Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces.

Previously, we demonstrated unique insertion/deletion polymorphisms of equine histidine-rich glycoprotein (eHRG) with five genotypes composed of 45-bp or 90-bp deletions in the histidine-rich region of eHRG in Thoroughbred horses. Although leukocytes are typically used to collect DNA for genotyping, blood sampling from animals is sometimes difficult and invasive. Moreover, the method for extracting DNA from blood leukocytes involves complicated steps and must be performed soon after blood sampling for sensitive gene analysis. In the present study, we performed eHRG genotyping using DNA, isolated from oral mucosa swabs collected by rubbing the mucosa on the underside of the upper lip of horses and 100 mg of freshly excreted feces obtained by scraping their surface. In the present study, we performed eHRG genotyping using DNA isolated from oral mucosa swabs and feces of horses (18 Thoroughbreds, 17 mixed breeds, 2 warm bloods), and compared the accuracy of this method with that of the method using DNA from leukocytes. The DNA derived from oral mucosa swabs was sufficient in quantity and quality for eHRG genotyping. However, DNA derived from fecal samples requires a more sensitive detection system because of contamination with non-horse DNA, and the test quality is low. Collection of oral mucosa swabs is less invasive than blood sampling; further, oral swabs can be stored for a longer period in a specified high-quality solution. Therefore, collecting DNA samples from oral mucosa swabs is recommended for the genetic analysis of not only horses but also other animals that are not accustomed to humans.

Unique insertion/deletion polymorphisms within histidine-rich region of histidine-rich glycoprotein in Thoroughbred horses.

Histidine-rich glycoprotein (HRG) is abundant plasma protein with various effects on angiogenesis, coagulation, and immune responses. Previously, we identified the base and amino acid sequences of equine HRG (eHRG) and revealed that eHRG regulates neutrophil functions. In this study, we first conducted a large-scale gene analysis with DNA samples extracted from 1700 Thoroughbred horses and identified unique insertion/deletion polymorphisms in the histidine-rich region (HRR) of eHRG. Here we report two types of polymorphisms (deletion type 1 [D1] and deletion type 2 [D2]) containing either a 45 bp or 90 bp deletion in the HRR of eHRG, and five genotypes of eHRG (insertion/insertion [II], ID1, ID2, D1D1, and D1D2) in Thoroughbred horses. Allele frequency of I, D1, and D2, was 0.483, 0.480, and 0.037 and the incidence of each genotype was II: 23.4%, ID1: 46.2%, ID2: 3.6%, D1D1: 23.1%, and D1D2: 3.7%, respectively. The molecular weights of each plasma eHRG protein collected from horses with each genotype was detected as bands of different molecular size, which corresponded to the estimated amino acid sequence. The nickel-binding affinity of the D1 or D2 deletion eHRG was reduced, indicating a loss of function at the site. eHRG proteins show a variety of biological and immunological activities in vivo, and HRR is its active center, suggesting that genetic polymorphisms in eHRG may be involved in the performance in athletic ability, productivity, and susceptibility to infectious diseases in Thoroughbred horses.

Period of excretion of equine herpesvirus 3 (EHV-3) from a stallion before showing clinical signs of equine coital exanthema and the effect of acyclovir treatment on the duration of EHV-3 excretion.

In 2017, two Thoroughbred stallions, A and B in Farms A and B, respectively, in Hokkaido in Japan showed clinical signs of equine coital exanthema (ECE). In 2020, stallion C in Farm B showed clinical signs of ECE. Eighteen mares were mated within five days before stallion A developed ECE. Ten mares that mated within 3 days before onset showed clinical signs of ECE on the external genitalia. Equine herpesvirus 3 (EHV-3) was isolated from vaginal swabs from three mares that mated within 2 days before onset. Swabs from 12 mares that mated within 4 days before onset were real-time PCR (rPCR)-positive and nine of those mares had an increased EHV-3 antibody titer. The three stallions were administered valaciclovir orally and topical acyclovir ointment was applied. Treatment started on the next day after onset in stallion A and on the day of onset in stallions B and C. EHV-3 was firstly isolated from penis swabs of stallions A and B before treatment and from penis swabs of stallion C 2 days after treatment. EHV-3 was not isolated after 8, 5 and 8 days from onset in stallions A, B and C, respectively. However, swabs were rPCR-positive for at least 12, 9 and 15 days after onset of stallions A, B and C, respectively. EHV-3 was excreted from the stallions at least within 4 days before the onset of ECE, and acyclovir treatment resulted in the termination of excretion within 8 days after onset.

Isolation of an Equine Foamy Virus and Sero-Epidemiology of the Viral Infection in Horses in Japan.

An equine foamy virus (EFV) was isolated for the first time in Japan from peripheral blood mononuclear cells of a broodmare that showed wobbler syndrome after surgery for intestinal volvulus and the isolate was designated as EFVeca_LM. Complete nucleotide sequences of EFVeca_LM were determined. Nucleotide sequence analysis of the long terminal repeat (LTR) region, gag, pol, env, tas, and bel2 genes revealed that EFVeca_LM and the EFV reference strain had 97.2% to 99.1% identities. For a sero-epidemiological survey, indirect immunofluorescent antibody tests were carried out using EFVeca_LM-infected cells as an antigen against 166 sera of horses in five farms collected in 2001 to 2002 and 293 sera of horses in eight farms collected in 2014 to 2016 in Hokkaido, Japan. All of the farms had EFV antibody-positive horses, and average positive rates were 24.6% in sera obtained in 2001 to 2002 and 25.6% in sera obtained in 2014 to 2016 from broodmare farms. The positive rate in a stallion farm (Farm A) in 2002 was 10.7%, and the positive rates in two stallion farms, Farms A and B, in 2015 were 40.9% and 13.3%, respectively. The results suggested that EFV infection is maintained widely in horses in Japan.

Evaluation of the chemiluminescent enzyme immunoassay system for the measurement of testosterone in the serum and whole blood of stallions.

Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs.

Occurrence of equine coital exanthema (ECE) in stallions in Japan and effectiveness of treatment with valacyclovir for ECE.

Equine coital exanthema (ECE) has been reported in many countries, but equine herpesvirus 3 (EHV-3) has been isolated only once in Japan. In 2015, symptoms of ECE were found, and EHV-3 was isolated in two stallions. Valacyclovir, an anti-herpesvirus agent, was administered orally. The stallions rested from mating for more than two weeks, causing enormous financial losses because of their high fees. This is the first study in which valacyclovir was administered for ECE. Though valacyclovir treatment did not shorten the duration of healing, the affected area did not expand after administration of valacyclovir. Valacyclovir therefore seems to be effective for suppression of EHV-3 infection. Further investigation about the administration protocol might be required.

Isolation of equine herpesvirus 3 (EHV-3) from equine coital exanthema of two stallions and sero-epidemiology of EHV-3 infection in Japan.

In the spring of 2015, two stallions reared in Farms A and B in Hokkaido in Japan showed symptoms of equine coital exanthema. Equine herpesvirus 3 (EHV-3) was isolated from penis swab samples of both stallions, and the isolates from each stallion in Farms A and B were designated as SS-1 and YS-1 strains, respectively. BamHI restriction profiles of SS-1 and Japanese reference strain Iwate-1 were indistinguishable, but the BamHI-A fragment of YS-1 was larger than those of SS-1 and Iwate-1 by 1.9 kbp because of the lack of two BamHI sites. Nucleotide sequence analyses of glycoprotein G (gG), gB, gC and VP13/14 coding regions revealed that SS-1 and YS-1 had 99.77% to 100% identities to each other. These results suggested that the origins of SS-1 and YS-1 were different. For a sero-epidemiological survey, serum neutralizing tests using SS-1 against 319 sera of horses from eight farms in Hokkaido were conducted. Six of the eight farms were EHV-3 antibody-positive, and positive rates ranged from 2.6% to 17.6%. To determine the infection time of four EHV-3 antibody-positive horses, a retrospective study was conducted. Infection time of the four horses was in the breeding season, and re-infection or reactivation of latently infected EHV-3 might have occurred in one horse. However, these four horses had never shown any clinical symptoms. The results suggested that several EHV-3 strains are distributed in Japan and that infection is maintained widely in horses without clinical symptoms.

PATHFAST, a novel chemiluminescent enzyme immunoassay for measuring estradiol in equine whole blood and serum.

A novel chemiluminescent enzyme immunoassay system, PATHFAST, for the measurement of estradiol in horses was evaluated. The concentrations of estradiol in the whole blood and serum of mares were measured using PATHFAST and the estradiol concentrations measured by PATHFAST were compared with those measured by a time-resolved fluoro-immunoassay (FIA). To monitor physiological changes, serum estradiol concentrations in mares were measured using PATHFAST throughout the gestation period. The serum estradiol concentrations correlated highly with those in whole blood samples. The serum concentrations of estradiol measured by PATHFAST also correlated well with FIA. Circulating estradiol increased during mid-gestation and high levels of serum estradiol were maintained in late gestation, followed by an abrupt decline to term. These results demonstrate the utility of PATHFAST in equine clinics as an accurate diagnostic tool for the rapid assay of estradiol within 26 min using unextracted whole blood.

Evidence for a PGF2α auto-amplification system in the endometrium in mares.

In mares, prostaglandin F2α (PGF2α) secreted from the endometrium is a major luteolysin. Some domestic animals have an auto-amplification system in which PGF2α can stimulate its own production. Here, we investigated whether this is also the case in mares. In an in vivo study, mares at the mid-luteal phase (days 6-8 of estrous cycle) were injected i.m. with cloprostenol (250 µg) and blood samples were collected at fixed intervals until 72 h after treatment. Progesterone (P4) concentrations started decreasing 45 min after the injection and continued to decrease up to 24 h (P < 0.05). In turn, 13,14-dihydro-15-keto-PGF2α (PGFM) metabolite started to increase 4h after an injection and continued to increase up to 72 h (P < 0.05). PGF receptor (PTGFR) mRNA expression in the endometrium was significantly higher in the late luteal phase than in the early and regressed luteal phases (P < 0.05). In vitro, PGF2α significantly stimulated (P < 0.05) PGF2α production by endometrial tissues and endometrial epithelial and stromal cells and significantly increased (P < 0.05) the mRNA expression of prostaglandin-endoperoxide synthase-2 (PTGS2), an enzyme involved in PGF2α synthesis in endometrial cell. These findings strongly suggest the existence of an endometrial PGF2α auto-amplification system in mares.

Evaluation of the PATHFAST Chemiluminescent Enzyme Immunoassay for Measuring Progesterone in Whole Blood and Serum of Mares.

Evaluation of a new chemiluminescent enzyme immunoassay, the PATHFAST assay system (PATHFAST), for measurement of circulating progesterone in mares was performed. Five mares at the mid-luteal stage were administrated a single i.m. injection of prostaglandin F2α analog (PGF2α; cloprostenol 250 µg/ml), and then blood samples were collected from the jugular vein at 0, 15, 30 and 45 min, at one-hour intervals until 24 and at 48 hr via a catheter in the jugular vein. To monitor the physiological changes in circulating progesterone in mares after induced luteolysis, concentrations of progesterone in whole blood and serum samples were measured by PATHFAST. In addition, concentrations of progesterone in serum samples measured by PATHFAST were compared with those measured by radioimmunoassay (RIA) and enzyme immunoassay (EIA). Using PATHFAST, the serum concentrations of progesterone in mares correlated highly with those of whole blood samples (r=0.9672, n=88). The serum concentrations of progesterone as measured by PATHFAST correlated well with RIA (r=0.9654, n=88) and EIA (r=0.9323, n=112). An abrupt decline in circulating progesterone in whole blood samples was observed within 2 hr (50%), followed by a gradual decline until 48 hr later. The results for progesterone in whole blood samples correlated highly with those in serum samples, and the declining pattern paralleled that of the serum samples. These results demonstrated that PATHFAST is useful in the equine clinic as an accurate diagnostic tool for rapid assay of progesterone within 26 min, using unextracted whole blood.

Annual Changes in Day-length, Temperature, and Circulating Reproductive Hormones in Thoroughbred Stallions and Geldings.

Changes in follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, immunoreactive(ir)-inhibin, testosterone, estradiol-17ß, and insulin-like growth factor (IGF)-I in Thoroughbred stallions along with changes in prolactin secretion in geldings were studied. The correlations of day-length with changes in the concentrations of these hormones were also studied. Five stallions and thirteen geldings were employed to draw blood samples in monthly basis and radioimmunoassay was performed to measure these hormones. All hormones showed a seasonal pattern, the levels being highest during the breeding season and lowest during the winter months. Most of the hormones were at their highest concentration during the month of April, the mid of spring in northern hemisphere. The concentration of circulating IGF-I also demonstrated seasonality, the peak lying on the month of April. The plasma concentration of prolactin also increased during the breeding season. This phenomenon was similar both in stallions and geldings although geldings had lower concentration than that of stallions. The changes in concentration of prolactin in stallions and geldings correlated more towards the day-length than towards the temperature. These results clearly indicate the seasonality of pituitary and gonadal hormones of Thoroughbred stallions, the activity being highest during the month of April and May of the breeding season.