Joao Pedro Cavasin

Objective: To adapt and partially validate the accuracy and precision of serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations as an analytical prerequisite to investigate the clinical utility of relative exchangeable copper and urinary small copper carrier-to-creatinine ratio as noninvasive biomarkers in dogs with copper-associated hepatitis. Methods: Serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations of surplus samples of dogs submitted for diagnostic purposes from October 2023 through March 2024 were measured by adapting a previously described ultracentrifugation method. Copper concentrations were quantified using inductively coupled plasma mass spectrometry. The optimal relative centrifugal force was selected based on minimizing the variability among replicates. Analytical performance was evaluated through spike recovery and intra- and interassay repeatability. Results: Optimal parameters were identified as 5 g/L EDTA and 5,000 X g for serum exchangeable copper and 4,000 X g for 3-kDa ultrafiltered urinary copper. Spike recovery ranged from 97.9% to 108.5% for serum exchangeable copper and from 96.7% to 120% for ultrafiltered urinary copper. The median intra- and interassay coefficients of variation were 8.5% and 10.8% for exchangeable copper and 2.6% and 5.6% for ultrafiltered urinary copper, respectively. Conclusions: Ultrafiltration-based methods for measuring serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations were successfully adapted for dogs, with spike recovery and intra- and interassay coefficients of variation within predefined acceptance criteria. Clinical Relevance: The results provide the analytical foundation for future investigation of the diagnostic utility of relative exchangeable copper and urinary small copper carrier protein as noninvasive biomarkers for copper-associated hepatitis in dogs.

Objectives: Serial measurement of serum feline pancreatic lipase immunoreactivity (fPLI) and trypsin-like immunoreactivity (fTLI) concentrations in cats with chronic enteropathy (CE) has not been previously reported. The aim of the current observational study was to serially evaluate serum fPLI and fTLI concentrations in cats with different forms of CE. Methods: A total of 60 cats with CE were included, diagnosed either with immunosuppressive-responsive enteropathy (IRE; n = 22), food-responsive enteropathy (FRE; n = 7) or low-grade intestinal T-cell lymphoma (LGITL; n = 32, one diagnosed previously as FRE progressed to lymphoma). Serum fPLI and fTLI concentrations were measured in 252 and 245 serum samples, respectively, from 60 cats with CE at initial presentation and during re-examination. Results: Serum fPLI concentrations indicative of pancreatitis were found in 3/60 cats (one with IRE and two with LGITL) at initial presentation and in an additional 8/60 cats (four with IRE and four with LGITL) during treatment (24/252 serum samples). Serum fTLI concentrations indicative of exocrine pancreatic insufficiency were found in 2/60 cats (both with IRE) at initial presentation and during re-examination (n = 3/245). None of the cats with FRE had fPLI concentrations above or fTLI concentrations below the respective reference intervals (RIs). Conclusions and relevance: Serum fPLI concentrations may fluctuate during treatment in a subset of cats with IRE or LGITL and, in some cats, are indicative of pancreatitis. Serum fTLI concentrations may vary within and outside the RI in a relatively small proportion of cats with IRE. The long-term clinical importance of these findings requires further study.

A nine-year-old, 10.5 kg, female-spayed Poodle-mix dog presented with a five-day history of vomiting and regurgitation. Thoracic radiographs revealed an area with increased opacity within the esophageal lumen. A ductal esophageal lesion with intraluminal extension and mild left axillary lymphadenopathy were detected on computed tomography. Esophagoscopy revealed a large vascular, obstructive mass with a smooth surface, in the mid part of the esophagus. Endoscopic biopsies were collected, and histopathologic findings were consistent with an inflammatory polyp. Surgical excision of the mass via sternotomy was performed and the mass was sent for histopathologic evaluation. Histopathological examination of the mass revealed an esophageal plasmacytoma with perivascular amyloid deposition, which was confirmed by immunohistochemical staining. There was no evidence of regrowth until 18 months after surgical removal, when evidence of regrowth was identified. The dog had only one episode of vomiting and regurgitation which was resolved after symptomatic treatment.

Antibiotics are commonly used to aid in the remission of gastrointestinal diseases, but usage may lead to prolonged dysbiosis. The objective of this study was to evaluate the effects of metronidazole on fecal microbiota, fecal metabolites, and serum bile acids and uremic toxins of healthy adult cats. Twelve healthy adult cats (4.7 ± 0.4 yr) received metronidazole (20 mg/kg BW PO BID) for 14 days (day 0–14) and were monitored during a 28-day recovery period (day 15–42). Fecal and blood samples were collected at baseline (day 0), after metronidazole (day 14), and weekly during recovery (on days 21, 28, 35, and 42). Fecal samples were analyzed for microbiota and bacterial metabolites. Serum samples were analyzed for bile acids and uremic toxins. Metronidazole increased dysbiosis index and fecal lactate concentrations (p < 0.0001) and decreased fecal propionate, butyrate, and secondary bile acid concentrations (p < 0.0001) for up to 28 days. Prolonged dysbiosis and Peptacetobacter (Clostridium) hiranonis reductions were observed in 10/12 (83%) cats. Serum uremic toxins were also reduced (p < 0.0001) after metronidazole administration. The observed changes after metronidazole administration illustrate how changes in the gut microbiome alter microbial metabolism and its relation to host dysmetabolism. In conclusion, metronidazole is a potent antibiotic with persistent effects observed in the microbiome and metabolome, even up to one month after administration.

Bile acids (BAs) are important signaling molecules in the gastrointestinal (GI) tract and are associated with health and disease in humans and animals. Intestinal bacteria transform BA through deconjugation, dehydroxylation, and epimerization reactions, producing various isoforms, many of which have not been investigated in companion animal diseases. We aimed to develop and analytically validate a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of 30 BAs in dog feces, with a simple extraction procedure and on-line solid-phase extraction. Validation demonstrated good accuracy, precision, sensitivity, spiking recovery, dilution, and stability for 29 BAs. The method was applied to fecal samples from healthy dogs (H; n = 121) and dogs with chronic enteropathy (CE; n = 58). The immediate and downstream products of bacterial 7α-dehydroxylation reactions with cholic acid were lower in concentration in dogs with CE when compared to healthy dogs (deoxycholic acid, 3-oxo-deoxycholic acid, and 12-oxo-lithocholic acid; q < 0.001). Across all fecal samples, the products of hydroxysteroid dehydrogenase (including oxo- and iso-BA) made up an average of 30% of the total measured fecal BA pool (glycine-BA, 0.1%; taurine-BA, 2.2%; unconjugated BA, 53%).

Antibiotics are commonly used to aid in the remission of gastrointestinal diseases, but usage may lead to prolonged dysbiosis. The objective of this study was to evaluate the effects of metronidazole on fecal microbiota, fecal metabolites, and serum bile acids and uremic toxins of healthy adult cats. Twelve healthy adult cats (4.7±0.4 yr) received metronidazole (20 mg/kg BW PO BID) for 14 days (day 0–14) and were monitored during a 28-day recovery period (day 15–42). Fecal and blood samples were collected at baseline (day 0), after metronidazole (day 14), and weekly during recovery (days 21, 28, 35, and 42). Fecal samples were analyzed for microbiota and bacterial metabolites. Serum samples were analyzed for bile acids and uremic toxins. Metronidazole increased dysbiosis index and fecal lactate concentrations (P<0.0001), and decreased fecal propionate, butyrate, and secondary bile acid concentrations (P<0.0001) for up to 28 days. Prolonged dysbiosis and Peptacetobacter (Clostridium) hiranonis reductions were observed in 10/12 (83%) cats. Serum uremic toxins were also reduced (P<0.0001) after metronidazole administration. The observed changes after metronidazole administration illustrate how changes in the gut microbiome alter microbial metabolism and its relation to host dysmetabolism. In conclusion, metronidazole is a potent antibiotic with persistent effects observed in the microbiome and metabolome, even up to one month after administration.

Several recent studies have reported a significantly greater prevalence of Clostridium perfringens encoding the novel pore-forming netF toxin gene in dogs with acute hemorrhagic diarrhea syndrome. However, the presence of netF in other canine diarrheal diseases remains poorly characterized. This retrospective, cross-sectional study aimed to describe the prevalence and abundance of netF-positive C. perfringens in fecal samples from 352 dogs with acute and chronic gastrointestinal diseases. Dogs were divided into five groups: acute hemorrhagic diarrhea syndrome (AHDS), acute diarrhea (AD), chronic enteropathy (CE), exocrine pancreatic insufficiency (EPI), and healthy controls (HCs). The abundances of C. perfringens 16S rRNA, the C. perfringens enterotoxin gene and the C. perfringens netF gene in fecal samples were analyzed by quantitative polymerase chain reaction. In total, 7 of 15 (46.7%) dogs with AHDS, 10 of 75 (13.3%) dogs with AD, 2 of 120 (1.7%) dogs with CE, 1 of 12 (8.3%) dogs with EPI, and 1 of 130 (0.8%) HC dogs tested positive for netF. This study provides further evidence that NetF may be a significant contributor to the etiology of AHDS and potentially to a subset of acute nonhemorrhagic diarrhea cases, while it was only rarely detected in chronic gastrointestinal disease phenotypes.

We are grateful to the authors for providing additional data to demonstrate the presence of domestic cat hepadnavirus in lymphoma tissues

While shifts in gut microbiota have been studied in diseased states, the temporal variability of the microbiome in cats has not been widely studied. This study investigated the temporal variability of the feline dysbiosis index (DI) and the abundance of core bacterial groups in healthy adult cats. The secondary aim was to evaluate the relationship between the fecal abundance of Clostridium hiranonis and the fecal concentrations of unconjugated bile acids. A total of 142 fecal samples collected from 17 healthy cats were prospectively included: nine cats with weekly collection over 3 weeks (at least four time points), five cats with monthly collection over 2 months (three time points), and three cats with additional collections for up to 10 months. The DI remained stable within the reference intervals over two months for all cats (Friedman test, p > 0.2), and 100% of the DI values (n = 142) collected throughout the study period remained within the RI. While some temporal individual variation was observed for individual taxa, the magnitude was minimal compared to cats with chronic enteropathy and antibiotic exposure. Additionally, the abundance of Clostridium hiranonis was significantly correlated with the percentage of fecal primary bile acids, supporting its role as a bile acid converter in cats.