A QPCR ASSAY AND TESTING GUIDELINES FOR THE MOLECULAR DIAGNOSIS OF SYSTEMIC ISOSPOROSIS (FORMERLY ATOXPLASMOSIS) IN PASSERINE BIRDS.

Systemic isosporosis (formerly atoxoplasmosis), is a protozoal infection that causes death in nestling and fledgling passerine birds impacting ex situ breeding and reintroduction programs. Because current antemortem diagnostic tests lack sensitivity, a qPCR was developed for detection of Isospora spp. using primers and a fluorescent-tagged MGB probe targeting the large subunit (28s) ribosomal RNA gene (assay efficiency = >100%; sensitivity = <1 dsDNA copy). The assay was used to screen postmortem frozen or formalin-fixed paraffin-embedded tissue samples from passerine birds (n = 24; 12 with confirmed systemic isosporosis), whole blood and feces (n = 38) from live passerines, and other tissues infected with phylogenetically similar protozoa. The qPCR identified Isospora sp. DNA in tissues from 21/24 birds including 12/12 birds with cytologically-histologically confirmed infection (100% sensitivity) and 9/12 birds lacking microscopic organisms. The assay also amplified Eimeria sp. DNA; however, sequence analysis ruled out infection in the passerine cases. Blood and/or feces were positive in 30/38 birds, and in only 7/38 birds, blood and feces both contained Isospora sp. DNA. Finally, the qPCR was utilized to screen 30 consecutive daily fecal samples from live passerines (n = 20) to determine optimal sampling protocols. One or more of the daily fecal samples were positive in all 20 birds. In individual birds, the interval between positive qPCR amplification results ranged from 0 to 23 days, with an average of 5.85 days. Simulated application of 13 potential sample collection schedules was used to identify the sensitivity of repeated testing for identification of infected birds. Increased sampling days resulted in higher sensitivity but increased both cost and animal handling requirements. Based on statistical analysis and clinical considerations, the testing recommendation for detection of fecal shedding was collection and assay of five consecutive daily fecal samples, which had an average diagnostic sensitivity of 0.86.

In situ hybridization to detect Escherichia coli in canine granulomatous colitis.

Canine granulomatous colitis (histiocytic ulcerative colitis) is an uncommon disease, predominantly of young French Bulldogs and Boxer dogs, that manifests from a dysregulated immune response, primarily to adherent-invasive Escherichia coli (AIEC). In conjunction with histopathology and periodic acid-Schiff staining, the diagnosis of granulomatous colitis currently relies on fluorescence in situ hybridization (ISH) or immunohistochemistry to identify and localize AIEC organisms within macrophages in the mucosa and/or submucosa. We investigated the utility of ISH for E. coli using formalin-fixed, paraffin-embedded specimens collected from 29 cases of suspected granulomatous colitis. Most confirmed cases of granulomatous colitis were in French Bulldogs (12 of 20; 60%) and Boxers (3 of 20; 15%), and the mean age was 25 ± 6 mo with no sex predilection. E. coli ISH signal localized bacterial genetic material within the mucosa in 20 of 29 (69%) cases, supporting the diagnosis. ISH signal was limited to the lumen in 8 of 29 (28%) cases, which did not support the identification of these organisms as AIEC. The remaining case had no hybridization signal, and the diagnosis of granulomatous colitis was not supported. Our results revealed that ISH is a quick and specific detection method that can effectively confirm the diagnosis of canine granulomatous colitis.

Comparison of Alfaxalone and Tricaine Methanesulfonate Immersion Anesthesia And Alfaxalone Residue Clearance In Rainbow Trout (Oncorhynchus Mykiss).

Alfaxalone, a synthetic neuroactive steroid, has been tested as an immersion anesthetic in ornamental fish, but its safety and efficacy in sport fish have not been investigated. In the current study, we compared the physiologic and behavioral effects of alfaxalone with those of tricaine methanesulfonate (MS222) for anesthesia of rainbow trout (Oncorhynchus mykiss) via water immersion. We also analyzed alfaxalone-exposed tissues to determine residue clearance times. Fish were anesthetized for 10 min by immersion in low-dose alfaxalone (Alow; 5 mg/L induction, 1 mg/L maintenance), high-dose alfaxalone (Ahigh; 5 mg/L induction, 2 mg/L maintenance), or MS222 (MS; 150 mg/L induction, 100 mg/L maintenance). Fish received all 3 treatments, separated by a washout period of at least 18 d in a blinded, complete crossover design. We hypothesized that immersion in Alow or Ahigh would provide a stable plane of anesthesia in rainbow trout, with dose-dependent time to recovery, and that opercular rates and depths of anesthesia would be equivalent to that of MS222. The time to anesthesia induction was longer for alfaxalone than MS222 but averaged less than 100 s. The time to recovery from anesthesia was also longer for alfaxalone than MS222, with significantly shorter recovery time for Alow than for Ahigh. All treatments decreased opercular rate and response to noxious stimuli. Alfaxalone residue clearance was greater than 80% from all tissues within 1 h, greater than 99% from muscle within 4 h, and 100% from all tissues within 36 h after exposure. We conclude that alfaxalone immersion at 5 mg/L for induction and 2 mg/L for maintenance provides a safe, viable alternative to MS222 for the anesthesia of rainbow trout.