Co-occurrence of ochratoxin A and citrinin in unprocessed cereals established during a three-year investigation period.

The aim of this study was to investigate ochratoxin A (OTA) and citrinin (CIT) co-occurrence in different unprocessed cereals (n = 189) originating from Croatia during a three-year investigation period (2014-2016) using validated enzyme immunoassay (ELISA) methods. CIT and OTA were determined in 49% and 7% of samples, respectively. Significantly higher (p < 0.05) overall mean concentrations were determined for CIT (66.8 ± 76.0 µg/kg) in comparison to OTA (5.2 ± 1.1 µg/kg). Based on the analysis of all investigated cereals, CIT was found about 15 times more frequently than OTA and in similarly (15-fold) higher concentrations, irrespective of the cultivation year. The results revealed a moderately positive correlation between OTA and CIT concentrations in maize (rs = 0.44) and wheat (rs = 0.59), whereas in barley and oat this correlation (p > 0.01) was not significant.

Natural incidence of zearalenone in Croatian pig feed, urine and meat in 2014.

The aim of this study was to determine the levels of zearalenone (ZEN) in different feed materials and feedstuffs for pigs, as well as in pig urine and pig meat following contaminated feed consumption. In total, 253 feed material and feedstuff samples were collected from Croatian pig farms. The results revealed the presence of ZEN in significant concentrations, the maximal being found in maize (5522 µg/kg), wheat (3366 µg/kg) and pig fattening feed (1949 µg/kg). In farms in which high feed contamination and pig hyperestrogenism were observed, samples of pig urine (n=30) and meat (n=30) were retrieved as well. The mean ZEN concentrations in pig urine and pig meat were 206±20.6 µg/L and 0.62±0.14 µg/kg, respectively. Despite high contamination of feedstuffs responsible for farmed pigs' intoxication, ZEN levels determined in pig meat were shown to be of little significance for human safety.

Analysis of naturally occurring zearalenone in feeding stuffs and urine of farm animals in Croatia.

The aim of this study was to determine feed and urinary levels of zearalenone. A total of 114 samples, 64 feeding stuffs (commodities, pig and cattle feed), and 50 urine samples were analyzed by the use of enzyme-linked immunosorbent assay (ELISA). Zearalenone was detected in 68.7% of feeding stuffs, while all urine samples except for four yearling samples were positive for zearalenone. The maximum zearalenone concentration in feeding stuffs and urine was 577 ng/g and 241.1 ng/mL, respectively. Although zearalenone concentrations in some samples were high, the risk for humans was negligible since the calculated concentrations in meat were below the tolerable daily intake (TDI).

Correlation of deoxynivalenol and fumonisin concentration determined in maize by ELISA methods.

Maize (Zea mays) is one of the most common crops in Croatia that is often contaminated with mycotoxins. The enzyme linked immunosorbent assay (ELISA) method as a screening immunological method has become very popular in the determination of mycotoxins contamination of cereals, as it has many advantages such as a rapid and simple determination of their concentration. In this present study, 40 maize samples collected from animal feed factories were analyzed for the concentration of deoxynivalenol (DON) and fumonisin (FUM) using the validated ELISA method of two different kit manufacturers to investigate the correlation of the determined mycotoxins concentration. The correlation coefficients for the concentration of DON and FUM, obtained using different kit manufacturers, were r = 0.9698 and r = 0.9303, respectively, yielding a high correlation between the applied ELISA methods. Concerning the legislation, in one maize sample, the DON concentration higher than the maximum recommended value was determined (16.24 mg/kg), whereas the FUM concentration in all the analyzed samples, without exception, were in accordance with recommended values.

Biochemical changes in pig serum after ochratoxin A exposure.

The objective of this study was to investigate the effect of ochratoxin A (OTA) on serum biochemical parameters of pigs during subchronic treatment with 300 µg OTA/kg of feed for 30 days. OTA treatment resulted in significantly higher (p < 0.05) serum levels of creatinine, urea, potassium and alkaline phosphatase, and significantly lower levels of glucose and total protein. These changes in serum biochemical parameters in treated pigs were indicative of impaired liver and kidney function caused by OTA exposure.

The natural occurrence of T-2 toxin and fumonisins in maize samples in Croatia.

Maize (Zea mays) considered to be one of the most frequent crops in Croatia, is often contaminated with Fusarium mycotoxins. The aim of this study was to investigate the possible contamination of maize with T-2 toxin and fumonisins on 46 samples from different regions of Croatia. The highest concentrations of T-2 toxin and fumonisins were 210 and 25,200 ng/g, with mean values of 110 and 4,509 ng/g, respectively, pointing to maize contamination with these mycotoxins after the period of the year with extremely high rainfall.

Mould and mycotoxin contamination of pig feed in northwest Croatia.

The aim of this study was to investigate the contamination of pig feed with moulds and the occurrence of mycotoxins. A total of 30 feed samples were collected at different animal feed factories in the north-western part of Croatia. Mycological analysis showed that the total number of moulds ranged from 1 × 10(3) to 1 × 10(5) cfu/g with samples contaminated with Aspergillus spp. (63 %), Penicillium spp. (80 %), and Fusarium spp. (77 %). A determination of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T-2 toxin (T-2) and fumonisin (FUM) concentration was done using the validated ELISA method. The mean concentrations of AFB1 (0.5 ± 0.6 µg/kg), OTA (1.53 ± 0.42 µg/kg) and FUM (405 ± 298 µg/kg) were below the maximum levels or recommended values in the EU in all the investigated samples. The observed results indicated an increased contamination of pig feed with Fusarium mycotoxins DON and ZEA with mean concentrations of 817 ± 447 and 184 ± 214 µg/kg, higher than recommended in 40 and 17 % of the analysed samples, respectively.

Determination of clenbuterol residues in retinal tissue of food-producing pigs.

The aim of the present study was to assess the persistence of clenbuterol residues in retinal tissue of pigs after repeated administration in a growth-promoting dose, using enzyme-linked immunosorbent assay (ELISA) as a screening method for quantitative determination. A growth-promoting dose of clenbuterol (20 µg/kg body mass per day) was administered orally to the experimental group (n = 6) for 21 days, whereas control animals (n = 3) were left untreated. Clenbuterol-treated pigs were randomly sacrificed (n = 3) on days 0 and 45 of treatment discontinuation, and clenbuterol residues were determined in retinal tissue dissected from the eye. ELISA was found to be acceptable for quantitative determination of clenbuterol in retinal samples because previous method validation yielded mean recovery values of 84.3-96.5% with variation coefficients < 14%. The mean (± SD) retinal clenbuterol concentration was 1874 ± 114 ng/g immediately upon clenbuterol withdrawal (day 0) and 73 ± 4 ng/g on the last day post-withdrawal (day 45). Study results pointed to a very high potential of clenbuterol accumulation in retinal tissue and marked persistence of clenbuterol residues upon anabolic dose administration, suggesting retinal tissue to be a very useful matrix for effective control of residual clenbuterol in food-producing pigs.

Accumulation of the beta(2)-adrenergic agonist clenbuterol in mouse dark hair.

The aim of the present study was to evaluate the suitability of dark hair as a matrix for determination of the beta(2)-adrenergic agonist clenbuterol residues using previously validated enzyme-linked immunosorbent assay (ELISA) as a screening method for its quantitative determination. The experimental group of mice (n = 60) were treated with two different anabolic dosages of clenbuterol for 15 days, whereas the control group of animals (n = 30) was left completely untreated. Hair samples were collected on days 0, 5, 10, and 15 of treatment. Validation of the ELISA analytical procedure showed good recovery (mean recovery 74%) with an acceptable intra-assay variation in individual measurements for all hair samples to which 5, 10, and 50 ng/g clenbuterol were added (CV < 10%). Low blank levels of clenbuterol (2.4 +/- 0.6 ng/g) were measured in hair of untreated mice, whereas significantly higher clenbuterol concentrations rising proportionally with the time of treatment were recorded in hair of mice treated with lower (6.5 mg/kg body mass) and higher (12.5 mg/kg body mass) dose of clenbuterol. The peak hair concentration of clenbuterol measured on the last day of treatment (day 15) was 1553.9 +/- 140.1 ng/g and 6248.3 +/- 589.4 ng/g in the lower and higher dose group, respectively. Study results clearly indicated dark hair as a pigmented tissue to have a high accumulation potential for clenbuterol residues, thus being the target matrix of choice for detection of clenbuterol abuse as an anabolic in meat production.