In vitro antileishmanial activity of resveratrol and its hydroxylated analogues against <Emphasis Type="Italic">Leishmania major</Emphasis> promastigotes and amastigotes

Dermatitis caused by penetrating bird schistosome cercariae is an emerging global public health problem. Infections may be prevented by the use of topical formulations that inhibit cercarial skin penetration. We evaluated nine water resistant formulations by exposing treated arms of volunteers to Trichobilharzia szidati cercariae. Six formulations protected from cercarial invasion. However, after immersion of the treated skin in water (2 × 20 min), only two formulations offered full protection: (1) Safe Sea™, a cream protecting against jelly fish, (2) niclosamide in water resistant sun protecting cream formulations at concentrations as low as 0.05%. In an in vitro system Safe Sea™ and a 0.1% niclosamide formulation caused a high damage rate in T. szidati (92% and 99% after 5 min; only niclosamide with lethal effect) but not in Schistosoma mansoni (1% and 72%; both formulations with lethal effect). However, a 1% niclosamide formulation damaged S. mansoni sufficiently (100% after 5 min) and might offer full penetration protection.     

The presence of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> in isolates of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> impacts significantly on DNA fingerprinting results

The genetic characterization of Trichomonas vaginalis (Protista: Trichomonadidae), the causative agent of trichomoniasis in humans, is central to understanding the epidemiology, treatment, drug resistance, and virulence as well as the diagnosis and control of this parasite. Various molecular approaches, including DNA fingerprinting, have been employed for this purpose, and random amplification of polymorphic DNA (RAPD) continues to be utilized. However, little attention has been paid to the fact that some T. vaginalis populations can harbor symbiotic Mycoplasma hominis and/or other agents, which could cause artifacts in the RAPD results. In the present study, we demonstrate clearly that the presence of M. hominis from T. vaginalis isolates impacts significantly on RAPD results and on the subsequent analyses and interpretation of data sets. Moreover, symbiotic M. hominis displays an isolate-to-isolate variability in RAPD profile before elimination, suggesting a variability of M. hominis infection. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Trichobilharzia</Emphasis> spp. in natural conditions in Annecy Lake,France

Annecy Lake is a well-known focus of human cercarial dermatitis in France. Identification of the parasites, however, was not performed in the past. Previous studies suspected two species, Trichobilharzia franki and Trichobilharzia regenti, based on the presence of parasites in mallards and/or morphological identification of snails emitting ocellate furcocercariae. Following a standardized molecular approach, we studied snails and furcocercariae and compared their haplotypes with those deposited in GenBank. The selected markers were the second internal transcribed spacer ITS-2 for the snails and ITS-2 and D2 domain of the ribosomal DNA for the parasites. Our results confirm the presence of T. franki and T. regenti and two probable new species that could be potential agents of cercarial dermatitis. All the snails emitting the ocellate furcocercariae belong to the same species identified as Radix peregra (=Radix ovata = Radix balthica). Parasite–host relationships between species of the genus Trichobilharzia and snails of the genus Radix do not seem to be as specific as supposed previously.     

Genetic differentiation between two species of the medicinal leech,<Emphasis Type="Italic"> Hirudo medicinalis</Emphasis> and the neglected<Emphasis Type="Italic"> H. verbana</Emphasis>, based on random-amplified polymorphic DNA

The medicinal leech is one of the few parasitic invertebrates widely used in medicine and as a scientific model object. Because of a dramatic decline in its natural populations, it is subject to considerable conservation effort. Despite all attention, there is confusion regarding the taxonomic status of different morphological forms. The prevailing view is that all varieties of medicinal leech in Europe represent the same species, Hirudo medicinalis. However, the present study based on RAPD molecular markers demonstrates that a second European taxon, H. verbana, forms a distinct species. Phenetic clustering and principal coordinate analysis of eight populations revealed the same basic structure, reflecting taxonomic rather than geographic subdivision. Variation between species explained 60% of the total molecular variance (_CT=0.60, P<0.001). Both taxa displayed a significant number of specific RAPD markers. Conversely, no specific fragment supporting the geographic association of both taxa was found. Since the stronghold of commercially exploited medicinal leech populations in southeastern Europe and Turkey belongs to H. verbana, most medicinal and scientific applications probably use this species, not H. medicinalis. Appropriate taxonomic correction of international conservation conventions and legislation is essential.     

Molecular identification of<Emphasis Type="Italic"> Trichinella spiralis</Emphasis> and<Emphasis Type="Italic"> Trichinella britovi</Emphasis> by diagnostic multiprimer large mitochondrial rRNA amplification

Trichinella parasites with different epidemiological features still occur in Europe and four species of genus Trichinella have been identified: T. spiralis, T. britovi , T. nativa and T. pseudospiralis. Until now, two of them, T. spiralis and T. britovi, have been identified in Poland. In our studies we selected sequence coding for large mitochondrial rRNA (mt LrDNA) as a genetic marker and developed a sensitive LrDNA multiprimer PCR assay allowing for rapid identification of T. spiralis and T. britovi, parasites present in wild and domestic animals in Poland.     

Decreased humoral and pathologic responses in undernourished mice infected with<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis>

Studies of selected species of Lymnaeidae and Planorbidae showed that infection with bird schistosomes occurred in 14 of the 26 Polish lakes studied. In Planorbarius corneus larvae of Bilharziella polonica were found, and in Lymnaea stagnalis and Radix auricularia larvae of Trichobilharzia ocellata. In spite of their comparatively low prevalence, cercariae, potentially pathogenic to humans, may have caused dermatitis in patients registered in one of the regions under study. The low prevalence of infection among the host snails may have been compensated for by the enormous number of cercariae released by them, which, moreover, showed a long-lasting viability, particularly at low ambient temperature.     

Population sub-structuring among <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> stocks

To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei’s genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). The mean index of association (I _A = 0.92) significantly deviated from zero, and the average number of multilocus genotypes (G/N ratio) was 0.21. Twenty-four multilocus genotypes were defined from the combination of alleles at the three loci. The Kenyan sub-populations showed F _st = 0.28 and analysis of molecular variance showed significant divergence (22.7%) between the Laikipia, Kulal and Galana regions. The regional and host distribution of multi-locus genotypes significant population differentiation and high Nei’s genetic distances suggest existence of genetic sub-structuring within T. evansi stocks while the few multi-locus genotypes and deviation of association index from zero indicate the lack of recombination. In conclusion, this study reveals that some genetic sub-structuring does occur within T. evansi, which has a clonal population structure.     

Genetic variability and population structure of endangered <Emphasis Type="Italic">Panax ginseng</Emphasis> in the Russian Primorye

Background  

The natural habitat of wild P. ginseng is currently found only in the Russian Primorye and the populations are extremely exhausted and require restoration. Analysis of the genetic diversity and population structure of an endangered species is a prerequisite for conservation. The present study aims to investigate the patterns and levels of genetic polymorphism and population structures of wild P. ginseng with the AFLP method to (1) estimate the level of genetic diversity in the P. ginseng populations in the Russian Primorsky Krai, (2) calculate the distribution of variability within a population and among populations and (3) examine the genetic relationship between the populations.     

DNA markers define plastid haplotypes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

To identify genetic markers in the Arabidopsis thaliana plastid genome (ptDNA), we amplified and sequenced the rpl2-psbA and rbcL-accD regions in 26 ecotypes. The two regions contained eight polymorphic sites including five insertions and/or deletions (indels) involving changes in the length of A or T mononucleotide repeats and three base substitutions. The 27 alleles defined 15 plastid haplotypes, providing a practical set of ptDNA markers for the Columbia, Landsberg erecta and Wassilewskija ecotypes that are commonly used in genetic studies and also for the C24 and RLD ecotypes that are the most amenable for cell culture manipulations.     

Molecular profiles of<Emphasis Type="Italic"> Trypanosoma brucei</Emphasis>,<Emphasis Type="Italic"> T. evansi</Emphasis> and<Emphasis Type="Italic"> T. equiperdum</Emphasis> stocks revealed by the random amplified polymorphic DNA method

A total of 20 random primers (10-mers) were used to amplify RAPD markers from the genomic DNA of four Trypanosoma brucei stocks from East and West Africa, four T. evansi stocks from Africa, Asia and South America and one T. equiperdum stock from Asia. Between 65 and 88 reproducible fragments ranging from 0.25 to 2.15 kb were generated from these stocks depending on the stock/primer combination. The similarity coefficient (SC) among the stocks of T. brucei from Kenya, Nigeria, Tanzania and Zambia ranged from 62.9% to 74.0% (average: 67.6%). The SC among the stocks of T. evansi from Kenya, China and Brazil was 76.4%–95.5% (average: 86.4%), while the SC between T. evansi stock from China and Brazil was 95.5%. For T. evansi and T. equiperdum, the SC among the stocks ranged from 81.2% to 94.4% (average: 87.6%). As for the SC among the stocks of T. brucei and T. evansi, it was found to be from 54.7% to 80.3% (average: 68.0%) and the SC among stocks of T. brucei and T. equiperdum was from 59.4% to 76.9% (average: 68.1%). Our results indicate that the stocks of T. evansi from China and from Brazil are more closely related to the stock of T. equiperdum from China than to the stocks of T. evansi isolated from Kenya and to the stocks of T. brucei. In addition, our results further support the hypothesis that T. evansi stocks from China and Brazil could have arisen from a single lineage. The possible evolution of T. evansi and T. equiperdum is also discussed.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Comparative analyses among the <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>, <Emphasis Type="Italic">Trichomonas tenax</Emphasis>, and <Emphasis Type="Italic">Tritrichomonas foetus</Emphasis> 5S ribosomal RNA genes

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers FJ492747, FJ492748, FJ492749, FJ492750, and FJ492751.     

Effects of <Emphasis Type="Italic">Schistosoma mansoni</Emphasis> and <Emphasis Type="Italic">Schistosoma haematobium</Emphasis> infections on calcium content in their intermediate hosts

Flame atomic absorption spectrometry was performed to determine the alteration of calcium concentration in the soft parts and shells of Biomphalaria alexandrina and Bulinus truncatus due to the infection with Schistosoma mansoni and S. haematobium, respectively. The results showed significant lowering in the calcium content of the shells of cercariae shedding B. alexandrina and B. truncatus relative to the calcium content in the shells of uninfected ones. In contrast, the calcium content in the soft parts of cercariae shedding snails was higher than in the soft parts of uninfected snails; the differences were statistically significant. Generally, calcium content was significantly higher in the shells than in the soft parts of the snails, regardless infected or uninfected. The results obtained and the hypothesis of hypercalcification in shells of infected snails were discussed.     

Genetic and phylogeographic structure of populations of<Emphasis Type="Italic"> Pulex simulans</Emphasis> (Siphonaptera) in Peru inferred from two genes (<Emphasis Type="Italic">CytB</Emphasis> and<Emphasis Type="Italic"> CoII</Emphasis>)

In this paper we discuss the potential usefulness of determining the phylogeographic and phylogenetic patterns of a vector for understanding the spread of pathogens or insecticide resistance. We do so using the example of Pulex simulans in Peru. Six populations from six different localities were investigated. Mitochondrial DNA sequences were obtained and branching patterns were inferred using phylogenetic reconstruction methods and nested clade analyses. Ten different haplotypes were discovered. Phylogenetic analysis revealed P. simulans in Peru as a monophyletic group, containing clades that were generally not geographically correlated. The data suggest that P. simulans is not a single genetic entity but rather that this species shows a high degree of intraspecific variation. Restricted gene flow with long distance dispersal coupled with range expansion and long distance colonization are likely to have contributed to the observed patterns of variation.     

Molecular identification of <Emphasis Type="Italic">Trichuris vulpis</Emphasis> and <Emphasis Type="Italic">Trichuris suis</Emphasis> isolated from different hosts

Trichuris suis was isolated from the cecum of two different hosts (Sus scrofa domestica—swine and Sus scrofa scrofa—wild boar) and Trichuris vulpis from dogs in Sevilla, Spain. Genomic DNA was isolated and internal transcribed spacers (ITS)1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced using polymerase chain reaction techniques. The sequence of T. suis from both hosts was 1,396 bp in length while that of T. vulpis was 1,044 bp. ITS1 of both populations isolated of T. suis was 661 nucleotides in length, while the ITS2 was 534 nucleotides in length. Furthermore, the ITS1 of T. vulpis was 410 nucleotides in length, while the ITS2 was 433 nucleotides in length. One hundred fifty-four nucleotides were observed along the 5.8S gene of T. suis and T. vulpis. Intraindividual and intraspecific variations were detected in the rDNA of both species. The presence of microsatellites was observed in all the individuals assayed. Sequence analysis of the ITSs and the 5.8S gene has demonstrated no sequence differences between T. suis isolated from both hosts (S. scrofa domestica—swine and S. scrofa scrofa—wild boar). Nevertheless, clear differences were detected between the ITS1 and ITS2 of T. suis and T. vulpis. Furthermore, a comparative molecular analysis between both species and the previously published ITS1-5.8S-ITS2 sequence data of Trichuris ovis, Trichuris leporis, Trichuris muris, Trichuris arvicolae, and Trichuris skrjabini was carried out. A common homology zone was detected in the ITS1 sequence of all species of trichurids.     

Polymorphic<Emphasis Type="Italic"> Alu</Emphasis> insertions and the genetic structure of Iberian Basques

Eight Alu sequences (ACE, TPA25, PV92, APO, FXIIIB, D1, A25 and B65) were analyzed in two samples from Navarre and Guipúzcoa provinces (Basque Country, Spain). Alu data for other European, Caucasus and North African populations were compiled from the literature for comparison purposes to assess the genetic relationships of the Basques in a broader geographic context. Results of both MDS plot and AMOVA revealed spatial heterogeneity among these three population clusters clearly defined by geography. On the contrary, no substantial genetic heterogeneity was found between the Basque samples, or between Basques and other Europeans (excluding Caucasus populations). Moreover, the genetic information obtained from Alu data conflicts with hypotheses linking the origin of Basques with populations from North Africa (Berbers) or from the Caucasus region (Georgia). In order to explain the reduced genetic heterogeneity detected by Alu insertions among Basque subpopulations, values of the Wright’s F_ST statistic were estimated for both Alu markers and a set of short tandem repeats (STRs) in terms of two geographical scales: (1) the Basque Country, (2) Europe (including Basques). In the Basque area, estimates of Wahlund’s effect for both genetic markers showed no statistical difference between Basque subpopulations. However, when this analysis was performed on a European scale, F_ST values were significantly higher for Alu insertions than for STR alleles. From these results, we suggest that the spatial heterogeneity of the Basque gene pool identified in previous polymorphism studies is relatively recent and probably caused by a differential process of genetic admixture with non-Basque neighboring populations modulated by the effect of a linguistic barrier to random mating.     

Detection and molecular characterization of <Emphasis Type="Italic">Babesia caballi</Emphasis> and <Emphasis Type="Italic">Theileria equi</Emphasis> isolates from endemic areas of Brazil

Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterhouse located in Araguari, Minas Gerais State, Brazil. For each blood sample, the packed cell volume (PCV) was determined, and Giemsa-stained smears were microscopically examined for the presence of hemoparasites. The plasma was examined by the indirect fluorescent antibody test for detection of antibodies against Babesia caballi and Theileria equi. In addition, DNA was extracted and analyzed by a multiplex real-time polymerase chain reaction (PCR), specific for B. caballi and T. equi. Products of PCR were sequenced and compared with each other and with known sequences. The serological results showed a total prevalence of 91.0% for T. equi and 83.0% for B. caballi, while by PCR, prevalences of 59.7% for T. equi and 12.5% for B. caballi were observed. However, no correlations were seen between positivity (neither by serology nor by PCR) and PCV values. As expected, the microscopic examination of blood smears showed low sensitivity in detecting the infections when compared to the PCR. Only 35 out of 570 blood smears were positive, with parasitemias below 0.1%. No congenital transmission was detectable. As far as sequencing is concerned, no differences were seen among the isolates of each species nor among them and known sequences available. These results confirm, by molecular methods, the high prevalence rates of T. equi and B. caballi infections in carrier horses in Brazil. However, no diversity was observed among the isolates within the studied regions.     

Haplotype architecture of the norepinephrine transporter gene <Emphasis Type="Italic">SLC6A2</Emphasis> in four populations

The norepinephrine transporter (NET) regulates levels of monoamine neurotransmitters integral to a variety of behaviors and autonomic functions. Two SLC6A2 polymorphisms have been used in genetic association studies, generating intriguing but nondefinitive results on traits such as hypertension and mood. One of these SLC6A2 variants is functional but rare. The other is common but not informative over the entire 48 kb SLC6A2 region and is insufficient to capture the functional diversity potentially contained within any SLC6A2 region. To elucidate SLC6A2 haplotype structure and define markers sufficient to capture haplotype diversity within detected haplotype blocks, 26 single-nucleotide polymorphisms (SNPs) were genotyped in 384 individuals evenly divided across Finnish Caucasian, US Caucasian, Plains American Indian, and African American populations. Three conserved blocks, 13.6, 12.5, and 25 kb in size and showing little evidence for historical recombination were observed in all populations. Haplotype diversity in block 1 and numbers of common haplotypes were highest in African Americans, among whom 5–6 optimal markers were sufficient to maximize diversity of each block. For other populations, 2–3 markers/block sufficed, but the optimal markers differed across populations. The SLC6A2 haplotype map and 25-marker panel (excluding the monomorphic one) is a comprehensive tool for genetic linkage studies on phenotypes related to NET function.Parts of this work were presented at the 53rd Annual Meeting of American Society of Human Genetics, Los Angeles, CA, USA, November 2003.