparent that the immune responses of the human host, as well as factors from the parasite and the mosquito, determined the final outcome of the mosquito infection. The age of the mosquito, crowding of parasites inside the peritrophic membrane and the quantity and particularly the quality of the gametocytes ingested all influence the chance of successful transmission. Cytokines and/or other mediators of inflammation from the human host can also reduce transmission, probably by promoting phagocytosis of the freshly emerged gametes by leucocytes in the bloodmeal.

An automated large-scale culture system of Plasmodium falciparum using tangential flow filtration for medium change.

Asexual stages and mature gametocytes of Plasmodium falciparum have been produced in a 500 ml suspension culture system containing 6-10% red cells. Medium change was automated and was accomplished using a tangential flow filtration unit. The rate of multiplication of the parasite (isolate NF 54) was consistently high when compared with static cultures and ranged from 6.4 X to 15.3 X per cycle in 8 experiments. Mature gametocytes were present in up to 2% of red cells in 14-day-old cultures. Only minor modifications would be required to further scale-up the volume of the culture.

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.

Transmission-blocking antibodies against multiple, non-variant target epitopes of the Plasmodium falciparum gamete surface antigen Pfs230 are all complement-fixing.

We have studied the properties of 16 newly derived monoclonal antibodies (MoAbs) against Pfs230, a gamete surface protein of Plasmodium falciparum and a target of transmission-blocking antibodies. All 16 MoAbs recognized Pfs230 by immunoprecipitation from non-ionic detergent extracts of the protein radio-labelled with 125Iodine. The MoAbs also recognized this protein on Western blots under non-reducing conditions but none of them recognized the protein under reducing conditions. Using an immunoradiometric assay the MoAbs appear to define nine different epitope regions. The MoAbs were tested for their ability to lyse extra-cellular female gametes of P. falciparum isolate 3D7. Eight of the MoAbs, all of isotype IgG2a, mediated complement-dependent lysis of the gametes; seven of the MoAbs, all isotype IgG1, failed to lyse the gametes in the presence of active complement. The eight complement-fixing MoAbs mediated almost total suppression of infectivity of gametocytes of P. falciparum 3D7 to mosquitoes; where tested this suppression was mainly complement-dependent. The seven non-complement-fixing MoAbs had no significant effect on the infectivity of gametocytes of P. falciparum 3D7 to mosquitoes. When tested by immunofluorescence the target epitopes of all the MoAbs were conserved in each of the five different isolates of P. falciparum which were tested.

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)

Plasmodium falciparum ookinetes migrate intercellularly through Anopheles stephensi midgut epithelium.

The migration of Plasmodium falciparum and P. berghei ookinetes through the midgut epithelium in Anopheles 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 parasite P. berghei appeared to take an intracellular position, as previously suggested by other authors. The intra- or intercellular ookinete migration of P. berghei or P. falciparum, respectively, can perhaps be related to the higher mortality of P. 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.

Leukocytes in a Plasmodium falciparum-infected blood meal reduce transmission of malaria to Anopheles mosquitoes.

Mosquitoes are infected with Plasmodium falciparum by taking a blood meal from a gametocyte carrier. Since a mosquito takes a volume of 1 to 2 microl, a blood meal may contain 1 x 10(4) to 3 x 10(4) leukocytes (WBC). The majority of WBC are composed of neutrophils which may phagocytose and kill developing gametes inside the mosquito midgut. Phagocytosis was measured in vitro by a luminol-dependent chemiluminescence (CL) assay. In the presence of P. falciparum gametes, sera from areas of endemicity had an increased CL response compared to controls. In mosquito membrane feeding experiments some such sera showed a transmission reduction which was related to the presence of viable WBC. The results of this study suggest that phagocytosis of opsonized gametes inside the mosquito midgut occurs and can contribute to a reduction in the transmission of P. falciparum parasites.

DNA synthesis in gametocytes of Plasmodium falciparum.

The DNA content of Plasmodium falciparum gametocytes during intra-erythrocytic development and during gametogenesis was established by cytophotometric methods. Intraerythrocytic micro- and macrogametocytes (Stage I-Stage VB) contain about twice the amount of DNA of haploid sporozoites and ringstages, indicating that DNA is synthesized during transformation of ringforms into Stage I gametocytes. Microgametocytes, after activation at pH 8, rapidly duplicate their genome several times, while the DNA content of macrogametocytes remains constant during gametogenesis.

Transmission blocking antibody of the Plasmodium falciparum zygote/ookinete surface protein Pfs25 also influences sporozoite development.

The Plasmodium falciparum zygote/ookinete surface protein, Pfs25, persists in the oocyst wall throughout its development. Anti-25 kD transmission blocking antibody, given to infected Anopheles stephensi or A. gambiae mosquitoes in an additional bloodmeal, 3-6 days after being fed gametocyte infected blood, penetrated the oocyst and reacted with the 25 kD protein within it. This reaction caused a significant reduction in the number of developing sporozoites. Mouse serum containing antibodies raised by immunization with a recombinant 25 kD yeast product showed a similar effect.

Large-scale production of Plasmodium vivax sporozoites.

Mass-scale production of Plasmodium vivax sporozoites in Anopheles stephensi was achieved using the chimpanzee (Pan troglodytes) as a source of infective blood. Membrane feeding was as successful as feeding mosquitoes directly on the animal so long as the time between drawing the blood and feeding was restricted to 45 min. Longer delays such as 2-3 h resulted in loss of infectivity in terms of oocyst production. The selected strain of A. stephensi was highly susceptible to P. vivax (Chesson strain). A strain of A. stephensi relatively refractory to P. falciparum showed no cross-refractoriness to P. vivax. Mixed infections of P. falciparum and P. vivax did not interfere with each other in their development in A. stephensi. A second normal blood meal to mosquitoes infected with P. vivax increased the yield of salivary gland sporozoites.