Characterization of subpopulations (clones and subclones) of the 21 SF strain of Trypanosoma cruzi after long lasting maintenance in the laboratory

Several studies have shown a clonal structure of Trypanosoma cruzi and its possible correlation with the behavioral heterogeneity of the parasite strains. In the present study, the 21 SF strain, that have been maintained in laboratory by successive passages in mice, for more than 15 years, showing a stability of biological and isoenzymic characteristics has been cloned, with the objective of establishing the characters of its clones and subclones. With the technique of isolation of a single parasite from the blood of infected mice, 5 clones and 14 subclones have been obtained. After four passages into mice, inoculum of 10(5) was obtained for each clone and subclone and inoculated into mice weighing 10 to 12 g. These were used for the study of the biological behavior of the clones: evolution of parasitemia, morphology of blood forms and host mortality. For isoenzymic characterization, the clones and subclones were analyzed for ALAT, ASAT, GPI and PGM enzymes. Results have shown that the 5 clones and the 14 subclones disclosed a biological behavior similar to the parental strain, with minor variability of the parasitemic profiles and also the same isoenzymic patterns. These results confirm the stability of the 21 SF strain and indicate a clonal homogeneity of its populations. This is compatible with the hypothesis that the T. cruzi strains represent an equilibrium of either homogenous or heterogeneous populations.

Morphological Aspects of the Larval Instars of Chrysomya albiceps (Diptera, Calliphoridae) Reared in the Laboratory

In order to study the morphology of young Chrysomya albiceps forms, newly hatched larvae were collected at 2 hr intervals, during the first 56 hr; after this time the collection was made at 12 hr intervals. For identification and drawing, larvae were placed between a slide and a coverslip. The cephalopharyngeal skeletons along with the first and last segments were cut off for observation of their structures and spiracles. The larvae present microspines, which are distributed randomly throughout the 12 segments of the body surface; the cephalopharyngeal skeleton varies in shape and extent of sclerotization according to larval instar; the second and third instars have relatively long processes (tubercles) on the dorsal, lateral and ventral surfaces, with microspine circles on the terminal portion

Helminth Parasites of Conventionally Maintained Laboratory Mice: II- Inbred Strains with an Adaptation of the Anal Swab Technique

Worm burdens recovered from inbred mice strains, namely C57Bl/6, C57Bl/10, CBA, BALB/c, DBA/2 and C3H/He, conventionally maintained in two institutional animal houses in the State of Rio de Janeiro, RJ, Brazil, were analyzed and compared, regarding their prevalences and mean intensities.Three parasite species were observed: the nematodes Aspiculuris tetraptera, Syphacia obvelata and the cestode Vampirolepis nana. A modification of the anal swab technique is also proposed for the first time as an auxiliary tool for the detection of oxyurid eggs in mice

Report of the Expert Group on Medical Laboratory Technician / Technologist Grades

The Labour Relations Commission (Ref. CC97/566) in May 1997, recommended the establishment of an Expert Group for medical laboratory technicians/technologists as part of a set of proposals to settle their pay claims under the PCW. Download the Report here

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.

Molluscicidal action of the latex of Euphorbia splendens var. hislopii N.E.B. ("Christ's Crown") (Euphorbiaceae) against Lymnaea columella (Say, 1817) (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica Linnaeus, 1758 (Trematode: Fasciolidae): 1- test in laboratory

The latex action of Euphorbia splendens var. hislopii (Christ's Crown) against snails Lymnaea columella, intermediate host of Fasciola hepatica, derived from irrigation ditches of the Station of Pisciculture at Universidade Federal Rural do Rio de Janeiro, was studied in the laboratory. Lab bioassays, using aqueous solutions of the latex, varying between 0.1 and 10 mg/l, have proven molluscicidal activity of the product collected on the same day the tests were performed, during the four seasons of the year, finding the following lethal concentrations (LC90): 1.51 mg/l in the spring; 0.55 mg/l in the summer; 0.74 mg/l in the fall and 0.93 mg/l in winter, after 24 h exposure of the snails, showing significant differences among the seasons of the year (ANOVA test, F = 11.01, G.L.= 3/33, p < 0.05), as well as among the concentrations (ANOVA test, F = 27.38, G.L.= 11/33, p < 0.05). In the summer, mortality reached 100% from concentration at 0.6 mg/l, the same during fall and in winter as of 1 mg/l, while in spring it only reached 100% mortality as of 2 mg/l. Mortality in the controls was low, reaching 5% in the summer and winter and 10% in the fall and spring. None of the samples died. During the assay, with an aqueous solution of the latex at a concentration of 5 mg/l, in order to check the time of duration of the product effect, in the laboratory, it was observed that the molluscicidal activity remained stable up to the 15th day after the beginning of the test with 100% mortality of L. columella, gradually losing its effect until the 23rd day, when we no longer observed animal mortality. In the control group, there was a random daily variation in mortality rate ranging 0-50% after 48 h of observation for 30 days.

Biology of the first generation of a laboratory colony of Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae)

The phlebotomine sand flies Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) are very close and may be involved in the transmission of Leishmania spp. Ross, 1903 in Brazil. The biology of the first laboratory-reared generations of these species, descended from insects captured in Além Paraíba (N. intermedia) and Corinto (N. neivai) in the Brazilian state of Minas Gerais, is described here. The captured females were fed on hamsters and maintained individually in rearing pots. Laboratory temperature and relative humidity were maintained at 25-26ºC and 80% respectively. The productivity of the first generation of N. intermedia was greater than that of N. neivai, and its development time clearly shorter, particularly for the second and third larval instars.

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.

Laboratory evaluation of the effects of triflumuron on the development of Rhodnius prolixus nymph

Studies were carried out to evaluate the efficacy of the growth regulator, triflumuron (TFM) (Starycide® sc 480 Bayer), for disrupting the development of Rhodnius prolixus fifth-instar nymph by oral, topical or continuous treatment. All treatments were able to induce high levels of mortality, delay development and molt inhibition. Oral treatment induced molt inhibition in all insects that survived at doses of 0.25, 0.50 and 5.0 mg/mL of a blood meal. The highest levels of both mortality in 24 h and molt inhibition were always observed after topical treatment. The lowest doses needed to obtain considerable biological effects were always observed after continuous treatment. In this way, the highest levels of mortality within 30 days were detected after continuous treatment, which also induced an extended inter-molting period, a lower number of over-aged nymphs and the highest level of molting in nymphs that survived. Moreover, the effects of TFM on insects were often displayed in a dose response manner. These results indicate that TFM acts as a potent growth inhibitor of R. prolixus nymphs and has the potential to be used in integrated vector control programs against hematophagous triatomine species.

Quantification of the genetic change in the transition of Rhodnius pallescens Barber, 1932 (Hemiptera: Reduviidae) from field to laboratory

Previous studies have reported genetic differences between wild-caught sylvatic, domestic and laboratory pop-ulations of several Triatominae species. The differences between sylvatic and laboratory colonies parallel are similar to the differences observed between sylvatic and domestic populations. Laboratory colonies are frequently used as references for field populations, but the consequences of founder events on the genetic makeup of laboratory or domestic populations are rarely quantified. Our goal was to quantify the genetic change in Rhodnius pallescens populations artificially submitted to founder effects via laboratory colonization. We compared the genetic makeup of two sylvatic populations and their laboratory descendants using a panel of 10 microsatellite markers. Both sylvatic populations were initially collected from palm trees, but the colonies differed in the number of founder insects and amount of time kept in the laboratory. We evaluated allelic polymorphism, differences between expected and observed heterozygosity, estimates of population differentiation (Fst) and inbreeding (Fis, Fit) and cluster analyses based on Nei's distances. We found a unique genetic structure for each sample population, with significant differentiation between the field insects and each of the laboratory generations. These analyses showed strong founder effects and showed that genetic drift had led to a genetic equilibrium over several generations of isolation. Our results suggest that laboratory colonies of R. pallescens have a different genetic structure than their wild relatives and similar processes likely affect other Triatominae laboratory stocks.

Occurrence of hybrids and laboratory evidence of fertility among three species of the Phyllosoma complex (Hemiptera: Reduviidae) in Mexico

In seven studied communities of Western Mexico, triatomine specimens were sympatrically collected, some with atypical morphological characteristics in contrast to pure specimens, which were presumed to be hybrids. More than 200 specimens of Meccus pallidipennis and Meccus longipennis with brown-yellow markings on dorsal connexival segments were collected in Ahuacapán and Quitupan. In La Mesa, more than 60 specimens similar to Meccus picturatus in most morphological characteristics (including size) were collected, although they presented a largely yellowish corium like M. pallidipennis. Interfertility was proven between all of the studied wild hybrid specimens, as well as between all the experimental laboratory hybrids. Two different phenotypes (M. picturatus and M. longipennis) were obtained from crosses between M. picturatus x M. picturatus and M. longipennis x M. longipennis from the three studied localities in state of Nayarit as from La Mesita. Results support the hypothesis that the subspecific ranking of those triatomines may, therefore, be more appropriate because reproductive isolation has not been developed and complete interbreeding was recorded.

Bromeliad-inhabiting mosquitoes in an urban botanical garden of dengue endemic Rio de Janeiro - Are bromeliads productive habitats for the invasive vectors Aedes aegypti and Aedes albopictus?

Immatures of both Aedes aegypti and Aedes albopictus have been found in water-holding bromeliad axils in Brazil. Removal of these plants or their treatment with insecticides in public and private gardens have been undertaken during dengue outbreaks in Brazil despite uncertainty as to their importance as productive habitats for dengue vectors. From March 2005-February 2006, we sampled 120 randomly selected bromeliads belonging to 10 species in a public garden less than 200 m from houses in a dengue-endemic neighborhood in Rio de Janeiro. A total of 2,816 mosquito larvae and pupae was collected, with an average of 5.87 immatures per plant per collection. Culex (Microculex) pleuristriatus and Culex spp of the Ocellatus Group were the most abundant culicid species, found in all species of bromeliads; next in relative abundance were species of the genus Wyeomyia. Only two individuals of Ae. aegypti (0.07%) and five of Ae. albopictus(0.18%) were collected from bromeliads. By contrast, immatures of Ae. aegypti were found in manmade containers in nearly 5% of nearby houses. These results demonstrate that bromeliads are not important producers of Ae. aegypti and Ae. albopictus and, hence, should not be a focus for dengue control. However, the results of this study of only one year in a single area may not represent outcomes in other urban localities where bromeliads, Ae. aegypti and dengue coincide in more disturbed habitats.

Comparison of the efficacy of long-lasting insecticidal nets PermaNet® 2.0 and Olyset® against Anopheles albimanus under laboratory conditions

Insecticide-treated nets provide a reduction in human-vector contact through physical barrier, mortality and/or repellent effects that protect both users and non-users, thereby protecting the wider community from vector-borne diseases like malaria. Long-lasting insecticide-treated nets (LLINs) are the best alternative. This study evaluated the bioefficacy of LLINs PermaNet® 2.0 and Olyset® under laboratory conditions with Anopheles albimanus. The laboratory strain was evaluated for insecticide susceptibility with selected insecticides used for malarial control. Regeneration time and wash resistance were evaluated with the standard bioassay cone technique following WHO guidelines. Heat assistance was used for Olyset® nets; the nets were exposed to four different temperatures to speed the regeneration process. The regeneration study of PermaNet® 2.0 showed that efficacy was fully recovered by 24 h after one and three washes and wash resistance persisted for 15 washes. Regeneration of Olyset® nets was not observed for nets washed three times, even with the different temperature exposures for up to seven days. Thus, for Olyset® the wash resistance evaluation could not proceed. Differences in response between the two LLINs may be associated with differences in manufacturing procedures and species response to the evaluated LLINs. PermaNet® 2.0 showed higher and continuous efficacy against An. albimanus.

Comparison of the insecticide susceptibilities of laboratory strains of Aedes aegypti and Aedes albopictus

A susceptible strain of Aedes albopictus derived from the Gainesville strain (Florida, USA) was established in our laboratory. The larvicidal efficacies of the neurotoxic insecticides temephos, permethrin and the pure cis and trans-permethrin isomers and the microbial insecticide Bacillus thuringiensis israelensis (Bti) against Ae. albopictus were estimated and compared to a susceptible strain of Aedes aegypti. The larvicidal effect of insect growth regulator pyriproxyfen was also evaluated in both mosquito strains. The median lethal concentration/median emergency inhibition values for Ae. aegypti and Ae. albopictus, respectively, were: temephos, 3.058 and 6.632 ppb, permethrin, 3.143 and 4.933 ppb, cis-permethrin, 4.457 and 10.068 ppb, trans-permethrin, 1.510 and 3.883 ppb, Bti, 0.655 and 0.880 ppb and pyriproxyfen, 0.00774 and 0.01642 ppb. Ae. albopictus was more tolerant than Ae. aegypti to all six larvicides evaluated. The order of susceptibility for Ae. aegypti was pyriproxyfen > Bti > trans-permethrin > temephos > permethrin > cis-permethrin and for Ae. albopictus was pyriproxyfen > Bti > trans-permethrin > permethrin > temephos > cis-permethrin. Because both species can be found together in common urban, suburban and rural breeding sites, the results of this work provide baseline data on the susceptibility of Ae. albopictus to insecticides commonly used for controlling Ae. aegypti in the field.

Laboratory and field evaluation of the effects of the neonicotinoid imidacloprid on the oviposition response of Aedes (Stegomyia) aegypti Linnaeus (Diptera: Culicidae)

In this paper, we assessed the suitability of using the neonicotinoid imidacloprid with standard ovitraps by evaluating the ovicidal properties of imidacloprid and its influence on the oviposition response of gravid females of Aedes (Stegomyia) aegypti Linnaeus (Diptera: Culicidae). First, we calculated the imidacloprid lethal dose 99 (LD99) by exposing third instar larvae of the target species to different concentrations of the insecticide. Next, Ae. aegypti eggs were exposed to the imidacloprid LD99 for 24 h and hatching inhibition was recorded. Finally, we investigated any potential repellent effect of the imidacloprid solution on the oviposition response of gravid Aedes females in field and laboratory conditions. The LD99 obtained from larvae tests proved to be sufficient to keep any exposed eggs from hatching. No repellent effect was observed; females laid as many eggs in imidacloprid-treated ovitraps as in traps containing either clean water or temephos-treated water in both field and laboratory conditions. Our results indicate that imidacloprid is a suitable insecticide for treating ovitraps against Ae. aegypti.