ABSTRACT
Plasmodium vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections likely initiated by activation of dormant liver stage parasites-hypnozoites. The lack of tractable P. vivax animal models constitutes an obstacle in examining P. vivax liver stage infection and drug efficacy. To overcome this obstacle, we have used human liver-chimeric (huHep) FRG KO mice as a model for P. vivax infection. FRG KO huHep mice support P. vivax sporozoite infection, liver stage development, and hypnozoite formation. We show complete P. vivax liver stage development, including maturation into infectious exo-erythrocytic merozoites as well as the formation and persistence of hypnozoites. Prophylaxis or treatment with the antimalarial primaquine can prevent and eliminate liver stage infection, respectively. Thus, P. vivax-infected FRG KO huHep mice are a model to investigate liver stage development and dormancy and may facilitate the discovery of drugs targeting relapsing malaria.
Subject(s)
Disease Models, Animal , Liver/pathology , Liver/parasitology , Malaria, Vivax/pathology , Malaria, Vivax/parasitology , Plasmodium vivax/physiology , Animals , Antimalarials/administration & dosage , Chemoprevention/methods , Chimera , Humans , Malaria, Vivax/drug therapy , Malaria, Vivax/prevention & control , Mice, Knockout , Mice, SCID , Plasmodium vivax/growth & development , Primaquine/administration & dosage , Treatment OutcomeABSTRACT
BACKGROUND: Malaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes. METHODS: Here is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants. RESULTS: This method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax) and rodent (Plasmodium yoelii) infective species with excellent recovery rates. CONCLUSIONS: This novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.
Subject(s)
Centrifugation, Density Gradient/methods , Parasitology/methods , Plasmodium/isolation & purification , Sporozoites/cytology , Animals , Anopheles/parasitology , Disease Models, Animal , Female , Humans , Liver/parasitology , Malaria/parasitology , Mice , Plasmodium/pathogenicity , Plasmodium falciparum/isolation & purification , Plasmodium vivax/isolation & purification , Plasmodium yoelii/isolation & purification , VirulenceABSTRACT
The human malaria parasite Plasmodium falciparum causes the most deadly parasitic disease worldwide, necessitating the development of interventions that block infection. Yet, preclinical assays to measure inhibition of infection date from the 1980s and are based on microscopy. Here, we describe the development of a simple flow cytometric assay that can be used to quantitatively assess P. falciparum sporozoite infection in vitro in low and medium throughput. We demonstrate the utility of this assay for assessing both drug inhibition of infection and measuring efficacy of antibodies in blocking parasite infection. This methodology will aid in assessing functional antibody responses to vaccination and novel drugs that prevent mosquito-to-man transmission of malaria.
Subject(s)
Flow Cytometry/methods , Plasmodium falciparum/cytology , Sporozoites/cytology , Antibodies, Protozoan/chemistry , Antigens, Protozoan/immunology , Antimalarials/pharmacology , Cells, Cultured , Cytochalasin D/pharmacology , Drug Evaluation, Preclinical/methods , Hepatocytes/cytology , Hepatocytes/parasitology , Humans , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Sporozoites/drug effects , Sporozoites/immunologyABSTRACT
The effect of NO(3)(-) addition on dissimilatory SO(4)(2-) reduction and sulfide conversion in organic-rich sludge from the digestion basin of a recirculating marine aquaculture system was studied. SO(4)(2-) reduction could only explain a minor fraction (up to 4-9%) of the observed total sulfide production (up to 35 mmol L(-1) day(-1)), indicating that the main source of sulfide in the sludge was not SO(4)(2-) reduction, but desulfuration during the decomposition of organic matter. Although NO(3)(-) inhibited SO(4)(2-) reduction, but not desulfuration, the primary NO(3)(-) mitigation effect was the onset of NO(3)(-)-mediated sulfide oxidation (up to 75 mmol L(-1) day(-1)), partially to elemental sulfur (S(0)). Above NO(3)(-) concentrations of 0.6 mM in the bulk water, the net sulfide production and oxidation zones were moved deeper into flocs and sludge cores, which effectively prevented sulfide from entering the water column. However, the sulfide efflux from the sludge instantly recovered after NO(3)(-) depletion. Thus, the NO(3)(-) level in the water column controls the zonation and magnitude of sulfur transformations in the sludge. The effect of NO(3)(-) relies therefore on its sustained presence in the water column, which in turn depends on a well-functioning nitrification in the mariculture system.
