Trials to infect Anopheles stephensi with Plasmodium yoelii nigeriensis by the membrane feeding technique.

The aim of this study was to find optimal conditions for the membrane feeding technique to obtain maximum infection rates of mosquitoes with Plasmodium yoelii nigeriensis. The results show that the malaria parasite Plasmodium yoelii nigeriensis is most infective to Anopheles stephensi mosquitoes on day 3 of the infection in the mice, 1 day before the peak of parasitaemia. The mortality rate of the mosquitoes fed on mice on day 3 after infection was the highest as compared to mosquitoes fed on other days after infection. Gametocytes from mice 3 days after infection were fed to mosquitoes by three different membrane feeding methods. The results indicate that feeding during the first 10 min after blood collection gave the highest infection rates. Keeping the blood meal at a pH of 7.2 yields higher infection rates than keeping it at pH of 8.5. Stirring of the blood and supplying it with CO2 is not necessary when feeding of the mosquitoes is completed within the first 10 min after collection of the blood.     

The effects ofNosema algerae on the development ofPlasmodium yoelii nigeriensis inAnopheles stephensi

Experimental simultaneous infections ofAnopheles stephensi (Diptera: Culicidae) withNosema algerae (Microsporida: Nosematidae) andPlasmodium yoelli nigeriensis under standardized laboratory conditions showed partial suppression of the malaria parasite. At 9 days after an infective bloodmeal, the oocysts in the midgut were counted; 12.1%–66.6% of the double-infected mosquitoes exhibited no oocysts, whereas only 4.5%–12% of the control group showed no oocysts. The mean reduction in oocyst numbers under the influence ofNosema was 84.68%. At 14 days after infection withPlasmodium, the amount of sporozoites was examined; their mean reduction in eight experiments was 70%.     

Monoclonal antibody MG96 completely blocks Plasmodium yoelii development in Anopheles stephensi

In spite of research efforts to develop vaccines against the causative agent of human malaria, Plasmodium falciparum, effective control remains elusive. The predominant vaccine strategy focuses on targeting parasite blood stages in the vertebrate host. An alternative approach has been the development of transmission-blocking vaccines (TBVs). TBVs target antigens on parasite sexual stages that persist within the insect vector, anopheline mosquitoes, or target mosquito midgut proteins that are presumed to mediate parasite development. By blocking parasite development within the insect vector, TBVs effectively disrupt transmission and the resultant cascade of secondary infections. Using a mosquito midgut-specific mouse monoclonal antibody (MG96), we have partially characterized membrane-bound midgut glycoproteins in Anopheles gambiae and Anopheles stephensi. These proteins are present on the microvilli of midgut epithelial cells in both blood-fed and unfed mosquitoes, suggesting that the expression of the protein is not induced as a result of blood feeding. MG96 exhibits a dose-dependent blocking effect against Plasmodium yoelii development in An. stephensi. We achieved 100% blocking of parasite development in the mosquito midgut. Preliminary deglycosylation assays indicate that the epitope recognized by MG96 is a complex oligosaccharide. Future investigation of the carbohydrate epitope as well as gene identification should provide valuable insight into the possible mechanisms of ookinete attachment and invasion of mosquito midgut epithelial cells.     

The action of metabolic inhibitors on microgametogenesis in Plasmodium yoelii nigeriensis

Summary Mature gametocytes of Plasmodium yoelii nigeriensis were incubated in vitro with a variety of antibiotics whose activities were directed against DNA, RNA, protein and microtubule assembly. Following incubation for periods between 5 and 90 min, exflagellation was induced and the degree of inhibition produced by each drug was assayed microscopically.The results obtained show that de novo synthesis of axonemes and proteins is essential to the events of microgametogenesis. The inhibitory effect of actinomycin-D suggests that de novo RNA synthesis is also required. However, the inactivity of 8-azaguanine, a drug also effective against m-RNA metabolism, casts some doubt on the requirement for m-RNA synthesis.DNA synthesis is not required during the three successive mitotic divisions of the parasite genome during microgametogenesis. The mature microgametocyte therefore contains the 8c DNA required for microgamete production.     

Status of hepatic heme and heme oxygenase during Plasmodium yoelii nigeriensis infection in mice

Plasmodium yoelii nigeriensis infection in albino mice caused a significant increase in hepatic heme level, the increase being concomitant with a rise in parasitemia. This elevated heme was found to be associated with all the subcellular fractions except the cytosol, where its content remained unaltered. Activity of heme oxygenase, the key enzyme responsible for catabolism of heme, also increased progressively with rise in parasitemia. Treatment of normal mice with cobalt chloride [60 mg (kg body wt)-1; subcutaneously] brought about a 150% increase in the level of heme oxygenase; similar treatment of infected mice at low parasitemia could induce the enzyme activity while at high parasitemia the enzymic activity remained unaltered as compared to untreated infected mice. In spite of an increased level of heme oxygenase in the cobalt-treated mice, the level of heme did not show any noticeable change. Oral administration of chloroquine [64 mg (kg body wt)-1 x 4 days] brought about a 56% reduction in the level of heme oxygenase of normal animals but there was no change in infected animals when compared with the corresponding untreated infected mice. However, the amount of chloroquine present in livers of normal and infected animals was not significantly different.     

Aberrations in cerebral vascular functions due to Plasmodium yoelii nigeriensis infection in mice.

Plasmodium yoelii nigeriensis infection in mice caused an increase in uptake of 125I-labeled bovine serum albumin, 51Cr-labeled erythrocytes and Evans blue dye from peripheral circulation into the brain. Isolated cerebral microvessels which were characterized in terms of their morphology under scanning electron microscope and enhancement of the specific activities of biochemical markers, viz. alkaline phosphatase, gamma-glutamyl transpeptidase, and monoamine oxidase, showed significant decrease in these activities due to P. yoelii nigeriensis infection. On the other hand, relatively minor (statistically insignificant) changes occurred in the first two enzyme specific activities in the cerebral cortex and monoamine oxidase registered an increase in this tissue due to infection. Histological examination of the cerebral tissue of infected animals by light and electron microscopy showed broken blood vessel walls and leakage of erythrocytes into extravascular space, some of which contained intraerythrocytic malarial parasite in a state of cell division.     

Effect of Plasmodium yoelii nigeriensis infection and chloroquine on the hepatic mixed function oxidase system of mice

Plasmodium yoelii nigeriensis infection in albino mice significantly altered the hepatic microsomal mixed function oxidase system. Cytochrome P-450 (the terminal monooxygenase) and other monooxygenases, viz. aniline hydroxylase, aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase were significantly lowered while microsomal heme showed 4-fold increase at 80% parasitaemia. Noticeable impairment in the other components like NADH:cytochrome b5 reductase, NADPH:cytochrome c reductase, cytochrome b5 and glucose-6-phosphatase was also observed. Oral treatment of normal and P. y. nigeriensis infected mice with chloroquine (64 mg per kg body weight for 4 days) caused lowering of mixed function oxidase activities which however showed a recovering trend, a week after cessation of treatment.     

Plasmodium yoelii nigeriensis circumsporozoite gene structure and its implications for the evolution of the repeat regions

The circumsporozoite (CS) gene encodes the most immunogenic component of the plasmodial sporozoites. The immunodominant epitope-encoding domain of the CS gene shows sequences that are repeated in tandem. A detailed analysis of the CS repeats of certain closely related malaria parasites (strains of Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium vivax) showed that they evolve rapidly yet are well conserved within the gene. We were interested in studying whether the CS repeats of Plasmodia more distantly related to these species evolve in a similar manner. To this end, we isolated and characterized the Plasmodium yoelii nigeriensis CS gene. A comparative analysis of its sequence with that of Plasmodium yoelii yoelii shows that both have three sets of repeats, termed PR, R1, and R2. The R1 and basically also the R2 sequences show the features observed in most CS repeats, i.e., they evolve rapidly and are nearly perfectly tandemly repeated. In contrast, the PR repeats are not internally conserved nor divergent in sequence. The implications of these findings for the evolution of the CS repeats are discussed.     

Development of larval thermotolerance and its impact on adult susceptibility to malathion insecticide and Plasmodium vivax infection in Anopheles stephensi

The effect of the range of temperature on the thermal adaptation in Anopheles stephensi Liston 1901 was evaluated in the laboratory. Late third instar larvae of An. stephensi were exposed to variable temperatures viz. 37°C, 39°C, 41°C, 43°C and 45°C, and their lethal time to cause 50% mortality (LTM₅₀) values were calculated. All larvae survived up to 39°C of exposure. However, at 45°C, they died within 30 min of exposure. Pre-exposure to variable temperatures and re-exposure to higher temperatures conferred adaptive thermotolerance. The larvae pre-adapted at 41°C that were re-exposed to 43°C, and larvae pre-adapted at 39°C that were re-exposed to 45°C, were found more thermotolerant than the thermally non-adapted larvae. Adaptive cross-tolerance to malathion was also induced by pre-exposing them to 37°C and 39°C. It suggests that temperature stress also play an important role in the development of adaptive cross-tolerance to other stress conditions. Similarly, the oocyst rate was relatively more in adults that emerged from larvae pre-adapted at 40°C as compared to adults that emerged from thermally non-adapted larvae and with lower oocyst load.     

Penetration of the mosquito midgut wall by the ookinetes ofPlasmodium yoelii nigeriensis

The penetration route of ookinetes ofPlasmodium yoelii nigeriensis in the midgut of a mosquito,Anopheles omorii, was investigated by electron microscopy. Within 15–18 h after an infective blood meal, ookinetes could be seen in the midgut lumen in the process of entering the midgut wall, or lodged between the basement membrane and the basal lamina. The morphology of the ookinetes and their transformation into early oocysts were found to be similar to those previously reported. Ookinetes penetrated the midgut wall by the intercellular route; however, the intracellular occurrence of the parasite was also observed. Vacuoles appeared around the penetrating ookinetes during the penetration process, but no change in the electron density of the rhoptry-microneme complex was noted.Abbreviations in the figures bl Basal lamina - bm basement membrane - c cristalloid - cj cellular junction - d ductuli - dm disrupted mitochondrion - f pedicular fold - ic intestinal content - im intercellular membrane - l labyrinth - mtoc microtubule organizing centre - mc midgut cell - mv microvilli - n nucleus - o ookinete - ow oocyst wall - p pigment bar - pm peritrophic membrane - rm rhoptry-microneme complex - v vacuole     

Regional immunosuppression induced by Plasmodium berghei yoelii infection in mice.

Plasmodium berghei yoelii infection in mice severely depressed the splenic antibody response to sheep erythrocytes but had lettle effect on antibody formation in lymph nodes.     

Effect of Plasmodium yoelii infection on GABA metabolism of mouse brain

Plasmodium yoelii infection in albino mice decreased the activity of brain glutamic acid decarboxylase (GAD) by about 30 and 48% in crude homogenate and its synaptosomal fraction, respectively. The decrease was evident from 20% parasitemia and remained more or less constant up to 80% parasitemia. The Km values of GAD in normal and infected animals were 1.2 x 10(-2) and 3.3 x 10(-2) mM, respectively, indicating a decrease in enzyme substrate affinity due to infection. The lowered GAD activity rose to slightly above normal by treatment of infected animals with chloroquine. Decrease in GAD activity reflected lower gamma-aminobutyric acid (GABA) levels in the infected brain; however, GABA-transaminase activity was not significantly influenced by infection. It has been proposed that impaired GABA synthesis may be due to hypoxia induced by malarial infection.     

Transgenic mice expressing C-reactive protein are susceptible to infection with Plasmodium yoelii sporozoites.

Human and rat C-reactive proteins, major acute-phase reactants, bind to sporozoites and inhibit their in vitro development in hepatocytes (A. Nussler, S. Pied, M. Pontet, F. Miltgen, L. Renia, M. Gentilini, and D. Mazier, Exp. Parasitol. 72:1-7, 1991, and S. Pied, A. Nussler, M. Pontet, F. Miltgen, H. Matile, P.-H. Lambert, and D. Mazier, Infect. Immun. 57:278-282, 1989). We show here that rabbit C-reactive protein has identical properties. Nevertheless, infection by Plasmodium yoelii sporozoites was not prevented in transgenic mice engineered to express rabbit C-reactive protein following induction of gluconeogenesis.     

Experimental transmission of Chikungunya virus by Anopheles stephensi mosquitoes

The Ades aegypti mosquito has been considered the principal vector of Chikungunya (CHIK) virus. As CHIK epidemics usually occur in urban regions and Anopheles stephensi is another highly endophilic and anthropophilic mosquito, there is a very high probability of this mosquito to feed on CHIK virus-infected patients, to pick up and transmit the virus. Therefore the present study was conducted to test the CHIK virus transmission capability for the A. stephensi mosquito. The obtained results showed that this mosquito species is capable of transmitting CHIK virus. It is surmised that during any epidemic of febrile illness CHIK virus isolation attempts should also be made from this mosquito species.     

Malaria: influence of Anopheles mosquito saliva on Plasmodium infection

Malaria is caused by Plasmodium protozoa that are transmitted by anopheline mosquitoes. Plasmodium sporozoites are released with saliva when an infected female mosquito takes a blood meal on a vertebrate host. Sporozoites deposited into the skin must enter a blood vessel to start their journey towards the liver. After migration out of the mosquito, sporozoites are associated with, or in proximity to, many components of vector saliva in the skin. Recent work has elucidated how Anopheles saliva, and components of saliva, can influence host–pathogen interactions during the early stage of Plasmodium infection in the skin. Here, we discuss how components of Anopheles saliva can modulate local host responses and affect Plasmodium infectivity. We hypothesize that therapeutic strategies targeting mosquito salivary proteins can play a role in controlling malaria and other vector-borne diseases.     

Ultrastructural studies on the interaction of Plasmodium falciparum ookinetes with the midgut epithelium of Anopheles stephensi mosquitoes

The interaction of Plasmodium falciparum ookinetes with the midgut epithelium of Anopheles stephensi is described by electron microscopy. Plasmodium falciparum ookinetes have been traced during the early stages of invasion and localization beneath the basal lamina of the midgut. It is generally assumed that ookinetes break through two membranes to reach the basal lamina of the midgut epithelium by an intracellular route. In the present study evidence is presented that the ookinete takes an intercellular route in traversing the epithelial layer. Compared to the mode of penetration and intracellular development of sporozoites and merozoites, an intercellular route for penetration of the ookinete is not inconsistent with its further extracellular development.     

Effect of chloroquine, methylene blue and artemether on red cell and hepatic antioxidant defence system in mice infected with Plasmodium yoelii nigeriensis

Reactive oxygen species are mediators of tissue injury and are involved in malaria infection. In this study, the status of red cell and hepatic oxidative stress and antioxidant defence indices were investigated during Plasmodium yoelii nigeriensis (P. yoelii) infection, and treatment with chloroquine (CQ), methylene blue (MB) or artemether (ART) in mice. P. yoelii infection caused a significant (p?<?0.05) increase in oxidative stress as evidenced by the elevated level of malondialdehyde. This was followed by a significant decrease (p?<?0.05) in hepatic antioxidant defence indices, viz. reduced glutathione (GSH) and glutathione-S-transferase (GST). Also, the red cell catalase activity was significantly (p?<?0.05) lower in malaria infection, while there was no significant difference (p?>?0.05) in the superoxide dismutase (SOD) activity of infected mice when compared to untreated normal. Treatment of infected mice with the three antimalarials showed that the drugs suppressed the parasitaemia in the order CQ?>?ART?>?MB. CQ, MB and ART treatment of infected mice caused a significant (p?<?0.05) increase in the levels of hepatic GSH and GST. Specifically, CQ, MB and ART increased the levels of hepatic GSH by 108, 124 and 98 %, respectively, at day 6. Also, ART treatment of infected mice significantly (p?<?0.05) elevated the red cell SOD level by 200 % at day 3. Taken together, the findings suggest that the antimalarial effect of CQ, MB and ART countered the P. yoelii-induced oxidative stress leading to the elevation of enzymatic and non-enzymatic antioxidants in the host system.