Clinical, pathologic, and epidemiologic features of nocardioform placentitis in the mare.

Placentitis is the leading cause of infectious abortion in the horse and contributes to roughly 19% of all abortions in the United States. A type of placental infection, nocardioform placentitis (NP) is associated with gram-positive branching actinomycetes localized within the ventral body of the feto-maternal interface to create a lymphoplasmacytic mucoid lesion. While the etiology of this disease is poorly described, this placental infection continues to cause episodic abortions in addition to weak and/or growth retarded neonates. The goal of the present study was to perform a comprehensive analysis of pregnancies associated with a nocardioform-affected placenta and make inferences into the epidemiology of this elusive disease. To do so, 264 mares were enrolled in the study, with 145 as having suspected disease (n = 145; NP) either based on pregnancy-related complications or postpartum placental evaluation, while an additional 119 were enrolled as healthy pregnancies (n = 119; CON). Following diagnosis as either NP or CON based on gross and histopathology at the University of Kentucky Veterinary Diagnostic Laboratory, information was gathered on the mares and neonates for comparisons between diseased and healthy pregnancies. Clinically, a significant portion of diseased mares had clinical indications of NP, including premature mammary gland development, thickening of the placenta noted on transrectal ultrasonography, and separation between the chorioallantois and endometrium noted on abdominal ultrasonography, while vulvar discharge was not commonly noted. Additionally, NP was correlated with increased mare age, decreased gestational length, and decreased neonatal weight, although neonatal IgG and WBC were comparable to CON. Incidence of NP was not correlated with last breeding date, pre- and post-breeding therapeutics, parity, prophylactic medications, or housing. Additionally, NP did not affect postpartum fertility. While NP was associated with a poor neonatal outcome (abortion and/or growth retarded neonate), this did not appear to be influenced by the bacteria isolated (Amycolatopsis spp. vs. Crossiella equi), and mares diagnosed with NP do not appear to be infectious to other pregnant mares nor have repetitive years of the disease. Interestingly, lesion size was positively correlated with last breeding date, as mares bred later in the breeding season correlating with a larger placental lesion. In conclusion, while the etiology of NP continues to elude researchers, the epidemiology of this disease has gained clarity, providing inferences into the management of suspect mares.

Alterations of Circulating Biomarkers During Late Term Pregnancy Complications in the Horse Part I: Cytokines.

Equine abortions are attributed to both infectious and noninfectious causes. Clinical extrapolations are often made from the experimental model for ascending placentitis towards other causes of fetal compromise, including various markers of inflammation, including the cytokines IL-2, 5, IL-6, IL-10, IFNγ, and TNF. It is unknown if these cytokine changes are noted under field conditions, or if they increase preceding other pregnancy related complications. To assess this, Thoroughbred mares (n = 702) had weekly blood obtained beginning in December 2013 and continuing until parturition. Fetal membranes were submitted to the UKVDL for complete gross and pathologic assessment and classified as either ascending placentitis (n = 6), focal mucoid placentitis (n = 6), idiopathic abortion (n = 6) or control (n = 20). Weekly serum samples were analyzed via immunoassay for concentrations of IL-2, IL-5, IL-6, IL-10, IFNγ, and TNF. For both focal mucoid placentitis and ascending placentitis, an increase (P < .05) in the concentrations of IL-2, IL-5, IL-6, IL-10, IFNγ, and TNF was noted preceding parturition in comparison to controls. Cytokine profiles preceding idiopathic abortion did not differ from controls. In conclusion, serum cytokines may be considered potential biomarkers for the prediction of placental infection, while no changes in cytokine profiles were noted when noninfectious causes of abortion occurred. Additionally, this is the first study to report an increase in cytokines during the disease process of focal mucoid placentitis, the etiology of which includes Nocardioform placentitis.

The imbalance of the Th17/Treg axis following equine ascending placental infection.

Ascending placentitis is a leading cause of abortion in the horse, but adaptive immune response to this disease is unknown. To evaluate this, sub-acute placentitis was experimentally-induced via trans-cervical inoculation of S. zooepidemicus, and endometrium and chorioallantois was collected 8 days later (n = 6 inoculated/n = 6 control). The expression of transcripts relating to Th1, Th2, Th17, and Treg maturation was assessed via RNASeq. IHC of transcription factors relating to each subtype in the same tissues (Th1: TBX21, Th2: GATA3, Th17: IRF4, Treg: FOXp3). An immunoassay was utilized to assess circulating cytokines (Th1: IFNg, IL-2; Th2: IL-4, IL-5; Th17: IL-17, IL-6; Treg: IL-10, GM-CSF). An increase in Th1 and Th17-related transcripts were noted in the chorioallantois, although no alterations were seen in the endometrium. Th2 and Treg-related transcripts altered in a dysregulated manner, as some transcripts increased in expression while others decreased. Immunolocalization of Th1, Th2, and Th17 cells was increased in diseased chorioallantois, while no Treg cells were noted in the diseased tissue. Secreted cytokines relating to Th1 (IFNg, IL-2), Th17 (IL-6), Th2 (IL-5), and Treg (IL-10) populations increased in maternal circulation eight days after inoculation. In conclusion, the Th1/Th17 response to ascending placentitis occurs primarily in the chorioallantois, indicating the adaptive immune response to occur in fetal derived placental tissue. Additionally, ascending placentitis leads to an increase in the helper T cell populations (Th1/Th17/Th2) while decreasing the Treg response. This increase in Th17-related responses alongside a diminishing Treg-related response may precede or contribute to fetal demise, abortion, or preterm labor.

Alterations in T cell-related transcripts at the feto-maternal interface throughout equine gestation.

INTRODUCTION: The tolerance of pregnancy by the maternal immune system is balanced between recognition and protection. In the human this is controlled by balancing helper T cell populations (Th1, Th2) in addition to immune suppression from the regulatory arm (Tregs), but this has not been evaluated in the horse. METHODS: RNA sequencing was performed on chorioallantois and endometrium of mares at 120, 180, 300 and 330 days of gestation (n = 4/stage), as well as 45-day chorioallantois (n = 4) and diestrus endometrium (n = 3). Transcripts were selected for relativity to Th1, Th2, or Treg-associated. qPCR and immunohistochemistry were used to confirm the results of select differentially expressed genes. RESULTS: In the endometrium, Th1 transcripts were highest in the diestrus mare and decreased as gestational length progressed. In contrast, Th2 transcripts were upregulated in comparison to the diestrus mare and highest in mid gestation. Treg transcripts were found increased in comparison to the diestrus mare, but decreased prepartum. In the chorioallantois no Th1 transcripts changed. The majority of Th2 transcripts increased from 45 to 300 days gestation, and then decreased prepartum. Treg-related transcripts trended down in the chorioallantois from 45 days to 120 days gestation, followed by an upregulation to 300 days and a secondary decline prepartum. DISCUSSION: The mare experiences a complex and evolving immune profile within the tissues of the feto-maternal interface. This consists of a balance between the Th1 and Th2 response, and a dynamic Treg response that is hypothesized to regulate overall events within the immune system.

The anti-inflammatory effect of exogenous lactoferrin on breeding-induced endometritis when administered post-breeding in susceptible mares.

The deposition of semen into the uterus of the horse induces a transient innate immune response that lasts 24-36 h in the normal mare. There exists a subset of mares that are unable to resolve this inflammation in a timely manner, and are classified as susceptible to the disease of persistent breeding-induced endometritis (PBIE). Lactoferrin is a protein of interest as a potential therapeutic for this persistent inflammation due to its anti-inflammatory and bactericidal properties. The addition of human recombinant lactoferrin (hrLF) to the insemination dose was previously shown to suppress mRNA expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α at 6 h after insemination, but no studies have shown the effect of lactoferrin when infused post-breeding. Therefore, the objectives of this study were to (1) assess the safety of intra-uterine infusion of hrLF, (2) evaluate the effect of intrauterine infusion of hrLF post-breeding as a modulator of the immune response to breeding in the susceptible mare, and (3) determine the most effective concentration of hrLF. For the first experiment four normal mares received an intrauterine infusion of 500 µg/mL hrLF resuspended in 10 mL lactated Ringer's solution (LRS) and heart rate, rectal temperature, respiration, and endometrial quality were evaluated. For the second experiment, six mares classified as susceptible to PBIE were bred during estrus with 500 × 106 progressively motile sperm comprised of the ejaculates from two stallions, which were centrifuged over Androcoll-E to remove seminal plasma. Each insemination dose was resuspended in 30 mL LRS. Six hours after breeding, a 1L LRS uterine lavage was performed prior to treatments. Four treatments were administered over four consecutive estrous cycles in randomized order of: 10 mL LRS (vehicle control), 50 µg/mL hrLF resuspended in 10 mL LRS, 250 µg/mL hrLF resuspended in 10 mL LRS, and 500 µg/mL hrLF resuspended in 10 mL LRS. Twenty-four hours after breeding the mares were evaluated via transrectal ultrasonography for fluid retention. A low volume uterine lavage (250 mL LRS) was performed and the effluent was evaluated for polymorphonuclear neutrophils (PMNs). Finally, an endometrial biopsy was obtained for qPCR analysis of selected inflammatory cytokines. Lactoferrin had no significant overall effect on vital signs or endometrial quality. The addition of hrLF (50 µg/mL, 250 µg/mL, 500 µg/mL) did not significantly affect the amount of fluid detected post-breeding, but suppressed the ratio of PMNs to epithelial cells at all three concentrations compared to controls. In addition, all three concentrations of hrLF increased the mRNA expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN), while the 50 µg/mL dose significantly suppressed mRNA expression of the pro-inflammatory cytokine interferon gamma (IFNγ). In conclusion, the infusion of hrLF post-breeding was found to modulate the inflammatory response to breeding in the mare, and appears to be most effective at the 50 µg/mL concentration.

The effect of select seminal plasma proteins on endometrial mRNA cytokine expression in mares susceptible to persistent mating-induced endometritis.

In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating-induced endometritis PMIE Select seminal plasma proteins cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP-3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six mares classified as susceptible to PMIE were inseminated during four consecutive oestrous cycles with treatments in randomized order of: 1 mg/ml CRISP-3, 150 µg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution (LRS; negative control) to a total volume of 10 ml combined with 1 × 109 spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis of selected genes associated with inflammation (pro-inflammatory cytokines interleukin (IL)-1ß, IL-8, tumour necrosis factor (TNF)-α, interferon (INF)-γ, anti-inflammatory cytokines IL-1RN and IL-10, and inflammatory-modulating cytokine IL-6). Seminal plasma treatment increased the mRNA expression of IL-1ß (p = .019) and IL-8 (p = .0068), while suppressing the mRNA expression of TNF (p = .0013). Lactoferrin also suppressed the mRNA expression of TNF (p = .0013). In conclusion, exogenous lactoferrin may be considered as one modulator of the complex series of events resulting in the poorly regulated pro-inflammatory response seen in susceptible mares.

Expression and localization of cysteine-rich secretory protein-3 (CRISP-3) in the prepubertal and postpubertal male horse.

The seminal plasma protein, cysteine-rich secretory protein-3 (CRISP-3), has been correlated with increased fertility and first-cycle conception rates, and has been suggested to be involved in the modulation of polymorphonuclear neutrophil and phagocytosis of spermatozoa during the inflammatory response to breeding in the horse. Previous research demonstrated that equine CRISP-3 is located in both the ampulla of the vas deferens and the seminal vesicles. However, this was done with nonquantitative laboratory techniques. In humans and rodents, CRISP-3 has been described as an androgen-dependent protein, but the effect of androgens on the expression of CRISP-3 has not been investigated in the horse. The objectives of this study were to (a) confirm and quantify the expression of CRISP-3 in the male equine reproductive tract, (b) describe the localization of CRISP-3 within the specific tissues which express it, and (c) determine if expression of CRISP-3 increases after puberty. We hypothesized that expression of CRISP-3 would be expressed in both the ampulla of the vas deferens and the seminal vesicles, and expression would increase after puberty. Tissues were collected postmortem from three prepubertal colts (<6 months) and six postpubertal stallions (>3 years). Tissue samples were collected from the ampulla of vas deferens, seminal vesicles, bulbourethral gland, prostate gland, testis, as well as the cauda, corpus, and caput aspects of the epididymis. Quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) were performed using an equine-specific CRISP-3 designed primer and monocolonal antibody. A mixed linear additive model was used to compare mRNA expression between age groups, and significance was set to P < 0.05. There was a significant interaction between maturity and tissue type (P < 0.0001). Expression of CRISP-3 mRNA was found primarily in the ampulla of vas deferens with lesser expression in the seminal vesicles. Expression of CRISP-3 was higher in the postpubertal stallion when compared with the prepubertal colt for the ampulla (P < 0.0001) and seminal vesicles (P = 0.0013). IHC showed that equine CRISP-3 is primarily located in the glandular aspects of both the ampulla of vas deferens and the seminal vesicles, with staining concentrated in the cytoplasm of the epithelial cells that surrounded the glands of the mucosa. CRISP-3 was only observed in the postpubertal male horse suggesting that puberty plays a role in the activation of equine CRISP-3 expression.

Reversible downregulation of the hypothalamic-pituitary-gonadal axis in stallions with a novel GnRH antagonist.

The GnRH antagonist, acyline, has not yet been investigated in the stallion. Our study aimed to: (1) evaluate the downregulation of the stallion hypothalamic-pituitary-gonadal axis by acyline through assessment of seminal parameters, testicular volume, and sexual behavior; (2) assess hormonal response of acyline-treated stallions to GnRH stimulation; and (3) verify reversibility after treatment. Stallions were assessed pretreatment and subsequently treated (every five days) for 50 days: acyline (n = 4; 330 µg/kg acyline) or control (n = 4, vehicle). The stallions were then monitored for 62 days after the last day of treatment. Treatment-induced declines (P < 0.05) in FSH, LH, testosterone, and estrone sulfate. Gonadotropins and testosterone returned to control values within 9 days, and estrone sulfate by 14 days, after discontinuation of treatment. Acyline-treated stallions failed to respond with a rise in FSH, LH, and testosterone after exogenous GnRH stimulation (gonadorelin) at Day 46 of treatment compared to pretreatment stimulation and control stallions. Decreases (P < 0.05) were observed in total sperm numbers and motility (week 2) in acyline-treated stallions, as well as total seminal plasma protein (week 2) and testicular volume (week 5). Over the course of the study, the time to erection, time to ejaculation, and number of mounts increased (P < 0.0001) across both groups of stallions; however, there was no effect of treatment or treatment by time interactions on these parameters. Testicular volume, and most seminal parameters regained normal levels within 62 days after treatment ended; on follow-up, sperm output of acyline-treated stallions was regained within 7 months after the end of experiment. In conclusion, acyline reversibly suppresses the stallion hypothalamic-pituitary-gonadal axis.