Occurrence of zearalenone, alpha- and beta-zearalenol in bile of breeding sows in relation to reproductive performance

Although zearalenone-induced reproductive disorders and the clinical appearance of hyperestrogenism were reproduced and documented quite often the role of zearalenone-contaminated fodder as a cause for fertility problems in sow breeding is still discussed controversial. Therefore the correlation of zearalenone and zearalenone-derivatives in bile (n = 794) and feed (n = 158) with fertility problems of unknown origin was investigated in this study. For the analysis of zearalenone and its derivatives in bile a HPLC/EIA combination was used. On the one hand, this procedure guaranteed the quantitatively reproducible detection, on the other hand, the investigation expenditure could be kept small with regard to a later effort in the routine diagnostics. The detection limits for zearalenone, alpha- and beta-zearalenol in bile were at 1.0 ng/ml, 1.0 ng/ml, and 3.0 ng/ml, respectively. Results were confirmed by GC-MS. Zearalenone and zearalenone-derivatives were detected in almost every bile analysed. The contamination rate was 96.2%. In opposition to recent investigations beta-zearalenol was perceived as a relevant metabolite in swine. The contamination rate of feeding stuffs was 25.9%. Incubation of samples with beta-glucosidase did not elevate the detected amounts of zearalenone. As the measurable concentrations in bile and fodder were only slightly correlated the analysis of bile represents a reasonable alternative for fodder investigation. However, a correlation between the occurrence of zearalenone, alpha- and beta-zearalenol in bile of sows and non-infectious reproductive disorders could not be established at the loading level found. These results are in line with those statements obtained in feeding experiments regarding the risk evaluation of zearalenone in sow reproduction.     

The occurrence of Fusarium varieties and their mycotoxins in silo corn. 3. The effect of silaging on the zearalenone content of CCM corn

The influence of silaging on zearalenone already formed and on the growth of Fusarium culmorum in naturally contaminated CCM maize was studied. The zearalenone content remained approximately constant (13.35 +/- 2.94 mg/kg, n = 59) over the whole test period (12 weeks), whereas Fusarium culmorum could not longer be detected after 11 days. Thus the hypothesis that zearalenone survives the silaging process unchanged is experimentally strongly supported. The study confirms the view that zearalenone detected in maize silages is probably already formed in the field or during intermediate storing. Relations between the fermentation process and the toxin content of the silage could not be ascertained.     

Investigation of suspected cases of mycotoxicosis in farm animals in Britain.

Mycotoxins were detected in 13 out of 131 feed samples examined over two years. Screening of feeds associated with cases of suspected mycotoxicosis occurring in farm animals over a further 12 month period showed that most incidents occurred during the winter and involved mainly cattle and pigs fed concentrates. A haemorrhagic syndrome in cattle and abortions in sows were most frequently connected with mouldy food. One or more known toxins (the aflatoxins, ochratoxin A, sterigmatocystin and zearalenone) were detected in three out of 65 cases and a wide variety of fungi were isolated. Toxicity to experimental animals was demonstrated in four out of 22 samples.     

The levels of zearalenone and its metabolites in plasma,urine and faeces of horses fed with naturally,Fusarium toxin–contaminated oats

Concentration profile of zearalenone (ZON) and its metabolites in plasma, urine and faeces samples of horses fed with Fusarium toxin–contaminated oats is described. In plasma, β‐zearalenol (β‐ZOL) was detected at high levels on day 10 of the study (3.21–6.24 μg/l). β‐Zearalenol and α‐zearalenol were the major metabolites in urine. Zearalenone, α‐ZOL and β‐ZOL were predominantly found in faeces. Zearalanone could also be detected in urine (1.34–5.79 μg/l) and faeces (1 μg/kg). The degree of glucuronidation was established in all sample types, approximately 100% in urine and plasma. Low per cent of glucuronidation (4–15%) was found in faeces samples. The results indicate the main conversion of ZON into β‐ZOL in horse. This finding could explain why horse is not susceptible to ZON in comparison with swine which produce α‐ZOL as a predominant metabolite.     

Mycotoxins as a risk factor for the origin of diseases and production decreases in swine facilities--an epidemiologic study]

Feed samples checked for the mycotoxins zearalenone and ochratoxin A from the harvest 1987 were positive at a markedly higher percentage (37.5%) compared to previous years, which is explained by the especially unfavourable harvesting conditions of 1987. In certain herd problems affecting the digestive or respiratory tract, mycotoxins could be detected with a much higher frequency (64.7% and 50.0% respectively). The mean level detected in feed samples by thin layer chromatography ranged within 30.3 ppb for zearalenone and within 58.3 ppb for ochratoxin A. In most cases there was a history of infertility. Considering the clinical situation, which is presented comparatively in herds with positive mycotoxin results, the possible involvement of mycotoxins in the disease, even at very low concentrations, is pointed out. In this context, zearalenone is incriminated of being an indicator of a multitoxic process besides its own direct effects. According to own experiences low levels of zearalenone in the range of 20-50 ppb in the feed have to be considered hazardous. If changing of pig feed in cases of herd problems will be recommended, a level of less than 10 ppb of zearalenone, especially in sow and piglet rations, should not be exceeded. Same may be valid to ochratoxin A.     

郑州某粮库小麦中玉米赤霉烯酮含量的检测

为了解动物饲料小麦中是否含有玉米赤霉烯酮毒素,应用竞争性酶免疫分析法快速定量测定43个小麦样品中玉米赤霉烯酮毒素的含量,并进行了分析。结果显示,在被检的43个小麦样品中,含有玉米赤霉烯酮的有5个(5/43),检出率占11.6%,玉米赤霉烯酮含量超过国家粮食卫生标准的有3个(3/43),超标率占7.0%,说明作为畜禽饲料原料的小麦,存在引发动物玉米赤霉烯酮毒素中毒的潜在危险。本法适宜于大批量小麦样品中玉米赤霉烯酮含量的快速测定,这对于选择品质优良的小麦作为饲料、避免玉米赤霉烯酮对畜禽生长危害有重要意义。     

奶牛场饲料霉菌毒素感染现状的调查与分析

随机抽取3家不同规模奶牛场饲料原料及自配料32份,采用进口ELISA试剂盒检测黄曲霉毒素、玉米赤霉烯酮、赭曲霉毒素和呕吐毒素等4种常见霉菌毒素含量。结果表明：所检测的4种霉菌毒素在所有样品中的检出率为100％,并同时含有2～3种霉菌毒素。其中,除豆粕、苜蓿干草、稻草、豆腐渣外,几乎在所有的饲料样本中玉米赤霉稀酮含量都超标,青贮玉米最高,超标4倍,TMR饲料中玉米赤霉酮含量超出了2倍以上;玉米副产品GGDS中赭曲霉毒素、玉米赤霉酮含量超标严重,呕吐霉素存在微量或少量超标;黄曲霉毒素在青贮玉米、稻草、豆腐渣中有超标,占总样品的15．63％。     

The effects of rumen fluid on the content of zearalenone in animal fodder

The object of the study was to clarify whether rumination has a bearing on the decomposition of zearalenone in the metabolism of the toxin or not. The experiment was performed using rumen fluid in vitro. Rumen fluid samples containing various amounts of zearalenone were incubated in a carbon dioxide atmosphere at 38 degrees C for two days. The zearalenone content was determined by liquid chromatography after 0, 4, 24 and 48 hours incubation periods. The incubation of 4 hours decreased the toxin content by 11%, that of 24 h by 29% and that of 48 h by 37.5% on average. These results indicate that the quantity of zearalenone and the quality of rumen fluid have effects on the ratio of the toxin decomposed.     

The effects of "Gasol" grain preservative dosages on the growth of Fusarium graminearum and the quantity of the toxin zearalenone

In earlier experiments regarding the effects of preservative "Gasol" (containing various acids and additional compounds and intended for whole grain preservations) in recommended dosages, prevents the growth of Fusarium graminearum and reduces the amount of zearalenone when added to milled grain. It was proved in the tests carried out that the smaller dosages of "Gasol" were sufficient for the prevention of growth of the fungus and reduction in quantity of zearalenone. The degree to which the toxin was destroyed depended on the dosage of the preservation. A dosage twice as strong as that recommended in the instructions for practice destroyed all the toxin formed in the grain. A very small dosage of "Gasol" (1/8 of recommended dosage) prevented the growth of the fungus and only slightly reduced the amount of toxin only at the beginning of the test. Later, the growth of the fungus was more vigorous and significantly more toxin was present in the treated grain than in the grain which had never been treated with the preservatives, it has been proved that the preservative     

Characterization of effects of zearalenone in swine during early pregnancy

Mature gilts (n = 16) were hand mated and randomly assigned to 1 of 4 groups of 4 gilts each. Treated gilts had 108 mg of purified zearalenone added to their diet on postmating days (PMD) 2 to 6, 7 to 10, or 11 to 15. Control gilts were given the same diet without added zearalenone. On PMD 6, 10, and 15, control gilts had venous cannulas placed in the jugular vein, and blood samples were taken at 20-minute intervals for 4 hours before feeding and 4 hours after feeding. Samples were collected from treated gilts on the last day that zearalenone was consumed. Samples were analyzed for follicle stimulating hormone, luteinizing hormone (LH), and prolactin. Single blood samples were taken by venipuncture on PMD 8, 12, 16, 20, 24, and 28 and at euthanasia and were analyzed for serum concentration of progesterone and estradiol-17 beta. All gilts were euthanatized 30 to 32 days after mating, and fetal development was assessed. Three gilts that were given zearalenone on PMD 7 to 10 were not pregnant and had regressing corpora lutea on the ovaries at euthanasia. All other treated and control gilts were pregnant. Serum samples from treated gilts on PMD 10 and 15 had lower mean prolactin concentrations than did those from controls. The number of LH spikes were fewer (P less than 0.05) in gilts that were given zearalenone on PMD 15 compared with those in controls on PMD 15. Serum progesterone concentrations indicated that corpora lutea regressed between PMD 20 and 28 in nonpregnant gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of zearalenone (F2) on estrous activity and reproduction in gilts

The effects of zearalenone on swine reproduction were investigated in two trials involving a total of 82 gilts which were allotted into three groups at puberty, mated at second estrus and slaughtered 80 d postbreeding. A control diet without mycotoxin (group 1) or an experimental diet containing 3.61 ppm (first trial) or 4.33 ppm zearalenone (second trial) were fed at a mean daily level of 2 kg/animal. The experimental diet was fed from puberty to mating (group 2) or during pregnancy (group 3). No difference was observed between the two trials. When fed to nonpregnant gilts, zearalenone induced a pseudopregnancy state in 45% of the animals; no estrus was detected within 50 d following puberty and corpora lutea developed at puberty were maintained. The uterine horns were edematous. Reproductive performance measured at 80 d postmating (ovulation rate, weight of corpora lutea, number of normal and abnormal fetuses, embryonic mortality) were not affected by zearalenone intake. But when zearalenone was fed during pregnancy, weights of uterus, placental membranes and fetuses were significantly decreased in comparison with those of control gilts and heterogeneity of fetuses in the same litter was increased. Hematocrit and erythrocyte count were lower in fetuses from gilts ingesting zearalenone, but hematology of the dams remained unaffected. No mycotoxin residue could be detected in gilts or fetal tissues despite the great consequences observed on cyclicity of the females or on the development of embryos. This experiment showed evidence of the estrogenic properties of zearalenone in mature gilts.     

The occurrence of Fusarium varieties and their mycotoxins in silo corn. 2. The formation of zearalenone in the field by artificial infection of silo corn with Fusarium culmorum (W. G. Smith) Sacc

The formation of zearalenone in a maize plot artificially infected with Fusarium culmorum was studied. The zearalenone concentration steeply increased only in the 8th week after inoculation and reached a maximum value of ca. 7 ppm, whereas zearalenone could not be detected in the control variants. The crude nutrient and dry matter content was not significantly influenced by the fungal infection. The infected crop showed average ear dry weights distinctly lower than that of the control variants (P less than or equal to 0.001). Apart from zearalenone, the mycotoxin deoxynivalenol was qualitatively detected in the infected maize. The toxicological relevance of the ascertained zearalenone content with regard to the health of dairy cattle and pigs was discussed.     

Mycotoxins in fungal contaminated samples of animal feed from western Canada, 1982-1994.

Feed samples from 94 cases involving fungal contamination and suspected mycotoxicosis of farm animals in western Canada were examined during 1982-1994 to assess the incidence of mycotoxins. Samples were analyzed for aflatoxins, ochratoxin A, citrinin, sterigmatocystin, and the fungal estrogen zearalenone. Samples infected with Fusarium fungi were additionally assayed for nivalenol, deoxynivalenol, fusarenone-x, 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin. Mycotoxins were found in 21 feed samples from 17 cases (18% of the reported cases), generally at levels far below those needed to induce symptoms under laboratory conditions. HT-2 toxin and other type-A trichothecenes were detected in 5 samples, deoxynivalenol and other type-B trichothecenes in 13, ochratoxin A in 5, and citrinin in 2. In 9 cases, symptoms observed in the animals were consistent with the known effects of the mycotoxin(s) found in the particular feed samples.     

甘草次酸对青年母猪生长性能、抗氧化能力及生殖激素浓度的影响

本试验旨在研究甘草次酸对青年母猪生长性能、抗氧化能力及生殖激素浓度的影响。选用(124±7)日龄、平均体重为(61.00±3.03)kg的健康长×大二元青年母猪64头,按日龄、体重相近的原则随机分为4个组,分别为对照组(基础饲粮)、玉米赤霉烯酮组(基础饲粮+1 mg/kg玉米赤霉烯酮)、甘草次酸组(基础饲粮+400 mg/kg甘草次酸),玉米赤霉烯酮+甘草次酸组(基础饲粮+1 mg/kg玉米赤霉烯酮+400 mg/kg甘草次酸),每组4个重复,每个重复4头母猪。预试期5 d,正试期28 d。结果表明:1)与对照组相比,玉米赤霉烯酮组青年母猪阴户发育异常,阴户长、宽及面积极显著增加(P<0.01)。2)甘草次酸组和玉米赤霉烯酮+甘草次酸组青年母猪的平均日采食量和平均日增重均显著高于对照组和玉米赤霉烯酮组(P<0.05)。3)与对照组相比,玉米赤霉烯酮组青年母猪的粗脂肪消化率显著增加(P<0.05),钙和磷消化率显著降低(P<0.05);甘草次酸组的粗纤维和粗脂肪消化率显著增加(P<0.05),钙和磷消化率极显著增加(P<0.01);玉米赤霉烯酮+甘草次酸组的粗脂肪消化率显著增加(P<0.05)。4)与对照组相比,玉米赤霉烯酮组青年母猪的血清吻素(Kp)、雌二醇(E2)、促黄体素(LH)浓度极显著增加(P<0.01),甘草次酸组的血清Kp和E2浓度显著增加(P<0.05),玉米赤霉烯酮+甘草次酸组的血清Kp、E2和LH浓度极显著增加(P<0.01)。5)玉米赤霉烯酮组青年母猪的血清丙二醛(MDA)含量显著高于对照组和甘草次酸组(P<0.05);与玉米赤霉烯酮组相比,玉米赤霉烯酮+甘草次酸组的血清MDA含量显著降低(P<0.05),血清乳酸脱氢酶(LDH)活性极显著降低(P<0.01)。综上所述,饲粮中添加甘草次酸可改善青年母猪的生长性能和抗氧化能力,影响生殖激素浓度,具有缓解玉米赤霉烯酮对青年母猪繁殖危害的作用。     

Prepubertal exposure to dietary zearalenone alters hypothalamo-hypophysial function but does not impair postpubertal reproductive function of gilts

At an average age of 70 d, 60 Yorkshire gilts born either in July (Trial 1; n = 30) or August (Trial 2; n = 30) received a diet containing zearalenone for 0 (control), 45 or 90 d. The concentration of zearalenone in diets was 2 ppm for 2 wk and 1.5 ppm for the remainder of the study. Vulval swelling and reddening was evident within 7 d after zearalenone was first fed. Zearalenone consumption had no effect on BW or backfat depth. Puberty occurred in Trial 1 at 219 +/- 6 d and was not influenced by zearalenone. Gilts in Trial 2 were divided into two groups; blood samples were taken from 12 gilts to assess pulsatile LH patterns and LH response to estradiol benzoate (EB) and 18 were handled similarly to those in Trial 1. Of this latter subgroup, age at puberty was younger (P less than .05) with zearalenone (217 +/- 7.0, 193 +/- 9.1 and 185.6 +/- 8.2 d for 0-, 45-, and 90-d treatments). Prepubertal consumption of zearalenone did not affect conception rates, ovulation rates, number of fetuses or percentage of embryo survival following mating at pubertal estrus. Two days before the 90-d experimental period ended for Trial 2, blood samples were taken from 12 gilts (four/treatment) every 15 min for 4 h prior to injection of EB (10 micrograms/kg) and every 6 h for 108 h after EB. Analysis of pulsatile patterns of LH revealed no influence of zearalenone on the number of peaks/4 h, baseline concentration or peak height.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of mycotoxins in forage of winter pasture during prolonged utilisation of sward

The concentration of mycotoxins in sward depending on time of pre-utilisation in summer and date of harvest in winter was analysed during the 2000-2002 vegetation seasons. Additionally, the yield of pasture sward was estimated. Higher concentrations of ochratoxin A--respectively: 0.55 and 050 ng/g DM--were found in the pasture sward harvested in December and January when compared to the sward collected in November (0.36 ng/g DM). The average concentration of zearalenone varied from 2.74 ng/g DM in December, to 6.00 ng/g DM in November and 9.77 ng/g DM in January. The highest concentration of ochratoxin A was 1.82 ng/g DM and zearalenone 47.89 ng/g DM. The highest number of samples in which ochratoxin A exceeded the level of 0.3 ng/g DM was noted in sward harvested in January (61.1%), while in the case of zearalenone, the number of samples containing this mycotoxin at a level exceeding 3.0 ng/g DM varied from 55.6% in December to 66.7% in November and January. It seems that the percentage of ochratoxin A positive samples and the toxin concentration level in the sward of winter pastures increased during the time of sward regrowth.     

Destruction of Trichinella spiralis spiralis during the preparation of the "dry cured" pork products proscuitto, proscuittini and Genoa salami

Genoa salami, proscuittini and proscuitto were prepared from pork carcasses that were heavily infected experimentally with Trichinella spiralis spiralis. Genoa salami was prepared with salt concentrations of 2.0%, 2.75% and 3.3%. Proscuitto was prepared by two procedures approved by Agriculture Canada. At various times postpreparation, samples of the various cured products were taken and examined by pepsin digestion and rat bioassay for the presence of viable trichinae. Water activity and pH of the cured meat were also determined. Curing of the various products was shown to destroy the Trichinella larvae. Pepsin digestion revealed that larvae progressively became loosely coiled, uncoiled and more subject to digestion (ghost larvae) during the curing process. Rat bioassay revealed the presence of viable trichinae in the proscuitto prepared using a sodium chloride salt mixture at day 34 but not at day 48 postpreparation. All other bioassays carried out on Genoa salami between 13 and 42 days postpreparation, on proscuittini between days 27 and 69 and on proscuitto between days 34 and 69 were negative for viable trichinae. Under the conditions of this study, preparing Genoa salami with salt concentrations as low as 2% did not appear to affect the destruction of Trichinella larvae.     

A sensitive bioassay for detection of dietary estrogens in animal feeds

Estrogen-responsive proliferation in the MCF-7 cell line was used as a bioassay for detection of dietary estrogens. The bioassay procedure was adapted to screen for estrogenic activity in feedstuffs that have been associated with hyperestrogenism in livestock. Methanolic feed extracts were added to the cell culture medium at microliter/ml concentrations for 4 days, after which the cell proliferation response was measured as DNA content. The half-maximal response for estradiol occurred at 2 pM, or 0.54 pg/ml. For zearalenone, a weaker estrogen, the half-maximal response occurred at approximately 200 pM, or 64 pg/ml. The bioassay was calibrated against a number of known estrogens (estradiol, diethylstilbestrol, zearalenone, zearalanol [cattle implant], beta-zearalenol, zearalane), including the naturally occurring phytoestrogens (formononetin, genistein, daidzein, biochanin A, and coumestrol). The estrogenic activity of feed samples was expressed as equivalents of zearalenone (ppm zearalenone) that would have to be present to equally stimulate proliferation of the MCF-7 cells. The sensitivity of the bioassay was 0.05-0.1 ppm equivalents of zearalenone in feed, well below the threshold level associated with reproductive problems. The feed additive melengestrol acetate (MGA) showed no estrogenic activity in this assay. Estrogenic activity of feed extracts was confirmed by competitive inhibition with the antiestrogens tamoxifen or LY156758 (keoxifene) to show that stimulation of growth by feed extracts was through an estrogenic mechanism. Confirmation of known estrogens was by tandem mass spectroscopy. The assay is a sensitive and reliable screening procedure for detecting estrogenic activity in feedstuffs.     

Detection of mixed infections with "Candidatus Mycoplasma haemominutum" and Mycoplasma haemofelis using real-time TaqMan polymerase chain reaction.

The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with "Candidatus Mycoplasma haemominutum" (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.     

Survey of vomitoxin-contaminated feed grains in midwestern United States, and associated health problems in swine

During the 1981 corn harvest season in Illinois and surrounding states, cold wet weather enhanced the growth of Fusarium graminearum, with resulting contamination by vomitoxin and, to a lesser extent, zearalenone. Of 342 feed samples analyzed, 274 contained vomitoxin at a concentration ranging from 0.1 to 41.6 ppm (mean, 3.1 ppm) and 40 samples contained zearalenone at a concentration ranging from 0.1 to 8 ppm (mean, 0.66 ppm). Animal health problems and reduced growth performance were observed mainly in swine fed vomitoxin-contaminated rations. The predominant clinical complaints, in decreasing frequency were: reproductive problems (50%), feed refusal (43%), reduced weight gain (25%), diarrhea (17%), death (14%), and emesis (11%).