Deoxynivalenol in Serum of Pigs

Poster presented at the BIOMIN World Nutrition Forum 2006



Introduction


Fusarium fungi are capable of producing mycotoxins to a variable degree. The major Fusarium toxins are fumonisins, A- and B-trichothecenes, and zearalenone (1). By definition, mycotoxins are toxic for vertebrates already at low concentrations and especially pigs react very sensitively (7).


DON intake exhibits acute toxicity and causes vomiting and feed refusal, inhibits protein synthesis and mitochondrial function, and shows immunosuppressive effects (6). The inhomogeneous growth of moulds in feeding stuff and different storage methods render interpretation of results difficult.


On the other hand biological samples such as serum are complex matrices and thus prone to exert interferences when analyzed by diverse analytical methods. Serum is routinely in use for diagnostic purposes (e.g. infectious diseases) by the veterinarians and can be sampled with little effort.


Additionally analysis of serum provides the veterinarian with information about actual mycotoxin exposure and thus may be more appropriate than analyzing only feeding stuff. The aim of the project is to establish extraction, clean-up, and analytical methods (HPLC, ELISA) with respect to DON-analysis, which are just partly validated for serum.


Additionally, the validity of the ELISA technique should be tested in comparison to HPLC, as ELISAs are cheaper, simple, fast, and require less material and equipment efforts.



Materials and methods


Feeding trial:

DON concentrations in serum samples out of a “negative” control group (DON-concentration of swine feed 106 μg/kg, equivalent to “low contaminated feed”) were compared to those of the DON-group fed with a concentration of 2.5 mg DON/kg. Feed amount was 4 % of the body weight (BW), which was 0.6 - 0.8 kg feed/day for pigs with 15 - 20 kg BW. After an adaptation phase in the clinic’s stable for 2 weeks and in the metabolism cages for 2 days with feeding of low contaminated feed one control pig (I) was fed with low contaminated feed and a group of 5 pigs (II) was fed with DON-contaminated feed for 2 days with a feeding interval of 12 hours. After that all groups were fed again low contaminated feed.


Sample collection:

In total 150 serum samples were collected over venous catheters. The first blood sample was drawn before the first feeding on the first trial day. Further samples were collected 1, 3, 4, 6, and 11 hours after the respective feeding.


Clean-up and analysis:

2 ml of serum were incubated over night at room temperature with 3 ml phosphate buffer (pH 6.8) and 80 μl ß-glucuronidase. 5 ml incubated serum were purified using immunoaffinity columns. The immuno affinity columns were washed once with 14 ml PBS-buffer and twice with 2 ml Aqua dest. Elution was carried out with 2 x 2 ml methanol. Samples were evaporated under nitrogen flow and reconstituted in 150 μl mobile phase (82 H2O : 9 methanol : 9 acetonitrile) for HPLC and in 500 μl Aqua dest. for ELISA application. Liquid chromatography was performed by using a reverse phase column (Phenomenex, 250 x 4.6mm, 5μm), UV detection was done at 220nm. The ELISA (Romer Labs® AgraQuant® Deoxynivalenol, Herzogenburg, Austria) was carried out according to manufacturer’s specifications. Limit of detection (LOD) was 10 ng/ml for the ELISA and 2 ng/ml for HPLC analysis.

Results


The feeds of the two different trial groups were analysed prior to the feeding trial for the following mycotoxins: DON (106 μg/kg), zearalenone (25 μg/ kg), acetyl-DON (neg.), nivalenol (neg.), and fusarenon X (neg.). The testing of DON-contaminated feed showed a constant DON-concentration of 2.5 mg/kg. DON serum concentrations after feeding pigs with contaminated and low contaminated feeds are shown in figure 1.



Discussion


The described feeding trial has to date not been carried out in this form. Most studies (2, 3, 4, 5) are endpoint studies with samples collected at the slaughterhouse. The present one is a kinetic study, which also allows some conclusions on (I) cumulative effects of DON, as DON-concentration in serum falls below the limit of detection (LOD) not until 15 hours after mycotoxin uptake (2), and (II) the ideal time point for sample collection.





Our data show distinct differences concerning the results obtained by ELISA and HPLC with the ELISA results being clearly settled in a higher range. Most probably this is due to the incomplete elimination of interfering metabolites, which are detected by the ELISA. Previous studies (data not shown) showed that the measurement of serum samples spiked with DON showed exactly the amount of the added DON whereas metabolized samples always showed higher results. The interacting metabolites could not be determined by HPLC.


The kinetic curve of the DON-group showed a peak 3 to 4 hours after the DON-intake. This timeframe can be defined as optimal for sample collection.


In the serum samples of the control group a certain DON-concentration could also be detected because the used feeding stuff was contaminated by DON at a concentration of 106 μg/kg. Remarkably, even this low concentration resulted in a measurable serum effect.


DON can be detected in serum by HPLC as well as by ELISA, but the interfering effects of metabolites must be considered at the interpretation of ELISA results. The requirement for the derivation of reliable results is the choice of an adequate time frame for sample collection based on the last feeding time. For the determination of disruptive factors especially concerning the ELISA further examinations should be performed.

References


1. Biselli, S. et al. (2005): Spectroscopy 20, 20-28.

2. Dänicke, S. et al. (2004a): Arch Anim. Nutr. 58, 169-180.

3. Dänicke, S. et al. (2004b): Anim. Feed Sc. Tech. 114, 141-157.

4. Dänicke, S. et al. (2004c): Arch. Anim. Nutr. 58, 1-17.

5. Döll, S. et al. (2003): Arch. Anim. Nutr. 57, 311-334.

6. Eriksen, G.S. (2003): Doctoral thesis, Swed. Univ. Agric. Sci., Uppsala, 1-38.

7. Razzazi-Fazeli, et al. (2003a): Wien. Tierärztl