Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein

The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

Species Diversity and Flagellate Infections in the Sand Fly Fauna near Porto Grande, State of Amapá, Brazil (Diptera: Psychodidae. Kinetoplastida: Trypanosomatidae)

Forty-six species of Lutzomyia and one species of Brumptomyia were identified among 20,008 sand flies collected in central Amapá. L. squamiventris maripaensis, L. infraspinosa, L. umbratilis and L. ubiquitalis accounted for 66% of the specimens caught in light traps, and L. umbratilis was the commonest of the 16 species found on tree bases. Seven species of Lutzomyia including L. umbratilis were collected in a plantation of Caribbean pine. Sixty out of 511 female sand flies dissected were positive for flagellates. Among the sand flies from which Leishmania was isolated, promastigotes were observed in the salivary glands and foregut of 13 out of 21 females scored as having very heavy infections in the remainder of the gut, reinforcing the idea that salivary gland invasion may be part of the normal life cycle of Leishmania in nature. Salivary gland infections were detected in specimens of L. umbratilis, L. whitmani and L. spathotrichia. Parasites isolated from L. umbratilis, L. whitmani and also from one specimen of L. dendrophyla containing the remains of a bloodmeal, were compatible with Le. guyanensis by morphology and behaviour in hamsters.

Cercarial Chaetotaxy and Sex Differentiation of Schistosoma mansoni Deriving from Humans and Nectomys squamipes (Muridae: Sigmondontinae) in Brazil

A comparative study was made between sympatric isolates of Schistosoma mansoni: one from a wild rodent (R) Nectomys squamipes and another one from humans (H) isolated from a low endemic schistosomiasis transmission area in Brazil. Our purpose was to detect differences between them concerning chaetotaxy (number and pattern of distribution of the argentophilic papillae) of the cercariae by means of silver impregnation. No significant difference (x > 0.05) between isolates was noted. Nevertheless, a significant difference (x < 0.05) was observed in the cercarial index (ratio of the distance between the first and the second preacetabular papillae and the distance between the first and the second dorsal preacetabular papillae) of male and female cercariae in both isolates. Males presented a greater cercarial index than females. By means of multivariate analysis, male cercariae were distinguished from female cercariae through the following characteristics: average number of dorsal papillae on the right quadrant, average number of ventral middle papillae on the right quadrant (H isolate) and average number of dorsal middle papillae on the left quadrant (R isolate). The results suggest that R and H isolates belong to the same population that could complete its life cycle in rodent-snail-rodent fashion.

Experimental Infection of Aedes albopictus (Diptera: Culicidae) Larvae with the Xiphidiocercariae of a Hematolechid

Aedes albopictus larvae were exposed, either individually or in groups, to different concentrations of xiphidiocercariae of Haematoloechus sp. for parasitological studies. It was observed the acute lethal effect and some aspects of the host-parasite relationship, such as delay or progress in the host life cycle, the number and location of the metacercariae in the host, adult host malformations and the amount of metacercariae required to cause death. A delay in the cycle and a high mortality rate was in general observed. Inside the larvae, the metacercariae were found predominantly in the thorax, abdominal segments and in the head, along with a reduced number in the anal lobe and cervix. It was shown that in addition to the quantity of metacercariae present, their location in the larvae was also relevant in the determination of mortality and anomalies. Malformed adults developed from larvae containing from one to three metacercariae.

Biology of Triatoma klugi Carcavallo, Jurberg, Lent & Galvão 2001 (Heteroptera: Reduviidae) under Laboratory Conditions: Effects of Distinct Blood Sources and Susceptibility to Trypanosoma cruzi and Trypanosoma rangeli

The life cycle of Triatoma klugi Carcavallo, Jurberg, Lent & Galvão 2001 was compared under laboratory conditions using two groups of the F1 generation obtained from field-collected bugs. Among the 100 nymphs weekly fed on mice (Group A) or chicken (Group B), 77% of Group A and 67% of Group B reached the adult stage, and the mean time from the first nymphal stage to adult was 190.08 ± 28.31 days and 221.23 ± 40.50, respectively. The average span in days for each stage per group and the number of blood meals required for each stage were also evaluated. The overall mortality rate was 23% and 33% for Groups A and B, respectively. The mean number of eggs laid per month in a three-month period was of 56.20, 51.70 and 73.20 for Group A, and 64.50, 53.50 and 38.71 for Group B. Despite the blood source, comparative analysis revealed no statistically significant differences in the life cycle of T. klugi under laboratory conditions. Infection rates over 60% were observed for both Trypanosoma cruzi strains tested. Even revealing high infection rates of the hemolymph by T. rangeli strains, T. klugi revealed no salivary gland infections and was not able to transmit the parasite.

Biology of Amblyomma aureolatum (Pallas, 1772) (Acari: Ixodidae) on some laboratory hosts in Brazil

The ixodid Amblyomma aureolatum is suspected to play a role in the epidemiology of wild life-cycle hemoparasites, which frequently infect dogs in rural and hunting areas in Brazil. Little is known about its bionomics. The objective of the present study was to evaluate some bionomic aspects of A. aureolatum ticks in Brazil. One engorged female, collected from a dog (Canis familiaris) in São Sebastião das Águas Claras, State of Minas Gerais, was used to establish a colony in the laboratory. Subsequently its parasitic stage progeny were fed on domestic dogs and laboratory animals. The free-living stages were incubated at 27ºC ± 2°C and minimum 70% relative humidity in a BOD incubator. The egg incubation period ranged from 31 to 34 days; the parasitic period of larvae ranged from 4 to 6 days and ecdysis to nymphs occurred from day 19 up to day 22. The parasitic period of nymphs ranged from 5 to 8 days and the period of ecdysis to adults from 31 to 33 days. The parasitic period of adults ranged from 11 to 15 days, the pre-oviposition period from 6 to 12 days, and the oviposition period from 9 to 38 days. The total duration of the life cycle ranged from 116 to 168 days.

Dynamics of evolution and resistance to starvation of Triatoma vitticeps (Stal 1859) (Reduviidae: Triatominae), submitted to two different regimens of food deprivation

The resistance to starvation of Triatoma vitticeps has been analyzed comparatively, according to different regimens of food deprivation under laboratory conditions. One cohort, composed of 100 specimens of each evolutionary nymphal stage, was submitted to continuous fasting until death; the second group, arranged in the same way, was fed once on chicken. Through this work, it was possible not only to compare the results obtained from the first group with other authors' results but, also, to analyze the dynamics of evolution, molting, longevity and the increase in insect longevity, in the second group. The average values recorded for survival time revealed statistical differences between the two groups. Among the important results detected, there is one that deserves to be emphasized: the incredible increase in longevity among insects that received only one feed - an average increase in survival time that reached 2.95 to 3.30 times in nymphs of 3rd and 4th stages, respectively. One 5th stage nymph survived for up to 350 days and the females may triplicate their survival rate, what represents an important epidemiological factor. The knowledge about this type of biological characteristic of T. vitticeps may contribute to prevent the domiciliation of this species, what seems to be incipient in some municipal districts, in Brazil.

Development of Hepatozoon caimani (Carini, 1909) Pessôa, De Biasi & De Souza, 1972 in the Caiman Caiman c. crocodilus, the frog Rana catesbeiana and the mosquito Culex fatigans

The sporogony of Hepatozoon caimani has been studied, by light microscopy, in the mosquito Culex fatigans fed on specimens of the caiman Caiman c. crocodilus showing gametocytes in their peripheral blood. Sporonts iniciate development in the space between the epithelium of the insect gut and the elastic membrane covering the haemocoele surface of the stomach. Sporulating oocysts are clustered on the gut, still invested by the gut surface membrane. Fully mature oocysts were first seen 21 days after the blood-meal. No sporogonic stages were found in some unidentified leeches fed on an infected caiman, up to 30 days following the blood-meal. When mosquitoes containing mature oocysts were fed to frogs (Leptodactylus fuscus and Rana catesbeiana), cysts containing cystozoites developed in the internal organs, principally the liver. Feeding these frogs to farm-bred caimans resulted in the appearance of gametocytes in their peripheral blood at some time between 59 and 79 days later, and the development of tissue cysts in the liver, spleen, lungs and kidneys. Transmission of the parasite was also obtained by feeding young caimans with infected mosquitoes and it is suggested that both methods occur in nature. The finding of similar cysts containing cystozoites in the semi-aquatic lizard Neusticurus bicarinatus, experimentally fed with infected C. fatigans, suggests that other secondary hosts may be involved.

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.

Trypanosoma (Herpetosoma) rangeli Tejera, 1920: an updated review

Trypanosoma rangeli, a parasite generally considered non-pathogenic for man, is the second species of human trypanosome to be reported from the New World. The geographical distribution of T. rangeli often overlaps with that of T. cruzi, the same vertebrate and invertebrate hosts being infected. Their differentiation thus becomes of real, practical importance, particularly as they share approximately half the antigenic determinants recognized by the humoral response. Little is known about the life cycle of T. rangeli in the vertebrate host, although thousands of human and wild animal infections have been reported. Recent studies have revealed 2 major phylogenetic lineages in T. rangeli having different characteristics, thus leading to better understanding of the epidemiology and interactions with this parasite's vertebrate hosts and triatomine vectors. Based on further genetic characterization analysis, the authors have proposed 2 alternative hypotheses and consider that T. rangeli could have had clonal evolution or have been subjected to speciation processes.

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.

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.

Ciclo biológico em laboratório de Rhodnius brethesi Matta, 1919 (Hemiptera, Reduviidae, Triatominae), potencial vetor silvestre da doença de Chagas na Amazônia

R. brethesi is a sylvatic species from the Amazon region; it has been incriminated as responsible for the transmission of Chagas disease in collectors of piaçaba in this region. The aim of present study was to investigate the efficiency of these insects as potential vectors of Trypanosoma cruzi. Aspects related with feeding and defecation patterns, life time, and mortality had been observed in each instar of R. brethesi. We use 5th instar nymphs to get adults virgins, after the moulting 3 groups with 6 females and 2 males each were created to obtain eggs. After hatching, 1st instar nymphs had been weighed and kept in bottles until the next moult. Insects were fed once a week in mice. Results showed that the average period of incubation was 17 days, the number of blood meal was increasing from the 1st to the 5th instar nymph with 7 (average) to become adult, a significative numbers of the defecations occurring immediately after the bloodmeals. The total percentual of mortality was 16%. This results suggests that this species presents a good exploitation of blood meals and a brief nymphal development in laboratory conditions reflecting its behavior in sylvatic environments.

From genomes to vaccines via the proteome

An effective vaccine against schistosomiasis mansoni would be a valuable control tool and the high levels of protection elicited in rodents and primates by radiation-attenuated cercariae provide proof of principle. A major obstacle to vaccine development is the difficulty of identifying the antigens that mediate protection, not least because of the size of the genome at 280Mb DNA encoding 14,000 to 20,000 genes. The technologies collectively called proteomics, including 2D electrophoresis, liquid chromatography and mass spectrometry, now permit any protein to be identified provided there is extensive DNA data, and preferably a genome sequence. Applied to soluble (cytosolic) proteins from schistosomes, proteomics reveals the great similarity in composition between life cycle stages, with several WHO vaccine candidates amongst the most abundant constituents. The proteomic approach has been successfully applied to identify the secretions used by cercaria to penetrate host skin, the gut secretions of adult worms and the proteins exposed on the tegument surface. Soluble proteins can also be separated by 2D electrophoresis before western blotting to identify the full range of antigenic targets present in a parasite preparation. The next step is to discover which target proteins represent the weak points in the worm's defences.

The Genus Cyclospora (Apicomplexa: Eimeriidae), with a description of Cyclospora schneideri n.sp. in the snake Anilius scytale scytale (Aniliidae) from Amazonian Brazil: a review

A review is made of the recorded species of the coccidian genus Cyclospora and major events leading up to the discovery of C. cayetanensis, which is responsible for serious outbreaks of diarrhoea in man and is one of the aetiological agents of "traveller's diarrhoea". Humans appear to be the specific hosts, with the entire life-cycle in the intestine: to date there is no convincing evidence that the disease is a zoonosis. A description is given of oocysts and endogenous stages of C. schneideri n.sp., in the snake Anilius scytale scytale. Sporulation is exogenous and completed after about one week at 24-26º. Mature oocysts 19.8 × 16.6 (15.1 × 13.8-25.7 × 20.1), shape-index 1.2 (1.0-1.3): no oocyst residuum or polar bodies. Oocyst wall a single colourless, smooth layer with no micropyle: it is rapidly deformed or broken. Sporocysts 13.6 × 9.4 (11.3 × 8.3-15.1 × 9.9), shape-index 1.4 (1.2-1.5) with an inconspicuous Stieda body. Sporozoites 11-13 × 2.5-3. Endogenous stages are intracytoplasmic in the epithelial cells of the small intestine and with the characters of the Eimeriorina.