Black soldier fly (Diptera: Stratiomyidae) larvae reduce cyathostomin (Nematoda: Strongylidae) eggs but develop poorly on horse manure.

Cyathostomins are common digestive tract parasites of grazing horses that spread through contact with horse feces. Horse feces are colonized by a variety of organisms, some of which could serve to reduce parasite loads in horse pastures. Black soldier fly (Hermetia illucens L.; Diptera: Stratiomyidae) larvae (BSFL) could be an ideal candidate for biological control of cyathostomins, due to their near-global distribution, low risk of pathogen transmission, ability to develop on a variety of nutrient-poor substrates (including horse manure), and dramatic effect on microbial communities that cyathostomins depend on. Here, using controlled feeding bioassays, we evaluated the effect of BSFL on cyathostomin egg densities in horse manure while also tracking BSFL performance on manure relative to standard grain-based diets. We found that BSFL consumed less substrate, were slower to reach the prepupal stage, and ultimately yielded less biomass when reared on horse manure compared to grain-based diets. However, BSFL reduced average cyathostomin egg densities in horse manure by over 3-fold. Overall, our results suggest that despite horse manure being a poor substrate for BSFL growth, BSFL effectively reduce cyathostomin egg loads in infected horse manure, though the mechanisms by which they do this are uncertain. While BSFL are known to transform the microbial communities within a diversity of rearing substrates, their effect on larger, parasitic organisms in animal manures may be underappreciated. Promoting the decomposition of infected horse manure with BSFL might be a promising approach to managing parasite populations among grazing horses.

Fecal Microbiota, Forage Nutrients, and Metabolic Responses of Horses Grazing Warm- and Cool-Season Grass Pastures.

Integrating warm-season grasses into cool-season equine grazing systems can increase pasture availability during summer months. The objective of this study was to evaluate effects of this management strategy on the fecal microbiome and relationships between fecal microbiota, forage nutrients, and metabolic responses of grazing horses. Fecal samples were collected from 8 mares after grazing cool-season pasture in spring, warm-season pasture in summer, and cool-season pasture in fall as well as after adaptation to standardized hay diets prior to spring grazing and at the end of the grazing season. Random forest classification was able to predict forage type based on microbial composition (accuracy: 0.90 ± 0.09); regression predicted forage crude protein (CP) and non-structural carbohydrate (NSC) concentrations (p < 0.0001). Akkermansia and Clostridium butyricum were enriched in horses grazing warm-season pasture and were positively correlated with CP and negatively with NSC; Clostridum butyricum was negatively correlated with peak plasma glucose concentrations following oral sugar tests (p ≤ 0.05). These results indicate that distinct shifts in the equine fecal microbiota occur in response different forages. Based on relationships identified between the microbiota, forage nutrients, and metabolic responses, further research should focus on the roles of Akkermansia spp. and Clostridium butyricum within the equine hindgut.

Fecal microbiome of horses transitioning between warm-season and cool-season grass pasture within integrated rotational grazing systems.

BACKGROUND: Diet is a key driver of equine hindgut microbial community structure and composition. The aim of this study was to characterize shifts in the fecal microbiota of grazing horses during transitions between forage types within integrated warm- (WSG) and cool-season grass (CSG) rotational grazing systems (IRS). Eight mares were randomly assigned to two IRS containing mixed cool-season grass and one of two warm-season grasses: bermudagrass [Cynodon dactylon (L.) Pers.] or crabgrass [Digitaria sanguinalis (L.) Scop.]. Fecal samples were collected during transitions from CSG to WSG pasture sections (C-W) and WSG to CSG (W-C) on days 0, 2, 4, and 6 following pasture rotation and compared using 16S rRNA gene sequencing. RESULTS: Regardless of IRS or transition (C-W vs. W-C), species richness was greater on day 4 and 6 in comparison to day 0 (P < 0.05). Evenness, however, did not differ by day. Weighted UniFrac also did not differ by day, and the most influential factor impacting ß-diversity was the individual horse (R2 ≥ 0.24; P = 0.0001). Random forest modeling was unable to accurately predict days within C-W and W-C, but could predict the individual horse based on microbial composition (accuracy: 0.92 ± 0.05). Only three differentially abundant bacterial co-abundance groups (BCG) were identified across days within all C-W and W-C for both IRS (W ≥ 126). The BCG differing by day for all transitions included amplicon sequence variants (ASV) assigned to bacterial groups with known fibrolytic and butyrate-producing functions including members of Lachnospiraceae, Clostridium sensu stricto 1, Anaerovorax the NK4A214 group of Oscillospiraceae, and Sarcina maxima. In comparison, 38 BCG were identified as differentially abundant by horse (W ≥ 704). The ASV in these groups were most commonly assigned to genera associated with degradation of structural carbohydrates included Rikenellaceae RC9 gut group, Treponema, Christensenellaceae R-7 group, and the NK4A214 group of Oscillospiraceae. Fecal pH also did not differ by day. CONCLUSIONS: Overall, these results demonstrated a strong influence of individual horse on the fecal microbial community, particularly on the specific composition of fiber-degraders. The equine fecal microbiota were largely stable across transitions between forages within IRS suggesting that the equine gut microbiota adjusted at the individual level to the subtle dietary changes imposed by these transitions. This adaptive capacity indicates that horses can be managed in IRS without inducing gastrointestinal dysfunction.

A Standard Scale to Measure Equine Keeper Status and the Effect of Metabolic Tendency on Gut Microbiome Structure.

Thriftiness in horses has been associated with more efficient nutrient harvesting in digestion, absorption and/or utilization, but the relative contribution of the gut microbiome to host metabolic tendency is not well understood. Recognizing the unreliability of owner reported assignment of keeper status, this research describes a novel tool for calculating whether a horse is an easy (EK) or hard (HK) keeper and then characterizes microbiome differences in these groups. The Equine Keeper Status Scale (EKSS) was developed and validated based on data gathered from 240 horses. Estimates of dietary energy intakes and requirements to achieve the optimal BCS score of 5 were used in EKSS assignments. Sixty percent of owners' characterizations disagreed with EKSS identified keeper assignments. Equine fecal 16S rRNA profiles (n = 73) revealed differences in α and ß diversities and taxa abundances based on EKSS assignments. EK communities had more Planctomycetes and fewer Euryarcheaota, Spirochaetes and Proteobacteria than HK indicating functional differences in nutrient harvesting between groups. Differences in the gut microbiomes of horses based on keeper assignment point to host/microbial interactions that may underlie some differences in metabolic tendency. The EKSS enables robust, repeatable determination of keeper status which can be used by researchers and horse owners.

The Use of Molecular Profiling to Track Equine Reinfection Rates of Cyathostomin Species Following Anthelmintic Administration.

Cyathostomins are a multispecies parasite ubiquitous in Equids. Cyathostomins have developed resistance to all but one class of anthelmintics, but species-level sensitivity to anthelmintics has not been shown. This study measured reinfection rates of cyathostomin species following the administration of three commercial dewormers. Nine treated horses were compared with 90 untreated controls during June-September 2017-2019. Ivermectin (IVM) (n = 6), Moxidectin (MOX) (n = 8) or Pyrantel (PYR) (n = 8) were orally administered. Fecal samples were collected every 14 d for 98 d. Fecal egg count reductions (FECR) were calculated using a modified McMaster technique. Nineteen cyathostomin species were identified by 5.8S-ITS-2 profiling using amplicon sequencing. Data were analyzed in QIIME1 and R statistical software using presence/absence methods. MOX had the lowest numbers of species present over the time course, followed by PYR then IVM (7.14, 10.17, 11.09, respectively); however, FECR was fastest for PYR. The presence of seven species: Coronocyclus labiatus, Cyathostomum catinatum, Cyathostomum tetracanthum, Cylicocylus elongatus, Cylicodontophorus bicoronatus, Cylicostephanus minutus, and Cylicostephanus goldi were unaffected by treatment (p > 0.05) points to species-specific differences in dewormer sensitivity and environmental persistence. Identifying resistance patterns at the species level will enable mechanistic understandings of cyathostomin anthelmintic resistance and targeted approaches to control them.

Lack of Association Between Barometric Pressure and Incidence of Colic in Equine Academic Ambulatory Practice.

Anecdotal accounts correlate equine colic onset to changing weather conditions; however, atmospheric effects on colic have not been studied extensively. We hypothesized that changes in barometric pressure would increase the likelihood of a colic diagnosis compared with other noncolic sick events. Multivariable logistic regression analysis was used to look for associations between colic diagnosis and barometric pressure. The University of Pennsylvania Field Service electronic medical records were searched by identifiable examination type via billing procedure codes collecting 3,108 emergent and nonemergent medical events along with corresponding weather data from the National Weather Service from January 1, 2005, through January 1, 2017. Barometric pressure values and changes were not found to be statistically associated with a diagnosis of colic (P = .1). Horses that did not survive were almost 12 times more likely to have a diagnosis of colic (odds ratio [OR]: 11.97; P < .0001). Horses with disease recurrence were 30% more likely to have a diagnosis of colic (OR: 1.29; P = .006). The likelihood of colic diagnosis increased with increasing latitude (OR: 2.43; P = .04). Horses were more likely to be diagnosed with colic in the fall (OR: 1.72; P < .0001), spring (OR: 1.29; P = .04), and summer (OR: 1.85; P < .0001), compared with winter. Stallions were 48% less likely to colic compared with mares (OR: 0.52; P = .016) and Quarter Horses were 32% less likely to colic compared with Thoroughbreds and Arabians (OR: 0.68; P = .047). This study provided evidence that changes in barometric pressure were not a contributing risk factor for colic, although seasons with changing weather and latitude may play a role.

Development of the equine hindgut microbiome in semi-feral and domestic conventionally-managed foals.

BACKGROUND: Early development of the gut microbiome is an essential part of neonate health in animals. It is unclear whether the acquisition of gut microbes is different between domesticated animals and their wild counterparts. In this study, fecal samples from ten domestic conventionally managed (DCM) Standardbred and ten semi-feral managed (SFM) Shetland-type pony foals and dams were compared using 16S rRNA sequencing to identify differences in the development of the foal hindgut microbiome related to time and management. RESULTS: Gut microbiome diversity of dams was lower than foals overall and within groups, and foals from both groups at Week 1 had less diverse gut microbiomes than subsequent weeks. The core microbiomes of SFM dams and foals had more taxa overall, and greater numbers of taxa within species groups when compared to DCM dams and foals. The gut microbiomes of SFM foals demonstrated enhanced diversity of key groups: Verrucomicrobia (RFP12), Ruminococcaceae, Fusobacterium spp., and Bacteroides spp., based on age and management. Lactic acid bacteria Lactobacillus spp. and other Lactobacillaceae genera were enriched only in DCM foals, specifically during their second and third week of life. Predicted microbiome functions estimated computationally suggested that SFM foals had higher mean sequence counts for taxa contributing to the digestion of lipids, simple and complex carbohydrates, and protein. DCM foal microbiomes were more similar to their dams in week five and six than were SFM foals at the same age. CONCLUSIONS: This study demonstrates the impact of management on the development of the foal gut microbiome in the first 6 weeks of life. The higher numbers of taxa within and between bacterial groups found in SFM dams and foals suggests more diversity and functional redundancy in their gut microbiomes, which could lend greater stability and resiliency to these communities. The colonization of lactic acid bacteria in the early life of DCM foals suggests enrichment in response to the availability of dams' feed. Thus, management type is an important driver of gut microbiome establishment on horses, and we may look to semi-feral horses for guidance in defining a healthy gut microbiome for domestic horses.

Effects of a chemical additive on the fermentation, microbial communities, and aerobic stability of corn silage with or without air stress during storage.

We evaluated the effects of a chemical additive on the microbial communities, fermentation profile, and aerobic stability of whole-plant corn silage with or without air stress during storage. Whole-plant corn was either untreated or treated with a chemical additive containing sodium benzoate, potassium sorbate, and sodium nitrite at 2 or 3 liters/t of fresh forage weight. Ten individually treated and replicated silos (7.5 liters) were made for each treatment. Half of the silos remained sealed throughout a 63-d storage period, and the other half was subjected to air stress for 2 h/wk. The composition of the bacterial and fungal communities of fresh forage and silages untreated or treated with 2 liters/t of fresh forage weight was analyzed by Illumina Miseq sequencing. Treated silage had greater (P < 0.05) aerobic stability than untreated, even when subjected to air stress during storage, but the numbers of yeasts culturable on selective agar were not affected. However, the additive reduced the relative abundance (RA) of the lactating-assimilating yeast Candida tropicalis (P < 0.01). In air-stressed silages, untreated silage had a greater (P < 0.05) RA of Pichia kudriavzevii (also a lactate assimilator) than treated silage, whereas treated silage was dominated by Candida humilis, which is usually unable to assimilate lactate or assimilates it slowly. The additive improved the aerobic stability by specifically preventing the dominance of yeast species that can consume lactate and initiate aerobic spoilage. To the best of our knowledge, this is the first work that identifies the specific action of this additive on shifting the microbial communities in corn silage.

Microbiome and Blood Analyte Differences Point to Community and Metabolic Signatures in Lean and Obese Horses.

Due to modern management practices and the availability of energy dense feeds, obesity is a serious and increasingly common health problem for horses. Equine obesity is linked to insulin resistance and exacerbation of inflammatory issues such as osteoarthritis and laminitis. While the gut microbiome is thought to play a part in metabolic status in horses, bacterial communities associated with obesity have yet to be described. Here we report differences in metabolic factors in the blood of obese, normal and lean horses correlated with differences in gut microbiome composition. We report that obese horses had higher levels of leptin, triglycerides, glucose, and cortisol in their blood, and more diverse gut microbiome communities with higher relative abundance of Firmicutes, and lower numbers of Bacteroidetes and Actinobacteria. Network analyses of correlations between body condition, blood analytes, and microbial composition at the genus level revealed a more nuanced picture of microbe-host interactions, pointing to specific bacterial species and assemblages that may be signatures of obesity and leanness in the horse gut. In particular, bacteria groups positively associated with two blood analytes and obesity included Butyrivibrio spp., Prevotellaceae, Blautia spp., two members of Erysipelotrichaceae, and a Lachnospiraceae taxa. These results are an important first step in unraveling the metabolic differences between obese and lean horse gut communities, and designing targeted strategies for microbial intervention.

Comparison of a modified phenol/chloroform and commercial-kit methods for extracting DNA from horse fecal material.

There are many choices for methods of extracting bacterial DNA for Next Generation Sequencing (NGS) from fecal samples. Here, we compare our modifications of a phenol/chloroform extraction method plus an inhibitor removal solution (C3) (ph/Chl+C3) to the PowerFecal® DNA Isolation Kit (MoBio-K). DNA quality and quantity coupled to NGS results were used to assess differences in relative abundance, Shannon diversity index, unique species, and principle coordinate analysis (PCoA) between biological replicates. Six replicate samples, taken from a single ball of horse feces manually collected from the rectum, were subjected to each extraction method. The Ph/Chl+C3 method produced 100× higher DNA yields with less shearing than the MoBio-K method. To assess the methods, the two method samples were sent for sequencing of the bacterial V3-V4 region of 16S rRNA gene using the Illumina MiSeq platform. The relative abundance of Bacteroidetes was greater and there were more unique species assigned to this group in MoBio-K than in Ph/Chl+C3 (P<0.05). In contrast, Firmicutes had greater relative abundance and more unique species in Ph/Chl+C3 extracts than in MoBio-K (P<0.05). The other major bacterial phyla were equally abundant in samples using both extraction methods. Alpha diversity and Shannon Weaver indices showed greater evenness of bacterial distribution in Ph/Chl+C3 compared with MoBio-K (P<0.05), but there was no difference in the OTU richness. Principle coordinate analysis (PCoA) indicated a distinct separation between the two methods (P<0.05) and tighter clustering (less variability) in Ph/Chl+C3 than in MoBio-K. These results suggest that the Ph/Chl+C3 may be preferred for research to identify specific Firmicutes taxa such as Clostridium, and Bacillus. However; MoBio-K may be a better choice for projects focusing on Bacteroidetes abundance. The Ph/Chl+C3 method required less time, but has some safety concerns associated with exposure and disposal of phenol and chloroform. While the MoBio-K may be better choice for researchers with less access to safety equipment like a fume hood.

The complete genome sequence of Clostridium indolis DSM 755(T.).

Clostridium indolis DSM 755(T) is a bacterium commonly found in soils and the feces of birds and mammals. Despite its prevalence, little is known about the ecology or physiology of this species. However, close relatives, C. saccharolyticum and C. hathewayi, have demonstrated interesting metabolic potentials related to plant degradation and human health. The genome of C. indolis DSM 755(T) reveals an abundance of genes in functional groups associated with the transport and utilization of carbohydrates, as well as citrate, lactate, and aromatics. Ecologically relevant gene clusters related to nitrogen fixation and a unique type of bacterial microcompartment, the CoAT BMC, are also detected. Our genome analysis suggests hypotheses to be tested in future culture based work to better understand the physiology of this poorly described species.

An in vitro model of the horse gut microbiome enables identification of lactate-utilizing bacteria that differentially respond to starch induction.

Laminitis is a chronic, crippling disease triggered by the sudden influx of dietary starch. Starch reaches the hindgut resulting in enrichment of lactic acid bacteria, lactate accumulation, and acidification of the gut contents. Bacterial products enter the bloodstream and precipitate systemic inflammation. Hindgut lactate levels are normally low because specific bacterial groups convert lactate to short chain fatty acids. Why this mechanism fails when lactate levels rapidly rise, and why some hindgut communities can recover is unknown. Fecal samples from three adult horses eating identical diets provided bacterial communities for this in vitro study. Triplicate microcosms of fecal slurries were enriched with lactate and/or starch. Metabolic products (short chain fatty acids, headspace gases, and hydrogen sulfide) were measured and microbial community compositions determined using Illumina 16S rRNA sequencing over 12-hour intervals. We report that patterns of change in short chain fatty acid levels and pH in our in vitro system are similar to those seen in in vivo laminitis induction models. Community differences between microcosms with disparate abilities to clear excess lactate suggest profiles conferring resistance of starch-induction conditions. Where lactate levels recover following starch induction conditions, propionate and acetate levels rise correspondingly and taxa related to Megasphaeraelsdenii reach levels exceeding 70% relative abundance. In lactate and control cultures, taxa related to Veillonellamontpellierensis are enriched as lactate levels fall. Understanding these community differences and factors promoting the growth of specific lactate utilizing taxa may be useful to prevent acidosis under starch-induction conditions.