Immunoenzyme detection of the mycotoxin, ochratoxin A]

Indirect, enzymoimmunological assays of the mycotoxin ochratoxin A were developed. In this technique a polyclonal (rabbit) antibody to ochratoxin A was used, along with the other, peroxidase-labelled (pig anti rabbit) antibody. The sensitivity of this method ranged around 75 pg of ochratoxin A per pit. The range of calibration curve was from 10 to 1000 pg per pit. The cross reactions with other ochratoxins made 1.4% (ochratoxin C). The ELISA test of ochratoxin A can be used as an expeditious screening method for a preliminary examination of the greater number of samples.     

Subacute toxicity testing of ochratoxin A and citrinin in swine.

Fourteen pigs were fed ochratoxin A and citrinin through a stomach tube at daily doses of 0.02 and 0.01 mg/kg body mass for 57 days. These toxin doses correspond to the average toxin contamination level of feeds in Central Europe. The clinical status of the pigs was monitored and clinical laboratory, haematological and mycotoxin-analytical examinations were performed throughout the trial. At the end of the experiment gross and histopathological examinations were carried out. The results of ochratoxin A and citrinin determination in the blood, obtained by high-performance liquid chromatography (HPLC), are important from the food hygienic point of view. The sensitivity of the method was 2 and 10 ng/ml for ochratoxin A and citrinin, respectively. The recovery rate of the mycotoxins was above 60%.     

A screening method for simultaneous immunochemical detection of aflatoxins and ochratoxin A in cereals and feed]

An immunochemical method was proposed for simultaneous determination of aflatoxin and ochratoxin A with the use of mixed solutions of the following reagents: standards of both mycotoxins, antiserums against the mycotoxins, and radioligands of 125I-aflatoxin B1 and 125I-ochratoxin A. The result of the analytical procedure is the value of concentration of the aflatoxin + ochratoxin A sum in the sample. The procedure needs half the amount of reagents as separate determination of each of the two mycotoxin, and is far less laborious. The proposed simultaneous immunoanalysis is suitable for large-are inspection of grain and feed safety from the viewpoint of aflatoxin and ochratoxin A levels.     

Ochratoxin A as a suppressor of mitogen-induced blastogenesis of porcine blood lymphocytes

The in vitro effect of ochratoxin A on pig lymphocytes stimulated by the mitogen Concanavalin A was studied by measuring the rates of ³H-thymidine incorporation in DNA.Ochratoxin A inhibited the mitogenic response to Concanavalin A in a dose dependent way. An almost total inhibition was obtained with ≥ 1 mg ochratoxin A/1, approximately 60 % inhibition was produced by 0.5 mg ochratoxin A/1 and approximately 10 % inhibition by 0.06 mg ochratoxin A/1.The immunosuppressive effect of ochratoxin A was not altered much by different contents of bovine serum albumin, 0.1 or 0.3 %, in the cell culture medium.     

Ochratoxin A in swine blood used for evaluation of cereal handling procedures

In a survey during the years 1985, 1986 and 1987 the quality of Swedish feeding grain was followed by the analysis of ochratoxin A in blood collected from swine at slaughter. The swine herds sampled were selected on feed handling procedures used. From information about the feed used, risk parameters for ochratoxin A contamination were identified. The results showed annual variation in the content of ochratoxin A in the grain and that ochratoxin A increased during storage of grain, particularly in the harvest of 1985. Drying of the grain with forced ambient air was found to be inferior to the use of heated forced air. It was also noticed that more than 9% of the grain was contaminated with ochratoxin A regardless of handling. The pronounced difference between the samples studied was seen mainly as a function of geographical origin, with the island of Gotland having a much higher frequency of positive samples than the rest of Sweden. No correlation between ochratoxin A in swine feed and post mortem signs of infectious diseases in the swine herds was found.     

Ochratoxin A and citrinin induced nephrosis in Beagle dogs. III. Terminal renal ultrastructural alterations

The extent and type of renal ultrastructural changes in Beagle dogs varied with the administration of ochratoxin A and citrinin alone and in the two dosage combinations. The three predominant changes were cytoplasmic vacuolation, myelin figure formation and lesions designated as cytoplasmic disarray. These changes were mainly of the endomembane system of the tubular epithelial cells. Cytoplasmic vacuoles were within proximal and distal tubules and collecting ducts and were most numerous in dogs given 10 mg/kg critrinin. Vacuolation of similar distribution, but less severe, was seen in renal tubular cells of dogs given the higher dose of the combined mycotoxins (0.2 mg/kg ochratoxin A + 10 mg/kg citrinin). This damage was limited to the proximal tubular cells in dogs given only ochratoxin A (0.1 or 0.2 mg/kg). Myelin figures were in proximal epithelial cells of dogs given ochratoxin A alone or combined with citrinin. There was cytoplasmic disarray in dogs of all groups except for dogs given 5 mg/kg citrinin. This lesions was usually limited to the proximal tubules. The lesions, however, was found in cells of the distal tubules of dogs given 10 mg/kg citrinin alone.     

Survey of pigs' kidneys with lesions consistent with PMWS and PDNS and ochratoxicosis. Part 2: pathological and histological findings

One thousand condemned pigs' kidneys were collected in February 2002 from two pig abattoirs in England to assess the lesions due to postweaning multisystemic wasting syndrome (pmws) and porcine dermatitis and nephropathy syndrome (pdns) and the possible contribution of ochratoxicosis; 174 of the kidneys were pale, 295 were swollen and 81 were abnormally firm with the gross appearance of fibrosis. The main macroscopic finding was the presence of multifocal pale cortical lesions, observed in 446 of the kidneys, and there were large cysts in 266 of them. Histopathological lesions of non-suppurative tubulointerstitial nephritis, with degeneration and fibrosis of renal tubules, were identified in 213 of 250 (85.2 per cent) of the kidneys examined. These lesions were consistent with those reported in cases of pmws and pdns. The tubular degeneration and fibrosis were also consistent with ochratoxicosis. A higher mean concentration of ochratoxin A was significantly (P=0.020) associated with the presence of multifocal pale cortical lesions consistent with ochratoxicosis, but a causal relationship was not confirmed because histochemistry was not used to detect ochratoxin in the lesions directly. There was no significant correlation between the microscopic lesions and the concentration of ochratoxin. The degenerative lesions may have been caused by previous exposure to ochratoxin that had subsequently been excreted, but the microscopic lesions also included non-suppurative interstitial nephritis, which was unlikely to have been caused by ochratoxicosis.     

Preparation of the mycotoxin ochratoxin A for research and diagnostic purposes

The following fungal strains were tested for the production of the mycotoxin ochratoxin A: Aspergillus melleus (CCM F 802), Aspergillus ochraceus (CCM 8002) and Aspergillus ochraceus (CCM F 803). The strain Aspergillus melleus (CCM F 802) proved to be very suitable for the laboratory preparation of ochratoxin A. The mould was multiplied on a natural substrate for 14 days at the temperature of 28 degrees C and at 100% relative humidity. Ochratoxin A was extracted into organic solvents (chloroform + acetic acid), purified with aqueous alkaline solutions and by preparative thin-layer chromatography. The isolated mycotoxin was identified by physico-chemical methods. The amount of pure ochratoxin A isolated from 600 g of substrate was 305 mg.     

Testing the screening method for assaying ochratoxin in grains

To detect ochratoxin in grains and some feed mixtures the method described by Nesheim et al. (1973) was chosen out of a large number of analytical procedures. No ochratoxin was assayed in any of 41 samples (barley, corn, wheat, oats, complete feed mixtures, degraded straw) which were obtained from the current operation of feed-processing plants, did not contain any macroscopically detectable molds, were taken from storehouses with safe store conditions and they were evaluated by agricultural enterprises as safe. This method can be recommended to assay grains for ochratoxin in Czechoslovakia.     

Potential Aflatoxin and Ochratoxin A Production by <Emphasis Type="Italic">Aspergillus</Emphasis> Species in Poultry Feed Processing

Poultry feeds are prone to fungal growth and mycotoxin production during processing. The identification of biota with the ability to produce mycotoxins is essential. The aims of this study were (1) to monitor the mycobiota counts at different stages of poultry feed processing; (2) to determine the occurrence of Aspergillus species; (3) to evaluate the natural incidence of aflatoxins and ochratoxin A. The ability of Aspergillus spp. and its teleomorphs isolated here to produce these toxins was also investigated. Samples (144) were collected at random from a factory in Brazil. The occurrence of Aspergillus and Eurotium species was demonstrated on DRBC and DG18 media and the production of aflatoxins and ochratoxin A and their natural incidence were determined by TLC and HPLC methods. A. flavus and E. chevalieri were the most prevalent species isolated. Fungal contamination was not found after the pelleting process, though Aspergillus and Eurotium species were recovered from trough samples. High levels of aflatoxin and ochratoxin A producers were found at all stages of poultry feed processing. Also, high natural contamination with aflatoxins and ochratoxin A was found in the samples. Contact of feed with remainder poultry feed could lead to fungal contamination, so the risk of aflatoxin and/or ochratoxin A contamination of feed must be taken into account.     

Survey of pigs' kidneys with lesions consistent with PMWS and PDNS and ochratoxicosis. Part 1: concentrations and prevalence of ochratoxin A

One thousand condemned pigs' kidneys were collected in February 2002 from two pig abattoirs in England to assess the possible contribution of ochratoxicosis to postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS); 250 of the kidneys with macroscopic lesions consistent with nephrosis/nephritis (pale or white cortical lesions) were selected, and the concentration of ochratoxin A was measured in samples of renal cortex by high-performance liquid chromatography (HPLC). Low concentrations were detected in 230 (92 per cent) of the kidneys tested, and in 41 (16.4 per cent) of them the concentration was below the limit of quantification of 0.2 microg/kg. In 187 (74.8 per cent) of the kidneys, the concentration was more than 0.2 microg/kg, and the highest concentration detected was 2.3 microg/kg. The mean (sd) concentration was 0.31 (0.33) microg/kg. The identification of ochratoxin A was confirmed by mass spectrometry. The concentrations of ochratoxin A did not exceed the threshold assessed by the Food Standards Agency to be safe for human food.     

Studies on some feed additives and materials giving partial protection against the suppressive effect of ochratoxin A on egg production of laying hens

The protective effects of various feed supplements against the harmful effect of ochratoxin A on egg production and sexual maturation of two-weeks old Plymouth Rock female chicks designed for laying hens were studied. A significant protective effect of the feed additives or materials: water extract of artichoke (WEA), sesame seed (SS), Roxazyme-G (RG) and l-β phenylalanine (PHE) against the suppressive effect of ochratoxin A (OTA) on egg production of laying hens was found. A similar protection was also seen on the toxic effect of OTA on various internal organs of the same hens. A significant protection was found against the decrease of the weight or the quantity of eggs as well as against the delay of the beginning of the laying period of chicks, both of which were provoked by ochratoxin A. These protective effects were strongest in chicks treated with SS or WEA, but were slightest in chicks treated with l-β PHE.     

Ochratoxin A in blood of slaughter pigs

The global ochratoxin A contamination of Swedish feed cereals was studied by analysis of pig blood samples from 122 different herds. The samples were collected at seven Swedish slaughterhouses. The ochratoxin A analysis showed 21% of the samples to contain greater than or equal to 2 ng ochratoxin A per ml. Samples from Visby showed a significantly higher frequency of contamination compared with the rest of the country.     

Ameliorative effects of L-carnitine and vitamin E (α-tocopherol) on haematological and serum biochemical parameters in White Leghorn cockerels given ochratoxin A contaminated feed

1. L-carnitine is a quaternary ammonium compound biologically synthesised from the amino acids methionine and lysine while vitamin E (α-tocopherol) is an important antioxidant. The objective of the present study was to evaluate the ameliorative effects of L-carnitine and vitamin E upon haematological and serum biochemical parameters in ochratoxin A intoxicated birds.

2. Day-old White Leghorn cockerels were acclimatised for 2 d, divided in 12 groups with 20 birds in each group. From d 3 of age, they were given different combinations of ochratoxin A (1.0 and 2.0 mg/kg), L-carnitine (1 g/kg) and vitamin E (200 mg/kg) in feed. Haematological (erythrocyte count, leucocyte count, haemoglobin concentration and haematocrit percentage) and serum biochemical parameters (serum urea, creatinine, albumin, total proteins and alanine aminotransferase) were evaluated.

3. Results confirmed that L-carnitine and vitamin E given alone or combined with 1.0 mg/kg ochratoxin A ameliorated toxin induced alterations in haematological and serum biochemical parameters. This amelioration, however, did not occur when ochratoxin of 2.0 mg/kg was given.

4. L-carnitine and vitamin E in combination have the ability to ameliorate ochratoxin altered haematological and serum biochemical parameters. However, the optimum ratio of L-carnitine + vitamin E, to be used to assure such mitigation of ochratoxin A altered changes in haematological and serum biochemical parameters in cockerels, has yet to be determined. The combination used in this study was indeed sufficient to ameliorate the alterations induced by ochratoxin A up to 1.0 mg/kg feed.     

Application of the polymerase chain reaction to detect fowl adenoviruses.

The possibility of using the polymerase chain reaction (PCR) for the detection of fowl adenoviruses (FAdV) was tested. The optimal reaction parameters were evaluated and defined for purified genomic DNA of type 8 fowl adenovirus (FAdV-8), and then the same conditions were applied for nucleic acid extracted from infected cells. One hundred picograms of purified viral DNA, or 250 FAdV-8-infected cells, were detected by ethidium bromide staining of the PCR products in agarose gels. The sensitivity was increased to 10 pg purified viral DNA, or 25 infected cells, when the PCR products were hybridized with a specific labeled probe. Several field isolates of FAdV and the CELO virus (FAdV serotype 1) could be amplified by the same primers and conditions, but the size of the amplicons was smaller than that for the FAdV-8 PCR product. Other avian viruses and uninfected cell cultures tested negative.     

The influence of contamination with separate mycotoxins (aflatoxins, ochratoxin A, citrinin, patulin, penicillic acid or sterigmatocystin) on the in vitro dry matter and organic matter digestibilities of some roughages (berseem hay and wheat straw).

In vitro study on berseem hay and wheat straw was undertaken to investigate the the effect of mycotoxin contamination on dry matter and organic matter digestibilities. The data revealed a negative effect of most studied mycotoxins on the materials digestibility. Among the investigated mycotoxins, penicillic acid with its two concentrations (5 and 10 nmol) was the most negative, affecting digestibilities of both feed materials. Wheat straw digestibility was more influenced than berseem hay by the ochratoxin A, citrinin and sterigmatocystin (besides the penicillic acid) particularly with their high level (10 nmol). Yet, some mycotoxins act as antibiotics which may affect only the harmful flora but encourage the rumen microflora resulting in slight improvement of digestibility. The rumen conditions were able to metabolize or deform the used levels of all mycotoxins studied. Thus, there were no detectable residues of these mycotoxins in the digestion media after the in vitro fermentation.     

Use of the radioimmunologic determination of ochratoxin A mycotoxin in the screening of foods and animal feed of plant origin--cereal type

Trials were conducted to verify a simple procedure of preparing food and feed samples of plant origin for the radioimmunological assay of ochratoxin A. All 27 food samples subjected to testing met the general hygienic regulations for foods (NPK - ochratoxin A 20 micrograms.kg-1), and so did all 23 samples of the tested feeds. The proposed method of sample preparation is not suitable for the examination of animal-origin foods because some proteins (albumin) might interfere.     

Mycotoxins as a risk factor for the origin of diseases and production decreases in swine facilities--an epidemiologic study]

Feed samples checked for the mycotoxins zearalenone and ochratoxin A from the harvest 1987 were positive at a markedly higher percentage (37.5%) compared to previous years, which is explained by the especially unfavourable harvesting conditions of 1987. In certain herd problems affecting the digestive or respiratory tract, mycotoxins could be detected with a much higher frequency (64.7% and 50.0% respectively). The mean level detected in feed samples by thin layer chromatography ranged within 30.3 ppb for zearalenone and within 58.3 ppb for ochratoxin A. In most cases there was a history of infertility. Considering the clinical situation, which is presented comparatively in herds with positive mycotoxin results, the possible involvement of mycotoxins in the disease, even at very low concentrations, is pointed out. In this context, zearalenone is incriminated of being an indicator of a multitoxic process besides its own direct effects. According to own experiences low levels of zearalenone in the range of 20-50 ppb in the feed have to be considered hazardous. If changing of pig feed in cases of herd problems will be recommended, a level of less than 10 ppb of zearalenone, especially in sow and piglet rations, should not be exceeded. Same may be valid to ochratoxin A.     

高效液相色谱法测定癸氧喹酯原料及制剂含量的研究

建立了癸氧喹酯原料药、固体分散体含量测定的高效液相色谱一紫外检测法。采用DiamonsilC18（4．6mm×150mm,5μm）反相色谱柱,以甲醇一水（95：5）溶液（用稀盐酸调pH值至3．5）为流动相,检测波长330nm,流速0．6mL／min,外标法定量。结果显示该方法最低检测限为0．0025p,g／mL,在0．5～10斗μmL浓度范围内,线性关系良好（A=11405C＋15821,R=0．999）,加样平均回收率99．61％,RSD为0．90％。此方法可用于癸氧喹酯的含量测定,适用于该药品固体分散体生产的质量控制。     

Rapid and specific differentiation of enterotoxin-producing Escherichia coli strains from other gram-negative enteric bacteria using multiplex PCR

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile (LT) and heat-stable (STI or STII) enterotoxins. Differentiation between ETEC and other pathogenic and non-pathogenic E. coli as well as other Gram-negative bacteria responsible for induction of diarrhoea, requires isolation, biochemical identification and determination of toxins (or their genes--elt, estI, estII). A multiplex polymerase chain reaction (PCR) system for the rapid and specific detection of enterotoxin-gene-positive E. coli was developed. The primers described by other authors, specific for the universal stress protein A (UspA) of E. coli and enterotoxin genes were used and allowed a simultaneous amplification of the E. coli-specific uspA and the respective toxin genes. The specificity of this multiplex PCR system was confirmed by testing ETEC, non-ETEC and other non-E. coli bacteria. The specific 884 bp uspA gene and 280 bp (eltI), 166 bp (estI) or 278 bp (estII) amplification products were generated with the respective ETEC strains whereas no amplification was detected with non-E. coli bacteria. The multiplex PCR developed allowed the rapid and specific identification of enterotoxin-producing E. coli colonies directly grown from faecal samples of pigs with diarrhoea. The test may be used as a method for the determination of ETEC among other pathogenic groups of E. coli and other Gram-negative enteric isolates.