Sequence analysis of the second internal transcribed spacer of ribosomal DNA in Anopheles oswaldoi (Diptera: Culicidae).

Sequence divergence in the second internal transcribed spacer (ITS2) of ribosomal DNA was examined for female specimens of Anopheles oswaldoi Peryassu from 7 localities in South America. The lengths of ITS2 for all mosquitoes ranged from 348 to 356 nucleotides. After alignment of these sequences, similarity ranged from 87 to 100%. Divergence was within the range of inter-specific differences for members of anopheline species complexes. Therefore, specimens were placed into 4 groups that may correspond to at least 4 cryptic species. One is probably related to An. oswaldoi sensu stricto and another to Anopheles konderi Galvão & Damasceno. The other 2 groups may correspond to species for which morphological identification remains to be clarified. These data provide evidence that An. oswaldoi comprise a complex of cryptic species and that DNA identification may help to resolve the taxonomic questions related to this group.

Influence of male gonadal hormones on the parasitemia and humoral response of male Calomys callosus infected with the Y strain of Trypanosoma cruzi.

Effects of orchiectomy on male Calomys callosus infected with the Y strain of Trypanosoma cruzi were studied. Male C. callosus of the same age and weight were divided into three groups: intact, sham operated, and castrated. After 1 month they were inoculated (i.p.) with 4000 blood trypomastigotes. Parasitemia was lower in orchiectomized animals than in the intact and sham groups. Hormone replacement with decanoate testosterone raised the parasitemia of castrated animals to levels similar to those of their intact and sham counterparts. Antibody levels were monitored by complement-mediated lysis. The trypomastigote lysis percentage varied through the course of infection, according to hormonal status and number of parasites during the acute phase. The most significant differences were found on the 30th day after infection, when lytic antibodies of intact males were high compared to the orchiectomized and sham groups. Higher resistance with lower lysis indexes were observed after orchiectomy, compared to intact and sham males.

Comparative susceptibility of two members of the Anopheles oswaldoi complex, An. oswaldoi and An. konderi, to infection by Plasmodium vivax.

We compared the susceptibility of Anopheles oswaldoi and An. konderi to infection by Plasmodium vivax based on the proportion of mosquitoes presenting oocysts and sporozoites. Anophelines were captured in the State of Acre and Rondônia, Brazilian Amazon, and used to obtain F1 progenies. After emergence of adults, male genitalia of mosquitoes of each family were dissected in order to identify them as either An. oswaldoi or An. konderi. F1 progenies of field-captured An. oswaldoi, An. konderi and An. darlingi (used as control) were fed simultaneously on P. vivax-infected blood. Mosquitoes were dissected on day 10-12 after feeding and examined for the presence of oocysts and sporozoites. Both An. oswaldoi and An. konderi developed oocysts in the midguts, however, the percentage of oocyst-positive mosquitoes for An. oswaldoi (13.8%) was higher than for An. konderi (3.3%), and only An. oswaldoi developed salivary infection with sporozoites (6.9% of positivity). Infection rates in An. darlingi ranged from 22.5% to 30.0% for both oocysts and sporozoites. These results indicate that An. oswaldoi can transmit P. vivax and suggest that it is more susceptible than An. konderi. Although An. oswaldoi is an exophilic and zoophilic species, it may be involved in malaria transmission as possibly occurred in the State of Acre.

Correlation between positive serology for Plasmodium vivax-like/Plasmodium simiovale malaria parasites in the human and anopheline populations in the State of Acre, Brazil.

Antibodies against the Plasmodium vivax-like/P. simiovale malaria parasite circumsporozoite repeat peptide (APGANQEGGAA)3 were determined by enzyme-linked immunosorbent assay (ELISA) in 120 sera randomly collected in 1994 from adults in 3 localities of the malaria endemic area in the State of Acre, Brazil; antibody was detected in 18 (15%). A 'sandwich' ELISA using monoclonal antibody (mab) Pam 172, directed against the same peptide, was carried out on 1207 Anopheles oswaldoi, 12 of which (1.0%) were positive, and 168 A. deaneorum, 2 of which (1.2%) were positive. This is the first report of serological detection of the P. vivax-like parasite in anophelines and the first report linking anopheline to human serology for this parasite in the same geographical area. It is an additional indication that A. oswaldoi is a malaria vector in Acre.

Prostaglandin and nitric oxide regulate TNF-alpha production during Trypanosoma cruzi infection.

The mechanisms that control TNF-alpha production by macrophages during Trypanosoma cruzi infection are still unknown. Destruction of intracellular forms by cytokine activated macrophages is considered to be a major mechanism of parasite elimination. Although in vitro TNF-alpha contributes to enhanced parasite destruction by macrophages, previous work in vivo has shown that as the parasite burden increases, serum TNF-alpha levels decline. In this report we show that TNF-alpha production by peritoneal adherent cells is elevated at the initial phase of T. cruzi infection. As infection progresses TNF-alpha production decreases. The observed reduction is partly due to inhibition, largely exerted by endogenous PG and secondarily by NO. Inhibition of their synthesis partially restored the ability to produce high levels of TNF-alpha to macrophages upon stimulation by LPS. Neither endogenous IL-10 nor TGF-beta seem to be involved in the negative regulation of TNF-alpha production.

Influence of female gonadal hormones on the parasitemia of female Calomys callosus infected with the "Y" strain of Trypanosoma cruzi.

Calomys callosus is a wild rodent found infected with Trypanosoma cruzi in nature. Groups of female C. callosus were subjected to ovariectomy or sham operation or served as intact controls. At 1 month after surgery, animals were inoculated intraperitoneally with 4000 blood trypomastigotes of the "Y" strain of T. cruzi. Parasitemia during the course of infection was significantly higher in ovariectomized animals as compared with sham-operated rodents and controls. On steroid hormone replacement the parasitemia of ovariectomized animals dropped to levels close to those of controls. High or low doses of progesterone, estrogen, or a combination of both exerted similar effects. Splenocyte proliferation of ovariectomized animals was unresponsive to stimuli with concanavalin A and lipopolysaccharide as compared with that of control and sham-operated groups. The results show that gonadal hormones play a fundamental role in the defense against T. cruzi infection. The influence of these procedures on the immune defense in experimental Chagas' disease is being further investigated.

Antibodies anti bloodstream and circumsporozoite antigens (Plasmodium vivax and Plasmodium malariae/P. brasilianum) in areas of very low malaria endemicity in Brazil.

During 1992-1994, 33 malaria cases were reported in two regions in Brazil where few sporadic atypical cases occur, most of them in home owners, who are weekenders, while home caretakers live there permanently. Indirect Fluorescent Antibody Test (IFAT), with Plasmodium vivax, and Enzime Linked Immunosorbent Assay (ELISA) with repeat peptides of the circumsporozoite (CS) proteins of the 3 known P. vivax variants and P. malarie/P. brasilianum, were performed on 277 sera, obtained within a 5 to 10 km range of malaria cases. Very rarely did any of these donors recall typical malaria episodes. Blood smears of all but 5 were negative. One of the 5 malaria cases included in our serology was of a home owner, I of a permanent resident, 3 from Superintendência de Controle de Endemias employees who went there to capture mosquitoes. In Region 1 the prevalence of IFAT positive sera was 73% and 28% among caretakers, 18% and 9.6% among home owners. In Region 2 (3 localities) no distinction was possible between caretakers and home owners, IFAT positivity being 38%, 28% and 7%. The relative percentage of positive anti-CS repeats ELISA, differed for each of the peptides among localities. Dwellings are in the vicinity of woods, where monkeys are frequently seen. The origin of these malaria cases, geographical differences and high seropositivity is discussed.

Seasonal variation of anti-Plasmodium falciparum antibodies directed against a repetitive peptide of gametocyte antigen pfs2400 in inhabitants in the State of Amapá, Brazil.

Antibodies to the Pfs2400 gametocyte antigen have been shown to inhibit the development of Plasmodium falciparum in anophelines and therefore this antigen is a candidate for a transmission-blocking vaccine. To test seasonal variation of these antibodies under field conditions, sera from 72 individuals were collected twice, first during the long-rains season with low malaria transmission and then, 6 months later, during the short-rains season, when transmission is high. This study was conducted in several localities in the State of Amapá, Brazil. All but three individuals had a positive indirect fluorescent antibody test (IFAT) with asexual forms of P. falciparum. Most of them did not report malaria attacks during the period between the first and second sampling. Their sera were tested by IFAT with P. falciparum gametocytes. The overall positivity of this test did not vary between seasons, and was 47.2 (34/72) and 48.6% (35/72), respectively. The sera were also tested by ELISA with the Pfs2400 repeat peptide. The positivity rate dropped from 29.2 (21/72) to 15.3% (11/72) and the mean absorbancies from 0.623 to 0.354, when we compared the results of the long-rains and short-rains seasons. Fifteen out of the 21 ELISA positive sera turned negative, with no change of geometric mean of titres (GMT) of asexual IFAT, while five negatives became ELISA positive on second sampling, with increase of GMT. Soon after the second sampling a malaria outbreak was reported in one of the localities. These results point toward a relatively short persistence of anti-Pfs2400 repeat peptide antibodies, under natural field conditions. A gametocyte antigen booster before a high transmission period might contribute towards lowering malaria incidence by eliciting a partially effective antibody response.

Nitric oxide involvement in experimental Trypanosoma cruzi infection in Calomys callosus and Swiss mice.

Nitric oxide (NO) production by peritoneal macrophages was evaluated in Calomys callosus and Swiss mice during the course of infection with two strains of Trypanosoma cruzi. In C. callosus, no NO production was detected throughout the period of observation in animals infected with either parasite strain, except for a very low amount measured on day 40 in animals infected with strain M226 and on the 28th day in animals infected with strain F after in vitro stimulation with interferon gamma (IFN-gamma). Macrophages of Swiss mice produced large amounts of NO, the highest values being observed on the 40th day in mice infected with the F strain. Induced nitrogen oxide synthase (iNOS) was not detected in macrophages of infected C. callosus but was detected in mice. The i.p. inoculation of thioglycolate, bacille Calmette-Guérin (BCG) and periodate, nonspecific macrophage activators, did not induce NO production in C. callosus, but high levels were observed in Swiss mice after secondary in vitro IFN-gamma plus lipopolysaccharide (LPS) stimulation. However, H2O2 release was induced in macrophages stimulated with phorbol myristate acetate (PMA) in both experimental models. Serum NOx(NO2 + NO3) levels were low in C. callosus infected with strain M226, which was originally isolated from this animal species. Strain-F-infected animals had higher serum NOx levels in the initial period of infection, which dropped to noninfected control values on the 40th day. In Swiss mice, both strains induced the production of higher levels of NOx throughout the period of observation, with the increase being more pronounced in mice infected with the F strain. Daily treatment of F-strain-infected C. callosus with the arginine analogue L-nitro-arginine drastically reduced NOx levels, with no influence on parasitemia or mortality being observed. The results obtained suggest that C. callosus shows a distinct behavior with regard to resistance to T. cruzi infection.

[Infection of Anopheles (Kerteszia) cruzii by Plasmodium vivax and Plasmodium vivax variant VK247 in the municipalities of São Vicente and Juquitiba, São Paulo]. / Infecção do Anopheles (Kerteszia) cruzii por Plasmodium vivax e Plasmodium vivax variante VK247 nos Municípios de São Vicente e Juquitiba, São Paulo.

Sporadic cases of autochthonous malaria have been recorded in São Paulo State, located in the Southeast region of Brazil. These cases are characterized by their benign course, low parasitemia, and mild symptomatology and have been identified as vivax malaria. Little is known about the symptoms and immune response elicited in humans by the variants Plasmodium vivax VK247 and P. vivax-like human malaria parasites. These variants are transmitted by Anopheles (Kerteszia) cruzii, one of the most common species of mosquitoes in the Southeast of Brazil. The objective of the study described in this paper was to investigate infection in anophelines using ELISA immunoenzymatic assay with specific monoclonal antibodies directed against the repetitive regions of the circumsporozoite protein in classic P. vivax, P. brasilianum/P. malariae, and P. vivax VK247. Between 1991 and 1993, mosquitoes were collected in São Vicente and Juquitiba, municipalites located in a remnant of the Brazilian Atlantic forest in São Paulo State, an ecosystem rich in plants of the Bromeliaceae family. These plants function as nurseries for immature forms of anophelines of the subgenus Kerteszia. Of 1,117 An. (Ker.) cruzii captured in São Vicente, 0.179% were positive for classic P. vivax. In Juquitiba, of 1,161 An. (Ker.) cruzii, 0.086% were positive for P. vivax VK247, confirming the presence of this variant in the region. Although the infection rate is low, the high density of these mosquitoes and their voracity (they exhibit 24-h biting activity) could compensate for the low percentage of infected specimens.

Reactivity of Trypanosoma cruzi strains with peanut agglutinin (PNA) correlates with number of in vitro infected host cells.

Reactivities of 4 lectins with intact trypomastigote forms derived from 8 different Trypanosoma cruzi strains were compared with their capacity to infect in vitro cultured LLC-MK(2)cells. A sensitive and reproducible titration method for lectin binding sites (ELLA: Enzyme Linked Lectin Assay) was employed, in which reactivities were scored through optical densities in an ELISA reader. Tissue culture trypomastigotes from the strains Y, CL, SC4, SC24, SC25, SC28, SC32 and SC33 were investigated for expression of different cell surface carbohydrate residues using Concanavalin A (ConA), Peanut agglutinin (PNA), Soybean agglutinin (SBA) and Wheat germ agglutinin (WGA) conjugated to peroxidase. The reactivity of the strains to PNA lectin was SC28 > SC32 > SC33 > SC25> SC24 > Y> CL> SC4. The optical density values obtained were highly correlated (r2=0.986, p< 10(-4)) with the number of parasitized LLC-MK(2) cells 24 hours after infection by trypomastigotes from each corresponding strain. We concluded that galactose and N-acetyl-D-galactosamine residues that are present on the surface of trypomastigotes are important in host-cell recognition.

In situ indirect fluorescent antibody: a new specific test to detect ongoing chagasic infections.

In situ immunofluorescence assay (ISIFA) was developed for the selection for treatment of chronically infected chagasic patients and their follow-up. Trypomastigotes flash fixed in situ on microscopic slides with 0.025% glutaraldehyde were used as antigen. ISIFA results were compared with complement-mediated lysis (CoML), membrane immunofluorescence (MbIFA), immunofluorescence assay (IFA) with epimastigotes, and xenodiagnosis (XENO). ISIFA was able to distinguish nontreated chagasic patients (geometric mean titer [GMT] = 180) and treatment failures (GMT = 160) from those considered successfully treated (GMT = 25). ISIFA revealed a high sensitivity and titers of 80 or higher detected 98.6% of patients with active infections, even in those with negative XENO, CoML, or MbIFA. Specificity evaluated in 63 sera from other infections, including leishmaniasis and autoimmune diseases, was 98%. IFA used in routine diagnostic procedures exhibited similar results in all groups, irrespective of therapy.

Detection of anti-Plasmodium falciparum antibodies directed against a repetitive peptide of the gametocyte antigen Pfs2400 in malaria patients in Brazil.

Sera collected from 164 individuals who had clinical Plasmodium falciparum malaria and came from several areas of Brazil where malaria is endemic were tested for the presence of anti-gametocyte antibodies. Antibodies directed against P. falciparum gametocytes were detected, by IFAT, in the sera of 67.1% of these patients. The prevalence of these antibodies was significantly higher in patients who had undergone multiple attacks of malaria than in those who were experiencing their first attack at the time of serum collection. Although circulating gametocytes were detected in 22% of the patients at this time, there was no difference in the percentages of IFAT positivity between apparent gametocyte 'carriers' and 'non-carriers'. All sera were also tested by ELISA, using a dimer of the nonamer peptide [PEE(L/V)VEEV(I/V)]2, which represents a tandem consensus repeat of the P. falciparum gametocyte antigen, Pfs2400, a target of transmission-blocking antibodies. ELISA demonstrated that 32.9% of the patients had antibodies that reacted with this peptide. Positive ELISA reactions were significantly more frequent amongst the sera of patients who had had multiple malaria attacks than in those undergoing their first malaria episode; positivity was lower in the gametocyte 'carriers' than in their 'non-carriers'. These results demonstrate that anti-gametocyte antibodies, which have already been shown to have potential transmission-blocking activity, are naturally elicited in Brazilian patients, the highest rates of seropositivity occurring after multiple malaria attacks.

Normal hematologic values of various age groups of Calomys callosus (Rodentia Cricetidae).

Hematologic values for Calomys callosus, a wild rodent, were established. Males and females from a colony raised in Animal Housing of the Institute of Tropical Medicine of São Paulo were examined individually. For the red blood cell series, the number of erythrocytes, hemoglobin concentration, hematocrit, and platelet and reticulocyte numbers were measured. All values except hemoglobin concentration were slightly higher in aged animals than in young animals. The white blood cell series had a predominance of lymphocytes, with no age-associated changes.

Interferon-gamma levels during the course of Trypanosoma cruzi infection of Calomys callosus (Rodentia-Cricetidae) and Swiss mice.

Serum levels of interferon-gamma (IFN-gamma) were evaluated in Calomys callosus and Swiss mice during the course of infection by four strains of Trypanosoma cruzi. All strains stimulated the production of this interleukine; however, the timing of its onset and permanence varied among strains and between the two animal models. When chronically infected animals with no detectable serum IFN-gamma were challenged with the homologous strain, they produced quantities comparable with those obtained during the acute phase of infection. In C. callosus there was a correlation between H2O2 liberation by peritoneal macrophages and serum IFN-gamma levels, whereas no such correlation was found in mice. C. callosus had a higher capacity to heal histopathological lesions, whereas lesions in mice were progressive. The results obtained suggest that C. callosus develops well-adapted immune mechanisms that may be important for its role as a reservoir of T. cruzi.

Gamma-IFN and macrophage respiratory burst in Calomys callosus challenged with Trypanosoma cruzi bloodstream and metacyclic forms.

Parasitemia levels of Calomys callosus inoculated with a high dose (HBT) of 4 x 10(3) Trypanosoma cruzi strain M226 bloodstream trypomastigotes (BT) exceeded those with the same inoculum of metacyclic trypomastigotes (MT) while a similar parasitemia was obtained with a low dose (LBT) of 5 x 10(2) of BT. Serum IFN-gamma levels during the acute phase of infection were higher in the LBT inoculated group when compared with the group inoculated with HBT, while the IFN-gamma levels in MT inoculated animals were close to uninfected controls. Spontaneous liberation of H2O2 of peritoneal macrophages explanted from animals on days 21 and 28 after infection was comparable to that of controls for HBT and LBT groups while that of the MT inoculated group was significantly higher. Phorbol Myristate Acetate (PMA) stimulation resulted in high H2O2 liberation specially in the infected groups. In vitro challenge with BT suppressed the small amount of spontaneous H2O2 release, while MT challenge stimulated this release to a limited degree in infected groups. In this animal model, interacting with a parasite strain isolated from the same host, macrophage activation as measured by H2O2 release was low, while the same strain had been previously observed to result in hyperactivation of mouse macrophages. We suggest that this distinctive behavior may be due to a host-parasite adaptation.

Sera from chronic Chagasic patients and rodents infected with Trypanosoma cruzi inhibit trans-sialidase by recognizing its amino-terminal and catalytic domain.

We investigated whether sera from chronic Chagasic patients and animals infected with Trypanosoma cruzi inhibit the removal of sialic acid from human erythrocytes and the transfer of sialic acid from sialyllactose to [14C]lactose in the reactions catalyzed by the parasite trans-sialidase. Sera from Swiss mice and Calomys callosus animals infected with three different T. cruzi strains inhibit both reactions. Inhibition increases during the infection, reaching maximal levels when the parasitemia decreases. Among 44 sera of untreated chronic Chagasic patients, 40 inhibit both reactions. Inhibition is observed with total, defatted sera or with purified immunoglobulins. Whereas most of the inhibitory antibodies from Chagasic patients react with the papain fragment of trans-sialidase in immunoblots, a few patients have noninhibitory antibodies that react only with the entire trans-sialidase. These findings may be relevant for the pathology of Chagas' disease.