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The high prevalence of the Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZON) in animal feeds in mild climatic zones of Europe and North America results in considerable economic losses, as these toxins affect health and productivity particularly of pigs from all age groups. The use of mycotoxin adsorbents as feed additives is one of the most prominent approaches to reduce the risk for mycotoxicoses in farm animals, and to minimise carry-over of mycotoxins from contaminated feeds into foods of animal origin. Successful aflatoxin adsorption by means of different substances (phyllosilicate minerals, zeolites, activated charcoal, synthetic resins or yeast cell-wall-derived products) has been demonstrated in vivo and in vitro. However, attempts to adsorb DON and ZON have been less encouraging. Here we describe the adsorption capacity of a variety of potential binders, including compounds that have not been evaluated before, such as humic acids. All compounds were tested at realistic inclusion levels for their capacity to bind ZON and DON, using an in vitro method that resembles the different pH conditions in the gastro-intestinal tract of pigs. Mycotoxin adsorption was assessed by chemical methods and distinct bioassays, using specific markers of toxicity as endpoints of toxicity in cytological assays. Whereas none of the tested substances was able to bind DON in an appreciable percentage, some of the selected smectite clays, humic substances and yeast-wall derived products efficiently adsorbed ZON (>70%). Binding efficiency was indirectly confirmed by the reduction of toxicity in the in vitro bioassays. In conclusion, the presented test protocol allows the rapid screening of potential mycotoxin binders. Like other in vitro assays, the presented protocol combining chemical and biological assays cannot completely simulate the conditions of the gastro-intestinal tract, and hence in vivo experiments remain mandatory to assess the efficacy of mycotoxin binders under practical conditions.

Forty newly hatched unsexed Japanese quail (Coturnix coturnix japonica) chicks were fed diets containing 3 ppm aflatoxin (AF) and 4 ppm T2 toxin either singly and in combination for 35 days. Sera samples were collected from six birds in each group at the end of the trial to study the effect of certain serum biochemical parameters. Highly significant (P < 0.01) differences were observed for serum total protein, albumin, globulin, glucose, cholesterol, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) between control and toxin treated groups. Toxin treated groups did not reveal any significant difference for serum urea nitrogen, creatinine, uric acid, calcium, phosphorus, sodium and potassium. There was a reduction in the levels of serum total protein, albumin, globulin, glucose, cholesterol, ALT and elevation of AST and GGT and variable ALP levels observed in toxin treated groups.

A total of 180 samples of poultry feeds were collected during 1996 and 1997 from different factories in the south of the province of Cordoba-Argentina. They were examined for the occurrence of Penicillium spp. and Aspergillus group species. Likewise, the capacity to produce aflatoxins by the Aspergillus section flavi group was determined. The predominant species of Aspergillus were A. flavus and A. parasiticus. For Penicillium spp., P. brevicompactum, P. purpurogenum and P. oxalicum were identified. Less frequently isolated were A. candidus, A. fumigatus, A. niger, A. orizae, A. parvulus, A. tamarii, A. terreus, and P. expansum, P. funiculosum, P. minioluteum, P. pinophylum, P. restrictum, P. variable and others. The mean value counts ranged from 1 x 10(3) to 9.5 x 10(4) CFU/g for the Aspergillus spp. and from 1.2 x 10(3) to 2.5 x 10(5) CFU/g for the Penicillium spp. When cultured on autoclaved rice kernels for 1 week in the dark at 25 degrees C, mycotoxin production by strains of A. flavus was as follows: 21 of the 45 assayed strains (47%) produced aflatoxins. From them, 24% of the isolates produced AFB1 and AFB2 with levels from 181 to 14545 and 6 to 3640 micrograms/kg respectively. Only 10 strains produced AFB1 with levels from 10 to 920 micrograms/kg. Fifty percent of the A. parasiticus strain was toxicogenic; six aflatoxicogenic profiles were identified. Only 10% of the strains produced all of the aflatoxins. These results showed that a potential exists for the production of mycotoxins by the Aspergillus section flavi and the Penicillium spp. They also suggested an association of mycotoxicosis with poultry feeds in Argentina.

The feeding of a shipment of imported corn was associated with a severe reduction in growth and increased mortality in geese, and increased mortality in broilers. Pathological examinations revealed hepatopathy, visceral gout and mild nephropathy in geese, and in broilers an hepatopathy, which was often severe, and ascites. Samples of feed from affected geese farms were examined for up to 24 mycotoxins, and ochratoxin was found in 6 of 15 samples at levels up to 930 ng/g. The syndrome was experimentally reproduced by feeding geese and broilers suspect feeds with the natural ochratoxin contamination. It is believed that another, unidentified, mycotoxin was the major cause of the hepatotoxicity, and that ochratoxin served in this case as an indicator of a multi-mycotoxin involvement.

In the European coastal dunes, marram grass (Ammophila arenaria) is planted in order to control sand erosion. In the years 1986 to 1991, workers on the Wadden islands in the Netherlands planting marram grass showed lesions of skin and mucous membranes, suggesting a toxic reaction. Fusarium culmorum dominated the mycoflora of those marram grass culms that were used for planting. This plant material had been cut and stored for more than one week in the open. The Fusarium toxin deoxynivalenol (DON) was detected in the suspect marram grass culms. Isolated F. culmorum strains were able to produce DON in vitro in liquid culture as well as in experimentally inoculated wheat heads. Pathogenicity tests, toxin test as well as RAPD analysis showed that the F. culmorum strains were not specialized for marram grass but may form part of the West-European F. culmorum population infecting cereals and grasses. Storage on old sand-dunes with plant debris may have led to the high occurrence off: culmorum and contamination with DON. Marram grass culms should be obtained from young plantings on dunes on the seaward slopes and cut culms should not be stored.

The adverse health effects of indoor mold exposure gained much public attention in recent years. Although many studies support the involvement of fungi in building-related illnesses and emphasize the importance of mold exposure as an emerging public health issue, the role of toxigenic species such as Stachybotrys chartarum (SC) remains highly controversial [1]. Widespread litigation is making it more contentious, and the existing scientific information does not offer sufficient evidence that could lead to a better understanding of the problem and development of comprehensive public health policies. The article by Nagayoshi et al. [2] published in this issue of Mycopathologia describing for the first time the remodeling of pulmonary arteries in mice as a result of inhalation exposure to the spores of S. chartarum significantly extends our knowledge of the pathologic effects of this fungus. The ability to produce potent trichothecene mycotoxins became a source of great concern about S. chartarum. Approximately one-third of the isolates from the United States and Europe represent a chemotype that synthesizes macrocyclic trichothecenes [3]. Macrocyclic trichothecenes are not found in SC isolates belonging to the second chemotype which produces atranones. However, both chemotypes can synthesize much less potent simple trichothecenes. Molds can affect human health through mycotoxicosis, infection, allergy, and inflammation. The first category is often disputed, especially in the context of residential indoor air exposure [1, 4]. Arguments against the role of S. chartarum in building-related diseases are mostly based on the projection that the relatively low concentrations of the SC spores found in most indoor environments are insufficient to cause severe systemic toxicoses. Such opinions do not take into account local toxic effects at the site of deposition of fungal particles where the toxins can reach millimolar concentrations. Reports focusing on mycotoxins tend to present the ‘‘toxic black mold,’’ as S. chartarum is known to the public, as a species with unique properties compared with other fungi. While its high toxigenic potential clearly sets it apart, the effects of different fungal compounds common to all fungi such as b-D-glucan, proteins including hemolysin and proteases or various low-molecular-weight metabolites are likely to play a significant role in pathophysiology of this species [5–9]. In recent years, a number of animal studies of pulmonary effects of S. chartarum have been published, utilizing different animals/strains, different fungal isolates, and experimental protocols. The common finding was lung injury and inflammation. Some of those effects were paralleled in the experiments conducted with pure trichothecene toxins or atranones but toxin-independent mechanisms were also postulated [1, 10, 11]. It is I. Yike (&) D. Dearborn Department of Environmental Health Sciences, Case Western Reserve University, School of Medicine, Location Code 4940, Room WG19, 2109 Adelbert Rd., Cleveland, OH 44106, USA e-mail: ixy@case.edu

In recent years cases of often fatal pulmonary hemorrhage in infants have been associated with water damaged homes and the toxigenic fungus Stachybotrys chartarum. The fungal spores contain mycotoxins which could be injurious to the rapidly developing lung. In order to understand the developmental pathophysiology of this disease we developed an infant rat model of stachybotrytoxicosis describing the effects of fungal spores on survival, growth, histopathology of the lung and respiration. Conidia of S. chartarum were instilled intratracheally (1.0-8.0 x 10(5)/gm wt.) in 4-d old Sprague-Dawley rat pups. Two control groups received either sterile PBS or a suspension of spores extensively extracted with ethanol to remove toxins. Lethal dose response was determined (LD50 = 2.7 x 10(5) spores/gm wt.). All dead pups had extensively hemorrhagic lungs. Growth of surviving animals was impaired in a dose-dependent manner. Changes of pulmonary function parameters in rats treated with 1.1 x 10(5) spores/g were consistent with an increased respiratory resistance. Histology of lungs revealed fresh hemorrhage, sparse hemosiderin-laden macrophages, and evidence of inflammation including thickened alveolar septa infiltrated by lymphocytes and mononuclear cells and intra-alveolar macrophages. Significant increases (p = 0.001) in numbers of macrophages (2-fold), lymphocytes (5-fold) and neutrophils (7-fold) were found in BAL fluid. Hemoglobin was elevated 2-fold (p = 0.004). Proinflammatory mediator IL-1beta increased more than 6-fold and TNF-alpha 30-fold (p = 0.001). Extracted spores had a minimal effect on all examined parameters in BAL fluid indicating that mycotoxins are primarily responsible for the hemorrhagic and inflammatory response.

The mycoflora of 39 feed samples associated with 29 Equine Leukoencephalomalacia (ELEM) outbreaks was studied from 1988 to 1990, in Brazil. Microbiological examination indicated Fusarium spp. as the most frequent mold which occurred in 97.4% of samples followed by Penicillium spp. in 61.5% and Aspergillus spp. in 35.9%. The moisture content of feed implicated in death of horses was above 15% which can favor the development of Fusarium spp. From the genus, F. moniliforme was the predominant species with an occurrence of 82.0%. Two additional species, not commonly associated with animal toxicosis, were isolated in low frequency, F. proliferatum (12.8%) and F. subglutinans (2.6%). It is important to emphasize that the isolation of F. proliferatum and F. subglutinans from feed obtained from the epizootic areas has not been documented previously in Brazil.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium moniliforme and F. proliferatum, induces liver damage and pulmonary edema in swine. We examined the temporal and dose-response features of FB1 toxicosis in male weanling crossbred pigs fed nutritionally balanced diets, containing corn screenings naturally contaminated with fumonisins, for 14 days. Total fumonisins (FB1 and FB2) in diets 1 through 6 were assayed at 175, 101, 39, 23, 5, and < 1 ppm (below detectable concentrations), respectively. Clinical signs, serum biochemical alterations, and morphologic changes were evaluated. Pigs were weighed, and bled for hematologic and clinical chemistry evaluation on days 5 and 14. They were euthanized on day 14, or earlier if respiratory distress was observed. Respiratory distress developed in 3/5 pigs fed diet 1 between days 4 and 6 due to severe pulmonary edema and pleural effusion. Histologic evidence of hepatic injury was present in all pigs fed diets 1 and 2, 3/5 on diet 3, and 1/5 on diet 4. Serum bilirubin and cholesterol concentrations, gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and arginase (ARG) activities were elevated in pigs fed diets 1 and 2. Based on liver histopathology, the no observed adverse effect level (NOAEL) for fumonisin toxicity in swine was < 23 ppm total fumosins for the 14-day period. Based on regression analyses of the clinical chemistry profiles at 14 days, the NOAEL was < 12 ppm, with ALP being the most sensitive parameter. In conclusion, pulmonary edema occurred only at the highest fumonisin concentration (175 ppm), while liver damage occurred at much lower concentrations with a NOAEL of < 12 ppm.

Sprouted barley grains, the waste product of malt extract production, were incriminated as the cause of a lethal (96% mortality) neurotoxic syndrome in sheep fed the grains. Clinical manifestations, comprising tremors, lameness, abnormal gait, paralysis and death indicated a tremorgenic mycotoxicosis. Whilst 50% of the flock died within 17 days, mortality continued over more than 5 months. Pathological findings were limited to neuronal degeneration and necrosis in the midbrain. Germinating grains were shown to be contaminated with growth of Aspergillus clavatus.

Mycotoxicoses are intoxications caused by ingestions of foodstuffs contaminated with mycotoxins, i.e. toxic secondary metabolites of microscopic filamentous fungi (moulds). By field observations and by experimental testing, toxins or toxic strains of more than 100 species of fungi have been encountered. However, causal associations have so far only been established for a small number of mycotoxicoses in farm animals, and the more important mycotoxicoses are aflatoxicosis, facial eczema, mycotoxic nephropathy, and estrogenic syndrome. A full assessment of the impact of mycotoxins on the health of farm animals can hardly be made at present, because the amount of surveillance data from mycotoxicoses is very limited, due mainly to inadequate diagnostic criteria. A new set of criteria for the diagnosis of mycotoxicosis is proposed, which has been successfully applied in the causative study of mycotoxic porcine nephropathy.