Studies on Equine Placentitis

Two types of placentitis were studied: ascending and nocardioform placentitis. Although the first diagnosis of nocardioform placentitis was made three decades ago, little is known about the disease, due to the lack of an experimental model. In attempt to develop a research model, Crossiela equi was inoculated through intrauterine, intravenous, intrapharyngeal, and oral routes, but none of the routes resulted in nocardioform placentitis. This may indicate that unidentified factors may play a role in disease pathogenesis and that simple presence of bacteria is not sufficient to induce nocardioform placentitis. The second and major component of this dissertation involved the identification of diagnostic markers for placentitis. Because ascending bacterial placentitis is readily and predictably induced using existing experimental models, this model was used to identify diagnostic markers for placentitis in maternal plasma and fetal fluids. Three potential biomarkers were examined: acute phase inflammatory proteins, steroid hormones produced by the fetoplacental unit, and protein composition of the fetal fluids. Of the three acute phase proteins investigated, serum amyloid A and haptoglobin but not fibrinogen increased in association with experimentally induced ascending placentitis. Androgens and progestins appear to be poor markers for placentitis. Serum estradiol 17β concentrations were reduced in mares with experimentally induced placentitis and appear to be a good marker for placentitis in mares. Different methods were used to study the protein composition of the fetal fluids. Alpha-fetoprotein was characterized as a major protein present in the equine fetal fluids, and this protein was elevated in plasma of mares with placentitis. In another study, using a high-throughput proteomic technique several new proteins were characterized in the amniotic and allantoic fluids of mares carrying normal pregnancies, and several previously uncharacterized proteins were detected in the allantoic fluid of mares with placentitis. Three secreting proteins were elevated in allantoic fluid of mares with experimentally induced ascending placentitis

High-Risk Pregnancy Is Associated With Increased Alpha-Fetoprotein Concentrations in the Amniotic Fluid and Foal Plasma

This study aimed to determine alpha-fetoprotein (AFP) concentrations in amniotic fluid, plasma of mares and respective foals: carrying normal pregnancies and delivering healthy foals (n = 20; Group 1); carrying apparently normal pregnancies and delivering sick foals (n = 15; Group 2); carrying high-risk pregnancies and delivering sick foals (n = 14; Group 3). High-risk pregnancy was defined by a history of premature udder development/lactation or increased of the combined thickness of the uterus and placenta, or vulvar discharge and/or mares' systemic illness. Sick foals were affected by neonatal encephalopathy, sepsis, prematurity/dysmaturity, or hypoxic-ischemic encephalopathy. Based on histological examination of the chorioallantois, AFP trend was analyzed in pregnancies with pathologic (PFM) and normal fetal membranes (NFM). Concentrations of AFP were measured using a commercially available immunoassay previously validated for horses. Mares' plasma AFP did not change during the last 15-20 days of pregnancy in the three groups, and there was no difference among them. Amniotic fluid AFP was higher in Group 3 (P = .014). Foals' plasma AFP concentration was higher from birth to 72hours in foals of Group 2 and 3 than in healthy ones, and foals of Group 3 had the highest value. The strong association (r = 0.84; P < .0001) between AFP in amniotic fluid and foals' plasma at birth is likely due to the presence of AFP in fetal urine. AFP was higher in pregnancy with PFM than with NFM in mare's plasma at admission (P = .031), amniotic fluid (P = .004), foal's plasma at birth (P = .002), at 24 (P = .005) and at 72 hours of life (P = .004). AFP is higher in pregnancy with histopathological lesions of the chorioallantois providing the evidence of the differences between pregnancy with a normal placental barrier and the more compromised ones. The increased AFP concentration in the amniotic fluid and plasma of high-risk foals suggests upregulation

Steroidogenic Enzyme and Steroid Receptor Expression in the Equine Accessory Sex Glands

The expression pattern and distribution of sex steroid receptors and steroidogenic enzymes during development of the equine accessory sex glands has not previously been described. We hypothesized that equine steroidogenic enzyme and sex steroid receptor expression is dependent on reproductive status. Accessory sex glands were harvested from mature stallions, pre-pubertal colts, geldings, and fetuses. Expression of mRNA for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), androgen receptor (AR), 3β-Hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD), P450,17α hydroxylase, 17–20 lyase (CYP17), and aromatase (CYP19) were quantified by RT-PCR, and protein localization of AR, ER-α, ER-β, and 3βHSD were investigated by immunohistochemistry. Expression of AR, ESR2, CYP17, or CYP19 in the ampulla was not different across reproductive statuses (p \u3e 0.1), while expression of ESR1 was higher in the ampulla of geldings and fetuses than those of stallions or colts (p \u3c 0.05). AR, ESR1 and ESR2 expression were decreased in stallion vesicular glands compared to the fetus or gelding, while AR, ESR1, and CYP17 expression were decreased in the bulbourethral glands compared to other glands. ESR1 expression was increased in the prostate compared to the bulbourethral glands, and no differences were seen with CYP19 or 3β-HSD. In conclusion, sex steroid receptors are expressed in all equine male accessory sex glands in all stages of life, while the steroidogenic enzymes were weakly and variably expressed

Dynamic regulation of the transcriptome and proteome of the equine embryo during maternal recognition of pregnancy

During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non-coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤5 mm and the second with higher expression in embryos ≥7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine-tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare

Uterine extracellular vesicles as multi-signal messengers during maternal recognition of pregnancy in the mare

In contrast to other domestic mammals, the embryo-derived signal(s) leading to maternal recognition of pregnancy (MRP) are still unknow in the mare. We hypothesize that these embryonic signals could be packed into uterine extracellular vesicles (uEVs), acting as multi-signal messengers between the conceptus and the maternal tract, and contributing to MRP. To unveil these signals, the RNA and protein cargos of uEVs isolated from uterine lavages collected from pregnant mares (P; day 10, 11, 12 and 13 after ovulation) and cyclic control mares (C; day 10 and 13 after ovulation) were analyzed. Our results showed a fine-tuned regulation of the uEV cargo (RNAs and proteins), by the day of pregnancy, the estrous cycle, and even the size of the embryo. A particular RNA pattern was identified with specific increase on P12 related to immune system and hormonal response. Besides, a set of proteins as well as RNAs was highly enriched in EVs on P12 and P13. Differential abundance of miRNAs was also identified in P13-derived uEVs. Their target genes were linked to down- or upregulated genes in the embryo and the endometrium, exposing their potential origin. Our study identified for first time specific molecules packed in uEVs, which were previously associated to MRP in the mare, and thus bringing added value to the current knowledge. Further integrative and functional analyses will help to confirm the role of these molecules in uEVs during MRP in the mare

Sexual differentiation and primordial germ cell distribution in the early horse fetus

It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.Fil: Scarlet, Dragos. University of Veterinary Medicine Vienna; Suiza. Universitat Zurich; SuizaFil: Handschuh, Stephan. University of Veterinary Medicine Vienna; SuizaFil: Reichart, Ursula. University of Veterinary Medicine Vienna; SuizaFil: Podico, Giorgia. University of Illinois. Urbana - Champaign; Estados UnidosFil: Ellerbrock, Robyn E.. University of Illinois. Urbana - Champaign; Estados UnidosFil: Demyda Peyrás, Sebastián. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Canisso, Igor F.. University of Illinois. Urbana - Champaign; Estados UnidosFil: Walter, Ingrid. University of Veterinary Medicine Vienna; SuizaFil: Aurich, Christine. University of Veterinary Medicine Vienna; Suiz

Milk microbiome and bacterial load following dry cow therapy without antibiotics in dairy cows with healthy mammary gland

Made available in DSpace on 2018-11-26T17:40:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-08-14Department of Veterinary Clinical Medicine, College of Veterinary MedicineUniversity of Illinois at Urbana-ChampaignFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Preventive infusion of antibiotics in the mammary gland of cows consumes 11 tons/year of medically relevant antimicrobials, yet, this practice might not be critical to prevent new infections in the healthy mammary gland of cows. Here, we used next-generation sequencing and quantitative real-time PCR to determine the impact of dry cow therapy without antibiotics on milk microbiome and bacterial load, respectively. Cows diagnosed as negative for mastitis at dry off were randomly allocated to receive antibiotic (intramammary ceftiofur hydrochloride) and teat sealant or just teat sealant. Firmicutes was the most abundant phylum, and Corynebacterium, Acinetobacter, and Staphylococcus, often involved in mastitis cases, were the most abundant genera across treatments and time. However, there were no effects of antimicrobial on milk microbiome and bacterial load. Bacterial load was greater at seven days postpartum than at dry off. Dry cow therapy based on teat sealant without antibiotics can be used with no detrimental impacts on milk microbiome and bacterial load in cows with a healthy mammary gland.Univ Illinois, Dept Vet Clin Med, Champaign, IL 61820 USASao Paulo State Univ, Dept Microbiol & Immunol, Inst Biosci, Botucatu, SP, BrazilCornell Univ, Dept Populat Med & Diagnost Sci, Ithaca, NY USAUniv Liverpool, Dept Epidemiol & Populat Hlth, Inst Infect & Global Hlth, Leahurst, Neston, EnglandSao Paulo State Univ, Dept Microbiol & Immunol, Inst Biosci, Botucatu, SP, BrazilFAPESP: 2015/15208

Equine Arteritis Virus Long-Term Persistence Is Orchestrated by CD8\u3csup\u3e+\u3c/sup\u3e T Lymphocyte Transcription Factors, Inhibitory Receptors, and the CXCL16/CXCR6 Axis

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a “hub” gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract

Intrauterine Blood Plasma Platelet-Therapy Mitigates Persistent Breeding-Induced Endometritis, Reduces Uterine Infections, and Improves Embryo Recovery in Mares

Microorganisms, including pathogenic or opportunistic bacteria and fungi, may gain access to the uterus during breeding, and infectious endometritis plays a major role in equine subfertility. This study aimed to assess the post-breeding inflammatory response, endometrial culture, and embryo recovery of mares susceptible to persistent breeding-induced endometritis (PBIE) treated with plasma-rich (PRP) or -poor (PPP) plasma. Mares (n = 12) susceptible to PBIE had three cycles randomly assigned to receive intrauterine infusions of lactate ringer solution (LRS, control), or autologous PRP or PPP pre- (−48 and −24 h) and post-breeding (6 and 24 h). Mares were bred with fresh semen from one stallion. Intrauterine fluid accumulation (IUF) and endometrial neutrophils were assessed every 24 h up to 96 h post-breeding. Uterine cytokines (Ilβ, IL6, CXCL8, and IL10) were evaluated before (0 h), 6, and 24 h post-breeding, and endometrial culture three and nine days after breed. Embryo flushing was performed 8 days post-ovulation. Data were analyzed with mixed model, Tukey’s post-hoc test, and multivariate regression. PRP treatment reduced endometrial neutrophils, post-breeding IUF, and pro-inflammatory cytokines when compared to control-assigned cycles, but not significantly different than PPP. Controls had a significantly higher percentage of positive bacterial cultures (33%) in comparison to PRP-assigned cycles (0%), whereas cycles treated with PPP were not significantly different from the other groups (25%). The PRP-assigned cycles had significantly greater embryo recovery rates (83%) than the control (33%), though not significantly different than PPP (60%). Plasma infusion reduced the duration and intensity of the post-breeding inflammatory response and improved embryo recovery in mares susceptible to PBIE. Platelets incrementally downregulate PBIE and appear to have a dose-dependent antimicrobial property

Equine arteritis virus long-term persistence is orchestrated by CD8+ T lymphocyte transcription factors, inhibitory receptors, and the CXCL16/CXCR6 axis

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8(+) T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a "hub" gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8(+) T lymphocyte response and unique lymphocyte homing in the reproductive tract