Article

Rapid identification of Leishmania species by specific hybridization of Kintoplast DNA in cutaneous lesions

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Abstract

Kinetoplast DNA (kDNA) was isolated from various species of the protozoic parasite Leishmania and analyzed by nucleic acid hybridization to detect species-related heterogeneity of kDNA. Purified DNA isolated from L. mexicana and L. braziliensis displayed no homology in nucleic acid hybridization studies. These results confirmed that rapid kDNA sequence change and evolution is occurring in New World species of Leishmania and suggested that such isolated kDNA could be used as a specific hybridization probe for the rapid identification of Leishmania species by using whole organisms. This work further demonstrates that such species-specific identification is feasible on isolated Leishmania promastigotes and, more important, directly on tissue touch blots derived from the cutaneous lesion. Thus, specific hybridization of isolated kDNA provides the basis for a rapid, accurate method for the diagnosis of human leishmaniasis directly from infected tissue.

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... El DNA se inmovilizó durante 2 horas a 80 o C con vacío. Los procedimientos de prehibridación y de hibridación se realizaron de acuerdo con lo descrito (9). Sin embargo, las condiciones estándares de la hibridación y de los lavados posthibridación se modificaron para compensar la Tm más baja que resulta del uso de sondas biotinadas, como sigue: la concentración de formamida se redujo de 50% a 45%, se hibridó a 42 o C y se añadieron dos lavados finales con 0.16X SSC a 50 o C durante 15 minutos cada uno. ...
... Nosotros mostramos aquí la identificación de 134 aislados diferentes de Leishmania correspondientes a enfermos con leishmaniasis cutánea de la Península de Yucatán. Hemos identificado las especies de estos parásitos aislados de los enfermos empleando metodología convencional para la purificación de DNA total de los parásitos y para el análisis de hibridación de ácidos nucleicos con sondas no radiactivas de kDNA (9,12). ...
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IDENTIFICATION OF PROTOZOAN FROM THE GENUS LEISHMANIA BY BIOTINYLATED kDNA PROBES IN THE PENINSULA OF YUCATAN, MEXICO. Antecedents.- Recent epidemiological studies have shown that the sylvatic region of the Peninsula of Yucatan in Mexico is an important endemic area of cutaneous leishmaniasis, known as “chiclero’s ulcer”. Parasitological identification is essential for diagnosis and to establish appropiate treatment. Objective.- In this paper, we report the identification of the parasites isolated from patients with “chiclero’s ulcer” in the Yucatan, Mexico by using DNA hybridization techniques with biotinylated kDNA probes. Material and Methods.- All the DNA from 134 different strains of Leishmania was purified, kDNA of two reference strains was isolated and labeled by nick translation. We analysed the 134 DNA preparations by dot blot DNA hybridization with the biotinylated probes and identified the hybrids by streptavidin and a biotin-conjugated alkaline phosphatase. Results.- By using this methodology, we identified 3 strains of Leishmania braziliensis and 131 strains of Leishmania mexicana. Conclusions.- The presence of L. braziliensis has been demonstrated in the Peninsula of Yucatan.
... We sequenced whole genomes of twelve species of Leishmania and one species of Endotrypanum (Table 1). Organisms were grown as promastigotes (Wirth and Pratt, 1982) at 22°C in Schneider's Medium supplemented with 20% heat inactivated FCS and 17.5 mg/mL gentamycin. The cells were pelleted and washed twice in phosphatebuffered saline (PBS). ...
... The cricetids (sigmodontines) encompass approximately 40 genera and more than 200 species that evolved within approximately 2.5 MYA [141]. A similarly rapid rate of evolution is observed in New World Leishmania [141,151]. ...
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The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale.Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate?We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.
... We sequenced whole genomes of twelve species of Leishmania and one species of Endotrypanum (Table 1). Organisms were grown as promastigotes (Wirth and Pratt, 1982) at 22°C in Schneider's Medium supplemented with 20% heat inactivated FCS and 17.5 mg/mL gentamycin. The cells were pelleted and washed twice in phosphatebuffered saline (PBS). ...
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The leishmaniases are a group of diseases caused by different species of the protozoan genus Leishmania and transmitted by sand fly vectors. They are a major public health problem in almost all continents. There is no effective control of leishmaniasis and its geographical distribution is expanding in many countries. Great effort has been made by many scientists to develop a vaccine against leishmaniasis, but, so far, there is still no effective vaccine against the disease. The only way to generate protective immunity against leishmaniasis in humans is leishmanization, consisting of the inoculation of live virulent Leishmania as a means to acquire long-lasting immunity against subsequent infections. At present, all that we know about human immune responses to Leishmania induced by immunization with killed parasite antigens came from studies with first generation candidate vaccines (killed promastigote extracts). In the few occasions that the T cell-mediated immune responses to Leishmania induced by infection and immunization with killed parasite antigens were compared, important differences were found both in humans and in animals. This review discusses these differences and their relevance to the development of a vaccine against leishmaniasis, the major problems involved in this task, the recent prospects for the selection of candidate antigens and the use of attenuated Leishmania as live vaccines.
... 4 Since then, the developments brought by electronic microscopy, molecular biology, biochemistry and immunology have opened new prospects in the taxonomy of leishmaniae. 5 The new methods that started to be employed in the characterization of leishmaniae include especially the study on the development of promastigotes in the phlebotomine vector intestine, 6 the morphometric study of amastigote and promastigote forms at electronic microscopy, 7-9 the electrophoretic motility of isoenzymes, 10 determination of the fluctuating density of nucleus and kinetoplast DNA, 11,12 analysis of DNA degradation products by restricting enzymes, 13 radiospirometry, 14 characterization of specific antigens of external membrane by monoclonal antibodies, 15 DNA/RNA hybridization techniques 16,17 and analysis of kinetoplast DNA by means of amplification technique using polymerase chain reaction. 12,18 Currently, the most used classifications follow the taxonomic model proposed by Lainson & Shaw (1987), 19 which divides the leishmaniae in subgenera of Bahia, among others. ...
... The classification of Leishmania was initially based on ecobiological criteria such as vectors, geographical distribution, tropism, antigenic properties, and clinical manifestations [38][39][40][41]. However, biochemical and molecular analysis showed that pathological and geographical criteria were often inadequate and thus, other criteria such as the patterns of polymorphism exhibited by kinetoplastid DNA (k-DNA) markers, proteins, or antigens came to be used to classify Leishmania [42][43][44][45][46][47][48][49]. ...
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Leishmaniasis ranks the third in disease burden in disability-adjusted life years caused by neglected tropical diseases and is the second cause of parasite-related deaths after malaria; but for a variety of reasons, it is not receiving the attention that would be justified seeing its importance. Leishmaniasis is a diverse group of clinical syndromes caused by protozoan parasites of the genus Leishmania. It is estimated that 350 million people are at risk in 88 countries, with a global incidence of 1-1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Improvements in diagnostic methods for early case detection and latest combitorial chemotherapeutic methods have given a new hope for combating this deadly disease. The cell biology of Leishmania and mammalian cells differs considerably and this distinctness extends to the biochemical level. This provides the promise that many of the parasite's proteins should be sufficiently different from hosts and can be successfully exploited as drug targets. This paper gives a brief overview of recent developments in the diagnosis and approaches in antileishmanial drug discovery and development.
... Hybridization applications include detection of various viruses and viroids (Bar-Joseph and Rosner, 1984;Hull, 1984;Diener and Owens, 1985;Lakshman et al., 1986;Haber, Polston and Bird, 1987;Kingsbury. 1987;Mills, 1987;Varveri, Ravelonandro and Dunez, 1987), fastidious prokaryotes (Borkhsenius, et al., 1987;Kirkpatrick et al.. 1987aKirkpatrick et al.. , 1987bNur, Leblanc and Tully, 1987;Razin etal., 1987;Roberts etal., 1987;Santha etal., 1987;Gobel et al.. 1987), as well as bacteria (Wirth and Piatt, 1982;Fest!. Ludwig and Schleifer, 1986;Palva, 1986;Lazo, Roffey and Gabriel, 1987). ...
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In this review, ways of detecting plant pathogens are divided into specific and non-specific methods. Specific methods are those which are used to detect a particular species or group of pathogens after preliminary diagnosis suggests the presence of that pathogen. Specific methods are also used when the assay involves a known pathogen, for instance as in epidemiological surveys. On the other hand, non-specific methods include those used to detect unknown pathogens or when detection of a number of pathogens is desired
... This kinetoplast DNA (kDNA) consists of two components: the maxicircle, which is a DNA strand of 20,000-40,000 base pairs and is the genetic material encoding mitochondrial genes, and the minicircle, which is a small DNA molecule (an average length of 2,500 base pairs), which has no known coding function but is thought to be involved in the structure or replication (or both) of the kDNA. Previous analysis of the kDNA has demonstrated species-related heterogeneity in the sequence of the kDNA and such differences for new world species and others have demonstrated such differences for old world species of Leishmania and other kinetoplastida [19] [20]. An excellent target for a sensitive and rapid detection method is the kinetoplastminicircle DNA, which is present at thousands of copies per cell. ...
Research
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This study was evaluated the effectiveness of molecular diagnostic assay for leishmaniasis by using PCR test targeting kinetoplastDNA (kDNA). Then it was assessed the efficacy of thIs diagnostic technique for detection the early stage of infection by determining the sensitivity and specificity for the test. In this study five hundred and eighty seven children aged 1-60 months were admitted to paediatric ward of Pediatric and Maternal Hospitals of Al-Najaf, Babil, Karbala, and Al-Qadisiyyah provinces with confirmed or clinically suspected visceral leishmaniasis were included; most of them had clinical manifestation of fever, hepatosplenomegaly, weight loss, Leukopenia, and anemia. The Bone marrow aspiration was carried out for just 38 cases under the supervision of pediatricians at private hospitals in each province. Microscopic examination method applied and detected the Leishmaniadonovani in their Pathological Lab, and then the results of aspirated bone marrow were sent. Only 34 cases were positive of Kala-azar by the demonstration of the parasite-the amastigotes-from direct smear of bone marrow, while 4 cases were negative, LeishmaniainfantumkDNA was detected in 25.21% of patients, and the sensitivity and specificity of PCR test were 85.29 %, 100%, respectively.
... We sequenced whole genomes of twelve species of Leishmania and one species of Endotrypanum (Table 1). Organisms were grown as promastigotes 25 at 22°C in Schneider's Medium supplemented with 20% heat inactivated FCS and 17.5 mg/mL gentamycin. The cells were pelleted and washed twice in phosphate-buffered saline (PBS) and gDNA extracted according to established methods. ...
Preprint
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Article
A molecular tool was developed for L. chagasi identification and its differentiation from L. infantum. Both species have been considered within the Zymodeme MON-1 and are difficult to distinguish from each other using conventional techniques such as Multilocus Enzyme Electrophoresis (MLEE). However a tremendous effort has been done to develop species-specific molecular markers for the diagnosis and identification of such parasites. Here, we used two oligonucleotides designed from the Ca+2ATPase gene in a PCR reaction with genomic DNA from L. donovani complex isolates. Further digestion of the PCR products with Hae III revealed a 600 bp band in L. chagasi. The 600 bp band was excised from the gel, the DNA was purified and used as molecular marker to hybridize a panel of DNA from Leishmania isolates. Results after southern blotting of total DNA digested with different restriction enzymes hybridized to the 600 bp probe labeled with biotin showed 3 hybridizations bands only in L. chagasi. Dot blot hybridization of the PCR product indicated that the 600 bp DNA fragment only hybridized with L(L.) chagasi. This molecular marker is new evidence supporting the status of L. chagasi as a different species in the Leishmania sub genus. No cross hybridization was observed with L.(L.) infantum nor with any other Leishmania species. These results provide a new tool for epidemiological studies since they allow differentiation between two closely related species.
Chapter
Protozoans are an extremely diverse group of unicellular animals. As free-living organisms they are found in virtually every ecological niche, such as bottoms of the deepest marine trenches, salt lakes, edges of ice flows, and even certain thermal springs. As parasites, they infect all species of vertebrates and many invertebrates, in which they have adapted to life in nearly all available sites within the body of the host.
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Chapter
The cutaneous leishmaniases of the Old World are socially and medically important conditions caused by species of the genus Leishmania transmitted by sandflies of the family Phlebotominae. They can be highly disfiguring diseases, and rapid initial diagnosis and identification of the causative organism are extremely important to the subseguent treatment and follow-up of infected individuals. Likewise in any leishmaniasis control programme the rapid and accurate identification of organisms is of paramount importance, especially as most of the leishmaniases are zoonoses (Lainson and Shaw, 1979). Often, species of Leishmania causing different forms of the disease, are endemic to the same geographical region, for example those from a country such as Saudi Arabia where L. major, L. arabica, L. tropica and L. donovani are all found (Peters et al., 1985, 1986).
Chapter
Any of the 15 subspecies of Leishmania at present identified as infective to man can cause the single cutaneous lesion which is the commonest clinical sign of the disease. The wide spectrum of symptoms which may result from the progression of the disease can, at least partially, be related to the subspecies of Leishmania. However the well known problems [1] of genuine or apparent self healing, metastisizing complications of cutaneous manifestations of visceral or secondary infections and immunopathology make clinical diagnosis difficult. The epidemiologists have the time consuming tasks of dissection of sandflies and patient culturing of organisms from the infected tissue of reservoir hosts to incriminate vectors and hosts in transmission. Both the clinical diagnosis and the epidemiologists’ problems could be alleviated by a rapid, cheap and accurate method of identification.
Chapter
DNA hybridization probes are potentially powerful tools to identify pg quantities of bacterial DNA in pure or mixed cultures or directly from infected tissues. Colony hybridization techniques allow in situ application of the probes to identify colonies without prior DNA isolation and purification. The versatility offered by improvements in non-radioactive labeling methodologies may allow widespread and routine uses.
Chapter
Infection with Entamoeba histolytica (amebiasis) is worldwide, extending from the tropics to the subarctic. However, its prevalence is higher, and the disease it produces more serious, in developing countries such as India. Infection with E. histolytica occurs following ingestion of the cyst stage of the parasite. The normal habitat of the ameba is the large bowel, and in most infections the relation between host and parasite is believed to be one of commensaiism. However, in some infections, for reasons poorly understood, the ameba becomes invasive and enters the colonic mucosa, producing colitis and in the worst cases bloody dysentery. Extraintestinal infection of vital organs such as the liver, lung, and brain occurs through hematogenous spread. Both intestinal and extraintestinal disease are life- threatening. Strains of E. histolytica exhibit a broad spectrum of virulence in laboratory animals. At present there are no markers for distinguishing clearly between these strains. None of the indices devised to date, e.g., collagenase activity (3), erythrophagocytosis (10), or isoenzyme patterns (9), provide anything more than the crudest separation of these strains.
Chapter
Leishmaniasis is an infectious disease caused by the protozoan parasite Leishmania. The three common clinical manifestations of the disease are cutaneous leishmaniasis, which is associated with chronic ulcers of the skin; mucocutaneous leishmaniasis, which is caused by the metastatic spread of Leishmania from a primary cutaneous source to the mucous membranes of the nose, mouth, or pharynx, where it causes destructive lesions; and visceral leishmaniasis or kala-azar, which is associated with fever, chronic weight loss, anorexia, marked splenomegaly, and pancytopenia.
Chapter
The leishmaniases are zoonoses caused by intracellular protozoan parasites of the genus Leishmania which are transmitted to man by phlebotomine sandflies. When man is infected a spectrum of clinical conditions may occur, varying from localized or disseminated cutaneous and mucocutaneous lesions to generalized visceral leishmaniasis. The leishmaniases are endemic in many areas of the world, and it has recently been estimated that 12 million individuals have different forms of this protozoan infection.
Chapter
In previous chapters I have referred to the remarkable changes in morphology and especially in physiology exhibited in the life cycles of the African trypanosomes (see Diagram V), causative agents of sleeping sickness in humans and of various diseases of cattle and other domestic animals. Especially striking is the switch in metabolism from the glycerophosphate oxidase system of the bloodstream form to the cytochrome-mediated respiration of the insect form (procyclic), accompanied by loss of infectivity. These changes seem to be mediated by changes in the single mitochondrion. This mitochondrion is unique in having a special region, always immediately adjacent to the basal body of the flagellum, that contains “the most unusual DNA in nature” (as P. Borst has aptly termed it). There is so much DNA that it can be seen by light microscopy after staining with Feulgen or Giemsa stain. Indeed, this was the first extranuclear DNA ever noted, long before DNA had been demonstrated in mitochondria and long before anyone suspected that the kinetoplast is actually a part of the mitochondrion of trypanosomes.
Chapter
The leishmaniases are a group of enzootic and zoonotic diseases caused by morphologically similar parasites in the genus Leishmania (Protozoa: Trypanosomatidae). Mammal reservoirs, of which there are many species (76), may or may not show signs of infection (24, 52, 71). Furthermore, some of the leishmanial species are host-specific and have not been reported in humans (e.g., Le. hertigi of porcupines). Those species that infect man cause an estimated 400,000 new cases each year throughout the world (178). Clinical symptoms of cutaneous, mucocutaneous, and visceral disease in man vary considerably, depending on the species of Leishmania, immunological responses of the individual, and other factors (178). Putative vectors of these diseases are sand flies in the genera Lutzomyia (New World) and Phlebotomus (Old World), but incrimination of specific vectors and mammal reservoirs remains undetermined in many foci (66).
Chapter
Gene transfer in bacteria occurs by three main processes: transformation and transfection, which involve the direct uptake of free DNA by bacteria; transduction, which involves bacteriophage mediated transmission of DNA from one bacterium to another; and conjugation, which involves the direct transmission of genetic material from one bacterium to another and requires cell: cell contacts. These mechanisms, in conjunction with recombination processes, are largely responsible for the rapid evolution of bacteria as a consequence of mutation, variation, and reassortment of genetic traits. The exploitation by geneticists of all three systems of gene transfer has been of crucial importance for the construction of new strains and the analysis and manipulation of a wide range of biological processes. In this chapter, each system is introduced and a typical procedure described. The following publications provide useful background information, Smith et al. (1981); bacterial systems available for transformation and transfection, Suzuki and Szalay (1979); P1 -mediated transduction of markers in E. coli, Willetts et al. (1969), and conjugal transfer of plasmids, Clark and Warren (1979).
Chapter
Parasitic diseases remain major causes of morbidity and mortality throughout the tropical world. Diagnosis of many parasitic diseases is cumbersome and often has inherent difficulties such as serological cross-reactivity with other infections. People living in endemic areas often have more than one infection simultaneously and it is necessary to have accurate diagnostic techniques for specific infections. In addition to diagnosis of overall infection, it is now desirable to be able to distinguish parasites with a particular characteristic, for example, drug resistance.
Chapter
Detection and classification of Leishmania parasites is still laborious and time-consuming. Detection of Leishmania infections depends largely on microscopic screening of smears and tissue sections, whereas classification relies on geographical area, pathology of the infection and more recently immunological and biochemical properties of the parasite. At this moment isoenzyme analysis is the most satisfactory method for the determination of Leishmania parasites. The main disadvantage of this method is that one has to culture the parasites.
Chapter
Leishmania species are among the most fascinating of the protozoal pathogens that infect humans. They are single-celled parasites that are capable of flourishing in widely disparate intracellular and extracellular environments. The Leishmania successfully take sanctuary in mononuclear phagocytes, host defense cells responsible for the ingestion and killing of microbes and the processing and presentation of antigens. The Leishmania bind to a number of macrophage receptors, are ingested by the phagocytes, avoid death by macrophage oxidative and nonoxidative killing mechanisms, and grow and multiply in the acidic environ-ment of the phagolysosome as amastigotes. Not only are the Leishmania able to survive in an intracellular environment that is lethal for other microbes, but in many instances they effectively delay or prevent the development of protective T-cell-mediated immune responses. Leishmanial infections have emerged as model systems for the study of cell-mediated immunity (Wilson and Pearson, 1990).
Article
Leishmania lainsoni has recently been recognized as a new peripylarian species belonging to the subgenus Viannia and the L. braziliensis complex. It has been isolated from its sandfly vector, reservoir host and cutaneous lesions of human patients. Microscopical examination has shown characteristics which are different from those of other members of the L. braziliensis complex. Nuclear deoxyribonucleic acid (DNA) hybridization patterns with a beta tubulin probe and kinetoplast DNA buoyant density measurements show close similarities with other species of the L. braziliensis complex. However, kinetoplast DNA restriction enzyme fragment patterns of L. (V.) lainsoni isolates show similarities to L. mexicana complex species as well as weak cross hybridization. L. (V.) lainsoni is also amplified with L. braziliensis complex specific polymerase chain reaction (PCR) primers but it requires a lower annealing temperature and gives a 300 base pair PCR product. A possible model for the binding of PCR primers to the L. (V.) lainsoni kinetoplast DNA minicircle is proposed.
Chapter
This chapter discusses the host–parasite interactions in leishmaniasis. Parasites have evolved many specific adaptations that enable them to interact with the host. Leishmania are obligatory intracellular parasites in a very narrow range of cells, and therefore, have great specificity for their host cell, the macrophage. This chapter also tries to point out how studies undertaken in order to understand fundamental processes of host–parasite interaction have opened the way for immediate practical applications, which could not have been possible otherwise. The identification of gp63 and LPG as parasite receptors for host has marked these molecules as potential vaccine candidates. The elucidation of the LPG structure has identified novel sugars, which provide unforeseen targets for rational antiparasite drug design.
Article
Full-text available
DNA hybridisation, using probes labelled with 32P, was used to type Leishmania samples isolated from patients living in endemic areas of Mato Grosso State (Brazil), and clinically diagnosed as having tegumentary leishmaniasis. kDNA cloned mini-circle probes specific for the Leishmania mexicana and Leishmania braziliensis complexes were used. The results showed that L.braziliensis is the predominant group infecting human patients in the state. Sixty-eight samples were typed, 64 samples (94.1%) belonging to the L. braziliensis complex and only four (5.9%) belonging to the L. mexicana complex. Accurate identification of the Leishmania permits better orientation of the medical follow-up, since clinical manifestations may vary depending on the complex to which the parasite belongs. The epidemiological information furnished by the identification of the Leishmania in given endemic area is also essential for the design of appropriate control measures
Article
Full-text available
A simple protocol was developed for the routine preparation of a kinetoplast DNA fraction from trypanosomatids. The digestion of this DNA with selected restriction endonucleases, followed by the electrophoretic analysis of the fragments on polyacrylamide gradient gels, yielded characteristic patterns that could be used for the intrinsic characterization of stocks (populations derived by serial passage in vivo and/or in vitro from a primary isolation, without any implication of homogeneity or characterization), strains (sets of populations originating from a group of trypanosomes of a given species or subspecies present at a given time in a given host or culture, and defined by the possession of one or more designated characters), and clones (trypanosomes derived from a single individual by binary fission) of certain pathogenic hemoflagellates.
Article
Full-text available
A simple method was developed for the characterization of different strains of Trypanosoma cruzi. T. cruzi stocks isolated from vectors or by hemoculture from patients with Chagas disease could be grouped in subpopulations having similar patterns of restriction endonuclease products of kinetoplast DNA minicircles. We designate such subpopulations by the term "schizodemes." Furthermore, it is shown that, from a given T. cruzi strain, clones with different biological properties can be isolated and identified by their restriction patterns.
Article
We have constructed a restriction map of the maxicircle component of the kinetoplast DNA of Leishmania tarentolae for the enzymes EcoRI, BamHI, HaeIII, HpaII, SalI, BglII and HindIII. the 9 and 12S kinetoplast RNAs were localized on this map. Two fragments of this maxicircle molecule were cloned in the bacterial plasmid, pBR322, including a 4.4 · 106 dalton EcoRI/BamHi fragment which contains the 9 and 12S RNA genes.
Article
The successful cultivation of a variety of haemoflagellates in three different liquid media is reported. These media include medium 199, Grace's insect tissue-culture medium and Schneider's drosophila medium, each in combination with 30% (v/v) foetal calf serum. These media were used to cultivate Old and New World species of visceral and cutaneous human Leishmania , as well as Leishmania species isolated from sandflies, rodents, and reptiles. Four strains of Trypanosoma cruzi , an isolate of T. rangeli and an isolate of T. lewisi have also been cultivated in these media. One or more of these media have been used to cultivate 121 strains of haemoflagellates, including at least 14 different species (11 Leishmania and 3 Trypanosoma ) and many geographic isolates or strains. The Leishmania include L. braziliensis , L. peruviana , L. mexicana , L. tropica , L. donovani , L. chagasi , L. enriettii , L. hertigi , L. hoogstraali , L. adleri , and L. agamae . Using the Schneider's based medium, we have obtained primary isolates of both cutaneous and visceral Leishmania of man and of experimentally infected laboratory rodents and canines. Freeze-dried preparations of the Schneider's based medium that were reconstituted with distilled water after 24 months of storage at ambient temperature have proven to be suitable cultivation media. This feature makes the media valuable field tools. The various species of human Leishmania cultivated in these media have in our experience demonstrated no differences in growth rate, viability after liquid nitrogen preservation, or infectivity for laboratory animals and tissue-culture cells compared with promastigotes derived from blood-agar cultivation.
Article
Kinetoplast DNA from the mitochondria of Crithidia is in the form of a two-dimensional network of thousands of minicircles each containing about 2.5 kb, and a small number of maxicircles each containing about 40 kb. Fractionation of kinetoplast DNA by equilibrium centrifugation in a CsCl-propidium diiodide gradient resolves it into three types of networks. Form I networks band at high density and contain minicircles which are covalently closed; form II networks band at low density and contain minicircles which are nicked or gapped; and replicating networks band at intermediate density and contain some minicircles of each type. Form I networks contain about 5000 minicircles; form II networks contain about 11,000; and replicating networks contain an intermediate number. When cells are pulse-labeled with 3H-thymidine, radioactivity in mitochondrial DNA is preferentially incorporated into replicating networks, but after a chase it appears first in form II networks and finally in form I. Examination of replicating networks by electron microscopy in the presence of ethidium bromide reveals that minicircles in the central region of the network are twisted and therefore covalently closed, whereas those in the peripheral region are not twisted and therefore must be nicked or gapped. The pulse-label is incorporated into the nicked or gapped minicircles of the replicating networks. These results indicate that replication of form I networks begins in peripheral minicircles and that progeny minicircles remain nicked or gapped. As replication proceeds, the size of the network increases, and the peripheral zone of nicked or gapped minicircles enlarges. Finally, when all minicircles have replicated, the network, now form II is double the size of form I and contains only nicked or gapped minicircles. The final step in replication presumably includes both the cleavage of the network into two form I species and the covalent closure of all the minicircles.
Article
Organisms of genus leishmania exist in two forms: an amastigote form in the human host and a flagellated promastigote in the insect vector. Temperature triggers change from one form to the other. Three clinical types of leishmaniasis are associated with specific organisms: visceral leishmaniasis (Leishmania donovani); mucocutaneous leishmaniasis (L. braziliensis) and cutaneous leishmaniasis (L. tropica and L. mexicana). L. donovani multiples in all parts of the reticuloendothelial system (liver, spleen, bone marrow). L. braziliensis involves skin and mucous membranes; L. tropica is restricted to the integument. Visceral leishmaniasis (Kala-Azar) is characterized by intermittent fever, anemia and marked hepatosplenomegaly in a patient who comes from an endemic area. There is usually an enormous rise in IgG globulin whereas in tropical splenomegaly which closely mimicks kala-azar, the excess globulin occurs in IgM. Amastigote forms may be found in bone marrow smears by means of the Giemsa stain, and the indirect fluorescent antibody test may be useful when parasites are not found in bone marrow smears. The major complications of kalza-azar are pulmonary and intestinal superinfections that are often fatal in the absence of treatment. The most common sites for granuloma formation in mucocutaneous leishmaniasis are the cartilaginous part of the nose, the mouth and the pharynx. The nasal septum is often perforated and sometimes the destruction invades the mouth and lower lids. Over 90% of the patients have a history of cutaneous leishmaniasis years before nasal involvement. In an endemic area studied by the research group of the University of Brasilia, 30% of the active cases had mucous membrane involvement. The mucosal lesions never heal spontaneously in contrast to the lesions of cutaneous leishmaniasis. The isolation of the organism is difficult, since direct smears and culture of biopsies fail to reveal the disease. A positive result from a leishmanin skin test occurs in 90% of the cases and is valuable evidence of infection. Cutaneous leishmaniasis produce skin ulcers on the legs, arms and face and parasites are found mainly in early, closed lesions. The leishmanin test is usually positive 4 to 6 weeks after onset. Pentavalent antimonials are the most effective drugs for all forms of leishmaniasis. Two compounds are in common use: sodium antimony gluconate (pentosam, Burroughs Wellcome) and antimony-N-methyl-glutamine (Glucantime, Rhodia). Both are given by slow i.v. injection or by i.m. injection during a 10-day course. Resistant cases may require other drugs, such as pentamidine or amphotericin B. (Mattar - Sao Paulo)
Article
SYNOPSIS. The occurrence and levels of activity of various enzymes of carbohydrate catabolism in culture forms (promastigotes) of 4 human species of Leishmania (L. brasiliensis, L. donovani, L. mexicana, and L. tropica) were compared. These organisms possess enzymes of the Embden-Meyerhof pathway but lack lactate dehydrogenase. No evidence could be found for the production of lactic acid by growing cultures and lactic acid could not be detected either in cell-free preparations or after incubation of cell-free extracts with pyruvate and NADH under appropriate conditions. All 4 species possess α-glycerophosphate dehydrogenase and α-glycerophosphate phosphatase which together could regenerate NAD, thus compensating for the absence of lactate dehydrogenase. The oxidative and nonoxidative reactions of the hexose monophosphate pathway are present in all 4 species. Cell-free extracts have pyruvate dehydrogenase activity which allows the entry of pyruvate into and its subsequent oxidation through the tricarboxylic acid cycle. All enzymes of this cycle, including a thiamine pyrophosphate dependent α-ketoglutarate dehydrogenase are present. Both NAD and NADP-linked malate dehydrogenase activities are present. The isocitrate dehydrogenase is NADP specific. There is an active glutamate dehydrogenase which could compete with α-ketoglutarate dehydrogenase for the common substrate (α-ketoglutarate). Replenishment of C4 acids is accomplished by heterotrophic CO2 fixation catalyzed by pyruvate carboxylase. All 4 species have high levels of NADH oxidase activity. Several enzymes thus far not found in any species of Leishmania have been demonstrated. These are: phosphoglucose isomerase, triose phosphate isomerase, fructose-1, 6-diphosphatase, 3-phosphoglycerate kinase, enolase, α-glycerophosphate dehydrogenase, α-glycerophosphate phosphatase, pyruvate dehydrogenase complex, citrate synthase, aconitase, α-ketoglutarate dehydrogenase, glutamate dehydrogenase, and NADH oxidase.
Article
1. Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields "extra" bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of "extra" bands. The "extra" bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weight, calculated from mobility in gels, are around 2 X 10(7). Double digests with two restriction endonucleases yield a new set of "extra" bands, showing that the "extra" bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the "extra" bands. 2. Treatment of kDNA networks with the single-strand-specific S1 nuclease of Aspergillus oryzae preferentially releases a linear DNA with a molecular weight of 26 X 10(6), calculated from mobility in gels. We present evidence that the 'extra' bands obtained with restriction endonucleases are derived from this component. 3. DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 X 10(7), making up less than 10% of the total kDNA. 4. From these results we conclude that 3--5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 X 10(6) and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.
Article
We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 X 10(6), i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts. DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 X 10(6), i.e. twice the molecular weight on one mini-circle. The delta tm of native and renatured duplexes is about 1 degree C, showing that the sequence heterogeneity is a micro-heterogeneity. Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the 'rosette' structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini-circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together. Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the mini-circles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence. Possible mechanisms that could account for the micro-heterogeneity in sequence of mini-circles are discussed.
Article
A large number of Leishmania isolates were examined for variation in electrophoretic mobility of malate dehydrogenase (MDH), using a disc polyacrylamide gel method. Numerous MDH variants were demonstrated. The results are correlated with those from DNA buoyant density determinations on the same isolates, and a preliminary summary of the biochemical taxonomy of Leishmania is presented.
Article
The buoyant density of total cell DNA was determined for a large number of Leishmania isolates by isopycnic centrifugation in CsCl. A wide spectrum of values was found and the results are briefly discussed in terms of the taxonomy of Leishmania. Isolated K DNA was examined by electron microscopy using the Kleinschmidt technique and measurements made of free minicircles. No difference in minicircle contour length was found between the species studies.
Article
The frequent difficulty of establishing a parasitologic diagnosis in American leishmaniasis has established the need for a reliable serologic test, which has not been available up to this time. Preliminary attempts to adapt the indirect fluorescent antibody test were unsuccessful when promastigote forms of a recent human isolate of Leishmania braziliensis from culture were used as antigen. However, the use of intracellular amastigote forms. freed from Vero tissue culture cells by osmotic disruption, resulted in reproducible specific reactions in 89% of 75 sera of leishmaniasis patients. Reactions up to a dilution of 1:1,024 were obtained, with a mode at 1:16. In comparison, 93% of control sera from 100 healthy persons and 75 hospital patients with other diseases gave completely negative reactions, and the great majority of those nonspecific reactions which did occur were at a dilution of 1:8. This test has proved to be a useful adjunct to other diagnostic procedures.
Article
We have used the method of cytological hybridization with complementary RNA to study the base sequence homology between kinetoplast DNAs of different hemoflagellates. 3H-labelled complementary RNA (spec. act. 20 × 106 dpm/μg) was synthesized on purified kinetoplast DNA with RNA polymerase of Escherichia coli. Smears of hemoflagellates were fixed with a formaldehyde-containing fixative, the DNA was heat-denatured in situ in the presence of 50% formamide and hybridization with complementary RNA was carried out with 20 μl RNA at 0.6 μg/ml. Unspecifically bound RNA was removed by treatment with ribonucleases and hybridization was quantitatively evaluated on autoradiograms with a microphotometer. In intraspecific hybridization reactions with Crithidia luciliae and Trypanosoma mega, RNA was bound only to the kinetoplast and not to the nucleus. In the interspecific hybridization reaction no cross-hybridization was found at all, either on cytological preparations or with kinetoplast DNA fixed to nitrocellulose filters. This shows that the evolution of a major part of kinetoplast DNA is not highly restricted. Cytological hybridization may, therefore, provide a useful method to differentiate closely related species of pathogenic hemoflagellates.
Article
The major kinetoplast DNA complement of Leishmania tarentolae promastigotes has been isolated as a single sheet of interconnected molecules on the basis of its relative stability to shear forces and its high sedimentation coefficient. Two successive differential centrifugations were sufficient to recover 50 ± 10% of the total kinetoplast DNA free of nuclear DNA contamination. The term 'network' is used to describe this unusual type of DNA configuration. Leishmania networks have a molecular weight of 1010 daltons and an S(20,W) in neutral sucrose gradients of 1729 ± 189 [n = 19] and exhibit an extremely low specific viscosity due to the compactness of packing of the DNA. The networks were visualized in the electron microscope, and in the light microscope either by fluorescence in solution after staining with acridine orange or in dried smears after staining with Giemsa. Purified networks from stationary phase cells banded in the position characteristic of closed monomeric minicircles in ethidium bromide CsCl equilibrium gradients, and were stable in alkaline sucrose. Treatment of the closed networks with RNase and pronase had no effect on the ethidium bromide CsCl banding pattern. However, treatment of closed networks with DNase I or II, X irradiation or γ irradiation changed the banding pattern by introducing single strand and double strand breaks, yielding an upper band and in some cases an intermediate band.
Article
An immunoenzymatic diagnostic technique applicable to cutaneous leishmaniasis is described. The antigen used (Leishmania tropica major) was equally useful in diagnosing visceral and mucocutaneous forms of the disease. The criteria for positivity were defined by using groups of negative controls, and the specificity of the reaction was evaluated by using sera from patients with various diseases. Among these, sera from patients with lepromatous leprosy, tuberculosis, or African trypanosomiasis strongly cross-reacted with leishmania antigen. Examining serial dilutions of the sera facilitated the interpretation of the results and eliminated a significant percentage of false positives.
Article
We have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved by any of the eight enzymes and that varies in size over 1 kilobase pair in the strains analysed. Mini-circles from these five strains, digested with endonuclease TaqI or MboII, yield very complex fragment patterns, showing that extensive mini-circle sequence heterogeneity is a common characteristic of these T. brucei strains. The size distribution of mini-circle fragments in these digests was identical for different clones of the 427 strain, but very different for mini-circles from different strains. These results show that maxi-circle sequence is conserved, whereas mini-circle sequence is not. Restriction digests of maxi-circles could be useful in determining how closely two Trypanosoma strains are related, whereas mini-circle digests can serve as sensitive tags for individual strains.
Article
Kinetoplast DNA (kDNA) has been isolated from the human cutaneous Leishmania isolates, L. tropica major, L. aethiopica and an unknown Kenyan isolate, Leishmania SP48. DNA sequence relationships among these isolates have been studied by restriction enzyme digestion and two phase hybridisation to Southern blots of kDNA covalently coupled to diazobenzyloxymethyl (DBM) paper. The results of this analysis confirm that rapid kDNA sequence evolution is occurring in the Old World leishmanias although some sequence conservation in defined regions of the mini-circle sequence is present. These results emphasise the danger of constructing a rigid Leishmania classification on buoyant density data alone. The covalent binding of kDNA electrophoretic separations to DBM paper permits the construction of a DNA sequence "library' which can be used in the classification and diagnosis of unknown Leishmania isolates.
Article
Bacterial clones containing inserted DNA sequences specific for α-tubulin, β-tubulin, β-actin and γ-actin have been constructed from mRNA of embryonic chick brain. Plasmids containing approximately 75, 90 and >90%, respectively, of the sequences present in α-tubulin, β-tubulin and β-actin mRNAs have been isolated as well as clones containing parts of the extensive 3′ untranslated regions of the β- and γ-actin mRNAs. The sequences for the two tubulins do not cross hybridize. Hybridization of labeled, cloned probes for each of the tubulins with chicken DNA digested with several restriction endonucleases reveals about four fragments for α- and four for β-tubulin. This seems to be the number of genes, since both the 5′ and 3′ ends of either cloned tubulin cDNAs hybridize to at least four common fragments in genomic DNA which has been digested with restriction endonucleases. The tubulin probes are able to hybridize under stringent conditions to DNA of all vertebrate genomes tested, as well as to sea urchin DNA, but not to yeast DNA. In digested sea urchin sperm DNA there are more than 20 different fragments which hybridize to both the 5′ and 3′ ends of the tubulin cDNAs. A full-length β-actin cDNA clone hybridizes to 4–7 bands in restricted chicken DNA and cross hybridizes to DNA from every other species tested, including sea urchin and yeast. Hybridization to chicken DNA of cloned probes specific for the 3′ untranslated regions of β- and γ-actin mRNA indicates that the β sequence is present only once in the genome and the γ is present in at most three copies. Neither 3′ untranslated sequence is conserved evolutionarily.
Article
Kinetoplast DNA of Trypanosoma brucei is composed of a network of about 10,000 interlocked minicircle DNA molecules (1.0 kilobase) that are catenated with about 50 maxicircle DNA molecules (23 kilobases). Several different DNA . DNA hybridization techniques using individual minicircle DNA sequences cloned in Escherichia coli have indicated that each minicircle molecule contains about one-fourth of its sequence in common with most other minicircles and the remaining three-fourths in common with about 1 out of every 300 minicircles. We have determined the complete sequence of two cloned minicircle DNA molecules that were released from the total kinetoplast DNA network by different restriction enzymes; one minicircle is 1004 base pairs long, the other is 983 base pairs. Both are about 72% dA + dT. They share about 27% of their sequences; the largest continuous region in common is 122 base pairs of near-perfect homology. Twelve other regions of perfect homology equal to or greater than 10 base pairs are also present. Both sequences contain a large number of translation termination codons in all potential translation reading frames. The largest oligopeptide potentially specified by one minicircle sequence is 52 amino acids; the largest by the other minicircle sequence is 71 amino acids. One minicircle contains a decanucleotide sequence that is repeated in tandem five times. It is proposed that massive recombination among the interlocked minicircles in the kinetoplast DNA network may account for much of the homology observed in the two minicirce sequences.
Article
Ultrastructural analysis has been carried out on three Leishmania isolates which are proven causal agents of human cutaneous Leishmaniasis, L. tropica major, L. aethiopica and a unidentified species, Leishmania SP48. No significant differences in submicroscopic morphology have been found in thin-sectioned organisms from the three isolates. Extensive plate cristae have been observed within the mitochondria and connections between the rim of the kinetoplast nucleoid and the inner mitochondrial membrane noted. Kinetoplast DNA (kDNA) has been isolated from these isolates and from L. tarentolae and examined by protein monolayer spreading and darkfield electronmicroscopy. The basic molecular arrangement of isolated kDNA in the form of 5 micrometers networks of 0.28--0.3 micrometer mini-circles with long looped DNA in the interior and at the periphery of networks is similar in all isolates. Minor differences between L. aethiopica and SP48 compared with L. tropica major have been observed. The kDNAs of L. aethiopica and SP48 are identical morphologically. Buoyant density analysis has shown that kDNA from L. aethiopica and SP48 have identical values and these are different from the values for L. tropica major. The finding of similar buoyant densities for kDNA from L. tropica major and L. tarentolae also imply a sequence homology by this criteria which is refuted by the results given in the following paper. The results given in this and the following paper (Arnot, D.E. and Barker, D.C.(1981) Mol. Biochem. Parasitol. 3, 47--56 indicate that the unknown Leishmania SP48 is very closely related to, if not identical with, L. aethiopica. This finding is consistent with the clinical and ecological facts known for the organism SP48.
Article
A total of 125 wild mammals (14 different species) were examined for evidence of infection with Leishmania in an area of primary forest highly endemic for "pian-bois", due to Leishmania braziliensis guyanensis, in north Pará State, Brazil. Parasites isolated were characterized biologically, and biochemically on enzymic profiles. L. b. guyanensis was isolated from the viscera of one lesser anteater (Tamandua tetradactyla) and one opossum (Didelphis marsupialis), and the skin of one rodent (Proechimys guyannensis). The isolates were indistinguishable from 10 others previously made from the sandfly vectors Lutzomyia umbratilis (five) and Lu. whitmani (five), and nine isolates from field-workers who became infected during these studies. Leishmania mexicana amazonensis was obtained from the skin of 21 animals, including three species of opossums (D. marsupialis, Philander opossum and Metachirus nudicaudatus) and two species of rodents (proechimys guyannensis and Dasyprocta sp.). A peripylarian Leishmania isolated from the viscera of two armadillos (Dasypus novemcinctus) was shown to be different, biologically and biochemically, from L. b. guyanensis and L. m. amazonensis. Four other isolates of Leishmania, from the rodents Rhipidomys leucodactylus (one) and P. guyannensis (three) have yet to be characterized owing to their very poor growth in both hamster skin and in vitro culture: they appear closest, however, to L. braziliensis braziliensis. The complexity of Amazonian leishmaniasis is discussed, and attention drawn to the importance of edentates as reservoir hosts of some leishmanias in the New World. Whereas L. mexicana subspecies appear largely restricted to the skin of their natural hosts, subspecies of L. braziliensis are commonly found in the viscera.
Article
30 Brazilian stocks of Leishmania mexicana amazonensis and 13 stocks of subspecies of Leishmania hertigi were characterized by starch-gel electrophoresis, using 18 enzymes selected from a total of 36 investigated. L. m. amazonensis was separable from subspecies of L. hertigi by enzymic profiles of 11 enzymes. The L. m. amazonensis stocks, which were from a wide range of hosts in a large geographical area, were enzymically extremely homogeneous, and could only be subdivided on two enzymes; sub-groups did not relate to each other or to any differences in epidemiological characters, including the clinical form of the human disease. 12 stocks regarded as L. hertigi deanei, that were isolated from Coendou prehensilis prehensilis and Coendou sp. in Pará State, Brazil, were separable into two sub-groups by three enzymes. A single stock of L. hertigi hertigi from Panama was separable from both enzymic sub-groups of L. h. deanei, in each case by three enzymes. The significance of these and other characters of diversity is discussed, together with the use of enzymes for the identification of the leishmaniae.
  • N Masuda
  • L Simpson
  • H Rosenblatt
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