Additional file 2: of Occurrence of health-compromising protozoan and helminth infections in tortoises kept as pet animals in Germany

Table S2. Isolated bacteria in pet tortoises from Germany; origin and species of tortoises regarding to the infestation with potentially health-critical endoparasites, performed microbiology and aetilogical death reason/ reported clinical signs. (DOCX 18 kb

Additional file 1: of Occurrence of health-compromising protozoan and helminth infections in tortoises kept as pet animals in Germany

Table S1. Data obtained from necropsies and faecal samples such as date of receive, post code and information collected from pet owners such as age, husbandry conditions, gender, length, weight, species and diagnosed parasites. (XLSX 394 kb

Resistance profiles of <i>Ae</i>. <i>aegypti</i> from Jakarta.

<p>Resistance profiles of <i>Ae</i>. <i>aegypti</i> from Jakarta.</p

Association of V1016G and F1534C with resistance to 0.75% permethrin.

<p>Association of V1016G and F1534C with resistance to 0.75% permethrin.</p

Allele-specific PCR exemplaries on agarose gel.

<p>(A) V1016G mutation, (B) F1534C mutation. Wt: Wild-type, Mh: Mutant homozygote, MHt: Mutant heterozygote, M: 50-bp DNA marker.</p

Co-localization of DNA with histones (H3), NE and MPO in tachyzoite-induced NET structures.

<p>Co-cultures of bovine PMN and <i>B. besnoiti</i> tachyzoites were fixed, permeabilized, stained for DNA using Sytox Orange (red: B, E, H) and probed for MPO (green: A), histones (green: D) and NE (green: G) using anti-MPO, anti-histone (H3) and anti-NE antibodies and adequate conjugate systems. Areas of respective co-localization (merges) are illustrated in C, F, I. The arrow in (C) indicates delicate globular structures within NETs. Arrows in (F) and (I) indicate tachyzoites being trapped in NET structures. Photomicrographs are of representative cells from 3 independent experiments. The time culture in this experiment was 60 min.</p

ROS production and enzymatic activities of NE and MPO in tachyzoite-exposed bovine PMN.

<p>Bovine PMN were exposed to <i>B. besnoiti</i> tachyzoites, zymosan (positive control) or plain medium (negative control) for 30 min. Thereafter, enzymatic activities of NE and MPO as well as ROS production were measured in the supernatants via the NE-chromogenic substrate MeoSuc-Ala-Ala-Pro-Val-chloromethyl ketone, Amplex red and the oxidation of DCFH-DA to fluorescent DCF, respectively. All experiments were performed in triplicates. Arithmetic means of three PMN donors, minimum and maximum. Differences were regarded as significant at a level of <i>p</i>≤0.05.</p

Additional file 2: of Differential intracellular calcium influx, nitric oxide production, ICAM-1 and IL8 expression in primary bovine endothelial cells exposed to nonesterified fatty acids

Effects of BAPTA and EGTA treatments on the slope of the Fura-2/AM signal in cells treated with myristic acid (MA) palmitic acid (PA), stearic acid (SA), linoleic acid (LA), or oleic acid (OA). Data are means ± standard deviations; NS: not significant. (DOC 29 kb

Infectivity of <i>B. besnoiti</i> tachyzoites after exposure to bovine PMN.

<p>Vital <i>B. besnoiti</i> tachyzoites were co-cultured for 3 h with bovine PMN ( =  PMN + <i>B.b</i>.) allowing for effective NET formation. To dissolve potential NET structures, DNase was supplemented 15 min before the end of the incubation period ( =  <i>B.b.</i> + PMN + DNase). Incubation of tachyzoites in plain medium served as PMN-free, infection control ( =  <i>B.b.</i> only). After incubation, samples were transferred to confluent BUVEC monolayers for 1 h. Thereafter, the cell layers were thoroughly washed and infection rates were estimated. Arithmetic means and standard deviations of three PMN donors, minimum and maximum. Differences were regarded as significant at a level of <i>p</i>≤0.05.</p

Dose-dependency of tachyzoite-triggered NET formation.

<p>PMN and <i>B. besnoiti</i> tachyzoites were incubated at different ratios (PMN:tachyzoites = 1∶1, 1∶2, 1∶3). After incubation, samples were analysed for extracellular DNA by quantifying PicoGreen-derived fluorescence intensities. Each condition was performed in duplicates. Arithmetic means of three PMN donors, minimum and maximum. Differences were regarded as significant at a level of <i>p</i>≤0.05.</p