Background/Objectives: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers.Methodology/Principal findings: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis.Conclusions/Significance: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains. Leishmania (Viannia) which can cause a variety of clinical symptoms ranging from single or few lesions, disseminated CL with multiple lesions, to disfiguring forms of mucocutaneous leishmaniasis. This study has used a multilocus microsatellite typing approach for exploring the genetic diversity and population structure among 120 strains representing different subgenus L. (Viannia) species and different Brazilian CL foci. Genetic diversity within the subgenus was much higher than expected, especially within L. (V.) braziliensis, L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni which were all from the north of Brazil. These strains could not be assigned to well-defined populations, but presented a rather loosely associated group. Strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil and strains of L. (V.) guyanensis formed, however, two clearly separated populations exhibiting remarkable levels of sexual exchange. The latter finding is in contrast to previous studies suggesting clonal modes of propagation or inbreeding for natural populations of Leishmania parasites and might explain the genetic heterogeneity and limited population structure for Brazilian strains of subgenus L. (Viannia) observed in this study.Cutaneous leishmaniasis (CL) constitutes a significant public health problem in all federal states of Brazil. Most cases are caused by parasites of the subgenus  Leishmania (Viannia) Lainson and Shaw, 1987, are exclusively endemic in the New World (NW) and infections of humans with these protozoan parasites constitute a significant public health problem in at least 18 countries of Latin America L. (Viannia) parasites are capable of causing a variety of clinical symptoms ranging from cutaneous leishmaniasis (CL) with single or few lesions that may heal spontaneously, disseminated CL (DL) with multiple lesions, to disfiguring forms of mucocutaneous leishmaniasis (MCL) that may occur concomitantly or after remission of CL Leishmania parasites is thought to be influenced by the immune status of the host and virulence of the infecting parasite Leishmania and was employed in most of the classification schemes, although MLEE is likely to be partially superseded by multilocus sequence typing (MLST). The application of numerical taxonomy and cladistic techniques to electrophoretic data has resulted in the identification of two species complexes in the subgenus L. (Viannia), namely the L. (V.) braziliensis complex comprising L. (V.) braziliensis and L. (V.) peruviana, and the L. (V.) guyanensis complex comprising L. (V.) guyanensis, L. (V.) panamensis and L. (V.) shawi, and of at least four single species, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) lindenbergi and L. (V.) utingensis . The disease is caused by six species of the subgenus L. (Viannia), L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) shawi, L. (V.) lainsoni, L. (V.) naiffi and L. (V.) lindenbergi plus one species of the subgenus L. (Leishmania), L. (L.) amazonensis. Around 6–7% of the CL patients will develop symptoms of MCL L. (V.) braziliensis and, to a lesser extent, also with L. (V.) guyanensisL. (V.) braziliensis, and only sporadic cases with L. (V.) guyanensisL. (L.) amazonensis infections In Brazil, CL is endemic in all federal states and an annual mean of 27,250 CL cases has been registered from 1990–2010 (L. (Viannia) involves different species of phlebotomine sand flies and a wide variety of wild and domestic animals have been implicated as reservoir hosts. Some species have a more restricted transmission cycle, whereas others are more complex with several different vectors and hosts in different ecological and geographical regions L. (Viannia) species has been reported particularly in the Amazon region Leishmania strains can be maintained in both sylvatic cycles, involving wild animals and sylvatic sand flies, and urban cycles involving domestic animals and peridomestic sand flies L. (Viannia) parasites are characterized by tremendous genetic diversity and the described species vary enormously in their degree of such diversity L. (V.) braziliensis and L. (V) naiffi, and it has been suggested that the genetic diversity of the parasites is most probably related to the sand fly vector(s) and/or animal reservoir(s) involved in the transmission cycles. On the other hand, L. (V.) guyanensis, particularly strains circulating in the Brazilian Amazon region, and L. (V.) shawi have been found to be rather homogenous by MLEE and ITS-RFLP typing Transmission of species of the subgenus Leishmania (reviewed in L. (Viannia) with special emphasis on L. (V) braziliensis and L. (V.) guyanensisL. (Viannia) Multilocus microsatellite typing (MLMT) is currently the method of choice for molecular epidemiological and population genetic studies of different species of L. (Viannia) from different Brazilian foci endemic for CL. Strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil and strains of L. (V.) guyanensis formed two clearly separated populations. Evidence for significant levels of recombination was obtained for both of these populations, and in Minas Gerais the emergence of an epidemic clone of L. (V.) braziliensis was identified. A third loosely associated group comprised L. (V.) braziliensis strains and several other subgenus L. (Viannia) species, all from northern Brazil.In the present study we investigated microsatellite variation in strains of the subgenus Leishmania strains isolated from human and animal hosts and analysed in this microsatellite analysis, were received from the “Coleção de Leishmania do Instituto Oswaldo Cruz – CLIOC , and from the collection hosted by the Universidade Federal de Minas Gerais in Belo Horizonte. Only previously gathered samples from animals have been used in this study. All human strains of Leishmania had been isolated from patients as part of normal diagnosis and treatment with no unnecessary invasive procedures and with written and/or verbal consent recorded at the time of clinical examination. Data on human isolates were coded and anonymised.Research in this study was subject to ethical review by the European Commission and approved as part of contract negotiation for Project LeishEpiNetSA (contract 01547): the work conformed to all relevant European regulations. The research was also reviewed and approved by the ethics committee of the London School of Hygiene and Tropical Medicine . The L. (Viannia) strains from Brazil that were used in this study are listed in L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. (V.) naiffi hybrid. Most of the strains were isolated from human CL cases, three from DL cases, and three strains from patients suffering from MCL. The reference strains were cloned, but all other strains represented uncloned material. Seven strains were isolated from sand fly vectors, and 18 from different animals, such as opossums (4), rodents (3), armadillos (3), dogs (2), sloths (2), pacas (2), a capuchin monkey (1) and a porcupine (1). Sources, designation, geographical origins, MLEE identification, if known, and clinical manifestation for the 120 subgenus Leishmania collection . Seventeen strains of L. (V.) braziliensis, 15 from Minas Gerais and two from Pará, were obtained from the collection of the Universidade Federal de Minas Gerais, Belo Horizonte. Sample preparation and MLEE typing, based on the electrophoretic mobility of 11 enzymes in agarose gel electrophoresis, were performed as previously described Most strains were obtained from the FIOCRUZ DNA was isolated using proteinase K- phenol/chloroform extraction L. (Viannia), was used for amplification of microsatellite containing fragments, as previously described L. braziliensis) and MHOM/SR/87/TRUUS1 (L. guyanensis), which were included as reference strains in every experiment, and for which the repeat numbers had been determined by sequencing. These repeat numbers were then multiplied by two, since we have used dinucleotide microsatellites throughout, after which the size of the flanking region was added, as determined by sequencing of the reference strains. This rigorous normalization process was applied to correct for small size differences that could occur due to the use of different sequencing machines and/or fluorescent dyes during the analyses. These normalized fragment sizes for all markers were assembled into a multilocus microsatellite profile for every strain under study. In 1.7% of all loci three or four peaks were observed of which only the two most prominent bands were included in the microsatellite profiles. The microsatellite profiles of all strains analysed in this study are given in The standard set of 15 primer pairs , specific for Population structure was investigated using the STRUCTURE software Factorial correspondence analysis (FCA) implemented in GENETIX v 4.03 software http://bioinformatics.org/~tryphon/populations) for the numbers of repeats within each locus using the Chord-distance Phylogenetic analysis was based on microsatellite genetic distances, calculated with the program POPULATIONS 1.2.28 (A), expected (gene diversity) and observed heterozygosity  applying GDA (http://hydrodictyon. eeb.uconn.edu/people/plewis/software. php). FIS is a measure of heterozygosity that assesses the level of identity within individuals compared to that between individuals. It ranges between −1 and 1, where a negative value corresponds to an excess of heterozygotes, and a positive value to heterozygote deficiency. FIS = 0 indicates Hardy-Weinberg allele proportions. Mean FIS estimates over loci in each population were calculated with the software FSTAT (version 2.9.3.2) F-statistics P-values (confidence test) using the MSA software P-values for multiple tests were corrected using a sequential Bonferroni correction to minimize the likelihood of Type 1 errors Microsatellite markers as well as populations were analysed with respect to diversity of alleles  and the dr statistic were calculated in MULTILOCUS 1.3b P values were derived through comparison to a null distribution of 1,000 randomizations; median values were taken from 1,000 diploid resamplings of the multiallelic dataset.To assess the level of multilocus linkage disequilibrium, the Index of Association (L. (Viannia) analysed here  although the existence of a considerable amount of inbreeding cannot be ruled out. A total of 107 strains had unique MLMT profiles (L. braziliensis isolated between 1986 and 1992 from human CL cases in Minas Gerais presented indistinguishable MLMT profiles (Lbra26). Only two of the Minas Gerais strains were different from the predominating genotype  analysed in this study. Twenty-three of the 120 strains presented such possibly aneuploid loci. Strain The multilocus microsatellite profiles consisting of the repeat numbers for 15 markers were processed using both model-based and distance-based methods and the results of both analyses are compared in L. (Viannia) to three main populations  guyanensis from the state of Amazonas (n = 36). Only one strain of L. (V.) guyanensis, strain L-2493 from Acre, was not part of this population. This strain was previously identified as a new enzymatic variant, Z110, of L. (V.) guyanensis by MLEE (unpublished results) and presented few differences in the hsp70 sequence and RFLP L. (V.) braziliensis isolated in the eastern states of the country, namely Pernambuco, Bahia, Minas Gerais, Espírito Santo, Rio de Janeiro, and Paraná. Population 3 (n = 41) presented a mixture of 20 strains of L. (V.) braziliensis, the single strain of L. (V.) guyanensis L-2493 from Acre mentioned above, all strains of L. (V.) shawi, L. (V.) lainsoni, L. (V.) naiffi, the L. (V.) lainsoni/L. naiffi hybrid and the single strains of L. (V.) utingensis and L. (V.) lindenbergi. Thirty-five of these strains were isolated in the north of Brazil, five in the northeast and one in Paraná. Several strains of L. (V.) braziliensis had mixed membership coefficients for Populations 2 and 3, especially those from Pará  lainsoni and of L. (V.) naiffi, including the L. (V.) lainsoni/L. (V.) naiffi hybrid, were assigned to distinct genetic groups only when Population 3 was re-analysed by STRUCTURE in order to check for hidden substructures within Population 3 (L. (V.) shawi, however, did not present a separate entity in this analysis. When STRUCTURE was performed on Populations 1 and 2 separately, Population 1 did not show meaningful subdivision, but Population 2 was split in two sub-populations with the majority of L. (V.) braziliensis strains from Pernambuco and Bahia (northeast Brazil) found in 2A and strains from Minas Gerais, Espírito Santo, Rio de Janeiro and Paraná (southeast Brazil) in 2B (The strains of lation 3 . Strainsl) in 2B .L. (V.) lainsoni were found in a small well-separated cluster. A distinct cluster was also obtained for the strains of L. naiffi plus the single strains of L. (V.) utingensis and L. (V.) lindenbergi, albeit with long branches. Interestingly, these strains were most closely related to five strains of L. (V.) braziliensis, three from Pernambuco and the two from Acre with mixed memberships for sub-populations 3A and 3D (L. (V.) shawi grouped together with the L. (V.) braziliensis strain L-0326 from Pará. The L. (V.) guyanensis strain L-2493 did not group with the other strains of L. (V.) guyanensis, but was closest to the strains of L. (V.) shawi. Another interesting cluster consisted of six relatively diverse L. (V.) braziliensis strains from Acre. The remaining strains of Population 3 were intermediate between the “Acre” cluster and Population 2. Most of these strains had mixed memberships in Populations 2 and 3 in the STRUCTURE analyses  excess of heterozygosity indicated by negative FIS values was detected for only two loci, locus CSg47 in population 1 (−0.551) and locus B3H in population 2. Another 8 loci presented significant deficit of heterozygosity in a least one of the two populations. Both global FIS values were significant  and therefore panmixia must be rejected. Testing HWE within population 1 and 2 supported this scenario since both populations significantly departed from the null-hypothesis . The Maynard Smith index of association, AI, dr, was implemented because the AI tends to increase with the number of loci, a trend corrected by this statistic. To summarise, both populations displayed positive AI and dr values  departing significantly (P<0.001) from panmixia (A and rdI = 0).The difference in the degree of microsatellite polymorphism between the three main populations was also reflected by mean number of alleles (MNA) which fluctuated from 4.1 to 4.9 in Populations 1 and 2, respectively, up to 14.2 in population 3 . In Population 2 . SignifiFIS values gathered from populations 1 and 2 are consistently lower than those observed in previous reports Viannia species present in the Amazon Basin and along the Atlantic coast and to a lesser extent westward from the Andes. In contrast, in Population 3 the observed heterozygosity was much less than the expected resulting in a high FIS value, most probably due to population subdivision (Wahlund effect) although high rates of gene conversion or inbreeding cannot be excluded. This group was not tested for all population genetic parameters, since it represents a composite and artificial unit.However, the L. braziliensis from Minas Gerais shared an identical genotype, Lbra26, and further four strains had highly related genotypes, Lbra 28, 29, 30 and 31. These strains most probably represent an epidemic clone and only one Lbra 26 strain was therefore included in the data matrix for linkage disequilibrium calculations. The Chi-square test and the exact test revealed 30 and 49 significant associations (28 and 45 after Bonferroni correction), respectively for population 2. The three populations detected according to the Bayesian algorithm were also significantly separate from each other with highly significant stF values ranging from 0.249 to 0.521  tropica, L. (L.) major and the L. (L.) donovani complex (for review see L. (V.) braziliensis and L. (V.) guyanensisL. (L.) infantum, which has been recently imported multiple times from southwest Europe to the New World L. (Viannia)Because of its high resolution potential, its reproducibility and the possibility of data storage, multilocus microsatellite typing (MLMT) is currently the most widely used approach for strain level differentiation in the genus F statistics revealed the existence of two well-defined populations in the sample set, namely Population 1 consisting of all but one strain of L. (V.) guyanensis from the Amazonas state and Population 2 comprising 43 strains of L. (V.) braziliensis from Eastern Brazil.Different types of population genetic analyses, including Bayesian inference (as implemented in STRUCTURE), distance-based (NJ and Neighbor Net in SplitsTree) and factorial correspondence analysis as well as L. (V.) braziliensis, one of L. (V.) guyanensis and the strains belonging to other species, most of which were isolated in the north of the country. When the strains of L. (V.) lainsoni, L. (V.) naiffi, L. (V.) shawi, L. (V.) utingensis and L. (V.) lindenbergi were excluded from the data set, the strains of L. (V.) braziliensis were assigned to the same Populations 2 and 3 (data not shown). The Population 3 identified in the complete data set was, however, not well supported by the distance and FCA analyses which showed that the strains of this population are highly diverse and only distantly related to each other as can be clearly seen in the NeighborNet network produced in SplitsTree (L. (V.) lainsoni (3C) and L. (V.) naiffi (3D) were assigned to separate sub-populations. Two strains of L. (V.) braziliensis from Acre (L-2492 and L-2498) were found to be putative L. (V.) braziliensis/L. (V.) naiffi hybrids, as previously suggested L. (V.) naiffi” sub-population. However, strains of L. (V.) shawi grouped together with ten strains of L. (V.) braziliensis, mainly from Acre, strain L-2493 of L. (V.) guyanensis and the strains of L. (V.) utingensis and L. (V.) lindenbergi (3A), and the fourth sub-population (3B) consisted of L. (V.) braziliensis from Pernambuco, Ceará, Pará, Paraná and Amazonas. This last group seems, however to be rather artificial, since seven of the ten strains have hybrid genotypes sharing alleles that are specific for Population 2 and 3, respectively. Strains of L. (V.) braziliensis of population 3 are considerably different from each other and very distinct from those that were assigned to Population 2, except those having mixed memberships to Populations 2 and 3. Whether such strains represent outliers, as stated in a different study that used AFLP for typing strains of L. (V.) braziliensis and L. (V.) peruviana mainly from Peru and Bolivia STRUCTURE identified a third population including 20 strains of litsTree . When relitsTree  and straNeighborNet analyses provide a snapshot overview of the general structure in the data and are useful as a guide for further analysis L. (V.) lainsoni which seem to represent a distinct lineage in the network. When the NeighborNet analysis was performed for Population 3 only, strains of L. (V.) lainsoni, L. (V.) shawi and L. (V.) naiffi were assigned to distinct clusters although conflicting signals were still detectable (L. shawi and its apparent affiliation with L. (V.) braziliensis by this high resolution microsatellite analysis does not agree with results of MLST analysis, in which L. (V.) shawi and L. (V.) guyanensis are not resolved as separate entities hsp70 had already previously suggested that L. (V.) guyanensis and L. (V.) shawi were closely related L. (V.) braziliensis from northeastern Brazil belonging to zymodeme Z75 were however, found to be related to L. (V.) shawiL. shawi thus warrants further investigation with more extensive DNA sequence comparisons. All strains of population 3 and only those are found on long branches in the overall network. Because NeighborNet is prone to long-branch attraction, rapidly evolving lineages can be inferred as being closely related regardless of their evolutionary relationships. Whether these strains have a high mutation rate leading to numerous homoplasies or to convergence, which could be misinterpreted as having evolved once in a common ancestor, remains to be established.For the strains assigned by STRUCTURE to Population 3 no clear structuring is seen in the NeighborNet network obtained for the whole data set , maybe wtectable . The indGiven its high genetic diversity, Population 3 could represent the ancestral lineage and might have given rise to two new populations through bottleneck events (Populations 1 and 2). The Amazon forest seems to be the central distribution area with secondary spreads to the northeast, east and south. This would be consistent with the hypothesis of an Amazon origin of CL in Brazil, with later spread to other regions, most probably through human migrations Almost all strains investigated in this study presented unique microsatellite profiles, except 13 strains from Minas Gerais that had identical or highly similar microsatellite profiles and might have been isolated during an outbreak of CL in this area.L. (V.) braziliensis is much more polymorphic than L. (V.) guyanensis. Furthermore, strains of L. (V.) braziliensis from the Amazon region and from Pernambuco were shown to present the highest level of genetic diversity whereas those from Rio de Janeiro were more homogeneous L. (V.) guyanensis (Population 1) and of L. (V.) braziliensis from east Brazil (Population 2) indicating a similar level of genetic diversity for these groups of strains. For L. (V.) braziliensis strains of Population 3, MNA and thus genetic heterogeneity was however, considerably higher. This confirms that the previously described greater diversity of L. (V.) braziliensis is related to the genetically heterogeneous strains from the north of Brazil.Previous studies using isoenzyme typing L. (V.) guyanensis investigated herein were more diverse than expected considering that they all belonged to the same zymodeme Z23 which is in agreement the results of a recent MLST study L. (V.) guyanensis sub-populations in the Amazonas state (data not shown) was not supported by the genetic distance analyses. Our MLMT approach confirmed however, the existence of a new L. (V.) guyanensis genotype in Acre, where this species is not commonly found. This strain was very closely related to L. (V.) shawi as previously suggested L. (V.) guyanensis had been studied using a PCR-RFLP approach targeting ribosomal DNA sequences and found to have originated from two ecologically different regions and to differ in clinical manifestations of CL L. (V.) guyanensis from Brazil, Peru, Suriname and French Guyana, the strains grouped according to their geographical origin L. (V.) guyanensis The strains of L. (V.) guyanensis, despite all being of zymodeme Z23, could be individualised by MLMT but were all grouped in Population 1, with the exception of the single strain from Acre. Strains of L. (V.) braziliensis with identical isoenzyme patterns were assigned to different populations or genetic groups by MLMT. The majority of the strains presenting the predominant zymodeme Z27 grouped in Population 2 but those with hybrid genotypes were found in Population 3B. This implies that zymodeme Z27 is paraphyletic and does not reflect the genetic diversity of the strains which was also shown by the MLST study We found only weak correlations between the MLMT profiles and the results of previous isoenzyme typing . As alreL. (Viannia) parasites is also influenced by host genetic and/or immune factors L. (Viannia) strains studied herein was that to their geographical origin. Similar observations were published earlier for L. (V.) braziliensisL. (L.) infantum in Brazil We did not find any correlation between a particular MLMT profile and the clinical presentation of the disease. The two strains isolated from MCL patients in Bahia grouped together with strains from CL cases from the same area, although the long term outcome of those CL cases is not known. This is consistent with previous studies which suggested that the clinical outcome of the disease caused by Leishmania has been proved to occur in vitro in the sand fly hosts Leishmania species are still considered as predominantly clonal organisms L. (Viannia) this hypothesis has been challenged by the frequent detection of hybrids involving different species of the L. (Viannia) subgenus indicating that recombination events are much more frequent in these parasites than previously thought L. (V.) braziliensis/L. (V.) peruviana hybrids have been identified by microsatellite typing and found to be quite common in Peruvian foci where both species can occur sympatrically L. (V.) braziliensis/L. (V.) guyanensis hybrids are not uncommon L. (V.) laisoni/L. (V.) naiffi, L. (V.) braziliensis/L. (V.) naiffi, L. (V.) braziliensis/L. (V.) guyanensis in Brazil L. (Viannia) strains has also provided evidence for recombination occurring in both L. braziliensis and L. guyanensisDespite the fact that recombination in L. braziliensis isolated between 1986 and 1992 from human CL cases in Minas Gerais. Nine of these strains shared an identical microsatellite profile (Lgua26) and the profiles of the other four strains differed from profile Lgua26 in only one locus each.Strong linkage disequilibrium, or non-random association of genotypes at different loci, and a distinct phylogenetic signal are the criteria for the identification of clonality L. (Viannia). The substantial heterozygote deficiency and extreme inbreeding found by MLMT analysis of L. (V.) braziliensis from Bolivia and Peru was consistent with a predominantly endogamic mode of reproduction (mating with relatives) with occasional recombination events between individuals of different genotypes L. (V.) guyanensis from French Guyana suggesting a high level of sexual recombination and substantial endogamy Fis values. In our study, mild linkage disequilibrium was observed together with relatively low Fis values, compared to those previously published Fis values observed for the L. guyanensis strains from the Brazilian Amazon region (0.184) and for the L. braziliensis strains from Eastern Brazil (0.088) are below the values observed in former publications (0.278 and 0.307) and this is without partitioning steps to correct for a potential Wahlund effect, which might deflate this index. Consequently, the selfing rates for the considered populations are likely to be ≪0.50. This implies that clonality, selfing and random union of gametes contribute to the shaping of Viannia's natural populations. However, one of the future challenges is to understand why the contribution of sex is more significant in the Amazon Basin.Different levels of clonality versus recombination have been earlier suggested to occur within some bacterial and protozoan species due to variation in geographic sampling LeishmaniaL. braziliensis strain, MHOM/BR/75//M2904, that has been fully sequenced so far, 30 of the 35 chromosomes were clearly triploid, three were tetrasomic and one hexasomic L. braziliensis show similar or different ploidy patterns, as shown for all other Leishmania species examined so far L. major chromosomal content varies not only from strain to strain but also from cell to cell creating ‘mosaic aneuploidy’. This leads to high karyotypic diversity and conserved intra-strain genetic heterogeneity combined with loss of heterozygosity per cell. The total number of alleles can, however be maintained in a strain. As a consequence, DNA-based typing methods, including the microsatellite typing approach used herein, cannot decide if a cell population (or strain) consists of heterozygous cells or of homozygous cells presenting different allelic and ploidy content The analyses used for calculations of heterozygosity are based on the assumption of diploidy. Recently, whole genome sequencing and FISH analyses have, however confirmed significant chromosomal copy number variations for different species of L. (Viannia) and indicated that L. (V.) braziliensis and, to a lesser extent, L. (V.) guyanensis exhibit features indicative of both clonality and recombination. Recombination could explain the tremendous genetic diversity and limited population structure. The genetic heterogeneity of Brazilian strains of different species of the subgenus L. (Viannia) was found to be higher than previously shown by techniques such as isoenzyme typing and PCR-RFLP approaches. The different clustering approaches used in this study identified two different genetic groups or populations, one consisting of L. (V.) guyanensis strains from the Amazon region and the other of L. (V.) braziliensis strains from the southeast of Brazil, clearly differentiated from the other investigated strains. L. (V.) braziliensis strains from the north of Brazil did not group with those from the Atlantic coast but were found to be very polymorphic. These strains seemed to be more closely related to the strains of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni also isolated in northern Brazilian CL foci. All findings concerning the strains from northern Brazil may, however, be subject to bias due to an inadequate sampling strategy. More strains need to be sampled from this region in order to fine tune the population structure of these parasites and their mode of reproduction.In conclusion, this study showed the extensive microsatellite diversity present in the subgenus Figure S1L. (Viannia) strains. A midpoint rooted Neighbour-joining (NJ) tree (rectangular version) was calculated for the MLMT profiles of 120 strains of different species of the subgenus L. (Viannia), based on 15 microsatellite markers and using the Chord distance measure. The assignment of these strains to three main populations by the Bayesian model-based clustering approach implemented in STRUCTURE is indicated by colored branches: population 1 (green), population 2 (red) and population 3 (blue). Strains belonging to these populations are listed in L. (V.) guyanensis analysed in this study. Population 2 consists of 43 strains of L. (V.) braziliensis mainly from east Brazil. Population 3 is very diverse and includes all investigated strains of L. (V.) lainsoni, L. (V.) naiffi, L. (V.) shawi, L. (V.) utingensis, L. (V.) lindenbergi, 20 strains of L. (V.) braziliensis mainly from the north of Brazil as well as one strain of L. (V.) guyanensis from Acre. Putative hybrids are indicated by red or blue circles, according to their population assignment. Strain origins are indicated in the window alongside.Rectangular NJ tree showing the populations and subpopulations of 120 Brazilian (TIF)Click here for additional data file.Figure S2L. (Viannia) to three populations. Population 1 (green) comprises all but one strain of L. (V.) guyanensis analysed in this study. Population 2 (red) consists of 43 strains of L. (V.) braziliensis mainly from eastern Brazil. Population 3 (blue) is very diverse and includes all investigated strains of L. (V.) lainsoni, L. (V.) naiffi, L. (V.) shawi, L. (V.) utingensis, L. (V.) lindenbergi, 20 strains of L. (V.) braziliensis mainly from the north of Brazil as well as one strain of L. (V.) guyanensis from Acre. Four sub-populations are distinguished in Population 3 when STRUCTURE was re-run separately for the strains of this population. Sub-population 3A comprises all strains of L. (V.) shawi, eight strains of L. (V.) braziliensis , the single strain of L. (V.) guyanensis from Acre, L. (V.) utingensis and L. (V.) lindenbergi; 3B ten strains of L. (V.) braziliensis ; 3C all strains of L. (V.) lainsoni; and 3D all strains of L. (V.) naiffi and two putative L. (V.) braziliensis/L. (V.) naiffi hybrids.Population structure of the 120 strains inferred by Bayesian analysis with STRUCTURE. The Bayesian algorithm assigned the 120 Brazilian strains of subgenus (TIF)Click here for additional data file.Figure S3Choloepus didactylus and four from Didelphis marsupialis, and one from a sand fly, Lutzomyia anduzei, all other strains were isolated from human CL cases.NeighborNet network of Population 1 as inferred by STRUCTURE. Six strains were isolated from animal hosts, two from (TIF)Click here for additional data file.Figure S4Nectomys sp. and Mesocricetus auratus, three from human MCL and three from human DL cases, all other strains were isolated from human CL cases. The assignment of the strains to the two sub-populations of Population 2, A and B, is indicated. Strains presenting mixed membership coefficients in two sub-populations are highlighted in grey. The two sub-populations are largely confirmed by the phylogenetic network albeit some strains occur at intermediate positions.NeighborNet network of Population 2 as inferred by STRUCTURE. Four strains were isolated from animal hosts, two from dogs and one each from (TIF)Click here for additional data file.Figure S5Dasypus sp., two from Cuniculus paca and one each from Coendou sp., Cebus apella and Rattus rattus, five from sand flies, three from Lutzomyia whitmani and one each from L. tuberculata, L. squamiventris and Lutzomyia sp., all other strains were isolated from human CL cases. The four sub-populations of Population 3, A, B, C and D, are indicated. The phylogenetic network confirms the assignment of strains of L. (V.) lainsoni to sub-population C and that of strains of L. (V.) naiffi to sub-population D. The sub-populations A and B are not well supported in this NeighborNet network. Strains of L. (V.) shawi were found on a separate branch together with one strain of L. (V.) braziliensis from Pará, L-0326, and the single strain of L. (V.) guyanensis from Acre, L-2493.NeighborNet network of Population 3 as inferred by STRUCTURE. Eight strains were isolated from animal hosts, three from (TIF)Click here for additional data file.Figure S6isF values were calculated for each of the 15 loci and over all loci. Strains with identical genotypes were excluded from the analyses to avoid medical driven sampling bias and clones over-representation. For each locus, 95% confidence intervals (CI) were obtained by bootstrapping over loci (GENETIX). P values are indicated by stars (P<0.05).Calculation of inbreeding coefficients for Populations 1 and 2. (PDF)Click here for additional data file.Table S1Leishmania (Viannia) used in this study. T – reference strain of the species; 1 – zymodemes according to the CLIOC system – IOC/Z 2 – population assignment according to STRUCTURE analysis; 3 – normalization of microsatellite fragment sizes in described as Designation, characteristics and MLMT profiles of the Brazilian strains of subgenus (XLSX)Click here for additional data file.Table S2Distribution of zymodemes and animal hosts for the populations and sub-populations found by MLMT.(DOCX)Click here for additional data file.


Toxoplasmosis, a zoonotic disease distributed worldwide, is an infection caused by the ubiquitous obligatory intracellular coccidian protozoan organism, Toxoplasma gondii. It is a major public health concern because the disease is serious in terms of mortality or physical and /or psychological sequellae in patients with HIV disease. The aim of the study was to assess the seroprevalence of Toxoplasma gondii IgG and IgM antibodies and associated risk factors in HIV infected and non-infected individuals attending Felege Hiwot referral hospital, Bahir Dar, Northwest Ethiopia.Toxoplasma gondii IgG and IgM antibodies using a commercially available ELISA kit. Socio-demographic and associated risk factors for Toxoplasmosis from each individual were also obtained and the data was analyzed using SPSS version 18.A cross sectional study was conducted at Felege Hiwot referral hospital, Bahir Dar, Amhara National Regional State. Venous blood samples were collected from 103 HIV infected pre anti-retroviral therapy patients at Felege Hiwot referral hospital and 101 HIV negative apparently healthy voluntary blood donors at the blood bank. Serum samples were analyzed for anti-T. gondii IgG and IgM antibodies, respectively. Multivariate analysis using logistic regression showed that anti-T. gondii seropositivity was independently significantly associated with undercooked or raw meat consumption  and having contact with cat  in HIV positive individuals. In HIV negative apparently healthy blood donors, prevalence of anti-T. gondii antibodies were 70.29% and 2.97% for IgG and IgM, respectively. Multivariate analysis showed that undercooked or raw meat consumption  and sex  were independently significantly associated with anti-T. gondii IgG seropositivity, with a significantly higher number of males affected than females.Of the examined HIV seropositive individuals, 87.4% (90/103) and 10.7% (11/103) were positive for anti-T. gondii antibodies in HIV infected pre-ART and HIV non-infected apparently healthy blood donors in Bahir Dar. Consumption of undercooked or raw meat might greatly contribute towards acquiring T. gondii infection in HIV infected pre-ART and HIV non-infected apparently healthy blood donors. It may be appropriate to include routine serological screening test for determination of anti-T. gondii antibodies in HIV infected pre-ART individuals and HIV negative apparently healthy blood donors. In addition, health education towards avoiding eating undercooked and raw meat, and avoiding contact with cats were recommended.The present findings showed a high sero-prevalence of anti- Toxoplasma gondii that infects a wide range of animals, man and birds [T. gondii is an opportunistic parasitic infection in immune compromised hosts [Toxoplasmosis is a globally distributed zoonosis  caused bd birds ,3. T. god hosts ,5 and esd hosts . Prevaled hosts . The pard hosts  and up td hosts .T. gondii is transmitted to humans by eating raw or inadequately cooked infected meat [T. gondii infection ranges from 10 to 23 days [ed meat ; throughed meat . In adul23 days .Toxoplasmosis is a major public health concern because the disease is serious in terms of mortality or physical and /or psychological sequellae in patients with HIV disease . In the T. gondii are generally considered to represent reactivation of latent infection [Toxoplasma antibodies are of a great interest in HIV-infected patients. Ethiopia is one of the high HIV epidemic countries [Toxoplasmosis serologic status and risk factors of infection among HIV-infected patients and HIV non-infected apparently healthy individuals remain unknown. Therefore, this study aimed to determine the seroprevalence of anti-T. gondii IgG and IgM antibodies in HIV infected and non-infected individuals and to identify the possible risk factors associated with toxoplasmosis in Bahir Dar, northwest Ethiopia.In the immunocompromised patients, diseases due to fection . Detectiuntries  with app′N 37°23′E11.6°N 37.383°E11.6; 37.383 coordinates and an elevation of 1840 meters above sea level. Based on the 2007 Census conducted by the Central Statistical Agency of Ethiopia (CSA), this city has a total human population of 221,991 [A cross sectional study was conducted to determine the seroprevalence and risk factors of toxoplasmosis in HIV infected and non-infected individuals at Felege Hiwot referral hospital, Bahir Dar, Amhara National Regional State, northwest Ethiopia. Bahir Dar is the capital of Amhara National Regional state, located approximately 578 km Northwest of Addis Ababa, having a latitude and longitude of 11°36The study participants were individuals who visited the hospital to know their HIV sero-status and to donate blood for their families. Thus, the study subjects were HIV sero-positive pre-ART individuals attending the pre-ART clinic of Felege Hiwot referral hospital (Group1), and HIV sero-negative apparently healthy adults who came to the hospital for blood donation (Group 2). The convenient sampling method was followed.Treponema pallidum sero-negatives); 2) aged 18–55 years; in both cases individuals who accepted to participate in the study were considered.Inclusion criteria of the study subjects in Group 1 were: 1) HIV sero-positive ART naive individuals attending the pre-ART clinic for further management; 2) aged 18 years and older; 3) not taking chemoprophylaxis for toxoplasmosis; and in Group 2 were: 1) voluntary blood donors  according to the manufacturers’ instructions. Positive and negative controls were used with each series of anti T. gondii IgG/IgM test ; results were obtained by comparison with a cut-off value measured at 450nm absorbance.Serum samples were screened for anti-Ethical clearance to conduct the study was obtained from Aklilu Lemma Institute of Pathobiology (ALIPB), Addis Ababa University (AAU) and Amhara National Regional State Health Bureau (ANRSHB). The purpose and procedures of the study were explained and a written informed consent was obtained from all study participants. Study participants with positive test result for toxoplasmosis were treated following standard protocols at Felege Hiwot referral hospital, following the FMOH , guidelines .P values were determined and taken as a level of significance when found less than 0.05.Data was entered into MS Excel, cleaned and analyzed using SPSS software, with Version 18.0. Pearson Chi-square, Odds Ratio and 95% confidence interval were used for group comparison and association of variables. T. gondii IgG antibodies in HIV positives pre-ART individuals and apparently healthy blood donors were found to be 87.4% (90/103) and 70.29% (71/101), respectively. The difference between the two groups in anti-T. gondii IgG seropositivity was statistically significant (p=0.003). Similarly, the overall anti-T. gondii IgM seropositivity was 10% (11/103) among HIV infected Pre ART and 2.97 % (3/101) in HIV non-infected study participants; the difference was statistically significant (p=0.02). Overall, the prevalence of anti-T. gondii antibodies in HIV positives pre-ART individuals was significantly higher than HIV non-infected apparently healthy blood donors . In addition, the prevalence of anti-T. gondii IgG, among the HIV positive pre-ART individuals was not statistically influenced by the sex and age categories tested. In the study there were only three individuals with history of blood transfusion, and all of them were HIV positive.In HIV infected pre-ART individuals studied, where the majority reported having contact with cats 72/103 (69.9%) and the habit of eating raw or undercooked meat 86/103 (83.5%), the bivariate analysis showed a significant association between these factors and seropositivity to anti-T. gondii IgM seropositivity and all the risk factors evaluated.In HIV infected pre-ART individuals, no statistically significant association was detected between anti-T. gondii IgG seropositivity. The rate of anti-T. gondii IgG seropositivity was significantly higher in males than females, and in those eating raw or undercooked meat compared to those who did not  and being male  were independently significantly associated with anti-T. gondii IgG seropositivity .As shown in Table T. gondii[Toxoplasma specific IgG and IgM antibody levels were analyzed using ELISA, one of the standard procedures for detection of antibodies [T. gondii and the chronological status of such exposure. In the present study the prevalence rate of anti-T. gondii IgG antibodies in HIV positive pre-ART individuals was significantly higher than in HIV negative blood donors . Similar finding of significantly higher Toxoplasma seropositivity in HIV infected individuals than in HIV-uninfected people was also demonstrated in Addis Ababa [Toxoplasmosis refers to the disease caused by T. gondii. In the ibodies . Detectis Ababa . The fins Ababa  and in Ns Ababa . These ds Ababa . In addiToxoplasma IgG antibodies using the Sabin-Feldman test and 80% seroprevalence was reported [T. gondii IgG antibody in the HIV-positive adult population [T. gondii antibodies could be due to differences in geographical distribution and/ or possible risk factors and socioeconomic conditions [The overall finding of a high seroprevalence of IgG in HIV positive pre-ART individuals was comparable with the study done in Akaki town, suburban of Addis Ababa, in which sera were screened for anti-eported . Our finulation . Howeverulation . It alsoulation . The obsditions  contribuT. gondii IgM seropositivity rate was 10.7% in HIV infected pre-ART individuals. A similar study in India reported 6% anti-Toxoplasma IgM positivity using double sandwich ELISA [T. gondii IgG in HIV infected pre-ART individuals. Similar results were demonstrated by studies done in Nazaret town, Ethiopia [T. gondii seropositivity. It is worth mentioning that the number of participants with a history of blood transfusion was only three and all of them were HIV positive. Nonetheless, a similar study in Malaysia has documented the absence of a significant association between a history of blood transfusion and T. gondii seropositivity [In the present study, anti-h ELISA . The conthiopia  and at Lthiopia . On the itivity . Beef, lT. gondii antibodies. The exception to this is the age group 21–30 in the HIV negatives where individuals are 5.58 times more likely to be seropositive for anti-T. gondii antibodies. This is in agreement with previous studies on the prevalence of anti-T. gondii IgG antibodies from blood donors in Yucatan [T. gondii antibodies in a healthy population from Slovakia [T. gondii IgG and IgM antibodies in HIV negative blood donors were 70.29% and 2.97%, respectively. Seroprevalence of IgM that we found was comparable to the 2.4% and 1.9% reported from Czech Republic and Mexico blood donors, respectively [T. gondii IgG antibodies in blood donors is consistent with the 75% prevalence reported from Brazil [T. gondii IgG in HIV negative individuals. Undercooked meat consumption has been found to be an important factor in parasite transmission in several studies [T. gondii IgG from male healthy blood donors was significantly higher than female HIV negative blood donors (p=0.001).According to the present findings, no statistically significant difference was observed among different age groups and inhabitants of rural and urban areas of HIV infected and non-infected study participants, and the associated sero-reactivity of anti-Yucatan  and serolovakia . The prectively ,35. The  Brazil  and 69%  Brazil . Consumpstudies ,35. A siToxoplasma IgG antibodies and education level [In the present study no significant association was found between the seroprevalence of toxoplasmosis and different educational status. Similar findings were recorded between anti n level  and the n level .T. gondii IgG antibodies could indicate a chronic infection [T. gondii IgM/IgG antibodies suggests the potential risk of parasite transmission by blood transfusion practices.Generally, IgM antibodies are detectable early after infection and can persist for prolonged times after infection. Therefore, the presence of IgM does not necessarily indicate an acute infection and its presence in subjects with anti fection . Hence, T. gondii seroprevalence (3%–22%) [T. gondii specific IgG in HIV-infected patients compared to non-infected individuals [Toxoplasma seroprevalence in both HIV infected and non-infected individuals that is not statistically significantly different between the two groups [Toxoplasma infection in HIV patients have been previously proposed [Toxoplasma infected individuals that leads to increased exposure to HIV infection. Such a change in behavior could be due to parasite-driven personality changes, as described in Toxoplasma-infected individuals by others [Toxoplasma infection is a marker of exposure to risky social contacts or habits, which correlates with early HIV infection.Findings on the association of HIV and toxoplasmosis seroprevalence are varied in different parts of the world. Previous studies involving HIV-infected individuals have reported wide variations in 3%–22%) ,39. Someviduals ,41, wherviduals . Serologviduals . Previou groups . On the  groups  found siroposed . One pos others . AnotherToxoplasma infection in males than females in the bivariate analysis, which disappeared in the multivariate analysis. In our case the difference is maintained in the multivariate model and may be attributed to differences in socialization behavior or environmental exposure of males compared to females.Regarding the higher prevalence in males than females, Shimelis et al.,  observedT. gondii antibodies in HIV infected pre-ART and HIV non-infected apparently healthy blood donors in Bahir Dar. Consumption of undercooked or raw meat and contact with cat might greatly contribute towards acquiring T. gondii infection. It may be appropriate to include a routine serological screening test for determination anti-T. gondii antibodies in HIV infected pre ART individuals and HIV negative apparently healthy blood donors. Those HIV infected individuals, positives for anti-T. gondii antibodies should be considered for chemoprophylactic treatment. In addition, health education particularly not to eat undercooked and raw meat, and avoiding contact with cats should be considered.In conclusion, the present findings showed a high seroprevalence of anti-This study was financially supported by Addis Ababa University and additional funds from Amhara National Regional State Health Bureau.The authors declared that they have no competing interest.FW conceived the study, undertook statistical analysis and drafted the manuscript. NK, AT and TK initiated the study and made major contributions to the study design and statistical analysis. All authors contributed to the writing of the manuscript and approved the submitted version of the manuscript.


Pseudomonas fluorescens species complex and ten strains of Stenotrophomonas sp., in order to assess intraspecific REP diversity and to gain insight into long-term REP evolution.Repetitive extragenic palindromic elements (REPs) constitute a group of bacterial genomic repeats known for their high abundance and several roles in host cells´ physiology. We analyzed the phylogenetic distribution of particular REP classes in genomic sequences of sixty-three bacterial strains belonging to the rayt-flanking REPs, or RAYT loss. RAYTs of both studied bacterial groups are monophyletic, and their cognate REPs show species-specific characteristics, suggesting shared evolutionary history of REPs, RAYTs and their hosts.Based on proximity to RAYT (REP-associated tyrosine transposase) genes, twenty-two and thirteen unique REP classes were determined in fluorescent pseudomonads and stenotrophomonads, respectively. In stenotrophomonads, REP elements were typically found in tens or a few hundred copies per genome. REPs of fluorescent pseudomonads were generally more numerous, occurring in hundreds or even over a thousand perfect copies of particular REP class per genome. REP sequences showed highly heterogeneous distribution. The abundances of REP classes roughly followed host strains´ phylogeny, differing markedly among individual clades. High abundances of particular REP classes appeared to depend on the presence of the cognate RAYT gene, and deviations from this state could be attributed to recent or ancient mutations of The results of our large-scale analysis show that REP elements constitute intriguingly dynamic components of genomes of fluorescent pseudomonads and stenotrophomonads, and indicate that REP diversification and proliferation are ongoing processes. High numbers of REPs have apparently been retained during the entire evolutionary time since the establishment of these two bacterial lineages, probably because of their beneficial effect on host long-term fitness. REP elements in these bacteria represent a suitable platform to study the interplay between repeated elements, their mobilizers and host bacterial cells. Haemophilus and related bacteria [Genomes of many higher eukaryotes are known to teem with repetitive DNA elements. By contrast, bacteria are notorious for their high coding density , which lbacteria . Similarbacteria . Repeatebacteria . Finallybacteria ,7.Escherichia coli and related enterobacteria [Pseudomonas putida[Pseudomonas fluorescens[Stenotrophomonas maltophilia[Xanthomonas campestris and others [A/GG) and downstream complementary  region with variable base composition. REP elements are mostly arranged into repeats of higher order. REPINs (REP doublet forming hairpin) are composed of two closely spaced REPs in inverted orientation [P. fluorescens[P. putida[S. maltophilia[E. coli, consist of tandemly repeated REPIN-like doublets [E. coli, three significant proteins interact with REPs or BIMEs: integration host factor [E. coli[S. maltophilia[REP  elements have now been known for over 30 years , originabacteria . They weas putida, Pseudomuorescens,12, Stenltophilia, Xanthomd others , each spd others  to refleentation  and wereuorescens,15, P. pP. putida and S. mltophilia. BIMEs  . Relatede family ,22, RAYTmentally , and thementally . The strOwing to rapid expansion of Next-generation DNA sequencing methods, increasing numbers of new genomic sequences are reported each year. These provide great opportunity to conduct comparative analyses. We explored the distribution of REP elements and their associated RAYTs in sequenced genomes of sixty-three fluorescent pseudomonads and ten stenotrophomonads, two groups of omnipresent environmental bacteria with biotechnological and biocontrol applications ,25. Our Pseudomonas fluorescens complex and in Stenotrophomonas sp. (data not shown). Comprehensive mining of bacterial genomic databases recovered 63 genomes affiliated to Pseudomonas fluorescens (fluorescent pseudomonads) and 10 genomes affiliated to Stenotrophomonas maltophilia (stenotrophomonads). Among fluorescent pseudomonads, species of P. agarici, P. brassicacearum, P. chlororaphis, P. extremaustralis, P. fragi, P. fuscovaginae, P. mandelii, P. protegens, P. psychrophila, P. synxantha and P. tolaasii, previously shown to belong to the P. fluorescens complex [Pseudomonas sp. isolates, unassigned to any species. For stenotrophomonads, Pseudomonas geniculata, synonym for S. maltophilia[Stenotrophomonas sp. SKA14. To resolve the evolutionary relationship between the strains, phylogenetic trees were constructed from three housekeeping genes  genes and REP elements. This approach (see Methods) yielded twenty-two and thirteen unique classes of REP elements in fluorescent pseudomonads PF1 – PF22) and stenotrophomonads SM1 – SM13), respectively , single clade  or several clades . Various other patterns in distribution can also be detected. REP numbers typically reach hundreds of occurrences of particular REP classes, and are typically more numerous in fluorescent pseudomonads. Here, in four cases, REP numbers exceed a thousand of copies per genome . Typically, several REP classes occur in a single host strain.The copy numbers of particular REP element classes were determined and compared in genomes of related bacterial strains. Table P. fluorescens R124, P. sp. UW4 and P. sp. GM78) contained two RAYTs associated with two different REPs belonging to the same orthogroup IV. In these cases, one RAYT gene is always located at a novel chromosomal site. This indicates different evolutionary origins of these RAYTs/REPs, for example RAYT duplication followed by mutation of flanking REPs into another REP class of orthogroup IV, or horizontal acquisition and integration of RAYT gene into a novel locus.Finally, we examined if the presence of RAYTs influenced REP abundance. In most cases, RAYTs associated with abundant REP classes were indeed present in host bacterial strains Table . On averi.e. at least once in every orthogroup, Table The instances when particular REPs were overrepresented while their cognate RAYTs were absent appeared quite often. However, for a great majority of these cases, one of the following was also observed: i) related strains possessed RAYTs associated with REP sequences in question, or ii) RAYTs in given strain were associated with different REP classes, belonging to the same orthogroup  and downstream palindrome. REP copy numbers were determined using pDRAW32 [Bacterial genomic sequences were downloaded from the NCBI Genome database . RAYTs wia RAYTs  as queryia RAYTs . Identifia RAYTs . REP seq pDRAW32 .rpoB), DNA gyrase beta subunit (gyrB) and RNA polymerase sigma subunit (rpoD) as well as RAYT protein sequences were processed with MEGA5 package [Concatenated complete nucleotide sequences of genes coding for RNA polymerase beta subunit  and ancient (right) RAYT loss, B – recent (left) and ancient (right) RAYT duplication and diversification, C – recent (left) and ancient (right) REP orthoswitch. REP elements (dots) and their associated rayt genes (arrows) are denoted with the same color. The host chromosomes are represented as black circles. The REP numbers are approximate.The model is based on two premises: i) RAYTs specifically disseminate their cognate REP elements throughout their host genome, ii) REP copies are getting slowly but constantly lost from host genome, due to accumulation of mutations. See Results and Discussion for further explanation.Click here for file


Kaposi sarcoma is a mesenchymal tumor associated to a human herpes virus-8. It often occurs in human immunodeficiency virus-positive subjects. Colorectal localization is rare. We report the case of a colorectal Kaposi sarcoma complicating a refractory ulcerative colitis treated with surgery after the failure of immunomodulator therapy in a human immunodeficiency virus-negative heterosexual man. Kaposi's sarcoma (KS) is a mesenchymal tumor, arising predominantly in the skin but which can affect any organ system. It's associated to human herpes virus-8 (HHV8) , 2. Pati2. Abdominal exam was normal. There was no skin lesion. Iron deficiency anemia (11g/dL) and signs of inflammation: erythrocyte sedimentation rate (ESR) of 37mm and C - reactive protein (CRP) of 74mg/L were noted. Albumin level was 25g/L. Ileocolonoscopy showed large superficial ulcerations, spontaneous bleeding and pseudo-polyps. Lesions were mainly located in the rectosigmoid colon and the colitis was extended to the hepatic flexure. Histology found clear signs of active UC with no signs of malignancy. Oral corticosteroids were prescribed with a good initial response. At week 4 of corticosteroid therapy, we noted a relapse of bloody diarrhea, inflammation and aggravation of anemia . A left-sided colonoscopy didn't show severe endoscopic signs. Copro-parasitological examinations were negative. Cytomegalovirus testing was negative too. Detection of Clostridium difficile toxins was not done. Intravenous corticosteroids and parenteral nutrition were prescribed during 1week. At week 5 of corticosteroid therapy, another relapse occurred  and we had considered that it was a refractory severe UC. As the patient was reticent to surgery, medical treatment with immunomodulators was indicated. Assessment before immunosuppressive therapy was normal. He received blood transfusion, albumin infusion, parenteral nutrition and intravenous corticosteroids (with progressive decreasing doses). After improvement in health status, anemia (9g/dL) and albumin concentration (37g/L), azathioprine  and IFX  were prescribed with unchanged disease activity. Drug failure led to a surgical treatment. A subtotal colectomy with double stomy of the ileum and of the sigmoid colon was performed. Colon macroscopic examination revealed multiple polypoid red lesions associated with large ulcerations. Histologic examination of polypoid lesions showed fusocellular spindle cells proliferation with a slit like vascular channels and extravased red blood cells consistent with the diagnosis of KS, associated with typical features of UC . He was initially treated with oral and local mesalamine with a good response. In 2011, he was treated 3 times with steroids (1mg/kg/day of prednisone) for a moderately active disease without a complete relief of diarrhea. In December 2011, at physical exam, his body mass index was 17kg/mes of UC . Immunoles of UC . InvolveThere are four clinical variants of KS with distinct clinical and epidemiological characteristics: classic, endemic, acquired immunodeficiency syndrome and iatrogenic . IatrogeAlthough it is rare, it is important to consider a concomitant KS in patients with refractory severe ulcerative colitis, on immunosuppressor therapy, independently of HIV status.


Compensatory changes in cis and trans are revealed when these promoters are used to drive expression in the other species. Functional changes in the C. briggsae promoter, which has experienced accelerated sequence evolution, did not lead to alteration of gene expression in its endogenous environment. Coevolution among promoter elements suggests that complex epistatic interactions within cis-regulatory elements may facilitate their divergence. Our results offer a detailed picture of regulatory evolution in which subtle, lineage-specific, and compensatory modifications of interacting cis and trans regulators together maintain conserved gene expression patterns.Phenotypes that appear to be conserved could be maintained not only by strong purifying selection on the underlying genetic systems, but also by stabilizing selection acting via compensatory mutations with balanced effects. Such coevolution has been invoked to explain experimental results, but has rarely been the focus of study. Conserved expression driven by the  cis-regulatory DNA of one species is used to drive expression in another species. In this way, we identified regions of the C. elegans and C. briggsae unc-47 promoters that have coevolved with the lineage-specific trans-regulatory environments of these organisms. The C. briggsae promoter experienced accelerated sequence change relative to related species. All of this evolution occurred without changing the expression pattern driven by the promoter in its endogenous environment.Some phenotypes, including gene expression patterns, are conserved between distantly related species. However, the molecular bases of those phenotypes are not necessarily conserved. Instead, regulatory DNA sequences and the proteins with which they interact can change over time with balanced effects, preserving expression patterns and concealing regulatory divergence. Coevolution between interacting molecules makes gene regulation highly species-specific, and it can be detected when the  However, considerable sequence divergence has been documented in orthologous regulatory elements Since evolutionary biologists are interested in species divergence, most studies of regulatory evolution focus on gene expression differences between two species or strains One possibility is that purifying selection preserves functional elements, which are nestled within functionless and divergent sequences cis-regulatory elements in a common trans background. This can be done in several ways. Diverged regulatory elements can be introduced into a hybrid trans environment on the genome-wide scale by interspecific crosses, after which allele-specific expression can be measured by microarray One way to document this phenomenon is to perform functional comparisons of orthologous cis and trans, and allowing experimental manipulations of regulatory sequence and genetic background to isolate mechanisms of action cis-regulatory element swaps can be performed Transgenic methods provide an approach to studying the loci of regulatory evolution in a controlled, experimentally manipulable way. Single regulatory elements from the genome of one species can be introduced into a host of a pure-species, rather than hybrid, background. While this method can only be used to dissect one regulatory element at a time, it has the advantages of being tractable in non-hybridizing species, isolating particular molecular interactions between Caenorhabditis elegans and C. briggsae, two nematodes with considerable sequence divergence cis-regulatory element of the unc-47 gene, which has a simple and quantifiable expression pattern cis-trans and cis-cis interactions have evolved.This necessary quality is found in C. elegans contains 26 GABAergic neurons  in around one-third of individuals . The trans environment of a C. elegans host animal always drives stronger expression in these cells than the trans environment of a C. briggsae host . Only when the cis and trans regulators of unc-47 from different species are combined experimentally can their different functions be observed.We therefore propose an explanation  for our cis and trans regulators, we searched for transcription factors that are expressed in SDQR/L. The gene ahr-1, which encodes a bHLH transcription factor C. elegans ahr-1 (ia03) mutants, expression of a C. briggsae unc-47 promoter in SDQR/L was completely abolished  and coding sequence differences between the species that imply, but do not prove, that the function of ahr-1 has diverged between C. elegans and C. briggsae and could affect unc-47 regulation.To identify the coevolved bolished , even thbolished . This extrans-regulatory differences are more difficult to identify than cis-regulatory differences. A causal cis-regulatory change must be located within the DNA that has divergent function . On the other hand, evidence for change in trans potentially implicates the entire genome. The causal nucleotide changes between C. elegans and C. briggsae could potentially reside in coding or regulatory sequence of ahr-1, an upstream regulator of ahr-1, a binding partner or antagonist of ahr-1, or in multiple interacting loci. However, we can use the ahr-1 clue to dissect the mechanism of cis-regulatory divergence.However, unc-47 . We interpreted this to mean that endogenous expression of unc-47 in DVB is controlled via this motif as well as one or more additional sequences. The distal promoters of unc-47 are highly divergent in their sequences and contribute to the robustness of expression in DVB cis-regulatory change that maintains expression in DVB via the conserved core consensus motif and has pleiotropic effects on expression in SDQR/L when the promoters are swapped between species.Worms carrying the mutated promoter showed significantly less intense expression in DVB  or vice versa . Whereas cis element evolutionary rate accelerations associated with phenotypic change are well-documented We compared the  species  in orderbriggsae . This pa derived , while Cunc-47 regulation in Caenorhabditis nematodes. Although the endogenous patterns of expression remain unchanged, the mechanisms responsible for maintaining them seem to have diverged in the C. briggsae lineage, possibly as a consequence of substantial divergence in the regulatory sequence. Because the C. briggsae promoter does not drive strong expression in SDQR/L in its endogenous trans environment despite its dramatic sequence evolution, a compensatory trans change must be inferred.Comparative functional and sequence data yielded a picture of evolution of trans-regulatory environments, both C. elegans and C. briggsae promoters of unc-47 drive similar weak expression in SDQR and SDQL. This similarity cannot be due to conservation of the underlying regulatory system, given the difference in expression patterns of reciprocally swapped promoters. The C. elegans promoter directed virtually no expression in SDQR/L of C. briggsae, while the C. briggsae promoter was strongly expressed in these cells when placed in C. elegans. At least two lineage-specific changes must have occurred since the divergence of C. elegans and C. briggsae: one in the unc-47 promoter and another, possibly in a transcription factor that controls its expression. Similar cases have been documented in yeast A simple model suggests the types of coevolutionary changes that were involved . In theicis-regulatory elements of unc-47 revealed that the same sequences responsible for misexpression of the C. briggsae promoter in C. elegans also control expression in other cells, such as AVL, RIS, and DVB  into temperature-sensitive C. elegans pha-1 (e2123) strain C. briggsae strains carrying extrachromosomal arrays were produced by injecting a mixture  into YR91 Cbr-unc-119 (nm67) strain. To examine the function of transcription factor ahr-1 we used ahr-1 (ia03), a loss-of-function allele C. briggsae unc-47 promoter fusion was integrated into the YR91 strain of C. briggsae through bombardment To generate reporter transgenes, promoter sequences were PCR amplified from genomic DNA and cloned upstream of GFP into the Fire lab vector pPD95.75. In all cases, the start codon of the C. elegans carrying transgenes were grown with abundant food and L4-stage worms were selected. These were immobilized on agar slides with 10 mM sodium azide in M9 buffer. The slides were examined on a Leica DM5000B compound microscope under 400-fold magnification. Presence/absence of GFP expression in a cell was recorded only if the cell was clearly visible, unobstructed by the intestine. Worms without any visible GFP expression were assumed to have lost the transgene. Fluorescence measurements were carried out as previously described Mixed-stage populations of CACGCTATTAAC to ATCACTATTAAC (putative binding site underlined). To test the roles of the nonconserved regions upstream of the conserved motif, we swapped these sequences between the C. elegans and C. briggsae unc-47 promoters via a two-step site-directed mutagenesis.Two types of mutagenized promoters were generated using the QuickChangeII kit (Stratagene). To test the role of the AHR-1 core consensus motif in regulating expression in SDQR/L, we introduced two point mutations in the conserved AHR-1 consensus motif Figure S1C. elegans promoter in C. elegans, (B) C. briggsae promoter in C. elegans, (C) C. elegans promoter in C. briggsae, (D) C. briggsae promoter in C. briggsae.Consistency of SDQR/L expression between independent strains carrying extrachromosomal arrays. The distribution of expression intensity in SDQR and SDQL relative to D-type neurons is plotted. The fraction of individuals showing expression over individuals scored is indicated underneath. Individuals were only scored if their cell was clearly visible, unobstructed by the intestine. Two independent strains carrying extrachromosomal arrays were measured for (A) (PDF)Click here for additional data file.Figure S2trans-regulatory environment, expression in SDQR and SDQL is presented. C. elegans is represented by straight lines, C. briggsae by wavy lines. Frequency of expression is represented by the width, and intensity of expression relative to D-type neurons by the height of black boxes. Compare with C. elegans promoter in C. elegans, (C) C. briggsae promoter in C. elegans, (D) C. elegans promoter in C. briggsae, (E) C. briggsae promoter in C. briggsae.Expression driven by integrated transgenes is consistent with expression driven by extrachromosomal arrays and between independent strains. (A) For each combination of promoter and (PDF)Click here for additional data file.Figure S3C. elegans and C. briggsae promoters in C. elegans. Frequency of expression is represented by the width, and intensity of expression relative to D-type neurons by the height of black boxes. Compare with C. elegans promoter in C. elegans and (C) C. briggsae promoter in C. elegans.Expression driven by MosSCI single-copy integrated transgenes is consistent with expression driven by extrachromosomal arrays and between independent strains. (A) Expression in SDQR and SDQL is presented for both (PDF)Click here for additional data file.Figure S4C. elegans and C. briggsae promoters have a single AHR-1 core consensus motif. Sequences of the C. elegans and C. briggsae promoters of unc-47. A single, conserved AHR-1 core consensus motif (highlighted in red) is present in both promoters.(PDF)Click here for additional data file.Figure S5unc-47 promoters. (A) An alignment of the sequences flanking the conserved AHR-1 consensus motif (boxed in red). Regions conserved between all four species are shaded in gray. In (B–J) bold face letters represent C. briggsae sequence, regular font represents C. elegans. All transgenes were tested in C. elegans. Full-length C. elegans promoter (B) drives inconsistent expression in SDQR, no expression in SDQL, and consistent expression in DVB. Full-length C. briggsae promoter (C) drives consistent expression in SDQR, SDQL and DVB. C. elegans promoter with C. briggsae Region A (D) or Region B (E) drives C. briggsae-like expression in SDQR, C. elegans-like expression in SDQL, while expression in DVB is unaffected. Additionally, expression in AVL and RIS is either severely reduced or abolished. Partial replacement of C. elegans Region B by 10 nucleotides of C. briggsae Region B (F) completely abolishes expression in SDQR/L and reduces the intensity of expression in DVB. This phenotype is similar to that observed with the C. briggsae promoter mutated in the conserved AHR-1 consensus motif (C. briggsae promoter with C. elegans Region A (G) or Region B (H) drives C. briggsae-like expression in SDQR, C. elegans-like expression in SDQL, while expression in DVB is unaffected. These chimeric promoters also drive strong ectopic expression in several head neurons and the pharyngeal-intestinal valve. A partial replacement of C. briggsae Region B by 10 nucleotides of C. elegans Region B (I) does not affect expression in SDQR/L in the context of the full-length promoter. However, in the context of the proximal promoter (J), the percentage of individuals expressing in SDQR is reduced and expression in SDQL is completely eliminated. Compare to Expression patterns driven by chimeric (PDF)Click here for additional data file.Figure S6unc-47. (A) VISTA plot of primary sequence conservation in the unc-47 cis-regulatory regions from C. briggsae, C. remanei, and C. brenneri aligned to C. elegans. Window size = 20 bp, threshold = 70%. (B) In the alignment of the proximal promoters from C. briggsae, C. remanei, C. brenneri, and C. elegans, conserved nucleotides are shaded in gray. Position -1 is the first nucleotide upstream of the translation start site. The conserved AHR-1 core consensus motif is boxed in red. (C) Insertions, deletions, and substitutions on each lineage are depicted as black boxes. The number of lineage-specific changes was counted by two methods. In the less stringent method, for C. briggsae, C. remanei, and C. brenneri, sites conserved between two species but divergent in the third were counted as branch-specific. The number of affected sites and events calculated in this way are reported in C. elegans and two other species were counted as species-specific. Using this method, C. briggsae has 14 substitutions, one insertion of 12 nucleotides, and a single deletion. C. remanei has 2 substitutions and one deletion of 2 nucleotides. C. brenneri has 6 substitutions and 3 insertions affecting a total of 6 sites. Eight sites for which the polarity of mutations could not be determined are not represented. In the region extending upstream of position -122, C. briggsae has 10 substitutions, 6 insertions affecting 26 nucleotides, and one deletion of 4 nucleotides. In contrast, in this region, there were no C. remanei or C. brenneri specific events.Pattern of sequence conservation in the proximal promoter of (PDF)Click here for additional data file.Table S1Consistency of independent strains.(PDF)Click here for additional data file.


AbstractKapsa acuminata, Kapsa quadrispina, Kapsa puerensis and Kapsa yanheensisspp. n. from southwest China are described and illustrated, and a key to the species recorded from China is provided.In the present paper, four new species,  KapsaErythroneurini (Typhlocybinae) with Typhlocyba furcifrons Jacobi, 1941 as its type species. Recent taxonomic work on the genus includes Kapsa have been recorded from China in PageBreakthe above studies. In this paper, four new species from southwest China are described and illustrated and a key to males of Chinese Kapsa is given. All specimens examined are deposited in the collection of the Institute of Entomology, Guizhou University, Guiyang, China (GUGC) and British Museum Natural History (BMNH).The leafhopper genus Dworakowskahttp://species-id.net/wiki/KapsaKapsa Dworakowska, 1972: 402; Typhlocyba furcifrons Jacobi, 1941Dorsum beige, yellow or white. Vertex unicolorous or with pair of preapical spots or with large median apical spot. Scutellum pale with or without dark lateral triangles or entirely dark.Head narrower than pronotum, fore margin weakly produced, broadly rounded. Forewing with outer apical cell short; hind wing submarginal vein not extended to wing apex.Male pygofer lobe with oblique dorsolateral internal ridge, usually with sparse long fine setae on lateral surface; dorsal appendage movably articulated; ventral appendages absent. Subgenital plates with 2-6 basal macrosetae. Style apex with extension; preapical lobe prominent. Connective Y-shaped, with central lobe well developed. Aedeagus with or without processes, gonopore apical on ventral surface. Anal tube usual with basal processes.Tautoneura Anufriev, 1969 externally  but the forewing lacks the red dots found in Tautoneura. It also differs in having the male pygofer lobe with basolateral setae not distinctly enlarged, and the pygofer without ventral appendages. The genus is also similar to Empoascanara Distant, 1918 in the male genitalia although differing in having the subgenital plate microsetae on the dorsal margin not in groups and the style with a 2nd extension. It differs externally from Emopoascanara in its narrower head.The genus is similar India; Nepal; Sri Lanka; China ; Vietnam; Indonesia; New Guinea.urn:lsid:zoobank.org:act:C3213F38-E901-470D-BF1C-0A6D218E5B5Fhttp://species-id.net/wiki/Kapsa_acuminataDorsum beige. Vertex with pair of milky yellow preapical spots; pronotum with anterior margin and median area ivory-white . ForewinAbdominal apodemes nearly reaching posterior margin of 4th sternite .Male pygofer with dorsal appendage bifurcate far from base . Anal tuBody length males 2.3~2.5 mm.Holotype, male, China: Guizhou Province, Mayanghe National Nature Reserve, 4 Oct. 2007, coll. Yue-hua Song. Paratypes: five males, same date as holotype.Kapsa biprocessa Song & Li (2008), but can be distinguished mainly by the aedeagus with pair of basal processes close to shaft; the more acute apex of shaft in ventral view with the gonopore longer , but the aedeagus has a pair of atrial processes and apical processes , but the aedeagus has branched apical processes, the lower one much longer than upper one  (see also The new species is similar to see also , but thesee also  and the see also .The new species is named for its type locality: Yanhe.