Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry (MS)-based proteomics technology has been extensively used in biological fluids and offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins. We utilized surplus lithium-heparin plasma from 30 healthy dogs, which were subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Our goal was to identify and quantify plasma proteins via label-free quantification LC-mass spectrometry. Additionally, we employed different methods to deplete the most abundant proteins. Firstly, we used a commercial kit for the depletion of high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation Factor V. However, neither of the depletion techniques achieved significant depletion of high-abundant proteins. Nevertheless, the two different depletion methods exhibited substantial differences in the fold-change of many proteins, suggesting partial depletion that did not contribute to an increase in the number of detected proteins. Despite this limitation, we were able to detect and quantify many clinically relevant proteins. The determination of the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. This reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

CASE DESCRIPTION A 9-year-old spayed female Maine Coon cat was presented at the University of Veterinary Medicine Vienna for further investigation of chronic nonpruritic bilateral ear disease and unilateral Horner syndrome. CLINICAL FINDINGS Physical examination and otoscopy findings included right sided Horner syndrome, a right head tilt of approximately 20° and a small pink nodule in the right and several smaller nodules in the left proximal horizontal external ear canal. Computed tomography and magnetic resonance imaging revealed soft tissue opacity material in both middle ear cavities, the caudal portion of the nasal cavity, the left nasopharyngeal meatus and the right frontal sinus. Via videootoscopy, 2 multilobular and several flat nodules were detected in the proximal right horizontal external ear canal and in the left tympanic bulla, respectively. Histopathological examination confirmed the diagnosis of cholesterol granulomas. TREATMENT AND OUTCOME All otic cholesterol granulomas (CGs) were removed via video-otoscopy (VO), and topical treatment was initiated in addition to oral prednisolone. After the histopathological confirmation, negative microbial cultures from the middle ear cavities, and the remission of the symptoms by the first recheck, topical, and systemic treatment were discontinued. A follow-up 6 months later, did not reveal any recurrence of the CGs. CLINICAL RELEVANCE To our knowledge, this is the first case of bilateral CGs diagnosed with a combination of CT, MRI, VO, and histopathology and removed minimal invasively via VO, without a need for ventral bulla osteotomy, which led to complete remission of all signs and no relapse until the follow up 6 months later.

The role of the gut microbiome in developing Inflammatory Bowel Disease (IBD) in humans and dogs has received attention in recent years. Evidence suggests that IBD is associated with alterations in gut microbial composition, but further research is needed in veterinary medicine. The impact of IBD treatment on the gut microbiome needs to be better understood, especially in a breed-specific form of IBD in Yorkshire Terriers known as Yorkshire Terrier Enteropathy (YTE). This study aimed to investigate the difference in gut microbiome composition between YTE dogs during disease and remission and healthy Yorkshire Terriers. Our results showed a significant increase in specific taxa such as Clostridium sensu stricto 1, Escherichia-Shigella, and Streptococcus, and a decrease in Bacteroides, Prevotella, Alloprevotella, and Phascolarctobacterium in YTE dogs compared to healthy controls. No significant difference was found between the microbiome of dogs in remission and those with active disease, suggesting that the gut microbiome is affected beyond clinical recovery.

The role of the faecal microbiome in the pathogenesis of both human and canine inflammatory bowel disease (IBD) has been in the spotlight in recent years. While trying to unravel the mechanism of IBD, clear alterations in intestinal microbial composition in individuals with IBD have been reported in the past. However, data in veterinary medicine are still scarce and inconsistent. Currently, the exact role of the gut microbiota alterations in the different phenotypes of canine chronic enteropathies and the impact of treatment on it are not well established. The objective of this study was to investigate the difference in the gut microbiome composition of Yorkshire Terriers with breed specific IBD, or Yorkshire Terrier Enteropathy (YTE), during disease and remission compared to healthy Yorkshire Terriers. Microbiome analysis of YTE was mainly characterised by a significant relative enrichment in taxa including Clostrodium sensu stricto 1, Escherichia-Shigella, Streptococcus and decrease in Bacteroides, Prevotella, Alloprevotella and Phascolarctobacterium compared to healthy controls. There was no significant difference in the microbiome composition of dogs in remission and dogs with active disease, indicating a longer lasting perturbation even beyond clinical recovery.

Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry (MS)-based proteomics technology has been extensively used in biological fluids and offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins. We utilized surplus lithium-heparin plasma from 30 healthy dogs, which were subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Our goal was to identify and quantify plasma proteins via label-free quantification LC-mass spectrometry. Additionally, we employed different methods to deplete the most abundant proteins. Firstly, we used a commercial kit for the depletion of high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation Factor V. However, neither of the depletion techniques achieved significant depletion of high-abundant proteins. Nevertheless, the two different depletion methods exhibited substantial differences in the fold-change of many proteins, suggesting partial depletion that did not contribute to an increase in the number of detected proteins. Despite this limitation, we were able to detect and quantify many clinically relevant proteins. The determination of the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. This reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.