Quinidine has long been used for the pharmacological treatment of atrial fibrillation (AF) in horses; however, the plasma concentration required for conversion to sinus rhythm remains unclear. Dominant frequency (DF), derived from surface electrocardiograms, reflects atrial activation rate during AF. This study aimed to quantify the relationship between plasma quinidine concentration and DF using a pharmacokinetic/pharmacodynamic (PK/PD) approach. Ten Thoroughbred horses with naturally occurring AF received quinidine sulfate via a nasogastric tube. Plasma quinidine concentrations and DF values, calculated from digitized atrial fibrillatory segments, were measured during treatment. PK/PD was analyzed using a sigmoid inhibitory Emax model, with typical parameters including a baseline DF of 6.27 Hz, a maximal DF reduction of 3.63 Hz, a theoretical minimum DF of 2.64 Hz, an EC50 of 0.78 μg/mL, and a Hill coefficient of 1.81. Nine horses converted to sinus rhythm, with median DF decreasing from 6.4 Hz to 3.0 Hz immediately before conversion. Model simulations indicated a pharmacodynamic plateau, with an increase in plasma quinidine concentration from 4 to 5 μg/mL resulting in only a 0.06-Hz reduction in DF. A model-predicted plasma quinidine concentration of 2.6 μg/mL achieved a DF of 3.0 Hz and may represent a target concentration for conversion.
Gray is a dominant coat color phenotype in horses caused by a 4.6 kb tandem triplication within intron 6 of syntaxin 17 (STX17). The copy number variation (CNV) of the duplicated segment influences the graying rate. The rare G2 allele (CNV = 2) is associated with a slower graying rate compared to the common G3 allele (CNV = 3) and is also relevant to melanoma risk. Current assays are limited because long and accurate PCR (LA-PCR) detects only the presence or absence of duplications, while droplet digital PCR (ddPCR) cannot reliably distinguish certain genotypes such as G3/g and G2/G2. We constructed a stepwise workflow combining (i) multiplex real-time PCR targeting the duplication junction for rapid gray/non-gray screening, (ii) ddPCR for copy number estimation, and (iii) LA-PCR for confirmatory genotyping of ambiguous copy-number classes. Using real-time PCR, we screened 4596 Japanese Thoroughbreds aged 2–7 years, of which 4374 were classified as non-gray and 222 as gray. Based on age and coat appearance, 23 Gy candidates were prioritized for slow-gray evaluation and analyzed by ddPCR; one was classified as G2/g, and 22 as G3/g or G2/G2. LA-PCR detected a g-derived band in all 22 cases, confirming that they were G3/g. Pedigree analysis suggested that the G2 allele was transmitted through the maternal line and that this lineage was distinct from the previously reported Japanese slow-gray family. This workflow enables practical molecular discrimination among non-gray, common gray, and slow-gray genotypes, supporting the surveillance of rare G2 alleles in the Japanese Thoroughbred population.
Equine herpesvirus myeloencephalopathy (EHM), caused by equine herpesvirus type 1 (EHV-1), poses a major threat to the equine industry because of its devastating impact on animal welfare and athletic performance. A single-nucleotide polymorphism (SNP), G2254, in ORF30 (UL30), which encodes the viral DNA polymerase, has been used as a marker of neuropathogenic EHV-1 strains, although its contribution to EHM pathogenesis remains controversial. In addition, other ORF30 SNPs have been reported in association with EHM, but their functional significance is unclear. Clinical observations indicate that fever and high levels of viremia are closely associated with EHM onset. Here, we investigated EHV-1 replication under elevated-temperature conditions that mimic febrile states using a fetal horse kidney cell line, equine peripheral blood mononuclear cells (PBMCs), and equine vascular endothelial cells (EVECs). EHV-1 isolates derived from horses with EHM consistently retained the ability to replicate at elevated temperature in permissive cells and remained competent for infection despite restricted replication in PBMCs, whereas replication of many non-EHM-derived isolates was suppressed. Specific ORF30 SNPs were associated with replication at elevated temperatures, and a molecular epidemiological association analysis of these SNPs suggested an association with EHM. Together, these findings suggest that replication at febrile temperature is a viral property relevant to EHV-1 pathogenesis and EHM risk. IMPORTANCE:Equine herpesvirus myeloencephalopathy (EHM), caused by equine herpesvirus type 1 (EHV-1), represents a serious threat to the equine industry. Here, we show that EHV-1 field isolates and recombinant viruses harboring EHM-associated UL30 variants retain replication capacity at elevated temperatures in fetal horse kidney cells and equine vascular endothelial cells, and that peripheral blood mononuclear cells (PBMCs) infected with EHM-associated viruses can mediate cell-to-cell transfer under these conditions. In contrast, replication of many non-EHM-derived isolates and recombinant viruses in these cells, as well as PBMC-mediated cell-to-cell transfer of EHV-1, is suppressed at elevated temperatures. Notably, we show that single nucleotide polymorphism (SNPs) in ORF30, which encodes the viral DNA polymerase UL30, and which have been implicated in EHM, are linked to the ability of EHV-1 to replicate at elevated temperatures. These findings suggest that replication at febrile temperature is a viral property relevant to EHV-1 pathogenesis and EHM risk.
Synephrine is a sympathomimetic alkaloid of regulatory interest in both horse racing and equestrian sports, where feed‑related exposure may lead to its detection in equine blood and urine. This study characterized plasma and urinary concentrations of synephrine in horses following controlled oral administration. Six Thoroughbred horses received single oral doses of 100 mg or 800 mg synephrine, and blood and urine samples were collected sequentially. Each sample was analyzed both before and after β‑glucuronidase hydrolysis, demonstrating that synephrine was present predominantly as conjugated metabolites in both plasma and urine, and that hydrolysis substantially increased measurable concentrations. Non‑compartmental analysis was used to determine selected pharmacokinetic parameters, including half‑life, Cmax, Tmax, and AUC. These pharmacokinetic results indicated that synephrine was rapidly absorbed, with dose‑related increases in both systemic and urinary exposure. The findings provide initial quantitative information on circulating and urinary synephrine following defined oral exposure in horses. The concentration ranges and detection patterns described here may assist in interpreting field detections and support future studies aimed at understanding exposure arising from feed‑borne sources.
Medetomidine, an α₂-adrenergic agonist, is widely used as a sedative in horses. While its pharmacological effects are established, limited data exist on elimination of its metabolites, 3'-hydroxy medetomidine (HMD) and 3'-carboxy medetomidine (CMD), which are of regulatory interest. HMD is currently targeted in plasma and urine under International Screening Limits (ISLs) established by the International Federation of Horseracing Authorities (IFHA). In this study, seven Thoroughbreds received 6.3 μg/kg of medetomidine intravenously. Blood and urine were collected for 96 h and analyzed by LC-MS/MS after solid-phase extraction with and without enzymatic hydrolysis. Parent drug was detectable in plasma up to 4 h but absent in urine. HMD was quantifiable in plasma to 48 h and urine to 72 h, while CMD, with a distinct profile, was measurable in plasma to 32 h and urine to 72 h. Based on the ISLs, the detection times (DTs) for both plasma and urine were 48 h. Pharmacokinetic modeling estimated clearance at 2.15 L/h/kg and steady-state distribution volume at 1.90 L/kg. These findings show rapid clearance of medetomidine and extended metabolite excretion. The DT of HMD aligns with loss of pharmacological effect, supporting current ISLs and reinforcing the rationale for targeting HMD, with CMD providing supplementary regulatory insight.
