Cyclone: an accessible pipeline to analyze, evaluate, and optimize multiparametric cytometry data

In the past decade, high-dimensional single-cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation, which are computationally intense and difficult to evaluate and optimize. Here, we present Cytometry Clustering Optimization and Evaluation (Cyclone), an analysis pipeline integrating dimensionality reduction, clustering, evaluation, and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full-spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification but also enables the unsupervised identification of lymphocytes and mononuclear phagocyte subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on a variety of cytometry datasets, which will further power immunology research and provide a scaffold for biological discovery

Aflatoxicosis in nine dogs after exposure to contaminated commercial dog food

The purpose of this study was to characterize light and electron microscopic findings from 9 dogs that had consumed aflatoxin-contaminated commercial dog food from recalled batches. Four dogs died and 5 were euthanized after signs of liver failure. Analysis of feed and liver samples confirmed exposure to aflatoxin. Of the 9 dogs, 8 had classic signs of liver failure, and 1 had signs of liver failure. Enlarged, pale yellow livers were seen macroscopically at necropsy in the dogs with subacute hepatopathy, and cirrhosis was noted in the dog with chronic hepatopathy. Histopathologic findings included hepatic lipidosis, portal fibroplasia, and biliary hyperplasia, which supported a diagnosis of subacute toxic hepatopathy in the 8 symptomatic animals. Marked lobular atrophy, bridging portal fibrosis, and regenerative hepatocellular nodules characterized the dog with chronic hepatopathy. Electron microscopy revealed marked hepatocellular lipid vacuolation and early fibroplasia in the dogs with acute hepatopathy and marked fibrosis and regeneration in the dog with chronic hepatopathy. Analysis of feed for aflatoxin consistently revealed high levels of aflatoxin B1 (range of 223-579 ppb), and hepatic tissue contained elevated levels of aflatoxin B1 metabolite M1 (0.6-4.4 ppb). Although dogs are not commonly affected by aflatoxicosis, they are highly susceptible and can present with classic signs of acute or chronic hepatopathy. Characteristic gross, histologic, and electron microscopic changes help pathologists determine a presumptive toxic insult. Detecting aflatoxins or their metabolites in feed or liver specimens can help confirm the diagnosis of aflatoxicosis

Prevalence of urovirulence genes cnf, hlyD, sfafoc, and papGIII in fecal Escherichia coli from healthy dogs and their owners

Objective¡ªTo determine the prevalence of 4 urovirulence genes in fecal Escherichia coli isolates from healthy dogs and their owners and to determine whether detection of E coli strains with these genes was associated with a history of urinary tract infection (UTI). Sample Population¡ª61 healthy dog-owner pairs and 30 healthy non¨Cdog owners. Procedures¡ªA fecal specimen was obtained from each participant, and 3 colonies of E coli were isolated from each specimen. A multiplex PCR assay was used to detect 4 genes encoding virulence factors: cytotoxic necrotizing factor (cnf), hemolysin (hlyD), s-fimbrial and F1C fimbriae adhesin (sfa/foc), and pilus associated with pyelonephritis G allele III (papGIII). Human participants completed a questionnaire to provide general information and any history of UTI for themselves and, when applicable, their dog. Results¡ª26% (16/61) of dogs, 18% (11/61) of owners, and 20% (6/30) of non¨Cdog owners had positive test results for ¡Ý 1 E coli virulence gene. One or more genes were identified in fecal E coli isolates of both dog and owner in 2% (1/61) of households. There was no difference in the detection of any virulence factor between dog-owner pairs. Female owner history of UTI was associated with detection of each virulence factor in E coli strains isolated from their dogs\u27 feces. Conclusions and Clinical Relevance¡ªDogs and humans harbored fecal E coli strains possessing the genes cnf, hlyD, sfa/foc, and papGIII that encode urovirulence factors. It was rare for both dog and owner to have fecal E coli strains with these virulence genes