Disseminated nocardiosis in a Japanese raccoon dog (Nyctereutes procyonoides viverrinus)

Abstract

A captive male Japanese raccoon dog died after self-mutilation by biting its tail and developing anorexia. At necropsy, ulceration of the tail root and pelvic region, an abdominal mass, multifocal nodules in the lungs, and thrombi in the heart were observed. Histopathological examinations revealed multifocal pyogranulomatous inflammation in the lungs, abdominal mass, spleen, brain, liver, and kidneys. Gram-positive bacterial aggregates were observed in the lungs, abdominal mass, and spleen. These organisms were partially acid-fast and periodic acid-Schiff-positive, and branching filaments were observed on Grocott’s methenamine silver staining. Nocardia aobensis was identified in the abdominal mass by PCR and sequence analysis. These findings suggest that N. aobensis should be considered a potential cause of disseminated pyogranulomatous disease in animals.

Nocardia spp. are Gram-positive, nonmotile, aerobic, facultatively intracellular actinomycetes that may cause infectious diseases similar to other actinomycetes, such as Mycobacterium and Actinomyces [14, 16, 17]. Nocardia spp. are environmental bacteria, and nocardiosis is considered an opportunistic infection in several species [1, 14, 16, 17], including cats [2, 3, 5, 8, 9], dogs [6, 11, 13, 19, 21], and raccoons [7]. Infection typically occurs via subcutaneous transmission, inhalation, or ingestion [6], leading to three main clinical presentations: cutaneous, pulmonary, and disseminated (systemic) [1, 14, 17]. In cats, subcutaneous transmission associated with inoculation through puncture wounds, foreign bodies, or scratches is the primary route of infection, whereas pulmonary nocardiosis caused by inhalation is common in dogs [17].

The Nocardia asteroides complex has been reclassified based on the results of molecular examinations and drug susceptibility into Nocardia abscessus (drug pattern I), Nocardia brevicatena/Nocardia paucivorans (drug pattern II), Nocardia nova complex (drug pattern III), Nocardia transvalensis complex (drug pattern IV), Nocardia farcinica (drug pattern V), and Nocardia cyriacigeorgica (drug pattern VI). The N. nova complex includes species such as N. nova, N. veterana, N. africana, and N. aobensis [4]. While there are reports of N. aobensis infections in humans [10, 12], N. aobensis infections in animals have not been reported. The present study describes the pathological findings of disseminated nocardiosis caused by N. aobensis in a Japanese raccoon dog.

A male Japanese raccoon dog (Nyctereutes procyonoides viverrinus) was captured in 2021 and kept at a zoological facility. Self-mutilation by biting its tail had been observed since 2023, resulting in tail loss and exposure of the perianal subcutaneous tissue and skeletal muscle. As no specific underlying conditions were identified, a psychogenic disorder was tentatively diagnosed, and treatment with an Elizabethan collar and selective serotonin reuptake inhibitor (SSRI) was initiated. The animal subsequently showed signs of recovery. In 2025, it exhibited anorexia, and due to the potential side effects, the SSRI was tapered and discontinued. Subsequently, the animal exhibited recurrent ulceration of the tail root and pelvic region due to self-mutilation. Despite surgical irrigation and suturing under anesthesia, the animal subsequently died. At necropsy on the day of death, the root of the tail and the pelvic region were severely ulcerated with suppurative exudate (Fig. 1A). A white mass, measuring 55 × 110 mm in diameter, was observed in the abdominal cavity, adjacent to the pylorus (Fig. 1B). Multifocal white nodules were observed in the lungs and diaphragm (Fig. 1C). A pale-yellow discoloration was observed on the capsule of the spleen. White spots were observed in the liver, and hemorrhage was noted on the renal capsule. The brain showed no remarkable gross lesions. Thrombi were observed in the right atrium and ventricle of the heart (Fig. 1D). Klebsiella pneumoniae and Enterobacter cloacae were cultured from the lung nodule; Klebsiella pneumoniae and Escherichia coli were cultured from the diaphragm nodule. Bacterial culture was not performed on other lesions. Tissue samples of lungs, heart, abdominal mass, liver, spleen, kidneys, adrenal glands, and brain were fixed in 10% neutral-buffered formalin and embedded in paraffin for histopathological examinations.

Fig. 1.

(A–D) Japanese raccoon dog, gross findings. (A) Severe ulceration in the pelvic region. (B) Abdominal mass. A mass (asterisk) adjacent to the pylorus. (C) Lungs. Multifocal white nodules. (D) Heart, formalin-fixed. Thrombi in the right atrium and ventricle (arrows).

Histopathological examinations revealed multifocal inflammation consisting of severe infiltration of neutrophils and macrophages with scattered multinucleated giant cells in the white nodules of the lungs, abdominal mass, and splenic capsule. Numerous bacterial aggregates were observed within these inflammatory foci (Fig. 2A–C). Extensive necrosis was observed in the abdominal mass. The center of the abdominal mass appeared to be adipose tissue; however, because the pancreas was not identified at necropsy, pancreatic involvement could not be ruled out. In the surrounding pulmonary parenchyma, neutrophil and macrophage infiltration with fibrin exudation was observed in the bronchioles, and thrombi were observed in the pulmonary arterioles. The thrombi in the right atrium and ventricle of the heart consisted of fibrin without bacterial aggregates. No remarkable changes were observed in the cardiac valves or myocardium. In the liver, mild centrilobular necrosis was observed. Fibrin exudates were observed on the splenic capsule, and extramedullary hematopoiesis and numerous hemosiderin-laden macrophages were observed in the parenchyma. Multifocal infiltration of neutrophils and macrophages was also observed in the cerebrum, liver, and kidneys, but no obvious bacteria were detected (Supplementary Fig. 1). Nodular hyperplasia of the cortex and thrombi in the venules were observed in the adrenal glands.

Fig. 2.

(A–D) Japanese raccoon dog. (A) Abdominal mass, hematoxylin and eosin (HE) staining. Multifocal bacterial colonies and extensive necrosis. Bar, 100 µm. (B) Lung, HE staining. Multifocal bacterial colonies and inflammatory cell infiltration. Bar, 100 µm. (C) Lung, HE staining. Infiltration of neutrophils and macrophages surrounding bacterial colonies. Multinucleated giant cell (arrow). Bar, 20 µm. (D) Lung, periodic acid-Schiff (PAS) reaction. Bacterial colonies are PAS-positive. Bar, 20 µm.

Sections of the lungs, abdominal mass, and spleen were used for periodic acid-Schiff reaction (PAS; Fig. 2D), Gram staining (Brown-Hopps method; Fig. 3A), Fite-Faraco staining (FFS; Fig. 3B), and Grocott’s methenamine silver staining (GMS; Fig. 3C). Rod- and coccoid-form Gram-positive bacteria, intermixed with Gram-negative bacilli, were detected in the white nodules of the lungs, abdominal mass, and splenic capsule. The Gram-positive organisms were partially acid-fast, PAS-positive, and branching filaments were observed on GMS staining. Based on these findings, disseminated nocardiosis was suspected.

Fig. 3.

(A–C) Japanese raccoon dog, abdominal mass. Bars, 10 µm. (A) Gram staining. Rod- and coccoid-form Gram-positive bacteria. (B) Fite-Faraco staining. The organisms are partially acid-fast. (C) Grocott’s methenamine silver staining. Filamentous, branching bacteria.

PCR was conducted using formalin-fixed paraffin-embedded (FFPE) tissues of the lungs and abdominal mass. Total DNA was extracted using the QIAamp^® DNA FFPE Tissue Kit (QIAGEN, Venlo, Netherlands). The groEL gene, which encodes hsp65, was amplified using a specific primer pair for Nocardia spp. (MN1 and MN2) [15], and the PCR master mix (KOD One^® PCR Master Mix -Blue-; TOYOBO, Osaka, Japan) was used. The target bands were cut out and purified using the Wizard^® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and sequenced using the Sanger method. Sequence data were queried using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi). Electrophoresis of the groEL PCR products revealed a 422 bp band in the lungs and abdominal mass samples. The sequence of the PCR product from the abdominal mass sample showed 99.69% identity with N. aobensis and 99.09% identity with N. nova (Table 1).

Table 1. National Center for Biotechnology Information Basic Local Alignment Search Tool analysis results of the groEL gene sequence for bacterial species identified with 98.5% sequence identity.

Species	Query cover	E-value	Identity (%)
Nocardia aobensis	76%	3.00E-163	99.69%
Nocardia nova	78%	2.00E-164	99.09%
Nocardia africana	80%	1.00E-167	98.53%
Nocardia kruczakiae	80%	1.00E-167	98.53%
Nocardia veterana	80%	1.00E-167	98.53%

Multiple hits for the same species are consolidated.

Because a high sequence identity was observed for both species, additional primers (forward: 5′-CGTGGGTAGCGAACAGGATT-3′ and reverse: 5′-AGGGGCATGATGACTTGACG-3′) were designed to target polymorphic regions within the 16S rRNA gene of both N. aobensis and N. nova for definitive species identification. Partial amplification and subsequent sequencing of the 16S rRNA gene were performed on the abdominal mass sample using the same protocol described above, except for the primer set. This procedure yielded a 443 bp product from the abdominal mass sample. Subsequent sequencing analysis showed 98.99% identity with N. aobensis and 98.74% identity with both N. africana and N. cerradoensis (Table 2), while the identity with N. nova was <98%.

Table 2. National Center for Biotechnology Information Basic Local Alignment Search Tool analysis results of the 16S rRNA gene sequence for bacterial species identified with 98.5% sequence identity.

Species	Query cover	E-value	Identity (%)
Nocardia aobensis	99%	0	98.99%
Nocardia cerradoensis	99%	0	98.74%
Nocardia africana	99%	0	98.74%

Multiple hits for the same species are consolidated.

In the present case, systemic pyogranulomatous inflammation with bacterial aggregates was observed, and N. aobensis was identified using PCR and sequence analysis. Although bacteria were not histopathologically observed in the cerebrum, liver, and kidneys, the inflammatory lesions in these organs were considered to be associated with disseminated nocardiosis based on their similar inflammatory changes to the lesions in the white nodules of the lungs and abdominal mass. The distribution of the lesions and histopathological findings in this case were consistent with those of nocardiosis in domestic animals [1,2,3, 5,6,7,8,9, 11, 13, 14, 17, 19, 21]. Nocardiosis is an opportunistic infectious disease, and although it may affect dogs without underlying conditions, immunosuppression caused by canine distemper virus (CDV) infection has been reported as a predisposing factor [1, 14]. In the present case involving a Japanese raccoon dog, no findings suggestive of immunosuppression or CDV infection were observed.

Several case reports of nocardiosis in dogs and raccoons have described abdominal masses adjacent to the pylorus and pancreas [7, 11], suggesting that this region may represent a predilection site for Nocardia infection. Previous reports have suggested that penetrating gastrointestinal trauma could be a potential route of infection leading to mass formation in this area [7, 11]. However, it has also been noted in the raccoon case that the mass was not attached to the intestine, leaving the exact infection process unclear [7]. Nocardiosis is known to have a propensity for vascular invasion and hematogenous dissemination [6, 14, 17]. Therefore, lesion formation in this area may result from hematogenous spread originating from primary respiratory or subcutaneous infections. In this case, chronic ulceration of the tail root and pelvic region, or the pulmonary lesion, was clinically suspected as the primary entry site for N. aobensis, followed by subsequent dissemination. However, because histopathological examinations of the skin lesions, pylorus, and duodenum were not performed, the definitive route of entry could not be determined.

Nocardia spp. are slow-growing organisms, and culture-based detection can take days to weeks, often resulting in low sensitivity and specificity [8, 17]. In the present case, N. aobensis was likely not cultured because it was overgrown by other rapidly growing bacteria, such as Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli. Molecular techniques, such as PCR and sequence analysis, offer high sensitivity and specificity and can shorten the time to diagnosis [14, 17, 20]. However, these targeted methods require a prior morphological examination to include nocardiosis in the differential diagnosis. In human medicine, next-generation sequencing is increasingly utilized for its comprehensive coverage, rapid turnaround time, and unbiased approach to the detection of Nocardia spp. [20].

N. aobensis, originally isolated from human clinical specimens in Japan, belongs to the N. nova complex and is closely related to N. nova and N. africana [10]. N. nova is a common pathogen in dogs and cats. However, despite its rarity, N. africana has been reported more frequently in cats [3, 5, 9] than in dogs [21], suggesting a potential trend in host susceptibility across Nocardia species. Further investigation is needed to clarify the host range and prevalence of N. aobensis. Furthermore, it is possible that N. aobensis has been involved in some cases previously diagnosed broadly as N. asteroides complex (including N. nova complex) infections. Moreover, the fact that N. aobensis, originally isolated in Japan, has now been identified in an animal in Japan may be epidemiologically meaningful, as some Nocardia species are more prevalent in specific geographical locations and climates [18].

N. nova has been reported to cause pyogranulomatous dermatitis, panniculitis, and systemic infection in cats [2, 17], as well as cutaneous abscesses, pneumonia, and encephalitis in dogs [3]. In contrast, N. africana has been documented to cause mycetoma, osteomyelitis, and cellulitis in cats [3, 5, 9], and skin lesions characterized by ulcerated nodules with fistulous tracts in dogs [21]. These species exhibit clinicopathological features similar to those identified in the present case, suggesting comparable pathogenicity. Consequently, N. aobensis should be recognized as a clinically significant pathogen with the potential to cause severe systemic infections, similar to other members of the N. nova complex.

In conclusion, this is the first report of disseminated nocardiosis in a Japanese raccoon dog, and the pathological findings were consistent with those in humans and domestic animals. In addition, N. aobensis should be considered a potential cause of disseminated pyogranulomatous disease in animals.

CONFLICT OF INTEREST

The authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this manuscript.