CASE REPORT: Disseminated nocardiosis caused by Nocardia abscessus in a dog

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Veterinary Clinical Pathology ISSN 0275-6382


Disseminated nocardiosis caused by Nocardia abscessus in a dog Amy L. MacNeill1, James C. Steeil2, Olivier Dossin2, Patricia S. Hoien-Dalen3, Carol W. Maddox1 Departments of 1Pathobiology and 2Veterinary Clinical Medicine and 3Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, IL, USA

Key Words Actinomycetes, filamentous bacteria, pyogranulomatous, 16S ribosomal DNA, systemic infection Correspondence Amy L. MacNeill, Department of Pathobiology, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave., Urbana, IL 61802, USA E-mail: [email protected] DOI:10.1111/j.1939-165X.2010.00247.x

Abstract: A 4-year-old female spayed Bichon Frise dog that had been receiving cyclosporine A per os 3 times per week for 2 months to control allergic dermatitis developed lethargy, anorexia, fever, and multiple firm subcutaneous masses. Pyogranulomatous inflammation with branching nonseptate filamentous organisms approximately 2 mm in diameter, presumptively fungal organisms, was diagnosed by cytologic evaluation of fine-needle aspirates from several masses. A partially acid-fast actinomycete was cultured from 2 of the masses. The organism was identified as Nocardia abscessus (formerly Nocardia asteroides type 1) based on 16S ribosomal DNA sequencing of samples extracted from cultures and unstained cytologic smears. Immunosuppression caused by long-term administration of cyclosporine A likely predisposed the dog to disseminated infection. To our knowledge, this is the first report of N. abscessus infection in a dog. This case demonstrates that N. abscessus may be mistaken for a fungal organism based on its cytologic appearance and underscores the importance of using molecular techniques for the diagnosis of suspected fungal diseases.

Case Presentation A 4-year-old female spayed Bichon Frise was presented to the University of Illinois, Veterinary Teaching Hospital (VTH) for evaluation of anorexia, lethargy, acute growth of multiple subcutaneous masses, and pain associated with these masses. Two weeks earlier the referring veterinarian had removed a similar mass, which was submitted for histopathologic evaluation and was diagnosed as pyogranulomatous inflammation with plant material. A few days later the dog was treated with amoxicillin–clavulanate (14 mg/kg per os [PO] twice daily) for a urinary tract infection; however, the dog developed signs of gastrointestinal upset and amoxicillin–clavulanate was discontinued and treatment changed to marbofloxacin. At presentation to VTH the dog was febrile with a temperature of 104.61F. Current medications included 4.7 mg/kg cyclosporine A 3 times per week PO for the past 2 months to control allergic dermatitis, 9.5 mg/kg marbofloxacin PO daily, and 0.1 mg/kg meloxicam PO daily for pain. Three firm, subcutaneous masses sensitive to touch were detected on the right caudal abdomen

(8.8  18.4 cm), right hip (2 cm in diameter), and right hind limb (3 cm in diameter). The dog was hospitalized for further evaluation of the subcutaneous masses and additional diagnostic testing, including a CBC, serum biochemical profile, total thyroxine concentration, urinalysis, ultrasonographic examination of the abdominal and right hind limb, and computed tomographic (CT) examination of the abdomen and pelvis with and without contrast. Hematologic abnormalities included leukocytosis (24,400/mL, reference interval 6000–17,000/mL) consisting of a mature neutrophilia (19,300/mL, reference interval 3000–11,500/mL) and monocytosis (2680/mL, reference interval 200–1400/mL). Significant abnormalities were not detected in the serum biochemical profile or urinalysis. Serum thyroxine concentration was decreased (8.98 nmol/L, reference interval 15.0–48.0 nmol/L), which was attributed to euthyroid sick syndrome given the presence of severe underlying disease and lack of clinical signs associated with hypothyroidism. Enlarged right inguinal and right medial iliac lymph nodes, multiple contrast-enhancing masses within the abdomen, right inguinal area, and along the

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Figure 1. Computerized tomogram (CT) of the cranial abdomen of a dog. Note 2 soft tissue-enhancing masses (arrows) in the caudal left lung lobe visable on the crainal aspect of the abdominal CT.

dorsal lateral aspect of the right hind limb, and at least 3 soft tissue-enhancing masses within the subpleural region of the left caudal lung lobe (Figure 1) were identified by CT. Differential diagnoses for the masses included disseminated fungal infection, atypical bacterial infection, and metastatic neoplasia. During the first day of hospitalization, lactated Ringer’s solution containing 16 mmol/L KCl was administered intravenously at a rate of 24 mL/h. Additionally, the dog received an intravenous infusion of fentanyl at 3 mg/kg/min and intravenous 5 mg/kg enrofloxacin every 12 hours. The following day the dog began to lunge at the cage door and was progressively more aggressive, presumably due to increasing severity of pain or possibly secondary to spread of infection to the central nervous system. Fluid therapy and fentanyl were discontinued and replaced with intravenous morphine at 0.6 mg/kg/h, lidocaine at 1.5 mg/kg/h, and ketamine at 0.12 mg/kg/h. After 12 hours the rates of morphine, lidocaine, and ketamine infusions were doubled because her pain score had not decreased at the initial rates. Intravenous administration of 22 mg/ kg cephazolin was initiated every 12 hours for further broad-spectrum antibacterial coverage. Ophthalmic examination showed no evidence of ocular manifestation of systemic disease. After 3 days the masses, especially the right hip lesion, began to ooze a purulent exudate. Fine-needle aspirates were obtained from the subcutaneous masses for cytologic evaluation, aerobic and anaerobic bacterial cultures, and fungal culture.


Figure 2. Fine-needle aspirate of a subcutaneous mass on the right hind limb of a dog. Wright–Giemsa. (A) Note neutrophils and epthithelioid macrophages (arrowhead).  50 objective. (B) Note the branching filamentous organisms (arrows) and several degenerate neutrophils.  100 objective.

Smears were stained with Wright–Giemsa, and some unstained smears were saved for additional testing, if indicated. The cytologic samples were highly cellular with rare RBCs in the background and contained many severely degenerate neutrophils and moderate numbers of macrophages, including epithelioid forms and multinucleate giant cells (Figure 2A). Several mats of nonstaining or basophilic, branching, filamentous structures were observed (Figure 2B). Filaments were approximately 2 mm in diameter and varied in length, and most had tapered ends, but a few ended in small, bulbous formations. Septa were not observed. The cytologic interpretation was suppurative to pyogranulomatous inflammation with filamentous microorganisms suspected to be fungal organisms. Additional smears for cytologic evaluation were stained with Grocott’s methenamine silver (GMS), which greatly enhanced microscopic detection of the organism (Figure 3) and further increased the suspicion of fungal infection. Concurrently, there was a

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Figure 4. Bacterial colony formation on a Sabouraud dextrose agar culture plate. Fine-needle aspirates from subcutaneous masses were incubated at 371C for approximately 1 week. Note colony morphology consistent with an actinomycete.

Figure 3. Fine-needle aspirate of a subcutaneous mass on the right hind limb of a dog. After applicaton of a silver stain, the organism stains strongly positive (black) against the nonstaining cells and light green background. Grocott’s methenamine silver,  100 objective.

preliminary report of positive growth in the fungal culture of an isolate from the subcutaneous mass on the abdomen. Therefore, a tentative diagnosis of disseminated fungal infection was made while awaiting the final results of bacterial and fungal cultures of aspirates from the subcutaneous masses on the abdomen and hip. The dog’s condition did not improve over 3 days and her demeanor, suggestive of pain, was poorly responsive to treatment. Therefore, for humane and financial reasons, the dog was euthanized. A necropsy was not performed. Light growth of small white colonies composed of vegetative filaments, thought to be fungal organisms, was seen on blood agar plates after incubation at 371C for 48 hours. A strong smell of soil was produced by the organism 72 hours postinoculation. Slides prepared from the filamentous growth on blood agar plates indicated that the gram-positive long filamentous rodshaped organism was a partially acid-fast actinomycete. The organisms also began to grow on mycology culture plates after incubation for 6 days. More than 1 week after inoculation at 371C on Sabouraud dextrose agar, the colonies formed by the organism had the appearance of an actinomycete and were composed of white to buff colonies, 3 mm in diameter with a velvety surface, that were embedded in the agar (Figure 4). No growth was detected in samples cultured under anaerobic conditions. DNA was extracted from specimens on the unstained smears and from the aerobic cultures of the original fine-needle aspirates using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). Oligonucleotide primers were used to amplify PCR products of 16S (bacterial) and 18S (fungal) ribosomal DNA (rDNA) for

sequencing. The 16S primers were modified from those previously reported,1 whereas the 18S intervening sequence primers were used as described previously2: 16S forward primer (16S Uni-L), 5 0 -AGA GTT TGA TCA TGG CTC AG-3 0 ; 16S reverse primer (16S Uni-R), 5 0 -GTG TGA CGG GCG GTG TGT AC-3 0 ; 18S forward primer (IT4), 5 0 -TCC TCC GCT TAT TGA TAT GC-3 0 ; 18S reverse primer (ITS5), 5 0 -GGA AGT AAA AGT CGT AAC AAG G-3 0 . Similarly sized 16S rDNA PCR products were amplified from samples taken from both smears and cultures; 18S rDNA could not be detected. Sequencing analysis of the 16S rDNA segments identified the organism as Nocardia abscessus based on 99% sequence similarity to the reference sequence (GenBank accession number AY544980). The bacterium was susceptible to amoxicillin–clavulanate, gentamicin, trimethoprim–sulfamethoxazole, cephalothin, ceftiofur, and cephazolin, but resistant to fluorinated quinolones, chloramphenicol, and rifampicin.

Discussion Actinomycetes (Nocardia and Actinomyces spp.) are typically described as long (up to 20 mm), thin (0.5 mm), filamentous, lightly basophilic rods with intermittent eosinophilic beaded areas along the length of the organism.3,4 The Nocardia isolate in this report had a thicker (approximately 2 mm in diameter), basophilic, branching filament. When the organism was stained with Wright–Giemsa, it had uniform deep basophilic edges and light basophilic centers. Many organisms were not stained. These characteristics are often attributed to fungal organisms, although in contrast to most fungi, the filamentous structures were relatively thin. The organism also stained strongly with GMS, which is often used to distinguish fungal organisms from plant material; however, GMS also stains Nocardia spp.5,6 The unusual microscopic morphologic appearance along with the initial growth of vegetative filaments

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on blood agar plates led to a presumptive diagnosis of systemic fungal infection. Accurate identification is important, because although the prognosis is poor for both disseminated fungal and nocardial infections, antibiotic treatment for nocardiosis is less expensive than antifungal therapy and may be an affordable option for clients. For human cases of nocardiosis, sulfamethoxazole–trimethoprim treatment for at least 6 months is recommended.7 Similarly, sulfamethoxazole–trimethoprim, with or without an additional antibiotic, is usually administered for several months to treat nocardial infection in dogs.5 Interestingly, N. abscessus strains are reported to be highly susceptible to amoxicillin–clavulanate.8 The first report of nocardiosis was published in 1888 following isolation of Nocardia asteroides from cattle diagnosed with ‘‘bovine farcy.’’9 Since then, nocardial organisms have been associated with disease in several mammalian, avian, and fish species. Nocardia spp. are aerobic actinomycetes that are present in soil and have worldwide distribution. Infection is thought to occur by inhalation of the bacteria or direct subcutaneous inoculation of soil that contains organisms. Underlying immunosuppression is documented in over 60% of people with nocardial infection; longterm corticosteroid therapy is a significant risk factor for invasive nocardiosis.7 Most human cases of nocardiosis have pulmonary involvement alone; disseminated cases are rare.7,10 A mortality rate as high as 50% is reported in people with nocardial infection.10 In contrast, disseminated nocardiosis is more common in companion animal species.11 Signs in dogs include fever, high pain scores, depression, anorexia, and weight loss, with or without subcutaneous swelling, respiratory distress, cough, and seizures.11–13 The disseminated form is frequently associated with lung involvement,5 as was likely in this dog given the presence of subpleural nodules. Leukocytosis, neutrophilia, and monocytosis are frequently detected in infected dogs11,12 and were noted in this case. Interestingly, coinfection with canine distemper virus was documented in 55.5% of dogs in a recent study from Brazil.13 This dog was on long-term immunosuppressive therapy for atopy, which likely was a predisposing factor to developing systemic infection. Bacteria could have been inoculated with plant material via a cutaneous wound as suggested by the observation of plant material in the histologic sections of the first excised mass. Dissemination was probably a consequence of immunosuppressive treatment; cyclosporine A is a strong suppressor of T-lymphocyte function14 required to clear nocardial infection.5 Migration of a grass awn


is another possible cause of disseminated infection, but was not documented in this case. The signs and diagnostic findings correspond with those reported previously for dogs with nocardiosis.11–13 The diagnosis of N. abscessus infection has not been reported in dogs previously. However, N. abscessus was not defined as separate species until 200015 and was formerly known as N. asteroides type 1 based on the antimicrobial susceptibility profile.16 Infection with species that formerly belonged to N. asteroides complex account for approximately 60% of human cases of nocardiosis7 and are common isolates from dogs.5 Since its reclassification, N. abscessus has accounted for approximately 5% of human nocardial infections.7 The recent availability of 16S ribosomal DNA sequencing will allow veterinarians to accurately determine the incidence of N. abscessus infection in companion animals. Sequencing analysis is especially valuable because the inciting organism can be identified more quickly than with conventional culture of slowgrowing organisms. This is particularly true when DNA can be extracted directly from a clinical sample as in this case, but often this is not possible owing to contaminating DNA from nonpathogenic bacteria present on the surface of skin lesions. When an adequate sample cannot be isolated from the initial aspirate, DNA isolated from the cultured organism can be utilized for species identification permitting more rapid results than obtained by traditional biochemical analyses. In conclusion, this report underscores the importance of using molecular techniques for the diagnosis of suspected fungal diseases, particularly when the organism has an atypical morphologic appearance on cytologic examination. The slide and plate culture sequences of the organism isolated from this patient were deposited into GenBank under accession #GU471234 and #GU471235, respectively.

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4. Cowell RL, Tyler RD, Meinkoth JH, DeNicola DB. Selected infectious agents. In: Cowell RL, Tyler RD, Meinkoth JH, DeNicola DB, eds. Diagnostic Cytology and Hematology of the Dog and Cat. 3rd ed. St. Louis, MO: Mosby Elsevier; 2008:48–51.

10. Portola O, Guitart R, Olona M, Vidal F, Castro A. Epidemiology and clinical manifestations of infection due to Nocardia species in Tarragona, 1997–2008; Nocardia cyriacigeorgica is an emerging pathogen. Enferm Infecc Microbiol Clin. 2009;27:585–588.

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6. Wada R, Itabashi C, Makayama Y, Ono Y, Murakami C, Yagihashi S. Chronic granulomatous pleuritis caused by nocardia: PCR based diagnosis by nocardial 16S rDNA in pathological specimens. J Clin Pathol. 2003;56: 966–969. 7. Minero MV, Marin M, Cercenado E, Rabadan PM, Bouza E, Munoz P. Nocardiosis at the turn of the century. Medicine (Baltimore). 2009;88:250–261. 8. Wauters G, Avesani V, Charlier A, Janssens M, Vaneechoutte M, Delmee M. Distribution of Nocardia species in clinical samples and their routine rapid identification in the laboratory. J Clin Microbiol. 2005;43:2624–2628. 9. Nocard ME. Note sur la maladie des boeufs de la Guadeloupe: connue sous le nom de farcin [Note on the disease of cattle of Guadeloupe, known as farcy]. Ann Inst Pasteur. 1888;2:293–302.

12. Kirpensteijn J, Fingland RB. Cutaneous actinomycosis and nocardiosis in dogs: 48 cases (1980–1990). J Am Vet Med Assoc. 1992;201:917–920. 13. Ribeiro MG, Salerno T, De Mattos-Guaraldi AL, et al. Nocardiosis: an overview and additional report of 28 cases in cattle and dogs. Rev Inst Med Trop Sao Paulo. 2008;50:177–185. 14. Horsburgh T, Wood P, Brent L. Suppression of in vitro lymphocyte reactivity by cyclosporin A: existence of a population of drug-resistant cytotoxic lymphocytes. Nature. 1980;286:609–611. 15. Yassin AF, Rainey FA, Mendrock U, Brzezinka H, Schaal KP. Nocardia abscessus sp. nov. Int J Syst Evol Microbiol. 2000;50:1487–1493. 16. Kong F, Chen SCA, Chen Z, et al. Assignment of reference 50 -end 16S rDNA sequences and species-specific sequence polymorphisms improves species identification of Nocardia. Open Microbiol J. 2009;3:97–105.

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