Genetic Variation among Natural and Laboratory Colony Populations of Lutzomyia longipalpis (Lutz & Neiva, 1912)(Diptera:Psychodidae) from Colombia

Genetic diversity among three field populations of Lutzomyia longipalpis in Colombia was studied using isozyme analysis. Study sites were as much as 598 km apart and included populations separated by the eastern Cordillera of the Andes. Genetic variability among populations, estimated by heterozygosity, was within values typical for insects in general (8.1%). Heterozygosity for field populations were compared with a laboratory colony from Colombia (Melgar colony) and were only slightly lower. These results suggest that establishment and long term maintenance of the Melgar colony has had little effect on the level of isozyme variability it carries. Genetic divergences between populations was evaluated using estimates of genetic distance. Genetic divergence among the three field populations was low (D=0.021), suggesting they represent local populations within a single species. Genetic distance between field populations and the Melgar colony was also low (D=0.016), suggesting that this colony population does not depart significantly from natural populations. Finally, comparisons were made between Colombian populations and colonies from Brazil and Costa Rica. Genetic distance values were high between Colombian and both Brazil and Costa Rica colony populations (D=0.199 and 0.098 respectively) providing additional support for our earlier report that populations from the three countries represent distinct species

Lutzomyia sand flies (Diptera: Psychodidae) from middle and lower Putumayo department, Colombia, with new records to the country

A total of 4,840 phlebotomine sand flies from 54 localities in Putumayo department (=state), in the Colombian Amazon region, were collected in Shannon traps, CDC light traps, resting places and from human baits. At least 42 Lutzomyia species were registered for the first time to the department. Psychodopygus and Nyssomyia were the subgenera with the greatest number of taxa, the most common species being L. (N.) yuilli and L. (N.) pajoti. They were sympatric in a wide zone of Putumayo, indicating that they should be treated as full species (new status). Among the anthropophilic sand flies, L. gomezi and L. yuilli were found in intradomiciliar, peridomestic, urban or forest habitats. L. richardwardi, L. claustrei, L. nocticola and L. micropyga are reported for the first time in the Colombian Amazon basin. L. pajoti, L. sipani and L. yucumensis are new records for Colombia.

Effect of a second bloodmeal on the oesophagus colonization by Leishmania mexicana complex in Lutzomyia evansi (Diptera: Psychodidae)

Migration and colonization of the oesophagus by Leishmania mexicana parasites were enhanced after digestion of a second bloodmeal intake in Lutzomyia evansi. This event has epidemiological significance since it affects the infection susceptibility of this sand fly species, which is a proven vector of L. chagasi in Colombian and Venezuelan visceral leishmaniasis foci. Also, it may explain the host seeking behaviour displayed by some partially bloodfed flies found inside houses.

Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains

In this work, the susceptibility to benznidazole of two parental Trypanosoma cruzi strains, Colombian and Berenice-78, was compared to isolates obtained from dogs infected with these strains for several years. In order to evaluate the susceptibility to benznidazole two groups of mice were infected with one of five distinct populations isolated from dogs as well as the two parental strains of T. cruzi. The first group was treated with benznidazole during the acute phase and the second remained untreated controls. The animals were considered cured when parasitological and serological tests remained persistently negative. Mice infected with the Colombian strain and its isolates Colombian (A and B) did not cure after treatment. On the other hand, all animals infected with Berenice-78 were cured by benznidazole treatment. However, 100%, 50% and 70% of cure rates were observed in animals infected with the isolates Berenice-78 B, C and D, respectively. No significant differences were observed in serological profile of infected control groups, with all animals presenting high antibody levels. However, the ELISA test showed differences in serological patterns between mice inoculated with the different T. cruzi isolates and treated with benznidazole. This variability was dependent on the T. cruzi population used and seemed to be associated with the level of resistance to benznidazole.

Fibrogenesis and collagen resorption in the heart and skeletal muscle of Calomys callosus experimentally infected with Trypanosoma cruzi: immunohistochemical identification of extracellular matrix components

Intense inflammatory lesions and early development of interstitial fibrosis of the myocardium and skeletal muscle with spontaneous regression, have been described in Calomys callosus infected with Trypanosoma cruzi. The genetic types of collagen present in this model were investigated through immunohistochemistry using specific antibodies, combined with histopathology and Picro-Sirius staining of collagen. Thirty-five calomys were infected with the Colombian strain of T. cruzi and sacrificed at 24, 30, 40, 60 and 90 days post-infection. Inflammatory lesions and fibrogenesis were prominent at the early phase of infection and significantly decreased during late infection. Immunoisotyping of the matrix components was performed by indirect immunofluorescence on 5 µm thick cryostat sections using specific antibodies against laminin, fibronectin and isotypes I, III and IV of collagen. In the early phase, positive deposits of all the matrix components were present, with predominance of fibronectin, laminin and collagens types I and III in the myocardium and of types III and IV in the skeletal muscles. From the 40th day, type IV collagen predominates in the heart. At the late phase of infection (60th to 90th day), a clear fragmentation and decrease of all the matrix components were detected. Findings of the present study indicate that a modulation of the inflammatory process occurs in the model of C. callosus, leading to spontaneous regression of fibrosis independent of the genetic types of collagen involved in this process.

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.

Morphological and polymerase chain reaction-restriction fragment lenght polymorphism characterization of Biomphalaria kuhniana and Biomphalaria amazonica from Colombia

In Colombia, five Biomphalaria planorbid species are known: B. kuhniana, B. straminea, B. peregrina, B. canonica and B. oligoza(var. B. philippiana). Among them, B. straminea is intermediate host of Schistosoma mansoni and B. peregrina has been found to be experimentally susceptible to this parasite. B. straminea is commonly confused with B. kuhniana and they have been clustered together with B. intermedia in the complex named B. straminea. The difficulties involved in the specific identification, based on morphological data, have motivated the use of new techniques as auxiliary tools in cases of inconclusive morphological identification of such planorbid. In the present study, five Biomphalaria populations from the Colombian Amazon region and from Interandian Valleys were morphologically identified and characterized by polymerase chain reaction-restriction fragment lenght polymorphism directed at the internal transcribed spacer region of the rRNA gene, followed by digestion of the generated fragment with restriction enzymes (DdeI, AluI, RsaI, MvaI and HaeIII). Known profiles of the Brazilian species B. straminea, B. peregrina, B. kuhniana, B. intermedia and B. amazonica, besides B. kuhniana from Colombia, were used for comparison. The five populations under study were morphologically and molecularly identified as B. kuhniana and B. amazonica.

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae) from Western and Northeastern Colombia

Random amplified polymorphic DNA (RAPD) markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8) but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8). According to molecular variance analysis, the genetic distance between populations was significant (phiST 0.1131, P < 0.001). The variation among individuals within populations (phiST 0.8869, P < 0.001)was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.

Isozyme analysis of Taenia solium isolates from Mexico and Colombia

Mexican and Colombian Taenia solium cysticerci and some species of Taenia adults were assayed using cellulose acetate electrophoresis to distinguish between isolates. Isozyme patterns for ARK, GOT, G3PD, GPI, and MPI were identical in all cysticerci suggesting homozygotic profiles. G6PD and MDH showed different patterns between Mexican and Colombian cysticerci, suggesting regional differences. ME activity was mainly detected in the adult stage suggesting that this enzyme is active in anaerobic environment, while MDH, detected in cysticerci, could be related to an environment that contains oxygen. Finally, the species of taeniid adults analyzed showed different patterns among them.

Late Jurassic oceanic crust and Upper Cretaceous Caribbean plateau picritic basalts exposed in the Duarte igneous complex, Hispaniola

Four distinct rock units have been recognized near El Aguacate, in the Janico-Juncalito-La Vega area of the Duarte complex (Dominican Republic): (1) serpentinites crosscut by numerous diabasic dikes, (2) basalts interbedded with Late Jurassic ribbon cherts, (3) picrites and ankaramites relatively enriched in incompatible trace elements, and (4) amphibolites and gneissic amphibolites chemically similar to Oceanic Plateau Basalts. Similar Ar-Ar ages of late magmatic amphibole from a picrite, and hornblende from an amphibolite (86.1 +/- 1.3 Ma and 86.7 +/- 1.6 Ma, respectively), suggest that the Duarte picrites are contemporaneous with the Deep Sea Drilling Program Leg 15 and Ocean Drilling Program Leg 126 basalts drilled from the Caribbean oceanic plateau. These basalts are associated with sediments containing Late Cretaceous faunas. Sr, Nd, and Pb data show that enriched picrites and amphibolites are isotopically similar to mafic lavas from previously described Caribbean plateau and Galapagos hotspot basalts. Major element, trace element, and lead isotopic features of Late Jurassic basalts and diabases are consistent with those of normal oceanic crust basalt. However, these basalts differ from typical N-MORB because they have lower epsilon Nd ratios that plot within the range of Ocean Island Basalts. These rocks appear to represent remnants of the Caribbean Jurassic oceanic crust formed from an oceanic ridge possibly close to a hotspot. Later, they were tectonically juxtaposed with Late Cretaceous slices of the Caribbean-Colombian plateau.

Changes associated with laboratory rearing in antennal sensilla patterns of Triatoma infestans, Rhodnius prolixus, and Rhodnius pallescens (Hemiptera, Reduviidae, Triatominae)

We examined changes in the array of antennal sensilla of three species of Triatominae (Triatoma infestans, Rhodnius prolixus, and R. pallescens) following their establishment for different periods in laboratory culture. In each case, the laboratory colonies were compared with conspecific samples taken directly from the field, by quantitative analysis of the sensilla arrays on the three distal segments of the antenna in terms of the densities of three types of chemoreceptors (basiconics and thick and thin walled trichoids) and one type of mechanoreceptor (bristles). Sensilla densities were compared by ANOVA or non-parametric tests, and by multivariate discriminant analysis. Strains of the same species reared in different laboratories showed significant differences in their sensilla arrays, especially when compared to field-collected material from the same geographic origin. A Bolivian strain of T. infestans reared in the laboratory for 15 years and fed at monthly intervals, showed greatest differences from its conspecific wild forms, especially in terms of reductions in the number of chemoreceptors. By contrast, an Argentine strain of T. infestans reared for 25 years in the laboratory and fed weekly, showed a relative increase in the density of mechanoreceptors. A Colombian strain of R. prolixus reared for 20 years and fed weekly or fortnightly, showed only modest differences in the sensilla array when compared to its wild populations from the same area. However, a Colombian strain of R. pallescens reared for 12 years and fed fortnightly, did show highly significant reductions in one form of chemoreceptor compared to its conspecific wild populations. For all populations, multivariate analysis clearly discriminated between laboratory and field collected specimens, suggesting that artificial rearing can lead to modifications in the sensory array. This not only supports the idea of morphological plasticity in these species, but also suggests caution in the use of long-established laboratory material for experimental studies designed to extrapolate the natural behaviour and physiology of these species.

Infection of Calomys callosus (Rodentia Cricetidae) with strains of different Trypanosoma cruzi biodemes: pathogenicity, histotropism, and fibrosis induction

The influence of different Trypanosoma cruzi biodemes on the evolution of the infection and on the histopathological lesions of the heart and skeletal muscles, during the experimental infection of Calomys callosus, was investigated. Three groups of C. callosus were infected, respectively, with parasite strains representative of three different Biodemes: Type I (Y strain), Type II (21 SF strain), and Type III (Colombian strain). For each group, normal C. callosus were also used as controls. Marked differences have been detected in the responses of C. callosus to the infection with the three strains in this model. The strains Types I and II (Y and 21 SF) determined moderate lesions, mostly in the myocardium, with low parasitism, a rapid course, and total regression of the lesions by the 60th day of infection. Differently, Type III strain (Colombian), was more pathogenic for C. callosus and induced necrotic-inflammatory lesions in skeletal muscles and myocardium, in correspondence to intracellular parasitism. Proliferation of fibroblasts and amorphous matrix deposits, followed by interstitial fibrosis were present. Progressive regression of the inflammatory changes and collagen deposits occurred spontaneously. The progression and regression of both inflammation and fibrosis induced by the Colombian strain were further submitted to quantitative evaluation by morphometry. Results of the morphometric studies presented good correlation with the histopathological findings. The results confirm the importance of the different biodemes in the determination of tissue lesions and the peculiarities of response of C. callosus to infection with T. cruzi.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Participation of cytokines in the necrotic-inflammatory lesions in the heart and skeletal muscles of Calomys callosus infected with Trypanosoma cruzi

Calomys callosus, a sylvatic reservoir of Trypanosoma cruzi, when infected with the Colombian strain (Biodeme Type III, T. cruzi I ) develops necrotic-inflammatory lesions and intense early fibrogenesis in the heart and skeletal muscles, that spontaneously regress. Participation of pro-inflammatory and pro-fibrogenic cytokines, such as tumor necrosis factor-alpha (TNF-alpha), gamma interferon (IFN-gamma) , and tumor growth factor-beta (TGF-beta), in the pathogenesis of the lesions is herein studied. Eighty C. callosus weighing 20 to 30 g were used. Seventy of them were inoculated with the Colombian strain (10(5) blood forms) and 10 were maintained as intact non-infected controls. After infection, C. callosus were sacrificed at different time-points from 15 to 70 days. The heart and skeletal muscle were processed for histopathology and cryopreserved for immunohistochemistry. Early necrotic lesions of parasitized skeletal muscle and myocardium with intense inflammatory lesions were present. Search for the in situ presence of TNF-alpha and IFN-gamma, was performed using rat-IgG anti-mouse antibodies against these cytokines. For the in situ search of TGF-beta, rabbit IgG anti-mouse antibodies were used. Immunolabeling of the cytokines in tissues of infected C. callosus was successful. The cytokines TNF-alpha, IFN-gamma , and TGF-beta were detected in the cytoplasm of macrophages and in the necrotic material from 15 to 45 days post-infection, decreasing their intensity until complete disappearance by the 65th day, which correlated with subsiding histopathological lesions. These findings suggest the participation of these cytokines in the control of parasite multiplication, in the development of an early fibrogenesis and in the regression of fibrotic-inflammatory lesions observed in C. callosus.