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Abstract

Objective—To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms.

Sample Population—Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results.

Procedures—Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry.

Results—Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect.

Conclusions and Clinical Relevance—Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate the role of the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPKERK) pathway in the interaction between Mycobacterium avium subsp paratuberculosis (MAP) organisms and bovine monocytes.

Sample Population—Monocytes obtained from healthy adult Holstein dairy cows that were not infected with MAP organisms.

Procedures—Monocytes and MAP organisms were incubated together with or without a specific inhibitor of the MAPKERK pathway (PD98059), and the capacity of monocytes to express tumor necrosis factor alpha (TNF)-α and interleukin (IL)-10 and -12, produce nitric oxide, acidify phagosomes, kill MAP organisms, and undergo apoptosis was evaluated.

Results—The MAPKERK pathway was activated within 10 minutes after addition of MAP organisms to monocytes. Addition of PD98059 to monocyte-MAP mixtures decreased monocyte TNF-α and IL-12 mRNA expression but had no effect on IL-10 mRNA expression. Treatment with PD98059 failed to induce significant alterations in phagosome acidification, organism killing, nitric oxide production, or apoptosis of MAP-exposed monocytes.

Conclusions and Clinical Relevance—Results indicated that the MAPKERK pathway was activated during the interaction of MAP organisms with monocytes, which initiated TNF-α and IL-12 mRNA expression but failed to initiate antimicrobial activity. The MAPKERK pathway may be involved in initiating proinflammatory and proimmune responses in MAP infection in cattle.

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine the role of tissue factor (TF) in the coagulation events leading to intra-alveolar fibrin deposition and intravascular thrombosis associated with pneumonic pasteurellosis in cattle.

Animals

Healthy 2- to 4-week-old male Holstein calves.

Procedures

Blood and bronchoalveolar lavage samples were collected before and at 1, 2, 4, and 6 hours after inoculation of saline solution or Pasteurella haemolytica. Total leukocyte count, platelet count, plasma total protein concentration, prothrombin time, and partial thromboplastin time were measured in blood samples. Total nucleated cell count, total protein concentration, and procoagulant activity were measured in bronchoalveolar lavage samples. Additionally, platelet survival in blood, platelet accumulation in affected lung tissue, and gross and microscopic lung lesions were determined.

Results

Administration of TF monoclonal antibodies (MAB) TF1-1F7 prevented the decrease in platelet survival and the increase in bronchoalveolar lavage fluid TF-dependent procoagulant activity observed in calves not treated with MAB TF1-1F7 antibody, but did not attenuate the increase in lavage fluid neutrophil numbers and total protein concentration. MAB TF1-1F7 administration reduced the percentage of lung affected by pneumonic lesions from 51.81 % to 10.40% and attenuated intra-alveolar deposition of fibrin, neutrophils, and erythrocytes.

Conclusion

Intra-alveolar fibrin deposition and activation of coagulation in cattle with pneumonic pasteurellosis is, at least in part, mediated by TF.

Clinical Relevance

Treatments that neutralize TF activity may attenuate lung injury in cattle with pneumonic pasteurellosis (Am J Vet Res 1997;58:28–33)

Free access
in American Journal of Veterinary Research

Summary

A flow cytometric platelet immunofluorescence assay (fc-pifa) was compared with a previously developed microscopic platelet immunofluorescence assay (mi-pifa) for detection of circulating platelet antibody. Both assays were performed on serum from 10 healthy dogs with normal platelet count, and on serum from 27 thrombocytopenic dogs—18 had primary immune-mediated thrombocytopenia (imt), and 9 had imt in addition to other immune-mediated disease (secondary imt). Both assays yielded negative results for all control dogs. The mi-pifa and fc-pifa results were in agreement in 23 dogs with imt (14 positive and 9 negative). There was linear correlation between mi-pifa scores and fc-pifa results (r = 0.873). Positive results were obtained for 55.5% of the dogs with suspected imt, using the mi-pifa, compared with 67%, using the fc-pifa; however, the difference was not statistically significant. Use of fresh or frozen fixed donor platelets as the antigen source yielded similar results in the fc-pifa.

Free access
in American Journal of Veterinary Research