RESUMEN
OBJECTIVES: To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with susceptibility to DDT and pyrethroid insecticides in this vector. METHODS: Female mosquitoes were collected in two villages, Miyobo and Ngonamanga, of mainland Equatorial Guinea. Insecticide susceptibility tests were performed following WHO procedures. Anopheles gambiae complex specimens were identified to species and molecular form by PCR. Genotyping of the kdr locus was performed by allele-specific PCR and direct sequencing in a subset of samples. RESULTS: Both M and S molecular forms of A. gambiae were found in Ngonamanga whereas only the S-form was identified in Miyobo. The two kdr mutations were detected in S-form samples of both villages, with a higher frequency of the kdr-e (Leu-1014-Ser) allele (Miyobo: 16%; Ngonamanga: 40%). The kdr-w (Leu-1014-Phe) mutation was also detected in 3% of the M-form. All individuals tested for pyrethroids were susceptible. A mortality rate of 86% was obtained for DDT. An overall kdr allele frequency (i.e. kdr-e + kdr-w) of 22% was detected in DDT resistant individuals, whereas susceptible individuals had a kdr frequency of 6%. CONCLUSION: The co-occurrence of both kdr mutations and reduced susceptibility to DDT found in A. gambiae highlights the importance of implementing efficient surveillance of insecticide resistance in Equatorial Guinea.
Asunto(s)
Anopheles/efectos de los fármacos , DDT/farmacología , Insectos Vectores/efectos de los fármacos , Insecticidas/farmacología , Piretrinas/farmacología , Animales , Anopheles/genética , Guinea Ecuatorial , Femenino , Resistencia a los Insecticidas/efectos de los fármacosRESUMEN
In Colombia, Plasmodium resistance to antimalarials such as chloroquine and antifolates is a serious problem. As a result, the national Colombian health authorities are monitoring the efficacy of alternative drugs and schemes. The study of genetic polymorphisms related with drug resistance is required in the region. In vitro responses to chloroquine, quinine, mefloquine, amodiaquine, desethylamodiaquine, artesunate and dihydroartesunate were carried out by HRP ELISA. SNP analysis in Pfcrt and Pfmdr1 genes was performed by PCR-RFLP in 77 samples from the North West region of Colombia. In vitro resistance to chloroquine was high (74%), followed by mefloquine (30%) and desethylamodiaquine (30%). A positive correlation between the IC(50) of paired drugs was also detected. The allele Pfmdr1 N86 (wild) was present in 100% of the samples and 1246Y (mutant) in 92%. However, their presence did not correlate with in vitro drug resistance. Presence of the mutations K76T and N75E in Pfcrt was confirmed in all samples. Analysis of 4 codons (72, 74, 75 and 76) in pfcrt confirmed the presence of the haplotypes CMET in 91% and SMET in 9% of the samples.
Asunto(s)
Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Proteínas de Transporte de Membrana/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Plasmodium falciparum/efectos de los fármacos , Polimorfismo de Nucleótido Simple , Proteínas Protozoarias/genética , Amodiaquina/análogos & derivados , Amodiaquina/farmacología , Animales , Cloroquina/farmacología , Colombia/epidemiología , Humanos , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Mefloquina/farmacología , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
In the major malaria vector Anopheles gambiae Giles, two point mutations at the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides. Simple allele-specific polymerase chain reaction (PCR) assays to detect these single-nucleotide polymorphisms are prone to lack of specificity and therefore alternative techniques have been proposed. However, these may not be easily implemented in many laboratories from malaria endemic regions. Here, we describe a primer-introduced restriction analysis (PIRA)-PCR method to detect kdr mutations in An. gambiae. This method unambiguously identified all six genotypes for the kdr locus in a sample of 113 field-collected mosquitoes for which kdr genotypes had been confirmed by DNA sequencing. Co-occurrence of both kdr alleles was found in sites from Equatorial Guinea and Gabon and the L1014F mutation was detected in M-form individuals from Angola. The PIRA-PCR proved to be a reliable, robust, and simpler alternative for the detection of kdr mutations in this malaria vector.
Asunto(s)
Anopheles/genética , Insecticidas , Piretrinas , Canales de Sodio/genética , Sustitución de Aminoácidos , Animales , Análisis Mutacional de ADN/métodos , Cartilla de ADN , Femenino , Resistencia a los Insecticidas/genética , Reacción en Cadena de la PolimerasaRESUMEN
Antibodies are known to play an important role in the control of malaria infection. However, they can modulate parasite development enhancing infection. The effect of anti-Plasmodium antibodies on the expression of circumsporozoite protein gene (csp) was investigated. Plasmodium falciparum 3D7 in vitro cultures were submitted to: i) anti- circumsporozoite protein monoclonal antibody (anti-CSP-mAb) [1microg/ml, 0.1microg/ml, 0.01microg/ml and 0.001microg/ml] and ii) purified IgG Fab fragment from a pool of malaria patients [1mg/ml and 1microg/ml]; and compared to control cultures. After 24h the number of ring infected erythrocytes was determined in order to calculate invasion efficacy. At 48h culture supernatant was collected, and the amount of circumsporozoite protein determined by ELISA, parasitaemia was calculated and cells were processed for RNA preparation. Expression of csp gene was quantified using Real time RT-PCR. There was an increase in parasite growth when treated with lower anti-CSP-mAb concentration, which was associated with lower csp expression, while 1mug/ml anti-CSP-mAb treatment presented a growth inhibitory effect accompanied by high csp expression.
RESUMEN
Chloroquine has been described to increase Plasmodium infectivity to the mosquito vector and is known to affect the vertebrate host immune response including during malarial infection. Although knowledge of the mosquito immune response has recently improved, nothing is known about the impact of chloroquine on mosquito immunity. In order to characterize the influence of chloroquine on the mosquito immune system, we have analyzed the effect of chloroquine on Anopheles gambiae (i) serine proteases and (ii) antimicrobial peptide gene expression, in uninfected and Plasmodium berghei infected mosquitoes, using real-time PCR. We have demonstrated for the first time that mosquitoes fed on chloroquine-treated mice showed a significant down regulation of some immune-related genes. This effect was independent of midgut bacterial burden. These results suggest that chloroquine might act on the Anopheles serine proteases cascade, interfering with signal transduction pathways and at a transcriptional activation level.
Asunto(s)
Anopheles/inmunología , Anopheles/parasitología , Antimaláricos/farmacología , Cloroquina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Plasmodium berghei/patogenicidad , Plasmodium/patogenicidad , Serina Endopeptidasas/genética , Animales , Secuencia de Bases , Cartilla de ADN , Sistema Digestivo/efectos de los fármacos , Sistema Digestivo/microbiología , Sistema Digestivo/parasitología , Combinación de Medicamentos , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Plasmodium/efectos de los fármacos , Plasmodium berghei/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Serina Endopeptidasas/efectos de los fármacos , Trimetoprim/farmacologíaRESUMEN
Passage of rat antibodies induced by Plasmodium falciparum circumsporozoite protein (anti-CS IgG) from the bloodmeal into the hemocoel of uninfected Anopheles stephensi mosquitoes was quantified using enzyme-linked immunosorbent assay (ELISA) techniques. Anti-CS IgG were present in hemolymph immediately upon cessation of mosquito feeding. Titers peaked at 3 hr post-ingestion then declined steadily, becoming negligible at 18 hr. Substantial titers were present in the bloodmeal at 24 hr post-ingestion. By 48 hr, anti-CS IgG in both mosquito hemolymph and bloodmeal were virtually absent. Estimated quantities of anti-CS IgG in the hemolymph at 3 hr post-ingestion were 905-958 ng/ml, representing approximately 0.5% of that present in the host serum. Rat IgG subclasses 1, 2a, and 2b passed into hemolymph more readily than did IgM and possibly IgG2c. Hemolymph volume of unengorged mosquitoes (0.53 microliters) increased after a bloodmeal (0.73 microliters at 3 hr post-ingestion), suggesting that anti-CS IgG may move into the hemocoel in an aqueous solution.
Asunto(s)
Anopheles/inmunología , Anticuerpos Antiprotozoarios/análisis , Hemolinfa/inmunología , Inmunoglobulinas/análisis , Plasmodium falciparum/inmunología , Proteínas Protozoarias , Animales , Antígenos de Protozoos/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Cinética , RatasRESUMEN
Pyrimethamine resistance in cultivated laboratory isolates of Plasmodium falciparum is linked to the dihydrofolate reductase mutation Asn-108, a mutation that acts by interrupting drug binding within the active site of the enzyme. To determine the prevalence of this mutation in endemic regions harboring pyrimethamine-resistant malaria, we used a mutation-specific polymerase chain reaction assay to survey P. falciparum strains from a wide section of the Brazilian Amazon. Mutations were identified directly from blood samples without intervening steps of in vitro cultivation. Of 42 samples collected from four states in Brazil, 38 (90%) contained the Asn-108 codon AAC that confers pyrimethamine resistance, four samples contained only the wild-type Ser-108 codon AGC, and none contained the Thr-108 codon ACC found in cycloguanil-resistant pyrimethamine-sensitive strains. These findings indicate that a very high incidence of the Asn-108 DHFR mutation is responsible for pyrimethamine resistance in the Amazon, and they are consistent with recent failure rates reported for Fansidar (pyrimethamine-sulfadoxine). We suggest that limited use of proguanil be evaluated as an alternative to pyrimethamine.
Asunto(s)
Plasmodium falciparum/efectos de los fármacos , Pirimetamina/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Animales , Secuencia de Bases , Brasil , Codón/química , ADN Protozoario/química , Resistencia a Medicamentos/genética , Eritrocitos/parasitología , Humanos , Datos de Secuencia Molecular , Mutación , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Reacción en Cadena de la PolimerasaRESUMEN
Five days after receiving a Plasmodium falciparum NF54 infectious blood meal, Anopheles stephensi mosquitoes were fed rat anti-P, falciparum sporozoite or rabbit anti-R32tet32 antibodies. Sporozoites isolated from salivary glands were tested by the inhibition of sporozoite invasion (ISI) assay using monoclonal antibody (Mab) 2A10 to P. falciparum circumsporozoite protein or sera from human volunteers immunized with P. falciparum R32tet32 or (NANP)3-TT vaccines. Whereas sporozoites from control mosquitoes were neutralized by Mab 2A10 and vaccine sera, only the Mab and not the vaccine sera neutralized sporozoites from immune-fed mosquitoes. The implications of these results in vaccine design and the impact on transmission are discussed.
Asunto(s)
Anopheles/parasitología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias , Animales , Anticuerpos Antiprotozoarios/administración & dosificación , Interacciones Huésped-Parásitos , Humanos , Sueros Inmunes , Insectos Vectores/parasitología , Malaria/inmunología , Pruebas de Neutralización , Plasmodium falciparum/crecimiento & desarrollo , Ratas , Vacunas Sintéticas/inmunologíaRESUMEN
A method based on the polymerase chain reaction (PCR) for highly sensitive detection and identification of human malaria parasites was applied to blood and mosquito samples obtained from a village in Guinea Bissau. The prevalence of parasites in the human population was shown to be greatly underestimated by microscopical examination. In particular, a high incidence of Plasmodium malariae and P. ovale parasites was revealed only by the PCR assay. Preliminary evidence was obtained to show that the distribution of P. malariae infections within the village was non-random. This was supported by analysis of the parasite species infecting the mosquito vector. The implication of these results for the design and interpretation of epidemiological surveys is discussed.
Asunto(s)
Anopheles/parasitología , Insectos Vectores/parasitología , Malaria/parasitología , Plasmodium/aislamiento & purificación , Animales , Secuencia de Bases , ADN Protozoario/química , Guinea Bissau/epidemiología , Humanos , Malaria/epidemiología , Malaria Falciparum/diagnóstico , Datos de Secuencia Molecular , Plasmodium falciparum/aislamiento & purificación , Plasmodium malariae/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , PrevalenciaRESUMEN
Sera from human volunteers immunized with either synthetic peptide (NANP)3-TT or recombinant protein R32tet32 Plasmodium falciparum CS vaccines were tested in the inhibition of sporozoite invasion (ISI) assays using human hepatoma (HepG2-A16) cells or primary human hepatocytes. Sera or purified immunoglobulin (Ig) from volunteers who were completely protected against P. falciparum sporozoite challenge had higher ISI activity than sera from non-protected volunteers, or the highest titre endemic serum. However, Ig from protected and non-protected volunteers did not block sporozoite invasion of human hepatocytes, suggesting that P. falciparum sporozoites invade hepatocytes by mechanisms which differ from those concerned with invasion of HepG2-A16 cells.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Malaria/prevención & control , Oligopéptidos/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Animales , Carcinoma Hepatocelular/inmunología , Células Cultivadas , Humanos , Inmunoglobulinas/inmunología , Hígado/inmunología , Células Tumorales Cultivadas/inmunologíaRESUMEN
Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.
Asunto(s)
Malaria Falciparum/genética , Análisis de Varianza , Animales , Antígenos de Protozoos/genética , Genotipo , Humanos , Proteína 1 de Superficie de Merozoito/genética , Plasmodium falciparum/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Protozoarias/genéticaRESUMEN
We compared the palpal ratio method with the polymerase chain reaction (PCR) to distinguish between Anopheles gambiae s.s. and A. melas. At the end of the rainy season of 1995, female mosquitoes were collected indoors in the Antula area of Bissau, Guinea Bissau. A subsample of 354 mosquitoes were identified first with PCR and then with the palpal ratio method (study A). Subsequently, another 195 mosquitoes were identified first with the palpal ratio method and then with PCR (study B). The highest percentage (100%, n = 16) of correctly identified A. melas was obtained at the palpal ratio cut-off point of 0.83. However, at this point 4.0% (14/347) and 11.3% (21/186) of the A. gambiae were erroneously identified as A. melas in study A and B, respectively. This suggests that the palpal ratio method is not sufficiently reliable to distinguish between A. gambiae and A. melas from the Bissau area.
Asunto(s)
Anopheles/clasificación , Entomología/métodos , Reacción en Cadena de la Polimerasa , Animales , Anopheles/anatomía & histología , Anopheles/genética , Femenino , Guinea BissauRESUMEN
Malaria parasite species circulating in immune inhabitants of the Democratic Republic of São Tomé e Principe (DRSTP) and those acquired by non-immune travellers returning from this country have been compared. Using sensitive detection and species identification by PCR, Plasmodium parasites were found in the blood of 16 of the 43 travellers, who reported during the first 8 months of 1995 to a clinical diagnosis laboratory in Lisbon. Plasmodium vivax was found in four (25%) and Plasmodium ovale in ten (63%) of these patients. The observed prevalence of these two species in infected patients of DRSTP during 1995/1996 was <2%.
Asunto(s)
Malaria Vivax/inmunología , Malaria Vivax/transmisión , Malaria/inmunología , Malaria/transmisión , Adolescente , Adulto , Anciano , Islas del Atlántico/epidemiología , Niño , Preescolar , Enfermedades Endémicas , Humanos , Inmunidad Activa , Lactante , Malaria/epidemiología , Malaria/parasitología , Malaria Vivax/epidemiología , Malaria Vivax/parasitología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Prevalencia , ViajeRESUMEN
A cross-sectional survey was carried out in 16 localities on the island of São Tomé and three on the island of Príncipe, at the end of the rainy season of 1997, to determine malaria prevalence and vector densities. Blood samples from 664 inhabitants of all ages were examined by optical microscopy (OM) and PCR. Mosquito collections were made by outdoor landing captures from 21:00-23:00 h. Great differences were found between OM and PCR readings. OM had a sensitivity of 66%, a specificity of 79% and failed to reveal any mixed-infections. Overall prevalence, determined by PCR, was higher in São Tomé (53%) than in Príncipe (35%). It was highest in children below 16 years-old. All four human Plasmodium species occurred in São Tomé but P. ovale was not detected in Príncipe. The human population was largely asymptomatic. Bednet users had lower prevalence than did non-users. The FOREST form of Anopheles gambiae s.s., identified by PCR and cytogenetics, was the only vector on the islands. The sporozoite rate in São Tomé, assessed by ELISA, was 0.5%. Parasite prevalence and vector densities were positively correlated in São Tomé, where malaria transmission must occur predominantly in the more populated coastal areas.
Asunto(s)
Anopheles/crecimiento & desarrollo , Insectos Vectores/crecimiento & desarrollo , Malaria Falciparum/epidemiología , Plasmodium falciparum/aislamiento & purificación , Adolescente , Animales , Anopheles/clasificación , Anticuerpos Monoclonales , Islas del Atlántico/epidemiología , Niño , Preescolar , Estudios Transversales , Cartilla de ADN/química , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Incidencia , Lactante , Insectos Vectores/clasificación , Malaria Falciparum/sangre , Masculino , Parasitemia/epidemiología , Plasmodium falciparum/genética , Prevalencia , ARN Ribosómico/química , Lluvia , Estudios Seroepidemiológicos , Encuestas y CuestionariosRESUMEN
The host source and human blood index (HBI) of an exophilic population of the "forest" cytoform of Anopheles gambiae Giles sensu stricto, from a peri-urban area of the island of São Tomé were assessed. Blood meals of 434 An. gambiae females from all-night indoor light-trap collections, 193 from indoor and 422 from outdoor resting collections, were determined by ELISA. Significant differences were found in the HBI estimates from insects collected indoors (0.93) and outdoors (0.27). Blood-fed insects collected resting outdoors provided the most representative sample for host determination. Dogs were the predominant hosts, followed by humans and pigs. Of all human feeds, it was estimated that 81.5% were taken inside houses. The low HBI of 0.27 for the An. gambiae population explains the low sporozoite rate and the meso-endemicity of malaria in the island.
Asunto(s)
Anopheles , Conducta Apetitiva , África Occidental , Animales , Islas del Atlántico , Perros , Femenino , Humanos , MasculinoRESUMEN
Attempts to infect the New World marmot Callithrix penicillata with Plasmodium falciparum were unsuccessful. Attempts were also made to infect red blood cells of C. penicillata and Saimiri sciureus with P. falciparum in vitro, and these too were unsuccessful due to a high rate of hemolysis produced by apparently adverse culture conditions. It is concluded that modifications to the existing culture conditions will need to be made before successful parasitemia can be induced in vitro in simian erythrocytes.
Asunto(s)
Callithrix/parasitología , Callitrichinae/parasitología , Eritrocitos/parasitología , Plasmodium falciparum/patogenicidad , Ácido 4-Aminobenzoico , Animales , Ciclofosfamida , Susceptibilidad a Enfermedades , Hemólisis , Técnicas In Vitro , EsplenectomíaRESUMEN
The role of monoamine oxidase (MAO) and cytochrome P450 (P450) in the oxidative deamination of primaquine by rat liver fractions was studied. Rat liver fractions including liver homogenate, mitochondria, microsomes and 100,000 g supematant fractions were prepared from a pool of rat livers and characterised using benzylamine as a probe for MAO activity and N,N-dimethylbenzamide as a probe for P450 N-dealkylation activity. Incubation of all fractions with primaquine yielded carboxyprimaquine as the only metabolite detectable by HPLC. The mitochondrial fraction, which contained MAO activity but not P450 activity, presented the highest Vmax/K(M) value for the formation of carboxyprimaquine (8.5 x 10(-6) dm3mg(-1)h(-1). A substantially lower Vmax/K(M) value (1.3 x 10(-6) dm3mg(-1)h(-1)) was obtained in the microsomal fraction, which contained P450 but not MAO activity. The liver homogenate fraction presented a similar value (1.8 x 10(-6) dm3mg(-1)h(-1), though it contained both enzyme systems. Incubations of all the fractions that presented MAO activity, in presence of the MAO inhibitor pargiline, resulted in a marked inhibition of primaquine oxidation. P450 inhibitor SKF 525-A effectively inhibited primaquine metabolism in the microsomal fraction but inhibition in the liver homogenate was less effective. The results are consistent with an important role for MAO in primaquine biotransformation, though clearly metabolism by P450 has a contribution role.
Asunto(s)
Antimaláricos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/enzimología , Monoaminooxidasa/metabolismo , Primaquina/metabolismo , Animales , Fraccionamiento Celular , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos del Citocromo P-450 , Combinación de Medicamentos , Medicamentos Herbarios Chinos/farmacología , Inhibidores Enzimáticos , Glycyrrhiza , Hígado/ultraestructura , Masculino , Microsomas Hepáticos/enzimología , Mitocondrias Hepáticas/enzimología , Inhibidores de la Monoaminooxidasa/farmacología , Paeonia , Pargilina/farmacología , Ratas , Ratas WistarRESUMEN
Highly polymorphic regions of the circumsporozoite protein (CSP) of Plasmodium falciparum are associated with cellular immune responses. One of these regions, the TH3R polymorfic region of the csp gene codes for known T-cell epitopes. The present study tested the use of SSCP to determine sequence variations of the TH3R regions of four clones of P. falciparum (3D7, HB3, Dd2 and K1) which are known to have different TH3R regions. Single-strand conformation polymorphism (SSCP) technique was performed on amplified products labelled with fluorescent primer (both strands) and electrophoresed in an automated sequencer. Various gel compositions and electrophoresis conditions were tested. Even if some electrophoretogram differences were observed between clones, they could not distinguish between the alleles.
Asunto(s)
Antígenos de Protozoos/genética , ADN Protozoario/genética , Epítopos/genética , Plasmodium falciparum/genética , Polimorfismo Conformacional Retorcido-Simple , Proteínas Protozoarias/genética , Alelos , Animales , Variación Antigénica/genética , Antígenos de Protozoos/inmunología , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Plasmodium falciparum/inmunología , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/inmunología , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
Basic knowledge of the sporogonic development of malarial parasites is crucial when evaluating the sporontocidal activity of antimalarial drugs or when determining why certain vectors are refractory to a particular parasite while others are competent vectors. We have developed a model which we have used to i) assess the sporogonic development of Plasmodium berghei ANKA in Anopheles stephensi and A. freeborni mosquitoes and ii) determine the effect of chloroquine on the sporogony of P. falciparum NF-54 in A. stephensi. Criteria used to assay sporogonic development include: i) number of oocysts present, ii) percentage of mosquitoes with oocysts, iii) time of release of sporozoites from the oocysts into the hemolymph, iv) time and degree of sporozoite invasion of salivary glands, and v) transmission (P. berghei) into vertebrate hosts. Parasite development in the mosquito is evaluated every other day, commencing on ca. day 7 post-feed (PF) and continuing until ca. day 22 PF. These detailed observations allow us to delineate the chronology of sporogonic development.
Asunto(s)
Anopheles/parasitología , Plasmodium berghei/crecimiento & desarrollo , Plasmodium falciparum/crecimiento & desarrollo , Animales , Cloroquina , Femenino , Hemolinfa/parasitología , Larva , Ratones , Plasmodium falciparum/efectos de los fármacos , Glándulas Salivales/parasitología , Especificidad de la EspecieRESUMEN
After reviewing the use of non-human primates of the Old and New Worlds for human malaria research, we concluded that another experimental animal which is easily available to use and possible to rear indoors is needed. Thus, we studied the susceptibility of the marmoset Callithrix penicillata to Plasmodium falciparum erythrocytic infections. The marmosets received various P. falciparum human isolates, directly from a patient and from continuous cultures. The Palo Alto strain, which has been adapted to the night monkey Aotus trivirgatus and further maintained in the squirrel monkey Saimiri sciureus was also used. In a total of 20 marmosets we performed 31 inoculations, with 10(5) to 10(9) parasites, intraperitoneally, intracardiacly or intravenously. Blood samples from each animal were examined daily up to day 90 post-inoculation. None of the intact marmosets developed patent infections. Four out of 19 C. penicillata, previously splenectomized, showed circulating parasites for up to five days after intravenous inoculation with the Palo Alto strain, becoming negative thereafter. Neither the addition to the simian diet of p-aminobenzoic acid, essential for the parasite metabolism, nor drug-immunosuppression, improved the marmoset susceptibility to P. falciparum.