Eisaburo Deguchi

Measurement of urinary zearalenone concentrations for monitoring natural feed contamination in cattle herds: On-farm trials1

The aims of the present study were to investigate the efficacy of measuring bovine urinary zearalenone (ZEN) concentrations by using a commercially available ELISA method in cattle kept under different feeding conditions to monitor the natural contamination of feeds at the farm level, and to investigate the effects of supplementation of a mycotoxin adsorbent (MA) product in the feed based on urinary ZEN concentration. First, Japanese Black cattle herds kept for breeding (4 herds) and fattening (4 herds) purposes were provided with similar feeding conditions. Then, urinary samples from 5 cows in each herd were collected and analyzed. Second, dairy cows from 1 herd fed with total mixed rations (TMR) were selected. After thorough mixing of the MA (40 g/d) with TMR, the supplemented TMR was fed according to the following schedule: with MA for 2 wk, without MA for 3 wk; then with MA for 2 wk and without MA for 6 wk. Urine samples were collected from cows (n = 6 to 7) and examined before and after each interval. Zearalenone concentrations were measured by the ELISA and liquid chromatography-tandem mass spectrometry methods. The concentration of ZEN and its metabolites was expressed after creatinine (Crea) correction [ZEN or metabolites (pg/mL)/Crea (mg/dL); pg/mg of Crea]. In the first experiment, the urinary concentrations of ZEN and its metabolites were variable in all herds, and significant differences were observed between herds. In 1 fattening herd, in particular, urinary ZEN concentrations were greater (P < 0.001) than in the other 3 herds. This might reflect significant natural ZEN contamination of the feed at the farm level. In Exp. 2, urinary ZEN concentrations displayed peculiar trends after supplementation with MA. After 2 wk of supplementation, a significant decrease of ZEN (P < 0.05) was observed. Zearalenone concentrations remained at a reduced amount during 3 wk without MA supplementation and 2 wk with MA supplementation. When MA was not added to the feed for the next 6 wk, the concentrations increased to the original quantity. These findings indicate the usefulness of measuring concentrations of urinary ZEN and its metabolites not only for monitoring the natural ZEN contamination of cattle feed at the farm level but also for in vivo evaluation of MA function after supplementing feeds with MA.

Detection of zearalenone and its metabolites in naturally contaminated follicular fluids by using LC/MS/MS and in vitro effects of zearalenone on oocyte maturation in cattle

Zearalenone (Zen) and its metabolites are estrogenic and may be important factors involved in reproductive disorders in domestic animals. We aimed to (1) simultaneously detect Zen and its metabolites in bovine follicular fluids (FFs) by liquid chromatography-tandem mass spectrometry and (2) examine the in vitro effects of Zen on bovine oocytes. Zen and its metabolites were detected in 6 of 32 normal follicles and 7 of 20 cystic follicles. Bovine oocytes were cultured in a maturation media containing various Zen concentrations (0 [control], 1, 10, 100, and 1000microg/L), fertilized, and cultured further. Maturation rates decreased dose-dependently. Further, maturation of 62 (50%) of 124 oocytes examined in the 1000-microg/L group was arrested in metaphase I, without affecting the fertilization rate. Blastocyst-formation rates did not significantly differ among the groups. Zen and its metabolites were detectable in bovine FFs. High Zen concentration may adversely affect meiotic competence but not the fertilization and development rates.

Natural contamination of dietary rice straw with zearalenone and urinary zearalenone concentrations in a cattle herd

The present study was conducted to 1) identify the natural source of feed contamination by zearalenone (ZEN), which was suspected to have caused persistently increased urinary ZEN concentrations in one of our experimental cattle herds, and 2) evaluate the effects of intervention against this source of contamination. As an experimental model, a fattening Japanese Black cattle herd showing persistently increased urinary ZEN concentrations was identified. Urinary ZEN concentrations of cows fed with new rice straw (experimental group, n = 6) vs. cows that continued to feed on the old rice straw (control group, n = 4) were measured at the start (d 1) and at 2 wk (d 14) after the onset of feeding with straw. In addition, the ZEN concentration in feed and water samples was measured by using both the ELISA and HPLC methods. Furthermore, isolation and identification of fungi from rice straw and concentrate feed samples were performed. The urinary ZEN concentration [ZEN (pg/mL)/creatinine (mg/mL) = pg/mg of creatinine] of cows fed with new rice straw was significantly (P < 0.05) less (843 pg/mg of creatinine) than that of cows fed with old rice straw (15,951 pg/mg of creatinine). On both d 1 and 14, the ZEN concentrations of old rice straw were greater than those of new rice straw. In addition, fungal colonies were observed in the culture media that was obtained from the old rice straw suspected of ZEN contamination, but not in the culture media from new rice straw or other feed samples. In conclusion, our field trials clearly indicate that the rice straw fed to the cows was naturally contaminated with ZEN, and that the monitoring of urinary ZEN concentrations could prove to be a useful tool for detecting the exposure of cattle to ZEN contamination at the farm level.

Measurement of Sterigmatocystin Concentrations in Urine for Monitoring the Contamination of Cattle Feed

This study aimed (1) at determining the levels of the fungal toxin sterigmatocystin (STC) in the feed and urine of cattle and (2) at evaluating the effects of supplementing the feed with a mycotoxin adsorbent (MA) on STC concentrations in urine. Two herds of female Japanese Black cattle were used in this study. The cattle in each herd were fed a standard ration containing rice straw from different sources and a standard concentrate; two groups of cattle from each herd (n = six per group) received the commercial MA, mixed with the concentrate or given as top-dressing, whereas a third group received no supplement and served as control. Urine and feed samples were collected at various time points throughout the experiment. STC concentrations were measured using liquid chromatography-tandem mass spectrometry (LC-TMS). STC concentrations in straw were higher in Herd 1 (range 0.15–0.24 mg/kg DM) than in Herd 2 (range <0.01–0.06 mg/kg DM). In Herd 1, STC concentrations in urine significantly declined 2 weeks after replacing the contaminated feed, whereas MA supplementation had no effect. In conclusion, mycotoxins in urine samples are useful biological markers for monitoring the systemic exposure of cattle to multiple mycotoxins, as well as evaluating the effectiveness of interventions.

Efficient Isolation of Swine Influenza Viruses by Age-Targeted Specimen Collection

ABSTRACT The control of swine influenza virus (SIV) infection is paramount for increasing the productivity of pig farming and minimizing the threat of pandemic outbreaks. Thus, SIV surveillance should be conducted by region and on a regular basis. Here, we established a microneutralization assay specific for SIV seroprevalence surveillance by using reporter gene-expressing recombinant influenza viruses. Growth-based SIV seroprevalence revealed that most sows and piglets were positive for neutralizing antibodies against influenza viruses. In contrast, the 90-day-old growing pigs exhibited limited neutralizing activity in their sera, suggesting that this particular age of population is most susceptible to SIV infection and thus is an ideal age group for SIV isolation. From nasal swab specimens of healthy pigs in this age population, we were able to isolate SIVs at a higher incidence (5.3%) than those of previous reports. Nucleotide sequencing and phylogenetic analysis of the hemagglutinin (HA) genes revealed that the isolated SIVs have circulated and evolved in pigs but not have been recently introduced from humans, implying that a large number of SIV lineages may remain “undiscovered” in the global porcine populations. We propose that the 90-day-old growing pig-targeted nasal swab collection presented in this study facilitates global SIV surveillance and contributes to the detection and control of SIV infection.

Application of mycotoxin adsorbent to cattle feed contaminated with zearalenone: urinary zearalenone excretion and association with anti-Müllerian hormone

This study investigated (1) protective effects of a commercially available mycotoxin adsorbent (MA) and (2) endocrine effects of in vivo exposure to zearalenone (ZEA) in cattle. The sample included a Japanese Black female cattle herd (MYT herd) that displayed persistently high urinary ZEA concentrations. A second herd (NM herd) was used as a control. Three groups from each herd were assessed: MX (n=6; MA mixed with concentrate), TD (n=6; MA applied as topdressing with the concentrate), and a positive control (n=6; no MA application). Urine and blood samples were collected at the start of MA supplementation (day 0), on the final day of supplementation (day 16), and on the final day of the sampling period (day 58 for MYT herd and day 50 for NM herd). Urinary ZEA concentrations (pg/mg of creatinine) were measured by ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Haematological and serum biochemical analyses were performed to monitor hepatic, renal, nutritional, and mineral in...

Relationship between urinary zearalenone concentration and embryo production in superovulated cattle

Abstract. This field study aimed to investigate the relationships between the urinary zearalenone (ZEN) concentration, which reflects dietary ZEN intake, and the numbers of total and transferable embryos in superovulated cattle. A total of 38 cows (Japanese Black, n=16; Holstein, n=22) were superovulated for commercial embryo production. Urine samples were collected from all cows at the time of embryo flushing and the urinary ZEN concentration was measured. The ZEN concentration was corrected for the creatinine (Crea) concentration as follows: ZEN (pg/mL)/Crea (mg/dL); the corrected ZEN concentration was expressed in pg/mg Crea. The cows were divided into two groups according to whether the urinary ZEN level was less than (group 1) or more than (group 2) the mean value for each breed (Japanese Black: 97.4 pg/mg Crea; range 44.5–91.3 pg/mg Crea; Holstein: 155.5 pg/mg Crea; range 32.7–146.9 pg/mg Crea). The embryo flushing results were compared between the two groups within each breed. Overall, the total number of embryos collected and the number of transferable embryos did not differ significantly between the groups. These results suggest that natural ZEN contamination resulting in urine levels below the threshold value (i.e. below the maximal permissible urinary ZEN concentration) does not affect embryo production in Japanese Black and Holstein cows undergoing superovulation.

Effects of dietary contamination by zearalenone and its metabolites on serum Anti-Müllerian hormone: Impact on the reproductive performance of breeding cows

We investigated the effects of in vivo exposure to low zearalenone levels on the anti-Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (<1 ppm) at 30 days after calving. Urinary zearalenone, α-zearalenol and β-zearalenol concentrations were measured by chromatography-tandem mass spectrometry, and serum anti-Müllerian hormone concentrations were determined along with serum biochemical parameters. Urinary concentrations of α-zearalenol were significantly higher (p < 0.05) in cattle in Herd 1 than in cattle in Herd 2, reflecting the different amounts of zearalenone in the diet of the two herds. Although the number of 5-mm and 10-mm follicles of the herds and their fertility after artificial insemination were similar, the serum anti-Müllerian hormone concentrations in herds 1 and 2 were 438.9 ± 48.6 pg/ml and 618.9 ± 80.0 pg/ml, respectively, with a trend towards a significant difference (p = 0.053), which may indicate differences in the antral follicle populations between herds. Thus, zearalenone intake from dietary feed, even when below the threshold zearalenone contamination level permitted in Japan, may affect the ovarian antral follicle populations, but not the fertility, of post-partum cows.

Monitoring natural feed contamination in beef cattle by measurements of urinary zearalenone concentrations after oral administration of mycotoxin adsorbents as a top dressing

To investigate the effects of supplementation of mycotoxin adsorbents (MAs) in top dressing of cattle feed with respect to concentrations of urinary zearalenone (ZEN) and its metabolites, α-zearalenol and β-zearalenol, Japanese Black cattle herds for breeding (2 herds) and fattening (2 herds) were provided with similar feeding conditions. Two types of MAs were tested, and the maximal recommended dose of each MAs was supplemented in the feed as a top dressing for 2 weeks. Urine samples were collected from cows (n = 6–7) on day 0 and 14. The urinary concentrations of ZEN and its metabolites were found to be variable in all herds. This might reflect significant natural ZEN contamination of the feed at the farm level. However, the urinary concentrations of ZEN and its metabolites after supplementation with MAs for 2 weeks were not significantly different. Additionally, our results suggest the possibilities that supplementation of the feed with MA may affect the absorptivity of mycotoxins from the gastrointestinal tract or limit the binding of MA to mycotoxins.

Three cases of idiopathic eosinophilic enteritis with chronic obstinate diarrhea in Japanese Black fattening cattle.

Eosinophilic enteritis (EOE) is a type of inflammatory bowel disease and is characterized clinically by chronic obstinate diarrhea. Three Japanese Black (JB) fattening cattle (2 males and 1 female) on different cattle farms presented with chronic episodic diarrhea without fever or dehydration. Soft reddish spherical carneous tissues (1−3 cm) were occasionally excreted within the diarrheic feces. Administration of antibiotics, antidiarrheal drugs and vermicides had no therapeutic effect, but dexamethasone improved the fecal characteristics. The symptoms persisted until the animals were slaughtered at 27–30 months of age. Histopathological examination of the intestines revealed marked eosinophilic infiltration in the lamina propria and submucosa. From these findings, we diagnosed these cattle as the first cases of EOE in JB cattle.