Emiko Kokushi

Zearalenone (ZEN), an estrogenic mycotoxin affecting cow reproduction, and deoxynivalenol (DON) are cytotoxic and immunotoxic, reducing protein synthesis. Contamination with ZEN and DON decreases the productivity of breeding cows. We evaluated the feasibility of commercial lateral flow immunochromatographic strip tests (LFISTs) as a rapid first-line screening approach for urinary ZEN and DON exposure in cattle. Using 81 samples from 13 Japanese Black (JB) and 14 Holstein Friesian (HF) herds, line intensity ratios were quantified by analyzing photographs of test and control lines within regions of interest (ROIs). LFIST correlated significantly with ELISA (ZEN/Cre: p < 0.001 and τ = 0.35; DON/Cre: p < 0.001 and τ = 0.31) and LC-MS/MS (ZEN/Cre: p = 0.002 and r = 0.35; α-ZOL/Cre: p = 0.013 and r = 0.27; β-ZOL/Cre: p = 0.007 and r = 0.30; ΣZEN/Cre: p = 0.005 and r = 0.31). The relationship between parameters was explored using the Weibull accelerated failure time model analysis. LFIST-ZEN and LFIST-DON were significantly associated with ELISA ZEN/Cre (p = 0.00057) and ELISA DON/Cre (p = 0.00061), respectively. To test whether LFISTs could predict mycotoxin contamination in feed, data from nine other herds (n = 38), with known feed mycotoxin levels, were analyzed. Estimation equations related feed ZEN and DON concentrations to urinary ZEN and DON levels in the herds. Our findings support the potential utility of LFIST as a rapid first-line herd-level screening tool for ZEN and DON exposure, providing screening information with LFIST results obtainable within approximately 15 min after initiation of the assay, while confirmatory laboratory analysis remains necessary when exposure is suspected.

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are a group of PAHs derivatives formed by chemical or microbial oxidation that can negatively affect benthic organisms. However, their distribution in coastal sediments remains poorly understood. In the present study, we examined the distribution and ecological risks of oxy-PAHs in sediments collected from 18 sites in Tokyo Bay, Japan. Concentrations of target oxy-PAHs ranged from 10.2 to 1299 ng/g dry weight, generally exceeding previously reported values. Strong correlations were found between oxy-PAHs and sediment carbon content and between oxy-PAHs and parent PAHs, possibly showing common sources or transformation pathways. Lethal and developmental effects were observed in Java medaka (Oryzias javanicus) embryos exposed to the artificial sediments spiked with two oxy-PAHs (7,12-benz(a)anthraquinone and 1,4-naphthoquinone) found at several locations in Tokyo Bay. Both risk quotient indices exceeding 1 were shown at more than 10 sites, and suggest that these 2 substances may pose potential threats to benthic organisms at a number of sites in Tokyo Bay.

Mycotoxin contamination in animal feed can cause acute or chronic adverse effects on growth, productivity, and immune function in livestock. This study aimed to evaluate the impact of difructose anhydride III (DFA III) supplementation on serum biochemical parameters and intestinal environment in Japanese Black (JB) breeding cows under low-level chronic dietary exposure to zearalenone (ZEN). Using urinary ZEN concentration as an indicator of exposure, 25 JB cows were selected from a breeding farm with confirmed natural feed contamination. Blood samples were collected before DFA III supplementation (day 0), and on days 20 and 40 post-supplementation. Serum biochemical parameters and short-chain fatty acid concentrations were measured. During the studies, dietary ZEN concentration increased, yet improvements were observed in liver function, nutritional status, immune response, and inflammatory markers. Notably, serum butyrate concentration significantly increased following DFA III administration. These findings suggest that DFA III may positively influence intestinal microflora and enhance intestinal barrier function, which could contribute to improved health and nutritional status in cattle exposed to low-level chronic dietary ZEN contamination. DFA III supplementation may represent a promising strategy for mitigating the effects of low-level mycotoxin exposure in livestock production systems.

The widespread presence of Fusarium mycotoxins in animal feed is a global issue, not only for the health of livestock but also for ensure the safety of food as an end product. High concentrations of zearalenone (ZEN) and deoxynivalenol (DON) have been detected in the diets of Japanese Black (JB) and Holstein Friesian (HF) breeding herds. Consequently, we monitored serum biochemical parameters over a long time in both herds, focusing on anti-Müllerian hormone (AMH) levels and acute-phase inflammation. Additionally, urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and progesterone levels were measured in the HF herd. The JB herd, a ZEN-dominant model with low DON contamination, demonstrated ZEN levels that exceeded the Japanese limit in the purchased total mixed rations (TMR). Conversely, the HF herd, which primary consumes DON-dominant feed with low ZEN contamination, had high DON levels in the dent corn silage. Specifically, the JB herd’s TMR contained 1.79 mg/kg ZEN and 0.58 mg/kg DON, whereas the HF herd’s silage had 15.3 mg/kg DON (dried sample) and 0.1 mg/kg ZEN. Enzyme-linked immunoassay were used to measure urinary ZEN-DON levels following confirmation through liquid chromatography-tandem mass spectrometry. Urinary ZEN-DON levels measured were significantly correlated (p < 0.05, r > 0.6) in both herds. In the HF herd, AMH levels increased (p = 0.01) and serum amyloid A (SAA) levels decreased (p = 0.02) when contaminated and at the end of the monitoring period. Additionally, urinary ZEN and DON levels were significantly correlated with SAA levels (ZEN: p = 0.00, r = 0.46; DON: p = 0.03, r = 0.33), with an increase in ZEN and DON levels resulting in higher SAA levels. The JB herd showed no significant differences. Additionally, in the HF herd, 8-OHdG/Cre levels increased significantly during major contamination periods (p < 0.05). Clinical data from the HF herd indicated an increase in mastitis cases and treatment rates during periods of major contamination. Abortion rates in the HF herd decreased from 22.9% (before monitoring) to 8.9% (during the high contamination period) and finally to 1% (at the end of the monitoring period), with corresponding increases in progesterone levels. ZEN-DON contamination adversely affects breeding cattle’s productivity, reproductive performance, and health. Therefore, monitoring urinary ZEN-DON is valuable for detecting contaminants and ensuring the safety of food products.

We previously reported the mitigation effects of difructose anhydride III (DFA III) on mycotoxins, such as zearalenon and sterigmatocystin, based on the urinary concentrations of these molecules in calves.

In this study, a herd of Japanese Black (JB) breeding cattle with sporadic reproductive disorders was continuously monitored for an additional year to assess the effects of the urinary zearalenone (ZEN) concentration and changes in parameters (AMH and SAA) with time-lag variables and herd fertility (reproductive performance). This herd had high (exceeded the Japanese dietary feed regulations) urinary ZEN and rice straw ZEN concentrations (1.34 mg/kg). Long-term data of the herd with positive ZEN exposure revealed a decreasing ZEN concentration in urine and a gradual decrease in the AMH level with age. The AMH level was significantly affected by the ZEN value 2 months earlier and the AMH level in the previous month. The changes in ZEN and SAA values were significantly affected by the ZEN and SAA values in the previous month. Additionally, calving interval data between pre-monitoring and post-monitoring showed a significantly different pattern. Furthermore, the calving interval became significantly shorter between the time of contamination (2019) and the end of the monitoring period (2022). In conclusion, the urinary ZEN monitoring system may be a valuable practical tool for screening and detecting herd contamination in the field, and acute and/or chronic ZEN contamination in dietary feeds may affect herd productivity and the fertility of breeding cows.

This study addresses an advantageous application of a urinary zearalenone (ZEN) monitoring system not only for surveillance of ZEN exposure at the production site of breeding cows but also for follow-up monitoring after improvement of feeds provided to the herd. As biomarkers of effect, serum levels of the anti-Müllerian hormone (AMH) and serum amyloid A (SAA) concentrations were used. Based on the results of urinary ZEN measurement, two cows from one herd had urinary ZEN concentrations which were two orders of magnitude higher (ZEN: 1.34 mg/kg, sterigmatocystin (STC): 0.08 mg/kg in roughages) than the levels of all cows from three other herds (ZEN: not detected, STC: not detected in roughages). For the follow-up monitoring of the herd with positive ZEN and STC exposure, urine, blood, and roughage samples were collected from five cows monthly for one year. A monitoring series in the breeding cattle herd indicated that feed concentrations were not necessarily reflected in urinary concentrations; urinary monitoring assay by ELISA may be a simple and accurate method that reflects the exposure/absorption of ZEN. Additionally, although the ZEN exposure level appeared not to be critical compared with the Japanese ZEN limitation in dietary feeds, a negative regression trend between the ZEN and AMH concentrations was observed, indicating that only at extremely universal mycotoxin exposure levels, ZEN exposure may affect the number of antral follicles in cattle. A negative regression trend between the ZEN and SAA concentrations could also be demonstrated, possibly indicating the innate immune suppression caused by low-level chronic ZEN exposure. Finally, significant differences (p = 0.0487) in calving intervals between pre-ZEN monitoring (mean ± SEM: 439.0 ± 41.2) and post-ZEN monitoring (349.9 ± 6.9) periods were observed in the monitored five cows. These preliminary results indicate that the urinary ZEN monitoring system may be a useful practical tool not only for detecting contaminated herds under field conditions but also provides an initial look at the effects of long-term chronic ZEN/STC (or other co-existing mycotoxins) exposure on herd productivity and fertility.

The potential effect of difructose anhydride III (DFA III) supplementation in cattle feed was evaluated using a previously developed urinary-zearalenone (ZEN) monitoring system. Japanese Black cattle from two beef herds aged 9–10 months were used. DFA III was supplemented for two weeks. ZEN concentrations in feed were similar in both herds (0.27 and 0.22 mg/kg in roughage and concentrates, respectively), and below the maximum allowance in Japan. ZEN, α-zearalenol (α-ZOL), and β-ZOL concentrations in urine were measured using LC/MS/MS the day before DFA III administration, 9 and 14 days thereafter, and 9 days after supplementation ceased. Significant differences in ZEN, α-ZOL, β-ZOL, and total ZEN were recorded on different sampling dates. The concentration of inorganic phosphate in DFA III-supplemented animals was significantly higher than in controls on day 23 (8.4 vs. 7.7 mg/dL), suggesting a possible role of DFA III in tight junction of intestinal epithelial cells. This is the first evidence that DFA III reduces mycotoxin levels reaching the systemic circulation and excreted in urine. This preventive effect may involve an improved tight-junction-dependent intestinal barrier function. Additionally, our practical approach confirmed that monitoring of urinary mycotoxin is useful for evaluating the effects of dietary supplements to prevent mycotoxin adsorption.

The objective of this study was to evaluate the metabolic profile of cattle fed with or without zearalenone (ZEN) and sterigmatocystin (STC)-contaminated diets using a gas chromatography-mass spectrometry metabolomics approach. Urinary samples were collected from individual animals (n = 6 per herd) from fattening female Japanese Black (JB) cattle herds (23 months old, 550–600 kg). Herd 1 had persistently high urinary ZEN and STC concentrations due to the presence of contaminated rice straw. Herd 2, the second female JB fattening herd (23 months old, 550–600 kg), received the same dietary feed as Herd 1, with non-contaminated rice straw. Urine samples were collected from Herd 1, two weeks after the contaminated rice straw was replaced with uncontaminated rice straw (Herd 1N). Identified metabolites were subjected to principal component analysis (PCA) and ANOVA. The PCA revealed that the effects on cattle metabolites depended on ZEN and STC concentrations. The contamination of cattle feed with multiple mycotoxins may alter systemic metabolic processes, including metabolites associated with ATP generation, amino acids, glycine-conjugates, organic acids, and purine bases. The results obtained from Herd 1N indicate that a two-week remedy period was not sufficient to improve the levels of urinary metabolites, suggesting that chronic contamination with mycotoxins may have long-term harmful effects on the systemic metabolism of cattle.