Biological and genetic aspects of crosses between phylogenetically close species of Mexican triatomines (Hemiptera: Reduviidae).

Reproductive isolation between Triatoma mexicana (Herrich-Scaeffer) and the six species of the Phyllosoma complex, belonging to the genus Meccus, was examined by analyzing the fertility of experimental hybrids. The percentage of couples with offspring was highest in the set of crosses between Meccus phyllosomus (Burmeister) and T. mexicana, and lowest in those between Meccus pallidipennis (Stål) and Meccus bassolsae (Alejandre-Aguilar, Nogueda-Torres, Cortés-Jiménez, Jurberg, Galvão, and Carcavallo) with respect to T. mexicana. Crosses between Meccus mazzottii (Usinger), Meccus picturatus (Usinger), and Meccus longipennis (Usinger) and T. mexicana had scarce offspring, whereas offspring from crosses between M. phyllosomus and T. mexicana were abundant. However, all F1 nymphs died in different instars before reaching sexual maturity. The biological evidence in this study suggests that different degrees of reproductive isolation exist between T mexicana and the different species of Meccus. Our data also support the current status of T. mexicana as a separate species.

Mexican Trypanosoma cruzi isolates: in vitro susceptibility of epimastigotes to anti-trypanosoma cruzi drugs and metacyclic forms to complement-mediated lysis.

Trypanosoma cruzi has a clonal organization with an ample array of genetic and phenotypic features and probably anaploid constitution. Consequently, the biological behavior, biochemistry, and molecular attributes may be distinctive for each parasite strain in different geographical regions. As far as we know, there is no published information on the susceptibility of Mexican T. cruzi stocks to anti-T. cruzi drugs such as benznidazole and gentian violet, or on its resistance to complement-mediated lysis. We studied 10 Mexican T. cruzi isolates from different geographical areas, such as the pacific coast (Oaxaca, Guerrero, and Nayarit States), central part of Mexico (Guanajuato State), Gulf of Mexico (Veracruz State), and the Yucatan Peninsula (Campeche State). We searched for the natural resistance to drugs in in vitro assay against the 10 Mexican isolates using epimastigote forms and the complement-mediated lysis using metacyclic trypomastigotes insect-derived in three of them (one for each geographic region). In general, we observed high resistance to benznidazole in all the Mexican isolates tested, but in the complement-mediated lysis test, they showed moderate to high susceptibility. Although it is necessary to expand this study by using trypomastigotes and the intracellular form to verify its biological role, we suggest that Mexican T. cruzi parasites may have a variable susceptibility to antibody-mediated lysis and high resistance to benznidazole.

Taxonomic study of the Phyllosoma complex and other triatomine (Insecta: Hemiptera: Reduviidae) species of epidemiological importance in the transmission of Chagas disease: using ITS-2 and mtCytB sequences.

The purpose of this work was to clarify the taxonomy and phylogenetic relationship of the Phyllosoma complex and other important vectors in Mexico. The internal transcribed spacer 2 (ITS-2) of rDNA and the cytochrome B gene of mtDNA (mtCytB) were analyzed for the following species of triatomine: Triatoma bassolsae, T. longipennis, T. mazzottii, T. mexicana, T. pallidipennis, T. picturata, and T. phyllosoma belonging to the Phyllosoma complex, as well as T. dimidiata, T. rubida, T. infestans, and Rhodnius prolixus. The results obtained with the analysis of the ITS-2 sequences showed that the Phyllosoma complex species could not be phylogenetically separated, since T. bassolsae and T. pallidipennis, as well as T. phyllosoma and T. mazzottii were indistinguishable. In contrast, the mtCytB gene separates each one of these triatomine species. The results support the proximity of all seven species currently included in the Phyllosoma complex as well as the exclusion of T. dimidiata. For the first time T. lecticularia and T. rubida were analyzed and were also shown to be related to the Phyllosoma complex.

Development of Triatoma rubida sonoriana, Triatoma barberi, and Meccus mazzottii (Heteroptera, Reduviidae) under laboratory conditions.

The objective of this study was to determine some of the most important bionomic parameters related to the capacity of transmission of Trypanosoma cruzi and to estimate the potential transmission capacity and role of 3 groups of recent colonized Mexican Triatoma barberi, T. rubida sonoriana, and Meccus (formerly Triatoma) mazzottii in the prevalence of Chagas disease cases based on the distribution area of each species. Among the studied Triatominae species, the life cycle was shorter in T. r. sonoriana and longer in T. barberi, by as much as 4 times. The 1st and 5th instars had the highest rates of mortality in most of the studied species. Statistically significant shorter duration of feeding of instars was observed for T. r. sonoriana and longer duration was found for M. mazzottii when the species were compared. The mean number of blood meals per nymphal stadium was statistically greater in T. barberi than in T. r. sonoriana and M. mazzottii. Triatoma r. sonoriana and M. mazzotti had shorter defecation delay than in T. barberi and the delay was uniform in all instars. Most of the studied parameters showed that T. r. sonoriana and M. mazzotti could have an important potential role in the prevalence of Chagas disease cases where these species are commonly found.

Description of inflammation and cytokine profile at the inoculation site and in heart tissue of mice re-infected with Trypanosoma cruzi vector derived-metacyclic trypomastigotes.

We studied the role of Trypanosoma cruzi reinfection in regard to inflammatory and cytokine response at the inoculation site, lymph node and heart. We reinfected Balb/c mice intradermically into the hind foot-pad with natural infective metacyclic trypomastigotes. They were followed from 24 h to 30 days after the last reinfection. At the inoculation site 24 h after the last re-infection, the infiltrating inflammatory cells increased dramatically with respect to baseline inflammation, reaching maximum infiltrates for the third day. In contrast, parasite DNA was undetectable 24 h after inoculation, despite poor cytokine induction, only IFN-gamma, IL-12 and TGF-beta were noticeable on days 7 and 15, whereas in the lymph nodes draining the inoculation site positive expression of IL-2, IL-4, IL-12 and TGF-beta were found to be induced as soon as 24 h after re-entry of parasite. In the heart, the inflammatory response increased immediately 24 h after re-entry of parasites, reaching its maximum on the 7th day and returning to baseline on day 30. In conclusion, although the inflammatory response is triggered in both compartments by re-entry of parasites, the inflammatory process returns almost to baseline after 30 days, leaving a persistent low-grade inflammation.

Seroprevalence of human Trypanosoma cruzi infection in diferent geografic zones of Chiapas, Mexico.

A serologic survey was carried out in four different geographic zones of Chiapas, Mexico. A total of 1,333 samples were collected from residents of thirteen communities located on the Coast, Central Mountain, Lacandon Forest and a zone called Mesochiapas. One hundred and fifty one seropositive individuals (11.3%) were identified. Human Trypanosoma cruzi infection was influenced by geography. In the Lacandon Forest and Central Mountains there was a higher seroprevalence 32.1 and 13.8% respectively, than on the coast (1.2%). In Mesochiapas there were no seropositive individuals among the 137 persons tested. An active transmission is probably continuing because seropositive cases (13.8%) were detected in children under 10 years of age. The vector recognized on the Coast was Triatoma dimidiata while in the Lacandon Forest it was Rhodnius prolixus.

Role of two Triatoma (Hemiptera: Reduviidae: Triatominae) species in the transmission of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) to man in the west coast of Mexico.

From August 1997 to August 1998, 334 specimens of Triatoma longipennis and 62 of T. picturata were collected in four groups of localities placed in the zone from Guadalajara, Jalisco to Tepic, Nayarit, in the West Coast of Mexico. Most T. longipennis were collected outdoors (69.2%) while most T. picturata (58.1%) were collected indoors. All collected specimens were examined for Trypanosoma cruzi infection, which was detected on 98 (29.3%) T. longipennis and 17 (27.4%) T. picturata. This study confirms the role of T. longipennis and T. picturata as some of the main T. cruzi vectors to humans in Mexico. Habitation Infestation Rate with T. longipennis was of 0.09 and with T. picturata was of 0.03 and the predominating ecotopes were pile of blocks, chicken coops, pigsties, wall crawls and beds.

Vectorial importance of triatominae bugs (Hemiptera: Reduviidae) in Guaymas, Mexico.

The study was conducted in Guaymas city, Sonora, in Northwestern Mexico. Triatomines were collected manually during daytime, within and around houses selected randomly, with one person searching for bugs during one hour per house. Collected bugs were identified and analyzed for Trypanosoma cruzi infection. From a total of 279 collected specimens there were 123 females, 65 males and 91 nymphs (entomological indexes: 63% infestation, 68.4% colonization, 8.5% density and 13.5% stacking). There were 251 (90%) triatominae bugs infected with T. cruzi, one of the highest natural infestation levels recorded in Mexico. The insects collected were identified as Triatoma rubida (91%) and T. recurva (9%). Previous reports for the same locality (1959) indicated the presence of T. rubida in the wild, we found T. rubida within houses, and T. recurva as a peridomestic bug. Unplanned housing developments in originally wild areas may have favored T. cruzi transmitters to migrate from the wild and into human dwellings.

Maintenance of Acanthamoeba culbertsoni by cryopreservation.

In order to determine the viability of trophozoites of Acanthamoeba culbertsoni under cryopreservation conditions, cultures in serum-casein-glucose-yeast extract medium were subject to 5%, 7.5%, and 10% concentrations of dimethylsulfoxide (DMSO). With the methodology followed, the percentages of recovery varied between 75.6% and 86.6% with DMSO at 10%, between 54.5% and 73.5% with DMSO at 7.5%, and between 43.6% and 68.5% with DMSO at 5%. The amebae were kept in liquid nitrogen for 30-210 days. The highest viability of trophozoites was founded when DMSO was used at a final concentration of 10% and an equilibrium temperature of 4 C. Gross cultural or morphological changes were not noted in trophozoites thawed from frozen suspensions.

American trypanosomosis: in situ and generalized features of parasitism and inflammation kinetics in a murine model.

American trypanosomiasis: In situ and generalized features of parasitism and inflammation kinetics in a murine model. Experimental Parasitology 83, 267-274. Mimicking the natural conditions of mammalian infection, metacyclic trypomastigote forms of a Mexican isolate (Ninoa) of Trypanosoma cruzi were inoculated into mice in order to study inflammation kinetics and parasite clearance at the inoculation site, parasite tropism to different organs, and local inflammatory cell infiltrates. Polymorphonuclear cells were detected at the inoculation site as early as 1 hr after inoculation. Peak cell infiltrate was observed at 24 hr; at 96 hr polymorphonuclear cells had disappeared. Mononuclear cell infiltrates began at 24 hr, peaking at Day 15, and then stared disappearing by Day 30. Antigens and parasites were detected by conventional techniques up to 15 min and thereafter became undetectable. Amplification of the hypervariable region of kinetoplast minicircle DNA by polymerase chain reaction was positive from 24 hr to Day 15, and the reaction became negative on Day 30. Myositis was observed in skeletal muscle from Days 7 to 180, it progressed from slight to severe, with an inflammation process which included macrophages, plasmatic cells, and a few eosinophils, the phenotype of the infiltrating cells included LyT2+ and LyT1+ on Day 30, and both cell populations decreased in parallel on Day 180. Antigen and parasite nests were present from Day 15 to 180; in muscle the earliest time at which minicircle DNA was detected was Day 7 and it was present until Day 180. Myocarditis was also observed; it developed from slight to severe in between Days 7 and 30, then gradually decreased, and cleared up. Mononuclear cell infiltrates in the myocardium were present from Days 7 to 180. Antigen and parasite nests were detected at Days 15 and 30 and disappeared at Day 180, although minicircle DNA was detected until the last day of observation. Both skeletal and heart muscles showed inflammatory reaction foci containing T. cruzi antigen. There was twice the number of foci in heart as in skeletal muscle. This ratio was maintained by Day 30; later skeletal muscle showed persistent inflammation which was practically cleared up in the heart. Parasites or antigen were not detected by Day 180 in both skeletal and cardiac muscle; however, minicircle DNA was amplified, indicating that an small proportion of parasites evaded immune response. According to these data, Mexican Ninoa Strain has a classification as biodeme 3.

Frequency of triatomines infected with Trypanosoma cruzi collected in Cuernavaca city, Morelos, México.

In order to study the frequency of infection with Trypanosoma cruzi in the triatomines from Cuernavaca city (Morelos, Mexico), 1,060 triatomines were captured with the support of the population, from september 1990 to february 1992; 1,035 were Triatoma pallidipennis adults, 88% presented flagellated protozoa with morphological characteristics corresponding to Trypanosoma spp. Later, in the laboratory, their ability to infect NIH mouse confirmed the isolates identities as Trypanosoma cruzi. The other 25 triatomines captured were adults of Triatoma barberi, 70% of them were infected with Trypanosoma cruzi. The results show the need to increase the study of the Chagas' disease in the urban and suburban areas as well as showing the need to provide further information to the population regarding the prevention of this parasitosis.

[Experimental Trypanosoma cruzi infection via contaminated water and food]. / Infección experimental con Trypanosoma cruzi a través de agua y alimentos contaminados.

In order to determine the efficiency of different foods and water to maintain the infectivity of T. cruzi, the percentage of animals that resulted infected when they were ingested was registered. The materials were contaminated with metacyclic trypomastigotes from triatomine bugs feces, the infection in the mice were registered by directed observation of the parasite in the blood and corroborate by xenodiagnosis. Pasteurized milk infected the highest number of mice and the infectivity lasted longer than any other item tested. The efficacy of infectivity of fresh cheese and rice lasted after three hours and the percentage of infected mice was lower than with milk. Cooked and raw beefmeal and water resulted in the lowest, although similar number of infected mice. The infective capacity lasted only for a short time. It appears that the main differences obtained in infectivity depended on the different contents of moisture and nutrients in the solution.

[Comparative study of the susceptibility of 5 triatomine species (Insecta: Reduviidae) to Trypanosoma cruzi infection]. / Estudio comparativo de la susceptibilidad de cinco especies de triatominos (Insecta: Reduviidae) a la infección con Trypanosoma cruzi.

In order to determine the most suitable species of triatomine for xenodiagnosis of the Chagas disease, five species triatomine (Triatoma barberi, T. infestans, T. pallidipennis, T. phyllosoma, and Rhodnius prolixus) in third and fourth instars were used to evaluate their susceptibility to one T. cruzi strain isolated in Mexico. The susceptibility was determined by: a) percentage of infected triatomines, b) amount of flagellates in bug feces and c) amount of blood ingested. We found that in most species of triatomines tested the third instar was more susceptible than fourth instar. The order of susceptibility to T. cruzi was: T. pallidipennis > T. barberi > T. phyllosoma > T. infestans > R. prolixus.

[The oral route: an access port for Trypanosoma cruzi]. / La vía oral: una puerta de acceso para Trypanosoma cruzi.

Rats and mice were inoculated by oral route with T. cruzi from: 1. Infected mice blood, 2. Experimentally infected triatomines feces, 3. Experimentally infected alive triatomines, and 4. Free cell cultures. Mice showed to be more susceptible to the infection than rats. Mice exhibited to be infected: 30% with blood forms, 85% with triatomines feces, 75% with alive triatomines and 35% with culture forms. Rats exhibited to be infected: 10%, with blood forms, 45% with triatomines feces, 60% with alive triatomines and 25% with culture forms. In both species we observed that metacyclic trypomastigotes originated higher infection percentage than blood and culture forms. With these results we confirm that oral route is a probable way of transmission of T. cruzi and under standardized conditions, it represents a better experimental inoculation route than intraperitoneal route.