Transmission of Leishmania in coffee plantations of Minas Gerais, Brazil

Transmission of Leishmania was studied in 27 coffee plantations in the Brazilian State of Minas Gerais. Eighteen females and six males (11.6% of the people tested), aged between 7-65 gave a positive response to the Montenegro skin test. Awareness of sand flies based on the ability of respondents to identify the insects using up to seven predetermined characteristics was significantly greater among inhabitants of houses occupied by at least one Mn+ve individual. Five species of phlebotomine sand fly, including three suspected Leishmania vectors, were collected within plantations under three different cultivation systems. Four of these species i.e., Lu. fischeri (Pinto 1926), Lu. migonei (França 1920), Lu. misionensis (Castro 1959) and Lutzomyia whitmani (Antunes & Coutinho 1939) were collected in an organic plantation and the last of these was also present in the other two plantation types. The remaining species, Lu. intermedia (Lutz & Neiva 1912), was collected in plantations under both the "adensado" and "convencional" systems. The results of this study indicate that transmission of Leishmania to man in coffee-growing areas of Minas Gerais may involve phlebotomine sand flies that inhabit plantations.

Search for Borrelia sp. in ticks collected from potential reservoirs in an urban forest reserve in the State of Mato Grosso do Sul, Brazil: a short report

A total of 128 ticks of the genus Amblyomma were recovered from 5 marsupials (Didelphis albiventris) - with 4 recaptures - and 17 rodents (16 Bolomys lasiurus and 1 Rattus norvegicus) captured in an urban forest reserve in Campo Grande, State of Mato Grosso do Sul, Brazil. Of the ticks collected, 95 (78.9%) were in larval form and 22 (21.1%) were nymphs; the only adult (0.8%) was identified as A. cajennense. Viewed under dark-field microscopy in the fourth month after seeding, 9 cultures prepared from spleens and livers of the rodents, blood of the marsupials, and macerates of Amblyomma sp. nymphs revealed spiral-shaped, spirochete-like structures resembling those of Borrelia sp. Some of them showed little motility, while others were non-motile. No such structures could be found either in positive Giemsa-stained culture smears or under electron microscopy. No PCR amplification of DNA from those cultures could be obtained by employing Leptospira sp., B. burgdorferi, and Borrelia sp. primers. These aspects suggest that the spirochete-like structures found in this study do not fit into the genera Borrelia or Leptospira, requiring instead to be isolated for proper identification.

Phlebotomine sand flies (Diptera: Psychodidae) associated with the appearance of urban leishmaniasis in the city of Sincelejo, Colombia

Although once associated only with rural areas, the American leishmaniasis vectors now appear to be associated also with urban and suburban areas of the Neotropics. Following the appearance of the first autochthonous visceral and cutaneous leishmaniasis cases in the urban area of the city of Sincelejo, Colombia, a preliminary entomological survey of the sand fly species composition was performed using Shannon and CDC light traps. A total of 486 sand flies representing six Lutzomyia species were collected. L. evansi, L. panamensis and L. gomezi, known vectors of Leishmania spp. were the predominant sand fly species around dwellings. The finding of these species in relation to the appearance of the first cases of leishmaniasis in the city mentioned is discussed.

Changes in nuclear phenotype frequencies following sequential cold shocks in Triatoma infestans (Hemiptera, Reduviidae)

The nuclear phenotypes of Malpighian tubule cells in fifth instar nymphs of Triatoma infestans, one of the most important vectors of Chagas disease, were studied following sequential shocks at 0ºC, separated by intervals of 8 h and 24 h at 30ºC, under conditions of moderate fasting and full nourishment. The insects pertained to colonies reared in the laboratory and originated from domestic specimens collected in the Brazilian states of São Paulo (north) and Minas Gerais (south). Since nuclear phenotypes in this species are affected by single cold shocks, it was expected that these phenotypes could also be changed by sequential shocks. Nuclear phenotypes indicative of mechanisms of cell survival (nuclear fusion and heterochromatin decondensation) and cell death (apoptosis and necrosis) were observed concomitantly in all the conditions tested. Nuclear fusion and heterochromatin decondensation were not found relevant for the presumed acquisition of the cold-hardening response in T. infestans. The decreased frequency of apoptosis and necrosis following sequential cold shocks including under fasting conditions, indicated that tolerance to sequential cold shocks occurred in T. infestans of the mentioned origin.

Susceptibility of spiny rats (Proechimys semispinosus) to Leishmania (Viannia) panamensis and Leishmania (Leishmania) chagasi

The role of Proechimys semispinosus as reservoir of Leishmania (Viannia) panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID) in the snout or feet with 10(7) promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7) promastigotes) or ID in the ear (10(8) promastigotes). PCR-hybridization showed that 15% of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i.) and became more resistant upon reinfection. Infectivity to sand flies was low (1/20-1/48 infected/fed flies) and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs.

Feeding and defaecation behaviour of Triatoma patagonica (Del Ponte, 1929) (Hemiptera: Reduviidae)

Among the vectors of Chagas disease, Triatoma patagonica is a species in the process of adaptation to the human environment being recently registered in urban and suburban zones. However, its importance as a vector of Chagas disease is unknown. The aim of this work was to evaluate two aspects of vectorial competence: the feeding behaviour and the defaecation pattern. These processes were studied in females of T. patagonica fed ad libitum on a restrained pigeon. The results showed that the blood meal size was negatively correlated with the time of first defaecation (r = -0.42). The first defaecation was emitted before the first 10 min and defaecations during feeding were frequent. A total of 73% of females, defaecated during the first 30 min post-feeding. These results suggest that if this species subsequently colonizes the domicile, it would be capable of transmitting Trypanosoma cruzi.

Epidemiological and immunological aspects of human visceral leishmaniasis on Margarita Island, Venezuela

Sixty-five patients were diagnosed with visceral leishmaniasis (VL) on Margarita Island in the decade from 1990 to1999; 86.2% were <= 3 years old. All were leishmanin-negative at diagnosis. Evaluation of 23 cured patients in 1999 revealed that 22/23 had converted to leishmanin-positive; five had persisting antibodies to rK39 antigen, with no clinical evidence of disease. Leishmanin tests were positive in 20.2% of 1,643 healthy individuals from 417 households in endemic areas. Of the positive reactors, 39.8% were identified in 35 (8.4%) of the households, 15 of which had an antecedent case of VL, a serologically positive dog or both. Weak serological activity to rK39 antigen was detected in 3 of 488 human sera from the endemic areas. The presence of micro-foci of intense peri-urban transmission and the apparent absence of other Trypanosomatidae causing human disease offer a unique opportunity for the study of reservoirs, alternative vectors and evaluation of control measures on the Island.

Improved immunogenicity against HIV-1 clade C antigens by using NYVAC-C with restored replication competence

Background: In order to improve the immunogenicity of currently available non-replicating pox virus HIV vaccine vectors, NYVAC was genetically modified through re-insertion of two host range genes (K1L and C7L), resulting in restored replicative capacity in human cells. Methods: In the present study these vectors, expressing either a combination of the HIV-1 clade C antigens Env, Gag, Pol, Nef, or a combination of Gal, Pol, Nef were evaluated for safety and immunogenicity in rhesus macaques, which were immunized at weeks 0, 4 and 12 either by scarification (conventional poxvirus route of immunization), intradermal or by intramuscular injection (route used in previous vaccine studies). Results: Replication competent NYVAC-C-KC vectors induced higher HIV-specific responses, as measured by IFN-g ELISpot assay, than the replication defective NYVAC-C vectors. Application through scarification only required one immunization to induce maximum HIV-specific immune responses. This method simultaneously induced relatively lower anti-vector responses. In contrast, two to three immunizations were required when the NYVAC-C-KC vectors were given by intradermal or intramuscular injection and this method tended to generate slightly lower responses. Responses were predominantly directed against Env in the animals that received NYVAC-C-KC vectors expressing HIV-1 Env, Gag, Pol, Nef, while Gag responses were dominant in the NYVAC-C-KC HIV-1 Gag, Pol, Nef immunized animals. Conclusion: The current study demonstrates that NYVAC replication competent vectors were well tolerated and showed increased immunogenicity as compared to replication defective vectors. Further studies are needed to evaluate the most efficient route of immunization and to explore the use of these replication competent NYVAC vectors in prime/boost combination with gp120 proteinbased vaccine candidates. This study was performed within the Poxvirus T-cell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Morphometric discrimination of females of five species of Anopheles of the subgenus Nyssorhynchus from Southern and Northwest Colombia

The most important vectors of human Plasmodium in the neotropics belong to the subgenus Nyssorhynchus. These species are generally sympatric in terms of their geographical distributions. Some are difficult to identify based solely on examination of adult females using the available morphological keys, in these cases examination of immature stages and male genitalia is required to make correct determinations. However, in epidemiological studies it is necessary to identify the species of adult females which are found near humans, i.e. in studies of malaria transmission or evaluation of control measures. The purpose of the present study was to evaluate the discrimination of adult females of different species of Nyssorhynchus isolated mainly from Southern Colombia (department of Putumayo), using morphometric analysis. Adult females were obtained after rearing larvae collected in natural breeding places and from the progeny of females collected on humans. The morphological characteristics of the immature stages allowed the identification of four species of the subgroup Oswaldoi from Southern Colombia: Anopheles rangeli Gabaldon, Cova Garcia & Lopez, An. oswaldoi (Peryassu), An. benarrochi Gabaldon, Cova Garcia & Lopez and An. triannulatus (Neiva & Pinto). The species An. nuneztovari (Gabaldon) from the Northwest of Colombia was included for comparison. Morphometric analysis allowed differentiation of the females of all species to a confidence level approaching 90% using principal components analysis of 10 wing and leg variables, followed by canonical variate analysis of the first four principal components. We conclude that morphometrics may represent a useful taxonomic tool for this group and that its use should be further studied.

Schistosomiasis mansoni in Bananal (State of São Paulo, Brazil): II. Intermediate hosts

We conducted monthly snail captures in Bananal, State of São Paulo, Brazil, between March 1998 and February 2001, to identify Schistosoma mansoni vectors, estimate seasonal population changes, and delimit foci. We also evaluated the impact of improvements in city water supply and basic sanitation facilities. We identified 28,651 vector specimens, 28,438 as Biomphalaria tenagophila, 49 of them (0.2%) infected with S. mansoni, and 213 as B. straminea, none of the latter infected. Vectors predominated in water bodies having some vegetation along their banks. Neither population density nor local vegetation could be linked to vector infection. We found the first infected snails in 1998 (from March to May). Further captures of infected snails ocurred, without exception, from July to December, when rainfall was least. Irrespective of season, overall temperature ranged from 16.5ºC to 21ºC; pH values, from 6.0 to 6.8. Neither factor was associated with snail population density. Frequent contact of people with the river result from wading across it, extracting sand from its bottom, fishing, washing animals, etc. Despite a marked reduction in contamination, cercaria shedding persists. Whatever the location along its urban course, contact with river Bananal, particularly of the unprotected skin, entails risks of infection.

Should Trypanosoma cruzi be called "cruzi" complex? A review of the parasite diversity and the potential of selecting population after in Vitro culturing and mice infection

Morpho-biological diversity of Trypanosoma cruzi has been known since Chagas' first works in 1909. Several further studies confirmed the morphological differences among the parasite strains, which were isolated from different reservoirs and vectors, as well as from human beings. In the early sixties, antigenic differences were found in the parasite strains from various sources. These differences, coupled to the observation of regional variations of the disease, led to the proposal of the term cruzi complex to designate the taxon T. cruzi. Since then this protozoan has been typed in distinct biodemes, zymodemes and lineages which were consensually grouped into T. cruzi I, T. cruzi II and into non-grouped strains. T. cruzi genotypic characterization, initially carried out by schizodeme analysis and more recently by various other techniques, has shown a great diversity of the parasite strains. In fact, T. cruzi is formed by groups of heterogeneous sub-population, which present specific characteristics, including distinct histotropism. The interaction of the different infecting clones of the cruzi complex and the human host will determine the morbidity of the disease.

Diversity of biting midges of the genus Culicoides Latreille (Diptera: Ceratopogonidae) in the area of the Yacyretá Dam Lake between Argentina and Paraguay

The Culicoides communities have been analyzed between 1993/1998 in the area influenced by the Yacyretá Dam Lake (Paraná River, Argentina-Paraguay). Adults of Culicoides were collected monthly by using CDC light traps exposed for 24 h in 9 sampling sites located at both margins of the river; 21 species were recorded. Highest values of species richness were recorded during 1993/1994, being Quiteria and Corpus the sites with the higest number of species (10 and 11, respectively). The species diversity was elevated in Quiteria, Zaimán, Candelaria, Santa Tecla, Capitán Meza and Corpus (Shannon's diversity index 1.0-1.9) while Corateí, Ituzaingó and Aguapey showed less richness and diversity. The more abundant species were C. insignis, C. venezuelensis, C. leopoldoi, C. limai, C. flinti, C. debilipalpis, C. paraensis and C. guttatus. C. insignis, potential vector of bluetongue virus (BTV) to domestic and wild rumiants in the Neotropical region, is the predominant species in the area and was the only species widely distributed. C. paraensis, a proven vector of Oropouche virus to humans, is a common and abundant species. C. pusillus and C. lahillei, potential vectors of BTV and a filarial parasite, respectively, were occasionally collected. The taxonomic structure of communities was constant during the study period. The occasional species were not characteristic to one particular site and their presence could be related to non-intrinsic conditions.

Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System.

Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.

Impact of residual spraying on Rhodnius prolixus and Triatoma dimidiata in the department of Zacapa in Guatemala

As a vector control program to control Chagas disease in Guatemala, residual spraying of Rhodnius prolixus and Triatoma dimidiata was performed, and its impact was measured in the department of Zacapa. In order to identify infested villages and determine the degree of infestation, a baseline entomological survey to identify municipalities infested with vectors followed by an additional vector survey in areas known to be infested was conducted. Residual spraying using pyrethroid insecticides was performed at all the villages identified as being infested with the vectors. The residual spraying was shown to be highly effective against both vectors by the decrease in infestation indices after spraying. Analysis of the cost-effectiveness of the spraying showed that the average cost of insecticides per house is high when compared with that in Southern Cone countries.

Epidemiology of Chagas disease in Guatemala: infection rate of Triatoma dimidiata, Triatoma nitida and Rhodnius prolixus (Hemiptera, Reduviidae) with Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida, Trypanosomatidae)

A five-year domiciliary collection in the 22 departments of Guatemala showed that out of 4,128 triatomines collected, 1,675 were Triatoma dimidiata (Latreille, 1811), 2,344 were Rhodnius prolixus Stal 1859, and only 109 were T. nitida Usinger 1939. The Chagas disease parasite, Trypanosoma cruzi, was found in all three species. Their natural infection rates were similar in the first two species (20.6%; 19.1%) and slightly lower in T. nitida(13.8%). However there was no significant difference in the infection rates in the three species (p = 0.131). T. dimidiata males have higher infection rates than females (p = 0.030), whereas for R. prolixus there is no difference in infection rates between males and females (p = 0.114). The sex ratios for all three species were significantly skewed. More males than females were found inside houses for T. dimidiata (p < 0.0001) and T. nitida (p = 0.011); a different pattern was seen for R. prolixus (p = 0.037) where more females were found. Sex ratio is proposed as an index to show the mobility of T. dimidiata in different populations. T. dimidiata is widely distributed in the country, and is also the main vector in at least ten departments, but R. prolixus with higher vectorial capacity is an important vector in at least two departments.