An international collaborative study on a method for determination of formaldehyde in veterinary vaccines.

An international collaborative study of a quantitative colorimetric method for determination of formaldehyde in veterinary vaccines was conducted on a series of replicate, blinded veterinary vaccine products by 15 laboratories in three regions: North America, Europe and Japan. Participants conducted determinations using a modification of a method from the European Pharmacopoeia, a colorimetric method based on the reaction of formaldehyde with methylbenzothiazolone hydrazone hydrochloride. For this study, three licensed vaccine products containing formaldehyde were revialed, randomly numbered, tested for uniformity and distributed by one of the participating laboratories through regional coordinators to collaborators. One of the revialed products was spiked with a known amount of formaldehyde and included in the test series. Results along with all raw data were returned to the distributing laboratory for consolidation and statistical treatment. For the modified method spike recovery was 101% and reproducibility (inter-laboratory variation expressed as relative standard deviation) ranged from 18.0 to 8.0% for respective formaldehyde concentrations of 0.28 to 1.07 g/l. Based on the study, the method was proposed by the Biologicals Working Group of the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) as a candidate for the VICH Guideline standard method for residual formaldehyde.

Fumonisin B1 promotes aflatoxin B1 and N-methyl-N'-nitro-nitrosoguanidine-initiated liver tumors in rainbow trout.

Laboratory studies have described the carcinogenicity of fumonisin B1 (FB1) in rodents and epidemiological evidence suggests an association between FB1 (a mycotoxin produced by Fusarium moniliforme) and cancer in humans. This study was designed to reveal in rainbow trout, a species with very low spontaneous tumor incidence, if FB1 was (i) a complete carcinogen, in the absence of an initiator; (ii) a promoter of liver tumors in fish initiated as fry with aflatoxin B1 (AFB1); and (iii) a promoter of liver, kidney, stomach, or swim bladder tumors in fish initiated as fry with N-methyl-N'-nitro-nitrosoguanidine (MNNG). FB1 was not a complete carcinogen in trout. No tumors were observed in any tissue of fish fed diets containing 0, 3.2, 23, or 104 ppm FB1 for a total of 34 weeks (4 weeks FB1 exposure, 2 weeks outgrowth on control diet, followed by 30 weeks FB1 diet) in the absence of a known initiator. FB1 promoted AFB1 initiated liver tumors in fish fed > or = 23 ppm FB1 for 42 weeks. A 1-week pretreatment of FB1 did not alter the amount of liver [3H]AFB1 DNA adducts, which suggests that short-term exposure to FB1 will not alter phase I or phase II metabolism of AFB1. In MNNG-initiated fish, liver tumors were promoted in the 104 ppm FB1 treatment (42 weeks), but FB1 did not promote tumors in any other tissue. Tumor incidence decreased in kidney and stomach in the 104 ppm FB1 treatment of MNNG-initiated trout. The FB1 promotional activity in AFB1-initiated fish was correlated with disruption of sphingolipid metabolism, suggesting that alterations in associated sphingolipid signaling pathways are potentially responsible for the promotional activity of FB1 in AFB1-initiated fish.

Serum copper concentrations in beef cows and heifers.

OBJECTIVE: To characterize serum copper status of cows and heifers in beef cow-calf herds throughout the United States and to evaluate use of copper supplements in those herds. DESIGN: Cross-sectional survey. ANIMALS: 2,007 cows and heifers from 256 herds in 18 states. PROCEDURES: Producers participating in a health and management survey conducted as part of the National Animal Health Monitoring System voluntarily allowed serum samples to be obtained from cows and heifers for determination of copper concentration. Results were categorized as deficient, marginally deficient, or adequate. The proportion of cattle and herds (on the basis of mean value of the tested cattle) in each category was determined. Copper concentrations were compared between herds that reportedly used copper supplements and those that did not. RESULTS: Overall, 34 of 2,007 (1.7%) cows and heifers were deficient in copper, and 781 (38.9%) were marginally deficient. In each region, at least a third of the cattle were deficient or marginally deficient. For herds, 92 of 256 (35.9%) were marginally deficient, and 22 (0.8%) were deficient. Approximately half of the producers reported use of copper supplements, but a sizeable proportion of those producers' cattle and herds were classified as marginally deficient or deficient. CONCLUSIONS AND CLINICAL RELEVANCE: Copper deficiency is not restricted to a single geographic region of the United States. Copper deficiency can persist despite reported use of supplements by producers. Veterinarians dealing with beef cow-calf herds that have problems consistent with copper deficiency should not rule out copper deficiency solely on the basis of geographic region or reported use of copper supplements for the herd.

Fusarium mycotoxins in corn and corn products in a high-risk area for gastric cancer in Shandong Province, China.

Consumption of fermented, but not unfermented, corn pancakes has been linked with elevated stomach cancer mortality rates in rural Linqu County in Shandong Province, China. Previous surveys of fungal contamination of corn in China have detected fumonisins, which are mycotoxins produced by Fusarium moniliforme. To determine whether mycotoxins might account for the increased risk of cancer among those consuming fermented pancakes, we obtained specimens of corn, cornmeal, unfermented and fermented pancake batter, and cooked fermented pancakes from each of 16 households in Linqu County for analysis by the U.S. Department of Agriculture. Fumonisins B1, B2, and B3 were detected (> or = 0.5 microgram/g) in 19, 25, and 6% of the corn specimens, respectively, as well as in various corn products. No type A trichothecenes were detected; however, the type B trichothecenes deoxynivalenol and 15-acetyldeoxynivalenol were detected (> or = 0.5 microgram/g) in 58 and 17% of the corn specimens, respectively, and zearalenone was detected (> or = 0.5 microgram/g) in 15% of the cornmeal specimens. The mycotoxins were detected only at low levels (< 10 micrograms/g), which did not increase with fermentation. These findings do not support the hypothesis that mycotoxin contamination increases the risk of gastric cancer among those who consume fermented Chinese pancakes.

Screening nitrate in forages with a test strip: collaborative study.

A collaborative study was conducted for screening nitrate in forages with a commercially available test strip. The method involves extracting a finely ground sample with deionized water. The test strip is dipped in the sample extract. The color of the reaction zone on the test strip changes from white to pink or purple depending on the nitrate concentration in sample extract. The nitrate present in the extract is determined by comparing the color of the test strip to the color scale on the test strip container. Six blind quintuplicates of forage samples were analyzed by 20 collaborators. Nitrate concentrations in forage samples tested ranged from < 1000 ppm nitrate to > 10,000 ppm nitrate on dry matter basis. Each collaborator was asked to assign each sample to one of the 4 following nitrate concentration ranges: (1) < 1000 ppm, (2) 1000 to 5000 ppm, (3) > 5000 ppm to 10,000 ppm, and (4) > 10,000 ppm. Nineteen of 20 collaborators reported results. Results from 2 laboratories were rejected as outliers by inspection and chi 2 test. Sensitivity rates (p+) ranged from 0.965 to 0.998, with standard errors of 0.006 to 0.16. Specificity rates (p-) ranged from 0.991 to 0.997 for the 4 ranges, with standard errors of 0.003 to 0.006. False-positive rates (pf+) ranged from 0.006 to 0.046, with standard errors of 0.006 to 0.025. False-negative rates (pf-) ranged from 0.003 to 0.007, with standard errors of 0.003 to 0.006. Screening nitrate in forages with a test strip has been adopted first action by AOAC INTERNATIONAL.

Thin-layer chromatographic determination of monensin in feeds: screening method.

Monensin is extracted from feed with methanol and purified by solvent-partitioning solid-phase extraction. After solvent reduction, monensin is separated by thin-layer chromatography on silica gel and visualized by color development with vanillin. No false-positive results were obtained in validation studies by submitting or peer laboratories when blank samples were analyzed. Three of 20 samples spiked with 5 ppm monensin were reported as containing no monensin. All samples spiked with 10 ppm monensin were reported positive for monensin.

Disruption of sphingolipid metabolism and induction of equine leukoencephalomalacia by Fusarium proliferatum culture material containing fumonisin B(2) or B(3).

Fumonisin B(1), B(2), and B(3) are inhibitors of ceramide synthase, a key enzyme in the pathway for de novo sphingolipid biosynthesis. Corn, naturally contaminated with either predominantly fumonisin B(1) or pure fumonisin B(1), has been shown to cause equine leukoencephalomalacia (ELEM). It has been hypothesized that fumonisin-induced disruption of sphingolipid metabolism is an early event in the development of ELEM. Recently, it was shown that Fusarium proliferatum corn culture diets containing predominantly fumonisin B(2), but not diets which were predominantly fumonisin B(3), at 75 ppm (0.75 mg/kg BW/day) caused hepatotoxicity and ELEM. Analysis of free sphingoid bases and complex sphingolipids in serum, liver, and kidney, revealed that both the fumonisin B(2) and B(3) diets caused significant disruption of sphingolipid metabolism, however, the fumonisin B(2) culture material diet was significantly more effective than the fumonisin B(3) culture material diet at disrupting sphingolipid metabolism and in causing hepatotoxicity and clinical signs indicative of the onset of ELEM. A significant increase in the ratio of free sphinganine to free sphingosine in serum was first evident at day 4 and 11 with the fumonisin B(2) and B(3) diets, respectively. Increase in serum enzymes indicative of liver toxicity was first evident at day 34 in ponies fed the fumonisin B(2) diet and clinical signs (head shaking, gait problems, and muscle tremors) were first observed at day 48. Ponies fed the fumonisin B(3) diets showed no increase in serum enzymes or clinical signs for as long as 65 days when the study with fumonisin B(3) was stopped. The results support the conclusion fumonisin B(2) is more effective than fumonisin B(3) in disrupting sphingolipid metabolism and induction of ELEM and liver injury in ponies.

Blood selenium concentrations in cows and heifers on 253 cow-calf operations in 18 states.

This study was conducted to determine the geographic distribution of selenium deficiency among beef cows and heifers in selected states. Whole blood selenium concentrations were determined for cows and heifers on 253 cow-calf operations in 18 states. Overall, 7.8% of the samples were severely deficient, and another 10.4% of the samples were considered marginally deficient for selenium. Blood selenium concentrations varied by geographic region. Cattle from the southeastern states were more commonly considered severely or marginally deficient (18.6% and 23.8%, respectively) than cattle from other regions. Herds from the southeastern states were also more commonly considered severely or marginally deficient (14.9% and 20.9%, respectively) than herds from other regions. Blood selenium concentration also varied by whether selenium had been supplemented to the herd. We conclude that blood selenium levels for cattle vary by geographic region and selenium supplementation of the herd.

Natural occurrence of gliotoxin in turkeys infected with Aspergillus fumigatus, Fresenius.

Thirteen samples of infected turkey lung tissue from cases of 'airsacculitis' were collected either at the processing plant or from a local turkey farm and subjected to cultural and gliotoxin analysis. Aspergillus fumigatus was isolated from 6 of the 13 samples; all isolates were determined to be gliotoxin producers when grown in laboratory culture and assayed by HPLC procedures. Gliotoxin was isolated from 5 of the 13 tissue but was not isolated from all tissues that were infected with A. fumigatus. Gliotoxin was isolated from which no A. fumigatus was isolated and it was not detected in three tissues from which gliotoxin-producing isolates of A. fumigatus were obtained. The ability of this pathogenic fungs to produce this immunomodulating compound in naturally infected turkeys provides further evidence that gliotoxin may be involved in the pathogenesis of the disease, aspergillosis of turkeys.

Absence of detectable fumonisins in the milk of cows fed Fusarium proliferatum (Matsushima) Nirenberg culture material.

Fumonisins, a group of mycotoxins produced by the ubiquitous fungi Fusarium moniliforme and F. proliferatum, were first identified about eight years ago. They have been shown to cause a variety of health effects in animals, including epidemiological evidence of esophageal cancer in humans. Cattle are less sensitive to ill effects than horses and swine. Fumonisins are common contaminants of low quality grain fed to cattle. Culture material containing fumonisins (FB1, FB2, and FB3) was mixed into the total diet and fed for 14 days to two midlactation Jersey cows to determine if fumonisins are excreted in milk. The dietary equivalent of fumonisin was approximately 75 ppm and the two cows consumed an average of 3 mg fumonisin B1/kg body weight (bwt)/day. Fumonisins were not detected in any of the milk samples by two analytical laboratories using methods with a sensitivity of 5 ng/ml. Except for transient diarrhea at the beginning of the contaminant feeding period and an increase in serum cholesterol, clinical and hematologic changes were not observed in the animals. The appearance or carry over of fumonisins from feed to milk in dairy cows does not appear to be significant and likely not a hazard or food safety concern for humans.

Quality assurance initiative at the U.S. Department of Agriculture National Veterinary Services Laboratories.

The National Veterinary Services Laboratories (NVSL) provides laboratory support and technical assistance for animal health programs of the United States Department of Agriculture. During this past year, the NVSL completed strategic planning activities and identified improved quality assurance (QA) as one of the key elements. The specific QA goal was the establishment of an internationally recognized QA program at the NVSL by the year 2000. This article provides a brief summary of the mission of the NVSL and the short- and long-term goals of the QA initiative.

Effects of feeding fumonisin B1 in lactating sows and their suckling pigs.

The mycotoxin fumonisin B1 (FB1) produced by Fusarium moniliforme in corn causes pulmonary edema in finishing swine. Effects of lower nonlethal amounts and effects in lactating sows with suckling pigs are unknown. An initial study was conducted to determine a nonlethal concentration of FB1 for lactating sows; whether ingested FB1 could be detected in the milk; and whether toxicosis could be detected in the pigs, as determined by necropsy. Another study was conducted to determine toxicosis in the pigs by measuring liver sphinganine-to-sphingosine ratio, and whether ingested FB1 affected T-lymphocyte function in sows and their pigs. Furthermore, sows of this study were maintained in controlled hot (27 to 32 C, 50 to 70% relative humidity) and thermoneutral (21 C, 55% relative humidity) environments to determine whether high temperature exacerbated the effects of FB1. In the first study, 100 micrograms of FB1/g of corn soybean meal diet was found to be nonlethal when fed for 14 days. Fumonisin B1 was not detected in the milk at 30 ppb and lesions were not found in the necropsied pigs, including 1 from a sow that died of porcine pulmonary edema syndrome after ingesting FB1 at a concentration of 175 ppm. In the second study, differences in liver sphinganine-to-sphingosine ratio of pigs were not found. Expressions of cell surface antigens on blood lymphocytes and lymphocyte proliferation response to various mitogens were not affected by FB1 or high temperature in sows or their pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a liquid chromatographic method for the determination of fumonisins in corn, poultry feed, and Fusarium culture material.

The performance of a liquid chromatographic method for determining fumonisins in corn, animal feeds, and culture material was evaluated. Efficiencies of extractions with the following solvent systems were determined: acetonitrile-water (50 + 50, v/v), methanol-water (75 + 25, v/v), and 100% water. The acetonitrile solvent gave both higher extraction efficiencies and faster extraction times than the other 2 solvents. Extraction was followed by C18 solid-phase extraction column cleanup. Fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) were measured by precolumn derivatization with o-phthalaldehyde followed by isocratic separation on a C18 reversed-phase column with a mobile phase of 50 mM potassium dihydrogen phosphate (pH 3.3)-acetonitrile (60 + 40). Commercially prepared poultry feed, corn, and Fusarium spp. corn cultures were analyzed at the following levels: FB1, 1.5 to 15,000 micrograms/g; FB2, 0.5 to 4000 micrograms/g; FB3, and 0.17 to 1,500 micrograms/g. Recoveries were 91-94%, 90-100%, and 81-93% for FB1, FB2, and FB3, respectively. Precision (coefficient of variation) was determined with pooled field samples and ranged from 2% at 19 micrograms/g for FB1 to 9% at 0.17 microgram/g for FB3. Time and pH studies of the formation of the fluorescent derivative and its stability were conducted. Complete reaction occurred at pHs above 7.9, with optimal pH for chromatography between 8.0 and 8.5. No statistically significant response differences were detected for reaction times ranging from 4 to 40 min; however, the detector signal was significantly reduced when reaction times were shorter than 4 min. Chromatograms of samples were free of interferences for all feeds, corn, and culture material tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of total nitrogen in urine by pyrochemiluminescence: collaborative study.

Twelve collaborating laboratories analyzed 5 blind duplicate samples of human urine for total nitrogen using a pyrochemiluminescence method. The nitrogen content ranged from low (650 mg/L) to high levels (8800 mg/L) in urine samples of people under moderate to severe stress. In addition to test samples, collaborators also received a certified standard (sodium nitrite in water) as an external control. The pyrochemiluminescence assay was performed on urine samples diluted in water within a range of 1:50 to 1:100. The method detects total nitrogen by reaction of the product of high temperature oxidative pyrolysis and ozone. Repeatability standard deviation values (RDSr) ranged from 1.49 to 3.91% and reproducibility standard deviation values (RSDR) ranged from 3.66 to 9.57%. The average recovery of total nitrogen was 99.9%. The pyrochemiluminescence method for determination of total nitrogen in urine was adopted first action by AOAC INTERNATIONAL.

Analysis of naturally occurring mycotoxins in feedstuffs and food.

Aflatoxins, zearalenone, deoxynivalenol, fumonisins, and their respective metabolites require specific procedures for their determination because of their diverse chemistry and occurrence in complex matrices of feedstuffs and foods. Major sources of error in the analysis of these mycotoxins arise from inadequate sampling and inefficient extraction and cleanup procedures. The determinative step in the assay for each of these toxins is sensitive to levels below those that are considered detrimental to humans and animals. Aflatoxins can be determined in grains and animal fluids and tissues by TLC, HPLC, gas chromatography-mass spectrometry (GC-MS), and ELISA procedures. Zearalenone, an estrogenic mycotoxin, can readily be determined in cereal grains and foods by HPLC (50 ng/g) and by TLC (300 ng/g). No incurred levels of zearalenone or its metabolites have been detected in animal tissues destined for human consumption. Deoxynivalenol can be determined in wheat and corn at 300 ng/g by a rapid TLC procedure and at 325 ng/g by a GC method. Although not tested collaboratively, an HPLC procedure and an ELISA screening procedure are capable of detecting deoxynivalenol at low (nanograms/gram) levels in feedstuffs and foods. The recently characterized fumonisins can be detected by TLC, HPLC, and GC-MS at levels below those now considered harmful. Thin-layer chromatography and HPLC (with fluorescence detection of derivatives) procedures can detect fumonisins at approximately 100 ng/g; GC-MS is required for detection at lower levels.

An HPLC method for the detection of ergot in ground and pelleted feeds.

A high performance liquid chromatography (HPLC) method is described for detecting ergot in ground or pelleted forages and grains. Samples were extracted with alkaline chloroform, filtered, and applied to silica gel/organic binder cleanup columns. Following elution of pigments with acetone: chloroform, ergopeptine alkaloids were eluted with methanol and analyzed by HPLC with fluorescence detection. Average recovery of ergotamine, the major ergopeptine alkaloid produced by Claviceps, was 93%, with a relative standard deviation of 4.9%. The detection limit of ergotamine was approximately 50 ppb in all feedstuffs. Confirmation of ergopeptine alkaloids was accomplished by treating the parent ergopeptine alkaloids with 0.2% acetic acid to produce their -inine isomers and reexamining by HPLC with fluorescence detection or silica gel/organic binder column cleanup in combination with tandem mass spectroscopy. The method described is a valid alternative to microscopic inspection for detecting ergot contamination in ground or pelleted feedstuffs.

Effects of fumonisin-contaminated corn screenings on growth and health of feeder calves.

Fumonisins are myocotoxins produced by Fusarium moniliforme and F. proliferatum, common molds of corn in North America. The toxin is at especially high concentrations in corn screenings. Fumonisins are toxic to swine and horses, but effects of these toxins in cattle have not been evaluated. This experiment was conducted to determine the effects in cattle of feeding fumonisins at levels known to be toxic to swine and horses. A total of 18 crossbred feeder calves were fed diets containing fumonisins at 15, 31, or 148 micrograms/g for 31 d. Feed consumption, weight gain, complete blood count, serum clinical chemistries, and an immune function profile were done on d -3, 4, 10, 17 and 31 relative to the start of fumonisin feeding. There was no treatment-related effect on feed intake or weight gain, but feed containing 148 micrograms/g of fumonisins seemed to be less palatable than other feeds. Significant increases in serum aspartate amino transferase, gamma glutamyl transpeptidase, lactate dehydrogenase, bilirubin, and cholesterol occurred from d 10 through 31. Mild microscopic liver lesions were present in two calves fed at the highest fumonisin level. Lymphocyte blastogenesis was significantly impaired at the end of the feeding period in the group given the highest dose. Other measures of immune function were not affected significantly. Fumonisins are capable of causing changes in liver function and in some measures of immune function. However, cattle seem to be relatively less susceptible to fumonisins present naturally in grains than either swine or horses.

Experimental equine leukoencephalomalacia, toxic hepatosis, and encephalopathy caused by corn naturally contaminated with fumonisins.

A study to evaluate the effects of dietary fumonisin B1 was conducted using 6 ponies (4 test and 2 control). A ration naturally contaminated with fumonisin B1 was fed in 3 phases: 1) 44 ppm fumonisin B1, 2) less than 1 ppm fumonisin B1, and 3) 88 ppm fumonisin B1. All ponies were monitored daily, weighed weekly, and limit fed at a rate of 0.8% body weight plus hay. Feed intake was measured daily, and a serum chemistry panel was completed once or twice weekly. Four to 7 days after initiation of the trial (Phase 1), all 4 test ponies had decreased feed consumption, and selected serum chemistry parameters were markedly elevated. On day 9, 1 pony died acutely with mild encephalopathy and hepatic necrosis. Another pony, euthanized on day 45, also had mild encephalopathy and hepatic necrosis. The remaining 2 test ponies continued the 44 ppm fumonisin B1 diet for 98 days. Phase 2 consisted of a diet with < 1 ppm fumonisin B1 for 120 days. During this phase, the serum chemistry values of the 2 ponies returned to normal. Following Phase 2, the 2 ponies were fed a diet containing 88 ppm fumonisin B1. After 75 days, 1 animal died of equine leukoencephalomalacia with mild hepatic necrosis. On day 78, the remaining pony was euthanized after showing distress; it also had leukoencephalomalacia and hepatic lesions.

Increases in serum sphingosine and sphinganine and decreases in complex sphingolipids in ponies given feed containing fumonisins, mycotoxins produced by Fusarium moniliforme.

Consumption of food contaminated with Fusarium moniliforme causes leucoencephalomalacia and hepatotoxicity in horses, pulmonary edema in pigs and liver cancer in rats, and has been correlated with esophageal cancer in humans. The causative agents are thought to be a family of compounds called fumonisins, which have recently been shown to be potent inhibitors of sphingosine (sphinganine) N-acyltransferase. Because inhibition at this step blocks the formation of complex sphingolipids while leading to accumulation of sphinganine, we hypothesized that exposure of animals to fumonisin-contaminated feed might be detected by analyses of serum sphingolipids. Within days of giving ponies feed contaminated with 15 to 44 micrograms/g fumonisin B1, there was an increase in the amount of free sphinganine (and sometimes sphingosine) and a reduction in complex sphingolipids. Free sphinganine and sphingosine decreased when ponies consumed less of the contaminated feed, and increased again when they consumed more fumonisin. When toxicosis was evident as indicated by other serum markers, complex sphingolipids as well as free sphingosine and sphinganine were elevated, probably due to loss of sphingolipids from dying cells. These findings establish that consumption of fumonisin-contaminated feed disrupts sphingolipid metabolism. Because the changes in sphinganine and sphingosine were seen before liver enzymes were noticeably elevated, they may be an early marker of exposure to fumonisins.