RESUMEN
Eimeria zuernii and E. bovis are host-specific apicomplexan parasites of cattle causing haemorrhagic typhlocolitis in young animals worldwide. During first merogony, both Eimeria species form giant macromeronts (>300⯵m) in host endothelial cells containing >120,000 merozoites I in vivo. During the massive intracellular replication of macromeronts, large amounts of cholesterol and fatty acids are indispensable for enormous merozoite I-derived membrane production. From a metabolic perspective, host endothelial cells might be of advantage to the parasite, as transcription of several genes involved in both, cholesterol de novo biosynthesis and low density lipoprotein (LDL)-mediated uptake, are up-regulated in Eimeria macromeront-carrying host endothelial cells. In order to analyse further influence of E. zuernii/E. bovis infections on the metabolism of cholesterol, fatty acids, and glycolysis of the host endothelial cells, suitable in vitro cell culture systems are necessary. So far, in vitro cell culture systems based on primary bovine umbilical vein endothelial cells (BUVEC) are available for E. bovis-macromeront I formation, but have not been evaluated for E. zuernii. A novel E. zuernii (strain A), initially isolated from naturally infected calves in Antioquia, Colombia, was used for sporozoite isolation. Primary BUVEC monolayers were concomitantly infected with E. zuernii- and E. bovis-sporozoites, resulting in large sized macromeronts whose morphological/morphometric characteristics were compared. BUVEC carrying E. zuernii-macromeronts resulted in the release of viable and highly motile merozoites I. Overall, E. zuernii-merozoites I differed morphologically from those of E. bovis. The new E. zuernii (strain A) will allow detailed in vitro investigations not only on the modulation of cellular cholesterol processing (i. e. cholesterol-25-hydroxylase and sterol O-acyltransferase) but also on the surface expression of LDL receptors during macromeront formation.
Asunto(s)
Enfermedades de los Bovinos/parasitología , Coccidiosis/veterinaria , Eimeria/crecimiento & desarrollo , Células Endoteliales/parasitología , Técnicas In Vitro/métodos , Animales , Bovinos , Coccidiosis/parasitología , Eimeria/citología , Eimeria/fisiología , Merozoítos/citología , Merozoítos/crecimiento & desarrollo , Merozoítos/fisiología , Esporozoítos/citología , Esporozoítos/crecimiento & desarrollo , Esporozoítos/fisiologíaRESUMEN
In vitro culture of Plasmodium vivax liver stages underlies key understandings of the fundamental biology of this parasite, particularly the latent, hyponozoite stage, toward drug and vaccine development. Here, we report systematic production of Plasmodium vivax sporozoites in colonized Anopheles darlingi mosquitoes in the Peruvian Amazon. Human subject-derived P. vivax-infected blood was fed to Anopheles darlingi females using standard membrane feedings assays. Optimizing A. darlingi infection and sporozoite production included replacement of infected patient donor serum with naïve donor serum, comparing anticoagulants in processing blood samples, and addition of penicillin-streptomycin and ATP to infectious blood meals. Replacement of donor serum by naïve serum in the P. vivax donor blood increased oocysts in the mosquito midgut, and heparin, as anticoagulant, was associated with the highest sporozoite yields. Maintaining blood-fed mosquitoes on penicillin-streptomycin in sugar significantly extended mosquito survival which enabled greater sporozoite yield. In this study, we have shown that a robust P. vivax sporozoite production is feasible in a malaria-endemic setting where infected subjects and a stable A. darlingi colony are brought together, with optimized laboratory conditions.
Asunto(s)
Anopheles/parasitología , Parasitología/métodos , Plasmodium vivax/crecimiento & desarrollo , Esporozoítos/crecimiento & desarrollo , Animales , Femenino , Tracto Gastrointestinal/parasitología , Perú , Plasmodium vivax/aislamiento & purificación , Esporozoítos/aislamiento & purificaciónRESUMEN
Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.
Asunto(s)
Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Plasmodium yoelii/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Eritrocitos/parasitología , Femenino , Humanos , Inmunización , Malaria/inmunología , Malaria/mortalidad , Malaria/parasitología , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/química , Vacunas contra la Malaria/genética , Masculino , Merozoítos/química , Merozoítos/crecimiento & desarrollo , Merozoítos/inmunología , Ratones , Ratones Endogámicos C57BL , Plasmodium yoelii/química , Plasmodium yoelii/genética , Plasmodium yoelii/crecimiento & desarrollo , Estructura Terciaria de Proteína , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Esporozoítos/química , Esporozoítos/crecimiento & desarrollo , Esporozoítos/inmunologíaRESUMEN
Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites.
Asunto(s)
Aedes/parasitología , Plasmodium gallinaceum/crecimiento & desarrollo , Aedes/genética , Aedes/inmunología , Aedes/fisiología , Animales , Pollos/parasitología , Modelos Animales de Enfermedad , Ecdisteroides/sangre , Femenino , Fertilidad , Regulación de la Expresión Génica , Aptitud Genética , Hemolinfa/parasitología , Hemolinfa/fisiología , Interacciones Huésped-Parásitos , Inmunidad Innata , Proteínas de Insectos/genética , Metabolismo de los Lípidos , Lipoproteínas/genética , Malaria Aviar/parasitología , Plasmodium gallinaceum/fisiología , Esporozoítos/crecimiento & desarrollo , Esporozoítos/fisiología , VitelogénesisRESUMEN
Most medicinal plants used against malaria in endemic areas aim to treat the acute symptoms of the disease such as high temperature fevers with periodicity and chills. In some endemic areas of the Brazilian Amazon region one medicinal plant seems to be an exception: Ampelozyziphus amazonicus, locally named "Indian beer" or "Saracura-mira", used to prevent the disease when taken daily as a cold suspension of powdered dried roots. In previous work we found no activity of the plant extracts against malaria blood parasites in experimentally infected animals (mice and chickens) or in cultures of Plasmodium falciparum. However, in infections induced by sporozoites, chickens treated with plant extracts were partially protected against Plasmodium gallinaceum and showed reduced numbers of exoerythrocytic forms in the brain. We now present stronger evidence that the ethanolic extract of "Indian beer" roots hampers in vitro and in vivo development of Plasmodium berghei sporozoites, a rodent malaria parasite. Some mice treated with high doses of the plant extract did not become infected after sporozoite inoculation, whereas others had a delayed prepatent period and lower parasitemia. Our data validates the use of "Indian beer" as a remedy for malaria prophylaxis in the Amazon, where the plant exists and the disease represents an important problem which is difficult to control. Studies aiming to identify the active compounds responsible for the herein described causal prophylactic activity are needed and may lead to a new antimalarial prophylactic.