Real-time PCR strategy for rapid discrimination among main lymnaeid species from Argentina.

Snails of the Family Lymnaeidae act as an intermediate hosts of Fasciola hepatica worldwide. The taxonomy of lymnaeid species is relevant for epidemiological studies and molecular strategies are increasingly used for that purpose. This work presents the first report of a real-time PCR approach used to identify the most important lymnaeid species in the Southern Cone of South America. Species discrimination is based on the sequence polymorphism located within the helix E10-1 of the variable region V2 of the 18S rRNA genes, which yields amplicons with clearly different melting temperatures. This procedure minimises the risk of carry-over contamination because it does not require post-PCR manipulations, and the whole protocol can be completed in less than 4h with a single snail foot as starting material. This method was successfully carried out in a blind study that included a panel of 20 Galba truncatula, 5 Lymnaea viatrix, 5 Lymnaea diaphana and 5 Pseudosuccinea columella specimens from different endemic areas for fasciolosis. This molecular approach constitutes a key laboratory tool complementing ecological studies that ultimately will promote more efficient control strategies.

Comparison of two coprological methods for the veterinary diagnosis of fasciolosis

Compararam-se a sensibilidade dos métodos padrão de sedimentação fecal (MPSF) e modificado da tamisação e coloração das fezes (MTCF)-quatro tamises, ambos empregados no diagnóstico de infecção por Fasciola hepatica. Amostras de fezes foram obtidas de 51 bovinos oriundos de uma área endêmica por fasciolose no Sudoeste da Argentina. Cada amostra foi colocada em um recipiente contendo formalina 5%. Oito mililitros da suspensão, equivalente a 2g de fezes, foram usados em cada método testado, registrando-se o número de ovos por amostra. A porcentagem de amostras positivas pelo MTCF (27/51) foi maior que a apresentada pelo MPSF (11/51), o que representou 60% de amostras falso-negativas. A porcentagem de concordância pelos dois métodos foi 41%. A complexidade do MPSF pode diminuir sua sensibilidade pela perda de ovos durante o processamento. Estes resultados confirmaram que o MPSF subestima a contagem de ovos e que o método MTCF é mais confiável.

Characterization of Triatoma guasayana biotopes in a hardwood forest of Santiago del Estero, Argentina.

Triatoma guasayana is a silvatic triatomine species distributed in Argentina, Bolivia and Paraguay. The study was performed in a secondary forest of Santiago del Estero, Argentina. The abundance of T. guasayana was evaluated by census in the following wild biotopes: quimiles (Opuntia quimilo), chaguares (dry bromeliads), logs and underground burrows. Ten biotopes of each type were dismantled in winter (August) and another 40 in summer (January); all fauna was recorded. The biotopes most infested by T. guasayana were quimiles (65%), followed by chaguares (55%), and logs (25%). Quimiles and chaguares were infested in both seasons, whereas logs were positive only in summer and burrows were never infested. Infestation and abundance were higher in summer than in winter. The biotope structure is a key factor for T. guasayana colonization. The larger number of refuges, the constant presence of blood sources and suitable inner microclimatic conditions offered by quimiles may favour the persistence of T. guasayana colonies. The richness of invertebrate fauna per type of biotope was ranked in the same order as that of T. guasayana, suggesting similar microhabitat requirements for all studied arthropods.

Triatoma infestans in Greater Buenos Aires, Argentina.

The Health Administration Agencies of many municipalities in Greater Buenos Aires (GBA) receive frequent reports on triatomines in houses. The aim of this work was to identify and describe the dispersal foci of Triatoma infestans in an urban neighborhood of GBA, and contribute to the knowledge of the epidemiological situation in the region. In June 1998, potentially infested places were entomologically evaluated. T. infestans was only detected in a hen building for egg production, which housed approximately 6,000 birds. A total of 2,930 insects were collected. Density was about 9 triatomines/m(2). The proportions of fifth instar nymphs and adults were significantly higher than those of the other stages (p<0.001). The number of triatomines collected largely exceeded the highest domestic infestation found in one house from rural endemic areas of Argentina. Though triatomines were negative for Trypanosoma cruzi, they could acquire the parasite by coming in contact with infected people living in GBA. Besides, the numerous and widely distributed places housing hens and chickens, would favor the settlement of the vector. Together, both facts may constitute a risk of parasitic vectorial transmission. It is recommended to intensify systematic activities of vector search and case detection in GBA.

Triatoma infestans in Greater Buenos Aires, Argentina

The Health Administration Agencies of many municipalities in Greater Buenos Aires (GBA) receive frequent reports on triatomines in houses. The aim of this work was to identify and describe the dispersal foci of Triatoma infestans in an urban neighborhood of GBA, and contribute to the knowledge of the epidemiological situation in the region. In June 1998, potentially infested places were entomologically evaluated. T. infestans was only detected in a hen building for egg production, which housed approximately 6,000 birds. A total of 2,930 insects were collected. Density was about 9 triatomines/m². The proportions of fifth instar nymphs and adults were significantly higher than those of the other stages (p<0.001). The number of triatomines collected largely exceeded the highest domestic infestation found in one house from rural endemic areas of Argentina. Though triatomines were negative for Trypanosoma cruzi, they could acquire the parasite by coming in contact with infected people living in GBA. Besides, the numerous and widely distributed places housing hens and chickens, would favor the settlement of the vector. Together, both facts may constitute a risk of parasitic vectorial transmission. It is recommended to intensify systematic activities of vector search and case detection in GBA

[Prevalence of Trypanosoma cruzi infection in opossum (Didelphis albiventris) in Santiago del Estero, Argentina]. / Estudio de la prevalencia de la infección por Trypanosoma cruzi en zarigüeyas (Didelphis albiventris) en Santiago del Estero, Argentina.

The opossum of the genus Didelphis is one of the principal wild reservoirs of Trypanosoma cruzi and is widely distributed in the Western Hemisphere. Didelphis albiventris is the most common marsupial in Amamá and Trinidad, two communities in the province of Santiago del Estero, Argentina. The D. albiventris population is replaced every year, and the opossum normally has two reproductive periods, one at the beginning of the spring and another at the beginning of the summer. The two litters are weaned, and they leave the mother's marsupial pouch to join the population, the first (G1) at the beginning of the summer and the second (G2) at the beginning of the fall. Between 1988 and 1991 409 D. albiventris opossums were studied, and xenodiagnoses showed that 35% of them were infected with T. cruzi. Annual cycles of renewed infection were observed, with prevalences that ranged between 22% and 43%. The acquisition of the parasite occurred over the entire year, from the summer through the spring. The prevalence of infection increased with age. The G1 individuals tended to present higher prevalences than the G2 individuals, probably from being exposed to transmission for a longer period of time. In the first two (younger) age categories for the opossums, G2 individuals showed higher prevalences than did the G1 individuals. This indicates a significant increase in transmission intensity during the fall. Opossums should be regarded as a potential source of T. cruzi entry to the domestic transmission cycle.

Comparative meiotic studies in Triatoma sordida (Stål) and T. guasayana Wygodzinsky & Abalos (Reduviidae, Heteroptera).

Triatoma sordida and T. guasayana are competent Trypanosoma cruzi vectors, with overlapping distribution areas in Argentina. Both species are morphologically similar, and their immature stages are hard to discriminate. Cytogenetic studies in the genus Triatoma reveal scarce karyotypic variations, being 2n = 20 + XY the most frequent diploid number in males. In the present work the meiotic behaviour of different Argentinian populations of T. sordida and T. guasayana has been analyzed; the meiotic karyotype of both species has also been compared. The species differ in total chromosome area and in the relative area of the sex chromosomes. These meiotic karyotypic differences constitute an additional tool for the taxonomic characterization of T. sordida and T. guasayana. The analysis of an interpopulation hybrid of T. sordida (Brazil x Argentina) reveals a regular meiotic behaviour; despite the presence of heteromorphic bivalents. Our observations support the hypothesis that karyotype variations through the gain or loss of heterochromatin can not be considered as a primary mechanism of reproductive isolation in Triatoma.

Spatial distribution of Triatoma guasayana (Hemiptera:Reduviidae) in hardwood forest biotopes in Santiago del Estero, Argentina.

In the study area Triatoma guasayana Wygodzinsky & Abalos is the only wild triatomine found sympatric with Triatoma infestans (Klug) in peridomestic premises. The Trypanosoma cruzi Chagas wild cycle is centered around the same biotopes occupied by T. guasayana, which are also visited mainly by opossums with annual prevalences of 29-50%. Twelve hectares were sampled for 4 consecutive years during all seasons. During that time, 420 T. Guasayana individuals were collected in 11.3% of 1,188 biotopes of 4 types, which included quimiles (the cactus Opuntia quimilo), chaguares (several species of bromeliads), trees, and logs. Quimiles had the highest percentage of positive biotopes (31.5%) followed by chaguares (22.3%), whereas 5% of the logs were found infested. During all seasons, 9.6-15.2% of biotopes were found infested. Distance to artificial biotopes was not statistically significant when comparing the frequency of triatomine numbers per biotope in all biotope-season combinations. With the exception of quimiles in the fall, the mean number of triatomines was higher in chaguares during all seasons. Triatomine abundance by biotope and season strata showed a clumped distribution, except for the quimiles biotope during the summer. When pooling by seasons, the mean number of triatomines in chaguares and quimiles biotope was higher than in logs and trees, with all biotopes showing a strong clumped distribution. When pooling by biotopes, the mean number of T. guasayana was relatively similar for all seasons, with a strong clumped distribution. The strong contagious distribution of T. guasayana in the hardwood forest biotopes may explain the maintenance of the wild cycle of T. cruzi, despite the low number and the low prevalences of the insect vector.

Potential colonization of the peridomicile by Triatoma guasayana (Hemiptera:Reduviidae) in Santiago del Estero, Argentina.

Sylvatic triatomines might use the peridomicile as a 1st step in the process of domiciliation. Therefore, we evaluated the capability of sylvatic species to colonize the peridomicile of a rural area in the Province of Santiago del Estero, Argentina. The research was carried out in 6 houses in the village of Trinidad. The person per hour capture method was employed to determine the presence of triatomines in all the buildings (n = 44). Dispersing adults were collected by means of light traps and by villagers when approaching their houses. Triatoma infestans (Klug) was the most abundant species followed by the sylvatic Triatoma guasayana Wygodzinsky & Abalos. The branch pens, which included cacti, Opuntia quimilo, and bromeliads in their structure, were significantly associated with T. guasayana. Most of these insects had fed on domestic blood sources. With the exception of 1 Triatoma sordida (Stål), dispersing adults were T. guasayana; among those approaching houses, 12 were females (2 of which were infected with Trypanosoma cruzi Chagas) and 3 were males. T. guasayana was found to be capable of intensively invading the intradomicile and the peridomicile, showing a high tendency to settle in the ecotopes which included nontransformed raw material from the wild and where T. infestans was less abundant.

Interaction between Didelphis albiventris and Triatoma infestans in relation to Trypanosoma cruzi transmission.

This paper attempts to prove if a high Trypanosoma cruzi prevalence of opossums might be reached with few potential infective contacts. One non-infected Didelphis albiventris to T. cruzi and 10 infected nymphs of Triatoma infestans were left together during 23 hr in a device that simulated a natural opossum burrow. Twenty-six replicates were performed using marsupials and triatomines only once. Potentially infective contacts occurred in all the trials. From the 26 opossums used in trials, 54% did not eat any bug. Of the 260 bugs used, 21% were predated. In the 25 trials involving 205 surviving bugs, 36% of them did not feed. In 15/25 cases, > or = 60% of the triatomines were able to feed. The parasitological follow-up of 24 opossums showed that among 10 that had eaten bugs, 4 turned out infected and among the 14 that had not predate, 3 (21%) became positive. In sum, 7/24 (29%) of the marsupials acquired the infection after the experiment. This infection rate was similar to the prevalences found for the opossum population of Santiago del Estero, Argentina, suggesting that the prevalences observed in the field might be reached if each marsupial would encounter infected bugs just once in its lifetime.

Laboratory comparison of feeding success among Triatoma infestans, T. guasayana, and T. sordida (Hemiptera: Reduviidae).

Success in obtaining a blood meal and rapid access to hiding places after feeding are the principal requirements triatomines have as they colonize artificial ecotopes. Feeding success and postfeeding location of 3rd and 5th instars of Triatoma sordida (Stål), of T. guasayana Wygodzinsky & Abalos, and T. infestans (Klug) were studied in an experimental box in which triatomines and a chicken were left to interact overnight. The bird was enclosed in a glass cube, slightly raised to allow triatomines to get in and out, turning the space outside into an extensive refuge area. Four bunches of dry corn husks and a wooden panel were also offered as shelters. The number of live and dead insects and their locations at the end of the experiment were recorded. Predation--as the percentage of missing insects--and success--as the percentage of insects alive and fed--were calculated. Interactions with the host were different among species and sometimes also between individuals of different life stages of the same species. Predation was always > 20%. T. sordida was the best exploiter of the blood source, because 3rd instars were more successful and 5th instars were as successful as the corresponding T. infestans stages. Performance of T. guasayana was equivocal because survival and feeding success were different for both instars. T. infestans showed a pronounced tendency to climb, and 3rd instars of T. guasayana were equally distributed in the upper half of the box and in the corn husks, whereas the majority of T. sordida and 5th instars of T. guasayana sheltered in the husks.

The interaction between poultry and Triatoma infestans Klug, 1834 (Hemiptera: Reduviidae) in an experimental model.

This paper compares the predation pressure that ducks and chickens exert on triatomines. For the tests, these birds were placed in individual boxes together with a known number of Triatoma infestans and left to interact from 6 p.m. till the next morning, involving a long lasting period of complete darkness limited by two short-term periods of semi-darkness. There was a shelter which could prevent the bugs from being predated. The number of live and dead triatomines was recorded, considering missing bugs as predated by the birds. Ducks exhibited a greater predatory activity than chickens, that could be due to a long term active period at night while chickens sleep motionless from sunset to dawn. Surviving triatomines that had fed on chickens outnumbered those fed on ducks suggesting that these were less accessible to the triatomine biting. If ducks are better than chickens to detect and eat bugs and to interfere with their feeding in the field, an increase in duck number might help to diminish triatomine density. Further research is needed to determine the feasibility of application of these experimental results.

[The relationship between Triatoma infestans, poultry, and humans in a rural settlement in Santiago del Estero, Argentina]. / Relação entre Triatoma infestans, aves domésticas e o homem num povoado de Santiago del Estero, Argentina.

An association was determined between Triatona infestans, poultry, and humans in Trinidad, in the Province of Santiago del Estero, Argentina. To collect triatomines, four samples were taken at the area immediately surrounding six houses by the one hour/man capture method (December 1991 - October 1992). Peridomiciliary ecotopes were classified as arthropic (where humans carry out daily activities) or non-anthropic. Tratomine feeding habits were also determined. Of 134 biotopes, 21% had T. infestans, 22% had poultry, and 54% were anthropic. Some 25% of the latter harboured both poultry and T. infestans. Poultry were the only domestic animals associated with T. infestans, a finding that was exclusive to anthropic ecotopes. The proportion of feedings on individual fowl (61/146) was highly significant. Chicken coops are not used in Trinidad, and poultry brood in anthropic structures. Due to the materials used for making their nests and their repeated use, a periodic bug flow can be established from the intra- to the peridomiciliary area and vice versa, through passive transport. The close relationship among T, infestans, poultry, and humans in anthropic biotopes where other T. cruzi reservoirs such as dogs are also present contributes to the maintenance of domestic triatomine colonies and transmission of Chagas' disease to humans.

Dispersive flight and house invasion by Triatoma guasayana and Triatoma sordida in Argentina.

Flight activity and invasion of houses by Triatoma sordida and T. guasayana were studied in the Province of Santiago del Estero, Argentina. Spontaneous findings of both species in houses were recorded from 1982 to 1989. Light trap collections were performed in 1982, 1983 and 1984, at the woods surrounding the settlements of Amamá (43 houses) and Trinidad (19 houses). Most of the 101 triatomines collected, were unfed and negative for Trypanosoma cruzi. T. guasayana predominated over T. sordida, and both appeared on the lighted screens between 19-31 min (mean 24) after dusk and the catch time was 30-45 min. Although entomological evaluation of 41 houses at Amamá performed in September 1985, just before insecticidal spraying, showed that Triatoma infestans predominated, adults of T. guasayana were collected in sleeping places, in 7 houses (17%). Most triatomines invading houses from then up to 1990 were flying T. guasayana (20/27) and females outnumbered males. Three non-infected T. guasayana females were fed on man and two T. guasayana males positive for "T. cruzi like" trypanosomes were unfed. Therefore, visiting hungry adults could transmit T. cruzi to people and introduce wild parasites to the domestic cycle. T. guasayana stands as the main potential substitute of T. infestans in the studied area, and it might play there the same role as T. sordida in Brazil.

A new host of Trypanosoma cruzi from Jujuy, Argentina: Octodontomys gliroides (Gervais & D'Orbigny, 1844) (Rodentia, Octodontidae).

To identify wild hosts of Trypanosoma cruzi, surveys were conducted in the subandean valleys of Jujuy Province, Argentina, between June 1986 and March 1987. Seventy two mammals from 13 different species were examined by xenodiagnosis. Fifty two of them were mostly rodents trapped at the localities of Maimará, León and Tilcara, and the remainder had been kept in captivity at the Estación Biológica Experimental, in Jujuy. Trypanosoma cruzi infection was detected only in 2 Octodontomys gliroides (2 pos./8 exam. 25%) from all 72 examined mammals. Isolates were called Octodontomys Argentina 1 and 2 (OA1 and OA2). Both infected animals were caught at the archaelogical ruin of Pucará, at Tilcara. Repeated searches for triatomines in the ruin itself and in neighbour houses rendered negative results. Groups of mice inoculated with either OA1 or OA2 isolates became infected between 7 (OA1) to 12 days (OA2) postinoculation PI. Parasitemia peaks were observed between day 12th-14th PI. Scarce amastigote nests were found in myocardium and skeletal muscle. Mortality was observed only for mice inoculated with OA1. Isoenzyme patterns of OA1 and OA2 were identical to one found in dogs and slightly different from that of human parasites in Argentina. Bones from Octodontomys sp., were recently found in a cave, dated 10200-8600 BC, in Pumamarca, near Tilcara, Jujuy. There are evidences that O. gliroides cohabited with man in ancient times and was associated to the domestic cycle of T. cruzi transmission, playing a role like that of domestic caves in Bolivia.

Sylvatic American trypanosomiasis in Argentina. Trypanosoma cruzi infection in mammals from the Chaco forest in Santiago del Estero.

Trypanosoma cruzi infection in sylvatic mammals of the quebracho woods of the eastern part of Santiago del Estero province, Argentina, was studied from October 1984 to December 1987. 301 mammals of 20 different species were caught. T. cruzi, characterized biologically and biochemically, was isolated by xenodiagnosis from 23 of 72 (32%) Didelphis albiventris opposums, 2/36 (5.5%) Conepatus chinga skunks, and one ferret (Galictis cuja). 53 opossum refuges were located and triatomine bugs were found in 2 of them: one male Triatoma infestans, infected with T. cruzi, and 5 uninfected nymphs of T. sordida, had all fed on opossum blood. Electrophoretic zymogram patterns of the T. cruzi populations isolated from opossums and skunks were similar to isoenzyme profiles already described for populations isolated from infected humans in Argentina. The small number of triatomines found in the opossum refuges seems inadequate to account for the prevalence of T. cruzi infection recorded for these mammals, so other possible contaminative routes of infection should be investigated.

The skunk Conepatus chinga as new host of Trypanosoma cruzi in Argentina.

We report the first systematic epidemiological research carried out in Argentina on the skunk Conepatus chinga. Forty-nine animals were captured in the settlements of Amamá, Trinidad, and nearby forested areas located in the Department of Moreno, Province of Santiago del Estero, between April 1985 and May 1989. Isolation of parasites was done through xenodiagnosis, and their identification as Trypanosoma cruzi was achieved by biological and biochemical criteria. The isolate was highly virulent and pathogenic in inoculated C3H mice. Prevalence was 4.1% (2 of 49). Two facts account for a possible domestic source of infection: both infected skunks were captured near Trinidad, in an area that had never been treated with insecticides, and electrophoretic isoenzyme patterns of the parasites isolated from the skunks were identical to those found in humans. Because extensive deforestation probably would increase the distribution area of C. chinga, further investigation should be performed to evaluate the epidemiological role of this wild mammal.