Evaluation of an Electrolyte Analyser for Measurement of Concentrations of Ionized Calcium and Magnesium in Cats

The goal of this study was to evaluate the Nova CRT 8 electrolyte analyser for determination of concentrations of ionized calcium (Cai) and magnesium (Mgi) in cats, todetermine the effects of sample handling and storage and to establish reference ranges. The precision and analytical accuracy of the Nova CRT 8 analyser were good. The concentrations of Cai and Mgi were significantly lower in aerobically handled serum samples than in those handled anaerobically. The concentrations of Cai and Mgi differed significantly among whole blood, plasma and serum. In anaerobically handled serum, the concentration of Cai was stable for 8 h at 22°C, for 5 days at 4°C and for 1 week at −20°C. The concentration of Mgi was stable for 4 h at 22°C but for less than 24 h at 4°C and for less than 1 week at −20°C. In serum from 36 cats, the reference ranges were 1.20-1.35 mmol/L for Cai and 0.47-0.59 mmol/L for Mgi. The Nova CRT 8 electrolyte analyser is suitable for determination of Cai and Mgi concentrations in cats. Anaerobically handled serum samples are recommended and, stored at room temperature, they yield accurate results when analysed within 4

The Effect of Metronidazole versus a Synbiotic on Clinical Course and Core Intestinal Microbiota in Dogs with Acute Diarrhea

The usefulness of antibiotics in dogs with acute diarrhea (AD) is controversial. It is also unclear what effect metronidazole has on potential enteropathogens such as Clostridium perfringens and Escherichia coli. Thus, the aim of this study was to evaluate the effect of metronidazole vs. a synbiotic on the clinical course and core intestinal bacteria of dogs with AD. Twenty-seven dogs with AD were enrolled in this prospective, randomized, blinded clinical trial and treated with either metronidazole (METg) or a synbiotic (SYNg; E. faecium DSM 10663; NCIMB 10415/4b170). The Canine Acute Diarrhea Severity (CADS) index was recorded daily for eleven days. Bacteria were quantified using qPCR. Data were analyzed using mixed models with repeated measures. A higher concentration of E. coli was observed in the METg group vs. the SYNg group on Day 6 (p < 0.0001) and Day 30 (p = 0.01). Metronidazole had no effect on C. perfringens. C. hiranonis was significantly lower in the METg group than in the SYNg group on Days 6 and 30 (p < 0.0001; p = 0.0015). No significant differences were observed in CADS index, fecal consistency, or defecation frequency between treatment groups (except for the CADS index on one single day). In conclusion, metronidazole negatively impacts the microbiome without affecting clinical outcomes. Thus, synbiotics might be a preferred treatment option for dogs with AD

The fecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease.

Recent molecular studies have revealed a highly complex bacterial assembly in the canine intestinal tract. There is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (IBD). The aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders. Fecal samples from healthy dogs (n = 32), dogs with acute non-hemorrhagic diarrhea (NHD; n = 12), dogs with acute hemorrhagic diarrhea (AHD; n = 13), and dogs with active (n = 9) and therapeutically controlled idiopathic IBD (n = 10) were analyzed by 454-pyrosequencing of the 16S rRNA gene and qPCR assays. Dogs with acute diarrhea, especially those with AHD, had the most profound alterations in their microbiome, as significant separations were observed on PCoA plots of unweighted Unifrac distances. Dogs with AHD had significant decreases in Blautia, Ruminococcaceae including Faecalibacterium, and Turicibacter spp., and significant increases in genus Sutterella and Clostridium perfringens when compared to healthy dogs. No significant separation on PCoA plots was observed for the dogs with IBD. Faecalibacterium spp. and Fusobacteria were, however, decreased in the dogs with clinically active IBD, but increased during time periods of clinically insignificant IBD, as defined by a clinical IBD activity index (CIBDAI). Results of this study revealed a bacterial dysbiosis in fecal samples of dogs with various GI disorders. The observed changes in the microbiome differed between acute and chronic disease states. The bacterial groups that were commonly decreased during diarrhea are considered to be important short-chain fatty acid producers and may be important for canine intestinal health. Future studies should correlate these observed phylogenetic differences with functional changes in the intestinal microbiome of dogs with defined disease phenotypes

Evaluation of an electrolyte analyser for measurement of concentrations of ionized calcium and magnesium in cats

The goal of this study was to evaluate the Nova CRT 8 electrolyte analyser for determination of concentrations of ionized calcium (Cai) and magnesium (Mgi) in cats, todetermine the effects of sample handling and storage and to establish reference ranges. The precision and analytical accuracy of the Nova CRT 8 analyser were good. The concentrations of Cai and Mgi were significantly lower in aerobically handled serum samples than in those handled anaerobically. The concentrations of Cai and Mgi differed significantly among whole blood, plasma and serum. In anaerobically handled serum, the concentration of Cai was stable for 8 h at 22°C, for 5 days at 4°C and for 1 week at −20°C. The concentration of Mgi was stable for 4 h at 22°C but for less than 24 h at 4°C and for less than 1 week at −20°C. In serum from 36 cats, the reference ranges were 1.20-1.35 mmol/L for Cai and 0.47-0.59 mmol/L for Mgi. The Nova CRT 8 electrolyte analyser is suitable for determination of Cai and Mgi concentrations in cats. Anaerobically handled serum samples are recommended and, stored at room temperature, they yield accurate results when analysed within 4

Effect of amoxicillin‐clavulanic acid on clinical scores, intestinal microbiome, and amoxicillin‐resistant Escherichia coli in dogs with uncomplicated acute diarrhea

Background Despite limited evidence of efficacy, antibiotic treatment is still frequently prescribed in dogs with uncomplicated acute diarrhea (AD). Objective To assess whether amoxicillin‐clavulanic acid has a clinical benefit, an effect on the fecal microbiome, and the proportion of amoxicillin‐resistant Escherichia coli in dogs with AD. Animals Sixteen dogs with AD of <3 days duration. Methods Prospective, placebo‐controlled, double‐blinded study. Clinical scores were compared between client‐owned dogs randomly assigned to an antibiotic (AG) or a placebo (PG) group. The intestinal microbiome was analyzed using quantitative PCR assays. Amoxicillin‐resistant fecal E. coli were assessed semiquantitatively with microbiological methods. Results There was no difference in clinical recovery between treated dogs or controls (CADS index day 10: AG group median: 2 (range: 1‐3; CI [1.4; 2.6]); PG group median: 1.6 (range: 1‐3; CI [1.1; 2.4]); P > .99). All dogs gained normal clinical scores (CADS index ≤3) after 1 to 6 days (median 2 days) after presentation. There was no significant difference in the fecal dysbiosis index (during treatment: AG mean −2.6 (SD 3.0; CI [−5.1; 0.0]); PG mean −0.8 (SD 4.0; CI [−4.2; 2.5]; P > .99) or its bacterial taxa. The proportion of resistant fecal E. coli increased (to median: 100%; range: 35%‐100%) during treatment with amoxicillin‐clavulanic acid and was still increased (median: 10%; range 2%‐67%) 3 weeks after treatment, both of which were significantly higher proportions than in the placebo group for both time points (during treatment AG median 100% versus PG median 0.2% (P < .001); after treatment AG median 10% versus PG median 0.0% (P = .002)). Conclusions and Clinical Importance Our study suggests that treatment with amoxicillin‐clavulanic acid confers no clinical benefit to dogs with AD, but predisposes the development of amoxicillin‐resistant E. coli , which persist for as long as 3 weeks after treatment. These findings support international guideline recommendations that dogs with diarrhea should not be treated with antimicrobials unless there are signs of sepsis

Long-term effects of canine parvovirus infection in dogs

Background Canine parvovirus (CPV) is the most important viral cause of acute canine enteritis leading to severe damage of the intestinal barrier. It has been speculated that dogs might develop chronic disorders after surviving CPV infection. However, no studies regarding the longterm implications of CPV infection have been published to date. The aim of this study was to evaluate whether dogs that have survived CPV infection will have an increased risk for developing chronic gastroenteritis, atopic dermatitis, or cardiac disease. Methodology/Principal findings Dogs that had been treated at the Clinic of Small Animal Medicine, LMU Munich, for CPV infection for which a follow-up of at least 12 months was available, were included in the study. Owners completed a questionnaire on the presence of chronic gastrointestinal and cutaneous signs, cardiac disease, and other potential disorders. An identical questionnaire was sent to owners of matched control dogs during the same time period. Seventy-one questionnaires of dogs with CPV infection and 67 of control dogs were analyzed. Significantly more CPV-infected dogs (30/71) compared to control dogs (8/67) had developed chronic gastrointestinal signs later in their lives (P < 0.001). No significant differences were observed regarding skin diseases (P = 1), cardiac problems (P = 0.160), or any other diseases (P = 0.173) later in life. Conclusions: Results of this study suggest that dogs that survive CPV infection have a significantly higher risk (odds ratio = 5.33) for developing a chronic gastrointestinal disease. Further prospective studies to identify the trigger for the development of chronic diarrhoea and possible targeted treatment strategies are needed

Prevalence of Clostridioides difficile in Canine Feces and Its Association with Intestinal Dysbiosis

The role of Clostridioides (C.) difficile as an enteropathogen in dogs is controversial. In humans, intestinal bile acid-dysmetabolism is associated with C. difficile prevalence. The relationship between fecal qPCR-based dysbiosis index (DI) and especially the abundance of bile acid-converting Clostridium hiranonis with the presence of C. difficile in dogs was explored across the following 4 cohorts: 358 fecal samples submitted for routine diagnostic work-up, 33 dogs with chronic enteropathy, 14 dogs with acute diarrhea, and 116 healthy dogs. Dogs that tested positive for C. difficile had significantly higher DI (median, 4.4 (range from 0.4 to 8.6)) and lower C. hiranonis (median, 0.1 (range from 0.0 to 7.5) logDNA/g) than dogs that tested negative for C. difficile (median DI, −1 (range from −7.2 to 8.9); median C. hiranonis abundance, 6.2 (range from 0.1 to 7.5) logDNA/g; p < 0.0001, respectively). In 33 dogs with CE and 14 dogs with acute diarrhea, the treatment response did not differ between C. difficile-positive and -negative dogs. In the group of clinically healthy dogs, 9/116 tested positive for C. difficile, and 6/9 of these had also an abnormal DI. In conclusion, C. difficile is strongly linked to intestinal dysbiosis and lower C. hiranonis levels in dogs, but its presence does not necessitate targeted treatment

Intestinal lesions in dogs with acute hemorrhagic diarrhea syndrome associated with netF-positive Clostridium perfringens type A

Acute hemorrhagic diarrhea syndrome (AHDS), formerly named canine hemorrhagic gastroenteritis, is one of the most common causes of acute hemorrhagic diarrhea in dogs, and is characterized by acute onset of diarrhea, vomiting, and hemoconcentration. To date, histologic examinations have been limited to postmortem specimens of only a few dogs with AHDS. Thus, the aim of our study was to describe in detail the distribution, character, and grade of microscopic lesions, and to investigate the etiology of AHDS. Our study comprised 10 dogs with AHDS and 9 control dogs of various breeds, age, and sex. Endoscopic biopsies of the gastrointestinal tract were taken and examined histologically (H&E, Giemsa), immunohistochemically (Clostridium spp., parvovirus), and bacteriologically. The main findings were acute necrotizing and neutrophilic enterocolitis (9 of 10) with histologic detection of clostridia-like, gram-positive bacteria on the necrotic mucosal surface (9 of 10). Clostridium perfringens isolated from the duodenum was identified as type A (5 of 5) by multiplex PCR (5 of 5). In addition, each of the 5 genotyped isolates encoded the pore-forming toxin netF. Clostridium spp. (not C. perfringens) were cultured from duodenal biopsies in 2 of 9 control dogs. These findings suggest that the pore-forming netF toxin is responsible for the necrotizing lesions in the intestines of a significant proportion of dogs with AHDS. Given that the stomach was not involved in the process, the term acute hemorrhagic diarrhea syndrome seems more appropriate than the frequently used term hemorrhagic gastroenteritis

Fecal Microbial and Metabolic Profiles in Dogs With Acute Diarrhea Receiving Either Fecal Microbiota Transplantation or Oral Metronidazole

The aim was to characterize differences in fecal consistency, and fecal microbiota and metabolome profiles in dogs with acute diarrhea (AD) treated with either fecal microbiota transplantation as enema (FMT;n = 11) or oral metronidazole (MET;n = 7) for 7 days. On days 0, 7, and 28 fecal samples were obtained. Fecal samples from healthy dogs (HC;n = 14) were used for comparison. Samples were analyzed by the previously validated qPCR based canine Dysbiosis Index (DI;increased values indicate microbiota dysbiosis) and 16S rRNA gene sequencing. The fecal metabolome was analyzed using a previously validated targeted canine assay for fecal unconjugated bile acids, and untargeted metabolomics. Fecal consistency improved significantly in dogs treated with FMT and MET by day 7 and day 28 (p < 0.01) compared to day 0. However, on day 28 fecal consistency was significantly better in FMT compared to MET (p = 0.040). At day 0, dogs with AD had an altered microbiota indicated by significantly increased DI, decreased alpha-diversity, and altered beta-diversity. In the FMT group, the DI decreased over time, while MET led to a significant increase in the dysbiosis index at day 7 and 28 compared to FMT. Sequencing data revealed that in FMT microbial diversity and beta-diversity was similar to HC at day 28, while in MET these parameters were still significantly different from HC. In dogs treated with FMT, a decrease in cholic acid and the percentage of primary bile acids was observed, whereas treatment with metronidazole led to an increase in cholic acid at day 7 and an increase in percentage of primary bile acids over time. Based on untargeted metabolomics, dogs with AD had an altered fecal metabolome compared to HC. Dogs treated with FMT clustered closer to HC at day 28, while dogs treated with MET did not. In this pilot study, dogs with AD had significant differences in fecal microbiota and metabolome profiles. Dogs treated with MET still had altered microbial and metabolic profiles at day 28 compared to dogs treated with FMT or healthy dogs

Effect of probiotic treatment on the clinical course, intestinal microbiome, and toxigenic Clostridium perfringens in dogs with acute hemorrhagic diarrhea

Introduction The impact of probiotics on dogs with acute hemorrhagic diarrhea syndrome (AHDS) has not been evaluated so far. The study aim was to assess the effect of probiotic treatment on the clinical course, intestinal microbiome, and toxigenic Clostridium perfringens in dogs with AHDS in a prospective, placebo-controlled, blinded trial. Methods Twenty-five dogs with AHDS with no signs of sepsis were randomly divided into a probiotic (PRO;Visbiome, ExeGi Pharma) and placebo group (PLAC). Treatment was administered for 21 days without antibiotics. Clinical signs were evaluated daily from day 0 to day 8. Key bacterial taxa, C. perfringens encoding NetF toxin and enterotoxin were assessed on days 0, 7, 21. Results Both groups showed a rapid clinical improvement. In PRO a significant clinical recovery was observed on day 3 (p = 0.008), while in PLAC it was observed on day 4 (p = 0.002) compared to day 0. Abundance of Blautia (p<0.001) and Faecalibacterium (p = 0.035) was significantly higher in PRO on day 7 compared to day 0, while in PLAC the abundance of Faecalibacterium was not significantly higher on any study day and Blautia (p = 0.016) was only significantly higher on day 21 compared to day 0. Abundance of C. perfringens was significantly lower on day 7 (p = 0.011) compared to day 0 in PRO but not in PLAC. Enterotoxin genes were significantly lower in PRO on day 21 (p = 0.028) compared to PLAC. Fecal samples of 57% of all dogs were positive for netF toxin genes on day 0 and the abundance was significantly lower on day 7 compared to day 0 in PRO (p = 0.016) and PLAC (p = 0.031). Conclusion The probiotic treatment was associated with an accelerated normalization of the intestinal microbiome. Dogs with aseptic AHDS showed a rapid decrease of netF toxin genes and fast clinical recovery in both groups under symptomatic treatment without antibiotics