Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies.

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.     

Ochratoxin A in blood from slaughter pigs in Sweden: use in evaluation of toxin content of consumed feed.

Samples of pig blood, intended for ochratoxin A analysis, were collected from pigs of 279 randomly selected herds. The samples were obtained at nine different slaughterhouses from different areas of Sweden. Pigs from 47 herds (16.8% of the total) exhibited ochratoxin A in amounts of greater than or equal to 2 ng of ochratoxin A per ml of blood. One sample each from a single pig per herd identified herds contaminated with ochratoxin A in amounts exceeding three times the detection limit of the method (3 x 2 ng of ochratoxin A per ml of blood = 6 ng of ochratoxin A per ml of blood). There was a good agreement between ochratoxin A concentrations in the blood from different pigs within the same herd (correlation coefficient = 0.80). The ochratoxin A concentration in pig blood was used as an estimate of the ochratoxin A content of the consumed feed. This method showed that feed from grain produced on-farm contained higher concentrations of ochratoxin A than commercial feed preparations. No geographical variation of ochratoxin A occurrence within Sweden was detected.     

Penicillium verrucosum in feed of ochratoxin A positive swine herds

Ochratoxin A contamination of cereal feed grain was monitored during October 1989–September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2–45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (2 ng/ml blood) and ochratoxin A negative herds (<2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p < 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p < 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5% ) of the feed samples collected from the ochratoxin A negative herds.     

A French monitoring programme for determining ochratoxin A occurrence in pig kidneys

Ochratoxin A is a carcinogen and nephrotoxin which can enter the food chain resulting in human exposure. As pig herds are exposed to ochratoxin A through their feed, their kidneys, livers and pork meat are considered as a possible route of exposure for humans. France, an important producer of pork and pork products, set up a national monitoring programme which included the training of six routine public laboratories in the analysis of ochratoxin A using an immunoaffinity step followed by a HPLC-fluorimetric detection. The programme randomly sampled 300 healthy and 100 nephropathic pig kidneys in 1997 and 710 healthy pig kidneys in 1998. Less than 10% of samples were significantly contaminated by ochratoxin A : in the 1997 survey, 1% of samples contained 0.40-1.40 microg kg(-1) of ochratoxin A and in the 1998 survey 7.6 % exhibited ochratoxin A levels in the range 0.5-5 microg kg(-1). In the case of nephropathic kidneys, only traces of ochratoxin A (0.16 to 0.48 microg kg(-1)) were detected in six samples out of 100. Even if not a major route of exposure for humans, pigs are clearly exposed to this mycotoxin and monitoring of pork products and of feed for swine is necessary.     

Ochratoxin A in porcine blood and in consumed feed samples

The aim of the study was to estimate occurrence of ochratoxin A (OA) in feeds and the metabolite residues in porcine blood serum in Poland. Samples were collected in the period from February to May, 1999, in the southern Wielkopolska region. Altogether 40 and 45 samples of feed and porcine blood serum, respectively, were analyzed for OA. Percentage of samples contaminated with OA, both in case of feeds and blood, collected in the winter season was considerably higher than that for the spring season. The percentages for feeds were as follows: 47.6 and 26.3 %, while for porcine serum: 66.7 and 50.0 %, respectively winter and spring. In 25 % of cases ochratoxin A was present in both types of investigated material (feed, blood), whereas in 27.5 % of samples this metabolite was detected in blood only, or in 7.5 % only in the feed. The presence of OA was found neither in the feed nor in the serum in 40 % of all cases. In subgroups (feed, blood) the concentration in the whole collective of positive samples were in the range 0.3–13.5 ng/g and 0.3–69.5 ng/ml, respectively, while median values were 2.3 ng/g and 6.0 ng/ml. Only one feed and three porcine serum samples, were found to be contaminated at concentration levels higher than 10 ng/g or 10 ng/ml.     

Evidence of ochratoxin A-detoxification activity of rumen fluid,intestinal fluid and soil samples as well as isolation of relevant microorganisms from these environments

Dietary ochratoxin A (OTA) has a negative impact on performance of chickens and pigs. To avoid losses in animal production through intake of this mycotoxin and to prevent carry over to humans, strategies for counteracting have to be developed. In contrast to physical and chemical detoxification methods inactivation of ochratoxins by enzymatic reactions represent a very specific and gentle process. For the development of a new feed additive various environments have been screened for microorganisms with the capability of degrading or of cleaving the phenylalanine-moiety of ochratoxin A. Two OTA-degrading bacterial strains were isolated from rumen fluid and four pure cultures capable of cleaving ochratoxin A were obtained from pig intestine. The highest number of ochratoxin A degrading strains were found amongst aerobic bacteria which have mainly been isolated from soil.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

赭曲霉毒素A和玉米赤霉烯酮-二联胶体金免疫层析试纸条的制备及应用

【背景】真菌毒素为真菌的有毒次级代谢产物,混合污染时毒性显著增强,可对人类和动物健康造成严重伤害。制备二联胶体金免疫层析试纸条,实现对常见真菌毒素混合污染的快速监测,具有重要意义。【目的】制备赭曲霉毒素A (Ochratoxin A,OTA)和玉米赤霉烯酮(Zeralenone,ZEN)金标单克隆抗体,基于免疫层析原理,采用竞争反应模式,建立二联胶体金免疫层析检测法用于污染样品中OTA和ZEN的同时快速检测。【方法】采用柠檬酸钠还原法制备胶体金颗粒,并标记获得两种真菌毒素金标单克隆抗体,通过优化相关条件,建立稳定的二联胶体金免疫层析检测方法,用于同时检测谷物和饲料样品中的OTA和ZEN。【结果】制备的OTA和ZEN二联胶体金试纸条对OTA和ZEN的检测限分别为0.625 ng/mL和1.25 ng/mL,且与谷物和饲料中其它真菌毒素(黄曲霉毒素B1、伏马毒素B1、桔青霉毒素、展青霉毒素和呕吐毒素)均无交叉反应,人工添加试验结果准确。对天然样本检测结果表明该方法与LC-MS/MS一致性良好。【结论】本研究制备的二联胶体金试纸条可用于实际样品中OTA和ZEN的同时快速筛查。     

Ochratoxin A as the cause of spontaneous nephropathy in fattening pigs.

At a number of slaughters nephropathy and high ochratoxin A contents in kidneys have been observed in fattening pigs from two Swedish farms. In one herd the source of contamination was barley grown on the home farm and stored under such conditions that the growth of fungal species (Penicillium verrucosum var. verrucosum) producing ochratoxin A occurred, with the subsequent formation of the toxin. In this case high ochratoxin A levels in fattening pigs were found during a period of about 18 months. In the second herd, where compounded feed was used, it was impossible to locate the source of contamination. It was presumed that a consignment of feed was damaged by rain during storage at the farm. Ochratoxin A was found in fattening pigs from this herd for a period of about 2 months. Ochratoxin A appeared in the kidneys of all investigated pigs. In some animals the livers, whole blood, and plasma were analyzed, too. The livers contained somewhat lower amounts of ochratoxin A than the kidneys, whereas the content in whole blood and plasma, respectively, was 5 and 13 times greater. Kidneys spontaneously contaminated with ochratoxin A, when stored for 10 months at -70 degrees C, showed no systematic decrease in toxin content.     

DNA damage in the spleen, liver and kidneys of mice treated with ochratoxin A

Ochratoxin A a natural contaminant of feed and food has been shown to induce experimental liver and kidney tumors. Since there is a good correlation between the carcinogenic potency of chemicals and the DNA damages induced in mammalian cells treated either in vivo or in vitro by these compounds, we have measured single-strand breaks induced by ochratoxin A in DNA of liver, spleen and kidney. Our data clearly showed that ochratoxin A induced DNA damages in vitro as well as in vivo. Damages were dose-dependent, reversible and vary upon the time according to the tissue. In spite there is no report up to now on experimental leukemia induced by ochratoxin A, our results indicate that this possibility have to be considered.     

A new validated HPLC-FLD method for detecting ochratoxin A in dry-cured meat and in blue cheese

In the present study, a fast and sensitive method for the quantification of ochratoxin A in two lipidicproteic food matrices has been developed. In particular, the sample preparation procedure has been optimized for dry-cured meat products and blue cheeses and tested for several validation parameters (LOD, LOQ, recovery, repeatability and within-laboratory precision). The procedure has been then applied to several dry-cured meat products and blue cheeses from the market. Ochratoxin A has been occasionally found in dry-cured and smoked ham from the market and the contamination occurred both in the outer and in the inner part of the products. Concerning the blue cheese, the occurrence of ochratoxin A is reported for the first time: OTA was occasionally found at low levels (0.1–3 μg/kg) in commercial samples of Roquefort from France and Gorgonzola from Italy, opening a new issue for risk assessment and quality control. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007 Financial support: This work was partially granted by Emilia-Romagna region (projects: SIQUAL and “Safety and quality of typical pork meat production chain”)     

A procedure to quantify the feed intake response of growing pigs to perturbations

Improving robustness of farm animals is one of the goals in breeding programmes. However, robustness is a complex trait and not measurable directly. The objective of this study was to quantify and characterize (elements of) robustness in growing pigs. Robustness can be analysed by examining the animal’s response to perturbations. Although the origin of perturbations may not be known, their effect on animal performance can be observed, for example, through changes in voluntary feed intake. A generic model and data analysis procedure was developed (1) to estimate the target trajectory of feed intake, which is the amount of feed that a pig desires to eat when it is not facing any perturbations; (2) to detect potential perturbations, which are deviations of feed intake from the estimated target trajectory; and (3) to characterize and quantify the response of the growing pigs to the perturbations using voluntary feed intake as response criterion. The response of a pig to a perturbation is characterized by four parameters. The start and end times of the perturbation are ‘imposed’ by the perturbing factor, while two other parameters describe the resistance and resilience potential of the pig. One of these describes the immediate reduction in daily feed intake at the start of the perturbation (i.e., a ‘resistance’ trait) while another parameter describes the capacity of the pig to adapt to the perturbation through compensatory feed intake to rejoin the target trajectory of feed intake (i.e., a ‘resilience’ trait). The procedure has been employed successfully to identify the target trajectory of feed intake in growing pigs and to quantify the pig’s response to a perturbation.     

Liquid chromatography tandem mass spectrometry applied to quantitation of the organophosphorus nerve agent VX in microdialysates from blood probes

VX (O-ethyl-S-[2(di-isopropylamino)ethyl] methylphosphonothiolate) is a low volatility organophosphorus (OP) nerve agent and therefore the most likely route of exposure is via percutaneous absorption. Microdialysis has been used as a tool to study percutaneous poisoning by VX in the anesthetised guinea pig. A liquid chromatography tandem mass spectrometry (LC–MS–MS) method using positive electrospray ionisation (ESI) was used to quantitate VX in microdialysate samples collected from microdialysis probes, implanted into a blood vessel of anesthetised guinea pigs. The method resulted from modification of a LC–MS–MS method previously developed for the analysis of dermal microdialysates. Modification increased the sensitivity of the method, allowing quantitation of the trace levels of VX in blood microdialysates, over the range 0.002–1 ng/ml, with linear calibration. Quantitative results have been used to determine the time course of VX concentrations in the blood of guinea pigs following percutaneous poisoning.     

Implications of fungal infections and mycotoxins in camel diseases in Saudi Arabia

Natural feed ingredients (corn, barley and wheat bran) and compound feed (manufactured pellet) are two types of fodder used for animal feeding, especially camel in Saudi Arabia. Twenty samples of each type of fodder were collected from seven different regions and screened for the presence of fungi, aflatoxins, ochratoxin and zearalenone. Fungal isolation of natural feed ingredients yielded 10 genera and 38 species of different fungi. Compound fodder samples were contaminated with 16 genera and 32 species of fungi. Total counts of Aspergillus, Penicillium and Fusarium in the animal feed samples were ranged from 54 to 223 × 10³, 31.9 to 60 × 10³ and 18 to 29 × 10³ CFU/g, respectively. These isolates when tested for aflatoxin, ochratoxin and zearalenone producing ability, revealed this property in only four isolate, identified as Aspergillus flavus, A. parasiticus, A. ochraceus and Fusarium graminaerum. The percentage of toxigenic fungi was ranged from 5.5% to 30% for natural feed ingredients and from 4.5% to 20% for compound feed. The incidence of aflatoxins (AFT) in samples of natural feed ingredients was found to be ranged from 1 to 24.8 ppb, ochratoxin A (OTA) ranged from 1 to 44 ppb and zearalenone (ZON) ranged from 1 to 23 ppb. Contamination of compound feed with aflatoxin and ochratoxin A was ranged from 1 to 6.4 ppb and 1 to 4.7 ppb, respectively. All samples collected were found contaminated with fungi or their toxins and natural feed samples were more contaminated compared to compound feed samples. The concentrations detected were in the allowed limit (<20 ppb) except four samples of natural feed ingredients which were above the allowed limit of the tested mycotoxins. In conclusion, feed samples were contaminated with fungi and some toxigenic isolates which were responsible about mycotoxin production. Some samples had exceeded amount of AFT, OTA and ZON and may be contaminated with other mycotoxins which mean implication of fungi in camel health problems and death in Saudi Arabia.     

Entwicklung eines hochempfindlichen Enzymimmuntests zum Nachweis von Ochratoxin A

Antibodies against ochratoxin A were produced in rabbits after immunization with an ochratoxin A-keyhole limpet hemocyanine conjugate. The immunogen was found to be very efficient, and high antibody titers were detected in the sera of all immunized rabbits. In a competitive enzyme immunoassay using ochratoxin A-horseradish peroxidase as the labelled antigen, low levels of ochratoxin A in buffer solution could be detected. The mean standard curve detection limit and 50% inhibition level of the optimized assay were at 15 pg/ml and 50 pg/ml, respectively. Relative cross-reactivity of ochratoxin B was found to be 2%. With these characteristics, this novel enzyme immunoassay should be useful for the routine detection of ochratoxin A in food and in biological samples at levels well below 1 ng/g.     

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?×?10³ to 1?×?10⁵?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???g/kg), OTA (1.53?±?0.42???g/kg) and FUM (405?±?298???g/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???g/kg, higher than recommended in 40 and 17?% of the analysed samples, respectively.     

The occurrence of ochratoxin A in dust collected from a problem household

Accumulated dust samples were collected from the heating ducts in a household where signs resembling ochratoxin poisoning in animals occurred. Several Penicillium spp. and Aspergillus ochraceous had been identified previously from air samples taken from this house. A composite sample from six collected samples was examined by HPLC, and it was determined that 58 ppb of ochratoxin A was present in this sample. A second set of six samples was collected and determinations were made by HPLC of the ochratoxin content in each sample. All samples, including one sample of dirt from a crawl space, yielded at least a trace of ochratoxin A; however, one sample of dust collected from the heating ducts yielded over 1500 ppb of ochratoxin A, and another sample of dust from a different heating duct yielded 306 ppb of ochratoxin A. Ochratoxin A was confirmed in all samples by LC-MS, and ochratoxin was evident in the samples by TLC analysis. This is believed to be the first report of finding ochratoxin inhouse dust. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mycotoxic porcine nephropathy and spontaneous occurrence of ochratoxin A residues in kidneys and blood of Polish swine.

Kidneys showing renal changes characteristic for mycotoxic porcine nephropathy were collected during the period 1 April 1982 to 31 March 1983 from 225,000 swine processed in a large slaughterhouse in the district of Poznań, Poland. Of 113 kidneys suspected of mycotoxic porcine nephropathy, 27 exhibited ochratoxin A levels from traces to 23 ng/g. In 17 kidneys the level of the toxin was lower than 2 ng/g. Increased frequency of ochratoxin A presence and its level in kidneys were observed during the spring. Of 195 porcine blood samples collected at random, 36 exhibited toxin levels from 3 to 270 ng/ml.     

Occurrence of trichothecenes, zearalenone and ochratoxin A in cereals and mixed feed from central Lithuania

A total of 92 samples — 23 winter wheat, 12 summer barley, 5 oats and 52 mixed feed — were collected from a state factory in Kaunas, Lithuania and were analysed for the presence of trichothecenes, zearalenone (ZEN) and ochratoxin A (OA) using gas chromatography with electron capture detection and immunoaffinity column/high performance liquid chromatography with fluorescence and UV detections. Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin and HT-2 toxin were detected at concentrations above 10 μg/kg in 68%, 48%, 38% and 8% of cereal samples, respectively, and in 98%, 88%, 12% and 8% of samples of mixed feed for swine and poultry. More than 10 μg/kg of zearalenone and ochratoxin A were found in 58% and 92% of the mixed feed samples, respectively. The highest concentrations of all analysed trichothecenes in Lithuanian mixed feed and cereal grains, with an exception of T-2 toxin in one oat lot and one sample of mixed feed and OA in two mixed feed samples, were lower than those reported as Lithuanian advisory or tolerance limits.