Kinetoplast DNA and molecular karyotypes of Trypanosoma evansi and Trypanosoma equiperdum from China

We compared 12 stocks of Trypanosoma evansi and 1 recently isolated stock of Trypanosoma equiperdum from different regions of China by analysis of kinetoplast DNA (kDNA), nuclear DNA and molecular karyotypes. The T. equiperdum stock was remarkably similar to the T. evansi stocks, except for the possession of kDNA maxi-circles, suggesting a very close evolutionary relationship between T. evansi and T. equiperdum. The maxi-circles of the Chinese T. equiperdum stock were approximately 14.3 kb in size, i.e., about half the size of those of Trypanosoma brucei. This stock is thus similar to an old laboratory stock of T. equiperdum, which also has maxi-circles with a sizeable deletion. Both T. equiperdum and T. evansi kDNA mini-circles hybridised with a T. evansi-specific mini-circle fragment isolated from a Kenyan T. evansi stock. Our results extend the generality that T. evansi and T. equiperdum mini-circles are microheterogeneous rather than homogeneous. Molecular karyotypes obtained by pulsed field gradient gel electrophoresis provided a more sensitive way of distinguishing the T. evansi stocks than isoenzymes or restriction fragment length polymorphisms in kDNA mini-circles, genes for ribosomal RNAs and variant surface glycoproteins. Our results fit the general idea that T. evansi stocks worldwide have a single origin.     

Specific probes for Trypanosoma (Trypanozoon) evansi based on kinetoplast DNA minicircles

Trypanosoma evansi is difficult to distinguish from other members of subgenus Trypanozoon, save for its inability to develop cyclically in the tsetse fly and its characteristic kinetoplast DNA (kDNA). We have used cloned kDNA minicircle fragments as specific probes to distinguish T. evansi from other trypanosomes of subgenus Trypanozoon. Two probes were required, each specific for one of the subgroups of T. evansi previously described. Probe A reacted only with the major isoenzyme group of T. evansi stocks, which have minicircle type A and occur in South America, Kenya, Sudan, Nigeria and Kuwait. The probe did not hybridise with various Trypanosoma brucei spp. stocks, Trypanosoma vivax, Trypanosoma congolense or Trypanosoma simiae, nor with trypanosomes of the minor isoenzyme group of T. evansi stocks found in Kenya with type B minicircles. Probe B was specific for the latter. The probes were sensitive down to a level of 100 trypanosomes in a dot blot. These probes thus provide a simple means of distinguishing T. evansi from T. brucei spp. using comparatively few trypanosomes and without resort to tsetse transmission experiments.     

Kinetoplast DNA from Trypanosoma vivax and T. congolense

We have analysed kinetoplast DNA (kDNA) of the African trypanosomes Trypanosoma vivax and T. congolense. The maxi-circles from these organisms resemble those of T. brucei in size, but only to a limited extent in sequence as judged from restriction enzyme digests and DNA X DNA hybridization. The kDNA networks of T. vivax have three distinguishing features: they contain the highest maxi-circle concentration of any kDNA (at least twice that of T. brucei); they contain the smallest mini-circles (465 bp) yet found thus far and the width of the kDNA nucleoid in thin sections is correspondingly small (55 nm against 91 nm for T. brucei); they contain a substantial fraction of mini-circle dimers.     

Kinetoplast DNA and RNA of Trypanosoma brucei

Kinetoplast DNA (kDNA) and kinetoplast RNA (kRNA) were isolated from bloodstream and procyclic culture forms of two clonal strains of Trypanosoma brucei. No differences were observed in kDNA (maxicircle) restriction profiles between bloodstream or procyclic culture forms of the same strain. Some differences were observed in kDNA maxicircle restriction sites between the two strains. Buoyant density analysis of Pst I digested kDNA showed the release of a minor low density band representing unit length linearized maxicircle DNA. Pst I or Bam H1-linearized maxicircle DNA was isolated by the Hoechst 33258 dye--CsCl method and a restriction enzyme map of the maxicircle was constructed. Closed monomeric minicircles released from kDNA networks by sonication sedimented with a molecular size of around 1100 base pairs. A substantial minor length heterogeneity was evident in acrylamide gel electrophoresis of once cut minicircles. Several minicircle sequence classes and two Hind III maxicircle fragments representing approx. 50% of the maxicircle were cloned in the bacterial plasmid, pBR322, in Escherichia coli. A purified kinetoplast-mitochondrion fraction was isolated from procyclic culture forms by the Renografin flotation method. The major kRNA components were two small RNAs which comigrated with Leishmania tarentolae 9 and 12 S kRNAs in denaturing gels. These RNAs hybridized to the maxicircle component of the kDNA, specifically to the smaller cloned Hind III maxicircle fragment. This cloned fragment had substantial sequence homology with the cloned maxicircle fragment from L. tarentolae which contains the 9 and 12 S RNA genes, implying an evolutionary conservation of the 9 and 12 S gene sequences. Identical kRnAs were observed in cultured bloodstream forms of T. brucei.     

Kinetoplast DNA analysis of four Trypanosoma evansi strains.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a network of thousands of topologically interlocked DNA minicircles and about 50 maxicircles. In this study, we have analysed the kDNA molecules of 6 strains of Trypanosoma evansi from different geographical areas. 2 strains were found to be dyskinetoplastic mutants and other 4 kinetoplastic strains absent of maxicircles. The electrophoretic analysis of the minicircles digested with various restriction endonucleases clearly shows that all of the kinetoplastic strains lack profound minicircle heterogeneity typical of T. brucei. However, a slight restriction fragment length polymorphism could be observed with 2 enzymes (Dde I and HinfI) within the minicircle population of each cloned strain. We propose that this sequence diversity is the result of point mutations. Further analysis of the minicircles by nucleotide sequencing revealed that the 4 minicircles of T. evansi strains share extensive regions of homology with each other but only about 50% homology with other species. This homogeneity of T. evansi minicircle sequences may provide a useful tool for classification and identification.     

Dominant IgM synthesis against the soluble form of the prevailing variant surface glycoprotein from TeAp-N/D1 Trypanosoma equiperdum throughout the experimental acute infections of horses with non-tsetse transmitted Trypanozoon parasites

ABSTRACT

Two horses were infected with distinct non-tsetse transmitted Trypanozoon Venezuelan stocks, namely TeAp-N/D1 Trypanosoma equiperdum and TeAp-El Frio01 Trypanosoma evansi. Preceding reports have revealed that a 64-kDa antigenic glycopolypeptide (p64), which is the soluble form of the predominant variant surface glycoprotein from TeAp-N/D1 T. equiperdum, can be used as a good antigen for immunodiagnosis of animal trypanosomosis. Here, the course of the experimental acute infection in both horses was monitored by evaluating total anti-p64 IgG and particular anti-p64 γ-specific IgG and μ-specific IgM isotypes in sera using indirect enzyme-linked immunosorbent assays. Both equines showed a maximum of whole anti-p64 antibody generation, which dropped to readings below the maximum but always above the positive cutoff point. Levels of specific IgG and IgM isotypes oscillated throughout the course of the experiments. Essentially, the γ-specific IgG response remained very close to the cutoff point, whereas the μ-specific IgM response displayed values that were mostly above the positive cutoff point, showing a major peak that coincided with the maximum of complete anti-p64 IgG production. These results showed that horses infected with non-tsetse transmitted Trypanozoon parasites developed an immune reaction characterized by a dominant IgM generation against the p64 antigen.     

直接PCR法检测血液中伊氏锥虫感染

为建立伊氏锥虫动基体DNA(KinetoplastDNA,kDNA)的PCR扩增技术,血液直接PCR法检测伊氏锥虫的感染,本文以伊氏锥虫kDNA片段为靶扩增DNA设计引物,确定最适PCR反应条件,建立kDNA片段的PCR扩增技术,应用直接扩增缓冲液(Ampdirect)从感染小鼠的滤纸血标本直接PCR检测伊氏锥虫。结果发现,PCR扩增伊氏锥虫kDNA片段分子量为364bp,能检测的最小DNA量是0·06pg,与布氏锥虫、活动锥虫没有交叉反应。应用直接扩增缓冲液不需进行样本的DNA抽提,直接PCR能检测感染小鼠的早期滤纸血样本,敏感性高于镜检法。本实验建立的直接PCR法检测伊氏锥虫具有较高的敏感性和特异性,方法快捷,可进一步应用于伊氏锥虫病的早期诊断以及现场调查。     

Immunological identification of a cAMP-dependent protein kinase regulatory subunit-like protein from the Trypanosoma equiperdum TeAp-N/D1 isolate

Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified R^II-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to the Sus scrofa R^IIα isoform. In order to evaluate whether Trypanosoma equiperdum possessed PKA R-like proteins, parasites from the Venezuelan TeAp-N/D1 strain were examined using the generated anti-R^II IgY antibodies. Western blot experiments revealed a 57-kDa polypeptide band that was distinctively recognized by these antibodies. Likewise, polyclonal antibodies raised in mice ascites against the recombinant T. equiperdum PKA R-like protein recognized the PKA R^II-subunit purified from porcine heart and the recombinant human PKA R^Iβ-subunit by immunoblotting. However, a partially purified fraction of the parasite PKA R-like protein was not capable of binding cAMP, implying that this protein is not a direct downstream cAMP effector in T. equiperdum. Although the function of the S. scrofa PKA R^IIα and the T. equiperdum PKA R-like protein appear to be different, their cross-reactivity together with results obtained by bioinformatics techniques corroborated the high level of homology exhibited by both proteins. Moreover, its presence in other trypanosomatids suggests an important cellular role of PKA R-like proteins in parasite physiology.     

Specific probes for Trypanosoma (Trypanozoon) evansi based on kinetoplast DNA minicircles

Trypanosoma evansi is difficult to distinguish from other members of subgenus Trypanozoon, save for its inability to develop cyclically in the tsetse fly and its characteristic kinetoplast DNA (kDNA). We have used cloned kDNA minicircle fragments as specific probes to distinguish T. evansi from other trypanosomes of subgenus Trypanozoon. Two probes were required, each specific for one of the subgroups of T. evansi previously described. Probe A reacted only with the major isoenzyme group of T. evansi stocks, which have minicircle type A and occur in South America, Kenya, Sudan, Nigeria and Kuwait. The probe did not hybridise with various Trypanosoma brucei spp. stocks, Trypanosoma vivax, Trypanosoma congolense or Trypanosoma simiae, nor with trypanosomes of the minor isoenzyme group of T. evansi stocks found in Kenya with type B minicircles. Probe B was specific for the latter. The probes were sensitive down to a level of 100 trypanosomes in a dot blot. These probes thus provide a simple means of distinguishing T. evansi from T. brucei spp. using comparatively few trypanosomes and without resort to tsetse transmission experiments.     

Susceptibility of dyskinetoplastic Trypanosoma evansi and T. equiperdum to isometamidium chloride

Isometamidium chloride (ISM) is an effective trypanocide with curative and prophylactive activity in husbandry animals. The mode of action of ISM against pathogenic trypanosomes is not fully understood, but there is evidence in the literature that kinetoplastic topoisomerase type II is selectively inhibited by the drug. This prompted the hypothesis that dyskinetoplastic trypanosomes would express a reduced level of susceptibility to ISM. From parental Trypanosoma evansi and T. equiperdum populations we generated clones containing only dyskinetoplastic organisms and clones containing organisms with kinetoplasts. The susceptibility of these clones to ISM was quantified by in vitro assays. The susceptibility of all clones was in the same range. Minor differences in drug susceptibility found between the clones showed that the dyskinetoplastic T.␣evansi and T. equiperdum clones were even more susceptible to ISM than their kinetoplastic counterparts. Thus, the hypothesis that the dyskinetoplastic trypanosomes would be less susceptible to or tolerant of ISM was clearly disproved. The previously demonstrated inhibition of kinetoplastic topoisomerase type II by ISM cannot be the primary toxic effect of the drug on trypanosomes, and the mode of action of ISM needs to be reassessed. Received: 2 April 1997 / Accepted: 14 May 1997     

Beyond replication: Division and segregation of mitochondrial DNA in kinetoplastids

The mitochondrial genome of kinetoplastids, called kinetoplast DNA (kDNA) is a complex structure that must be faithfully duplicated exactly once per cell cycle. Despite many years of thorough investigation into the kDNA replication mechanism, many of the molecular details of the later stages of the process, particularly kDNA division and segregation, remain mysterious. In addition, perturbation of several cellular activities, some only indirectly related to kDNA, can lead to asymmetric kDNA division and other segregation defects. This review will examine unifying features and possible explanations for these phenotypes in the context of current models for kDNA division and segregation.     

Nucleoside analysis of DNA from Trypanosoma brucei and Trypanosoma equiperdum

We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Supelcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.     

Cloning and characterization of a gene coding for a protein (KAP) associated with the kinetoplast of epimastigotes and amastigotes of Trypanosoma cruzi.

We have cloned and characterized a gene of Trypanosoma cruzi which encodes a protein, KAP (kinetoplasts-associated protein), expressed in the kinetoplasts of epimastigotes and amastigotes, the replicative stages of the parasite, but not in kinetoplasts of trypomastigotes. The single-copy gene is transcribed into a 3900-nt polyadenylated mRNA. Its trans-splicing acceptor site is preceded by a run of 15 adenosine residues. An open reading frame of 1052 codons is followed by a 3′ untranslated region containing short sequences characteristic of rapidly degradable RNAs. The potential translation product of the KAP gene contains a central region composed of four blocks of repeats of a 9-amino-acid motif. Rabbit antibodies raised against three synthetic peptides containing KAP sequence recognized a 175-kDa protein in epimastigotes and amastigotes which appears by indirect immunofluorescence to be associated with their kinetoplasts. The antibodies do not recognize the kinetoplast of trypomastigotes. The amino terminus of KAP contains features compatible with mitochondrial topogenic sequences.     

DNA contents and molecular karyotypes of hybrid Trypanosoma brucei

We have used restriction fragment length polymorphism markers to characterise parental and hybrid trypanosome stocks. Unexpected differences in the intensities of Southern hybridisation banding patterns led us to suspect that the hybrid organisms contained more DNA than the parental stocks. This has been confirmed using flow cytofluorimetry (FCF). Hybrids contained significantly more DNA than the parents, both as procyclic organisms (1.5 fold) and as bloodstream forms (1.5-1.6 fold). The DNA contents of both forms were stable through prolonged culture (procyclics), or serial passage (bloodstream forms), although limited data indicated that falls in DNA content could occur in bloodstream forms. FCF analysis of purified nuclei revealed that the increased DNA content of hybrids could be wholly ascribed to nuclear DNA. Our methods are able to detect hybrid organisms with elevated DNA contents in uncloned isolates following cyclical mixed transmission. We have used alternating field electrophoresis techniques to investigate whether the inheritance by the hybrids of the smaller chromosomes could account for their elevated DNA contents. Hybrids lacked the single 500 kb chromosome from one of the parents but appeared to have virtually double the amount of minichromosomes. However, this increase could only account for about 20% of the additional DNA. We are unable at present to distinguish between models for hybrid formation based on the fusion of predominantly diploid cells, and models in which the diploid chromosomes participate in conventional meiosis.     

High immunological response against a Trypanosoma equiperdum protein that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases

Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this protein was specifically recognized by the hyperimmune mice sera. Cellular proliferation assays using splenic B cells from the immunized mice showed higher values when the recombinant T. equiperdum R-like protein was employed than when concanavalin A was utilized as an unspecific mitogen. Two healthy horses that were experimentally infected using either T. equiperdum or Trypanosoma evansi showed a curve response characterized by the appearance of anti-T. equiperdum PKA R-like protein antibody production in sera using indirect ELISA. The recombinant parasite PKA R-like protein was also recognized by sera from naturally trypanosome-infected horses using western blotting. These findings demonstrated that the T. equiperdum PKA R-like protein is an antigen that exhibits cross-reaction with T. equiperdum and T. evansi.     

Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. b. brucei infection

Actin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi actin shows 100%, 98.7%, and 93.1%, homology with Trypanosoma equiperdum, Trypanosoma brucei brucei, and Trypanosoma cruzi. Recombinant actin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant actin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818, and T. b. brucei STIB 940, showing 63.3%, 56.7%, and 53.3% protection, respectively. Serum collected from the rabbit immunized with recombinant actin inhibited the growth of T. evansi, T. equiperdum, and T. b. brucei in vitro cultivation. Serum from mice and rabbits immunized with recombinant actin only recognized T. evansi actin but not mouse actin. The results of this study suggest that the recombinant T. evansi actin induces protective immunity against T. evansi, T. equiperdum, and T. b. brucei infection and may be useful in the development of a vaccine with other cytoskeletal proteins to prevent animal trypanosomiasis caused by these three trypanosome species.     

Kinetoplast DNA signatures of Trypanosoma cruzi strains obtained directly from infected tissues.

We report here a polymerase chain reaction (PCR)-based DNA profiling technique that permits Trypanosoma cruzi strain characterization by direct study of infected tissues. This is based on application of a recently developed method of DNA fragment identification, called low-stringency single specific primer PCR (LSSP-PCR), to the study of the variable region of kinetoplast DNA (kDNA) minicircles from T. cruzi Thus, we can translate the intraspecific polymorphism in the nucleotide sequence of kDNA minicircles into a specific and highly reproducible kDNA signature. Comparison with the phenogram obtained by DNA fingerprinting analysis of a set of T. cruzi strains showed good qualitative correlation between the degree of divergence of the LSSP-PCR profiles and the genetic distance between the strains. kDNA signatures of heart tissue from acutely or chronically infected animals revealed perfect concordance with the patterns obtained from cultured parasites for the CL and Colombiana strains but not for the Y strain, which is known to be multiclonal. However, the match was perfect for studies with two clones of the Y strain. We take this as evidence that in some multiclonal strains there is heterogeneity among the clones in the degree of tropism for the heart tissue. Finally, we showed that it is possible to obtain a T. cruzi kDNA signature from the heart of a human patient with chronic Chagasic myocardiopathy. kDNA signatures obtained by LSSP-PCR of sequences amplified from infected tissues constitute a new tool to study the molecular epidemiology of Chagas' disease.     

The maxicircle of Trypanosoma brucei kinetoplast DNA encodes apocytochrome b

The region of the maxicircle of Trypanosoma brucei kinetoplast DNA which hybridizes at low stringency with the apocytochrome b gene of Saccharomyces cerevisiae has been identified and cloned. The nucleotide sequence of a 1.7 kb segment of this region is reported. This segment contains a single long open reading from capable of coding for a 350 amino acid protein with substantial homology to apocytochromes b of other species. The trypanosome protein is considerably more distantly related to other apocytochromes b than they are to each other. Several short unassigned open reading frames (300 nucleotides or shorter) also are described. If polypeptides are synthesized from these regions, they are more hydrophilic than known mitochondrially coded proteins.     

Experimental Trypanosoma evansi infection in the goat. I. Clinical signs and clinical pathology

A strain of Trypanosoma evansi isolated from an equine case of surra in Mindanao, Philippines was used to infect intravenously two groups (A and B) of five male goats aged 8-10 months. Animals of groups A and B received 5000 and 50 000 trypanosomes, respectively, and five further animals (group C) served as uninfected controls. Four of the 10 infected goats died 8-78 days after inoculation. Group C goats gained weight (mean 22.8 g/day) while infected goats in groups A and B lost weight (means of 21.4 and 45.0 g/day, respectively). Parasitaemia fluctuated regularly between peaks and troughs, with repeated periods of about 6 days during which no trypanosomes were detected in the blood. Clinical signs and clinico-pathological changes in infected goats were not pathognomonic in the absence of parasites in the blood, and leucocytosis was not a reliable indicator of infection. It was concluded that in endemic areas fluctuating fever, progressive emaciation, anaemia, coughing, testicular enlargement and diarrhoea are suggestive of surra; confirmation, however, may necessitate examination of blood every few days for trypanosomes, and possibly other diagnostic tests.