Identification and expression of maebl, an erythrocyte-binding gene, in Plasmodium gallinaceum.

Avian malaria is of significant ecological importance and serves as a model system to study broad patterns of host switching and host specificity. The erythrocyte invasion mechanism of the malaria parasite Plasmodium is mediated, in large part, by proteins of the erythrocyte-binding-like (ebl) family of genes. However, little is known about how these genes are conserved across different species of Plasmodium, especially those that infect birds. Using bioinformatical methods in conjunction with polymerase chain reaction (PCR) and genetic sequencing, we identified and annotated one member of the ebl family, merozoite apical erythrocyte-binding ligand (maebl), from the chicken parasite Plasmodium gallinaceum. We then detected the expression of maebl in P. gallinaceum by PCR analysis of cDNA isolated from the blood of infected chickens. We found that maebl is a conserved orthologous gene in avian, mammalian, and rodent Plasmodium species. The duplicate extracellular binding domains of MAEBL, responsible for erythrocyte binding, are the most conserved regions. Our combined data corroborate the conservation of maebl throughout the Plasmodium genus and may help elucidate the mechanisms of erythrocyte invasion in P. gallinaceum and the host specificity of Plasmodium parasites.

Transcriptome analysis of Aedes aegypti transgenic mosquitoes with altered immunity.

The mosquito immune system is involved in pathogen-elicited defense responses. The NF-κB factors REL1 and REL2 are downstream transcription activators of Toll and IMD immune pathways, respectively. We have used genome-wide microarray analyses to characterize fat-body-specific gene transcript repertoires activated by either REL1 or REL2 in two transgenic strains of the mosquito Aedes aegypti. Vitellogenin gene promoter was used in each transgenic strain to ectopically express either REL1 (REL1+) or REL2 (REL2+) in a sex, tissue, and stage specific manner. There was a significant change in the transcript abundance of 297 (79 up- and 218 down-regulated) and 299 (123 up- and 176 down-regulated) genes in fat bodies of REL1+ and REL2+, respectively. Over half of the induced genes had predicted functions in immunity, and a large group of these was co-regulated by REL1 and REL2. By generating a hybrid transgenic strain, which ectopically expresses both REL1 and REL2, we have shown a synergistic action of these NF-κB factors in activating immune genes. The REL1+ immune transcriptome showed a significant overlap with that of cactus (RNAi)-depleted mosquitoes (50%). In contrast, the REL2+ -regulated transcriptome differed from the relatively small group of gene transcripts regulated by RNAi depletion of a putative inhibitor of the IMD pathway, caspar (35 up- and 140 down-regulated), suggesting that caspar contributes to regulation of a subset of IMD-pathway controlled genes. Infections of the wild type Ae. aegypti with Plasmodium gallinaceum elicited the transcription of a distinct subset of immune genes (76 up- and 25 down-regulated) relative to that observed in REL1+ and REL2+ mosquitoes. Considerable overlap was observed between the fat body transcriptome of Plasmodium-infected mosquitoes and that of mosquitoes with transiently depleted PIAS, an inhibitor of the JAK-STAT pathway. PIAS gene silencing reduced Plasmodium proliferation in Ae. aegypti, indicating the involvement of the JAK-STAT pathway in anti-Plasmodium defense in this infection model.

A microfluidic platform to isolate avian erythrocytes infected with Plasmodium gallinaceum malaria parasites based on surface morphological changes.

This paper reports on a microfluidic platform to isolate and study avian red blood cells (RBCs) infected to various degrees by the malaria parasite Plasmodium gallinaceum. The experimental findings point to the feasibility of using the morphological changes on the surface of the malaria infected avian RBC (miaRBCs) as biomarkers for diagnosis. A glass substrate with a controlled surface roughness was used as part of a polydimethylsiloxane (PDMS) microfluidic channels. When whole-blood samples were introduced into the channels, the miaRBCs would be preferentially slowed and eventually become immobilized on the roughened surface. The surface lesions and furrow-like structures on the miaRBC surfaces offered a markedly higher probability to interact with the roughened substrate and allowed the cells to become imobilized on the surface. The captured miaRBCs were from blood samples at various degrees of infection at 3.2%, 3.9%, 9.1%, 13.4%, 20.1%, 28%, and 37%. It was observed that the miaRBCs could be selectively captured under a wall shear rate between 2.1 to 3.2 s(-1), which was directly proportional to the flow rate through the channels. This capture rate could be improved by increasing the channel length and finer flow control. It was also found that a roughened glass substrate with ten-point-height larger than the depth of surface lesions and furrow-like structures of miaRBCs showed a substantial enhancement on the number of immobilized infected RBCs. These findings indicated that surface morphologies, including surface lesions and furrow-like structures, can serve as an alternative biomarker for malaria diagnosis.

Inbreeding depression affects life-history traits but not infection by Plasmodium gallinaceum in the Asian tiger mosquito, Aedes albopictus.

Emerging and re-emerging vector-borne diseases represent an increasingly significant public health challenge. While geographic variation among populations of vector species for susceptibility to pathogen infection and vector competence has been thoroughly documented, relatively little attention has been devoted to understanding the ultimate evolutionary causes of this intraspecific variation. Local genetic drift is known to influence genetic differentiation among populations for a variety of container-inhabiting mosquito species, including Aedes albopictus. Because genetic drift is expected to reduce genetic variation and lead to the accumulation of (partially) recessive deleterious alleles, we hypothesized that reduced genetic variation might affect susceptibility to pathogen infection in a model pathogen-vector system. We therefore created replicate inbred (two generations of full-sib mating, expected f=0.375) and control (expected f approximately 0.07) lines of Ae. albopictus and measured life-history traits including larval survivorship, adult longevity, and female wing length (body size) as well as susceptibility to infection by a model pathogen, Plasmodium gallinaceum. Inbred mosquitoes had significantly reduced larval survivorship and female adult longevity but inbreeding did not affect male adult longevity or female wing length (body size). Furthermore, there was no effect of inbreeding on susceptibility to infection by P. gallinaceum. Therefore, while our results did not support the hypothesis that reduced genetic variation influences susceptibility to pathogen infection in this system, we did find evidence for an effect of reduced genetic variation on female adult longevity, an important component of vectorial capacity. We suggest that additional research is needed to elucidate the genetic underpinnings of intraspecific variation in traits related to disease transmission and discuss the implications of our results for the efficacy of creating transgenic strains refractory to disease transmission.

Plasmodium gallinaceum: clinical progression, recovery, and resistance to disease in chickens infected via mosquito bite.

Historically, in vivo experiments of Plasmodium gallinaceum in chickens have caused high mortality. Perhaps because of this high mortality, it remains to be demonstrated whether recovered birds will resist a second episode of illness when re-exposed to infected mosquitoes. In the current study, groups of 10 chicks were infected with P. gallinaceum via mosquito bite. Parasitemia and anemia were followed by recovery in all birds, although they had persisting, low levels of parasitized erythrocytes (0.007 +/- 0.019%). Twenty-three days after the initial exposure, 10 recovered chicks were rechallenged with P. gallinaceum via mosquito bite; none of them developed clinical or hematological evidence of malaria, in contrast to matched control birds, which all became diseased (P < 0.001). Unlike previous studies, the current experiment had no mortality in chickens infected with P. gallinaceum by mosquito bite. Recovered birds resisted disease from re-exposure to the same organism. The duration and nature of immunity or premunition remain to be determined.

Experimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections.

BACKGROUND: Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections. RESULTS: Whereas there was a trade-off between transmission success and virulence as defined by host mortality, contradictory clone-specific patterns occurred when defining virulence by anaemia. A negative relationship between anaemia and transmission success was found for one of the parasite clones, whereas there was no relationship for the other. Notably the two parasite clones also differed in a transmission phenotype (gametocyte sex ratio) that has previously been shown to respond adaptively to a changing blood environment. In addition, as predicted by evolutionary theory, mixed infections resulted in increased anaemia. The increased anaemia was, however, not correlated with any discernable parasite trait (e.g. parasite density) or with increased transmission. CONCLUSIONS: We found some evidence supporting the hypothesis that there is an adaptive basis correlating virulence (as defined by host mortality) and transmission success in malaria parasites. This confirms the validity of applying evolutionary virulence theory to biomedical research and adds support to the prediction that partially effective vaccines may select for increasingly virulent malaria parasite strains. By contrast, there was no consistent correlation between transmission and sub-lethal anaemia, a more common outcome of malaria infection. However, overall, the data are not inconsistent with the recent proposal that sub-lethal effects may impose an upper limit on virulence. Moreover, clone specific differences in transmission phenotypes linked to anaemia do suggest that there is considerable adaptive potential relating anaemia and transmission that may lead to uncertain consequences following intervention strategies.

Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for parasite invasion of the mosquito midgut.

The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding the blood meal in the mosquito midgut. Since the peritrophic matrix is a physical barrier that the parasite must cross to invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions. We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene, PgCHT1, encoding it. PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases. Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone derivatives of chitin oligosaccharides. Allosamidin inhibited recombinant PgCHT1 with an IC(50) of 7 microM and differentially inhibited two chromatographically separable P. gallinaceum ookinete-produced chitinase activities with IC(50) values of 7 and 12 microM, respectively. These two chitinase activities also had different pH activity profiles. These data suggest that the P. gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion.

Parasitismo de leucocitos y trombocitos de gallus gallus l. por plasmodium (novyella) juxtanucleare (apicomplexa: plasmodiidae) / Parasitism of leukocytes and thrombocytes of gallus gallus L. by plasmodium (novyella) juxtanucleare (apicomplexa: plasmodiidae)

Se realizó un investigación del parasitismo de plasmodium juxtanucleare en gallinas sin razas definida, provenientes de criaderos rústicos en el municipio de Seropédica, estado de Río de Janeiro, de Brasil. Se realizaron frotis sanguíneo periféricos, los cuales fuero coloreados con giemsa diluido en tampón sorensen pH6,8. En el examen hemoscópico se puede observar en aves con alto índice de (< 10 por ciento ) formas parasitarias de trofozoítas y esquizontes en el citoplasma de células de la línea leucocitica y trofozoítas en células de la línea trombocítica. Las observaciones en el presente estudio hacen inferir que la cepa de P. juxtanuclear que ocurre en Seropédica realiza esquizogonia fanerozóica. Este trabajo constituye el primer hallazgo de formas de P. juxtanucleares en leucocitos

[The effect of antihormones on the susceptibility of Anopheles sacharovi Favre. mosquitoes to the causative agent of malaria Plasmodium gallinaceum Brumpt]. / Vliianie antigormonov na vospriimchivost' kpmarov Anopheles sacahrovi Favre. k vozbuditeliu maliarii Plasmodium gallinaceum Brumpt.

A laboratory model of circulation of the malaria causative agent P. gallinaceum has been used to show that the effect of precocene (antijuvenoid) leads to a statistically significant reduction in the proportion of infected females developing eggs after blood suction. The females failing to develop eggs are not infected. Trichopol (antiexdisone) inhibits vitellogenesis The females undeveloping eggs become susceptible to the causative agent though to a lesser degree than those developing them. The findings suggest that there is an association of the mosquito susceptibility to the malaria causative agent with the balance of hormones in the body of disease the carrier.

Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti.

Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F2 populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs[2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filarial nematode Brugia malayi and the yellow fever virus.

[The effect of iuvemon on oogenesis in female Anopheles sacharovi Favre mosquitoes and their infection with the malarial causative agent Plasmodium gallinaceum Brumpt]. / Vliianie iuvemona na oogenez samok komarov Anopheles saccharovi Favre i ikh zarazhaemost' vozbuditelem maliarii Plasmodium gallinaceum Brumpt.

The paper provides evidence that the An. sacharovi females which do not develop mature eggs after blood-sucking on the malaria-infected donor could not be infected by the bird malaria agent P. gallinaceum. The addition of juvemon (an analogue of juvenile hormone) to glucose solution (mosquito carbohydrate diet) before blood meal stimulates the vitellogenesis of the mosquito after blood digestion, as clearly demonstrated on the female with an incomplete portion of the infected blood. This study has demonstrated that the juvemon does not exert a direct effect on mosquito susceptibility to the bird malaria agent, but it increases the number of females with mature eggs, thus promoting the increase in the percentage of the infected specimens and the number of oocysts.

[The susceptibility of mosquitoes for Plasmodium gallinaceum in the joint use of biologically active substances]. / Vospriimchivost' komarov k maliariinym plazmodiiam pri sochetannom primenenii biologicheski aktivnykh veshchestv.

Making use of a model pair Aedes aegypti--Plasmodium gallinaceum, the authors assess the susceptibility of mosquito female survivors to malaria agent after treatment of larvae with various bioactive substances. Eight binary combinations of 6 preparations have been tried: dimilin and uvemon, insect development regulators; fundosol and copper sulfate, fungicides; phytobacteriomicin (PBM), a larvicidal antibiotic; bactoculicide, a bacterial agent. Combinations of PBM with compounds differing by their mechanisms of action were found to inhibit the specific effect of PBM on the vector, PBM specific effect consisting in depression of mosquito susceptibility to P. gallinaceum. PBM combinations with some agents may alter other parameters of the vector potential: combinations of copper sulfate or uvemon with low concentrations of PBM potentiated the larvicidal effect, and PBM mixtures with fungicides reduces the activity of female attacks.

Developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host.

Sporozoites are an invasive stage of the malaria parasite in both the mosquito vector and the vertebrate host. We developed an in vivo assay for mosquito salivary gland invasion by preparing Plasmodium gallinaceum sporozoites from infected Aedes aegypti mosquitoes under physiological conditions and inoculating them into uninfected female Ae. aegypti. Sporozoites from mature oocysts were isolated from mosquito abdomens 10 or 11 d after an infective blood meal. Salivary gland sporozoites were isolated 13 or 14 d after an infective blood meal. Purified oocyst sporozoites that were inoculated into uninfected female mosquitoes invaded their salivary glands. Using the same assay system, sporozoites derived from salivary glands did not reinvade the salivary glands after inoculation. Conversely, as few as 10 to 50 salivary gland sporozoites induced infection in chickens, while only 2 of 10 chickens inoculated with 5,000 oocyst sporozoites were infected. Both sporozoite populations were found to express a circumsporozoite protein on the sporozoite surface as determined by immunofluorescence assay and circumsporozoite precipitation test using a circumsporozoite protein-specific monoclonal antibody. We conclude that molecules other than this circumsporozoite protein may be responsible for the differential invasion of mosquito salivary glands or infection of the vertebrate host.

[The relation between changes in the intestinal microflora of mosquito larvae under the action of phytobacteriomycin and female susceptibility to the causative agent of malaria]. / Sviaz' mezhdu izmeneniiami mikroflory kishechnika lichinok komarov pod deistviem fitobakteriomitsina i vospriimchivost' samok k vozbuditeliu maliarii.

A relationship was found between Pseudomonas suppression by phytobacteriomycin (PBM) in the mosquito larval gut and the mosquito vectorial capacity. The suppression of Pseudomonas bacteria in larvae caused a decrease in the vectorial capacity of emerged imagines. The results of the in vivo tests were verified in vitro with the culture of bacteria isolated from mosquito larva in liquid medium with pH 8.4 which is equal to intestinal pH of Ae. aegypti larva inside the peritrophic membrane. The laboratory Ae. aegypti fed on sugar with PBM addition (titer 100 U/ml) were 18.9% lees sensitive to malaria parasite. Without additional blood meal, mosquito sensitivity to malaria parasite sharply decreases at day 14, without substantially changing their intestinal microflora.

[New models of the circulation of the causative agent of malaria Plasmodium gallinaceum using malarial mosquitoes in the fauna of the USSR]. / Novye modeli tsirkuliatsii vozbuditelia maliarii Plasmodium gallinaceum s ispol'zovaniem maliariinykh komarov fauny SSSR.

The capacity of An. sacharovi and An. pulcherrimus to be infected with P. gallinaceum and to transmit the agent to the vertebrate host, a chick, has been established. Sufficient differences have been found in the extensiveness and intensity of infection of different species of mosquitoes and in other characteristics reflecting the agent-vector relationships.

[The characteristics of Aedes togoi susceptibility to Plasmodium gallinaceum]. / Osobennosti vospriimchivosti Aedes togoi k Plasmodium gallinaceum.

A high level of mutual adaptation has been established between Ae. togoi and P. gallinaceum strains. The level of sensitivity of Ae. togoi to the above agent is higher than that of Ae. aegypti in the proportion of the individuals infected and the number of agents in them. No correlation in the infectivity of the species compared has been observed.

[Further comment on the role of sugars for the successful infection of blood-sucking Diptera by protozoa that are the causative agents of human diseases]. / Eshche raz o roli sakharov dlia uspeshnogo zarazheniia krovososushchikh dvukhkrylykh prosteishimi-vozbuditeliami boleznei cheloveka.

Possible reasons of a successful development of Plasmodium gallinaceum in Aedes aegypti, which were not given sugar feeding, are analysed. Such reasons are assumed to be high contents of sugars (up to 200 mg/% in the blood of chickens-donors) and retention of sugar in the crop of mosquitoes, which were fed from a tampon, in control.

[The effect of the conditions for the development of Aedes aegypti L. mosquitoes on their infectivity with Plasmodium gallinaceum Brumpt]. / Vliianie uslovii razvitiia komarov Aedes aegypti L. na ikh zarazhaemost' Plasmodium galinaceum Brumpt.

Relationship between the conditions of larvae development and mosquito infection with malaria agent was studied. Using the Plasmodium gallinaceum--Aedes aegypti L. model, it was stated that alteration in temperature, number of specimens, and quantity of forage do not change the index of specimen morbidity. Worsening conditions in respect of every one of the enumerated factors lead to a decrease in quantity of agents in carrier.