Immunisation against a serine protease inhibitor reduces intensity of Plasmodium berghei infection in mosquitoes

The mosquito innate immune response is able to clear the majority of Plasmodium parasites. This immune clearance is controlled by a number of regulatory molecules including serine protease inhibitors (serpins). To determine whether such molecules could represent a novel target for a malaria transmission-blocking vaccine, we vaccinated mice with Anopheles gambiae serpin-2. Antibodies against Anopheles gambiae serpin-2 significantly reduced the infection of a heterologous Anopheles species (Anopheles stephensi) by Plasmodium berghei, however this effect was not observed with Plasmodium falciparum. Therefore, this approach of targeting regulatory molecules of the mosquito immune system may represent a novel approach to transmission-blocking malaria vaccines.     

Energy metabolism affects susceptibility of Anopheles gambiae mosquitoes to Plasmodium infection

Previous studies showed that Anopheles gambiae L3-5 females, which are refractory (R) to Plasmodium infection, express higher levels of genes involved in redox-metabolism and mitochondrial respiration than susceptible (S) G3 females. Our studies revealed that R females have reduced longevity, faster utilization of lipid reserves, impaired mitochondrial state-3 respiration, increased rate of mitochondrial electron leak and higher expression levels of several glycolytic enzyme genes. Furthermore, when state-3 respiration was reduced in S females by silencing expression of the adenine nucleotide translocator (ANT), hydrogen peroxide generation was higher and the mRNA levels of lactate dehydrogenase increased in the midgut, while the prevalence and intensity of Plasmodium berghei infection were significantly reduced. We conclude that there are broad metabolic differences between R and S An. gambiae mosquitoes that influence their susceptibility to Plasmodium infection.     

Apoptosis in the malaria protozoan,Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito

Death by apoptosis regulates cell numbers in metazoan tissues and it is mediated by activation of caspases and results in characteristic morphological and biochemical changes. We report here that the malaria protozoan, Plasmodium berghei, exhibits features typical of metazoan apoptotic cells including condensation of chromatin, fragmentation of the nuclear DNA and movement of phosphatidylserine from the inner to the outer lamellae of the cell membrane. In addition, proteins with caspase-like activity were identified in the cytoplasm of the ookinete suggesting that the cellular mechanism of cell death may be similar to that of multicellular eukaryotes. Our data show that more than 50% of the mosquito midgut stages of the parasite die naturally by apoptosis before gut invasion. Cell death was prevented by a caspase inhibitor, treatment resulting in a doubling of parasite intensity. All these features also occur in vitro. Cell suicide thus plays a major and hitherto unrecognised role in controlling parasite populations and could be a novel target for malaria control strategies.     

Alterations in membrane permeability of malaria-infected human erythrocytes are related to the growth stage of the parasite

During the intraerythrocytic growth of Plasmodium falciparum in culture, marked changes are observed in the permeability properties of the host cell membrane. Anionic substances otherwise impermeant to normal cells, become highly permeant to infected cells. These changes in permeability become apparent as rings mature into trophozoites and remain throughout schizogony. The permeability changes to anionic substances are not manifested as degradation of band 3, the purported erythrocyte anion transporter. They probably reflect alterations of a more general nature.     

Effects of in vivo exposure to DEET on blood feeding behavior and fecundity in Anopheles quadrimaculatus (Diptera: Culicidae)

This study determined the effects of contact with DEET on guinea pig skin on mortality, probing time, blood feeding rate, engorgement time, and fecundity responses in female Anopheles quadrimaculatus Say. Exposure, in this manner, to 10% DEET (in ethanol) for 5 min, resulted in 98% mortality in mosquitoes after 24h. The median probing time (PT(50)) required by females, when exposed to 0.1%, 1.0%, and 10% DEET, was significantly (P<0.0001) longer (12.5, 12.1, and 19.1s, respectively) than the 6.8s required by females to probe ethanol-treated skin (control). Similarly, mean blood feeding rates in populations of females exposed to 1.0% DEET for < or = 5 min (14.4%) was 6x lower (P<0.001) (85.5%) than in females exposed to ethanol-treated skin, whereas the mean engorgement time on skin treated with 1.0% DEET (66.3s) was significantly shorter (P<0.0001) than for females feeding on the control guinea pigs (105.9s). The mean number of mature o?cytes per female (fecundity) in treatment (1.0% DEET) and control mosquitoes was not significantly different. The responses to DEET observed in this study suggest that repeated exposure of female A. quadrimaculatus populations to this repellent, in laboratory bioassays, could result in confounding of toxicant and repellent effects and inaccurate estimates of DEET repellency.     

Susceptibility of adult mosquitoes to insecticides in aqueous sucrose baits

Mosquitoes characteristically feed on plant‐derived carbohydrates and honeydew just after emergence and intermittently during their lives. Development of toxic baits focusing on this carbohydrate‐seeking behavior may potentially contribute to localized control. In the present study, ten insecticides were fed to female Culex quinquefasciatus, Anopheles quadrimaculatus, and Aedes taeniorhynchus in a 10% sucrose solution. Active ingredients representative of five classes of insecticides (pyrethroids, phenylpyroles, pyrroles, neonicotinoids, and macrocyclic lactones) were selected for comparison with commercial formulations used to facilitate incorporation of active ingredients into aqueous sucrose solutions. Sucrose as a phagostimulant significantly enhanced mortality to toxicants. In general, the most effective active ingredients were fipronil, deltamethrin and imidacloprid, followed by spinosad, thiamethoxam, bifenthrin, permethrin, and cyfluthrin. The least effective ingredients were chlorfenapyr and ivermectin. For some of the ingredients tested, Cx. quinquefasciatus was the least susceptible species. One‐day‐old male Cx. quinquefasciatus were more susceptible than females; however, no differences existed between one‐ and seven‐day‐old mosquitoes. There were no differences in susceptibility between unfed and gravid ten‐day‐old female Cx. quinquefasciatus to bifenthrin. In conclusion, several pesticides from different classes of compounds have potential for use in development of toxic baits for mosquitoes.     

Adenovirus vector production using low-multiplicity infection of 293 cells

The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001–0.1 transductional unit (TU) cell⁻¹. The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected at low MOIs (<1 TU cell⁻¹), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells. When 293-F cells at a relatively low density of 1 × 10⁵ cells cm⁻³ were infected with Ad EGFP at a low MOI of 0.001 TU cell⁻¹, the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell⁻¹) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium.     

Plasmodium gallinaceum: Erythrocyte factor essential for zygote infection of Aedes aegypti

Zygotes of Plasmodium gallinaceum, fertilized in vitro and fed to Aedes aegypti mosquitoes through a membrane, formed oocysts only when a substance in the cytoplasm of uninfected erythrocytes was present. The relation between erythrocyte volume and infectivity was linear (1:1.2) up to a 50% hematocrit. The intraerythrocytic substance was both nondialyzable and poorly soluble in plasma. By carboxymethyl cellulose chromatography, cytoplasmic constituents eluted at pH 8.6 supported the same infection as control blood did; but higher and lower pH eluates supported none. Dialyzable factors present in the plasma, but absent from M199, enhanced infection but were not essential. Zygotes developed normally to ookinetes in the gut of plasma-fed mosquitoes, or when cultured in plasma or M199. Ookinetes from culture formed normal oocysts when fed to mosquitoes in blood or when injected with M199 into the hemocoels of unfed females. Mosquitoes fed infected blood containing lima bean or soybean trypsin inhibitor were unable to digest the erythrocytes and, although normal ookinetes developed, no oocysts formed. It appears from this and histological evidence that an erythrocyte substance, released by mosquito digestion, is needed for ookinete invasion of the gut epithelium.     

Larval competition alters susceptibility of adult Aedes mosquitoes to dengue infection

Dengue, the most important human arboviral disease, is transmitted primarily by Aedes aegypti and, to a lesser extent, by Aedes albopictus. The current distributions of these invasive species overlap and are affected by interspecific larval competition in their container habitats. Here we report that competition also enhances dengue infection and dissemination rates in one of these two vector species. We determined the effects of competition on adult A. aegypti and A. albopictus, comparing their susceptibility to infection with a Southeast Asian strain of dengue-2 virus. High levels of intra- or interspecific competition among larvae enhanced the susceptibility of A. albopictus to dengue virus infection and potential for transmission, as indicated by disseminated infections. Doubling the number of competing larvae (A. albopictus or A. aegypti), led to a significant (more than 60%) increase in the proportion of A. albopictus with disseminated dengue-2 infection. Competition-enhanced vector competence appears to result from a reduction in 'barriers' (morphological or physiological) to virus infection and dissemination and may contribute to the importance of A. albopictus in dengue transmission. Similar results for other unrelated arboviruses suggest that larval competition, common in mosquitoes, should be considered in estimates of vector competence for pathogens that infect humans.     

Co-occurrences of parasite clones and altered host phenotype in a snail-trematode system

The frequent co-occurrence of two or more genotypes of the same parasite species in the same individual hosts has often been predicted to select for higher levels of virulence. Thus, if parasites can adjust their level of host exploitation in response to competition for resources, mixed-clone infections should have more profound impacts on the host. Trematode parasites are known to induce a wide range of modifications in the morphology (size, shell shape or ornamentation) of their snail intermediate host. Still, whether mixed-clone trematode infections have additive effects on the phenotypic alterations of the host remains to be tested. Here, we used the snail Potamopyrgus antipodarum-infected by the trematode Coitocaecum parvum to test for both the general effect of the parasite on host phenotype and possible increased host exploitation in multi-clone infections. Significant differences in size, shell shape and spinosity were found between infected and uninfected snails, and we determined that one quarter of naturally infected snails supported mixed-clone infections of C. parvum. From the parasite perspective, this meant that almost half of the clones identified in this study shared their snail host with at least one other clone. Intra-host competition may be intense, with each clone in a mixed-clone infection experiencing major reductions in volume and number of sporocysts (and consequently multiplication rate and cercarial production) compared with single-clone infections. However, there was no significant difference in the intensity of host phenotype modifications between single and multiple-clone infections. These results demonstrate that competition between parasite genotypes may be strong, and suggest that the frequency of mixed-clone infections in this system may have selected for an increased level of host exploitation in the parasite population, such that a single-clone is associated with a high degree of host phenotypic alteration.     

Population diversity and multiplicity of infection in Theileria annulata

The tick-borne apicomplexan parasite Theileria annulata is endemic in many sub-tropical countries and causes the bovine disease tropical theileriosis. Although the parasite is known to be highly diverse, detailed information is lacking on the genetic structure of natural populations and levels of multiplicity of infection in the cattle host. With the widespread deployment of live attenuated vaccines and the emergence of drug-resistant parasites in the field, it is vital to appreciate the factors which shape genetic diversity of the parasite both within individual hosts and in the wider population. This study addresses these issues and represents an extensive genetic analysis of T. annulata populations in two endemic countries utilising a high-throughput adaptation of a micro- and mini-satellite genotyping system. Parasite material was collected from infected cattle in defined regions of Turkey and Tunisia to allow a variety of analyses to be conducted. All animals (n = 305) were found to harbour multiple parasite genotypes and only two isolates shared an identical predominant multi-locus profile. A modelling approach was used to demonstrate that host age, location and vaccination status play a measurable role in determining multiplicity of infection in an individual animal. Age was shown to positively correlate with multiplicity of infection and while positive vaccination status exerted a similar effect, it was shown to be due not simply to the presence of the immunising genotype. Importantly, no direct evidence was found for the immunising genotype spreading or recombining within the local parasite community. Genetic analysis confirmed the tentative conclusion of a previous study that the parasite population appears to be, in general, panmictic. Nevertheless, evidence supporting linkage disequilibrium and a departure from panmixia was uncovered in some localities and a number of explanations for these findings are advanced.     

Baculovirus production for gene therapy: the role of cell density,multiplicity of infection and medium exchange

One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used, with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers as high as 2.6 × 10¹⁰ IP.mL⁻¹ were obtained in cultures infected at 3.5 × 10⁶ cells.mL⁻¹, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange.     

The intestinal parasite infection status of inhabitants in the Roxas city,The Philippines

We carried out a small-scale survey to investigate the status of intestinal protozoa and helminthes infection of inhabitants in Roxas city, Mindoro, the Philippines. Total 301 stool samples were subjected to the formalin-ether concentration method for the detection of helminth ova and protozoan cysts. The overall positive rate was 64.5%, and that of male and female were 56.6% and 72.5%, respectively. The highest infected helminth was Ascaris lumbricoudes (51.2%), followed by Trichuris trichiura (27.6%), hookworm (8.0%) and Enterobius vermicularis (0.3%). The protozoa infection status revealed that Entamoeba coli was the most frequent (15.0%). Iodoamoeba buetschlii and E. histolytica were found but few. The multiple infection more than two parasites was 29.6%, and double infection with A. lumbricoides and T. trichiura was most common. The intestinal helminth infections were highly prevalent in this area, according to this result, and we concluded that anthelminthic drugs should be given to inhabitants, especially to children of 1 to 15 years-old.     

Plasmodium berghei: mechanisms and sites of resistance to sporogonic development in different mosquitoes

An experimental study of the mechanisms and patterns of resistance to Plasmodium berghei in different mosquito species revealed a diversity of factors which prevent or inhibit sporogonic development in its different phases and in different sites in the mosquito vector. The experiments showed that Culex salinarius was a totally resistant species in which exflagellation and ookinete formation are prevented. In Aedes aegypti, ookinetes in small or moderate numbers are formed but penetration of the mosquito midgut wall is blocked and oocysts are not formed. In the three experimental vectors, Anopheles quadrimaculatus, Anopheles aztecus, and Anopheles stephensi grades of enhanced susceptibility are recognized. They are expressed in lesser numbers of abnormal and degenerative oocysts, in higher numbers of sporozoites in the salivary gland, and greater viability and invasiveness of these sporozoites. In Anopheles dureni, the natural vector of rodent malaria, one observes both in nature and under experimental conditions the highest adaptation and most pronounced grade of susceptibility to P. berghei.     

Sugar digestion in mosquitoes: identification and characterization of three midgut alpha-glucosidases of the neo-tropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

Dietary carbohydrates provide an important source of energy for flight, and contribute to longevity and fecundity of mosquitoes. The most common sugar mosquitoes ingest is sucrose, and digestion of this substance is carried out mainly by alpha-glucosidases. In the current work, we tested the efficiency of sucrose on Anopheles aquasalis female diet. The best longevity (days) was reached when sugar was available in the diet, whereas most only blood fed females were dead 6 days after emergence. Three alpha-glucosidase isoforms were detected in the adult female midgut, named alphaGlu1, alphaGlu2 and alphaGlu3. These are acidic alpha-glucosidases with optima pH around pH 5.5. alphaGlu1 and alphaGlu2 are present in both secreted and membrane-bound forms, whereas alpha-Glu3 only in anchored to membranes. The alpha-glucosidase activity is concentrated mainly in the posterior midgut (70%), both in non-fed or 10% sucrose fed females. The single form of these alpha-glucosidases seemed to be approximately 70 kDa polypeptides, although alphaGlu2 is presented in >or=600 kDa self-aggregates. Km values of alphaGlu1, alphaGlu2 and alphaGlu3 differed significantly from each other, supporting the statement that three alpha-glucosidases are produced in the female midgut. Together, all data suggest that sugar is an essential component of A. aquasalis female diet. In addition, alpha-glucosidases are synthesized in the same place where sucrose is digested and absorbed, the midgut.     

Two glycosylation alterations of mouse intestinal mucins due to infection caused by the parasite Nippostrongylus brasiliensis

The glycosylation alterations of mouse small intestinal mucins during a 12-day infectious cycle caused by the parasite Nippostrongylus brasiliensis have been studied. The guanidinium chloride insoluble mucins were isolated at day 0 to 12 from the small intestine of infected and non-infected C57BL/6 mice. The O-linked oligosaccharides were released by reductive -elimination from the mucins and separated into neutral, sialylated and sulfated fractions. All fractions were analyzed by monosaccharide composition analysis and the neutral oligosaccharides were structurally characterized by gas chromatography/mass spectrometry. Two oligosaccharides containing blood group H-type epitopes (Fuc1-2Gal-) were transiently expressed with a maximum at day 6. Additional oligosaccharides with the common structure HexNAc-Gal-3GalNAcol were transiently induced with a maximum at day 10. Northern blot analysis on total RNA showed a transient expression at day 4–6 of the Fut2 gene encoding a Fuc1-2 fucosyltransferase, probably responsible for the detected blood group H-type epitopes. Comparisons with the corresponding infection in rat studied previously, revealed structurally different alterations, although occurring as transient events in both species. Both showed an induced blood group-type transferase halfway through the infection (a blood group A transferase in rat) and an induced transferase adding a terminal GalNAc (to a sialic acid- containing epitope in rat) towards the end of the infection. These differences between closely related species suggest rapid evolutionary alterations in glycosyltransferase expression.     

Salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection

Abstract Susceptibility to Salmonella typhimurium infection was compared in H (high Ab responder) and L (low Ab responder) mice obtained by several selective breeding experiments (Selections I, II, III, IV and IV A) [10,19,22]. H mice were always much more susceptible to infection than their L mice counterparts within a continuous LD 50 variation range. In three of the selections (I, II and IV A) the low responsiveness character is known to result mainly from rapid Ag degradation in L mice macrophages. It was hypothesized that resistance to multiplication of intracellular pathogens could be related to an increased catabolic activity towards Ag. This was actually demonstrated, in F₂ segregant hybrids of selection IV A, by the significant inverse correlation between capacity for Ab production and resistance to infection.