Paramonostomum deseado n. sp. (Digenea: Notocotylidae) parasitizing the South American Black Oystercatcher and their atypical life cycle from the Patagonian coast.

By way of morphological and molecular analysis we describe a new species of notocotylid, Paramonostomum deseado n. sp., parasitizing Haematopus ater from Argentina and we contribute to elucidate its life cycle. Within this genus, 4 groups can be morphologically distinguished according to body shape: 'Oval', 'Pyriform', 'Elongate', 'Overlong'. The new species belongs to the 'Elongate group', which presents a wide variation in body length (597­4500 µm). The new species, Paramonostomum caeci from Australia, Paramonostomum actitidis from the Caribbean and Paramonostomum alveoelongatum from Russia share the smallest range of body size in this group (<1130 µm). The new species more closely resembles P. actitidis but differs from it by cirrus-sac length, which is shorter in the new species (97­146 vs 280­430 µm in P. actitidis), and egg size which is larger in the new species (25­33 vs 18­20 µm in P. actitidis). Paramonostomum deseado n. sp. uses the limpet Nacella magellanica as both first and second intermediate hosts in which metacercariae encyst inside the redia. This is the first abbreviated cycle described for notocotylid species. Ribosomal RNA sequences provided for adults (ITS1, ITS2 and 28S) and metacercariae inside the rediae (ITS1) support the species identification and the correspondence among stages. Phylogenetic analysis based on 28S placed P. deseado n. sp. close to other Notocotylus spp. and Paramonostomum anatis. Molecular results demonstrate that the hosts involved in the life cycles and the habitat more than morphological differences are determining the phylogenetic relationships in members of Notocotylidae.

The life cycle of Magnivitellinum saltaensis n. sp. (Digenea: Alloglossidiidae) in Salta Province, Argentina.

We describe the alloglossiid trematode Magnivitellinum saltaensis n. sp., a parasite of the characiform fish Psalidodon endy, and its life cycle from Salta, northwest of Argentina. This is the first life cycle described for a species belonging to the genus Magnivitellinum. Cercariae emerged naturally from Biomphalaria tenagophila snails and infected experimentally exposed larvae of Diptera and Ephemeroptera as second intermediate hosts. These larvae in turn were exposed to commercially raised fish, and adults were recovered from characiform albino fish Gymnocorymbus ternetzi. Molecular analysis of natural and experimental adults showed the same genetic sequence for the partial region of 28S rDNA, thus confirming conspecificity. Comparison of these sequences with those published for M. simplex from Mexico showed 1.45% divergence, indicating that the specimens found in Salta belong to a different species, the third described of Magnivitellinum, in agreement with morphological data, geographical location, and host species composition. The new species is distinguished by its small body, vitelline follicles extending from the mid-level of the ventral sucker, Y-shaped excretory vesicle, and presence of papillae around the mouth.

Sand fly typing: a simple and morphologically-supported method based on polymorphism of 18S rRNA gene in a Leishmaniasis endemic area of Argentina.

Leishmaniases are vector-borne diseases that in the Americas are distributed from southern United States to northern Argentina. The vectors for this disease are small dipterans known as sand flies that are usually identified morphologically by observing structures with taxonomic value; but it is time-consuming, laborious, and requires entomological expertise. Then, this work was aimed at identifying sand flies with molecular techniques, using the morphological identification as a reference technique, in an endemic area of American Tegumentary Leishmaniasis (ATL) located in northern Argentina. For this, sand flies were caught at two patches of vegetation adjacent to rural areas in Orán department, Salta Province. Females were dissected with sterile needles; the head and last abdominal segments were analyzed for morphological identification. The remaining thorax and abdominal segments were used to extract DNA, which was amplified by PCR of the small subunit (SSU), 18S rRNA gene. PCR products were digested with CviQI and DdeI enzymes to identify sand fly species by Restriction Fragment Length Polymorphism (RFLP) analysis. Thus, the restriction pattern of each caught species was defined according to morphological identification. A total of 1501 females, belonging to four sand fly species, were captured. Nyssomyia neivai (1347/1501) was the most abundant species, followed by Migonemyia migonei (90/1501). From the total, 801 females were morphologically and molecularly identified, while 700 females were characterized only molecularly. For those females analyzed by both methods, there was total coincidence in the achieved result. Besides, the 5% (38/801) of females that could not be determined morphologically due to inadequate mounting were molecularly identified. All the females characterized just by PCR-RFLP, were successfully identified. Our results indicate that the explored method is capable of identifying the sand fly species that circulate in an ATL endemic area. Since this method is based on the analysis of markedly different patterns, the identification process might be more easily reproduced, as the bias introduced by the technician's lack of experience is removed.

Development of a Multilocus sequence typing (MLST) scheme for Pan-Leishmania.

Since the description of the Leishmania genus, its identification and organization have been a challenge. A high number of molecular markers have been developed to resolve phylogenetic differences at the species level and for addressing key epidemiological and population genetics questions. Based on Multilocus enzyme electrophoresis (MLEE), Multilocus sequence typing (MLST) schemes have been developed using different gene candidates. From 38 original gene targets proposed by other authors, 27 of them were chosen. In silico selection was made by analyzing free access genomic sequence data of 33 Leishmania species, one Paraleishmania representative, and one outgroup, in order to select the best 15 loci. De novo amplifications and primers redesign of these 15 genes were analyzed over a panel of 20 reference strains and isolates. Phylogenetic analysis was made at every step. Two MLST schemes were selected. The first one was based on the analysis of three-gene fragments, and it is suitable for species assignment as well as basic phylogenetic studies. By the addition of seven-genes, an approach based on the analysis of ten-gene fragments was also proposed. This is the first work that two optimized MLST schemes have been suggested, validated against a phylogenetically diverse panel of Leishmania isolates. MLST is potentially a powerful phylogenetic approach, and most probably the new gold standard for Leishmania spp. characterization.

Morphological and ITS2 Molecular Characterization of Ribeiroia Cercariae (Digenea: Psilostomidae) from Biomphalaria spp. (Gastropoda: Planorbidae) in Northern Argentina.

Species of Ribeiroia use planorbid snails as intermediate host. Since there is little information about these digenean parasites in South America, we aimed to assess whether Ribeiroia cercariae from 3 north Argentina locations belonged to the same species and differed from Ribeiroia cercariae described elsewhere. Specimens were obtained from Biomphalaria tenagophila and Biomphalaria orbignyi (Salta Province), and Biomphalaria occidentalis (Corrientes Province). Morphological traits of cercariae were analyzed, as well as their sequence of the ribosomal internal transcribed spacer 2 (ITS2). The ITS2 region consisted of 426 nucleotides identical in all samples, suggesting that all specimens belong to the same species in spite of their morphological differences and first intermediate host species. Comparison of the ITS2 region with GenBank database records showed that specimens from Argentina were different from Ribeiroia ondatrae (0.9% divergence), Ribeiroia marini (0.7% divergence), and Cercaria lileta (0.2% divergence). In summary, morphological, ecological, and ITS2 molecular data suggest that specimens from Argentina belong to a different species.

Optimized multilocus sequence typing (MLST) scheme for Trypanosoma cruzi.

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.

How often do they have sex? A comparative analysis of the population structure of seven eukaryotic microbial pathogens.

The model of predominant clonal evolution (PCE) proposed for micropathogens does not state that genetic exchange is totally absent, but rather, that it is too rare to break the prevalent PCE pattern. However, the actual impact of this "residual" genetic exchange should be evaluated. Multilocus Sequence Typing (MLST) is an excellent tool to explore the problem. Here, we compared online available MLST datasets for seven eukaryotic microbial pathogens: Trypanosoma cruzi, the Fusarium solani complex, Aspergillus fumigatus, Blastocystis subtype 3, the Leishmania donovani complex, Candida albicans and Candida glabrata. We first analyzed phylogenetic relationships among genotypes within each dataset. Then, we examined different measures of branch support and incongruence among loci as signs of genetic structure and levels of past recombination. The analyses allow us to identify three types of genetic structure. The first was characterized by trees with well-supported branches and low levels of incongruence suggesting well-structured populations and PCE. This was the case for the T. cruzi and F. solani datasets. The second genetic structure, represented by Blastocystis spp., A. fumigatus and the L. donovani complex datasets, showed trees with weakly-supported branches but low levels of incongruence among loci, whereby genetic structuration was not clearly defined by MLST. Finally, trees showing weakly-supported branches and high levels of incongruence among loci were observed for Candida species, suggesting that genetic exchange has a higher evolutionary impact in these mainly clonal yeast species. Furthermore, simulations showed that MLST may fail to show right clustering in population datasets even in the absence of genetic exchange. In conclusion, these results make it possible to infer variable impacts of genetic exchange in populations of predominantly clonal micro-pathogens. Moreover, our results reveal different problems of MLST to determine the genetic structure in these organisms that should be considered.

Reassessment of MLST schemes for Leptospira spp. typing worldwide.

Leptospirosis is a neglected zoonosis of global importance. Several multilocus sequence typing (MLST) methods have been developed for Leptospira spp., the causative agent of leptospirosis. In this study we reassessed the most commonly used MLST schemes in a set of worldwide isolates, in order to select the loci that achieve the maximum power of discrimination for typing Leptospira spp. Global eBURST algorithm was used to detect clonal complexes among STs and phylogenetic relationships among concatenated and individual sequences were inferred through maximum likelihood (ML) analysis. The evaluation of 12 loci combined to type a subset of strains rendered 57 different STs. Seven of these loci were selected into a final scheme upon studying the number of alleles and polymorphisms, the typing efficiency, the discriminatory power and the ratio dN/dS per nucleotide site for each locus. This new 7-locus scheme was applied to a wider collection of worldwide strains. The ML tree constructed from concatenated sequences of the 7 loci identified 6 major clusters corresponding to 6 Leptospira species. Global eBURST established 8 CCs, which showed that genotypes were clearly related by geographic origin and host. ST52 and ST47, represented mostly by Argentinian isolates, grouped the higher number of isolates. These isolates were serotyped as serogroups Pomona and Icterohaemorrhagiae, showing a unidirectional correlation in which the isolates with the same ST belong to the same serogroup. In summary, this scheme combines the best loci from the most widely used MLST schemes for Leptospira spp. and supports worldwide strains classification. The Argentinian isolates exhibited congruence between allelic profile and serogroup, providing an alternative to serological methods.