Transmission potential of paromomycin-resistant Leishmania infantum and Leishmania donovani.

OBJECTIVES: Former studies demonstrated quick selection of paromomycin resistance for Leishmania infantum and Leishmania donovani accompanied by increased fitness. The present study aimed to interpret these findings in an epidemiological context by comparing infection of WT and experimentally derived paromomycin-resistant strains in the sand fly vector. METHODS: Depending on the Leishmania species, Lutzomyia longipalpis and Phlebotomus perniciosus or Phlebotomus argentipes sand flies were artificially infected with procyclic promastigotes of WT and paromomycin-resistant L. infantum (MHOM/FR/96/LEM3323-cl4) or L. donovani (MHOM/NP/03/BPK275/0-cl18). The infection rate and gut/stomodeal valve colonization were determined to monitor parasite phenotypic behaviour within the vector. The impact of the previously described gain of fitness in the vertebrate host on infectivity for the vector was assessed by feeding L. longipalpis on Syrian golden hamsters heavily infected with either WT or paromomycin-resistant parasites. RESULTS: WT and paromomycin-resistant Leishmania of both species behaved similarly in terms of infection and parasite location within the studied sand fly species. Blood feeding on infected hamsters did not reveal differences in acquisition of WT and paromomycin-resistant parasites, despite the higher organ burdens observed for the paromomycin-resistant strain. Strains remained resistant after passage in the vector. CONCLUSIONS: Although paromomycin-resistant parasites show an increased parasite fitness in vitro and in laboratory rodents, the intrinsic infection potential of paromomycin-resistant parasites remains unaltered in the sand fly. Of importance is the fact that paromomycin-resistant Leishmania are able to complete development in the natural vectors and produce stomodeal infection with metacyclic forms, which clearly suggests their potential to spread and circulate in nature.

Multiple radiations of spiny mice (Rodentia: Acomys) in dry open habitats of Afro-Arabia: evidence from a multi-locus phylogeny.

BACKGROUND: Spiny mice of the genus Acomys are distributed mainly in dry open habitats in Africa and the Middle East, and they are widely used as model taxa for various biological disciplines (e.g. ecology, physiology and evolutionary biology). Despite their importance, large distribution and abundance in local communities, the phylogeny and the species limits in the genus are poorly resolved, and this is especially true for sub-Saharan taxa. The main aims of this study are (1) to reconstruct phylogenetic relationships of Acomys based on the largest available multilocus dataset (700 genotyped individuals from 282 localities), (2) to identify the main biogeographical divides in the distribution of Acomys diversity in dry open habitats in Afro-Arabia, (3) to reconstruct the historical biogeography of the genus, and finally (4) to estimate the species richness of the genus by application of the phylogenetic species concept. RESULTS: The multilocus phylogeny based on four genetic markers shows presence of five major groups of Acomys called here subspinosus, spinosissimus, russatus, wilsoni and cahirinus groups. Three of these major groups (spinosissimus, wilsoni and cahirinus) are further sub-structured to phylogenetic lineages with predominantly parapatric distributions. Combination of alternative species delimitation methods suggests the existence of 26 molecular operational taxonomic units (MOTUs), potentially corresponding to separate species. The highest genetic diversity was found in Eastern Africa. The origin of the genus Acomys is dated to late Miocene (ca. 8.7 Ma), when the first split occurred between spiny mice of eastern (Somali-Masai) and south-eastern (Zambezian) savannas. Further diversification, mostly in Plio-Pleistocene, and the current distribution of Acomys were influenced by the interplay of global climatic factors (e.g., Messinian salinity crisis, intensification of Northern Hemisphere glaciation) with local geomorphology (mountain chains, aridity belts, water bodies). Combination of divergence dating, species distribution modelling and historical biogeography analysis suggests repeated "out-of-East-Africa" dispersal events into western Africa, the Mediterranean region and Arabia. CONCLUSIONS: The genus Acomys is very suitable model for historical phylogeographic and biogeographic reconstructions of dry non-forested environments in Afro-Arabia. We provide the most thorough phylogenetic reconstruction of the genus and identify major factors that influenced its evolutionary history since the late Miocene. We also highlight the urgent need of integrative taxonomic revision of east African taxa.

Development of Leishmania parasites in Culicoides nubeculosus (Diptera: Ceratopogonidae) and implications for screening vector competence.

Biting midges of the genus Forcipomyia (Diptera: Ceratopogonidae) have recently been implicated as vectors of kinetoplastid parasites in the Leishmania enrietti complex. This study assesses susceptibility of one of the few successfully colonized Ceratopogonidae, Culicoides nubeculosus Meigen, to infection with Leishmania parasites infecting humans. While Leishmania infantum initially developed in the midgut of C. nubeculosus until 2 d postfeeding, parasite populations on day 3 were considerably reduced. Despite this, a polymerase chain reaction-based assay continued to indicate presence of L. infantum for up to 7 d after the bloodmeal. These findings are discussed within the wider context of implicating arthropods as vectors of Leishmania and it is suggested that conventional polymerase chain reaction use in vector-competence studies should be accompanied by direct microscopical observations.

Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival.

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.

Blocked stomodeal valve of the insect vector: similar mechanism of transmission in two trypanosomatid models.

The regurgitation of metacyclic stages from the sand fly cardia is thought to be the prevailing mechanism of Leishmania transmission. This regurgitation may result through damage of the stomodeal valve and its mechanical block by the parasites. We found this phenomenon in three sand fly-Leishmania models and also in avian trypanosomes transmitted by Culex mosquitoes. Phlebotomus duboscqi, Phlebotomus papatasi, Lutzomyia longipalpis, and Culex pipiens were membrane-fed on blood containing Leishmania major, Leishmania chagasi (syn. infantum) and an unidentified avian Trypanosoma from Trypanosoma corvi clade, respectively. Females with the late-stage infections were processed for the optical and transmission electron microscopy. Localization of the parasites and changes to the stomodeal valve were in some aspects similar in all vector-parasite pairs studied: (i) a large plug of flagellates was observed in cardia region, (ii) parasites were attached to the chitin lining of the stomodeal valve by the formation of zonal hemidesmosome-like plaques. Leishmania promastigotes were found both attached to the valve as well as unattached in the lumen of midgut. The stomodeal valve of infected sand flies was opened, its chitin lining was destroyed and the unique filamentous structures on the apical end of cylindrical cells were degraded. In the Culex-Trypanosoma model, the whole population of epimastigotes was found in close contact with the chitin lining, and degenerative changes of the valve were less pronounced. We suggest that the phenomenon involving a blocked valve facilitating the regurgitation of parasites into the vertebrate host may occur generally in heteroxenous trypanosomatids transmitted by the bite of nematoceran Diptera.

Occurrence of Leishmania major in sandfly urine.

Promastigotes of Leishmania major were frequently detected in the urine droplets discharged by infected Phlebotomus papatasi and P. duboscqui females during feeding. Parasites were present in the urine of 37.5% P. papatasi and 16.1% P. duboscqi females, even in those with low intensity gut infections. Free-swimming forms (elongated nectomonads, short slender promastigotes and metacyclic forms) predominated in excreted droplets. Viability of excreted parasites was proved by cultivation on blood agar, and the presence of metacyclic forms in urine droplets was confirmed by specific fluorescence assay with 3F12 antibodies. While the release of promatigotes from the anus of the sandfly was frequent, these were rarely egested from the mouth-parts of sandfly females (1.3% for P. duboscqi and 0% for P. papatasi) fed on microcapillaries, even if the females were heavily infected. The possible role and significance of the discharge of parasites in sandfly urine are discussed.

Phlebotomus (Adlerius) halepensis vector competence for Leishmania major and Le. tropica.

In Eurasia, phlebotomine sandflies of the subgenus Adlerius (Diptera: Psychodidae) comprise about 20 known species. Some are suspected vectors of visceral leishmaniasis (VL) and at least one species has been implicated as a vector of cutaneous leishmaniasis (CL). We tested Phlebotomus (Adlerius) halepensis Theodor (Jordan strain) for CL vector competence, compared with three standard vectors: Phlebotomus (Phlebotomus) duboscqi N-L. from Senegal, Phlebotomus (Paraphlebotomus) sergenti Parrot from Turkey and the Neotropical Lutzomyia longipalpis (L. & N) (Jacobina strain). Sandfly females were membrane-fed on amastigote suspensions of Leishmania major Y. & S. and Le. tropica (Wright) (Kinetoplastida: Trypanosomatidae) and examined for parasite development 3, 6 and 10 days post-infection. Phlebotomus halepensis showed high susceptibility to both leishmanias, supporting typical suprapylarian parasite development similar to the other vectors. Phlebotomus halepensis infection rates were approximately 90% for Le. major and approximately 80% for Le. tropica, with high parasite densities. Development of infections was relatively fast, colonizing the thoracic midgut by 6 days post-bloodmeal in every case and reaching the stomodeal valve in >80% of flies. In late-stage infections, 10 days post-bloodmeal, nearly all P. halepensis females had cardia and stomodeal valve filled with very high numbers of parasites and some Le. tropica-infected females had promastigotes in the pharynx and proboscis. Host choice experiments in the laboratory showed that P. halepensis females fed readily on rat or rabbit and preferred the human forearm. In view of its vector competence and partial anthropophily, we infer that P. halepensis is a potential vector of cutaneous as well as visceral leishmaniases.

Leishmania major: effect of repeated passages through sandfly vectors or murine hosts.

Virulence for BALB/c mice, infectivity for Phlebotomus papatasi, haemagglutination activity and expression of metacyclic lipophosphoglycan (LPG) were studied in four strains of Leishmania major (LV561, FV1, L119 and Neal) and various lines of the LV561 strain. Attenuated line LV561/AV was passaged five times through sandflies or mice and the resulting lines (AVS5 and AVM5, respectively) and two of the earlier sandfly passages (AVS1 and AVS2) were used for further study. The highly virulent line LV561/V served as a control. Virulence for mice was not regained during passaging of LV561/AV in sandflies or mice (none of the mice infected with AVM5, AVS1, AVS2 or AVS5 displayed overt lesions) and the success rate in cultivating parasites, from lymph-node samples from inoculated mice, was not significantly higher for any of these lines than for the original line (LV561/AV). However, AVM5 and AVS5 developed better than LV561/AV in P. papatasi and the intensity and localisation of their infections were similar to those of the virulent control. In smears of the infected guts of P. papatasi, the AVS5 parasites resembled the virulent line (LV561/V) morphologically whereas the AVM5 parasites were similar to the avirulent LV561/AV. Haemagglutination activity increased as a result of passaging, the most pronounced difference being observed in AVM5, which had 60-fold higher activity than LV561/AV. Expression of metacyclic LPG was not increased by passaging. The proportion of forms reacting positively with 3F12 antibodies was high (about 17%) in the virulent LV561/V but low (2%-3%) in the avirulent lines LV561/AV, AVS5 and AVM5. A defect in LPG is not, however, likely to be the only reason for the avirulence observed, as the avirulent lines of LV561 still produced about 10 times as many metacyclic promastigotes as the strain L119, which caused delayed lesions in mice.

Comparative cytogenetics of hamsters of the genus Calomyscus.

Karyotypes of Calomyscus from different regions of Turkmenistan, Iran, and Azerbaijan were studied using chromosome banding (G- and C-banding) and analyses of meiosis in laboratory hybrids. Extensive variation in the diploid number and the number of autosomal arms (FNa) was revealed (2n = 30, FNa = 44; 2n = 32, FNa = 42; 2n = 44, FNa = 46; 2n = 44, FNa = 58; 2n = 37, FNa = 44; 2n = 50, FNa = 50; 2n = 52, FNa = 56). Centric and tandem fusions and heterochromatin changes were identified as the major modes of karyotype evolution in this group. Natural hybrids between individuals with different karyotypes were recorded, and regular chromosome pairing in meiosis was observed in laboratory hybrids. Fluorescence in situ hybridization with a 353-bp BspRI complex tandem repeat indicated that chromosomal repatterning occurred recently within the genus. There is no unequivocal evidence suggesting the role of chromosomal change in the speciation of the populations of Calomyscus examined.