Association among results of serum ELISA, faecal culture and nested PCR on milk, blood and faeces for the detection of paratuberculosis in dairy cows.

Paratuberculosis is a chronic, infectious disease of ruminants that entails a serious concern for the cattle industry. One of the main issues relates to the efficiency of diagnosis of subclinically infected animals. The objective of this field study was to analyse the association among results of a serum enzyme-linked immunosorbent assays (ELISA), faecal culture and nested PCR tests on milk, blood and faeces for Mycobacterium avium subsp. paratuberculosis detection in dairy cows. Faeces, blood and milk samples were collected from 328 lactating dairy cows in four known infected herds. Results were analysed to determine associations and levels of agreement between pairs of tests. A total of 61 animals (18.6%) tested positive when all the tests were interpreted in parallel. The agreement between results in different pairs of tests was poor, slight and fair in two, five and three of the 10 possible combinations respectively. Faecal culture and faecal polymerase chain reaction (PCR) resulted in the highest kappa coefficient (0.39; fair agreement), with the lowest agreement being for ELISA and blood PCR (-0.036; poor agreement). Fisher's exact test resulted in statistically significant associations (P < or = 0.05) between the following test pairs: ELISA : faecal culture; ELISA : faecal PCR; milk PCR : faecal PCR, blood PCR : faecal PCR and faecal culture : faecal PCR. Enzyme-linked immunosorbent assays showed the highest complementary sensitivity values for all the possible two-test combinations, followed by faecal PCR. The combined use of ELISA and faecal PCR has the potential to increase the overall sensitivity for the diagnosis of paratuberculosis infection.

Tolerance of inorganic selenium by range-type ewes during gestation and lactation.

The objectives of this 72-wk study were to evaluate and compare the effects of 6 dietary levels of inorganic Se on serum, whole blood, wool, and tissue Se concentrations and to determine the maximum tolerable level of Se for mature ewes during lamb production. Forty-one, 4-yr-old, range-type ewes (57.4 +/- 5.7 kg) were used in a completely randomized design with 6 dietary treatments. Sodium selenite was added to a corn and soybean meal-based diet to provide 0.2 (control), 4, 8, 12, 16, or 20 mg of dietary Se/kg to ewes during lamb production. Serum Se and ewe BW were measured at 4-wk intervals; whole blood Se and wool Se were measured every 12 wk; and samples of brain, diaphragm, heart, hoof, kidney, liver, and psoas major were collected at the termination of the experiment. Dietary Se did not affect ewe BW during the study (P = 0.69), and there was no treatment x time interaction. Serum Se increased linearly as dietary Se level increased (P < 0.001) and responded cubically (P = 0.02) over time. Selenium in whole blood increased linearly (P < 0.001) as supplemental Se increased. Wool Se increased linearly (P < 0.001) as dietary Se increased, and the response over time was quadratic (P < 0.001). Brain, diaphragm, heart, and psoas major Se increased (P < 0.05) linearly as dietary Se increased, liver Se responded quadratically (P < 0.05), and hoof and kidney Se increased cubicically (P < 0.05) as supplemental Se increased. In general, serum, whole blood, and tissue Se concentrations of ewes receiving 12, 16, or 20 mg of dietary Se/kg were greater (P < 0.05) than those of controls and ewes receiving less dietary Se. Although they were elevated in ewes receiving increased dietary Se, at no time did serum, whole blood, or wool Se concentrations reach levels previously reported as toxic, nor were clinical signs of Se toxicosis observed. Histopathological evaluation of liver, kidney, diaphragm, heart, and psoas major did not reveal evidence of Se toxicosis in ewes at any dietary Se level. Ewes under our experimental conditions and during the stresses of production were able to tolerate up to 20 mg of dietary Se/kg as sodium selenite for 72 wk. These findings suggest that the maximum tolerable level of inorganic Se for sheep is much greater than 2 mg/kg as was suggested previously. Experiments of longer duration and utilizing greater dietary Se concentrations are necessary to clearly define the maximum tolerable level.

Nested PCR on blood and milk for the detection of Mycobacterium avium subsp paratuberculosis DNA in clinical and subclinical bovine paratuberculosis.

OBJECTIVE: To determine the potential of PCR on blood and milk to detect cattle infected with Mycobacterium avium subsp paratuberculosis. PROCEDURE: A nested PCR method probing for IS900 was developed and compared to ELISA serology in 11 clinically infected and 46 subclinically infected, lactating Holstein cows from a herd with confirmed paratuberculosis (Johne's disease). RESULTS: When compared to serum ELISA the nested blood- and milk PCRs were equal in identifying DNA from clinically infected animals. The PCR procedures also gave positive DNA results with some subclinically infected animals when these only gave suspicious or negative results in the ELISA test. Most clinically and subclinically infected animals were detected with milk PCR. CONCLUSION: Since there may well be a haematological phase in paratuberculosis, nested PCR testing of blood and milk samples shows potential to detect animals subclinically infected with M a paratuberculosis. More subclinically infected animals need to be tested and confirmed infected before estimates of sensitivity and specificity can be made.

Causes of mortality in the Florida panther (Felis concolor coryi).

Panthers necropsied at the University of Florida ranged between 2 weeks and 14 years of age; there were 38 males and 17 females in the cohort. Main categories of causes of death included trauma inflicted from either vehicular collisions (43%) or territorial fights (16%). Specific endogenous diseases involved the respiratory system in 13%, the urinary system in 4%, and the central nervous system in 2%. Ostium secundum atrial septal defects (ASD) were diagnosed in 11% of the panthers necropsied. Seventeen (54%) of the 38 male panthers had either unilateral or bilateral cryptorchidism. Cause of death remained undetermined in 11% of the total cohort.

Porcine ileitis model induced by TNBS-ethanol instillation.

Nine young pigs were used to evaluate the ability of an trinitrobenzene sulfonic acid-ethanol (TNBS-EtOH) mixture, in varying combinations, to induce ileitis comparable to that caused by intraintestinal instillation in other species. The distal ileum was accessed via laparotomy in anesthetized animals and the TNBS-EtOH was instilled via hypodermic needle. In three pigs in which the instillate was not held within an ileal segment, there were no ileal lesions noted upon necropsy at two weeks after instillation. In all six pigs in which the instillate was confined to a 10-cm length of distal ileum for 10-15 min, there was definite gross and histologic evidence of severe ileitis upon necropsy at one week after instillation. The histopathology was more consistent with Th-1- than Th-2-mediated inflammation.

Postprandial venous ammonia concentrations in the diagnosis of hepatobiliary disease in dogs.

A postprandial ammonia tolerance test (PPATT) was performed on normal dogs and dogs with signs that suggested they may have liver disease. All dogs underwent transcolonic scintigraphy, liver biopsy, or both and were assigned to extrahepatic disease, primary hepatocellular, and congenital portosystemic vascular anomalies (PSVA) groups. Each dog was fed a chicken and rice diet providing 25% of its estimated daily metabolizable energy requirement (MER) as an ammonia challenge. This is practical in patients with liver disease because ammonium chloride administration often causes vomiting or ammonia toxicity. Venous ammonia concentrations were measured before feeding and every 2 hours after feeding for 8 hours. No difference in mean ammonia concentrations between dogs with extrahepatic disease and control dogs was found. Therefore, the specificity of the PPATT was 100%. Dogs with hepatocellular disease showed no change in mean ammonia concentration at any time point, before or after feeding, but sensitivity was greatest when venous ammonia was measured 6 hours after feeding (sensitivity before feeding, 28%, and after feeding, 36%). Among dogs with congenital PSVA, mean ammonia concentrations were higher than the reference range at all time points before and after feeding, and peak mean ammonia concentration occurred 6 hours after feeding. In this group, the sensitivity of the PPATT was 81% before feeding and 91% 6 hours after feeding. This study demonstrates that the measurement of venous ammonia concentration is a useful test to detect congenital PSVA, and the sensitivity of the test may be improved by sampling 6 hours after feeding. The PPATT has poor sensitivity in detecting primary hepatocellular disease.

The histopathologic diagnosis of subclinical Johne's disease in North American bison (Bison bison).

The morphologic changes of subclinical Johne's disease in North American Bison (Bison bison) are characterized by microgranulomas composed of epithelioid macrophages and individual multinucleate giant cells of Langhans'-type occasionally containing individual cytoplasmic acid-fast bacilli compatible with Mycobacterium avium paratuberculosis. The microgranulomas are best visualized in the mesenteric lymph nodes of infected subclinical animals. Macrophages that can be confused with infection-associated epithelioid macrophages in the mesenteric lymph nodes are pigment-carrying cells from the intestinal tract. Mesenteric lymph node biopsy may be a useful diagnostic tool for detection of mild subclinical infection in individual ruminants from herds of unknown infection status. The biopsy may also be useful for Johne's disease surveillance during test-and-cull programs.

The pathology of spontaneous paratuberculosis in the North American bison (Bison bison).

Gross and histopathologic examinations were performed on 70 North American bison (Bison bison) from a Mycobacterium avium paratuberculosis culture-positive herd. The bison examined were part of a breeding herd totaling 2,800 animals. Eight of 70 (11%) animals had gross findings of intestinal mucosal thickening, and 16 of 70 (23%) of the animals had enlarged mesenteric lymph nodes. Histologic lesions compatible with Johne's disease were diagnosed in 30 of 70 (43%) bison on the basis of the demonstration of noncaseating granulomatous inflammatory infiltrates and of one or more acid-fast bacilli characteristic of Mycobacterium avium paratuberculosis. A suspicious diagnosis of Johne's disease was obtained in 11 of 70 (16%) bison on the basis of the observation of noncaseating granulomatous inflammatory infiltrates without demonstrable acid-fast bacteria. Twenty-nine of 70 (41%) animals were assessed as histologically paratuberculosis free. Histologic results were compared to Johne's disease tests such as culture, serology, and polymerase chain reaction, which were performed on some of the cohort animals.

Bilateral renal oncocytoma in a Greyhound dog.

A bilateral, locally invasive renal oncocytoma was diagnosed in a 10-year-old spayed female Greyhound dog. The diagnosis was based on positive staining of the tumor with the periodic acid-Schiff reaction prior to diastase treatment, on the immunohistochemical expression of cytoplasmic cytokeratin, and on the prominence of mitochondria in the tumor cells.

Atrial septal defects in Florida panthers.

Ostium secundum atrial septal defects (ASDs) were observed in six (3 M, 3 F) of 33 (20 M, 13 F) (18%) Florida panthers (Puma concolor coryi) necropsied by veterinary pathologists between 1985 and 1998. A seventh ASD was found in a female panther necropsied in the field and is included in the pathological description but not the prevalence of ASDs in Florida panthers. One panther (FP205) with severe ASD also had tricuspid valve dysplasia (TVD). Atrial septal defects and/or TVD are believed to have caused or contributed to the deaths of three (9%) Florida panthers in this study. Mean diameter +/- SD of ASDs was 9.0 +/- 4.7 mm (range 3 to 15 mm). Gross pathological changes attributed to ASDs/TVD in severely affected panthers (ASD > or = 10 mm) (n = 4) included mild right ventricular dilatation (n = 3) and hypertrophy (n = 2), mild to severe right atrial dilatation (n = 2), and acute pulmonary edema (n = 3). Panthers with mild ASDs (ASD < or = 5 mm) (n = 3) had no other detectable gross pathological changes associated with the ASDs. Histological examination of lungs of three panthers with severe ASDs revealed mild to moderate dilatation with fibrosis and smooth muscle atrophy of the tunica media of medium to large caliber arteries (n = 2), interstitial and/or pleural fibrosis (n = 2), perivascular fibrosis (n = 1), and acute to chronic edema (n = 3). Twenty-six necropsied panthers were examined one or more times while living; medical records were retrospectively evaluated. Antemortem radiographic, electrocardiographic, and echocardiographic examinations were performed on two panthers with severe ASDs (FP20 and FP205). Thoracic radiographic abnormalities in both included right heart enlargement, and in FP205 (severe ASD and TVD), mild pulmonary overperfusion. Electrocardiographic examination of FP205 revealed a right ventricular hypertrophy pattern, while FP205 had a normal electrocardiogram. Echocardiographic examination of FP20 revealed marked right atrial dilatation; a bubble contrast study indicated regurgitation across the tricuspid valve. Echocardiographic abnormalities in FP20 included right atrial and ventricular lilatation, atrial septal drop-out, and severe tricuspid regurgitation; non-selective angiography revealed significant left to right shunting across the ASD. All panthers with severe ASDs ausculted (n = 3) had systolic right or left-sided grade I-V/VI murmurs loudest at the heart base. All male panthers with ASDs (n = 3) (100%) and 9 of 17 (53%) male panthers without ASDs in this study were cryptorchid.

Cranial edema associated with a protein-losing nephropathy in a golden-mantled flying fox (Pteropus pumilus).

An adult golden-mantled flying fox (Pteropus pumilus) was diagnosed with nephrotic syndrome on the basis of the findings of proteinuria, hypoalbuminemia, hypercholesterolemia, and cranial edema. Membranoproliferative glomerulitis and interstitial nephritis were confirmed antemortem by renal biopsy. The bat had received seven injections of oxytocin in the period immediately prior to presentation. The possible role of oxytocin in the development of the nephropathy is discussed. Supportive care and treatment with a single plasma transfusion, furosemide, and prednisone led to a gradual but complete resolution of the nephrotic syndrome in this animal.

Nutritional myodegeneration associated with dorsal scapular displacement in beef heifers.

Scapular displacement due to bilateral rupture of the serratus ventralis muscles ("flying scapula") occurred in four heifers from a large beef herd. Two of the four affected animals were necropsied. Additional animals on the farm developed intermittent lameness when enclosed on certain pastures. The lameness and scapular displacement were attributed to selenium deficiency in the feed and pasture environment. This is the first account of "flying scapula" in the United States.

Fibrosing granulomas in the equine liver and peritoneum: a retrospective morphologic study.

Fibrosing granulomas were found in the liver and occasionally on the intestinal and diaphragmatic serosae and in the lung of 11 horses submitted for necropsy. Although these granulomas were considered incidental findings in most of the horses, they had caused liver failure in 1 horse. The granulomas typically were characterized by a dense collagenous core that frequently was mineralized. The periphery contained a rim of inflammatory cells. Only in 1 of the 11 horses was there evidence of an egg shell suggestive of schistosomes. Schistosomal eggs were not detected in the feces of the horses. The typical architecture of the granulomas combined with the occasional finding of a residual egg led to the circumstantial conclusion that the fibrosing granulomas were the result of chronic schistosomiasis of undeterminable origin.

Susceptibility of Balb/c, C57/B6 and C57/B10 mice to infection with Mycobacterium paratuberculosis.

Balb/C, C57/B10 and C57/B6 mice were examined for their susceptibility to disseminated Mycobacterium paratuberculosis infection after intraperitoneal inoculation with a suspension of organisms containing mineral oil. Animals were examined monthly by histopathology and bacterial tissue counts of liver and spleen over a 6-month period. Only Balb/c mice maintained a steady infection with an average of 4.1 x 10(5) +/- 7.8 x 10(3) and 8.1 x 10(5) +/- 2.6 x 10(4) colony forming units (cfu) per gram of liver and spleen, respectively, during the course of the study. In contrast, C57/B10 mice reduced the bacterial counts in the liver and spleen from 6.8 x 10(4) and 1.3 x 10(5) to 7.1 x 10(2) and 4.3 x 10(3), respectively during the first 120-150 days after infection. The reduction in cfu was associated with the development of caseous necrotic lesions. C57/B10 mice were of intermediate resistance, slowly reducing cfu in the liver, but not the spleen, during the 6-month period. Balb/c was found to be a suitable mouse strain for the study of chronic M. paratuberculosis infection.

Reproducibility of a commercial enzyme-linked immunosorbent assay for bovine paratuberculosis among eight laboratories.

Interlaboratory reproducibility of an absorbed enzyme-linked immunosorbent assay (ELISA) kit for detection of bovine serum antibodies to Mycobacterium paratuberculosis was evaluated. A panel of 30 bovine sera (15 positives and 15 negatives) was tested in triplicate microtiter wells on each of 2 days at 8 different laboratories. One laboratory had invalid results because of positive or negative serum control optical density (OD) readings beyond the acceptable range specified by the kit. The coefficient of variation (CV) for mean OD values was influenced by low ODs on test negative sera at 2 laboratories, thus the CVs on positive sera were considered a more representative measure of kit reproducibility. Between-well CVs averaged 6.7% +/- 2.8% (mean +/- standard deviation), and between-day CVs averaged 14.5% +/- 9.8% among the 7 laboratories with valid assays on the 15 positive sera. The OD values were converted to positive or negative classifications for each assay well, and the results were compared. Among 1,392 assays in 7 laboratories, 98.6% were in agreement. Eleven of 18 discrepant results were due to a sample that consistently gave OD values near the cutoff for a positive test. Exclusion of that serum from the analysis resulted in a 99.8% rate of agreement among laboratories. Results indicated that the absorbed ELISA kit provided reproducible results within and between laboratories.

Evaluation and comparison of automated biopsy devices. Work in progress.

The performance of four automated biopsy devices (Bard Biopty, Bard Monopty, Microvasive ASAP 18, Medical Device Technologies Ultra-Cut) was compared when they were used to obtain 96 liver and 96 kidney samples from eight dogs under ultrasound guidance. There was no significant difference in the lengths of the samples obtained with the four devices. The Monopty device yielded a significantly greater mean weight of both kidney (30.8%) and liver (31.6%) samples compared with the other devices. There were no significant differences between the four devices relative to cellular and histologic preservation, crush artifact, and number of renal glomeruli or liver lobules and portal triads. Renal subcapsular hematomas were identified in most instances, and there was no difference between the devices in the amount of renal trauma resulting from their use. There was only one instance of severe injury to the liver. The choice of instrument should remain one of personal preference, since all four devices were satisfactory and none produced significantly greater renal or hepatic injury.