Capillaria spp. eggs in Patagonian archaeological sites: statistical analysis of morphometric data

Discriminant analysis was used to identify eggs of Capillaria spp. at specific level found in organic remains from an archaeological site in Patagonia, Argentina, dated of 6,540 ± 110 years before present. In order to distinguish eggshell morphology 149 eggs were measured and grouped into four arbitrary subsets. The analysis used on egg width and length discriminated them into different morphotypes (Wilks' lambda = 0.381, p < 0.05). The correlation analysis suggests that width was the most important variable to discriminate among the Capillaria spp. egg morphotypes (Pearson coefficient = 0.950, p < 0.05). The study of eggshell patterns, the relative frequency in the sample, and the morphometric data allowed us to correlate the four morphotypes with Capillaria species.

Long-term storage and viability of Ochlerotatus albifasciatus (Diptera: Culicidae)

The viability of Ochlerotatus albifasciatus (Macquart) eggs stored at room temperature and at 5ºC was studied over 31 months. After 12, 18 and 31 months of storage, eggs were acclimatized at 22ºC for ten days, and then inundated twice every seven days. The effect of the storage period on the percentage of hatching was analyzed by one way ANOVA. Differences on the hatching response between the first and second flooding were analyzed by paired t-test. Differences on the hatching response between the two storage conditions were analyzed by Mann-Whitney rank test. Results showed that (1) Oc. albifasciatus eggs were able to survive and hatch over 31 months; (2) the percent hatching of eggs stored at 5ºC was higher than that of eggs stored at room temperature; and (3) low temperatures and long periods without water favor installment hatching.

Impact of moisture on survival of Aedes aegypti eggs and ovicidal activity of Metarhizium anisopliae under laboratory conditions

The effect of relative humidity (43%, 75%, 86% and > 98%) on Aedes aegypti eggs treated with Metarhizium anisopliae or water only was tested for up to a six months exposure at 25ºC. Survival of larvae inside eggs was clearly affected by the lowest humidity (43%) tested, and eclosion diminished at all humidities after increasing periods of exposure. M. anisopliae showed to have a strong ovicidal activity only at humidity close to saturation. No difference of activity was found between conidia and hyphal bodies tested. This fungus affected larvae inside eggs and has potential as a control agent of this important vector in breeding sites with high moisture.

Cheap, rapid and efficient DNA extraction method to perform multilocus microsatellite genotyping on all Schistosoma mansoni stages

Schistosomes are endoparasites causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand the schistosomiasis epidemiology and disease transmission patterns. In this paper, we propose a novel assay for a rapid, low costly and efficient DNA extraction method of egg, larval and adult stages of Schistosoma mansoni. One euro makes possible to perform 60,000 DNA extraction reactions at top speed (only 15 min of incubation and 5 handling steps).

Cytokine profile associated with chronic and acute human schistosomiasis mansoni

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma (IFN-³) was evaluated in 43 schistosomiasis patients with different clinical forms. Whole-blood cultures cytokine production in response to soluble egg antigen (SEA), soluble worm adult preparation (SWAP), mitogens, neutralizing antibodies or recombinant IL-13 were measured by ELISA. After SWAP stimulation, chronic patients, particularly hepatointestinals, produced higher levels of IL-4 in comparison with acute patients, suggesting the presence of a type 2 cytokine profile in these patients. Following SEA and SWAP stimulation, hepatosplenic (HS) patients showed increased levels of IFN-³ when compared with acute patients, indicating that HS disease in humans is associated with a type 1 cytokine response. The mechanisms of immune regulation are apparently different between the clinical stages of the disease, some of which are antigen-specific.

Life cycle, feeding and defecation patterns of Rhodnius ecuadoriensis (Lent & León 1958) (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Rhodnius ecuadoriensis is the second most important vector of Chagas Disease (CD) in Ecuador. The objective of this study was to describe (and compare) the life cycle, the feeding and defecation patterns under laboratory conditions of two populations of this specie [from the provinces of Manabí (Coastal region) and Loja (Andean region)]. Egg-to-adult (n = 57) development took an average of 189.9 ± 20 (Manabí) and 181.3 ± 6.4 days (Loja). Mortality rates were high among Lojan nymphs. Pre-feeding time (from contact with host to feeding initiation) ranged from 4 min 42 s [nymph I (NI)] to 8 min 30 s (male); feeding time ranged from 14 min 45 s (NI)-28 min 25 s (male) (Manabí) and from 15 min 25 s (NI)-28 min 57 s (nymph V) (Loja). The amount of blood ingested increased significantly with instar and was larger for Manabí specimens (p < 0.001). Defecation while feeding was observed in Manabí specimens from stage nymph III and in Lojan bugs from stage nymph IV. There was a gradual, age-related increase in the frequency of this behaviour in both populations. Our results suggest that R. ecuadoriensis has the bionomic traits of an efficient vector of Trypanosoma cruzi. Together with previous data on the capacity of this species to infest rural households, these results indicate that control of synanthropic R. ecuadoriensis populations in the coastal and Andean regions may have a significant impact for CD control in Ecuador and Northern Peru.

Association of oxamniquine praziquantel and clonazepam in experimental Schistosomiasis mansoni

The antischistosomal activity of clonazepam, when administered alone or in association with oxamniquine and praziquantel, was experimentally evaluated in mice infected with Schistosoma mansoni. The animals were treated 45 days post-infection with a single dose, by oral route, according to three treatment schedules: clonazepam 25 mg/kg and sacrificed 15 min, 1h or 4 h after treatment; clonazepam 1.0, 2.5 or 10.0 mg/kg and sacrificed 15 days post-treatment or with the dose of 10 mg/kg in association with oxamniquine 50 mg/kg or praziquantel 200 mg/kg, single dose, orally, every schedule with a control group. The efficacy of the drugs in vivo was assessed by means of worm counts and their distribution in mesentery and liver, mortality and oogram changes. In the chemotherapeutic schedules used, clonazepam did not present antischistosomal activity and the result of the association of this drug with oxamniquine or praziquantel was not significantly different from the one obtained when these two last drugs were administered alone. In the in vitro experiments, the worms exposed to 0.6 mg/mL clonazepam remained motionless throughout the 8-day-period of observation, without egg-laying, whereas the worms of the control group showed normal movements, egg-laying and hatching of miracidia on the last day of observation. The results obtained in the present study confirm the action of clonazepam on S. mansoni adult worm, in vitro, causing total paralysis of males and females. However, no additive or synergistic effects were observed when clonazepam were used in association with oxamniquine or praziquantel.

Embryonic development of Aedes aegypti (Diptera: Culicidae): influence of different constant temperatures

Despite its vector importance little attention is given to Aedes aegypti embryonic development. In this study, temperature influence on time course of Ae. aegypti larvae hatching and egg viability were evaluated. The dormancy state at the end of embryogenesis could be interrupted with a proper stimulus. Temperatures tested ranged between 12-36°C; the maximum temperature limit is 35°C and the minimum one is below 12°C. Egg viability between 16-31°C was above 80%. The definition of physiological embryonic parameters at this temperature range corroborates Ae. aegypti presence on tropical and subtropical world regions.

High fat diet has a prominent effect upon the course of chronic schistosomiasis mansoni in mice

This study investigated whether a long-term high-fat diet has an effect on the outcome of chronic murine schistosomiasis mansoni compared to a standard diet. Swiss Webster female mice (3 weeks old) were fed each diet for up to six months and were then infected with 50 Schistosoma mansoni cercariae. Their nutritional status was assessed by monitoring total serum cholesterol and body mass. Infected mice were examined 6-17 weeks post infection to estimate the number of eggs in faeces. Mice were euthanised the next day. Total serum cholesterol was lower in infected mice in comparison to uninfected controls (p = 0.0055). In contrast, body mass (p = 0.003), liver volume (p = 0.0405), spleen volume (p = 0.0124), lung volume (p = 0.0033) and faecal (p = 0.0064) and tissue egg density (p = 0.0002) were significantly higher for infected mice fed a high-fat diet. From these findings, it is suggested that a high-fat diet has a prominent effect on the course of chronic schistosomiasis mansoni in mice.

Use of a saline gradient for the diagnosis of schistosomiasis

The development of novel methods for parasitological diagnosis that are both highly sensitive and low in cost has been strongly recommended by the World Health Organization. In this study, a new technique for diagnosis of schistosomiasis mansoni is proposed based on the differential sedimentation of eggs when subjected to a slow continuous flux of 3% saline solution through a porous plaque. This influx suspends low-density faecal material, effectively cleaning the sample. The remaining sediment covering the porous plaque surface is then transferred to a glass slide and examined under a bright field microscope. Twelve Kato-Katz slides were used for comparison in the present study. Our results suggest that the saline gradient method detects a signifi-cantly higher number of eggs than the 12 Kato-Katz slides (p < 0.0001). We also found microscopic inspection to be quicker and easier with our newly described method. After cleaning the sample, the obtained sediment can also be conserved in a 10% formaldehyde solution and examined for at least 45 days later without statistically significant egg count differences.

Potential of laticifer fluids for inhibiting Aedes aegypti larval development: evidence for the involvement of proteolytic activity

It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.

Comparative descriptions of eggs from three species of Rhodnius (Hemiptera: Reduviidae: Triatominae)

The authors describe and compare the morphological and ultrastructural characteristics of eggs from the three most recent described species of the genus Rhodnius Stål, 1859, which have not previously been studied. These species are Rhodnius colombiensis (Mejia, Galvão & Jurberg 1999), Rhodnius milesi (Carcavallo, Rocha, Galvão & Jurberg 2001) and Rhodnius stali (Lent, Jurberg & Galvão 1993). The results revealed that there are similarities in the exochorial architecture of optical microscopy and scanning electron microscopy; these include the predominance of hexagonal cells that are common to all Rhodnius species and variable degrees of lateral flattening, which is common not only to species of this genus, but also to the Rhodniini tribe. Differences in overall colour, the presence of a collar in R. milesi, a longitudinal bevel in R. stali and the precise length of R. colombiensis can be useful distinguishing features. As a result of this study, the key for egg identification proposed by Barata in 1981 can be updated.

Morphological study of the eggs and nymphs of Triatoma dimidiata (Latreille, 1811) observed by light and scanning electron microscopy (Hemiptera: Reduviidae: Triatominae)

Eggs and nymphs of Triatoma dimidiata were described using both light and scanning electron microscopy. The egg body and operculum have an exochorion formed by irregular juxtaposed polygonal cells; these cells are without sculpture and the majority of them are hexagonal in shape. The five instars of T. dimidiatacan be distinguished from each other by characteristics of the pre, meso and metanotum. The number of setiferous tubercles increases progressively among instars. The sulcus stridulatorium of 1st instar nymphs is amorphous, showing median parallel grooves; from the 2nd instar on the sulcus is, progressively, elongate, deep and posteriorly pointed with stretched parallel grooves. All instars have a trichobothrium on the apical 1/3 of segment II of the antenna. The opening of the Brindley's gland is on the mesopleura. Fifth instar nymphs have an apical ctenidium on the ventral surface of the fore tibia. Dorsal glabrous patches are found on the lateral 1/3 of abdomen. Bright oval patches are found on the ventral median line of the abdomen, from segment IV-VI; 1st instar nymphs lack these patches. Abdominal dorsal plates are present from the 1st-5th instars; the 1st instar also contains a rectangular plate in segment IX. From the 2nd instar on, variably-shaped plates are present on segments VII to IX. Morphometric data were also obtained and proved to be useful for distinguishing T. dimidiata instars.

Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats

More sensitive methodologies are necessary to improve strongyloidiasis diagnosis. This study compared the sensitivities of the McMaster modified technique and polymerase chain reaction (PCR) assays, both performed in faecal samples. Lewis rats were subcutaneously infected with 4,000, 400 or 40 infective third-stage larvae, considered as high, moderate or low infection, respectively. Seven days later, they were euthanized to count adult nematodes recovered from the small intestine. Stool samples were used to count the number of eggs per gram (EPG) of faeces and to detect parasite DNA by PCR performed with a species and a genus primer pair. The sensitivity of these assays depended upon parasite burden and the primer specificity. All assays presented 100% sensitivity at the highest parasite load. In the moderate infection, EPG and PCR with the genus primer maintained 100% specificity, whereas PCR sensitivity with the species primer decreased to 77.7%. In low infection, the sensitivity was 60% for EPG, 0% for PCR with the species primer and 90% for PCR done with the genus primer. Together, these results suggest that PCR with a genus primer can be a very sensitive methodology to detect Strongyloides venezuelensisin faeces of Lewis rats infected with very low parasite burden.

Life cycle of Triatoma ryckmani (Hemiptera: Reduviidae) in the laboratory, feeding patterns in nature and experimental infection with Trypanosoma cruzi

A cohort initiated with 121 eggs, yielding 105 first instar nymphs (eclosion rate: 86.78%), allowed us to observe the entire life cycle of Triatoma ryckmani under laboratory conditions (24ºC and 62% relative humidity), by feeding them on anesthetized hamsters. It was possible to obtain 62 adults and the cycle from egg to adult took a mean of 359.69 days with a range of 176-529 days (mortality rate of nymphs: 40.95%). Mean life span of adults was of 81 days for females and 148 days for males. The developmental periods of 4th and 5th nymphs were longer than those of the other instars. This suggests that young siblings have a better chance of taking a hemolymph meal from older ones, in order to survive during fasting periods during prolonged absences of vertebrate hosts from natural ecotopes. The stomach contents of 37 insects showed blood from rodents (15 cases), lizards (7 cases), birds (6 cases) and insect hemolymph (7 cases). Out of 10 insects fed by xenodiagnosis on a Trypanosoma cruzi infected mouse, all but one became infected with the parasite.