M.W.F. Nielen

Potential of treatment-specific protein biomarker profiles for detection of hormone abuse in cattle.

Targeted protein biomarker profiling is suggested as a fast screening approach for detection of illegal hormone treatment in meat production. The advantage of using biomarkers is that they mark the biological response and, thus, are responsive to a panel of substances with similar effects. In a preliminary feasibility study, a 4-plex protein biomarker flow cytometric immunoassay (FCIA) previously developed for the detection of recombinant bovine somatotropin (rbST) was applied to cattle treated with steroids, such as estradiol, dexamethasone, and prednisolone. Each treatment resulted in a specific plasma biomarker profile for insulin-like growth factor-1 (IGF-1), IGF binding protein 2, osteocalcin, and anti-rbST antibodies, which could be distinguished from the profile of untreated animals. In summary, the 4-plex biomarker FCIA is, apart from rbST, also capable of detecting treatment with other growth-promoting agents and therefore clearly shows the potential of biomarker profiling as a screening method in veterinary control. It is proposed to perform additional validation studies covering high numbers of treated and untreated animals to support inclusion or adaptation of protein biomarker approaches in future monitoring regulations.

Development of a flow cytometric immunoassay for recombinant bovine somatotropin-induced antibodies in serum of dairy cows.

Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST-related biomarkers. In this study, a fast and easy-to-perform microsphere-based flow cytometric immunoassay (FCIA) for detection of rbST-induced antibodies in serum was developed. Until now, detection of rbST-induced antibodies was also problematic due to non-specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non-specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non-specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST-treated dairy cows. These rbST-induced antibodies were successfully detected for up to 4 weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle.

Value of alternative sample matrices in residue analysis for stanozolol

Detection of hormones and veterinary drugs in urine samples is limited to the short-term following illegal administration. Alternative sample matrices such as hair or retina are of interest for prolonged detectability of residues. In this preliminary study, we developed sensitive liquid chromatography–tandem mass spectrometry methods for the confirmatory analysis of stanozolol in different matrices and applied them to urine, retina and hair samples obtained from 11 suspect bovines at the slaughterhouse. Most of the stanozolol metabolite (16-hydroxystanozolol) levels in urine samples were below 0.5 ng ml −1 , but nevertheless in some urines traces of this metabolite could still be positively confirmed. Retina samples were all negative so the value of this matrix for stanozolol might be questioned. The hair samples, on the other hand, yielded many positive stanozolol results, and in some animals levels were up to 100-fold higher than the corresponding metabolite levels in urine thereby underlining the potential for prolonged detectability of stanozolol abuse.

Multiplex ready flow cytometric immunoassay for total insulin like growth factor 1 in serum of cattle

The European Union has banned the use of recombinant bovine somatotropins (rbST, growth hormones) to increase milk yield in dairy cattle. As direct detection of rbST in serum is problematic, methods based on the detection of changes in multiple rbST-dependent biomarkers have high potential for monitoring rbST abuse. In this study immunoassays were developed for total insulin-like growth factor 1 (IGF-1) in cow sera. Ultimately aiming at combination with other rbST-dependent biomarker assays two multiplex formats were studied and compared critically, a multi-channel surface plasmon resonance (SPR)-based biosensor and flow cytometry combined with color encoded microbeads. Moreover, a new dedicated sample pretreatment was developed for the dissociation of complexed IGF-1 in serum, while keeping other biomarkers in solution. Compared to the SPR biosensor immunoassay, the flow cytometric immunoassay (FCIA) was more sensitive, less antibody-consuming and less vulnerable to necessary but interfering reagents from the sample treatment. In an initial in-house validation study the developed FCIA showed to be fast, specific, robust, and a high repeatability and reliability, and generated realistic IGF-1 values for bovine serum, without compromising the potential for simultaneous detection of other biomarkers. Due to the xMAP technology, in which 100 different bead sets can be measured simultaneously, the total IGF-1 assay can easily be extended with other immunoassays for candidate biomarkers. Preliminary results about a FCIA for IGF-1 multiplexing with insulin-like growth factor binding protein 2 (IGFBP2) are presented which strongly supported both the FCIA multiplex format as well as the generic nature of the developed sample pretreatment.

Screening for estrogen residues in calf urine: Comparison of a validated yeast estrogen bioassay and gas chromatography-tandem mass spectrometry

Within the European Union, the control for residues of illegal hormones in food-producing animals is based on urine analysis for a few target analytes using gas chromatography/mass spectrometry and/or liquid chromatography–tandem mass spectrometry. Recently, we developed a robust yeast bioassay screening tool for estrogens, which was validated as a qualitative screening method in accordance with EC decision 2002/657/EC. In this study, we present long-term performance data and a comparison of urine data obtained with this bioassay, and data from an established gas chromatography–tandem mass spectrometry (GC/MS/MS) confirmatory analysis method. More than 120 calf urine samples from a controlled reference experiment were analysed using both protocols. According to the GC/MS/MS method, only the natural estrogens 17α-estradiol and estrone were present in the non-compliant samples. The bioassay was less sensitive than GC/MS/MS for the relatively weak estrogenic compound 17α-estradiol, in accordance with expectations. Assuming that application of the mass spectrometric method is considered beyond reasonable doubt, the bioassay performed very well: only 5.6% of the calf urine samples found compliant in GC/MS/MS were screened false suspect in the bioassay screening method. The bioassay results of non-compliant urine samples under routine conditions were as predicted, taking into account the relative estrogenicity of the natural estrogens 17α-estradiol and estrone vs. 17β-estradiol. Only one sample was screened false negative for 17α-estradiol and estrone. Application of this fast and simple estrogen bioassay in routine surveillance and control can significantly reduce GC/MS/MS sample workload and allow higher percentages of animals to be screened for potential hormone abuse.

Possible contamination with clenbuterol from treated veal calves to untreated pen mates.

To investigate whether clenbuterol-treated calves could contaminate untreated pen mates, three animal experiments were performed. (1) One calf of a pen of five was treated with clenbuterol by injection (Ventipulmin injection, REG NL 2532, 2.5 mL/100 kg) twice a day for 10 days. (2) In two pens, one animal was treated with clenbuterol via oral administration (Ventipulmin syrup, REG NL 2532, 4 mL/125 kg) for 4 weeks. (3) In two pens, one animal was treated with clenbuterol via the milk (Ventipulmin, REG NL 2532, 2.5 mL/100 kg body weight) twice a day for 10 days. Here, the animal was set apart during treatment, cleaned and put back into the group. Levels of clenbuterol were analysed in hair and urine with LC-MS/MS. Clenbuterol administered by injection could not be transferred from treated to untreated calves. In the second experiment, all pen mates were found positive for clenbuterol in the hair. This contamination was probably due to licking the mouth of the treated animal or saliva from the treated animal spoiling the floor. In the third experiment, no pen mates were found positive for clenbuterol in the hair. Clenbuterol was found in the urine and hair of only treated animals.

Veterinary treatment of cows with isoxsuprine for a caesarian section may temporarily lead to residues in hair of both cow and calf

Isoxsuprine is a beta-agonist that can be used for growth promotion in cattle, but it is also used as registered veterinary medicine. To investigate if veterinary treatment of cows could lead to residues of isoxsuprine in the hair of their newborn calves, an animal experiment was performed. Four cows, treated on veterinary indication with isoxsuprine lactate (Duphaspasmin) before a caesarian section, were included in the experiment. Hair samples from cows and from their calves were analyzed. The animals were shaved every week for 16 weeks and levels of isoxsuprine were measured in hair. In the cows, the levels of isoxsuprine were highest (>15 µg/kg) just after administration of the isoxsuprine lactate. After two weeks in two cows, a sort of plateau was reached and then the levels decreased. After approximately 10-15 weeks the levels were around the CCα level of the method used (0.5 µg/kg). In calves, for the first two weeks after birth, no isoxsuprine was found above CCα level in three of the four animals. At about 20-30 days old, a maximum concentration of 4 µg/kg was found. Then the levels dropped again under the CCα level, after 60 days no levels above CCα level were found. In one animal, the levels never reached CCα level. We conclude that veterinary treatment of cows with isoxsuprine may temporarily lead to low levels of isoxsuprine in the hair of their newborn calves which can be measured for a maximum of 60 days after birth.