Optimization of a Membrane Feeding Assay for Plasmodium vivax Infection in Anopheles albimanus

Abstract

IntroductionIndividuals exposed to malaria infections for a long time develop immune responses capable of blocking Plasmodium transmission to mosquito vectors, potentially limiting parasite spreading in nature. Development of a malaria TB vaccine requires a better understanding of the mechanisms and main effectors responsible for transmission blocking (TB) responses. The lack of an in vitro culture system for Plasmodium vivax has been an important drawback for development of a standardized method to assess TB responses to this parasite. This study evaluated host, vector, and parasite factors that may influence Anopheles mosquito infection in order to develop an efficient and reliable assay to assess the TB immunity.Methods/Principal FindingsA total of 94 P. vivax infected patients were enrolled as parasite donors or subjects of direct mosquito feeding in two malaria endemic regions of Colombia (Tierralta, and Buenaventura). Parasite infectiousness was assessed by membrane feeding assay or direct feeding assay using laboratory reared Anopheles mosquitoes. Infection was measured by qPCR and by microscopically examining mosquito midguts at day 7 for the presence of oocysts.Best infectivity was attained in four day old mosquitoes fed at a density of 100 mosquitos/cage. Membrane feeding assays produced statistically significant better infections than direct feeding assays in parasite donors; cytokine profiles showed increased IFN-γ, TNF and IL-1 levels in non-infectious individuals. Mosquito infections and parasite maturation were more reliably assessed by PCR compared to microscopy.ConclusionsWe evaluated mosquito, parasite and host factors that may affect the outcome of parasite transmission as measured by artificial membrane feeding assays. Results have led us to conclude that: 1) optimal mosquito infectivity occurs with mosquitoes four days after emergence at a cage density of 100; 2) mosquito infectivity is best quantified by PCR as it may be underestimated by microscopy; 3) host cellular immune response did not appear to significantly affect mosquito infectivity; and 4) no statistically significant difference was observed in transmission between mosquitoes directly feeding on humans and artificial membrane feeding assays.

Protective Efficacy of Plasmodium vivax Radiation-Attenuated Sporozoites in Colombian Volunteers: A Randomized Controlled Trial

“…vivax samples are successfully infective to An . albimanus mosquitoes due to numerous biological factors [ 25 ]. Nevertheless, seven immunization sessions provided a median of 434 Pv RAS bites for an efficacy of 42%.…”

Susceptibility of wild and colonized Anopheles stephensi to Plasmodium vivax infection

“…Laboratory-adapted mosquitoes offer significant advantages in logistics, ease of maintenance, flexibility of scaling up and reproducibility of experimental infections. Many Anopheles species, especially the Plasmodium vectors Anopheles gambiae , Anopheles dirus , Anopheles albimanus , and Anopheles darlingi have been colonized and are used for experimental Plasmodium infections [ 2 , 4 – 10 ]. However, it is also known that when colonized mosquitoes (and other insects) are maintained in the laboratory for generations, may not accurately reflect the genetic make-up of a wild population due to founder effects, inbreeding, genetic drift, and accumulation of traits that favour their survival in artificial breeding conditions [ 6 , 11 – 14 ].…”

A Multi-Stage Plasmodium vivax Malaria Vaccine Candidate Able to Induce Long-Lived Antibody Responses Against Blood Stage Parasites and Robust Transmission-Blocking Activity

“… NA, not applicable .a Number of mosquitoes infected (total number of mosquitoes dissected) .b Average calculated as the total of oocyst /total of mosquitoes dissected ± standard deviation .c Percent inhibition of mean mosquitoes with rabbit sera post-third immunization compared to pre-immune rabbit sera in each independent assay calculated as (1 – [mean mosquitoes in normal rabbit sera/mean mosquitoes post 3rd immunization sera] x 100). A pool of AB normal human sera was used as a negative control .d Pre-immune serum from this rabbit was not tested for TBA, antibody titer was 1280 .e Statistical significance P < 0.05 . Sera samples from immunized rabbits (cPvMSP1-Pvs25 and Pvs25) were tested in direct membrane-feeding assays (three different experiments) using blood samples collected from naturally infected patients as described (Vallejo et al, 2016) .…”

“…Sera samples from immunized rabbits (cPvMSP1-Pvs25 and Pvs25) were tested in direct membrane-feeding assays (three different experiments) using blood samples collected from naturally infected patients as described (Vallejo et al, 2016) .…”

“…The transmission-blocking activity of sera derived from rabbits immunized with Pvs25 or cPvMSP1-Pvs25 was measured by direct membrane feeding assays as described elsewhere (Arevalo-Herrera et al, 2005, 2015; Vallejo et al, 2016). Briefly, 150 μl of infected RBCs from P. vivax infected patients were washed twice with RPMI 1640 medium (Sigma Aldrich) and diluted in a 150 μl of sera from rabbits that were not heat-inactivated obtained after three immunizations with 20 μg of cPvMSP1-Pvs25 or Pvs25 to feed 100 adults (2–3 day old) An.…”