Clinical cases were initially reported in lots A (9 cases) and B (13 cases). The first two cases occurred during December 2018, 1 on January, 9 during February and 10 during March 2019. All the affected calves (24 out of 545; morbidity = 4.4%) showed progressive, subacute to chronic respiratory distress, coughing, hyperpnea, poor body condition, lethargy, dehydration and fever (39.5 to 41.3ºC). Thirty percent of the affected calves also showed lameness, grinding noise when they walk, and pain in one or more joints associated with visible swelling of the affected front (Fig. 1a) and hind leg joints (elbow or carpal, and knee or tarsal joints, respectively).
Post mortem examinations were performed in five affected calves. Fibrinonecrotic arthritis and tenosynovitis were detected in the affected joints (Fig. 1b). The lungs of the five calves showed cranioventral consolidation affecting 20 to 50% of the pulmonary parenchyma. All the examined lungs showed multifocal to coalescent white nodules, some of them protruding above the pleural surface (Fig. 1c). On section, white nodules ranged 0.3 to 2.5 cm and contained white-yellowish caseous exudate, encircled by fibrous tissue (Fig. 1d). Pleural fibrosis was observed in one of the examined animals (Fig. 1e) and chronic pleural adhesions in two calves. No other gross lesions were observed in the affected necropsied calves.
Histologically, multifocal necrotic areas in the pulmonary parenchyma were observed. These foci contained many necrotic inflammatory cells that retained their cellular outlines but had intensely eosinophilic cytoplasm and lysis of the nuclei (Fig. 2a and 2b). These foci were delineated by a band of neutrophils and macrophages, encircled by a layer of fibroblasts macrophages, lymphocytes, and plasma cells. The smaller bronchioles contained an accumulation of necrotic leukocytes in the bronchiolar lumen and the epithelium was discontinuous. The bronchiolar walls were thickened by edema and infiltrate of lymphocytes with fewer neutrophils and macrophages. Similar foci of caseous necrosis that contained recognizable necrotic leukocytes were occasionally present in the alveoli. Macrophages and scarce plasmocytes were infiltrating the interlobular septa. Follicular hyperplasia was observed in mediastinal lymph nodes. In joint capsule, fibrin admixed with many neutrophils was adherent to the synovium, with areas of hyperplasia alternating with areas of necrosis or denudation of synoviocytes. The subsynovial stroma had severe infiltration of many neutrophils, macrophages, lymphocytes, and plasma cells; and prominent fibroblast hyperplasia (Fig. 2c). No other microscopical lesions were detected in the affected calves.
Immunohistochemistry showed abundant M. bovis antigen in the lungs and joint capsule of calves. In lungs, the positive staining was observed mainly at the margin of the necrotic lesions, and to a lesser extent in the center of the necrotic foci. In bronchioles containing caseous debris, antigen was present within the debris and adjacent to bronchiolar epithelial cells (Fig. 2d). Antigen of M. bovis was identified in synovium and stroma subsynovial within the debris and adjacent neutrophils and macrophages. Staining for M. bovis antigen was not visible in the sections of negative controls.
Typical Mycoplasma fried-egg-shaped colonies were observed in all the lung samples from the five calves (Fig. 2e). In none of the synovial fluid samples compatible-Mycoplasma colonies were observed. Lung and synovial fluid sampled during the necropsies of calves #1, #2, #3 and #4 resulted negatives for the isolation of aerobic and microaerophilic bacteria using the routine diagnostic procedures. Trueperella pyogenes and H. somni were isolated from calf #5 lung sample. No AFB were observed in the ZN staining of lung smears.
The lung samples from all calves and synovial samples from calves #1, #3, and #4 rendered PCR positive results. The presence of the agent was also confirmed in the lung cultures. The 16S-23S rRNA ITS consensus sequence obtained showed 100 % similarity with the same region of M. bovis strains NADC59 (CP042939.1), MJ1 (CP042938.1), KG4397 (AP019558.1), NADC61 (CP022599.1), NADC67 (CP022596.1), NADC62 (CP022595.1), NADC58 (CP022594.1), NADC57 (CP022593.1), NADC56 (CP022592.1), NADC55 (CP022591.1), NADC54 (CP022590.1), NADC18 (CP022589.1), MJ4 (CP022588.1), MJ3 (CP022587.1), MJ2 (CP022586.1), JF4278 (LT578453.1), Ningxia-1 (CP023663.1), 08M (CP019639.1), 72242 (KX687011.1), 393B08 (KX687010.1), 268B07 (KX687009.1), HB0801-P115 (CP007589.1), NM2012 (CP011348.1), 1982-M6152 (CP058969.1), 2019-043682 (CP058968.1), PG45 (CP002188.1), 70-213 (AY779747.1) and ATCC 25523 (AY729934.1).
BoHV-1, BPIV-3, BRSV and BVDV isolation resulted negative in the lung samples tested.