Wirkung des Fusarientoxins Deoxynivalenol beim wachsenden Schwein in Abhängigkeit von der Darreichungsform

In der Literatur finden sich zahlreiche widersprüchliche Angaben zur Wirkung des Mykotoxins Deoxynivalenol (DON) bei Schweinen, wobei meist für natürlich mit DON kontaminiertes Futter (DONnat) stärkere Wirkungen beobachtet wurden als für künstlich mit DON-Reinsubstanz kontaminiertes Futter (DONrein). In dieser Arbeit wurde der Einfluß von Deoxynivalenol (DON) auf die Entwicklung wachsender Schweine untersucht. Von besonderem Interesse war hierbei die Frage, inwieweit für natürlich kontaminiertes Futter beobachtete Wirkungen (DONnat) auch durch Verfütterung einer mit DON-Reintoxin künstlich kontaminierten, getreidefreien Futtermatrix (DONrein) reproduziert werden können. Hierzu wurden männliche Läuferschweine einerseits mit einer natürlich kontaminierten Getreideration und andereseits mit einer getreidefreien Ration auf Kartoffelbasis unter Zusatz von Reintoxin gefüttert. Aufgrund der baulichen Gegebenheiten sowie der tierschutzrechtlichen Bestimmungen, wurde das Projekt in Teilabschnitten umgesetzt. Neben den Leistungsparametern Futteraufnahme und Gewichtsentwicklung wurden ferner Parameter wie Blut, Darmenzymatik, Gewebeveränderungen und DON-Metabolisierung im Kot untersucht. Zur Abschätzung der erforderlichen Toxingehalte für ein sicheres Auftreten eines Toxineffektes wurden in einem Vorversuch (Durchgang A) jeweils 5 Tiere parallel mit 2000 mg/kg und 4000 mg/kg DONnat bzw. DONrein belastet. Das Fütterungsregime entsprach einer restriktiven Futtervorlage, welche so bemessen war, dass sie einer ad libitum-Fütterung entsprechen sollte. Zu jeder Belastungsgruppe in jeder Futtervariante wurde eine Kontrollgruppe mitgeführt. Die Ergebnisse aus dem ersten Durchgang (A) zeigten lediglich Trends hinsichtlich einer möglichen Toxinwirkung auf. Insbesondere Tiere der natürlichen Belastungsgruppe wiesen Gewichtseinbußen auf. Demgegenüber waren in der Gruppe DONrein, trotz der hohen Toxinbelastung, keine Unterschiede der Leistungsparameter festzustellen. In einem zweiten Durchgang (B) wurde daraufhin jeweils 5 Tieren ausschließlich eine kontaminierte Weizenration mit einer DON-Belastung von 4000 mg/kg und 6000 mg/kg verabreicht, und im Anschluß daran in einem dritten Durchgang (C) wiederum jeweils 5 Tiere ausschließlich mit DONrein in Höhe von 4000 mg/kg und 6000 mg/kg in einer getreidefreien Futtermatrix belastet. Auch das Fütterungsregime wurde in diesen beiden Abschnitten an eine ad libitum-Fütterung adaptiert. Das variierte Versuchsverfahren in Durchgang B ließ signifikante Unterschiede in Gewichtsentwicklung und Futteraufnahme der Tiere erkennen, im gleichermaßen gestalteten Durchgang C konnte jedoch kein Einfluß des zugesetzten reinen DON in der getreidefreien Diät abgeleitet werden. Die Untersuchung der Blutparameter lieferte keinen Anhaltspunkt auf einen systemischen Toxineffekt. Veränderungen einzelner Parameter traten sporadisch und inkonstant auf. Die Thyroxingehalte stiegen nur in der Versuchsgruppe mit reinem Toxin regelmäßig in den Durchgängen A und C gegen Versuchsende an. In den Durchgängen A und B lagen die T4-Werte der getreidehaltig gefütterten Gruppen deutlich höher, als die der getreidefrei gefütterten Tiere, was allerdings der Diät zuzuschreiben war. In Versuchsdurchgang B fiel der Blut-Triglyceridgehalt mit einem signifikanten Anstieg auf, allerdings nur in der mittleren Belastungsgruppe 4000 mg/kg DONrein. Dagegen konnte in diesem Abschnitt ein signifikanter SDH-Anstieg in der Gruppe DONnat gefunden werden. Bezüglich der IgA-Gehalte im Serum waren zwischen den Behandlungen keine Unterschiede zu erkennen. Mit zunehmendem DON-Gehalt im Futter ließ sich lediglich ein Trend zu höheren IgA-Gehalten feststellen, der bei Verabreichung von DONnat deutlich ausgeprägter erschien. Die Fähigkeit der Darmmikroflora (aus dem Rektum), DON zu dem Metaboliten Deepoxy-Deoxynivalenol (DOM-1) zu transformieren war sowohl von der Darreichungsform und der Toxinmenge als auch vom Fütterungsregime abhängig. Der Anteil transformierender Mikroorganismen im Kot nahm unabhängig von der Darreichungsform mit steigender Toxinkonzentration im Futter zu. Bei den Kontrolltieren dagegen war kein einheitliches Muster abzuleiten. Ein Einfluß des Toxins auf den Proteingehalt der Darmmukosa sowie der ALT- und -KGDH-Aktivität der Enterozyten war nicht eindeutig zu bestimmen. Histologisch ließen sich vereinzelt deutlich Veränderungen der Mukosa von Magen und Darm finden, allerdings traten diese Veränderungen ebenfalls unabhängig von der Behandlung auf. Diese Arbeit zeigt den grundsätzlichen Unterschied bezüglich der Efffekte von DON als Reinsubstanz und als natürlich gebildetes Toxin in kontaminiertem Getreide auf. Die bislang festgestellten toxischen Wirkungen von DON sind allein durch Verabreichung der Reinsubstanz ohne natürliche Matrix nicht reproduzierbar. Das heißt, dass im natürlich kontaminierten Futter ein oder mehrere andere toxische Agentien zu den Vergiftungssymptomen beitragen oder diese sogar ausschließlich verursachen. Andererseits ist bei Vergiftungsfällen in der Praxis immer auch DON in entsprechenden Mengen nachzuweisen, DON könnte somit als Leitsubstanz benannt werden. Um die Zusammenhänge und auch um eine sichere Einschätzung der Gefährdung durch diese Substanz gewährleisten zu können, sind hierzu weitere Untersuchung erforderlich. Aber sowohl hinsichtlich der Kosten und des Aufwandes als auch unter Tierschutzaspekten sind die aufzustellenden Versuchskonzepte nur sehr schwer umsetzbar.Publications show a considerable amount of inconsistant information for effects of mycotoxin deoxynivalenol in pigs. Naturally contaminated feeds (DONnat) seem to cause more severe effects than pure DON in artificially contaminated feed (DONpure). This study examined the development of growing pigs under DON-influence. Most interestingly was the question, wether effects of DON-contaminated feed (DONnat) could be replicated using a grainless diet containing pure DON (DONpure). Therefore a group of male pigs were fed a diet containing naturally contaminated wheat and compared to another group fed a grainless diet based on potato supplemented with DONpure. Due to the building capacity and for reasons of animal welfare, the project had to be divided in several parts. Beside the performance parameters feed intake and weight development other parameters (blood, intestinal enzymes, tissue alterations and DON-metabolisation in feces) were examined. To estimate the required DON dose to provide certain toxic effects a preceding study (Part A) was drawn consisting of 4 groups with 5 animals each. The treatment was both naturally contaminated wheat diet and pure DON in grainless potato diet. The contents in both diets were 2000 mg/kg and 4000 mg/kg DONnat respectively DONpure. The amount of food was calculated corresponding to ad libitum feeding. Every treatment group was compared to a control group. The results of Part A only showed slight trends concerning a possible toxic effect. Especially the naturally contaminated group demonstrated weight loss. In contrast, there was no evidence of any toxic effect in the DONpure –group concerning performance. In a second study (Part B) 3 groups comprising 5 animals each received wheat diet, exclusively, containing 4000 mg/kg and 6000 mg/kg DONnat and control group, followed by Part C, altered by feeding grainless potato diet with corresponding amounts of DONpure. Also the feeding regime was changed to a real ad libitum feeding. The trial variation in Part B showed significant differences in weight gain and feed intake. These were not reproducible in Part C, no effect of admitted DONpure in grainless diet was derived. The examination of blood parameters gave no evidence of a systemic toxic effect. Alterations of single parameters were inconstant and intermittent. Only the thyroxin levels increased in the grainless group during Parts A and C at the end of each trial. In Part A and B the levels in the wheat diet groups increased, indicating an effect of the diet. In Part B, the blood triglycerides showed a significant rise, but only in the group with medium exposure of pure DON (4000 mg DONpure /kg). In contrast, a significant rise of SDH contents was found in the contaminated wheat diet group (DONnat). Regarding the serum IgA-levels no differences between the treatments could be diagnosed. With higher DON-levels in food a distinct trend to higher IgA-levels, esp. in the naturally contaminated group (DONnat), could be assessed. The ability of Intestinal flora (rectum) for DON-degradation (DOM-1) depended on both, sort of food (ingredients) and dosage and also the feeding regime. The fraction of transforming microorganisms in faeces rose with increasing toxin contents independent of diet. In contrast, the control animals showed no consistent pattern. The influence of protein content of intestinal mucosa and activity of ALT and -KGDH in enterocytes could not be identified clearly. Several mucosal variations of stomach and intestine were determined in histological examination. These changes also appeared independent of treatment. This study showed basic differences of pure DON and DON from a naturally contaminated source, referring to toxic effects. Only pure DON without natural material cannot bring out any toxic effect, which was described up to now. That means, there must be ne or more further agents in naturally contaminated material, supporting or just releasing an intoxication. On the other hand, in cases of intoxication DON is detected regularly. Therefore the conclusion for DON as leading substance may be established. For connections and a reliable estimation of the risks through this substance, further examinations are necessary. But expenses, complexity and also animal welfare reasons make the realisation of required trials very difficult

Uncoupling of mitochondrial oxidative phosphorylation stimulates biofilm matrix production.

<p>The wild type stain DAY185 was used for these experiments. (A) qPCR analysis of the expression of the indicated mitochondrial biogenesis genes in 48 h grown biofilm samples upon treatment with CCCP. Error bars are ± standard errors of the average of three biological replicates. <i>P</i> values are as follows: ** <0.01, * < 0.05. (B) SEM of biofilms in the presence or absence of 20 μM CCCP. The ECM is indicated with black arrows. Scale bar = 10 μm. (C) The susceptibility to zymolyase of CCCP-treated biofilms grown in RPMI-MOPS was determined by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. <i>P</i> value is * < 0.05.</p

Biofilm mRNA targets of the Pumilio RNA binding protein Puf3.

<p>(A) Analysis of the gene expression data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref014" target="_blank">14</a>] revealed that 162 genes down-regulated in biofilms are annotated to GO “Mitochondrion”, and 36/162 contain a Puf3 recognition element in their 3’ UTR. The putative biofilm-regulated Puf3 targets include several important mitochondrial biogenesis factors, such as mitochondrial ribosomal subunits, proteins required for respiratory chain function and assembly, and proteins that belong to the mitochondrial protein import machinery. Gene Ontology analysis was performed using the tools in the Candida Genome Database. (B) Cartoon of the Puf3 RNA binding domain from <i>S</i>. <i>cerevisiae</i> showing binding to the core recognition element and the interaction with the -2 cytosine (based on the structure reported in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref039" target="_blank">39</a>]. (C) Alignment of the PUM domains of the <i>C</i>. <i>albicans</i> and <i>S</i>. <i>cerevisiae</i> Puf3 proteins containing the 8–8’ repeat. The LAS motif of the Puf3 binding pocket that interacts with the -2 cytosine is boxed in blue, and this binding pocket is conserved in the <i>C</i>. <i>albicans</i> protein (see also [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref066" target="_blank">66</a>]).</p

The <i>C</i>. <i>albicans</i> Puf3 regulon and mitochondrial roles.

<p>(A) Venn diagram showing the number of mRNAs with a Puf3 binding motif in the 3′ UTRs in <i>S</i>. <i>cerevisiae</i> and <i>C</i>. <i>albicans</i> (only genes which contain orthologs in both species are depicted here). (B) Functional groupings of the Puf3 regulon in <i>C</i>. <i>albicans</i>. The data used to produce this chart is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.s014" target="_blank">S4 Dataset</a>. (C) Growth of <i>C</i>. <i>albicans</i> wild type and <i>puf3Δ/Δ</i> mutant on plates supplied with glucose, glycerol or lactate. Ten-fold serial dilutions were made starting from OD₆₀₀ = 0.5, and plates were photographed after 2 days of growth. (D) Mitochondria in the indicated strains were stained with MitoTracker and imaged as described in Materials and Methods. (E) <i>C</i>. <i>albicans</i> growth on plates was tested as in (C), in the presence or absence of CCCP. Growth was observed on glycerol plates where mitochondrial function is essential.</p

Effects of <i>CCR4</i> on the extracellular matrix and biofilm stability.

<p>(A) Quantification of total extracellular matrix (ECM) in <i>C</i>. <i>albicans</i> biofilms. Biofilms were grown in Spider medium and collected after 48 h. The proportion of ECM was calculated relative to the total biofilm biomass (cells + ECM), as determined by dry weight measurement (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#sec010" target="_blank">Materials and Methods</a>). Results were calculated from three biological repeats in technical triplicates. Shown are the averages and the standard errors. <i>P</i> value of the difference between WT and <i>ccr4Δ/Δ</i> is shown as * <0.05. (B) Biofilms were grown in 96-well microtiter plates in RPMI-MOPS for 24 h and then exposed to zymolyase (20T) prepared in RPMI-MOPS + 0.9% NaCl (1:1 ratio) for 24 h. The remaining biomass was quantified by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. <i>P</i> value of the difference between WT and <i>ccr4Δ/Δ</i> is shown as * < 0.05. (C) Matrix 1,3 β-glucan was quantified from biofilms grown in Spider medium as described in Materials and Methods. The yield of 1,3 β-glucan was calculated as the weight of 1,3 β-glucan (pg) per 1 μg of biofilm cells. Results were calculated from 6 biological repeats. Shown are the averages and the standard errors. Differences: WT versus <i>ccr4Δ/Δ</i>, <i>p</i> = 0.067; <i>ccr4Δ/Δ</i> + <i>CCR4</i> versus <i>ccr4Δ/Δ</i>, <i>p</i> = 0.062. (D) qPCR analysis for the indicated transcripts was performed on biofilms grown for 48 h in Spider medium and data normalized to <i>SCR1</i> RNA. Error bars are ± standard errors of the average of 3 biological replicates. <i>P</i> values are as follows: ** <0.01 or * <0.05.</p

Posttranscriptional regulation of the mitochondrial ribosome and the <i>COX</i> genes by carbon source and Puf3 in <i>S</i>. <i>cerevisiae</i>.

<p>Decay of the indicated mRNAs was measured in wild type and <i>puf3Δ</i> strains grown in glucose (A) or lactate (B) following transcriptional repression at 37°C. The decay curves and half-life (T1/2) were calculated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.g004" target="_blank">Fig 4</a>. The data are shown as the average and standard error of 2–4 biological replicates.</p

The 3′ UTR landscape of the <i>C</i>. <i>albicans</i> transcriptome.

<p>(A) Comparison of 3′ UTRs as determined by our study and Bruno <i>et al</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref042" target="_blank">42</a>]. Of the 3′ UTR that are called by both technologies, 84.5% are within 100 bases (r = 0.4684 (<i>p</i> much < 0.001, n = 2697. Of note, the correlation is highly significant because of the high numbers and would be classed as of moderate strength). Where there is a difference, it is due to filtering differences: we have used the most abundant 3′ UTR, whereas Bruno <i>et al</i> used the longest 3′ UTR for which there was evidence, including minor alternative 3′ UTR isoforms. (B) Graph showing 3′ UTRs are overall shorter in <i>C</i>. <i>albicans</i> than <i>S</i>. <i>cerevisiae</i>. The global positions of polyadenylation in the forward direction and, where it exists, the position of any anti-parallel overlapping adenylated RNA running in the reverse direction. Note any effect of filtering is avoided by this approach as all adenylation sites are utilized in this comparison (370997 and 201547 sites in <i>C</i>. <i>albicans</i> and <i>S</i>. <i>cerevisiae</i> respectively). (C) Comparison between the 3′ UTR lengths of the 3552 orthologous genes between <i>C</i>. <i>albicans</i> and <i>S</i>. <i>cerevisiae</i>. Genes annotated to GO “Mitochondrion” are labeled in red. (D) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between <i>S</i>. <i>cerevisiae</i> and <i>C</i>. <i>albicans</i> relative to the closest coding sequence (CDS). (E) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between <i>S</i>. <i>cerevisiae</i> and <i>C</i>. <i>albicans</i> relative to the polyadenylation site (PA).</p

Model for the role of posttranscriptional gene regulation and mitochondrial activity in biofilm matrix production and stress protection.

<p>As the biofilms mature, environmental changes, such as hypoxia and nutrients, lead to lowering of mitochondrial activity. Lower mitochondrial activity might be sensed as a stress signal and drive the production of protective extracellular matrix. Cell wall integrity pathways are known to function in matrix production [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref015" target="_blank">15</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref016" target="_blank">16</a>], and mitochondrial function has been linked to pathways of fungal cell wall integrity (reviewed in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref027" target="_blank">27</a>]), thus providing a plausible mechanism of mitochondrial control over biofilm matrix production. Mitochondrial dysfunction could also lead to weaker cell walls and cell lysis, further contributing to extracellular matrix deposition. Posttranscriptional regulators, such as the Ccr4-NOT mRNA deadenylase and Puf3, coordinate biofilm maturation pathways by responding to nutrients and hypoxia to adjust mitochondrial biogenesis, as well as the expression of genes needed for biofilm matrix production.</p

<i>C</i>. <i>albicans</i> Puf3 is a repressor of mRNA stability in glucose and lactate growth conditions.

<p><b>(</b>A) A cartoon depicting the location of primers used for specific amplification of the <i>MET3p</i>-driven <i>COX23</i> and <i>MRPL25</i> genes. Detection of this allele was specific, as demonstrated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.s003" target="_blank">S3 Fig</a>. (B) qPCR showing time dependent decay of <i>COX23</i> and <i>MRPL25</i> genes following transcriptional shutdown of <i>MET3p</i> in glucose. Fold expression was represented as ratio of expression levels for each time point after dividing with the expression levels at time 0. Decay curves were obtained with the nonlinear regression (curve fit) method using the exponential, one phase decay equation in the GraphPad “Prism 6” software. The half-life (T1/2) was also calculated using this equation by plotting the decay curve of 3–4 biological replicates separately, and is shown as the average ± standard error. (C) The experiments were performed as described in (B), but with lactate as the carbon source. Data represent the average and standard error from 4 biological replicates. (D) The cartoon depicts mutations in the core and -2C positions of the Puf3 binding motif in the <i>COX23</i> 3′ UTR. Decay curves are from wild type <i>C</i>. <i>albicans</i> strains expressing either a <i>COX23</i> construct with either the wild type or the mutant Puf3 recognition element. The experiments were performed as in (B). Strains were grown in lactate media and shown are results of 2 biological replicates. (E) The experiment was performed as in (C), but concomitant with addition of methionine and cysteine to inhibit transcription, the temperature was raised to 37°C. Shown are results of 3 biological repeats.</p

The mRNA deadenylase Ccr4 regulates extracellular matrix production in biofilms.

<p>(A) qPCR analysis of the expression of mitochondrial biogenesis genes in biofilms grown for 48 h in Spider medium. <i>SCR1</i> RNA was used for normalization. Error bars are ± standard errors of the average of 3 biological replicates. <i>P</i> values are as follows: *** <0.001, ** <0.01, * <0.05. Additional genes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.s007" target="_blank">S7 Fig</a>. (B) Scanning electron microscopy of biofilms formed by <i>C</i>. <i>albicans</i> wild type (DAY185), <i>ccr4Δ/Δ</i> and <i>pop2Δ/Δ</i> mutants and their respective complemented strains. Mature biofilms (48 h) grown in Spider medium were assessed. Experiments were repeated at least twice and inset boxes are regional amplifications. Scale bar = 10 μm. Similar results were obtained when biofilms were grown in RPMI-MOPS or YNB (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.s009" target="_blank">S9 Fig</a>). (C) The data for RPMI-MOPS are from the control samples in the experiments performed to assess biofilm susceptibility to zymolyase (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.g007" target="_blank">Fig 7B</a>). Biofilms were grown for 48 h in the indicated media. Total biofilm biomass was determined by staining with crystal violet. Bars represent averages ± standard errors from three biological replicates. <i>P</i> value of the difference between WT versus <i>ccr4Δ/Δ</i> is shown as ** <0.01 in RPMI-MOPS and *<0.05 in Spider media. (D) Metabolic activity of <i>C</i>. <i>albicans</i> biofilms was determined using the XTT reduction assay. Results were calculated from three independently grown biofilms for each of the strains assayed in technical duplicates. The error bar represents standard errors. <i>P</i> value of the difference between WT and <i>ccr4Δ/Δ</i> is shown as * <0.05.</p