The Genetic Relationship between Leishmania aethiopica and Leishmania tropica Revealed by Comparing Microsatellite Profiles

Background Leishmania (Leishmania) aethiopica and L. (L.) tropica cause cutaneous leishmaniases and appear to be related. L. aethiopica is geographically restricted to Ethiopia and Kenya; L. tropica is widely dispersed from the Eastern Mediterranean, through the Middle East into eastern India and in north, east and south Africa. Their phylogenetic inter- relationship is only partially revealed. Some studies indicate a close relationship. Here, eight strains of L. aethiopica were characterized genetically and compared with 156 strains of L. tropica from most of the latter species' geographical range to discern the closeness. Methodology/Principal Findings Twelve unlinked microsatellite markers previously used to genotype strains of L. tropica were successfully applied to the eight strains of L. aethiopica and their microsatellite profiles were compared to those of 156 strains of L. tropica from various geographical locations that were isolated from human cases of cutaneous and visceral leishmaniasis, hyraxes and sand fly vectors. All the microsatellite profiles were subjected to various analytical algorithms: Bayesian statistics, distance-based and factorial correspondence analysis, revealing: (i) the species L. aethiopica, though geographically restricted, is genetically very heterogeneous; (ii) the strains of L. aethiopica formed a distinct genetic cluster; and (iii) strains of L. aethiopica are closely related to strains of L. tropica and more so to the African ones, although, by factorial correspondence analysis, clearly separate from them. Conclusions/Significance The successful application of the 12 microsatellite markers, originally considered species-specific for the species L. tropica, to strains of L. aethiopica confirmed the close relationship between these two species. The Bayesian and distance-based methods clustered the strains of L. aethiopica among African strains of L. tropica, while the factorial correspondence analysis indicated a clear separation between the two species. There was no correlation between microsatellite profiles of the eight strains of L. aethiopica and the type of leishmaniasis, localized (LCL) versus diffuse cutaneous leishmaniasis (DCL), displayed by the human cases

Multilocus Microsatellite Typing reveals intra-focal genetic diversity among strains of Leishmania tropica in Chichaoua Province, Morocco

AbstractIn Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is a major public health threat. Strains of this species have been shown to display considerable serological, biochemical, molecular biological and genetic heterogeneity; and Multilocus Enzyme Electrophoresis (MLEE), has shown that in many countries including Morocco heterogenic variants of L. tropica can co-exist in single geographical foci. Here, the microsatellite profiles discerned by MLMT of nine Moroccan strains of L. tropica isolated in 2000 from human cases of CL from Chichaoua Province were compared to those of nine Moroccan strains of L. tropica isolated between 1988 and 1990 from human cases of CL from Marrakech Province, and also to those of 147 strains of L. tropica isolated at different times from different worldwide geographical locations within the range of distribution of the species. Several programs, each employing a different algorithm, were used for population genetic analysis. The strains from each of the two Moroccan foci separated into two phylogenetic clusters independent of their geographical origin. Genetic diversity and heterogeneity existed in both foci, which are geographically close to each other. This intra-focal distribution of genetic variants of L. tropica is not considered owing to in situ mutation. Rather, it is proposed to be explained by the importation of pre-existing variants of L. tropica into Morocco

Investigations on the epidemiology and diversity of Leishmania tropica and L. aethiopica and the differentiation of their sand fly vectors

Leishmania tropica ist der Auslöser von kutaner Leishmaniose beim Menschen und kommt in Afrika über den Mittleren Osten bis nach Nordindien vor. Mittels Mikrosatellitentypisierung (MLMT) wurde die weltweite Populationsstruktur dieser Spezies und der nahe verwandten Spezies L. aethiopica aufgedeckt, indem sowohl Methoden angewandt wurden, die auf genetischen Distanzen beruhen als auch solche, die auf der Analyse von Allelfrequenzen basieren. Die 195 Stämme von L. tropica sowie die acht Stämme von L. aethiopica gruppierten hauptsächlich gemäß ihrer geographischen Herkunft. Die Stämme von L. aethiopica stellten eine eigene Gruppe dar, die allerdings innerhalb der afrikanischen Stämme von L. tropica gruppierte. Vorläufige Ergebnisse einer genomweiten SNP-Analyse haben die Ergebnisse der Mikrosatellitenanalyse weitgehend bestätigt. Um die Gründe für die hohe genetische Variabilität innerhalb der Spezies L. tropica herauszufinden, wurde eine Funktionelle Klonierung durchgeführt, in der N-methyl-N’-nitro-N-nitrosoguanidin (MNNG) als Indikator für ein funktionierendes oder eingeschränktes Mismatch Repair (MMR)-System eingesetzt wurde. Dafür wurde ein Akzeptorstamm (hohe MNNG-Toleranz) mit einer Cosmidbibliothek, die genomische DNA eines Donorstammes (niedrige MNNG-Toleranz) enthielt, transfiziert. Die erhaltenen Transfektanten wurden dann auf ihre MNNG-Toleranz getestet. Die zeit- und kosteneffiziente Identifizierung von großen Mengen an Sandmücken ist wichtig für Feldstudien, in denen mehrere Tausend Mücken gefangen werden. Hier wird eine multiplexe Technik zur ligationsabhängigen Sondenamplifizierung vorgestellt, die die Identifizierung von Sandmücken-Spezies im Mittleren Osten ermöglicht. Die Spezies Phlebotomus syriacus, P. arabicus und P. papatasi können durch diese Methode mit spezies-spezifischen Sonden eindeutig identifiziert werden. Außerdem kann diese Methode dazu genutzt werden, weitere Spezies zu diskriminieren und auf gepoolte Sandmücken angewandt werden.Leishmania tropica is the causative agent of human cutaneous leishmaniasis in foci ranging from Africa through the Middle East to northern India. By multilocus microsatellite typing (MLMT), the world-wide population structure of this species and its closely related species L. aethiopica has been revealed applying methods based on both genetic distances and allele frequencies. The 195 strains of L. tropica and eight strains of L. aethiopica largely clustered according to their geographical origins. The strains of L. aethiopica formed a distinct group, although clustering among other African strains of L. tropica. Preliminary data obtained through a whole genome sequencing approach including strains of L. tropica, L. aethiopica and L. major have largely corroborated the results of the MLMT approach. To reveal the reasons for the high genetic variability among strains of L. tropica, a Functional Cloning approach was conducted using N-methyl-N’-nitro-N-nitrosoguanidin (MNNG) as an indicator for a functioning or impaired mismatch repair (MMR) system. The transfectants retrieved from the transfection of an acceptor strain exhibiting high MNNG tolerance with a cosmid library bearing the genomic DNA of a donor strain with a reduced MNNG tolerance were screened for the restored phenotype of the donor strain. The time- and cost-efficient identification of a large amount of sand flies is important since several thousands are caught during field studies. Here, a multiplex ligation-dependent probe amplification approach (MLPA) for the identification of sand flies endemic to the Middle East is introduced. The unambiguous identification of Phlebotomus syriacus, P. arabicus and P. papatasi was possible with this approach using species-specific probes. Furthermore, this technique has the potential to discriminate more species and to be applied to pooled sand fly specimens

Descriptive statistics per population.

<p>A, number of alleles; <i>H</i>_o, observed heterozygosity; <i>H</i>_e, expected heterozygosity; <i>F</i>_IS, inbreeding coefficient (-1 = outcrossing, 0 = random mating, +1 = inbreeding)</p><p>Descriptive statistics per population.</p

Descriptive statistics per locus.

<p>A, number of alleles; <i>H</i>_o, observed heterozygosity; <i>H</i>_e, expected heterozygosity; <i>F</i>_IS, inbreeding coefficient (-1 = outcrossing, 0 = random mating, +1 = inbreeding)</p><p>Descriptive statistics per locus.</p

Factorial correspondence analysis (FCA) of the nine sub-populations.

<p>FCA displays the calculated genetic distances 3-dimensionally based on allele similarities. Each square represents one genotype. The colours correspond to clustering based on Bayesian statistical results. The software requires pre-assignment to single populations, therefore strains were assigned to the nine sub-populations proposed by STRUCTURE.</p

Neighbour Joining tree displaying the phylogenetic relationship between strains of <i>L</i>. <i>aethiopica</i> and <i>L</i>. <i>tropica</i>.

<p>Bootstrap values >50 are indicated at the nodes. For clarity, only the WHO codes of the strains in population Africa/Galilee and of the four ' intermediate ' strains IL/1959, IN/1979, IL/1980, TR/1995 are given. The partial WHO codes specify the host, the country of origin and the year of isolation: I = insect, SER = <i>P</i>. <i>sergenti</i>, ARA = <i>P</i>. <i>arabicus</i>, ROS = <i>P</i>. <i>rossi</i>, M = mammal, PRV = <i>Procavia</i> (hyrax). If not indicated otherwise, the strains were isolated from human cases. The full WHO codes of all the strains are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131227#pone.0131227.s004" target="_blank">S1 Table</a>. The different colours indicate the clustering based on the Bayesian statistical results at the sub-population level for the population Africa/Galilee; the main populations Turkey/old strains/Palestine and Israel/Palestine are in grey and black, respectively.</p

Network presenting the genetic relationship between strains of <i>L</i>. <i>aethiopica</i> and <i>L</i>. <i>tropica</i>.

<p>Cross connections indicate probable reticulation events like hybridisation, recombination and horizontal gene transfer between the strains. The sub-populations within the population Africa/Galilee resulting from clustering based on Bayesian statistical results are in different colours; the main populations Turkey/old strains/Palestine and Israel/Palestine are highlighted in grey and black, respectively.</p

Map showing the geographical distribution of the six sub-populations in the main population Africa/Galilee.

<p>The six colours in the circles represent the six sub-populations where the colours correspond with those in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131227#pone.0131227.g001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131227#pone.0131227.g003" target="_blank">3</a>. The numbers in the circles indicate the number of strains of each sub-population from the country specified. Reprinted from and modified after <a href="http://commons.wikimedia.org/wiki/Maps_of_the_world#/media/File:BlankMap-World-v2.png" target="_blank">http://commons.wikimedia.org/wiki/Maps_of_the_world#/media/File:BlankMap-World-v2.png</a> under a CC BY license.</p