Evaluation of anti-malaria potency of wild and genetically modified Enterobacter cloacae expressing effector proteins in Anopheles stephensi.

BACKGROUND: Malaria is one of the most lethal infectious diseases in tropical and subtropical areas of the world. Paratransgenesis using symbiotic bacteria offers a sustainable and environmentally friendly strategy to combat this disease. In the study reported here, we evaluated the disruption of malaria transmission in the Anopheles stephensi-Plasmodium berghei assemblage using the wild-type (WT) and three modified strains of the insect gut bacterium, Enterobacter cloacae. METHODS: The assay was carried out using the E. cloacae dissolvens WT and three engineered strains (expressing green fluorescent protein-defensin (GFP-D), scorpine-HasA (S-HasA) and HasA only, respectively). Cotton wool soaked in a solution of 5% (wt/vol) fructose + red dye (1/50 ml) laced with one of the bacterial strains (1 × 109cells/ml) was placed overnight in cages containing female An. stephensi mosquitoes (age: 3-5 days). Each group of sugar-fed mosquitoes was then starved for 4-6 h, following which time they were allowed to blood-feed on P. berghei-infected mice for 20 min in the dark at 17-20 °C. The blood-fed mosquitoes were kept at 19 ± 1 °C and 80 ± 5% relative humidity, and parasite infection was measured by midgut dissection and oocyst counting 10 days post-infection (dpi). RESULTS: Exposure to both WT and genetically modified E. cloacae dissolvens strains significantly (P < 0.0001) disrupted P. berghei development in the midgut of An. stephensi, in comparison with the control group. The mean parasite inhibition of E. cloacaeWT, E. cloacaeHasA, E. cloacaeS-HasA and E. cloacaeGFP-D was measured as 72, 86, 92.5 and 92.8 respectively. CONCLUSIONS: The WT and modified strains of E. cloacae have the potential to abolish oocyst development by providing a physical barrier or through the excretion of intrinsic effector molecules. These findings reinforce the case for the use of either WT or genetically modified strains of E. cloacae bacteria as a powerful tool to combat malaria.

Experimental Study on Plasmodium berghei, Anopheles Stephensi, and BALB/c Mouse System: Implications for Malaria Transmission Blocking Assays.

BACKGROUND: Plasmodium berghei is a rodent malaria parasite and has been very valuable means in the progress of our understanding of the essential molecular and cellular biology of the malaria parasites. Availability of hosts such as mice and vectors such as Anopheles stephensi has made this parasite a suitable system to study the parasite-host and vector-parasite relationships. METHODS: This study was performed at Medical Entomology and Parasitology laboratories of the School of Public Health, Tehran University of Medical Sciences, Iran in 2016. The investigation was carried out to describe life cycle and parameters influencing maintenance of the parasite within the mice or the mosquito. RESULTS: Results have revealed details and addressed some parameters and points influence maintenance of various life stages of the parasite including merozoites, macrogametocytes, ookinetes, oocysts and sporozoites in the laboratory model P. berghei-A. stephensi-BALB/c mouse. Injection of fresh infected blood results in higher gametocytemia in the animals. The more injected parasites result in earlier and higher parasitemia and exfelagellation centers in the mice blood. However, the highest number of infected mosquitoes and oocysts formation were observed when the parasitemia and exflagellation centers per microscopic field were 9% and 3.6 in the infected mice respectively. The infected mosquitoes should be maintained on 8% (w/v) fructose, 0.05% (w/v) PABA at 20±1 °C and 50%-80% relative humidity. CONCLUSION: This study helps to understand the biology of vertebrate-parasite and mosquito-malaria interactions that may aid in the development of a new generation of drug/vaccine and vector-based measures for malaria control.