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.     

Guttation droplets of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> and <Emphasis Type="Italic">Penicillium verrucosum</Emphasis> contain high concentrations of the mycotoxins ochratoxin A and B

Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10–14 days at 25°C. Parallel cultivation of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25°C. Guttation droplets were carefully collected between day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between 92.7–8667.0 ng OTA and 159.7–2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were found in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB/g) and fungal free agar (15.3–417.0 ng OTA and 12.7–151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets.     

Spontaneous occurrence of ochratoxin A residues in porcine kidney and serum samples in Poland.

During the period 1 April 1983 to 31 July 1984, 214,700 swine were processed in a slaughterhouse in Poznań, Poland. Of these pigs, 122 (0.057%) exhibited macroscopical kidney changes typical for mycotoxic porcine nephropathy. Ochratoxin A was found in kidneys from 52 of these pigs. Porcine serum samples not biased for nephropathy were collected at random in the same slaughterhouse. Of 388 samples, 148 exhibited ochratoxin A residues from 1 to 520 ng/ml. Significant increases in nephropathy and ochratoxin A frequencies were observed during the spring of 1984.     

Spontaneous occurrence of ochratoxin A residues in porcine kidney and serum samples in Poland

During the period 1 April 1983 to 31 July 1984, 214,700 swine were processed in a slaughterhouse in Poznań, Poland. Of these pigs, 122 (0.057%) exhibited macroscopical kidney changes typical for mycotoxic porcine nephropathy. Ochratoxin A was found in kidneys from 52 of these pigs. Porcine serum samples not biased for nephropathy were collected at random in the same slaughterhouse. Of 388 samples, 148 exhibited ochratoxin A residues from 1 to 520 ng/ml. Significant increases in nephropathy and ochratoxin A frequencies were observed during the spring of 1984.     

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.     

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.     

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.     

The level of ochratoxin a in patients after nephrectomy

Ochratoxin A (OTA) was analyzed in human serum and kidney samples, collected in Poland in the Pomeranian region from March to September 2005. OTA was determined using reversed phase liquid chromatography with fluorescence detection. The collected samples were from patients after nephrectomy (9 from men and 11 from women) and control serum samples from people without kidney diseases (3 and 3, respectively). The mean concentration OTA in serum of the healthy group was 0.37 ng/ml in both men and women. In patients subjected to nephrectomy it reached 1.06 ng/ml in men and 0.94 ng/ml in women, the mean content of ochratoxin A in kidneys was 0.23 ng/g and 0.20 ng/g, respectively. The highest concentration of OTA in serum among the patients subjected to nephrectomy was 3.77 ng/ml in men and 2.27 ng/ml in women while in kidneys 0.45 ng/g and 0.39 ng/g, respectively. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Occurrence of the toxigenic fungi (producers of aflatoxins and ochratoxin A) in foodstuffs in the Czech Republic 1999–2000

The occurrence of toxigenic fungi producing aflatoxins and ochratoxin A in foodstuffs was studied in the Czech Republic. Twenty five commodities were collected at twelve collection places in the Czech Republic (300 food samples). The presence of potentially toxigenicAspergillus flavus was observed in 28% of the sampled foods (black pepper, caraway seeds, fruit tea, black tea, oat flakes, fine flour, rolled oat flakes and semolina) in the year 1999, and in 25% of the sampled foods (black pepper, black tea, fine flour) in the year 2000.A tamarii (aflatoxins producer) was found in 3 black pepper samples (25%) in both years. Aflatoxins were detected in black pepper and caraway seed samples in the year 1999 and in sweet red pepper in the year 2000.A parasiticus andA nomius were not isolated. Aspergillus section Nigri (potential producer of ochratoxin A) was detected in some foodstuffs. Ochratoxin A was detected in raisins.Penicillium verrucosum andA ochraceus were not isolated from foodstuffs.     

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.     

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.     

Untersuchungen Zur Kühllagerung Von Weizen

Abstract

Saatweizen wurde ohne vorhergchende Sterilisation mit einem Ochratoxin A und Citrinin bildenden Stamm von Penicillium verrucosum beimpft und bei Wassergehalten von 18, 20, 22, 24 und 26% bei 4 und 10°C gelagert. Die Produktion von Ergosterin, eines chemischen Indikators für die von Pilzen gebildete Biomasse, setzte innerhalb der untersuchten Lagerdauer (240 Tage) ein. Lediglich bei 18% H₂O/4°C war keine Zunahme des Ergosteringehaltes zu beobachten. Ochratoxin A und Citrinin waren bei 18% H₂O/4°C und 20% H₂O/4°C über 240 Tage nicht zu finden (Nachweisgrenze: 10 bzw. 25 μg/kg). Bei den übrigen Kombinationen von Wassergehalt und Temperatur begann die Anhäufung der beiden Toxine etwa gleichzeitig mit der Ergosterinproduktion. Mit steigendem Wassergehalt und steigender Temperatur nahm die Zeit bis zum Beginn der Ergosterinproduktion ab, während die Geschwindigkeit der Produktion von Ergosterin, Ochratoxin A und Citrinin zunahm. Beide Toxine wurden während einer ersten Phase der Anhäufung etwa mit gleicher Rate gebildet. Bei 20–26% H₂O hatten der Wassergehalt und die Temperatur keinen Einfluß auf die Beziehung zwischen dem Toxingehalt und dem gleichzeitig erreichten Ergosteringehalt. Es wird empfohlen, stark mit Penicillium verrucosum kontaminierten Weizen nicht über den Beginn der Ergosterinproduktion hinaus zu lagem.

INVESTIGATIONS ON REFRIGERATED STORAGE OF WHEAT

1. Ergosterol, ochratoxin A and citrinin after inoculation with Penicillium verrucosum

Seed wheat was innoculated without having been sterilized with an ochratoxin A and citrinin forming strain of Penicillium verrucosum and stored at moisture contents of 18, 20, 22, 24, and 26% at 10 and 4°C. The production of ergosterol, a chemical indicator of fungal biomass, started within the storage time investigated (240 days). Only at 18% H₂O/4°C an increase of the ergosterol content was not observed. Ochratoxin A and Citrinin were not detected at 18% H₂O/4°C and 20% H₂O/4°C within 240 days (detection limit: 10 and 25 μg/kg, respectively). At the other combinations of moisture content and temperature the first detection of the two toxins approximately coincided with the onset of ergosterol production. With increasing moisture content and temperature the time up to the start of ergosterol production decreased, whereas the production rates of ergosterol, ochratoxin A and citrinin increased. Both toxins were produced with about the same rate during a first phase of accumulation. At 20–26% H₂O there was no influence of moisture content and temperature on the relation between toxin content and the simultaneously reached ergosterol content. It is recommended that wheat highly contaminated with Penicillium verrucosum should not be stored beyond the start of ergosterol production.     

Kinetics of ochratoxin A production in different Aspergillus species

Kinetics of ochratoxin A production was examined in a number of ochratoxin producing isolates representing different sections of the Aspergillus genus. Both weak and high ochratoxin producers were tested using immunochemical or high-performance liquid chromatograhic methods. All isolates were found to produce the highest amounts of ochratoxin A after 7-10 days of incubation. Ochratoxin production varied between 30 - 5 x l0(5) ng ml(-1) among the Aspergillus isolates tested. The A. albertensis and A. melleus isolates examined were found to produce ochratoxin A constitutively. A. albertensis produced the highest amounts of ochratoxin A at 30 degrees C after 7 days' incubation in YES liquid medium. Ergosterol content and ochratoxin production of A. albertensis cultures were in good correlation.     

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.     

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.     

<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> occurrence and Ochratoxin A contents in organically cultivated grain with special reference to ancient wheat types and drying practice

This study addresses the relationship between the ochratoxigenic strains of Penicillium verrucosum and ochratoxin A (OTA) contents in organically cultivated grain. It included 37 combined, non-dried grain samples from farmers with no drying facilities as well as 19 non-dried and 22 dried samples from six farms with on-farm drying facilities (Case studies 1–6). The study focused on the ancient wheat type spelt but also included samples of wheat, rye, barley, oats, triticale, emmer, and einkorn. All 78 samples were analysed for moisture content (MC) and occurrence of P. verrucosum. The latter was assessed by plating non-disinfected kernels on DYSG agar and counting those contaminated by the fungus. Fifty-five samples were analysed for OTA. Most of the combine harvested samples (82%) were contaminated with P. verrucosum prior to drying. This was ascribed to difficult harvest conditions and many samples of spelt, which was significantly more contaminated by P. verrucosum than oats, wheat and barley. Though not statistically significant, the results also indicated that spelt was more contaminated than rye, which is usually regarded the most sensitive small grain cereal. No correlation was found between number of kernels contaminated by P. verrucosum and OTA content. Despite many non-dried samples being contaminated by P. verrucosum, only two exceeded the EU maximum limit for grain (5 ng OTA g^–1), both being spring spelt with 18 and 92 ng g^–1, respectively. The problems were most likely correlated to a late harvest and high MC of the grain. The case studies showed exceedings of the maximum limit in a batch of dried oats and spring wheat, respectively, probably to be explained by insufficient drying of late harvested grain with high MC. Furthermore, our results clearly indicate that OTA is not produced in significant amounts in samples with MCs below 17%. All dried samples with MCs above 18% exceeded the 5 ng OTA g^–1 limit in grain. However, no correlation between MC and the amount of OTA produced was found.     

Ochratoxin A and fumonisins (B1 and B2) in maize from Balkan nephropathy endemic and non endemic areas of Croatia

The occurrence of ochratoxin A, fumonisin B1 and B2 has been investigated in maize samples collected in 1996 (105 samples) and 1997 (104 samples) in 14 counties of Croatia, including Brodsko-Posavska county, the main area of Balkan endemic nephropathy in Croatia. Ochratoxin A and fumonisins co-occurred in 21% of the examined samples. In particular, ochratoxin A (OTA) was found in 10 samples (10%) of the 1996 and 36 samples (35%) of the 1997 crops with mean concentrations of positive samples of 37.9 ng/g and 57.1 ng/g, and highest concentrations at 223.6 ng/g and 613.7 ng/g, respectively. Similar incidence of OTA contamination was observed in 1996 samples from both endemic and non endemic areas of Balkan nephropathy, whereas a significant difference (P<0.01) was found between the two areas in 1997, with 50% and 20% incidence of contamination in the endemic and non endemic area, respectively, and relevant OTA mean concentration of positive samples of 73.4 ng/g and 20.2 ng/g. High incidence of infection byPenicillium spp. (potential OTA producers) was found in all tested samples, with mean values of 88% and 93% in samples of 1996 and 1997, respectively. With respect to fumonisin B1 (FB1) and B2 (FB2) all but one of the 1996 samples were contaminated, with highest and mean concentrations of positive samples (FB1+FB2) at 11661 ng/g and 645 ng/g, respectively. Similar incidence of positive samples (93%), but lower contamination levels (mean 134 ng/g, maximum 2524 ng/g) were found in 1997 samples. The results of fumonisin analysis were in agreement with the mycological analysis showing higher incidence of Fusarium infection in samples of 1996 with respect to those of 1997. These data provide additional information on the occurrence of ochratoxin A in Balkan endemic nephropathy areas and, for the first time, its co-occurrence with other nephrotoxic compounds, such as fumonisins, that may contribute to the disease development. However the finding of these mycotoxins in the non-endemic areas, also at high levels, do not allow to draw a conclusion about their role in the etiology of the disease.     

Ochratoxin Production by the Aspergillus ochraceus Group and Aspergillus alliaceus

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30°C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 μg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 μg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.     

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.