Using mycobacterium cell wall fraction to decrease equine chorionic gonadotropin after abortion.

BACKGROUND: Equine embryonic loss following the development of endometrial cups delays return to cyclicity due to the production of equine chorionic gonadotropin (eCG). Natural degradation of endometrial cups coincides with an influx of immune cells at 100-120 days of gestation, but therapeutic stimulation of reduced eCG production has been relatively unsuccessful. Recently, we observed an increase in pro-inflammatory cytokine production following the use of the immunostimulant mycobacterium cell wall fraction (MCWF). OBJECTIVES: To evaluate the efficacy of hysteroscopic-guided injection of MCWF on the accelerated decline of eCG secretion. STUDY DESIGN: In vivo experiment. METHODS: Mares were pharmacologically aborted at 40-45 days of gestation, and then divided into groups: MCWF-treated (6 mg MCWF suspended in 20 mL LRS; n = 10) and Control (20 mL LRS; n = 6). Five days after abortion, hysteroscopic-guided injection of endometrial cups was performed, with 1 mL of volume placed into each visible endometrial cup. This was repeated 7 days later. Trans-rectal ultrasonography was performed to monitor ovarian activity, and serum was obtained to assess eCG and cytokine concentrations. RESULTS: Concentrations of eCG decreased in the MCWF-treated group (p < 0.01) with a significant suppression noted as early as 14 days after onset of treatment and remained suppressed for the duration of the study. This coincided with an increase in peripheral IFN-γ (p < 0.01) and IL-1ß (p < 0.01) concentrations. Eight out of ten MCWF-treated mares (80%) developed pre-ovulatory follicles, in comparison to 2/6 controls (33%). A pre-ovulatory follicle was noted 23 ± 4 days after onset of treatment. MAIN LIMITATIONS: No pregnancy data was obtained following treatment. CONCLUSIONS: This is the first report of a treatment for the accelerated reduction of eCG following abortion. Stimulation of this process allowed mares to develop a pre-ovulatory follicle within a month of MCWF treatment onset, granting repeat attempts at breeding within the confines of a single breeding season.

The role of equine seminal plasma derived cysteine rich secretory protein 3 (CRISP3) in the interaction between polymorphonuclear neutrophils (PMNs) and populations of viable or dead spermatozoa, and bacteria.

Breeding-induced endometritis is a physiological reaction to clear the uterus from excess spermatozoa and bacteria after breeding. Cysteine rich secretory protein 3 in seminal plasma (spCRISP3) protects spermatozoa from binding and destruction by uterine PMNs, but it is not clear if this involves all sperm and bacteria, or if it is selective to a sub-population of live sperm. The objective of this report was to determine if spCRISP3 (1) is selective in its suppression of PMN-binding to sperm based on viability of spermatozoa, (2) protects bacteria from binding to PMNs, and (3) to determine the localization pattern of spCRISP3 on viable and dead sperm. Semen was collected from five stallions and each ejaculate was divided into (1) live and (2) snap frozen (dead) sperm. Two distinct sperm populations were confirmed by DNA fragmentation and membrane integrity assays. CRISP3 was purified from pooled seminal plasma, and binding of PMNs (isolated from peripheral blood) to the two sperm populations and E. coli was evaluated with flow cytometry in the presence of spCRISP3. In addition, localization of spCRISP3 on live and dead spermatozoa was determined by immunocytochemistry. Comparisons between treatments were analyzed using a one-way-ANOVA and Bonferroni's comparison test, or Kruskal-Wallis ANOVA if not normally distributed. spCRISP3 significantly suppressed binding of PMNs to live spermatozoa (p < 0.0001) but had no effect on dead sperm or bacteria (p > 0.05). Immunocytochemistry confirmed binding of spCRISP3 to live, but not dead spermatozoa. It was concluded that a selective interaction between spCRISP3 and live spermatozoa may be part of a biological mechanism that allows safe transport of viable spermatozoa to the oviducts, while enabling dead spermatozoa and bacteria to be eliminated in a timely fashion after breeding.

Galectins in Equine Placental Disease.

Galectins are proteins that bind to glycans in targeted cells and function in cell-to-cell signaling throughout the body. Galectins have been found to be involved in various reproductive processes, including placental dysfunction, but this has not been investigated in the horse. Therefore, the objective of this study was to assess alterations in galectin expression of the abnormal placenta in pregnant mares. Next-generation RNA sequencing was performed on the postpartum chorioallantois of two placental pathologies following clinical cases of ascending placentitis (n = 7) and focal mucoid placentitis (n = 4), while chorioallantois from healthy postpartum pregnancies (n = 8; 4 control samples per disease group) served as the control. When evaluating ascending placentitis, both galectin-1 (p < 0.001) and galectin-3BP (p = 0.05) increased in the postpartum chorioallantois associated with disease, while galectin-8 (p < 0.0001) and galectin-12 (p < 0.01) decreased in the diseased chorioallantois in comparison with those in the control. In mares with focal mucoid placentitis, numerous galectins increased in the diseased chorioallantois, and this included galectin-1 (p < 0.01), galectin-3BP (p = 0.03), galectin-9 (p = 0.02), and galectin-12 (p = 0.04), in addition to a trend toward increases in galectin-3 (p = 0.08) and galectin-13 (p = 0.09). In contrast, galectin-8 expression decreased (p = 0.04) in the diseased chorioallantois in comparison with that of the controls. In conclusion, galectins alter in abnormal placentae with variations observed among two forms of placental pathologies. These cytokine-like proteins may further our understanding of placental pathophysiology and warrant attention as potential markers of placental inflammation and dysfunction in the horse.

Tumor necrosis factor signaling during equine placental infection leads to pro-apoptotic and necroptotic outcomes.

Ascending placentitis is the leading cause of abortion in the horse. The pleiotropic cytokine tumor necrosis factor (TNF) is an upstream regulator of this disease, but little is understood regarding its function in pregnancy maintenance or placental infection. To assess this, RNA sequencing was performed on chorioallantois and endometrium of healthy pregnant mares at various gestational lengths (n = 4/gestational age), in addition to postpartum chorioallantois, and diestrus endometrium to assess expression of TNF, TNFR-1, and TNFR-2. Additionally, ascending placentitis was induced via trans-cervical inoculation of S. equi spp. zooepidemicus in pregnant mares (n = 6 infected / n = 6 control) and tissues and serum were collected to evaluate TNF-related transcripts. IHC was performed to confirm protein localization of TNFR-1 and TNFR-2. In healthy pregnancy, TNFR-1 appears to be the predominant TNF-related receptor. Following induction of disease, TNF concentrations increased in maternal serum, but expression did not alter at the tissue level. While both TNFR-1 and TNFR-2 increased following induction of disease, alterations in downstream pathways indicate that TNFR-1 is the dominant receptor in ascending placentitis, and is primarily activated within the chorioallantois, with minimal signaling occurring within the endometrium. In conclusion, TNF appears to be involved in the pathophysiology of ascending placentitis. An increase in this cytokine during disease progression is believed to activate TNFR-1 within the chorioallantois, leading to various pro-apoptotic and necroptotic outcomes, all of which may signal for fetal demise and impending abortion.

Development and Use of an Enzyme-Linked Immunosorbent Assay to Determine Temporal Exposure Patterns to Putative Agents of Nocardioform Placentitis.

Cases of nocardioform placentitis are characterized by focal, mucoid placentitis resulting in late-term abortion, premature birth, or small, full-term foals, occur sporadically, and are most commonly associated with Crossiella equi and Amycolatopsis spp. infection. The goal of this project was to develop an enzyme-linked immunosorbent assay (ELISA) for quantifying antibodies against Crossiella equi and Amycolatopsis spp. and utilize the ELISA to determine when exposure occurs. Serum samples collected during the 2020 foaling season from Crossiella equi (n = 8) and Amycolatopsis spp. (n = 32) infected mares, as well as nonaffected mares (n = 51 mares), were used to develop and optimize bacteria-specific ELISAs. Following development of the ELISAs, banked serum samples from a single, central Kentucky Thoroughbred farm collected during 2012 to 2013 (n = 104 mares) and 2013-14 (n = 82 mares) were analyzed. Differences in various groups were analyzed using one-way analysis of variance (ANOVA). Crossiella equi-infected mares had significantly higher ELISA unit (EU) values on the Crossiella equi ELISA near parturition when compared to the other two groups (P < .001). Using the Amycolatopsis spp. ELISA, EU values were not significantly different between Amycolatopsis spp. infected and non-affected mares, suggesting this ELISA is not specific for Amycolatopsis spp. During 2013 to 2014, there were significant increases in EU values between June and late September for the Crossiella equi ELISA, suggesting exposure in the summer and early fall months. Data from the Crossiella equi ELISA may help provide a better understanding of the epidemiology of nocardioform placentitis, guide the development of a successful experimental challenge model, and allow for further refinement of these ELISAs.

Binding of Equine Seminal Lactoferrin/Superoxide Dismutase (SOD-3) Complex Is Biased towards Dead Spermatozoa.

Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To evaluate this, we attempted to purify equine seminal LF from five stallions (n = 5), biotinylate LF, and evaluate potential binding site(s) on spermatozoa. LF was consistently associated with superoxide dismutase (SOD-3), and all attempts to separate the two proteins were unsuccessful. Flow cytometric and microscopic analyses were used to compare LF/SOD-3 binding to viable and nonviable spermatozoa. Additionally, various methods of biotinylation were assessed to optimize this methodology. Biotinylation of seminal plasma protein was an effective and efficient method to study seminal plasma protein properties, and the binding site for LF/SOD-3 was found to be broadly localized to the entire sperm cell surface as well as selective towards nonviable/defective sperm. Although we were not able to determine if the binding to equine spermatozoa was through LF or SOD-3, we can conclude that equine seminal LF is tightly bound to SOD-3 and this protein complex binds selectively to nonviable spermatozoa, possibly to mark them for elimination by neutrophil phagocytosis.

Galectinology of Equine Pregnancy.

Galectins are a family of proteins that bind to glycans, acting in a cytokine-like manner throughout the body. In the majority of mammalians, galectins have been found to be involved in pregnancy maintenance, but few studies have evaluated this in the horse. Therefore, the objective of this study was to examine the expression of various galectins in pregnant and nonpregnant mares. Next-generation RNA sequencing was performed on the chorioallantois and endometrium of healthy pregnant mares at 120, 180, 300, and 330 days of gestation (n = 4/stage), as well as 45-day chorioallantois (n = 4), postpartum chorioallantois (n = 3), and diestrus endometrium (n = 3). In the endometrium, galectin-1 and galectin-13 were found in the highest expression in the nonpregnant mare, with decreasing levels of expression noted throughout gestation. In contrast, galectin-8 and galectin-12 were found to be the lowest in the nonpregnant mare and reached the highest expression levels in mid-gestation before declining as parturition neared. In the chorioallantois, galectin-1, galectin-3, and galectin-3BP were found to have heightened expression levels at 45 d of gestation, with lesser expression levels noted throughout gestation. In contrast, galectin-9, galectin-12, and galectin-13 experienced the highest expression levels in the late-term chorioallantois (300 d/330 d), with lesser expression noted in early- to mid-gestation. Of note, galectin-1, galectin-3BP, galectin-9, galectin-12, and galectin-13 all experienced the lowest expression levels in the postpartum placenta, with heightened expression noted during gestation. In conclusion, galectins appear to be involved in equine pregnancy, and this is dependent on both the tissue within the feto-maternal interface and the specific galectin involved.

Transcriptomic analysis of equine chorioallantois reveals immune networks and molecular mechanisms involved in nocardioform placentitis.

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. The CA were collected from mares with confirmed NP based upon histopathology, microbiological culture and PCR for Amycolatopsis spp. Samples were collected from the margin of the NP lesion (NPL, n = 4) and grossly normal region (NPN, n = 4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n = 4). Transcriptome analysis identified 2892 differentially expressed genes (DEGs) in NPL vs. CRL and 2450 DEGs in NPL vs. NPN. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways are involved in NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Alterations of Circulating Biomarkers During Late Term Pregnancy Complications in the Horse Part II: Steroid Hormones and Alpha-Fetoprotein.

Preterm labor and/or abortion causes considerable economic impact on the equine industry. Unfortunately, few experimental models exist for the induction of various pregnancy-related complications, and therefore extrapolations are made from the experimental model for ascending placentits, although inferences may be minimal. Certain steroid hormones (progestogens, estrogens) and fetal proteins (alpha-fetoprotein; AFP) might improve the diagnostics for abnormal pregnancy, but the utility of these markers in the field is unknown. To assess this, thoroughbred mares (n = 702) were bled weekly beginning in December 2013 until parturition/abortion. Following parturition, fetal membranes were assessed histopathologically and classified as either ascending placentitis (n = 6), focal mucoid placentitis (n = 6), idiopathic abortion (n = 6) or no disease (n = 20). Weekly serum samples were analyzed for concentrations of progesterone, estradiol-17ß, and AFP. Samples were analyzed retrospectively from the week of parturition/abortion in addition to the preceding four weeks. For both ascending and focal mucoid placentitis, a significant increase in progesterone and AFP was noted, alongside a significant decrease in estradiol-17ß and the ratio of estradiol-17ß to progesterone in comparison to controls. In contrast, idiopathic abortions experienced a decrease in progesterone concentrations alongside an increase in AFP, and this was only noted in the week preceding parturition/abortion. In conclusion, spontaneous placental infection in the horse altered both endocrine and feto-secretory markers in maternal circulation, while minimal changes were noted preceding noninfectious idiopathic abortion. Additionally, this is the first study to report an alteration in steroid hormones and AFP during the disease process of focal mucoid placentitis, the etiology of which includes Nocardioform placentitis.

Interleukin-6 pathobiology in equine placental infection.

PROBLEM: Ascending placentitis is the leading cause of abortion in the horse. Interleukin (IL)-6 is considered predictive of placental infection in other species, but little is understood regarding its role in the pathophysiology of ascending placentitis. METHOD OF STUDY: Sub-acute ascending placentitis was induced via trans-cervical inoculation of S zooepidemicus, and various fluids/serum/tissues collected 8 days later. Concentrations of IL-6 were detected within fetal fluids and serum in inoculated (n = 6) and control (n = 6) mares. RNASeq was performed on the placenta (endometrium and chorioallantois) to assess transcripts relating to IL-6 pathways. IHC was performed for immunolocalization of IL-6 receptor (IL-6R) in the placenta. RESULTS: IL-6 concentrations increased in allantoic fluid following inoculation, with a trend toward an increase in amniotic fluid. Maternal serum IL-6 was increased in inoculated animals, while no changes were noted in fetal serum. mRNA expression of IL-6-related transcripts within the chorioallantois indicates that IL-6 is activating the classical JAK/STAT pathway, thereby acting as anti-inflammatory, anti-apoptotic, and pro-survival. The IL-6R was expressed within the chorioallantois, indicating a paracrine signaling pathway of maternal IL-6 to fetal IL-6R. CONCLUSION: IL-6 plays a crucial role in the placental response to induction of sub-acute equine ascending placentitis, and this could be noted in amniotic fluid, allantoic fluid, and maternal serum. Additionally, IL-6 is acting as anti-inflammatory in this disease, potentially altering disease progression, impeding abortion signals, and assisting with the production of a viable neonate.

The Effect of Mycobacterium Cell Wall Fraction on Histologic, Immunologic, and Clinical Parameters of Postpartum Involution in the Mare.

Maintaining yearly foal production is important for the economic success of the broodmare, and this requires breeding to occur as quickly postpartum as possible. The initial postpartum estrus occurs within 5-20 days postpartum, whereas the uterus is still undergoing repair from tissue alterations during pregnancy and parturition, a process known as involution. Attempts have been made to hasten this process, but with minimal success. Mycobacterium cell wall fraction (MCWF) is an immunomodulator that has been shown to reduce bacterial growth and alter aspects of the immune response to breeding, but it is unknown if MCWF hastens the process of involution. Therefore, the objectives of this study were to (1) investigate the effect of MCWF on tissue remodeling, (2) assess the effect of MCWF on the local immune system of the uterus, and (3) determine the optimal treatment interval needed for these processes to occur. We hypothesize that repeated treatments of MCWF postpartum will hasten the process of involution. To study this, 16 pregnant mares of mixed breeds were evaluated postpartum. Control mares (n = 4) received 1.5 mL lactated Ringer's solution intravenously on Day 1 (Day 0 = day of parturition) postpartum and again on Day 7, whereas treated mares either received 1.5 mL Settle intravenously on Day 1 and Day 7 (TX1; n = 6) or 1.5 mL Settle intravenously on Day 1 and then every 3 days until ovulation was detected (TX2; n = 6) and then evaluated until 15 days postpartum. Mares were assessed every 3 days for clinical, immunologic, and histologic parameters. Clinical parameters were assessed with transrectal ultrasonography and included ovarian activity, uterine fluid retention, and measurement of the uterine diameter, in addition to endometrial culture. Immunologic parameters included endometrial biopsies for quantitative polymerase chain reaction for expression of various cytokines (interleukin [IL]-1ß, IL-1RN, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor [TNF], interferon [IFN]-γ, and granulocyte-macrophage colony-stimulating factor) in addition to endometrial cytology. Formalin-fixed endometrial biopsies were histologically assessed for the retention of microcaruncles, dilation of endometrial glands, and inflammation of the mucosa, stratum compactum, and spongiosum. Statistics were performed using SAS 9.4, using a mixed model for repeated measures with mare and treatment as a random effect. All post-hoc analysis was done using a Tukey's honestly significant difference test. Involution was considered complete by Day 15 postpartum in all mares, and the day postpartum had a significant effect on almost all parameters investigated, indicating the immunologic process of involution. Treatment with MCWF decreased the magnitude of bacterial growth in addition to time to negative culture. In addition, MCWF increased the expression of IL-1ß, IFNγ, and TNF. Although minimal treatment effect was noted histologically, a decrease in mucosal inflammation was seen in MCWF-treated mares. In conclusion, involution appears to be influenced by the immune system. In addition, MCWF appears to have a bactericidal effect on the postpartum mare, and this may be because of an increase in proinflammatory cytokines. It is unknown if this bactericidal property will improve fertility on the first estrous cycle postpartum, and future studies are needed to determine this.

Persistent Breeding-Induced Endometritis in Mares - a Multifaceted Challenge: From Clinical Aspects to Immunopathogenesis and Pathobiology.

Post-breeding endometritis (i.e., inflammation/infection of the endometrium), is a physiological reaction taking place in the endometrium of mares within 48 hours post-breeding, aimed to clear seminal plasma, excess sperm, microorganisms, and debris from the uterine lumen in preparation for the arrival of an embryo. Mares are classified as susceptible or resistant to persistent breeding-induced endometritis (PBIE) based on their ability to clear this inflammation/infection by 48 hours post-breeding. Mares susceptible to PBIE, or those with difficulty clearing infection/inflammation, have a deficient immune response and compromised physical mechanisms of defense against infection. Molecular pathways of the innate immune response known to be involved in PBIE are discussed herein. The role of the adaptive uterine immune response on PBIE remains to be elucidated in horses. Advances in the pathobiology of microbes involved in PBIE are also revised here. Traditional and non-traditional therapeutic modalities for endometritis are contrasted and described in the context of clinical and molecular aspects. In recent years, the lack of efficacy of traditional therapeutic modalities, alongside the ever-increasing incidence of antibiotic-resistant microorganisms, has enforced the development of non-traditional therapies. Novel biological products capable of modulating the endometrial inflammatory response are also discussed here as part of the non-traditional therapies for endometritis.

The feto-maternal immune response to equine placentitis.

PROBLEM: Ascending placentitis is one of the leading causes of abortion in the horse. Minimal work has focused on its effect on fetal fluids or the antenatal immune response of the fetus. METHODOLOGY: Placentitis was induced via transcervical inoculation of Streptococcus equi ssp Zooepidemicus, and fluids/serum/tissues were collected 4-6 days later following euthanasia. Cytokine concentrations were detected using a multiplex immunoassay within fetal fluids (amniotic and allantoic) and serum (maternal and fetal) in inoculated and control mares. In addition, tissues from fetal (spleen, liver, lung, umbilicus, amnioallantois) and maternal (spleen, liver, lung, chorioallantois, endometrium) origin were analyzed in inoculated and control mares utilizing qPCR for expression of cytokines. RESULTS: No difference in cytokine concentrations in maternal or fetal serum was noted between inoculated and control mares. Concentrations of IL-1ß, IL-6, IL-10, and GRO were upregulated in the amniotic fluid following inoculation, with a trend toward higher IL-6 concentration in allantoic fluid. The amnioallantoic tissue separating the two fluids had higher expression of IL-1ß and IL-6 following inoculation, while chorioallantois and endometrium upregulated IL-1ß and IL-8 expression. IL-1ß was upregulated in the maternal spleen following inoculation. Fetal spleens were upregulated in expression of IL-1ß, GRO, and IL-6, while IL-6 was higher in fetal liver after inoculation than in controls. CONCLUSION: The maternal response to placentitis is primarily pro-inflammatory while the fetus appears to play a regulatory role in this inflammation. Additionally, amniotic fluid sampling may be more diagnostic of ascending placentitis than circulating cytokines.

New Treatment for Urethral Rent in Stallions.

The aim of this report is to describe a new methodology to successfully treat stallions diagnosed with urethral rent. Four stallions of ages ranging from 7 to 12 years (median 9) with hemospermia were admitted for clinical evaluation, breeding soundness examination, and urethroscopy for inspection of the urethra and vesicular glands. Once the presence of urethral rent was identified and/or other sources of hemorrhage were excluded, a topical treatment was performed with 4% Policresulen solution (Albocresil). The treatment was carried out by infusing 100 mL of the solution into the lumen of the urethra through a catheter placed up to the region of the ischial arch. This procedure was repeated once daily, or at 48 hours intervals, resulting in a total of 4-7 infusions. In all cases, chemical cauterization was efficient in the healing of the urethral rent. However, due to masturbation during treatment, one animal did not completely heal, and the treatment with the Policresulen was prolonged. It is believed that the low pH of the solution resulted in urethritis, which was treated with systemic therapy of antibiotic and anti-inflammatory nonsteroidal. Topical treatment with 4% Policresulen was found to be efficient in the chemical cauterization of urethral rent in stallions. This treatment was efficient, practical, less invasive, and less costly than the alternative of surgical methods, which are more invasive and require longer recovery time of the animal. However, sexual rest and the elimination of sexual stimuli from the environment are essential management in association with this therapeutic method.