In vitro ovicidal activity of Brongniartia montalvoana against small ruminant gastrointestinal nematodes.

Several plants of the Fabaceae family have been assessed regarding their high nutritional value and anthelmintic properties. The ovicidal effect of the hydroalcoholic extract (Bm-HAE) and subfractions from the aerial parts of Brongniartia montalvoana (Fabaceae) against a mixed strain of gastrointestinal nematodes (GIN) (Haemonchus spp., Trichostrongylus spp. and Oesophagostomum spp.) resistant to albendazole sulfoxide, ivermectin and levamisole was evaluated by the egg hatch test (EHT). The Bm-HAE was subjected to liquid-liquid chemical separation with ethyl acetate giving two fractions, an aqueous (Bm-Aq) and an organic (Bm-EtOAct). The purification of the bioactive fraction (Bm-EtOAct) through chromatographic separation resulted in four bioactive subfractions (BmR6, BmR7, BmR8 and BmR10). The treatments were designed as follows: Bm-HAE at 800, 1,500, 3,000 and 6,000 µg/mL, and Bm-Aq, Bm-EtOAct and subfractions (BmR6, BmR7, BmR8 and BmR10) at 100, 200, 400 and 800 µg/mL. Two properly negative controls (distilled water and 2% methanol) and thiabendazole (100 µg/mL) as a positive control were used for each bioassay. The chemical identification of the extract, fractions and subfractions was performed through chromatographic processes like open column chromatography, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC-PDA). Additionally, the GIN eggs exposed to the bioactive compounds were observed through confocal laser scanning microscopy (CLSM). The Bm-HAE showed 99.5% egg hatching inhibition (EHI) at 6,000 µg/mL with a lethal concentration (LC50) of 1110 µg/mL. The Bm-EtOAc fraction displayed 99.1% EHI at 800 µg/mL with LC50 = 180 µg/mL. The ovicidal activity of the four subfractions was similar at 800 µg/mL: BmR6 (92% EHI); BmR7 (100% EHI); BmR8 (97.8%); and BmR10 (99.1%). The HPLC-PDA analysis of the bioactive subfractions allowed identification of p-coumaric acid, ferulic acid and coumarin derivatives as major compounds. The CLSM analysis allowed observation of morphological alterations in unhatched larvae caused by bioactive compounds present in the Bm-EtOAc and BmR10. In addition, the flavonoids eriodyctiol, luteolin and cynaroside were described for the first time for B. montalvoana.

In vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts against Haemonchus contortus (Nematoda) and their anthelmintic effect in gerbils.

The in vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts (E-Cham and E-Cato, respectively) on Haemonchus contortus infective larvae (L3) and the anthelmintic effect of these extracts against the pre-adult stage of the parasite in gerbils were evaluated using both individual and combined extracts. The in vitro confrontation between larvae and extracts was performed in 24-well micro-titration plates. The results were considered 24 and 72 h post confrontation. The in vivo nematicidal effect was examined using gerbils as a study model. The extracts from the two assessed plants were obtained through maceration using n-hexane as an organic agent. Gerbils artificially infected with H. contortus L3 were treated intraperitoneally with the corresponding extract either individually or in combination. The results showed that the highest individual lethal in vitro effect (96.3%) was obtained with the E-Cham extract at 72 h post confrontation at 40 mg/ml, followed by E-Cato (78.9%) at 20 mg/ml after 72 h. The highest combined effect (98.7%) was obtained after 72 h at 40 mg/ml. The in vivo assay showed that the individual administration of the E-Cato and E-Cham extracts reduced the parasitic burden in gerbils by 27.1% and 45.8%, respectively. Furthermore, the anthelmintic efficacy increased to 57.3% when both extracts were administered in combination. The results of the present study show an important combined nematicidal effect of the two plant extracts assessed against L3 in gerbils.

The TcTASV proteins are novel promising antigens to detect active Trypanosoma cruzi infection in dogs.

In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans.

Anthelmintic effects of Prosopis laevigata n-hexanic extract against Haemonchus contortus in artificially infected gerbils ( Meriones unguiculatus).

The anthelmintic effect of Prosopis laevigata (mezquite) n-hexanic extract was evaluated against Haemonchus contortus endoparasitic stages in artificially infected gerbils (Meriones unguiculatus). Prosopis laevigata leaves were collected from the Sierra de Huautla, Ecological Reserve of the Biosphere, in Morelos State, Mexico; dehydrated under shade and macerated with n-hexane for 3 days, followed by distillation for 8 h. This procedure was repeated three times and the final extract was kept at 4 degrees C. The in vivo effect of the plant extract was evaluated in gerbils artificially infected with H. contortus. Plant extract concentration was 40 mg/ml. Three groups of gerbils were as follows: group 1 (n = 7), P. laevigata extract at 100 microl intraperitoneally (IP); group 2 (n = 6), control--Tween 20 in water at a single dose of 100 microl IP; group 3 (n = 8) also served as a control, receiving water only, to determine the mortality due to causes other than the plant extract. An additional group of seven gerbils (group 4) was administered fenbendazole, as a positive control. Five days later the animals were euthanized and stomach and mucosa removed to quantify the nematodes. Data were analysed using the Student's t-test to compare the mean of nematodes obtained in groups 1, 2 and 3. The parasite population in the plant extract treated group 1 was reduced by 42.5% (P < 0.05) with respect to the control group 2; and when control group 3 was used for comparison the parasitic reduction was estimated as 53.11%. This study shows the in vivo anthelmintic effect of P. laevigata n-hexane extract for the first time, using gerbils as an in vivo model, with potential use in sheep.

Two simultaneous hepatitis B virus epidemics among injecting drug users and men who have sex with men in Buenos Aires, Argentina: characterization of the first D/A recombinant from the American continent.

Previous studies have revealed that hepatitis B virus (HBV)/D and HBV/F predominate among blood donors from Buenos Aires, Argentina. In the present study, blood samples from two high-risk groups were analysed: 160 corresponding to street- and hospital-recruited injecting drug users [81.2% showing the 'anti-hepatitis B core antigen (anti-HBc) only' serological pattern] and 20 to hepatitis B surface antigen (HBsAg)(+)/anti-HBc(+) men who have sex with men. HBV genotypes were assigned by polymerase chain reaction amplification followed by restriction fragment length polymorphism and confirmed by nucleotide sequencing of two different coding regions. HBV DNA was detected in 27 injecting drug users (16.9%, occult infection prevalence: 7.7%), and 14 men who have sex with men (70%). HBV/A prevailed among injecting drug users (81.8%) while HBV/F was predominant among men who have sex with men (57.1%). The high predominance of HBV/A among injecting drug users is in sharp contrast to its low prevalence among blood donors (P = 0.0006) and men who have sex with men (P = 0.0137). Interestingly, all HBV/A S gene sequences obtained from street-recruited injecting drug users encoded the rare serotype ayw1 and failed to cluster within any of the known A subgenotypes. Moreover, one of the HBV strains from a hospital-recruited injecting drug user was fully sequenced and found to be the first completely characterized D/A recombinant genome from the American continent. Data suggest that two simultaneous and independent HBV epidemics took place in Buenos Aires: one spreading among injecting drug users and another one sexually transmitted among the homosexual and heterosexual population.

Dynamics of a hepatitis B virus e antigen minus population ascribed to genotype F during the course of a chronic infection despite the presence of anti-HBs antibodies.

The in vivo evolution of genotype F HBV variants was recorded in a chronically infected patient throughout a 3-year observation period. Fluctuating levels of HBs Ag and anti-HBs antibodies were recorded, both of them cocirculating in peripheral blood samples at given times. Fifty S gene derived clones were sequenced and phylogenetically analyzed. As expected, some amino acid replacements within the S ORF were also observed within the P ORF while others were silent for the former. Such change was statistically significant for both S and P overlapping genes, which clearly indicates the appearance of a positive selection pressure. Supporting this notion, amino acid replacements were documented at both B and T cell epitopes in samples from 1997 and 1998. Several mutations were documented within and outside the "a" determinant in the major hydrophilic region. Such substitutions might have resulted from the attempt of HBV to evade both humoral and/or cellular immune response. To the best of our knowledge this unusual profile of HBV variants in presence of usually "neutralizing" anti-HBs antibodies was examined in vivo for the first time.

Homogeneous and heterogeneous mini-circle subpopulations in Trypanosoma cruzi kinetoplast DNA.

The small circular components (mini-circles) from Trypanosoma cruzi kinetoplast DNA (kDNA) were cloned in the plasmid vector pBR325. These clones have been used before to demonstrate the rapid evolution of mini-circle subpopulations (S anchez , D.O., Frasch , A.C.C., Carrasco , A.E., Gonzalez Cappa , S.M., Isola , E. and Stoppani , A.O.M. (1984) Mol. Biochem. Parasitol ., in the press). We have now analyzed the cloned molecules and used them to study some structural characteristics of T. cruzi mini-circles and their distribution in total kDNA restriction endonuclease digests. Most molecules partially conserved TaqI, HaeIII and HapII site clusters (constant regions) separated by one-quarter of the total mini-circle length, also detected in total kDNA digests. In addition, in one of the cloned mini-circles, the constant region was present only once, instead of four times as expected. Outside the conserved regions, the mini-circles diverged enough so that no cross-hybridization took place even under relaxed conditions. The recombinant molecules were used to probe total kDNA digests from T. cruzi. Some of them hybridized with most restriction endonuclease kDNA fragments, while one cloned mini-circle ( pTck -14) detected only its homologous subpopulation. The mini-circles detected with the latter probe proved to be nearly homogeneous, and were present in the proportion of 1/20 molecules. These results suggest that some of the generated molecules might have acquired a higher replication rate, giving rise to the homogeneous subpopulation detected. Further mutations, insertions and/or deletions, together with recombination between molecules, would bring this process to an end.

A putative pyruvate dehydrogenase alpha subunit gene from Trypanosoma cruzi.

A full-length DNA clone encoding a putative pyruvate dehydrogenase alpha subunit (E1 alpha) gene was isolated from a Trypanosoma cruzi (RA strain) DNA library. Sequencing of this clone revealed it to encode a 378 amino acid protein (M(r) 42774) with high sequence similarity to E1 alpha obtained from different sources. The highest score is obtained with human E1 alpha: 43,3% similarity. Southern blot analysis is consistent with the existence of a single copy of this putative T. cruzi E1 alpha gene per haploid genome in different parasite strains. Expression of this gene was demonstrated by Northern blot analysis and its trans-splicing acceptor site was identified by Polymerase Chain Reaction-mediated amplification of its cDNA.

A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases.

Several genes encode members of the Trypanosoma cruzi (Tc) trans-sialidase (TS) family. These proteins contain an enzymatic domain on the N terminus, the only one required for TS activity, and an antigenic domain (SAPA (shed acute phase antigen) amino acid (aa) repeats) on the C terminus. Only some members of this glycoprotein family are enzymatically active. The complete sequence of two clones encoding the enzymatic domain of active and inactive protein from each of two Tc strains has now been obtained. Comparison of these sequences showed a limited divergence among them: 20 out of the 642 deduced aa in the enzymatic domain were found to differ. From these 20 aa, only one was found to be essential for enzymatic activity. A Tyr342 residue is deduced in both active proteins while a His342 is present in both inactive ones. This naturally occurring Tyr342-->His substitution completely abolished the TS activity. In addition to Tyr342, a second deduced aa, Pro231, was found to be necessary for full enzymatic TS activity; a Pro231-->Ala change rendered the TS protein partially active. Fourteen aa residues, including Tyr342, out of the 16 aa in the active site of a sialidase from Salmonella typhimurium are present at the same or very similar positions in the Tc TS.

Rapid identification of Trypanosoma cruzi isolates by 'dot-spot' hybridization.

The presence of isolate-specific Trypanosoma cruzi minicircles has been shown in the kinetoplast DNA of this parasite. This led to the rapid identification of isolates and clones of trypanosomes by means of 'dot-spot' hybridizations with molecularly cloned minicircle probes. Unexpectedly, whole kDNAs were also suitable as probes for this purpose, provided that filters were washed under stringent conditions. This was attributed to the presence of the above-mentioned isolate-specific minicircle sequences. The fact that parasites could be directly spotted onto nitrocellulose filters simplified the rapid routine screening of a large number of samples.

Polymorphisms within minicircle sequence classes in the kinetoplast DNA of Trypanosoma cruzi clones.

Four minicircle classes were analyzed using cloned minicircles as probes and single-cell cloned Trypanosoma cruzi parasites. The hybridization conditions used allowed identification of minicircle classes within kinetoplast DNA that were non-homologous to each other. Two of these minicircle classes, detected with probes pTckAWP-2 and -3, were present together in several of the CA 1 and Miranda clones, in spite of the fact that either pTckAWP-2 or both minicircle classes were undetectable in other isolates and clones of the parasite. The other two minicircle classes (pTckM-84 and -88) were located in some Miranda cloned parasites which were characterized by the simple restriction endonuclease pattern of their minicircles. Both pTckM-84 and -88 minicircle classes represented 52-71% of the kinetoplast DNA in the latter group of trypanosomes. Restriction endonuclease mapping allowed the identification of polymorphic minicircles in two of the four minicircle classes analyzed (pTckAWP-2 and pTckM-88). The polymorphisms were observed in part of the molecules of one minicircle class within a single trypanosome clone, as well as when different clones or even some of those obtained from the same isolate were compared. In addition, a different proportion of pTckM-88 type of minicircle sequence class was observed in the kinetoplast DNA from two of the Miranda clones analyzed. These observations demonstrated that similar molecules may evolve independently in sequence in each parasite. The polymorphic minicircles detected may arise from sequence variations before expansion of a future homogeneous minicircle sequence class.

Sequence diversity in the kinetoplast DNA minicircles of Trypanosoma cruzi.

Minicircles are the most abundant component of the mitochondrially located kinetoplast DNA in the members of the order Kinetoplastida. Minicircle sequences differ among most trypanosomatid species. To learn about the molecular mechanisms that give rise to this diversity, we sequenced a complete minicircle (pTckAWP-2) and two homologous but polymorphic minicircle fragments isolated from different Trypanosoma cruzi clones. Comparison of these sequences revealed 23 point mutations, 19 of which were transitions. A single base pair insertion was also detected in one of the two minicircle fragments sequenced. Analysis of pTckAWP-2 sequence showed the following features: the presence of four internal 118 base pairs conserved regions with 80% or higher homology; the fact that these four conserved regions also differed mainly by point mutations, although in this case a bias in favor of transversions was observed; the existence in each of these four regions of the highly conserved 13 bp sequence 5'GGGGTTGGTGTAA3', detected in all trypanosomatid minicircles, which is thought to be the origin of replication; and the presence of several direct and inverted repeat sequences of 8 base pairs or longer, scattered throughout the minicircle molecule. Comparison of the T. cruzi conserved minicircle region with that of other trypanosomatids showed a higher homology of T. cruzi with T. lewisi, another stercorarian trypanosome, than with African trypanosomes or Leishmania.

Repetitive sequences scattered throughout the genome of Trypanosoma cruzi.

A clone bank from Trypanosoma cruzi DNA was constructed in the plasmid vector pBR325 and screened with total labelled DNA from the same parasite. The experimental conditions used enable recombinant clones containing repetitive sequences to be detected. 2% of the clones gave a strong positive signal. Half of them carried mini-circle sequences but the other half contained repetitive sequences from the nuclear genome. 50% of all colonies showed up more weakly suggesting that half of the trypanosome DNA fragments carried few repetitive elements. One family of repeats, present in two clones from different genomic regions, hybridized with a broad range of nuclear DNA fragment sizes. Moreover, one of these clones had at least two kinds of elements with no common sequences. A third clone, detected under the same conditions, hybridized with distinct nuclear DNA bands. The number of copies estimated for the latter was much lower than the number of homologous sequences detected in nuclear DNA with the former two. This third recombinant plasmid proved useful to differentiate among closely related trypanosome stocks. Neither poly(A)+ or poly(A)- RNA, nor the 50 kilobase pair band corresponding to the satellite DNA already described in trypanosomes, contribute to the repeats present within these recombinant DNAs. Sequences with some degree of homology were found in the nuclear genome of T. brucei and Crithidia fasciculata.

Trypanosoma cruzi isolates from Argentina and Chile grouped with the aid of DNA probes.

Fifty-two isolates and several clones from Trypanosoma cruzi, the agent of Chagas' disease, were analyzed using cloned minicircles or total kinetoplast DNA as probes. Isolates were obtained from triatomines, guinea pigs and infected humans in the Central and Northern regions of Argentina and the North of Chile. 35% of all the randomly selected isolates could be identified with one cloned minicircle probe. This widely distributed T. cruzi group was detected on both sides of the Andes mountain range (Argentina and Chile) in Triatoma infestans as well as in human infections. Most of the other isolates could be grouped with four kinetoplast DNAs as probes, but their geographical distribution seems to be restricted as compared with the one mentioned above. These results confirm the heterogeneity of T. cruzi subspecies in nature and the usefulness of DNA probes to group them.

Rapid evolution of kinetoplast DNA mini-circle subpopulations in Trypanosoma cruzi.

Nine Trypanosoma cruzi isolates not examined previously for kDNA structure were characterized by (a) endonuclease restriction analysis of mini-circles, followed by agarose gel-electrophoresis of digests, and (b) hybridization of mini- and maxi-circle fragments with four 32P-labeled cloned mini-circles from T. cruzi (pTck-1, 12, 13 and 14) or with 32P-labeled maxi-circles from T. brucei, respectively. The gel electrophoresis patterns demonstrated significant differences between isolates, which were confirmed and extended by the hybridization assay. When using pTck-1 and pTck-12 as probes, widely distributed heterogeneous mini-circle subpopulations were demonstrated in all the examined isolates, despite the occurrence of extensive homologies. pTck-14, assayed under high stringent conditions, detected an almost homogeneous mini-circle subpopulation in only three isolates, although under relaxed conditions, pTck-14 shared sequence homologies with most of the mini-circle subpopulations from all isolates. Rapidly evolving mini-circle regions were also detected using as probe pTck-13, a small mini-circle fragment. Preliminary maxi-circle characterization revealed polymorphic restriction endonuclease sites in the different T. cruzi isolates. These results were consistent with those obtained with mini-circles subjected to the same treatment.

Mapping the Trypanosoma cruzi genome: analyses of representative cosmid libraries.

In order to generate contiguous cosmid coverage of the genome of the protozoan parasite Trypanosoma cruzi for large-scale sequence analysis, a cosmid library of 36864 individual, primary clones was generated. Total genomic DNA of the reference strain CL Brener was fragmented both by partial digestion with MboI and by physical shearing. For cloning, a modified cosmid vector was used that simplifies analyses such as restriction mapping. The library's representation is about 25 genome equivalents, assuming a size of 55 Mb per haploid genome. No chimerism of inserts in the clones could be detected. The colinearity between cosmid inserts and genomic DNA was verified. Also, hybridizations to the gel-separated karyotype of the organism were carried out as a quality check. Gridded onto two nylon filters, the library was analyzed with a variety of probes. Apart from being used for combined physical and transcriptional mapping of the genome, library filters and clones are also available to interested parties.

Genetic diversity of GBV-C/HGV strains among HIV infected-IVDU and blood donors from Buenos Aires, Argentina.

GBV-C/HGV RNA was investigated in serum samples from 70 HIV(+) intravenous drug users (IVDU), as well as from 200 blood donors from Buenos Aires, Argentina. Viral RNA was demonstrated in 21 IVDU by reverse transcription-nested PCR of the 5' UTR. c-DNA amplified products were analyzed and their sequences compared with those downloaded from GenBank. A phylogenetic tree based on 171 sequences demonstrated the presence of three major genogroups, including two subgroups, within local samples, i.e. group 1 (n=1), 2a (n=11), 2b (n=4) and 3 (n=5). These results agreed entirely with those obtained by a novel RFLP (J. Clin. Microbiol. 37, 1340-1347, 1999) of the same 5' UTR amplicons. As expected, GBV-C/HGV RNA prevalence was significantly higher among IVDU than among blood donors (P<0.0001), although within the latter group an unexpectedly high rate was also detected, since 11 of 200 sera (5.5%) proved positive. These viral isolates were ascribed either to subgroup 2a (n=5), subgroup 2b (n=5) or genogroup 3 (n=1). Briefly, this partial view of GBV-C/HGV molecular epidemiology in Argentina shows: (i) different rates of GBV-C/HGV infection within both IVDU and blood donors; (ii) a high prevalence of viral RNA among blood donors; and (iii) a predominant circulation of genogroup 2, with minor contribution of groups 3 and 1.

[Organization of the network for the study of the Trypanosoma cruzi genome]. / Organización de la red para el estudio del genoma de Trypanosoma cruzi.

Five years ago the Special Programme for Research and Training in Tropical Diseases (TDR) from the World Health Organization (WHO) launched the Parasite Genome Project. The aims were to obtain information on genome organization and gene discovery in five parasites, namely, Schistosoma, Filaria, Leishmania and Trypanosomas brucei and cruzi. Organization of research networks for each parasite under study, promotion of international collaboration and training of researchers in developing countries, were also main objectives of the programme. After five years, a large amount of information has been obtained, which is now available to researchers in the field.

Trypanosoma cruzi: immunobiological studies on epimastigotes of several strains.

Epimastigotes from six Trypanosoma cruzi strains showed similar growth pattern and duplication time in a biphasic culture medium as well as similar capability to differentiate into metacyclic forms when grown in the special GM-TIIH culture; interactions of epimastigotes and Concanavalin A, Soy bean agglutinin or Wheat germ agglutinin were also similar for all the strains. On the other hand, differences in the antigenic patterns were detected by double diffusion tests. At least one precipitogen, undetectable in the other strains, was shared by the Y and RA strains. In the RA a second precipitogen was shared with Tul strain. The three strains mentioned, as identified by their IEPh pattern belonged to the C group described by Nussenzweig and Goble in 1966. However, the differences between Y and Tul precipitogens proved that the C group originally described might be made up by several subgroups. As Tul strain had been previously classified twice in the A group and once in the C group, the classic A, B and C antigenic groups seem to be relatively unstable. Due to this, and because of the differences in shape, infective capability surface saccharides, etc. already established for the trypomastigote stage, which correlate making strain grouping feasible, we consider the latter might prove a better stage to search for a strain marker.