API 20E and invA PCR were evaluated for the identification of Salmonella enterica isolates from swine farms. API 20E had the highest agreement with other tests at the 99.9% likelihood level. Both tests had 100% sensitivity and 96% specificity compared to 16S rRNA sequencing. Compared to serotyping, both tests had 96% sensitivity; specificity was 86% for API 20E and 79% for invA PCR.The API 20E diagnostic, which detects 20 biochemical reactions, is a traditional method for the identification of Salmonella enterica and other Enterobacteriaceae (11). Previous studies of API 20E have reported both good (14,16,20) and inaccurate (1, 2, 9, 18) sample classifications. Genetic identification systems may improve Salmonella identification (8,22). An alternative is the highly sensitive and specific invA PCR (5, 13). invA PCR accuracy for S. enterica detection needs to be confirmed for animal and environmental samples. The present study compared API 20E and invA PCR in the identification of S. enterica, using isolates from swine farms. Test results were validated by comparison with serotyping and 16S rRNA gene sequencing.Three Illinois swine farms were each visited twice over a 3-month period (January to March 2003). The samples collected included feces from pigs and captured rodents, insects, pen floor contents, boot scrapings, feed, and water. Samples were cultured for Salmonella by using tetrathionate (Remel, Lenexa, KS) and then Rappaport-10 (Remel, Lenexa, KS) enrichment broths, plated on xylose-lysine-tergitol-4 (Remel, Lenexa, KS), and brilliant green (Remel, Lenexa, KS) selective and differential media. Suspect colonies were subcultured to tryptic soy agar plates (Difco, Detroit, MI).For each isolate, an API 20E strip (bioMérieux, Inc., Hazelwood, MO) was inoculated and incubated according to the manufacturer's instructions. The likelihood of S. enterica was calculated, using the manufacturer's coding system, based on reactions to reagents in the 20 compartments.Template DNA for amplification was prepared by adding a tryptic soy agar plate colony sample to 100 l sterile Millipore water and boiling for 5 min. An oligonucleotide primer set, producing a 244-bp amplified fragment, was selected from a published S. enterica invA gene sequence (5). For all assays, 2 l of template was added to a 23-l PCR mixture, containing 0.4 M of each primer (Integrated DNA Technologies, Inc., Coralville, IA), 250 M deoxynucleoside triphosphates (TaKaRa Bio Inc., Otsu, Shiga, Japan), 1ϫ PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl 2 ), and 0.5 units of TaKaRa recombinant Taq (TaKaRa Bio Inc., Otsu, Shiga, Japan). Amplification conditions were as described previously (5).A 16S rRNA primer (rrs) set (Integrated DNA Technologies, Inc.), amplifying a 320-bp fragment common to all bacteria (21), served as the DNA template quality control. A 0.1 M concentration of each rrs primer was added to each PCR. Amplicons were separated by gel electrophoresis and photographed under UV illumination.For comparison, two additional primer set...

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 μm 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.

An 8-year-old dog presented with several dermal excoriations. Lesion cytology revealed pyogranulomatous inflammation with branching, septate hyphae. A mold identified as Mycoleptodiscus indicus by morphology and sequencing was cultured from fine-needle aspirates. This is the first report of a Mycoleptodiscus species as an etiologic agent in a dog. CASE REPORTAn 8-year-old, outdoor, male, castrated pointer dog was presented to the University of Illinois Veterinary Teaching Hospital in April 2009 for recheck blood work 2 months after diagnosis of immune-mediated hemolytic anemia. Immunosuppressive treatment included daily oral administration of 2.1 mg prednisone/kg of body weight and 10.4 mg/kg cyclosporine (Atopica; Novartis Animal Health), along with 20 mg aspirin to prevent clot formation and 20 mg famotidine to limit gastrointestinal upset associated with the steroid administration. In addition, 200 mg doxycycline had been given twice daily for several weeks to treat for a possible underlying tick-borne illness.The general physical exam revealed a potbellied appearance, hepatomegaly, and moderate to marked cachexia. The left rear leg was swollen, with pitting edema and a draining tract on the lateral aspect of the hock, the left popliteal lymph node was markedly enlarged, and several areas of minor dermal excoriations were present along the nasal planum. In addition, the dog's weight had decreased from 38.5 kg to 35 kg over the 2-month time period. Clinical differentials for the draining tract included phaeohyphomycosis, zygomycosis, pythiosis, lagenidiosis, sporotrichosis, and atypical bacterial infections, such as nocardiosis, actinomycosis, and mycobacteriosis.Fine-needle aspirates were acquired from the left popliteal lymph node and from lesions on the bridge of the nose and the distal aspect of the left hind leg. Aspirates were submitted for cytologic evaluation, aerobic bacterial cultures, and fungal cultures. All cytology samples were Wright-Giemsa stained and were highly cellular, with a mixed population of severely degenerate neutrophils and reactive macrophages, including epithelioid macrophages and multinucleated giant cells. Bacterial cocci were noted extracellularly and within neutrophils. Aspirates from all sites contained basophilic, septate hyphae which measured 30 to 100 m in length and 3 to 6 m in width (Fig. 1). Some hyphae terminated in bulbous ends. The sample from the popliteal lymph node also contained several lymphocytes and rare, large, round yeastlike structures which rarely exhibited a narrow-base bud. The samples were interpreted as marked pyogranulomatous inflammation with bacterial and fungal sepsis.Pending results of bacterial and fungal cultures, itraconazole was started at 5 mg/kg once daily, in addition to 32 mg/kg terbinafine once daily and 29 mg/kg cephalexin twice daily. The aerobic bacterial culture revealed a Staphylococcus pseudintermedius strain that was resistant to cephalexin but susceptible to clindamycin, amoxicillin-clavulanate, and enrofloxacin. Cephalexin was di...

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