Plasmodium Berghei Sporozoites Research Articles

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.

Freeze‐Fracture Study of Malaria Sporozoites: Antibody‐Induced Changes of the Pellicular Membrane*

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane, IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.

Antisporozoite antibodies in mice immunized with irradiation-attenuated Plasmodium berghei sporozoites

Sera from NMRI/NIH mice were tested for the presence of IgM and IgG anti-sporozoite antibodies using the indirect fluorescent antibody test (IFAT). Both IgM and IgG antibody titres were related to the number of immunizations with irradiation-attenuated Plasmodium berghei sporozoites, and protection from challenge with subsequent non-attenuated sporozoites correlated with the pre-challenge antibody titre. Sera taken five days following challenge showed marked reductions in antibody titres, except for the group receiving the maximum (four) immunizations. Groups immunized with frozen sporozoites or mosquito tissue antigen developed neither antibodies to sporozoites nor protective immunity; nor did animals infected with parasitized blood. However, sera from mice immunized four times with attenuated sporozoites demonstrated IFA titres to blood-stage antigens. The results show that both IgM and IgG anti-sporozoite antibodies could be detected in mice immunized with attenuated-sporozoites by IFAT, and that the antibody titres correlated with protective immunity. Cross reaction with blood-stage antigens occurred, but the test should still prove useful.

Plasmodium berghei: Preparation of rat hepatic cell suspensions that include infective exoerythrocytic schizonts

Employing an enzymatic method to dissociate rat liver, we prepared suspensions of liver cells from rats infected with sporozoites of Plasmodium berghei 3 to 10, 18 to 28, or 29 to 36 hr prior to liver dissociation. These suspensions of liver cells included hepatocytes, Kupffer cells, fibroblasts, and unidentified cells, as well as hepatocytes infected with exoerythrocytic schizonts (HEX) of P. berghei. These HEX were infective for recipient rodents when inoculated intraperitoneally into the recipients. The number of infective HEX present in the liver cell suspensions was quantitated by varying the number of HEX inoculated into recipients. This infectivity assay made it possible to compare the numbers of HEX in suspensions of liver cells from different donor rats. Infective HEX were obtained from donor rats in 35 of 41 experiments. The greatest number of infective HEX was obtained from donors injected with sporozoites 18 to 28 hr prior to liver dissociation. For morphological observation of mature HEX in cell suspensions, hepatic cells were prepared from donors infected with sporozoites 48 hr prior to liver dissociation. For experimental purposes, the preparation of infective HEX in suspensions of liver cells is superior to the preparation of infective HEX in liver fragments, because it is possible to quantitate the number of HEX which are present either visually or by means of the infectivity assay.

Plasmodium berghei: Energy metabolism of sporozoites

The energy metabolism of Plasmodium berghei sporozoites was studied by using their motility as an indicator of energy production and consumption. Sporozoites suspended in medium without sugars or amino acids ceased to move. Motility was restored by the addition of any of several sugars or amino acids to the medium. Inhibition of sporozoite motility, under otherwise favorable conditions, was induced by fluoride, malonate, cyanide, amytal, rotenone, antimycin A, arsenate, 2,4-DNP, and diphenylamine. The results suggest that these sporozoites utilize glycolysis, the Krebs' cycle, and conventional electron transport through the cytochrome chain.

Rodent Malaria: BCG-Induced Protection and Immunosuppression

One dose of 10(7) viable units of Mycobacterium bovis, strain BCG, protected a significant number of Swiss mice from a primary challenge with 10(4) thoracic sporozoites of Plasmodium berghei. Immunization with irradiated sporozoites induced greater protection than that observed in BCG-treated with BCG and surviving a primary sporozoite challenge were not protected from rechallenge, whereas mice immunized with irradiated sporozoites and surviving initial challenge of sporozoites were solidly immune to further challenge. Immunizing mice with BCG and irradiated sporozoites simultaneously resulted in a synergistic effect of increased protection against a primary challenge of sporozoites only if the two immunogens were administered on the same day and if the mice were challenged 1 to 3 days later. Mice given BCG and irradiated sporozoites and surviving a primary challenge of sporozoites were unable to survive rechallenge. BCG given to mice previously immunized with irradiated sporozoites suppressed their protective immunity against sporozoite challenge.

Sporozoites of Rodent and Simian Malaria, Purified by Anion Exchangers, Retain their Immunogenicity and Infectivity

Sporozoites of rodent malaria, Plasmodium berghei, and simian malaria, Plasmodium knowlesi and Plasmodium cynomolgi, were partially separated from mosquito debris and microbial contaminants by passage of Anopheles material through a DEAE-cellulose column. In addition to eliminating most of the contaminants (80-90%), this simple technic has made it possible to recover rapidly large numbers of viable sporozoites (55-75% yield), which have retained their infectivity, immunogenicity, and capacity to react with known antisera. Mice injected with varying doses of column-purified sporozoites (CS) of P. berghei produced infections which paralleled those seen in the controls. Total protection against challenge with a potentially lethal dose of viable sporozoites was acquired by mice inoculated twice with irradiated CS of P. berghei CS of P. berghei and P. cynomolgi gave positive circumsporozoite precipitation (CSP) reactions, upon inoculation with the respective immune sera. The preservation of the surface antigens of CS was documented by immunofluorescence. It was shown that differences in elution behavior exist among sporozoites of certain species of Plasmodium as well as among sporozoiters of the same species derived from different organs of the mosquito. These results may be attributed to differences in the surface charge of the sporozoites or conditions in sample media. Purified sporozoites obtained by the method described in this report provide an adequate source of parasites for a variety of in vitro studies.

Plasmodium berghei: T cell dependence of sporozoite-induced immunity in rodents

Immunization of mice with irradiated or viable sporozoites of rodent malaria induces protection against an otherwise lethal challenge with sporozoites of Plasmodium berghei, as well as the formation of antisporozoite antibodies. The effect of T cell depletion on the sporozoite-induced immune response was investigated in thymectomized, X-irradiated, bone marrow-reconstituted A J mice, and also in congenitally athymic nude (nu/nu) mice. In both instances, the T cell-deprived mice failed to develop protection after immunization. In addition, their sera failed to present any detectable sporozoite-neutralizing activity (SNA), and only about 50% of the animals developed circumsporozoite (CSP) antibodies, late in the immunization and in reduced amounts. The capacity to develop protection, as well as sporozoite-neutralizing and CSP antibodies, was fully restored when 10 7 or 10 8 thymocytes were given to these mice prior to immunization. The data demonstrate that the formation of sporozoite-neutralizing antibodies, as well as the development of protection, is T cell-dependent in this rodent malaria system.

Plasmodium berghei: Immunization of mice against the ANKA strain using the unaltered sporozoite as an antigen

Mice were immunized with normal sporozoites of Plasmodium berghei. Test mice were placed on suppressive doses of chloroquine throughout the immunization period and were given curative courses of primaquine prior to challenge. All mice survived sporozoite challenge but succumbed to challenge with infected erythrocytes. It is concluded that the normal infective sporozoite is immunogenic and will stimulate a degree of protection against sporozoite challenge comparable to that obtained by immunization with X-ray irradiated sporozoites. A biphasic density gradient for separating sporozoites from mosquito tissue is described.

Plasmodium berghei: Quantisation of sporozoites injected by mosquitoes feeding on a rodent host

Attempts were made to determine the numbers of viable Plasmodium berghei sporozoites injected by Anopheles stephensi mosquitoes feeding on rodent hosts. Assessment of the numbers injected was made by counting the numbers of exoerythrocytic forms produced by known numbers of infected mosquitoes. The results showed the numbers of viable sporozoites injected to be quite small, on the order of about 1% of the total number of sporozoites in the salivary glands. Mosquitoes with larger numbers of sporozoites in their salivary glands injected more sporozoites. Thus, heavily infected mosquitoes may be more effective vectors. The relative numbers of sporozoites injected by the mosquitoes varied with the host being fed upon. Because of this, it is speculated that host skin factors may be an important determinant of the number of infective sporozoites able to reach the blood after being deposited in the skin.

Antibody-Induced Ultrastructural Changes of Malarial Sporozoites

Immunization with irradiated sporozoites produces a considerable degree protection against rodent, simian, and human malaria. This protection is in part antibody mediated. Antibodies neutralize sporozoites (SNA), i.e. abolish their infectivity, and cause, in vitro, the formation of a thread-like precipitate on the parasites (CSP reaction). The present study was undertaken to characterize the ultrastructural aspects of antibody-sporozoite interaction.

Intramuscular immunization of mice with irradiated Plasmodium berghei sporozoites. Enhancement of protection with albumin.

Abstract These studies have confirmed a previous report which demonstrated that intramuscularly injected irradiated sporozoites of Plasmodium berghei are far less effective than those injected intravenously in protectively immunizing mice against sporozoite-induced malaria. Because albumin has been shown to induce motility in Plasmodium sporozoites, attempts were made to determine whether the use of albumin in immunizing inocula given intramuscularly might enhance the protection thus obtained. The results showed that albumin did indeed significantly enhance the development of protective immunity, though this protection was still considerably less than that obtained by intravenous immunization. This effect of albumin may be due to its ability to induce motility in the injected sporozoites, thereby permitting them to reach and enter blood vessels more readily.

Development of Infectivity by the Plasmodium berghei Sporozoite

Studies were done on the development of infectivity during ontogeny of the sporozoite of the rodent malaria parasite, Plasmodium berghei. Populations of sporozoites were separated from the oocysts, the hemocoel, and the salivary glands, with special precautions being taken to avoid cross-contamination between the different populations. The results indicated that populations of salivary gland sporozoites were more than 10,000 times as infective as populations of oocyst sporozoites from the same mosquitoes. The development of this infectivity appears to be asynchronous, in some cases taking place in the hemocoel, while in other cases not occurring until after the sporozoites have invaded the salivary glands. Thus, the development of infectivity seems to be time-dependent rather than site-dependent. There is also a continued increase in sporozoite infectivity during their residence in the salivary gland. The development of infectivity may be associated with other aspects of sporozoite maturation, including changes in their antigenicity and motility.

Labeling of Sporozoites of Plasmodium berghei with Tritiated Purines

Sporozoites were labeled in vivo. Mosquitoes (Anopheles stephensi) infected with the NK-65 strain of P. berghei were allowed to feed ad lib. for 14 days subsequent to an infective blood meal on 5% Karo syrup solution containing different levels of tritium-labled purines and pyrimidines. Sporozoites were harvested on day 16 by dissection of mosquito salivary glands. Isotope incorporation in sporozoites was determined by autoradiography. Each of the purines tested, including adenine-8-3H, adenosine-3H (G), deoxyadenosine-3H (G), and deoxyguanosine-3H (G), was incorporated into both nuclear and cytoplasmic regions of sporozoites when fed to mosquitoes at 5 to 100 ,uCi/ml. Similar levels of the pyrimidines tested, including thymine-methyl-3H, thymidine-methyl-3H, or orotic-5-3H acid, failed to label sporozoites. The incorporation of preformed purines, but not pyrimidines, by developing sporozoites suggests that the parasite may require exogenous sources of purine during its development in the mosquito but relies on de novo synthesis of pyrimidines. The adenine8-3H-labeled sporozoites were shown to be infective by intravenous inoculation into A/J mice. These results demonstrate the feasibility of labeling developing sporozoites and should provide a means for further studies on the developmental cycle of malaria in the vertebrate host. After the discovery of exoerythrocytic bodies of malaria in the liver of monkeys and man (Shortt and Garnham, 1948) it was generally assumed that relapses were caused by a continual cycling of histiotrophic merozoites in the liver. More recently, Garnham (1967) has proposed that relapses may be caused by sporozoites which remain latent in parenchymal cells of the liver for various periods of time before undergoing development. To date, these latent forms have not been demonstrated, possibly because of their small size and similarity to the cytoplasm of liver cells in stained preparations. Radionuclide labeling of sporozoites would provide a means for recognizing these latent forms in tissue if, indeed, they exist. In the present study a method for labeling sporozoites in vivo with tritiated purines was developed. In addition, the labeled sporozoites were shown to retain their infectivity when inoculated into susceptible hosts. MATERIALS AND METHODS Anopheles stephensi mosquitoes were allowed to feed on hamsters infected with the NK-65 strain of Plasmodium berghei and maintained at Received for publication 18 September 1973. * This work was presented in part at the 57th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey (1973, Fed. Proc. 32: 704). 21 C as described by Yoeli et al. (1965). Subsequent to the infective blood meal, mosquitoes were allowed to feed from the mouth of a plastic dropping pipette that contained tritium-labeled purines or pyrimidines in 5% Karo syrup solution. The mouth of the pipette was mounted in contact with the mesh on top of the cage. Fresh isotope solution was supplied every other day for a period of 16 days. Sporozoites were harvested on day 18 by dissection of mosquito salivary glands into a 1:1 mixture of saline and mouse plasma. The glands were gently homogenized in a 3-ml tissue grinder fitted with a Teflon pestle in an ice bath. Sporozoite yield was estimated from counts in a Neubauer hemocytometer. Viability of adenine-8-3H-labeled sporozoites was evaluated by the intravenous inoculation of approximately 10,000 sporozoites into 6to 8-weekold A/J mice (Jackson Laboratories, Bar Harbor, Maine) within 1 hr after dissection of salivary glands. Infectivity and prepatent period were determined by daily Giemsa-stained blood films. Isotope incorporation by sporozoites was determined by autoradiography using Kodak NTB-2 nuclear track emulsion (Eastman Kodak Co.). Infected salivary glands were crushed with a glass cover slip on slides previously coated by dipping in 1% bovine serum albumin. Sporozoites were then fixed with methanol and allowed to air-dry. Slides were coated by dipping into undiluted photographic emulsion at 40 C. After exposure to the emulsion for various intervals at 4 C, slides were processed with Kodak D-19 developer and fixer. The preparation was then stained through the emulsion with 7.5% Giemsa for 1 hr at pH 7.2. The degree of labeling was determined microscopically under oil immersion by counting silver grains associated with individual sporozoites.

Sporozoite and normal salivary gland induced immunity in malaria.

STUDIES using sporozoites as antigenic material to induce protection against malaria have been relatively scarce compared with those employing antigens of the blood stages. Many workers1–5 have reported considerable protection against challenging homologous sporozoites of Plasmodium gallinaceum. Richards4, using three injections of sporozoites exposed to ultraviolet, dried or treated with formalin, found at least eighteen birds out of twenty were protected, but with freeze-thawed sporozoite material he found only fifteen out of twenty protected. By using six to seven injections of X-irradiated sporozoites of Plasmodium berghei, Nussenzweig et al.6,7 obtained 90–100% protection when the animals were challenged with 2,000 sporozoites two weeks after the last injection.

Plasmodium berghei: Accelerated clearance of sporozoites from blood as part of immune-mechanism in mice

The maximum circulation time, as well as the rate of clearance, of viable, infective, Plasmodium berghei sporozoites was determined after intravenous injection into immunized and normal mice. In animals actively immunized by the previous injection of X-irradiated sporozoites, the circulation time of infective sporozoites was considerably reduced as a consequence of an increased clearance rate of the parasites. The transfer of large volumes of immune serum into normal mice also resulted in a considerably increased disappearance rate of infective sporozoites from their peripheral blood. Exo-erythrocytic forms (EEF) of the parasite were either not detectable or greatly reduced in number in the livers of both groups of actively and passively immunized animals, as compared to controls. However, in spite of the apparent absence of EEF in some of these animals and the reduced number of EEF in all the others, protection was never complete in the passively immunized animals, all of them developing parasitemia after a prolonged prepatent period. This differed markedly from the results obtained in the actively immunized animals, the majority of which was totally protected. Together with other experimental evidence, this seems to indicate that in addition to the important role of immune serum, another, as yet undetermined mechanism might be necessary to bring about complete protection to malaria.

Sporozoite-induced infections of Plasmodium berghei administered by the oral route.

Administration of suspensions of Plasmodium berghei sporozoites by the oral route to groups of susceptible animals (A/J mice, young rats, hamsters, and Thamnomys) resulted in high rates of infection (66 to 100 percent). Control animals each given a suspension of 200,000 to 500,000 erythrocytes infected with P. berghei by the same oral route did not develop parasitemia. Direct intubation of sporozoite suspensions into the stomach failed to produce parasitemia. In vitro studies showed that sporozoites kept in medium M 199 acidified to pH 3.6 (the acid environment of the rat stomach) for 5 to 15 mtinutes lost their viability and infectivity. We believe that sporozoites of P. berghei find their way to the bloodstream during their brief sojourn in the esophagus.

Protective Immunity Produced by the Injection of X-Irradiated Sporozoites of Plasmodium berghei

Protective Immunity Produced by the Injection of X-Irradiated Sporozoites of Plasmodium berghei

Protective Immunity Produced by the Injection of X-Irradiated Sporozoites of Plasmodium berghei

Protective Immunity Produced by the Injection of X-Irradiated Sporozoites of Plasmodium berghei

Specificity of protective immunity produced by x-irradiated Plasmodium berghei sporozoites.

VACCINATION against malarial infections remains a goal to be achieved. Even if it were feasible, it might be difficult to obtain necessary amounts of the parasitic antigen, and only homologous species or strains of the parasite may be immunologically effective. Protective immunity is often a highly strain specific phenomenon as has been shown in experiments using the blood stages of the parasite as the immunizing agent1,2. Strain specificity of the immune response also occurs in natural human malaria3. Our aim was to determine whether a strict strain and species specificity occurs in animals vaccinated with sporozoites, or if a much broader spectrum of protective immunity develops.