Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

Experimental American leishmaniasis and Chagas'' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi

, and 1988. Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi. International Journal for Parasitology 18: 1053–1059. Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) previously infected with either Leishmania braziliensis braziliensis or L. b. panamensis were challenge infected with blood-form trypomastigotes of Trypanosoma cruzi (Brazil strain). Monkeys previously infected with T. cruzi were challenged with stationary phase promastigote forms of L. b. braziliensis. Monkeys were examined during the course of challenge for evidence of infection, electrocardiographic alterations and parasite-specific antibody responses. T. cruzi epimastigotes were cultured from the blood of monkeys up to 3 months after challenge with this parasite. Unulcerated cutaneous lesions appeared and persisted in monkeys challenged with L. b. braziliensis. The formation of satellite lesions was observed in one monkey. Increased QRS intervals were not observed in T. cruzi challenged monkeys without prior cardiac irregularities and QRS left axis shifts were observed in only two of these monkeys. Elevated titers of parasite binding IgM and IgG specific for both T. cruzi and L. braziliensis were observed in all monkeys following challenge. These results indicate that prior infection with T. cruzi or L. braziliensis does not protect against heterologous challenge infection with these organisms. However, prior infection with Leishmania parasites may provide some protection against chagasic cardiopathies.     

A panel of microsatellite and minisatellite markers for the characterisation of field isolates of Theileria parva

Mini- and microsatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis of parasites. Herein we describe the identification of a panel of 11 polymorphic microsatellites and 49 polymorphic minisatellites of the protozoan haemoparasite Theileria parva. The PCR products were run on high resolution Spreadex gels on which the alleles were identified and sized. The sequences of the mini- and microsatellites were distributed across the four chromosomes with 16 on chromosome 1, 12 on chromosome 2, 14 on chromosome 3 and 18 on chromosome 4. The primers from the 60 sequences were tested against all the Theileria species that co-infect cattle in East and Southern Africa and were found to be specific for T. parva. In order to demonstrate the utility of these markers, we characterised eight tissue culture isolates of T. parva isolated from cattle in widely separated regions of Eastern and Southern Africa (one from Zambia, one from Uganda, two from Zimbabwe, four from Kenya) and one Kenyan tissue culture isolate from Cape buffalo (Syncerus caffer). The numbers of alleles per locus range from three to eight indicating a high level of diversity between these geographically distinct isolates. We also analysed five isolates from cattle on a single farm at Kakuzi in the central highlands of Kenya and identified a range of one to four alleles per locus. Four of the Kakuzi isolates represented distinct multilocus genotypes while two exhibited identical multilocus genotypes. This indicates a high level of diversity in a single population of T. parva. Cluster analysis of multilocus genotypes from the 14 isolates (using a neighbour joining algorithm) revealed that genetic similarity between isolates was not obviously related to their geographical origin.     

The circadian system of Trypanosoma cruzi-infected mice

The effects of Chagas disease on the mammalian circadian system were studied in Trypanosoma cruzi-infected C57-Bl6J mice. Animals were inoculated with CAI or RA strains of T. cruzi or vehicle, parasitism confirmed by blood specimen visualization and locomotor activity rhythms analyzed by wheel-running recording. RA-strain infected mice exhibited significantly decreased amplitude of circadian rhythms, both under light-dark and constant dark conditions, probably due to motor deficiencies. CAI-treated animals showed normal locomotor activity rhythms. However, in these mice, reentrainment to a 6h phase shift of the LD cycle took significantly longer than controls, and application of 15min light pulses in DD produced smaller phase delays of the rhythms. All groups exhibited light-induced Fos expression in the suprachiasmatic nuclei. We conclude that the main effect of T. cruzi infection on the circadian system is an impairment of the motor output from the clock toward controlled rhythms, together with an effect on circadian visual sensitivity.     

Trypanosoma cruzi infection by oral route: how the interplay between parasite and host components modulates infectivity

Trypanosoma cruzi infection by oral route constitutes the most important mode of transmission in some geographical regions, as illustrated by reports on microepidemics and outbreaks of acute Chagas' disease acquired by ingestion of food contaminated with parasites from triatomine insects. In the mouse model, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium, a unique portal of entry for systemic infection. High efficiency of metacyclic forms in establishing infection by oral route is associated with expression of gp82, a stage-specific surface molecule that binds to gastric mucin and to epithelial cells. Gp82 promotes parasite entry by triggering the signaling cascades leading to intracellular Ca²⁺ mobilization. T. cruzi strains deficient in gp82 can effectively invade cells in vitro, by engaging the Ca²⁺ signal-inducing surface glycoprotein gp30. However, they are poorly infective in mice by oral route because gp30 has low affinity for gastric mucin. Metacyclic forms also express gp90, a stage-specific surface glycoprotein that binds to host cells and acts as a negative regulator of invasion. T. cruzi strains expressing gp90 at high levels, in addition to gp82 and gp30, are all poor cell invaders in vitro. Notwithstanding, their infectivity by oral route may vary because, unlike gp82 and gp30, which resist degradation by pepsin in the gastric milieu, the gp90 isoforms of different strains have varying susceptibility to peptic digestion. For instance, in a T. cruzi isolate, derived from an acute case of Chagas' disease acquired by oral route, gp90 is extensively degraded by gastric juice in the mouse stomach and this renders the parasite highly invasive towards target cells. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of the disease reported in outbreaks of oral infection.     

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes

One of the most widely studied simple sequences in the mammalian genome is the (TG)_n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)₁₀ probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)_n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.     

Differential detection of Hammondia hammondi from Toxoplasma gondii using polymerase chain reaction

Hammondia hammondi and Toxoplasma gondii are two related coccidian parasites, with cats as definitive hosts and warm-blooded animals as intermediate hosts. It is difficult to differentiate them by morphological and serological parameters. In the present study, primers were designed to specifically amplify the ITS-1 region of H. hammondi to differentiate it from T. gondii. Attempts were made to detect the presence of H. hammondi DNA in the tissues of mice infected with H. hammondi alone, as well as from mixed infections with T. gondii, using the newly designed primers. The de novo primers effectively amplified the H. hammondi-specific target fragment from all samples containing H. hammondi, including those with concomitant T. gondii infection. Further, the primers did not amplify any fragment from the related parasites like T. gondii, Neospora caninum and Hammondia heydorni. The new primers provide simple and efficient means to differentially diagnose H. hammondi from T. gondii even in samples containing both parasites, thus obviating the need for other labourious techniques like mouse bioassay and in vitro cultivation.     

Trypanosoma cruzi: skin-penetration kinetics of vector-derived metacyclic trypomastigotes

In order to investigate the natural route of infection of nude and normal BALB/c mice with Trypanosoma cruzi via the skin, a drop of vector faeces/urine containing metacyclic trypomastigotes was placed onto the puncture site of a bite from Triatoma infestans. The periods of exposure, i.e. until removal of flagellates from the skin, and the time elapsed until surgical removal of the skin around the puncture were varied. After 15 min of exposure, T. cruzi developed in all nude mice without surgery, and in four of 10 mice if the puncture region of the skin was removed directly after exposure. In a shaved puncture region, 5 min of exposure were sufficient to infect all normal BALB/c mice without surgery and one of four mice with direct removal of the puncture region. Longer periods of exposure or time until removal of the skin only sometimes resulted in higher infection rates. Prepatent periods and the development of parasitaemia varied irrespective of the period of exposure or the period until skin removal at the puncture site. The importance of these findings is that they clearly prove that T. cruzi can rapidly invade the host via the puncture site of the bite of the vector and that at least some parasites are immediately transported away from this site.     

Two hybridization events define the population structure of Trypanosoma cruzi

Genetic variation in Trypanosoma cruzi is likely a key determinant in transmission and pathogenesis of Chagas disease. We have examined nine loci as markers for the extant T. cruzi strains. Four distinct alleles were found for each locus, corresponding to the sequence classes present in the homozygous discrete typing units (DTUs) I, IIa, IIb, and IIc. The alleles in DTUs IIa and IIc showed a spectrum of polymorphism ranging from DTU I-like to DTU IIb-like, in addition to DTU-specific sequence variation. DTUs IId and IIe were indistinguishable, showing DTU homozygosity at one locus and heterozygosity with DTU IIb and IIc allelic sequences at eight loci. Recombination between the DTU IIb and IIc alleles is evidenced from mosaic polymorphisms. These data imply that two discrete hybridization events resulted in the formation of the current DTUs. We propose a model in which a fusion between ancestral DTU I and IIb strains gave rise to a heterozygous hybrid that homogenized its genome to become the homozygous progenitor of DTUs IIa and IIc. The second hybridization between DTU IIb and IIc strains that generated DTUs IId and IIe resulted in extensive heterozygosity with subsequent recombination of parental genotypes.     

Interaction with host factors exacerbates Trypanosoma cruzi cell invasion capacity upon oral infection

Outbreaks of severe acute Chagas’ disease acquired by oral infection, leading to death in some cases, have occurred in recent years. Using the mouse model, we investigated the basis of such virulence by analyzing a Trypanosoma cruzi isolate, SC, from a patient with severe acute clinical symptoms, who was infected by oral route. It has previously been shown that, upon oral inoculation into mice, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium by engaging the stage-specific surface glycoprotein gp82, whereas the surface molecule gp90 functions as a down-modulator of cell invasion. We found that, when orally inoculated into mice, metacyclic forms of the SC isolate, which express high levels of gp90, produced high parasitemias and high mortality, in sharp contrast with the reduced infectivity in vitro. Upon recovery from the mouse stomach 1 h after oral inoculation, the gp90 molecule of the parasites was completely degraded, and their entry into HeLa cells, as well as into Caco-2 cells, was increased. The gp82 molecule was more resistant to digestive action of the gastric juice. Host cell invasion of SC isolate metacyclic trypomastigotes was augmented in the presence of gastric mucin. No alteration in infectivity was observed in T. cruzi strains CL and G which were used as references and which express gp90 molecules resistant to degradation by gastric juice. Taken together, our findings suggest that the exacerbation of T. cruzi infectivity, such as observed upon interaction of the SC isolate with the mouse stomach components, may be responsible for the severity of acute Chagas’ disease that has been reported in outbreaks of oral T. cruzi infection.     

大蹄蝠全基因组微卫星分布特征分析

脊椎动物基因组含有丰富的微卫星信息。本研究对翼手目动物中的大蹄蝠全基因组及其基因的微卫星分布特征进行分析,并对含有微卫星编码序列的基因进行注释分析。结果表明,大蹄蝠全基因组大小为2.24 Gb,共含有497 883个微卫星,其中,数量和比例最多的是单碱基和二碱基重复类型,分别有173 953个(34.94%)和222 591个(44.71%),相对丰度分别为77.78 loci/Mb和99.52 loci/Mb。微卫星数量从单碱基重复到六碱基重复单元最多的类型分别为(A)n、(AC)n、(TAT)n、(TTTA)n、(AACAA)n和(TATCTA)n,比例分别为95.14%、55.25%、38.41%、22.17%、48.68%和20.30%。不同基因区和基因间区的数量及丰度不同,其中基因间区的微卫星数量及其丰度最大,分别为322 666个和2 541.57 loci/Mb,编码区的微卫星数量及其丰度最小,分别为1 461个和461.98 loci/Mb。基因间区和全基因组的微卫星的分布特征相似。编码区最多的微卫星类型为三碱基重复单元,外显子最多的微卫星类型为单碱基、二碱基和三碱基重...     

Evaluation of the trypanocidal and leishmanicidal in vitro activity of the crude hydroalcoholic extract of Pfaffia glomerata (Amarathanceae) roots

Three different concentrations (1, 10 and 50 μg/ml) of lyophilized hydroalcoholic crude extract of Pfaffia glomerata roots were assayed in vitro against strains of Trypanosoma cruzi (Y) and Leishmania braziliensis. It was observed that P. glomerata hydroalcoholic extract was relatively active within the tested concentrations for L. (V.) braziliensis, but inactive against T. cruzi. Despite the fact that both protozoans belong to the Trypanosomatidae family, we suggest that the difference observed for activity should be related to the biological differences between the two parasite species.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

Detection of polymorphism in the Trypanosoma cruzi TcP2β gene family by single strand conformational analysis (SSCA)

Single strand conformation analysis (SSCA) is a technique that has been used to detect point mutations. We explored its usefulness in the analysis of four different members of the Trypanosoma cruzi TcP2β gene family and its suitability for detection of polymorphism in different parasite strains. The availability of primers covering a 97-bp sequence at the 5′ end of the genes allowed assessment of the effect of a single base substitution, while the analysis of a 321 bp long sequence permitted the evaluation of sequences differing in several bases. PCR products were analysed under four different electrophoretic conditions: with or without the addition of 10% glycerol in a 6% polyacrylamide gel run at room temperature or at 4°C. Shifts in mobility were radically dependent on the migration condition. Both 97-bp and 321-bp amplicons were best resolved at 4°C, without glycerol. Amplification products derived from total genomic DNA showed a pattern that resembled closely a combination of the products derived from the cloned genes. The results herein demonstrate the usefulness of SSCA to differentiate forms of a complex protozoan gene family, and to scan its polymorphic nature. Furthermore, due to the remarkable sensitivity of the technique it can generate genomic markers, such as Sequence Tagged Sites (STS), of great need in the T. cruzi genome project     

Anti-plasmodial and anti-trypanosomal activity of synthetic naphtho[2,3-b]thiophen-4,9-quinones

Naphtho[2,3-b]thiophen-4,9-quinone and five derivatives were prepared using the Friedel-Crafts reaction and tandem-lithiation of aromatic diethylamides. These quinones were evaluated for their trypanocidal and anti-plasmodial activities by their effects on: (1) growth of epimastigote forms of Trypanosoma cruzi in vitro, (2) lysis of trypomastigote forms of T. cruzi in murine blood, (3) growth of Plasmodium falciparum in vitro, and (4) inhibition of the recombinant enzyme trypanothione reductase. The parent compound, naphtho[2,3-b]thiophen-4,9-quinone (3a), was among the most active quinone tested in vitro against P. falciparum at 0.2 μM. However, it was inactive against P. berghei-infected mice treated with 2.3 mmol/kg daily for 5 days. Most of the quinones prepared were active against T. cruzi epimastigotes in culture but exhibited weak activity at 4 °C against trypomastigotes in murine blood as well against the enzyme trypanothione reductase. Further structural modifications will be necessary to improve the in vivo activity of the naphthothiophenquinones.     

西北干旱区孑遗濒危植物四合木(Tetraena mongolica)叶绿体基因组特征研究及比较分析

四合木(Tetraena mongolica)是我国特有的蒺藜科(Zygophyllaceae)强旱生小灌木,因其起源古老、抗逆性强,所以可作为生物多样性起源和环境演变研究的理想对象,具有重要的学术研究价值。本研究采用Illumina双末端测序技术对四合木叶绿体基因组进行建库测序和分析。选取蒺藜目及牻牛儿苗目共计30个物种叶绿体基因组,与四合木进行系统发育关系分析探讨。结果表明:四合木叶绿体基因组长度为106259bp,其中反向重复区(IR区中)有7种基因,包括4种PCG基因,3种tRNA基因。叶绿体基因组共编码98种基因,包括65种蛋白编码基因、29种tRNA基因与4种rRNA基因。生物信息学表明,在四合木中共搜到92个SSR位点,其中包括74个单核苷酸重复基序,7个二核苷酸重复基序,1个三核苷酸重复基序,9个四核苷酸重复基序和1个五核苷酸基序。没有发现六核苷酸,其中单核苷酸重复在四合木的叶绿体基因组SSR中占比为80.1%。通过MEGA软件采用近邻结合法(neighbor-joining,NJ)对四合木等31个物种的叶绿体基因组进行聚类分析,发现四合木与蒺藜科三齿拉雷亚灌木为最近的姐妹种,其次为牻牛儿苗科智利白桦植物亲缘关系较近,与牻牛儿苗科天竺葵属和牻牛儿苗科高桂花属亲缘关系最远,说明四合木属于蒺藜科物种,这对于四合木的研究等具有一定的参考价值。     

An integrated pipeline for the development of novel panels of mapped microsatellite markers for Leishmania donovani complex, Leishmania braziliensis and Leishmania major

A panel of microsatellites mapped to the Leishmania genome might make it possible to find associations between specific loci and phenotypic traits. To identify such loci, a Perl programme was written that scans the sequence of a genome and writes all loci containing microsatellites to a MySQL database. The programme was applied to the sequences of the L. braziliensis, L. infantum and L. major genomes. The database is publicly available over the internet: http://www.genomics.liv.ac.uk/tryps/resources.html 'Microsatellite Locus Extractor', and allows the selection of mapped microsatellites that meet user-defined criteria from a specified region of the selected genome. The website also incorporates a primer design pipeline that will design primers to amplify the selected loci. Using this pipeline 12 out of 17 primer sets designed against the L. infantum genome generated polymorphic PCR products. A tailed primer protocol was used to label all microsatellite primers with a single set of labelled primers. To avoid the culture of parasites prior to genotyping, sets of nested PCR primers were developed to amplify parasite DNA eluted from microscope slides. The limit of detection was approximately 1.6 parasite equivalents. However, only 6/56 DNA from slides stored at ambient temperature for over 6 months gave positive PCR results.     

Genomic heterogeneity in the density of noncoding single-nucleotide and microsatellite polymorphisms in Plasmodium falciparum

The density and distribution of single-nucleotide polymorphisms (SNPs) across the genome has important implications for linkage disequilibrium mapping and association studies, and the level of simple-sequence microsatellite polymorphisms has important implications for the use of oligonucleotide hybridization methods to genotype SNPs. To assess the density of these types of polymorphisms in P. falciparum, we sampled introns and noncoding DNA upstream and downstream of coding regions among a variety of geographically diverse parasites. Across 36,229 base pairs of noncoding sequence representing 41 genetic loci, a total of 307 polymorphisms including 248 polymorphic microsatellites and 39 SNPs were identified. We found a significant excess of microsatellite polymorphisms having a repeat unit length of one or two, compared to those with longer repeat lengths, as well as a nonrandom distribution of SNP polymorphisms. Almost half of the SNPs localized to only three of the 41 genetic loci sampled. Furthermore, we find significant differences in the frequency of polymorphisms across the two chromosomes (2 and 3) examined most extensively, with an excess of SNPs and a surplus of polymorphic microsatellites on chromosome 3 as compared to chromosome 2 (P=0.0001). Furthermore, at some individual genetic loci we also find a nonrandom distribution of polymorphisms between coding and flanking noncoding sequences, where completely monomorphic regions may flank highly polymorphic genes. These data, combined with our previous findings of nonrandom distribution of SNPs across chromosome 2, suggest that the Plasmodium falciparum genome may be a mosaic with regard to genetic diversity, containing chromosomal regions that are highly polymorphic interspersed with regions that are much less polymorphic.