2.2.2. At 3 dpi

The extent of pulmonary lesions varied from 11% to 31% (Figure 1). Lesions were firm, grey-red marbled, had a dry cut-surface and were associated with fibrinous pleuritis.

By histology, the fibrinopurulent pneumonia and severe vasculopathy had a lobar distribution and were more extensive than at 2 dpi (Figure 3A,B). Foci of necrosis were more frequent in bronchiolar epithelium and vascular walls (Figure 3B,C). There were small areas of complete necrosis of pulmonary tissue. In these areas, diffuse labeling of fibrin using immunohistochemistry for factor VIII was seen (Figure 3C), but no epithelial cells were labeled for cytokeratin.

Lesions in interlobular septa and pleura had increased in severity. Chlamydial inclusions were numerous in neutrophils and macrophages (Figure 3E), but rare in alveolar epithelial cells.

**Figure 3.** Pulmonary lesions at 3 dpi. (**A**) Lobular fibrinopurulent bronchopneumonia (BP) and fibrin thrombi (F) in interlobular lymphatics. Inset: higher magnification of an alveolus filled with fibrin and neutrophils. HE-stain. (**B**) Severe exudation throughout the wall and in the perivascular space of an arteriole (A). HE-stain. (**C**) Diffuse precipitates of fibrin (brown) in the wall and perivascular space of an arteriole (A) extending into the alveoli. IHC, factor VIII. (**D**) Necrosis (arrows) in the wall of a bronchiolus (B). The lumen is filled with exudate (E). HE-stain. (**E**) Numerous small chlamydial inclusions (brown) in neutrophils and macrophages. Inset: higher magnification of multiple chlamydial inclusions. IHC, chlamydia.

#### 2.2.3. At 4 dpi

Pulmonary lesions varied between 20% and 27% (Figure 1). They were firm, grey-red marbled, bulging and associated with fibrinous pleuritis (Figure 4A).

By histology, lesions in airways, blood vessels and pleura were as described at 3 dpi. Areas of necrosis had expanded (Figure 4B). The inflammatory exudate consisted of neutrophils, fibrin and cellular detritus (Figure 4B). There was a more extensive loss of alveolar epithelial cells than at 3 dpi (Figure 4C,D). Labeling of factor VIII revealed massive accumulations of fibrin in the walls and around arterioles and in the capillaries of interalveolar septa (Figure 4E,F). Several bronchioles and blood vessels were completely obstructed by fibrin. Chlamydial inclusions were numerous in neutrophils, macrophages and detritus in areas of necrosis.

**Figure 4.** Pulmonary lesions at 4 dpi. (**A**) Section through a basal lobe. The lobular lesion (BP) bulges above the surface of the surrounding pulmonary tissue (PT). The cut surface is drey and marbled grey-red. Interlobular septae and subpleural space are dilated by fibrinous exudate (arrows). (**B**) A large area of necrosis (N) is demarcated by neutrophils and cellular detritus (N+D). Inset: higher magnification of necrosis. HE-stain. (**C**) Epithelial cells are labelled only in segments of the bronchiolar wall (B). There is no labelling of epithelial cells in the surrounding alveoli. IHC, cytokeratin. (**D**) Labelling of epithelial cells in a bronchiolus (B) and surrounding alveoli in the healthy lung of a control calf. Inset: higher magnification of AEC1 in alveolar septae. IHC, cytokeratin. (**E**) Accumulation of fibrin in the wall and perivascular space of an arteriole (arrow). Interalveolar capillaries are dilated by fibrin thrombi (brown). IHC, factor VIII. (**F**) Labeling of factor VIII (brown) is restricted to endothelial cells (arteriole as example, arrow) and thrombocytes in the healthy lung of a control calf. Inset: higher magnification capillaries in alveolar septae. IHC, factor VIII.

### 2.2.4. At 7 dpi

Gross pulmonary lesions were as described at 4 dpi, but only 14–22% of the pulmonary volume was affected (Figure 1). Pleural lesions had progressed from fibrinous to fibroblastic.

By histology, lesions were characterized by multiple confluent areas of necrosis (Figure 5A). A few weakly cytokeratin-positive cells were present multifocally at the periphery of the necrotic tissue (Figure 5A,B). These epithelial cells were single or formed small aggregates. Airways were filled with macrophages and neutrophils. Compared to 4 dpi, the number of neutrophils was lower and the number of macrophages increased. Moderate infiltrates of lymphocytes and plasma cells were present around bronchi, bronchioles, altered arterioles and in pulmonary tissue adjacent to interlobular septa (Figure 5A). Fibroblasts and delicate collagen fibers had formed in the thickened interlobular septa and subpleural space. Chlamydial inclusions were predominantly present in areas of necrosis (Figure 5C).

**Figure 5.** Pulmonary lesions at 7 dpi. (**A**) A few, small groups of epithelial cells (arrows, examples) form a small zone of regeneration (R) at the periphery of a necrosis (N). There is an infiltrate (I) of macrophages, lymphocytes and plasma cells in the adjacent interlobular septum. Inset: higher magnification of a rudimentary alveolar wall. HE-stain. (**B**) Higher magnification of the zone of regeneration. A few cells are weakly positive for cytokeratin (brown). IHC, cytokeratin. (**C**) Chlamydial inclusions (brown) predominate in the necrosis (N) and are rare (arrow) in the zone of regeneration (R). Inset: higher magnification of a chlamydial inclusion in the zoe of regeneration. IHC, chlamydia.

#### 2.2.5. At 10 dpi

Pulmonary lesions were grey, demarcated by hyperemia and firm (Figure 6A). They amounted to 8% in two calves and 11% of pulmonary volume in one calf (Figure 1) Pleural lesions were fibroblastic.

By histology, wide zones of regenerating pulmonary tissue were surrounding areas of necrosis (Figure 6B). The zone of regeneration was characterized by multifocal epithelial cell aggregates forming irregular tubular and alveolar structures (Figure 6C). Epithelial cells had the cuboidal shape of AEC2 and mitotic figures were frequent (Figure 6B inset). Factor VIII-positive endothelial shoots originated from interlobular septa and passed into the zone of regeneration (Figure 6D). Infiltrates of macrophages, lymphocytes and plasma cells were present throughout the zone of regeneration, especially along interlobular septa, and around arterioles, bronchioles and bronchioli (Figure 6A). They formed multifocal lymphoid aggregates. The exudate in bronchioli and bronchi was lined by

epithelial cells resulting in bronchiolitis obliterans. A few fibroblasts and delicate collagen fibers were observed in the bronchiolitis obliterans, perivascular space and interalveolar septa. The distribution of chlamydial inclusions was as described at 7 dpi, but their number was lower.

**Figure 6.** Pulmonary lesions at 10 dpi. (**A**) Section through a basal lobe. The greyish lesion centered round a bronchus (arrow) is demarcated by hyperemia. (**B**) A broad zone of regeneration (R) with multifocal epithelial proliferates surrounds the necrosis (N). Infiltrates of macrophages, lymphocytes and plasma cells are especially prominent next to the interlobular septum (S). Inset: higher magnification of the zone of regeneration. HE-stain. (**C**) Multiple foci of epithelial proliferates (brown) in the zone of regeneration (R) form alveolar structures lined with cuboidal cells. Inset: higher magnification cuboidal epithelial cells. IHC, cytokeratin. (**D**) Capillaries (brown) originating from the interlobular septum (S) pass into the zone of regeneration (R). Fibrinous exudate (brown) is present within the necrosis (N). Inset: higher magnification of capillaries. IHC, factor VIII.

#### 2.2.6. At 14 dpi

Pulmonary lesions were reduced to 3% of pulmonary volume in one and less than 1% in two calves (Figure 1). They were firm, white, poorly demarcated (Figure 7A) and frequently accompanied by fibrous adhesions between pulmonary lobes as well as between pulmonary lobes and thoracic wall.

By histology, lesions were characterized by extensive regeneration of pulmonary tissue and only small foci of necrosis (Figure 7B). In areas of regeneration, cuboidal epithelial cells formed irregular alveolar spaces (Figure 7C). Lesions in airways were organized by bronchiolitis and bronchitis obliterans (Figure 7D). Alveolar spaces were separated by thick interalveolar tissue, which contained capillaries originating from subpleural tissue and interlobular septa, and diffuse infiltrates of macrophages, lymphocytes and plasma cells (Figure 7A,B,D). Organized lymphoid tissue forming tertiary lymphoid tissue in interlobular septa and bronchus-associated lymphoid tissue (BALT) around airways and blood vessels was more abundant than at 10 dpi (Figure 7B). There was no leakage of fibrin through arteriolar walls (Figure 7E). The fibrinous exudate in the wall and around arterioles was replaced by several

layers of collagen fibres, which compressed the vascular lumen (Figure 7E,F). An increased amount of collagen fibers was also observed in interalveolar and interlobular septa, and in the bronchiolitis obliterans. Chlamydial inclusions were seen in very few alveolar macrophages.

**Figure 7.** Pulmonary lesions at 14 dpi. (**A**) Section through a middle lobe. The small lesions (white) are centered around a bronchus (arrow) and not well demarcated. (**B**) Extensive regeneration (R) of pulmonary tissue with thick interalveolar septa and narrow alveolar spaces; multifocal organized lymphoid tissue (stars) in the interlobular septum. HE-stain. (**C**) Cuboidal epithelial cells (brown) form irregular alveolar spaces; an area with more progressed regeneration is indicated (star). Bronchiolitis obliterans is seen in most airways (arrows). Inset a: higher magnification of cuboidal epithelial cells forming alveolar spaces. Inset b: higher magnification of delicate, elongated epithelial cells in the area with progressed regeneration. IHC, cytokeratin. (**D**) Higher magnification of more regular alveolar structures lined by type 2 alveolar epithelial cells (star) and the exudate (E) within a bronchiolus (B) lined by cuboidal epithelial cells. IHC, cytokeratin. (**E**) Factor VIII labels endothelial cells in blood vessels (arrows, examples). There is no exudation of fibrin into and through vascular walls. IHC, factor VIII. (**F**) Fibrinous exudate within and around arteriolar walls (arrows) is replaced by collagen fibers (blue). Increased collagen is present in interalveolar septa in the area of regeneration (R) and in the exudate (E) organized by bronchiolitis obliterans. Azan stain.
