Healthy Dogs Research Articles

The Molecular Signature Related to Local Inflammatory and Immune Response in Canine Cutaneous Hypersensitivity Reactions: A Preliminary Study.

Cutaneous hypersensitivity reactions (CHRs) are complex inflammatory skin disorders that affect humans and dogs. This study examined the inflammatory and immune responses leading to skin damage, inflammation, and irritation by investigating gene expression through quantitative PCR (qPCR) and protein localization through the immunohistochemistry (IHC) of specific receptors and molecules involved in CHRs. Formalin-fixed paraffin-embedded (FFPE) samples from canine CHR skin (n = 20) and healthy dog skin (n = 3) were analyzed for expression levels of eight genes, including members of the pattern recognition receptor (PRR) family, CD209 and CLEC4G, the Regakine-1-like chemokine, and acute phase proteins (APPs), LBP-like and Hp-like genes. Additionally, we examined the local involvement of IL-6, Janus Kinase 1 (JAK1), and the signal transducer activator of transcription 3 (STAT3) in the CHR cases. The study demonstrated statistically significant increases in the expression levels of CD209, Hp-like (p < 0.01), LBP-like, Regakine-1-like, and CLEC4G (p < 0.05) genes in CHRs compared to healthy controls. Conversely, IL-6, JAK1, and STAT3 showed no significant difference between the two groups (p > 0.05). Protein analysis revealed JAK1 and STAT3 expression in CHR hyperplastic epithelial cells, dermal fibroblasts, and endothelial cells of small capillaries, indicating a possible involvement in the JAK/STAT pathway in local inflammatory response regulation. Our findings suggest that the skin plays a role in the development of CHRs.

Characterization of faecal microbiota and serum inflammatory markers in dogs diagnosed with chronic enteropathy or small-cell lymphoma: a pilot study

Dogs diagnosed with chronic enteropathy (CE) or small-cell lymphoma (SCL) exhibit marked differences in faecal microbiota and organic acid profiles compared with healthy dogs, as well as immune abnormalities in intestinal mucosal tissue. However, few studies have analysed trace organic acids, such as succinic acid, which have been suggested to be associated with IBD in humans. Therefore, in this study, we compared the faecal microbiota and organic acid profiles as well as serum inflammatory markers between dogs with disease (n = 11; 6 with CE and 5 with SCL) and healthy controls (n = 16). We also performed machine learning and correlation analysis to obtain more detailed insights into the characteristics of affected dogs. These results revealed that dogs with CE and SCL had lower levels of Erysipelotrichaceae (e.g. Turicibacter and Allobaculum), exhibited abnormalities in the succinic acid metabolism (i.e. succinic acid accumulation and decreased levels of Phascolarctobacterium as succinic acid-utilising bacteria) and increased levels of pathobiont bacteria such as Escherichia-Shigella. Additionally, the presence of Dubosiella was significantly negatively correlated with Canine Inflammatory Bowel Disease Activity Index scores. These findings are expected to aid the development of microbiome-based medications and/or supplements, although further verification is needed.

Enterohemorrhagic Escherichia coli (EHEC) disrupts intestinal barrier integrity in translational canine stem cell-derived monolayers.

This study develops a new model to better understand Enterohemorrhagic E. coli (EHEC) infections, a serious bacterial disease affecting both dogs and humans, characterized by symptoms such as hemorrhagic diarrhea and hemolytic uremic syndrome. Traditional research models have fallen short of mimicking how this disease manifests in patients. Our research used intestinal tissues from healthy dogs to create layers of cells, known as colonoid-derived monolayers, which we then exposed to EHEC. We assessed the damage caused by the bacteria using several techniques, observing significant changes similar to those seen in actual cases of the disease. The model proved effective in replicating the interaction between the host and the pathogen, marking an important step toward understanding EHEC's effects and developing treatments. This canine colonoid-derived monolayer system not only bridges a crucial gap in current research but also offers a promising platform for studying other enteric pathogens affecting both canine and human health.

Future perspectives of lactic acid bacteria isolated from the urinary tract of healthy dogs: a comment.

Future perspectives of lactic acid bacteria isolated from the urinary tract of healthy dogs: a comment.

Evaluation of right ventricular function using conventional and real-time three-dimensional echocardiography in healthy dogs and dogs with myxomatous mitral valve disease

Introduction/ObjectivesTo compare conventional and three-dimensional (3D) echocardiographic indices of right ventricular (RV) systolic function in dogs with various stages of myxomatous mitral valve disease (MMVD), classified according to the 2009 guidelines of the American College of Veterinary Internal Medicine (ACVIM), with those from normal dogs. AnimalsSeventy-eight unsedated dogs (22 healthy controls, 23 ACVIM stage B1 MMVD, 20 ACVIM stage B2 MMVD, and 13 ACVIM stage C MMVD) were included in the study. Materials and methodsAll dogs underwent conventional and 3D echocardiography. Three-dimensional RV end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and ejection fraction (EF) were recorded. Right ventricular EDV, ESV, and SV were indexed to bodyweight. Echocardiographic variables were compared across groups using a Kruskal–Wallis test with subsequent post hoc analysis using Dunn's method for multiple comparisons between groups. A P-value of <0.05 was considered significant. ResultsRight ventricular EDV was smaller in stage B1 (P=0.012), stage B2 (P=0.035), and stage C (P=0.004) dogs than in controls. Stage B2 (P=0.003) and stage C (P<0.001) dogs had smaller RV ESV than controls. Stage B1 dogs had smaller RV SV than controls (P=0.012). Right ventricular EF was greater in stage C dogs than in controls (P=0.003) and in stage B1 (P=0.017) dogs. ConclusionsSeveral 3D echocardiographic indices of RV systolic function differ between dogs with advanced MMVD when compared with normal dogs. Further investigation is required to determine if these differences have clinical implications.

Assessment of Novel Haematological Inflammatory Markers (NLR, SII, and SIRI) as Predictors of SIRS in Dogs with Canine Monocytic Ehrlichiosis

Canine monocytic ehrlichiosis (CME) is a tick-borne and fatal disease that induces a systemic inflammatory response syndrome (SIRS), which underscores the importance of assessing the level of inflammation. In this study, we aimed to evaluate changes in the SII and SIRI indexes in dogs with CME based on the presence of SIRS criteria. A total of 53 dogs affected with CME and 13 healthy dogs were included in the study. Haematological data, including neutrophil to lymphocyte ratio (NLR), systemic inflammatory index (SII), and systemic inflammatory response index (SIRI), were collected and statistically compared between groups based on SIRS status. The NLR and SIRI indexes were significantly higher in dogs with CME that meet the criteria for SIRS compared to dogs without SIRS and healthy controls. In summary, monitoring the SIRI index can aid in evaluating SIRS criteria in dogs with CME, which can help improve their treatment and overall prognosis.

Plasma concentration of thrombopoietin in dogs with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a common cause of severe thrombocytopenia in dogs. The pathogenesis of nonassociative, primary ITP (pITP) appears complex, with ill-defined thrombopoietic response. Develop an immunoassay to measure plasma canine thrombopoietin (TPO) concentration and characterize TPO concentrations in dogs with pITP. Forty-one healthy dogs, 8 dogs in an induced ITP model (3 control, 5 ITP), and 58 pITP dogs. Recombinant canine TPO (rcTPO) was purchased and its identity confirmed by mass spectrometry. Monoclonal antibodies were raised to rcTPO and used to configure a sandwich ELISA using streptavidin-biotin detection. Assay performance, coefficients of variability, and healthy dog plasma TPO reference interval (RI) were determined, followed by assay of ITP samples. Assay dynamic range was 15 pg/mL (lower limit of detection) to 1000 pg/mL TPO, with limit of quantitation of 62 pg/mL. Plasma TPO RI was 0 to 158 pg/mL, with plasma TPO <62 pg/mL for 35/41 healthy dogs. All dogs with induced ITP developed marked increases in plasma TPO concentration. Peak values ranged from 515 to >6000 pg/mL. In contrast, only 2/58 pITP dogs had TPO values above RI. Plasma TPO concentration is paradoxically low at diagnosis for most dogs with pITP. This finding suggests that ineffective thrombopoiesis contributes to thrombocytopenia in pITP dogs and supports evaluating TPO receptor agonist treatment as used for pITP in humans. The TPO assay provides a new tool to study thrombopoiesis in pITP and other thrombocytopenic syndromes in dogs.

Serological survey of Toxoplasma gondii infection in a breeding kennel with reproductive disorders

IntroductionToxoplasma gondii (T. gondii) infection in dogs is distributed globally. Given the potential impact of T. gondii on human reproductive health and its zoonotic potential, focusing on infected dogs with breeding disorders may provide valuable new insights into the role of toxoplasmosis on reproductive disorders. This study aimed to elucidate the potential role of T. gondii on reproductive disorders by conducting a serosurvey on a kennel with breeding disorders in Kerman, Iran. Materials and methodsPurebred dogs with American Kennel Club-certified Pedigrees were randomly selected in both genders from a breeding kennel with a history of reproductive problems such as neonatal deaths, abortion stillbirths, and male infertility. Blood samples were collected from selected dogs, and serosurvey was done with a Toxo-Screen kit using the modified agglutination test (MAT). FindingsFrom 46 selected breeding dogs, 20 (43.5 %) were positive in the Toxoplasma MAT test, consisting of 24 samples (52.17 %) with different reproductive disorders and 22 (47.83 %) healthy dogs. However, the presence of reproductive disorder had no significant relationship with seropositivity (p = 0.067). Seroprevalence of toxoplasmosis had no significant relationship with sex (p = 0.28) and age (p = 0.18) in the studied population. ConclusionScreening with the standard MAT method reduces the risk of transmitting toxoplasmosis by preventing the entry of infected dogs into the kennel, identifying the infected cases to start specific treatments, optimizing the proper nutritional conditions, and ensuring the hygiene of the environment. Fresh/refrigerated raw meat, unpasteurized milk feeding, rodent infestation of grass-covered dog runs, and the main dry food storage facilities were the primary risk factors in kennels.

Impact of acute stress on the canine gut microbiota

There is growing evidence that a relationship exists between mental and emotional wellbeing and the gut microbiota. Little is known regarding how the microbiota reacts to repeated acute stress events in dogs, and whether it is a predictor of stress response. In this study, we explored the impact on the gut microbiota and digestive health with two common events many pet dogs find stressful. Twenty healthy adult dogs, living within a colony, were exposed to either car travel or separation three times across eight-week intervals. Faecal samples were collected 24 h before, within 24 h, and 24–48 h after. Faecal quality and pH, and microbiota diversity and composition were analysed in context with wider published work on physiological stress measures. No significant changes were observed in faecal quality or pH with either stress event at any timepoint, indicating all pets remained in good digestive health. Microbiota analysis demonstrated no significant impact on alpha or beta diversity with either stressor. Microbial signatures previously linked to stress were not identified in these dogs and no changes were observed in the functional gut composition. Irrespective of whether the pet was considered “stressed” (i.e., exhibited an increase in serum cortisol), there was no effect on the microbiota and no taxa were predictive of stress response. Collectively, this work demonstrates, for this population, certain acute stress events have no meaningful impact on the canine gut microbiota, and it has no impact on the associated stress response.

Analysis of the immunomodulatory properties of mycobacterium cell wall fraction on the cytokine production of peripheral blood mononuclear cells of healthy dogs.

Mycobacterium cell wall fraction (MCWF) is derived from nonpathogenic Mycobacterium phlei and is used as an immunomodulatory compound in clinical practice, yet its mode-of-action requires further research. To evaluate the host response to MCWF in canine peripheral blood mononuclear cells (PBMCs) by using enzyme-linked immunosorbent assays (ELISA) and quantitative reverse transcription (qRT)-PCR for assessment of cytokines. Eight healthy Labrador retrievers. PBMCs were isolated from whole blood using density centrifugation. The cells were cultured with different concentrations of MCWF or a potent stimulator of cytokine production, phorbol 12-myristate 13-acetate/ionomycin, or left in cell culture medium for 24, 48 and 72 h. Cytokines were measured by ELISA for interleukin (IL)-4, IL-10 and interferon-gamma (IFN-γ), and by qRT-PCR for IL-4, IL-10, IL-13, IFN-γ, tumour necrosis factor alpha (TNF-α) and transforming growth factor-beta. A significant increase of IL-10 messenger ribonucleic acid (mRNA) was detected at all time points for all concentrations of MCWF (p < 0.05). Protein analysis reflected this finding, with a maximum IL-10 concentration of 300.6 ± 38.3 μg/mL. Compared to the negative control, post-stimulation elevation of IFN-γ mRNA was noted at 24 h with all concentrations of MCWF (p < 0.01), and TNF-α mRNA was increased for 0.5 μg/dL MCWF only at 72 h (p < 0.05). MCWF stimulation of PBMCs results in the elevation of both proinflammatory and regulatory cytokine mRNA. Further research into the role of MCWF as a systemically administered regulatory immunomodulator or adjuvant to allergen-specific immunotherapy should be considered.

Ultrasonographic measurement of the prostatic dimension-to-aortic luminal diameter in healthy intact male dogs and dogs with benign prostatic hyperplasia.

To establish ultrasonographic measurements of the prostatic dimension-to-aortic luminal diameter ratios including the prostatic volume-to-aortic luminal diameter ratio in healthy, intact male dogs and compare these ratios between healthy dogs and dogs affected by benign prostatic hyperplasia. Healthy dogs (n = 75) and dogs affected by benign prostatic hyperplasia (n=104) were included in the study. Prostate length, width, average height and aortic luminal diameter were measured from ultrasonographic images. In addition, prostatic volume was calculated, and prostatic volume-to-aortic luminal diameter were also compared. In healthy dogs, aortic luminal diameter was significantly associated with bodyweight and the prostatic width while body condition scores, prostate length and prostatic average height were not associated. Prostatic dimension-to-aortic luminal diameter ratios and prostatic volume-to-aortic luminal diameter ratio was not significantly different among dogs with different body condition scores either in healthy dogs or dogs affected by benign prostatic hyperplasia, except prostatic volume-to-aortic luminal diameter ratio in benign prostatic hyperplasia. Prostatic dimension-to-aortic luminal diameter ratios were significantly higher in benign prostatic hyperplasia while prostatic volume-to-aortic luminal diameter ratio significantly higher in healthy dogs. Prostatic dimension-to-aortic luminal diameter ratios demonstrated medium to high sensitivity and specificity when distinguishing between healthy dogs and dogs affected by benign prostatic hyperplasia. Prostatomegaly, should be considered when the prostatic dimension-to-aortic luminal diameter ratios increase. The calculated cut-off values were 3.68, 4.46 and 3.06 for prostate length-to-aortic luminal diameter, prostatic width- to-aortic luminal diameter and prostatic average height-to-aortic luminal diameter, respectively. The prostatic dimension-to-aortic luminal diameter ratios could be useful to evaluate the size of the prostate in dogs, as it is unaffected by the body condition scores.

Intranasal atomized dexmedetomidine alone or in combination with ketamine or midazolam to sedate healthy dogs

A prospective, randomized, blinded experiment was conducted to compare the effects of intranasal (IN) dexmedetomidine (Dex, 10 µg/kg; n=12) alone or combined with midazolam (DexM, 0.3 mg/kg; n=12) or ketamine (DexK, 2 mg/kg; n=12) in healthy dogs. Ease of administration (EA1), total administration time (TAT), time for first (TA1) and second nostril administration (TA2), and adverse events during atomization were recorded. Two days later, EA2 was assessed by IN atomization of injectable water as an additional outcome variable. Onset of sedation was evaluated, along with behavioral scores and physiological parameters from T0 (baseline) to T120. Statistical analyses included Chi-square, one-way ANOVA or Kruskal-Wallis, repeated measures or Friedman’s ANOVA, and Wilcoxon’s tests. Significance was p≤0.05.Onset of sedation was 12.9 ± 4.1, 18.2 ± 7.5, and 9.9 ± 4.3 mins (mean ± SD) for Dex, DexM, and DexK, respectively. Onset was shorter in DexK compared to DexM (p=0.002), explaining the lower behavioral scores in DexM at T15. All dogs in Dex and DexK reached adequate sedation, with peak sedation occurring at T30, while some dogs in DexM never reached adequate sedation and this group peaked at T45. Adverse events such as saliva drooling and pawing at the nose were significantly higher in DexM and DexK, explaining their differences in TA2, TAT, and EA1 comparing to Dex. EA2 was also higher in Dex compared to DexM and DexK. In conclusion, Dex was better tolerated in dogs and DexK showed faster and more profound sedative effects. Due to paradoxical excitement, unpredictable sedation, and nasal irritation, DexM is not recommended.

Quantification of respiratory sinus arrhythmia in control dogs and in dogs with various severity classes of myxomatous mitral valve disease

IntroductionLoss of respiratory sinus arrhythmia (RSA) is a negative prognostic factor in dogs with myxomatous mitral valve disease (MMVD). The aim of this study was to calculate the percentage (%) of RSA in healthy dogs and dogs in various MMVD classes. AnimalsControl and MMVD dogs were prospectively included in the study. Materials and methodsRespiratory sinus arrhythmia was calculated from a dual channel electrocardiography and breathing curve recording using the peak-to-trough method, in percent of the average heart rate. ResultsOne hundred and forty-nine dogs were studied, including 24 control and 125 MMVD dogs of different severity classes. An overall %RSA decrease was documented with increasing disease severity up to the Ca class along with a relative %RSA increase in the Cc class. The %RSA magnitude differed between B2 and Ca (P<0.001), and between Ca and Cc (P = 0.001) groups, respectively. The %RSA showed a medium negative correlation with the La:Ao ratio (r2 = −0.568, P<0.001) and with the E-wave velocity (r2 = −0.561, P<0.001). DiscussionsA decrease in %RSA was shown with increased disease severity up to acute congestive heart failure (CHF). Dogs receiving cardiac therapy leading to stabilized CHF might restore their ability to exhibit RSA, often revealing a higher %RSA compared to those in acute CHF. Study limitationsLow number of respiratory cycles for analysis. Therapy effect not evaluated. ConclusionsThe findings of this study can serve as the basis for future risk stratification and carry the potential of proving an additional clinical marker for diagnostic and therapeutic decisions making when managing MMVD dogs.

Host-microbe interactions in the nasal cavity of dogs with chronic idiopathic rhinitis.

Chronic rhinitis (CR) is a frustrating clinical syndrome in dogs and our understanding of the disease pathogenesis in is limited. Increasingly, host-microbe interactions are considered key drives of clinical disease in sites of persistent mucosal inflammation such as the nasal and oral cavities. Therefore, we applied next generation sequencing tools to interrogate abnormalities present in the nose of dogs with CR and compared immune and microbiome profiles to those of healthy dogs. Host nasal cell transcriptomes were evaluated by RNA sequencing, while microbial communities were assessed by 16S rRNA sequencing. Correlation analysis was then used to identify significant interactions between nasal cell transcriptomes and the nasal microbiome and how these interactions were altered in animals with CR. Notably, we observed significant downregulation of multiple genes associated with ciliary function in dogs with CR, suggesting a previously undetected role for ciliary dysfunction in this syndrome. We also found significant upregulation of immune genes related to the TNF-α and interferon pathways. The nasal microbiome was also significantly altered in CR dogs, with overrepresentation of several potential pathobionts. Interactome analysis revealed significant correlations between bacteria in the genus Porphyromonas and the upregulated host inflammatory responses in dogs with CR, as well as defective ciliary function which was correlated with Streptococcus abundance. These findings provide new insights into host-microbe interactions in a canine model of CR and indicate the presence of potentially causal relationships between nasal pathobionts and the development of nasal inflammation and ciliary dysfunction.

Hypoxia-Mediated Long Non-Coding RNA Fragment Identified in Canine Oral Melanoma through Transcriptome Analysis.

Hypoxia contributes to tumor progression and metastasis, and hypoxically dysregulated RNA molecules may, thus, be implicated in poor outcomes. Canine oral melanoma (COM) has a particularly poor prognosis, and some hypoxia-mediated miRNAs are known to exist in this cancer; however, equivalent data on other hypoxically dysregulated non-coding RNAs (ncRNAs) are lacking. Accordingly, we aimed to elucidate non-miRNA ncRNAs that may be mediated by hypoxia, targeting primary-site and metastatic COM cell lines and clinical COM tissue samples in next-generation sequencing (NGS), with subsequent qPCR validation and quantification in COM primary and metastatic cells and plasma and extracellular vesicles (EVs) for any identified ncRNA of interest. The findings suggest that a number of non-miRNA ncRNA species are hypoxically up- or downregulated in COM. We identified one ncRNA, the long ncRNA fragment ENSCAFT00000084705.1, as a molecule of interest due to its consistent downregulation in COM tissues, hypoxically and normoxically cultured primary and metastatic cell lines, when compared to the oral tissues from healthy dogs. However, this molecule was undetectable in plasma and plasma EVs, suggesting that its expression may be tumor tissue-specific, and it has little potential as a biomarker. Here, we provide evidence of hypoxic transcriptional dysregulation for ncRNAs other than miRNA in COM for the first time and suggest that ncRNA ENSCAFT00000084705.1 is a molecule of interest for future research on the role of the transcriptome in the hypoxia-mediated progression of this aggressive cancer.

Metabolomic profiling of pheochromocytomas in dogs: Catecholamine phenotype and tricarboxylic acid cycle metabolites.

In humans with pheochromocytomas (PCCs), targeted metabolomics is used to determine the catecholamine phenotype or to uncover underlying pathogenic variants in tricarboxylic acid (TCA) cycle genes such as succinate dehydrogenase subunits (SDHx). To analyze catecholamine contents and TCA cycle metabolites of PCCs and normal adrenals (NAs). Ten healthy dogs, 21 dogs with PCC. Prospective observational study. Dogs diagnosed with PCC based on histopathological and immunohistochemical confirmation were included. Tissue catecholamine contents and TCA metabolites in PCCs and NAs were measured by liquid chromatography with mass spectrometry or electrochemical detection. Compared to NAs, PCCs had significantly higher tissue proportion of norepinephrine (88% [median: range, 38%-98%] vs 14% [11%-26%]; P < .001), and significantly lower tissue proportion of epinephrine (12% [1%-62%] vs 86% [74%-89%]; P < .001). Pheochromocytomas exhibited significantly lower fumarate (0.4-fold; P < .001), and malate (0.5-fold; P = .008) contents than NAs. Citrate was significantly higher in PCCs than in NAs (1.6-fold; P = .015). One dog in the PCC group had an aberrant succinate : fumarate ratio that was 25-fold higher than in the other PCCs, suggesting an SDHx mutation. This study reveals a distinct catecholamine content and TCA cycle metabolite profile in PCCs. Metabolite profiling might be used to uncover underlying pathogenic variants in TCA cycle genes in dogs.

Imaging of experimental venous thrombus by means of Doppler and CEUS techniques in dogs.

Venous thrombosis has been widely studied in humans, but not in dogs. This study was designed to evaluate a venous thrombus in dogs, from creation to solution, by means of various ultrasonographic techniques. Nine healthy Beagle dogs were included in the study. The venous thrombus was formatted by puncturing the lumen of the external jugular veins and then, the veins were examined with B-mode, color Doppler, pulsed-wave Doppler, and contrast-enhanced ultrasound (CEUS) techniques, at regular intervals, within 210-270min after venipuncture. Haemodynamic parameters were calculated at two different locations, before and after the site of the thrombus formation. The existence of a thrombus was confirmed by CEUS technique. Thrombus volume and echogenicity were evaluated. The results showed that the visualization of the venous thrombus by color Doppler modality was not feasible in some veins. The blood volume was the parameter that could more precisely indicate the presence or absence of a thrombus. In cases where thrombus volume was less than 0.001 cm3, it was impossible to detect its presence using haemodynamic parameters. The CEUS imaging depicted accurately the size and shape of an anechoic venous thrombus, even when its volume was 0.001cm3.

Effects of Yeast-Enriched Functionalized Canola Meal Supplementation on Apparent Total Tract Macronutrient Digestibility and Fecal Characteristics, Fecal Microbiota, and Immune Function of Healthy Adult Dogs.

Brewer's dried yeast has a high nutritional value and has long been utilized by the animal feed industry as a source of protein, B-complex vitamins, and minerals. Brewer's dried yeast is also rich in bioactive compounds and may thereby be used as a functional ingredient, providing benefits beyond that of its nutrient content. Canola meal is a high-fiber ingredient that also has unique properties, especially when it is wetted and dried using a proprietary drying system that creates a "functionalized" canola meal. The objective of this experiment was to evaluate the effects of a yeast-enriched functionalized canola meal (FCM) on apparent total tract digestibility (ATTD) and the fecal quality, metabolite concentrations, and microbiota populations, and immune function of healthy adult dogs. Twelve adult female beagles [body weight (BW) = 7.6 ± 0.7kg; age = 5.8 ± 1.3] were used in a replicated 4×4 Latin square design with 28-d periods. Each experimental period consisted of a 22-d adaptation phase, 5 days of total and fresh fecal collection, and blood collection on the last day. To start, all dogs were fed a basal diet to maintain BW for 14 d. Following fecal and blood collections at baseline (-1 d) to confirm health status, experimental periods began testing the following dietary treatments using a Latin square design experiment: 1) FCM only (no yeast inclusion), 2) FCM + low yeast dose, 3) FCM + medium yeast dose, and 4) FCM + high yeast dose. All treatments were top-dressed onto the basal diet at a rate estimated to be 1% of daily intake (as-is basis). Statistical analysis was performed using the PROC MIXED procedure of SAS with the main effect of treatment and the random effect of dog. Significance was declared at P ≤ 0.05, and trends reported if 0.05 < P ≤ 0.10. Supplementation with yeast-enriched FCM had no significant effect on the ATTD of macronutrients or energy or the fecal characteristics, metabolite concentrations, and microbiota populations of dogs. Additionally, no significant differences were observed in circulating immune cell counts or response to Toll-like receptor agonists among treatments. Our results suggest that the yeast-enriched FCM could be included in canine diets without negatively affecting stool quality, fecal metabolite concentrations, or ATTD. Further research is necessary to determine the effective dose of yeast-enriched FCM, potential mechanisms of action, and other potential implications it has on canine health.

Evaluating urine volume and host depletion methods to enable genome-resolved metagenomics of the urobiome.

The gut microbiome has emerged as a clear player in health and disease, in part by mediating host response to environment and lifestyle. The urobiome (microbiota of the urinary tract) likely functions similarly. However, efforts to characterize the urobiome and assess its functional potential have been limited due to technical challenges including low microbial biomass and high host cell shedding in urine. Here, to begin addressing these challenges, we evaluate urine sample volume (100 ml - 5 mL), and host DNA depletion methods and their effects on urobiome profiles in healthy dogs, which are a robust large animal model for the human urobiome. We collected urine from seven dogs and fractionated samples into aliquots. One set of samples was spiked with host (canine) cells to model a biologically relevant host cell burden in urine. Samples then underwent DNA extraction followed by 16S rRNA gene and shotgun metagenomic sequencing. We then assembled metagenome assembled genomes (MAGs) and compared microbial composition and diversity across groups. We tested six methods of DNA extraction: QIAamp BiOstic Bacteremia (no host depletion), QIAamp DNA Microbiome, Molzym MolYsis, NEBNext Microbiome DNA Enrichment, Zymo HostZERO, and Propidium Monoazide. In relation to urine sample volume, 3 3.0 mL resulted in the most consistent urobiome profiling. In relation to host depletion, individual (dog) but not extraction method drove overall differences in microbial composition. DNA Microbiome yielded the greatest microbial diversity in 16S rRNA sequencing data and shotgun metagenomic sequencing data, and maximized MAG recovery while effectively depleting host DNA in host-spiked urine samples. As proof-of-principle, we then mined MAGs for core metabolic functions and environmental chemical metabolism. We identified long chain alkane utilization in two of the urine MAGs. Long chain alkanes are common pollutants that result from industrial combustion processes and end up in urine. This is the first study, to our knowledge, to demonstrate environmental chemical degradation potential in urine microbes through genome-resolved metagenomics. These findings provide guidelines for studying the urobiome in relation to sample volume and host depletion, and lay the foundation for future evaluation of urobiome function in relation to health and disease.

Evaluation of hydroelectrolytic, energetic supplementation, and clinical laboratory parameters in search and rescue dogs

Establishing methods and actions to improve performance and reduce changes during physical activity is essential in search and rescue dogs. Studying the changes in search and rescue dogs during working activities and how to correct them can help improve their performance and avoid complications. Six healthy adult dogs, which had participated in search and rescue operations for at least one year, were used. Animals had no evidence of clinical disease nor received any supplement or medication prior to the assessment. Dogs were evaluated before, during, and after exercise and submitted to volume replacement with mineral water (T1) and hydroelectrolytic supplementation (T2). Body temperature (BT), parameters of hydration (body weight, erythrogram, and total protein (TP)), energy indicators (glucose, lactate), electrolytes (K+, Na+, Cl-, P+, Ca2+, Mg2+), and hormone levels (cortisol, aldosterone, insulin) were determined. After exercise, isotonic dehydration was detected in both treatments, accompanied by erythrocytosis and weight loss. During the recovery phase, in both treatments, dogs presented a significant increase in BT and lactate and a significant decrease in insulin, TP, and P+. BT and lactate increased after exercise and returned to basal upon recovery. Insulin decreased after exercise without changes in glucose. The maintenance of cortisol indicated the adjustability of dogs to environmental stimuli and stress resistance, and aldosterone did not change during exercise. Both volume replacement with water or hydroelectrolytic and energetic supplementation can correct the isotonic dehydration exhibited by dogs.