History
In late March to mid-April 2015, 2 young adult Angus-cross heifers were separately submitted for postmortem examination. Both heifers were from the same beef herd in southern Georgia. In this herd, multiple losses due to respiratory tract disease had occurred over a period of 1 month.
Clinical and Gross Findings
At necropsy, the heifers were in good body condition with minimal to mild postmortem autolysis. The heifers weighed 205.0 kg (451.0 lb; heifer A) and 205.5 kg (452.1 lb; heifer B). Gross findings were similar for both animals. Bilaterally, the nasal cavity contained mild to moderate amounts of white to light yellow, suppurative exudate. A similar exudate was present overlying the tracheal mucosa. Within the thoracic cavity, the lungs were mottled dark red to purple. The ventral surfaces of the right and left cranial lung lobes were nodular with a firm texture. Nodular foci in the pulmonary parenchyma were 0.5 to 3.0 cm in diameter; on cut surface, they were filled with marked amounts of yellow- to light-green–tinged purulent and caseous material (Figure 1). Sections of the lungs sank in neutral-buffered 10% formalin. In heifer B, the tracheobronchial lymph nodes were prominent and mildly enlarged. No additional notable findings were observed in either carcass. Samples from all major organ systems were fixed in neutral-buffered 10% formalin and routinely processed for histologic examination. Fresh lung and spleen tissue samples were submitted for virologic analysis; fresh lung tissue and trachea samples were submitted for microbial culture.
Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page →
Cross-sectioned portion of a cranial lung lobe of 1 from 2 young adult Angus-cross heifers that were submitted for postmortem examination. Both animals were from a beef herd in which there had been multiple deaths as a result of respiratory tract disease during the preceding 1-month period. Notice that the lung tissue is expanded by a suppurative exudate and a central area of caseous necrosis.
Citation: Journal of the American Veterinary Medical Association 252, 3; 10.2460/javma.252.3.291
Histopathologic Findings
Formalin-fixed sections of paraffin-embedded tissue from all major organ systems of both heifers were routinely stained with H&E stain. The most notable tissue changes involved the respiratory tract. In all lung tissue sections, the pulmonary parenchyma was disrupted by multifocal coalescing areas of necrosis and inflammation (Figure 2). These areas were centered on medium to large airways. In these areas and spreading out from the airways were lakes (often massive) of eosinophilic granular material admixed with necrotic cellular debris and moderate to high numbers of degenerate neutrophils (Figure 3). These necrotic foci obscured large regions of the parenchyma and associated alveoli. The foci were frequently bordered by thin layers of fibrous connective tissue and a low- to moderate-density rim of macrophages and epithelioid macrophages. Larger areas of necrosis contained small lakes of mineralized debris. In the lesser affected regions, lumina of small to medium-sized bronchioles were distended with similar accumulations of necrotic cellular debris and degenerate neutrophils (Figure 4). Nearby alveolar spaces were filled with moderate to high numbers of degenerate neutrophils and macrophages. Lesser-affected alveoli contained eosinophilic fibrillar to hyalinized material (compatible with fibrin). The interlobular septa were separated by eosinophilic material admixed with small amounts of karyorrhectic debris.
Photomicrograph of a section of the affected lung from the heifer in Figure 1. Multifocal areas of inflammation and necrosis extend into small airways and alveoli. H&E stain; bar = 500 μm.
Citation: Journal of the American Veterinary Medical Association 252, 3; 10.2460/javma.252.3.291
Photomicrograph of the same section of the affected lung from the heifer in Figure 1. In this view, there is a necrotic region bordered by neutrophilic inflammation. H&E stain; bar = 100 μm.
Citation: Journal of the American Veterinary Medical Association 252, 3; 10.2460/javma.252.3.291
Photomicrograph of the same section of the affected lung from the heifer in Figure 1. A bronchiole is filled with degenerate neutrophils, macrophages, and karyorrhectic debris. H&E stain; bar = 200 μm.
Citation: Journal of the American Veterinary Medical Association 252, 3; 10.2460/javma.252.3.291
In sections of the nasal cavity, the submucosa was expanded by a mild, multifocal, perivascular inflammatory infiltrate of lymphocytes, plasma cells, and neutrophils. Among the areas of inflammation were low numbers of individual necrotic cells. The deeper submucosa contained moderate amounts of maturing granulation tissue with occasional islands of woven bone. Similar submucosal inflammatory changes were observed in tracheal sections.
Other minor histologic changes included mild to moderate lymphoid hyperplasia of the tracheobronchial lymph node and mild lymphocytic and neutrophilic portal hepatitis in the liver of heifer B. No other noteworthy histologic findings were observed in heifer A.
Microbial Culture Results
For both heifers, aerobic bacterial culture of lung and trachea samples yielded heavy growth of Mannheimia hemolytica and, for cow B, a few colonies of Pasteurella multocida. Mycoplasma culture of lung tissue samples from both heifers yielded growth of Mycoplasma sp. Results of PCR analysis of these colonies confirmed Mycoplasma bovis in both cases.
Immunohistochemical Findings
Immunohistochemical analysis to detect M bovis antigen in lung sections revealed weak positivity for M bovis among necrotic cellular debris and moderate cytoplasmic positivity in macrophages. Immunohistochemical analysis of trachea sections revealed weak to moderate positivity for M bovis in the airway epithelium, submucosal glands, and macrophages and among necrotic cellular debris.
Virological Analysis Results
Fluorescent antibody testing was performed on lung and spleen tissue samples from both heifers to detect bovine herpesvirus 4, bovine herpesvirus 1 (causative agent of infectious bovine rhinotracheitis), bovine respiratory syncytial virus, parainfluenza-3 virus, and bovine viral diarrhea virus. Heifer B was negative for all viral agents, whereas heifer A was positive for bovine herpesvirus 4 and parainfluenza-3 virus.
Morphologic Diagnosis and Case Summary
Morphologic diagnosis: severe, subacute to chronic, multifocal to coalescing, caseonecrotic, neutrophilic, and histiocytic bronchopneumonia; and mild to marked, subacute to chronic, multifocal, necro-ulcerative lymphocytic-plasmacytic, and neutrophilic rhinotracheitis (affecting the nasal cavity and trachea).
Case summary: Mycoplasma bronchopneumonia in 2 heifers.
Comments
In the heifers of the present report, the gross and histologic patterns of caseonecrotic bronchopneumonia were characteristic of M bovis infection.1,2 The diagnosis was confirmed on the basis of the results of microbial culture, immunohistochemical analysis, and PCR assay testing of lung tissue and trachea samples. Mycoplasma bovis is an important respiratory pathogen in bovids, predominantly affecting young calves and feedlot cattle1,3; it is an emerging pathogen of American bison.4,5 Along with pneumonia, this organism is a causative agent of arthritis, tenosynovitis, otitis, mastitis, and conjunctivitis.1,2,4,6 Indeed, in southern Georgia, this mycoplasma is a common component of the bovine respiratory disease complex; between 1999 and 2013, M bovis was detected immunohistochemically in 235 of 378 (62.1%) lung sections from cattle with bronchopneumonia.a Young calves typically become infected via ingestion of contaminated milk, whereas feedlot cattle often acquire M bovis infection through direct contact (eg, nasal secretions) with infected animals.1
Mycoplasma bovis is capable of adhering to the epithelium of the upper respiratory tract and is adept at evading the host immune response. The organisms adhere to epithelial cells via variable surface lipoproteins. In the presence of specific host antibodies to such proteins, these bacteria are capable of altering expression of these surface lipoproteins, thereby evading the humoral immune response by the host.1–3,5 In addition, M bovis has the capability to induce lymphocyte apoptosis and inhibit neutrophil activity, allowing for further immune system evasion.1–3 The exact mechanism of M bovis–induced host tissue injury is not fully elucidated, but may be associated with oxidative injury from peroxides produced by the pathogen.1,6
Clinically, M bovis–infected animals are often dyspneic and febrile (rectal temperature, > 40°C [> 104°F]) and have a persistent cough with a mucopurulent nasal discharge and signs of depression. Lameness and swollen joints can be evident in animals with concurrent arthritis.1,2
In cattle infected with M bovis, the classic gross respiratory tract lesion is a suppurative bronchopneumonia that predominantly involves the cranial and ventral areas of the lungs with variably sized, nodular foci of caseous necrosis in the lung parenchyma, as was detected in the heifers of the present report. Other concurrent gross lesions include suppurative exudates in the nasal cavity and trachea, enlargement of tracheobronchial lymph nodes, and possible evidence of arthritis or tenosynovitis.1,2 Differential diagnoses for these gross pulmonary lesions would be infection with other bacteria commonly associated with the bovine respiratory disease complex such as M hemolytica, P multocida, Histophilus somni, and Trueperella pyogenes.
Histologically, these gross lesions correspond to foci of necrosis with neutrophilic infiltrates originating from the airways. Eventually these areas spread into the parenchyma to form large, often massive, lakes of necrotic cellular debris admixed with degenerate neutrophils. Such necrotic foci are typically bordered by a thin rim of fibrous tissue intermingled with macrophages and neutrophils.1,2 Upper respiratory tract lesions typically consist of superficial areas of neutrophilic and lymphocytic inflammation, often with areas of mucosal erosion or ulceration and associated fibrinonecrotic exudates.
If M bovis infection is suspected clinically, grossly, or histologically, various diagnostic tests are available for confirmation. Cultures for Mycoplasma spp can be performed on lesion samples, and the culture results can be augmented with PCR assay findings to determine the species of Mycoplasma involved. Additional diagnostic tests include immunohistochemical analysis and PCR assay of fresh tissue samples.1,2 Also, assessment of serum concentration of antibody against M bovis (eg, by use of an indirect ELISA) can be performed to detect exposure in individuals or groups of animals.1,5
Mycoplasma bovis can act as a primary lung pathogen. However, in most cases, it is present in conjunction with other respiratory pathogens, either viral (eg, bovine herpesvirus 1, bovine respiratory syncytial virus, or parainfluenza-3 virus) or bacterial (eg, M hemolytica, P multocida, or H somni). It is suspected that M bovis may act synergistically with M hemolytica, in that M hemolytica initiates the pulmonary lesions and M bovis exacerbates the foci of caseous necrosis.1,5 In the 2 heifers of the present report, bacterial culture of lung tissue samples yielded heavy growth of M hemolytica, indicative of such a co-infection. Whether M bovis was the primary pathogen with secondary M hemolytica involvement, or vice versa, could not be determined. In heifer A, parainfluenza-3 virus was detected in the lung tissue samples by fluorescent antibody testing; such a viral infection may have predisposed this animal to bacterial pneumonia. Rhinotracheitis in both heifers raised concern for an initiating herpesvirus infection (bovine herpesvirus 1), which could not be fully excluded. However, no intranuclear inclusions were identified histologically, and bovine herpesvirus-1 was not detected by fluorescent antibody testing, thereby decreasing the likelihood of an active herpesviral infection. In addition, the immunohistochemical findings for the sections of trachea from both animals indicated that M bovis was actively contributing to the tracheitis. Because bacteria in the Mycoplasma group lack cell walls, they are resistant to many antimicrobials commonly used for bacterial respiratory tract infections in cattle, namely those in the β-lactam group (eg, ampicillin and ceftiofur).1,5 Also, M bovis has been shown to be resistant to florfenicol, tilmicosin, and oxytetracycline.1,2 Therefore, animals with M bovis pneumonia often do not respond well to treatment. Some antimicrobials (eg, enrofloxacin and spectinomycin) have excellent in vitro activity against M bovis1,2; however, such antimicrobials may have reduced tissue penetration in large foci of caseous necrosis within the lungs.1
Mycoplasma bovis cannot be grown with routine culture techniques. Therefore, for animals with caseonecrotic bronchopneumonia or clinical signs suggestive of M bovis infection (eg, chronic pneumonia with polyarthritis), Mycoplasma-specific culture of appropriate samples should be requested to confirm or possibly exclude M bovis involvement. One should be aware that molecular diagnostic testing (ie, PCR assay) is typically required to determine the species of Mycoplasma involved.
Acknowledgments
The authors thank Dr. Murray E. Hines II for technical assistance and Dr. Alison H. Thornhill for case submissions.
Footnotes
Turnquist S, Rajeev S. A retrospective analysis of Mycoplasma bovis lung infection in dairy and beef cattle with pneumonia (abstr), in Proceedings. Am Assoc Vet Lab Diagnosticians Annu Conf 2014;181.
References
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