P. D. Ready

Mitochondrial haplotypes and phylogeography of Phlebotomus vectors of Leishmania major

Haplotypes of eight phlebotomine species were characterized by cycle sequencing a mitochondrial (mt) DNA fragment (cytochrome b to NADH1) amplified from single sandflies by PCR. Phlebotomus (Phlebotomus) papatasi displayed little variation throughout its large geographical range. We conclude that this vector of Leishmania major suffered a population bottleneck late in the Pleistocene and then radiated out from the eastern Mediterranean subregion. There was no support for a recent domestic lineage of P. papatasi. The mtDNA molecular clock in phlebotomines (subgenera Phlebotomus and Larroussius) was calibrated by reference to palaeogeographical events in Africa and the Mediterranean subregion. It fitted a pairwise nucleotide sequence divergence rate of 1.0–2.5% per million years. Co‐evolution of L. major, its Phlebotomus vectors and mammalian reservoirs is discussed.

Sandflies of the Phlebotomus perniciosus complex: mitochondrial introgression and a new sibling species of P. longicuspis in the Moroccan Rif

Abstract.  The bloodsucking adult females of Phlebotomus perniciosus Newstead and P. longicuspis Nitzulescu (Diptera: Psychodidae) are important vectors of the protozoan Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae) in western Mediterranean countries. The species status of the two phlebotomine sandflies was assessed, along with the epidemiological implications. Individual sandflies from three Moroccan Rif populations were characterized morphologically, isoenzymatically (by the isoelectrofocusing of alleles at the polymorphic enzyme loci of HK, GPI and PGM), and by comparative DNA sequence analysis of a fragment of mitochondrial Cytochrome b (mtDNA). By reference to the character profiles of specimens from other locations, including southern Spain and the type‐locality countries, the Moroccan flies were placed in three lineages: first, the lineage of P. perniciosus, which contained two mtDNA sublineages, one (pnt) widely distributed and associated with the morphology of the male types from Malta, and the other (pna) associated with a P. longicuspis‐like male morphology; second, the lineage of P. longicuspis sensu stricto, including typical forms from Tunisia; and third, a new sibling species of P. longicuspis. The mtDNA sublineage (pnt) of typical P. perniciosus was also found in some P. longicuspis from Morocco, indicating interspecific hybridization. The typical race of P. perniciosus occurs in Italy as well as in Malta, Tunisia and Morocco. It is replaced in southern Spain by the Iberian race (with the pni mtDNA sublineage). The discovery of interspecific gene introgression and a new sibling species mean that previous records of the two morphospecies do not necessarily reflect their true vectorial roles or geographical and ecological distributions.

Reproductive isolation between different forms of Lutzomyia longipalpis (Lutz & Neiva), (Diptera: Psychodidae), the vector of Leishmania donovani chagasi Cunha & Chagas and its significance to kala-azar distribution in South America

The males of the sandfly Lutzomyia longipalpis occur in two forms, one which bears a single pair of pale spots on tergite 4 and another in which an additional pair of spots characterizes tergite 3. In crosses between laboratory reared stocks of the two forms originating from allopatric and sympatric sites in Brazil nearly all males of one form fail to inseminate females of the other. In addition, insemination failure between some allopatric populaytions of Lu. longipalpis with similar tergal spot patterns is recorded, indicating the existence of additional forms in an apparent species complex. The possibility that Lu. longipalpis sensu latu represents more than a single taxon is discussed and the relevance of these findings to future epidemiological studies on kala-azar is considered.

Environmental risk mapping of canine leishmaniasis in France.

BackgroundCanine leishmaniasis (CanL) is a zoonotic disease caused by Leishmania infantum, a Trypanosomatid protozoan transmitted by phlebotomine sandflies. Leishmaniasis is endemic in southern France, but the influences of environmental and climatic factors on its maintenance and emergence remain poorly understood. From a retrospective database, including all the studies reporting prevalence or incidence of CanL in France between 1965 and 2007, we performed a spatial analysis in order to i) map the reported cases in France, and ii) produce an environment-based map of the areas at risk for CanL. We performed a Principal Component Analysis (PCA) followed by a Hierarchical Ascendant Classification (HAC) to assess if the locations of CanL could be grouped according to environmental variables related to climate, forest cover, and human and dog densities. For each group, the potential distribution of CanL in France was mapped using a species niche modelling approach (Maxent model).ResultsResults revealed the existence of two spatial groups of CanL cases. The first group is located in the Cévennes region (southern Massif Central), at altitudes of 200-1000 m above sea level, characterized by relatively low winter temperatures (1.9°C average), 1042 mm average annual rainfall and much forest cover. The second group is located on the Mediterranean coastal plain, characterized by higher temperatures, lower rainfall and less forest cover. These two groups may correspond to the environments favoured by the two sandfly vectors in France, Phlebotomus ariasi and Phlebotomus perniciosus respectively. Our niche modelling of these two eco-epidemiological patterns was based on environmental variables and led to the first risk map for CanL in France.ConclusionResults show how an ecological approach can help to improve our understanding of the spatial distribution of CanL in France.

Molecular identification of vectors of Leishmania in Colombia: mitochondrial introgression in the Lutzomyia townsendi series.

The identity of the sandfly vectors of Leishmania braziliensis in Valle del Cauca Department, Colombia, was originally given as Lutzomyia townsendi, but then changed to L. youngi, another member of the L. townsendi series (Verrucarum group) with isomorphic females. To identify members of this series in Valle del Cauca, we analyzed the nuclear gene elongation factor-alpha (EF-alpha) and the mitochondrial gene cytochrome b (Cyt b). DNA sequences from the L. verrucarum series (L. columbiana, L. evansi and L. ovallesi) were used as outgroups. Flies from two locations on the western cordillera of the Andes were identified as L. townsendi s.s., according to male morphology and distinctive gene lineages. In the third location, on the central cordillera of the Andes, most specimens were identified as belonging to a geographical population of L. youngi, according to male morphology, an EF-alpha lineage shared with L. youngi from the Venezuelan-type locality, and a distinctive Cyt b sub-lineage. All other specimens were identified as L. youngi with the introgressed Cyt b sequences of L. townsendi. Such interspecific introgression implies that vectorial traits and ecological associations may no longer be viewed as fixed properties of different morphospecies.

Integrated Mapping of Establishment Risk for Emerging Vector-Borne Infections: A Case Study of Canine Leishmaniasis in Southwest France

Background Zoonotic visceral leishmaniasis is endemic in the Mediterranean Basin, where the dog is the main reservoir host. The diseases causative agent, Leishmania infantum, is transmitted by blood-feeding female sandflies. This paper reports an integrative study of canine leishmaniasis in a region of France spanning the southwest Massif Central and the northeast Pyrenees, where the vectors are the sandflies Phlebotomus ariasi and P. perniciosus. Methods Sandflies were sampled in 2005 using sticky traps placed uniformly over an area of approximately 100 by 150 km. High- and low-resolution satellite data for the area were combined to construct a model of the sandfly data, which was then used to predict sandfly abundance throughout the area on a pixel by pixel basis (resolution of c. 1 km). Using literature- and expert-derived estimates of other variables and parameters, a spatially explicit R 0 map for leishmaniasis was constructed within a Geographical Information System. R 0 is a measure of the risk of establishment of a disease in an area, and it also correlates with the amount of control needed to stop transmission. Conclusions To our knowledge, this is the first analysis that combines a vector abundance prediction model, based on remotely-sensed variables measured at different levels of spatial resolution, with a fully mechanistic process-based temperature-dependent R 0 model. The resulting maps should be considered as proofs-of-principle rather than as ready-to-use risk maps, since validation is currently not possible. The described approach, based on integrating several modeling methods, provides a useful new set of tools for the study of the risk of outbreaks of vector-borne diseases.

MITOCHONDRIAL AND WOLBACHIA MARKERS FOR THE SANDFLY PHLEBOTOMUS PAPATASI: LITTLE POPULATION DIFFERENTIATION BETWEEN PERIDOMESTIC SITES AND GERBIL BURROWS IN ISFAHAN PROVINCE, IRAN

Abstract.  In Iran, Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) is the main vector of Leishmania major Yakimoff & Schokhor (Kinetoplastida: Trypanosomatidae), the causative agent of rural zoonotic cutaneous leishmaniasis. This sandfly is abundant both in villages and in the burrows of the main reservoir host, the gerbil Rhombomys opimus (Licht.) (Rodentia: Gerbillidae). Populations of P. papatasi were sampled from the edges of villages in Isfahan province, using CDC miniature light traps in peridomestic sites and sticky papers placed at the entrances to gerbil burrows. Single peridomestic sites in two northern provinces were also sampled. Individual sandflies were characterized by PCR amplification and sequencing of fragments of their mitochondrial cytochrome b gene and of the wsp gene of endosymbiotic Wolbachia pipientis Hertig (alpha‐Proteobacteria: Rickettsiaceae). The distributions of the haplotypes of these two maternally inherited genes were analysed to assess the population differentiation of P. papatasi, knowledge of which will be needed for planning control measures. For the first time these markers were used to characterize P. papatasi from gerbil burrows, and they indicated the absence not only of sympatric cryptic species but also of any long‐term differentiation of lineages in different habitats. A single lineage of cytochrome b haplotypes was found, and both sexes in all populations had a high infection rate of the same A‐group strain of Wolbachia (wPap). The distributions of cytochrome b haplotypes were consistent with females dispersing more than males, which has been reported for P. papatasi in other countries. The widespread distribution of wPap suggests that Wolbachia could be used to spread transgenes between populations of P. papatasi in different habitats.

Amplification and analysis of human DNA present in mosquito bloodmeals

Abstract. DNA fingerprinting should permit the identification of individual human hosts of haematophagous arthropods, providing epidemi‐ologically useful information, for example, the biting rates on different people and the impact of insecticide‐impregnated bednets.

Variation between geographical populations of Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho, 1939) sensu lato (Diptera:Psychodidae:Phlebotominae) in Brazil

Phylogenetic analysis of morphometric and biological characters indicated that there are two distinct forms of Lutzomyia whitmani in Brazil: one is present both north and south of the River Amazonas in the State of Pará while the other occurs in northeast Brazil, in the State of Ceará, and further south, including the type locality in State of Bahia. The Amazonian form is reportedly neither strongly anthropophilic nor synanthropic, and it is the vector of Leishmania shawi; whereas the southern form is often collected peridomestically, while biting man, and has been found infected with Le.(V.) braziliensis. The ratio of the length of the genital filaments to that the genital pump was found to be consistently smaller in males of the Amazonian populations. A middle repetitive DNA element was isolated by differentially screening a genomic library made using Amazonian material, and the sequence was diagnostic for this form of Lu. whitmani (being absent or occurring in low copy number in the southern form). The total evidence suggests there are at least two, geographically-isolated forms of Lu. whitmani, which may represent different cryptic species.

Predicting the distribution of canine leishmaniasis in western Europe based on environmental variables.

The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971-2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (<5%, 5-20%, >20%). Both the four-country and France-only models performed reasonably well for predicting correctly the <5% and >20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5-20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping.