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 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.     

Transmission of Yersinia enterocolitica 4/O:3 to pets via contaminated pork

AIMS: This study was conducted to investigate sources of Yersinia enterocolitica 4/O:3 infections in dogs and cats. METHODS AND RESULTS: Transmission of Y. enterocolitica 4/O:3 to pets via contaminated pork was studied using PFGE with NotI, ApaI and XhoI enzymes. A total of 132 isolates, of which 16 were from cat and dog faeces and 116 from raw pork samples, were recovered in Finland during 1998-99. Cat 1, whose diet consisted mostly of raw pig hearts and kidneys, excreted Y. enterocolitica 4/O:3 of genotype G4. This predominant genotype was also found in isolates recovered from the pig heart, liver, kidney, tongue and ear, and minced pork samples. Dog 2, which was fed raw minced pork, excreted Y. enterocolitica of genotype G13. This genotype was also identified in isolates recovered from the pig heart, kidney and tongue, and minced pork samples. CONCLUSION: These results show that raw pork can be an important source of Yersinia enterocolitica 4/O:3 infections in dogs and cats. Significance and Impact of the Study: Raw pork should not be given to pets.     

Comparison of ochratoxin A levels in edible pig tissues and in biological fluids after exposure to a contaminated diet

The aim of this study was to compare ochratoxin A (OTA) levels in pig tissues and biological fluids after animal exposure to contaminated diet (250 μg OTA/kg of feed) during 4 weeks of fattening. OTA concentrations were quantified using a validated immunoassay method (ELISA) and high-performance liquid chromatography with fluorescence detector (HPLC-FD). The highest mean OTA concentration in pig tissues was determined in kidneys of exposed animals (13.87?±?1.41 μg/kg), followed by lungs (10.47?±?1.97 μg/kg), liver (7.28?±?1.75 μg/kg), spleen (4.81?±?0.99 μg/kg), muscle tissue (4.72?±?0.86 μg/kg), fat tissue (4.11?±?0.88 μg/kg), heart (3.71?±?1.09 μg/kg), and brain (3.01?±?0.25 μg/kg). Furthermore, on the last day of exposure (day 28), significantly higher mean OTA levels were determined in urine (16.06?±?3.09 μg/L) in comparison to serum (4.77?±?1.57 μg/L) showing that OTA urine analysis could be a good marker to identify elevated levels of this contaminant in porcine tissues used for human consumption. This study gave guidelines for the most efficient OTA control in pig-derived biological materials that can be exercised at slaughterhouses.     

Natural contamination with mycotoxins in forage maize and green coffee in Nayarit State (Mexico)

The presence of mycotoxins in forage maize (zearalenone, fumonisin B1, T-2 toxin and diacetoxyscirpenol) and green coffee (ochratoxin A) from Nayarit State (Mexico) has been studied. All maize samples analyzed showed fumonisin B1 contamination, with an average concentration of 2,541 microg/kg. Fifteen percent of the samples contained zearalenone, with an average concentration of 1,610 microg/kg. Only one sample showed T-2 toxin contamination (7 microg/kg), and no diacetoxyscirpenol was detected. Sixty-seven per cent of green coffee samples were contaminated with ochratoxin A, with an average concentration of 30.1 microg/kg. This is the first study about mycotoxins developed in Nayarit and it has shown that mycotoxin contamination is a real problem in both foodstuffs studied.     

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.     

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。     

A survey of porcine kidneys and chicken liver for ochratoxin A in Spain

Contamination studies by ochratoxin A on pork kidney and chicken liver has been carried out in Catalonia (Spain). 73% of the pork kidney samples analyzed did not contain an amount of ochratoxin A over our detection limit (0.5 ng/g) whereas only 7% had contamination higher than 1 ng/g. None of the chicken samples analyzed were contaminated by this toxin above the detection limit. All contamination levels found are below the maximum levels accepted by several countries for this kind of material. A confirmative test is necessary before discarding false positive samples.     

Mycotoxins in laboratory rodent feed

Twenty-one batches of fixed-formula rodent diets from three feed manufacturers were tested for the presence of five mycotoxins: deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin, T-2 toxin and ochratoxin A (OTA). Five batches were also tested for the presence of zearalenone (ZEN) and six batches for aflatoxins. Detectable levels of DON (up to 298 microg/kg), NIV (up to 118 microg/kg), OTA (up to 3.1 microg/kg) or ZEN (up to 26.7 microg/kg) were found in samples from all manufacturers. Three batches contained two (DON or NIV and OTA or ZEN) and one batch contained three (DON, OTA and ZEN) different mycotoxins. Aflatoxins, T-2 and HT-2 were not detected in any of the batches. The concentrations of mycotoxins detected in the feed were low, but indicated that feed ingredients, probably the cereal ingredients, were contaminated by mycotoxins. Since mycotoxins are known to have toxic and/or immunosuppressive effects, non-contaminated ingredients should be used for production of laboratory animal feed. The results imply that an improved quality control of ingredients used for laboratory rodent feed should be implemented.     

Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction

A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C(18) bonded silica column with a deionized water-methanol-acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 microg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1-20 microg/kg were in the range of 71.4-99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 microg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 microg/kg were in the range of 6.2-13.9% and 4.0-8.7%, respectively. The intra-day precision (n=5) for nitroimidazoles residues in chicken spiked at 20 microg/kg is 6.9%, and the inter-day precision for 5 days (n=25) is 11%. The method is capable of identifying nitroimidazole residues at > or =0.7 microg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.     

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.     

Ochratoxinogenicity of Aspergillus ochraceus strains from nephropathic and non-nephropathic areas in Yugoslavia

Mycological analyses of 855 samples of stored grains and dried meat collected in period 1980–1987 from individual households in the nephropathic and wider non-nephropathic area in SR Croatia in Yugoslavia showed 10% of samples to be contaminated with Aspergillus ochraceus. Ability to produce ochratoxin A (OA) was tested in 70 samples (27 from nephropathic areas and 43 from non-nephropathic areas). The detection was carried out under UV-light (365 nm) (light blue fluorescence) and 6 OA-producers were found.A biosynthetic procedure on liquid nutritional substrate with sacharose and yeast extract as well as a method using wet crushed wheat revealed that 37% of the samples from a nephropathic area, and 35% of the samples from a non-nephropathic area produce OA.In the nephropathic area 1/10 strains was a strong producer of OA (concentration crushed wheat 135 mg/kg, and 240 mg/l on YES liquid substrate), 1/10 strains was a moderate producer (concentration 16.6 mg/l and 0.07–7.0 mg/l and 0.1–10.4 mg/kg).Among the strains isolated from a wider non-nephropathic area no strong producers of OA were found, but 2/15 strains were moderate producers of OA (concentration of OA 20.4–27.0 mg/l and 15.0–33.7 mg/kg). The other strains, 8/10 on the crushed wheat and 13/15 on the liquid substrate, were weak producers of OA with concentrations of OA between 0.2–9.0 mg/l and 0.2–10.0 mg/kg with the two methods respectively.     

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.     

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.     

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.     

Predicting noncompliant levels of ochratoxin A in cereal grain from Penicillium verrucosum counts

AIMS: To model the probability of exceeding the European legislative limit of 5 microg ochratoxin A (OTA) per kilogram grain in relation to Penicillium verrucosum levels and storage conditions, and to evaluate the possibilities of using P. verrucosum colony counts for predicting noncompliant OTA levels. METHODS AND RESULTS: Cereal samples were inoculated with P. verrucosum spores and stored for up to 9 months at temperatures and water activities ranging from 10-25 degrees C and aw 0.77-0.95. A logistic regression analysis showed that the probability of exceeding 5 microg OTA kg(-1) grain was related to colony counts of P. verrucosum and water activity. The sensitivity and specificity of various P. verrucosum count thresholds for predicting noncompliant OTA levels were estimated, using data from the storage trial and natural cereal samples. CONCLUSION: The risk of exceeding 5 microg OTA kg(-1) grain increased with increasing levels of P. verrucosum, and with increasing water activities. A threshold of 1000 CFU P. verrucosum per gram grain is suggested to predict whether or not the legislative limit is exceeded. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided a tool to evaluate the levels of P. verrucosum in grain in relation to OTA levels. Hence, mycological analyses can be used to identify cereal samples with high risk of containing OTA levels above the legislative limit.     

Feeding OTA to pigs: Analytical evaluation of a trial

Methods were developed to analyse the concentrations of ochratoxin A (OTA) and its main metabolite ochratoxin α in blood plasma of pigs and in their kidneys. By the use of these methods blood and kidneys of pigs that were fed contaminated feedstuff containing 1500 μg/kg ochratoxin A for the whole trial period of 42 days were analysed.High levels of 1500 – 2000 μg/I OTA were found in the plasma, while the concentration of OTA in the kidneys did not exceed 250 μg/kg at the end of the trial. Neither in the plasma nor in the kidneys of the animals fed contaminated feedstuff ochratoxin a could be detected.     

A survey on the occurrence of ochratoxin A in feeds for swine and laying hens

Results of a 2-year (2009–2010) survey on the occurrence of ochratoxin A (OTA) in swine feed and in feed for laying hens in Portugal are reported. A total of 664 samples (478 swine feed, 186 feed for laying hens) were analyzed by a HPLC method using fluorescence detection with 2 μg kg^?1 as detection limit. In swine feed, 31 samples (6.49%) were positive for OTA. In feed for laying hens, 12 samples (6.45%) were OTA-positive. The average levels of contamination were low, with median values of positive samples at 3–4 μg kg^?1 in both years and both commodities, although a few samples contained exceptionally high levels (maximum 130 μg kg^?1). Only the maximum level sample (swine feed) contained OTA at a concentration exceeding the European Commission guidance value. The remaining OTA concentrations found in feed samples were much lower than the guidance values.     

Metabolism of ochratoxin A by rats.

Albino rats were given ochratoxin A (6.6 mg/kg body weight) intraperitoneally or per os. Independent of route administration, 6% of a given dose was excreted as the toxin, 1 to 1.5% as (4R)-4-hydroxyochratoxin A, and 25 to 27% as ochratoxin alpha in the urine. The metabolite (4S)-4-hydroxyochratoxin A, which is formed by rat liver microsomes in the presence of NADPH, was not detected. Only traces of ochratoxins A and alpha were found in feces. Identical experiments were carried out with brown rats, since the Km value for the formation of the 4S epimer was considerably lower when brown rat microsomes were used. About the same ratios of metabolites and metabolite recoveries as those found for albino rats were found for brown rats. Brown rats were also given the two hydroxylated metabolites and ochratoxin alpha (0.66 mg/kg body weight) intraperitoneally. The three compounds were excreted in the urine; within 48 h, 90% recovery of ochratoxin alpha and 54 and 35%, respectively, of the 4R and 4S isomers were observed.