Systemic Staphylococcus pseudintermedius infection in an arctic fox (Vulpes lagopus) with severe multifocal suppurative meningoencephalitis and nephritis.

A 2-year-female arctic fox (Vulpes lagopus) developed anorexia, dehydration, and emaciation during the quarantine period for importation from Norway, and died 17 days later. At necropsy, a fistula was observed on the left gluteal region, and the left eye, left brain, and kidneys were discolored. Histologically, severe diffuse suppurative meningoencephalitis and renal abscesses were detected. Numerous Gram-positive cocci were detected in these lesions. Multidrug-susceptible Staphylococcus pseudintermedius were isolated from the lesions. These results suggest that S. pseudintermedius can cause severe multifocal suppurative meningoencephalitis and nephritis in foxes. This is the first report of multidrug-susceptible S. pseudintermedius meningoencephalitis and nephritis in a fox.

Staphylococcus pseudintermedius is a Gram-positive, facultative anaerobic coccus; its colonies are medium-sized, raised, and unpigmented, and display large incomplete β- and small complete δ-hemolysis either alone or in combination (double hemolysis) on sheep or bovine blood agar [3]. In the past, S. aureus and S. hyicus have been recognized as principal pathogenic staphylococci in veterinary medicine. S. intermedius was first described by Hajek in 1976, based on isolates from pigeons, dogs, minks and horses [9]. For over 30 years, S. intermedius had been considered a common cause of skin and soft tissue infections in dogs [1, 2, 11].

Recent work has shown that isolates phenotypically identified as S. intermedius are indeed differentiated into three distinct species: S. intermedius, S. delphini, and S. pseudintermedius, which are together referred to as the Staphylococcus intermedius group (SIG) [4, 6, 14]. The newly described S. pseudintermedius species, and not S. intermedius, is a common cause of canine cutaneous infections [4, 14] and occasionally causes infection in cats [10] and humans [12]. The natural hosts of S. delphini were recognized as Mustelidae such as minks, ferrets, and badgers [7], and those of S. pseudintermedius were not only dogs, but also foxes [7]. S. pseudintermedius infection has been sporadically reported in other hosts, such as horses, including cases of fatal infection [8]. Moreover, the incidence of methicillin-resistant S. pseudintermedius (MRSP) has been increasing in dogs, and is primarily associated with skin, surgical site, and wound infections [13]. However, information regarding S. pseudintermedius infection in foxes is still limited [7]. There are no previous reports of fatal S. pseudintermedius infection in foxes.

This report describes the clinical, microscopic, and bacteriological finding in an imported arctic fox (Vulpes lagopus) with severe multifocal suppurative meningoencephalitis and nephritis.

Four pregnant arctic foxes were imported from Norway by air for exhibition. At the time of arrival, all foxes appeared healthy and no clinical abnormalities were present until the sixteenth day of legal quarantine for importation. However, on the seventeenth day, a 2-year-old female fox displayed anorexia, dehydration, and weakness, and the animal was treated from day 20 of quarantine onward with acetic acid Ringer’s solution, 25% glucose solution, fursultiamine, primperan, and ursodeoxycholic acid by intravenous injections. On the twenty-second day, it had an abortion, and on the thirty-fourth day, it was found dead in the cage. No clinical abnormalities were observed in the remaining 3 foxes until the end of the quarantine period of 180 days.

A necropsy was performed, and tissue samples from the liver, kidney, heart, lung, stomach, intestines and left brain (cerebrum, cerebellum, midbrain, pons and medulla oblongata) were fixed in 10% neutral-buffered formalin and embedded in paraffin wax. The hole right brain was not histologically examined because it was refrigerated and used as diagnostics samples for rabies (The result was negative). Tissue sections (approximately 3 µm thick) were stained with hematoxylin and eosin (HE) and Gram stained for histological examination.

For immunohistochemical analysis, a rabbit anti-bovine glial fibrillary acidic protein (GFAP) serum (Biomedical Technologies, Inc., Stoughton, MA, U.S.A.) was used at a dilution of 1 in 1,024 with a commercial kit (N-Histofine Simple Stain MAX-PO®; Nichirei Bioscience, Tokyo, Japan).

Samples from the subcutaneous abscess at the costal region, cerebrum, cerebrospinal fluid, and kidney were used for bacterial isolation.

For genetic tests, genomic DNA was extracted from the bacterial colonies using a DNA extraction kit (InstaGene Matrix; Bio-Rad Laboratories, Hercules, CA, U.S.A.). A ~500-bp region of the 16S ribosomal RNA gene (16S rDNA) region was amplified and sequenced using a MicroSeq 500 16S rDNA PCR/Sequencing Kit (Applied Biosystems Life Technologies, Carlsbad, CA, U.S.A.). Therefore, S. pseudintermedius identification was confirmed by pta PCR-restriction fragment length polymorphism (RFLP) analysis using MboI [5].

The minimum inhibitory concentration (MIC) of antibiotics against the isolate was determined by the broth micro-dilution technique (Dry plate; Eiken Chemical Co., Ltd., Tokyo, Japan). The tested antibiotics were ampicillin, cefazolin, cefotaxime, kanamycin, tetracycline, gentamycin, ciprofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole.

After the skin was removed at necropsy, a fistula connected to the subcutaneous abscess of the left costal region was observed on the left gluteal region. Liquid pus colored in whitish-yellow was flowed from the fistula to the surrounding subcutaneous connective tissue and the subcutaneous tissue showed gelatinous appearance with edema throughout the left side of the trunk (Fig. 1a). There was no abnormality in appearance on the surface of the cerebrum hemisphere. After fixation, Paramedian cut surfaces of the left corpus callosum, fornix and cerebellum meninges at central lobule showed greyish in color. The renal papilla was pale brown in color and there was a white spot in the renal cortex at the cut surface of the kidney. The left eye showed corneal opacity. No gross abnormalities were found in the other organs.

Fig. 1.

a. The subcutaneous tissues of the left costal region. Pus was detected in the fistula (arrow). Bar=2 cm. b. HE staining of the cerebellum showing severe suppurative meningitis. Bar=500 µm. c. HE staining of white matter of the piriform lobe at endbrain showing abscess. Bar=200 µm. The inserted figure is astrocytes hyperplasia by GFAP Bar=50 µm. d. HE staining of the renal papilla showing abscess. Bar=500 µm. The inserted figure is Gram staining of marginal region of the renal abscess showing infiltrations of macrophages with numerous Gram-positive cocci. Bar=20 µm.

Histologically, severe diffuse suppurative meningitis were detected in the pons, cerebral peduncles, and cerebellum meninges at central lobule (Fig. 1b). Suppurative encephalitis with multifocal abscesses were also detected in the white matter of the piriform lobe at cerebrum hemisphere (Fig. 1c), corpus callosum, and fornix. Immunohistochemically, proliferation of GFAP-positive astrocytes was observed in the neural tissue surrounding abscesses (Fig. 1c inset). In the kidney, abscesses in the renal papilla (Fig. 1d), microabscesses in the renal cortex, and mild infiltration of lymphocytes and plasma cells in the interstitium around the glomeruli were observed. Numerous Gram-positive cocci were detected in those lesions and some were detected in the macrophages in the renal lesions (Fig. 1d inserted figure) and the blood vessels of the lung.

Gram-positive cocci were isolated from the subcutaneous abscess, the cerebrum, cerebrospinal fluid, and kidney, and were β-hemolytic, catalase-positive, and oxidase-negative. The sequencing of the amplified 16S rDNA region of the isolate confirmed that it was Staphylococcus sp., and it was identified as S. pseudintermedius by RFLP analysis using MboI. Furthermore, it demonstrated sensitivity to all tested antibiotics.

The present case was diagnosed as septicemic infection with severe diffused suppurative meningoencephalitis and nephritis caused by S. pseudintermedius because Gram-positive cocci were observed in the suppurative lesions and in the macrophages. Furthermore, the bacteria were isolated from the subcutaneous abscess, cerebrum, cerebrospinal fluid, and kidney, and the isolate was identified as S. pseudintermedius by 16S rDNA sequence and RFLP analysis [5].

The present isolate demonstrated sensitivity to all tested antibiotics. On the other hand, the worldwide spread of strains resistant to virtually all antimicrobial agents available in veterinary medicine, namely MRSP, has complicated treatment considerably [13, 16]. It appears that MRSP did not spread out to the arctic foxes imported for exhibition in this case.

Foxes as well as dogs have also been considered as natural hosts of S. pseudintermedius [7]. Therefore, opportunistic infections may occur in foxes with weakened immune systems resulting from long-distance transportation, environmental changes and/or pregnancy. An injury leading to the subcutaneous abscess was considered as the probable initial site of S. pseudintermedius entry. Subsequently, the bacteria spread throughout the whole body including the kidney and left cerebrum. S. pseudintermedius infection has sporadically been reported in other hosts, such as horses and humans [8, 12]. The first case of human infection with S. pseudintermedius from a dog bite wound was described over 20 years ago [15]. Systemic S. pseudintermedius infection in the organs of foxes is rare and has not been previously reported globally.

In conclusion, this is the first report of multidrug-susceptible S. pseudintermedius meningoencephalitis and nephritis in a fox. Further investigation is necessary to clarify the cause of the systemic infection and the virulence factor. In any case, more attention will be necessary to develop control measures to prevent opportunistic infectious diseases caused by bacteria such as S. pseudintermedius in animals procured for exhibition purposes such as foxes.