Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors.

Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.

Stem Cell Conditioned Medium Treatment for Canine Spinal Cord Injury: Pilot Feasibility Study.

Spinal cord injury (SCI) involves nerve damage and often leads to motor, sensory and autonomic dysfunctions. In the present study, we have designed a clinical protocol to assess the feasibility of systemic delivery of allogenic canine bone marrow tissue-derived mesenchymal stem cell conditioned medium (BMMSC CM) to dogs with SCI. Four client-owned dogs with chronic SCI lasting more than six months underwent neurological and clinical evaluation, MRI imaging and blood tests before being enrolled in this study. All dogs received four intravenous infusions with canine allogenic BMMSC CM within one month. Between the infusions the dogs received comprehensive physiotherapy, which continued for three additional months. No adverse effects or complications were observed during the one, three and six months follow-up periods. Neither blood chemistry panel nor hematology profile showed any significant changes. All dogs were clinically improved as assessed using Olby locomotor scales after one, three and six months of BMMSC CM treatment. Furthermore, goniometric measurements revealed partial improvement in the range of joint motion. Bladder function improved in two disabled dogs. We conclude that multiple delivery of allogenic cell-derived conditioned medium to dogs with chronic SCI is feasible, and it might be clinically beneficial in combination with physiotherapy.

Oral administration of bacteriocin-producing and non-producing strains of Enterococcus faecium in dogs.

The current effort to incorporate microbial cultures in canine nutrition and thus intake of them on daily base increases our interest in careful and more complex study of their effects in dogs. Many of the commercially used strains have not been tested in dogs and are incorporated only on the base of beneficial effects observed in humans with specific disorders. Moreover, no information on the effects of bacteriocin-producing strains in dogs is available. Therefore, we decided to test and to compare overall effect of bacteriocin non-producing Enterococcus faecium DSM 32820 and enterocin B-producing E. faecium LMG 30881 strain (both of canine origin). Dogs were divided into three treatment groups of ten dogs each: control; DSM 32820 group; and LMG 30881 group, dosing 109 CFU/day/dog. The experiment lasted 35 days with a 14-day treatment period (sample collection at days 0, 7, 14, 35). Despite bacteriocin production is believed that may provide a competitive advantage over neighbouring sensitive strains within shared environment, results indicated somewhat better survival for the DSM 32820 compared to the LMG 30881 group. Furthermore, dogs of DSM 32820 group had optimal faecal consistency throughout the experiment, significantly stimulated phagocytic activity (days 7 and 14) and metabolic burst activity of leukocytes (days 14 and 35) and lower serum glucose concentration (day 35). In contrast, dogs of LMG 30881 group showed higher faecal count of Gram-negative bacteria (day 35), lower haemoglobin and glucose concentration (day 35), and higher metabolic burst activity (days 14 and 35). These results are further evidence of the existence of inter-strain differences in efficacy despite the same origin.

Experimental application of Lactobacillus fermentum CCM 7421 in combination with chlorophyllin in dogs.

Chlorophyll belongs in a larger class of phytochemical plant pigments currently receiving more attention as a physiologically active dietary component. Although most research has focused on its biological activities such as its antioxidant, antimutagenic, anti-inflammatory or apoptotic effects in humans or rodents, there is limited knowledge at this time about the combinative possibilities of chlorophyll with probiotic bacteria. Our aim was to test the growth characteristics of canine-derived probiotic strain Lactobacillus fermentum CCM 7421 in the presence of different concentrations of chlorophyllin in vitro. Antimicrobial activity of chlorophyllin against canine indicator bacteria was also detected. In the in vivo study, chlorophyllin, L. fermentum CCM 7421 and the combination of both additives on faecal microbiota, faecal organic acid concentrations, haematological and immunological parameters in dogs were tested. Forty dogs were divided into 4 treatment groups; control (C); receiving chlorophyllin (60 mg/day/dog, CH group); L. fermentum CCM 7421 (10(8) CFU/day/dog, LF group); and both additives (CH + LF group), 10 dogs in each group. The experiment lasted for 28 days with a 14-day treatment period (sample collection at days 0, 7, 14 and 28). Results showed no growth inhibition of strain CCM 7421 by 0.05-0.25 % of chlorophyllin in broth after 24 h. Reduced growth of staphylococci, Listeria monocytogenes and Citrobacter freundii was observed at 1 % chlorophyllin (P < 0.05). In dogs, lower coliform bacteria numbers and higher concentration of propionic acid in faeces of the CH group during the treatment compared to baseline were detected (P < 0.01). Phagocytic activity of leukocytes was stimulated in all three treated groups of dogs (P < 0.05).

Oral administration of inosine promotes recovery after experimental spinal cord injury in rat.

Inosine, a purine nucleoside, is one of the novel substances, which can preserve the neuronal and glial viability and stimulate intact neurons to extend axons. We, herein, evaluated the effect of oral inosine treatment on spinal cord injury (SCI) recovery by means of locomotor and bladder function, quantification of neurons and spinal cord tissue sparing. Rats after compression SCI were divided into groups-SCI-Aqua and SCI-Inosine (daily application of aqua for injection or inosine)-locomotion of hind limbs (BBB score) and urinary bladder function were evaluated from day 1 to 28 after SCI. The neuronal profile was determined by immunohistochemistry with NeuN antibodies and tissue sparing by Luxol fast blue staining method. SCI affected the functional movement of hind limbs in both groups with gradual improvement (increased BBB score) during survival. However, we found a significant difference in BBB score and recovery of bladder function between SCI-Aqua and SCI-Inosine groups during the second week of survival following SCI. In addition, the number of NeuN positive cells and percentage of tissue sparing was also significantly higher in SCI-Inosine group when compared with the SCI-Aqua group. Daily oral administration of inosine after SCI throughout the survival was beneficial for locomotion and micturition, neuronal survival and tissue sparing. This indicates that inosine may represent one of the co-stimulatory factors for treatment strategies to promote neuronal plasticity after SCI.

Who fans the flames of Alzheimer's disease brains? Misfolded tau on the crossroad of neurodegenerative and inflammatory pathways.

Neurodegeneration, induced by misfolded tau protein, and neuroinflammation, driven by glial cells, represent the salient features of Alzheimer's disease (AD) and related human tauopathies. While tau neurodegeneration significantly correlates with disease progression, brain inflammation seems to be an important factor in regulating the resistance or susceptibility to AD neurodegeneration. Previously, it has been shown that there is a reciprocal relationship between the local inflammatory response and neurofibrillary lesions. Numerous independent studies have reported that inflammatory responses may contribute to the development of tau pathology and thus accelerate the course of disease. It has been shown that various cytokines can significantly affect the functional and structural properties of intracellular tau. Notwithstanding, anti-inflammatory approaches have not unequivocally demonstrated that inhibition of the brain immune response can lead to reduction of neurofibrillary lesions. On the other hand, our recent data show that misfolded tau could represent a trigger for microglial activation, suggesting the dual role of misfolded tau in the Alzheimer's disease inflammatory cascade. On the basis of current knowledge, we can conclude that misfolded tau is located at the crossroad of the neurodegenerative and neuroinflammatory pathways. Thus disease-modified tau represents an important target for potential therapeutic strategies for patients with Alzheimer's disease.

Detection of Periodontal Pathogens from Dental Plaques of Dogs with and without Periodontal Disease.

Dental plaque bacteria are one of the main factors responsible for the development of a periodontal disease, which is the most common infectious disease in dogs. The aim of this study was to identify the presence of periodontal disease-related bacteria in the dental plaque of dogs. Plaque samples were taken from dogs with and without periodontal disease. Samples were analyzed for the presence of Porphyromonas gulae, Tannerella forsythia and Treponema denticola using a PCR technique amplifying 16S rRNA genes of P. gulae and T. forsythia and flaB2 genes of Treponema species, including T. denticola. The presence of T. forsythia was confirmed in all samples. P. gulae was detected in all dogs with periodontal disease and in 71.43% of dogs without periodontal disease. Treponema spp. were detected in 64.29% of the samples. Based on Sanger sequencing and Basic Local Alignment Search Tool algorithm, Treponema spp. were identified as T. denticola and Treponema putidum. T. denticola was present in 28.57% of dogs with periodontal disease, while T. putidum was present in 42.86% of dogs with periodontal disease and in 57.14% of dogs without periodontal disease. T. putidum was positively correlated with both P. gulae and T. forsythia, suggesting that it may be involved in the development of periodontal disease.

A Survey of Intestinal Helminths of Dogs in Slovakia with an Emphasis on Zoonotic Species.

Dogs are the most popular pets worldwide; however, close contact with people increases the risk of transmission of different zoonotic parasites. This study aims to determine the prevalence of gastrointestinal helminths in dogs in Slovakia. A total of 495 faecal samples collected from pet, shelter, guard, working (police), and hunting dogs, as well as dogs from segregated Roma settlements between 2016 and 2021, were examined using flotation and molecular methods. Eggs of intestinal helminths were detected in 134 (27.1%) samples. Microscopically, six different species/genera/families, namely, Toxocara canis (14.7%), Toxascaris leonina (1.6%), Trichuris vulpis (6.3%), Capillaria spp. (1.4%), Ancylostoma/Uncinaria spp. (8.3%), and taeniid eggs (4.0%), were recorded. Molecular analyses revealed infection with Echinococcus multilocularis in 2.2% of dogs and 0.4% of the animals were infected with Taenia hydatigena. The results showed a correlation between the occurrence of intestinal helminths and the availability of veterinary care, as dogs from Roma settlements and shelter dogs were the most often infected (66.7% and 39.2%, respectively). On the other hand, working animals were in the best health condition, with only 2.5% being positive. The relatively frequent occurrence of zoonotic species points to the constant need for preventive measures and regular deworming of dogs.

Effect of Hydrolyzed Yeast Administration on Faecal Microbiota, Haematology, Serum Biochemistry and Cellular Immunity in Healthy Dogs.

Fungal microorganisms are regularly found in the gastrointestinal tract of healthy and diseased dogs especially from the phyla Ascomycota and Basidiomycota; however, it is necessary to better understand their role in host health. One of the most commonly studied yeast species in humans or animals is Saccharomyces cerevisiae especially in its live cell form. Scarce knowledge on its hydrolysate product effects in dogs forced us to test diet supplemented with hydrolyzed brewery S. cerevisiae (at a dose 0.3% of the diet) for 14 days to healthy adult dogs. Twenty German Shepherds were randomly divided into 2 groups: control and experimental, ten dogs in each. The experiment lasted 42 days (blood and faeces sample collection at days 0, 14, 28 and 42). The results of this straighforward experiment showed significant increase in the abundance of bifidobacteria (day 14), lactic acid bacteria (day 42) and clostridia (day 42). The faecal pH was significantly increased at day 28. In blood serum, the concentration of triglyceride and cholesterol decreased (day 42) while activities of alanine aminotransferase (at day 14) and aspartate aminotransferase significantly increased (at days 28 and 42). Activities of these enzymes were above reference range top in 7 dogs (ALT) and 4 dogs (AST). Haematological paramaters and activity of phagocytes as well as on percentage of lymphocyte subsets CD4+, CD8+, CD4+CD8+ and CD21+ were not changed during the experiment. The important point of these results is their onset mostly in the post-supplementation period. The observation of some unexpected effects emphasizes the need for reassessment to use yeasts products for dogs but further studies using different doses are necessary.

Study of microbiocenosis of canine dental biofilms.

Dental biofilm is a complex microbial community influenced by many exogenous and endogenous factors. Despite long-term studies, its bacterial composition is still not clearly understood. While most of the research on dental biofilms was conducted in humans, much less information is available from companion animals. In this study, we analyzed the composition of canine dental biofilms using both standard cultivation on solid media and amplicon sequencing, and compared the two approaches. The 16S rRNA gene sequences were used to define the bacterial community of canine dental biofilm with both, culture-dependent and culture-independent methods. After DNA extraction from each sample, the V3-V4 region of the 16S rRNA gene was amplified and sequenced via Illumina MiSeq platform. Isolated bacteria were identified using universal primers and Sanger sequencing. Representatives of 18 bacterial genera belonging to 5 phyla were isolated from solid media. Amplicon sequencing largely expanded this information identifying in total 284 operational taxonomic units belonging to 10 bacterial phyla. Amplicon sequencing revealed much higher diversity of bacteria in the canine dental biofilms, when compared to standard cultivation approach. In contrast, cultured representatives of several bacterial families were not identified by amplicon sequencing.

Evaluation of Probiotic Lactobacillus fermentum CCM 7421 Administration with Alginite in Dogs.

There are growing efforts to find applications for various naturally occurring organo-mineral rocks. They have so far been preferentially used in agriculture and forestry; however, medicine and nutrition may also be interesting areas for their application. This study investigates the effects of dietary supplementation with canine-derived probiotic strain Lactobacillus fermentum CCM 7421 in combination with alginite in dogs. Alginite is a loam-like material of volcanic origin composed of clay minerals and fossilised unicellular algae. The effects of these additives on faecal microbiota, faecal characteristics, short-chain fatty acid profile, haematology, serum biochemistry and cellular immunity parameters were monitored. Forty dogs were randomly divided into four treatment groups: control group (C), alginite-supplemented group (A; 1% diet), probiotic group (LF; L. fermentum CCM 7421 at a dose of 109 cfu/day/dog) and combined group (A + LF group); 10 dogs in each group. The experiment lasted for 35 days with a 14-day treatment period (sample collection at days 0, 7, 14 and 35). The results of this straightforward experiment showed beneficial effects in the combined A + LF group. In detail, a decrease in faecal coliforms and clostridia and an increase in lactic acid bacteria, haemoglobin and serum magnesium levels compared to baseline were observed in the A + LF group (P < 0.05). In contrast, sole application of alginite (A group) led to several unexpected effects such as an increase in clostridial population and serum alanine aminotrasferase and a decrease in haemoglobin concentration (P < 0.05). The addition of alginite prevented a decrease in faecal pH and serum mineral content observed in the LF group. This indicates the possibility of applying alginite also in the nutrition of dogs as a combinative additive with probiotic bacteria for restoring optimal acid-alkali balance without affecting positive probiotic effects.

Risk factors for canine cognitive dysfunction syndrome in Slovakia.

BACKGROUND: Increasing prevalence of cognitive impairment in an aging canine population poses a serious health problem. Identifying risk factors, which may influence the onset of cognitive decline, is becoming increasingly important. Here we investigated whether age, sex, weight, nutrition, dogs' housing and reproductive state were associated with increased risk of canine cognitive dysfunction syndrome (CCDS) in Slovakia. RESULTS: Age was associated with cognitive decline and nutrition emerged as a significant predictor variable. Dogs fed controlled diets had 2.8 times lower odds of developing CCDS when compared with dogs fed uncontrolled diets. Sex, weight, reproductive state and dogs' housing were not significantly associated with cognitive decline. Further, the prevalence of CCDS was similar in both small and medium/large sized dogs aged 8-11 years, but differed in dogs at an age of 11-13 years. CONCLUSION: Age was found to be the most prominent risk factors of CCDS. Nutrition may influence the cognitive state of dogs. This finding suggests that nutritional interventions may modify canine cognitive functions.

Tau hyperphosphorylation in synaptosomes and neuroinflammation are associated with canine cognitive impairment.

Canine cognitive impairment syndrome (CDS) represents a group of symptoms related to the aging of the canine brain. These changes ultimately lead to a decline of memory function and learning abilities, alteration of social interaction, impairment of normal housetraining, and changes in sleep-wake cycle and general activity. We have clinically examined 215 dogs, 28 of which underwent autopsy. With canine brains, we performed extensive analysis of pathological abnormalities characteristic of human Alzheimer's disease and frontotemporal lobar degeneration, including ß-amyloid senile plaques, tau neurofibrillary tangles, and fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP43) inclusions. Most demented dogs displayed senile plaques, mainly in the frontal and temporal cortex. Tau neurofibrillary inclusions were found in only one dog. They were identified with antibodies used to detect tau neurofibrillary lesions in the human brain. The inclusions were also positive for Gallyas silver staining. As in humans, they were distributed mainly in the entorhinal cortex, hippocampus, and temporal cortex. On the other hand, FUS and TDP43 aggregates were not present in any of the examined brain samples. We also found that CDS was characterized by the presence of reactive and senescent microglial cells in the frontal cortex. Our transcriptomic study revealed a significant dysregulation of genes involved in neuroinflammation. Finally, we analyzed tau phosphoproteome in the synaptosomes. Proteomic studies revealed a significant increase of hyperphosphorylated tau in synaptosomes of demented dogs compared with nondemented dogs. This study suggests that cognitive decline in dogs is related to the tau synaptic impairment and neuroinflammation. J. Comp. Neurol. 524:874-895, 2016. © 2015 Wiley Periodicals, Inc.

Tau-mediated synaptic damage in Alzheimer's disease.

Synapses are the principal sites for chemical communication between neurons and are essential for performing the dynamic functions of the brain. In Alzheimer's disease and related tauopathies, synapses are exposed to disease modified protein tau, which may cause the loss of synaptic contacts that culminate in dementia. In recent decades, structural, transcriptomic and proteomic studies suggest that Alzheimer's disease represents a synaptic disorder. Tau neurofibrillary pathology and synaptic loss correlate well with cognitive impairment in these disorders. Moreover, regional distribution and the load of neurofibrillary lesions parallel the distribution of the synaptic loss. Several transgenic models of tauopathy expressing various forms of tau protein exhibit structural synaptic deficits. The pathological tau proteins cause the dysregulation of synaptic proteome and lead to the functional abnormalities of synaptic transmission. A large body of evidence suggests that tau protein plays a key role in the synaptic impairment of human tauopathies.