Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.

The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.

Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme-linked immunosorbent assay.

The use of lipoarabinomannan (LAM; obtained from Mycobacterium paratuberculosis) in and ELISA (LAM-ELISA) to test 75 sheep sera from a paratuberculosis-infected flock resulted in an approximate threefold increase in sensitivity (from 23.5% to 70.6%), compared with the use of Annau's polysaccharide in a complement fixation test (P-CFT). Even after manipulation of the LAM-ELISA cut-off value to produce a specificity of 100% to match that of the P-CFT, the sensitivity still was approximately twofold greater than that of the P-CFT. Anti-bovine monoclonal antiglobulin-enzyme conjugates matched commercially available anti-ovine polyclonal antiglobulin-enzyme conjugates with respect to sensitivity and specificity. False-positive results were found to be less frequent after combining 2 serodiagnostic tests, LAM-ELISA and D antigenagar gel immunodiffusion, resulting in an increase in specificity from 88.1% to 95.2%. The repeatability of true seropositive and seronegative results was found to be 89.5% and 91.1%, respectively, for sera obtained less than or equal to 1 month prior to slaughter and 91.7% and 95.5%, respectively, for reanalysis of sera obtained at the time of slaughter.

Evaluation of the serological response of sheep in one flock to Mycobacterium paratuberculosis by crossed immunoelectrophoresis.

Sera from 74 sheep culled from one flock on the basis of performance and response to immunological tests for paratuberculosis or maedi visna were used to evaluate the serological response to a sonicated antigen of Mycobacterium paratuberculosis by crossed immunoelectrophoresis. A total of seven precipitating components was demonstrated. Four components (A,C,V,W) were detected in low frequency only with sera from animals with paratuberculosis while two components (X,Y) were detected in high frequency with sera from animals with or without paratuberculosis. One component (D) was observed in high frequency with sera from animals with paratuberculosis. The magnitude of the serological response to the D component as measured by crossed immunoelectrophoresis correlated well with bacterial load and generally agreed with the quantitative assessment by agar gel immunodiffusion. A development time for crossed immunoelectrophoresis of 24-72 hours after electrophoresis was required to achieve correlation with agar gel immunodiffusion.

Testing a short-term feeding trial to assess compositional and histopathological changes in hearts of rats fed vegetable oils.

Male, female and castrated rats, three wk of age, were fed a low-fat diet for 14 wk followed by high-fat diets (20% by weight) for one wk containing graded levels of erucic acid from 1 to 50%, to evaluate the effect of short-term feeding and interaction of male sex hormones on formation of heart lesions. Some rats within each group were returned to the low-fat diet for one wk after the test period. For comparison, one group of three-wk-old male rats was fed the high fat 50% erucic acid diet for 15 wk. Erucic acid depressed growth rate and food consumption and increased cardiac lipidosis and triglycerides proportional to the erucic acid content of the diet. There were no sex differences, and the effects disappeared once rats were returned to the low-fat diet for one week. There was a significance (P less than 0.05) in the incidence of myocardial necrosis among male rats fed increased levels of erucic acid for one week, but the response was not linear to the increase in dietary erucic acid. Furthermore, the response was much less than in males fed the 50% erucic acid diet continually for 15 weeks. These results suggest that the short-term model is not a suitable substitute for the long-term feeding trial to test the cardiopathogenicity of a vegetable oil. The significantly lower incidence in myocardial lesions in female and castrated male rats compared with male rats suggests involvement of sex hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathological changes associated with the feeding of soybean oil or oil extracted from different rapeseed cultivars to single comb white Leghorn cockerels.

A total of 384 Single Comb White Leghorn cockerels of the Hyline strain were fed either a basal (control) diet containing no added oil or a diet supplemented with 20% by weight of soybean oil, rapeseed oil from cultivars Tower, Candle, a mixture of Echo and Arlo high erucic acid rapeseed (HEAR), or R-500. Levels of erucic acid (22:1) in the rapeseed diets varied from .03 to 10.31%. Three birds from each unit were killed at 28, 56, 84, and 112 days and a wide range of tissues were examined histologically. A number of birds in all dietary groups had healed lesions of avian encephalomalacia. Two cockerels fed HEAR oil and 12 fed R-500 developed marked ascites, firm shrunken livers, hydropericardium, and cachectic muscular atrophy. Marked periacinar necrosis was present in birds dying with ascites. A significantly higher number of birds fed the rapeseed oils developed hepatic sinusoidal distention than birds fed the basal or soybean oil diets. Degenerative myocardial changes were seen only in R-500 and HEAR oil-fed birds. This, coupled with hepatic changes, producing shrunken firm livers, led to development of ascites, cachectic muscular atrophy, and periacinar hepatic necrosis. These changes were probably produced by the high erucic acid content of the HEAR and R-500 oils and the resultant grossly imbalanced diet.

Some aspects of the composition of avian ascitic fluid.

Ascitic fluid from male Single Comb White Leghorn chickens fed a diet containing 20% by weight rapeseed oil rich (51.6 weight percent) in erucic (22:1n9) acid contained 2.84 +/- .09 g/dl protein and 2.55 +/- .22 micrograms/ml DNA. A characteristic feature of ascitic fluid as compared to liver and plasma was its high polyunsaturates, low saturates, and low saturate to unsaturate ratio. In general, the fatty acid profile of ascitic fluid is more comparable to the fatty acid profile of plasma lipids than to that of liver lipids. These data suggest that there is no direct absorption of fat and transfer to the ascitic fluid.

The methionine and choline status of rat diets and their effects on nutrition and myocardial lesions.

This study evaluated for rats the nutritional adequacy of casein-based diets routinely used to test the cardiopathogenicity of vegetable oils. Diets were formulated containing 20% by weight casein, 20% soybean oil and graded levels of choline with and without methionine and were fed to male Sprague-Dawley rats for 16 weeks. Rats fed methionine-supplemented diets had improved growth and food consumption, increased liver lipids mainly in the form of triglycerides and normal amino acid metabolism. On the other hand, choline supplementation reduced liver lipids but had no effect on growth and feed consumption. The results would indicate that diets including 20% casein and 20% oil require methionine supplementation to assure the nutritional adequacy of this high caloric diet. However, in this study there was no evidence to indicate that choline or methionine supplementation affected the heart lesion incidence in male rats. It was therefore concluded that the amount and type of fat in the diet, not the choline and methionine status of the diet, is related to heart lesions in male rats.

Growth, heart weight, cardiac lipid, and pathology of chickens fed soybean oil or oil extracted from different rapeseed cultivars.

A total of 384 male Single Comb White Leghorn chicks of the Hyline strain were randomized in 24 Petersime battery units of 16 birds per unit. Four replicate pens were fed either a basal (control) diet containing no added oil or a diet supplemented with 20% by weight of either soybean oil or rapeseed oil (RSO) from cultivars Tower, Candle, Regular, or R-500. Levels of crucic acid (22:1) in the RSO's ranged from .1 to 51.6%. Four birds from each unit were killed at 4, 8, 12, and 16 weeks for cardiac lipid analysis and pathology. Feeding RSO had no consistent effect on body weights, heart weights, or the heart-to-body weight ratio. Only the diet containing R-500 (51.6% 22:1) consistently increased cardiac lipid levels as measured gravimetrically. Maximum deposition of the long-chain monoenes (22:1) and eicosenoic acids (20:1) occurred in the cardiac lipids by 8 weeks, and the levels remained high throughout the 16-week trial. The relative concentration of these long-chain monoenes in the cardiac lipids was positively correlated to the dietary levels of these acids. The incidence of lesions found in heart, liver, and skeletal muscle was significantly higher in chickens fed RSO's high in 22:1 than in chickens fed control or soybean oil, and the incidence significantly correlated to the level of 22:1 in the diet. The incidence of heart and muscle lesions and sinusoidal distention in chickens fed RSO's low in 22:1 was not significantly different from that of chickens fed the control diet or soybean oil, but the incidence of hepatic necrosis was significantly higher for chickens fed RSO's low in 22:1 compared to those fed the control diet or soybean oil.

Reduction of myocardial necrosis in male albino rats by manipulation of dietary fatty acid levels.

A comprehensive statistical analysis had shown a significant correlation between the incidence of myocardial lesions in male albino rats and the concentration of certain dietary fatty acids. To test this result under controlled conditions, male rats were fed for 16 weeks diets containing 20% by weight soybean oil or a low erucic acid rapeseed (LEAR) oil. Both dietary oils contained substantial amounts of linolenic acid, and both groups developed a high incidence of myocardial necrosis. The addition of dietary saturated fatty acids to the oil in the form of cocoa butter significantly lowered the incidence of heart lesions in both groups. The addition of cocoa butter resulted in increased absorption of saturates and increased growth. Replacement of the cocoa butter by at least an equal amount of synthetic triolein resulted in no significant changes in the cardiopathogenic response compared to the original oils, thus ensuring that the reduction in heart lesions associated with the addition of cocoa butter was not due to dilution of cardiopathogenic compounds in the original vegetable oils. These results support the hypothesis that myocardial lesions in male rats are related to the balance of dietary fatty acids and not to cardiotoxic contaminants in the oils. Changes in the dietary fatty acids did not appear to influence the proportion of the cardiac phospholipids, but their fatty acid composition was markedly influenced. Dietary linolenic acid affected the C22 polyunsaturated fatty acids (PUFA) and dietary saturates increased the level of saturates in cardiac phospholipids. The level of arachidonic acid and total C22 PUFA did not appear to be affected by diet.

Role of dietary saturated fatty acids on lowering the incidence of heart lesions in male rats.

Male weanling rats were fed soybean or low erucic acid rapeseed oils alone or in combination with cocoa butter (a source high in saturates) or triolein for 16 weeks. All diets contained 20% by weight of the test oils. The apparent digestibility of all diets and test oils increased with the age of the rat. The apparent digestibility of saturated fatty acids was lower in rats fed the diets containing cocoa butter. The relative organ weights, however, were not affected by diet, but growth was improved by supplementing the vegetable oils with cocoa butter. This growth difference was significant for the addition of cocoa butter to low erucic acid rapeseed oil. After 16 weeks all groups of rats developed myocardial necrosis. A dramatic lowering of myocardial lesion incidence was observed in rats fed diets enriched with saturated fatty acids. The results of the present experiment suggest that enriching a vegetable oil with saturated fatty acids affects both nutritional and cardiopathological properties of the oil.

Bovine neoplasms encountered in Canadian slaughterhouses: a summary.

During the past five and one half years veterinarians of the Food Production and Inspection Branch of Agriculture Canada submitted tissues from 1535 cases of suspected bovine neoplasia for diagnosis to the Animal Diseases Research Institute, Nepean. Neoplasia was confirmed in 1370 cases. Lymphosarcoma was diagnosed in 738 (53.9%) of these cases, but it was not possible to accurately assess the prevalence of this condition at slaughter. Other commonly found neoplasms in order of frequency were neurofibroma, squamous cell carcinoma, uterine carcinoma, adrenal tumors, ovarian tumors and primary liver tumors. Many metastatic anaplastic carcinomas involving lymph nodes were not specifically identified as to tissue of origin. Nonneoplastic lesions were found in 165 submissions.

An investigation of the etiology of Brucella abortus singleton reactors.

Single animals in a herd that react serologically to Brucella abortus for no apparent reason are a problem. A number of such reactors from Ontario and Quebec were gathered for extensive clinical, serological, pathological and bacteriological examination in an attempt to investigate the etiology of these single serological reactions. While a variety of pathological changes were found, there was no apparent correlation to the serological, clinical or bacteriological findings.

Bovine paratuberculosis I. A herd study using complement fixation and intradermal tests.

A dairy herd (102 cattle) which had been enrolled under a paratuberculosis control program for two years utilizing a complement fixation test (carbohydrate antigen) and intradermal skin test (johnin PPD) was subjected to two further herd tests and followed to slaughter to determine infection status by culture and histology. Mycobacterium paratuberculosis infection was demonstrated in 37 of the animals of which only five were considered reactors on the basis of the last two herd tests applied. Cultural and histopathological evaluation indicated the testing procedures had eliminated heavily infected animals. The limitations of these testing procedures under free stall housing conditions are discussed.

Cardiopathogenicity of soybean oil and tower rapeseed oil triglycerides when fed to male rats.

The triglycerides of soybean oil were purified by molecular distillation and those of Tower rapeseed oil by molecular distillation and adsorption chromatography. The original oils and the purified triglycerides were incorporated in semisynthetic diets at 20% by weight and fed for 16 weeks to weanling male Sprague-Dawley rats to compare the nutritional and pathological effects of the oils and their triglyceride fractions on rats. The study was carried out at two independent laboratories. No significant differences were observed between the results of the two establishments. The incidence of myocardial lesions was significantly higher in rats fed Tower rapeseed oil than in those fed soybean oil. Purification of the triglycerides by molecular distillation and adsorption chromatography appeared to have no major effect on the incidence of myocardial lesions. This supports our previous findings that the cardiopathogenicity appeared to have no major effect on the incidence of myocardial lesions. This supports our previous findings that the cardiopathogenicity of the test oils to rats resides in the triglycerides of these oils.

Maedi-visna in Canadian sheep.

Lesions of maedi-visna were seen in sheep from the institutional research flock of the Animal Research Institute, Research Branch, Agriculture Canada in Ottawa. Viral particles demonstrated by electron microscopy in tissue culture cells and serological results confirm the diagnosis of maedi-visna. The extent of the problem in this flock will be described in a future paper.

Growth, lipid metabolism and pathology of two strains of rats fed high fat diets.

Studies were carried out on Sprague-Dawley (SD) and Chester Beatty (CB) rats to determine whether the difference in incidence of myocardial lesions can be related to dietary factors and parameters known to be affected in SD rats fed rapeseed oils. The two strains of young, male rats were fed diets which contained 20% by weight of either corn, LEAR (low erucic acid) or HEAR (high erucic acid rapeseed) oils for a period of up to 16 weeks. A significantly lower incidence of focal myocardial necrosis was observed in CB rats than in SD rats. The incidence of this heart lesion in CB rats was similar between all diets; in SD rats a higher incidence was observed in the groups fed rapeseed oils. In both strains the growth rates of rats fed LEAR and corn oils were similar; growth rates with HEAR oil diets were much lower than the other oils. Severe myocardial lipidosis was only evident in rats fed HEAR oil, but no strain differences were observed. The pattern and extent of lipidosis, including cardiac and hepatic lipid levels and fatty acid compositions, were similar in both strains at the time periods studied. Cardiac triglycerides and free fatty acids increased in rats fed HEAR oil, but no strain differences were evident. Strain differences were found in the levels of a few cardiac phospholipids of rats fed HEAR oil, however, the relative fatty acid compositions of each phospholipid were remarkably similar. Microscopic examination of the livers showed no evidence that feeding rapeseed oil caused any specific effect which could be related to differences in heart lesion response. There was no evidence to indicate that differences in heart lesion response between the two rat strains could be related to cardiac triglycerides, free fatty acids or phospholipids.

Heterotopic calcification in swine.

Pigs on nutritional studies developed heterotopic calcifications in the left atrial endocardium, submucosa of the gastric fundus and to a lesser extent in other sites. Light and electron microscopy of mineralized tissue showed early edema and connective tissue change, deposition of calcium, granulomatous reaction and repair. There was no alkaline phosphatase activity at the sites of calcification and the distribution of this enzyme activity was normal in other tissues. Increased acid phosphatase activity was demonstrated only in the lysosomes of macrophages in areas of granulomatous inflammation. Calcium and magnesium were within normal range in serum and femoral bone. The character and distribution of the lesions suggested the possibility of protracted low grade hypervitaminosis D3.

Myocardial lesions in rats fed rapeseed oil. I. Influence of strain of rats.

The influence of strain of rat on the development of myocardial lesions was investigated in an experiment which included two factors: strain (Wistar, Sherman, Chester Beatty (Hooded), and Sprague-Dawley from two sources designated Sprague-Dawley A, Sprague-Dawley C) and diet (5% corn oil, 20% corn oil, and 20% Brassica napus var. Zephyr rapeseed oil). Groups of 30 rats, housed 2 per cage, from each of three different strains of rats and groups of 10 rats, housed 2 per cage, from one strain of rats (Hooded) were fed semisynthetic diets containing the test oils for 16 weeks on an ad libitum basis. Rats of the Hooded strain consumed considerably less feed and grew at a significantly (P less than 0.01) slower rate than did rats of the Sprague-Dawley C strain, which in turn ate less and grew significantly (P less than 0.01) slower than rats of the other three strains. No marked differences were observed in the fatty acid composition of total cardiac lipids among strains. Rats from all strains except Hooded fed the diet containing 20% Zephyr RSO had a significantly (P less than 0.001) higher incidence of myocardial lesions than rats fed the 20% corn oil diet which in turn had a significantly (P less than 0.001) higher incidence than rats fed 5% corn oil. Similarly, significantly (P less than 0.05) more Sprague-Dawley C rats had myocardial lesions than Sprague-Dawley A rats regardless of diet. None of the Hooded rats fed the diet containing 20% Zephyr RSO developed myocardial lesions whereas all other strains fed this diet developed a high incidence of myocardial lesions.