J.H.E.Th. Meuwissen

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.

Developmental biology of Pneumocystis carinii, an alternative view on the life cycle of the parasite.

SummaryIn this paper we present, based on elaborate ultrastructural studies, data on the existence of both intracellular and extracellular stages of Pneumocystis carinii, which result in a proposal of a new life cycle of the parasite. Up to now the formation of daughter cells in thick-walled pneumocysts is supposed to be the only way of multiplication. The present study shows that in rats treated with cortisone acetate the formation of daughter cells also takes place within thin-walled pneumocysts. In our opinion this way of multiplication is important for the understanding of the rapid increase in number of the parasites in an infected lung.The presence of pneumocysts inside the alveolar epithelial cells suggests that intracellular development of the parasites can occur, but the method of cell penetration, intracellular multiplication and parasite liberation is still unknown.Moreover our observations for the first time indicate a direct pathogenicity of the parasites in host cells.

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.

Transformation of sporozoites of Plasmodium berghei into exoerythrocytic forms in the liver of its mammalian host

SummaryIntrahepatocytic transformation in vivo of the rodent malaria sporozoite of Plasmodium berghei, into the young trophic exoerythrocytic tissue stage was studied by immunofluorescence, light- and electron microscopy. The first 20 h of intracellular life were involved entirely in dedifferentiation with limited proliferation of organelles. From about 20 h onwards nuclear division commenced, rough endoplasmic reticulum became markedly expanded, and mitochondria increased in numbers. However, remains of the sporozoite pellicle (i.e., inner membranes and subpellicular microtubules) persisted for at least 28 h, which correlates with the persisting reaction of young exoerythrocytic forms with antisporozoite antibodies. In general, the basic mechanism of transformation resembles that of the ookinete into oocyst and that of the merozoite into erythrocytic trophozoite.

The dual role of macrophages in the sporozoite-induced malaria infection. A hypothesis.

Abstract Macrophages were thought to act as a barrier to sporozoite entry in liver cells. Evidence is presented here for a possible dual role of macrophages under different sets of conditions. Before activation macrophages might act as transporting agents of sporozoites to the parenchymal cells of the liver. Activated macrophages however might take the opposite role of preventing parasite entry. Various possible mechanisms involved are reviewed.

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.

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.