Supranutritional Selenium-Yeast Supplementation of Beef Cows during the Last Trimester of Pregnancy Results in Higher Whole-Blood Selenium Concentrations in Their Calves at Weaning, but Not Enough to Improve Nasal Microbial Diversity.

We previously reported that feeding Se-biofortified alfalfa hay to weaned beef calves in a preconditioning program increases whole-blood Se (WB-Se) concentrations and nasal microbiome abundance and diversity during the preconditioning period, decreases morbidity and mortality during the feedlot period, and increases carcass weight and quality at slaughter. The objective of the current study was to see whether similar improvements can be achieved through Se supplementation of dams during various pregnancy trimesters. In a two-year experimental study, 80 Angus-cross cows received once-weekly Se-yeast boluses containing 105 mg of Se, during either the first (TR-1), second (TR-2), or third (TR-3) pregnancy trimester, or were not bolused (CTR). Whole-blood Se concentrations were higher from CTR, to TR-1, to TR-2, and to TR-3 in newborn calves (all p < 0.01). At weaning, only calves from TR-3 mothers had higher WB-Se concentrations compared with calves from CTR mothers (p = 0.02), and no significant differences in nasal microbiome abundance and diversity or nasal microbiota were observed. In the feedlot period, morbidity was low, and no differences were observed. At slaughter, no differences in carcass weight and quality were observed. In conclusion, Se supplementation of pregnant cows is effective for increasing WB-Se concentration of newborn calves, and the increase can be sustained until weaning for calves born to TR-3 dams. However, the increase in WB-Se concentrations is small and does not result in beneficial changes in the nasal microbiome. Thus, calves should be fed Se-biofortified forages again at weaning in a preconditioning program in order to diversify the nasal microbiome prior to entering the feedlot.

Feeding selenium-biofortified alfalfa hay during the preconditioning period improves growth, carcass weight, and nasal microbial diversity of beef calves.

We previously reported that feeding Se-biofortified alfalfa hay to weaned beef calves in a preconditioning program decreases morbidity and mortality during the feedlot period. To understand the mode of action by which supranutritional Se supplementation supports calf health, we examined the effect of agronomic Se-biofortification on nasal microbiome and fecal parasites. Recently weaned Angus-cross beef calves (n = 30) were randomly assigned to two groups and fed an alfalfa hay-based diet for 9 weeks in a preconditioning program. Alfalfa hay was harvested from fields fertilized with sodium selenate at a rate of 0 or 90 g Se/ha. Calculated Se intake from dietary sources was 1.09 and 27.45 mg Se/calf per day for calves consuming alfalfa hay with Se concentrations of 0.06 and 3.47 mg Se/kg dry matter, respectively. Feeding Se-biofortified alfalfa hay for 9 weeks was effective at increasing whole-blood Se concentrations (556 ± 11 vs 140 ± 11 ng/mL; P < 0.001) and increasing body weight (PTreatment, = 0.03) in weaned beef calves. Slaughter yield grades were higher for calves that had been fed Se-enriched alfalfa hay during the preconditioning period (PTreatment = 0.008). No significant differences were observed in fecal parasite load, which remained low. The nasal microbiome and microbiota diversity within calves and across calves expanded from weaning (week 0) to the feedlot period (week 12), which was promoted by feeding Se-biofortified alfalfa hay. Especially concerning was the expansion of nasal Mycoplasmataceae in the feedlot, which reached over 50% of the total microbiota in some calves. In conclusion, we identified dietary Se-biofortified alfalfa hay as a potential promoter of nasal microbiome genome and microbiota diversity, which may explain in part high-Se benefits for prevention of bovine respiratory disease complex in beef calves.

The cecal and fecal microbiomes and metabolomes of horses before and after metronidazole administration.

Antibiotic administration can be a cause of gastrointestinal disease in horses, creating a disruption in the normal population and function of bacteria found in the hindgut. The objective of this study was to describe the changes in the cecal and fecal microbiomes and metabolomes of clinically healthy horses before and after metronidazole administration. Metronidazole (15 mg/kg BID PO) was given to five horses with cecal cannulas. The study was suspended on Day 3 due to adverse gastrointestinal effects. Cecal and fecal samples were obtained before (Days minus52, m28, m14, and 0) and after (Days 7, 14, 28, and 52) metronidazole administration. DNA was extracted from the cecal and fecal samples, and 16S rRNA genes were sequenced. Richness and evenness indices were significantly decreased by metronidazole administration in both cecal and fecal samples, but the overall composition was only significantly changed in fecal samples on Day 3 (ANOSIM, p = 0.008). The most dominant phyla were Bacteroidetes and Firmicutes in all groups examined. In fecal samples, significant changes of the phyla Actinobacteria, Spirochaetes, Lentisphaerae, and Verrucomicrobia occurred on Day 3, which correlated with clinical signs of gastrointestinal disease. The metabolome was characterized by mass spectrometry-based methods and only named metabolites were included in the analysis. Fecal, but not cecal, metabolites were significantly affected by metronidazole. The fecal metabolites affected represent diverse metabolic pathways, such as the metabolism of amino acids, carbohydrates, lipids, nucleic acids and cofactors and vitamins. Metronidazole administration has potential to cause adverse effects in horses, alters the bacterial composition of the horse's cecal and fecal content, and the metabolome of fecal samples.

The effect of diet on the gastrointestinal microbiome of juvenile rehabilitating green turtles (Chelonia mydas).

Threatened and endangered green turtles (Chelonia mydas) are unique because as juveniles they recruit from pelagic to near-shore waters and shift from an omnivorous to primarily herbivorous diet (i.e. seagrass and algae). Nevertheless, when injured and ill animals are admitted to rehabilitation, animal protein (e.g. seafood) is often offered to combat poor appetite and emaciation. We examined how the fecal microbiome of juvenile green turtles changed in response to a dietary shift during rehabilitation. We collected fecal samples from January 2014 -January 2016 from turtles (N = 17) in rehabilitation at the Georgia Sea Turtle Center and used next generation sequencing to analyze bacterial community composition. Samples were collected at admission, mid-rehabilitation, and recovery, which entailed a shift from a mixed seafood-vegetable diet at admission to a primarily herbivorous diet at recovery. The dominant phyla changed over time, from primarily Firmicutes (55.0%) with less Bacteroidetes (11.4%) at admission, to primarily Bacteroidetes (38.4%) and less Firmicutes (31.8%) at recovery. While the microbiome likely shifts with the changing health status of individuals, this consistent inversion of Bacteroidetes and Firmicutes among individuals likely reflects the increased need for protein digestion, for which Bacteroidetes are important. Firmicutes are significant in metabolizing plant polysaccharides; thus, fewer Firmicutes may result in underutilization of wild diet items in released individuals. This study demonstrates the importance of transitioning rehabilitating green turtles to an herbivorous diet as soon as possible to afford them the best probability of survival.

Preliminary evaluation of fecal fatty acid concentrations in cats with chronic kidney disease and correlation with indoxyl sulfate and p-cresol sulfate.

BACKGROUND: Straight- and branched-chain (BCFA) short-chain fatty acids (SCFAs) are produced by colonic microbiota and have both beneficial and deleterious effects in humans with chronic kidney disease (CKD). Fecal SCFAs in cats with CKD have not been described. OBJECTIVE: To characterize fecal SCFA concentrations in cats with CKD as compared to healthy geriatric cats and correlate SCFA to serum indoxyl sulfate (IS) and p-cresol sulfate (pCS) concentrations. ANIMALS: Twenty-eight cats with CKD (International Renal Interest Society [IRIS] stages 2, 3, and 4) and 11 older (≥ 8 years) healthy geriatric cats. METHODS: Prospective, cross-sectional study. Voided feces were analyzed using stable isotope dilution gas chromatography-mass spectrometry to determine fecal concentrations of SCFAs. Serum concentrations of IS and pCS were measured using liquid chromatography tandem mass spectrometry. RESULTS: Fecal isovaleric acid concentrations were significantly higher in CKD cats(P = .02) Cats with IRIS CKD stage 3 and 4 had significantly higher fecal isovaleric acid concentrations compared to healthy geriatric cats (P = .03), but not compared to IRIS CKD stage 2 cats. Total fecal concentrations of BCFAs were found to correlate weakly with serum creatinine concentration (rho, 0.33; P = .05), blood urea nitrogen concentration (rho, 0.40; P = .01), and pCS concentration (rho, 0.35; P = .04). CONCLUSIONS AND CLINICAL IMPORTANCE: Fecal isovaleric acid concentrations were higher in CKD cats, particularly in late stage disease, compared to healthy geriatric cats. Fecal BCFA concentrations correlated with pCS and were higher in cats with muscle wasting, providing evidence for malassimilation of protein in CKD cats.

Fecal short-chain fatty acid concentrations and dysbiosis in dogs with chronic enteropathy.

BACKGROUND: Accumulating evidence shows an important relationship between the gastrointestinal (GI) microbiota and host health. Microbial metabolites are believed to play a critical role in host-microbial interactions. Short-chain fatty acids (SCFAs) are major end products of bacterial carbohydrate fermentation in the intestinal tract. Decreased concentrations of SCFAs have been observed in humans with GI disease. However, large-scale clinical data in dogs are lacking. HYPOTHESIS/OBJECTIVE: To evaluate fecal concentrations of SCFAs and the fecal microbiota in healthy control (HC) dogs and dogs with chronic enteropathy (CE). ANIMALS: Forty-nine privately owned HC dogs and 73 dogs with CE. METHODS: Prospective cohort study. Fecal concentrations of SCFAs were measured using gas chromatography/mass spectrometry. Illumina sequencing and quantitative real-time polymerase chain reaction were utilized to evaluate the fecal microbiota. RESULTS: Fecal concentrations (median [range] µmol/g of dry matter) of acetate were lower (P = .03) in dogs with CE (185.8 [20.1-1042.1]) than in HC dogs (224.0 [87.7-672.8]). Propionate were also lower (P < .001) in dogs with CE (46.4 [0.4-227.9]) than in HC dogs (105.9 [1.6-266.9]). Moreover, total SCFAs were lower (P = .005) in dogs with CE (268.1 [21.8-1378.2]) than in HC dogs (377.2 [126.6-927.0]). Dysbiosis in dogs with CE was characterized by decreased bacterial diversity and richness, distinct microbial community clustering compared with that in HC dogs, and a higher dysbiosis index. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with CE had an altered fecal SCFA concentration accompanied by significant changes of the fecal microbiota.

Gastrointestinal pythiosis with concurrent presumptive gastrointestinal basidiobolomycosis in a Boxer dog.

A 2-year-old female spayed Boxer dog was presented for a 1-month history of progressive hemorrhagic diarrhea with tenesmus and weight loss despite trial courses of antibiotics and diet change. Abdominal ultrasound revealed severe, focal thickening, and loss of normal architecture of the colonic wall with abdominal lymphadenomegaly. Dry-mount fecal cytology, performed on several consecutive days, consistently revealed numerous, round, 16-20 µm structures with basophilic, granular content, and a thin cell wall. Transmission electron microscopy identified these structures as fungi. Culture, polymerase chain reaction (PCR), and sequencing of the internal transcribed spacer, D1/D2 regions, and DNA-directed RNA polymerase II core subunit (RPB2) confirmed the presence of Basidiobolus microsporus in the feces. Biopsies collected via ileocolonoscopy revealed marked, multifocal, chronic, neutrophilic, and eosinophilic ileitis and colitis with ulceration, granulation tissue, and intralesional hyphae (identified with Gomori methenamine silver stain). A Pythium enzyme-linked immunosorbent assay and Pythium-specific PCR performed on the formalin-fixed paraffin-embedded biopsy specimens were positive while Basidiobolus-specific PCR was negative, thus confirming a diagnosis of pythiosis. This report describes a fatal case of colonic and intestinal pythiosis with the presence of fecal Basidiobolus sp. spores, suggestive of concurrent gastrointestinal basidiobolomycosis.

The fecal microbiome and serum concentrations of indoxyl sulfate and p-cresol sulfate in cats with chronic kidney disease.

BACKGROUND: Intestinal dysbiosis has been documented in humans with chronic kidney disease (CKD) and is thought to contribute to production of the uremic toxins indoxyl sulfate (IS) and p-cresol sulfate (pCS). Characteristics of the fecal microbiome in cats with CKD and correlation to serum concentrations of uremic toxins are unknown. OBJECTIVES: To characterize the fecal microbiome and measure serum IS and pCS concentrations of cats with CKD in comparison to healthy older cats. ANIMALS: Thirty client-owned cats with CKD (International Renal Interest Society stages 2-4) and 11 older (≥8 years) healthy control cats. METHODS: Prospective, cross-sectional study. Fecal samples were analyzed by sequencing of 16S rRNA genes and Escherichia coli quantitative PCR (qPCR). Serum concentrations of IS and pCS measured using liquid chromatography tandem mass spectrometry. RESULTS: Cats with CKD had significantly decreased fecal bacterial diversity and richness. Escherichia coli qPCR showed no significant difference in bacteria count between control and CKD cats. Cats with stage 2 (P = .01) and stages 3 and 4 (P = .0006) CKD had significantly higher serum IS concentrations compared to control cats. No significant difference found between stage 2 and stages 3 and 4 CKD. The pCS concentrations were not significantly different between CKD cats and control cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Decreased fecal microbiome diversity and richness is associated with CKD in cats. Indoxyl sulfate concentration is significantly increased with CKD, and cats with stage 2 CKD may suffer from a similar uremic toxin burden as do cats with later stage disease.

Comparison of the intestinal mucosal microbiota in dogs diagnosed with idiopathic inflammatory bowel disease and dogs with food-responsive diarrhea before and after treatment.

We report the first study to evaluate the intestinal mucosal microbiota of dogs with inflammatory bowel disease (IBD) and dogs with food-responsive diarrhea (FRD) before and after treatment. It was hypothesized that differences in the microbial composition exist between both disease groups and within groups pre- vs. post-treatment. Duodenal and colonic biopsies were obtained endoscopically from 24 dogs (15 FRD, 9 IBD) before and after treatment. The intestinal microbiota was evaluated by Illumina sequencing of the bacterial 16S rRNA gene. The global bacterial composition did not differ between IBD and FRD dogs, nor between treatment status. However, several bacterial taxa showed a difference in abundance. Comparing disease groups, an unclassified genus of Neisseriaceae was abundant in the duodenum in the IBD group, whereas Bilophila occurred more frequently in the duodenum and Burkholderia in the colon of FRD dogs. Comparing the microbiota pre- and post-treatment revealed Enterococcus, Corynebacterium and Proteobacteria to be enriched in the duodenum of FRD dogs pre-treatment, while Bacteroides was abundant in the colon post-treatment. In dogs with IBD, Bacteroides also reached significant abundance in the colon post-treatment. In conclusion, some differences in individual bacterial taxa were identified between IBD and FRD dogs and between treatment status.

Characterization of the nasal and oral microbiota of detection dogs.

Little is known about physiological factors that affect the sense of olfaction in dogs. The objectives of this study were to describe the canine nasal and oral microbiota in detection dogs. We sought to determine the bacterial composition of the nasal and oral microbiota of a diverse population of detection canines. Nasal and oral swabs were collected from healthy dogs (n = 81) from four locations-Alabama, Georgia, California, and Texas. Nasal and oral swabs were also collected from a second cohort of detection canines belonging to three different detection job categories: explosive detection dogs (SP-E; n = 22), patrol and narcotics detection dogs (P-NDD; n = 15), and vapor wake dogs (VWD-E; n = 9). To understand if the nasal and oral microbiota of detection canines were variable, sample collection was repeated after 7 weeks in a subset of dogs. DNA was extracted from the swabs and used for 454-pyrosequencing of the16S rRNA genes. Nasal samples had a significantly lower diversity than oral samples (P<0.01). Actinobacteria and Proteobacteria were higher in nasal samples, while Bacteroidetes, Firmicutes, Fusobacteria, and Tenericutes were higher in oral samples. Bacterial diversity was not significantly different based on the detection job. No significant difference in beta diversity was observed in the nasal samples based on the detection job. In oral samples, however, ANOSIM suggested a significant difference in bacterial communities based on job category albeit with a small effect size (R = 0.1079, P = 0.02). Analysis of the composition of bacterial communities using LEfSe showed that within the nasal samples, Cardiobacterium and Riemerella were higher in VWD-E dogs, and Sphingobacterium was higher in the P-NDD group. In the oral samples Enterococcus and Capnocytophaga were higher in the P-NDD group. Gemella and Aggregatibacter were higher in S-PE, and Pigmentiphaga, Chryseobacterium, Parabacteroides amongst others were higher within the VWD-E group. Our initial data also shows that there is a temporal variation in alpha diversity in nasal samples in detection canines.

Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal microbiota compared with healthy controls.

Selenium (Se) is an essential trace mineral important for immune function and overall health of cattle. The nasopharyngeal microbiota in cattle plays an important role in overall respiratory health, especially when stresses associated with weaning, transport, and adaptation to a feedlot affect the normal respiratory defenses. Recent evidence suggests that cattle diagnosed with bovine respiratory disease complex have significantly less bacterial diversity. The objective of this study was to determine whether feeding weaned beef calves Se-enriched alfalfa (Medicago sativa) hay for 9 weeks in a preconditioning program prior to entering the feedlot alters nasal microbiota. Recently weaned beef calves (n = 45) were blocked by sex and body weight, randomly assigned to 3 treatment groups with 3 pens of 5 calves per treatment group, and fed an alfalfa hay based diet for 9 weeks. Alfalfa hay was harvested from fields fertilized with sodium selenate at a rate of 0, 45.0 or 89.9 g Se/ha. Blood samples were collected biweekly and analyzed for whole-blood Se concentrations. Nasal swabs were collected during week 9 from one or two calves from each pen (total n = 16). Calculated Se intake from dietary sources was 3.0, 15.6, and 32.2 mg Se/head/day for calves consuming alfalfa hay with Se concentrations of 0.34 to 2.42 and 5.17 mg Se/kg dry matter, respectively. Whole-blood Se concentrations after 8 weeks of feeding Se-fertilized alfalfa hay were dependent upon Se-application rates (0, 45.0, or 89.9 g Se/ha) and were 155, 345, and 504 ng/mL (PLinear < 0.0001). Microbial DNA was extracted from nasal swabs and amplified and sequenced. Alpha rarefaction curves comparing the species richness (observed OTUs) and overall diversity (Chao1, Observed OTU, and Shannon index) between calves fed selenium-biofortified alfalfa hay compared with control calves showed that Se-supplementation tended to be associated with an enriched nasal microbiota. ANOSIM of unweighted UniFrac distances showed that calves fed high Se-biofortified alfalfa hay clustered separately when compared with control calves in the PCoA plot (R = 0.216, P = 0.04). The bacterial orders Lactobacillales and Flavobacteriales were increased in healthy control calves compared with Clostridiales and Bacteroidales being increased in calves fed Se-biofortified alfalfa hay. Although there were strong trends, no significant differences were noted for any of the bacterial taxa. Based upon these findings, we suggest that weaned beef calves fed Se-biofortified hay tend to have an enriched nasal microbiota. Feeding Se-biofortified alfalfa hay to weaned beef calves prior to entering the feedlot is a strategy for increasing nasopharyngeal microbial diversity.

The fecal microbiome of dogs with exocrine pancreatic insufficiency.

Exocrine pancreatic insufficiency (EPI) in dogs is a syndrome of inadequate synthesis and secretion of pancreatic enzymes. Small intestinal bacterial dysbiosis occurs in dogs with EPI, and is reversed with pancreatic enzyme therapy. However, there are no studies evaluating the fecal microbiome of dogs with EPI. The objective of this study was to evaluate the fecal microbiome of dogs with EPI. Three day pooled fecal samples were collected from healthy dogs (n = 18), untreated (n = 7) dogs with EPI, and dogs with EPI treated with enzyme replacement therapy (n = 19). Extracted DNA from fecal samples was used for Illumina sequencing of the bacterial 16S rRNA gene and analyzed using Quantitative Insights Into Microbial Ecology (QIIME) and PICRUSt was used to predict the functional gene content of the microbiome. Linear discriminant analysis effect size (LEfSe) revealed significant differences in bacterial groups and functional genes between the healthy dogs and dogs with EPI. There was a significant difference in fecal microbial communities when healthy dogs were compared to treated and untreated dogs with EPI (unweighted UniFrac distance, ANOSIM P = 0.001, and 0.001 respectively). Alpha diversity was significantly decreased in untreated and treated EPI dogs when compared to the healthy dogs with respect to Chao1, Observed OTU, and Shannon diversity (P = 0.008, 0.003, and 0.002 respectively). The families Bifidobacteriaceae (P = 0.005), Enterococcaceae (P = 0.018), and Lactobacillaceae (P = 0.001) were significantly increased in the untreated and treated dogs with EPI when compared to healthy dogs. In contrast, Lachnospiraceae (P < 0.001), and Ruminococcaceae (P < 0.01) were significantly decreased in dogs with EPI. Dogs with EPI (before treatment) had significant increases in functional genes associated with secretion system, fatty acid metabolism, and phosphotransferase system. In contrast, healthy dogs had a significant increase in genes related to phenylalanine, tyrosine and tryptophan biosynthesis, transcription machinery and sporulation. In conclusion, this study shows that the fecal microbiome of dogs with EPI (both treated and untreated) is different to that of healthy dogs.

Mitigation of colitis with NovaSil clay therapy.

BACKGROUND/AIMS: Five million people currently live with Crohn's disease (CD) or ulcerative colitis, the two major forms of inflammatory bowel disease. Available treatments frequently result in side effects that compromise the immune health of the patient. Consequently, alternative therapies that cause fewer systemic effects are needed. Dioctahedral smectite clays have been utilized to treat medical conditions, including diarrheal and enteric disease. Herein, we report the ability of a refined dioctahedral smectite (NovaSil, NS) to sorb inflammatory proteins and reduce inflammation in a TNBS (2,4,6-trinitrobenzenesulfonic acid) mouse model of CD. We also investigated whether NS could rescue gut microbial diversity in TNBS-induced mice. METHODS: ELISA, X-ray diffraction, and transmission electron microscopy were employed to characterize the NS-cytokine interaction in vitro. A TNBS mouse colitis model was utilized to study the efficacy of NS supplementation for 4 weeks. The three treatment groups included control, TNBS, and TNBS + NS. DNA was extracted from feces and sorted for bacterial phylogenetic analysis. RESULTS: Results suggest that NS binds TNFα in vitro. In TNBS-treated mice, supplementation with NS significantly reduced weight loss, and serum proinflammatory cytokine levels (IL-2, IL-6, and IL-12, TNFα, IFNγ) compared with the TNBS group. TNBS-treated mice demonstrated a significant reduction in gut microbiota species richness when compared with the TNBS + NS group and control group. CONCLUSIONS: NovaSil mitigated the effects of TNBS-induced colitis based on reduction in systemic markers of inflammation, significant improvement in weight gain, and intestinal microbial profile.

Faecal microbiota of cats with insulin-treated diabetes mellitus.

Microorganisms within the gastrointestinal tract significantly influence metabolic processes within their mammalian host, and recently several groups have sought to characterise the gastrointestinal microbiota of individuals affected by metabolic disease. Differences in the composition of the gastrointestinal microbiota have been reported in mouse models of type 2 diabetes mellitus, as well as in human patients. Diabetes mellitus in cats has many similarities to type 2 diabetes in humans. No studies of the gastrointestinal microbiota of diabetic cats have been previously published. The objectives of this study were to compare the composition of the faecal microbiota of diabetic and non-diabetic cats, and secondarily to determine if host signalment and dietary factors influence the composition of the faecal microbiota in cats. Faecal samples were collected from insulin-treated diabetic and non-diabetic cats, and Illumina sequencing of the 16S rRNA gene and quantitative PCR were performed on each sample. ANOSIM based on the unweighted UniFrac distance metric identified no difference in the composition of the faecal microbiota between diabetic and non-diabetic cats, and no significant differences in the proportions of dominant bacteria by phylum, class, order, family or genus as determined by 16S rRNA gene sequencing were identified between diabetic and non-diabetic cats. qPCR identified a decrease in Faecalibacterium spp. in cats aged over ten years. Cat breed or gender, dietary carbohydrate, protein or fat content, and dietary formulation (wet versus dry food) did not affect the composition of the faecal microbiota. In conclusion, the composition of the faecal microbiota was not altered by the presence of diabetes mellitus in cats. Additional studies that compare the functional products of the microbiota in diabetic and non-diabetic cats are warranted to further investigate the potential impact of the gastrointestinal microbiota on metabolic diseases such as diabetes mellitus in cats.