Erythroid progenitor cells and stimulating factors during murine embryonic and fetal development.

Murine embryonic and fetal yolk-sacs, peripheral blood, and livers were assayed for hemopoietic multipotential and progenitor cell content between days 6 and 13 of gestation. Multipotential cells (Mix-CFC), erythroid-committed progenitor cells (BFU-E), and nonerythroid progenitor cells (predominantly GM-CFC) were assayed by their ability to form hemopoietic colonies in vitro when stimulated by pokeweed-mitogen-stimulated spleen-cell-conditioned media (as a source of Multi-CSF) and either human or murine erythropoietin. Late erythroid progenitor cells (CFU-E) were stimulated to form colonies by erythropoietin. Mix-CFC, BFU-E, and nonerythroid cells were first detected on day 8 in yolk-sacs, day 9 in peripheral blood, and day 11 in liver. Maximum absolute numbers of yolk-sac Mix-CFC (182), BFU-E (331), and non-erythroid CFC (1358) occurred at 11 days of gestation. The maximum frequency of peripheral blood mix-CFC (24/10(5) cells) and BFU-E (55/10(5) cells) occurred at ten days of gestation. The absolute numbers of hepatic Mix-CFC, BFU-E, nonerythroid CFC, and CFU-E increased exponentially from 11 to 13 days' gestation. CFU-E were first detected at nine days in peripheral blood, at ten days in yolk-sac, and 11 days in liver and at all ages were equally responsive to erythropoietin. The maximum frequency (151/10(5) cells) of CFU-E in the peripheral blood and the maximum number per yolk-sac (1699) both occurred on day 11 of gestation. In confirmation of previous studies, yolk-sac fluid was found to contain a macrophage colony-stimulating activity. In addition, an activity capable of stimulating fetal liver CFU-E was also detected in yolk-sac fluid. However, no activity (Multi-CSF) capable of stimulating Mix-CFC or BFU-E was detected in either yolk-sac fluid or fetal plasma.

The ATPase subunit 6 gene sequence predicts that RNA editing is conserved between lizard- and human-infecting Leishmania.

Here we investigate the similarities in the kinetoplastid RNA editing process between human- and lizard-infecting Leishmania species. We present the sequence of the maxicircle-encoded ATPase subunit 6 gene from L. (V.) panamensis, L. (L.) mexicana and L. (L.) donovani species of human-infecting Leishmania. These represent the first available sequences of this gene from Leishmania species other than the lizard-infecting L. tarentolae. The gene sequences are highly conserved, both over the edited and unedited parts of the gene, implying that the RNA editing process is likely to be highly conserved between Leishmania species. Indeed, the first editing domain is absolutely conserved in all three Leishmania species studied and L. tarentolae. A phylogeny based on part of the ATPase subunit 6 gene placed the lizard-infecting Leishmania within the monophyletic Leishmania genus, supporting previous data which suggest that lizard- and human-infecting Leishmania species are closely related.

Analysis of the genomic organisation of a small chromosome of Leishmania braziliensis M2903 reveals two genes encoding GTP-binding proteins, one of which belongs to a new G-protein family and is an antigen.

Leishmania braziliensis M2903 contains a highly amplified small chromosome. This work is aimed at resolving its structural organization and determining whether this unusual chromosome contains specific genes encoding proteins with important functions in disease pathology or drug resistance. Our results show that the M2903 250-kb small chromosome contains LD1 sequences and has an inverted repeat structure. The LD1 sequences and two cDNAs (cDNA2 and cDNA53) were mapped on a cosmid contig, and the two cDNAs and the corresponding genomic fragments from the small chromosome were sequenced. The gene encoding cDNA2 predicts a putative GTP-binding protein with homology to other GTP-binding proteins only in the G-1 domain region; however, four other conserved motifs can be recognized. Sequence similarity to cDNA53 is located in at least five chromosomes, and its small chromosome copy is a pseudogene. An open reading frame downstream of the cDNA53 pseudogene predicts another GTP-binding protein that belongs to a new G-protein family with an unusual conserved GTP-binding domain and a newly characterized conserved sequence motif. A portion of this GTP-binding protein gene was studied previously in L. aethiopica as a recombinant antigen that reacts with human antibodies.

Biochemical identification of cutaneous leishmanias by analysis of kinetoplast DNA. II. Sequence homologies in Leishmania kDNA.

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.

Biochemical identification of cutaneous leishmanias by analysis of kinetoplast DNA. I. Ultrastructural and buoyant density analysis.

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.

The origin and evolution of the Leishmania donovani complex as inferred from a mitochondrial cytochrome oxidase II gene sequence.

Members of the Leishmania donovani complex are parasites of the reticulo-endothelial system that are often associated with serious epidemics of a life threatening disease known as visceral leishmaniasis or kala-azar. Twenty-two Leishmania isolates representative of the geographical range of the parasite were analysed for sequence variations in their cytochrome oxidase II gene. In performing phylogenetic analysis, the maximum parsimonious, neighbour joining and maximum likelihood trees were congruent and produced a tree that differentiated between two clades conforming to the current classification of the species complex into two species: Leishmania donovani and Leishmania infantum. Furthermore, the molecular haplotypes were concordant, in general, with the isoenzyme data of the complex. The donovani isolates from the Sudan that possessed the most ancestral sequence were of a single haplotype that significantly resembled the sequence of Leishmania major. Our sequence data tallied with a general neutral model of sequence evolution with manifestations of weak selection. The data allowed an approximate dating of the origin of the complex to a period contemporary to or predating the spread of modern humans out of Africa.

Identification of the C-terminal region of 70 kDa heat shock protein from Leishmania (Viannia) braziliensis as a target for the humoral immune response.

A Leishmania (Viannia) braziliensis (Lb) promastigote cDNA library in lambda gt11 was screened with patients' sera with the aim of identifying antigens specifically related to mucocutaneous leishmaniasis (MCL). One of the clones isolated, 133P, consistently reacted with MCL sera; it was sequenced and found to encode the C-terminal three-quarters of a protein belonging to the highly conserved Hsp70 family. Since Hsp70 proteins from different species tend to be less conserved through the C-terminal end, it was predicted that this region would be more antigenic and was likely to bear the discriminatory epitopes. In order to test this hypothesis, the N- and C-terminal halves of the polypeptide encoded by 133P, 133P-N and 133P-C, respectively, were expressed in Escherichia coli. Immunoblotting analysis demonstrated that 133P-C reacted more strongly with a pool of MCL sera than 133P-N, and both recombinant proteins reacted faintly with pools of cutaneous (CL) and visceral (VL) leishmaniasis sera. These results confirmed the predicted epitope location in the C-terminal region. The 133P-C fragment was also expressed as a fusion protein with glutathione-S-transferase (GST-133P-C), affinity-purified with glutathione-agarose and tested by ELISA with individual sera. From 46 Lb-infected patients, 41 sera (89%) were positive, no cross-reactivity was observed with healthy, Trypanosoma cruzior L. amazonensis-infected individuals. Despite a relatively high cross-reactivity with VL sera, the enhanced humoral response of MCL as compared with CL patients might be interesting for studies on disease aggravation.

Cardiovascular and Valsalva responses during parabolic flight.

We investigated the integrated cardiovascular responses of 15 human subjects to the acute gravitational changes (micro- and hypergravity portions) of parabolic flight. Measurements were made with subjects quietly seated and while subjects performed controlled Valsalva maneuvers. During quiet, seated, parabolic flight, mean arterial pressure increased during the transition into microgravity but decreased as microgravity was sustained. The decrease in mean arterial pressure was accompanied by immediate reflexive increases in heart rate but by absent (or later-than-expected) reflexive increases in total vascular resistance. Mean arterial pressure responses in Valsalva phases IIl, III, and IV were accentuated in hypergravity relative to microgravity (P < 0.01, P < 0.01, and P < 0. 05, respectively), but accentuations differed qualitatively and quantitatively from those induced by a supine-to-seated postural change in 1 G. This study is the first systematic evaluation of temporal and Valsalva-related changes in cardiovascular parameters during parabolic flight. Results suggest that arterial baroreflex control of vascular resistance may be modified by alterations of cardiopulmonary, vestibular, and/or other receptor activity.

Geographic distribution and clinical description of leishmaniasis cases in Peru.

Studies were conducted from 1986 through 1993 to further define the geographic distribution and relative importance of different species of Leishmania as a cause of leishmaniasis in Peru. Patients with a clinical diagnosis of cutaneous and/or mucosal or diffuse cutaneous leishmaniasis were enrolled at the Naval Medical Research Institute Detachment (NAMRID) Laboratory in Lima, the Tropical Disease Clinic at San Marcos University Daniel A. Carrión, the Central Military Hospital, and a Ministry of Health hospital in Cusco, Peru. Clinical features, lesion aspirates, and biopsy tissue were obtained from each patient. All specimens were collected and assayed separately, including multiple specimens from some of the same patients for Leishmania parasites by inoculating aliquots of either aspirates or biopsy tissue suspensions onto Senekji's blood agar medium. Stocks of Leishmania isolates were used to prepare promastigotes to produce extracts for identifying the Leishmania species by the cellulose acetate electrophoresis enzyme technique. A total of 351 isolates of Leishmania were obtained from 350 patients who were infected primarily in the low and high jungle of at least 15 different Departments of Peru. Of the 351 isolates, 79% were identified as L. (V.) braziliensis, 7% as L. (V.) guyanensis, 10% as L. (V.) peruviana, 2% as L. (V.) lainsoni, and 1.7% as L. (L.) amazonensis. The clinical form of disease varied depending on the species of Leishmania, with L. (V.) braziliensis being associated most frequently with cutaneous, mucosal ulcers and mixed cutaneous and mucosal disease, and L. (V) peruviana, L. (V.) guyanensis, L. (V.) lainsoni with cutaneous lesions. Leishmania (L.) amazonensis was isolated from six patients, three with cutaneous lesions, one with mucosal lesions, and two with diffuse cutaneous lesions. Among all of the leishmaniasis cases, males were affected more frequently, and cases occurred among patients less than 10 to more than 51 years of age. These data further defined the geographic distribution and the relative frequency of Leishmania species associated with different clinical forms of leishmaniasis in Peru.

Kala-azar in a high transmission focus: an ethnic and geographic dimension.

In 1994-1996, we studied a group of 58 game wardens stationed in an area known to be highly endemic for visceral leishmaniasis (kala-azar) for evidence of infection with Leishmania donovani. Leishmania DNA was detected by the polymerase chain reaction in the peripheral blood of cases of active kala-azar, former patients with visceral leishmaniasis, patients, and asymptomatic subjects. Using the cloned antigen rk39, antibodies were detected in 44.2% of the game wardens while leishmanin skin test result was positive in 77% of our sample. It was shown that certain tribes from northern Sudan were more likely to develop subclinical infections, while those of the Baria tribe from southern Sudan and those of the Nuba tribe from western Sudan were more likely to develop visceral leishmaniasis. Whether this is due to genetic factors or previous exposure to Leishmania parasites remains to be elucidated.

Analysis of minicircle classes in Leishmania (Viannia) species.

This paper reports the analysis of minicircle sequence classes from 4 Leishmania species, all belonging to the 'New World' species of the subgenus Viannia: Leishmania braziliensis, L. guyanensis, L. panamensis and L. peruviana. A minicircle library was constructed for each species, and clones were analysed by restriction enzyme digest and sequence analysis. 319 minicircles from the 4 species were examined and 96 of these were wholly or partially sequenced. The sequences of 41 whole minicircles--21 from L. panamensis, 8 from L. guyanensis and 6 each from L. braziliensis and L. peruviana are presented. Sequence classes were identified within which sequences were highly conserved, with only a small number of single base pair changes between them. In contrast, minicircles from different classes differed significantly, displaying sequence homology only over the minicircle conserved region. Some minicircle classes were identified which were shared between species. These minicircles displayed sequence variation which was potentially species-specific, and were analysed phylogenetically. These results question the hypothesis that minicircle sequence is rapidly evolving and also suggest that an as yet unknown selective pressure maintains sequence class conservation over the entire minicircle molecule even in different species, not only over the conserved region and the guide ribonucleic acid gene. A novel hypothesis is proposed to explain these results.

The use of DNA probes in the identification of leishmanias: discrimination between isolates of the Leishmania mexicana and L. braziliensis complexes.

Kinetoplast DNA (kDNA), initially characterized by buoyant density, from ten reference isolates of the Leishmania braziliensis and L. mexicana complexes has been radio-actively labelled and used as hybridization probes. Filters containing endonuclease digested, electrophoresed, Southern transferred fragments of kDNA from reference and other isolates sent to us for DNA typing have been tested for kDNA sequence homology. We record a complete lack of sequence homology between kDNA of any isolate of the L. braziliensis complex and kDNA of any isolate of the L. mexicana complex. L. b. braziliensis, L. b. guyanensis and L. b. panamensis have kDNA sequences in common with each other and with a number of test isolates from Brazil, Panama, Venezuela and Peru. L. b. panamensis (1.695 g/ml) can be separated from L. b. braziliensis or L. b. guyanensis (1.691-1.693 g/ml) by differences in buoyant density of kDNA. L. m. mexicana and L. m. pifanoi have kDNA sequences in common with each other but kDNA of L. m. amazonensis has insignificant homology with kDNA of other reference isolates of the L. mexicana complex. We conclude that the kDNAs of species of the L. mexicana complex are sufficiently different from kDNA of species of the L. braziliensis complex to make kDNA sequence homology identification a feasible proposition.

Detection of Leishmania braziliensis in naturally infected individual sandflies by the polymerase chain reaction.

The natural infection of sandflies by Leishmania in wild-caught specimens was studied, using the polymerase chain reaction (PCR)-hybridization technique. The PCR was carried out using 2 oligonucleotides (primers 3J1 and 3J2) derived from a repetitive nuclear DNA sequence. The primers support the enzymatic amplification of a fragment of approximately 500 bp, present in the nuclear DNA of Leishmania braziliensis. The expected band was observed in 5 of 65 sandflies containing flagellates. After hybridization with a species-specific probe, we confirmed natural infection by L. braziliensis. The technique allowed the identification of Lutzomyia gomezi and Lu. panamensis as vectors of L. braziliensis in an endemic area of cutaneous leishmaniasis in Urama, Puerto Cabello district in Venezuela. As far as we are aware, this work constitutes the first report of natural infection of Lu. panamensis with L. braziliensis in the study area. We also demonstrate that PCR-hybridization is a suitable approach to establish the Leishmania-sandfly relationship and will be useful in epidemiological studies of leishmaniasis in endemic areas.

Leishmania (Viannia) lainsoni occupies a unique niche within the subgenus Viannia.

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.

Diagnosis of leishmaniasis in Maltese dogs with the aid of the polymerase chain reaction.

Visceral leishmaniasis due to infection with Leishmania infantum (a member of the L. donovani complex) has been known in Malta since the beginning of the century. In 1946, when human diseases became compulsorily notifiable on the islands, the leishmaniasis figures were 1264 visceral cases, 36 cutaneous cases and 5 unspecified. Five cases of cutaneous infection were reported in 1997 and 23 cases of cutaneous and 3 of visceral infection in January-October 1998. There may be considerable under-reporting of the disease. Figures of between 18% and 47% have been reported for canine leishmaniasis. This large discrepancy between reservoir and human hosts suggests that the canine reservoir could be a serious threat and is worthy of careful examination. This pilot study was carried out to determine the proportion of dogs serologically positive for leishmaniasis in order to assess the necessity for a possible control programme in Malta. Using 60 canine blood samples from the Maltese islands, we tested for deoxyribonucleic acid (DNA) of the L. donovani complex using the polymerase chain reaction (PCR). The samples had all been subjected to the indirect immunofluorescent antibody test (IFAT) and a direct comparison was made. DNA was extracted using the phenol/chloroform method and amplified with primers specific for kinetoplast mini-circle DNA of the L. donovani complex and L. major, Southern blotted and hybridized with a radio-labelled probe specific for the L. donovani complex. Twelve of the samples gave positive results in the IFAT, whilst 37 (62%) were positive by PCR and hybridization. All samples from 36 dogs from a non-endemic area in the UK were negative by PCR. Five of the 12 samples positive by IFAT gave negative PCR results.

Genes coding structural proteins in the Leishmania braziliensis complex.

Acidic ribosomal P1 and P2b proteins, referred to as P proteins, and histone H3 are reported for first time in the Leishmania braziliensis complex. Deoxyribonucleic acid analysis and multiple sequence alignment suggest that both P proteins may maintain their structural function in the ribosomal stalk, in spite of the high rate of mutations detected. The deduced amino acid sequence of protein P1 showed 51% identity with Trypanosoma cruzi protein P1 and protein P2b showed 61% identity with T. cruzi protein P2b. Another conserved protein, L. (Viannia) braziliensis histone H3, showed 82% and 70% identity with histone H3 of L. (Leishmania) infantum and T. cruzi, respectively. The N-terminal end of this histone is divergent in comparison with the consensus eukaryotic sequence. Their predicted tridimensional structure was designed.

A kinetoplast DNA minicircle probe (B18) specific for the Leishmania subgenus Viannia.

This paper reports the development, sequence and specificity of probe B18, which hybridizes only to the kinetoplast deoxyribonucleic acid (kDNA) minicircle of Leishmania species of the subgenus Viannia. This probe was developed in 1985 and has been used extensively since, on whole kDNA, Leishmania dot-blots and kDNA minicircles amplified by the polymerase chain reaction.

Recombinant antigens for specific and sensitive serodiagnosis of Latin American tegumentary leishmaniasis.

The diagnostic potential of recombinant leishmanial antigens for Latin American tegumentary leishmaniasis (LATL) was examined. Two Leishmania (Viannia) peruviana recombinant proteins, T26-U2 and T26-U4, were assessed by their reactivity to detect specific anti-leishmanial antibodies. Seventy-eight individual sera from persons with LATL, 39 from those with other diseases, and 10 negative control sera were tested by Western blotting and enzyme-linked immunosorbent assay. The sensitivity of the test using T26-U2 plus T26-U4 was similar to that obtained with whole parasite extract (92%). However, the specificity obtained using both recombinant antigens (87%) was higher than that of the whole parasite extract (65%). All tests using recombinant proteins (T26-U2, T26-U2 plus T26-U4 or T26-U4) had a higher positive predictive value (89%, 92% and 98%, respectively) than the value obtained using total parasites (81%). Eleven Colombian sera were also tested, and the results indicated that T26-U2 plus T26-U4 could be used successfully in Peru and in other Latin American countries.

Molecular tracking of infections by Leishmania infantum.

Leishmania infantum is a major opportunistic parasite in patients with acquired immune deficiency syndrome and is very variable in these subjects. Isoenzyme characterization is not able to explain this variability, since half of the stocks isolated from patients co-infected with human immunodeficiency virus and Leishmania belong to zymodeme MON-1. Amplification of L. infantum minicircles by the polymerase chain reaction (PCR) and digestion of the amplified product to reveal restriction fragment length polymorphisms (RFLP) has proved very useful in distinguishing between relapses and reinfections in co-infected, treated patients. We have confirmed the existence of a leishmaniasis outbreak among intravenous drug users in north-east Spain, previously detected by isoenzymatic analysis. We have documented persistence of the same strain of Leishmania in 2 treated co-infected patients throughout several years, regardless of the theoretical rapid evolution ascribed to kinetoplast deoxyribonucleic acid minicircle sequences. We suggest using this PCR-RFLP technique to detect reinfections in treated co-infected subjects.

A focus of mucocutaneous leishmaniasis in Três Braços, Bahia, Brazil: characterization and identification of Leishmania stocks isolated from man and dogs.

The characterization and identification to species and subspecies of 20 stocks of Leishmania isolated from the region of Três Braços, Bahia, Brazil, are described: 17 stocks were from patients and three from dogs. The following techniques were used (i) biological (growth in culture, hamster tissues and phlebotomine gut), (ii) biochemical (isoenzyme and kinetoplast DNA analysis) and (iii) immunological (using monoclonal antibodies). All except two stocks belong to the L. braziliensis complex. One of these two corresponded to L. mexicana amazonensis but the other, while clearly in the mexicana complex, showed slight differences from the L. mexicana amazonensis reference strain on isoenzyme analysis. Two stocks from different lesions in the same patient and with different growth characteristics in hamster tissues were both identified as L. braziliensis braziliensis. All the fully characterized stocks of the L. braziliensis complex were identified as L. braziliensis braziliensis. L. braziliensis guyanensis was not identified. Dog and human stocks of L. braziliensis braziliensis were indistinguishable. From these findings and other evidence, L. braziliensis braziliensis seems to be the predominant species transmitted in Três Braços.