The elimination of seropositive dogs in Brazil has been used to control zoonotic visceral leishmaniasis but with little success. To elucidate the reasons for this, the infectiousness of 50 sentinel dogs exposed to natural Leishmania chagasi infection was assessed through time by xenodiagnosis with the sandfly vector, Lutzomyia longipalpis. Eighteen (43%) of 42 infected dogs became infectious after a median of 333 days in the field (105 days after seroconversion). Seven highly infectious dogs (17%) accounted for >80% of sandfly infections. There were positive correlations between infectiousness and anti-Leishmania immunoglobulin G, parasite detection by polymerase chain reaction, and clinical disease (logistic regression, r2=0.08-0.18). The sensitivity of enzyme-linked immunosorbent assay to detect currently infectious dogs was high (96%) but lower in the latent period (<63%), and specificity was low (24%). Mathematical modeling suggests that culling programs fail because of high incidence of infection and infectiousness, the insensitivity of the diagnostic test to detect infectious dogs, and time delays between diagnosis and culling.

Infectiousness in a Cohort of Brazilian Dogs: Why Culling Fails to Control Visceral Leishmaniasis in Areas of High Transmission

In this review article the history of leishmaniasis is discussed regarding the origin of the genus Leishmania in the Mesozoic era and its subsequent geographical distribution, initial evidence of the disease in ancient times, first accounts of the infection in the Middle Ages, and the discovery of Leishmania parasites as causative agents of leishmaniasis in modern times. With respect to the origin and dispersal of Leishmania parasites, the three currently debated hypotheses (Palaearctic, Neotropical and supercontinental origin, respectively) are presented. Ancient documents and paleoparasitological data indicate that leishmaniasis was already widespread in antiquity. Identification of Leishmania parasites as etiological agents and sand flies as the transmission vectors of leishmaniasis started at the beginning of the 20th century and the discovery of new Leishmania and sand fly species continued well into the 21st century. Lately, the Syrian civil war and refugee crises have shown that leishmaniasis epidemics can happen any time in conflict areas and neighbouring regions where the disease was previously endemic.

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The history of leishmaniasis

Trypanosoma cruzi is the protozoan agent of Chagas disease, and the most important parasitic disease in Latin America. Protozoa of the genus Leishmania are global agents of visceral and cutaneous leishmaniasis, fatal and disfiguring diseases. In the 1970s multilocus enzyme electrophoresis demonstrated that T. cruzi is a heterogeneous complex. Six zymodemes were described, corresponding with currently recognized lineages, TcI and TcIIa-e--now defined by multiple genetic markers. Molecular epidemiology has substantially resolved the phylogeography and ecological niches of the T. cruzi lineages. Genetic hybridization has fundamentally influenced T. cruzi evolution and epidemiology of Chagas disease. Genetic exchange of T. cruzi in vitro involves fusion of diploids and genome erosion, producing aneuploid hybrids. Transgenic fluorescent clones are new tools to elucidate molecular genetics and phenotypic variation. We speculate that pericardial sequestration plays a role in pathogenesis. Multilocus sequence typing, microsatellites and, ultimately, comparative genomics are improving understanding of T. cruzi population genetics. Similarly, in Leishmania, genetic groups have been defined, including epidemiologically important hybrids; genetic exchange can occur in the sand fly vector. We describe the profound impact of this parallel research on genetic diversity of T. cruzi and Leishmania, in the context of epidemiology, taxonomy and disease control.

/pdf/the-molecular-epidemiology-and-phylogeography-of-trypanosoma-5g32fd9pkm.pdf

The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania : looking back and to the future

Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis (VL) in the New World (NW) with endemic regions extending from southern USA to northern Argentina. The two hypotheses about the origin of VL in the NW suggest (1) recent importation of L. infantum from the Old World (OW), or (2) an indigenous origin and a distinct taxonomic rank for the NW parasite. Multilocus microsatellite typing was applied in a survey of 98 L. infantum isolates from different NW foci. The microsatellite profiles obtained were compared to those of 308 L. infantum and 20 L. donovani strains from OW countries previously assigned to well-defined populations. Two main populations were identified for both NW and OW L. infantum. Most of the NW strains belonged to population 1, which corresponded to the OW MON-1 population. However, the NW population was much more homogeneous. A second, more heterogeneous, population comprised most Caribbean strains and corresponded to the OW non-MON-1 population. All Brazilian L. infantum strains belonged to population 1, although they represented 61% of the sample and originated from 9 states. Population analysis including the OW L. infantum populations indicated that the NW strains were more similar to MON-1 and non-MON-1 sub-populations of L. infantum from southwest Europe, than to any other OW sub-population. Moreover, similarity between NW and Southwest European L. infantum was higher than between OW L. infantum from distinct parts of the Mediterranean region, Middle East and Central Asia. No correlation was found between NW L. infantum genotypes and clinical picture or host background. This study represents the first continent-wide analysis of NW L. infantum population structure. It confirmed that the agent of VL in the NW is L. infantum and that the parasite has been recently imported multiple times to the NW from southwest Europe.

/pdf/comparative-microsatellite-typing-of-new-world-leishmania-3586mp4mg6.pdf

Comparative microsatellite typing of new world leishmania infantum reveals low heterogeneity among populations and its recent old world origin.

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Cukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.

/pdf/genomic-confirmation-of-hybridisation-and-recent-inbreeding-1ccyfrpc6h.pdf

Genomic Confirmation of Hybridisation and Recent Inbreeding in a Vector-Isolated Leishmania Population

The Leishmania donovani complex comprises four described species: L. 
donovani, L. archibaldi, L. infantum and L. chagasi. L. chagasi is the only New World 
species and has been considered similar to L. infantum, although some authors insist 
on maintenance of its independent species status. L. donovani has at least two major 
epidemiological subgroups whose relationships are poorly understood. 
In this thesis, molecular biological techniques were used to investigate the 
taxonomy and phylogenetic relationships within the L. donovani complex, with 
isoenzyme analysis (lEA) as reference technique. Random amplification of 
polymorphic DNA (RAPD) was used to provide anonymous genetic markers which 
allowed overall comparisons of genomes. Selected target genes and intergenic 
regions were also amplified by the polymerase chain reaction (PCR), namely the 
major surface protease (msp or gp63), the mini-exon and the ribosomal internal 
transcribed spacer (ITS). PCR products of intergenic regions between msp genes 
(ITG/CS and ITG/L), mini-exon and ITS were analysed by restriction fragment length 
polymorphism (RFLP). Phylogenies generated from each of the methods were 
compared with that of IEA. 
L. infantum and L. chagasi were found to be synonymous, whilst L. donovani 
was found to be more polymorphic than L. infantum and a fourth possible species in 
the complex, L. archibaldi, was not supported. Six genetic groups of strains were 
identified in the L. donovani complex, based on all DNA based analyses, which 
agreed with IEA typing. Pooled data from RFLP and RAPD analyses generated 
robust phylogenies which were congruent with ITG/CS RFLP and msp DNA 
sequence based phylogenies, but not with lEA phylogenies. The evolutionary history 
of the L. donovani complex is analysed in the light of the present results. The diverse 
typing methods were also evaluated and genetic markers suggested, that are 
applicable to classification and typing of L. donovani species and strains.

Isabel Larguinho Mauricio

Papers

Genetic diversity in the Leishmania donovani complex.