Karen J. McDowell

A simple and highly sensitive spectrophotometric method for the determination of cyanide in equine blood.

An epidemiological association among black cherry trees (Prunus serotina), eastern tent caterpillars (Malacosoma americana), and the spring 2001 episode of mare reproductive loss syndrome in central Kentucky focused attention on the potential role of environmental cyanogens in the causes of this syndrome. To evaluate the role of cyanide (CN −) in this syndrome, a simple, rapid, and highly sensitive method for determination of low parts per billion concentrations of CN − in equine blood and other biological fluids was developed. The analytical method is an adaptation of methods commonly in use and involves the evolution and trapping of gaseous hydrogen cyanide followed by spectrophotometric determination by autoanalyzer. The limit of quantitation of this method is 2 ng/mL in equine blood, and the standard curve shows a linear relationship between CN − concentration and absorbance (r >. 99). The method throughput is high, up to 100 samples per day. Normal blood CN − concentrations in horses at pasture in Kentucky in October 2001 ranged from 3-18 ng/mL, whereas hay-fed horses showed blood CN − levels of 2-7 ng/mL in January 2002. Blood samples from a small number of cattle at pasture showed broadly similar blood CN − concentrations. Intravenous administration of sodium cyanide and oral administration of mandelonitrile and amygdalin yielded readily detectable increases in blood CN − concentrations. This method is sufficiently sensitive and specific to allow the determination of normal blood CN − levels in horses, as well as the seasonal and pasture-dependent variations. The method should also be suitable for investigation of the toxicokinetics and disposition of subacutely toxic doses of CN − and its precursor cyanogens in the horse as well as in other species.

Invited review: the role of caterpillars in mare reproductive loss syndrome: a model for environmental causes of abortion.

A new abortigenic disease, now known as mare reproductive loss syndrome (MRLS), significantly affected the horse industry in the Ohio River Valley of the United States in late April and early May of 2001 and 2002. In 2001, approximately 25% of all pregnant mares aborted within several weeks (over 3,000 mares lost pregnancies), and abortion rates exceeded 60% on some farms. Mare reproductive loss syndrome struck hard and without warning, it was caused by something in the environment, it was not transmitted between animals, and it was not associated with any known abortigenic agent or disease. These experiments demonstrated that horses will inadvertently consume Eastern tent caterpillars (ETC) when the insects are present in the pasture or other feedstuffs, and MRLS-type abortions were induced in experimental animals (mares and pigs) by mixing ETC with the feed of the animals. Eastern tent caterpillars are hirsute (hairy) caterpillars, and the only part of the caterpillar that caused MRLS abortions was the cuticle. The experiments revealed that the setae (hairs) embed into the submucosa of the alimentary tract creating microgranulomatous lesions. It is hypothesized that the alimentary tract lesions allow bacteria from the alimentary tract of the mare, principally streptococci, actinobacilli, and to a lesser extent enterococci, to invade the circulatory system of the mare. The bacteria then establish infections in tissues where the immune surveillance of the mare is reduced, such as the fetus and placenta. Fetal and placental fluid bacterial infections lead to fetal death and abortion characteristic of MRLS. Inadvertent ingestion of ETC by pregnant mares causes MRLS. Currently the only known means to prevent MRLS is to avoid exposure of horses, particularly pregnant mares, to ETC and probably most hirsute caterpillars.

Vasoconstriction in horses caused by endophyte-infected tall fescue seed is detected with Doppler ultrasonography.

The hypotheses that endophyte (Neotyphodium coenophialum)-infected tall fescue (TF) seed causes vasoconstriction in horses in vivo and that ground seed would cause more pronounced vasoconstriction than whole seed were tested. Ten horses each received 1 of 3 treatments: endophyte-free ground (E-G; n = 4 horses) seed, endophyte-positive whole (E+W; n = 3) seed, or endophyte-positive ground (E+G; n = 3) seed. There were two 14-d periods, P1 and P2. During P1, animals were adapted to a concentrate (0.2% BW, as fed, twice daily) and alfalfa cubes. During P2, the seed was mixed into the concentrate portion of the diet and alfalfa cubes were offered ad libitum. Fescue seed was fed in increasing amounts ranging from 0.02% BW on d 1 (averaging 76 ug/kg ergovaline + ergovalinine) to 0.22% BW on d 11 to 14 (averaging 713 ug/kg ergovaline + ergovalinine). The distal palmar artery of the left foreleg of each horse was scanned via Doppler ultrasonography for 4 d during each period, with 5 replicate scans performed on each scanning day. The measurements taken at each scan included artery luminal diameter, area, and circumference, peak systolic velocity, end diastolic velocity and blood flow variables. Animal temperature, heart rate, and respiration rate and ambient temperature and humidity were also recorded. Blood samples were taken on each scanning day to measure inflammatory cytokine mRNA abundances, and blood samples were collected on d 0, 4, 8, and 14 of P2 to measure prolactin concentrations. Consumption of E+G TF seed caused decreased artery lumen diameter (P = 0.0033), area (P = 0.0406), and circumference (P = 0.0480) compared with E-G seed, and E+W seed produced an intermediate response. Blood flow volume was reduced (P < 0.05) during P2 in horses receiving E+G seed compared with horses receiving E-G seed. Other ultrasound variables were not different (P > 0.05) among treatment groups, and neither were cytokine mRNA or prolactin concentrations. Treatment did not alter (P > 0.05) animal temperature, heart rate, or respiration rate, and neither ambient temperature nor relative humidity was consistently correlated with any response variable measured. Taken together, these data confirm that consumption of E+G fescue seed caused vasoconstriction in horses, which could be readily measured by Doppler ultrasonography. Use of Doppler ultrasound to monitor the diameter of the palmar artery of horses grazing endophyte-infected (E+) fescue pastures may provide a convenient and satisfactory biomarker to determine premonitory signs of fescue toxicosis.

CHARACTERISATION OF PROTEINS IN THE SEMINAL PLASMA OF STALLIONS, GELDINGS AND GELDINGS SUPPLEMENTED WITH TESTOSTERONE

Abstract The major proteins in stallion seminal plasma were characterised by two-dimensional polyacrylamide gel electrophoresis, and compared with the patterns of proteins in normal geldings (castrated males) and geldings supplemented with testosterone. The major proteins or groups of proteins identified according to their approximate relative molecular weight in kilodaltons (kDa) and apparent isoelectric point (pl) were: 1) 60 kDa, pl 7; 2) 23 kDa, pl 4–5; 3) 25–30 kDa, pl 5·5–6; 4) 23 kDa, pl 7–8; and 5) 15–20 kDa, pl 6–7·5. Protein groups 1 and 2 were more prominent in the seminal plasma from the stallions and supplemented geldings than that from the unsupplemented geldings, while protein groups 3, 4 and 5 were more prominent in the seminal plasma from the unsupplemented geldings.

The Toxicokinetics of Cyanide and Mandelonitrile in the Horse and Their Relevance to the Mare Reproductive Loss Syndrome

The epidemiological association between black cherry trees and mare reproductive loss syndrome has focused attention on cyanide and environmental cyanogens. This article describes the toxicokinetics of cyanide in horses and the relationships between blood cyanide concentrations and potentially adverse responses to cyanide. To identify safe and humane blood concentration limits for cyanide experiments, mares were infused with increasing doses (1-12 mg/min) of sodium cyanide for 1 h. Infusion at 12 mg/min produced clinical signs of cyanide toxicity at 38 min; these signs included increased heart rate, weakness, lack of coordination, loss of muscle tone, and respiratory and behavioral distress. Peak blood cyanide concentrations were about 2500 ng/mL; the clinical and biochemical signs of distress reversed when infusion stopped. Four horses were infused with 1 mg/min of sodium cyanide for 1 h to evaluate the distribution and elimination kinetics of cyanide. Blood cyanide concentrations peaked at 1160 ng/mL and then declined rapidly, suggesting a two-compartment, open model. The distribution (alpha) phase half-life was 0.74 h, the terminal (beta phase) half-life was 16.16 h. The mean residence time was 12.4 h, the steady-state volume of distribution was 2.21 L/kg, and the mean systemic clearance was 0.182 L/h/kg. Partitioning studies showed that blood cyanide was about 98.5% associated with the red cell fraction. No clinical signs of cyanide intoxication or distress were observed during these infusion experiments. Mandelonitrile was next administered orally at 3 mg/kg to four horses. Cyanide was rapidly available from the orally administered mandelonitrile and the C max blood concentration of 1857 ng/mL was observed at 3 min after dosing; thereafter, blood cyanide again declined rapidly, reaching 100 ng/mL by 4 h postadministration. The mean oral bioavailability of cyanide from mandelonitrile was 57% ± 6.5 (SEM), and its apparent terminal half-life was 13 h ± 3 (SEM). No clinical signs of cyanide intoxication or distress were observed during these experiments. These data show that during acute exposure to higher doses of cyanide (~600 mg/horse; 2500 ng/mL of cyanide in blood), redistribution of cyanide rapidly terminated the acute toxic responses. Similarly, mandelonitrile rapidly delivered its cyanide content, and acute cyanide intoxications following mandelonitrile administration can also be terminated by redistribution. Rapid termination of cyanide intoxication by redistribution is consistent with and explains many of the clinical and biochemical characteristics of acute, high-dose cyanide toxicity. On the other hand, at lower concentrations (<100 ng/mL in blood), metabolic transformation of cyanide is likely the dominant mechanism of termination of action. This process is slow, with terminal half-lives ranging from 12-16 hours. The large volume of distribution and the long terminal-phase-elimination half-life of cyanide suggest different mechanisms for toxicities and termination of toxicities associated with low-level exposure to cyanide. If environmental exposure to cyanide is a factor in the cause of MRLS, then it is likely in the more subtle effects of low concentrations of cyanide on specific metabolic processes that the associations will be found.

Identification and initial characterization of calcyclin and phospholipase A2 in equine conceptuses

For development to proceed normally, the appropriate genes must be expressed in the correct tissues and in the correct time frame. Knowledge of gene expression during development provides information about the changes taking place within the conceptus as well as possible reasons for pregnancy failure. However, little is known about gene expression during development in the equine conceptus. In this study, we examined differences in gene expression between day 12 and day 15 equine conceptuses by suppression subtractive hybridization. This technique was used to isolate transcripts that are more abundantly expressed in day 15 conceptuses compared to day 12 conceptuses. Between day 12 and 15 of pregnancy in horses, maternal recognition of pregnancy occurs, gastrulation is taking place, and mesoderm is beginning to form.

Attempts to induce nocardioform placentitis (Crossiela equi) experimentally in mares

REASONS FOR PERFORMING STUDY Nocardioform placentitis in horses is poorly understood, and the development of an experimental model would be of help in understanding the pathogenesis of the disease. OBJECTIVES To investigate whether (1) intrauterine inoculation of Crossiela equi during the periovulatory period or (2) i.v., oral or intranasopharyngeal inoculation of C. equi during midgestation would result in nocardioform placentitis, and (3) before and after mating endometrial swabs present evidence of nocardioform placentitis-associated organisms (C. equi or Amycolatopsis spp.). METHODS In Study I, mares (n = 20) received an intrauterine inoculation of C. equi 24 h after artificial insemination. Endometrial swabs were obtained 24 h post inoculation for PCR analysis. In Study II, pregnant mares (at 180-240 days of gestation) were inoculated with C. equi by intranasopharyngeal (n = 5), oral (n = 4) or i.v. (n = 4) routes. Sixty contemporaneous pregnant mares maintained on the same farm served as control animals. In Study III, privately owned Thoroughbred mares (n = 200) had endometrial swabs collected before and within 24-48 h after mating for detection of nocardioform microorganisms. RESULTS In Study I, C.equi was identified by PCR in 3 of 20 mares following intrauterine inoculation. Pregnancy was established in 19 of 20 treated mares. There were 2 embryonic losses and one abortion at 177 days of gestation (undetermined cause). Sixteen mares delivered a normal foal and placenta. In Study II, one mare (oral inoculation) aborted at 200 days of gestation (unidentified cause). The remaining mares delivered a normal foal and placenta. In Study III, none of the mares yielded positive endometrial PCR for nocardioform microorganisms. CONCLUSIONS We were unable to induce nocardioform placentitis, and there was no evidence of nocardioform microorganisms in endometrial swabs of broodmares before or after mating. These findings suggest that nocardioform placentitis is not induced simply via the presence of nocardiform actinomycetes and that route, insufficient duration of exposure and dose may play a role in the development of disease. Additional predispositions may also be involved in the development of nocardioform placentitis.

Molecular cloning and sequencing of equine inhibin α cDNA

Abstract A cDNA probe for recombinant human inhibin α subunit was used to screen an equine testis cDNA library. Approximately 220,000 plaques were screened and 10 positive clones were identified. The largest of these clones, approximately 1 kb, was sequenced. The sequence (993 nucleotides [nt]) contained the ARG‐ARG proteolytic processing site that precedes the N‐terminus of the a subunit mature peptide. The sequence that followed this processing site was 402 nt in length and encoded for a protein of 134 amino acids. Nucleotide homology was 88%, 87%, 88%, 82% and 86% with the a subunit cDNA of cattle, pigs, sheep, mice and humans, respectively. Amino acid homology within the mature peptide region was 99%, 88%, 92%, 87% and 86% with the a subunit of cattle, pigs, sheep, mice and humans, respectively. The equine a subunit peptide contained 6 cysteine residues and one potential N‐glycosylation site (Asn‐Ile‐Ser). The estimated molecular weight of the amino acid sequence for the mature protein was 14.7 kDa. T...

Chorionic gonadotropin (α and β subunit RNA's are present in equine placental membranes by day 30 of pregnancy

Summary Northern analysis was used to investigate timing of transcrip tion of the genes for α and β subunits of chorionic gonadotropin (CG) by equine conceptuses. Two conceptuses were collected on each of days 26, 28, 30 and 32 after ovulation. Conceptus mem branes and embryos were separated and frozen immediately on dry ice. Total RNA was extracted, electrophoretically separated on agarose gels, and RNA was transferred to nitrocellulose membranes by osmotic blotting. Total RNA from equine liver, pituitary, testis and from a day 32 embryo sewed as control. The α and β subunits were both first detected in RNA extracted from day 30 conceptus membranes. The α subunit was detected in placental membrane RNA from both samples at day 30 and in both samples at day 32. The β subunit was detected in RNA in only one sample at day 30 and in both samples at day 32. Both α and β subunits were detected in equine pituitary RNA, but neither was detected in embryo, liver or testis RNA. These results indicate that transcription of the genes for α and β subunits of CG has begun by day 30 of gestation. This is well before the chorionic girdle cells invade the endometrium to form endometrial cups, and earlier than has been reported for presence of the hormone in serum or for in vitro production by girdle cells.

Profiles of strongyle EPG values for Thoroughbred mares on 14 farms in Kentucky (2012-2013)

The main purpose of the research was to determine if three weekly samplings would produce consistent results for low strongyle egg shedders, generally associated with older horses. Strongyle egg counts per gram of feces (EPG) were done for 661 Thoroughbred mares on 14 farms in 2012-2013 once a week for three consecutive weeks. None of the mares had been treated with ivermectin or moxidectin for more than 90 days and with fenbendazole, oxibendazole, or pyrantel pamoate for more than 60 days. Strongyle EPG count data indicated that 38.3% of the mares were positive at first and second sampling, and 37.8%--at third sampling. There were 5.6-63.3% of horses positive per farm. Correlations between EPG count data in all three weeks are quite high (Rs = 0.84, p<0.001). Level of horse infection was found to be dependent on horse age, type of farm and individual characters of horse. This study documented that strongyle egg counts (EPGs) from individual horses did not differ significantly between weeks. Thus, a single strongyle EPG count can be interpreted as indicative of the level of egg shedding for a given horse. These data support the current trend of not deworming all adult horses on a farm, so that high shedders can be identified and treated. This saves horse owners money and reduces development of potential drug resistance of parasites because of less frequent usage of antiparasitic compounds. Information from this study helps to conclude recommendations for parasite control in horses globally.