Development of a novel real-time PCR multiplex assay for detection of Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus.

Strangles is a contagious bacterial disease of horses caused by Streptococcus equi subspecies equi (SEE) that occurs globally. Rapid and accurate identification of infected horses is essential for controlling strangles. Because of limitations of existing PCR assays for SEE, we sought to identify novel primers and probes that enable simultaneous detection and differentiation of infection with SEE and S. equi subsp. zooepidemicus (SEZ). Comparative genomics of U.S. strains of SEE and SEZ (n = 50 each) identified SE00768 from SEE and comB from SEZ as target genes. Primers and probes for real-time PCR (rtPCR) were designed for these genes and then aligned in silico with the genomes of strains of SEE (n = 725) and SEZ (n = 343). Additionally, the sensitivity and specificity relative to microbiologic culture were compared between 85 samples submitted to an accredited veterinary medical diagnostic laboratory. The respective primer and probe sets aligned with 99.7 % (723/725) isolates of SEE and 97.1 % (333/343) of SEZ. Of 85 diagnostic samples, 20 of 21 (95.2 %) SEE and 22 of 23 SEZ (95.6 %) culture-positive samples were positive by rtPCR for SEE and SEZ, respectively. Both SEE (n = 2) and SEZ (n = 3) were identified by rtPCR among 32 culture-negative samples. Results were rtPCR-positive for both SEE and SEZ in 21 of 44 (47.7 %) samples that were culture-positive for SEE or SEZ. The primers and probe sets reported here reliably detect SEE and SEZ from Europe and the U.S., and permit detection of concurrent infection with both subspecies.

Disseminated Scytalidium infection in a German shepherd dog.

We report a systemic mycosis in a German shepherd dog caused by Scytalidium spp. The patient presented for progressive cervical pain and forelimb hemiparesis. Cervical computed tomography revealed lysis associated with multiple vertebrae and a soft tissue mass adjacent to the spinal cord, as well as prescapular lymphadenopathy. Fine needle aspirates of the lymph nodes yielded hyphae, and a subsequent culture obtained a Scytalidium spp. Itraconazole therapy was initiated, but the subject was euthanized three months later due to progressive neurologic disease and discomfort. This appears to be the first report of disseminated disease by this species in veterinary medicine.

Frequency of Corynebacterium pseudotuberculosis infection in horses across the United States during a 10-year period.

OBJECTIVE: To quantify the number of horses with Corynebacterium pseudotuberculosis infection identified in the United States from January 2003 through December 2012. DESIGN: Cross-sectional study. SAMPLE: State veterinary diagnostic laboratory records of 2,237 C pseudotuberculosis culture-positive samples from horses. PROCEDURES: 44 state veterinary diagnostic laboratories throughout the United States were invited by mail to participate in the study. Data requested included the number of C pseudotuberculosis culture-positive samples from horses identified per year, geographic location from which the C pseudotuberculosis culture-positive sample was submitted, month and year of sample submission, breed and age of horses, and category of clinical manifestation (ie, internal infection, external infection, or ulcerative lymphangitis). RESULTS: Of the 44 invited laboratories, 15 agreed to participate and provided data on affected horses from 23 states. The proportion of C pseudotuberculosis culture-positive samples submitted during 2011 through 2012 (1,213/2,237 [54%]) was significantly greater than that for the period from 2003 through 2010 (1,024/2,237 [46%]). Corynebacterium pseudotuberculosis was recovered from horses in states where the disease has not been previously recognized as endemic. Affected horses were identified year-round. The greatest proportion of C pseudotuberculosis culture-positive samples was identified during November, December, and January (789/2,237 [35%]). No significant association between the clinical form of disease and age or breed of horse was observed. CONCLUSIONS AND CLINICAL RELEVANCE: The occurrence of C pseudotuberculosis infection in horses increased during the 10-year period, and affected horses were identified throughout the United States. Further studies to determine changes in annual incidence and to identify potential changing climatic conditions or vector populations associated with disease transmission are warranted to help control the occurrence and spread of C pseudotuberculosis infection in horses.

Cutaneous atypical mycobacteriosis in a clouded leopard (Neofelis nebulosa).

A 16-yr-old male clouded leopard (Neofelis nebulosa) was presented for lethargy and anorexia. A cutaneous abdominal mass extending from the pubis to just caudal to the xiphoid process was present. A biopsy revealed histologic lesions consistent with an atypical mycobacterial infection consisting of diffuse, severe, pyogranulomatous dermatitis and panniculitis, with clear vacuoles and 3-5 microm, intravacuolar, faintly eosinophilic, filamentous bacilli that stained positively with FiteFaraco modified acid-fast stain. The clouded leopard had biochemical findings suggestive of chronic renal failure and euthanasia was elected. Histological evaluation of tissues collected at postmortem examination revealed multicentric B-cell lymphoma involving the oral cavity, liver, spleen, and multiple lymph nodes, bilateral testicular seminomas, thyroid follicular cell adenoma, thyroid C cell adenoma, and biliary cystadenomas. Bacterial culture and molecular sequencing identified the causative agent of the cutaneous abdominal mass as belonging to the Mycobacterium fortuitum group.

Evaluation of effects of high incubation temperatures on results of protozoal culture and real-time PCR testing for Tritrichomonas foetus inoculated in a commercially available self-contained culture media system.

OBJECTIVE: To evaluate effects of high incubation temperatures on results of protozoal culture and real-time PCR testing for Tritrichomonas foetus inoculated in a commercially available self-contained culture media system. DESIGN: In vitro experimental study. SAMPLE: 2 strains of T foetus (1 field isolate from the University of California-Davis and 1 field isolate from the Texas Veterinary Medical Diagnostic Laboratory). PROCEDURES: 2 sets of 36 dual-chamber media pouches were inoculated with T foetus (36 sample pouches/strain) and incubated at temperatures of 37.0°C (98.6°F), 46.1°C (115.0°F), or 54.4°C (130.0°F) for 1, 3, 6, or 24 hours. Six uninoculated media samples in pouches stored at 37.0°C for the entire treatment period were used as negative controls. Pouches were removed from incubators and stored at 22.2°C (72.0°F) until all treatments were complete. Samples were submitted to a diagnostic laboratory for protozoal culture and real-time PCR testing. RESULTS: T foetus was detectable microscopically in inoculated pouches incubated at 37.0°C regardless of exposure time, whereas those incubated at 46.1°C yielded T foetus after 1 and 3 hours only, and those incubated at 54.4°C yielded T foetus after 1 hour only. Testing via real-time PCR assay yielded positive results for all inoculated media samples and negative results for all uninoculated control samples. CONCLUSIONS AND CLINICAL RELEVANCE: Samples collected into the self-contained culture media system for T foetus testing via culture alone should be protected from high temperatures. Realtime PCR amplification may be a more reliable method for identification of the organism if storage and transport temperatures cannot be controlled.