Additional file 1: Table S1. of Circulation of Dirofilaria repens and Dirofilaria immitis in Moldova

Mosquitoes collected in Moldova between 2010 and 2015 with information on the coordinates of the sampling site, sampling date, mosquito species, pool size and Dirofilaria screening results. (XLSX 32 kb

Additional file 1: Figure S1. of A comparative study of the localization and membrane topology of members of the RIFIN, STEVOR and PfMC-2TM protein families in Plasmodium falciparum-infected erythrocytes

Characterization of α-VSA sera. In order to specify the exact target sequence of the polyclonal antisera, the semi-conserved and the variable domain of the RIFIN variants RIF40 and RIF50 and of the STEVOR proteins PFL2610w and MAL13P1.7 were separately expressed as recombinant proteins. The RIFIN antisera α-RIF40.2, α-RIF44 and α-RIF50 (A) as well as the STEVOR α-PFL2610w, α-MAL13P1.7, α-PFC0025c and α-PFA0750w (B), which were generated by immunization with a protein spanning the semi-conserved and the variable domain, were subsequently used for Western blot analysis. Furthermore, the α-PfMC-2TM-SC serum generated solely against the semi-conserved region of PFF1525c was included in the analysis to check the specificity for its protein family (C). Approximately 20 ng of each recombinant protein or lysate from 1x107 cell membranes were loaded in each lane as indicated. All four antisera, α-RIF40.2, α-RIF50, α-PFL2610w and α-MAL13P1.7, are specific to the semi-conserved region of their own antigen. Furthermore, all antisera used in this study were shown to recognize the semi-conserved region of their own protein family, but are not cross-reactive with other VSA families. Unspecific cross-reactions of the small VSA antisera with the His-tag were excluded using an unrelated Entamoeba histolytica His-tagged protein recombinantly expressed under the same conditions (His-tag control) and lysate from uninfected red blood cells (RBC lysate). The recombinant proteins of all three small VSA families tend to form multimer complexes; accordingly bands corresponding in size to monomers are labelled with a single *, dimers with ** and trimers with *** as calculated by the size of the recombinant His-tagged protein

Additional file 1: Figure S1. of A comparative study of the localization and membrane topology of members of the RIFIN, STEVOR and PfMC-2TM protein families in Plasmodium falciparum-infected erythrocytes

Characterization of α-VSA sera. In order to specify the exact target sequence of the polyclonal antisera, the semi-conserved and the variable domain of the RIFIN variants RIF40 and RIF50 and of the STEVOR proteins PFL2610w and MAL13P1.7 were separately expressed as recombinant proteins. The RIFIN antisera α-RIF40.2, α-RIF44 and α-RIF50 (A) as well as the STEVOR α-PFL2610w, α-MAL13P1.7, α-PFC0025c and α-PFA0750w (B), which were generated by immunization with a protein spanning the semi-conserved and the variable domain, were subsequently used for Western blot analysis. Furthermore, the α-PfMC-2TM-SC serum generated solely against the semi-conserved region of PFF1525c was included in the analysis to check the specificity for its protein family (C). Approximately 20 ng of each recombinant protein or lysate from 1x107 cell membranes were loaded in each lane as indicated. All four antisera, α-RIF40.2, α-RIF50, α-PFL2610w and α-MAL13P1.7, are specific to the semi-conserved region of their own antigen. Furthermore, all antisera used in this study were shown to recognize the semi-conserved region of their own protein family, but are not cross-reactive with other VSA families. Unspecific cross-reactions of the small VSA antisera with the His-tag were excluded using an unrelated Entamoeba histolytica His-tagged protein recombinantly expressed under the same conditions (His-tag control) and lysate from uninfected red blood cells (RBC lysate). The recombinant proteins of all three small VSA families tend to form multimer complexes; accordingly bands corresponding in size to monomers are labelled with a single *, dimers with ** and trimers with *** as calculated by the size of the recombinant His-tagged protein

Expression of peptidase genes of the isolate HM-1:IMSS as determined by microarrays

<p><b>Copyright information:</b></p><p>Taken from "The genome: primary structure and expression of proteolytic enzymes"</p><p>http://www.biomedcentral.com/1471-2164/8/170</p><p>BMC Genomics 2007;8():170-170.</p><p>Published online 14 Jun 2007</p><p>PMCID:PMC1913524.</p><p></p> Error bars represent the standard error of the mean of nine hybridizations (biological replicates)

Additional file 3: Table S3. of Host-feeding patterns of mosquito species in Germany

Chi-square tests on the differences in the frequencies of detected mammalian or avian hosts among all pairs of used trapping methods with adjusted P-values for multiple comparisons. (DOCX 13 kb

Additional file 2: Table S2. of Host-feeding patterns of mosquito species in Germany

Kruskal-Wallis tests on the differences of the percentages of detected birds, non-human mammals and humans between the three land use classes (natural, rural and urban) for the three most frequent mosquito species (Fig. 3). (DOCX 14 kb

Temporal Expression and Localization Patterns of Variant Surface Antigens in Clinical <em>Plasmodium falciparum</em> Isolates during Erythrocyte Schizogony

<div><p>Avoidance of antibody-mediated immune recognition allows parasites to establish chronic infections and enhances opportunities for transmission. The human malaria parasite <em>Plasmodium falciparum</em> possesses a number of multi-copy gene families, including <em>var</em>, <em>rif</em>, <em>stevor</em> and <em>pfmc-2tm,</em> which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on <em>in vitro</em> analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by <em>P. falciparum</em> in the human host. To investigate the expression of the <em>var</em>, <em>rif-A</em>, <em>rif-B</em>, <em>stevor</em> and <em>pfmc-2tm</em> family genes under conditions that mimic more closely the natural course of infection, <em>ex vivo</em> clinical <em>P. falciparum</em> isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns in the clinical isolates at various time points during the first intraerythrocytic developmental cycle <em>in vitro</em> were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families <em>rif-A</em>, <em>stevor</em> and <em>pfmc-2tm</em> at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen families were overexpressed in the clinical isolates relative to 3D7, with the exception of the <em>pfmc-2tm</em> family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and <em>Pf</em>MC-2TM. Proteins were exported into the infected erythrocyte at the young trophozoite stage, whereas they remained inside the parasite membrane during schizont stage and were subsequently observed in different compartments in the merozoite. These results reveal a complex pattern of expression of <em>P. falciparum</em> multi-copy gene families during clinical progression and are suggestive of diverse functional roles of the respective proteins.</p> </div

Expression of multi-copy gene families in the 3D7 laboratory strain during erythrocyte schizogony.

<p>Expression profiles of the multi-copy gene families <i>var</i> (red line, squares), <i>rif-A</i> (dark blue line, triangles), <i>rif-B</i> (light blue, upside-down triangles), <i>stevor</i> (green line, diamonds), and <i>pfmc-2tm</i> (yellow line, dots) in long-term <i>in vitro</i> cultivated strain 3D7 during erythrocyte schizogony. In contrast to the clinical isolates, 3D7 parasites exhibited a slight increase of <i>rif-A</i>, <i>stevor</i> and <i>pfmc-2tm</i> expression during the ring stage, and the main peak of expression of the multi-copy gene families was observed in mid-age trophozoites. Transcription of the <i>var</i> gene family was restricted to ring stage parasites. Expression was normalized to the reference gene <i>fructose-bisphosphate aldolase</i> and is represented by either ΔCt (left chart) or relative expression (RELATEXP, right chart). 3D7 time course experiments were performed twice and two samples were analysed for each time point at least in duplicates. Graphically shown are mean values and standard deviations of all qPCR runs performed for the respective time points Parasite developmental age (hpi) and the corresponding parasitic stage (shown as Giemsa staining) are plotted on the x-axis.</p

Proportion of infected erythrocytes with VSAs exported to host cell compartments in clinical isolates and 3D7 at different developmental stages.

1<p>Time point not determined for isolate #1.</p>2<p>Time point of 44 h for isolate #4.</p><p>hpi, hours post-infection.</p

Immunoblot analysis of VSA abundance in the clinical isolate #5 and the 3D7 strain.

<p><b>A, B</b>: Isolate #5 (h: time of <i>in vitro</i> cultivation) and 3D7 (hpi: hours post infection) at successive developmental stages were harvested (<b>A</b>), and differences in VSA abundance in the membrane fraction were assessed by immunoblot using α-RIF40, α-RIF44, α-RIF50, α-STEVOR-mix, α-<i>Pf</i>MC-2TM-SC, and α-<i>Pf</i>MC-2TM-CT antisera (<b>B</b>). As expected, RIFIN and STEVOR were present at higher levels in the clinical isolate; in contrast, <i>Pf</i>MC-2TM proteins were quantitatively increased in 3D7 parasites. Differences were most obvious in pigmented parasite stages (trophozoites, schizonts, left), which also exhibited the highest levels of protein during intraerythrocyic development, but upon longer exposure (*), similar results were also observed for ring stage parasites (right). The luminal endoplasmic reticulum (ER) protein BiP (HSP70) served as a loading control.</p