Ochratoxigenic moulds and ochratoxin A in forages and grain feeds.

The contamination of forages and grain feeds with ochratoxigenic moulds and ochratoxin A was examined. The investigations were carried out over a period of three years in all seasons. Feeds were found to be contaminated with moulds at a high level throughout the three research years. The highest percentage (95 to 100) of contaminated feed samples was noticed during the second year. Total viable counts of moulds established in 1 g of feed samples ranged from 0.5 to 7.8 x 10(6). Penicillium spp. were dominant in mycopopulations isolated from feeds. Ochratoxin-A producing moulds were present permanently. In the summer period of the second research year as much as 94% of the feed samples were contaminated by ochratoxigenic Penicillium species. P. verrucosum var. cyclopium P. verrucosum var. verrucosum, P. commune and P. chrysogenum, i.e. ochratoxin-producing moulds, were the most prevalent Penicillium species throughout the three-year investigation. Ochratoxin A was found in various feeds in all seasons, except in summer of the first research year. Concentrations of the toxin varied from traces to 400 micrograms/kg. It occurred consistently in the same types of feeds (hay, dried alfalfa, fresh alfalfa, concentrate, pelleted sugar beet pulp, corn silage).     

Cold storage of wheat. 1. Ergosterol, ochratoxin A and citrinin after inoculation with Penicillium verrucosum]

Seed wheat was inoculated 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 degrees C. The production of ergosterol, a chemical indicator of fungal biomass, started within the storage time investigated (240 days). Only at 18% H2O/4 degrees C an increase of the ergosterol content was not observed. Ochratoxin A and Citrinin were not detected at 18% H2O/4 degrees C and 20% H2O/4 degrees C within 240 days (detection limit: 10 and 25 micrograms/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.     

Effects of mould and toxin contaminated barley on laying hens performance and health.

Moulded and mycotoxin containing barley was incorporated into the diets for laying hens to study the effects on performance and health. Health indicators were different blood plasma parameters and liver vitamin A and E levels. A total of 30 hens were fed 3 diets, one supplemented with 30% of toxin-free and two with differently moulded barley from 1997 and 1998 for 7 weeks. The moulded diets contained low to moderate concentrations of ochratoxin A, zearalenone, deoxynivalenol and nivalenol. Inclusion of mouldy barley in the diets had an adverse effect on feed intake, feed conversion, digestibility of nutrients, egg production and egg quality. Plasma alkaline phosphatase was increased and certain biochemical blood parameters (bilirubin, uric acid, chlorine, protein, albumin, vitamin A) were also higher or changed compared to control. The ochratoxin A contamination although relatively low could have contributed to some of these effects as well as reduced intake of feed. The higher mould contamination and an unidentified cell-toxic constituent in the diet containing barley from 1998 can probably also explain the more marked effects from this diet.     

The role of Penicillia in ryegrass staggers

Tremorgenic strains of Penicillium verrucosum var cyclopium, P canescens, P janthinellum, P novaezeelandiae and P estinogenum were isolated from the faeces of 15 of 23 affected sheep and cattle in eight of nine field outbreaks of ryegrass staggers. One tremorgenic strain of P griseofulvum was isolated from the faeces of one of 25 sheep grazing in unaffected flocks. Tremorgenic strains of P verrucosum var cyclopium, P canescens, P janthinellum and P estinogenum were also isolated from the A horizon of New Zealand soils. Since a large proportion of experimentally dosed live P verrucosum var cyclopium died during passage through the gut, the faecal evidence from naturally staggering animals suggests that at least some outbreaks of ryegrass staggers are caused by tremorgenic Penicillia and that their source may be soil.     

Experimental bovine Trichophyton verrucosum infection. Comparison of the rate of epidermal cell proliferation and keratinisation in non-infected and reinoculated cattle.

The rate of epidermal cell renewal in normal bovine skin and that reinoculated with Trichophyton verrucosum was measured using a radioautographic technique. The transit times of both nucleated and fully keratinised cells were measured in sequential biopsy samples removed at predetermined periods after intradermal inoculation with radio-labelled isotopes. The total time taken for cells of the basal layer to travel to the point of desquamation in the stratum corneum was 18 days in normal cattle. In similar areas on cattle that had been reinoculated with T verrucosum the total epidermal cell renewal time was reduced to 12 days. Increased protein synthesis, as measured by incorporation of radio-labelled nucleoside was evident in basal cells within 24 h of reinoculation with the fungus. The nucleated epidermal cell thickness had almost doubled in areas of reinoculated skin within 72 h and increased cell proliferation was maintained for at least 10 days. Desquamation of the thickened stratum corneum had occurred within seven days of reinoculation with the fungus.     

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.     

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.     

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.     

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.     

Rejection by pigs of mouldy grain containing deoxynivalenol

SUMMARY: Weaner pigs on a farm near Beaudesert in south eastern Queensland refused to eat feed comprised largely of wheat and barley. Older pigs consumed small amounts and some prepubertal gilts subsequently displayed enlarged and reddened vulvas. Wheat, barley and triticale were grown on the farm during 1983, which was unusually and persistently wet. The wheat and triticale were harvested and stored for about 3 weeks with moisture contents above 14% before being fed. Samples of the wheat and triticale contained pale pink grains, which can indicate infection by the fungus Fusarium graminearum Schw. On analysis 2 mycotoxins known to be produced by F. graminearum were detected, deoxynivalenol (vomitoxin) which causes feed refusal and vomiting, and zearalenone which causes oestrogenic effects. Concentrations of deoxynivalenol in the wheat, triticale and barley were 34, 10, and < 0.1 mg/kg respectively. Concentrations of zearalenone were 6.2, 2.8 and 0.1 mg/kg respectively. Subsequently, F. graminearum was isolated from grains and crop residues. Although the wet weather contributed to F. graminearum infection of the crops before harvest, most of the toxins probably developed during storage.     

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.     

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.     

Ochratoxin A from a toxicological perspective

Ochratoxin A (OTA) is a widespread mycotoxin which is produced mainly by the mould fungi Aspergillus ochraceus and Penicillum verrucosum during the storage of cereals, cereal products and other plant-derived products such as herbs, spices, grapes, etc. By carry over from mouldy fodder, ochratoxin A is also found in pork meat, offal and sausages containing pork blood. When ingested as a food contaminant, OTA is very persistent in human beings with a blood half-life of 35 days after a single oral dosage due to unfavourable elimination toxicokinetics. This renders the toxin among the most frequent mycotoxin contaminants in human blood in the EU, the US, Canada, and elsewhere, where it has been investigated. OTA is neither stored nor deposited in the body, but heterogeneous body distribution may impose serious damage to the kidneys. The toxin was classified a 2B cancer compound, being possibly carcinogenic for humans. It was among the strongest carcinogenic compounds in rats and mice. As the toxicological profile also includes teratogenesis, nephrotoxicity, and immunotoxicity, legislation authorities are currently discussing maximal residue levels (MRL) for OTA in various foodstuffs. In the present article arguments are presented which suggest an acceptable daily intake (ADI) of 1.5 ng OTA/kg body weight and a much lower MRL than 5 microgram OTA/kg cereals and cereal products as has been postulated by the EU commission.     

Effect of feeding regime on the metabolism of ochratoxin A during the in vitro incubation in buffered rumen fluid from cows.

Pure ochratoxin A (OA) was added to buffered rumen fluid collected from two fistulated cows and incubated under anaerobic conditions. Both animals were fed six diets containing grass, grass silage or hay, and two different amounts of concentrate consisting of barley and soybean meal. Four incubations per animal and diet were carried out at consecutive days. The concentration of OA declined exponentially to a very low or non-detectable level under all conditions examined, with half-lives at 0.51 to 2.76 h. The disappearance of OA was accompanied by the appearance of ochratoxin alpha (O alpha) with an average amount of O alpha formed relative to the disappearance of OA near 100%, independent of diet and animal. Based on four incubations per animal and diet the rate of OA disappearance was affected (P < 0.1) by the origin of rumen fluid from two animals, as well as by the type of basic component and amount of concentrate in the diet, with interactions between these factors. The disappearance of OA mostly was accelerated (P < 0.1) by replacing grass silage or hay by fresh grass and by increasing the content of concentrate from 10 to 50% of dry matter. It is concluded that the capacity of the rumen to detoxify OA is not limited by the yield of Oa from OA but is strongly dependent on animal and diet.     

Experimental trichophytosis in calves caused by cultures of Trichophyton verrucosum and Trichophyton equinum

Tests were performed on 130 one-month-old calves. In 16 animals, treatment with a suspension of Trichophyton verrucosum culture at the dosage of 5 mil. conidia for an area of 10 x 10 of the clipped skin caused clinical trichophytic changes. The length of the incubation time was shorter in the calves scarified before inoculation of the infecting culture (10 to 13 days), as compared with the group of calves without scarification of the skin (11 to 19 days). Only 3 in 6 calves, infected by the same dose into non clipped skin, did contract trichophytosis, manifesting themselves as separate mycotic deposits, which appeared 26 to 35 days after inoculation. Trichophytic changes lasted longest in the group with scarification of the infected area. The ID50 in T. verrucosum and T. equinum cultures was about 1500 conidia per one calf in the case of the method of infecting into clipped scarified skin (area 100 sq cm). A 10 times lower dose (150 conidia per one calf) of the T. verrucosum suspension did not induce visible mycotic changes, but the T. equinum inoculation produced infection in one of 10 calves.     

Exposure of game birds to ochratoxin A through supplemental feeds.

Thirty randomly selected game bird feeders were sampled at 25-33-day intervals from November 1996 to March 1997 to quantitate ochratoxin A concentrations in supplemental feed. Monthly mean ochratoxin A concentration of grain in feeders was 8.3 +/- 0.8 ppb (n = 167). Ochratoxin A concentrations from individual feeders ranged from <5 to 109.9 ppb, levels that have not been demonstrated to negatively affect game birds in a laboratory environment. Stress may increase the chance of ochratoxin-induced mortality or morbidity for wild game birds. Only mean relative humidity was significantly correlated with monthly mean ochratoxin A concentration.     

In vitro release of interferon-gamma by trichophytin-stimulated whole blood cell cultures from ringworm-vaccinated and control calves experimentally inoculated with Trichophyton verrucosum

Of a group of seven calves, four were vaccinated against bovine ringworm with the vaccine Ringvac bovis LTF-130 (Alpharma, Oslo, Norway), while three calves were left unvaccinated. All calves were inoculated epicutaneously with a virulent strain of Trichophyton verrucosum . Clinical signs were recorded. In response to stimulation with trichophytin, in vitro interferon-γ (IFN-γ) production in whole blood cell cultures was assessed in samples obtained pre-and post-vaccination and pre- and post-inoculation. A commercial enzyme immunoas-say kit was used to measure IFN-γ levels (Bovigam, CSL, Victoria, Australia). Control calves developed typical ringworm lesions, whereas vaccinated calves did not. Following vaccination, release of IFN-γ in whole blood cell cultures indicated the presence of circulating trichophytin-specific lymphocytes. After inoculation with T. verrucosum , IFN-γ production was demonstrated in samples from both vaccinated and control calves. This study showed that vaccination with Ringvac bovis LTF-130 elicits a protective immune response suggesting involve-ment of the cellular branch of the immune system. Experimental infection of naïve nonvaccinated calves with T. verrucosum , also indicated stimulation of a cell-mediated immune response essential for resolution of lesions.     

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.     

Mycoflora of poultry feeds and ochratoxin-producing ability of isolated Aspergillus and Penicillium species

In Brazil, commercial feedstuffs are an important component in modern animal husbandry, but there is no information available about fungal contamination and ochratoxin A (OTA) production. The aims of this study were to determine the mycoflora incidence in poultry feeds and evaluate OTA production. In addition, the ability to produce OTA by several Aspergillus and Penicillium species was investigated. A total of 96 samples of poultry feeds were collected from four factories in Rio de Janeiro. Samples were examined for total moulds, for Aspergillus and Penicillium spp. occurrence and for their relative densities on dichloran rose bengal chloramphenicol and dichloran 18% glycerol media. The capacity to produce ochratoxin A by selected Aspergillus and Penicillium species was determined by HPLC. Total mould counts were generally higher than 1 x 10(5 )CFU ml(-1). Aspergillus and Penicillium species were isolated in the highest numbers. Aspergillus flovus and Penicillium citrinum were the most prevalent species. There was a high percentage of potential OTA producers (46%). The amount of OTA produced on this substrate was enough to cause adverse effects in animals. Several strains isolated from poultry feeds were able to produce high levels of OTA on chloramphenicol yeast medium. OTA in raw materials needs to be surveyed and storage practices must be investigated to determine occurrence and establish the livestock toxicological risk in poultry feed.     

Isolation and characterization of a haemolysin from Trichophyton mentagrophytes

Haemolytic activities of Trichophyton (T.) mentagrophytes were detected and characterized by qualitative and quantitative assays. On Columbia agar supplemented with blood from horses, cattle or sheep, T. mentagrophytes expressed a strong zone of complete haemolysis. No haemolytic activities could be detected in the closely related T. verrucosum var. ochraceum. The same results were obtained after cultivation of the fungi on sterile cellulose acetate filters placed on the surface on Columbia blood agar. After removal of the filter, complete haemolysis was detected below the colony of T. mentagrophytes. A soluble haemolysin from culture supernatant of this strain was isolated and partially purified. Specific haemolytic activity per mg protein was enriched 2.6-fold in filtrate F(1), a fraction obtained as filtrate after filtration through 3kDa cut-off membranes. The partially purified haemolysin was neither affected by proteinase K treatment, nor by high and low temperatures, suggesting that it represents a small peptide haemolysin. Accordingly, in a commercial enzymatic activity test only the crude culture filtrate, but none of the subsequent purification fractions showed reactivity. Evaluation of the specificity of the haemolysin using erythrocytes from different mammalian species revealed that sensitivity was highest to those of equines, followed by erythrocytes from sheep, cattle, swine, dogs and humans. None of the erythrocytes was lysed by filtrate F(1) from T. verrucosum var. ochraceum. Furthermore, different eukaryotic cell lines from different species were tested in their sensitivity to cytolytic activities of the haemolysin, but no membrane damage could be detected.