William F. Hall

Characterization of fumonisin toxicity in orally and intravenously dosed swine

Fumonisin B1 (FB1), a recently identified mycotoxin produced by Fusarium moniliforme in corn, has been shown to cause death in swine due to pulmonary edema, an apparently species specific effect, and to interfere with sphingolipid metabolism in vitro. Here we characterize the toxicity of fumonisins, using female cross-bred swine weighing 6 to 13 kg, and present a hypothesis regarding the mechanism of fumonisin-induced pulmonary edema in swine. FB1 was given daily intravenously (IV) to pig 1 for 9 days for a total of 72 mg (7.9 mg/kg) and to pig 2 for 4 days for a total of 67 mg (4.6 mg/kg). Pig 3 (control) was given saline IV for 9 days. Corn screenings naturally contaminated with FB1 (166 ppm) and FB2 (48 ppm) were fed to pigs 4, 5, and 6, and ground corn was fed to pigs 7 and 8 (controls). Pigs 4 and 7 were killed on day 5; pig 5 was found dead on day 6; and pigs 6 and 8 were killed on day 15. Pigs 4 and 5 had ingested 187 and 176 mg total fumonisins, respectively, while pig 6 had ingested 645 mg. Feed consumption had decreased in pigs fed corn screenings, with an additional sharp decrease prior to onset of clinical signs. Increases in serum liver enzymes, total bilirubin, and cholesterol were present, but electrocardiograms, heart rate, and body temperature were unaffected. Pigs dosed IV with FB1, developed mild intermittent respiratory abnormalities, while those fed screenings developed respiratory distress within 5 days. Mild interstitial pulmonary edema was observed in pig 1. Severe interstitial pulmonary edema, pleural effusion, and increased lung wet/dry weight ratio were observed in pigs 4 and 5. All pigs given fumonisin (either IV or orally) had hepatic changes characterized by hepatocyte disorganization and necrosis; pancreatic acinar cell degeneration was also observed. Ultrastructural changes in orally dosed swine included loss of sinusoidal hepatocyte microvilli; membranous material in hepatic sinusoids; and multilamellar bodies in hepatocytes, Kupffer cells, pancreatic acinar cells and pulmonary macrophages. Pulmonary intravascular macrophages (PIMs) contained large amounts of membranous material. Thus, the target organs of fumonisin in the pig are the lung, liver, and pancreas. At lower doses, slowly progressive hepatic disease is the most prominent feature, while at higher doses, acute pulmonary edema is superimposed on hepatic injury and may cause death. We hypothesize that altered sphingolipid metabolism causes hepatocellular damage resulting in release of membranous material into the circulation. This material is phagocytosed by the PIMs thus triggering the release of mediators which ultimately results in pulmonary edema.

Effect of orally administered epidermal growth factor on intestinal recovery of neonatal pigs infected with rotavirus

The effect of oral epidermal growth factor (EGF) on histological and biochemical changes in epithelium in the small intestine was studied in colostrum-deprived neonatal pigs. Forty-eight pigs were infected at 4 days of age with 2 ± 107 plaque-forming units of porcine group A rotavirus and orally fed a simulated sow-milk diet supplemented with 0.0, 0.5, or 1.0 mg/L recombinant human EGF. Sixteen noninfected pigs were fed a diet without EGF supplementation. Infected pigs developed severe diarrhea; they also consumed 25% less food and gained 60% less weight than noninfected pigs. Pigs were killed 8 days postinfection to collect samples at seven equidistant points in the small intestine. Rotavirus infection decreased villus height by 37% and reduced specific activity of lactase by 54%, of leucine aminopeptidase by 43%, and of alkaline phosphatase by 54% in the small intestine, compared with noninfected pigs. Only the supraphysiological dose of EGF (1.0 mg/L) consistently increased villus height in the proximal and mid-small intestine and lactase-specific activity in the mid-small intestine of rotavirus-infected pigs. However, this dose was only partially effective in restoring intestinal mucosal dimensions and enzyme activities. Supplemental EGF did not hasten the resolution of diarrhea. These data indicate that high physiological levels of EGF are beneficial in stimulating recovery of epithelium in the small intestine following rotavirus infection.

Temporal and dose-response features in swine fed corn screenings contaminated with fumonisin mycotoxins

Fumonisin B1 (FB1), a mycotoxin produced byFusarium moniliforme andF. 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.

Relationship between fumonisin contamination of feed and mystery swine disease

Fumonisin is a recently identified mycotoxin that has been shown to be the cause of pulmonary edema disease in swine and leukoencephalomalacia in horses. Mystery Swine Disease (MSD), is an economically devastating disease complex of unknown etiology that has been reported to have occurred in several swine producing states since 1988. To determine the relationship between MSD and fumonisin, a case-control study was carried out in Illinois in mid-1990. Feed samples collected from 12 case and 9 control farms were analyzed for fumonisin. Sera from swine on all farms was screened for titers against encephalomyocarditis (EMC) virus and concentrations of alpha-1 acid glycoprotein (an acute phase reactive protein). Fumonisin concentrations ⩾20 ppm were found on 1 control farm (1/9) and 8 case farms (8/12). Titers against EMC virus (⩾ 1∶16) were found on 5 control farms (5/9) and on 6 case farms (6/12). Farms with ⩾20 ppm fumonisin in the feed were at significantly increased risk (OR= 11.2, Fishers exact test p=0.037) for MSD. Furthermore, the π2 test for trend was significant (p= 0.017), meaning that as the level of fumonisin in the feed increased, the risk of MSD also increased. The presence of EMC virus titers in the sow herd was not a significant risk for MSD (OR =1.25, Fishers exact test p=0.75). Alpha-1 acid glycoprotein concentrations obtained from 2-week old nursing pigs differed significantly (p=0.0005) between MSD case and control herds.

Sensitivity of the Standardized Pseudorabies Virus Neutralization Test Varies with the Test Strain Used

The effect of altering the strain of the test virus used in the standardized pseudorabies virus neutralization (VN) test on the sensitivity of the assay was evaluated. Comparative VN tests were performed using 4 different strains: the avirulent Bartha parental, the avirulent recombinant Bartha gIIIKa, the moderately virulent Shope (currently used for the VN test at the National Veterinary Services Laboratory, Ames, IA), and the highly virulent P2208 (Funkhauser). A radioimmunoassay and a Western immunoblotting technique were employed to verify the presence of anti-pseudorabies virus (PrV) antibodies in sera. Statistical analysis indicated that replacement of the Shope strain by the Bartha gIIIKa or the P2208 strain resulted in VN titers that were 4.23- and 2.00-fold higher, respectively. Despite these differences, specificity with regard to PrV diagnosis was unaltered. This apparent enhancement of the sensitivity of the PrV VN test would be beneficial for the serologic identification of PrV-infected animals during an eradication effort.

Evaluation of the sensitivity and specificity of two diagnostic tests for antibodies to pseudorabies virus glycoprotein X.

The diagnostic performance of 2 enzyme-linked immunosorbent assays (gX-T, gX-H) for antibodies to pseudorabies virus (PRV) glycoprotein X (gX) were evaluated using 311 serum samples from a nonvaccinated quarantined herd. When the standardized virus neutralization (VN) test, which uses the Shope strain (VN Shope), was used as the comparative diagnostic standard, the gX-T test had a 7% false-negative rate and a 52% false-positive rate, and the gX-H test had a 19% false-negative rate and a 19% false-positive rate. When the VN test with a Bartha recombinant strain (VN Bartha gIIIKa) was used as the diagnostic standard, the gX-T test had a 9% false-negative rate and a 26% false-positive rate, and the gX-H test had a 24% false-negative rate and a 11% false-positive rate. Thus, the gX-T test was more sensitive and the gX-H test was more specific. Additional diagnostic tests on 79 serum samples from a noninfected herd did not produce false positives for the gX-H test, but there was an 8% false-positive rate for the gX-T test. Previous studies from our laboratory have demonstrated that VN Bartha gIIIKa has higher sensitivity than VN Shope, without losing specificity, and thus is a better comparative diagnostic standard. When adding a suspect range to the gX-T test, using the same criteria as the suspect range for the gX-H test, the false-positive rate of the gX-T test was reduced to 5% when evaluated versus VN Bartha gIIIKa in the infected herd and to 1% for the PRV-negative herd. However, 18% of the positive samples were classified as suspect (vs. 8% for the gX-H test). In PRV eradication programs, the cost of false negatives is greater than the cost of false positives; thus, the gX-T diagnostic used in this study is of greater diagnostic value.

Cross-reactivity of an automated human haptoglobin immunoturbidimetric assay for detection of haptoglobin in swine serum.

The acute phase response has been previously characterized in the pig.2,9 Haptoglobin (Hpt), an a-2 glycoprotein, is a normal serum protein that increases 5–7 fold within 24– 48 hours upon the advent of an acute inflammatory response.9 Serum Hpt increases as a result of natural infections and experimentally induced inflammation in pigs as in other species.1,2,6,9,11,12 Besides serum Hpt as a marker for inflammatory disease, it has been used as an indicator of weight gain4 and stress from environmental conditions.5 The serum Hpt concentration may therefore be useful as a monitor of overall health of pigs in a commercial production unit. Automated assays for measuring serum Hpt have been validated for use in dogs, horses, and sheep.7,11,13 A commercially available immunoturbidimetric assay for human Hpt has recently been validated for use in dogs and horses.13 The purpose of this study is to report on the cross-reactivity of this same assaya for use in determining serum Hpt concentrations in swine. Serum samples were obtained from pigs housed at the Veterinary Medicine Research Farm at the University of Illinois and from samples submitted to the Purina Mills Research Center for unrelated testing. Samples from the University of Illinois were collected by venipuncture into serum clot tubes, and after centrifugation, the serum was withdrawn and stored at 270 C until assayed. Those samples submitted to Purina Mills Research Center were treated in a similar fashion. Serum used for immunoelectrophoresis, western blot, and correlation studies were from the University of Illinois, and serum for all other experiments was from the Purina Mills Research Center. A cyanmethemoglobin (CHB) assay for serum Hpt was performed as previously described.3,4 Hpt was reported in milligrams of CHB binding capacity (HBC) per deciliter of serum. Samples with relatively low, medium, and high HBC were pooled and labeled respectively. The pooled samples were used for further experiments. Immunoelectrophoresis was performed as previously described13 using 1-ml aliquots of pooled swine serum containing various Hpt concentrations as determined by the CHB assay. Serum from a healthy human donor served as a control. Following standard electrophoresis on a 1.0% agarose gel film,b anti-human Hpt antiserum (anti-Hpt antibody) was added to diffusion wells on either side of each electrophoretic lane. The antiserum was allowed to diffuse for 24 hours at room temperature, forming immunoprecipitates. The film

Sulfamethazine residues in swine: comparison of on-farm monitoring methods

Abstract Three sulfamethazine-residue detection methods were used to evaluate samples collected from five swine farms over a 12-month period. All cooperating farms included sulfamethazine in swine diets at various stages of production, for growth promotion or disease control, and followed recommended drug withdrawal periods. Swine finishing ration, swine urine, and swine serum from market-weight animals were tested monthly for the presence of sulfamethazine. Thin-layer chromatograph (TLC) analysis of swine urine was the gold standard by which three other test method-sample combinations were compared. Samples were analyzed for sulfamethazine using TLC (feed), competitive enzyme immunoassay (serum), and agar-diffusion swab test (urine). The relative sensitivities and specificities of sulfamethazine-residue detection for the three combinations were: (1) TLC analysis (27%, 94%); (2) competitive enzyme immunoassay analysis (58%, 59%); (3) agar-diffusion swab test (78%, 12%). None of the three methods tested was individually adequate for on-farm monitoring of sulfonamide residues. Sulfamethazine residues in swine urine were found on 43.3% of the monthly farm visits and in 19.7% of all swine tested.