Necrotic pharyngitis associated with Mycoplasma bovis infections in American bison ( Bison bison )

Abstract

Mycoplasma bovis has emerged as a significant and costly infectious disease problem in bison, generally presenting as severe, caseonecrotic pneumonia. Three diagnostic cases in which M. bovis is associated with necrotic pharyngitis in bison are described in the current study. The bacterium was isolated from lesions of the pharynx or lung of 3 American bison (Bison bison), at 2 different locations in the upper Midwestern United States, with severe, necrotic pharyngeal abscesses. Chronic caseonecrotic inflammation typical of M. bovis infection in bovines was observed microscopically in the pharynxes of affected bison. A mixed population of bacteria was recovered from the pharyngeal lesions, and Trueperella pyogenes, a frequent secondary pathogen in ruminant respiratory disease, was consistently isolated from the affected animals. Distinctive histopathological features of the pharyngeal lesions favor causation by M. bovis, although a role for T. pyogenes in the clinical presentation cannot be excluded. Veterinarians and producers working with bison should be aware that M. bovis may be associated with pharyngitis in bison.

The major reported causes of necrotic pharyngitis in beef cattle include Fusobacterium necrophorum, Bovine viral diarrhea virus (BVDV), Bovine herpesvirus 1 (Infectious bovine rhinotracheitis virus), and trauma-induced infections of other etiologies.^4,5,7,10 A single case of pharyngitis in bison has been reported and was attributed, in part, to infection with Mycoplasma bovis. ³ Recent reports^3,6 of M. bovis infections in bison indicate that it is a significant pathogen that has a primary effect on the respiratory tract and musculoskeletal system. The current study describes a recent cluster of diagnostic cases from the upper Midwestern United States implicating M. bovis as a pathogen associated with necrotic pharyngitis in American bison (Bison bison).

The carcass of a 10-month-old bison bull calf (B1) weighing 306 kg was submitted to the Veterinary Diagnostic Laboratory at North Dakota State University (Fargo, ND) for postmortem examination in April 2012. Five animals out of 100 from this herd were reported dead. The submitting veterinarian made a tentative diagnosis of pneumonia. The calf was a purchased animal that had been dewormed and vaccinated with a 7-way clostridial vaccine. The calf was receiving pelleted feed and free choice hay. Antemortem clinical signs included shortness of breath and frothing at the mouth.

Gross postmortem observations were emaciation and abscessations in the oropharyngeal region and the liver. There was a prominent, irregular, partially encapsulated, 3 cm × 6 cm abscess containing purulent exudate in the caudal soft palate with a necrotic tract to the oral surface. Purulent exudate seeped from the tract opening. Smaller, variably sized abscesses were randomly distributed from the base of the tongue to the larynx. On the cut surface, both retropharyngeal lymph nodes were diffusely tan, necrotic, and friable. There was moderate, diffuse, bilateral pulmonary congestion. Three irregular adhesions measuring approximately 4 cm × 4 cm were present between the parietal surface of the liver and the diaphragm. Within the liver parenchyma there were several, variably sized, thick-walled abscesses containing green-tinged purulent exudate.

Salient histopathological findings included prominent, random zones of fibrosis surrounding foci of suppurative inflammation in the liver and chronic active, necrosuppurative pharyngitis. Specifically, the pharyngeal lesion was characterized by effacement of normal tissue by a distinct outer zone of variably dense fibrous connective tissue infiltrated with mononuclear inflammatory cells, a thinner middle bilayer of activated macrophages, plasma cells, and necrotic neutrophils, and an interior core of caseous necrosis. Mild lymphoplasmacytic interstitial inflammation was noted in the kidney and lung.

No ova or oocysts were noted on flotation of fecal material. Fluorescent antibody examination of frozen sections of lung was negative for Bovine herpesvirus 1 and Bovine respiratory syncytial virus. A BVDV-specific polymerase chain reaction (PCR) ⁸ was negative using nucleic acid purified from the spleen. Swabs of the lung, pharynx, and liver were each used to inoculate MacConkey II agar plates ^a and trypticase soy agar (TSA) plates containing 5% sheep’s blood. ^a Plates were incubated in a moist chamber at 37°C under atmospheric conditions. Two additional TSA plates per swab were similarly inoculated with lung, pharyngeal, or liver swabs and incubated under 5% CO₂ and 15% CO₂. Lung and pharyngeal swabs were also cultured on Mycoplasma agar ^b under 5% CO₂. Lung cultures yielded Pasteurella multocida, Bibersteinia trehalosi, Mycoplasma spp., and contaminants. Further analysis of the Mycoplasma isolate using PCR identified it as M. bovis. Pasteurella multocida, B. trehalosi, and contaminants were isolated from the pharynx and liver; Trueperella pyogenes (also known as Arcanobacterium pyogenes) was also cultured from the pharynx.

During April 2012, 2 animals, a bison heifer (B2) and a bison bull (B3) were submitted to the Animal Disease Research and Diagnostic Laboratory at South Dakota State University (Brookings, SD). The carcass of the heifer was moderately autolyzed. The primary lesion observed was a large, 7.5 cm in diameter, irregular ulcer at the base of the tongue, from which caseous exudate was expressed. In the surrounding pharyngeal tissue, there were several, variably sized abscesses, the largest of which was roughly 10 cm × 5 cm, containing caseonecrotic exudate. The carcass of the bison bull was severely autolyzed. Like the heifer, the bull had several variably sized peripharyngeal abscesses. A prominent abscess on the left tonsil, 8 cm × 4 cm, exuded purulent material into the oral cavity. No lung lesions were apparent in either B2 or B3.

Microscopic examination of lung sections from B2 showed mild, multifocal infiltration of bronchioles and alveolar spaces with neutrophils. Tissue sections from the pharynx displayed chronic, multifocal, pyogranulomatous lesions with strongly eosinophilic centers of caseonecrotic material, similar to M. bovis–induced lung lesions in cattle. Sections of the primary pharyngeal lesion showed effacement of native tissue with dense, partially encapsulated, neutrophilic aggregates (abscesses). There was locally extensive, full thickness loss of the overlying oral mucosa. No significant lesions were observed in the liver, spleen, kidney, heart, or brain. Microscopic examination of the pharyngeal lesion from B3 showed chronic abscess formation similar to that observed in the pharynx of B2. Gram-stained sections from the pharyngeal lesions of B2 and B3 showed large, rod-shaped, Gram-positive cadaver bacilli and, for B3 only, Gram-positive coccobacilli.

Fluorescent antibody analysis of lung, kidney, and spleen from B2 was negative for BVDV. Anaerobic cultures of the pharyngeal lesions from B2 and B3 yielded Clostridium perfringens and a possible Bacteroides sp. (B2) as well as possible Fusobacterium spp. (B3). Aerobic cultures of pharyngeal abscesses from B2 and B3 yielded T. pyogenes. Flotation of fecal material from both animals did not identify nematode ova or protozoal oocysts. A Mycoplasma sp. was isolated from pharyngeal abscesses of both B2 and B3. Further PCR analysis ¹ of the isolates identified them as M. bovis.

First passage M. bovis cultures from the lung of B1 and pharyngeal abscesses of B2 and B3 were forwarded to the National Animal Disease Center (NADC; Ames, IA) for further analysis. A filter-cloned isolate was derived from each culture, and all were identified as M. bovis using species-specific PCR primers. ² The PCR amplicons comprising a 770-bp fragment of the 16S ribosomal RNA (rRNA) gene from each isolate were obtained using previously reported genus-specific primers ⁹ and were sequenced at the NADC Genomics Center. Amplicon sequences of all 3 isolates were identical. The 12 highest-scoring hits from a BLAST search (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) of the National Center for Biotechnology Information nonredundant nucleotide database, all 100% identical to the query sequence, are from the 16S rRNA genes of other isolates of M. bovis.

In summary, M. bovis was recovered from 3 American bison showing pharyngeal inflammation and necrosis. The pharyngeal lesions were consistent with the caseonecrotic inflammation previously reported in bison mycoplasmal pneumonia. ³ Ancillary testing did not reveal an underlying viral cause for the pharyngeal lesions. Although M. bovis was not isolated from the pharynx of B1, its isolation from the lung and the similarity of the pharyngeal lesions to those of B2 and B3 (from which M. bovis was cultured) argues for involvement of M. bovis in the pharyngitis observed in B1. Coinfection with P. multocida in the lung of B1 may underlie the clinical signs of pneumonia observed in that calf.

Trueperella pyogenes, a common secondary opportunistic pathogen in chronic respiratory disease of ruminants, was also isolated from all 3 bison. The characteristics of the pharyngeal lesions, specifically, the caseous core and outer rim of inflammatory cells, are suggestive of causation by M. bovis rather than T. pyogenes. However, the possibility that T. pyogenes contributed to the clinical presentation, particularly as a secondary invader, cannot be ruled out.

It cannot be determined from these cases whether M. bovis was introduced as a respiratory pathogen or was a resident of the upper respiratory tract. A 2012 pilot study in which the tonsils of fifty 18-month-old slaughter bison bulls and heifers were cultured for pathogenic bacteria did not identify M. bovis (unpublished data). Although more work of this type is warranted, there is no indication that M. bovis is a normal resident of the upper respiratory tract in bison. The source of the M. bovis in the current cases, then, could be attributed to aerosol exposure or traumatic introduction to the pharynx. In either case, the bison upper respiratory tract appears to support colonization by M. bovis, with subsequent necrosis and inflammation. The nature of the lesions makes it difficult to ascertain whether M. bovis can colonize this area as a primary pathogen or requires compromise of the epithelium. There was no history of balling gun use in these animals that would have accounted for any predisposing pharyngeal trauma; however, it seems likely that epithelial erosion or ulceration would favor establishment of the organism. Producers and veterinarians should consider M. bovis as a possible contributor to diphtheritic lesions and abscesses of the larynx and pharynx in bison.