Debunking the Myth of Fusarium poae T-2/HT-2 Toxin Production.

Fusarium poae is commonly detected in field surveys of Fusarium head blight (FHB) of cereal crops and can produce a range of trichothecene mycotoxins. Although experimentally validated reports of F. poae strains producing T-2/HT-2 trichothecenes are rare, F. poae is frequently generalized in the literature as a producer of T-2/HT-2 toxins due to a single study from 2004 in which T-2/HT-2 toxins were detected at low levels from six out of forty-nine F. poae strains examined. To validate/substantiate the observations reported from the 2004 study, the producing strains were acquired and phylogenetically confirmed to be correctly assigned as F. poae; however, no evidence of T-2/HT-2 toxin production was observed from axenic cultures. Moreover, no evidence for a TRI16 ortholog, encoding a key acyltransferase shown to be necessary for T-2 toxin production in other Fusarium species, was observed in any of the de novo assembled genomes of the F. poae strains. Our findings corroborate multiple field-based and in vitro studies on FHB-associated Fusarium populations which also do not support the production of T-2/HT-2 toxins with F. poae and therefore conclude that F. poae should not be generalized as a T-2/HT-2 toxin producing species of Fusarium.

Chromosome-level draft genome sequences of three isolates of the toxigenic fungus Claviceps purpurea showing structural rearrangements.

The whole genomes of three Claviceps purpurea strains were sequenced using Oxford Nanopore Technologies' MinION and assembled into complete, chromosome-level assemblies. The C. purpurea genome consists of eight conserved chromosomes, with evidence of inter-chromosomal structural rearrangements between strains.

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors.

Untargeted metabolomics screening reveals unique secondary metabolite production from Alternaria section Alternaria.

Alternaria section Alternaria is comprised of many species that infect a broad diversity of important crop plants and cause post-harvest spoilage. Alternaria section Alternaria species, such as A. alternata and A. arborescens, are prolific producers of secondary metabolites that act as virulence factors of disease and are mycotoxins that accumulate in infected tissues-metabolites that can vary in their spectrum of production between individuals from the same fungal species. Untargeted metabolomics profiling of secondary metabolite production using mass spectrometry is an effective means to detect phenotypic anomalies in secondary metabolism within a species. Secondary metabolite phenotypes from 36 Alternaria section Alternaria isolates were constructed to observe frequency of production patterns. A clear and unique mass feature pattern was observed for three of the strains that were linked with the production of the dehydrocurvularin family of toxins and associated detoxification products. Examination of corresponding genomes revealed the presence of the dehydrocurvularin biosynthesis gene cluster associated with a sub-telomeric accessory region. A comparison of sequence similarity and occurrences of the dehydrocurvularin biosynthetic gene cluster within Pleosporalean fungi is presented and discussed.

Untargeted Metabolomic Profiling of Fungal Species Populations.

This chapter describes protocols for the development of consensus chemical phenotypes or "metabolomes" of fungal populations using ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS). Isolates are cultured using multiple media conditions to elicit the expression of diverse secondary metabolite biosynthetic gene clusters. The mycelium and spent culture media are extracted using organic solvents and profiled by ultra-high pressure chromatography coupled with a high resolution Thermo Orbitrap XL mass spectrometer with the ability to trap and fragment ions to general MS2 spectra. MS data preprocessing is explained and illustrated using the freely available software MZMine 2. Through data processing, binary matrices of mass features can be generated and then combined into a consensus secondary metabolite phenotype of all isolates grown in all media conditions. The production of consensus chemical phenotypes is useful for screening large fungal populations (both inter and intra-species populations) for isolates potentially expressing novel secondary metabolites or analogs of known secondary metabolites.

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Claviceps purpurea Sclerotia.

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.

A metabolomic study of vegetative incompatibility in Cryphonectria parasitica.

Vegetative incompatibility (VI) is a form of non-self allorecognition in filamentous fungi that restricts conspecific hyphal fusion and the formation of heterokaryons. In the chestnut pathogenic fungus, Cryphonectria parasitica, VI is controlled by six vic loci and has been of particular interest because it impedes the spread of hypoviruses and thus biocontrol strategies. We use nuclear magnetic resonance and high-resolution mass spectrometry to characterize alterations in the metabolome of C. parasitica over an eight-day time course of vic3 incompatibility. Our findings support transcriptomic data that indicated remodeling of secondary metabolite profiles occurs during vic3 -associated VI. VI-associated secondary metabolites include novel forms of calbistrin, decumbenone B, a sulfoxygenated farnesyl S-cysteine analog, lysophosphatidylcholines, and an as-yet unidentified group of lipid disaccharides. The farnesyl S-cysteine analog is structurally similar to pheromones predicted to be produced during VI and is here named 'crypheromonin'. Mass features associated with C. parasitica secondary metabolites skyrin, rugulosin and cryphonectric acid were also detected but were not VI specific. Partitioning of VI-associated secondary metabolites was observed, with crypheromonins and most calbistrins accumulating in the growth medium over time, whereas lysophosphatidylcholines, lipid disaccharide-associated mass features and other calbistrin-associated mass features peaked at distinct time points in the mycelium. Secondary metabolite biosynthetic gene clusters and potential biological roles associated with the detected secondary metabolites are discussed.

Apicidin biosynthesis is linked to accessory chromosomes in Fusarium poae isolates.

BACKGROUND: Fusarium head blight is a disease of global concern that reduces crop yields and renders grains unfit for consumption due to mycotoxin contamination. Fusarium poae is frequently associated with cereal crops showing symptoms of Fusarium head blight. While previous studies have shown F. poae isolates produce a range of known mycotoxins, including type A and B trichothecenes, fusarins and beauvericin, genomic analysis suggests that this species may have lineage-specific accessory chromosomes with secondary metabolite biosynthetic gene clusters awaiting description. METHODS: We examined the biosynthetic potential of 38 F. poae isolates from Eastern Canada using a combination of long-read and short-read genome sequencing and untargeted, high resolution mass spectrometry metabolome analysis of extracts from isolates cultured in multiple media conditions. RESULTS: A high-quality assembly of isolate DAOMC 252244 (Fp157) contained four core chromosomes as well as seven additional contigs with traits associated with accessory chromosomes. One of the predicted accessory contigs harbours a functional biosynthetic gene cluster containing homologs of all genes associated with the production of apicidins. Metabolomic and genomic analyses confirm apicidins are produced in 4 of the 38 isolates investigated and genomic PCR screening detected the apicidin synthetase gene APS1 in approximately 7% of Eastern Canadian isolates surveyed. CONCLUSIONS: Apicidin biosynthesis is linked to isolate-specific putative accessory chromosomes in F. poae. The data produced here are an important resource for furthering our understanding of accessory chromosome evolution and the biosynthetic potential of F. poae.

Accessory Chromosome-Acquired Secondary Metabolism in Plant Pathogenic Fungi: The Evolution of Biotrophs Into Host-Specific Pathogens.

Accessory chromosomes are strain- or pathotype-specific chromosomes that exist in addition to the core chromosomes of a species and are generally not considered essential to the survival of the organism. Among pathogenic fungal species, accessory chromosomes harbor pathogenicity or virulence factor genes, several of which are known to encode for secondary metabolites that are involved in plant tissue invasion. Accessory chromosomes are of particular interest due to their capacity for horizontal transfer between strains and their dynamic "crosstalk" with core chromosomes. This review focuses exclusively on secondary metabolism (including mycotoxin biosynthesis) associated with accessory chromosomes in filamentous fungi and the role accessory chromosomes play in the evolution of secondary metabolite gene clusters. Untargeted metabolomics profiling in conjunction with genome sequencing provides an effective means of linking secondary metabolite products with their respective biosynthetic gene clusters that reside on accessory chromosomes. While the majority of literature describing accessory chromosome-associated toxin biosynthesis comes from studies of Alternaria pathotypes, the recent discovery of accessory chromosome-associated biosynthetic genes in Fusarium species offer fresh insights into the evolution of biosynthetic enzymes such as non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs) and regulatory mechanisms governing their expression.

Transcriptome analysis implicates secondary metabolite production, redox reactions, and programmed cell death during allorecognition in Cryphonectria parasitica.

The underlying molecular mechanisms of programmed cell death associated with fungal allorecognition, a form of innate immunity, remain largely unknown. In this study, transcriptome analysis was used to infer mechanisms activated during barrage formation in vic3-incompatible strains of Cryphonectria parasitica, the chestnut blight fungus. Pronounced differential expression occurred in barraging strains of genes involved in mating pheromone (mf2-1, mf2-2), secondary metabolite production, detoxification (including oxidative stress), apoptosis-related, RNA interference, and HET-domain genes. Evidence for secondary metabolite production and reactive oxygen species (ROS) accumulation is supported through UPLC-HRMS analysis and cytological staining, respectively. Differential expression of mating-related genes and HET-domain genes was further examined by RT-qPCR of incompatible interactions involving each of the six vegetative incompatibility (vic) loci in C. parasitica and revealed distinct recognition process networks. We infer that vegetative incompatibility in C. parasitica activates defence reactions that involve secondary metabolism, resulting in increased toxicity of the extra- and intracellular environment. Accumulation of ROS (and other potential toxins) may result in detoxification failure and activation of apoptosis, sporulation, and the expression of associated pheromone genes. The incompatible reaction leaves abundant traces of a process-specific metabolome as conidiation is initiated.

The effect of curve running on distal limb kinematics in the Thoroughbred racehorse.

During racing, injury is more likely to occur on a bend than on a straight segment of track. This study aimed to quantify the effects of galloping at training speeds on large radius curves on stride parameters and limb lean angle in order to assess estimated consequences for limb loading. Seven Thoroughbred horses were equipped with a sacrum-mounted inertial measurement unit with an integrated GPS, two hoof-mounted accelerometers and retro-reflective markers on the forelimbs. Horses galloped 2-4 circuits anticlockwise around an oval track and were filmed at 120 frames per second using an array of ten cameras. Speed and curve radius were derived from GPS data and used to estimate the centripetal acceleration necessary to navigate the curve. Stride, stance and swing durations and duty factor (DF) were derived from accelerometer data. Limb markers were tracked and whole limb and third metacarpus (MCIII) angles were calculated. Data were analysed using mixed effects models with a significance level of p < 0.05. For horses galloping on the correct lead, DF was higher for the inside (lead) leg on the straight and on the curve. For horses galloping on the incorrect lead, there was no difference in DF between inside and outside legs on the straight or on the curve. DF decreased by 0.61% of DF with each 1 m s-2 increase in centripetal acceleration (p < 0.001). Whole limb inclination angle increased by 1.5° per 1 m s-1 increase in speed (p = 0.002). Limb lean angles increase as predicted, and lead limb function mirrors the functional requirements for curve running. A more comprehensive understanding of the effects of lean and torque on the distal limb is required to understand injury mechanisms.

Water-jet-assisted liposuction for the treatment of lipedema: Standardized treatment protocol and results of 63 patients.

BACKGROUND: Lipedema is a condition of painful increase in subcutaneous fat affecting almost exclusively women. Several studies have examined the effectiveness of liposuction in the treatment of lipedema, but none has focused on water-jet-assisted liposuction technique. METHODS: A standardized treatment protocol for liposuction in lipedema, which was established over the course of 15 years, is presented. Patients received questionnaires preoperatively and after operative treatment assessing characteristics and symptom severity on visual analog scales in a prospective manner. RESULTS: Pre- and postoperative questionnaires were available for 63 patients. Median age was 35 years and mean (body mass index) BMI 28.4 ± 0.6, all patients had stages I or II lipedema diagnosed by two separate specialists. After a mean follow-up of 22 months after operative treatment, all assessed symptom had decreased significantly in severity. All patients wore compression garments and/or received manual lymphatic drainage preoperatively; this could be reduced to only 44% of patients needing any conservative treatment postoperatively. CONCLUSION: Liposuction in water-jet-assisted technique using the presented treatment protocol is an efficient method of operative treatment of early-stage lipedema patients leading to a marked decrease in symptom severity and need for conservative treatment.

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall-degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

Enniatin Production Influences Fusarium avenaceum Virulence on Potato Tubers, but not on Durum Wheat or Peas.

Fusarium avenaceum is a generalist pathogen responsible for diseases in numerous crop species. The fungus produces a series of mycotoxins including the cyclohexadepsipeptide enniatins. Mycotoxins can be pathogenicity and virulence factors in various plant-pathogen interactions, and enniatins have been shown to influence aggressiveness on potato tubers. To determine the role of these mycotoxins in other F. avenaceum-host interactions, enniatin synthase 1 (ESYN1) disruption and overexpression mutants were generated and their ability to infect wheat and peas investigated. As a preliminary study, the transformants were screened for their ability to cause potato tuber necrosis and, consistent with a previous report, enniatin production increased necrotic lesion size on the tubers. By contrast, when the same mutants were assessed in their ability to cause disease in pea roots or durum wheat spikes, no changes in disease symptoms or virulence were observed. While it is known that, at least in the case of wheat, exogenously applied enniatins can cause tissue necrosis, this group of mycotoxins does not appear to be a key factor on its own in disease development on peas or durum wheat.

Assessment of clinical and computed tomographic findings for association with the outcome of intraoral cheek tooth extraction in horses and ponies.

OBJECTIVE: To describe clinical and CT findings for horses and ponies undergoing intraoral cheek tooth extraction and assess potential associations between these features and outcome of the procedure. ANIMALS: 74 horses and 7 ponies. PROCEDURES: Medical records were searched to identify horses and ponies that underwent CT and intraoral extraction of ≥ 1 cheek tooth with standing sedation. Signalment and clinical variables were recorded, and CT scans were reviewed. Anatomic location and measurements of affected teeth; abnormalities of the periodontium, pulp, infundibula, roots, and tooth shape; fracture presence and type; presence of sinusitis; and affected sinus cavities were assessed by a surgeon and a radiologist. Intraoral extraction outcome was recorded as successful (complete removal of the tooth in 1 intraoral extraction procedure) or unsuccessful. Univariable and multivariable logistic regression analyses were performed to assess associations between clinical or CT findings and outcome. RESULTS: 89 cheek teeth (80 maxillary and 9 mandibular) were included in the analyses. Sixty of 89 (67%) cheek teeth were extracted successfully (56/80 [70%] maxillary and 4/9 [44%] mandibular cheek teeth). Only presence of a simple fracture (vs no fracture) was associated with outcome on multivariable regression analysis; odds of successful intraoral extraction were significantly lower when this feature was present. CONCLUSIONS AND CLINICAL RELEVANCE: Most extractions of cheek teeth in the study sample were successful, and results may be useful for practitioners in refining cheek tooth extraction plans for horses and ponies. Further studies are required to assess whether specific CT findings can be used to predict the outcome of intraoral extraction in equids.

The Effect of Training on Stride Duration in a Cohort of Two-Year-Old and Three-Year-Old Thoroughbred Racehorses.

Objective gait monitoring is increasingly accessible to trainers. A more comprehensive understanding of 'normal' gait adaptations is required. Forty two-year-old thoroughbred racehorses were recruited when entering training and followed for 22 months. Gait analysis was performed by equipping each horse with an inertial measurement unit with inbuilt GPS (GPS-IMU) mounted on the dorsum. Horses were exercised as per their regular training regimen. Data were analysed using a linear mixed model. For two-year-old horses, there was a non-linear pattern of stride duration (SD) over time (p < 0.001) with SD decreasing initially and then 'flattening off' over time (linear and quadratic coefficients -0.29 ms/week and 0.006 ms/week2). Horses showed an increase in SD of 2.21 ms (p < 0.001) per 100 m galloped, and over time, SD decreased by 0.04 ms (p < 0.001) with each 100 m galloped per week. Three-year-old horses overall showed no change in SD over time (p = 0.52), but those that had a period of time off showed a decrease in SD of -0.59 ms per week (p = 0.02). They showed an increase in SD of 1.99 ms (p < 0.001) per 100 m galloped, and horses that had a period of time off showed an increase in stride duration of 1.05 ms per 100 m galloped (p = 0.01) compared to horses which did not have time off. Horses demonstrate an adaptation to high-speed exercise over time. SD decreases with training when other factors are controlled for in naïve horses.

Maxillary nerve blocks in horses: an experimental comparison of surface landmark and ultrasound-guided techniques.

OBJECTIVE: The aim of this preliminary proof-of-concept study was to evaluate and compare the success and complication rate of infiltration of the maxillary nerve of cadaver heads using previously described surface landmarks, standard ultrasound and a novel needle guidance positioning ultrasound system (SonixGPS). STUDY DESIGN: Prospective, anatomical, method-comparison study. ANIMALS: Thirty-eight equine cadaver heads. METHODS: Twenty-six veterinary students performed the three methods consecutively on cadaver heads using an 18 gauge, 8.9 cm spinal needle and 0.5 mL iodinated contrast medium. Computed tomography was used to quantify success (deposition of contrast in contact with the maxillary nerve) and complication rate (contrast identified within surrounding vasculature or periorbital structures) associated with each method. RESULTS: Perineural injection of the maxillary nerve was attempted 76 times, with an overall success rate of 65.8% (50/76) and complication rate of 53.9% (41/76). Success rates were 50% (13/26) with surface landmark, 65.4% (17/26) with standard ultrasound guidance and 83.3% (20/24) with SonixGPS guidance approaches (Fisher's exact test, p=0.046). No significant difference in complication rate was found between the three methods. CONCLUSIONS: Ultrasound-guided maxillary nerve blocks were significantly more successful than surface landmark approaches when performed by inexperienced operators, and the highest success rate was achieved with guidance positioning system (GPS) needle guidance. CLINICAL RELEVANCE: Local anaesthesia of the equine maxillary nerve in the fossa pterygopalatina is frequently used for diagnostic and surgical procedures in the standing sedated horse. Due to vague superficial landmarks with various approaches and the need for experience via ultrasound guidance, this block remains challenging. GPS guidance may improve reliability of maxillary and other nerve blocks, and allow a smaller volume of local anaesthetic solution to be used, thereby improving specificity and reducing the potential for side effects.

Imaging quasiperiodic electronic states in a synthetic Penrose tiling.

Quasicrystals possess long-range order but lack the translational symmetry of crystalline solids. In solid state physics, periodicity is one of the fundamental properties that prescribes the electronic band structure in crystals. In the absence of periodicity and the presence of quasicrystalline order, the ways that electronic states change remain a mystery. Scanning tunnelling microscopy and atomic manipulation can be used to assemble a two-dimensional quasicrystalline structure mapped upon the Penrose tiling. Here, carbon monoxide molecules are arranged on the surface of Cu(111) one at a time to form the potential landscape that mimics the ionic potential of atoms in natural materials by constraining the electrons in the two-dimensional surface state of Cu(111). The real-space images reveal the presence of the quasiperiodic order in the electronic wave functions and the Fourier analysis of our results links the energy of the resonant states to the local vertex structure of the quasicrystal.

Contribution of industry funded post-marketing studies to drug safety: survey of notifications submitted to regulatory agencies.

OBJECTIVES:  To investigate the practice of post-marketing studies in Germany during a three year period and to evaluate whether these trials meet the aims specified in the German Medicinal Products Act. DESIGN:  Survey of notifications submitted to German regulatory agencies before post-marketing studies were carried out, 2008-10. SETTING:  Notifications obtained through freedom of information requests to the three authorities responsible for registering post-marketing studies in Germany. MAIN OUTCOME MEASURES:  Descriptive statistics of post-marketing studies, including the products under study, intended number of patients, intended number of participating physicians, proposed remunerations, study plan and protocol, and availability of associated scientific publications and reports on adverse drug reactions. RESULTS:  Information was obtained from 558 studies, with a median of 600 (mean 2331, range 2-75 000) patients and 63 (270, 0-7000) participating physicians per study. The median remuneration to physicians per patient was €200 (€441, €0-€7280) (£170, £0-£6200; $215, $0-$7820), with a total remuneration cost of more than €217m for 558 studies registered over the three year period. The median remuneration per participating physician per study was €2000 (mean €19 424), ranging from €0 to €2 080 000. There was a broad range of drugs and non-drug products, of which only a third represented recently approved drugs. In many notifications, data, information, and results were, by contract, strictly confidential and the sole property of the respective sponsor. No single adverse drug reaction report could be identified from any of the 558 post-marketing studies. Less than 1% of studies could be verified as published in scientific journals. CONCLUSIONS:  Post-marketing studies are not improving drug safety surveillance. Sample sizes are generally too small to allow the detection of rare adverse drug reactions, and many participating physicians are strictly obliged to maintain confidentiality towards the sponsor. High remuneration and strict confidentiality clauses in these studies could influence the physicians' reporting behaviours of adverse drug reactions.

IMAGING DIAGNOSIS-IDIOPATHIC HYOID MALFORMATION IN AN 8-YEAR-OLD WELSH SECTION D HORSE.

An 8-year-old gelding was evaluated for intermittent dorsal displacement of the soft palate (DDSP) at exercise. Standing laterolateral radiographs demonstrated an abnormal crossed appearance of the stylohyoid bones. Computed tomography (CT) confirmed abnormal positioning of the left stylohyoid bone and articulations with both the left thyrohyoid and ceratohyoid bones. Surgical treatment consisted of combined left ceratohyoidectomy and thyrohyoidectomy. Postoperatively the patient's presenting complaint resolved completely, with the horse returning to full work. Standing laterolateral radiographs should be obtained prior to laryngeal or hyoid surgery to rule out hyoid anomalies, which could result in surgical failure or subsequent hyoid fracture.