Changes in the equine fecal microbiota associated with the use of systemic antimicrobial drugs.

BACKGROUND: The intestinal tract is a rich and complex environment and its microbiota has been shown to have an important role in health and disease in the host. Several factors can cause disruption of the normal intestinal microbiota, including antimicrobial therapy, which is an important cause of diarrhea in horses. This study aimed to characterize changes in the fecal bacterial populations of healthy horses associated with the administration of frequently used antimicrobial drugs. RESULTS: Twenty-four adult mares were assigned to receive procaine penicillin intramuscularly (IM), ceftiofur sodium IM, trimethoprim sulfadiazine (TMS) orally or to a control group. Treatment was given for 5 consecutive days and fecal samples were collected before drug administration (Day 1), at the end of treatment (Days 5), and on Days 14 and 30 of the trial. High throughput sequencing of the V4 region of the 16S rRNA gene was performed using an Illumina MiSeq sequencer. Significant changes of population structure and community membership were observed after the use of all drugs. TMS caused the most marked changes on fecal microbiota even at higher taxonomic levels including a significant decrease of richness and diversity. Those changes were mainly due to a drastic decrease of Verrucomicrobia, specifically the "5 genus incertae sedis". Changes in structure and membership caused by antimicrobial administration were specific for each drug and may be predictable. Twenty-five days after the end of treatment, bacterial profiles were more similar to pre-treatment patterns indicating a recovery from changes caused by antimicrobial administration, but differences were still evident, especially regarding community membership. CONCLUSIONS: The use of systemic antimicrobials leads to changes in the intestinal microbiota, with different and specific responses to different antimicrobials. All antimicrobials tested here had some impact on the microbiota, but TMS significantly reduced bacterial species richness and diversity and had the greatest apparent impact on population structure, specifically targeting members of the Verrucomicrobia phylum.

Clinical utility of serum biochemical variables for predicting acid-base balance in critically ill horses.

BACKGROUND: Profiles from serum biochemical analyzers include the concentration of strong electrolytes (including l-lactate), total carbon dioxide (tCO2 ), and total protein. These variables are associated with changes in acid-base balance. Application of physicochemical principles may allow predicting acid-base balance from serum biochemistry without measuring whole blood pH and pCO2 . OBJECTIVES: The purpose of the study was to determine if the acid-base status of critically ill horses could be accurately predicted using variables included in standard serum biochemical profiles. METHODS: Two jugular venous blood samples were prospectively obtained from critically ill horses and foals. Samples were analyzed using a whole blood gas and pH analyzer (BG) and a serum biochemistry multi analyzer system (AMAS). Linear regression, Deming regression, and Bland-Altman plots were used for method comparison and P < .05 was considered significant. RESULTS: Values from 70 horses and foals for Na, K, Cl, and total protein concentrations, and consequently the calculated variables used for acid base interpretation, were different between the AMAS and BG analyzer. Using physicochemical principles, BG results accurately predicted pH, whereas the AMAS results did not when a fixed value for pCO2 was used. CONCLUSIONS: Measurement of pCO2 is required in critically ill horses for accurate prediction of whole blood pH. Differences in the measured values of Na and Cl concentration exist when measured in serum by the AMAS and in whole blood or plasma by BG, indicating that the accurate prediction of whole blood pH is analyzer-dependent. Application of physicochemical principles to plasma or serum provides a practical method to evaluate analyzer accuracy.

False-Positive Clostridium difficile in Negative-Control Reactions Peak and Then Decrease with Repetitive Refrigeration of Immunoassay.

Aberrant false-positive reactions in negative-controls during ELISA testing for Clostridium difficile indicated the potential for false-diagnoses. Experiments with 96-well products showed a maximum peak of false-positive immunoassay reactions with the provided negative-control reagents after 5 refrigeration-to-room temperature cycles (P < 0.001), decreasing thereafter with additional refrigeration cycles. Because repetitive refrigeration causes a peak of false-positives, the use of single negative-controls per ELISA run might be insufficient to monitor aberrant preanalytical false-positives if immunoassays are subject to repetitive refrigeration.

Clinicopathologic variables associated with hypokalemia in lactating dairy cows with abomasal displacement or volvulus.

OBJECTIVE: To identify potential mechanisms for hypokalemia in dairy cows with left-displaced abomasum (LDA), right-displaced abomasum (RDA), or abomasal volvulus (AV). DESIGN: Retrospective analysis of clinicopathologic data from 2 convenience samples of cows. SAMPLE: 112 lactating dairy cows with AV (group 1); 1,332 lactating dairy cows (group 2) with LDA (n = 1,160) or RDA or AV (172). PROCEDURES: Data were analyzed via Spearman ρ and multivariate stepwise regression. RESULTS: 78 of 112 (70%) group 1 cows were hypokalemic (mean serum potassium concentration, 3.5 mEq/L; reference range, 3.9 to 5.8 mEq/L). For group 1 cows, serum chloride concentration had the strongest positive association with serum potassium concentration, and serum potassium concentration was negatively associated with plasma bicarbonate and serum glucose, creatinine, and urea concentrations. Six hundred thirty-six of 1,160 (55%) of group 2 cows with LDA were hypokalemic (mean serum potassium concentration, 3.7 mEq/L). Ninety-two of 172 (53%) group 2 cows with RDA or AV were hypokalemic (mean serum potassium concentration, 3.8 mEq/L). For group 2 cows, serum chloride concentration had the strongest positive association with serum potassium concentration, and serum potassium concentration was negatively associated with indices of feed intake (serum bilirubin concentration) and hydration status. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested hypokalemia was associated with hypochloremia, alkalemia, low feed intake with high amount of milk produced, hypovolemia, and hyperglycemia in lactating dairy cows. Treatment of hypokalemia should include surgical correction of abomasal displacement, increased dietary potassium intake via dietary dry matter intake or oral administration of KCl, and correction of hypochloremia, alkalemia, metabolic alkalosis, and dehydration.

Comparison of the fecal microbiota of healthy horses and horses with colitis by high throughput sequencing of the V3-V5 region of the 16S rRNA gene.

The intestinal tract houses one of the richest and most complex microbial populations on the planet, and plays a critical role in health and a wide range of diseases. Limited studies using new sequencing technologies in horses are available. The objective of this study was to characterize the fecal microbiome of healthy horses and to compare the fecal microbiome of healthy horses to that of horses with undifferentiated colitis. A total of 195,748 sequences obtained from 6 healthy horses and 10 horses affected by undifferentiated colitis were analyzed. Firmicutes predominated (68%) among healthy horses followed by Bacteroidetes (14%) and Proteobacteria (10%). In contrast, Bacteroidetes (40%) was the most abundant phylum among horses with colitis, followed by Firmicutes (30%) and Proteobacteria (18%). Healthy horses had a significantly higher relative abundance of Actinobacteria and Spirochaetes while horses with colitis had significantly more Fusobacteria. Members of the Clostridia class were more abundant in healthy horses. Members of the Lachnospiraceae family were the most frequently shared among healthy individuals. The species richness reported here indicates the complexity of the equine intestinal microbiome. The predominance of Clostridia demonstrates the importance of this group of bacteria in healthy horses. The marked differences in the microbiome between healthy horses and horses with colitis indicate that colitis may be a disease of gut dysbiosis, rather than one that occurs simply through overgrowth of an individual pathogen.

Application of strong ion difference theory to urine and the relationship between urine pH and net acid excretion in cattle.

OBJECTIVE: To develop an equation expressing urine pH in terms of independent variables, derive an equation relating urine pH to net acid excretion (NAE), and apply this new knowledge to determine the role that monitoring urine pH should play when diets with low cationanion difference are fed to dairy cattle. ANIMALS: 11 Holstein-Friesian cows. PROCEDURES: A physicochemical strong ion approach was used to develop a general electroneutrality equation for urine that involved urine pH and strong ion difference (SID [difference between strong cation and strong anion concentrations]), PCO(2), the concentration of ammonium ([NH(4)(+)]) and phosphate ([PO(4)]), and 3 constants. The general electroneutrality equation was simplified for use in bovine urine and applied to 321 data points from 11 cows fed different diets. RESULTS: Urine pH was dependent on 4 independent variables (urine SID, [NH(4)(+)], PCO(2), and [PO(4)]) and 3 constants. The simplified electroneutrality equation for bovine urine was pH approximately {pK(1)' - log(10)(S PCO(2))} + log(10)([K(+)] + [Na(+)] + [Mg(2+)] + [Ca(2+)] + [NH(4)(+)] - [Cl(-)] - [SO(4)(2-)]). The relationship between urine pH and NAE (in mEq/L) for cattle fed different diets was pH = 6.12 + log(10)(-NAE + [NH(4)(+)] + 2.6). CONCLUSIONS AND CLINICAL RELEVANCE: A change in urine SID, [NH(4)(+)], PCO(2), or [PO(4)] independently and directly led to a change in urine pH. Urinary [K(+)] had the greatest effect on urine pH in cattle, with high urine [K(+)] resulting in alkaline urine and low urine [K(+)] resulting in acidic urine. Urine pH provided an accurate assessment of NAE in cattle when pH was > 6.3.

Equine renal tubular disorders.

Renal tubular disorders have been sporadically reported in horses. Only three types of tubular defects have been recognized: (1) nephrogenic diabetes insipidus, attributable to unresponsiveness of the renal tubules to antidiuretic hormone; (2) distal renal tubular acidosis (RTA; type I); and (3) proximal RTA (type II). The following review focuses on RTA and nephrogenic diabetes insipidus.

Natural and experimental infection of neonatal calves with Clostridium difficile.

Clostridium difficile toxins were associated with calf diarrhea in a recent retrospective study; however, no causal relationship has been prospectively investigated. This infection study tested whether the oral inoculation of neonatal calves with a toxigenic strain of C. difficile (PCR-ribotype 077) results in enteric disease. Fourteen 6-24 h old male colostrums-fed Holstein calves, received either three doses of C. difficile (1.4 x 10(8) +/- 3.5 x 10(8) cfu) (n = 8) or sterile culture broth (n = 6). Calves were euthanized on day 6 or after the onset of diarrhea, whichever came first. Fecal and intestinal samples were blindly cultured for C. difficile, and tested for its toxin A/B (C. difficile TOX A/B II ELISA, Techlab). PCR-ribotyping was used to compare inoculated and recovered isolates. Diarrhea was observed in all control calves and 3/8 of inoculated calves (p = 0.03), but it did not occur in calves that tested positive for C. difficile toxins. Fecal toxins were identified only from two controls. PCR-ribotyping confirmed the presence of C. difficile PCR-ribotype 077 in samples of all inoculated calves, but not from controls. The identification of five other PCR-ribotypes in 3/8 (37.5%) and 2/6 (33.3%) of inoculated and control calves, respectively, indicated early natural infection (< or = 24h of age). Five of 14 cecal samples had C. difficile (p = 0.01). In conclusion, the oral administration of C. difficile PCR-ribotype 077 to neonatal calves resulted in fecal/intestinal colonization but not in detection of toxins, or signs of enteric disease. Further studies are required to investigate the clinical relevance of C. difficile in calves.

Potential role of Clostridium difficile as a cause of duodenitis-proximal jejunitis in horses.

Duodenitis-proximal jejunitis (DPJ) is an idiopathic condition in the horse characterized by inflammation and oedema of the duodenum and proximal jejunum. Clinical signs include colic, ileus, depression, fluid accumulation in the small intestine and stomach, and endotoxaemia. The objective of this study was to investigate prospectively the role of Clostridium difficile in this idiopathic disease. Nasogastric reflux from 10 consecutive cases with DPJ and 16 consecutive horses with other causes of nasogastric reflux was cultured for C. difficile, other Clostridium spp., and Salmonella. Toxigenic strains of C. difficile were isolated from 10/10 (100%) of horses with DPJ and 1/16 controls (P<0.0001). No other known pathogenic clostridia were isolated from either group. Results of this study suggest that C. difficile might be an important cause of this syndrome.

Clostridium difficile PCR ribotypes in calves, Canada.

We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans.

Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in canine plasma.

Acid-base abnormalities frequently are present in sick dogs. The mechanism for an acid-base disturbance can be determined with the simplified strong ion approach, which requires accurate values for the total concentration of plasma nonvolatile buffers (A(tot)) and the effective dissociation constant for plasma weak acids (K(a)). The aims of this study were to experimentally determine A(tot) and K(a) values for canine plasma. Plasma was harvested from 10 healthy dogs; the concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, L-lactate) and nonvolatile buffer ions (total protein, albumin, phosphate) were determined; and the plasma was tonometered with CO2 at 37 degrees C. Strong ion difference (SID) was calculated from the measured strong ion concentrations, and nonlinear regression was used to estimate values for A(tot) and K(a), which were validated with data from an in vitro and in vivo study. Mean (+/- SD) values for canine plasma were A(tot) = (17.4 +/- 8.6) mM (equivalent to 0.273 mmol/g of total protein or 0.469 mmol/g of albumin); K(a) = (0.17 +/- 0.11) x 10(-7); pK(a) = 7.77. The calculated SID for normal canine plasma (pH = 7.40; P(CO2) = 37 mm Hg; [total protein] = 64 g/L) was 27 mEq/L. The net protein charge for normal canine plasma was 0.25 mEq/g of total protein or 0.42 mEq/g of albumin. Application of the experimentally determined values for A(tot), K(a), and net protein charge should improve understanding of the mechanism for complex acid-base disturbances in dogs.

Use of a quantitative strong ion approach to determine the mechanism for acid-base abnormalities in sick calves with or without diarrhea.

Acid-base abnormalities are frequently present in sick calves. The mechanism for an acid-base disturbance can be characterized using the strong ion approach, which requires accurate values for the total concentration of plasma nonvolatile buffers (A(tot)) and the effective dissociation constant for plasma weak acids (K(a)). The aims of this study were to experimentally determine A(tot), K(a), and net protein charge values for calf plasma and to apply these values quantitatively to data from sick calves to determine underlying mechanisms for the observed acid-base disturbance. Plasma was harvested from 9 healthy Holstein-Friesian calves and concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, L-lactate) and nonvolatile buffer ions (total protein, albumin, phosphate) were determined. Plasma was tonometered with CO2 at 37 degrees C, and plasma P(CO2) and pH measured over a range of 15-159 mm Hg and 6.93-7.79, respectively. Strong ion difference (SID) was calculated from the measured strong ion concentrations, and nonlinear regression was used to estimate values for A(tot) and K(a) from the measured pH and P(CO2) and calculated SID. The estimated A(tot) and K(a) values were then validated using data from 2 in vivo studies. Mean (+/- SD) values for calf plasma were A(tot) = 0.343 mmol/g of total protein or 0.622 mmol/g of albumin; K(a) = (0.84 +/- 0.41) x 10(-7); pK(a) = 7.08. The net protein charge of calf plasma was 10.5 mEq/L, equivalent to 0.19 mEq/g of total protein or 0.34 mEq/g of albumin. Application of the strong ion approach to acid-base disturbances in 231 sick calves with or without diarrhea indicated that acidemia was due predominantly to a strong ion acidosis in response to hyponatremia accompanied by normochloremia or hyperchloremia and the presence of unidentified strong anions. These results confirm current recommendations that treatment of acidemia in sick calves with or without diarrhea should focus on intravenous or PO administration of a fluid containing sodium and a high effective SID.

Effect of extraction time and acid concentration on the separation of proglycogen and macroglycogen in horse muscle samples.

The objective of this study was to determine whether the concentrations of proglycogen (PG) and macroglycogen (MG) in biopsy samples of horse muscle are influenced by extraction time or perchloric acid (PCA) concentration. In study 1, individual muscle-biopsy samples from 10 horses were divided into 4 parts each and then randomly subjected to 4 periods of extraction (10, 20, 60, or 120 min) with 1.5 M PCA. In study 2, individual muscle-biopsy samples from 6 horses were divided into 24 pieces each and then randomly subjected to 12 combinations of extraction time (10, 20, 30, or 40 min) and PCA concentration (0.5, 1.5, or 3.0 M). The results from study 1 indicated that PG and MG concentrations are affected only after extraction for 120 min; the PG concentration decreased significantly (P < 0.05), and the MG concentration increased (not significantly). In study 2, extraction in 3.0 M PCA yielded significantly lower PG and higher MG concentrations (P < 0.05) than extraction in 0.5 or 1.5 M PCA with each of the extraction times. The results of this study further support the existence of 2 glycogen pools and demonstrate that they are not an extraction artifact. The study also suggests that the 2 pools are stable during extraction over a range of extraction times and acid concentrations. However, if the exposure to acid is very long and, or, the acid concentration is high, some of the insoluble PG appears to be hydrolyzed and to enter the MG pool.

Analysis of proglycogen and macroglycogen content in muscle biopsy specimens obtained from horses.

OBJECTIVE: To determine proglycogen (PG) and macroglycogen (MG) content in equine skeletal muscle and to compare 2 analytical methods (acid hydrolysis [AC] and PG plus MG determination) for measurement of total muscle glycogen content (Gly(tot)) in biopsy specimens. SAMPLE POPULATION: Muscle biopsy specimens obtained from 41 clinically normal horses. PROCEDURE: Forty-five muscle biopsy specimens obtained from the middle gluteal (n = 31) or triceps (14) muscle were analyzed, using AC and MG plus PG determination for Gly(tot). Variability within muscle biopsy specimens for each method was calculated from duplicate analyses of muscle specimens. In a second experiment, variation in MG and PG content between muscle biopsy specimens and the effect of sample collection depth on the concentration of MG and PG in the middle gluteal muscle was evaluated. RESULTS: There was a strong correlation (r = 0.99) between Gly(tot) values obtained by use of AC and MG plus PG determination. Coefficients of variation for within- and between-specimen variability of Gly(tot) were approximately 4% for each method. The PG fraction was always in excess of the MG fraction. Biopsy specimens obtained from the superficial part of the middle gluteal muscle contained significantly more Gly(tot) and PG than specimens obtained from deeper parts. CONCLUSIONS AND CLINICAL RELEVANCE: This study confirms that MG and PG exist in equine skeletal muscle and can be measured reliably in biopsy samples. This technique could be applied in future studies to investigate glycogen metabolism in exercising horses and horses with glycogen-storage diseases.