A. Schoster

Comparison of microbial populations in the small intestine, large intestine and feces of healthy horses using terminal restriction fragment length polymorphism

BackgroundThe composition of the microbiota of the equine intestinal tract is complex. Determining whether the microbial composition of fecal samples is representative of proximal compartments of the digestive tract could greatly simplify future studies. The objectives of this study were to compare the microbial populations of the duodenum, ileum, cecum, colon and rectum (feces) within and between healthy horses, and to determine whether rectal (fecal) samples are representative of proximal segments of the gastrointestinal tract. Intestinal samples were collected from ten euthanized horses. 16S rRNA gene PCR-based TRFLP was used to investigate microbiota richness in various segments of the gastrointestinal tract, and dice similarity indices were calculated to compare the samples.ResultsWithin horses large variations of microbial populations along the gastrointestinal tract were seen. The microbiota in rectal samples was only partially representative of other intestinal compartments. The highest similarity was obtained when feces were compared to the cecum. Large compartmental variations were also seen when microbial populations were compared between six horses with similar dietary and housing management.ConclusionRectal samples were not entirely representative of intestinal compartments in the small or large intestine. This should be taken into account when designing studies using fecal sampling to assess other intestinal compartments. Similarity between horses with similar dietary and husbandry management was also limited, suggesting that parts of the intestinal microbiota were unique to each animal in this study.

Consistent detection of bovine papillomavirus in lesions, intact skin and peripheral blood mononuclear cells of horses affected by hoof canker

REASONS FOR PERFORMING THE STUDYnEquine hoof canker is a chronic proliferative pododermatitis of as yet unknown aetiology. Like equine sarcoid disease, canker is a therapy-resistant disorder characterised by hyperkeratosis, acanthosis and a marked tendency to recur.nnnHYPOTHESISnThere is an association of sarcoid-inducing bovine papillomaviruses of types 1 and 2 (BPV-1, BPV-2) with hoof canker disease.nnnMETHODSnUsing PCR-based techniques, we assessed canker tissue, intact skin and/or peripheral blood mononuclear cells (PBMCs) of 25 canker-affected horses for the presence of sarcoid-associated BPV-1 and -2.nnnRESULTSnConventional PCR revealed BPV-1/-2 DNA in 24/24 canker, 12/13 skin and 10/11 PBMC DNA isolates. Using inverse PCR, full-length BPV episomes were detected in 1/5 canker specimens. Sequencing of viral early and late genes amplified from canker, intact skin and PBMC DNA of 2 cases revealed an overall identity of 98% to BPV-1. Viral DNA loads amounted to ≤16 copies per cell in canker tissue and intact skin, and to ≤0.35 copies per PBMC, as determined by quantitative PCR. Using RT-PCR, the viral major oncogene E5 was shown to be transcribed in 2/4 canker tissue specimens and 5/7 PBMC isolates. Immunocapture PCR from 7 canker and 6 skin extract supernatants revealed capsomere-associated viral DNA in one canker and one skin sample. Hoof tissue, skin and PBMCs collected from 13 individuals with no signs of canker or BPV-related malignancies scored negative throughout the experiments.nnnCONCLUSIONnThese findings suggest that the observed presence of BPV-1/-2 in canker-affected horses is not coincidental but indicative of an active contribution to hoof canker disease.nnnPOTENTIAL RELEVANCEnThe use of antivirals and/or immune modulators may help improving canker therapy.

Effect of a Probiotic on Prevention of Diarrhea and Clostridium difficile and Clostridium perfringens Shedding in Foals

Background Up to 60% of foals develop diarrhea within 6 months after birth. Preventive measures are limited but potentially probiotics could be used. Objective To evaluate the effect of a newly designed probiotic on the incidence of foal diarrhea in a randomized field trial. Animals Seventy‐two healthy neonatal foals. Methods Randomized, placebo‐controlled field trial. Foals were administered a placebo or probiotic for 3 weeks and monitored for an additional week. A total of 3 fecal samples were taken from each foal at biweekly intervals. Statistical modeling was applied for comparison of incidence and duration of diarrhea and fecal shedding of Clostridium perfringens and Clostridium difficile between treatment and age groups. Results The overall incidence of diarrhea was 41 of 72 (59%) and did not differ (P = 0.37) between treatment groups. Foals treated with probiotics were more likely to develop diarrhea requiring veterinary intervention (P = 0.007). Age had a significant effect on incidence of diarrhea (P < 0.001); foals 8–15 days old having the highest probability of developing diarrhea. Duration of diarrhea and soft feces were not significantly different between groups. The prevalence of C. perfringens shedding was 55% with no difference between treatment groups (P = 0.23). The prevalence of C. difficile shedding was 11%. Conclusion and Clinical Importance There was no benefit of administering a 3‐week course of probiotics, but potential adverse effects were noted. Whether the probiotics lacked a clinical effect, or the choice of strains or dose was inadequate, is unknown. Clostridial shedding was not influenced by probiotics despite in vitro activity of probiotics.

Probiotic Use in Horses – What is the Evidence for Their Clinical Efficacy?

The gastrointestinal microbiota is extremely important for human and animal health. Investigations into the composition of the microbiota and its therapeutic modification have received increasing interest in human and veterinary medicine. Probiotics are a way of modifying the microbiota and have been tested to prevent and treat diseases. Probiotics are proposed to exert their beneficial effects through various pathways. Production of antimicrobial compounds targeting intestinal pathogens, general immune stimulation, and colonization resistance are among these mechanisms. Despite widespread availability and use, scientific, peer‐reviewed evidence behind commercial probiotic formulations in horses is limited. Additionally, quality control of commercial over‐the‐counter products is not tightly regulated. Although promising in vitro results have been achieved, in vivo health benefits have been more difficult to prove. Whether the ambiguous results are caused by strain selection, dosage selection or true lack of efficacy remains to be answered. Although these limitations exist, probiotics are increasingly used because of their lack of severe adverse effects, ease of administration, and low cost. This review summarizes the current evidence for probiotic use in equine medicine. It aims to provide veterinarians with evidence‐based information on when and why probiotics are indicated for prevention or treatment of gastrointestinal disease in horses. The review also outlines the current state of knowledge on the equine microbiota and the potential of fecal microbial transplantation, as they relate to the topic of probiotics.

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

BackgroundClostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status.ResultsToxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, β2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good.ConclusionsClostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.

The longitudinal effect of a multi-strain probiotic on the intestinal bacterial microbiota of neonatal foals.

REASONS FOR PERFORMING THE STUDYnThe microbiota plays a key role in health and disease. Probiotics are a potential way to therapeutically modify the intestinal microbiota and prevent disease.nnnOBJECTIVESnThe aim of this study was to investigate the effects of probiotics on the bacterial microbiota of foals during and after administration.nnnSTUDY DESIGNnRandomised placebo controlled field trial.nnnMETHODSnThirty-eight healthy neonatal foals enrolled in a previous study were selected. The foals had received a multi-strain probiotic (four Lactobacillus spp. 3-4 × 103 colony-forming units (cfu)/g each, Bifidobacterium animalis spp. lactis, 1 × 103-4 cfu/g) or placebo once daily for 3 weeks. A total of 3 faecal samples were collected from each foal at 2-week intervals and assessed via metagenomic sequencing. The Wilcoxon test was used to compare data between treatment groups.nnnRESULTSnThere were no changes on the phylum, order or class level between treatment groups at any age (all P>0.05) but some significant changes in relative abundance of families. Probiotic administration did not result in an increased relative abundance of lactobacilli or bifidobacteria at any age (Lactobacillus: P = 0.9, P = 0.1 and P = 0.2, Bifidobacterium: P = 0.3, P = 0.6 and P = 0.1 for Weeks 2, 4 and 6, respectively). Lactobacillus was enriched in the probiotic group at Week 6 on LEfSe analysis (linear discriminant analysis score 0.34, P = 0 .02). There was no effect on alpha diversity (all P>0.2) or community structure when parsimony and unifrac analysis were applied (all P>0.6).nnnCONCLUSIONSnThere were limited effects of probiotic treatment on the bacterial microbiota of foals. The studied probiotic based on lactobacilli and bifidobacteria has a limited potential for therapeutic modification of the gastrointestinal microbiota.

Effects of transport, fasting and anaesthesia on the faecal microbiota of healthy adult horses.

REASONS FOR PERFORMING STUDYnThe intestinal microbiota is important for health and disease. Factors that disturb the equine intestinal microbiota need further investigation.nnnOBJECTIVESnTo determine the effects of transport, fasting and anaesthesia on the faecal microbiota of healthy adult horses using next-generation sequencing.nnnSTUDY DESIGNnExperimental trial.nnnMETHODSnFaecal samples were taken from 8 horses at baseline, after transport, 12xa0h of fasting and 24, 48 and 72xa0h after a 6xa0h anaesthesia. Next generation sequencing of the V4 region of the 16S rRNA gene was used to assess the microbial composition of faeces. Alpha diversity, phylogenetic structures and beta diversity were assessed.nnnRESULTSnThere were significant changes in the relative abundances of phyla, classes, orders and families after transport, fasting and anaesthesia. Most notably horses had a significantly lower abundance of Clostridiales after transport compared with baseline (Pxa0=xa00.03) and a decreased abundance of Rickettsiales after fasting (Pxa0=xa00.024). Alpha diversity was not significantly different between time points (all P>0.21). When parsimony analysis was applied, anaesthesia had a significant effect on community membership and structure (Jaccard index and Yue and Clayton index both Pxa0=xa00.02).nnnCONCLUSIONSnThere was some effect of transport, fasting and anaesthesia on the composition and structure of the microbiota of healthy horses. This indicates these are potentially stress factors for the equine intestinal microbiota. Further investigation is required to look at the potential impact of changes in the microbiota on the development of disease in the post anaesthetic period.

Antimicrobial prescribing practices by Swiss, German and Austrian equine practitioners

ANTIMICROBIAL use contributes to the emerging concern of antimicrobial resistance (Traub-Dargatz and Dargatz 2009, Guardabassi 2012). In equine medicine, antimicrobials are prescribed for the treatment of diseases with an unlikely bacterial aetiology (Weese and Sabino 2005, Hughes and others 2013). Recent data from a European-wide survey indicated that veterinarians prescribe critically important antimicrobials (CIAs) in 13–30 per cent of cases depending on species, country and disease (De Briyne and others 2013). Less than 1 per cent of veterinarians from Austria and Switzerland responded in the above survey; therefore, there are currently little data for these countries.nnVeterinarians are encouraged to use antimicrobial products according to the product license; however, originally licensed dosages are often no longer adequate (Burton and others 2013, Weese and others 2015). Subtherapeutic doses of antimicrobials could contribute to the development of antimicrobial resistance, and it is currently unknown whether veterinarians rather adhere to licensing regulations or scientifically published data.nnThe objective of this study was to survey the antimicrobial prescribing practices of equine practitioners in Germany, Austria and Switzerland.nnTarget audiences of the online cross-sectional survey were the members of the Swiss Equine Veterinary Association (n=252), Association of German Equine Practitioners (n=740) and the Association of Austrian Equine Practitioners (n=235). The questionnaire used was similar to the one of a previous study in England (Hughes and others 2013). It was distributed to the practitioners via the software www.surveymonkey.com (see online Supplementary Item 1). Aside from demographic questions and resources on antimicrobials, six hypothetical clinical scenarios were presented. Respondents were asked to state if and which antimicrobial class they would use, as well as brand name, dose and duration of therapy.nnLicensed dosages for antimicrobials were retrieved from each countrys licensing body (Swissmedic—Switzerland, Federal ministry for Health—Germany, and Austrian Agency for Health …

Comparison of the fecal bacterial microbiota of healthy and diarrheic foals at two and four weeks of life

BackgroundDiarrhea in foals affects up to 60% of foals during the first six months of life. The effect of diarrhea on the fecal bacterial microbiota in foals has not been investigated. Little is known on the fecal bacterial microbial richness and diversity of foals at a young age. The objective was to compare the fecal bacterial microbiota of healthy foals to foals with diarrhea at two and four weeks of life.MethodsFecal samples were collected from foals (nxa0=xa020) at 1–14 (T1) and 15–28 (T2) days of age and analyzed using high throughput sequencing. Differences in relative abundance of bacterial taxa, alpha diversity and beta diversity indices were assessed between age-matched foals with diarrhea (nxa0=xa09) and healthy foals (nxa0=xa011), and between time points.ResultsDifferences in microbial community composition based on time point and health status were observed on all taxonomic levels. Of 117 enriched species in healthy foals at T2, 50 (48%) were Lachnospiraceae or Ruminococcaceae. The Chao richness index was increased in healthy foals at T2 compared to T1 (pxa0=xa00.02). Foals with diarrhea had a significantly lower richness index than non-diarrheic foals at T2 (pxa0=xa00.04). Diarrhea had an inconsistent effect, while time point had a consistent effect on microbial community structure.ConclusionsPreventative and therapeutic measures for diarrhea should focus on maintaining bacterial microbiota richness. Lachnospiraceae and Ruminococcaceae were underrepresented in foals with diarrhea. These should be evaluated further as potential therapeutic options.

Epidemiology and Antimicrobial Resistance in Clostridium difficile With Special Reference to the Horse

Clostridium difficile is an important gastrointestinal pathogen in humans and animals. Disease occurs after intestinal colonization with toxigenic strains of C. difficile and subsequent proliferation due to disturbances of the microbiota. Several animal species can be affected, but mainly pigs and horses, and occasionally dogs, develop clinical disease. Pathogenesis appears to be similar in animals and humans. Initially, this disease was mainly considered to be a nosocomial infection after antimicrobial exposure; now, it often has a community onset without prior antimicrobial exposure. Therefore, reservoirs of C. difficile including animal feces, food, and the environment have recently been investigated. The bacterium has been isolated from the feces of healthy individuals from several species, including domestic and wild animals. Molecular types isolated from animals correlate with human ones, suggesting that animals could serve as an important reservoir. The use of antimicrobials appears associated with infection patterns and resistance profiles of C. difficile.