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Passive transfer of monoclonal antibodies (MAbs) against malaria circumsporozoite (CS) proteins protects animals against malaria. Active immunization with synthetic or recombinant peptides induces a level of polyclonal antibodies to sporozoites comparable to those found after passive immunization but does not provide comparable protection. In the Plasmodium yoelii system, synthetic or recombinant peptide-induced antibodies have never been shown to protect. The current studies were designed to determine whether immunogen structure (native protein versus synthetic peptide) or immunoglobulin G (IgG) subclass of antibodies was responsible for the absolute differences between protective, passively transferred MAbs and nonprotective, actively induced polyclonal antibodies. In this study we produced two MAbs, QGP-S1 (IgG1) and QGP-S2 (IgG2b), by immunization with a synthetic peptide based on the P. yoelii CS major repeat, (QGPGAP)4, conjugated to keyhole limpet hemocyanin. These MAbs were compared tp NYS1 (IgG3), an anti-CS protein MAb previously produced by immunization with irradiated P. yoelii sporozoites, which recognizes (QGP GAP)2. QGP-S1 and QGP-S2 passively transferred protection. However, when compared with NYS1, there was a hierarchy of protection, NYS1 > QGP-S1 > QGP-S2. There was no correlation between antibody level at challenge as determined by immunofluorescent antibody test against sporozoites or enzyme-linked immunosorbent assay against (QGPGAP)2 or apparent antibody avidity for (QGPGAP)2 by sodium thiocyanate elution assay. The data demonstrate that a synthetic peptide can induce protective antibodies and that a specific antibody subclass is not required for protection. Work to determine whether antibody affinity or fine specificity can explain the hierarchy of protection among the MAbs is under way.

Saimiri monkeys immunized with a recombinant protein containing 20 copies of the nine amino acid repeat of the Plasmodium vivax circumsporozoite (CS) protein developed high concentrations of antibodies to the repeat sequence and to sporozoites, but were not protected against challenge. After intravenous injection of an immunoglobulin G3 monoclonal antibody (NVS3) against irradiated P. vivax sporozoites, four of six monkeys were protected against sporozoite-induced malaria, and the remaining two animals took significantly longer to become parasitemic. Epitope mapping demonstrated that NVS3 recognizes only four (AGDR) of the nine amino acids within the repeat region of the P. vivax CS protein. The monkeys immunized with (DRAADGQPAG)20 did not produce antibodies to the protective epitope AGDR. Thus, determination of the fine specificity of protective immune responses may be critical to the construction of successful subunit vaccines.

One of the primary strategies for malaria vaccine development has been to design subunit vaccines that induce protective levels of antibodies against the circumsporozoite (CS) protein of malaria sporozoites. In the Plasmodium yoelii mouse model system such vaccines have been uniformly unsuccessful in protecting against sporozoite-induced malaria. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of Navy yoelii sporozoite 1 (NYS1), an IgG3 mAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, a synthetic peptide derived from the repeat region of the P. yoelii CS protein, indicating that the epitope on sporozoites recognized by this mAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by indirect fluorescent antibody test and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing (QGPGAP)2 but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not absolute concentration could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective mAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. Although the results of the conventional antibody assays did not correlate with protection, sera from the protected animals inhibited sporozoite development in mouse hepatocyte cultures significantly more than did the sera from the unprotected, subunit vaccine-immunized animals, correlating with protection. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective mAb and nonprotective polyclonal antibodies will be important in the further development of malaria vaccines.

Two colonized populations of Anopheles albimanus isolated from the Suchiate region, Chiapas State, Mexico, were compared for their susceptibility to coindigenous Plasmodium vivax. Groups of mosquitoes were fed in vitro with either autologous donor blood or the same blood cells substituted with serum negative for anti-gametocyte antibody. Significant differences in susceptibility between the 2 colonies were encountered if the autologous blood from a patient was fed to mosquitoes: mean infection rates of AnA2-positive groups was double that in AnA1 mosquitoes. Consistent for both colonies, only 23.6% of samples positive from malaria-negative serum-substituted blood were infected with an autologous blood feed. Vector competence in these mosquito populations was partially linked to the human populations's immune response to the parasite.

In Balb/c mice, the sterile protective immunity induced by immunization with radiation-attenuated Plasmodium yoelii sporozoites is eliminated by in vivo depletion of CD8+ T lymphocytes, suggesting that cytotoxic T lymphocytes (CTL) against malaria antigens expressed on infected hepatocytes are required for mediating this protective immunity. To produce a vaccine that would induce CTL against the P. yoelii circumsporozoite protein (CS), we constructed an attenuated pseudorabies virus (PRV) containing a gene encoding this protein. Balb/c mice that received three doses of 10(7) plaque-forming units (p.f.u.) of this vaccine intravenously at 3 week intervals developed high levels of antibodies to sporozoites (indirect fluorescent antibody titre = 4096) and CTL against a 16 amino acid epitope (SYVPSAEQILEFVKQI, amino acids 281-296) from the P. yoelii CS protein designated PYCTL1. The cytotoxic activity of the CTL was antigen-specific, MHC-restricted, and dependent on CD8+ T cells. Furthermore, these CTL eliminated P. yoelii-infected hepatocytes from in vitro culture, indicating that they recognize this peptide on the surface of infected hepatocytes. However, all nine mice that were challenged with 200 sporozoites developed a blood-stage malaria infection. We attribute this lack of protection to the great difficulty of inducing sterile immunity against this highly infectious parasite P. yoelii. We conclude that recombinant pseudorabies virus (PRV) worked successfully as a live vaccine vector to induce both antibodies and CTL, albeit non-protective in vivo, and the herpesviruses should be considered as subunit vaccines where T- and B-cell immunity is required.

Little is known about the immune response against liver stage antigens which were first described for Plasmodium falciparum. In order to provide a basis for experimental studies, we analysed antigenically the liver stages of Plasmodium yoelii using sera of restricted specificity. Several distinct fluorescence patterns could be described in maturing liver forms. One pattern was identified as corresponding to antigens specific to the liver phase which are also species specific. Another pattern corresponds to sporozoite surface antigens which were predominant in liver trophozoites. Trophozoite-like liver forms were detected at least 7 days after the injection of irradiated sporozoites suggesting that parasites may persist and contribute to the immunity induced by this procedure.

The circumsporozoite (CS) protein has been the target for development of malaria sporozoite vaccines for a decade. However, immunization with subunit vaccines based on the CS protein has never given the complete protection found after immunization with irradiated sporozoites. BALB/c mice immunized with irradiated Plasmodium yoelii sporozoites produced antibodies and cytotoxic T cells against a 140-kilodalton protein, sporozoite surface protein 2 (SSP2). Mice immunized with P815 cells that had been transfected with either SSP2 or CS genes were partially protected, and those immunized with a mixture of SSP2 and CS transfectants were completely protected against malaria. These studies emphasize the importance of vaccine delivery systems in achieving protection and define a multi-antigen sporozoite vaccine.

In an attempt to induce a protective cytotoxic T-cell mediated immunity against sporozoites of Plasmodium yoelii, the gene encoding the P. yoelii circumsporozoite (CS) protein was engineered into three live vectors: vaccinia, attenuated pseudorabies, and attenuated Salmonella typhimurium. Balb/c mice were immunized with 1-4 doses of 10(8) pfu of the vaccinia construct (IP), 3 doses of 10(5), 10(6) or 10(7) pfu of pseudorabies construct (IV), and 3 doses of 10(9) salmonella transformants (orally). In the case of vaccinia and pseudorabies constructs, an excellent immune response was obtained as measured by antibodies to sporozoites. No protection or delay in prepatent period was seen in any of the experimental animals when challenged with 200 (vaccinia, pseudorabies) or 100 (salmonella) sporozoites, although mice immunized with irradiation-attenuated sporozoites were consistently protected against challenge with greater than 10(4) sporozoites. Since other vaccinia, pseudorabies, and salmonella CS constructs have been shown to induce cytotoxic T lymphocytes (CTL) against the CS protein, it is likely that CTL against the CS protein were induced during these studies. It is currently unclear if the vaccines did not induce the appropriate CTL or inadequate numbers of CTL, or if CTL against the P. yoelii CS protein are inadequate to protect against sporozoite challenge.

Three subunit vaccines based on the major repeat, (QGPGAP)n, and flanking regions of the Plasmodium yoelii circumsporozoite protein were designed, produced, and tested. All were immunogenic, but none gave consistent protection against a 40-200 sporozoite challenge. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of NYS1, an IgG3 MAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, indicating that the epitope on sporozoites recognized by this MAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by IFAT and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing QGPGAP but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not the absolute concentration, could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective MAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective MAbs and non-protective polyclonal antibodies will be important in the further development of malaria vaccines.

ABSTRAcr: Two colonized populations of Anopheles albimanus isolated from the Suchiate region, Chiapas State, Mexico, were compared for their susceptibility to coindigenous Plasmodium vivax. Groups of mosquitoes were fed in vitro with either autologous donor blood or the same blood cells substituted with serum negative for antigametocyte antibody. Significant differences in susceptibility between the 2 colonies were encountered if the autologous blood from a patient was fed to mosquitoes: mean infection rates of AnA2-positive groups was double that in AnAl mosquitoes. Consistent for both colonies, only 23.6% of samples positive from malarianegative serum-substituted blood were infected with an autologous blood feed. Vector competence in these mosquito populations was partially linked to the human population's immune response to the parasite. The susceptibility of mosquito populations to coindigenous species of Plasmodium is an important variable in the determination of transmission levels in an endemic area. The real de

A method consisting of filtration of up to 100 macerated mosquitoes in a batch, followed by fixation with glutaraldehyde and concentration of filtrate by centrifugation has been developed to rapidly assess malaria infection in anopheline mosquitoes. Determination of the presence of sporozoites is made by observation of a sample of the final filtrate with a phase microscope. The method is simple and field adaptable, essential factors for the application of any technique to large scale field operations. Application of the technique in El Gancho, Chiapas, Mexico, in February 1984 yielded an infection rate for Anopheles albimanus of 0.9% for intradomicile-collected human bait mosquitoes and 0.1% for peridomicile-collected human bait mosquitoes.

Freeze-fracturing has been used to study the architecture of the pellicular complex of the gametocytes of Plasmodium falciparum. The gametocyte is surrounded by three membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes is split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on their surfaces. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. In gametocytes, unlike in sporozoites, the intramembranous particles are always distributed randomly and lack any definite pattern or orientations. A unique feature of gametocytes revealed by the freeze-fracturing technique is the presence of several transverse sutures on the middle membrane that encircle the gametocyte and give it a segmented appearance.

The specific identification of plasmodial sporozoites is not possible on morphological grounds. This study presents a serological method for the identification of sporozoite species, indicating the suitability of this approach for detection and determination of sporozoites in wild vectors collected from malaria endemic areas. Specific antisera and monoclonal antibodies prepared against each of two species of rodent malaria (Plasmodium berghei or P. yoelii) were evaluated for their ability to discriminate among sporozoites of different malaria species (P. vivax, P. gallinaceum, P. berghei, P. yoelii) from varied host types. Antisera produced by intravenous immunization of rabbits or mice and hybridoma-produced monoclonal antibodies reacted only with homologous sporozoites in an immunofluorescent antibody test. Antisera produced by intravenous challenge showed no significant difference in reactivity with fresh sporozoites as compared with sporozoites frozen at −80 °C for up to six months, whereas antisera produced by infective mosquito bites showed reduced sensitivity to frozen-stored homologous sporozoites and some cross-reactions with heterologous sporozoites. Antisera prepared against the erythrocytic stages of P. berghei or P. yoelii also cross-reacted with sporozoites of all four plasmodia tested, indicating that it is advantageous to use anti-sporozoite antibodies for the identification of malaria sporozoite species by means of serological tests.

With the use of a double staining technique that permits localization of the sporozoite during the process of entering a host cell, we studied the biologic effects of three mAb directed against determinants contained in the circumsporozoite of Plasmodium yoelii. These mAb, which included one IgM and two IgG3, were studied in primary cultures of rodent hepatocytes inoculated with sporozoites of P. yoelii. These results confirm previous reports of the extended action of antibodies on Plasmodium falciparum after entering hepatocytes by producing a strong intrahepatocyte inhibitory effect in addition to the inhibitory effect on sporozoite entry. As with P. falciparum the intracellular effects on P. yoelii liver stages are only observed when the antibodies are present at the time the sporozoite enters the cell. While carrying out experiments on this phenomenon, it was discovered that, at lowered antibody concentrations, an increase in number of maturing liver schizonts occurs, with the increase or enhancement of infection reaching up to 150% of that of controls. It was also observed that there was an inverse relationship between the antibody concentration that was inhibitory and that which enhanced parasite infectivity.

The exoerythrocytic stages of Plasmodium lophurae and P. fallax were grown in cell cultures derived from embryonic mouse livers. Liver cell monolayers were maintained in continuous culture with frequent subculturing for extended periods of time. Morphologically, the main cell type was a large, flat cell which closely resembled in shape the mouse parenchymal cell, although small colonies of spindle-shaped cells could also be found. Mammalian cells were labelled with thymidine- 3 H prior to infection with avian merozoites so they could be positively distinguished from any avian cells which might be present in the merozoite inoculum as contaminants. Merozoites were observed inside the labelled mammalian cells within three hours and large forms were seen at 48 hr. The mammalian cells supported parasite growth comparable to that observed in avian cell cultures.

Antibodies were raised in mice immunized with several recombinant and synthetic peptides of the circumsporozoite protein of Plasmodium falciparum. The antibodies were evaluated for protective activity in a human hepatocyte culture system. They exerted their protective effect against the parasite at three points: sporozoite attachment to the hepatocyte surface, entry, and subsequent intracellular development. Inhibition of attachment and entry were found to be related to the antibody titer against the authentic circumsporozoite protein on the sporozoite surface, especially when peptides were administered with alum or complete Freund's adjuvant. Even when invasion was not totally inhibited, the presence of abnormal trophozoites and a frequent inhibition of schizont development in long-term cultures suggested continued activity of antibodies at the intracellular level after sporozoite penetration had been completed.

The distribution of 3 H-primaquine within cultures of the exoerythrocytic stages of Plasmodium fallax was studied using electron microscope autoradiography techniques. 3 H-primaquine first becomes localized within the parasite mitochondria where it is often associated with mitochondrial swelling. In advanced stages of treatment, mitochondria overlaid by silver grains are invariably swollen, although exposed grains can also be detected overlying the electron-dense paired organelles.

White-tailed deer, Odocoileus virginianus, from central Pennsylvania were examined for helminth parasites by regular autopsy procedures supplemented by a direct centrifugal flotation technique. The two methods were compared and evaluated for suitability in diagnosis and survey work. The reliability and repeatability of the flotation method were studied. Prevalence of most nematodes encountered was increased as a direct result of supplementing autopsy routines with microscopic examination of fecal pellets by the DCF technique. The DCF technique was especially advantageous when dealing with small cryptic forms such as Ostertagia sp. and Capillaria sp.

The study compares parasite prevalence in two geographically separated populations of white-tailed deer in central Pennsylvania. Differences in prevalence were found for certain of the parasites studied. Characteristics of curves comparing prevalence with host-age group, however, were remarkably similar for individual parasites from both populations. Prevalence increased with host age for certain species of parasite and decreased or remained constant for others. Some of the underlying biological properties of the infections which may be responsible for the characteristics of the age specific prevalence curves are discussed.

Previous work demonstrated that corticosterone induces relapse of avian malaria, and that this effect varies markedly from winter to spring. In the present study, English sparrows with latent Plasmodium relictum infections were treated in winter with corticosterone, gonadotropin, corticosterone + gonadotropin, or a control regimen consisting of the oil vehicle. Gonadotropin neither induced relapse nor potentiated the induction of relapse by corticosterone. These data cast doubt on the hypothesis that spring relapse in malarial infections is mediated by seasonal changes in reproductive hormone levels.

Muskrats (Ondatra zibethicus) collected from three different habitats: river, marshes and streams were examined for helminth parasites. Muskrats were aged by a lens-weight technique. Using the age specific prevalence, transmission rats for Wardius zibethicus, Quinque-serialis quinqueserialis, Echinostomum revolutum and the strobilo-cercus stage of Hydatigera taeniaeformis were constructed for stream muskrats. Parasite burdens and the occurrence of multiple infections increased with host age. Differences in prevalence between habitats were observed.

An in vitro model was developed to study the hepatic phase of Plasmodium falciparum, the only malaria parasite lethal to man. Primary cultures of human hepatocytes were inoculated with sporozoites of Brazilian and African strains of P. falciparum. On days 1 through 7 after inoculation examination of fluorescence-labeled and Giemsa-stained preparations demonstrated the presence of many intracellular parasites. In three separate sets of experiments all cultures were found to be infected with as many as 650 liver schizonts measuring up to 40 micrometers. After the addition of red blood cells, intraerythrocytic forms of P. falciparum were detected on days 12 and 13 by an immunofluorescence assay, indicating that the hepatic cycle had been completed in vitro.

A procedure for the purification of very high numbers of sporozoites is described. As few as 25 or as many as 200A. stephensi mosquitoes can be processed at one time. Sporozoite yields have ranged as high as 10 to 12,000,000 sporozoites/100 mosquitoes. The sporozoites are contained within a visible band in the gradient and few are found outside this zone. This process is both a reliable and an efficient method for the recovery of sporozoites.

The method described has initiated sporozoite entry into host tissue culture cells. Prior to the development of this technique there was no evidence of intracellular parasites. The incidence of developing intracellular parasites has risen to 9.4% in Turkey brain, 14.1% in rat brain, and 3.0% in rat liver cultures, respectively. There has been no increase in contaminating microorganisms associated with this single, one-step operation.

A method is described wherebyP. berghei sprorozoites may be obtained from the salivary glands of infected femaleA. stephensi mosquitoes. The method is relatively easy to master, requires minimal handling of biological material, and is done with ordinary laboratory equipment. In addition, the sporozoites are rendered relatively free of contaminating micro-organisms and the entire operation may be performed aseptically.

A mouse liver perfusion model was adapted to evaluate the efficiency of the liver in retaining Plasmodium berghei sporozoites. Specific numbers of sporozoites were perfused into each liver via a portal vein cannula. The numbers of sporozoites in the perfusate effluent were counted and the percent sporozoite retention calculated. Over 95% of sporozoites suspended in medium with plasma were retained in a normal liver following a single passage. Sporozoites were seen in sinusoids of perfused livers using scanning electron microscopy. This liver perfusion model offers a valuable method to help clarify sporozoite interactions with elements of the liver.

Routine procedures for growing exoerythrocytic stages of avian malaria parasites in continuous culture have been available for many years. This system, adapted for the cultivation of mammalian forms, would have certain advantages in vaccine production, the great advantage being the potential for self-replication by both the host cell and the parasite. Many obstacles must be overcome, however, before efforts in this direction can be successful. The recent demonstration that merozoites of the exoerythrocytic cycle are immunogenic supports the view that efforts should be made to develop a mammalian culture system based on the exoerythrocytic stage.

Plasmodium berghei sporozoites successfully entered and developed into exoerythrocytic schizonts in a variety of cell types cultured in vitro, but segmentation and release of merozoites was only observed in human embryonic lung cells. Exoerythrocytic development was generally not influenced by the culture medium, and NCTC-135 was used routinely. In vitro infectivity of P. berghei sporozoites was unaffected by the serum type used for isolation.

Mice were protected against challenge with infective Sporozoites following immunization with X-ray irradiated Sporozoites. The immunity lasted at least 8 weeks. Mice immune against sporozoite challenge remained fully susceptible to challenge with erythrocytic stages. Immunization of mice with extracts of mosquito thorax failed to protect them, indicating that mosquito antigens were not directly responsible for the immunity observed in the basic experiments.

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.

A study spanning 10 years indicates that the erythrocytic stages of malarial parasites can be stored indefinitely in the vapour phase of liquid nitrogen without significant loss of infectivity. The results also suggest that loss due to the recovery procedures exceeds any loss that may have occurred during freezing and thawing. When feasible, large pools of material should be divided into smaller aliquots before storing the organisms at a low temperature, since freezing a second time produces significant losses of infectivity. Sporozoites and exoerythrocytic stages may be treated similarly, but the optimum conditions for freezing these forms have yet to be determined.

Ten monoclonal antibodies developed against Plasmodium falciparum sporozoites at four institutions were evaluated for use in an enzyme-linked immunosorbent assay (ELISA). Four of the antibodies were eliminated because of their low sensitivity or requirement for high concentrations of capture antibody, while an additional four were rejected because they exhibited cross-reactivity with P. berghei sporozoites. Of the two remaining monoclonal antibodies, that designated 2A10 had the highest sensitivity, a requirement for lower concentrations of capture antibody, and had been tested successfully against sporozoites from a wider range of geographical areas than the others. Use of this monoclonal antibody in a standardized ELISA method gave a test ten times more sensitive than previously reported for P. falciparum sporozoites and its detection limit was less than 100 sporozoites per mosquito.

The technique of freeze-fracturing has been used to study the architecture of the pellicular complex of the intraoocyst sporozoite of Plasmodium berghei . The sporozoite is surrounded by three plasma membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes can split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on the surface. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. Two of the fracture faces exhibit a unique arrangement of particles in well-organized parallel rows along the long axis of the sporozoite. This arrangement has not been reported in either the erythrocytic or the exoerythrocytic forms of Plasmodium spp. Another unique feature in the sporozoite revealed through freeze-fracturing is a single suture line that traverses the long axis of the inner two membranes of the parasite.

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.

Sporogonic development of Plasmodium berghei berghei is frequently ectopic, occurring deep within the tissue of the midgut with oocysts expelling sporozoites into its lumen. Inocula containing oocysts and sporozoites defecated with blood during the mosquito blood meal produced infections when introduced into mice. The fine structures and pellicle of luminal parasites appeared normal in all respects.

A rodent model of sporozoite immunization against malaria based on a single immunizing dose is described. The duration of protective immunity was measured as a function of dose, and the results suggest that the percentage of protection follows a bimodal distribution. The first peak occurs between days 7-12 after immunization, while the second peak occurs at approximately 28 days. Although the percentage of protection declines steadily after the second peak, some immunity was detectable as long as 140 days after immunization with the higher doses.

A battery of monoclonal antibodies against Plasmodium yoelii sporozoites was produced. Five of these (NYS1 through NYS5) were selected for characterization. All five were positive in the indirect immunofluorescent antibody test with P. yoelii sporozoites; however, each showed a different immunofluorescence pattern. Although NYS1 (immunoglobulin G3 [IgG3]), NYS2 (IgG3), and NYS3 (IgM) were positive in the circumsporozoite precipitation test, only NYS1 and NYS2 were able to neutralize sporozoite infectivity in mice. NYS4 (IgM) and NYS5 (IgG1) were not positive in the precipitation test and did not protect mice from sporozoite infection. All except NYS4 were species as well as stage specific. NYS4 cross-reacted with sporozoites of P. berghei. Electrophoretic immunoblotting analysis showed that these monoclonal antibodies detected sporozoite antigens of various molecular weights. Inhibition enzyme-linked immunosorbent assays indicated that each recognized a different antigenic epitope. The differences in their immunochemical and biological reactivity make them useful for screening a variety of P. yoelii antigens in recombinant DNA libraries. These antigens will be used in an animal model system for vaccine development.

Sporozoites of the rodent malaria, Plasmodium berghei, were obtained from infected Anopheles stephensi by grinding mosquitoes, prepurifying the material in a discontinuous Hypaque gradient and further purifying by means of continuous free-flow electrophoresis. Bacteria, debris, mitochondria, mitoplasts, and other contaminants were removed in the electric field. The isolated sporozoites were morphologically intact and were positive in indirect immunofluorescence assay. They were infective to mice prior to and following free-flow electrophoretic separation. The surface of the sporozoites exhibited a polysaccharide-rich layer. The predominant surface protein labelled after surface iodination had a molecular weight between 42,000 and 46,000 daltons.

Although studies on Plasmodium infections of wild birds have been reported frequently in the literature, our knowledge of the ecology of these parasites remains incomplete. A synthesis of data and ideas from these field studies, and recent experimental work led to the construction of the following hypothetical model for the ecology of avian malaria: During the late spring, summer, and early fall susceptible birds (young of the year or previously uninfected adults) contract the infection from the bite of an infected mosquito on the breeding ground. The birds migrate or remain in the area and infections become latent over the winter. In the spring, migratory birds return to the breeding area and all birds commence reproductive activity. With the onset of migration and breeding activity, parasite populations become elevated in the birds. This relapse of malarial infections coincides with emergence of vectors. The mosquitoes obtain the parasite, passing it on to susceptibles in the population (whose numbers a...

Primaquine-resistant strains of Plasmodium fallax and P. gallinaceum were produced by subjecting a large population of erythrocytic parasites to the selection pressure of continuous drug therapy. It was also demonstrated that the resistance acquired by drug pressure on the erythrocytic stages becomes a property of the exoerythrocytic parasite, that it is stable in the absence of drug, and that there is no cross resistance with pyrimethamine-resistant strains. An attempt to confirm asexual transfer of the property of drug resistance from one strain to another was unsuccessful.

Previous work has shown that latent infections of Plasmodium relictum in English sparrows become patent in the spring of the year. This spring relapse phenomenon may be ecologically important in reestablishing transmission of the parasite if the increase in parasitemia is associated with an increased infectivity to mosquitoes. The present study compared laboratory transmission rates during winter and spring of chronically infected sparrows. Results of the study indicated that birds were significantly more infective to mosquitoes during the spring relapse period than during the winter latent period.

An experiment was designed to study the effects of hibernation on the production and induction of antibody response to cysticerci of Taenia crassiceps in woodchucks ( Marmota monax ). Eight woodchucks were placed at 6 °C (hibernators) and eight at 20 °C (nonhibernators). The food was removed from all woodchucks to induce hibernation or lethargy. Four of each group were infected before hibernation and four after. Cysticerci were injected intraperitoneally. The response was judged by hemagglutination titration. The results showed no difference in antibody level among the four groups. Thus, it is concluded that antibodies to at least some helminths are produced during the period of hibernation. Whether or not production occurs only during the brief periods of arousal was not determined.