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.

Low-temperature preservation of sporozoites of Plasmodium berghei.

Large numbers of biologically active sporozoites are needed as a source of potential antigen in the development of a malaria vaccine and the most practical method of accumulating sufficient numbers of these forms would be to freeze and store them at low temperature. The purpose of this work was to determine the feasibility of preserving the infectivity of frozen and thawed sporozoites. The results indicate that sporozoites of Plasmodium berghei exhibit a typical response to freezing over a wide range of cooling rates; the distribution around the optimum was a normal one and both the magnitude and position of the peak of their infectivity depended upon the preservative used. The optimum cooling rate with preservatives of high relative molecular mass was between 20 degrees C and 60 degrees C per min, but varied with the preservative used. A new apparatus was designed and built to yield reproducible controlled cooling rates over the range studied. A comparison of various commonly used preservatives showed mouse serum alone to be effective, but the combination of serum and hydroxyethyl starch proved to be the best cryopreservative of those examined.

A quantitative long-term cryobiological study of malarial parasites.

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.

A comparative study of the parasites in two populations of white-tailed deer.

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.