Equine Feces Research Articles

Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses.

To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA. Healthy horses (n = 28) and horses with intestinal inflammation (n = 10). Feces were suspended in buffer to create fecal supernatant. Serum and fecal supernatant were analyzed using ELISA kits validated for the detection of MPO and CP in equine serum. Assay validation steps included intra- and interassay variability (coefficient of variation [CV]), dilution linearity, spike recovery, and sample type correlation. Variations in sample handling protocols (centrifugation speed, extraction buffer, and filtration) were evaluated. 17 paired fecal and serum samples were used for initial analysis (10 healthy horses, 7 colitis). Previously reported sample handling protocols resulted in detectable MPO and CP but poor CV, linearity, and spike recovery. There was a linear correlation between serum and fecal samples for CP but not MPO. There was a significant difference between the concentration and CV of alternative sample handling protocols for CP and MPO, with improved CV for CP (2.1% to 18.6%) but not MPO (14.4% to 53.4%). Processing fresh feces with a fecal extraction buffer and filtration of supernatant resulted in the best CV (0.5% to 3.8%) and recovery (45% to 64%) for CP. Detection of MPO was inconsistent regardless of method. There are few reliable diagnostic modalities for inflammation of the equine large colon. Findings support quantification of CP in equine feces using the described ELISA kit and protocol. With additional study to establish reference interval and clinical utility, the fecal inflammatory biomarker CP may allow for noninvasive quantification of intestinal inflammation in horses.

Dry matter concentration, particle size distribution and sand presence in faeces from horses with and without colic

A study comprising 74 colic and 74 control horses admitted to an animal hospital was performed. Faecal samples were collected and analysed for dry matter concentration, particle size distribution using wet-sieving, and sand presence through a sand sedimentation test. Data on horse breed, age, gender and basic feeding variables was collected and analysed using χ2-tests. Faecal dry matter concentration, particle size distribution and sand score was compared between colic and non-colic horses, and between horses with different colic types, using one-way ANOVA. Results showed that colic and non-colic horse groups were similar in breed, age, gender and basic feeding variables. Faecal dry matter concentration, particle size distribution and sand score were similar among colic and non-colic horses. Horses diagnosed with “unknown colic cause” had higher proportion of particles >0.5 <1.0 mm size compared to horses with colic due to impactions in caecum or colon, torsion or gas accumulation (P<0.05), but this difference was very small and most likely not of biological importance. Faecal dry matter concentration and sand score were similar among horses with different types of colic. Increased knowledge of the composition of particles of different size in equine faeces may enhance our understanding of digesta passage rate in colic and non-colic horses, which is needed to develop preventative measures of certain types of colic.

Validation of a commercial ELISA kit to measure 11-oxoetiocholanolone in equine and bovine feces.

Feces are a noninvasive and easily collectible matrix that may help determine cumulative hormone metabolite concentrations over medium-to-long times. To date, 11-oxoetiocholanolone, an important metabolite of cortisol, has been measured in equine and bovine feces solely by an in-house enzyme immunoassay (EIA). Therefore, we validated the use of a commercial ELISA kit (11-oxoetiocholanolone ELISA kit; Cayman Chemical), which had been validated on sheep feces and human urine, to measure 11-oxoetiocholanolone in feces from 42 horses and 32 bulls. The ELISA kit had good precision (intra- and inter-assay CVs: 5.8% and 11.2% for equine feces; 9.9% and 11.2% for bovine feces, respectively), analytical sensitivity (0.186 ng/mL for both equine and bovine feces), and accuracy (parallelism and recovery tests) in determining 11-oxoetiocholanolone concentrations in feces from both species. We found ranges of 11-oxoetiocholanolone concentrations of 1-109 ng/g in equine feces and 40-302 ng/g in feces of bulls. The Cayman Chemical ELISA kit offers a simple and accessible means of analysis of 11-oxoetiocholanolone in equine and bovine fecal samples.

Improved Equine Fecal Microbiome Characterization Using Target Enrichment by Hybridization Capture

GITDs are among the most common causes of death in adult and young horses in the United States (US). Previous studies have indicated a connection between GITDs and the equine gut microbiome. However, the low taxonomic resolution of the current microbiome sequencing methods has hampered the identification of specific bacterial changes associated with GITDs in horses. Here, we have compared TEHC, a new approach for 16S rRNA gene selection and sequencing, with conventional 16S rRNA gene amplicon sequencing for the characterization of the equine fecal microbiome. Both sequencing approaches were used to determine the fecal microbiome of four adult horses and one commercial mock microbiome. Our results show that TEHC yielded significantly more operational taxonomic units (OTUs) than conventional 16S amplicon sequencing when the same number of reads were used in the analysis. This translated into a deeper and more accurate characterization of the fecal microbiome when the samples were sequenced with TEHC according to the relative abundance analysis. Alpha and beta diversity metrics corroborated these findings and demonstrated that the microbiome of the fecal samples was significantly richer when sequenced with TEHC compared to 16S amplicon sequencing. Altogether, our study suggests that the TEHC strategy provides a more extensive characterization of the fecal microbiome of horses than the current alternative based on the PCR amplification of a portion of the 16S rRNA gene.

Equine fecal microbiota response to short term antibiotic administration

Though generally safe, research continues to demonstrate negative side effects of antibiotic administration on the gastrointestinal (GIT) microbiota across species. In horses, antibiotic associated diarrhea (AAD) is a life-threatening condition linked to the GIT microbiota. This study tested the hypothesis that short term antibiotic administration to healthy horses would negatively impact the fecal microbiota as measured by their ability to digest nutrients and through fecal shedding of disease-associated-bacteria. Twenty-four horses were assigned to one of four treatment groups: control (CO); potassium penicillin/gentamicin sulfate (KPG); ceftiofur crystalline free acid (EX); trimethoprim/sulfamethoxazole (SMZ); and treated for 4 days. Fecal samples were collected before treatment began (S0), the day after treatment conclusion (S5), and at 10, 14, 21, and 28 days after initiating treatment. Horses had highly individualized responses to antibiotic administration. All horses receiving antibiotics experienced significantly softer stool compared to controls. Lactobacillus spp. were dramatically reduced in all antibiotic treated S5 samples. Horses receiving antibiotics were significantly more likely to test positive for C. difficile or C. perfringens on fecal qPCR. In conclusion, response to antibiotic administration displays high inter-individual variability, but shows changes to the functions of fecal microbiota that may depend on the antibiotic used.

Estimation of In Vitro True Digestibility and Fiber Degradation from Feedstuff Fiber Composition When Incubated in Equine Fecal Inoculum

Neutral detergent fiber (NDF) and acid detergent fiber (ADF) composition have been shown to predict in vitro true digestibility (IVTD), in vitro NDF digestibility (IVNDFD), and in vitro ADF digestibility (IVADFD) in ruminants. This study’s objective was to estimate in vitro digestibility measures within the DaisyII incubator using equine fecal inoculum from feedstuff NDF and ADF composition. Analyzed feedstuffs included alfalfa hay (Medicago sativa), Coastal Bermudagrass hay, soybean meal, rice bran, hempseed meal, and Bluebonnet® Equilene® Pellets. Data were analyzed using Akaike’s information criterion (AIC) within the R Statistical Program©. The highest ranked model for IVTD was the interaction of NDF and ADF: 10003.32 – 0.2904 × NDF − 0.4220 × ADF − 0.0010 × NDF × ADF (Adjusted R2 = 0.959 and AICc = 474.97). Sample IVNDFD was moderately predicted by ADF: 855.15 – 1.5183 × ADF (Adjusted R2 = 0.749 and AICc = 560.82). Feedstuff ADF produced the highest ranked model for IVNDFD: 881.91 – 1.5952 × ADF (Adj. R2 = 0.835 and AICc = 541.33). These results indicate the effectiveness of using feedstuff NDF and ADF composition to predict IVTD, IVNDFD, and IVADFD within equine fecal inoculum. The findings of this study provide better understanding of feedstuff digestibility using equine fecal inoculum, but more research is warranted for validation of the models and the potential impact in vivo.

Homemade Nucleic Acid Preservation Buffer Proves Effective in Preserving the Equine Faecal Microbiota over Time at Ambient Temperatures.

The equine faecal microbiota is often assessed as a proxy of the microbial community in the distal colon, where the microbiome has been linked to states of health and disease in the horse. However, the microbial community structure may change over time if samples are not adequately preserved. This study stored equine faecal samples from n = 10 horses in four preservation treatments at room temperature for up to 150 h and assessed the resulting impact on microbial diversity and the differential abundance of taxa. Treatments included "COLD" (samples packaged with a cool pack), "CLX" (2% chlorhexidine digluconate solution), "NAP" (nucleic acid preservation buffer), and "FTA" (Whatman FTA™ cards). The samples were assessed using 16S rRNA gene sequencing after storage for 0, 24, 72, and 150 h at room temperature under the different treatments. The results showed effective preservation of diversity and community structure with NAP buffer but lower diversity (p = 0.001) and the under-representation of Fibrobacterota in the FTA card samples. The NAP treatment inhibited the overgrowth of bloom taxa that occurred by 72 h at room temperature. The COLD, CLX, and NAP treatments were effective in preserving the faecal microbiota for up to 24 h at room temperature, and the CLX and NAP treatments improved the yield of Patescibacteria and Fibrobacterota in some cases. The cold and CLX treatments were ineffective in preventing community shifts that occurred by 72 h at room temperature. These findings demonstrate the suitability of the COLD, NAP, and CLX treatments for the room temperature storage of equine faeces for up to 24 h and of NAP buffer for up to 150 h prior to processing.

Efficacy Evaluation of a Commercial Formulation With Duddingtonia Flagrans in Equine Gastrointestinal Nematodes

The indiscriminate use of antiparasitics for the treatment of helminths in horses has caused the ineffectiveness of commonly used chemical active principles, therefore, new alternatives such as the use of helminthophagous fungi have been studied. In this context, this study aimed to evaluate the in vitro efficacy of the commercial formulation Bioverm, composed of the fungus Duddingtonia flagrans strain AC001, in the reduction of gastrointestinal nematode larvae in equine feces. In coproculture, the genus Cyathostomum sp. was the most prevalent in the analyzed samples. The commercial formulation with D. flagrans demonstrated effectiveness in the predation of Cyathostomum sp. in tests. The recommended dose of 0.4 g, containing 105 chlamydospores per gram of product, reduced larvae by 44.23%, while the extrapolated dose of 1.0 g with the same concentrations of chlamydospores (105/g) resulted in a reduction of 57.20%, indicating the effectiveness of the product in controlling infective larvae.

Multi-Centered Field Evaluation of a Salmonella spp. Point-of-Care PCR Assay Using Equine Feces and Environmental Samples

The introduction of microfluidic card technology has opened the field for rapid point-of-care (POC) molecular assays, including fecal and environmental Salmonella spp. testing. The purpose of this study was to evaluate a novel POC PCR assay for the detection of Salmonella spp. in feces and environmental samples. A total of 143 fecal samples and 132 environmental samples were collected for POC PCR Salmonella spp. testing as well as qPCR testing. Each sample was inoculated into selenite broth and incubated for 18 to 24 hours. For the POC PCR assay, 14 μl of selenite broth were mixed with 126 μl of PCR reaction mix and pipetted into a microfluidic test card targeting the invA and ttrC gene of Salmonella enterica. For qPCR analysis, 200 µl of the selenite broth were processed for DNA purification and Salmonella spp. testing targeting the invA gene. The overall agreement between the POC PCR Salmonella spp. assay and qPCR assay was 88.1% for feces and 97.0% for environmental samples. Strong agreement and short turn-around-time make the POC device the first molecular diagnostic platform allowing detection of Salmonella spp. in a hospital setting without having to ship out samples to a veterinary diagnostic laboratory. The availability of an accurate POC PCR assay for the detection of Salmonella spp. will enhance the diagnostic capability of equine veterinarians to timely support a diagnosis of salmonellosis and also monitor the environment in order to reduce the risk of nosocomial infections.

39 An in-Vitro Model of the Equine Fecal Microbiome to Assess How Horse Population Affects Fermentation Following a Starch Challenge

Abstract The use of an in-vitro model of the equine microbiome is beneficial to assess how fermentation patterns differ based on a horse populations’ habitual diet. The purpose of this study was to determine the in-vitro fermentation patterns of the microbial community within different horse populations before and following a starch challenge. Fecal samples were taken from three different populations of horses: horses from the Shackleford Banks, a North Carolina feral horse population living on native grasses; horses from the NCSU Equine Educational Unit that are predominantly kept on cool season mixed pastures, though may be supplemented with hay and concentrates when warranted; and privately owned horses that were fed mixed diets consisting of grass hay, concentrates and some pasture. Horses were monitored and fecal samples were collected immediately following a void and were stored on dry ice and frozen until analysis. Fecal samples from individual horses were pooled to form a representative sample for each population and mixed with an anaerobic medium to prepare an inoculum. The inoculum was placed into bottles containing either a treatment substrate of alfalfa (A) or of alfalfa and starch (AS). Bottles were capped, purged with CO2 and placed in a water bath at 39°C to incubate for 0, 2, 4 or 24 hours. Culture samples were processed to measure methane and short chain fatty acids (SCFAs; acetate, propionate, butyrate and other isoacids) using gas chromatography. Results were analyzed using the Proc Mixed procedure in SAS to compare the effects of horse population, time and treatment. Methane was significantly greater after 24 hours within all populations with AS, as the inoculum compared with A alone (P = 0.03). Propionate was greater for AS (molar percentage, mean ± standard deviation; 2.21 ± 4.97%) versus the A treatment (11.95 ± 4.97%, P = 0.02). Acetate concentrations were significantly (P &amp;lt; 0.001) greater within the A treatment in the Shackleford and NCSU horses (60.31 ± 10.3% and 62.18 ± 11.47%, respectively) compared with the AS treatment (59.09 ± 11.21% and 59.77 ± 8.85%, respectively). Privately owned horses showed similar values of acetate concentration when comparing the treatment of A (63.42 ± 10.13%) versus AS (63.55 ± 10.77%). Butyrate concentrations were greater in the Shackleford Banks and NCSU horses (13.16 ± 1.92% and 2.64 ± 4.22%, respectively) compared with the privately owned horses (10.96 ± 2%). Isoacids were greater in the Shackleford Banks horses (10.19 ± 4.15%) than the NCSU horses (9.87 ± 3.98%) and the privately owned horses (9.52 ± 3.53%; P&amp;lt; 0.0001). It appears that fermentation of starch differs between these horse populations, likely due to their habitual diet.

6 Effect of sampling and storage conditions upon equine fecal microbial community

Equine feces offer a non-invasive sample which can provide an indication of the microbial community structure of part of the hindgut; however, microbial communities may be altered by technical artifacts introduced under field conditions. Four types of preservation method (preservation buffer (NAP), treatment with chlorhexidine digluconate, FTA card sample collection, cold (buffer-free) storage with insulated ice packs (COLD)) and time at ambient temperature (0, 24, 48, 72 h), were tested for their effect upon the microbial community structure of fecal samples from n = 5 mature, mixed breed horses, collected immediately post defecation. Sequencing of the 16S rRNA gene was performed using the Qiagen QIAseq 16S/ ITS screening panel and Illumina MiSeq sequencing platform. Alpha diversity was estimated using Shannon, Simpson, Chao1 and Observed diversity metrics, and compared using Kruskal-Wallis’ test (P < 0.05) and post hoc Dunn's test with Bonferroni adjustment. Bray Curtis distances and permutational multivariate ANOVA were used to assess β-diversity. There were significant differences in α-diversity with preservation method, but not with time at ambient storage or individual. Alpha diversity was significantly lower in FTA card samples compared with COLD samples for Shannon and Simpson, but not Chao1 or Observed metrics. All α-diversity metrics were significantly lower in FTA card samples compared with those in NAP (Table 1). No significant differences in α-diversity were identified for other comparisons. Regarding β-diversity, individual pony (P = 0.001), and time at ambient storage (P = 0.004) were significant and explained most of variance between samplesacross the study. Preservation method significantly influenced β-diversity as the second term in an additive model, after accounting for variance driven by individual pony (P = 0.011). These results highlight the strength of inter-individual variability and suggest that minimizing sample time at ambient temperature remains important for preserving the microbial composition of equine fecal samples. While preservation method may not have drastically affected the composition of dominant taxa across samples, microbial diversity was not fully preserved by FTA cards. This study identified a selection of comparably effective sample collection conditions which preserve microbial diversity in equine fecal samples.

Bioaccumulation and Elimination of Ivermectin by Eisenia foetida (Savigny 1826) Earthworms

Purpose: Bovine and equine faeces are commonly used to produce vermicompost of Eisenia foetida earthworms as a soil fertility enhancer. In animal health, ivermectin (IVM) is frequently used for parasite control. However, IVM is eliminated mainly by faeces, which raises environmental concerns. Little is known about the transfer of IVM by the earthworms´ activity. In this work the accumulation of IVM in E. foetida worms cultured in vermicompost containing IVM and the ability of the worms to release IVM to a drug-free substrate were evaluated.&#x0D; Methodology: The acute toxicity test (72 h) of IVM and two bioassays, accumulation (A) and elimination (B), with E. foetida earthworms were performed in the current work. In A, the vermicompost produced was a mixture of equine and bovine faeces, the latter added with IVM 3,000 ng/g. Worms and substrates were sampled between 1 and 28 days post treatment (dpt). In B, worms obtained at 28 dpt in A were transferred to a substrate without IVM and sampled between 1 and 14 days later. Samples of worms and substrates were analysed using High Performance Liquid Chromatography (HPLC).&#x0D; Findings: There was no worm mortality in the toxicity test. In assay A, E. foetida worms bio-accumulated up to 26.8% of the IVM present in the substrate. When worms from assay A, were moved to IVM-free substrate, they released 84% of the bio-accumulated IVM during the first day.&#x0D; Recommendations: This study highlights that IVM bio- accumulated by earthworms and releasing in residue-free substrates represents a contamination risk, especially in farms that are minimising the use of chemical compounds.&#x0D;

Impacts of Domestication and Veterinary Treatment on Mobile Genetic Elements and Resistance Genes in Equine Fecal Bacteria.

Antimicrobial resistance in bacteria is a threat to both human and animal health. We aimed to understand the impact of domestication and antimicrobial treatment on the types and numbers of resistant bacteria, antibiotic resistance genes (ARGs), and class 1 integrons (C1I) in the equine gut microbiome. Antibiotic-resistant fecal bacteria were isolated from wild horses, healthy farm horses, and horses undergoing veterinary treatment, and isolates (9,083 colonies) were screened by PCR for C1I; these were found at frequencies of 9.8% (vet horses), 0.31% (farm horses), and 0.05% (wild horses). A collection of 71 unique C1I+ isolates (17 Actinobacteria and 54 Proteobacteria) was subjected to resistance profiling and genome sequencing. Farm horses yielded mostly C1I+ Actinobacteria (Rhodococcus, Micrococcus, Microbacterium, Arthrobacter, Glutamicibacter, Kocuria), while vet horses primarily yielded C1I+ Proteobacteria (Escherichia, Klebsiella, Enterobacter, Pantoea, Acinetobacter, Leclercia, Ochrobactrum); the vet isolates had more extensive resistance and stronger PC promoters in the C1Is. All integrons in Actinobacteria were flanked by copies of IS6100, except in Micrococcus, where a novel IS5 family element (ISMcte1) was implicated in mobilization. In the Proteobacteria, C1Is were predominantly associated with IS26 and also IS1, Tn21, Tn1721, Tn512, and a putative formaldehyde-resistance transposon (Tn7489). Several large C1I-containing plasmid contigs were retrieved; two of these (plasmid types Y and F) also had extensive sets of metal resistance genes, including a novel copper-resistance transposon (Tn7519). Both veterinary treatment and domestication increase the frequency of C1Is in equine gut microflora, and each of these anthropogenic factors selects for a distinct group of integron-containing bacteria. IMPORTANCE There is increasing acknowledgment that a "one health" approach is required to tackle the growing problem of antimicrobial resistance. This requires that the issue is examined from not only the perspective of human medicine but also includes consideration of the roles of antimicrobials in veterinary medicine and agriculture and recognizes the importance of other ecological compartments in the dissemination of ARGs and mobile genetic elements such as C1I. We have shown that domestication and veterinary treatment increase the frequency of occurrence of C1Is in the equine gut microflora and that, in healthy farm horses, the C1I are unexpectedly found in Actinobacteria, while in horses receiving antimicrobial veterinary treatments, a taxonomic shift occurs, and the more typical integron-containing Proteobacteria are found. We identified several new mobile genetic elements (plasmids, insertion sequences [IS], and transposons) on genomic contigs from the integron-containing equine bacteria.

Evaluation of Bacterial Composition and Viability of Equine Feces after Processing for Transplantation

Fecal microbiota transplantation (FMT) has been used empirically for decades in equine medicine to treat intestinal dysbiosis but evidence-based information is scarce. This in vitro study aimed at assessing the effect of a commonly used pre-FMT processing method on the bacterial composition and viability of the fecal filtrate. Three samples of fresh equine manure (T0) were processed identically: the initial manure was mixed with 1 L of lukewarm water and chopped using an immersion blender to obtain a mixture (T1), which was left uncovered during 30 min (T2) and percolated through a sieve to obtain a fecal filtrate (T3). Samples were taken throughout the procedure (Tn) and immediately stored at 4 °C until processing. The 16S rDNA amplicon profiling associated with propidium monoazide treatment was performed on each sample to select live bacteria. Analyses of α and β diversity and main bacterial populations and quantitative (qPCR) analysis were performed and statistically compared (significance p < 0.05) between time points (T0-T3). No significant differences in ecological indices or mean estimated total living bacteria were found in the final fecal filtrate (T3) in regard to the original manure (T0); however, relative abundances of some minor genera (Fibrobacter, WCHB1-41_ge and Akkermansia) were significantly different in the final filtrate. In conclusion, the results support the viability of the major bacterial populations in equine feces when using the described pre-FMT protocol.

218 Hard and Easy Keeper Microbiomes Reveal Unique Microbial Responses to Carbohydrate and Protein Sources

Abstract This study reports differential responses of the equine gut microbiome to protein- and/or carbohydrate-based on keeper status [easy keeper (EK), medium keeper (MK), hard keeper (HK)]. Anaerobic microcosms were inoculated with equine feces from healthy animals on the same diets (n=12 total, n = 3 / EK, MK, HK of four breeds) with three dietary treatments [C = Carb (cornmeal), P = Protein (soybean meal), M = mix (50% C, 50% P), and F = fecal (no additive)]. Over 48 hours, fermentation products were measured using colorimetric assays and high performance liquid chromatography. Microbial populations were surveyed using 16S rRNA gene sequencing analyzed by QIIME2. Total SCFA production was not different between keeper status but the acetate: propionate ratio and isobutyrate production was greatest for HK and least for EK. Residual protein and carbohydrate at hour 48 was greatest in HK pointing to less efficient nutrient utilization. Diversity measures were not different between keeper statuses over time or within treatments (P &amp;gt; 0.05). Differential abundance (ANCOM and LEfSe), and Spearman correlations identified that while EK, MK and HK shared responses to treatment, groups had characteristic microbial responses to C and P. For example, Lactobacillales, a prominent lactate producer was differentially abundant for EK and MK across treatments but not a biologically significant marker in HK. Opportunistic Gamma proteobacteria bloomed in MK and HK by hour 48, but was only identified in EK at hour 8 indicating that EK communities can resist this growth. While the gut microbiome compositions of keeper groups were similar, they were functionally different in processing key nutrients. MK were more adaptive to dietary challenges and HK were less efficient at nutrient utilization. Analysis of the keeper status microbiomes may uncover novel functioning pathways and relationships that underlie the metabolic tendency of the host towards obesity or leanness.

Potential and Valorization of Salix babylonica Waste Leaf Extract to Mitigate Equine Fecal Production of Methane, Carbon Monoxide, and Hydrogen Sulfide

Potential and Valorization of Salix babylonica Waste Leaf Extract to Mitigate Equine Fecal Production of Methane, Carbon Monoxide, and Hydrogen Sulfide

Morphomolecular characterization of Strongylus vulgaris isolated from donkeys with special references to histopathological study on the affected organs.

Equine gastrointestinal tract is infected with Strongylus vulgaris (S. vulgaris) which is highly pathogenic parasite for its harmful effect on cranial mesenteric artery during its migration. So, this study was applied for identification of S. vulgaris in donkeys ultramorphologically and molecularly. In addition to, detection of the pathological effect of larval stage of S. vulgaris on the mesenteric arterial system using histopathology and immunohistochemistry. During the period from September to December; 2019, 60 male and 20 female donkeys at the Giza Zoo was postmortem examined. S. vulgaris adults and larvae were collected from the large intestine and cranial mesenteric arteries (CMAs), respectively. Ultramorphological examination of the collected adults was done using scanning electron microscope (SEM). DNA was extracted from 5 larvae and 6 adults for further conventional PCR studies and sequencing of the internal transcriped spacer 2 (ITS2) gene. The ITS2 gene were amplified and showed bands at 148 base pair (bp). The ITS2 gene nucleotide sequences of all isolates were aligned using Basic Local Alignment Search Tool (BLAST). Histological sections of S. vulgaris affected mesenteric arteries exhibited the presence of the parasite larvae either in the lumen with thrombus formation or attached to the intima. Most of the detected inflammatory cell populations were CD68-positive cells. From these results, it can be concluded that the ribosomal spacers genes could be used as markers for Strongylus species identification in eggs collected from equine feces as a beneficial method of diagnosis. Also, it could be important in disease surveillance, improving preventive measures and developing an effective control strategy.

Effect of Mid-Term Dietary Administration of the Caesalpinia coriaria Extract on the Sustainable Mitigation of Equine Fecal Methane, Carbon Monoxide and Hydrogen Sulfide Production

This study aimed to evaluate the dietary administration of the Caesalpinia coriaria (CC) extract for 30 days on in vitro fecal greenhouse gases production. Fecal samples, as inoculums, were collected from horses given daily 0- (Fecal 0), 60- (Fecal 60) and 120- (Fecal 60) mL CC aqueous extract per animal. The extract dose was mixed with the morning feeding diet at 6:00 h for each horse. During incubation, 0-, 0.6-, 1.2- and 1.8-mL CC extracts were added to the basal diet which was fed to horses (as subtract) and evaluated with each fecal type. Feces from the horses given no CC extract produced the lowest (P = .0014) methane while the fecal from horses given CC produced more methane . It was also observed that all CC doses linearly (P = .0457) produced more methane than the control. Furthermore, Fecal 0 was more efficient and produced less methane for every unit of metabolizable energy, organic matter, and short chain fatty acids while Fecal 60 was the least efficient. Production of H2S showed that feces of equine orally give 60 mL/day CC produced the highest while Fecal 0 and Fecal 120 were similar. Fecal type x dose showed that 0 mL/g DM produced the highest H2S while 1.8 mL/g DM produced the lowest. Thus, based on gas production, H2S, CO and CH4, feeding horses with 60 mL/day of CC with or without 0.6 mL/g DM of CC extract is recommended for the sustainable mitigation of greenhouse gases emission in horses.

Effects of Intravenous Antimicrobial Drugs on the Equine Fecal Microbiome.

Simple SummaryThe objectives of this study were to determine the effect of intravenous administration of antimicrobials (ceftiofur, enrofloxacin, oxytetracycline) on equine fecal bacterial communities over time. Healthy horses were treated for 5 days with enrofloxacin, ceftiofur sodium, oxytetracycline and saline solution, and fecal samples were collected over 30 days. Microbiome analysis was carried out via 16S rRNA gene sequencing. Microbial diversity and abundance were altered using ceftiofur and enrofloxacin. This study showed that antimicrobials alter gut bacterial communities, which could predispose horses to gastrointestinal inflammation, diarrhea and possibly systemic disorders.Alterations in the gastrointestinal microbiota after antimicrobial therapy in horses can result in loss of colonization resistance and changes in bacterial metabolic function. It is hypothesized that these changes facilitate gastrointestinal inflammation, pathogen expansion and the development of diarrhea. The objectives of this study were to determine the effect of intravenous administration of antimicrobial drugs (ceftiofur, enrofloxacin, oxytetracycline) on equine fecal bacterial communities over time, to investigate whether those changes are detectable after 5 days of treatment and whether they persist over time (30 days). Sixteen horses were randomly assigned into 4 treatment groups: group 1 (enrofloxacin, n = 4); group 2 (ceftiofur sodium, n = 4); group 3 (oxytetracycline, n = 4); group 4 (0.9% saline solution, placebo, n = 4). Antimicrobial therapy was administered for 5 days. Fecal samples were obtained before (day 0) and at 3, 5 and 30 days of the study period. Bacterial DNA was amplified using specific primers to the hypervariable region V1–V3 of the 16S rRNA gene using a 454 FLX-Titanium pyrosequencer. Antimicrobial therapy failed to cause any changes in physical examination parameters, behavior, appetite or fecal output or consistency throughout the study in any horse. There was a significant effect of treatment on alpha diversity indices (richness) over the treatment interval for ceftiofur on days 0 vs. 3 (p < 0.05), but not for other antimicrobials (p > 0.05). Microbial composition was significantly different (p < 0.05) across treatment group and day, but not for interactions between treatment and day, regardless of taxonomic level and beta-diversity distance metric. The most significant antimicrobial effects on relative abundance were noted after intravenous administration of ceftiofur and enrofloxacin. The relative abundance of Fibrobacteres was markedly lower on day 3 compared to other days in the ceftiofur and enrofloxacin treatment groups. There was an increase in Clostridia and Lachnospiraceae from day 0 to days 3 and 5 in ceftiofur and enrofloxacin treated groups. These findings showed the negative effect of antimicrobial drugs on bacterial communities associated with gut health (Fibrobacteres and Lachnospiraceae) and indicate that changes in specific taxa could predispose horses to gastrointestinal inflammation and the development of diarrhea.

Technical Note: A method for determination of titanium dioxide concentration in fecal samples.

Titanium dioxide has been used as a marker for determining diet digestibility indirectly, but some authors have expressed difficulty in measuring TiO2 concentrations in fecal material. We developed an accurate and precise method to determine TiO2 concentrations in equine feces. The method includes dry-ashing samples, digestion with (NH4)2SO4 in concentrated sulfuric acid, followed by the addition of H2O2 to produce a yellow to orange color that can be read spectrophotometrically. Accuracy was tested by spike recovery, and precision was tested by examining the coefficient of variation (CV) between duplicates of 449 individual samples. The method described here was compared with a previously published method by examining CV between duplicates of samples analyzed using both methods and comparing them using a paired t-test. Titanium dioxide spike recovery averaged 106%, and the CV between duplicates averaged 4.0%, with 79% of sample pairs having a CV of <5%. When compared with a previously published method, the method described here had a lower CV between duplicates (P < 0.0001). The method described here provides an accurate and precise quantitative analytical procedure for TiO2 in equine fecal samples.