Two (one male and one female) 3-week-old, Holstein calves (Bos taurus).These two calves had been housed in separate calf pens in a calf barn with 14 other calves. The calves were fed 2 liters of milk replacer twice per day and heifer calves received Deccox-ï¿½ in the milk daily. The calves received Calf-Guard-ï¿½, Colimune-ï¿½ and 3 to 4 liters of colostrum at birth. These two calves became ill at 7 to 10 days of age with mildly elevated temperature, poor drinking behavior and scours and subsequently developed abnormal breathing. Despite treatment with antibiotics (Nuflor-ï¿½) and tube feeding of milk and electrolytes, the calves became progressively weaker and died.
Both calves exhibited mildly sunken eyes with a moderate reduction of internal fat stores.Â The lungs of both calves exhibited dark red to plum purple lobular discoloration of the intermediate lobes, anteroventral aspect of the diaphragmatic lobes and ventral aspect of the anteroventral lobes.Â Affected lobules were depressed, firm on palpation, and on cut surface exhibited dark red to plum purple discoloration with purulent exudate in airways.Â The female calf also exhibited mild fibrin exudation over the pleural surface of the discolored lobes, fibrinous adhesions of these lobes to the pericardial sac and increased clear yellow fluid in the pericardial sac with fibrin strands.Â Intestinal content was watery.
There was moderate to marked pulmonary lobular consolidation characterized by macrophages and neutrophils intermixed with edema fluid, red blood cells and fibrin strands within alveolar spaces and airway lumina.Â Variably sized, multifocal aggregates of highly condensed leukocytes with nuclear streaming obscured parenchymal architecture and were observed throughout the sections.Â These foci were often associated with colonies of coccobacillary bacteria, hemorrhage and fibrin.Â Depending on the section examined, foci of bronchiolar epithelial necrosis and attenuation, with and without syncytical cells, were occasionally observed.Â Interlobular interstitium and lymphatics were mildly to markedly dilated with edema fluid, fibrin strands and a mild to moderate number of neutrophils and macrophages.Â Fibrin was focally present on the pleural surface and there were subpleural inflammatory infiltrates similar to the interlobular interstitium.Â There were scattered areas of atelectasis.
Lung, bronchopneumonia, anteroventral, marked, subacute, fibrinosuppurative with intralesional coccobacilli and focal bronchiolar necrosis with syncytial cells.
Mannheimia granulomatis, 4+, in lungs of both calves; Mannheimia granulomatis, few, in pericardial sac of female calf; bovine respiratory syncytial virus (BRSV) positive on lung by PCR and immunohistochemistry; rotavirus and bovine coronavirus positive on intestinal contents by PCR.
BRSV; Mannheimia granulomatis
Pasteurella multocida and Mannheimia hemolytica are the most common Pasteurellaceae bacteria isolated from bovine pneumonia.(3) These organisms are commensals of the bovine nasopharynx which, during periods of stress or viral infection, can overwhelm host defense mechanisms establishing infection in the lower respiratory tract.(3) M.Â granulomatis is one of five recognized species of the genus Mannheimia.(1) This bacterium has been reported in association with oral abscesses and suppurative bronchopneumonia in Australian cattle (1), purulent bronchopneumonia, pleuritis, stomatitis and abscesses in Danish Roe deer (2), purulent bronchopneumonia and conjunctivitis in European Brown hares (5) and a fibrogranulomatous panniculitis in Brazilian cattle known as lechiguana.(6)
The bronchopneumonia in the two Holstein calves reported here was similar to pneumonia described with M.Â haemolytica infection in that there was significant leukocyte necrosis as evidenced by oat cells, widespread accumulation of edema fluid, neutrophils, macrophages, fibrin, foci of hemorrhage and bacterial aggregates within alveolar spaces and airway lumina and interlobular septae were distended with serofibrinous exudate.(3) A similar lesion to M.Â haemolytica would be anticipated as it has been shown that M.Â granulomatis has leukotoxin activity in vitro.(2,7) However, large areas of coagulation necrosis surrounded by a thick rim of leukocytes, as is frequently observed with M.Â haemolytica pneumonia (3), were lacking in the M.Â granulomatis lesion.Â These observations suggest that, although both M.Â granulomatis and M.Â haemolytica share virulence factors (2,7), there may be subtle differences in pathological expression.Â However, more cases of M.Â granulomatis pneumonia would need to be examined before any conclusions could be drawn with regard to expression of virulence.Â
These calves also had focal infection with BRSV which was confirmed by PCR and immunohistochemistry.Â Viral BRSV lesions were not present in all tissue sections submitted.Â Pulmonary BRSV infection likely predisposed these calves to bacterial pneumonia.Â While P.Â multocida and M.Â haemolytica are reported to be commensal in the bovine nasopharynx (3), it is unclear how these two calves became infected with M.Â granulomatis.Â
Lung: Bronchopneumonia, fibrinosuppurative, multifocal to coalescing, marked, with edema, hemorrhage, and coccobacilli.
The conference moderator emphasized the importance of additional diagnostic testing when assessing the histomorphology present in this case; the two most likely etiologies include P.Â multocida and M.Â haemolytica, as noted by the contributor, and definitive determination necessitates additional diagnostics, such as microbial culture, immunohistochemistry, in situ hybridization, and PCR, as employed in this case.
Leukotoxin, mentioned by the contributor, is an important virulence factor of M.Â haemolytica.Â A member of the repeats in toxin (RTX) family, leukotoxin is secreted during the log phase of growth, and its receptor is CD18.(3) At low concentrations, leukotoxin activates bovine platelets and leukocytes or induces their apoptosis (4), while at high concentrations it induces leukocyte lysis, resulting in the characteristic nuclear streaming noted microscopically in areas of necrosis.(3) Lipopolysaccharide, another important virulence factor, is synergistic with leukotoxin because it induces increased expression of Î²2-integrins on leukocytes which contain the CD18 receptor.Â Other important bacterial virulence factors include a polysaccharide capsule that assists in attachment and impairs phagocytosis, iron-regulated outer membrane proteins that bind transferrin and alter neutrophil function, adhesins that mediate attachment, and neuraminidase that decreases both respiratory mucus viscosity and repellant negative charge on host cells.Â Cytokines TNF-Î±, IL-1Î², and IL-8 are particularly important components of the complex inflammatory milieu produced in pneumonic mannheimiosis.(8)
Bovine parainfluenza virus 3, bovine herpesvirus 1, and BRSV all increase susceptibility to M.Â haemolytica, and presumably other respiratory pathogens, by infecting ciliated epithelium and reducing mucociliary clearance; the latter two also infect and impair alveolar macrophages.Â Of note, bovine herpesvirus 1 is also thought to increase susceptibility to the effects of M.Â haemolytica by upregulating CD18 expression in neutrophils.(3)
As mentioned by the contributor, there is some slide variation with respect to the presence of viral syncytia.Â In a few sections, participants noted viral syncytia in bronchioles with multifocal bronchiolar epithelial necrosis, consistent with bronchointerstitial pneumonia due to BRSV infection.Â Additionally, lymphatic vessel lumina within interlobular septa are multifocally occluded by coagula of fibrin and mixed inflammatory cells.Â There are rare intravascular megakaryocytes in some sections, which participants speculated may be associated with terminal bone marrow hypoxia.Â Intravascular megakaryocytes in the human lung were first described in 1893, and it has since been demonstrated that under normal conditions, intact megakaryocytes leave the bone marrow and reach the pulmonary capillary bed, where they release platelets by fragmentation of their cytoplasm.(9)
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