Laboratory estimation of the effects of increasing temperatures on the duration of gonotrophic cycle of Anopheles albimanus (Diptera: Culicidae)

The increase of malaria transmission in the Pacific Coast of Colombia during the occurrence of El Niño warm event has been found not to be linked to increases in the density of the vector Anopheles albimanus, but to other temperature-sensitive variables such as longevity, duration of the gonotrophic cycle or the sporogonic period of Plasmodium. The present study estimated the effects of temperature on duration of the gonotrophic cycle and on maturation of the ovaries of An. albimanus. Blood fed adult mosquitoes were exposed to temperatures of 24, 27, and 30°C, held individually in oviposition cages and assessed at 12 h intervals. At 24, 27, and 30°C the mean development time of the oocytes was 91.2 h (95% C.I.: 86.5-96), 66.2 h (61.5-70.8), and 73.1 h (64-82.3), respectively. The mean duration of the gonotrophic cycle for these three temperatures was 88.4 h (81.88-94.9), 75 h (71.4-78.7), and 69.1 h (64.6-73.6) respectively. These findings indicate that both parameters in An. albimanus are reduced when temperatures rose from 24 to 30°C, in a nonlinear manner. According to these results the increase in malaria transmission during El Niño in Colombia could be associated with a shortening of the gonotrophic cycle in malaria vectors, which could enhance the frequency of man-vector contact, affecting the incidence of the disease.

Variability and genetic differentiation among Anopheles (Ano.) intermedius Chagas, 1908 and Anopheles (Ano.) mattogrossensis Lutz & Neiva, 1911 (Diptera: Culicidae) from the Brazilian Amazon

Anopheles (Anopheles) intermedius and Anopheles (Ano.) mattogrossensis are Brazilian anopheline species belonging to the scarcely studied Anopheles subgenus. Few studies have been done on the genetic differentiation of these species. Both species have been found infected by Plasmodium and are sympatric with other anopheline species from the Nyssorhynchus subgenus. Eighteen enzymatic loci were analyzed in larval specimens of An. intermedius and An. mattogrossensis aiming to estimate the variability and genetic differentiation between these species. An. mattogrossensis population showed higher genetic variability (P = 44.4 and Ho = 0.081 ± 0.031) than that of An. intermedius (P = 33.3 and Ho = 0.048 ± 0.021). Most analyzed loci showed genotypic frequencies according to Hardy-Weinberg equilibrium, except for LAP1 and LAP2 in An. intermedius, and EST1 and PGM loci in An. mattogrossensis. The genetic distance between these species (D = 0.683) was consistent with the inter-specific values reported for Anopheles subgenus. We verified that the polymorphism and heterozygosity percentile values found in both species and compared to those in the literature, showed no relation between the level of isozyme variability and geographical distribution. The low variability found in these two species is probably more related to the niche they occupy than to their geographic distribution.

The importance of Anopheles albitarsis E and An. darlingi in human malaria transmission in Boa Vista, state of Roraima, Brazil

In several districts of Boa Vista, state of Roraima, Brazil we found Anopheles (Nyssorhynchus) albitarsis E to be the primary vector of human malaria parasites, and during 2001-2002 it was significantly more abundant than An. darlingi (p < 0.001). Other species sampled were An. (Nys.) braziliensis, An. (Ano.) peryassui, An. (Nys.) nuneztovari, An. (Nys.) oswaldoi s.l., and An. (Nys.) triannulatus. As determined by the ELISA technique An. darlingi had a higher overall infection rate (2.1%) compared with An. albitarsis E (1.2%). However, a marginally higher proportion of An. albitarsis E was infected with Plasmodium vivax compared with An. darlingi, and the An. albitarsis E biting index was also much higher. These results suggest the importance of An. albitarsis E in malaria transmission in a savannah ecoregion of northern Amazonian Brazil, and reconfirm the importance of An. darlingi even if at lower abundance.

Eimeria lepidosirenis n.sp. (Apicomplexa:Eimeriidae) of the South American lungfish Lepidosiren paradoxa (Osteichthyes:Dipnoi) from Amazonian Brazil

The mature oocysts of Eimeria lepidosirenis n.sp. are described in faeces removed from the lower region of the intestine of a single specimen of the South American lungfish Lepidosiren paradoxa, from Belém, state of Pará, Amazonian Brazil. Oocysts with endogenous sporulation: spherical to slightly subspherical, 30.8 × 30.3 µm (28.1 × 25.9 -33.3 × 31.8), shape-index (ratio length/width) 1.0, n = 25. Oocyst wall a very thin, single layer approximately 0.74 µm thick, smooth, colourless, with no micropyle and rapidly breaking down to release the sporocysts. Oocyst residuum a bulky ovoid to spherical mass of approximately 20.0 × 15 µm, composed of fine granules and larger globules and enclosed by a very fine membrane: no polar bodies seen. Sporocysts 15.5 × 9.0 µm (14.5 × 8.0 16.0 × 9.0), shape index 1.7 (1.6-1.8), n = 30, ovoid, with one extremity rather pointed and with a very delicate Stieda body but no sub-Stieda body: sporocyst wall a single extremely thin layer with no valves. Sporocyst residuum a spherical to ovoid mass of approximately 5.0 × 4.0 µm, composed of fine granules and small globules and enclosed by a very fine membrane. Sporozoites strongly recurved at their ends and apparently with only a single refractile body. Site of development in the host uncertain: no evidence of endogenous stages was found in fresh scrapings and stained smears of the intestinal epithelium.

Three new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the lesser seed-finch, Oryzoborus angolensis (Passeriformes: Emberizidae) from Brazil

Three new coccidian (Apicomplexa: Eimeriidae) species are reported from the lesser seed-finch, Oryzoborus angolensis from Brazil. Sporulated oocysts of Isospora curio n. sp. are spherical to subspherical; 24.6 × 23.6 (22-26 × 22-25) mum, shape-index (SI, length/width) of 1.04 (1.00-1.15). Oocyst wall is bilayerd, ~ 1.5 mum thick, smooth and colourless. Micropyle and oocyst residuum are absent. The sporocysts are ovoid, 13.2 × 10.9 (15-17 × 10-13) mum, SI = 1.56 (1.42-1.71), with a small Stieda body and residuum composed of numerous granules scattered among the sporozoites. Sporozoites are elongated and posses a smooth surface and two distinct refractile bodies. Oocysts of Isospora braziliensis n. sp. are spherical to subspherical, 17.8 × 16.9 (16-19 × 16-18) mum, with a shape-index of 1.06 (1.00-1.12) and a smooth, single-layered wall ~ 1 mum thick. A micropyle, oocyst residuum and polar granules are absent. Sporocysts are ellipsoid and slightly asymmetric, 13.2 × 10.8 (12-14 × 9-12) mum, SI = 1.48 (1.34-1.61). Each sporocyst contains a barely visible Stieda body and a residuum composed numerous of granules. Sporozoites are elongated and each of them contains two distinct refractile bodies. Oocysts of Isospora paranaensis n. sp. are subspherical to broadly ellipsoid 24.3 × 19.8 (22-26 × 18-22) mum, SI = 1.22 (1.15-1.38) with smooth single-layered wall ~ 1.5 mum thick. A micropyle and oocyst residuum are absent, but one distinct ellipsoid polar granule (2.5-3.5 × 1.5-2.5 mum) is present. Sporocyst are ovoid, 15.7 × 10.1 (14-18 × 8-12) mum, SI = 1.46 (1.31-1.72), with distinct Stieda and sub-Stieda bodies. Each sporocyst contains a spherical sporocyst residuum, 4 mum in diameter. All described isosporan species represent a possible cause of acute coccidiosis for O. angolensis in captivity.

Effects of pyrimethamine-sulphadoxine, chloroquine plus chlorpheniramine, and amodiaquine plus pyrimethamine-sulphadoxine on gametocytes during and after treatment of acute, uncomplicated malaria in children

The effects of pyrimethamine-sulphadoxine (PS), chloroquine plus chlorpheniramine, a H1 receptor antagonist that reverses chloroquine resistance in Plasmodium falciparum in vitro and in vivo (CQCP), and amodiaquine plus pyrimethamine-sulphadoxine (AQPS) on gametocyte production were evaluated in 157 children with acute, symptomatic, uncomplicated falciparum malaria who were treated with these drugs. PS was significantly less effective than CQCP or AQPS at clearing asexual parasitaemia or other symptoms of malaria. Gametocyte carriage on days 3, 7, and 14 were significantly higher in those treated with PS. The ratio of the density (per µl blood) of peripheral young gametocyte (PYG), that is, < stage III to peripheral mature gametocyte (PMG), that is, stage IV and V, an index of continuing generation of gametocytes, rose to 1 by day 7 of treatment in those treated with PS, but remained consistently below 1 in the other treatment groups. PYG-PMG density ratio increased significantly from day 0-14 in those treated with PS and CQCP (chi2 = 76, P = 0.000001 and chi2 = 42.2, P = 0.00001, respectively) but decreased significantly in those treated with AQPS (chi2 = 53.2, P = 0.000001). Both PS-sensitive and -resistant infections generated PYG (18 of 29 vs 13 of 20, chi2 = 0.04, P = 0.93) but PYG was present only in those with resistant response to CQCP. Combination of PS with amodiaquine (AQ), that is, (AQPS) resulted in less production of PYG, but in this setting, PYG was not indicative of response to AQPS. These data indicate that PS enhanced production or release of young gametocytes when used alone, but generated less young gametocytes when used in combination with AQ. PYG may be used as an indicator of response to CQCP but not PS or PS-based combination drugs.

Immunization with synthetic peptides containing a defined malaria epitope induces a highly diverse cytotoxic T lymphocyte response. Evidence that two peptide residues are buried in the MHC molecule.

In the present study, we have explored ways of inducing a CTL response to a previously defined H-2Kd MHC class I restricted epitope in the circumsporozoite (CS) protein of Plasmodium berghei, and studied in detail the fine specificity of the response. We found that the s.c. injection of a variety of synthetic peptides emulsified in Freund's adjuvant efficiently induced a specific CTL response in (BALB/c x C57BL/6)F1 (H-2d x H-2b) mice. In contrast, BALB/c mice responded only marginally, consistent with the possible requirement for a concomitant Th response that would be provided by the C57BL/6 strain. Similar to our previous observations in analyzing CTL clones from sporozoite-immunized mice, the CTL response induced by peptide immunization was in part cross-reactive with an epitope from the Plasmodium yoelii species. The minimal P. berghei CS epitope, the octapeptide PbCS 253-260, was studied in detail by the analysis of a series of variant CS peptides containing single Ala substitutions. The relative antigenic activity for each variant peptide was calculated for 28 different CTL clones. Overall, the response to this P. berghei CTL epitope appeared to be extremely diverse in terms of fine specificity. This was evident among the CTL derived from sporozoite-immunized mice, as well as among those from peptide-immunized animals. The heterogeneity found at the functional level correlates with the highly diverse TCR repertoire that we have found for the same series of CTL clones in a study that is reported separately. The relative competitor activity for each Ala-substituted peptide was also determined in a quantitative functional competition assay. For the residues (Tyr253 and Ile260) within the 8-mer CS peptide, substitution with Ala reduced competitor activity by at least 40-fold, and for two others the reduction was 5- to 10-fold. When the relative antigenic activity for each CTL/peptide combination was normalized to the relative competitor activity of the peptide, a striking pattern emerged. The two residues that most affected competitor activity showed no additional effect on recognition beyond that observed for competition. In marked contrast, Ala substitutions at the other five positions tested varied widely, depending on the CTL/peptide combination. This pattern not only supports a model whereby the Tyr253 and Ile260 residues anchor the peptide to the Kd molecule, but also implies that they are virtually inaccessible to the TCR.

Urban malaria in the Brazilian Western Amazon Region I: high prevalence of asymptomatic carriers in an urban riverside district is associated with a high level of clinical malaria

Cross sectional studies on malaria prevalence was performed in 2001, 2002, and 2004 in Vila Candelária, an urban riverside area of Porto Velho, Rondônia, in the Brazilian Western Amazon, followed by longitudinal surveys on malaria incidence. Vila Candelária is a working class district, provided with electricity, water supply, and basic sanitation. Previous preliminary surveys indicated high malaria incidence in this community. At the end of year 2000 regular diagnostic and treatment measures for malaria were introduced, with active search of febrile cases among residents. Despite of both rapid treatment of cases and relative good sanitary and housing conditions, the malaria incidence persisted at high levels during the following years with an annual parasite index of 150 to 300/1000 inhabitants. Parasite surveys in 2001, 2002, and 2004 achieved through microscopy and polymerase chain reaction to diagnose malaria showed a constant high prevalence of asymptomatic carriers for both Plasmodium falciparum and P. vivax parasites. It was concluded that asymptomatic carriers represent an important reservoirs of parasites and that the carriers might contribute to maintaining the high level of transmission. Comparing our findings to similar geo-demographic situations found in other important urban communities of the Brazilian Amazon, we propose that asymptomatic carriers could explain malaria's outbreaks like the one recently observed in Manaus.

Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela

A longitudinal epidemiological and entomological study was carried out in Ocamo, Upper Orinoco River, between January 1994 and February 1995 to understand the dynamics of malaria transmission in this area. Malaria transmission occurs throughout the year with a peak in June at the beginning of the rainy season. The Annual Parasite Index was 1,279 per 1,000 populations at risk. Plasmodium falciparum infections accounted for 64% of all infections, P. vivax for 28%, and P. malariae for 4%. Mixed P. falciparum/P. vivax infections were diagnosed in 15 people representing 4% of total cases. Children under 10 years accounted for 58% of the cases; the risk for malaria in this age group was 77% higher than for those in the greater than 50 years age group. Anopheles darlingi was the predominant anopheline species landing on humans indoors with a biting peak between midnight and dawn. A significant positive correlation was found between malaria monthly incidence and mean number of An. darlingi caught. There was not a significant relationship between mean number of An. darlingi and rainfall or between incidence and rainfall. A total of 7295 anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite (CS) protein. Only An. darlingi (55) was positive for CS proteins of P. falciparum (0.42%), P. malariae (0.25%), and P. vivax-247 (0.1%). The overall estimated entomological inoculation rate was 129 positive bites/person/year. The present study was the first longitudinal entomological and epidemiological study conducted in this area and set up the basic ground for subsequent intervention with insecticide-treated nets.

Antimalarial drugs disrupt ion homeostasis in malarial parasites

Plasmodium chabaudi malaria parasite organelles are major elements for ion homeostasis and cellular signaling and also target for antimalarial drugs. By using confocal imaging of intraerythrocytic parasites we demonstrated that the dye acridine orange (AO) is accumulated into P. chabaudi subcellular compartments. The AO could be released from the parasite organelles by collapsing the pH gradient with the K+/H+ ionophore nigericin (20 µM), or by inhibiting the H+-pump with bafilomycin (4 µM). Similarly, in isolated parasites loaded with calcium indicator Fluo 3-AM, bafilomycin caused calcium mobilization of the acidic calcium pool that could also be release with nigericin. Interestingly after complete release of the acidic compartments, addition of thapsigargin at 10 µM was still effective in releasing parasite intracellular calcium stores in parasites at trophozoite stage. The addition of antimalarial drugs chloroquine and artemisinin resulted in AO release from acidic compartments and also affected maintenance of calcium in ER store by using different drug concentrations.