Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: characterization of lung pathology, persistence of variable surface protein antigens and local immune response

Abstract

Background: Mycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067.

Methods: Lung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4+ and CD8+ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC.

Results: Suppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4+ and CD8+ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II.

Conclusions: The results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins.

Figure 1

(A) Gross pathology of caseonecrotic pneumonia; cut surfaces of lung tissue with various sized caseonecrotic lesions (largest area depicted by asterisk). There is marked interlobular fibrosis and suppurative bronchopneumonia in the remaining lung parenchyma. Calf no. 5. Bar = 1 cm; (B) Histopathology of caseonecrotic pneumonia; caseonecrotic lung lesion. The eosinophilic centre (N) is demarcated by inflammatory cells and remnants of necrotic bronchiolar epithelium (arrows). Calf No. 3. H&E. Bar = 184 μm; (C) Immunohistochemical detection of wide spectrum cytokeratin-positive epithelial cells; immunohistochemical staining with antibodies (mAb AE1/AE3) to wide spectrum cytokeratin of the same bronchiole as shown in (B) reveals remnants of bronchiolar epithelial cells (arrows) surrounding the necrotic area (N). At the periphery, demarcating macrophages are present (asterisk). Calf No. 3. ABC method. Bar = 92 μm; (D) Histopathology of obliterative bronchiolitis; obliterative bronchiolitis with vacuolated epithelial cells (arrows) and intraluminal fibroblasts and inflammatory cells. Calf No. 5. H&E. Bar = 46 μm.

Figure 2

(A) Immunohistochemistry for M. bovis Vsp antigen in necrotic lesion; the centre of a caseonecrotic pulmonary lesions (N) is surrounded by mostly degenerate neutrophilic granulocytes and macrophages. Immunohistochemical labelling of extracellular accumulations of M. bovis antigens with mAb 1A1. Calf No. 5. ABC method. Bar = 46 μm; (B) Immunolabelling of M. bovis Vsp antigens with mAb 1A1 within the lumen of a bronchiole containing exudate. Calf No. 3. ABC method. Frozen section. Bar = 46 μm; (C) Immunolabelling of M. bovis variable antigen pMB67 with mAb I2. Sequential frozen section of the same location as in (B); (D) Numerous IgG1-positive plasma cells in the lamina propria of bronchial mucosa. Calf No. 8. ABC method. Bar = 92 μm; (E) Lower numbers of IgG2-positive plasma cells in the lamina propria of bronchial mucosa in a sequential paraffin section of the same location as in (D); (F) Immunohistochemistry for MHC class II in necrotic and perinecrotic lung area; caseonecrotic lung lesion with MHC class II immunoreactivity within the necrotic area (N) and within the perinecrotic region. The majority of macrophages surrounding the necrosis (asterisk) is negative for MHC class II. Calf No. 5. ABC method. Bar = 92 μm.