Fig 1.
Experimental design using Aotus lemurinus lemurinus monkeys.
Diagram showing the inoculations of the Aotus monkeys with P. vivax Sal-1. The percentages shown are for staging of the parasites at bleed (R, rings; T, trophozoites; S, schizonts; G, gametocytes). Table shows the experiments that each bleed was used for.
Fig 2.
Co-staining of reticulocytes and parasites reveals ex vivo P. vivax Sal-1 is found in reticulocytes of different maturity.
(A) Heilmeyer classification according to [19.22]. The youngest reticulocytes will have a dense reticulum network when stained with NMB (Heilmeyer I), older reticulocytes will have a mild reticulum network (Heilmeyer II-III) and the oldest reticulocytes will have weak reticulum network (Heilmeyer IV). Mature RBCs will not stain with NMB. (B) Two independent biological replicates, MN23009a and MN23009b were examined for the Heilmeyer classification of the host reticulocyte infected with P. vivax Sal-1. Smears were stained first with NMB for the reticule network, followed by Giemsa staining for the parasite’s nucleus and ribosomes. MN23009a was drawn at 100% rings while MN23009b was drawn at mostly trophozoites. For MN23009b, parasites were kept in culture for 20 hr and the resulting second-generation rings and trophozoites were assessed for host cell Heilmeyer classification. S1 Fig contains examples.
Fig 3.
Reticulocyte enrichment method can affect invasion of P. vivax Sal-1.
(A) Table showing methods used to enrich reticulocytes from peripheral blood derived either from hemochromatosis patients and Buffy Coat blood packs. The enrichment was performed according to either published studies or manufacturer’s instructions detailed in the Materials and Methods. The resulting total reticulocyte percentage was determined by staining with NMB. (B) The final fold change in parasitemia following invasion can be normalized to compare different invasion assays using the parasitized erythrocyte multiplication rate (PEMR) by dividing the final number of rings by the input number of schizonts [18]. (C) PEMR demonstrates that the hemochromatosis reticulocytes enriched by density using the [14] method resulted in the best invasion in two independent biological replicates. This invasion was better than the invasion in the same blood enriched by Percoll and significantly better than the reticulocytes from Buffy Coat enriched by density or CD71-microbeads. Parasites were from Aotus MN21014 and MN28016. Statistical significance was determined using Dunnett’s multiple comparison test. P value <0.05 (D) Final ring parasitemias of the invasion assays in B. Error bars represent the standard error.
Fig 4.
Leukodepeletion can affect P. vivax Sal-1 parasites from Aotus.
(A) Staging and parasitemia levels for four independent biological replicates from two Aotus monkeys. Decrease in parasitemia levels occurred with later stage parasites after Plasmodipur filtration compared to CF11 filtration in MN26032a (enriched in rings), MN26032b (mixed stages), MN23009a (enriched in rings) and MN23009b (mixed stages). (B). Parasites from the four independent biological replicates had similar parasitemias throughout their time in vitro. (C) However, differences were observed in the PEMR in parasites from CF11 and Plasmodipur. This was mostly contributed by one biological replicate, MN23009a and the trend was not statically significant (Mann Whitney). Error bars represent the standard error.
Fig 5.
Media supplementation can influence the health and growth of the parasite.
(A) Parasitemia graph showing the effect of GlutaMAX on parasitemia at different time points using three independent biologicals P. vivax Sal-1 parasites from MN23062a and b and MN23009a. Reticulocytes from hemochromatosis patients enriched by density were used for these experiments. (B) Histogram comparing parasite stages at different time points from the parasites in A: (C) Conversion percentages of rings to trophozoites and trophozoites to schizonts in media supplemented with or without GlutaMAX. Data from the three independent biological replicates in A. Error bars represent the standard error. While the parasitemia differences are not statically significant, we did observed a longer persistence of parasites in the cultures supplemented with GlutaMAX and we did observed reinvasion only in the GlutaMAX culture.
Fig 6.
Shaking and static format support cultures similarly.
(A) Glass T flask used for shaking conditions to replicate the conditions used in [14]. (B) Parasitemias of two biological replicates (MN23026b and MN23009b) grown in shaking or static conditions at equal haematocrits. (C) Conversion rates between rings to trophozoites or trophozoites to schizonts in the two biological replicates (D) Parasitized erythrocyte multiplication rate (PEMR) of the two biological replicates in either shaking or static conditions using reticulocytes from hemochromatosis patients enriched by density. Error bars represent the standard error. Results are not statically significant.
Fig 7.
Starting haematocrit (hct) can influence in vitro outcome of P.vivax Sal-1.
Cultures from two independent biological replicates, MN23009a and MN23009b, were initiated at different hcts. Reticulocytes enriched by density from patients with hemochromatosis were used. Cultures were initiated with the initial draw at 6% or 12% hct (iRBC, infected RBC) or were initiated with the 6% initial draw (iRBC) and hct was doubled to 12% hct with uninfected enriched reticulocytes (density hemochromatosis) (12% iRBC + RBC) to account for any differences due to the overall total parasite biomass. (A). Parasitemia over time from the two biological replicates. Survival was affected by the conversion differences in B as the two cultures were initiated at majority rings (MN23009a) or mixed rings and trophozoites (MN23009b) (B) Conversion rate differences in the two biological replicates in A between rings to trophozoites and trophozoites to schizonts. (C) Parasitized erythrocyte multiplication rate was only possible to measure in one biological replicate, MN23009a. Error bars represent the standard error. To set up a culture at 12% hct, we first set up the culture at 6% hct and doubled the hct with uninfected fresh, hemochromatosis human blood (12% iRBC + RBC). Results were not statically significant.