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Objective: To evaluate sensitivity and specificity of a new ELISA for antibodies against Mycobacterium avium subsp paratuberculosis., Design: Cross-sectional observational survey., Sample Population: Serum samples from 590 cattle that were infected with M avium subsp paratuberculosis and 723 cattle that were not infected., Procedure: Serum samples were tested by use of an ELISA for antibodies against M avium subsp paratuberculosis., Results: Sensitivity of the test varied from 15.4 to 88.1%, depending on the clinical stage and bacterial shedding status of the cattle., Conclusions and Clinical Relevance: Results obtained with use of the new ELISA agreed favorably with those of a previous ELISA. Practitioners must be aware of variability in the sensitivity of the test, which depends on the clinical and shedding status of the cattle, because this may affect interpretation of test results.

Objective: To compare cytokine secretion patterns of peripheral blood mononuclear cells (PBMC) from healthy cows and cows subclinically and clinically infected with Mycobacterium paratuberculosis., Animals: 5 noninfected cows, 6 cows with subclinical paratuberculosis, and 4 cows with clinical paratuberculosis., Procedure: PBMC were isolated, and concentrations or activities of secreted interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) were measured after in vitro stimulation of cells with concanavalin A (ConA), lipopolysaccharide (LPS), or a whole-cell sonicate of M paratuberculosis (MpS). Proliferative responses of PBMC were also determined after stimulation with ConA, phytohemagglutinin, pokeweed mitogen (PWM), or MpS., Results: After stimulation with ConA, cells from subclinically infected cows secreted significantly more, and cells from clinically infected cows secreted significantly less, IFN-gamma, compared with cells from control cows. Cells from cows with subclinical paratuberculosis produced significantly more TNF and IFN-gamma in response to MpS than cells from the other 2 groups. Stimulation of PBMC from subclinically infected cows with ConA or MpS resulted in significantly higher proliferative responses, compared with cells from control and clinically infected cows. In contrast, clinically infected cows had significantly higher proliferative responses to PWM than cells from the other 2 groups., Conclusions and Clinical Relevance: A decrease in T-cell responses to mitogens or MpS was observed in cows clinically infected with M paratuberculosis, compared with subclinically infected cows, suggesting that activated T cells may delay the progression of paratuberculosis.

Objective: To study the effects of 1,25(OH)2D3 and calcium (Ca) on splenocyte cytokine secretion during Mycobacterium paratuberculosis infection., Design: Mice were assigned to the following treatments: 1-noninfected, 2-infected, 3-noninfected/1,25(OH)2D3, 4-infected/1,25(OH)2D3, and 5-infected/low-Ca diet (0.15%). ANIMALS--Male beige mice averaging 6 weeks of age and 20 g in body weight., Procedure: After acclimation to their diets, mice in treatments 2, 4, and 5 were inoculated IV with 10(8) colony-forming units of M paratuberculosis. At 1, 6, and 12 months after infection, mice in treatment groups 3 and 4 had miniosmotic pumps implanted subcutaneously that delivered 13 ng of 1,25(OH)2D3/day for 14 days. Treatment 5 was included as a control for comparison with treatment 4 because low dietary Ca should increase endogenous 1,25(OH)2D3 values. Splenocytes were isolated from mice at 1, 6, and 12 months and stimulated in vitro with medium alone (nonstimulated), lipopolysaccharide (LPS), concanavalin A, and M paratuberculosis whole-cell sonicate (MpS)., Results: Production of interleukin 6 after stimulation with LPS, concanavalin A, or MpS was higher (P < 0.05) for splenocytes isolated from mice fed the low-Ca diet, compared with control infected mice 1 and 6 months after infection. Interleukin 1 and tumor necrosis factor activities were increased (P < 0.05) in splenocytes cultured with LPS and MpS after isolation from mice of the low-Ca group. Mice infused with 1,25(OH)2D3 had higher (P < 0.05) interleukin 1 secretion after stimulation of splenocytes with LPS and higher (P < 0.05) tumor necrosis factor production after incubation with MpS., Conclusion: 1,25(OH)2D3 and low dietary Ca increase cytokine secretion in mice infected with M paratuberculosis.

Objective: To determine whether Pasteurella multocida toxin (PMT) affects growth of the proximal portion of the humerus of young pigs., Animals: 20 colostrum-deprived, cesarean-derived pigs., Design and Procedure: 5 groups (n = 4/group) of pigs were formed. Group-1 pigs received 0.1 ml of phosphate-buffered saline solution for 4 weeks; group-2 pigs received 0.05 microgram of PMT/kg of body weight at 14 and 21 days; group-3 pigs received 0.05 microgram of PMT/kg at 28 and 35 days; group-4 pigs received 0.1 microgram of PMT/kg at 14 and 21 days; and group-5 pigs received hyperimmune serum (from a sow given purified toxin) on days 13, 20, 27, and 34, and 0.1 microgram of PMT/kg on days 14, 21, 28, and 35., Results: All pigs given 0.1 microgram of PMT/kg without serum died or were euthanatized, as were 4 pigs given 0.05 microgram of PMT/kg. These pigs had increased serum interleukin 1 and 6 bioactivities. Pigs surviving 0.05 microgram of PMT had decreased weight gain, rough coat, marked atrophy of the ventral concha (as determined by turbinate perimeter ratios), and small stature. The surviving pigs also had reduced area and decreased proliferation indices in physeal chondrocytes on the basis of bromodeoxyuridine immunoreactivity. Control and serum-treated pigs gained weight, had no clinical effects, had similar physeal areas, and had higher cell proliferation indices., Conclusions: PMT inhibits endochondral bone formation by reducing physeal area and chondrocyte proliferation in vivo. Hyperimmune serum neutralizes the effects of toxin on weight gain, clinical appearance, physeal area, and chondrocyte proliferation., Clinical Relevance: PMT may affect growth of the skeletal system. Antiserum to PMT is protective.

Protein expression profiles of 10 isolates of Mycobacterium paratuberculosis, M avium 18 (formerly M paratuberculosis 18), and 1 isolate each of M avium serotype 2, M avium serotype 8, and M bovis BCG were examined. Protein expression profiles of M paratuberculosis and M avium were similar. However, two-dimensional gel analysis of [35S]methionine-labeled cellular proteins resolved 4 proteins, with molecular mass of 28,000, 32,000, 32,000, and 42,000 daltons, which were expressed in greater amounts in M paratuberculosis than in M avium. Two proteins, with molecular mass of 43,000 and 60,000 daltons, were identified, which were expressed in greater amounts in M avium than in M paratuberculosis. Immuno (western)-blot analysis, using antiserum from 2 cows clinically infected with M paratuberculosis as the primary antibodies, suggested that the 42,000-dalton protein may be specific for M paratuberculosis. Comparison of protein expression profiles may be useful as a tool for differentiating isolates of M paratuberculosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled extracellular proteins revealed variability among the isolates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cellular proteins divided the M paratuberculosis isolates into 2 groups on the basis of a difference in the amount of expression of a 28,000-dalton protein. This information may be useful in epidemiologic studies.

Six nonpregnant, nonlactating Jersey cows, averaging 4 to 6 years old, were used to evaluate the immunomodulatory effects of recombinant bovine interleukin 1 beta (rBoIL-1 beta). Cows were given 166 ng of rBoIL-1 beta/kg of body weight at 8-hour intervals for 96 hours. Persistent leukocytosis was observed within 3 hours of rBoIL-1 treatment, peaking 24 hours after the first IL-1 beta injection and returning to baseline values within 72 hours after cessation of IL-1 beta treatment. Injection of cows with rBoIL-1 beta stimulated lymphocyte blastogenesis and mitochondrial methyl-thiazoltetrazolium cleavage activity in resting cell cultures. Increases in the aforementioned lymphocyte activities were also observed in stimulated blood mononuclear cell cultures during IL-1 beta administration. Change in IgM production in cell cultures was not observed during IL-1 beta treatment. Within 24 hours of the first IL-1 beta injection, IL-1 beta mRNA transcription in stimulated blood mononuclear cell cultures was markedly increased, suggesting that IL-1 beta upregulates its own production in mononuclear cells. These data provide evidence that administration of cytokines, such as rBoIL-1 beta, enhances immune cell function and, therefore, may be useful in alleviating immunosuppresion in cattle.