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.     

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.     

Risk assessment and the importance of ochratoxins

The multi-faceted and potent toxicity of ochratoxin A to experimental animals, its accepted role as disease determinant in mycotoxic porcine nephropathy, its putative role in the idiopathic human disease Balkan endemic nephropathy and associated urinary tract tumours, and its widespread occurrence usually in trace amounts in foodstuffs is focussing attention on minimising human intake. Conservative risk assessment based on porcine renal disease data has set tolerable intake values rather similar to actual average human intake. However, translating experimental rat tumour data to possible human risk is proving difficult partly because, in the author's view, the finding of a low incidence of DNA adducts could arise from an artificial surge in serum concentration following rapid uptake from aqueous intragastric administration, and this probably exaggerates what would occur during more natural slow uptake from food. The need for experimental data that can assist meaningful evaluation of risk of natural intake of ochratoxin A is emphasised, but prospective legislation should be balanced against socio-economic consequences of setting over-strict limits on contamination of relevant agricultural commodities, particularly in vulnerable specialist sectors such as coffee.     

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.     

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.     

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.     

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.     

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     

Natural occurrence of the mycotoxin viomellein in barley and the associated quinone-producing penicillia

In a batch of barley associated with field cases of mycotoxic porcine nephropathy and containing ochratoxin A and citrinin, the mycoflora were isolated by parallel incubation at 10 and 25 degrees C. Subsequently, the isolated cultures were checked for production of nephrotoxins (xanthomegnin, viomellein, ochratoxin, and citrinin). The nephrotoxin producers, all isolated by incubation at 10 degrees C, were comprised of one culture of Penicillium viridicatum, five cultures of Penicillium cyclopium, and one culture of Penicillium crustosum, all producing xanthomegnin and viomellein. One culture of P. cyclopium produced citrinin. Viomellein was detected in the barley at a concentration of approximately 1 mg/kg. The method of analysis for xanthomegnin and viomellein included extraction with chloroform, partitioning in hexane-acetone, and thin-layer chromatographic separation and identification. The identity of the xanthomegnin and viomellein produced by the isolated fungi and of viomellein detected in the barley was supported by infrared spectroscopy. This is the first report of viomellein as a natural contaminant of foodstuffs.     

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.     

Natural occurrence of the mycotoxin viomellein in barley and the associated quinone-producing penicillia.

In a batch of barley associated with field cases of mycotoxic porcine nephropathy and containing ochratoxin A and citrinin, the mycoflora were isolated by parallel incubation at 10 and 25 degrees C. Subsequently, the isolated cultures were checked for production of nephrotoxins (xanthomegnin, viomellein, ochratoxin, and citrinin). The nephrotoxin producers, all isolated by incubation at 10 degrees C, were comprised of one culture of Penicillium viridicatum, five cultures of Penicillium cyclopium, and one culture of Penicillium crustosum, all producing xanthomegnin and viomellein. One culture of P. cyclopium produced citrinin. Viomellein was detected in the barley at a concentration of approximately 1 mg/kg. The method of analysis for xanthomegnin and viomellein included extraction with chloroform, partitioning in hexane-acetone, and thin-layer chromatographic separation and identification. The identity of the xanthomegnin and viomellein produced by the isolated fungi and of viomellein detected in the barley was supported by infrared spectroscopy. This is the first report of viomellein as a natural contaminant of foodstuffs.     

Nephrotoxicity of Dietary Ochratoxin A in Broiler Chikens

Graded doses of pure ochratoxin A (0,0.5,1.0,2.0,4.0, and 8.0 mug of toxin per g of feed) were incorporated into a commercial diet which was fed to chicks from 1 day to 3 weeks of age, at which time the experiments were terminated. Growth was inhibited at 2.0 4,0, and 8.0 mug/g, whereas the kidneys were enlarged at doses of 1.0 mug/g and above. Renal function as measured by clearance of phenol red was decreased 15 and 31% by doses of 4.0 and 8.0 mug/g, respectively. Uric acid was increased 38 and 48% over the control values by doses of 4.0 and 8.0 mug/g, respectively. The plasma electrolytes Na, Cl,Ca, and K were measured; however, only K was significantly ( P smaller than 0.05) altered, showing a decrease at doses of 4.0 and 8.0 mug/g. The percentage dry weight of the kidneys decreased significantly at dose levels of 4.0 and 8.0 mug/g, indicative of edema. Histological examination of kidney sections gave the impression of edema and some tubular necrosis. Pathological changes were observed at all dose levels. These data demonstrate that ochratoxin A is a severe nephrotoxin in young broiler chickens.     

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.     

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.     

Fate of ochratoxin A in brewing.

The fate of ochratoxin A in brewing was investigated by adding (3H)ochratoxin A to the raw materials at 1- and 10-mug/g levels during mashing in a conventional microbrewing process. The results indicated that large portions (28 to 39%) of the added toxin were recovered in spent grains, with less recovery in the yeast (8 to 20%) and beer (14 to 18%). About 38 and 12% of the added toxin at levels of 1 and 10 mug/g, respectively, were degraded during brewing.     

Occurrence of ochratoxin A in herbal drugs of Indian origin — a report

This paper contains a report of occurrence of ochratoxin A in some common herbal medicines collected from different store-houses and shop-keepers of Bihar, India. Of 129 samples of 9 plants, 55 were found to be contaminated with various levels of ochratoxin A. The level of ochratoxin A was found maximal in barks ofHolarrhena antidysenterica (1.14 – 2.34 μg/g) whereas it was minimal in rhizomes ofTacca aspera (0.3 – 0.74 μg/g).Aspergillus ochraceus, A sulphureus and Penicillium viridicatum isolates obtained from drug samples were also examined for their toxigenic potentials. 19 isolates ofA ochraceus, 13 ofA sulphureus and 37 isolates ofP viridicatum were found to be toxigenic out of 67, 33, and 107 isolates, respectively. The ochratoxin A produced by Aochraceus was in the range of 0.09 to 2.44 μg/mL, byA sulphureus 0.1 to 1.76 μg/mL, and byP viridicatum 0.14 to 2.78 μg/mL of the culture filtrate.     

Monoclonal antibody technology for mycotoxins

Specific monoclonal antibodies (MABs) against aflatoxins, ochratoxin A, zearalenone, diacetoxyscirpenol and T-2 toxin have been prepared in various laboratories by the application of hybridoma technology to mycotoxins. These antibodies can be selected for sensitivity, reduced cross-reactivity, reliability and ease of production. When a suitable antibody is chosen it can then be used in a rapid immunological method such as an enzyme-linked or radio-immunoassay or immunoaffinity chromatography system. These assays have a lower limit of mycotoxin detection in the ng/ml range and have been applied to the determination of mycotoxins in samples such as maize, peanuts, peanut butter, milk and porcine kidneys. Using these immunoassay techniques, sample preparation has generally been simplified to a matter of solvent extraction of mycotoxins from the sample followed by dilution; under these conditions, levels of 1-5ug of mycotoxins/kg of sample can be found. The application and advantages of MABs to mycotoxins and the use of these antibodies in various assay techniques is discussed.     

Natural occurrence of Fusarium mycotoxins in field samples from the 1992 Wisconsin corn crop.

Analysis of 98 moldy corn samples collected in Wisconsin between November 1992 and January 1993 for Fusarium toxins by various immunochemical assays revealed overall average mycotoxin concentrations of 305.6, 237.7, and 904.3 ng/g for type A trichothecenes (TCTCs), deoxynivalenol (DON)-related type B TCTCs (total DON), and zearalenone (ZE), respectively. A small portion (5.1%) of the samples was found to be contaminated with high levels ( > 1 microgram/g) of type A TCTCs and total DON during the whole survey. Over 40% of the samples had 100 to 1,000 ng of total DON per g, while 17% of the samples had the same levels of type A TCTCs. The analytical data were consistent with those from mycological examinations for the samples in which various toxic Fusarium spp., including F. sporotrichioides, F. poae, and F. graminearum, were found. The samples received in November 1992 had relatively low concentrations of toxin; the average levels of type A TCTCs and total DON were 9.9 and 79 ng/g, respectively. The toxin concentrations became progressively higher in the samples received in December. The average levels for the type A TCTCs and total DON increased to 920 and 335 ng/g, respectively. However, the levels of ZE were higher in the samples collected earlier. The average levels for samples collected in November and late December were 1,195 and 242 ng/g, respectively. Analysis of selected samples by high-performance liquid chromatography monitoring with an enzyme-linked immunosorbent assay revealed that T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, and T-2 tetraol (T-2-4ol) were common in these samples. Statistical analysis revealed a weak correlation between the levels of total type A TCTCs and total DON in the samples (r = 0.18, P = 0.09), but a strong correlation between the levels of ZE and total type B TCTCs (r = 0.75, P < 0.0001) was found. The mycotoxin levels of total type A TCTCs, total DON-related type B TCTCs, and ZE in the cobs (5.2, 3.9, and 21 micrograms/g, respectively) were considerably higher than those in the kernels (1.0, 0.5, and 0.5 microgram/g, respectively). The type A toxin levels increased from a range of 14 to 35 ng/g to a range of 110 to 538 ng/g after the moldy corn samples were held at 5 degrees C for 8 days in the laboratory.