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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.

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

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.

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.

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.

Hybridoma cell lines, which secreted antibodies directed against either the surface of Plasmodium berghei sp or mosquito debris, were produced by fusion of spleen cells of P. berghei sporozoite- immunized mice with P3-X63-Ag8 myleoma cells. Four cloned antibody-secreting cell lines were successfully established. Two of these clones (F9 and G 10) were obtained from the fusion of spleen cells from mice that had undergone two immunizations. These clones produced an IgM antibody that did not produce a CSP response on the sporozoite and did not neutralize the infectivity of the sporozoite in mice. One clone (B6), produced by the fusion of spleen cells from mice that had been given three immunizations, secreted an IgG antibody that did produce a CSP response on the sporozoite and neutralized the infectivity of the parasite. A fourth clone (D5) directed against mosquito debris was also found to produce an IgG antibody, but did not show a CSP reaction or neutralization of the sporozoite infectivity.

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.

The developmental stages of plasmodia receiving the most attention in the search for a malaria vaccine are sporozoites, blood stages, and gametes. The liver stages have been the least studied. Nonetheless, recent evidence has shown that liver stages(LS) of Plasmodium falciparum have their own unique stage-specific antigens and conceivably the liver stages may eventually provide antigens with demonstrated capability of inducing protective immunity at the level of the liver. In addition, the presence of circumsporozoite (CS) antigen in trophozoites that develop from irradiated and nonirradiated sporozoites implicates the liver stages in the development of malarial immunity. The indirect fluorescent antibody test (IFAT) has been applied in assessing immune responses to malaria infections and in developing hybridomas secreting antibodies directed against stage-specific antigens. The IFA technique applied to LS relies on the use of either thin frozen liver sections or sections cut from blocks of liver fixed in Carnoy's solution as the antigen. The present report describes a rapid, simple method of preparing high-quality antigen slides of LS of Plasmodium yoelii. The method can also be applied to any rodent malaria parasite. Keywords: Reprints; Bioassay; Fluorescent antibody techniques.

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.