Fatal Systemic Infection of Clostridium tarantellae in a Wild Korean Raccoon Dog (Nyctereutes procyonoides koreensis)

The raccoon dog (Nyctereutes procyonoides) is endemic to East Asia (Kauhala and Kowalczyk 2011). The Korean raccoon dog (Nyctereutes procyonoides koreensis) is an endemic subspecies and is abundant throughout the Republic of Korea.

Clostridium tarantellae, formerly called Eubacterium tarantellae (Lawson and Rainey 2016), was first reported from dead and moribund striped mullets (Mugil cephalus) showing neurological signs (Udey et al. 1977). The bacterium has also been found in gut microflora of a Kazakhstani women (Kushugulova et al. 2014) and in a patient suffering from septic arthritis (Cointe et al. 2019). However, infection of C. tarantellae associated with free-living mammals, including the Korean raccoon dog, has not previously been reported.

A wild Korean raccoon dog, age 3–6 mo, was submitted to the Daejeon Wildlife Rescue Center, Daejeon, Republic of Korea. Physical examination of the animal at the time of admission revealed severe dehydration, loss of appetite, and decreased response to stimulation. Hematology showed low hematocrit (27.5%; reference interval 36.5–57.3%), indicating anemia. Despite supportive care with normal saline and intravenous supplementation with a commercial multivitamin and mineral complex (vitamins B1, B2, B3, and B6, taurine, glucose, and iron; Handong, Seoul, Korea), the anemia and loss of appetite continued. The animal's body weight gradually declined and its mobility deteriorated, and it died 2 mo after admission.

Gross necropsy revealed severe emaciation and marked pallor of the skin and mucous membranes. In the abdominal cavity, about 8 mL of clear white to yellow ascitic fluid was observed. On the heart, liver, kidneys, and spleen, multiple 3–5 mm–sized yellow-to-gray-colored foci were found, also visible on the cut surface. In particular, the kidneys were extremely pale. However, no specific pathogen related to the lesion of kidneys, liver, lungs, and ascites was detected using aerobic cultures.

The main histopathologic finding was multifocal necrosis in the liver, kidneys, and spleen (Fig. 1). Necrotic lesions were characterized by coagulative necrosis with a relatively minimal inflammatory response. The center of each necrotic focus contained 7–10-µm-long filamentous microbes, arranged haphazardly. These bacteria, thick and wormlike in appearance, were observed in the cytoplasm, especially at the border of the necrotic areas.

Silver staining (HT101, Sigma-Aldrich, St. Louis, Missouri, USA), performed according to the manufacturer's guidelines, showed clearer morphology of the microbes than H&E staining (Fig. 2). The bacteria were filamentous shaped, up to 10 µm in length, and straight or bent to almost 90 degrees. Some of these bacteria were thick, but thin organisms were also observed. The bacteria were aggregated in the necrotic foci or scattered around these foci and distributed in both intracellular and extracellular areas. The microscopic lesions were highly similar to those seen in previous reports of Clostridium piliforme infection (Tyzzer's disease) in animals (e.g., Wojcinski and Barker 1986; Brooks et al. 2006) in that the pathogens were long, slender bacilli and caused multifocal necrosis in the liver. However, unlike C. piliforme infection in other species, multifocal necrosis was observed in multiple organs and associated with minimal inflammation. Based on the gross and histopathologic examination, we presumed the causative agent to be C. piliforme, and further molecular analysis was performed in order to confirm this.

Tissue samples (liver, kidney, intestine) were assessed by indirect immunofluorescence, PCR against C. piliforme, and microbiome taxonomic profiling (MTP) test. Indirect immunofluorescence assays were performed by incubating acetone-fixed air-dried frozen tissue samples on slides with anti–C. piliforme antibody for 30 min, with green fluorescence indicating a positive reaction. In addition, PCR amplification of a 196-base pair DNA fragment specific for 16S ribosomal RNA of C. piliforme was positive for the microbes in the liver, intestine, and kidney. The MTP test is a sequencing technique that distinguishes among all the microbiomes collected from a sample (Yoon et al. 2017). This test was performed by Chunlab Inc. (Seoul, Republic of Korea) and showed that of 33,800 valid microbiomes from the kidney sample, 32,979 (97.57%) were C. tarantellae. Based on these results, we concluded that the major pathogen causing fatal systemic disease in this animal was C. tarantellae.

Until recently, the diagnosis of Tyzzer's disease has been based on histopathological characteristics, including multifocal necrosis with heavy inflammatory cell infiltration of the liver, and observation of the pathogen with filamentous morphology, as shown by H&E and/or silver staining (Brooks et al. 2006; Simpson et al. 2008; Headley et al. 2009; Mete et al. 2017). The major histopathological characteristics of the wild Korean raccoon dog infected with C. tarantellae were similar to those of observed in animals infected with C. piliforme, including the morphology of the pathogen and the presence of multifocal necrosis in the liver (Udey et al. 1977). Moreover, the antibody used for indirect immunofluorescence assays and the 196-base pair DNA fragment specific to 16S ribosomal RNA of C. piliforme both cross-reacted with C. tarantellae in our study. This cross-reactivity between the two species is probably due to the similarity of their 16S ribosomal RNA sequences, which are 81.7% identical using the lalign program (Pearson 2021) on sequences available in GenBank. In contrast to C. piliforme infection, C. tarantellae infection in our case was characterized by anemia, immunosuppression, and bone marrow atrophy. Bacterial infection sometimes occurs during the period in care, but the Daejeon Wildlife Rescue Center has comprehensive sanitation protocols, and the animal had shown clinical signs since admission to the Wildlife Rescue Center; thus we consider that the raccoon dog probably contracted the infection in the wild rather than during the period of care, but additional studies are required to confirm the pathogenesis of fatal systemic infection of C. tarantellae.

In conclusion, we diagnosed fatal systemic infection with C. tarantellae in a wild Korean raccoon dog based on MTP assays. Pathology, immunofluorescence, and PCR results showed high similarity with Tyzzer's disease. Multiple analytic methods are therefore required in the differential diagnosis between these two infections.