Signalment:  

Three six-month-old female SCID-Beige mice, Mus musculus.More than six Scid/Beige mice (C.B-lgh- Gbms Tac-Prkdcscid-Ly) from the same colony died during the course of one month. These mice had not been experimentally manipulated and presented weak, lethargic, hunched and losing weight before death. Three sick animals were submitted to the Laboratory of Comparative Pathology for diagnosis. They presented similarly to those that had died previously, except for one animal that had a head tilt to the left.

Gross Description:  

On necropsy, the skin covering the head and neck was removed and one to three, small, rounded, tan to yellow, firm but slightly fluctuant nodules measuring up to 0.5 cm in diameter (abscesses) were found at the base of the ear canal in two out of three mice. The left ear was affected by the largest of these abscesses on the mouse that presented with left head tilt, and the right ear was affected on another mouse. The third mouse did not have gross lesions but all three mice had similar microscopic changes. Purulent material from these abscesses, as well as blood and spleen, were collected for culture. No other gross lesions were detected in the three mice.

Histopathologic Description:

Coronal sections of head containing tympanic bulla, surrounding soft tissues and brain are submitted to conference participants. Microscopic appearance varies slightly from slide to slide, according to the depth of the section. In all slides the tympanic bulla is filled and obliterated by a cellular exudate composed of neutrophils, fibrin, proteinaceous and cellular debris, and colonies of small gram negative rods. There is extensive osteolysis of the bulla, with involvement of the surrounding soft tissue and abscess formation within the mandibular musculature locally. A rim of early fibroplasia and large number of rods, free or within macrophages, are noted at the periphery of these abscesses. The inflammation extends into the internal ear in some slides. There is focal lysis of the skull bone adjacent to the tympanic bulla with involvement of the meninges and cerebral parenchyma. The extent of cerebral involvement varies from slide to slide. In areas of cerebral abscess formation the surrounding parenchyma is compressed and a thick band of Gitter cells packed with bacterial rods is noted at the border between necrotic and healthy tissue. There is fibrinoid necrosis of small vessels in the affected cerebral parenchyma. The lesions in all three cases were unilateral. Other changes included miliary histiocytic inflammation of the liver in one of the mice, likely due to septicemia as indicated by the recovery of B. cepacia from cultures of blood and spleen.

Morphologic Diagnosis:  

1. Middle ear: Severe, chronic, suppurative, unilateral otitis media with extensive bulla osteolysis and intralesional gram negative rods.
2. Inner ear (depending on slide): Severe, chronic, suppurative, unilateral otitis interna.
3. Brain and meninges: Severe, chronic, focal, suppurative meningoencephalitis with focal cerebral abscess and intralesional gram negative rods (secondary to otitis).

Lab Results:  

Microbiological culture results of the purulent exudate from the abscesses, as well as from blood and spleen indicated Burkholderia cepacia. Cultures were sent to the Research Animal Diagnostic Laboratory at the University of Missouri for PCR to differentiate B. cepacia from B. gladioli. PCR results confirmed B. cepacia.

Condition:  

Burkholderia cepacia

Contributor Comment:  

Spontaneous otitis media in immunocompetent mice has been frequently associated with Mycoplasma pulmonis, Pasteurella pneumotropica, Pseudomonas aeruginosa, Streptococcus sp., and viral agents such as reovirus.(1) The inflammatory infiltrate can vary from neutrophilic ( in most of the bacterial infections ) to lymphoplasmacytic and serous and is usually unilateral. When bilateral, the inflammatory infiltrate can differ, i.e. suppurative in one side and serous on the other. Proliferative and papillary lesions, as well as fibrosis of the tympanic cavity are common findings in chronic cases. Causative agents such as bacterial rods or cocci are rarely seen histologically, despite the use of special stains. The incidence of otitis media in mice varies according to strain and age. It is very common in aging 129:B6 mice with a 79 -84% incidence, independent of gender. cBA/J mice are also susceptible to otitis media with a 90% incidence in animals older than one year.(2) However, otitis was found to be more common in aging 129S6 mice than in another CBA strain studied (CBA/Caj).(3)

The most common clinical presentation is head tilt, but neurologic signs such as circling and rolling have been reported in C3H mice and likely depend on the extent and severity of the lesion.(4) A history of previous chemically-induced otitis with ear damage has been found to predispose Rb/3 mice (non-susceptible strain) to audiogenic seizures.(5) In the ICR strain, otitis is manifested as mutilation of the external ear canal due to self-trauma.(6) In addition, Jeff (Jf) mutant mice are also predisposed to otitis, likely due to craniofacial abnormalities in the strain.(7)

In athymic Balb/C-derived nude mice, naturallyoccurring Sendai virus infection can cause a chronic respiratory disease characterized by rhinitis, laryngotracheitis, bronchitis/ bronchopneumonia and otitis media (usually suppurative).(8)

The pathogenesis of otitis media in the mouse is uncertain but since it is commonly found without associated otitis externa it may be a sequel of previous viral or bacterial infection of the nasopharynx with ascending infection via the Eustachian tube.(1) Acidification of the drinking water is an effective preventive measure. Tetracycline is recommended for treatment of affected animals.

Otitis media can be experimentally induced in mice by direct injection of the middle ear (often transtympanic) with human pathogenic organisms such as Streptococcus pneumonia, Haemophilus influenzae or Moraxella catharralis.9 Interleukin-8, as well as Salmonella typhimurium endotoxins can also be used to induce otitis media experimentally in mice.(10,11)

In 2004, an outbreak of otitis media associated with Burkholderia gladioli was reported in immunosuppressed mice. After an athymic nude mouse presented with head tilt and otitis, several other immunosuppressed mice in the facility presented with similar clinical and pathological findings. Culture of the middle ear of the affected mice initially yielded the phytopathogen Burkholderia cepacia, however the isolate was later identified as Burkholderia gladioli based on 16S rDNA PCR.(12)

SCID-beige mice are severely immunosuppressed because they lack mature T- and B- lymphocytes (prkdcscid mutation) and have a series of other defects affecting granulocytes, such as the lack of NK cells, reduced bacteriocidal activity of granulocytes, and decreased lysosomal enzymes in neutrophils (Beige or Bg mutation), as noted in the Jackson Laboratory Database. In light of the findings in these mice and the previously reported otitis outbreak in immunosuppressed mice caused by B.gladioli, we sent a subculture of the organism to a referral lab at the University of Missouri for PCR. B. cepacia was confirmed as the causative agent of otitis in the sick and dying Scid-Beige mice from our colony.

The genus Burkholderia was initially divided in four species: B. mallei, B. pseudomallei, B. gladioli and B.cepacia. B. mallei is the causative agent of glanders in horses, mules and donkeys; B. pseudomallei is the cause of melioidosis, a disease prevalent in Southeast Asia and Australia; B.gladioli is a primary plant pathogen but has also been isolated from the sputum of human patients with cystic fibrosis (CF); and B. cepacia causes respiratory failure in at least 20% of patients with cystic fibrosis (CF). Currently, the socalled Burkholderia cepacia-complex consists of nine genetically distinct species, all important to humans due to their ability to cause CF-related infections. B. cepacia is a small, gram negative, non-motile rod that can be transmitted from individual to individual. When patients with a mild form of cystic fibrosis are infected by B. cepacia there is a rapid decline of lung function and resulting respiratory failure with a poor outcome. Immunosuppressed CF patients that received a lung transplant are at high risk of infection with subsequent bacteremia that may result in death (cepacia syndrome).(12,14) In domestic animals, B. cepacia has only been reported as a cause of subclinical mastitis in sheep, but may have been reported in the past under the name Pseudomonas aeruginosa associated with reptile diseases and infections.(15) In the present cases, large numbers of gram-negative rods can be seen in the histologic sections submitted to conference participants. The culture of B. cepacia from blood and spleen confirms a bacteremia and explains the death of the many affected animals. Antibiotic sensitivity results indicated that the B. cepacia strain affecting these mice was resistant to some of the most common antibiotics, such as amoxicillin/clavulanic acid, ampicillin, penicillin, tetracycline, oxacillin, and erythromycin. The strain was susceptible to chloramphenicol, ciprofloxacin/ enrofloxacin, gentamicin, trimethoprim/sulfa and amikacin. The remaining colony was treated with Sulfatrim (sulfamethoxazole and trimethoprim) in the feed and recovered from the outbreak.

JPC Diagnosis:  

Head, sagittal section: Otitis externa, otitis media, labrynthitis, neuritis, myositis, osteomyelitis and meningo encephalitis, necrosuppurative, with intrahistiocytic bacilli.

Conference Comment:  

The contributor provides a thorough summary of otitis in mice as well a review of Burkholderia gladioli and B.cepacia infections in both humans and animals. The significant slide variation noted by the contributor was discussed by participants, and the moderator cautioned participants to always evaluate multiple sections when examining the structures of the ear, as a full visualization of the structures of the middle and inner ear requires multiple serial sections to visualize in toto. Additionally, in keeping with terminology used in human medicine, the morphologic diagnosis of labrynthitis was favored over otitis interna.

References:

1. Haines DC, Chattopadhyay S, Ward JM. Pathology of aging B6;129 mice. Toxicol Pathol. 2001;29(6):653-6.
2. McGinn MD, Bean-Knudsen D, Ermel RW. Incidence of otitis media in CBA/J and CBA/CaJ mice. Hear Res. 1992;59(1):1-6.
3. Rosowski JJ, et al. The aging of the middle ear in l29S6/SvEvTac and CBA/CaJ mice: measurements of umbo velocity, hearing function, and the incidence of pathology. J Assoc Res Otolaryngol. 2003;4(3):371-83.
4. Kohn DF, MacKenzie WF. Inner ear disease characterized by rolling in C3H mice. J Am Vet Med Assoc. 1980;177(9):815-7.
5. Niaussat MM. Experimentally induced otitis and audiogenic seizure in the mouse. Experientia. 1977;33:4734.
6. Harkness JE, Wagner JE. Self-mutilation in mice associated with otitis media. Lab Anim Sci. 1975;25(3): 315-8.
7. Hardisty RE, et al. The deaf mouse mutant Jeff (Jf) is a single gene model of otitis media. J Assoc Res Otolaryngol. 2003;130-8.
8. Ward JM, et al. Naturally-occurring Sendai virus infection of athymic nude mice. Vet Pathol. 1976;13 (1):36-46.
9. Ryan AF, et al. Mouse models of induced otitis media. Brain Res. 2006;1091(1):3-8.
10. Krekorian TD, et al. Endotoxin-induced otitis media with effusion in the mouse Immunohistochemical analysis. Acta Otolaryngol. 1990;109(3-4):288-99.
11. Johnson M, Leonard G, Kreutzer DL. Murine model of interleukin-8 induced otitis media. Laryngoscope. 1997;107(10):1405-8.
12. Foley PL, Lipuma JJ, Feldman SH. Outbreak of otitis media caused by Burkholderia gladioli infection in immunocompromised mice. Comp Med. 2004-53(1):93-9.
13. McDowell A, et al. Epidemiology of Burkholderia cepacia complex species recovered from cystic fibrosis patients: tissues related to patient segregation. J Med Microbiol. 2004;53(7):663-8.
14. Coenye T, LiPuma JJ. Molecular epidemiology of Burkholderia species. Front Biosci. 2003;8:55-67.
15. Berriatua E, et al. Outbreak of subclinical mastitis in a flock of dairy sheep associated with Burkholderia cepacia complex infection. J Clin Microbiol. 2001;39 (3):990-4.

Click the slide to view.

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4-1. Cranium

4-2. Cranium

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