Experiment to determine limits of tolerance for fumonisin B1 in weaned piglets

In Hungary almost 70% of mould-affected maize inspected since 1993 was found to be contaminated with fumonisin B1 (FB1) (mean 2.6-8.65 mg/kg; maximum 9.8-75.1 mg/kg), the degree of this contamination was found to increase from year to year (Fazekas et al., 1997b). In this experiment, in order to define tolerance limit values, the effect of exposing weaned piglets to FB1 in low doses over a 4-week period was examined. The experiment was performed with 20 weaned barrows of Danish Landrace breed. After a 5-day adaptation period cultures of the fungus Fusarium moniliforme were mixed into the animals' feed in concentrations that resulted in a daily intake of fumonisin B1 of 0, 10, 20 and 40 mg/kg feed. Feeding with the toxin was observed to exert no significant effect on body weight gain or feed consumption in the animals, no clinical signs were observed and no mortality traceable to toxic effects occurred. In computer tomography examinations performed in the second and fourth weeks mild and more severe pulmonary oedema was diagnosed in the experimental animals. The processes developing in the pulmonary parenchyma were corroborated by the mathematical and statistical evaluation procedures applied. The haematological parameters examined revealed no change attributable to toxic effects, while with respect to the biochemical parameters, an increase in aspartate aminotransferase (AST) activity dependent on dosage, indicating a pathological change in the liver, was ascertained in all three experimental groups. The free sphinganine to sphingosine ratio (SA/SO), which is regarded as the most sensitive bioindicator of fumonisin toxicosis, showed an increase proportionate to toxin concentration for all three dosages. Dissection revealed mild cases of pulmonary oedema in three of the animals given doses of 10 p.p.m. (n = 4), two mild and two severe cases in those exposed to 20 p.p.m. (n = 5), and severe cases in all five animals given 40 p.p.m. The oedema of non-inflammatory origin was confirmed by histopathological examinations. The findings of this experiment which indicate that in this study FB1 administered in substantially lower concentrations than those reported in the literature resulted in severe pathological changes, point to the importance of studies involving even lower doses.     

Protective effects of silymarin on fumonisin B1-induced hepatotoxicity in mice

The present study was conducted to investigate the effect of silymarin on experimental liver toxication induced by Fumonisin B₁ (FB₁) in BALB/c mice. The mice were divided into six groups (n = 15). Group 1 served as the control. Group 2 was the silymarin control (100 mg/kg by gavage). Groups 3 and 4 were treated with FB₁ (Group 3, 1.5 mg/kg FB₁, intraperitoneally; and Group 4, 4.5 mg/kg FB₁). Group 5 received FB₁ (1.5 mg/kg) and silymarin (100 mg/kg), and Group 6 was given a higher dose of FB₁ (4.5 mg/kg FB₁) with silymarin (100 mg/kg). Silymarin treatment significantly decreased (p < 0.0001) the apoptotic rate. FB₁ administration significantly increased (p < 0.0001) proliferating cell nuclear antigen and Ki-67 expression. Furthermore, FB₁ elevated the levels of caspase-8 and tumor necrosis factor-alpha mediators while silymarin significantly reduced (p < 0.0001) the expression of these factors. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expressions were significantly elevated in Group 4 (p < 0.0001). Silymarin administration alleviated increased VEGF and FGF-2 expression levels (p < 0.0001). In conclusion, silymarin ameliorated toxic liver damage caused by FB₁ in BALB/c mice.     

Immunohistochemical detection of swine influenza A virus in formalin-fixed and paraffin-embedded tissues.

An avidin-biotin complex (ABC) immunohistochemical method utilizing a commercially-available polyclonal antiserum to human influenza A virus was used to detect antigens of influenza A virus in formalin-fixed, paraffin-embedded tissues of swine. Influenza A antigens were immunohistochemically detected in 28/30 cases in which influenza A virus was demonstrated by virus isolation and in 5/22 cases suspected to be influenza A-infected by clinical and histological criteria, but from which the virus was not isolated. Viral antigen was not demonstrated in 30/30 cases not suspected clinically or histologically to be associated with influenza infection. This method is a convenient, sensitive, and specific means of influenza A virus detection and is applicable to both routine diagnosis of influenza A virus infection and to retrospective and prospective studies of the occurrence and the pathogenesis of this virus in pigs.     

Effects of prolonged exposure to low-dose fumonisin B1 in pigs

From the point of view of human exposure, fumonisins (FB1, FB2, FB1, FB4), a relatively recently (1988) discovered and identified group of mycotoxins, represent one of the five most important mycotoxin groups causing human disease. In an earlier experiment studying the effects of relatively low doses (10, 20 and 40 p.p.m.) of FB1 in weaned piglets, it was established that the 4-week feeding of 10 p.p.m. (mg/kg feed) FB1 produced mild pulmonary oedema. This suggested the importance of studies with even lower doses of the toxin to determine the tolerable limits. The objective of this experiment was therefore to study the effects of prolonged (8-week) exposure to still lower concentrations (0, 1, 5 and 10 mg/kg feed) of FB1. The 8-week feeding of FB1 in low concentrations (1-10 p.p.m.) did not cause clinical signs and significant performance impairment in pigs, but rendered irreversible the chronic changes that had already developed in the animals in a dose-dependent manner. Dissection revealed pathological alterations of the lungs in one of the animals given 1 p.p.m. (n = 4), in two animals exposed to 5 p.p.m. (n = 5), and in three animals given 10 p.p.m. (n = 4). In all three treatment groups, proliferation of the connective tissue fibres, primarily of those around the lymphatic vessels, in the subpleural and interlobular connective tissue of the lungs, extending to the peribronchial and peribronchiolar areas, was seen. The results of this experiment call attention to the risk of prolonged low-dose toxin exposure, which has very important public health implications.     

Pulmonary edema and hydrothorax in swine produced by fumonisin B1, a toxic metabolite of Fusarium moniliforme

Pulmonary edema and hydrothorax were observed in mature swine that died approximately 5 days after consuming corn screenings. These postmortem observations were reproduced in younger swine (16-24 kg) that died within 1 week when fed the corn screenings under experimental conditions. Additionally, pulmonary edema and hydrothorax occurred in a pig (7.1 kg) that died after receiving 4 daily intravenous injections of fumonisin B1. A fungus was isolated from the corn screenings that is identical to Fusarium moniliforme MRC-826 in colony morphology and under microscopic examination.     

Immunohistochemical detection of avian pneumovirus in formalin-fixed tissues.

An immunohistochemical staining technique (IHC) was developed to detect avian pneumovirus (APV) antigen in formalin-fixed, paraffin-embedded tissue sections using streptavidin-biotin immunoperoxidase staining. Samples of nasal turbinates and infraorbital sinuses were collected from 4-week-old poults experimentally inoculated with APV and from older turkeys infected during naturally occurring outbreaks of avian pneumovirus. Tissue was fixed in 10% buffered neutral formalin, embedded in paraffin, sectioned and stained. Inflammatory changes were observed microscopically in the mucosa and submucosa of the nasal turbinates and infraorbital sinuses of both experimentally inoculated poults and naturally infected birds. Viral antigen was detected by IHC in the ciliated epithelial cells of nasal turbinates and infraorbital sinuses.     

Use of a Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal sampling sites in swine.

A number of polymerase chain reaction (PCR)-based diagnostic tests have been developed for Mycoplasma hyopneumoniae, including one from this research group. This report presents further development, optimization, and standardization of a nested PCR test. Detection sensitivity was 1 fg of M. hyopneumoniae chromosomal DNA (approximately 1 organism). This exceeded the sensitivity of or compared favorably with other published PCR tests. Polymerase chain reaction primers to porcine beta2-microglobulin were included as internal controls for amplifiable chromosomal DNA from porcine samples. To standardize the test, a number of samples from experimentally infected pigs, including nasal, tonsil, tracheobronchial swabs, lung tissue, bronchial alveolar lavage (BAL) fluid, and tracheobronchial brush samples, were examined by PCR. Samples obtained from BAL fluid and tracheobronchial sites were most predictive of infection, whereas nasal swabs and lung tissue were not reliable indicators of experimentally induced infection. In conclusion, the nested PCR developed for this study was found to be a highly sensitive and specific diagnostic tool for M. hyopneumoniae, but the enhanced sensitivity may be unnecessary if the proper sites are sampled.     

Identification of monoclonal antibodies for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed paraffin-embedded tissues.

Commercially-available monoclonal antibodies to B lymphocytes were evaluated for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed, paraffin-embedded tissues using an avidin biotin complex immunoperoxidase immunohistochemical technique. Three monoclonal antibodies: F46A and F72A raised to "carnivore" B lymphocytes and RA3.6B2 raised to murine B lymphocytes, stained B lymphocyte-dependent areas of frozen feline lymphoid tissue. In addition, antibody RA3.6B2 stained B lymphocyte dependent areas in formalin-fixed, paraffin-embedded feline tissues. There was no staining of T lymphocyte-dependent areas in either frozen or formalin-fixed tissues. Dual parameter flow cytometry, using an anti-pan-T lymphocyte antibody, revealed that greater than 99% of the cells stained by RA3.6B2 are a population distinct from T lymphocytes. F46A was shown to stain a sub-population of those cells stained with RA3.6B2. These antibodies may be useful in the identification of feline B lymphocytes using immunohistochemistry and flow cytometry and thereby provide additional tools to study B lymphocyte ontogeny and the significance of lymphocyte phenotype in lymphoid neoplasia in cats.     

Effect of dietary fumonisin B1 on laying Japanese quail

1. A 28-d experiment was conducted to evaluate the effects of fumonisin B1 (FB1) on egg production and egg quality of young laying Japanese quail fed on fumonisin-contaminated rations. 2. To this end, 128 7-week-old birds were randomly distributed into 4 experimental groups (32 birds per group) and given rations containing 0 (control), 10, 50 and 250mg FB1/kg feed. Each treatment consisted of 4 replicates of 8 quail. Egg production and egg weight were checked daily. Feed consumption and feed conversion were determined weekly. Eggs laid on the last day of each 7-d period were collected and subjected to individual analysis for specific gravity, Haugh units and percentage eggshell. 3. Compared with controls, quail given > or = 50 mg FB1/kg had reduced feed intake and lower body weight gain. Feed conversion was reduced only in birds given 250 mg FB1/kg. 4. Mean egg production and egg weight were lower in birds given 250mg FB1/kg. Eggshell weight was reduced in birds given > or =50mg FB1/kg. However, mean specific gravity, Haugh units and percentage eggshell were not affected by FB1. 5. No histopathological changes were observed in liver, kidney or heart samples from any treatment group. 6. The results indicated that exposure to FB1 at concentrations > or = 50 mg/kg could adversely affect quail performance, emphasising the importance of controlling fumonisin contamination of quail rations.     

Leukoencephalomalacia in two horses induced by oral dosing of fumonisin B1

Leukoencephalomalacia (LEM) was induced by the oral administration of fumonisin B1 (FB1) to 2 horses: a filly received 59.5 mg/kg of a 50% preparation of FB1, administered in 21 doses of 1.25-4 mg/kg over 33 days; a colt, 44.3 mg/kg of 95% pure FB1 in 20 doses of 1-4 mg/kg in 29 days. Both animals developed nervous signs such as apathy, changes in temperament, inco-ordination, walking into objects, and one showed paralysis of the lips and tongue. Characteristic lesions of LEM were present in the brains. These trials proved conclusively that FB1 can induce LEM in horses.     

Intestinal barrier function is modulated by short-term exposure to fumonisin B1 in Ussing chambers

Veterinary Research Communications - Fumonisin B1 (FB1) alters intestinal epithelial cell cycle, absorptive, secretory and barrier properties of intestinal epithelial cells in vitro but data with...     

Cardiovascular changes associated with intravenous administration of fumonisin B1 in horses

OBJECTIVE: To determine whether cardiovascular dysfunction is evident in horses with leukoencephalomalacia experimentally induced by administration of fumonisin B1. ANIMALS: 11 healthy horses of various breeds (body weight, 252 to 367 kg). PROCEDURE: Horses were randomly assigned to 3 groups and administered fumonisin B1 daily. Horses received IV injections of 0 (control horses; n = 4), 0.01 (3), or 0.20 mg (4) of fumonisin B1/kg for 7 to 28 days. Horses were examined daily for evidence of neurologic disease. When neurologic signs consistent with leukoencephalomalacia were evident, horses were anesthetized, and catheters were inserted for evaluation of the cardiovascular system. After recovery from anesthesia, hemodynamic measurements were obtained. RESULTS: Fumonisin-treated horses with clinical signs of neurologic disease had evidence of cardiovascular dysfunction manifested as decreases in heart rate, cardiac output, right ventricular contractility (assessed by measuring the maximal rate of change of right ventricular pressure), coccygeal artery pulse pressure, and pH and base excess in venous blood as well as increases in systemic vascular resistance, compared with values for control horses. Fumonisin-treated horses with and without clinical signs of neurologic disease also had higher serum and right ventricular sphinganine and sphingosine concentrations than control horses. CONCLUSIONS AND CLINICAL RELEVANCE: An association was detected among fumonisin-induced neurologic disease, increased serum and myocardial sphinganine and sphingosine concentrations, and decreased cardiovascular function in horses. Fumonisin-induced decreases in cardiovascular function may contribute to the pathophysiologic development of leukoencephalomalacia in horses.     

Sequence of cardiovascular changes leading to pulmonary edema in swine fed culture material containing fumonisin

OBJECTIVES: To determine the sequence of cardiovascular and blood gas changes induced by ingestion of fumonisin-containing culture material in swine and to examine the temporal relationship of these changes to plasma sphinganine and sphingosine concentrations. ANIMALS: 12 healthy castrated pigs (38 to 50 kg). PROCEDURE: Pigs were instrumented to permit cardiovascular monitoring and collection of blood samples. Baseline values were obtained, and pigs were randomly assigned to 1 of 2 groups. Control pigs (n = 6) were fed a standard grower diet, whereas culture material that contained 20 mg of fumonisin B1/kg of body weight was added to the feed of treated pigs (n = 6) each day. Hemodynamic data, results of arterial and mixed venous blood gas analyses, and plasma sphinganine and sphingosine concentrations were recorded every 12 hours until treated pigs were euthanatized because of impending death from pulmonary edema. RESULTS: Sphinganine and sphingosine concentrations were increased in plasma of treated pigs within 24 hours of initial fumonisin exposure and continued to increase dramatically until euthanasia. Fumonisin-treated pigs had increased respiratory rate, mean pulmonary artery pressure, and pulmonary artery wedge pressure, along with decreased heart rate and cardiac output in the 12-hour period before euthanasia. Fumonisin-treated pigs also had systemic arterial hypotension, arterial and mixed venous hypoxemia, metabolic acidosis, decreased oxygen delivery, and increased oxygen consumption immediately before euthanasia. CONCLUSIONS AND CLINICAL RELEVANCE: Fumonisin-induced pulmonary edema in swine is probably caused by acute left-sided heart failure. Onset of hemodynamic changes was associated with plasma sphinganine concentration > or = 2.2 microM/L and plasma sphingosine concentration > or = 1 microM/L.     

Characterization of an epizootic of pulmonary edema in swine associated with fumonisin in corn screenings.

In 1989, corn screenings were associated with acute interstitial pulmonary edema, hydrothorax, and death in swine. Attack rate was 5-50%, case fatality rate was 50-90%, and clinical course was 1-2 days. Screenings from farms with pigs affected with pulmonary edema contained 20-330 micrograms fumonisin B1 per gram. Screenings containing 92 micrograms fumonisin B1 per gram fed to weanling pigs caused pulmonary edema and death. Sterilized corn inoculated with Fusarium moniliforme and diluted 1:1 with clean corn contained fumonisin B1 (17 micrograms/g) and caused acute pulmonary edema when fed for 5 days. Survivors developed subacute hepatotoxicosis with individual hepatocellular necrosis, hepatomegalocytosis, and increased numbers of mitotic figures. Similar liver lesions occurred in pigs given fumonisin B1 intravenously at 0.8 mg/kg body weight for 14 days.     

高效液相色谱法测定猪组织中维吉尼亚霉素M1的残留

建立了猪组织中维吉尼亚霉素M1残留的高效液相色谱测定方法.样品用甲醇-0.2mol/L NH4H2PO4溶液提取,正己烷洗涤,经C18固相萃取柱净化后做HPLC分析.选用二极管矩阵检测器,波长245 nm;色谱柱为ODS-3 C18柱(4.6 mm×250mm,5 μm);流动相为乙腈-水(37.5:62.5,V/V).维吉尼亚霉素M1标准曲线线性范围在0.1～2.0 μg/mL.对不同组织的3种加药浓度进行回收率测定,回收率在72%～85%,变异系数在10%以内.最低检测限为10 ng/g.     

A rapid, sensitive thin layer chromatography procedure for the detection of fumonisin B1 and B2.

A thin layer chromatography (TLC) method was developed for the detection of fumonisin B1 and B2 in corn and corn-based feedstuffs. Finely ground samples were extracted with acetonitrile:water (1:1), filtered, and applied to C18 cleanup columns. The columns were washed with 1% aqueous KCl followed by acetonitrile: 1% aqueous KCl (1:9), and the fumonisins were eluted with acetonitrile:water (7:3). The eluants were concentrated and spotted on reverse-phase C18 TLC plates along with fumonisin B1 and B2 standards, and the plates were developed in methanol: 4% aqueous KCl (3:2). The fumonisins were visualized by spraying the TLC plates successively with 0.1 M sodium borate buffer, fluorescamine, and 0.01 M boric acid. The plates were then dried and examined under longwave ultraviolet light. Fumonisin B1 and B2 appeared as bright yellowish-green fluorescent bands at Rfs of 0.5 and 0.1, respectively. The detection limit for the fumonisins on the TLC plate was 0.1 ppm in corn. Recoveries from spiked samples averaged greater than 80%. The identification of the fumonisins was confirmed by hydrolyzing the parent compounds of B1 and B2 to their respective C22 amino-alcohols and reexamining by TLC with the same visualizing reagents. This procedure was used to survey 193 corn samples collected from University of Missouri test plots in 1990 for fumonisin B1. Fumonisin B1 was detected in 15% of the corn samples.     

Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues.

A PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections.     

Leukoencephalomalacia in a horse induced by fumonisin B1 isolated from Fusarium moniliforme

Each of two horses was dosed by stomach tube with culture material on maize of Fusarium moniliforme MRC 826. One horse developed severe hepatosis and mild oedema of the brain after 6 doses of 2.5 g of culture material/kg body mass/day in 7 days. The second horse, in a similar experiment but at a dosage rate of 1.25 g/kg/day, developed mild hepatosis and moderate oedema of the brain. In both animals the brain oedema was particularly noticeable in the medulla oblongata. The mycotoxin fumonisin B1 was extracted and purified from the culture material of F. moniliforme MRC, 826 which contained approximately 1 g/kg of this compound. A horse was injected intravenously 7 times from Day 0-Day 9 with 0.125 mg of fumonisin B1/kg body mass/day. Clinical signs of neurotoxicosis, which appeared on Day 8, included nervousness followed by apathy, a wide-based stance, trembling, ataxia, reluctance to move, paresis of the lower lip and tongue, and an inability to eat or drink. Euthanasia was performed on the horse on Day 10 while the animal was in a tetanic convulsion. The principal lesions were severe oedema of the brain and early, bilaterally symmetrical, focal necrosis in the medulla oblongata. This report provides experimental evidence that fumonisin B1, produced by F. moniliforme, causes equine leukoencephalomalacia.