Plasmodium gallinaceum: antibodies to circumsporozoite protein prevent sporozoites from invading the salivary glands of Aedes aegypti.

A circumsporozoite protein-specific monoclonal antibody (N2H6D5) was injected into malaria-infected mosquitoes to determine its effect on the sporogonic cycle. After injection of antibody into mosquitoes (100 ng each), positive immunofluorescence (measured on air-dried sporozoites) reactions in hemolymph extracts were observed at a dilution of 1:1000. At 72 hr postinjection the levels dropped to 1:10. Sporozoites coinjected with antibody did not invade the salivary glands. In naturally infected mosquitoes, sporozoites were released over a period of 3 to 4 days. Therefore, mosquitoes were injected twice. The first injection was a day before the beginning of sporozoite release and the second, 2 days later. Sporozoite invasion of the salivary glands was assessed 3 days after the second injection, by microscopic examination of dissected glands. At this stage, all oocysts had completed maturation and released the sporozoites. Salivary gland infections were totally prevented in mosquitoes given two injections of 100 ng N2H6D5. Hence, sustained presence of anti-circumsporozoite antibodies in the hemolymph can render female Aedes aegypti refractory to Plasmodium gallinaceum.     

Defined medium supporting development of cleansed Plasmodium berghei ookinetes in Anopheles stephensi.

Hamsters blood infected with Plasmodium berghei was cultured in vitro for the development of ookinetes. The ookinetes were separated from blood components, suspended in various defined media and fed to Anopheles stephensi through a membrane. The development of the oocysts and infective sporozoites was recorded. Mosquitoes infected with ookinetes suspended in L15 formulated into L15-B, L15-D (a medium specially modified for this purpose), IPL-41 or 199 media with no proteins added, developed at least as many oocysts as the control mosquitoes fed ookinetes suspended in blood. Ookinetes suspended in the L15-B medium yielded more oocysts than after feeding ookinetes suspended in L15-B with 5% casein. Sporozoites from mosquitoes maintained on blood, L15-B, L15-D, or L15-B with 5% casein were shown to be infective to hamsters. Mosquitoes fed ookinetes suspended in sucrose solutions showed very few oocysts, but the yield was increased when a blood meal was given 2-4 days after the infective meal. Some of the oocysts which had developed from the ookinetes suspended in artificial media were found to have degenerated. The described system could be potentially useful for a study of the interaction between the vector physiology and the parasite. The possible use of the system to learn which media should be developed in the future for in vitro cultivation of oocysts is discussed.     

Gametocytocidal and sporontocidal effects of antimalarial drugs on malaria parasites. II. Action of the folic reductase inhibitors, chlorguanide, and pyrimethamine against Plasmodium cynomolgi

The antimalarial activity of the dihydrofolate reductase inhibitors, chlorguanide and pyrimethamine, against gametocytes and sporogony of the simian parasite, Plasmodium cynomolgi was tested. Plasmodium cynomolgi infected rhesus monkeys (Macaca mulatta) were treated orally with multiple doses of chlorguanide (5.6 or 11.3 mg base/kg/Day X5) or a single dose of pyrimethamine (3 mg base/kg). Batches of mosquitoes (Anopheles maculatus) were allowed to feed immediately prior to and at appropriate intervals after drug administration. The effects of the drugs on the developmental stages of the parasite were assessed within the mosquito host. The results indicated that sporogony was interrupted in mosquitoes fed 1 hr after initial dosing with chlorguanide. With pyrimethamine, a clear-cut sporontocidal action was shown as early as 15 min after the drug was given. Both compounds inhibited further development of young oocysts and successive batches of mosquitoes fed 1 hr or more after medication ware consistently negative for sporozoites. Administration of either compound in the indicated doses did not prevent exflagellation, zygote or ookinete formation. However, the behavior of the latter forms was apparently altered by the action of the drug. There was a marked retarding effect in the formation of ookinetes in mosquitoes fed 24 hr after initial medication and thereafter. The schizontocidal and gametocytocidal action of chlorguanide was more evident than it was in pyrimethamine.     

Comparative studies on the infectivity of Plasmodium berghei gametocytes and ookinetes for gnotobiotic and xenobiotic Anopheles stephensi

Previous studies indicated that gnotobiotic Anopheles stephensi mosquitoes were less susceptible to infection with Plasmodium berghei than xenobiotic ones (Munderloh and Kurtti, 1985). Groups of 100 to 200 mosquitoes were fed on infected hamsters, heparinized gametocytemic blood (via a membrane feeder), and in vitro-formed ookinetes suspended in blood (membrane feeder). Xenobiotic A. stephensi were readily infected by all 3 routes. Gnotobiotic mosquitoes consistently acquired infection after engorging on hamsters (average level of infected females in 8 experiments: 54.1%), but the parasite yield was low (average number of oocysts per infected female: 21.6). In 7 experiments where gnotobiotic A. stephensi were membrane-fed infected hamster blood, an average of only 8.8% of the females became infected, harboring a mean of 2.4 oocysts, and in 7 additional cases no infection was achieved. This pattern was reversed when gnotobiotic A. stephensi were fed ookinetes. A larger proportion of them became infected (mean level of infection in 8 experiments: 76.2%) and they acquired a higher mean number of oocysts per female (94.4) than did xenobiotic mosquitoes. Thus, gnotobiotic A. stephensi are as able as xenobiotic ones to support the sporogonic development of P. berghei, but are less able to support ookinete development.     

Eimeria tenella: local antibodies and interactions with the sporozoite surface

Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10(3) oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10(5) oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and immunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results.(ABSTRACT TRUNCATED AT 250 WORDS)     

True Intermediate Hosts for Eimeria funduli (Apicomplexa) from Estuarine Fishes1

Grass shrimp (Palaemonetes pugio) fed liver containing sporulated oocysts of Eimeria funduli permitted development of sporozoites that became infective to a variety of killifishes. The shrimp's gastric mill mechanically ruptured the oocysts. Sporozoites then excysted through an opening in the sporocyst, and by 12 and 13 h postinfection (p.i.) numerous empty sporocysts and free sporozoites occurred extracellularly in the intestine of the grass shrimp. Even at 5, 7, 8, 11, 46, 79, and 83 days p.i., and presumably for many months, numerous sporozoites still occurred free in the alimentary tract or between intestinal cells. The coccidium did not infect killifish at either 2 or 4 days p.i., but did at 5 days; after release from the sporocyst, it became more elongate with a distinct nucleus and two relatively large refractile bodies. Infections of E. funduli resulted in about one half of the fish that were fed either entire hepatopancreas or tips of hepatopancreas from experimentally infected shrimp. Feeding either the entire alimentary tract proximal to the first abdominal segment or any portion of that section from experimentally infected shrimp produced infections in nearly all tested fish. Feeding portions of the cephalothorax without any attached hepatopancreas or alimentary tract failed to produce an infection. Feeding killifish with wild grass shrimp from an enzootic area produced infections in only a fourth of the fish sample; however, feeding experimentally infected wild, laboratory-reared, and juvenile grass shrimp produced infections in nearly all fish. Palaemonid shrimps other than P. pugio also can serve as intermediate hosts for E. funduli, and these shrimps include Palaemonetes vulgaris, P. paludosus, P. kadiakensis, and Macrobrachium ohione. In contrast, a penaeid shrimp, mysidacean, amphipod, and crab fed liver with sporulated oocysts did not produce infections when fed to killifish.     

Motility and infectivity of Plasmodium berghei sporozoites expressing avian Plasmodium gallinaceum circumsporozoite protein

Avian and rodent malaria sporozoites selectively invade different vertebrate cell types, namely macrophages and hepatocytes, and develop in distantly related vector species. To investigate the role of the circumsporozoite (CS) protein in determining parasite survival in different vector species and vertebrate host cell types, we replaced the endogenous CS protein gene of the rodent malaria parasite Plasmodium berghei with that of the avian parasite P. gallinaceum and control rodent parasite P. yoelii. In anopheline mosquitoes, P. berghei parasites carrying P. gallinaceum and rodent parasite P. yoelii CS protein gene developed into oocysts and sporozoites. Plasmodium gallinaceum CS expressing transgenic sporozoites, although motile, failed to invade mosquito salivary glands and to infect mice, which suggests that motility alone is not sufficient for invasion. Notably, a percentage of infected Anopheles stephensi mosquitoes showed melanotic encapsulation of late stage oocysts. This was not observed in control infections or in A. gambiae infections. These findings shed new light on the role of the CS protein in the interaction of the parasite with both the mosquito vector and the rodent host.     

Malaria-induced reduction of fecundity during the first gonotrophic cycle of Anopheles Stephensi mosquitoes

Abstract. Anopheles stephensi mosquitoes which had fed upon mice infected with Plasmodium yoelii nigeriensis malaria parasites produced significantly fewer eggs than mosquitoes fed on an uninfected mouse. Fecundity reduction was more pronounced when the bloodmeal contained malaria gametocytes and the mosquitoes developed oocysts. Egg production and haematin excretion were correlated for uninfected bloodfed mosquitoes; the presence of P.y. nigeriensis in the blood affected this relationship. Reduced fecundity was associated with a significant reduction of bloodmeal size (measured by haematin excretion) in mosquitoes which ingested gametocytaemic blood. The bloodmeal size in mosquitoes fed on parasitaemic blood without gametocytes was not significantly reduced. The use of haematin assays for determination of bloodmeal size in mosquitoes is discussed.     

Susceptibility of species A, B, and C of Anopheles culicifacies complex to Plasmodium yoelii yoelii and Plasmodium vinckei petteri infections

The comparative susceptibilities of colonized species A, B, and C of Anopheles culicifacies complex and Anopheles stephensi were determined for 2 rodent malaria parasites Plasmodium vinckei petteri and Plasmodium yoelii yoelii. All the 3 members of the complex were found to support complete sporogony with varying success. Controls, A. stephensi, become readily infected, with >70% developing oocysts. Of the test groups, species A had the highest percentage of mosquitoes with oocysts (>25%) and sporozoites (>15%). Anopheles culicifacies species B were least susceptible; less than 10% had oocysts and sporozoites in the salivary glands. The results demonstrate that A. culicifacies species A is most susceptible and species B is least susceptible to infections with both the parasites.     

Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae)

Rabbits were immunized three times with extracts of Anopheles stephensi midgut. Immunized rabbits showed a high titer of antibodies when characterized by ELISA. We investigated the effect of anti-mosquito midgut antibodies on mosquito fecundity, longevity, mortality, engorgement, and the development of the malaria parasite in mosquitoes. Fecundity was reduced significantly (38%) and similarly hatchability by about 43.5%. There was no statistically significant effect on mortality, longevity, and engorgement. When the mosquito blood meal contained anti-midgut antibodies, fewer oocysts of Plasmodium vivax developed in the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. We also found that the midgut antibodies inhibit the development and/or translocation of the sporozoites. Antisera raised against midgut of A. stephensi recognized eight polypeptides (110, 92, 70, 45, 38, 29, 15, 13 kDa) by Western blotting. Cross-reactive antigens/epitopes present in other tissues of A. stephensi were also examined both by Western blotting and in vivo ELISA. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission blocking vaccine and/or anti-mosquito vaccine.     

Rapid, large-scale isolation of Plasmodium berghei sporozoites from infected mosquitoes.

The discontinuous gradient technique for recovery of malarial sporozoites from mosquitoes (Beaudoin et al., 1977) has been modified to speed up recovery and prevent sensitization of mice by components of the gradient which contaminate the sporozoites used as antigen. Mouse serum was substituted for BSA in the gradient because the latter produced hypersensitivity. Best results were obtained with gradients consisting of Medium 199, Renografin and mouse serum. Heavy and light solution of gradient components are layered in a centrifuge tube. Centrifugation of comminuted, infected mosquitoes applied to the top of the discontinuous gradient concentrates sporozoites at the interface. Sporozoites recovered from the gradient were infective, immunogenic, and relatively free of mosquito tissue. This improved method enables recovery of 100,000 sporozoites from each Anopheles stephensi infected with the ANKA strain of Plasmodium berghei. As many as 2,800 mosquitoes have been processed in 2 hr without a significant decrease in yield.     

Chesson strain Plasmodium vivax in Saimiri sciureus boliviensis monkeys

Nine Saimiri sciureus boliviensis monkeys were inoculated with sporozoites of Plasmodium vivax (Chesson strain) dissected from Anopheles stephensi mosquitoes infected by feeding on blood from infected chimpanzees. The animals were splenectomized 7 days after inoculation. Seven animals developed infections with prepatent periods ranging from 12 to 43 days (mean of 19.6 days). Parasitemias were low during the first 50 days. Maximum parasitemias in 5 animals in which the strain adapted ranged from 10,000 to 46,800 per mm3. Anopheles freeborni mosquitoes were infected by feeding on 4 of the monkeys.     

The effect of Plasmodium yoelii nigeriensis infection on the feeding persistence of Anopheles stephensi Liston throughout the sporogonic cycle.

Vector-borne parasites such as malaria have been shown to modify the feeding behaviour of their invertebrate hosts so as to increase the probability of transmission. However, evolutionary consideration of developmental changes in malaria within Anopheles mosquitoes suggests that the nature of altered feeding by mosquitoes should differ depending on the developmental stage of the parasite. We present laboratory evidence that the feeding persistence of female Anopheles stephensi towards a human host is decreased in the presence of Plasmodium yoelii nigeriensis oocysts (which cannot be transmitted), but increased when the malaria has developed into transmissible sporozoites in the salivary glands. In ten-minute trials, 33% of uninfected mosquitoes gave up their feeding attempt before the test period had ended, 53% of those harbouring oocysts had given up, but only 20% of those infected with sporozoites gave up by this time. We conclude that changes in feeding behaviour of mosquitoes mediated by parasite infection are sensitive to the developmental stage of the parasite and that these changes have important implications for malaria epidemiology.     

Susceptibility of 1- and 3-Day-Old Chicks to Infection with the Coccidium, Eimeria acervulina

SYNOPSIS. One-day-old chicks were less susceptible to experimental infection with E. acervidina than were 3-day-old chicks. Chicks fed intact oocysts when 3 days old produced 5.3, 6.7, and 42.7 times as many oocysts and had more extensive lesions than did those fed a similar number of oocysts when 1 day old. When oocyst suspensions that contained both liberated sporocysts and intact oocysts were administered, chicks infected when 3 days old produced only 1.8, 1.3, and 2.6 times as many oocysts as did those infected when 1 day old.
Examination of gizzard and intestinal contents of chicks killed 2–1½ hr after receiving massive numbers of intact oocysts showed that only a few sporocysts were liberated from oocysts in the gizzard of 1-day-old chicks, whereas more were liberated in the gizzard of 3-day-old chicks. Very few sporozoites were found in the duodenum of the 1-day-old chicks. but there was a linear increase in the percentages in samples from lower levels of the small intestine. In 3-day-old chicks, excystation in the duodenum was high and, instead of increasing, remained at about the same level in the jejunum.
The far smaller number of liberated sporocysts in the gizzards of 1-day-old chicks is attributed to less musculature and an incompletely developed grinding surface The delayed excystation of sporozoites in the intestine of 1-day-old chicks is thought to be due to suboptimal concentrations of trypsin and/or other pancreatic enzymes effecting excystation.
The lighter infections observed in 1-day-old chicks, as compared to those in chicks 3 days old, are attributed to (a) a smaller number of liberated sporocysts leaving the gizzard, (b) delayed excystation in the intestine, and (c) less opportunity for sporozoites to penetrate epithelial cells.     

Probing behaviour and sporozoite delivery by Anopheles stephensi infected with Plasmodium berghei

We observed that Plasmodium berghei sporozoite-infected Anopheles stephensi was not impaired in its ability to locate blood on a host. When probing rats, infected mosquitoes took as long as non-infected mosquitoes to locate blood. Contrary to previous suggestions, infective mosquitoes delivered sporozoites into mineral oil even after extensively probing a vertebrate host. We observed that, in mosquitoes having probed a host, both the mean number of sporozoites ejected over 3 min into oil (35.9 v. 31.7 sporozoites) and the proportion of mosquitoes delivering sporozoites (60% v. 50%) were similar to mosquitoes not having probed. We then developed a model of sporozoite delivery, taking into account observations that sporozoites are clumped in the lumen of the glands as well as upon delivery, and that output is uneven and inconsistent. We conclude that clumping optimizes transmission, if a threshold of infection exists and the mean number of sporozoites per clump is greater than the threshold.     

Analysis of the sporogonic development of Plasmodium falciparum and Plasmodium berghei in anopheline mosquitoes

Basic knowledge of the sporogonic development of malarial parasites is crucial when evaluating the sporontocidal activity of antimalarial drugs or when determining why certain vectors are refractory to a particular parasite while others are competent vectors. We have developed a model which we have used to i) assess the sporogonic development of Plasmodium berghei ANKA in Anopheles stephensi and A. freeborni mosquitoes and ii) determine the effect of chloroquine on the sporogony of P. falciparum NF-54 in A. stephensi. Criteria used to assay sporogonic development include: i) number of oocysts present, ii) percentage of mosquitoes with oocysts, iii) time of release of sporozoites from the oocysts into the hemolymph, iv) time and degree of sporozoite invasion of salivary glands, and v) transmission (P. berghei) into vertebrate hosts. Parasite development in the mosquito is evaluated every other day, commencing on ca. day 7 post-feed (PF) and continuing until ca. day 22 PF. These detailed observations allow us to delineate the chronology of sporogonic development.     

Life cycle of Calyptospora funduli (Apicomplexa: Calyptosporidae)

The taxonomic status of the extraintestinal piscine coccidium Calyptospora funduli is based in part on its requirement of an intermediate host (the daggerblade grass shrimp Palaemonetes pugio). In the present study, grass shrimp fed livers of Gulf killifish (Fundulus grandis) infected with sporulated oocysts of C. funduli exhibited numerous sporozoites suspended in the intestinal contents when fresh squash preparations were examined by light microscopy. Using this method, sporozoites were not seen in intestinal epithelial cells of the grass shrimp or in any other cell types. Ultrastructural examination, however, revealed sporozoites in the cytoplasm of the gut basal cells. Cross-sections of 1-13 sporozoites were seen within a single cell, and those sporozoites each appeared to be situated in individual membrane-bound vesicles, rather than in a single parasitophorous vacuole. These ultrastructural observations indicate that in the grass shrimp intermediate host, sporozoites that develop into an infective stage probably undergo that development in gut mucosal basal cells. Prior studies revealed that these sporozoites modified their structure over 4-5 days and that before that time, they were not infective to the fish host. Following ingestion of an infected shrimp by a killifish, the infective sporozoites apparently reach the liver of their killifish definitive hosts through the bloodstream. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hr after fish were fed experimentally infected grass shrimp. Additionally, coccidian trophozoites and early meronts were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hr postinfection. This study, in conjunction with previous findings, clearly confirms that a true intermediate host is required in the life cycle of C. funduli, that a developmental period of about 5 days in grass shrimp is necessary for sporozoites to become infective to killifishes, and that sporozoites do occur intracellularly in gut basal cells of the grass shrimp.     

Adaptation of a strain of Plasmodium vivax from India to New World monkeys, chimpanzees, and anopheline mosquitoes.

A strain of Plasmodium vivax from India was adapted to develop in splenectomized Saimiri boliviensis, Aotus lemurinus griseimembra, A vociferans, A. nancymai, A. azarae boliviensis, hybrid Aotus monkeys, and splenectomized chimpanzees. Infections were induced via the inoculation of sporozoites dissected from the salivary glands of Anopheles stephensi and An. dirus mosquitoes to 12 Aotus and 8 Saimiri monkeys; transmission via the bites of infected An. stephensi was made to 1 Aotus monkey and 1 chimpanzee. The intravenous passage of infected erythrocytes was made to 9 Aotus monkeys and 4 chimpanzees. Gametocytes in 13 Aotus monkeys and 4 chimpanzees were infectious to mosquitoes. Infection rates were markedly higher in mosquitoes fed on chimpanzees. PCR studies on 10 monkeys injected with sporozoites revealed the presence of parasites before their detection by microscopic examination. The India VII strain of P. vivax develops in Aotus and Saimiri monkeys and chimpanzees following the injection of parasitized erythrocytes, or sporozoites, or both. The transmission rate via sporozoites to New World monkeys of approximately 50% may be too low for the testing of sporozoite vaccines or drugs directed against the exoerythrocytic stages. However, the strain is highly infectious to commonly available laboratory-maintained anopheline mosquitoes. Mosquito infection is especially high when feedings are made with gametocytes from splenectomized chimpanzees.     

Gametogenesis and Sporogony of Hepatozoon Mocassini (Apicomplexa: Adeleina: Hepatozoidae) In an Experimental Mosquito Host, Aedes Aegypti

ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti , an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiatioe were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a panoitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporazoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endopbsmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.     

Effect of anti-fat body antibodies on reproductive capacity of mosquito Anopheles stephensi and transmission blocking of Plasmodium vivax

Effect of anti-mosquito-fat body antibodies on the development of the malaria parasite, Plasmodium vivax has been studied by feeding Anopheles stephensi mosquitoes with infected blood supplemented with serum from immunized rabbits. Immunogenic polypeptides were identified by western blot. Mosquitoes that ingested anti-fat body antibodies along with infectious blood meal had significantly fewer oocysts than the mosquitoes in the control group. Effect of anti-mosquito fat body antibodies on fecundity, hatchability, mortality and engorgement of mosquitoes has also been reported. A significant reduction in fecundity and hatchability was observed, however, effect on mortality and engorgement was variable and statistically insignificant. Results indicated that fat body antibodies have the potential to disrupt reproductive physiology of malaria vector An. stephensi.