Henry R. Stämpfli

Clostridium difficile PCR Ribotypes in Calves, Canada

C. difficile, including epidemic PCR ribotypes 017 and 027, were isolated from dairy calves in Canada.

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.

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.

Epidemiology of Clostridium difficile on a veal farm: prevalence, molecular characterization and tetracycline resistance.

Concern has been raised about the potential for Clostridium difficile to be a bovine and foodborne pathogen, yet limited study has been performed in cattle, and none in veal calves. This study evaluated the epidemiology and microbiology of C. difficile on one veal farm. Rectal swabs were obtained from calves within 48 h of arrival and at one, 17 and 21 weeks later. Selective culture for C. difficile was performed. Isolates were characterized by PCR ribotyping and PCR for tcdA, tcdB and cdtA. Tetracycline resistance and resistance genes were investigated. Multivariable logistic regression models were constructed to determine the relationship between shedding of the bacterium and specific ribotypes and the independent variables: time of sampling and area of housing. Calves were twice more likely to test positive 1 week after arrival (51%) when compared to initial results (32%). Shedding at 17 and 21 weeks was significantly lower (2% at both samplings). Ribotype 078 was the most common. Twelve different ribotypes were present initially with only three ribotypes found subsequently. Seventy-six percent (40/53) of isolates initially recovered were tetracycline resistant compared to 93% (81/87) from 2nd sampling. Tetracycline resistance genes were detected in 24% (13/53) of isolates during 1st and in 55% (50/91) during 2nd sampling. The high prevalence of pathogenic C. difficile in veal calves could be of zoonotic concern. The low prevalence before slaughter may be of importance for the evaluation of foodborne risks. Oxytetracycline administration to calves may have an impact on prevalence of C. difficile colonization.

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

BackgroundThe 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.ResultsTwenty-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.ConclusionsThe 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.

Natural and experimental infection of neonatal calves with Clostridium difficile

Abstract 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 6–24h old male colostrums-fed Holstein calves, received either three doses of C. difficile (1.4×108 ±3.5×108 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 (≤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.

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.

Development of the faecal microbiota in foals

REASONS FOR PERFORMING STUDY The intestinal microbiota is a complex polymicrobial ecosystem that exerts extremely important roles in the development and maintenance of health. Recently, as new sequencing technologies have become more available, there has been a revolution in the understanding of the equine intestinal microbiota. However, studies characterising the pioneer intestinal bacteria colonising foals and its development over time are still limited. OBJECTIVES The objectives of this study were to characterise the intestinal bacterial colonisation of newborn foals and to follow individual animals over time until age 9 months. STUDY DESIGN Longitudinal study. METHODS Eleven pregnant mares from one farm were enrolled and faecal samples were collected longitudinally from mares and foals during their first day post partum and again periodically until foals were age 9 months. The V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. RESULTS Newborn foals had a rich and diverse bacterial community, which was mainly comprised of the Firmicutes phylum with several low abundant genera being unique at this age. Foals aged 2-30 days had significantly decreased diversity compared to older animals, with the majority of organisms classified as Akkermansia spp. After 60 days of life, the intestinal microbiota structure tended to remain stable, but differences in community membership were still present between 9-month-old animals and mature mares. Several differences at the phylum level were observed between different ages, including a higher abundance of Fibrobacteres after weaning. CONCLUSIONS The intestinal microbiota of the equine newborn is already complex by the first day of life. Microbiota adaptation occurs during the first month and the microbiota of foals older than 60 days resemble the mothers microbiota, although differences in community membership are still present.

Contribution of Unmeasured Anions to Acid–Base Disorders and its Association with Altered Demeanor in 264 Calves with Neonatal Diarrhea

Background The quantitative effect of strong electrolytes, unmeasured anions (UAs), p CO 2, and plasma protein concentrations in determining plasma pH and bicarbonate (HCO 3 −) can be demonstrated using the physicochemical approach. Demeanor of calves with diarrhea is associated with acidemia, dehydration, and hyper‐d‐lactatemia. Hypothesis Unmeasured anions are a major factor influencing changes in plasma pH and HCO 3 − of calves with diarrhea and UAs and strong UAs, estimated by anion gap (AG) and strong ion gap (SIG), respectively, are more strongly associated with alteration of demeanor compared to other acid–base variables. Animals A total of 264 calves with diarrhea from two data sets (DS1 and DS2). Methods Retrospective study. Forward stepwise regression was performed to determine the relationship between measured pH or HCO 3 −, and physicochemical variables. A two‐way ANOVA was performed to investigate the association between acid–base variables and attitude (bright, obtunded, and stuporous), posture (standing, sternal or lateral recumbency), and strength of suckling reflex (strong, weak, or absent). Results Increased strong UAs estimated by SIG was the most important contributor to changes in measured pH and HCO 3 − (DS1: r 2 66 and 59%, DS2: 39 and 42%, P < .0001). SIG and AG were correlated to deteriorating calf demeanor for all three clinical scoring categories: attitude, posture, and suckle reflex (P < .0001). Conclusion and Clinical Relevance Elevated concentrations of strong UAs were the primary cause of acidemia and had an important influence on the demeanor of calves with diarrhea. These findings emphasize the importance of the calculation of UAs when evaluating acid–base abnormalities in calves.

Physicochemical Interpretation of Acid-Base Abnormalities in 54 Adult Horses with Acute Severe Colitis and Diarrhea

BACKGROUND The quantitative effect of strong electrolytes, pCO2 , and plasma protein concentration in determining plasma pH and bicarbonate concentrations can be demonstrated with the physicochemical approach. Plasma anion gap (AG) and strong ion gap (SIG) are used to assess the presence or absence of unmeasured anions. HYPOTHESES The physicochemical approach is useful for detection and explanation of acid-base disorders in horses with colitis. AG and SIG accurately predict hyperlactatemia in horses with colitis. ANIMALS Fifty-four horses with acute colitis and diarrhea. METHODS Retrospective study. Physicochemical variables were calculated for each patient. ROC curves were generated to analyze sensitivity and specificity of AG and SIG for predicting hyperlactatemia. RESULTS Physicochemical interpretation of acid-base events indicated that strong ion metabolic acidosis was present in 39 (72%) horses. Mixed strong ion acidosis and decreased weak acid (hypoproteinemia) alkalosis was concomitantly present in 17 (30%) patients. The sensitivity and specificity of AG and SIG to predict hyperlactatemia (L-lactate > 5 mEq/L) were 100% (95% CI, 66.4-100; P < .0001) and 84.4% (95% CI, 70.5-93.5 P < .0001). Area under the ROC curve for AG and SIG for predicting hyperlactatemia was 0.95 (95% CI, 0.86-0.99) and 0.93 (95% CI, 0.83-0.99), respectively. CONCLUSION AND CLINICAL RELEVANCE These results emphasize the importance of strong ions and proteins in the maintenance of the acid-base equilibria. AG and SIG were considered good predictors of clinically relevant hyperlactatemia.