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 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.     

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.     

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.     

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.     

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.     

Isolation of Penicillium strains producing ochratoxin A, citrinin, xanthomegnin, viomellein and vioxanthin from stored cereal grains

Ochratoxin A producing strains of Penicillium were isolated from two out of 18 cereal samples known to be positive for this mycotoxin. These isolates were identified as Penicillium verrucosum and all also produced citrinin. However, the storage fungi isolated most frequently in the study were those of the complex Penicillium aurantiogriseum group, most strains of which produced xanthomegnin, viomellein and vioxanthin in liquid and solid culture. Potential for the simultaneous occurrence of five nephrotoxic mycotoxins in poorly stored grain in the UK has therefore been shown. The less sensitive analytical method for xanthomegnin, viomellein and vioxanthin may result in under-estimation of their occurrence in practice in comparison to that for ochratoxin A and critinin.     

Co-occurrence of mycotoxins in swine feed produced in Portugal

A total of 404 samples of commercial swine feed from Portugal feed mills were analysed by HPLC methods for the presence of mycotoxins: 277 samples of feed for fattening pigs were analysed for ochratoxin A (OTA), zearalenone (ZEA), and deoxynivalenol (DON), and 127 samples of feed for sows were analysed for ZEA and fumonisins (FB₁ + FB₂). Concerning feed for fattening pigs, 21 (7.6%) samples were positive for OTA, (2–6.8 μg/kg), 69 (24.9%) were positive for ZEA (5–73 μg/kg), and 47 (16.9%) were positive for DON (100–864 μg/kg). In feed for sows, the results showed 29.9% of positive samples for ZEA (5–57.7 μg/kg) and 8.7% positive samples for FB₁ and FB₂ (50–391.4 μg/kg). Co-occurrence of DON/ZEA was found most frequently, but simultaneous contamination with OTA/ZEA and OTA/DON was also found.     

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.     

Effect of Ochratoxin and Aflatoxin on Serum Proteins, Complement Activity, and Antibody Production to Brucella abortus in Guinea Pigs

The effect of ochratoxin alone and in combination with aflatoxin and Brucella abortus antigen on complement activity, serum proteins, and antibody response in guinea pigs was investigated. Ochratoxin did not affect complement activity or antibody response and there was no interaction between ochratoxin and aflatoxin on any of the responses tested. Ochratoxin significantly lowered the level of beta-globulin in serum of guinea pigs. There was no significant interaction between aflatoxin and antigen on lowering of the serum albumin levels of guinea pigs.     

Production of Ochratoxins in Different Cereal Products by Aspergillus ochraceus

The effects of temperature and length of incubation on ochratoxin A production in various substrates were studied. The optimal temperature for toxin production by Aspergillus ochraceus NRRL-3174 was found to be around 28 C. Very low levels of ochratoxin A are produced in corn, rice, and wheat bran at 4 C. The optimal time for ochratoxin A production depends on the substrate, ranging from 7 to 14 days at 28 C. Ochratoxin B and dihydroisocoumaric acid, i.e., one of the hydrolysis products of ochratoxin A, were produced in rice but at levels considerably lower than ochratoxin A. No ochratoxin C was produced in rice at 28 C. When added to rice cereal or oatmeal, the toxin was found to be very stable over prolonged storage and even to autoclaving for 3 hr.     

Ochratoxin A: study of grain contamination

Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9-291.7 micrograms/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 micrograms/kg.     

Ochratoxin A: Contamination of grain

Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9–291.7 μg/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 μg/kg.     

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.     

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.