The Results of pathological anatomy
The results showed by dissect of sick sheep that the lesions were mainly in the lungs and trachea, with serious substantive fleshy lesions in the bilateral cranial lobe of the lungs (Fig. 1A), and there is a lot of foam-like mucus in the bronchus (Fig. 1B), and a lot of white mucus in the trachea (Fig. 1C). Light yellow effusion was found in the pericardium (Fig. 1D).
The Results of PCR identification and molecular evolution analysis of M. ovipneumoniae isolates
The 13 M. ovipneumoniae isolates obtained by isolation and culture were amplified by PCR using M. ovipneumoniae-specific primers, and the expected product was obtained (Fig. 2).
Phylogenetic tree based on M. ovipneumoniae-specific sequence (Fig. 3) showed that all 13 isolates belong to M. ovipneumoniae. There was the highest homology between 13 M. ovipneumoniae isolates with M. ovispneumoniae MYC022 (MK789496), M. ovipneumoniae NCTC10151 (LR215028), M. ovipneumoniae XJ-3f, M. ovipneumoniae Y-98 (NR025989). The relation between 13 M. ovipneumoniae isolates with M. bovoculi M165/69 (NR121721), M. bovoculi M165/69 (CP007154), M. conjunctivae HRC/583 (NR044781), and M. conjunctivae Goat 655 (FJ226571) was the most distant relatively.
Histopathological Results
The collected visceral tissues were paraffin sectioning and H·E staining for microscopic examination. The results were as follows:
Lung: local alveolar septum was thickened, part of bronchial epithelial cells was necrotic and exfoliated, exfoliated cell fragments and inflammatory cell infiltration were found in the bronchial lumen, mainly segmented neutrophils. A large number of inflammatory cells were infiltrated in the alveolar space, mainly neutrophils, with an increased number of macrophages, and a small number of alveolar epithelial cells with necrosis and hyperplasia. Epithelial cells proliferated around the bronchus, forming lumen-like structures, and a small amount of protein-like exudation was observed in local areas (Fig. 4A, 4B).
Trachea: mucosal epithelial cells were necrotic and shed, some epithelial cells disintegrated, the structure was blurred. mucosal lamina propria became edema with a small amount of inflammatory cell infiltration. The intercellular space widened, and there was an uneven amount of inflammatory cell infiltration in the interstitium, some of which formed inflammatory infiltration foci. The inflammatory cells were mainly lymphocytes, but plasma cells and a small number of neutrophils were also can be seen (Fig. 4C, 4D).
Bronchi: a few cells of the mucosa epithelium were necrotic, and the cytoplasm of the epithelium cells was vacuolated and the nucleus was contracted. A small number of inflammatory cells, mainly lymphocytes, can be seen locally in the superficial layer of lamina propria (Fig. 4E, 4F).
Immunohistochemical Test Results
Through immunohistochemical analysis of different tissues, the results showed that M. ovipneumoniae antigen was present in pathological sections of all visceral tissues, but the organs or tissues where M. ovipneumoniae was mainly distributed were lung, bronchus and trachea. Among them, the average proportion of M. ovipneumoniae positive area in the lung, bronchus and trachea was 5.30%, 3.87%, and 0.78% respectively (Fig. 5).
Immunohistochemical sections of lung showed alveolar cells, collapse, exfoliation and necrosis, and thickened alveolar septum. The area of lung tissue in intercellular and visual field was about 196603.10µm2, and the total area of M. ovipneumoniae positive staining was about 11704.44µm2. The positive area accounted for about 5.95% (Fig. 6A).
Immunohistochemical analysis of bronchial sections showed that the cells in the upper bronchial lumen were exhaled and necrotic, and a large amount of M. ovipneumoniae was attached to the bronchial lumen, and a large amount of M. ovipneumoniae was colonized in the cytoplasm of epithelial cells. The bronchial tissue area in the visual field was about 196607.90µm2, and the total area with M. ovipneumoniae positive staining was about 10946.44µm2, accounting for about 5.57% of the positive area (Fig. 6B).
Epithelial cells were exfoliated in immunohistochemical sections of the trachea, and M. ovipneumoniae was found both on the surface of the epithelial cells and in the cytoplasm. The tracheal tissue area in the visual field was about 196608µm2, and the total M. ovipneumoniae positive staining area was about 2796.5µm2, accounting for about 1.42% of the positive area (Fig. 6C).
Cytokine Transcription Level
By using qRT-PCR to detect the relative expression levels of cytokine mRNA in trachea, bronchus, lung and its different sites. The results showed that the expression levels of IL-1β mRNA in trachea, bronchus and lung were increased, and the increasing degree was on the rise from trachea to apical lobe of lung and on the decline from apical lobe to phrenic lobe of lung. IL-1β mRNA levels were positively correlated with the degree of lung lesions (Fig. 7A). The mRNA expression of IL-6 increased in trachea, bronchus and apical lobe, cardiac lobe of lung, with the most abundant expression in the bronchus. (Fig. 7B). The mRNA expression of IL-10 and IL-12 increased in trachea and bronchus, with the highest expression in trachea (Fig. 7C, 7D). The mRNA expression of TNF-α increased in bronchi and all lobe of lung, with the highest expression in apical lobe of lung (Fig. 7E). The expression of IFN-γ mRNA increased in trachea and bronchus, with the highest expression in bronchus (Fig. 7F). The mRNA of NF-κB increased in trachea, bronchus and lung, with the highest expression in bronchus (Fig. 7G).
After infection with M. ovispneumoniae, mRNA expression of up-regulated cytokine in tracheal tissues included IL-1β, IL-6, IL-10, IL-12, IFN-γ and NF-κB; TNF-α was down-regulated (Fig. 8A). Cytokines up-regulated in bronchus included IL-1β, IL-2, IL-10, IL-12, TNF-α, IFN-γ and NF-κB (Fig. 8B). IL-1β, TNF-α, IFN-γ and NF-κB were up-regulated expression in apical lobe of lung (Fig. 8C); IL-1β, IL-6, TNF-α, IFN-γ and NF-κB were up-regulated expression in cardiac lobe of lung (Fig. 8D); IL-1β, TNF-α and NF-κB were up-regulated in phrenic lobe of lung (Fig. 8E).