A rodent malaria, Plasmodium berghei, is experimentally transmitted to mice by merely probing of infective mosquito, Anopheles stephensi

We found that infection of a rodent malaria, Plasmodium berghei, occurred when the sporozoites were injected into the skin, the muscle, the peritoneal cavity and the tail end. Mice, which were injected with sporozoites in the tail end and had the site cut 5 min later, did not develop malaria. We also found that mice developed malaria when malaria infective mosquitoes, Anopheles stephensi, were forced not to take blood but only to probe into the skin. Moreover, the mice probed by the infective mosquitoes were protected from malaria infection if the site was treated with Kyu (heat treatment) after the mosquitoes had probed. These findings indicate that malaria infection occurs not only by blood feeding of the infective mosquito but also by probing of the mosquito. Sporozoites injected into the skin remain at the injected site for at least 5 min, then migrate to the blood vessels and invade into the blood stream. At present, the mechanism is not clear, although we propose here the existence of the skin stage of malaria parasites before the liver stage and the blood stage.     

Plasmodium falciparum and Plasmodium berghei: effects of ornithine decarboxylase inhibitors on erythrocytic schizogony

Five ornithine decarboxylase inhibitors: alpha-difluoromethylornithine (DFMO) (eflornithine); alpha-monofluoromethyl-3,4-dehydroornithine; alpha-monofluoromethyl-3,4-dehydroornithine methyl ester; alpha-monofluoromethyl-3,4-dehydroornithine ethyl ester; and (2R,5R)-delta-methyl-alpha-acetylenic putrescine were shown to inhibit erythrocytic schizogony of Plasmodium falciparum in vitro and reduced spermidine levels in infected erthrocytes. Only DFMO was effective at limiting erythrocytic schizogony of P. berghei in vivo. Administration of DFMO as a 2% solution in the drinking water for 4 days reduced parasitemia in mice by 50% in a 4-day suppression test but did not increase survival time of infected mice. This is the first demonstration of an effect of DFMO on plasmodial erythrocytic schizogony in vivo and suggests that interference with polyamine biosynthesis may, in fact, be a viable chemotherapeutic target in erythrocytic malaria.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

Malaria-induced reduction of fecundity during the first gonotrophic cycle of Anopheles Stephensi mosquitoes

Abstract. Anopheles stephensi mosquitoes which had fed upon mice infected with Plasmodium yoelii nigeriensis malaria parasites produced significantly fewer eggs than mosquitoes fed on an uninfected mouse. Fecundity reduction was more pronounced when the bloodmeal contained malaria gametocytes and the mosquitoes developed oocysts. Egg production and haematin excretion were correlated for uninfected bloodfed mosquitoes; the presence of P.y. nigeriensis in the blood affected this relationship. Reduced fecundity was associated with a significant reduction of bloodmeal size (measured by haematin excretion) in mosquitoes which ingested gametocytaemic blood. The bloodmeal size in mosquitoes fed on parasitaemic blood without gametocytes was not significantly reduced. The use of haematin assays for determination of bloodmeal size in mosquitoes is discussed.     

Comparison of bednets treated with alphacypermethrin, permethrin or lambdacyhalothrin against Anopheles gambiae in the Gambia

In the Gambian village of Saruja, where malaria is transmitted mainly by mosquitoes of the Anopheles gambiae complex, a trial was undertaken of the acceptability and efficacy of bednets treated with one of three pyrethroid insecticides – alphacypermethrin 40 mg/m², permethrin 500 mg/m² and lambdacyhalothrin 10 mg/m^2. Fewer mosquitoes were found alive under nets treated with insecticide than under control nets. Significantly more dead mosquitoes were found under nets treated with alphacypermethrin than under nets treated with permethrin or lambdacyhalothrin. Side-effects were reported by a proportion of the users of nets treated with each of the insecticides, but none were severe and their prevalence was similar between treatment groups. Unwashed nets treated with alphacypermethrin were more effective at killing anopheline mosquitoes in bioassays than nets treated with permethrin or lambdacyhalothrin. Killing activity was reduced when nets were washed, irrespective of which insecticide was used. Bednets treated with alphacypermethrin are well accepted, effectively killed anopheline mosquitoes and should therefore be evaluated for personal protection against malaria transmission.     

Mosquito mortality and the evolution of malaria virulence

Abstract Several laboratory studies of malaria parasites (Plasmodium sp.) and some field observations suggest that parasite virulence, defined as the harm a parasite causes to its vertebrate host, is positively correlated with transmission. Given this advantage, what limits the continual evolution of higher parasite virulence? One possibility is that while more virulent strains are more infectious, they are also more lethal to mosquitoes. In this study, we tested whether the virulence of the rodent malaria parasite P. chabaudi in the laboratory mouse was correlated with the fitness of mosquitoes it subsequently infected. Mice were infected with one of seven genetically distinct clones of P. chabaudi that differ in virulence. Weight loss and anemia in infected mice were monitored for 16–17 days before Anopheles stephensi mosquitoes were allowed to take a blood meal from them. Infection virulence in mice was positively correlated with transmission to mosquitoes (infection rate) and weakly associated with parasite burden (number of oocysts). Mosquito survival fell with increasing oocyst burden, but there was no overall statistically significant relationship between virulence in mice and mosquito mortality. Thus, there was no evidence that more virulent strains are more lethal to mosquitoes. Both vector survival and fecundity depended on parasite clone, and contrary to expectations, mosquitoes fed on infections more virulent to mice were more fecund. The strong parasite genetic effects associated with both fecundity and survival suggests that vector fitness could be an important selective agent shaping malaria population genetics and the evolution of phenotypes such as virulence in the vector.     

Malaria parasite growth is stimulated by mosquito probing

The ability of malaria parasites to respond positively to the presence of feeding mosquito vectors would clearly be advantageous to transmission. In this study, Anopheles stephensi mosquitoes probed mice infected with the rodent malaria parasite, Plasmodium chabaudi. Growth of asexual stages was accelerated and gametocytes appeared 1-2 days earlier than in controls. This first study, to our knowledge, of the effects of mosquitoes on 'in-host' growth and development of Plasmodium has profound implications for malaria epidemiology, suggesting that individuals exposed to high mosquito numbers can contribute disproportionately high numbers of parasites to the transmission pool.     

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.     

The role of Pvs28 in sporozoite development in Anopheles sinensis and its longevity in BALB/c mice

To develop a vivax malaria vaccine for blocking malarial transmission, the ookinete surface protein Pvs28 was cloned from Korean malaria patients using polymerase chain reaction. The Pvs28 gene consists of 726 bp and encodes 241 amino acids. It was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein, rPvs28, has a molecular weight of about 28 kDa in SDS–PAGE analysis. A monoclonal antibody against rPvs28 was produced using BALB/c mice. It inhibited sporozoite development in Anopheles sinensis mosquitoes (n = 81) which is one of the malaria vectors in Korea, with relatively high antibody titer against rPv28 persisting for more than 6 months. These results indicate that rPvs28 induces an immune response in mice that effectively blocks sporozoite development in mosquitoes. Therefore it could be a vaccine candidate for preventing vivax malaria in Korea.     

Host liver changes in leukemic mice: effect of alpha-difluoromethyl ornithine, an inhibitor of polyamine synthesis

Elevated levels of polyamines and gamma-Glutamyl Transpeptidase were seen in the liver of P388 leukemia bearing mice. There was also increase in specific activity of beta-Hexosaminidase and the B/A isoenzyme ratio. Administration of a 2% solution of alpha-Difluoromethylornithine (DFMO) immediately after inoculation of tumor cells prevented increases in polyamines in liver, but did not have any effect on gamma-Glutamyl Transpeptidase. Almost normal ratio of beta-Hexosaminidase B to A was maintained during treatment. Endogenous ornithine level was not altered both in treated and untreated mice. However, proline level was elevated in liver of untreated mice and DFMO prevented this increase. Glutathione levels were altered both by leukemia and DFMO in the host liver. The effect of drug was more prominent in the early stages rather than during terminal stages of leukemic growth.     

A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami

We investigated the parasitology, pathogenicity (virulence) and infectivity to mosquitoes of blood infections in mice, of two strains, DS and DK, of the rodent malaria parasite Plasmodium chabaudi adami. Blood infections of DS were found to be highly pathogenic; the asexual parasites in these infections were fast-growing and showed no evidence of selectivity in their infection of host erythrocytes. In contrast to DS, blood infections of DK were much less pathogenic; the asexual parasites were slower-growing and showed a moderate degree of selectivity to a subset of erythrocytes which were not reticulocytes. In both DS and DK infections, infectivity to mosquitoes was highest before the peak of asexual parasitaemia had occurred; usually this did not coincide with the time when gametocyte numbers in the blood were highest. Infections with the pathogenic DS strain in CBA mice produced fewer gametocytes than did the less pathogenic DK strain. The DS strain infections in both CBA and C57 mice were also significantly much less infective to mosquitoes than the DK strain. Investigations by others on the related rodent malaria parasite subspecies, Plasmodium chabaudi chabaudi, have indicated that the mosquito infectivity of blood infections in mice tended to be higher in the more pathogenic (virulent) and lower in the less pathogenic strains of this parasite subspecies. This is the converse of the finding of the present investigation of blood infections of P. c. adami in mice in which a more pathogenic, or virulent, strain (DS) of these parasites was significantly much less infective to mosquitoes than was a less pathogenic strain (DK).     

Analysis of recent data on a fauna and ranges of malaria mosquitoes (Diptera: Culicidae: Anopheles) from the territory of Russia

The lists of malaria mosquitoes in Russia includes 12 species, rather than 13, as it was formerly considered (Gornostaeva, 2000; Gornostaeva, Danilov, 2002), because Anopheles subalpinus is a junior synonym of An. melanoon. Data accumulated up to present are still not sufficient to characterize ranges of many species in Russia, specifically An. beklemishevi, An. maculipennis, and An. messea. Careful investigations of biodiversity and ranges of the malaria mosquitoes in different territories of Russia have not been carried out since 40-60th. In the last 50 years, a cytodiagnostic method appeared to be the most perspective method for the study of biodiversity and ranges of the malaria mosquitoes in Russia. Critical analysis of data obtained by this methods shows that the studies of biodiversity and ranges of the malaria mosquitoes should be performed in a contact of entomologist and genetic experts to avoid errors in a collection of field data and laboratory tests. The study of biodiversity and ranges of the malaria mosquitoes is a quite important field of investigations, because of increase of local cases of the malaria disease and unfavorable prognosis for nearest years in relation to the malaria.     

Epidemiological consequences of a newly discovered cryptic subgroup of Anopheles gambiae

A cryptic subgroup of Anopheles gambiae sensu stricto mosquitoes was recently discovered in West Africa. This 'GOUNDRY' subgroup has increased susceptibility to Plasmodium falciparum, the most deadly form of malaria. Unusual for this major malaria vector, GOUNDRY mosquitoes also seem to bite exclusively outdoors. A mathematical model is developed to assess the epidemiological implications of current vector control tools, bednets and indoor residual spray, preferentially suppressing the more typical indoor biting mosquitoes. It is demonstrated that even if the GOUNDRY mosquitoes have a decreased preference for human blood, vector controls which select for increased GOUNDRY abundance relative to their indoor biting counterparts risks intensifying malaria transmission. Given the widely observed phenomenon of outdoor biting by major malaria vectors, this behaviour should not be ignored in future modelling efforts and warrants serious consideration in control programme strategy.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Effects of DL-alpha-difluoromethylornithine, 4-deoxypyridoxine and methylglyoxal bis(guanylhydrazone) on allograft prolongation

DL-alpha-difluoromethylornithine (DFMO), 4-deoxypyridoxine (4-DOP), and methylglyoxal bis (guanylhydrazone) (MGBG) were tested as inhibitors of acute skin graft rejection. Proximal full thickness tail skins were exchanged between C57BL/6 and Balb/C mice. Distal autografts were placed to monitor healing. Inhibitors were given singly or in combination, either orally or by injection, in various schedules to 10 groups of mice. Compared to controls, singly treated mice had significant mean prolongation of allografts ranging from 126% to 141%. Likewise, DFMO plus MGBG extended mean time of complete rejection ranging from 172% to 206%. Autografts remained intact. Some grafts persisted after discontinued immunosuppression. Complete rejection was preceded by a decline in vascularity of the graft bed and/or gradual replacement by host tissue. Graft protection in such stringent circumstances i.e., the use of skin in strains with complete histoincompatibility at the H-2 MHC loci, clearly indicate the anti-rejection effects of polyamine synthesis inhibitors. Moreover, primary and secondary effects of DFMO establish the critical role of polyamine pathway activation in acute rejection. In doses and schedules used, toxicity was encountered when DFMO and 4-DOP were used in combination and when increased amounts of MGBG were administered.     

Dynamics of malaria transmission model with sterile mosquitoes

In this paper, a malaria transmission model with sterile mosquitoes is considered. We first formulate a simple SEIR malaria transmission model as our baseline model. Then sterile mosquitoes are introduced into the baseline model. We consider the case that the release rate of sterile mosquitoes is proportional to the wild mosquito population size. To investigate the impact of releasing sterile mosquitoes on the malaria transmission, the dynamics of the baseline model and the models with the sterile mosquitoes are discussed. We derive formulas of the reproductive numbers and explore the existence of endemic equilibrium as the reproductive number is more than unity for these models. It is shown that both the baseline model and the models with the sterile mosquitoes undergo backward bifurcations. Based on theoretical analysis and numerical simulation, we investigate the impact of releasing sterile mosquitoes on malaria transmission.     

The Dutch school of malaria research

An epidemic of tertian malaria in some coastal areas of The Netherlands resulted in the setting up of official measures in 1920. A scientific and a propaganda commission were charged with control. Efforts were made to reduce mosquito populations by adult and larval spraying. After the discovery that infected mosquitoes were to be found only inside houses, control operations were focussed against adult mosquitoes. Some later discoveries resulted in a more effective control. a) Spraying ditches with Paris green did not prevent adult mosquitoes from entering the control area. b) Anopheles maculipennis turned out to be a complex of species, with A. atroparvus as the vector. The latter preferred brackish water and did not go into full hibernation. The closing of the Zuyder Sea and the expected desalinization gave hope for less suitable conditions for the vector. c) Plasmodium vivax normally had an incubation period of 8 months. d) Pyrethrum was an effective but short-lasting insecticide. e) Healthy parasite carriers could infect mosquitoes. This knowledge was applied through an extensive system of investigation, including spleen examination of schoolchildren. Suspected houses were sprayed bimonthly from August to November, during which period infected mosquitoes were likely to be present. This system worked extremely well, and during the next epidemic from 1943 to 1947 the thus treated towns remained virtually free of malaria! DDT became available and was either sprayed in suspected houses as before, or through wide-spread coverage of all houses. The epidemic subsided whatever method employed and not only due to the use of DDT. The number of cases even went down to the point of no return and the last case of Dutch malaria was recorded in 1959. The wealth of experience on house-spray control, parasite and mosquito biology and experimental malaria of the Dutch malariologists has had its impact on the international bodies engaged in the battle against malaria.     

Stimulation of DNA synthesis in human epidermis by UVB radiation and its inhibition by difluoromethylornithine

The purpose of this study was to determine whether the rate of DNA synthesis in human skin could be increased by UVB radiation and to determine the potential for reversing the stimulatory effects of UVB radiation by alpha-difluoromethylornithine (DFMO). Split-thickness facial skin was grafted onto athymic CD-1 Nu/Nu mice on the anterolateral dorsal surface. Following graft healing for 6 weeks, grafts were treated with 0%, 2%, or 5% DFMO (a potent inhibitor of polyamine biosynthesis) and subsequently irradiated with 0.15 J/cm2 of UVB light. Two days after UVB exposure, [3H]thymidine was injected and the grafts were dissected and counted. Ultraviolet radiation significantly increased thymidine incorporation, indicating increased DNA synthesis. The stimulatory effects of UV radiation were significantly reduced by topical application of 5% DFMO. Thus administration of DFMO most likely decreased the polyamine level and decreased the rate of DNA synthesis, which may have caused a decreased rate of epidermal proliferation. Thus the topical application of DFMO may prove beneficial for UVB exposure and other hyperproliferative states where a decrease in the rate of cell turnover might be desirable.     

The effect of Plasmodium yoelii nigeriensis infection on the feeding persistence of Anopheles stephensi Liston throughout the sporogonic cycle.

Vector-borne parasites such as malaria have been shown to modify the feeding behaviour of their invertebrate hosts so as to increase the probability of transmission. However, evolutionary consideration of developmental changes in malaria within Anopheles mosquitoes suggests that the nature of altered feeding by mosquitoes should differ depending on the developmental stage of the parasite. We present laboratory evidence that the feeding persistence of female Anopheles stephensi towards a human host is decreased in the presence of Plasmodium yoelii nigeriensis oocysts (which cannot be transmitted), but increased when the malaria has developed into transmissible sporozoites in the salivary glands. In ten-minute trials, 33% of uninfected mosquitoes gave up their feeding attempt before the test period had ended, 53% of those harbouring oocysts had given up, but only 20% of those infected with sporozoites gave up by this time. We conclude that changes in feeding behaviour of mosquitoes mediated by parasite infection are sensitive to the developmental stage of the parasite and that these changes have important implications for malaria epidemiology.