Influence of the blood meal source on the biology of Meccus picturatus Usinger 1939 (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Aspects related to hatching, time-lapse between presenting the blood meal and beginning of feeding, feeding time, postfeed defecation delay,life time, mortality and fecundity for each stage of Meccus picturatus, life-cycle were evaluated and compared in two cohorts of M. picturatus fed on hens or rabbits. The hatching rate observed for each of the two studied groups of eggs was 78.1% (n = 2298) on the group fed on hens and 82.1% (n = 2704) on that fed on rabbits, and the average time of hatching was 20 days. Mean time-lapse for beginning feeding was under 3 min in nymphal stages and postfeed defecation delay was under 10 min in all stages, in both cohorts. Mean feeding time was significantly (P < 0.05) shorter in triatomines fed on hens than on rabbits. A similar number of nymphs of each cohort, 69 fed on hens (34.5%) and 68 fed on rabbits (34%), completed the cycle. No significantly (P > 0.05) differences were recorded among the average times from NI to adult in the cohort fed on hens (196.8 ± 15.8 days) and the average time in the cohort fed on rabbits (189.5 ± 22.9). The average span in days for each stage fed on hens was not significantly different to the average span for each stage fed on rabbits. The number of blood meals at each nymphal stage varied from 1 to 6 in both cohorts. The mortality rates were higher on fifth nymphal stage, in both cohorts. No significant (P > 0.05) differences were recorded on mortality rates on most nymphal stages of both cohorts. The average number of eggs laid per female from the cohort fed on hens in a 9-month period was 791.1, whereas the average number of eggs in the cohort fed on rabbits was 928.3.

Prevalence of infection with hantavirus in rodent populations of central Argentina

We studied hantavirus seroprevalence and virus variability in rodent populations in Diego Gaynor, northwest of Buenos Aires province, Argentina. Rodent samplings were conducted in railroads and cropfield borders in March and July 1999, September and December 2000, and March 2001. Antibody detection was performed by an enzyme link immunosorbent assay (ELISA), using the recombinant nucleoprotein of Andes (AND) virus as antigen. Tissue samples were taken from positive antibody individuals in order to confirm the presence of hantavirus genomic material and to identify virus genotypes. Akodon azarae was the most abundant species, followed by Oligoryzomys flavescens, while Calomys laucha and C. musculinus were rarely caught. We found a rate of seroprevalence of 9.3% for a total sample of 291 A. azarae and 13.5% for 37 O. flavescens. After molecular analyses of hantavirus, we confirmed the presence of hantavirus genomic material in 16 individuals with ELISA (+) results and two individuals with ELISA (-). Four amplimers for each species were sequenced and compared to the corresponding sequences of representative hantaviruses. We identified the AND Cent Lec from three O. flavescens, and the Pergamino virus from four A. azarae and from one O. flavescens. A. azarae males had higher seroprevalence than females, and heavier individuals showed higher seroprevalence than lighter ones. We did not find seroprevalence differences according to sex in O. flavescens, although this result may have been produced by the low sample size. The lowest seroprevalence was found in a period of high rodent density, when juveniles prevailed in the population. We found higher seroprevalences than those detected in previous studies for other localities of central Argentina where cases of hantavirus pulmonary syndrome (HPS) have been reported. The presence of AND Cent Lec virus in rodent populations of the study area, which is responsible of HPS cases in central Argentina, suggests that human populations are at risk of HPS disease, although there were not reported cases of this disease until today.

Feeding, defecation, and development times of Meccus longipennis Usinger, 1939 (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Aspects related to hatching, time-lapse between presenting the blood-meal and beginning of feeding, feeding time, postfeed defecation delay, mortality, and fecundity for each stage of Meccus longipennis life-cycle were evaluated. The bugs were maintained in a dark incubator at 27 ± 1ºC and 80 ± 5% rh, were fed weekly and checked daily for ecdysis or death. The hatching rate observed for 300 eggs was 76.7% and the average time of hatching was 19.8 days. Mean time-lapse between presentation of the blood meal and the beginning of feeding was under 5 min in nymphal stages and postfeed defecation delay was under 10 min in most stages, except in fourth and fifth stages. Mean feeding time was longer than 10 min in most stages, except in fourth stage. One hundred thirty-one nymphs (N) (65.5%) completed the cycle and the average time from NI to adult was 192.6 ± 34.8 days. The average span in days for each stage was 18.1 for NI, 21.4 for NII, 29.5 for NIII, 45.5 for NIV and 55.9 for NV. The number of bloodmeals at each nymphal stage varied from 1 to 5. The mortality rate was 3.29 for NI, 6.8 for NII, 2.92 for NIII 3.76 for NIV, and 10.16 for NV nymphs. The average number of eggs laid per female in a 9-month period was 615.6. Based on our results, we conclude that M. longipennis has some biological and behavioral characteristics which influence its capacity of becoming infected and transmitting Trypanosoma cruzi to human populations in those areas of Mexico where it is currently present.

Evaluation of four methods for detecting the beta-lactamase activity in Neisseria gonorrhoeae isolated in Cuba

Four methods (chromogenic, acidimetric, inhibition, and iodometric) for demonstration of the beta-lactamase production by 70 isolates of Neisseria gonorrhoeae, were evaluated in Cuba. There was 100% correlation between all beta-lactamase methods and the standardized penicillin dilution susceptibility test for penicillinase-non-producing N. gonorrhoeae. For penicillinase-producing N. gonorrhoeae strains, there was a perfect correlation between the chromogenic method and penicillin susceptibility testing, but one and two strains failed to give a positive result for beta-lactamase with the inhibition/acidimetric and the iodometric methods, respectively. There was a high concordance between the chromogenic method, considered as gold standard and the rest of penicillinase tests evaluated: Kappa Index (KI) = 0.98 for inhibition/acidimetric methods and KI = 0.97 for the iodometric method. The four methods evaluated were accurate, reproducible, easily readable, economical, and ease to use for screening primary isolates of N. gonorrhoeae in Cuba. We recommended the use of the inhibition method, when testing the penicillinase activity in gonococcal isolates in provincial and municipal reference laboratories.

Malaria during pregnancy in a reference centre from the Brazilian Amazon: unexpected increase in the frequency of Plasmodium falciparum infections

Malaria remains globally the most important parasitic disease of man. Data on its deleterious effects during pregnancy have been extensively documented in hyperendemic, holoendemic, and mesoendemic areas from Africa and Asia where Plasmodium falciparum is responsible for almost all infections. However, knowledge about malaria during pregnancy in areas where transmission is unstable and P. vivax is the most prevalent species, such as the Brazilian Amazon, is scarce. Here, we report a preliminary cross sectional descriptive study, carried out at the Fundação de Medicina Tropical do Amazonas, a reference centre for diagnosis and treatment of tropical diseases in the west-Amazon (Manaus, Brazil). A total of 1699 febrile childbearing age women had positive thick blood smears to Plasmodium species, between January and November 1997: 1401 (82.5%) were positive for P. vivax , 286 (16.8%) for P. falciparum and 12 (0.07%) carried mixed infections. From the malarious patients, 195 were pregnant. The ratio of P. falciparum to P. vivax infections in the group of non-pregnant infected women was 1:5.6 while it was 1:2.3 in that of pregnant infected ones. Similar rates or even proportionally more vivax infections during pregnancy were expected to occur, in function of the contraindication of primaquine with the resulting increased P. vivax relapse rates. Such an observation suggests that the mechanism of resistance/susceptibility to infection and/or malaria pathogenesis in pregnant women may differ according to Plasmodium species and that the extensively described increase in the frequencies of malaria infection during pregnancy may be specifically due to P. falciparum infection.

Abnormal expression of CD54 in mixed reactions of mononuclear cells from hyper-IgE syndrome patients

Hyper-IgE syndrome (HIES) is a rare multisystem disorder characterized by increased susceptibility to infections associated with heterogeneous immunologic and non-immunologic abnormalities. Most patients consistently exhibit defective antigen-induced-T cell activation, that could be partly due to altered costimulation involving accessory molecules; however, the expression of these molecules has never been documented in HIES. Therefore, we investigated the expression of CD11a, CD28, CD40, CD54, CD80, CD86, and CD154 in peripheral blood mononuclear cells from six patients and six healthy controls by flow cytometry after autologous and mixed allogeneic reactions. Only the allogeneic stimuli induced significant proliferative responses and interleukin 2 and interferon gamma production in both groups. Most accessory molecules showed similar expression between patients and controls with the exception of CD54, being expressed at lower levels in HIES patients regardless of the type of stimulus used. Decreased expression of CD54 could partly explain the deficient T cell activation to specific recall antigens in HIES patients, and might be responsible for their higher susceptibility to infections with defined types of microorganisms.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Ivermectin resistant and susceptible third-stage larvae of Haemonchus contortus: cholinesterase and phosphatase activities

Cholinesterase and acid phosphatase (AP), but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.

Naturally acquired immunity to Haemophilus influenzae type B in healthy Cuban children

We have evaluated the prevalence of antibody to immunogenicity of Haemophilus influenzae type b (Hib) in a group of 4 to 5 years old healthy children, who were too old to be included in the first vaccinated cohort when Hib vaccination begun in Cuba in 1999. Serum capsular polysaccharide specific IgG antibody concentrations were measured in 974 healthy children, between February and May 2002. The prevalence of Hib nasopharyngeal carriage was also estimated. The majority of children (99.7%) had more than 1 µg/ml of antibody. The preliminary report of the nasopharyngeal cultures was positive for H. influenzae in 16 children, but in only one was confirmed as Hib after serotyping (0.1% Hib nasopharyngeal carrier). These results provide evidence that in Cuba the natural active immunity to Hib can be acquired at an early age.

Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis

The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12), sera from dogs infected with helminths other than E. granulosus (n = 30), and helminth-free dog sera (n = 20). These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag). ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.

Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

Hydatid cyst fluid (HCF), somatic antigens (S-Ag) and excretory-secretory products (ES-Ag) of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.

Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela

The mosquito Aedes aegypti is the main vector of dengue in Venezuela. The genetic structure of this vector was investigated in 24 samples collected from eight geographic regions separated by up to 1160 km. We examined the distribution of a 359-basepair region of the NADH dehydrogenase subunit 4 mitochondrial gene among 1144 Ae. aegypti from eight collections. This gene was amplified by the polymerase chain reaction and tested for variation using single strand conformation polymorphism analysis. Seven haplotypes were detected throughout Venezuela and these were sorted into two clades. Significant differentiation was detected among collections and these were genetically isolated by distance.

Biology of three species of the Meccus phyllosomus complex (Hemiptera: Reduviidae: Triatominae) fed on blood of hens and rabbits

Aspects related to hatching, life time, number of blood meals to molt, mortality, feeding time and postfeed defecation delay for each instar of Meccus phyllosomus, M. mazzottii, and M. bassolsae, life-cycle were evaluated and compared in two cohorts of each of those three species, fed on hens or rabbits. No significant (p > 0.05) differences were recorded among cohorts fed on hens respect to cohorts fed on rabbits in M. phyllosomus and M. mazzottii and the average time of hatching was 21.5 days for cohorts fed on hens and 22.5 for cohorts fed on rabbits. Average egg-to-adult development times were no significant (p > 0.05) different between both cohorts of M. phyllosomus and M. mazzotti, independent of the blood meal source. The average span in days for each instar fed on hens was not significantly different to the average span for each instar fed on rabbits, when comparisons were made by species. The number of blood meals at each nymphal instar varied from 1 to 6 in both cohorts of each species. The mortality rates were higher on older nymphs, in both cohorts of M. phyllosomus and M. bassolsae, whereas they were higher on first instar nymphs on M. mazzottii. Mean feeding time was no significant (p > 0.05) different in triatomines fed on hens or fed on rabbits, when each species were compared separately. A similar number of nymphs of each cohort, completed the cycle. Defecation delay was no significant (p > 0.05) different when cohorts fed on hens and fed on rabbits were compared by species. Most of the studied parameters showed no significant (p > 0.05) differences among those cohorts fed on hens and for fed on rabbits, which could mean a high degree of association of those species with birds as much as mammals, under wild conditions, increasing their capacity to colonize human dwellings.

Trypanosoma cruzi ribosomal protein S4: characterization of its coding locus, analysis of transcripts, and antigenicity of the protein

Two allelic genomic fragments containing ribosomal protein S4 encoding genes (rpS4) from Trypanosoma cruzi (CL-Brener strain) were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an rpS4 gene. In the other, only one rpS4 gene is found. Sequence comparison to the accessed data in the genome project database reveals that our two-copy allele corresponds to a variant haplotype. However, the deduced aminoacid sequence of all the gene copies is identical. The rpS4 transcripts processing sites were determined by comparison of genomic sequences with published cDNA data. The obtained sequence data demonstrates that rpS4 genes are expressed in epimastigotes, amastigotes, and trypomastigotes. A recombinant version of rpS4 was found to be an antigenic: it was recognized by 62.5% of the individuals with positive serology for T. cruzi and by 93.3% of patients with proven chronic chagasic disease.

Biology of three species of North American Triatominae (Hemiptera: Reduviidae: Triatominae) fed on rabbits

Aspects related to hatching, lifetime, number of blood meals for molting, mortality, feeding time, and postfeeding defecation delay were evaluated and compared in each instar of three North American Triatominae: Triatoma gerstaeckeri, Triatoma lecticularia and Triatoma protracta, all of them fed on rabbits. No significant differences (p > 0.05) were found among the three species regarding mean hatching rate, which was close to 20 days. Egg-to-adult development times were significantly shorter (p < 0.05) for T. lecticularia. Number of blood meals for molting to next instar ranged from one to five for T. protracta, and from one to six for T. gerstaeckeri and T. lecticularia. Mortality rates were higher in younger nymphs of T. lecticularia and T. protracta, while rates in T. gerstaeckeri were higher in fifth-instar nymphs. Mean feeding time was longest in T. gerstaeckeri, followed by T. lecticularia. More than twice the number of T. gerstaeckeri nymphs completed the development process, if compared to the nymphs from the other two species. Defecation delay was less than 10 min for T. lecticularia, T. protracta and the youngest nymphs of T. gerstaeckeri. Results point out that these three species may be important potential vectors of Trypanosoma cruzi for human populations, in areas of Mexico where these species are currently present.