Background: Reliable biomarkers to differentiate gastrointestinal cancer (GIC) from chronic inflammatory enteropathy (CIE) in dogs are needed. Fecal and serum micro-RNAs (miRNAs) have been proposed as diagnostic and prognostic markers of GI disease in humans and dogs.Hypothesis/Objectives: Dogs with GIC have fecal and serum miRNA profiles that differ from those of dogs with CIE. Aims: (a) identify miRNAs that differentiate GIC from CIE, (b) use high-throughput reverse transcription quantitative real-time PCR (RT-qPCR) to establish fecal and serum miRNA panels to distinguish GIC from CIE in dogs.

Objectives To validate a new method for stabilization and quantitation of the clopidogrel active metabolite (CAM), clopidogrel, and inactive clopidogrel carboxylic acid and 2-oxo-clopiodgrel. Animals Feline plasma was used for assay validation. A pilot pharmacokinetic study was conducted with 2 healthy cats. Methods CAM stabilization was achieved by adding 2-bromo-3′methoxyacetophenone to blood tubes to form a derivatized CAM (CAM-D). CAM-D was quantified using high performance liquid chromatography and tandem mass spectrometry. Validation of the methodology included evaluation of calibration curve linearity, accuracy and precision, and stability using quality control samples spiked with CAM-D, clopidogrel, clopidogrel carboxylic acid, and 2-oxo-clopidogrel. In vivo utility of this assay was evaluated by conducting a pharmacokinetic study in cats receiving a single oral dose of 18.75mg clopidogrel. Results The 2-oxo-clopidogrel metabolite was unstable. CAM-D, clopidogrel, and clopidogrel carboxylic acid appear stable for 1 week at room temperature and 9 months at −80°C. Standard curves showed linearity for CAM- D, clopidogrel, and clopidogrel carboxylic acid (r>0.99). Between assay accuracy and precision was ≤2.6% and ≤2.8% for CAM-D and ≤17.9% and ≤11.3% for clopidogrel and clopidogrel carboxylic acid. Within assay precision for all three compounds was ≤7%. All three compounds were detected in plasma from healthy cats receiving clopidogrel. Conclusions This methodology is accurate and precise for simultaneous quantitation of CAM-D, clopidogrel, and clopidogrel carboxylic acid in feline plasma but unsuitable for 2-oxo-clopidogrel. Validation of this assay is the first step to more fully understanding the use of clopidogrel in cats in both a population and individual setting.

BackgroundGastrointestinal (GI) cancer accounts for 14% of feline malignancies. There is a great need for reliable noninvasive diagnostic biomarkers to reach a timely diagnosis and initiate treatment. Fecal microRNAs (miRNAs) could be such a biomarker and have shown great potential in colorectal screening in people but have yet to be investigated in cats.ObjectivesWe aimed to evaluate the presence and stability of feline fecal miRNA under different storage conditions (room temperature [RT], 4, and −20°C) and to evaluate the expression levels of specific fecal miRNAs collected on three separate days (days 1, 4, and 7) in healthy cats.MethodsHealthy cats were prospectively recruited. Fecal samples were collected, aliquoted, and stored for 24 hours at RT and then transferred to −20°C, stored for 24 hours at 4°C and then transferred to −20°C, or were immediately placed at −20°C on day 1 or at −20°C on days 4 and 7 postcollection. Expression of 22 miRNAs was investigated using quantitative real‐time PCR.ResultsTen miRNA assays worked well, and one, let‐7b, was used for normalization. No differences in miRNA expression were seen between the three storage temperatures for the nine miRNAs investigated. Only miR‐26a showed a significant increase in expression between samples of days 1 and 7. The rest of the miRNAs levels were stable over time.ConclusionsFecal miRNA can be isolated from healthy cats. The expression was stable at different temperatures and for most of the miRNAs over time. Prospective studies evaluating fecal miRNA as biomarkers in cats with GI neoplasia are warranted.

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Dirofilariasis is spreading among dogs and humans in Europe with infections being established in many countries. In Denmark, at least one to two generations of Dirofilaria spp. can occur per year. Here, we describe the first molecular biologically confirmed case of D. repens infection in a dog in Denmark.