Affected Ferrets

In April 2009, a 2-year-old, spayed female ferret at the ferret rescue and shelter, which had originated from the breeding facility in Canada and passed through the US distribution center, became acutely dyspneic and died within 15 minutes. The ferret had shown signs of respiratory disease since arrival at the shelter. Previously at the shelter, 2 ferrets, 4.5 years of age, had shown chronic cough; 1 died of dyspnea in October 2010, and the other was euthanized for humane purposes in November 2010. Both had originated from the breeding facility and passed through the distribution center. Postmortem examinations were performed on all 3 ferrets. After the 2-year-old ferret died in April 2009, BAL samples and ocular swabs were obtained in July 2009 from 3 other ferrets with a history of respiratory disease since their arrival at the ferret shelter. For further diagnostic investigation, BAL samples and ocular swabs were collected from 9 additional affected ferrets in January 2010; one of these was the ferret that died in October 2010.

Survey of Healthy Ferrets

At a large commercial breeding facility in which signs of respiratory disease had not been observed, BAL samples were obtained from 10 euthanized healthy male ferrets, 5 weeks to 5 years of age. Before postmortem examination, samples were collected from the euthanized ferrets by BAL through an incision in the caudal trachea. Nonbacteriostatic saline (10 mL/kg) was flushed into the caudal trachea and lungs and then recovered by aspiration into the syringe. That process was repeated 2× and the final flush fluid was submitted for bacterial culture. Complete postmortem examinations were performed, and sections of lung were collected for bacterial and mycoplasma culture. Additional tissue samples were collected from the lungs, trachea, nasal turbinates, brain, liver, kidneys, spleen, stomach, small and large intestine, thoracic and mesenteric lymph nodes, pancreas, and adrenal glands for routine histopathologic examination.

Histologic and Immunohistochemical Analyses and Confocal Microscopy

From the 3 ferrets that died April 2009–November 2010, postmortem tissue samples (lungs, trachea, nasal turbinates, brain, liver, kidneys, spleen, stomach, small and large intestine, thoracic and mesenteric lymph nodes, pancreas, and adrenal glands) were collected. They were fixed in neutral-buffered, 10% formalin solution and processed by standard methods for histopathologic examination.

For immunohistochemical examination, paraffin-embedded samples of lung from the 3 ferrets that died were cut into 5-μm sections. An Enhanced Alkaline Phosphatase Red Detection Kit (Ventana Medical Systems, Inc., Tucson, AZ, USA) and bulk buffers specifically designed for use on the BenchMark Automated Staining System (Ventana Medical Systems, Inc.) were used for immunolabeling. Slides were baked in a drying oven at 60°C for 20 min, barcode labeled, and placed in the BenchMark for deparaffinization and heat-induced epitope retrieval. Slides were then incubated with a mouse monoclonal antibody against mycoplasma (primary antibody) (Chemicon, Billerica, MA, USA) at a concentration of 1:100 for 30 min. The monoclonal antibody was raised against M. bovis strain M23, but it is known to cross-react with numerous other mycoplasma species.

The slides were counterstained by using hematoxylin (Ventana Medical Systems, Inc.), then dehydrated, cleared, and mounted. For a positive control, we used formalin-fixed, paraffin-embedded sections of lung from an M. bovis–positive cow (tested by bacterial culture). For negative controls, we replaced the primary antibody with homologous nonimmune serum.

A Zeiss 510 microscope (Jena, Germany) was used for confocal imaging to acquire fluorescent images, and the Zeiss LSM image analysis software was used for characterizations. The images represented a differential interference contrast/Nomarski image with green (488 nm, argon laser excitation, fluorescein isothiocyanate [FITC]) and red (543 nm, rhodamine, helium–neon excitation, tetramethylrhodamine-5- [and 6-] isothiocyanate [TRITC])–labeled overlay to demonstrate localization of labels, as described, with slight modified according to Ubels et al. (12).

Transmission and Scanning Electron Microscopy

For transmission electron microscopy, lung tissue samples that had been fixed in neutral-buffered, 10% formalin solution were trimmed into 2-mm pieces and postfixed in 1% osmium tetroxide in 0.1 M sodium phosphate buffer for 2 h. Tissues were serially dehydrated in acetone and embedded in Poly/Bed 812 resin (Polysciences Inc., Warrington, PA, USA) in flat molds. Sections were obtained with a Power Tome XL ultramicrotome (Boeckeler Instruments, Tucson, AZ, USA). To identify areas of interest, we stained semithin (0.5-μm) sections with epoxy tissue stain and examined them under a light microscope. Then we cut ultrathin (70-nm) sections, mounted them onto 200-mesh copper grids, stained them with uranyl acetate and lead citrate, and examined them under a 100 CXII transmission electron microscope (JEOL, Peabody, MA, USA).

For scanning electron microscopy, formalin-fixed lung tissues were trimmed into 2–4-mm pieces, postfixed for 1 h in 1% osmium tetroxide, and rinsed for 30 min in 0.1 M sodium phosphate buffer. Tissues were serially dehydrated in ethanol and dried in a critical point dryer (Model 010; Balzers, Witten, Germany) with liquid carbon dioxide as the transitional fluid. Samples were mounted on aluminum studs by using carbon suspension cement (SPI Supplies, West Chester, PA, USA). Samples were then coated with an osmium coater (NEOC-AT; Meiwa Shoji Co., Tokyo, Japan) and examined in a JSM-7500F (cold field emission electron emitter) scanning electron microscope (JEOL).

Bacterial Cultures

We submitted 12 BAL samples and 12 ocular swab samples for bacterial and mycoplasma culture by standard microbiologic techniques. The samples were from live ferrets that originated from the distribution center and showed clinical signs of respiratory disease, including coughing. We also submitted 10 BAL samples from 10 healthy ferrets from a different commercial breeding facility not affected by respiratory disease.

PCR and Sequence Analysis

Only mycoplasmas obtained from BAL samples were analyzed by PCR and nucleic acid sequencing. A plug of agar containing Mycoplasma spp. colonies was gouged from the surface of a mycoplasma agar plate by using a 10-μL disposable inoculation loop and transferred to a microcentrifuge tube. The agar plug was digested by addition of 200 μL of Buffer ATL (QIAGEN, Valencia, CA, USA) and 20 μL of proteinase K solution (QIAGEN), followed by overnight incubation at 56°C. DNA was extracted from the digest by using a DNeasy Blood and Tissue kit (QIAGEN) according to manufacturer’s instructions.

For PCR, we used 2 sets of primers selective for the bacterial 16S rDNA or the mycoplasma RNA polymerase B (rpoB) gene. The nucleic acid sequences for the 16s rDNA gene were 5′-AGAGTTTGATCMTGGCTCAG-3′ for the forward primer and 5′-GGGTTGCGCTCGTTR-3′ for the reverse primer; this primer set produced an amplicon of ≈1,058 bp. The nucleic acid sequences for the mycoplasma rpoB gene were 5′- GGAAGAATTTGTCCWATTGAAAC-3′ for the forward primer and 5′- GAATAAGGMCCAACACTACG-3′ for the reverse primer; this primer set produced an amplicon of ≈1,613 bp. The PCRs were performed by using Platinum Taq DNA Polymerase High Fidelity (Invitrogen Corp., Carlsbad, CA, USA). The reaction mixture consisted of 3 μL DNA; 1 unit of Platinum Taq DNA Polymerase High Fidelity; 60 mmol/L Tris-SO4 (pH 8.9); 18 mmol/L ammonium sulfate; 2 mmol/L magnesium sulfate; 0.2 mmol/L each of dATP, dCTP, dGTP and dTTP; 16.9 μL molecular biology grade water; and 0.5 µmol/L each of the PCR primer. The reaction conditions for the 16s rDNA gene were 1 cycle at 94°C for 4 min; 35 cycles at 94°C for 30 s, 58°C for 45 s, 68°C for 75 s; followed by a final extension step at 68°C for 5 min. The reaction conditions for the rpoB gene were 1 cycle at 94°C for 4 min; 40 cycles at 94°C for 45 s, 55°C for 45 s, 68°C for 90 s; followed by a final extension step at 68°C for 5 min.

The PCR products were stained with ethidium bromide and examined after electrophoresis through a 1.5% agarose gel. The PCR amplicons were excised from gels, purified by using the QIAquick Gel Extraction Kit (QIAGEN), and submitted to the Research Technology Support Facility at Michigan State University for nucleic acid sequencing. Several internal primers were designed to derive the complete sequences of the PCR amplicons. The derived sequences were edited by using Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA) and analyzed by using BLAST (www.ncbi.nlm.nih.gov/blast/Blast.cgi).

The nucleic acid sequences of the mycoplasma isolates and sequences from other Mycoplasma spp. obtained from GenBank were imported into the MEGA4 program (www.megasoftware.net), aligned by using ClustalW in the MEGA4 program, and subjected to phylogenetic analyses. For each isolate analyzed, 933 bp of the 16S rDNA gene sequence and 733 bp of the rpoB gene sequence were available. Phylogenetic trees were constructed by using the neighbor-joining method; data were resampled 1,000× to generate bootstrap percentage values.

Gross and Histologic Lesions

Figure 2

Figure 2. . . Lungs from a 2-year-old ferret that died of acute dyspnea, showing multifocal, tan to gray semifirm nodules centered on airways and severely narrowed lumina of affected airways.

Figure 3

Figure 3. . . Micrographs of a section of lung from a 2-year-old ferret that died of acute dyspnea. A) Image shows moderate bronchointerstitial pneumonia with severe hyperplasia of bronchiole-associated lymphoid tissue around...

Gross and histologic lesions from the 3 ferrets that died or were euthanized because of respiratory disease were similar and restricted to the lungs. The lungs were characterized by multifocal, tan to gray, somewhat firm nodules centered on airways randomly distributed throughout the pulmonary parenchyma (Figure 2). Hematoxylin and eosin–stained lung sections revealed a moderate bronchointerstitial pneumonia with severe bronchiole-associated lymphoid tissue (BALT) hyperplasia (Figure 3, panel A). BALT hyperplasia was commonly associated with marked narrowing of airway lumina. Additional findings included moderate perivascular lymphoid cuffing and diffuse pulmonary congestion. The lumina of some bronchi contained large amounts of mucus admixed with few sloughed epithelial cells and lymphocytes (catarrhal bronchitis).

Immunohistochemical examination (with antibodies against mycoplasmas) of affected lung tissue from all 3 ferrets that died exhibited strong labeling along the brush border of terminal respiratory epithelial cells (Figure 3, panel B). There was no penetration of organisms into the adjacent pulmonary parenchyma. With the same antibodies against mycoplasmas labeled with a fluorescent chromogen, confocal laser microscopy showed positive labeling along the apical border of the lining epithelium of terminal airways (Figure 3, panel C). Additional immunohistochemical examination and reverse transcription PCR for canine distemper and influenza A viruses, performed on samples of lung from all 3 ferrets that died, detected no virus.

Figure 4

Figure 4. . . Transmission electron micrograph of the lung from a 2-year-old ferret that died of acute dyspnea, showing loss of cilia in bronchial epithelial and cellular degeneration characterized by swelling of...

Transmission electron microscopy showed bronchial epithelial cells with loss of cilia and cellular degeneration characterized by swelling of endoplasmic reticulum, vacuolization of mitochondria with loss of christae, and intranuclear chromatin dispersement. Attached to the apical surface of a ciliated cell were pleomorphic, round to ovoid, ≈0.8-µm mycoplasma-like organisms (Figure 4).

Figure 5

Figure 5. . . Scanning electron micrographs of the lung from a 2-year-old ferret that died of acute dyspnea, showing A) marked loss of cilia with multifocal degenerative changes characterized by bulbous swelling...

Electron microscopy showed severe denudation of bronchial epithelial cells. Cilia were commonly lost or had undergone degenerative changes characterized by bulbous swelling (Figure 5, panel A). Many necrotic bronchial epithelial cells were adhered to the luminal surface, and many pleomorphic mycoplasma-like organisms were diffusely attached to the mucosal surface of bronchi and bronchioles (Figure 5, panel B). In some areas, focal loss of cilia and cell membrane damage and mycoplasma-like organisms were observed along the periphery of such lesions (Figure 5, panel C). In other areas, the mucosal surface was covered by many mycoplasma-like organisms that completely obscured the cilia (Figure 5, panel D). Among the 10 healthy ferrets, no gross or histologic lesions suggestive of mycoplasma infection were identified.

Bacteria

The 12 BAL samples from affected ferrets were all positive for fast-growing, glucose-fermenting mycoplasmas but negative for other bacteria. Ocular swabs from these ferrets were negative for bacteria. No bacteria or mycoplasmas were isolated from the 10 healthy ferrets.

PCR and Sequences

Figure 6

Figure 6. . . Phylogenetic analysis of A) partial 16S rDNA gene (933 bp) and B) partial RNA polymerase B gene (733 bp) for the new mycoplasma isolates and other closely related Mycoplasma...

Analyses of nucleic acid sequences from the 16S rDNA gene (GenBank accession nos. JQ910955– JQ910966) for each of the 12 mycoplasma isolates showed that the isolates were 99% similar to each other and segregated the isolates into 2 groups defined by nucleotide differences at 3 positions. Phylogenetic analysis with partial 16S rDNA gene sequences showed that the isolates were 96% to 97% similar to M. molare (isolated from a canid). Other closely related Mycoplasma spp. included M. lagogenitalium (isolated from Afghan pika), M. neurolyticum (isolated from mice and rats), M. sp. LR5794 (isolated from raccoons), M. collis (isolated from mice and rats), M. cricetuli (isolated from Chinese hamsters), and M. sp. EDS (isolated from house musk shrews) (Figure 6, panel A). On the basis of the 16S rDNA gene sequences, these mycoplasmas isolated from ferrets, along with the aforementioned closely related Mycoplasma spp., are in the hominis group of mycoplasmas.

Analyses of nucleic acid sequences from the rpoB gene (GenBank accession nos.. JQ91067–JQ91078) for each of the mycoplasma isolates from ferrets segregated the isolates into 2 groups of genetic variants (groups 1 and 2), which were 90% –91% similar to each other. Within a group, the isolates were 99%–100% or 98%–100% similar to each other. Although nucleotide differences were identified in as many as 12 positions within a group and 65 positions between groups, the corresponding amino acid sequences were 100% similar within a group and differed at only 2 aa positions between groups. Phylogenetic analysis showed that the partial rpoB gene sequences of the isolates were only 85%–86% similar with M. molare and 84%–86% similar to M. lagogenitalium, the most closely related Mycoplasma species. (Figure 6, panel B). Grouping of the isolates according to sequences of 16S rDNA and rpoB gene were in agreement for all but 1 isolate. Phylogenetic relatedness of these newly identified mycoplasmas to other Mycoplasma spp. was similar for the 16s rDNA and the rpoB genes.